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M.

Danish Faraz
Msc(Hons) Entomology
2nd semester
2014-ag-4951
Supervisor:Dr.Dildar Gogi
sb
Topic:

‘ Isolation ,identification and role of fruit


fly gut bacteria in its manangement.
Fruit Fly

 It is notorious pest of different fruit crops mainly Citrus & also different
vegetables.
 Our country is facing losses of at least $200 million annually owing to fruit fly
attacks which is serious threat to the export of fruits.
 Fruit fly can be Control through pheromonal traps like methyl uginol which js
majnly sex pheromonal trap .
 Most effective pesticide against fruit fly is Dipterex.which mainly consists of
Trichlorofon & methyl uginol.
 But we can also control fruit fly movement & we can manage it through
microflora present in the fruit fly gut.
 There are different genera of bacteria are present in the gut of fruit fly.
 We should isolate & identify these bacteria for proper study how these
bacteria are helpful in the management of fruit fly.
Isolation of bacteria

 Mexican fruit flies are attracted to the odor of Staphlycococcus aureus


cultures largely to find protein.
 It might be the construction pf plasmid Encoding a specific gene of inerest for
use in a manipulation of the gut flora,the utility bacteria in biological control
and a better understanding of reasons for resistannce of insect associated
bacteria.
 Mature gravid adult A.ludens were collected & then transferred at the
rearing facility
 The larvae are reared on an artificaial diet consisting mainly of wheat germ
and torula yeast.
 The adults are fed a diet of sucrose:brewers yeast hydolysate.
 Larvae of A.ludens were placed into 70% ethanol and gently swirled for 1.5 to
2min and then washed twice in sterile distilled water.
 Using standard aseptic techniques, surface sterilized flies were individually
dissected in sterile distilled water.
 Each gut will be removed,spread directly on a slant of Nutrient agar and
incubated at 36°C for 24 to 48hrs.
 At the end of incubation period,dicrete colonies were aseptically removed by
using inoculation loop,restreaked on nutrient agar.
 Individual colonies were isolated and subcultured twice to ensure purity.
 All bacterial isolates were initially Gram-stained for Gram-positive and Gram-
negative identification and tested for oxidase activity.
 Analytical profile indexes from kit API 20 E,API 20 NE,API 20 strep,API
staph,API CHB and API 50 CH systems were used for the identification of
bacteria.
 The ID profiles were from excellent to good offered by the API codes .
Identifcation of Bacteria

 Analytical profile indexes from kit API 20 E,API 20 NE,API 20 strep,API


staph,API CHB and API 50 CH systems were used for the identification of
bacteria.
 The members of the family Enterobacteriaceae are the most common micro
organisms
Associated with Tephritidae.
 These bacteria appeared to be an important adult food source & thus played
a significant role in the biology of fruit fly and possibly as a stimulus to
attract the foraging flies to the host fruit tree .
 Enterobacteriaceae is the predominant organism present in the different
sources of A.ludens.
 Through this technique we identified other species belonging to the families
pseudomonadaceae, Vibrionaceae, Micrococceae,Bacillaceae and the genus
Listeria.
Antibiotic assay:
 In this assay we observed the antibiotic sensitivity and resistance of six
isolates.
 Generally these isloates showed the highest frequency of resistance to
penicillin & ampicillin followed by novobiocin and erythromycin and lowest to
cephalothin.
 All strains showed high sensitivity to rifampin and streptomycin.
 Thia antiobiotic resitance can be conferred by various mechanisms and is
often associated with transferable plasmids (t-plasmid).
 The variability in antibiotic resistance paterns we found & indicates that this
characteristic might be useful for distinguishing variant of these bacteria.
 Antibiotic rrsistance that we found might have implications for the
construction of plasmids encoding specific gene of interest and for the utility
of different bacterial isloates in biological control of the alternative control
strategies such as paratransgenisis by using recombinant strains.
 Paratransgenesis is based on the use of symbiotic bacteria to express
effector molecules inside the target vector to eliminate the pathogen.
Management of Fruit Fly

 Fruit flies that have their natural gut microbes were less active than flies that
lack the gut bacterium Lactobacillus brevis.
 Enzyme modifies certain sugars, which leads, by an unknown process, to a
decrease in the level of the sugar trehalose in the body of flies in which this
bacterial enzyme is present.
 which a decrease in trehalose is accompanied by a decrease in the activity of
octopaminergic neurons (those that produce the neurotransmitter molecule
octopamine) that regulate fly locomotion flies that have their natural gut
microbes.
 Flies lacking Lactobacillus brevis have higher levels of the sugar trehalose in
their body and are proposed to have higher activity of octopaminergic neurons.
 These gut bacteria influence the behavior including movement.Fruit flies
which do not carry bacteria are hyperactive move faster over greater distance
& took shorter rests.
 Locomotion is important for a number of activities such as mating and
searching for food.
 When germ free flies received L.brevis their movement slow down.
 Through octopamine these flies can produce nerve cells which helps in the
normal movement but these bacteria turn off this octopamine so they cannot
move faster for foraging or mating .
Bacterial Bait preparation

 The predominant bacterial symbionts in the gut of fruit flies, Bactrocera spp.
Washed and fermented bacterial preparations of these symbionts were
evaluated for their attractancy to fruit fly species (Bactrocera cucurbitae) under
laboratory and field conditions.
 P. agglomerans (washed bacterial preparation) in combination with sugar
attracted maximum number (mean 11.33 adults/30 min) of B. cucurbitae, while
protein hydrolisate in combination with sugar .
 Combination of washed bacteria proved superior to control in terms of
attractancy for both species.
 Fermented bacterial prepration ,k.oxytoca In combination with jaggary attracted
to maximum fruit flies when applied in lab condition on potted plants.
 This fermented bacteria can also used in field condition on foliar application
and as bait in combination with different insecticides resulted in significance
reduction in fruit fly infestaion over 65%.