A New Approach of the Academic Research in Biology

Book of abstracts

Editors: Octavian Popescu, Mdlin Enache, Medana Zamfir, Sorin tefnu Text set: Sorin tefnu Cover: Lucian Apetre Scientific Committee: Prof. Dr. Octavian POPESCU Director of Institute of Biology Bucharest; Dr. Medana ZAMFIR Scientific Secretary of Institute of Biology Bucharest; Dr. Nicolae MIRANCEA Head of Plant and Animal Cytobiology Department; Dr. Sorin TEFNU Head of Ecology, Taxonomy and Nature Conservation Department; Dr. Mdlin ENACHE Institute of Biology Bucharest.

EDITURA ARS DOCENDI UNIVERSITATEA DIN BUCURETI Editur cu profil academic i cultural, recunoscut de CONSILIUL NAIONAL AL CERCETRII TIINIFICE DIN NVMNTUL SUPERIOR os. Panduri 90, sector 5, Bucureti Tel./Fax: +4 021 410 25 75 www.arsdocendi.ro office@arsdocendi.ro

Descrierea CIP a Bibliotecii Naionale a Romniei 50 YEARS OF ACADEMIC RESEARCH IN BIOLOGY. Conference ( 2010 ; Bucureti) 50 years of academic research in biology : the anniversary conference of the Institute of Biology : Bucharest, 2010 : book of abstracts / Institute of Biology Bucharest. - Bucureti : Ars Docendi, 2010 ISBN I. Institutul de Biologie (Bucureti) 061.62(498 Buc.):57

Copyright Institute of Biology Bucharest, 2012 Printed in Romania, Institute of Biology Bucharest

Institute of Biology Bucharest

A New Approach of the Academic Research in Biology

Book of abstracts

ARS DOCENDI

2012

UNIVERSITY OF BUCHAREST

CONTENTS
THE PROJECT - IBB INFRASTRUCTURE DEVELOPMENT FOR ENHANCING THE BIODIVERSITY INVESTIGATION CAPACITY, IN ACCORDANCE WITH GLOBAL CLIMATE CHANGES - DIBIOCLIM 11

INVITED LECTURES AND ORAL PRESENTATIONS

WHATS IN A NAME? THOUGHTS ABOUT SPECIES CONCEPTS, TAXONOMY AND NOMENCLATURE OF PROKARYOTES
A. OREN

17

UNRAVELING OF THE COCOA BEAN FERMENTATION PROCESS OPENS NEW PERSPECTIVES FOR CHOCOLATE PRODUCTION
LUC DE VUYST

18

SPECIES AND SPECIATION

O. POPESCU

22 23

PLANT CONSERVATION TOWARD SOCIETY

D. DIMITROVA, T. IVANOVA, Y. BOSSEVA, CH. GUSSEV, S. TSONEVA, V. GEORGIEV and T. STOEVA

PLANT BIOTECHNOLOGY DEVELOPMENT IN THE INSTITUTE OF BIODIVERSITY AND ECOSYSTEM RESEARCH, BAS FACILITIES, ACHIEVEMENTS, AND CHALLENGES
M. STANILOVA

25

CLONAL PROPAGATION AND GENETIC IMPROVEMENT OF SELECTED SMALL FRUIT SPECIES THROUGH BIOTECHNOLOGY APPROACHES
A. GAJDOOV, G. LIBIAKOV and M. LATEKOV

28

NUCLEOTYPIC EFFECTS IN VITRO CONDITION OF DIFFERENT CABBAGE GENOTYPES INFECTED WITH CAULIFLOWER MOSAIC VIRUS
L. ANDRONIC

30

MISSING-LINK CARBAMOYL PHOSPHATE SYNTHETASE

E. POPA, N. PERERA, C.Z. KIBDI-SZAB, H. GUY-EVANS, D.R. EVANS, C PURCAREA

33

HALOARCHAEA FROM SOME ROMANIAN MAN-MADE AND NATURAL HYPERSALINE ENVIRONMENTS

G. TEODOSIU and M. ENACHE

34

MICROBIAL DIVERSITY IN THE SUBTERRANEAN ICE DEPOSITS OF SCRIOARA CAVE

A.M. HILLEBRAND-VOICULESCU, C. ICU, A. RUSU, I. ARDELEAN, D. PASCU, A. PEROIU, T. BRAD, E. POPA, B.P. ONAC and C. PURCREA

37

DYNAMICS OF MICROBIOTA IN MARINE MICROCOSMS (INDOOR): RESULTS AND PERSPECTIVES

I.I. ARDELEAN and S. GHI

38

THE GROWTH OF Ulocladium chartarum IN MICROGRAVITY: ON RANDOM POSITIONING MACHINE AND ON BOARD OF INTERNATIONAL SPACE STATION
GOMOIU, S. VADRUCCI, I. WALTHER and E. CHATZITHEODORIDIS

39

DIVERSITY OF ARCHAEAL COMMUNITY IN THE CHEMOCLINE OF A HYPERSALINE LAKE IN THE TRANSYLVANIAN BASIN (ROMANIA)
.-A. ANDREI, A. BARICZ, V. MUNTEAN, C. COMAN, Z. G. KERESZTES, M. ALEXE and H. BANCIU

40

STATINS AS CANDIDATES FOR TUMOR-TARGETED NANOMEDICINES

M.C. ALUPEI, E. LICARETE, F.B. CRISTIAN and M. BANCIU

42

EX SITU APPROACH TO CONSERVATION OF LILIACEAE IN BULGARIA

T. IVANOVA, Y. BOSSEVA, CH. GUSSEV, S. TSONEVA, V. GEORGIEV, G. ANGELOV, D. DIMITROVA and T. STOEVA

44

PLANT IN VITRO SYSTEMS FOR EX SITU CONSERVATION AND BIOTECHNOLOGICAL APPLICATIONS

I. HOLOBIUC, A. AIFTIMIE-PUNESCU, C. BANCIU, R. CATANA, R. MIHAI, M.E. MITOI, C. VOICHITA, F. ALDEA, D. CRISTIAN, F.E. HELEPCIUC, I. ISTRATE and G. COGLNICEANU

46

TELOCYTES INFRASTRUCTURE AND FUNCTIONAL ROLE

N. MIRANCEA

49 51

INVESTIGATING THE IMPACT OF CYANOBACTERIAL BLOOMS ON AQUATIC ECOSYSTEMS OF THE DANUBE DELTA
P. SPAAK and C. SANDU

CONTRIBUTIONS TO THE KNOWLEDGE OF LITTER DECOMPOSITION RATES OF PLANT LITTERS OF Acer pseudoplantanus, Fraxinus excelsior AND Quercus robur IN FOREST ECOSYSTEMS UNDER THE INFLUENCE OF ANTHROPOGENIC PRESSURE
A. JERCA , E. PREDA and A. VDINEANU

54

BIODIVERSITY DATA TRANSFERRED IN ECOSYSTEM SERVICES STUDIES

M. ONETE, D.C. NICOLAE, M. MINODORA, V. HONCIUC and D. PURICE

55

PRELIMINARY ASSESSMENT ON THE EFFECT OF URBANIZATION ON ECTOMYCORRHIZAL DIVERSITY

D. MOGLDEA

56

CITES AND NATURE CONSERVATION IN REPUBLIC OF KENYA

S. MIHILESCU

57 59

PROJECT SOP ENVIRONMENT: CONSERVATION STATUS MONITORING OF SPECIES AND HABITATS FROM ROMANIA UNDER THE 17 ARTICLE OF THE HABITATS DIRECTIVE 2011-2012
S. MIHILESCU, M. ION, I. GHEORGHE and I. CRISTEA

TURBOVEG DATABASE USED IN BUCEGI MASSIF STUDIES

C.D. NICOLAE

61

ECOLOGY, TAXONOMY AND NATURE CONSERVATION POSTERS

LONG-TERM ECOLOGICAL RESEARCH OF ZOOPLANKTON COMMUNITY IN LAKE ROU (DANUBE DELTA)
L. PARPAL

65

SPATIAL DISTRIBUTION OF THE ROTIFERS DIVERSITY IN SFNTU GHEORGHE BRANCH

L. FLORESCU

67

LINKING THE STRUCTURE OF THE WEED-BED FAUNA COMMUNITIES AND THE ENVIRONMENTAL VARIABLES IN SFNTU GHEORGHE ARM OF THE RIVER DANUBE
E. RADU, A. COMAN and G. RNOVEANU

69

LONG-TERM DYNAMICS (1993-2011) OF THE BENTHIC MACROINVERTEBRATES COMMUNITIES IN THE INNER DANUBE DELTA
C.C. BRSAN, G. RNOVEANU, E. RADU, G. IGNAT and S. CRISTOFOR

71

THE DYNAMICS OF CYANOBACTERIAL BLOOMS IN LAKE ROU REFLECTED IN LONG-TERM STUDIES

M. MOLDOVEANU

73

THE DYNAMICS OF EXTRACELLULAR - GLUCOSIDASE AND ALKALINE PHOSPHATASE IN AQUATIC ECOSYSTEMS FROM THE DANUBE DELTA IN 2011-2012 INTERVAL
I. PCEIL

74

STRUCTURAL-FUNCTIONAL PARTICULARITIES OF THE ECOSYSTEMS OF A PLAIN CATCHMENT AREA (CASE STUDY)

O. CIOBOIU and G. BREZEANU

76

MODERN METHODS USED IN TAXONOMY AND ECOLOGY OF SOIL FAUNA

M. MANU

78

METHODS USED IN THE ECOLOGICAL RESEARCH OF SOIL MITES (ARACHNIDA - ACARI) HISTORY AND PERSPECTIVES
V. HONCIUC

79

THE COMMUNITIES STRUCTURE OF INVERTEBRATES FAUNA FROM RAPE AND ALFALFA CROPS
C. FIERA, S. MAICAN and D. PURICE

80

THE EPIGEIC INVERTEBRATES IN THE ECOLOGICAL STUDIES IN ROMANIA. METHODS OLD, CURRENT AND PERSPECTIVES
D. PURICE

82

THE ECOLOGICAL SUCCESSION IN TERMS OF THE TROPHIC STRUCTURES IN FOREST PLANTATIONS

D. PURICE and O. CIOBOIU

83

SPECIFIC DIVERSITY OF ENTOMOFAUNA (COLLEMBOLA, COLEOPTERA) FROM SRATA - MONTEORU FOREST (BUZU COUNTY)
S. MAICAN and C. FIERA

84

BIOGEOGRAPHYCAL DATA ON MOLLUSKS SPECIES IN DOBROGEA, ROMANIA

M. AXINI and FAWAZ AL-AZKI

85

THE GRAYLING (Thyallus thymallus L., 1758), A DECLINING SPECIES

I. CRISTEA

87 88

BIRD RINGING CAMP FROM LETEA, DANUBE DELTA BIOSPHERE IMPORTANT SITE FOR THE STUDY OF BIRDS MIGRATION
I. COBZARU, G. CHIAMERA and V. POCORA

THREATENED BRYOPHYTES IN ROMANIA

S. TEFNU

89 90

FIRST RECORD OF Draba dorneri HEUFF. (BRASSICACEAE) IN PARNG MOUNTAINS (ROMANIAN CARPATHIANS)
R. ION

BIODIVERSITY OF THE FUTURE IN MILLENIUM SEED BANK

M. ONETE

91 92

NON-NATIVE VASCULAR AQUATIC MACROPHYTES IN BULGARIA

V. GEORGIEV and S. TSONEVA

CONSERVATIONALLY IMPORTANT VASCULAR AQUATIC MACROPHYTES IN BULGARIA

S. TSONEVA and V. GEORGIEV

93

NATURA 2000 SITES IN THE LOWER OLT HYDROGRAPHIC BASIN KEY SPECIES AND HABITATS FOR THE INTEGRATED MANAGEMENT OF THE AREA
S. MIHILESCU, M. ION and I. COBZARU

95

THE ESTIMATION OF COMMUNITY INTEREST MAMMALS IN ROMANIA

O. IONESCU, M. ADAMESCU, C. CAZACU, G. IONESCU, A. COTOVELEA, M. FEDORCA, R. JURJ, M. POPA and A. GRIDAN

97

MICROBIOLOGY POSTERS
THE SELECTION OF THE ACIDOPHILIC BACTERIA BELONGING TO THE Acidiphilium GENUS WITH HIGHER ENZYMATIC ACTIVITY IN THE PRESENCE OF HEAVY METAL IONS
C.M. CISMASIU

103

LACTIC ACID BACTERIA FROM FERMENTED VEGETABLES WITH POTENTIAL APPLICATIONS IN FOOD INDUSTRY AND HEALTH
S.S. GROSU-TUDOR, M. ZAMFIR, D. WOUTERS and L. DE VUYST

104

STUDIES CONCERNING THE MORPHOLOGICAL AND FUNCTIONAL VARIATIONS OF SOME BACTERIAL CELLS UNDER CONTAMINATION STRESS IMPACT
M. TEFNESCU and D.M. CRSTEA

106

ORGANIC SOLVENT RESISTANCE MECHANISMS IN Vibrio alginolyticus IBBCT2

M.M. STANCU

108

HALOPHILIC BACTERIA FROM DIFFERENT HYPERSALINE HABITATS IN SLANIC PRAHOVA, ROMANIA AND THEIR BIOTECHNOLOGICAL POTENTIAL
R. COJOC, S. NEAGU, G. TEODOSIU and M. ENACHE

110

METALIC NANOPARTICLES CITOTOXICITY AGAINST PHOTOTROPHIC AND HETEROTROPHIC BACTERIA

I.I. ARDELEAN, M. CRNU , D. PASCU, I. SARCHIZIAN , V. DAMIAN, I. APOSTOL , I. IORDACHE and D. APOSTOL

112

STRUCTURE-FUNCTION INVESTIGATION OF Methanobrevibacter smithii FULL-LENGTH CARBAMOYL PHOSPHATE SYNTHETASE

E. POPA, N. PERERA, H. GUY-EVANS, D.R. EVANS and C. PURCREA

113

CELL GROWTH AND DGGE ANALYSIS OF MICROORGANISMS FROM SCRIOARA CAVE ICE SEDIMENTS
C. ICU, A.M. HILLEBRAND-VOICULESCU, D. PASCU, A. PEROIU, T. BRAD and C. PURCREA

114

PLANT AND ANIMAL CYTOBIOLOGY, BIOTECHNOLOGY POSTERS

FIBROLAMELLAR LIVER CARCINOMA LIKE TUMOR IN AN EXPERIMENTALLY IRON LOADED MOUSE
P. PRUNESCU and C.C. PRUNESCU

117

LONG-TERM CULTURES IN MINIMAL GROWTH CONDITIONS IN Dianthus trifasciculatus KIT SSP. parviflorus AS EX SITU PRESERVATION TOOL
I. HOLOBIUC, C. VOICHITA, M.E. MITOI and F.E. HELEPCIUC

119

GREEN SYNTHESIS OF SILVER NANOPARTICLES USING PLANT CELL CULTURES

G. COGLNICEANU, M.E. MITOI and G. HRISTEA

120

DEFENSE-RELATED ENZYMES GENE EXPRESSION IN Cucumis sativus L. PLANTLETS INDUCED BY MICROORGANISMS

F.E. HELEPCIUC, M. CIUC, M.E. MITOI, A. BREZEANU and C.P. CORNEA

122

DIRECT SOMATIC EMBRYOGENESIS AS POSSIBLE WAY OF VIRUS ELIMINATION FROM SEEDLESS GRAPEVINE
L. ANDRONIC, S. SMEREA and T. GRIGOROV

123

ANTIOXIDANT ACTIVITY OF POLYPHENOLS FROM Vitis vinifera CVS. isabelle LONG-TERM CALLUS CULTURE UNDER THE INFLUENCE OF SOME ELICITORS TREATMENT
R. MIHAI, F.E. HELEPCIUC and M.E. MITOI

125

AN EFFICIENT METHOD FOR SOMATIC EMBRYOGENESIS INDUCTION AND PLANT REGENERATION FOR Serratula bulgarica ACHTAROFF ET STOJ. EX SITU GERMPLASM CONSERVATION
A. AIFTIMIE-PUNESCU

127

GENETIC STABILITY ASSESSMENT OF Scilla autumnalis REGENERANTS

C. BANCIU and F.E. HELEPCIUC

128

COMPARATIVE STUDIES BETWEEN FERNS GAMETOPHYTE AND SPOROPHYTE BY BIDIMENSIONAL ELECTROPHORESIS AND POLYPLOIDY ANALYSIS
F. ALDEA, F.E. HELEPCIUC, C. BANCIU, L. DOMALSKA, S. SPANIEL, M.E. MITOI, A. BREZEANU and L.C. SOARE

129

ELECTRONOMICROSCOPICAL ANALYSIS OF THE Usnea barbata (L.) MOTT. (LICHENOPHYTA) SYMBIOTROPHIC FORMATIONS REGENERATED IN IN VITRO CULTURE
D. VOICU and A. BREZEANU

131

THE PROJECT - IBB INFRASTRUCTURE DEVELOPMENT FOR ENHANCING THE BIODIVERSITY INVESTIGATION CAPACITY, IN ACCORDANCE WITH GLOBAL CLIMATE CHANGES DIBIOCLIM
General data of the project: Beneficiary: Institute of Biology Bucharest of the Romanian Academy POS-CCE: O.2.2.1. Development of existent R-D infrastructure and creation of novel R-D infrastructures (laboratories, research center) Period of implementation: 01.03.2009 31.08.2012 Project budget: 42.428.052 lei FEDR: 25.874.785 lei National Budget: 7.860.267 lei Ineligible expenditure: 8.693.000 lei Target: Development of IBB infrastructure for enhancing the biodiversity investigation capacity, in accordance with global climate

Investment
The project contribute to develop research capacities by enhancing the scientific and technical potential, in view of the preservation and the stable management of natural resources and biodiversity, considering the global climate changes, and in accordance with the European Research Area. The main interest of all research departments of the Institute of Biology is the study of species biodiversity from all categories of organisms, from microorganisms to plants and animals. The activities developed in this project (acquisition of modern and performing research equipment, expansion and organization of laboratories and spaces for Research Development and Innovation (RDI), acquisition of RDI software and hardware) already started a direct contribution to the development of RDI studies of the

environment, and offer the possibility of new research domains and directions, in accordance with European strategies in research fields. The Institute of Biology has an excellent research tradition, marked by prestigious names of national and international recognition, and is known for its constant involvement in priority research directions considered worldwide.

Novelty and impact

The current project successfully implemented by the Institute of Biology directs to a very prominent and up-to-date scientific area, of high interest worldwide the quantitative and qualitative evaluation of species, population and ecosystem diversity, the monitoring of biomarkers, the analysis of space- and time- evolution of populations, of natural and human related ecosystems and socio-ecological complexes for the environment protection and durable development. European research areas are directed to the evaluation and prediction of biodiversity changes and the characterization of ecosystem dynamics, including terrestrial and marine environments. The successfully implementation of this project for infrastructure development started to determine a substantial research progress regarding species diversity, with important impact on the advancing of both scientific knowledge and applications in different economy areas (environment, industry, medicine). The major Ecological researches in our institute focused on taxonomical studies of invertebrates and plants isolated from terrestrial and aquatic ecosystems, evaluation of the diversity of species, ecosystems and complexes of natural and human related environments, the production and efficiency of these terrestrial and aquatic ecosystems, and the conservation of their biodiversity. Considering the global climate changes, the proposed studies concerning plant and invertebrates biodiversity will be directed in accordance with the international tendencies and strategies. Microbiology researches focuses on (1) the evaluation of taxonomical and physiological diversity of microorganisms isolated from environments characterized by normal and extreme conditions of temperature, hydrostatic

pressure, pH, high concentrations of salt, heavy metals and carbohydrates, and (2) the identification of cellular and molecular structures and metabolites with bio(nano)technology potential, as well as microbial biomarkers for polluted environments.

Period of implementation and costs

This Project has been implemented during a period of 42 months, starting in March, 2009. The total budget was 42.428.052 Lei (11.000.000 ), 90% of this costs representing a direct investment in infrastructure and RDI equipment. Major research equipments purchased into frame of DIBIOCLIM project - Q TRAPTM LC/MS/MS system and software - Genetic Analyzer ABIPrism 3500 (Applied Biosystems) and software - Atomic Absorption Spectrophotometer and software - Transmission Electron Microscopy and software - Scanning Electron Microscopy and software - Atomic Force Microscopy and software - X-ray Spectrophotometer and software - Ultracentrifuge and software

INVITED LECTURES AND ORAL PRESENTATIONS

WHATS IN A NAME? THOUGHTS ABOUT SPECIES CONCEPTS, TAXONOMY AND NOMENCLATURE OF PROKARYOTES
A. OREN
Department of Plant and Environmental Sciences, The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. E-mail: aharon.oren@mail.huji.ac.il

The number of different species of prokaryotes with names with standing in the nomenclature has recently surpassed ten thousand, and these are classified in about two thousand genera and more than three hundred families. These numbers represent only a very small fraction of the true number of different prokaryote taxa that exist in nature; based on culture-independent studies using molecular markers (in particular 16S rRNA genes) it is clear that most species of Bacteria and Archaea are still awaiting to be isolated and described. The nomenclature of the prokaryotes, as regulated by the rules of the International Code of Nomenclature of Prokaryotes ("The Bacteriological Code"), is extremely well-ordered. A new beginning was made in 1980 with the publication of the "Approved Lists of Bacteral Names". Thanks to central registration and indexing, the number of names of species, genera, families, orders and classes with standing in the nomenclature of prokaryotes is known at any moment. However, the names of the higher taxa of prokaryotes (phyla, kingdoms or domains) are currently not covered by the rules of the Code. The nomenclature of one group of prokaryotes is still highly problematic, and that is the Cyanobacteria, a group traditionally covered by the International Code of Botanical Nomenclature (now renamed as the International Code of Nomenclature for Algae, Fungi, and Plants). The two codes are not compatible in many aspects, and solutions are currently been sought for the problem. While we have exact information about the number of prokaryote species with validly published names described in the literature, there is no consensus on how to define and to delineate such species. No generally accepted species concept exists yet for the prokaryotes. Currently the description of new species is based on a polyphasic approach, taking into account phenotypic, chemotaxonomic and genotypic properties. Comparison of small-subunit ribosomal RNA sequences is insufficient to delineate species, and the operational definition of a species based on DNA-DNA hybridization is also not always satisfactory. To illustrate all this I intend to present the halophilic Archaea of the family Halobacteriaceae as an example, showing how species concepts and nomenclature have changed over the years and how the occurrence of lateral gene transfer events and possibly even the formation of recombinant hybrids complicate the delineation of species.
17

UNRAVELING OF THE COCOA BEAN FERMENTATION PROCESS OPENS NEW PERSPECTIVES FOR CHOCOLATE PRODUCTION
LUC DE VUYST
Research Group of Industrial Microbiology and Food Biotechnology, Department of Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

Cocoa beans originate from the fruit pods of the cocoa tree, Theobroma cacao L. They have an astringent and bitter flavor and taste and have to be processed before they can be roasted and converted into chocolate. This post-harvest processing includes fermentation and drying. Actually, it is the cocoa pulp surrounding the cocoa beans that undergoes a fermentation process, thereby being removed and hence facilitating drying. This combined fermentation and drying initiates physical and enzymatic processes inside non-germinating cocoa beans to develop color and flavor precursors. Cocoa bean fermentations are still spontaneous, uncontrolled, on-farm processes that differ in methods (heaps, boxes, platforms, etc.) and practices (agricultural and operational practices on the farm) and hence generally result in end-products of variable quality. Once the cocoa pods are opened, either manually or mechanically, the cocoa pulp-bean mass is inoculated by microbiota from the direct environment, for instance the cocoa pod surfaces. These microorganisms grow fast and selectively because of the nutritious environment and low-pH conditions of the cocoa pulp-bean mass, which is fermented for approximately four to six days. A particular succession of microbial activities carried out by yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) guarantees a successful cocoa bean fermentation process. Yeasts are important for a fast and consistent ethanol production through anaerobic carbohydrate (glucose) fermentation. They also contribute to pulp removal through pectinolysis. LAB that develop with the yeasts simultaneously are necessary for citric acid conversion, thereby producing lactic acid and acetic acid, next to lactic acid production by carbohydrate (fructose and glucose) fermentation and mannitol formation through fructose reduction under microaerophilic conditions, caused by air ingress due to pulp degradation. AAB are necessary for exothermic oxidation of ethanol (produced by the yeasts) and lactic acid (produced by the LAB) to acetic acid under aerobic conditions. Penetration of ethanol, acetic acid, and heat into the beans kills the embryos and disintegrates the beans internally, hence initiating enzymatic reactions involving glycosidases, proteases, and polyphenol oxidase. Diffusion of
18

polyphenols and alkaloids out of the beans reduces bitterness and astringency of well-fermented cocoa beans. Detailed investigation of the cocoa bean fermentation process in several cocoa-producing countries (Ghana, Ivory Coast, Brazil, Ecuador, and Malaysia) by means of a multiphasic approach, encompassing both culture-dependent and culture-independent microbiological analyses of temporal fermentation samples, temporal metabolite target analysis of cocoa pulp-bean mass and fermented dry beans, and sensory analysis of chocolates produced thereof, unraveled that a restricted species diversity of yeast, LAB, and AAB communities was necessary for a successful fermentation, independent of the fermentation method applied (heap, box, or platform) and the cocoa population used (Criollo, Forastero, Nacional), provided that good agricultural practices are applied under correct circumstances of fermentation and drying. For instance, it has been shown that high-quality fermented dry cocoa beans were obtained when mature, fresh and healthy cocoa pods and beans were used, in addition to a removal of the placenta before fermentation, a regular mixing of the cocoa pulp-bean mass during fermentation, a well-maintained and clean fermentation (baskets, boxes, etc.) and drying infrastructure, and a controlled operation and duration of the fermentation and drying processes. Although different new species were discovered when analyzing spontaneous cocoa bean fermentation processes, such as Lactobacillus cacaonum, Lactobacillus fabifermentans, Weissella fabaria, Weissella ghanensis, Acetobacter fabarum, Acetobacter ghanensis, and Acetobacter senegalensis, it turned out that always the same species of yeast (Saccharomyces cerevisiae), LAB (Lactobacillus fermentum), and AAB (Acetobacter pasteurianus) dominated, albeit that Hanseniaspora opuntiae, Pichia kudriavzevii, Fructobacillus pseudoficulneus, Leuconostoc paramesenteroides, Lactobacillus plantarum, A. ghanensis, and A. senegalensis as well as Enterobacteriaceae were present at the start of the fermentation process. This species diversity has been confirmed by constructing a 16S rRNA gene clone library as well as by metagenomic analysis of cocoa bean fermentation samples. Whereas fructophilic LAB contribute to the initial fructose fermentation and reduction, Enterobacteriaceae may contribute to pectinolysis, citric acid conversion and gluconic acid production early in the fermentation while Glucon(aceto)bacter may be responsible for late gluconic acid production. Several of the prevailing microorganisms were tested in appropriate cocoa pulp simulation media to unravel their functional roles as well as in small plastic vessels containing fresh cocoa pulp-bean mass as to evaluate their capacity to dominate the cocoa bean fermentation process. It turned out that the use of cocoa pulp simulation media allowed to simulate spontaneous cocoa bean fermentations and confirmed the functional roles of S. cerevisiae
19

(conversion of glucose into ethanol), L. fermentum (conversion of glucose, fructose and lactic acid into lactic acid and acetic acid and of fructose into mannitol), and A. pasteurianus (conversion of ethanol and lactic acid into acetic acid and further oxidation of lactic acid and acetic acid into carbon dioxide and water), the fermentation being finished within four days. Also, it has been shown that cocoa-specific LAB species could be distinguished into fructose/non-fructose lovers and citric acid/non-citric acid convertors, whereas cocoa-specific AAB species could be distinguished into preferential lactic acid oxidizers and simultaneous lactic acid and ethanol oxidizers. Furthermore, the vessel fermentations confirmed the dominance of these species when inoculated into fresh cocoa pulp-bean mass. Ultimately, triculture fermentations in a cocoa pulp simulation medium confirmed the interactions between S. cerevisiae, Lb. fermentum, and A. pasteurianus. Taken together, these data allowed developing a starter culture composed of appropriate strains of S. cerevisiae, Lb. fermentum, and A. pasteurianus to improve the cocoa bean fermentation process. Finally, different starter cultures were implemented on several farms in two different cocoa-producing regions (West Africa and Southeast Asia). Two starter culture mixtures were tested during cocoa bean heap and box fermentations, namely one composed of S. cerevisiae H5S5K23, L. fermentum 222, and A. pasteurianus 386B and another composed of L. fermentum 222 and A. pasteurianus 386B. In all starter culture-added cocoa bean fermentation processes, the inoculated starter culture species were able to outgrow the natural contamination of the cocoa pulp-bean mass and they prevailed during fermentation. The application of these starter cultures resulted in fermented dry cocoa beans that gave concomitant milk and dark chocolates with a reliable flavour, independent of cocoa-producing region or fermentation method. The addition of the LAB/AAB starter culture to the fermenting cocoa pulp-bean mass accelerated the cocoa bean fermentation process regarding citric acid conversion and lactic acid production through carbohydrate fermentation and resulted in chocolates with standard flavour profiles. For the production of a standard bulk dark chocolate, the addition of a yeast strain to the LAB/AAB starter culture was necessary. This enabled a fast and consistent ethanol production by yeasts. It resulted in chocolates with masked bitterness and/or without unfermented flavour, as yeasts produced not only ethanol but also volatiles that contributed to the final cocoa flavour. In conclusion, these research results ultimately led to the composition of a mixed-strain starter culture consisting of appropriate strains of S. cerevisiae, L. fermentum, and A. pasteurianus for reliably improved cocoa bean fermentation processes regarding speed of fermentation (from six to four days), uniform quality of the fermented dry cocoa beans (avoiding variations in fermentation degree and flavour deviations typically seen in the
20

case of spontaneous cocoa bean fermentation processes), and flavourful chocolates produced thereof. This starter culture is ready now as to test it for commercial exploitation in cocoa bean fermentation and chocolate production.

21

SPECIES AND SPECIATION

O. POPESCU
Institute of Biology Bucharest, Romanian Academy

The fundamental unit of biological diversity is the species. There are several distinct species concepts. Essentially, the species is a taxonomical rank represented by a group of individuals closely related to each other in terms of morphological, biological and phylogenetic properties. A species is designated binomial by gender and specific epithet name. The speciation is an evolutionary process through which is made a gradual transformation of one ancestral species into two or more new species, followed by the acquisition of some reproductive isolating mechanisms. Furthermore, this complex process, responsible for the significant amount of current biological diversity, is currently one of the most important biological challenge. In this context, my lecture will discuss certain species concepts and selected contemporary data concerning speciation study.

22

PLANT CONSERVATION TOWARD SOCIETY

D. DIMITROVA, T. IVANOVA, Y. BOSSEVA, CH. GUSSEV, S. TSONEVA, V. GEORGIEV and T. STOEVA
Institute of Biodiversity and Ecosystem research, Bulgarian Academy of Sciences, Bulgaria

Biodiversity loss is a major concern of the human society in the last decades. We are facing a global ecological crisis, which involves land and sea ecosystems and where the main actor who also bears the most responsibility, putting enormous pressure on natural habitats is Man. The loss of biodiversity does not only affect wild species, but also agrobiodiversity, which declines due to the globalization of agriculture. According to FAO estimates, 75% of agricultural crop varieties have disappeared and three quarters of the worlds food come from only 12 plant species. The conservation of biodiversity, including wild plant species and crops, has been a main focus of politicians, scientists, stakeholders and NGOs. Various strategies, action plans and other tools have been developed to preserve biological resources. The Convention on Biological Diversity and the two recent derivative documents: the updated Global strategy for Plant Conservation (2011-2020) and Nagoya Protocol on Access and Benefit sharing of genetic resources (2010) set out advanced targets and provides modern tools to ensure sound plant conservation policies on international and national level. The importance and urgency of the issues related to biodiversity conservation requires the strengthening of the link between scientific data and traditional knowledge preserved by the local communities and the finding of their present interpretation in the modern reality. Plant species, nature habitats and environment issues, which could capture social involvement, are main target of participatory approach in nature conservation. Flag species of conservation value, economically and/or socially importance are most suitable to attract public interest and engagement. Bulgaria is among the countries with richest biodiversity in Europe. Bulgarian native flora comprises about 4000 taxa of which 12.8 % are Balkan and Bulgarian endemics. According the Red List of Bulgarian Vascular Plants (2009) 176 endemics (108 Balkan and 68 Bulgarian) have been assigned an IUCN threat category CR, EN or VU. Among the endemic species, 165 are legally protected by the national Biological Diversity Act. Moreover Bulgaria is one of the top five countries in terms of crop wild relatives richness in Europe. Various examples for developed strategies and actions taken in Bulgaria that perform different levels of
23

efficiency in the preservation of wild and agro-biodiversity are provided so to represent current status of plant conservation in the country. The biodiversity-engaged community and especially its scientific part need to reinforce its identity and build up interdisciplinary groups to be able to launch more efficiently long-term and funding-supported measures.

24

PLANT BIOTECHNOLOGY DEVELOPMENT IN THE INSTITUTE OF BIODIVERSITY AND ECOSYSTEM RESEARCH, BAS FACILITIES, ACHIEVEMENTS, AND CHALLENGES
M. STANILOVA
Institute of Biodiversiy and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria

Plant biotechnology is a new research area in the Institute of Biodiversity and Ecosystem Research with the Bulgarian Academy of Sciences. Its development began eleven years ago with the establishment of the Biotechnological Laboratory of Medicinal Plants in the frame of the 5year project Alternative Approaches of Bioproduction of Alkaloids and Active Substances from Bulgarian Rare and Threatened Medicinal Plants (2001-2006) supported by the NATO Science for Peace Programme. The laboratory is compact, close to the GLP requirements, with modern equipment and furniture, consisting of several special sectors corresponding to the interdisciplinary scientific studies: laminar box for in vitro manipulations in sterile ambience, cultures room, microscopy for karyological and histological control, analytical for phytochemical analyses, and auxiliary rooms for media preparation, extraction of bioactive compounds, glass-ware cleaning, and so forth. Recently, the laboratory was extended with an extra room for ex vitro adaptation of the in vitro obtained plants, allowing their graduate adaptation to lower humidity, which was crucial for the successful completion of the whole procedure of plant propagation. The facilities were designed and organized to serve a new research direction, aiming at rapid plant multiplication by means of different in vitro techniques. The objects of the investigations are rare and threatened plant species from the Bulgarian flora, populations of which need to be strengthened. Among them, the most perspective for in vitro cultivation are some law-protected medicinal plants with market demand and resource deficit, which gathering from the wild populations is forbidden or limited. In vitro propagated medicinal plants are suitable planting material for an establishment of field plantations as an alternative way to ensure plant biomass for production of drugs. Besides, the biosynthesis of active substances by in vitro cultures of medicinal plants is desirable for many advantages. The first in vitro cultivated species in our laboratory was the medicinal plant Leucojum aestivum L. (Amaryllidaceae), which is used as raw material for extraction of the alkaloid galanthamine and production of the medicine Nivalin by the Bulgarian pharmaceutical factory Sopharma
25

AD. We developed successfully both approaches: rapid in vitro micropropagation and in vitro biosynthesis of alkaloids. Several in vitro clones were selected for their high propagation rate and galanthamine content over 1 mg/g DW, similar to that of the commercially important wild populations. The cultures features studied during 3 years of subcultivation revealed clone specific dynamics of the biosynthetic activity in spite of the unchanged cultivation conditions and medium composition. The acclimatized plants on the experimental field plot kept the features of the parental plants. Other successfully in vitro multiplied medicinal plants are the critically endangered in Bulgaria species Alchemilla mollis (Buser) Rothm. (Rosaceae) and three Bulgarian high-mountain endemics from the same genus: A. achtarowii Paw., A. jumrukczalica Paw and A. bundericensis Paw. Several hundreds of ex vitro adapted Alchemilla plants are well developing on the field plot and the first 80 A. mollis plants were easily acclimated on Vitosha Mt. last summer and distinguished with high antioxidant activity (IC50 13,11,9 g/mL, similar to the commercial antioxidant butylated hydroxytoluene, IC5012.65 g/ml). All of them survived the winter at 1500 m a. s. l., reached flowering stage and formed normal seeds. Seeds of many threatened plant species included in the Red Book of Bulgaria and protected by the Biodiversity Act have been in vitro germinated and multiplied as a first step of ex situ conservation. Among them are: Pancratium maritimum L. and some other psammophytes from the white and grey dunes along the Bulgarian Black Sea Coast, affected by the anthropogenic pressure; several endemics from the alpine area as Papaver degenii (Urum. & Jav.) and Geum bulgaricum Pancic; some critically endangered species according to the IUCN criteria with problematic seed germination as Fritillaria meleagroides Patrin. and Astragalus physocalyx Fisch. It is worth to note that the success of the next steps related to the ex vitro adaptation and acclimation of the plants to open air conditions increased significantly after the setting of the phytotron. Thus, plants of the endemics Centaurea davidovii Urum. C. pseudaxillaris Stef.&T.Georgiev, Astragalus physocalyx Fisch., Alchemilla achtarowii Paw. are ready to be transferred to their natural habitats as well as to the Botanical garden of BAS. Nowadays, the most important challenges, due to difficult financial situation, are related to the opportunities to keep young people in the Academy and to establish partnerships between the scientific team and the business world, which is not a common practice in our country. Until now, 2 MSc thesis and 3 PhD thesis have been developed in the Biotech lab in the frame of several projects with national or international funding. Scientific network has been established between biotechnologists from several Balkan
26

countries, including Romania, and exchange of experience is in progress. The biotechnological approach of in vitro plant multiplication in the institute is on the threshold of a new stage.

27

CLONAL PROPAGATION AND GENETIC IMPROVEMENT OF SELECTED SMALL FRUIT SPECIES THROUGH BIOTECHNOLOGY APPROACHES
A. GAJDOOV, G. LIBIAKOV and M. LATEKOV
Institute of Plant Genetics and Biotechnology of the Slovak Academy of Sciences, Slovak Republic

The species of Vaccinium and Rubus genus belong among commercially and biologically important small fruits because of their high content of vitamins, bioactive organic substances with antibacterial and anticancer effects, anthocyanin pigments, and also as an excellent source of antioxidants. They are widely cultivated in various parts of the world, therefore an increase in effort to establish the large-scale cultivation of productive cultivars is noticed. Increasing demand for berry fruits on the world market evocates also necessity to create new genotypes with improved attributes. Classical breeding is in fruit trees limited by their long reproductive cycle and high heterozygosity what make the breeding process very time-consuming. Tissue cultures represent the effective tools not only for rapid mass propagation of high quality planting material needed for large-scale cultivation, but also for germplasm conservation and improvement. Traditional vegetative propagation by cuttings has some limitations, such as rapid spreading of diseases, lack of initial material, dependence on seasonal growth or low propagation coefficient, which can be avoid by tissue cultures. Micropropagation via direct shoot regeneration from dormant buds is well suited to obtain genetically homogeneous planting material, identical with mother genotype. In vitro plant regeneration via adventitious organogenesis is an effective system for the production of plant material, as well as for genetic engineering. Utilization of genetic engineering could ensure relatively quick acquiring of desired useful agronomic traits without cardinal changes of cultivar genotype. In vitro adventitious organogenesis was studied in different Vaccinium and Rubus species and cultivars, however the results of these studies showed that development of efficient and widely applicable regeneration system in these species is complicated because the regeneration ability is highly genotype-specific and it is often impossible to regenerate plants from tissue derived from mature woody plants. Genetic transformation and following regeneration of transgenic plants is in Vaccinium and Rubus spp. considerably limited till now, mainly due to high sensitivity of their cells and tissues to selection antibiotics commonly used in genetic transformation.
28

The present paper is focused on description of reliable system for micropropagation and adventitious regeneration of Vaccinium and Rubus spp. selected cultivars, genetic transformation of selected cultivars by Agrobacterium tumefaciens and regeneration of transgenic plants. In our research the focus was put on testing of factors influencing successful micropropagation and regeneration of adventitious shoots (type of explants, basic culture medium, type and concentration of growth regulators), testing for influence of selection antibiotics commonly used in genetic transformation on regeneration ability of the respective cultivars. Transformation of plants was done by use of different strains of Agrobacterium tumefaciens and the transformation efficiency was evaluated with the aim to select the appropriate strain of Agrobacterium tumefaciens. For optimization of transformation and regeneration conditions for studied cultivars the duration of co-cultivation of plant tissue with Agrobacterium, influence of plant genotype, influence of age and type of explants on efficiency of genetic transformation and selection procedure were followed.

29

NUCLEOTYPIC EFFECTS IN VITRO CONDITION OF DIFFERENT CABBAGE GENOTYPES INFECTED WITH CAULIFLOWER MOSAIC VIRUS
L. ANDRONIC
Institute of Genetics and Plant Physiology of the ASM, Chisinau, Republic of Moldova

Impact of stress-derived stimuli, including viral pathogens, on perception structures in plants, result in a wide range of modification triggering local and global responses via multiple (frequently intercrossing) transduction pathways. It is known that the viral infections dramatically affect plant physiology, inducing photosynthesis decreasing, respiration increasing and carbohydrate levels alteration. On basis of studies has been established that viral infection can contribute to chromosome breaks, activation of transposing elements, chromatin condensation and cells proliferation, gene expression modification. Preservation of genome stability and integrity is the primary task for every organism, including plants. The development of viral infection influence correlates with selective methylation of different genomic DNA regions that leads to changes in their transcriptional activity (Boyko A., Kovalchuk I., 2011). These modifications would start with changes in DNA methylation and chromatin conformation patterns and, if the pathogen pressure continues over several generations, it could lead to a genetic mutation causing fixation of a new treats. Also, it was recognized the contribution of in vitro condition in induction of genetic instability and variability. The objective of this study is the morphometric evaluation of nuclear structures in vitro condition from cabbage calli cells derived from virus infected and non-infected plants. As biological materials, were the cultivated cabbage, Brassica oleracea var. capitata, cultivars Haricovscaia, Moldavscaia and Iuniscaia. Cabbage plants were grown in field following a standard technique. The experimental plants were infected with cauliflower mosaic virus (CaMV, isometric virions, DNA genomic nucleic acid component). For in vitro culture were used the leaf explants from plants presented positive results to viral particle presence and which developed a systemic infection. The stereologic technique adapted for electron microscopy was used for morphometric analysis. According the proposed objectives, the morphogenetic calli pieces were fixed for transmission electron microscopy, using solution of 2.5% glutaraldehyde on cacodylate buffer, pH 7. Postfixation was done in 1% OsO4. The tissue were dehydrated in series of ethanol and propylene oxide and embedded in Epons resin. Ultrathin
30

sections were stained with uranyl acetate and lead citrate. Sections were examined with transmission electron microscope (AM). As morphometric parameters were used: the relative volume of cells, nucleus, chromatin (membrane associeted and nucleplasmic), nucleo-cytoplasmic rate and its coefficient of variation. In study were used only the cells and nuclei in which a not denoted the viral particles. According the obtained data, the infection, inducted by the CaMV, in dependence of the host genotype, conducts to the emphasize in the chromatin distribution, generated and by the in vitro culture (table).
Table 1. Morphometric changes of nuclear parameters in vitro condition in noninfected and CaMV infected cabbage cells
NucleoGenotype Treatments cytoplasmic rate 0.261 Relative volume of nuclear structures Nucleus Membrane chromatin 0.1770.009 35.90 % 0.2150.013* 37.52 % 0.1850.105 36.25 % 0.2350.013** 37.49 % 0.1680.008 33.65 % 0.2190.011* 33.84 % 0.0320.0009 19.09 % 0.0440,0002** 33.85 % 0,0340,0007 13.81 % 0.0530,0028** 34.63 % 0.0330,0009 18.01 % 0.0480,0028** 37.25 % Nucleplasmic chromatin 0.0200.0006 22.08 % 0.0260,0019 47.61 % 0.0190,0007 21.94 % 0.0280,0020 ** 47.10 % 0.0180,0007 23.45 % 0.0250,0019 * 47.82 %

Haricovscaia

Control

Coef. of var. CaMV 0.318**

Coef. of var. Moldavscaia Control 0.268

Coef. of var. CaMV 0.334***

Coef. of var. Iuniscaia Control 0.252

Coef. of var. CaMV 0.330***

Coef. of var.

*; **; *** - significant at P 0.05; 0.01; 0.001

For all three cabbage cultivars were established the increase of the membrane chromatin relative volume, nucleo-cytoplasmic rate. Although the relative value of chromatin distributed in nucleplasm varied significantly in cells derived from virus-infected donors (47% to 22-24% in control), the modifications of means value were not been statistically confirmed for all analyzed genotypes. On basis of the obtained data, we assume that the observed deviations could result due to a position effect variation inducted by viral infection,
31

which mechanism is not clear. It is proposed that cauliflower mosaic virus triggers a rapid systemic response that precedes virus movement and results in a rapid local and systemic induction of reactive oxygen species (Love et al., 2005).

1. Boyko A., Kovalchuk I., 2011. Genetic and Epigenetic Effects of PlantPathogen Interactions: An Evolutionary Perspective, Molecular Plant, pp. 110. 2. Love A.J., Yun B.W., Laval V., Loake G.J., Milner J.J. , 2005. Cauliflower Mosaic Virus, a Compatible Pathogen of Arabidopsis, Engages Three Distinct DefenseSignaling Pathways and Activates Rapid Systemic Generation of Reactive Oxygen Species, Plant Physiol.,139: 935948. 32

MISSING-LINK CARBAMOYL PHOSPHATE SYNTHETASE

E. POPA1, N. PERERA2, C.Z. KIBDI-SZAB1, H. GUY-EVANS2, D.R. EVANS3, C PURCAREA1
1 Institute of Biology Bucharest, Romanian Academy, Department of Microbiology, Bucharest, Romania; 2Eastern Michigan University, Department of Chemistry, Ypsilanti, MI, USA; 3 Wayne State University School of Medicine, Department of Biochemistry and Molecular Biology, Detroit, MI, USA

The genome of Methanobrevibacter smithii, the major human intestinal archaeon, contains several genes coding for carbamoyl phosphate synthetase (CPSase), a major enzyme in the biosynthesis of arginine and pyrimidine nucleotides. In addition to a bacterial type full-length (120 kDa) synthetase subunits, this methanogen possesses a reduced size (41 kDa) homologous subunit (MS-s). The putative small size CPSase was isolated and characterized in order to identify a putative missing link for this class of enzymes. Structural analysis and 3-D molecular modeling reveal a similar folding with that of the carbamate-phosphorylating domain of the E. coli CPSase, inspite of a reduced primary sequence conservation. The recombinant MS-s was cloned, expressed in E. coli, and purified by affinity chromatography. Chemical cross-linking and size-exclusion chromatography indicate a dimeric and tetrameric structure. ATP-dependent partial reactions indicate that MS-s that could catalyzed a CPSase specific reaction that occurs in three steps. Kinetic parameters reveal a higher apparent affinity for ATP and ammonia as compared to other large CPSases, suggesting a particular role MS-s in ammonia trapping in the cell. MS-s represents the smallest active CPSase characterized so far that might correspond to the postulated ancestral kinase which evolved into fulllength CPSases, and appears to be a methanogen specific ammoniametabolizing enzyme.

33

HALOARCHAEA FROM SOME ROMANIAN MAN-MADE AND NATURAL HYPERSALINE ENVIRONMENTS

G. TEODOSIU and M. ENACHE
Institute of Biology Bucharest, Romanian Academy

Hypersaline environments (lakes, solar salterns, Dead Sea, salt deposits, etc.) are found in nature throughout the world, having natural or man-made origin. These hypersaline environments are populated by halophilic microorganisms, mainly haloarchaea, which require high salt amounts for growth (at least 2.02.5M NaCl) and are able to grow up to saturation (13). Haloarchaea, members of the family Halobacteriaceae, are a group of extremely halophilic microorganisms, forming a part of the domain Archaea. The physiological and biochemical features specific to haloarchaea, as well as their capacity to produce different compunds (biopolymers, bacteriorhodopsin, halorhodopsin, enzymes, carotenoid pigments, halocins, lipids, etc) of industrial interest, recommend them as a group of wide biotechnological potential (10, 12). Hypersaline environments are widely distributed also in Romania either in solid or liquid forms: salt lakes and salt mines located in Prahova county, Techirghiol lake nearby to Black Sea coast, Balta Alb lake in Buzu county, hypersaline lakes from Ocna Sibiului, etc. The salt deposits in Slnic Prahova and Telega (DoftanaTelega), formed in the Neogene period are located at around 100 km north of Bucharest. Salt exploitation in these areas started around the year 1685 by using the bell type exploitation technology. After the 18th century, some exploitation areas were abandoned resulting in various man-made salt lakes with different depths (3- 40 m) and widths known today as Green Bath, Shepherd Bath, Red Bath, Bride Cave and Telega (Palada) (1). The aim of our studies was to evaluate the diversity of haloarchaea present in Bride Cave, Shepherd Bath, Red Bath, Green Bath and Telega lakes and in the salt crystals taken from subterranean salt mine Unirea by a polyphasic approach using biochemical and molecular methods. Also, we studied some potential biotechnological applications of some haloarchaea strains: - the synthesis of exopolysaccharides, enzymes and halocins; - the response to some heavy metals and strains capacity to reduce their concentration; - the isolation and characterization of S-layer from haloarchaeal cell envelope and preliminary investigations related to the binding of S-layer to some porous silicon substrates. The results showed the predominant presence of Haloferax species in the investigated salt lakes. This suggests that members of this genus play an important role in ecology of salt lakes, even though the largest number of
34

Halorubrum species was identified in hypersaline environments (6, 7). The microbiota of subterranean rock salt from Slnic area were characterized by the presence of some Halorubrum and Haloarcula species, and some isolates appear to be closely related to Hbt. noricense. The Haloferax members, observable in hypersaline lakes from the surface of Slnic salt deposit, were identified also in subterranean rock salt, but with decreasing numbers (9). The exopolysaccharide biosynthesis activity was detected in some Haloferax strains isolated from Telega lake such as, Hfx. prahovense and Hfx. sp. TL5. In the case of Hfx. prahovense, the maximum EPS yield (0.475 g%) was obtained in medium with 3% glucose and 2M NaCl. According to data resulted from TLC and FT-IR analysis, the polymer of Hfx. prahovense was a heteropolysaccharide containing mainly glucose, fructose, galactose, mannose, uronic acids and sulfate and have a thermal stability until to 2070 C. The amylases were produced by some strains isolated from Bride Cave and Techirghiol lakes. The enzyme of Hfx. sp. GR1 showed maximum activity at 3.5M NaCl, pH 6.5 and 500 C and lost its activity bellow 1.5M NaCl. Amylases produced by Haloferax and Halorubrum strains isolated from Techirghiol lake, showed higher activity with increasing concentration of MgCl2 in the presence of 2.1M NaCl, but activity decreased with increasing Mg concentrations at 3.4M NaCl. Extracellular lipase activity was detected in some strains isolated from lakes Shepherd Bath, Green Bath, Red Bath and Bride Cave. The enzyme produced by Hfx. sp. GR1 was influenced by NaCl concentrations in the growth media and had a maximum activity at 3M NaCl. The activity was lost at NaCl concentrations below 2.5M (4, 8). Halocins were also detected in some strains isolated from Romanian salt lakes, and they showed a variety of action spectra. When compared as halocin producer and targets, some strains showed identical patterns, supporting the tight clustering of strains in phylogenetic tree reconstructed from 16S rRNA gene sequences (5). The investigated haloarchaea strains were susceptible to Zn and Hg but moderately resistant to Cr and Ni, being classified as tolerant. Hfx. sp. TL5 strain and the collection strain Hfx. mediterranei tolerated 5.0 mM Cr, 2.5 mM Ni and Pb. Hfx. sp. TL5 strain has a higher susceptibility for Zn ions compared with Hfx. mediterranei (2). Both strains showed the capacity to reduce the concentration of Pb, Cr, Zn and Ni ions from media with high salinity. The synthesis of EPS enhanced the reduction activity of Cr, Zn and Ni by the tested strains (11). The biochemical characterizations by protein content and chemical treatment have demonstrated the presence of S-layer in the Hfx. sp. GR2 strain isolated from the Bride Cave lake. The TEM examination of the isolated S-layer showed the existence of the monomolecular crystalline lattice with a highly ordered arrangement in the dense form, while in relaxed
35

form after treatment with 4M urea. The S-layer proteins attached to all investigated plates of porous silicon, on both hydrophilic and hydrophobic surfaces; but it seemed that the hydrophobic surface was more favorable. The treatment of silicon plates with hexamethyldisilazane increased the amount of attached S-layer protein (3).

1. 2. 3.

4. 5. 6. 7. 8. 9.

10. 11. 12. 13.

Drgnescu L., 1990. Dates from historic salt exploitation at Slanic-Prahova (in Romanian), Rev Muzeelor, 27: 6871. Dumitru L., Teodosiu G., Enache E., 2002, The tolerance of extremely halophilic archaea to heavy metals, Proc. Inst. Biol. 4: 261267. Dumitru L., Teodosiu-Popescu G., Enache M., Cojoc R., Kleps I., Ignat T., 2007. S-layer of Haloferax sp. GR 2 (JCM 13922): isolation, characterization and binding to silicon nanostructurated substrates. In: Kleps I., Ion A.C., Dascalu D. (eds) Progress in nanoscience and nanotechnologies, Seria Vol. 11, Micro and nanoengineering. Ed. Acad Romne, Bucharest, pp. 146152. Enache M., Teodosiu G., Dumitru L., Zarnea G., 2004a. The effect of NaCl concentrations on the growth and lipase activity at Haloferax sp. GR1, Proc. Inst. Biol., 6: 233236. Enache M., Faghi A.M., Dumitru L., Teodosiu G., Zarnea G., 2004b. Halocin HF 1 a bacteriocin produced by Haloferax sp. GR 1, Proc. Rom. Acad., Series B, 1: 2732. Enache M., Itoh T., Kamekura M., Teodosiu G., Dumitru L., 2007. Haloferax prahovense sp. nov., an extremely halophilic archaeon isolated from a Romanian salt lake, Int. J. Syst. Evol. Microbiol., 57: 393397. Enache M., Itoh T., Kamekura M., Popescu G., Dumitru L., 2008. Halophilic archaea isolated from man-made young (200 years) salt lakes in Slnic, Prahova, Romania, Cent. Eur. J. Biol., 3: 388-395. Enache M., Popescu G., Dumitru L., Kamekura M., 2009. The effect of Na+/Mg2+ ratio on the amylase activity of haloarchaea isolated from Techirghiol lake, Romania, a low salt environment, Proc. Rom. Acad., Series B, 11: 37. Enache M., Popescu G., Itoh T., Kamekura M., 2012. Halophilic microorganisms from man-made and natural hypersaline environments: physiology, ecology and biotechnological potential. In: Helga Stan-Lotter & Sergiu Fendrihan (eds.), Adaptation of Microbial Life to Environmental Extremes, Novel Research Results and Application, Springer, Wien, NewYork, pp. 173197. Oren A., 2010. Industrial and environmental applications of halophilic microorganisms, Environ. Technol., 31:825834. Popescu G., Dumitru L., 2009. Biosorption of some heavy metals from media with high salt concentrations by halophilic Archaea. Biotechnol. Biotechnol. Eq., 23: 791795. Rodriguez-Valera F., 1994. Biotechnological potential of halobacteria, Biochem. Soc. Symp., 58: 135147. Stan-Lotter H., 2012. Physico-chemical boundaries of life. In: Helga Stan-Lotter & Sergiu Fendrihan (eds.), Adaptation of Microbial Life to Environmental Extremes, Novel Research Results and Application, Springer, Wien, NewYork, pp. 119.

36

MICROBIAL DIVERSITY IN THE SUBTERRANEAN ICE DEPOSITS OF SCRIOARA CAVE

A.M. HILLEBRAND-VOICULESCU1, C. ICU2, A. RUSU2, I. ARDELEAN2, D. PASCU2, A. PEROIU3, T. BRAD4, E. POPA2, B.P. ONAC4 and C. PURCREA2
Emil Racovi Institute of Speleology, Bucharest, Romania; 2Institute of Biology Bucharest, Romanian Academy; 3tefan cel Mare University, Suceava, Romania; 4Emil Racovi Institute of Speleology, Cluj-Napoca, Romania
1

While extensive studies have been performed on various exposed ice deposits, very little is known about subterranean ice biotopes an isolated and light deprived cold environment. Scrioara Cave (Romania) hosts the oldest and largest underground perennial ice block in the world (2). Well characterized geo-chronologically (3, 4) it represents an important paleoclimatic archive. We are interested in characterizing the microbial diversity of this underground glacier, in order to correlate the composition of the microbial communities (prokaryotes and eukaryotes) trapped in different aged ice layers with the corresponding climate pattern. Ice samples of different ages were investigated by cultivation on several media and C-sources (BIOLOG Ecoplates) at 4, 10 and 15C. Genomic DNA was extracted from each sample and the corresponding amplified SSU-rRNA fragments were analysed by DGGE. Florescent microscopy was performed on light exposed ice samples to examine the presence of phototrophs. Here we report the presence of both prokaryotic and eukaryotic microorganisms in different aged ice layers growing within this range of temperatures. The diversity of these communities correlated with the age of the ice and the growth conditions. Moreover, viable cyanobacteria and phototrophic eukaryotes were found in the light exposed recent ice sediments.
1. Miteva V., Teacher C., Sowers T., Brenchley J., 2009. Comparison of the microbial diversity at different depths of the GISP2 Greenland ice core in relationship to deposition climates, Environ. Microbiol., 11(2): 640-656; 2. Peroiu A., Pazdur A., 2010. Ice genesis and its long-term dynamics in Scrioara Ice Cave, Romania, The Cryosphere Discuss, 4: 19091929. 3. Feurdean A., Peroiu A., Pazdur A., Onac B.P., 2011. Evaluating the paleoecological potential of pollen recovered from ice in caves: A case study from Scarisoara Ice Cave, Romania, Review of Paleobotany and Palynology, 165: 1-10. 4. Peroiu A., Onac B.P., Wynn J.G., Bojar A.-V., Holmgren K., 2011. Stable isotope behavior during cave ice formation by water freezing in Scarisoara ice cave, Romania, JGR Atmospheres, 116: D02111. 37

DYNAMICS OF MICROBIOTA IN MARINE MICROCOSMS (INDOOR): RESULTS AND PERSPECTIVES

I.I. ARDELEAN1 and S. GHI2
1

Institute of Biology Bucharest, Romanian Academy; 2Maritime University Constana

In this paper we present our results concerning microbial cell density dynamics [i-total cell count (acridine orange, Sybr GreenI, DAPI); ii-dead cells (propidium iodide positive cells); iii) cells able to grow and to multiply (Kogure method) over 24 hours] and metabolic activity (CBO5) in indoor (18 C) marine microcosms (non filtered or filtered [(0.45m) sea water)] supplemented in different concentrations with gasoline and inorganic nutrients. Special attention is devoted to the isolation and cultivation of phototrophic microorganisms growing together with heterotrophic microorganisms in gasoline supplemented microcosms. The perspectives, together with new co-workers, concerns: a) the increase in microcosms diversity by the use of dispersants; b) monitoring of metabolic activity by more diverse methods (resazurine reduction, CTC reduction, esterase activity etc.) both at population level and at single cell level by automated image analysis, c) quantification of pollutant consumption in microcosms and d) analysis of microbial diversity by molecular techniques. The relationships between basic and applicative aspects will be also taken into account.

38

THE GROWTH OF Ulocladium chartarum IN MICROGRAVITY: ON RANDOM POSITIONING MACHINE AND ON BOARD OF INTERNATIONAL SPACE STATION
GOMOIU1, S. VADRUCCI2, I. WALTHER2 and E. CHATZITHEODORIDIS3
1 Institute of Biology Bucharest, Romanian Academy; 2Space Biology Group, ETH Zrich, Switzerland; 3National Technical University of Athens, School of Mining and Metallurgical Engineering, Department of Geological Sciences, Athens, Greece

The effect of simulated microgravity during Experiment Sequence Test (EST), performed on Random Positioning Machine (RPM) and of microgravity during Space Experiment performed on board of International Space Station (ISS), on the growth of Ulocladium chartarum colonies, mycelium and spores viability as well as changes at the cellular level were investigated. Colonies of U. chartarum (provided by Institute of Biology, Bucharest) having different ages, grown on culture plates (provided by Institute for Space Science, Magurele) were integrated in three biocontainers (provided by European Space Agency) which were finally sealed. To assure a high protection during experiments, each biocontainer was introduced in a pouch of protective foam and NOMEX fabric. The duration of space experiment was 14 days, two days longer then EST due to NASA decision. On ISS, the Crew took pictures at FD5 (flight day) and FD9. On RPM, science team of IBB took pictures at FD4 and FD8. Photo sessions had been also performed by science team of IBB for ground controls. The study of Ulocladium chartarum colonies after de-docking and landing clearly suggested that the growth took place in microgravity. The positive effect of microgravity is better expressed on ISS experiment then on EST, for all colonies. Interestingly was that the strongest positive effect of microgravity was put in evidence on 1 day old colonies. In this case, culture plates were inoculated just before handover; launch was planned to take place one day after handover. All colonies developed sporulated aerial and submerged mycelium in both experiments but only submerged mycelium was viable after landing, due to the fact that secondary metabolites acted as toxic compounds in completely isolated environment. Spores and hyphae were not spread in microgravity due to the fact that they are attached on the nutrient. Ulocladium chartarum colonies developed as response to microgravity a complex colony morphotype, never mentioned in similar conditions. Results suggest that EST is very important as a step of strategy to perform space experiments with fungi but the real effect of microgravity can be studied only on board of ISS. In Spaceships and Space Stations fungal species can develop submerged mycelium in the depth of different substrates, including tools containing small amounts of organic compounds acting as biodeteriogens and risk factor for crew health.
39

DIVERSITY OF ARCHAEAL COMMUNITY IN THE CHEMOCLINE OF A HYPERSALINE LAKE IN THE TRANSYLVANIAN BASIN (ROMANIA)
.-A. ANDREI1, 2, A. BARICZ1, V. MUNTEAN1, C. COMAN3, Z. G. KERESZTES1, M. ALEXE4 and H. BANCIU1, 2
Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, BabeBolyai University, Cluj-Napoca, Romania; 2Institute for Interdisciplinary Research in Bio-NanoSciences, Babe-Bolyai University, Cluj-Napoca, Romania; 3National Institute of Research and Development for Biological Sciences (NIRDBS), Institute of Biological Research, Cluj-Napoca, Romania; 4Faculty of Geography, Babe-Bolyai University, Cluj-Napoca, Romania
1

The halite deposits of the Transylvanian Basin in Romania were formed as massive evaporites following the sea level drop in the former Paratethys basin during the Middle Miocene. As a consequence of their tectonic evolution the salts reached the surface in several lower pressure areas. Saline lakes in Transylvania have an almost circular distribution at the inner periphery of the Carpathian Mountains, and the majority was formed after the collapse of abandoned salt mines (Alexe, 2010). The strong salinity gradients of these hypersaline lakes prevent the mixing of water layers, thus leading to a density-based stratification in the water body. Microbial communities living in the chemocline, compared to those from the upper (mixolimnion) and lower (monolimnion) water layers are exposed to a variety of ecological niches on a small spatial scale, in which diverse microorganisms have the possibility to thrive. This study aims to investigate the diversity of Archaea that possibly inhabit the chemocline of the thalassohaline Ocnei Lake (Turda area, Cluj County, Romania; 463509 N, 234717 E). This anthropo-hypersaline lake has a surface of approx. 2000 m2 and a depth of 33.7 m. It has a conical contour due its placement in a former medieval bell-shaped salt mine (Alexe, 2010). The salinity recorded in the water column ranged from 67 g/l at the top to over 300 g/l at 4 m below the surface. To achieve the proposed objectives we used culture-dependent and culture-independent methods, together with bioinformatics and statistical analysis. In situ measurements (water temperature, pH, dissolved oxygen and conductivity) were determined using a portable water quality meter monitor. Samples were collected with a submersible electric pump and stored in sterile 2 L polypropylene bottles. They were used for: biomass fixation on polycarbonate filters, physico-chemical analysis (salinity, determination of major ions, total organic carbon, etc.), isolation of archaeal strains on selective media, chlorophyll extraction and total cell counts
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(Atlas, 2010; Wetzel and Likens, 1991; Antn et al., 2000). DNA was extracted from the filtered biomass, and used in the PCR amplification of partial 16S rRNA genes. A clone library was constructed and the sequences obtained were compared to those stored in GenBank (NCBI) for taxa identification. Based on the physico-chemical profiling of the water column in Ocnei Lake and the total cell counts, our results showed a direct relationship between the microbial abundance and salinity. The highest number of prokaryotic cells was recorded throughout the chemocline and in the hypolimnion. Measurements of chlorophyll-a and total organic carbon indicated that the primary productivity is most probably located in the upper layer (0.5-3 m) of water column. We found that genetic variability of Archaea (at the sampling site) is restricted to members of the Halobacteriaceae. Our clone library revealed phylotypes belonging to Halobacterium, Halorubrum and to the recently described genera Halonotius and Halolamina. The Halobacterium phylotype is the dominant member in the archaeal clone library (94% coverage) and is the only taxon that could be isolated by culture-based methods. Acknowledgements: This work was supported by a grant of the Romanian National Authority for Scientic Research, CNCS UEFISCDI, project number PN-II-ID-PCE-2011-3-0546.

1. Alexe M., 2010. A study on the salt lakes of the Transylvanian Basin. Cluj University Press, Cluj-Napoca (in Romanian). 2. Antn J., Rossello-Mora R., Rodriguez-Valera F., Amann R., 2000. Extremely halophilic bacteria in cristallizer ponds from solar salterns. Appl. Environ. Microbiol., 66 (7): 3052-3057. 3. Atlas R.M., 2010. Handbook of microbiological media, 4th Ed. CRC Press, Washington D.C.,. 4. Wetzel R.G., Likens G.E., 2000. Limnological Analyses, 3rd Ed. Springer-Verlag, New York. 41

STATINS AS CANDIDATES FOR TUMOR-TARGETED NANOMEDICINES

M.C. ALUPEI1,2, E. LICARETE2, F.B. CRISTIAN1 and M. BANCIU1,2
Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, BabesBolyai University, Cluj-Napoca, Romania; 2Molecular Biology Centre, Institute for Interdisciplinary Research in Bio-Nano-Sciences, Babes-Bolyai University, Cluj-Napoca, Romania
1

Statins as inhibitors of 3-hydroxy-3-methylglutharyl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in de novo cholesterol synthesis, can exert tumor cytotoxic actions. However, despite the increasing data regarding the antitumoral activities of statins, their complete mechanisms of action still remain obscure. Therefore, this study aims to investigate the underlying mechanisms of the cytotoxic effects of statins on B16.F10 murine melanoma cells in vitro and on established murine melanoma models in mice, after statin encapsulation in long-circulating liposomes. We have compared the effects of one lipophilic statin, simvastatin (SIM), with the effects induced by a hydrophilic statin, pravastatin (PRV), as free drugs as well as liposomal statin formulations. In vitro cytotoxicity was investigated in regard with B16.F10 melanoma cell proliferation and viability at concentrations of each statin ranging from 20-100 g/ml. To assess the pleiotropic effects of statins that can be responsible for statin-induced cytotoxicity in vitro, we studied the statin actions on the expression of the hypoxia-inducible factor 1 (HIF1), on antioxidant defense mechanisms of tumor cells, as well as on tumor cell capacity to support vital processes for tumor development in vivo, such as angiogenesis and inflammation. To study the anticancer potential of statins in vivo, statins were encapsulated in long-circulating liposomes. These liposomal formulations enable accumulation of the statins at sites of malignancy. Liposomal statins were prepared by lipid film-hydration method, followed by multiple extrusion steps. Mean particle size of the liposomes was determined by dynamic light scattering and found to be 0.1 m, with a polydispersity value lower than 0.1. To study the antitumor activity of liposomal statins and free statins, we used the subcutaneous B16.F10 murine melanoma model. Statin treatments started at day 11 after tumor cell inoculation. Free statins and liposomal statins were administered i.v. at a dose of 5 mg/kg at days 11 and 14 after tumor cell inoculation. On day 15, the mice were sacrificed and tumors were isolated. Our in vitro data have shown that only the lipophilic statin had strong inhibitory actions on proliferation and viability of B16.F10 melanoma cells. These cytotoxic effects were mainly mediated via suppressive actions of SIM on HIF-1 expression and nonenzymatic antioxidant levels, as well as
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due to the inhibition of superoxide dismutase activity in cancer cells. In addition, SIM induced the reduction of angiogenic/inflammatory capacity of tumor cells that can strengthen and support its cytotoxicity. The hydrophilic statin, PRV did not have any cytotoxic activity on B16.F10 murine melanoma cells. We noted only a delayed and lower suppressive action of PRV compared to the SIM on the expression of HIF-1 and on production of proangiogenic/ proinflammatory proteins in tumor cells. These limited antitumor effects of PRV might be explained by the hydrophilic nature of this drug and by the lack of a drug transporter in the membrane of B16.F10 melanoma cells. The strong cytotoxic capacity of SIM might be related to the inhibition of 3-hydroxy-3-methylglutharyl coenzyme A (HMG-CoA) reductase - the rate-limiting enzyme in de novo cholesterol synthesis. This leads to high reduction in isoprenoids levels which in turn determines an inhibition of the synthesis of several intracellular antioxidants, vital for membrane stabilization and suppression of HIF- 1 expression via quenching the activation of Ras proteins. Our in vivo data suggested that both liposomal statins had strong inhibitory activity on tumor growth, with a higher extent for liposomal SIM (85% inhibition of tumor growth) compared to liposomal PRV (60% inhibition of tumor growth). No antitumor actions were noted after statin administration as free forms. In conclusion, the antitumor activity of both statins was enabled by liposomal formulations that can allow passive accumulation of drugs at the sites of malignancy. These beneficial actions might be exploited in future in vivo experiments that will aim to investigate the antiangiogenic potency of liposomal statins on other cell types than tumor cells, such as inflammatory cells infiltrated in tumor mass. Altogether, our results offer promise for anticancer therapies with statins in combination with antitumor cytokines (IFN-, IL-12p70).

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EX SITU APPROACH TO CONSERVATION OF LILIACEAE IN BULGARIA

T. IVANOVA, Y. BOSSEVA, CH. GUSSEV, S. TSONEVA, V. GEORGIEV, G. ANGELOV, D. DIMITROVA and T. STOEVA
Institute of Biodiversity and Ecosystem research, Bulgarian Academy of Sciences, Sofia, Bulgaria

Family Liliaceae comprises 140 species of Bulgarian flora, most of them economically important with ornamental, edible and medicinal value. Most abundant genera are Allium (41), Gagea (13), Ornithogalun (12) and Colchicum (11). Ten of the genera are monotypic (fig. 1).

Figure 1. Richness of Liliaceae in Bulgarian flora.

Nearly half of Liliaceae species are of conservation importance and 20 are included in Bulgarian Red Data Book (2011). According to the IUCN criteria, 13 are critically endangered, 5 are endangered and 1 is vulnerable. Legally protected by the Bulgarian Biodiversity Act (2002) are 43 species and 17 are under the regulations of the Medicinal Plants Act (2000). Ex situ storage of germplasm in seed banks, living and in vitro collections are extensively used for the conservation Liliaceae species. Many of them are slow-growing and have reproduction impediments that require development of species-specific procedures. Propagation and conservation protocols are developed predominately for breeding programs of economically important
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species. However, for many other taxa, especially for local endemics, such protocols are missing or insufficient. Case studies for different conservation approaches applied to Liliaceae are presented. Seed collections of 22 species are deposited in Millennium Seed Bank and since 2011 a Research seed bank has been launched in IBER-BAS. In vivo and in vitro propagation have been applied to selected species from Tulipa, Lillium, Fritillaria and Ruscus and results for seed germination, cytogenetic and isozyme analysis are presented. The effectiveness of the applied procedures, scientific value of the obtained data and implications for further conservation measures are discussed.

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PLANT IN VITRO SYSTEMS FOR EX SITU CONSERVATION AND BIOTECHNOLOGICAL APPLICATIONS

I. HOLOBIUC, A. AIFTIMIE-PUNESCU, C. BANCIU, R. CATANA, R. MIHAI, M.E. MITOI, C. VOICHITA, F. ALDEA, D. CRISTIAN, F.E. HELEPCIUC, I. ISTRATE and G. COGLNICEANU
Institute of Biology Bucharest, Romanian Academy

The main objective of our team is to use plant in vitro systems for fundamental research and biotechnological applications. We focused during the last decade on plant cell differentiation and morphogenesis, electrophysiology, genetic transformation, in vitro stress phenomena, and apoptosis. Our current research topics include endangered plant ex situ conservation, biosynthesis of useful secondary metabolites by plant cell cultures, and the study of plant - microbe interactions. Biotechnological approaches for plant ex situ conservation. The continuous decline of biodiversity and the growing problem of climate change had led to an increasing interest for ex situ plant conservation. Despite existing in situ conservation activities Romania still does not have such a strategy, although the European Plant Conservation Strategy (GSPC target 8) recommends that 60% of threatened plant species should be introduced in ex situ collections, preferably in the country of origin. In this context, we have started an in vitro collection for endangered plant conservation. Our objective was to develop reproducible and efficient protocols for collecting, micropropagation and storage of endangered plant species for short, medium and long term. Analysis of the genetic diversity within native populations is a prerequisite for ex situ conservation being useful both for sampling and prioritizing strategies. Isoenzyme patterns (catalase, esterase, peroxidase, alkaline phosphatase) and RAPD analyses have been used to characterize intra-populational variability in some wild populations of Centaura pontica, Convolvulus persicus, Achillea schurii, and Marsilea quadrifolia. Knowledge about breaking dormancy and optimizing seeds germination is useful in plant conservation. Germination experiments on Nitraria schoberi L, an extreme halophile plant, revealed that the first and the compulsory step for their ex situ conservation have to be the dormancy breaking. We have established reproductive multiplication protocols (by somatic embryogenesis, axillary shoot proliferation, adventitious shoot regeneration) for several Vascular species listed in the Romanian Plant Red Book, belonging to Caryophyllaceae, Asteraceace, Scrophulareaceae, Papaveraceae, Primulaceae, Plumbaginaceae, Gentianaceae, Convolvulaceae, Cucurbitaceae, Liliaceae, Campanulaceae,
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Brassicaceae, Fabaceae families. For the first time in Romania, we succeeded to cultivate in vitro: lichens (Xanthoria parietina, Cetraria islandica, Usnea barbata), bryophytes (mosses: Bartramia halleriana, Atrichum undulatum, liverworts: Bucegia romanica, Athalamia hyalina, Reboulia hemisphaerica, Marchanthia polymorpha), and ferns (Marsillea quadrifolia, Asplenium trichomanes, Athyrium filix-femina, Polypodium vulgare). As a working principle for the lower plants, the in vitro methodologies were tested previously on common species. To assess the genetic stability of the in vitro regenerants, different strategies were used each of them with advantages and limitations. For example, we studied in vivo and in vitro variability of histological traits of aerial vegetative organs, for several species (Dianthus callizonus, Alyssum borzaeanum, Marsilea quadrifolia, Ruscus aculeatus, Scilla autumnalis). The identified anatomical differences represented reversible adaptive responses to the in vitro culture conditions. Isozyme analyses provided a convenient method for detecting genetic changes, but are influenced by ontogenic development and physiological status. In vitro regenerants from nine plant species were characterized by enzyme variability for detecting possible somaclonal variations. RAPD analyses are sensitive in detecting variations among individuals between and within species. The RAPD profiles of Marsilea quadrifolia showed a genetic stability among micropropagated plants. For any in vitro conservation program of rare and/or endangered plants a method for medium and long term storage is required. We successfully established active in vitro plant collection based on medium term preservation protocols in several species from Caryophillaceae, Gentianaceae and Scrophulariaceae families. Synthetic seeds defined as "encapsulated meristems or somatic embryos which functionally mimic seeds able to develop into seedlings under sterile conditions were also obtained in Dianthus and Gentiana spp. Long term preservation methods (encapsulation- dehydration, vitrification, droplet freezing) have been tested on plant material from our collection. Plant in vitro systems for secondary metabolites biosynthesis. Bioactive compounds extracted from plant cell cultures are already widely used. In our lab we have obtained callus cultures of Ecballium elaterium, Gentiana punctata, Fragaria x ananassa, Melissa officinalis, Glycine max. But the most spectacular and the most studied is a long-term (20 yrs) stable callus culture of Vitis vinifera which is producing anthocyanin pigments, resveratrol and pycnogenol. In vitro manipulation of plant cell cultures for screening of useful biocompounds is a research direction that will be developed in the future in our lab. Plant-microbe interactions. Microbial and fungal cultures or different fungal extracts were applied to activate plant defense systems. We used optic and electron microscopy analyses, isozyme analyses and reverse
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transcription-PCR (RT-PCR) methods to detect cellular and molecular responses. Defense-specific changes were observed in treated plants, like cell wall strengthening, activation of new isoforms, and expression of some defense-related genes.

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TELOCYTES INFRASTRUCTURE AND FUNCTIONAL ROLE

N. MIRANCEA
Institute of Biology Bucharest, Romanian Academy

A new type of interstitial (stromal) cell termed interstitial Cajal-like cells (ICLCs) because of their morphologic aspect, at first glance, similar to canonical (gastro-intestinal) interstitial cells of Cajal (ICCs) have been described by Popescus group (Popescu et al., 2005) in Bucharest (Romania). Recently, the term telocytes (TCs) was introduced as replacement for the ICLC. TCs were identified and described in different normal tissue types (Popescu, 2011; Gherghiceanu and Popescu, 2012; Rusu et al., 2011, 2012 a, b, c; Cantarero et al., 2011). TCs are represented by a special phenotype of interstitial (stromal) cells. A telocyte (TC) has a cell body where an ovoidal nucleus is located and two or more (maximum five) cell prolongations (Fig. 1).

Fig. 1. Diagramatic representation of telocyte ultrastructural phenotype. Two long telopodes originate from the cell body where the nucleus is located. Each telopode is represented by alternations of podomer-podome. A synapse is depicted (elipsoidal area) between the telocyte and an adjacent extension of another cell.

Cell-body prolongations termed telopodes are very long (tens up to hundreds of micrometers) with a moniliform aspect. Sometimes, telopodes follow a convoluted aspect (Popescu, 2011) as is also the case of such phenotype in stromal adipous tissue (personal ultrastructural unpublished data). Each telopode start abrouptly from the cell body as slender cell extension with moniliform aspect: thin segments less than 200 nm thickness (podomeres) alternate with dilated segments (podoms). As a general rule, inside of the perinuclear cytoplasm, a rough endoplasmic reticulum and, eventually, dyctiosomes of Golgi apparatus are located. Usually, podoms accommodate mitochondria, endoplasmic reticulum and caveolae (Ca2+ uptake/release units). Telopodes may perform homo- and/or hetero-cellular junctions. Moreover, by their telopodes, TCs release small microvesicles (mean diameter of 180 nm); the delivered macromolecules from these microvesicles are considered to play a paracrine role by sending signals to
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neighboring cells. In spite of the fact that TCs give positive immunohistochemical reaction for CD34, C-kit, and vimentin, unfortunately, so far there is no report about a reliable immunolabeling specific to TCs, so that, the ultrastructural cell phenotype identification remains the only possibility to discriminate TCs from other stromal cell mimicing the telocytic phenotype. Using TEM investigations, we describe for the first time TCs as interstitial cell phenotype inside of the human skin tumor stroma (manuscript in preparation). TCs establish homocellular junctions by specialized somatic synapses leading to a 3-D network inside of the tissue stroma. As stromal cells, TCs are located in strategic positions: often they are in close vicinity or in direct contact with endothelial cells, mast cells, naked nerve edges etc. Due to their heterocellular junctions (heterocellular connexions), TCs are considered to be involved in intercellular signalling in pathophysiology. In many respects, TCs from the tumor stroma share general ultrastructural characteristics to those described in normal human skin, but mention must be made, that some particular aspects are detectable, especially concerning the number of heterocellular junctions. The possible functional role of the TCs as tumorstroma cells remains largely unknown, being a matter of debate and, further investigations are required.

1. Cantarero C., Luesna B., Junquera E.C., 2011. J. Cell. Mol. Med., 15: 26-30. 2. Gherghiceanu M., Popescu L.M., 2012. Cell Tissue Res., 348: 265-279. 3. Popescu L.M., Gherghiceanu M., Creoiu D., Radu E., 2005. J. Cell Mol. Med., 9(3): 714-730. 4. Popescu L.M., 2011. Internat. J. Biol. Biomed. Engin., 5(2):83-92. 5. Rusu M.C., Pop F., Hostiuc S., Dermengiu D., Lal A.I., Ion D.A., Mnoiu V.S., Mirancea N., 2011. Ann. Anat., 193: 403-411. 6. Rusu M.C., Mirancea N., Mnoiu V.S., Vlcu M., Nicolescu M.I., Pduraru D., 2012a. Ann. Anat., 194(4): 359-367. 7. Rusu M.C., Jianu A.M., Mirancea N., Didilescu A.C., Mnoiu V.S., Pduraru D., 2012b. J. Cell Mol. Med., 16(2): 401-405. 8. Rusu M.C., Pop F., Hostiuc S., Gurca G.C., Jianu A.M., Paduraru D., 2012c. Histol. Histopathol., 27(6): 807-816. 50

INVESTIGATING THE IMPACT OF CYANOBACTERIAL BLOOMS ON AQUATIC ECOSYSTEMS OF THE DANUBE DELTA
P. SPAAK1 and C. SANDU2
1

Swiss Federal Institute of Aquatic Science and Technology, EAWAG, Dbendorf, CH; 2Institute of Biology Bucharest, Romanian Academy

The goal of this research project is to emphasize the impact of harmful cyanobacterial blooms on aquatic ecosystems of the Danube Delta and develop methods to predict future patterns of such blooms in order to identify protection strategies to reduce health hazards in the future. The nutrient pollution of the Danube River led to an accelerated eutrophication of the shallow lakes in the Danube Delta in the 80s, with drastic consequences for the trophic networks: massive cyanobacterial blooms occurred, affecting the seasonal succession of plankton and macrophytes communities, impacting the food web and having possible adverse effects on livestock- and human health. Although apparently between 1990-2000 the aquatic ecosystems seemed to recover slightly, the nutrients stored in the sediments and the increasing impact of climate change favored new episodes of cyanobacterial blooms, especially in the very dry and warm years occurred in the last decade. Their presence may not only affect drinking water quality and recreational use by reduced transparency, bad odour and taste, but over 40 species from the genera Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Microcystis, Nodularia, Nostoc and Planktothrix have been found to be able to produce toxic secondary metabolites that represent a health hazard to human and animal health. In eutrophic water bodies mass mortality of fish can occur as a consequence of oxygen depletion, principally by high microbial oxygen demand during bloom die-offs. Oxygen depletion can also occur after raise in pH due to increased photosynthesis, shifting the chemical equilibrium in the lake from harmless ammonium to highly toxic ammonia. However, mass mortality of fish can also be caused by cyanobacterial toxins. Cyanobacterial blooms, whether toxic or not, can have tremendous impact on aquatic ecosystems and wildlife. Field studies have shown that cyanobacteria negatively influence zooplankton biomass and community composition compared to other microalgae such as chlorophytes and flagellates, which influences the whole food web. Monitoring lakes to follow the pollution and recovery process takes decennia and requires large investments. Sediments are an excellent archive of biotic remains and biomolecules under cold and anaerobic conditions, and in the recent years, the history of pollutants and species compositions
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using DNA markers started to be deduced from lake sediments. The identification of a broad range of cyanobacterial genetic markers allow their use to detect, quantify and study structural, functional and evolutionary aspects of cyanobacteria and their communities in the field; moreover, the discovery of gene clusters responsible for the biosynthesis of cyanobacterial toxins (microcystins, nodularin, cylindrospermopsin, saxitoxin (STX), anatoxin-a and homoanatoxin-a) allows the identification of toxigenic strains in the environment. Quantitative real-time PCR (qPCR) has been increasingly applied to monitor potentially toxic cyanobacteria population in diverse aquatic environments, in some instances being also possible to derive a toxicity index based on the quantity of toxic genes. These methods can also be applied to ancient DNA in lake sediments, offering a novel window into past aquatic ecosystems and the environmental changes that triggered community dynamics. An alternative method to reconstruct past cyanobacterial dynamics in sediments targets lipid biomarkers, based on total lipid extracts from sediment layers and subsequent analysis by gas-chromatography-massspectrometry (GC-MS). The molecular distribution of lipid biomarkers can also reflect different aspects of the biogeochemical processes linked to lake contamination and cultural eutrophication. The detection of patterns associated with N-fixing cyanobacteria allows the reconstruction of historical changes in N-limitation of the aquatic ecosystem. The hydrogenisotopic composition of individual lipids from aquatic organisms can be related to the composition of their growth environment; also, contamination by petroleum-hydrocarbons may also emerge as molecular signature in lipid biomarkers. The goal of this research project is to reconstruct: i) the history of cyanobacterial blooms, ii) the occurrence of toxic genes from lake sediments, and iii) the effects of cyanobacteria on locally adapted zooplankton in the Danube Delta. This knowledge is crucial to predict the risk for harmful cyanobacterial blooms in the context of environmental change and their consequences for lake food webs. Hence, the project will focus on 4 major objectives: development of methods to reconstruct cyanobacteria dynamics from lake sediment cores relate the cyanobacterial blooms with other biotic and abiotic factors that may influence their development resurrect Daphnia and cyanobacteria from sediment samples to test for adaptations and eventual parasites prevalence

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 analyse the sediment cores of selected Danube Delta lakes and develop conceptual model to forecast future blooms and consequences based on climate, discharge and pollution scenarios The methods developed during the project and its results will represent valuable tools for water managers in Eastern European countries, where eutrophication of lakes and rivers is still of high concern, especially in the context of the expected increasing impact of climate change.

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CONTRIBUTIONS TO THE KNOWLEDGE OF LITTER DECOMPOSITION RATES OF PLANT LITTERS OF Acer pseudoplantanus, Fraxinus excelsior AND Quercus robur IN FOREST ECOSYSTEMS UNDER THE INFLUENCE OF ANTHROPOGENIC PRESSURE
A. JERCA1 , E. PREDA2 and A. VDINEANU3
Faculty of Biology, Bucharest Unicersity

Litter decomposition is a key component of nutrient cycling and of terrestrial productivity, particularly forest ecosystems and global carbon cycle. Litter decomposition resulting from trees and herbaceous layer reinforces the organic matter enrichment of soil and releases necessary nutrients for plants growth and soil CO2 fluxes. Determination of litter decomposition rates has a crucial role in identifying factors that influence nutrient availability. The aim of this paper is to make contributions in identifying, understanding and explaining the mechanisms involved in litter decomposition under the influence of anthropogenic pressure. The experimental research was conducted in Neajlov catchment, component of the national Long Term Socio-Ecological Research Network. Litter decomposition process was investigated in forest ecosystems using of plant litters Acer pseudoplantanus, Fraxinus excelsior and Quercus robur and the results of decomposition rates depending on anthropogenic pressure exerted, respectively different nutrients fertilization.

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BIODIVERSITY DATA TRANSFERRED IN ECOSYSTEM SERVICES STUDIES

M. ONETE, D.C. NICOLAE, M. MINODORA, V. HONCIUC and D. PURICE
Institute of Biology Bucharest, Romanian Academy

In the former time scientists used the terms species diversity and species richness. In our day biodiversity is defined at the organizational level of life as the totality of genetic, species and ecosystem diversity. All studies have been focused on the biodiversity of a region. In the last decades, studies of climate change especially showed that changes in biodiversity at all the organizational levels determined changes of the processes and the conditions from the local, to regional and world level. These processes and conditions of natural ecosystems that support and sustain human life are defined as ecosystem services. The biodiversity generates ecosystem services. More than the presence or absence of the species integrated in the ecosystem, the ecosystem characteristics and processes are determined by the non-additive interaction among species. For instance, trophic interactions influence the abundance of species that control and modify fluxes of materials and energy affecting the ecosystem processes. The food, fuel, structural materials, medicinal or genetic resources for human beings are directly reduced by changes in species diversity and abundance, leading to reduced control of the ecosystem processes, changes in organisms community composition and vulnerability to invasion, going finally to affect ecosystem goods or services and to alter controls on critical ecosystem processes. For human society, biodiversity has great impact on human values (cultural, intellectual, aesthetic and spiritual). Also, changes in ecosystems good and services provided to society have high economic impact. For maintaining these values people need conservative strategies for minimizing loss of biodiversity. Debates regarding both the form of the relationship between species richness and ecosystem processes and the mechanisms underlying these relationships have been along time. Developing the project RO1567IBB01/2012 Researches regarding the relationship between biodiversity and functions in some ecosystems from Romanian Carpathians we try to clarify some aspects of the relationships between biodiversity and ecosystem functions at regional level by transferring biodiversity data obtained in the last decades in the ecosystem services studies.
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PRELIMINARY ASSESSMENT OF THE EFFECT OF URBANIZATION ON ECTOMYCORRHIZAL DIVERSITY

D. MOGLDEA
Institute of Biology Bucharest, Romanian Academy

The conversion of natural ecosystems in urbanized ecosystems increase ecological footprint and have strong negative impacts on the ability of natural capital to produce resources and services. The urban ecosystems exhibit a simplified structure and reduced function compared to natural ecosystems. It is known that fungi are key organisms in nutrient cycling and ecosystem stability, but their function differ in ecosystems with different anthropization levels. The aim of this study is to investigate the ectomycorrhizal (ECM) diversity from areas affected by anthropogenic disturbance compared to those from less disturbed systems. This data are fundamental for understanding the differences between ECM communities functions in ecosystems with different degree of anthropization and for identification of the level of anthropogenic disturbance from which the ectomycorrhizae role is highly perturbed. Three sites, that have Quercus as dominant host tree, with various levels of disturbance, were identified. A reference site of a mature forest was selected for comparison. Quercus associating ectomycorrhizal fungi communities were sampled from each site. The preliminary data revealed that in the reference site, the highest frequency have the species belonging to genera of Russula, Tuber and Cenococcum, which have extramatrical mycelium of contact and short distance exploration type. The first data set regarding colonizing root tips revealed that in the stands of varying levels of anthropization the ECM species diversity is lower that in the reference site. The research was carried out in the framework of Synthesys Project GB-TAF-564/2012 and RO1567-IBB03/2012 Project.

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CITES AND NATURE CONSERVATION IN REPUBLIC OF KENYA

S. MIHILESCU
Institute of Biology Bucharest, Romanian Academy

The Convention on International Trade in Endangered Species of Wild Fauna and Flora CITES is the specific legal instrument for conservation of species that should be protected in natural habitats for their survival. In respect of CITES convention, the European countries and the African countries have common purpose for world species conservation. The important nature conservation developing in Kenya was start in 1975 with Environmental Law Protection, under the United Kingdome control. Developing the applied conservation measures according with ecological criterias for establish the rules for living closing to the animals is the main challenge. In the last fourteen years, a number of species have endangered status according with IUCN categories of species protection. In Africa, especially in Kenya, the many Mammals species request special management for their protection, for examples: African Elephant, White Rhino, Lion, Leopard, Roan and Sable Antelope. The conservation activities (training, species specific programmes such as Monitoring the Illegal Killing of Elephants - MIKE) must find external funding, mostly from donor countries and regional organizations such as the European Union. Main anthropic pressures on species and their habitats are: reduction and fragmentation the species habitat due to expansion of human population; increase the conflicts between human population and wild animals; decrease the area of wild natural habitats; increase the uncontrolled trade with ivory objects, jewels and rhino corn for medicinal purpose, especially in the countries from south-east Asia. The outcomes of species and their habitat protection projects are: increase the protected areas and wildlife sanctuary according with national and international regulation; developing the basic infrastructure and operations; developing the human elephant conflict resolution, law enforcement, management of species conservation oriented to research; increase the conservation education, increase the local and international community participation. Conclusions: Increasing the research studies about wild animals behavior is the proper mechanism for human animals conflict resolutions. The frequent reducing population of wild animals is related with illegal hunting, due to this reason, it is very important to use the scientific studies
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for analyzing the possible securing critical corridors and dispersal areas of the wild endangered animals. Future important step in conservation: developing the public awareness campaign to inform people both about the non-medicinal importance of rhino corn and about negative effect of developing the manufacturing of ivory objects and jewels. Acknowledgement: Project Implementation of CITES and EU Wildlife Trade Regulations in CEE region - financial and organizational support of the European Commission, CEEweb for Biodiversity, IFAW and Kenya Wildlife Service.

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PROJECT SOP ENVIRONMENT: CONSERVATION STATUS MONITORING OF SPECIES AND HABITATS FROM ROMANIA UNDER THE 17 ARTICLE OF THE HABITATS DIRECTIVE 2011-2012
S. MIHILESCU, M. ION, I. GHEORGHE and I. CRISTEA
Institute of Biology Bucharest, Romanian Academy

The Habitats Directive defines a six-year cycle for reporting. The deadline for submitting the 3rd report to the Commission is the 30 June 2013, being for Romania the first assessment of conservation status based on best available data for each species and each habitat type. The Reporting Format for 2007-2012 consists of: - Annex A: General reporting format - a structured brief report on the general implementation of the directive in each Member State, providing links to important information sources. - Annex B: Reporting format for the conservation status of a species it defines the information required for each species covered by Annex II, IV and V of the directive on biogeographic level. - Annex C: Evaluation matrix for the conservation status of a species defines the method and criteria on how to assess the conservation status of each species on biogeographic level. - Annex D: Reporting format for the conservation status of a habitat type - it defines the information required for each habitat type covered by Annex I of the directive on biogeographic level. - Annex E: Evaluation matrix for the conservation status of a habitat type - defines the method and criteria on how to assess the conservation status of each habitat type on biogeographic level. For all of the Member States the reporting format request the general description of the main measures taken (overview at national level, activities taken including legal measures, systematic studies, links to online resources, etc.). Regarding the criteria represented by the Method used /Population size, the following can be used: complete survey or a statistically robust estimate; estimate based on partial data with some extrapolation and/or modelling; estimate based on expert opinion with no or minimal sampling, or absent data can be reported. Main achievements under the Habitats Directive are represented by the reporting period with a special emphasis on the Natura 2000 network.

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The project goal is to protect biodiversity and natural heritage and to improve the natural environment through proper monitoring of the conservation status of species and habitats of Community interest in Romania, the preparation of the National Report according to Article 17 of the Habitats Directive 92/43/EEC, as well as the implementation of the Natura 2000 network nationwide, following the conclusions of the report and the information dissemination activities. Monitoring plans are made for all species and habitats of Community interest, differentiated for each biogeographical region, being developed by taking into account the requirements of the European Commission, the particularities of each species or habitat, like the distribution and biology. During 2011-2012, the project has recorded some progress which will contribute to the final objectives of the project, which aims by 2015, the following: database with existing information on the status of species and habitats of Community importance; distribution maps for species and habitats of Community importance in the standard format GIS ETRS, regular grid of 10x10 km, as follow -species of plants (105 species), marine (4 species of mammals and 9 species of fish), amphibians and reptiles (15 and 19 respectively), fish (35), mammals (terrestrial- 22), invertebrates (73), bats (Chiroptera, 28) and habitats, like coastal and marine (9), caves (1), halophytic/dunes/ grasslands/ freshwater (33), scrubs/ bogs and mires/ swamps/ rocky formations/ forest (44); database of publications on species and habitats of Community interest; assessments for species and habitats of Community interest -species of plants (102), marine (21), amphibians and reptiles (15 137), fish (93), mammals (72), invertebrates (192), bats (107) and habitats, like coastal and marine (10), caves (3), halophytic/dunes/ grasslands/ freshwater (79), scrubs/ bogs and mires/ swamps/ rocky formations/ forest (80); metafile database with records made in the field; sets of data in the reporting format, introduced in national monitoring system (SIMSHAB); monitoring guides for species and habitats, published in 500 copies each; Summary Report on the state the conservation of species and habitats of Community interest in Romania. Acknowledgement: Conservation status monitoring of species and habitats from Romania under the 17 Article of the Habitats Directive 20112015, project financed by SOP Environment

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TURBOVEG DATABASE USED IN BUCEGI MASSIF STUDIES

C.D. NICOLAE
Institute of Biology Bucharest, Romanian Academy

The computer software package TURBOVEG (for Microsoft Windows) was developed by two Dutch researchers (S. Hennekens and J. Schamine) in 2001. This is a very useful tool for the processing of phytosociological data, designed for the storage, selection, and export of vegetation data (relevs). Data can be imported manually (separate relevs or tables) and automatically (import of data files). On the other hand, there are filters available to export the selected relevs to enable further analysis with various programs (e.g. MS Access,Twinspan, Canoco, PC-ORD, Mulva, Syntax-5, Excel, Juice, ArcView, ArcGIS, GoogleEarth). There is a fixed standard structure of the table, but it easily to extend it by defining an extra field. In this way, there could extend both the header data (e.g. add altitude or total cover) and the species data. Each database can have its own extension. Since 1994, TURBOVEG has been accepted as the standard programme for the European Vegetation Survey. According to this, in Romania there are known in this moment five vegetation database: Vegetation Database of Dry Grasslands in the Southeast Romania, Vegetation Database of the Dry Grasslands from the Transylvanian Basin, Oak-Hornbeam Forest Database of the Transylvanian Basin, Database Beech Forest from Romanian Carpathians and Vegetation Database of Oak Forest of Romania. In the first of these, Vegetation Database of Dry Grasslands in the Southeast Romania, alpine grasslands of Bucegi Massif are included (RO1567-IBB01/2012). For the beginning they were included 72 releves of natural and seminatural grasslands of studied teritorry. There are releves from Koelerio-Corynephoretea (incl. Sedo- Scleranthetea, Festucetea vaginatae), Elyno-Seslerietea (Seslerietea albicantis, Kobresio myosuroidis-Seslerietea caerulea) and Festuco-Brometea classes.

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ECOLOGY, TAXONOMY AND NATURE CONSERVATION

POSTERS

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64

LONG-TERM ECOLOGICAL RESEARCH OF ZOOPLANKTON COMMUNITY IN LAKE ROU (DANUBE DELTA)

L. PARPAL
Institute of Biology Bucharest, Romanian Academy

The pressure of natural and anthropogenic factors on shallow lakes in the Danube Delta, resulted in the completion of several distinct stages of trophic status succession. The responsible factor for this process is the dynamics of nutrient concentrations. Their increased intake favored the replacement of macrophyte type producers dominance with algal type. The consequence was powerful "water bloom"phenomena due to excessive algal development. These processes, led to structural and functional changes of entire foodweb. In terms of trophic levels, the evolution of Danube Delta lacustrine ecosystems was characterized by three significant stages: the reference period (before 1980) characterized by meso-eutrophy; the hypertrophy period with maximum anthropogenic impact (1981-1990), and the recovery period (after 1990) defined by a decline in trophic level. Between 1975-2011, the zooplankton dynamics of Lake Rou, follows the general trend of deltaic shallow lakes. In the reference period the species richness was much higher than in eutrophic-hypertrophic period, while the values of abundance, biomass and productivity are lower (194 ind/l, 860 wet weight g/l, 104 wet weight g/l/24 h, respectively). In the second period, the species richness values were diminished. The abundance, biomass and productivity recorded significant increases (668 ind/l, 7231 wet weight g/l, 842 wet weight g/l/24 h, respectively). In 2011, the Lake Rou shows a special case. All quantitative parameters reached the highest levels (1008 ind/l, 19338 wet weight g/l, 2359 wet weight g/l/24 h, respectively). Future research will elucidate the causes. The question is: can this be a new stage of hypertrophy in the shallow lakes of Danube Delta? To detect the differences among years based on specific richness, abundance, biomass and productivity of zooplankton, a single factor ANOVA test was applied. The obtained values indicate statistically significant differences (p<0.05) for all parameters. This fact confirms the variations of zooplankton parameters in different trophic conditions, in 1975 2011 interval. The Bray Curtis analysis was used to assess the similarity degree among the annual values of zooplankton biomass. Between 2011 and 1983 (the eutrophy peak) a high similarity (68%) was noticed. By applying the simple correlation between planktonic producers and consumers in 2011, a strong relationship was found, while the Pearson correlation between phytoplankton and zooplankton abundance was very highly significant (R2=0.588778,
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P=0.000841). The zooplankton biomass is highly significant related to phytoplankton biomass (R2=0.550458, P=0.001541). Ecological research conducted between 1975-2011, lead us to conclude that the zooplankton dynamics is directly dependent on ecosystem trophic status.

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SPATIAL DISTRIBUTION OF THE ROTIFERS DIVERSITY IN SFNTU GHEORGHE BRANCH

L. FLORESCU
Institute of Biology Bucharest, Romanian Academy

Rivers are dynamic ecosystems, highly complex, which provide many goods and services. Due to their importance, the river systems played a fundamental role, being in permanent anthropogenic changes simultaneous with society development. During the 80s, in Sfntu Gheorghe branch (Danube River), major hydrotechnical changes in the original structural configuration occured. In order to meliorate the navigation, these works consisted in straightening out the branch, by cutting off six meanders (the bends areas of the river) and consequently shortening the river with around 32 km. This kind of human impact induced severe effects on the environmental conditions (e.g. water flow, turbidity, water residence time, riverbank morphology, erosion processes). Thus, the technical engineering changed the relationship between biocoenoses and hydrogeomorphological conditions by creating new areas (new canals) with different features from the rest of the branch. Our research conducted between 2008 and 2010 has shown the consequences of these works, such as decreasing of the species number, density and biomass. Earlier research results at Ceatal Sf Gheorghe (1981-1985, 1991-1992) emphasized 105 species of rotifers, while the recent results point out a decrease to 83 species. The annual K dominance analysis highlights the following species: Brachionus calyciflorus dorcas Gosse, Keratella quadrata Gosse, Synchaeta oblonga Ehrenberg; they have presented among the highest values of abundance in the entire branch. The seasonally graphic representation of K dominance curves for each area illustrates the diversity dynamics during the research. The highest diversity was found in the autumn of 2009, for all three types of ecosystems: meanders, natural sectors and canals, while the autumn 2008 was the most unfavourable season for rotifers development for the entire Sfntu Gheorghe branch. Despite the fact that the diversity indices are widely used in ecological research, comparing the different sites or samples may not be so accurate. One sample may contain a higher species richness while the other has a better Shannon Index. These issues can be solved by using in addition, a diversity ordering method, as Rnyi diversity profiles. Rnyi diversity profiles easily order from high to low the diversity for each area, giving a general view for the entire survey period. The lowest curve of the diversity profile belongs to the canals representing the areas with smalest diversity of the Sfntu Gheorghe branch. Instead, the natural sectors and meanders
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curves tend to overlap when reach the value of 1, making impossible to distinguish the diversity differences between them. For confirmation, we applied oneway ANOVA single factor analysis using the Shannon Wienner, Simpsons dominance and Evenness values. The results have shown no statistically significant difference between natural sectors and meanders (P>0.05).

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LINKING THE STRUCTURE OF THE WEED-BED FAUNA COMMUNITIES AND THE ENVIRONMENTAL VARIABLES IN SFNTU GHEORGHE ARM OF THE RIVER DANUBE
E. RADU1, A. COMAN1 and G. RNOVEANU2
1

Institute of Biology Bucharest, Romanian Academy; 2University of Bucharest, Faculty of Biology, Department of Systems Ecology and Sustainability

The Danube Delta an important ecological system belonging to the World Natural Heritage has been transformed over time by different types of human activities, including the development of hydrotechnical works along the Sfntu Gheorghe arm of the River Danube, particularly during the period 1983-1989. The aim was to shorten the navigation route with about 32 km and increase the fluid and the solid flows (Popa, 1996, Vdineanu, 2004). Along the Sfntu Gheorghe arm, six meanders were cut and new canals were built, modifying its morphology with important consequences on the structure and functioning of aquatic communities. The effects of the past hydrotechnical works are still poor documented in the scientific literature. The research project entitled The impact of hydrotechnical changes on the ecological systems of the Sfntu Gheorghe arm in the wider context of the sustainable development (2008-2010, Romanian Academy) has attempted to fill this gap in knowledge. Understanding the communities mechanisms of response and identifying the main factors that induce and control the structural differences of the trophodynamic modules of the weed-bed fauna in different habitats along the river arm represented important objectives of the research to which this paper is addressed. In order to capture and characterize the weed-bed fauna response to the particular conditions of the habitats in different morphological sections of the watercourse, the communities structure was characterized and related to the variation of physico-chemical parameters. Multivariate statistical analysis allowed the identification of key control variables explaining the spatial and temporal variability of the studied communities. The results have shown the following: numerical density, taxonomic richness and diversity of the weed-bed communities are generally higher in the free-flowing sections (reference) and in the cut meanders of the Danube arm, compared with those in the newly built canals; greater morphological complexity of the aquatic or riparian plant associations (as well as of their component taxa) in the reference sections and meanders could have been a better premise for the

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development of the associated weed-bed fauna communities in these areas; some of the monitored environmental variables are well explaining the inter-seasonal and interannual variations of the weed-bed fauna communities structure (e.g. the water temperature and the concentrations of the dissolved oxygen in the water column) while others are explaining - in certain proportions - the variability of these communities structure in space (e.g. the total phosphorus and total nitrogen, the dissolved organic carbon, the organic matter and the chlorophyll in the water column, and the water transparency).

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LONG-TERM DYNAMICS (1993-2011) OF THE BENTHIC MACROINVERTEBRATES COMMUNITIES IN THE INNER DANUBE DELTA
C.C. BRSAN1, G. RNOVEANU2, E. RADU1, G. IGNAT3 and S. CRISTOFOR3
1

Institute of Biology Bucharest, Romanian Academy; 2University of Bucharest, Faculty of Biology; 3 University of Bucharest, Brila Ecological Research Station

Benthic macroinvertebrates, consisting in sediment-dwelling species, are major, constant and dominant components in freshwater systems. They are significantly contributing to energy flow at the sediment-water interface as well as in the nutrients cycling, sustaining the ecosystem services provision. Benthic communities are also a cost-effective and commonly use indicators in long-term ecological research and water quality monitoring programs. They are particularly important in highly eutrophic systems, characterized by high densities of energy and mass flows from sediments to the benthic fauna and consequently, along the higher trophic levels. The Lower Danube Wetlands System (LDWS) that includes the Inner Danube Delta (IDD) is one of the largest and still well preserved wetlands systems in the world, covering a total area of 8.307 km2. Compared to other weighty wetlands, the boast of this area is given by the largest expanse of reed bed and pelican colonies. Nevertheless, in the last three decades of the 20th century, transition towards hypertrophy of the aquatic ecosystems due to increasing anthropogenic disturbance, has resulted in important changes of both water quality and structure of the food webs, and has affected the functions of the LDWS and the ecosystem services it provides (buffering capacity for nutrients and pollutants, part of regional and global biodiversity, production). Benthic samples were analyzed over the 19 years period (1993-2011) in two shallow lakes (lakes Chiriloaia and Fundu Mare), representative for the IDD. Communities taxonomic composition, macroinvertebrates total densities as well as diversity and similarity indexes were used in order to assess in time and space variations within the macroinvertebrate communities structure. Results revealed a simplified structure of the studied communities. Eleven taxa were identified (Nematoda, Gordiidae, Oligochaeta, Hirudinea, Gastropoda, Amphipoda, Ephemeroptera, Trichoptera, Chironomidae, Ceratopogonidae, Tipulidae). The total abundance recorded by the 11 taxa in L. Chiriloaia varied between 777 and 10.134 ind./m2. In L. Fundu Mare, the abundance of 9 taxa recorded ranged between 3.521 8.750 ind./m2. Oligochaetes and chironomids were frequently co-dominants in both,
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number and biomass. The low values of Shannon diversity index (0.70 to 1.17 in L. Chiriloaia and 0.37 to 0.91 in L. Fundu Mare) and Pielou equitability (0.38 to 0.67 in L. Chiriloaia and 0.26 to 0.63 in L. Fundu Mare) suggest an advanced simplification level of benthic communities under the pressure of hypertrophy.

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THE DYNAMICS OF CYANOBACTERIAL BLOOMS IN LAKE ROU REFLECTED IN LONG-TERM STUDIES

M. MOLDOVEANU
Institute of Biology Bucharest, Romanian Academy

Cyanobacteria became increasingly dominant as concentrations of total phosphorus and total nitrogen increased during the eutrophication of Danube Delta ecosystems. The temporal dynamics of Cyanobacteria blooms is variable, with a wide range of possible biological impacts including potentially toxic effects and impacts on food web functionality. As large, inedible algae, they induce a bottleneck in the carbon and energy flow of the plankton food web. Taking into account these reasons, our work aimed to highlight the dynamics of cyanobacterial blooms in Lake Rou at a wider temporal scale (1975-2002) and update the existing information by presenting the evolution of Cyanobacteria in the year 2011. The Lake Rou is representative for the lacustrian-type of ecosystems, being the largest lake (1375 ha) of the fluvio-maritime delta. The hypertrophy, a characteristic stage of the deltaic ecosystems in the critical period after 1980, involves increased concentration of nutrients and also the nitrogen factor limiting role of phytoplankton development. In the new environmental conditions, the chance of intense proliferation of cyanobacteria group increased, becoming dominant in the ecosystem. After 1980, yearly averages of biomass exceeded 10-30 times the "water blooms threshold. Monthly averages in July-September period exceeded 100-125 times the mentioned limit. The spectacular values of cyanobacterial abundance and biomass between 19801990 triggered also the dominance of potential toxic species. The Mc Naughton and Wolf dominance index of phytoplankton biomass between 1982-1985 exceeded the threshold of 0.5 in all seasons. In general, the dominant species belong to toxin-forming Cyanobacteria: Microcystis aeruginosa Ktzing, Microcystis flos-aquae (Wittr.) Kirchner, Microcystis pulverea (Wood) Migula, Anabaena hassalii (Ktz.) Wittrock, Anabaena scheremetievi Elenkin, Oscillatoria tenuis Agardh. The edibility degree of these species is very low, the herbivorous zooplankton being forced to feed on detrito-bacterial aggregates to be able to survive. While in 2001 the diatoms and cyanobacteria biomass decreased, especially in the warm seasons, and the ecosystem tended to reach a functional regime, more stable, due to a lower nutrient pressure, in 2011, the high values of phytoplankton biomass (78.72 wet weigh mg-l) have shown new eutrophication signals, including intense cianobacterial blooms episodes.
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THE DYNAMICS OF EXTRACELLULAR - GLUCOSIDASE AND ALKALINE PHOSPHATASE IN AQUATIC ECOSYSTEMS FROM THE DANUBE DELTA IN 2011-2012 INTERVAL
I. PCEIL
Institute of Biology Bucharest, Romanian Academy

In the aquatic ecosystems most part of the organic matter consists of polymeric macromolecules that, due their size, cannot be used directly by heterotrophic microorganisms (Chrst, 1991). In order to use the energy stored in this macromolecules, heterotrophic microorganisms produce extracellular hydrolytic enzymes able to catalyze dissociation of macromolecular compounds to their monomeric components outside of the cells. The resulted monomeric compounds can be directly transferred from the environment to microbial cells. Extracellular enzymatic hydrolysis is the first step in mineralization of most of the polymeric constituents included in the composition of the organic matter (polysaccharides, proteins, organoesters) by heterotrophic microorganisms, playing therefore an important role in the transfer of matter and energy through aquatic ecosystems (Harbott et al., 2005). Nowadays, the recording of enzymatic activities can be used as a method to assess the intensity of metabolic activity of the microbial communities. This paper presents some characteristics of two enzymatic activities -glucosidase and alkaline phosphatase - from water and sediments of two aquatic ecosystems from Danube Delta: Rou Lake and Mndra pond. -glucosidase is a specific enzyme that act in the final step of cellulose degradation (Nicolescu et al., 2000), while alkaline phosphatase is a nonspecific enzyme that catalyzes the hydrolysis of a large variety of phosphate esters (Janson et al., 1988). Both -glucosidase and phosphatase activities recorded high values in water column and sediment. In water column the average values for glucosidase activity were 855 nmol p-nitrophenol L-1h-1 in Rou Lake and 630 nmol p-nitrophenol L-1h-1 in Mndra pond, while for alkaline phosphatase the average values were 1080 nmol p-nitrophenol L-1h-1 in Rou Lake and 795 nmol p-nitrophenol L-1h-1 in Mndra pond. In sediment the high average values were, also, recorded in Rou Lake - 8330 nmol pnitrophenol g-1h-1 for -glucosidase and 6470 nmol p-nitrophenol g-1h-1 for alkaline phosphatase. The seasonal dynamics of -glucosidase activity shows maximum activity in autumn, after the fall of vegetation, while for alkaline phosphatase the maximum activity was reached in summer, both in 2011
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and 2012. In sediment, the highest values were recorded in autumn for both -glucosidase and alkaline phosphatase activities. Annual average values of the two studied enzymatic activities were higher in 2012 than in 2011 for both water and sediment samples. In aquatic ecosystems microbial enzymatic activity depends on a multitude of abiotic and biological factors (temperature, pH, oxygen content, phytoplankton blooms, macrophytes development, etc.) that may influence the amount of available substrate as well as the hydrolysis kinetics. Future investigations will be focused on the correlations between the extracellular enzymatic activity and the dynamics of the other environmental parameters.

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STRUCTURAL-FUNCTIONAL PARTICULARITIES OF THE ECOSYSTEMS OF A PLAIN CATCHMENT AREA (CASE STUDY)

O. CIOBOIU1 and G. BREZEANU2
1

The Oltenia Museum, Nature Sciences, Craiova; 2Institute of Biology Bucharest, Romanian Academy, Bucharest

Generally, a catchment area includes, due to its dimensions, a vast diversity of geographical, hydrological, physical, chemical, and ecological structures. Thus, taking into account the hierarchy of the river systems and the classification established by Strohler (apud, Ujvary, 1959), rivers can be of the 1st, 2nd, 3rd, and 4th order, defining in this way the complexity of the river system within a hydrographical basin. Starting from the definition of a river system, we bring to your attention a more or less particular case, due to its structural-functional characteristics, namely the case of the Preajba Valley system. Located within a plain area, the system that belongs to the Jiu hydrographical basin, covers a surface of 30 Km2. This small surface includes a great variety of ecosystems: hillocks covered by hay fields and pastures, agricultural fields and a complex hydrographical network springs, streams, rivulets, swamps, and small reservoirs. This complex organization induces the structural-functional characteristics and complexity of the ecosystems (Fig. 1).

Fig. 1. Draft of the Preajba Valley hydrographical basin from Oltenia Plain (Cioboiu 2002).

The composition and distribution of the biocoenotic and population structures of the Preajba Valley system functions in accordance with the
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particularities of the ecosystem organization. Thus, in the area of the rhitron (including the springs and rivulets), there have been identified species of Diatomeae, Ciliata, Testaceae, Ostracoda, Gamarida, Chironomida, Ephemeroptera, Heteroptera. In the area of potamon (the area that covers the hilly and plain sector of the Prejba Valley hydrographical basin), the structure of the biocoenosis is made up of representatives of all groups specific to the aquatic ecosystems (pelagic and benthic communities). In case of phytoplankton, there have been identified more than 70 alga species and in case of zooplankton, 65 species of Ciliata, Rotiphera, Cladocera, Copepoda. Zoobenthos is made up of 13 large groups of invertebrates, some comprising a great number of individuals (Chironomida 4,600 ind./m2, Ostracoda 1,750 ind./m2, Ephemeroptera 213 ind./m2, Plecoptera 226 ind./m2) (Table 1).
Table 1. Zoobenthos structure (mean values)

Taxonomic group Chironomidae Gamaridae Ostracoda Heteroptera Gastropoda Bivalvia Cladocera Copepoda Efemeroptera Plecoptera Isopoda Oligocheta Hirudinea TOTAL

No. of individuals/sq. m 2,746.5 1,753 353.5 186.5 293 26.5 113.5 160 213.5 226.5 53 26.5 20 6172

Abundance % 38.35 18.8 8.65 3.7 5.35 0.25 3.7 5.25 5.25 7.45 1.45 0.95 0.85 100.0

Frequency % 100 16.5 66 100 66 16.5 16.5 16.5 66 16.5 49.5 16.5 33

Gastropods represent one of the main groups of the benthos structure (there have been identified 18 species). In case of the reservoirs, the ichtyofauna is represented by 10 species, cyprinids being dominant. The Preajba Valley system is atypical due to its geographical location (plain area) and small surface (30 Km2), but it gathers all the structuralfunctional characteristic of a large dimension hydrographical system.

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MODERN METHODS USED IN TAXONOMY AND ECOLOGY OF SOIL FAUNA

M. MANU
Institute of Biology Bucharest, Romanian Academy

In order to clarify some systematic problems and to highlight the ecological role of the soil fauna groups, in the last decades, new research methods have been used. Traditional taxonomy techniques are now improved with modern methods as: field emission low temperature scanning electron microscopy (FE-LTSEM); digital illustration techniques; cybertaxonomy. Molecular applications have had particular success in facilitating the identification of taxonomically difficult species, in establishment of genetic diversity, in genomic mapping, in understanding of population structures, elucidating phylogenetic relationships. Modern ecological methods are based especially on molecular analysis and on statistical ones. Molecular analysis could be used in revealing the trophic preferences. Approaches with a polymerase chain reaction (PCR) step to amplify dietary DNA are highly effective. Analysing the trophic interrelationships in soil food web was made using modern techniques as: stable isotope analysis (N15/N14; C12/C13); fatty acids (FAs); the combination of stable isotope and fatty acid analysis with gas-chromatographycombustion-isotope-ratiomonitoring-mass-spectrometry (GC-C-IRM-MS); immunological techniques, which use the specifity of antibodies to detect an antigen; immuno assays and ELISA (enzyme linked immuno-sorbent assay), FISH (fluorescence in situ hybridization). Other modern molecular methods are used to establish indicators for soil quality such as: terminal-restriction fragment length polymorphism (T-RFLP) analysis and the metallothionein system, which is one of the best studied stress response systems in the context of heavy metals. Statistical modeling programmes as: Statistica, Canoco, Infostat, Mathcad, Systat, BiodiversityPro, SAS, SPSS, were the most used for the last ten years. A new used programme is R, which provides a wide variety of statistical and graphical techniques. All these modern research methods provide a more detailed and complex study on soil fauna, making possible a better highlight of their ecological role and of their ecological services.

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METHODS USED IN THE ECOLOGICAL RESEARCH OF SOIL MITES (ARACHNIDA - ACARI) HISTORY AND PERSPECTIVES
V. HONCIUC
Institute of Biology Bucharest, Romanian Academy

The work is part of the research project-Research on the relationship between biodiversity and ecosystem function in some of the Romanian Carpathians; phase 2012-Identify ecosystems which will be considered in the study and calibration methodology of work, reference database. In this context the work aimed at a presentation of the research methods used in environmental research, soil fauna in different ecosystems from Romania, with reference to the similar research carried out in similar areas in Europe. As is known, along with aspects of a task for the evaluation of the hypothesis as of suprasistems issues and biological systems that integrates and soil fauna, is using the most appropriate methods for quantitative and qualitative assessment of the taxonomic groups of soil fauna of both methods rely on statistical calculation. Through the use of quantitative methods in research verified made far get quantitative parameters as: numerical abundance, density, and biomass; and through the use of qualitative methods to obtain structural parameters: coefficient of variation, relative abundance, dominance, frequency, number the vertical distribution, dispersion, diversity, as well as functional indicators such as productivity, production and energy budget. These methods have been used in research achievement until now in all kinds of ecosystems in Romania. By using the methods he showed with the highest fidelity the structure of soil fauna in the ecosystems. On the basis of the results obtained in the research conducted so far, we plan to test them using the methods of modern ecology (mathematical modeling, molecular methods, etc.) that will elucidate questions concerning ecology and the evolution of biological system and aboveindividual, investigated, soil fauna.

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THE COMMUNITIES STRUCTURE OF INVERTEBRATES FAUNA FROM RAPE AND ALFALFA CROPS
C. FIERA, S. MAICAN and D. PURICE
Institute of Biology Bucharest, Romanian Academy

Our investigations have been conducted under a bilateral project between Romania and Republic of Moldova, focusing on invertebrates knowledge from rape (Brassica napus L.) and alfalfa crops (Medicago sativa L.) from two localities: Singureni (Giurgiu County) and Frumuani (Clrai County). This study aims to establish the structure of arthropod communities (Collembola, Coleoptera), to identify both the useful and crop pest arthropods and finally to characterize the invertebrate communities in terms of their numerical abundance. The entomofauna of alfalfa and rape crops was collected with an entomological net. Establishing the quantitative and qualitative structure of the epigean fauna was performed using pitfall (Barber) traps filled 2/3 with 4% formaldehyde solution. Springtails were sampled with a Macfadyen cylindrical corer. 19 families of beetles were identified in the studied crops. The largest number of families was encountered in the cultures of rape (at Frumuani, 12 families and 15 families at Singureni), while in the alfalfa crop from Singureni we found only 11 families. In the culture of rape we could determine species belonging to Phyllotreta and Chaetocnema genera (Chrysomelidae: Alticinae). Propylaea quatuordecimpunctata and Coccinella septempunctata (Coleoptera: Coccinellidae), identified in the alfalfa crop from Singureni are known to be the most common species of predatory coccinelids that are characteristic to this crop. Gonioctena fornicata (Coleoptera: Chrysomelidae), from alfalfa crop was also present in our samples, known as a crop pest. In terms of the average density number, the highest values were registered in the rape crops (16.5 ind./u.p at Frumuani and 48.22 ind./u.p. at Singureni), and in the alfalfa crop, at Singureni only 711. ind./u.p. In the rape culture from Frumuani, eudominant were: Carabidae (58.59%) and Staphylinidae (10.10%), dominant were: Dermestidae (8.08%), Scarabaeidae (7.07%) and Chrysomelidae (5.05%), the other left families of beetles, with the relative numerical abundance between 1.01% and 3.03% were classified as subdominant (Bostrychidae - 3.03%), recedent (Corylophidae and Endomychidae - each with 2.02%) and subrecedent (Cantharidae, Curculionidae, Coccinellidae, Byturidae - each with 1.01%). As we expected, most of the families of Coleoptera found in this culture, had a status of
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accidental groups, only the family of Carabidae (with a frequency of 83.33%) was constantly present, relatively constant were the families of Dermestidae, Staphylinidae and Scarabaeidae (each with 50% as frequency), the rest of beetles were accessory or had an incidental status in the integrating biocenosis. In the rape culture from Singureni, we could identify similar features as found in the rape culture from Frumuani, both in the structure of dominance and constance classes. Here the eudominant families were: Carabidae (32.99%), Dermestidae (18.89%), Chrysomelidae (16.58%), Silphidae (15.67%) and the left 11 families were subdominant (Byturidae - 4.6 % Curculionidae - 4.37%, Staphylinidae - 2.3%) and subrecedent (another 8 beetle families). Beetle fauna was within four classes of constancy; constant were Curculionidae (88.89% as frequency) and Carabidae (77.78% as frequency), relatively constant were Staphylinidae and Silphidae (with frequencies of 66.67%) and Byturidae (frequency - 55.56%). The accessory class was represented by 4 families and the accidental class by 5 families. Regarding the alfalfa crop, eudominant, in terms of their numerical abundance, were the families Curculionidae (39.09%), Carabidae (26.56%) and Byturidae (12.5%), the family Silphidae - dominant (9.37%) and the left 7 families were subdominant (Tenebrionidae) and recedent (5 families). 34 species of carabid beetles (Fam. Carabidae) have been identified in the studied crops. We notice the presence of predatory species, Agonum dorsale at Frumuani and Carabus glabratus at Singureni, these two species were eudominant in terms of their density number and frequency in the rape crops in both localities. In the alfalfa crop from Singureni we also identified Cicindela germanica, known as a predatory species, eudominant in number density, but with an accidental status, found together with Pterostichus cylindricus and Callistus lunatus, these species are also part of the predatory invertebrate epigean fauna. 15 species of Collembola were identified, belonging to 7 families (Hypogastruridae, Onychiuridae, Neanuridae, Isotomidae, Entomobryidae, Tomoceridae and Sminthuridae). Hemisotoma thermophyla (Isotomidae), Entomobrya handschini and Lepidocyrtus paradoxus (Entomobryidae) were the most dominant species of the alfalfa crop. Protaphorura sakatoi (Onychiuridae) and Orchesella flavescens (Entomobryidae) were dominant in the culture of rape from both localities. From the insects collected by sweeping the vegetation with nets, the most dominant were the beetles, from the rape culture and in the other one crop the most dominant, were the dipters, followed by Homoptera and Hymenoptera. Our acquired data, from numerical density of invertebrates populations point of view and their spectrum of activity represent a major economic importance in reducing the attacks caused by pests naturally.
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THE EPIGEIC INVERTEBRATES IN THE ECOLOGICAL STUDIES IN ROMANIA. METHODS OLD, CURRENT AND PERSPECTIVES
D. PURICE
Institute of Biology Bucharest, Romanian Academy

In the ecological studies, invertebrates are a major part both as structural and functional contribution in the integrating coenoses. This is why in the ecological studies; the chosen methods of study are decissive for the accuracy of studys purpose. There are two main directions of study for the structure of invertebrates populations: qualitative and quantitative approach. The quantitative approach involves fauna sampling, using various protocols, during a determied period of time (enough to provide a dataset signifficant statistically). This approach is used with restrictions in protected areas requiring non-destructive sampling methods. In the qualitative approach the methods are more subjected to the sampling error risk (it is about the time and intensity sampling, when more than one person is taking the samples). In this cathegory of methods, the light traps and feromone traps quite rare used, because the field trips are time limited and because they are quite costy. The same reasons are valid for the method of markingrecapture necessary to establish the dispersal power and the fauna potential in recolonisation of vairous disturbed areas. These methods cover the coenoses structure and the methods already used are classical ones but cover all the aspects necessay in the ecological studies. A lack in the ecological studies is represented by the ex-situ methods related to interspecific relationships, trophic structure and energetic budget. These methods require expensive equipment (respirometers, growing chambers) and are long-time studies. Another necessary approach in the functional ecology studies is the molecular analysis of invertebrates material, to confirm the species identity (in case the classical taxonomical tools are not enough) and to establish the gut content (the food spectrum) of some generalist consumers. These molecular analyses are also costy and require extra- training and knowledge in this domain. As a conclusion, the classical methods are still ued successfully and the lack of practice in the ecosystem functioning studies (energetic budget, secondary production & productivity, molecular analysis) are critical elements necessary in the ecological studies in order to draw some applicative conclusions for environmental protection and environmental management.
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THE ECOLOGICAL SUCCESSION IN TERMS OF THE TROPHIC STRUCTURES IN FOREST PLANTATIONS

D. PURICE1 and O. CIOBOIU2
1

Institute of Biology Bucharest, Romanian Academy; 2Muzeul Olteniei Craiova

It is known that plantations, as the implementation model they attempt to mimic as accurately as possible the patterns of the natural forests. But regarding as succession, the plantations have a go - the time at least different in comparison with the ecological structures they imitate. At the epigeous level, the invertebrate fauna answers / reflects in a particular way the succession processes at the local scale. To illustrate this idea, an example is the short study (during the summer season) on a group of plantations of 4-9 years old from the Cri and Mure basins, in comparison with a much older plantation from the Bucharest surroundings (Codrii Vlsiei). First information show that at the supraspecific level, the epigeic invertebrates have generally the main composition, regardless the plantations age. The groups of invertebrates exclusively fitophagous are dominant numerically and quite constant during the summer season. There are also more or less signifficant differences in the interspecific relationships (trophic), depending this time on the plantations age. It is about the predator-prey relationships (Carabidae-Gasteropoda, Carabidae-Isopoda) and interspecific competition Carabidae-Araneae. In this last case, the correlation coefficient (r) shows the interspecific trends at the middle of the lifecycle for the most of the studied invertebrates. The preliminary data allow some partial conclusions: the differences in the structure and relationships are hard to be noticed at short time intervals (4-9 years, talking about the sites from Cri and Mure basins). To have more precise and complex conclusions on the succesional evolution in plantations, further studies are necessary in similar sites but with different ages. For now, we can say that at a scale of 10 years, the evolutionary differences, even noticed, are far from being signifficant at coenotic level.

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SPECIFIC DIVERSITY OF ENTOMOFAUNA (COLLEMBOLA, COLEOPTERA) FROM SRATA - MONTEORU FOREST (BUZU COUNTY)
S. MAICAN and C. FIERA
Institute of Biology Bucharest, Romanian Academy

The paper presents data on specific diversity of the entomofauna (Collembola, Coleoptera) in the forest of Querco petraeae-Carpinetum from Srata-Monteoru (Buzu county), in the context of research on the taxonomy and chorology of the flora and fauna species from Romania (made in the project no. RO1567-IBB03/2012). The taxonomic studies on coleopterogical material collected between 2011and 2012 revealed the presence of some taxa belonging to Chrysomelidae, Cetoniidae, Cerambycidae, Buprestidae, Coccinellidae, Silphidae, Staphylinidae and Scarabaeidae families. The leaf beetles (Chrysomelidae family, exclusively phytophagous) were represented by species from the following subfamilies: Chrysomelinae (Gonioctena fornicata, Gastrophysa polygoni, Chrysolina varians common species on Hypericum sp., in the hilly and mountainous regions), Cassidinae (Cassida viridis, Cassida vibex), Cryptocephalinae (Cryptocephalus quinquepunctatus a rare species in the Romanian fauna) etc. From the Cerambycidae family (longhorned beetles) the following species were recorded: Xylotrechus antilope, Anisorus quercus, Anoplodera sexguttata ab. exclamationis, Judolia cerambyciformis and Cerambyx scopolii. According to IUCN Red List categories and European Red List of Saproxylic Beetles (Nieto & Alexander, 2010), the species Gnorimus nobilis (Cetoniidae) and Cerambyx scopolii (Cerambycidae) are classified as Least Concern (LC). The Class Collembola was represented by species belonging to Neaunuridae (Neanura minuta, Thaumanura carolii, Deutonura albella etc.), Entomobryidae (Lepidocyrtus paradoxus dominant species, Orchesella pannonica, O. multifasciata) and Isotomidae (Isotomiella minor) families. Among valuable faunistical elements, it is noted the presence of Lucanus cervus (Coleoptera: Lucanidae) - protected species listed in the Council Directive 92/43/EEC on the Conservation of natural habitats and of wild fauna and flora and in the Government Emergency Ordinance no. 57/2007 on the regime of natural protected areas, the conservation of natural habitats, wild fauna and flora. Also, Orchesella pannonica (Collembola) is recorded for the second time in the Romanian fauna.
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BIOGEOGRAPHYCAL DATA ON MOLLUSKS SPECIES IN DOBROGEA, ROMANIA

M. AXINI1 and FAWAZ AL-AZKI2
1

Monachus Group of Scientific Research and Ecological Education; 2 Faculty of Sciences, Department of Geology, University from Lattakia, Syria

Dobrogea region is located on the northern Balkan Peninsula in southeastern Central Europe (44017`03,77``N, 28021`53,27``E). It occupies an area of approximately 23.142 km2, of which 15.570 km2 are located in Romania (making up 6.52% of the total area of Romania) and 7.572 km2 in Bulgaria. By its characteristics different from the rest of the country (geographical position, presence and influence of Black Sea climate and soil structure, history and evolution of land with consequences for the history and evolution of its biodiversity), Dobrogea is an geographical "entity" characterized by a mixture of flora and fauna elements of Southern, Eurasian, Ponto-Caspian, European origin, etc.; many species are rare or endemic to this geographical region, or at limit of areal. The plenty and the diversity of original ecological and genetic fund which Dobrogea was so generous endowed, it is more expressive by the fact that Romania is only 2.36% of Europe surface. In this geological, geographical and ecological context, the species of mollusks have appeared and developed. Currently, 309 living species occurs in Dobrogea, which inhabit both terrestrial, aquatic, underground and marine environmentals. Of these, many species are endemic or relict. The Danubian and Pontic species of gastropods - Esperiana esperi (FRUSSAC 1823) and Esperiana acicularis (FRUSSAC 1829), both the Fam. Thiaridae (Order Mesogastropoda); they are Pontian-Southeast European relicts. Identification of these species in continental zones of Dobrogea, as Conacu-Negreti Valley, demonstrates that these areas are former branches of the Danube River which connects between it and Black Sea, or the presence of millions years ago of ParaThethys Sea, and later, of Sarmatian Sea. This is demonstrated by geology and geography data, as well as by our field observations (the form of the canyons and of the valleys). On limestone slopes with rocks to day, on plateaus, hills and canyons in the south-west of Dobrogea is worth mentioning many specimens of the gastropod Helicella obvia dobroudschae (CLESSIN 1886), Fam. Helicidae (Order Stylomatophora), xerophilous species, endemic to Dobrogea. Chondrula tridens (O. F. MLLER 1774), Zebrina detrita (syn. Helix detrita) (MLLER 1774), and Zebrina varnensis (L. PFEIFFER 1847), all
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three species of the Fam. Enidae (Order Stylomatophora), are found on the limestone walls with rocks to day, in less numerous specimens. The presence in areas without forests of Zebrina detrita (syn. Helix detrita) large snail, and Chondrula tridens (relatively common species in the foliage of oak forests) demonstrates the existence of pubescent oak forests (Quercus pubescent) in these zones of Dobrogea, which subsequently disappeared from various natural and man-made causes (for example, formation of the lake from the Conacu-Negreti Valley, recent geological time lake, formed by natural damming result of catastrophic fooding). These are xerophilous species belonging to the genera of West Asian origin. Zebrina varnensis is a xerophilous species, highly resistant to drought, found on sunny slopes covered with herbaceous vegetation, in sandy areas. This species is a regional endemism spread of the Black Sea coast of Romania and Bulgaria. Presence of this species in continental zones of Dobrogea demonstrates once again the same thing above mentioned for Esperiana genus. The analysis of the zoogeographical elements of mollusks from Dobrogea again demonstrates that this region is a mixture of Central and South European-Anatolian, European, Dobrogean Endemic, Regional Endemic, European-Turanian, Holarctic, Holopalearctic, North Mediterranean, North Mediterranean-Turanian, Mediterranean-Lusitanian, Pontian, Ponto-Mediterranean, Pontian-Southeast European, Caspian Relict, Preglacial Relict, South European-Turanian, Transpalaearctic, West and Central Eurosiberian, West and Central Palearctic, West-Palearctic, etc. elements. In ecological terms, living mollusks in Dobrogea are: calciphilous, crenobiont, eurybiont, hygrophilous, of freshwater, lithophilous, mesophilous, pelophilous, phytophilous, pomatophilous, rhithrophilous, stenoepibathic, of stagnant water, terrestrial, trogloxene, xerophilous, -mesosaprobic, - mesosaprobic, o--mesosaprobic, etc. species. The presence of millions years ago of ParaThethys Sea, and later, of Sarmatian Sea led to the formation of Dobrogea territory, an area predominantly limestone. Fossil marine species are present (for example, Mactra sp.) in the rocks from the lakes, in the canyons and ravines, on limestone walls and on Sarmatian coasts with limestone rocks to day. The presence of these species help us to reconstruct of paleo-environment and natural history of extinct and existing species of mollusks in Dobrogea.
T

86

THE GRAYLING (Thyallus thymallus L., 1758), A DECLINING SPECIES

I. CRISTEA
Institutul de Cercetari si Amenajari Silvice

The paper presents the results of the research made between 20092011 with the main purpose of carrying out estimate studies on the distribution area of the species in the Romanian rivers and of the habitats populated by the grayling. Although they have registered an increasingly decline in number and a continuous regress of the distribution area, the grayling populations still maintain in some rivers whose hydrological regime has not been modified by anthropic activities. However, recent observations on grayling populations and habitats certify the decline of the populations in many of the mountaineous rivers where this species was traditionally present. The main reason for the reduction in number or even the disappearance of the species is the hydropower works carried out in the upper segments of the rivers by which the adults migration route towards the breeding areas is interrupted. The regulation of the river streams and the embankment represent other reasons which cause important changes in the structure of ichtyofauna, reducing the area or access to the habitats favorable to the species and exposing the populations to additional selection pressures. Last, but not least, climate changes, and especially the dryness of the climate have caused the flow reduction in the majority of rivers and the restriction of the optimal habitat for the grayling. The site works undertaken have consisted in the inventory and monitoring of ichthyofauna species in the habitats populated by the grayling in order to estimate the population parameters. Works for the description of the aquatic habitats favorable to the grayling, monitoring of water quality, characterization of the trophic structure of the rivers, identification of the main sources and forms of water pollution have also been carried out. As a result of these activities, the distribution maps of the grayling populations in the Romanian rivers have been drafted and a database related to the habitats populated by this species has been created.

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BIRD RINGING CAMP FROM LETEA, DANUBE DELTA BIOSPHERE - IMPORTANT SITE FOR THE STUDY OF BIRDS MIGRATION
I. COBZARU1, G. CHIAMERA2 and V. POCORA3
1

Institute of Biology Bucharest, Romanian Academy; 2"Grigore Antipa National Museum of Natural History, 3Al. I. Cuza University, Iai

Scientific bird ringing is a research method which consists in the individual marking of birds. Any record of a ringed bird, through recapture or on the occasion of its final recovery as a dead bird, will bring a lot of information about its life. This technique is one of the most effective methods to study the biology, ecology, behavior, movement, breeding and population demography of birds. Danube Delta Biosphere is on the one of the most important migration route in Europe, the Eurasiatic pathway, offering the best conditions for birds on migration, for nesting and wintering. We perform a ringing camp in Letea, in autumn 2012, having 7 trapping station with 200 m of ornithological nets. We capture and ringed 260 birds of 32 species in 5 days. Most of the birds were Passeriformes-25 species, 4 stations being placed in reed beds. All the birds parameters related to physiognomic and physiologic characteristics were taken (measurements, weight, body fat index, sex, age) and an individual ring with code was place on the foot of every bird. All the birds were controlled for ectoparasites and released. We had two recaptures, one for Athene noctua and one for Parus major, bouth sedentay species, ringed by the same team in the past years. The most abundent species captured was Acrocephalus
schoebaenus (37 birds) and Parus caeruleus (32 birds), followed by Acrocephalus scirpaceus (31 birds) and Phylloscopus collybita (27 birds).

From conservation point of view, we captured 7 protected species by the Birds Directive (annex I): Acrocephalus melanopogon, Picus canus, Alcedo atthis, Ficedula parva, Luscinia svecica and Lanius collurio.

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THREATENED BRYOPHYTES IN ROMANIA

S. TEFNU
Institute of Biology Bucharest, Romanian Academy

Bryophytes are much more sensitive to air pollution than higher plants and disappear earlier than many phanerogams due to the increasing habitat destruction. Bryophytes are good indicators of climate changes. The area distribution of bryophytes is more rapidly changed than of the phanerogams. The assessing of the threatened bryophytes status in Romania is very important in achieving conservation strategies and designation of important national sites for bryophytes conservation (IBrA Important Bryophytes Area). Bryological researches in Romania have known a real progress in the last 20 years. Statistically, in this time, two new species of bryophytes have been published every year in Romania. Until now, 980 species of bryophytes were published from Romania, of which 4 are hornworts, 218 liverworts and 758 mosses species. A number of 979 species was published by tefnu S. & I. Goia (2012), and Marsupella apiculata was recently reported from Climani Mountains, Rchiti Peak, leg. & det. Erzberger P., Hhn M. & T. Pcs, 2011, conf. S. tefnu, 2012 [EGR, BUCA] (Erzberger, Hhn & Pcs 2012). In the present the Red list of Bryophytes from Romania includes 375 threatened species, 158 are Critically Endangered, 113 Endangered and 104 Vulnerable species (Table 1).
Table 1. Number of threatened bryophytes in Romania
IUCN category CR EN VU NT LC DD NE TOTAL
Threatened
(CR+EN+VU)

Hornworts No. of species 2 0 0 0 1 1 0 4 2 % 50,00 0,00 0,00 0,00 25,00 25,00 0,00 100,00 50,00

Liverworts No. of species 32 17 23 6 128 12 0 218 72 % 14,68 7,80 10,55 2,75 58,72 5,50 0,00 100,00 33,03

Mosses No. of species 124 96 81 73 333 30 21 758 301 % 16,36 12,66 10,69 9,63 43,93 3,96 2,77 100,00 39,71

Bryophytes No. of species 158 113 104 79 462 43 21 980 375 % 16,12 11,53 10,61 8,06 47,15 4,39 2,14 100,00 38,27

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FIRST RECORD OF Draba dorneri HEUFF. (BRASSICACEAE) IN PARNG MOUNTAINS (ROMANIAN CARPATHIANS)
R. ION
Institute of Biology Bucharest, Romanian Academy

Draba dorneri Heuff. (Brassicaceae) is an endemic plant restricted to Southern Carpathians (Romania). Known only from few localities, the discovery of a new population from Parng Mountains is reported here. It occurs in small crevices on the glacial caldera walls of Mndra Lake, on a north-eastern facing cliffs between 2250 and 2300 m elevation. Notes on habitat characteristics that provide habitat for D. dorneri and some conservation issues are also discussed. This work was supported by the national project Monitoring the conservation status of species and habitats from Romania under Article 17 of the Habitats Directive, project no. 130537 financed by European Fund of Regional Development and implemented by Institute of Biology Bucharest Romanian Academy with Ministry of Environment and Forest Biodiversity Department of Romania.

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BIODIVERSITY OF THE FUTURE IN MILLENIUM SEED BANK

M. ONETE
Institute of Biology Bucharest, Romanian Academy

Today, 60,000 to 100,000 species of plant are faced with the threat of extinction. Plants provide the air we breathe and clean water and we all rely on plants for food. Kew's Millennium Seed Bank Partnership aims to save plants world wide with a focus on plants most at risk and most useful for the future. The Millennium Seed Bank Project is an international conservation project coordinated by the Royal Botanic Gardens, Kew. Launched in the year 2000 and housed in the Wellcome Trust Millennium Building situated in the grounds of Wakehurst Place, West Sussex, its purpose is to provide an "insurance policy" against the extinction of plants in the wild by storing seeds for future use. The storage facilities consist of large underground frozen vaults preserving the world's largest collection of seeds. Where possible, collections are kept in the country of origin with duplicates being sent to the Millennium Seed Bank Project for storage. Major partnerships exist on all the continents, enabling the countries involved to meet international objectives such as the Global Strategy for Plant Conservation and the Millenium Developmental Goals of the United Nations Environment Programme, the letter and the spirit of the 1973 Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), the 1992 Convention on Biological Diversity (CBD), the Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity, the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGFRA), and relevant national and regional laws and regulations concerning biodiversity including laws relating to access to plant genetic resources, associated benefit sharing and traditional knowledge. The Millennium Seed Bank Partnership is the largest ex situ plant conservation project in the world. It is focused on global plant life faced with the threat of extinction and plants of most use for the future. The seeds that are saved are conserved outside their native habitat. Together with their partners in 50 countries worldwide, they have already successfully saved seeds from over 10% of the world's wild plant species. By 2020, their aim is to secure the safe storage of seed from 25% of the worlds plants. They target plants and regions most at risk from climate change and the everincreasing impact of human activities. They also save the seeds of the world's plant life faced with the threat of extinction, and those that could be of most use in the future.
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NON-NATIVE VASCULAR AQUATIC MACROPHYTES IN BULGARIA

V. GEORGIEV and S. TSONEVA
Institute of Biodiversity and Ecosystem research, Bulgarian Academy of Sciences,

This publication focuses on the group of non-native vascular aquatic macrophytes in Bulgaria. The term aquatic macrophytes refers to large plants visible to the naked eye and having at least their vegetative parts growing permanently or periodically in aquatic habitats. According to our recent estimates the following non-native vascular aquatic macrophyte species are occurring in Bulgaria: Acorus calamus, Arundo donax, Azolla filiculoides, Cyperus esculentus, C. strigosus, Eclipta prostrata, Elodea canadensis, E. nuttallii, Heteranthera rotundifolia, Lindernia dubia, Mimulus guttatus, Najas graminea, Paspalum distichum, Portulaca oleracea, Rumex maritimus, Sagittaria latifolia. One species of them is freefloating on the water surface, three species are submerged plants and the other 12 are emergent plants. As a result of field studies and an analysis of literary sources only two species (Elodea canadensis and E. nuttallii) are estimated as a potentially invasive species in Bulgaria.

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CONSERVATIONALLY IMPORTANT VASCULAR AQUATIC MACROPHYTES IN BULGARIA

S. TSONEVA and V. GEORGIEV
Institute of Biodiversity and Ecosystem research, Bulgarian Academy of Sciences,

This publication aims to highlight the conservation value of the group of vascular aquatic macrophytes in Bulgaria. The term aquatic macrophytes refers to large plants visible to the naked eye and having at least their vegetative parts growing permanently or periodically in aquatic habitats. The group of vascular aquatic macrophytes comprises about 9% of the total diversity of vascular plants in Bulgaria and is one of the most vulnerable parts of it. Presented and analyzed are summarised data about plants listed under European and national policy instruments and documents such as the Habitats Directive, Bern Convention, Bulgarian Biological Diversity Act (BBDA), Red List of Bulgarian vascular plants, and Red Data Book of the Republic of Bulgaria. Fig. 1 illustrates the count of species among the different protection documents.

Figure 1. Aquatic macrophytes with protection status in Bulgaria.

Fig. 2 shows the distributions of species amongst the different categories in the Red List of Bulgarian vascular plants. Under the threatened categories (Critically Endangered, Endangered and Vulnerable) are 59 species, Regionally Extinct are 5 species.
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The present work discusses also the major threats to the vascular aquatic macrophytes and gives some examples of successfully applied active protection measures.

Figure 2. Red List species counts of aquatic macrophytes in Bulgaria

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NATURA 2000 SITES IN THE LOWER OLT HYDROGRAPHIC BASIN - KEY SPECIES AND HABITATS FOR THE INTEGRATED MANAGEMENT OF THE AREA
S. MIHILESCU, M. ION and I. COBZARU
Institute of Biology Bucharest, Romanian Academy

The hydrographic basin of the river Olt is one of the largest in our country, covering more than 24 300 km2. The natural landscape of the river has been shifted dramatically, beginning in the 1970s and 1980s, with the construction of the water dams and regulations for electricity purposes. Now there are 15 water dams between Rmnicu Vlcea and Izbiceni with a complete regulation of the water course between the two localities. The mouth of the river, between Izbiceni and the flow in the Danube, is the last natural landscape, which, despite of economical interests for the pit run, it remains one of the most important site for wildlife and natural habitats.
Starting with the expansion of Natura 2000 network in Romania, in the lower basin of the river Olt were designated a number of areas of biodiversity conservation concern. There are 12 Sites of Community Importance (SCIs designated under Habitat Directive) and 3 Special Protection Areas (SPAs designated under Birds Directive) with a total surface of 55.080 ha for SCIs and 74.979 ha for SPAs. The SCIs network in the studied area comprise from small units of 66 ha, as the ROSCI0174 Pdurea Studinia which shelters a pure Eastern white oak wood (91AA) with Ponto-Sarmatic deciduous thickets (40C0*), to large areas like ROSCI0354 Platforma Cotmeana with a surface of 12.529 ha and a high fragmentation of habitats. Here more than 80% of the area is represented by community interest wood habitats considered to be in a favorable state.

Most of the woody habitats, shelters invertebrates like Lucanus cervus, Cerambyx cerdo, Morimus funereus, but their populations are threatened by habitat loss, landscape fragmentation and the loss of dead wood habitats. The avifauna from the Olt inferior basin is young and has been transformed together with the transformation of the landscape, changing from a dominant alluvial forest bird fauna to predominant open water and land fauna. Protected species like Phalacrocorax pygmaeus, Sterna albifrons, Himantopus himantopus, Burhinus oedicnemus, Coracias garrulus, Cionia nigra are nesting in the sites and more than 20.000 water birds are wintering at the flowing of the Olt river. We identified for the entire area from database and regular field trips the habitats and species which are most critical for conservation. We consider that a global view must be the first step in terms of management, the protected areas having a lot of common elements with the same
95

conservation needs. The second step is the individual approach where the specific conservation measures must be taken to be integrated in the management plans of the sites. Acknowledgement: Project RO1567-IBB04: Identificarea i cunoaterea habitatelor naturale i a speciilor endemice, rare i/sau periclitate din Romnia.

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THE ESTIMATION OF COMMUNITY INTEREST MAMMALS IN ROMANIA

O. IONESCU, M. ADAMESCU, C. CAZACU, G. IONESCU, A. COTOVELEA, M. FEDORCA, R. JURJ, M. POPA and A. GRIDAN
1

Department of Forest Sciences, Transilvania University of Brasov; 2Forest Research and Management Institute

The activity of monitoring the species and the habitats of conservative interest, is an obligation defined by the European Directives (Habitats and Birds), and the dates which will be reported must be compatible and comparable, allowing analyze at the European level. The monitoring activity, as an observation which is repeated on long term is needed in order to track the conservation status of species and their trends on long term. Monitoring and evaluating the conservation status can be achieved by processing the scientific information and the results will be aggregated and evaluated at different spatial scales. In this context, the monitoring activity is defined as the whole observation measures undertaken in order to ensure that the favorable conservation status are maintained in the monitoring period. Monitoring suppose the development of continue and permanent program in order to provide a series of observations in time, so that it can permit the establishment of the conservation status and the trends of species evolution. The species habitat in Romania will be considered and after a stratified and randomly analysis will result grids (10X10 km)for all species: RODENTIA: 1. Spermophilus (Citellus) citellus; 2. Mesocricetus newtoni; 3. Cricetus cricetus; 4. Microtus tatricus; 5. Sicista subtilis; 6. Sicista betulina; 7. Muscardinus avellanarius; 8. Dryomys nitedula; 9. Castor fiber; CARNIVORA: 10. * Canis lupus; 11. Canis aureus; 12. Ursus arctos 13. Lutra lutra; 14. Mustela lutreola; 15. Vormela peregusna; 16. Mustela eversmanni; 17. Martes martes; 18. Mustela putorius; 19. Lynx lynx; 20. Felis silvestris; ARTILODACTYLA 21. Bison bonasus (extincted in wild); 22. Rupicapra rupicapra;

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Figure 1. The landuse from CORINE LANDCOVER

As a starting point in this activity there will be used : a information from the hunting units database regarding the animals which have been hunted in the interval 2007-2012 (The Ministry of Environment and Forests). b. centralized information at country level concerning the damages created by large carnivore (The Ministry of Environment and Forests, Environmental Agencies, ITRSV. ICAS.). c. the reports of the projects in which studies and researches have been conducted (The National Authority of Management POS environment, The Ministry of Environment and Forests) In order to establish the population size/the number of individuals there will be used different methods as: a. Identifying the tracks when there is a fresh snow layer b. Direct observations c. Photo - trapping d. Capturing and recapturing of some individuals Selected grids will be covered in order to determine the conservation status of species habitat and data with GPS tracking will be collected. Points were individuals or tracks were observed will be marked with GPS point.

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History Period: 19502012 References Raportedpresence Huntingunits Distributionmapof speciesXatnationallevel

Actual

Potential

Specieshabitat (Peterken,1996)

Bioregions

SpecieshabitatXin BioregionY

SpecieshabitatXin BioregionY Gridwithdistribution[10x10km]

Grids stratifiedrandomly Whatdowewhantwiththefielddat? (basedonArticle17) 1. 2. 3. 4. 5. Area Population(size) Specieshabitat Perspectives Evaluationofconservation status

Parameters/variables

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Figure 2. Selected grids (stratified and randomized) for Rupicapra rupicapra

Photo 1. Rupicapra rupicapra

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MICROBIOLOGY

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THE SELECTION OF THE ACIDOPHILIC BACTERIA BELONGING TO THE Acidiphilium GENUS WITH HIGHER ENZYMATIC ACTIVITY IN THE PRESENCE OF HEAVY METAL IONS C.M. CISMASIU
Institute of Biology Bucharest, Romanian Academy

The uncontrolled discharge of industrial waste waters into the river systems containing metallic ions induce profound changes of water quality, which are translated in disrupting the ecosystems due to perturbation processes in the trophic chain, with inhibiting mineralization, accumulating of heavy metals in elevated concentrations in certain the aquatic organisms and are ultimately to humans. The present of metallic ions in the solution and the need for their removal has opened two important ways to approach of the research, namely: their extraction in order to recover of heavy metal ions and industrial waste water treatment to reduce the concentrations of heavy metal ions to values mentioned in international standards. The study of the interactions between acidophilic heterotrophic bacteria and heavy metal ions can occur through active processes, involving the metabolic sequences of living microorganisms or passive processes, independent of cellular metabolism. The residual biomass of heterotrophic bacteria belong to the Acidiphilium genus proved an affinity for a wide variety of heavy metal ions such as Cr6+, Cr3+, Cu2+, Zn2+, Fe3+ and Ni2+. In these contexts, it became necessary studying starch degradation capacities of the acidophilic heterotrophic bacteria cultures in the presence of heavy metal ions. Growth experiments were carried out at different species type, heavy metal ion type and contact times between cell and these ions. The comparative studies regarding the influence of the metallic ions solution on the growth and the starch degradation activity is developed with a maximum intensity up to 14 days of incubation periods at the same substrate concentration. High percentages degradation of substrate were obtained using populations of heterotrophic bacteria compared with purified strains, which confirms the adaptation of populations to the concentrations of heavy metal ions used.

1. Pandey B., Fulekar M.H., 2012. Bioremediation technology: A new horizon for environmental clean-up, Biology and Medicine, 4(1): 51-59. 2. Wang J., Chen C., 2009. Biosorbents for heavy metals removal and their future, Biotechnology Advances, 27: 195-226. 3. Johnson D.B., Hallberg K.B., 2008. Carbon, iron and sulfur metabolism in acidophilic micro-organisms, Advances in Microbial Physiology, 54: 201-255. 103

LACTIC ACID BACTERIA FROM FERMENTED VEGETABLES WITH POTENTIAL APPLICATIONS IN FOOD INDUSTRY AND HEALTH
S.S. GROSU-TUDOR1, M. ZAMFIR1, D. WOUTERS2 and L. DE VUYST2
1

Institute of Biology Bucharest, Romanian Academy; 2Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

Fermented vegetables represent a frequently used food in Romania, especially during the winter. The preservation is based on the property of lactic acid bacteria (LAB) to ferment sugars to lactic acid, causing an acidification and hence, a stabilization of the end product. The most common vegetables used are cucumbers, cabbage, and bell peppers. Green tomatoes, carrots, and cauliflower are used too, sometimes mixed with fruits (water melon, apples, or pears). The aims of our study were: to isolate and identify the LAB involved in these spontaneous fermentations of vegetables; to test the newly isolated LAB for the production of bacteriocins and exopolysaccharides; to purify and characterize these metabolites; to evaluate the probiotic characteristics of the new isolates, such as the bile salt resistance, salt tolerance, survival under low pH environment, and the antimicrobial activity in the presence and absence of prebiotics. Brine and vegetable samples were collected from 20 fermentations carried out at household level in the region of Vlenii de Munte (Chiojdu) and Bucharest. Samples were analysed for acidification (pH measurements) and plated on VRBG agar medium (for enumeration of enterobacteriaceae) and MRS agar medium (for enumeration and isolation of LAB). Colonies were randomly picked up, purified and tested for catalase production and Gram-staining. Gram-positive, catalase-negative isolates were stored at 75C in liquid MRS medium supplemented with 25% (v/v) of glycerol as cryoprotectant and used in our further experiments. LAB were prevalent in all end-samples, as represented by their MRS counts, whereas enterobacterial counts were low in most cases, indicating good fermentation quality. The LAB isolates (139) were identified through (GTG)5-PCR fingerprinting and 16S rRNA gene sequencing. The most frequently isolated LAB were Lactobacillus plantarum and Lactobacillus brevis, independent of the vegetable type. Also, Leuconostoc mesenteroides, Leuconostoc citreum, and Weissella kimchi were found. The strains were further screened for the ability to produce exopolysaccharides and for their antagonistic activity against a set of nine
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LAB strains, three Bacillus strains and four Gram-negative bacteria. Eighty five of the tested strains showed a variable antimicrobial activity against Listeria monocytogenes ATCC 1911, 35 of the strains showed a fade inhibition zone against Escherichia coli ATCC25922 and 26 strains against Salmonella enterica ATCC 14024, while 19 strains showed inhibition against one or all three Bacillus strains used as indicators. None of the tested strains showed an antimicrobial activity against Staphylococcus aureus ATCC 25923. Several strains showed antibacterial activity against more then one indicator strain. For instance, Lb. plantarum 307, Lb. brevis 308, and Lb. plantarum/pentosus 358 were active against 5 of the indicator strains used, while other 23 LAB were active against 3 indicator strains. In the case of two strains, namely Leuc. citreum 344 and Lb. brevis 183, the activity was maintained after neutralizing the pH of the cell free supernatant, being due, probably, to the production of bacteriocins. The gel permeation chromatography-based screening revealed seven EPS-producing LAB strains. Two of the positive strains, namely Leuc. citreum 177 and Leuc. citreum 52 have been shown to produce large amounts of EPS, of about 20 g/L. All isolated EPS have a high molecular mass, of above 1400 KDa, and a monomer composition dominated by the presence of glucose. All exept five isolates were shown to produce capsular polysaccharide and 31 developed mucoid colonies on sucrose-based media. To evaluate the probiotic potential, six selected strains were investigated for tolerance to acidity (pH values of 2.0, 3.0, and 4.0 respectively) and to bile salts (concentrations of 0.3% and 0.5%, w/v), respectively. All six strains tested were resistant to pH 3.0 and pH 4.0, after 24 h of incubation showing viability rates of 109 CFU/ml and 1012 CFU/ml, respectively. After 3 h of exposure to pH 2.0, four strains reached viability rates of at least 108 CFU/ml. Moreover, Lb. brevis 183 survived even after 24 h of exposure to pH 2.0, reaching a viability of 104 CFU/ml. Concerning the bile salt resistance, all six strains showed a high resistance to a concentration of 0.3% (w/v), reaching a cell viability of 105-108 CFU/ml after 24 h of treatment. After 2 h of exposure to 0.5% (w/v) of bile salts, the viability of strains Lb. brevis 183 and Leuc. mesenteroides 348, reached about 106 CFU/ml, a value still adequate for the use of these strains as probiotics. Romanian traditionally fermented vegetables can be regarded as important sources of LAB strains with functional properties, such as production of EPS or bacteriocins, with potential biotechnological application. The two strains producing high amounts of high molecular mass EPS might be used to control the rheological properties of fermented foods, while the strains showing antibacterial activity could be used in a probiotic complex, or to improve the shelf life of certain fermented foods.
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STUDIES CONCERNING THE MORPHOLOGICAL AND FUNCTIONAL VARIATIONS OF SOME BACTERIAL CELLS UNDER CONTAMINATION STRESS IMPACT
M. TEFNESCU and D.M. CRSTEA
Institute of Biology Bucharest, Romanian Academy

Areas contaminated by oil spills, incidental or as a result of historical activities that have induced chronic pollution to deeper layers of the soil, represent a permanent challenge for finding solutions to protect the affected environment. Last but not least, bioremediation appears to be a promising alternative for reducing harmful compounds that affect the ecosystem. Studies conducted so far have allowed us to select and identify groups of hydrocarbon-oxidizing microorganisms capable of surviving in extreme conditions of contamination stress, which possess the ability to metabolize the carbon that is found in the oil structures. The investigated microbial activity consisted in breaking down the complex compounds into simpler constituents, biodegradable, this being accompanied by the production of metabolites such as polyhydroxyalkanoates (PHA) - also known as the biopolymers, and rhamnolipids (RHL) - biosurfactants. These bacterial products have the capacity to "isolate" the contaminated area, reducing the spread of the pollutant, and emulsify the lipid constituents, favoring thus, by increasing the contact surface, the oxidative reactions, the bacteria acting either directly or complementary for the reduction of the contaminants. The researches have focused on the isolation and characterization of several bacterial strains from oil-contaminated areas and assessing both their tolerance to high concentrations of petroleum contaminants and their involvement in the bioremediation process. Their intra- and extra-cellular biosynthetic capacity of the above mentioned metabolites was also assessed. Among the tested microbial strains, the most representative turned out to be bacterial strains belonging to Pseudomonas genus, able to synthesize under contamination stress conditions, PHA and RHL, even simultaneously. The current research is a continuation, but also a novelty as concerns demonstration of the versatile character of these microorganisms, able to grow both in the presence and absence of pollutant, and extension of their characterization by electron microscopic studies approach. Changing the carbon source was found to be directly correlated to the ability of synthesis of metabolites. Nevertheless, the morphological and structural appearance of the bacterial cells growing under contamination stress should not be
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overlooked. The intracytoplasmic metabolic accumulations and the extracellular synthesis products entail characteristic cellular changes that can only be highlighted by electron microscopic observations. The paper submitted to your attention is the beginning of a research work aiming to argument the structural cellular versatility concept, i.e. modification and return of the cell to its initial shape, depending on variation of the perturbing factors, which result in changes in membrane ion pumps system. The bacterial strains to which we relate belong to Pseudomonas genera (figure 1). Cultivation was performed on mineral media supplemented with carbon sources such as: octanoate, glucose, nhexadecane, benzene, the tolerance reaching concentrations up to 10%. Grown on the same mineral medium, but without carbon sources, microorganisms did not develop. The captured microscopic images are relevant in this respect.

Acumulation within the cell or as exo-products entails structural differentiations If the activity of rhamnolipids was found to be comparable to that of detergents, polyhydroxyalkanoates mode of action is less demonstrated in the direct bioremediation process. The formation of these metabolites of bio-plastic type involves the use of carbon within the contaminants structure for producing biodegradable polyesters. The undertaken observations, using electron microscopy techniques, allowed visualization of the external structure of the selected cells, while the cell sections allow identification of endometabolite accumulations, such as PHA. A more complete characterization of bacterial strains for potential bioremediation use, is a prerequisite for understanding the complex mechanisms of microbial decontamination of affected environments.
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ORGANIC SOLVENT RESISTANCE MECHANISMS IN Vibrio alginolyticus IBBCT2

M.M. STANCU
Institute of Biology Bucharest, Romanian Academy

Constanta harbor has been contaminated from shipping activities and accidental fuel spills with petroleum and petroleum products, which contain various types of toxic organic solvents. Organic solvent tolerant bacteria are a relatively novel group of extremophilic microorganisms with novel tolerance mechanisms, which enable them to overcome the toxic and destructive effects of organic solvents in solvent-saturated environments. Isolation of strain IBBCt2 from Constanta seawater was carried out on artificial seawater medium, using the enriched cultures method, with organic solvents as the sole carbon source. The taxonomic affiliation of strain IBBCt2 was determined on the basis of phenotypic characteristics and the 16S rRNA gene sequence. The analysis of the 16S rRNA gene sequence located strain IBBCt2 (GenBank JN874640) within the genus Vibrio, showing 99% similarity to V. alginolyticus strain J608 (JF836167.1). The toxic effects of 1% organic solvents on V. alginolyticus IBBCt2 and the mechanisms behind its resistance have been studied in saline stress conditions. n-Hexane, ndecane and cyclohexane with logarithm of partition coefficient in n-octanol and water (log POW) > 3.35, were less toxic for V. alginolyticus IBBCt2, compared with toluene, m-xylene and ethylbenzene with log POW < 3.17. Exposure of V. alginolyticus IBBCt2 cells to salt stress decreases the organic solvents tolerance of this bacterium. The high organic solvent resistance of V. alginolyticus IBBCt2 could be due to the presence of some catabolic (alkB, alkB/alkB1, todC1, xylM, C23DO) and transporter (HAE1, acrAB) genes. The regions harboring catabolic and transporter genes are located on the chromosome in this bacterium. The adaptation mechanisms, underlying cyclohexane, n-hexane, n-decane, toluene, m-xylene, and ethylbenzene resistance in V. alginolyticus IBBCt2 showed a complex response of cells 60 min after solvent shock (i.e., modifications of the cell viability, changes in the membranes lipid and protein profile, modifications of the genomic fingerprinting). Moreover, the exposure for 60 min of V. alginolyticus IBBCt2 cells to organic solvents had different effects on the lipolytic and proteolytic enzymes production. The results from the present study suggest that the salt stress does not protect the bacterial cells against the toxic effects of 1% organic solvents (cyclohexane, n-hexane, n-decane, toluene, mxylene, ethylbenzene), and in the presence of salt the toxicity of the solvents seems to be intensified.
B

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The study was funded by project no. RO1567-IBB05/2012 from the Institute of Biology Bucharest of Romanian Academy. The author is grateful to Ana Dinu for technical support.

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HALOPHILIC BACTERIA FROM DIFFERENT HYPERSALINE HABITATS IN SLANIC PRAHOVA, ROMANIA AND THEIR BIOTECHNOLOGICAL POTENTIAL
R. COJOC, S. NEAGU, G. TEODOSIU and M. ENACHE
Institute of Biology Bucharest, Romanian Academy

Lately, research on extremophiles gained an increasing interest, mainly due to their prospective applicative roles in various biotechnological fields, for oil recovery, in the biodegradation of organic pollutants and forensics. Halophilic bacteria represent a category of extremophilic microorganisms remarkable by their ability to survive in environments with very high salt concentrations. Microorganisms in this group are physiologically different, being represented by Gram-negative or Grampositive bacteria, with an optimal growth in media containing 0,5 2,5M NaCl [1]. They are categorized as slight, moderate or extreme, based on the extent of their halotolerance. Because of their abundance in hypersaline habitats with different salt concentrations, they have an important role in the ecology of these extreme environments, representing a model of adaptation to frequent variations of the extracellular osmolarity. The present study reports the isolation, preliminary characterization and biotechnological potential of some halophilic bacterial strains isolated from hypersaline habitats in Prahova county, Romania. Samples were harvested from the wall of subterranean salt Mine Unirea (around 200 m underground), two water pounds inside Mine Unirea, Brides Cave, Green Bath and Shepherds Bath lakes. The investigated bacterial strains were isolated on MH selective medium containing 10% NaCl [2]. The results showed a low diversity of microorganisms in the samples prelevated from the salt wall of the subterranean mine (around 3000 CFU/g rock salt). Specific inhibitors (sodium deoxicolate and chloramphenicol) testing allowed the separation of the halophilic bacterial strains from the haloarchaean ones. The presence of bacterial 16S rRNA genes in the case of all investigated strains, demonstrated by PCR amplification [3] with bacterial specific primers 27 F and 1492 R [4] also proved the affiliation of the tested strains to halophilic bacteria. Microscopic investigations revealed that most of the isolated strains were represented by Gram-negative rods, having oxidizing and catalytic properties and being able to produce indole and reduce nitrates to nitrites. Qualitative determination of the capacity to extracellularly hydrolyze starch, gelatin, casein, Tween 80, olive oil, carboxymethyl cellulose and RBB-xylan revealed that all selected strains were able to degrade at least one of the tested substrates. Some of the
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isolates even showed combined hydrolytic activities [5], this characteristic recommending them as possible valuable candidates for use in biotechnological processes involving complex compound degradation in order to obtain desired final products (i.e. agriculture, production of fermented food and food supplements, pharmaceutical industry). Some of the selected halophilic bacterial strains harbored the capacity to synthesize extracellular polysaccharides [6]. In this case, both the carbon source and growth conditions influenced the dynamics of the culture development and the exopolymers synthesis. The results showed that there was no strict correlation between the optimum conditions for the exopolysaccharides synthesis and the demands for optimum growth.

1. Kushner D.J., Kamekura M., 1988. Physiology of halophilic Eubacteria. In: RodriguezValera F. (ed) Halophilic Bacteria, Vol. I. CRC Press, Inc. Boca Raton, Florida, pp. 109-138. 2. Ventosa A., Quesada E., Rodriguez-Valera F., Ruiz-Berraquero F., Ramos-Cormenzana A. 1982. Numerical Taxonomy of Moderately Halophilic Gram-negative Rods, J. Gen. Microbiol., 128: 1959-1968. 3. Wilson K.H., Blitchington R.B., Greene R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction, J. Clin. Microbiol., 28: 19421946. 4. Polz M.F., Cavanaugh M.C. 1998. Bias in Template-to-Product Ratios in Multitemplate PCR, Appl. Environ. Microbiol., 64: 3724-3730. 5. Cojoc R., Merciu S., Popescu G., Dumitru L., Kamekura M., Enache M., 2009. Extracellular Hydrolytic Enzymes of Halophilic Bacteria Isolated from a Subterranean Rock Salt Crystal, Romanian Biotechnological Letters, 14(5): 46584664. 6. Cojoc R., Merciu S., Oancea P., Pincu E., Dumitru L., Enache M., 2009. Highly Thermostable Exopolysaccharide Produced by the Moderately Halophilic Bacterium Isolated from a Man-Made young Salt Lake in Romania, Polish Journal of Microbiology, 58(4): 289-294. 111

METALIC NANOPARTICLES CITOTOXICITY AGAINST PHOTOTROPHIC AND HETEROTROPHIC BACTERIA

I.I. ARDELEAN1,3, M. CRNU1 , D. PASCU1, I. SARCHIZIAN3 , V. DAMIAN2, I. APOSTOL2 , I. IORDACHE2 and D. APOSTOL2
1

Institute of Biology Bucharest, Romanian Academy; 2National Institute for Laser, Plasma and Radiation Physics, Magurele; 3Ovidius University, Constaa

Cytotoxicity of gold, silver and aluminium was studied against heterotrophic bacteria, (S. aureus and E.coli), culture collection cyanobacteria (Synechocystis PCC 6803) and cyanobacterial strains isolated and purified in our laboratory (single cells strains and filamentous ones). The cytotoxicity was tested, in dark as well as in light, with respect to bacterial growth, multiplication and intermediary metabolism associated with the reduction of artificial electron acceptors such as resazurine or MTT. The screening part of the work was done in micro plates; for quantification of resazurine reduction the readings were done simultaneously at 570nm and 600nm. The main results show that i) nanoparticles, especially silver ones, affect bacterial growth and multiplication, as well as the ability to reduce resazurine and ii) the citotoxycity, depending evidently on the ratio between nanoparticles and bacteria, is different in light as compared with the dark regime. One working hypothesis to explain the results is based on :i) the ultrastructural differences between Gram-negative and Gram-positive bacteria and ii) cellular localization of main energetic processes in cyanobacteria. In cyanobacteria respiration occurs both at the cell membrane and at intracellular membranes- thylakoides, photosynthesis only at intracellular membranes and intermediary metabolism within cytoplasme, all of them interacting very deeply.

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STRUCTURE-FUNCTION INVESTIGATION OF Methanobrevibacter smithii FULL-LENGTH CARBAMOYL PHOSPHATE SYNTHETASE

E. POPA1, N. PERERA2, H. GUY-EVANS2, D.R. EVANS2 and C. PURCREA1
1

Department of Microbiology, Institute of Biology Bucharest, Romanian Academy; 2 Department of Chemistry, Eastern Michigan University, Ypsilanti, Mich. , and c Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Mich., USA

The genome of M. smithii, the predominant archaeal component of human gut, contains genes coding for two carbamoyl phosphate synthetases (CPSase). In addition to a reduced size enzyme (MS-s) that appears to be the smallest CPSase characterized so far (Popa et al., 2012), this methanogen also contains a putative full-length CPSase composed of a 40kDa glutaminase (GLN) subunit and a 118- kDa synthetase (SYN/MS-l) subunit. In view of determining the role of this complex system involved in ammonia-metabolism enzyme that appears to be characteristic for some intestinal methanogens, our investigation focused on determining the structural and functional properties of MS-l. Structural analysis indicated high sequence identity of both M. smithii GLN and MS-l with E. coli and other bacterial and archaeal CPSase corresponding subunits. Pair alignment of the primary structures of MSl and E. coli GLN and SYN subunits showed the conservation of active site residues. In addition, the residues involved in allosteric regulation in E. coli enzyme are conserved in MS-s, suggesting that this archaeal CPSase could be inhibited and activated by UMP and ornithine respectively. 3-D molecular modeling of MS-l based on the E. coli CPSase x-ray structure revealed a similar folding and the presence of all SYN domains and subdomains. The gene coding for MS-l was cloned and expressed in E. coli, and the recombinant enzyme fused with a His-tag polypeptide was purified in one step by affinity chromatography. Preliminary functional analysis showed an NH3-dependent CPSase activity for MS-l, measured at 370C. The kinetic parameters were calculated from the saturation curves determined for all three substrates. Further functional characterization of the reaction mechanism and enzyme allosteric regulation of the large M. smithii CPSase, and of the contribution of the GLN subunit to this reaction are currently under way to elucidate the role of M. smithii full-lenth enzyme and of the two-CPSase system in ammonia utilization by the human gut methanogen for arginine and pyrimidine nucleotides synthesis.
1. E. Popa, N. Perera, C.Z. Kibdi-Szab, H.Guy-Evans, D.R. Evans, C.Purcarea, 2012. J.Mol.Microbiol Biotechnol., 22:287299. 113

CELL GROWTH AND DGGE ANALYSIS OF MICROORGANISMS FROM SCRIOARA CAVE ICE SEDIMENTS
C. ICU1, A.M. HILLEBRAND-VOICULESCU2, D. PASCU1, A. PEROIU3, T. BRAD4 and C. PURCREA2
Institute of Biology Bucharest, Romanian Academy; 2Emil Racovi Institute of Speleology, Bucharest, Romania; 3tefan cel Mare University, Suceava, Romania; 4Emil Racovi Institute of Speleology, Cluj-Napoca, Romania
1

Cave glaciers represent an important environment for paleoclimate and exobiology studies. While up to date there are almost no reports on microorganisms biodiversity from underground ice sediments, we are interested in characterizing the prokaryotic and eukaryotic microorganisms communities in different aged ice layers from Scrioara Cave and determining their chronological distribution and climate biomarkers. Ice samples of different ages were collected from the perennial ice block of the Great Hall and Little Reserve, and the microbial diversity was investigated by culture-dependent and independent methods, consisting of (1) cell growth on various substrates at different temperatures, and (2) DGGE analysis of 16S/18S-rRNA amplified genes from each sample. Cultivation on 31 carbon sources was carried on using EcoPlates (BIOLOG). Each of the ice samples of 1, 400 and 900 years old showed a different substrate preference and selectivity, which is temperature dependent. Moreover, liquid cultivation at 4C and 15C on five different rich and supplemented mineral media suggests the prevalence of heterotrophs in all ice samples, with a higher cell growth occurring at 15C corresponding to psychrotolerant species predominance. Total DNA was extracted from each ice sample, and SSU-rRNA gene fragments were amplified by PCR using specific GC-clamp primers. DGGE analysis indicates for each sample a high variety of both bacterial and eukaryotic microorganisms, resulted from the different number and position of DNA amplicons. The microbial species isolated from recent and old perennial ice sediments will be further identified by SSU-rRNA 454 pyrosequencing.

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PLANT AND ANIMAL CYTOBIOLOGY, BIOTECHNOLOGY

POSTERS

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FIBROLAMELLAR LIVER CARCINOMA LIKE TUMOR IN AN EXPERIMENTALLY IRON LOADED MOUSE

P. PRUNESCU and C.C. PRUNESCU
Institute of Biology Bucharest, Romanian Academy

A fibrolamellar carcinoma like tumor was found in the liver of the last survivor of an experimental group of iron overloaded mice. This tumor was studied in order to make a comparison with the fibrolamellar liver carcinoma (FLC) in man (1), described for the first time by R. L. Peters (2), in 1975. A possible relation between the iron overloading and the occurrence of this tumor represented a subject of interest. Albino Swiss young adult male mice of 222 g weight were treated with Ferrum Hausmann, by intraperitoneal (ip) inoculations, to obtain the loading of 2 mg iron/g body weight of living animal. The last surviving mouse of the experimental group was sacrificed after seventeen months from the beginning of the experiment. An intra-hepatic spherical formation of about 8 mm diameter with white-yellow color was observed. The uninvolved liver presented red-brown color. Small liver and tumor fragments were fixed in 4% formaldehyde in saline, respectively in cold 2.5% glutaraldehyde in 0.1 M cacodylic buffer, pH 7.2. Histological sections of 5 m thickness were stained with Hemalum-Eosine (H-E), Perls method to make evident the ferric ions and Picro-Sirius red (PSR) stain specific for collagen fibers. Ultrathin sections double contrasted with uranyl acetate and lead citrate were observed at a Zeiss 100 EM. The histological observations revealed that in the tumor territory the histologic hepatic structure was no more preserved. The fibrolamellar carcinoma was constituted by short cellular cords, pseudoglandular structures like the cysts and fibrous stroma which invaded the tumor tissue. The tumor cells presented polygonal or spindle shapes. They have abundant, eosinophilic cytoplasm and nuclei with irregular outlines. Some nuclei seemed to be polyploid, other were pyknotic. Strands of multistratified collagen fibers made evident by PSR stain, surrounded the cellular pseudotrabeculae and formed branches into the tumor tissue. The EM observations showed the cytoplasm of the tumor cells full of relative uniform in shape mitochondria and numerous electron-light vesicles. Among the packed mitochondria there were dilated profiles of smooth and rough endoplasmic reticulum and free ribosomes. Golgi complexes were not frequent. Small Golgi vesicles were packed with electron-dense material. Glycogen and lipids were absent. In the vicinity of the nucleus were often observed bundles of fine parallel fibrilles which seemed to originate from
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this zone. Other similar bundles of fibrils run through the cytoplasm to get the intercellular space, where they can be concentrated in the thicker fascicles of the fibrous stroma. In the untransformed liver parenchyma the histological hepatic structure was relatively maintained. The great majority of the hepatocytes presented nuclear inclusions containing ferritin specifically stained in bright-green following Perls method (3). Such nuclear ferritin aggregates with a conspicuous ultrastructure were also scattered in the hepatocytes cytoplasm. In the portal spaces, hepatocytes cords, sinusoids lining and Glisson capsule siderosomes agglomerations were observed. - The main features of the fibrolamellar liver carcinoma (FLC) described in man were observed in the experimental fibrolamellar tumor occurred in mouse. - It is possible that the long time lapsed for the mouse iron overloaded liver to represent the major irritative condition which led to the tumorigenesis process. - The mouse hepatocytes nuclei capacity to concentrate ferritin molecule as nuclear inclusions following the treatment of iron loading, must be considerate about the biological significance of the possible perturbation of the cellular divisions.

1. Craig J.R., Peters R.L., Edmonson H.A., and Masao Omata, 1980. Fibrolamellar carcinoma of the liver: A tumor of adolescents and young adults with distinctive clinico-pathologic features, Cancer, 46: 372-379. 2. Peters R.L., Pathology of hepatocellular carcinoma, In: Okuda K., Peters R.L.,eds. Hepato Carcinoma, New York, Wiley, pp. 107-168. 3. Prunescu C-C., Prunescu P., 1991. Ferritin accumulation in nuclei of hepatocytes in polymaltosed iron injected mice, Rev. Roum. Biochim., 28(3-4): 169-172. 118

LONG-TERM CULTURES IN MINIMAL GROWTH CONDITIONS IN Dianthus trifasciculatus KIT SSP. parviflorus AS EX SITU PRESERVATION TOOL
I. HOLOBIUC, C. VOICHITA, M.E. MITOI and F.E. HELEPCIUC
Institute of Biology Bucharest, Romanian Academy

In the last decade, ex situ preservation strategies gained more importance in the frame of the plants biodiversity conservation increased concern. Besides traditional methods (seeds banks, field collection, botanical gardens), the use of biotechnology can help the stop or diminishing of the plant biodiversity loss. The main advantage of this approach is the possibility to supply healthy plant material in short time, in reduced space and protected against the biotic and abiotic agents. The preservation based on in vitro protocols can be made on different duration: short time based on multiplication in active collection, medium term preservation using growth retardation methods and long-term conservation using cryopreservation procedures using plant propagules with morphogenic potential (apexes, embryos, meristems). The taxon taken into study, Dianthus trifasciculatus ssp. parviflorus is cited in the Red Book of Vascular Plants from Romania, being common to Romania and Bulgaria also. We used as explants, stem fragments collected from a plant growing in the Al. Borza Botanical Garden, Cluj-Napoca. In the first step an optimized micropropagation protocol was established. For medium-term maintenance it was ellaborated an efficient method to preserve plant material in minimal cultures with growth retardation, for this purpose being tested several compounds. It is possible to modulate in vitro conditions as if to obtain a limited growth with the maintenance of the regeneration capacity at high level, to obtain a moderate growth retardation or even a growth stopping without regeneration. Based on the elaborated medium-term preservation protocol, this taxon was maintained in the collection during 4 years. Normal plants were regenerated from this cultures and were biochemically characterized.

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GREEN SYNTHESIS OF SILVER NANOPARTICLES USING PLANT CELL CULTURES

G. COGLNICEANU1, M.E. MITOI1 and G. HRISTEA2
1

Institute of Biology Bucharest, Romanian Academy; 2National Institute for Electrical EngineeringAdvanced Research, INCDIE-CA.SA,Bucharest,

The development of environmental-friendly technologies for the production of nanoparticles with controllable size, shapes, distribution, or uniformity represents a challenge in the nanotechnology field. Silver nanoparticles (AgNPs) have many applications: non-linear optics, spectrally selective coatings for solar energy absorption, bio-labeling, optical receptors, catalysis, cosmetics, medical and pharmaceutical applications. Performances of AgNPs biosynthesis are dependent on the different biological organisms/systems used: bacteria, yeast, fungi or plants. The biological compounds involved in reducing AgNO3 to Ago are different for each species or even strain. Plant cell cultures are closed systems that allow the possibility of manipulating important biological processes (e.g. cytodifferentiation, morphogenesis, proliferation, secondary metabolism) by applying specific physical or chemical signals. The absence of contaminants, the stability of the plant tissue culture, and its availability all year round represent additional advantages of plant in vitro systems. Our aim was to obtain AgNPs with useful characteristics, using callus (non-differentiated, non-morphogenetic, proliferative cells) of strawberry (Fragaria x ananassa Duch.). We tested three variants of callus raw extract: (i) grinding at room temperature; (ii) boiling; and (iii) freezing. All three extracts rapidly mediated biosynthesis of AgNPs from AgNO3, at light and 60oC conditions. The shift of color in the reaction mixture from white to orange, typical for AgNPs, as a result of surface plasmon vibration in the metal nanoparticles, was obtained in the range of minutes. The kinetics of AgNPs synthesis evidenced by the UV-Vis absorbance spectroscopy monitored every 30 min for 6 hrs was different for the three extracts. The boiling extract (ii) reduced AgNO3 faster than the other variants and the highest absorbance values were obtained in shorter time. The maximum absorbance peaks were achieved at wavelengths between 420- 440 nm. After 24 h of biosynthesis reaction in the same conditions, the absorbance registered for grinding (i) and freezing (iii) extracts was higher compared to the boiled extract (ii) which did not increased anymore.

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Scanning electron microscopy (SEM), EDAX, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) analysis revealed that the biosynthetised Ag nanoparticles were in the size range of 10-25 nm. We tested the growth inhibition effect of the synthesised AgNPs solutions against different bacteria strains (Staphilococcus aureus, Salmonella enterica, Salmonella panama, Escherichia coli, Pseudomonas aeruginosa and Bacillus licheniformis) with the agar disc-diffusion method. The AgNPs were more effective against S. aureus and P. aerugionosa. An intermediary effect exhibited on B. licheniformis and the lowest effect was against E. coli and two types of Salmonella. A similarly inhibition zone was observed for AgNO3 which suggest that the antimicrobial activity of AgNPs was due to Ag presence. Our method proves to be a simple and rapid biosynthesis of AgNPs from strawberry callus extract that is environmentally safe and has potential for various medical, cosmetic and industrial applications. To our knowledge this is the first report for the use of plant cell cultures to synthesize AgNPs.

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DEFENSE-RELATED ENZYMES GENE EXPRESSION IN Cucumis sativus L. PLANTLETS INDUCED BY MICROORGANISMS

F.E. HELEPCIUC1, M. CIUC2, M.E. MITOI1, A. BREZEANU1 and C.P. CORNEA3
Institute of Biology Bucharest, Romanian Academy; 2Department of Molecular Biology, National Agricultural Research and Development Institute, Fundulea; 3Departemnt of Biotechnology, University of Agronomical Sciences and Veterinary Medicine, Bucharest
1

As a response of interaction with microorganisms, plants use numerous defense mechanisms, which can be divided in structural, chemical and enzymatic (protein-based) defense mechanisms. Enzymatic defense mechanisms refer to different defense-related enzymes production, with various catalytic activities. Plant-microbe interaction can induce the expression of antioxidant enzymes like superoxide dismutases (SOD), catalases (CAT), peroxidases (POX), which contribute to reactive oxygen species (ROS) detoxification. Also, systemic or local expression of lytic enzymes can be triggered by microorganisms. Lytic enzymes like chitinases and glucanases are involved in breaking down polymers like chitin and glucan, which are a part of fungal cell wall, having an important role in fungal pathogen inhibition. Another important defense-related enzyme is phenylalanine ammonia lyase (PAL), involved in flavonoids and other phenolic derivatives synthesis. The purpose of this study was to evaluate gene expression of defenserelated enzymes in cucumber plantlets treated with antagonistic bacteria and fungal phytopathogens. Suspensions of Pythium debaryanum phytopathogen and antagonistic bacteria (Bacillus sp. B40, Bw, Bw1 and Pseudomonas sp. P14) were applied on the roots of two weeks old cucumber plantlets. Reverse transcription-PCR (RT-PCR) method was used to determine alterations in defense-related genes expression of treated plants, using specific primers. Preliminary results indicate that differences in gene expression appear in plants exposed to bacteria and fungi comparing with non-treated plants, indicating that simple treatment with antagonistic bacteria, but also mixed treatment with bacteria and phytopathogenic fungi, activate the defense system in plants.

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DIRECT SOMATIC EMBRYOGENESIS AS POSSIBLE WAY OF VIRUS ELIMINATION FROM SEEDLESS GRAPEVINE
L. ANDRONIC, S. SMEREA and T. GRIGOROV
Institute of Genetics and Plant Physiology of the ASM, Chisinau, Republic of Moldova

Viticulture is one of the most important branches of the agriculture complex in the Republic of Moldova, providing significant revenue for the national budget. Grapevine seedless varieties have major advantages compared with varieties with seeds as they having a broader range of industrial and food application and command a larger demand in international markets. Using conventional breeding procedures, it is not possible to obtain hybrid progeny in grape crosses involving seedless (stenospermocarpic) female parents. Modern biotechnology and especially, the application of in vitro techniques, offers the unique possibility of obtaining new forms of seedless grapevine vines, with rapid multiplication in controlled environmental conditions resulting in healthy material. The International Council for the Study of Virus and Virus-like Diseases of Grapevine recognizes over 70 infectious agents affecting grapevine. The recognized virological check for grapevine varieties from the Genofond of the Research-and-Practical Institute for Horticulture and Food Technologies Institute indicates that seedless genotypes demonstrated virus particles in 70-80% of analyzed samples. Assessment of the phytosanitary status of referenced plants confirmed the widespread prevalence of the degenerative and leafroll complex of viruses (Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus (GLRaV -3), Arabis mosaic virus (ArMV), and Grapevine virus B (GVB). Among the tested techniques (meristem culture aided with thermo- and chemotherapy), somatic embryogenesis seems to be a promising procedure for virus elimination. Regeneration through somatic embryogenesis permits obtaining plants free from some harmful viruses and allows recovery of a higher proportion of seedless progenies. Technologies associated with in vitro embryo development represent a single enabling method in a relatively short period of time to produce plants from new combinations of genotypes with different degree of seedlessness. Previous results indicate that the success of in vitro technique depends on the selected medium, hybrid combination and post-anthesis period (determines embryo age until the time of transfer onto culture medium). In conformity of the proposed objectives were made the crosses according to the scheme: seedless x seedless; seedless x seed; autopollination, selfpollination, using the maternal and paternal forms plants infected with viruses degenerative complex: GFLV, GLRaV-1, GLRaV-3 and ArMV. As biological
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materials were used the 5 maternal forms (Apiren roz Basarabean, Apiren Negru de Grozeti, Centinales seedless, 1-15-15 and TAD VOG) and 6 paternal genotypes (Apiren roz, Apiren roz Basareabean, Interlaikin, Romulus, I-2-24, I5-58). Viruses were detected using serologically test enzyme-linked immunosorbent assay (ELISA), immuno-electron microscopy (IEM) or negative staining (NS). For ELISA were used commercial antisera (Sediag, France) against GFLV, ArMV, GLRaV-1 and GLRaV-3, according to manufacturer instructions. The berries resulted from free crosses of the genotypes were harvested 3540 days after anthesis. Collected berries were cold pretreated and then surface sterilized with 70 % ethylic alcohol followed by 5.2 % calcium hypochlorite and washed with sterilized distillated water. The ovules were aseptically extracted and inoculated on cultivation medium in Petri dishes or pots. Two recovery medium were selected: Nitsch, Nitsch (1969) supplemented with 1.5 mg/l of IAA, 1 mg/l of zeatin and 0.2 mg/l of giberilinic acid (GA3) (NN1) and Nitsch, Nitsch (1969) with addition of 2.5 mg/l of IAA, 0.2 mg/l of GA3 and 0.3mg/l of putrescine (NN2). Ovules were cultivated for 30 days in total darkness conditions at 2520C, and then transferred in culture room in the same temperature regime and period of 16 hours of light. Every 30 days until seeds residuum were dissected to recover viable embryos. Viable embryos were placed on recovery mediums. The composition of mediums was the same: half strength of macroand microelements, and vitamins after Murashige Skoog (1962) with 15 g/l of sugar, 100 mg/l of mio-inositol, 3 mg/l of glicine and 6.8 g/l of agar. It was find that, the reaction of immature embryos to the in vitro cultivation depends essentially on genotype. The way of converting into plants the embryos recovered by in ovulo manipulations, must take into account the peculiarities of the varieties that were involved in the crosses and phytosanitary status. The number of required plants varied between 75-90% from viable embryos, depending on the cultivation mediums, genotypes used in crossings and the age of embryos at the time of transfer onto mediums, as well as phytosanitary status of parental forms. Considerable attention is paid to the selection of parental genitors, the requirements during grape harvest and viable embryos excising. More descendants with desired characteristics, especially with the seedless feature, are obtained from combinations where both parents are seedless. Experimental data shows the results of virus eradication are variable depending on the number and type of viruses, peculiarities of starting material. Primary results of virological check of grapevine plantlets before ex vitro adaptation show that somatic embryogenesis proved to be effective technique in eradicating phloem-limited viruses.

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ANTIOXIDANT ACTIVITY OF POLYPHENOLS FROM Vitis vinifera CVS. isabelle LONG-TERM CALLUS CULTURE UNDER THE INFLUENCE OF SOME ELICITORS TREATMENT
R. MIHAI, F.E. HELEPCIUC and M.E. MITOI
Institute of Biology Bucharest, Romanian Academy

In the last decade, it has been established that oxidative stress is among the major causative factors in induction chronic and degenerative diseases such as: artherosclerosis, diabetes mellitus, cancer, Parkinson`s disease and immune dysfunction. Antioxidants can be effective for the suppression of such disorders and in prevention of the free radical formation. Nowadays, a considerably increasing interest consists in finding naturally occurring antioxidants from plant sources used in food industry and in preventive medicine in order to replace synthetic antioxidants restricted due to their possible carcinogenic effect. Polyphenolic compounds play an important role in adsorbing and neutralizing free radicals, quenching singlet oxygen or decomposing peroxides. The antioxidant properties of polyphenols have been widely studied, but it has become clear that the mechanisms of action of polyphenols go beyond the modulation of oxidative stress. Their synthesis and accumulation is generally stimulated in response to biotic/abiotic stresses. In the present study, total phenolic content and the evaluation of antioxidant potential by DPPH test, were comparatively analyzed due to the influence of some biotic and abiotic elicitors in the long- term callus extracts of Vitis vinifera cvs Isabelle. This study was achieved by carrying out a quantitative analysis of the role of elicitors on resveratrol and total phenols production, compounds in general responsible for the antioxidant activities, in Vitis vinifera L callus culture. The elicitor treatment consisted in using eight medium variants in a two-stage experiment. For the first stage of the experiment, the long- term callus was grown on the medium variants supplemented with mannitol in concentration 2mM to ensure the callus proliferation. Further subcultivation of the callus during the second stage of the experiment ensured the biosynthetic capacity of the callus using in the medium the addition of elicitors such as Fusarium oxysporum extract, mannitol, abscisic, jasmonic acids, ethephon, in different concentrations. The highest antioxidant activity was achieved using as elicitors treatment, in the two-stage culture system, the combination of mannitol (2mM) and jasmonic acid (40 M), suggesting that this culture system could be used as a new method for improving natural antioxidants biosynthesis.
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The antioxidant activity may be attributed to the high content of phenolic compounds and resveratrol in the callus extract subject to the treatment of these combined elicitors. Moreover, this two-stage culture system for the Vitis vinifera L. cvs Isabelle callus extract can be used as an easily accessible and rich source of natural antioxidants.

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AN EFFICIENT METHOD FOR SOMATIC EMBRYOGENESIS INDUCTION AND PLANT REGENERATION FOR Serratula bulgarica ACHTAROFF ET STOJ. EX SITU GERMPLASM CONSERVATION
A. AIFTIMIE-PUNESCU
Institute of Biology Bucharest, Romanian Academy

Serratula bulgarica is a Balkan near-endemic and critically endangered plant species growing in the herbaceous plant communities from wet meadows to steppes from North-Western Bulgaria and Eastern Romania. Population are strongly fragmented and with low number of individuals. The main conservation measures already taken, concerns only in situ approaches. The proposed method relied on indirect morphogenesis pathway followed by the morphogenetic competent somatic cells. Meristemoidlike structures and non-organogenic calli were induced from explants derived from aseptically growing seedlings (leaf fragments and roots). After 30 days of culture on basal media (MS) supplemented with different combination of auxins and cytokinins the best morphogenetic response for both type of explants, were registered on MS media supplemented with 1mg/l 6-benzylaminopurine (BAP) and 0.1mg/l 1naphthylacetic acid (NAA). The results suggest that the response type was highly related with the explant source; the leaf epidermal and mesophyll cells showed embryogenic competence while root epidermal and parenchymal cells show callusogenic competence. Onto media supplemented with 1mg/l Kinetin (Kn) plantlets derived from leaf explants elongate and rooted at optimal rate. Root explants yield non-embryogenic callus which proliferate when subcultured on fresh media supplemented with 1mg/l BAP and 0.1mg/l NAA. The method presented is efficient for plant regeneration and subsequent natural population recovery or ex situ conservation. Likewise, non-organogenic callus yield from roots explants could be important as ecdydisteroids source, with multiple applications in medicine and plant defense.

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GENETIC STABILITY ASSESSMENT OF Scilla autumnalis REGENERANTS

C. BANCIU and F.E. HELEPCIUC
Institute of Biology Bucharest, Romanian Academy

Scilla autumnalis is a critically endangered plant species from Romanian flora of conservative, pharmacological and ornamental applications. The genetic stability of the regenerants was evaluated using the RAPD method by amplifying the DNA of regenerants with non-specific plant polymorphic primers. A number of 5 samples from individuals regenerated from a single plant were used to compare their amplicons. As a standard a sample from natural habitat individual was used. The results showed the identity of the amplicons on all five samples proving that the regenerants kept their genotype and the in vitro culture conditions did not affect the original genetic pattern of the plant species. This work was cofinanced from the Romanian Academy project no. RO1567-IBB06/2012 and from European Social Found through Sectoral Operational Progeamme Human Resources Development 2007-2013, project number POSDRU/89/1.5/S/63258 Postdoctoral school for zootechnical biodiversity and food biotehnology based on the eco-economy and the bioeconomy required by eco-san-genesys.

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COMPARATIVE STUDIES BETWEEN FERNS GAMETOPHYTE AND SPOROPHYTE BY BIDIMENSIONAL ELECTROPHORESIS AND POLYPLOIDY ANALYSIS
F. ALDEA1, F.E. HELEPCIUC1, C. BANCIU1, L. DOMALSKA2, S. SPANIEL3, M.E. MITOI1, A. BREZEANU1 and L.C. SOARE4
1

Institute of Biology Bucharest, Romanian Academy; 2Plant Biotechnology Department, Botanical Garden Center for Biological Diversity Conservation, Polish Academy of Sciences; 3Institute of Botany of Slovakian Academy of Sciences; 4University of Pitesti, Faculty of Science

Athyrium filix-femina, Polypodium vulgare and Asplenium trichomanes are three valuable ferns species due to their potential as ornamental plants and secondary metabolite production originate to Valsan Valley protected area. Our primary experiments were focused on developing an optimal in vitro multiplication method and medium term conservation for these species. The reactivity of each species has been evaluated on the basis of biometric (morfometric and gravimetric) and biochemical determinations. Current study shows the differences between gametophytes and sporophytes of the mentioned species by comparing the proteomic profiles and determination of ploidy levels by flow cytometry. We used gametophytes and sporophytes from Athyrium filix-femina, Polypodium vulgare and Asplenium trichomanes obtained by in vitro culture and multiplicated on MS 1/2 medium. The protein samples were separated by two-dimensional gel electrophoresis and stained with Coomassie Brilliant Blue 250. Proteins were isolated using to a phenol-based extraction procedure (Hurkman and Tanaka 1986). Although in previous studies using PAGE technique we have not observed significant differences between gametophyte and sporophyte, the patterns obtained by two-dimensional gel electrophoresis showed the existence of distinct spots for both gametophyte and sporophyte extracts. The differences in protein expression suggest that gametophyte and sporophyte have different metabolic pathways of protein synthesis. Ploidy level was determined using two step procedure with Otto I+II buffers and the fluorochrome DAPI. In case of Athyrium filix-femina and Asplenium trichomanes both sporophyte and gametophyte were diploid, while for Polypodium vulgare we detected that the gametophyte was haploid and sporophyte was diploid. It is possible that the diploides origin of Athyrium filix-femina and Asplenium trichomanes gametophytes to appeare by a false meiosys. This study presents a preliminary analysis of proteomics profiles and compares the ploidy level for detection of further changes that may occur
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during the process of cryopreservation at the molecular level. Proteomic analysis allows the observation of translationally manifested cell physiological response.

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ELECTRONOMICROSCOPICAL ANALYSIS OF THE Usnea barbata (L.) MOTT. (LICHENOPHYTA) SYMBIOTROPHIC FORMATIONS REGENERATED IN IN VITRO CULTURE
D. VOICU and A. BREZEANU
Institute of Biology Bucharest, Romanian Academy

The aim of the paper is to realize a ample cytological study of the symbiotrophic formations which are developed in in vitro culture, in order to elaborate an experimental system for lichens thalli resynthesis useful for both classical and modern cell biological researches as well as for biotechnological applications and for their conservation and repopulating strategies. Although symbiotrophic formations regenerated in in vitro culture seems to be similar to their natural correspondents, observations on their ultrastructure revealed some significant differences. The most affected cellular substructures seem to be chloroplasts of the phycobiont cells which presents a small number of thylakoids and pyrenoglobules randomly spreaded. Another characteristic is the presence of so - called storage droplets. These kinds of storage bodies are associated with the cytoplasm and may represent reserves of lipids, proteins and other metabolic substrates. Two categories of storage bodies were identified. One of them presents electronodense content and in other one electron light content, probably in relation with their composition. Their presence may be determinated by inhibitory effect of the culture conditions especially on the thylakoid system development. Possibly, they are involved in enabling of phycobiont to endure adverse environmental conditions, and may represent adaptative modification. The fine structure of the mycobiont is from many points of views similar with that of the old mycobiont from the native thallus. Concentric bodies or ellipsoidal bodies are visible and are present in the cytoplasm like small formations, singly or in clusters with a core body surrounded by electron transparent halos. The cell wall also consist of the cell layers and the thickness of the outer layer, varying according to the age. As a peculiarity is that the outer layer consists of an amorphous electronodense material and a fibrilar matrix and the plasmalemma is highly folded. Our investigations allowed us to appreciate that the experimental systems do not induce severe alterations of the inner structure of the cells and could represent an alternative methodology to obtain lichen biomass of the biotechnological interest namely secondary metabolites.
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Copyright Institutul de Biologie Bucureti Material editat de Institutul de Biologie Bucureti

11 decembrie 2012

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