Refractory Reasoning: Teasing Apart and Characterizing Refractory Mechanisms

to Dengue in Aedes aegypti

Heather Coatsworth , Paola Caicedo , Clara Ocampo , and Carl Lowenberger
1

http://aedes.caltech.edu

Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada, Centro Internacional de Entrenamiento e Investigaciones Mdicas, Cali, Colombia
2

INTRODUCTION
Dengue: A tropical infectious disease, primarily
transmitted by adult female Aedes aegypti, that infects 50100 million people per year1. There are four serotypes of
Dengue that are antigenically related, 1 infection causes
immunity to secondary infections with the same serotype. 2
infections with a different serotype can cause cross reactivity
and lead to hemorrhaging, and dengue shock syndrome 2.
Aedes Aegypti and Dengue: Ae. aegypti are
anthropophillic daytime biters. 24 hours after biting an
infected human, dengue can be found replicating in the
mosquito midgut. After 4 days the virus migrates into the
hemocoel, replicates again, and finally, after 12 days,
migrates to the salivary glands to be passed onto the next
host3.
Vector Control: There are no vaccines or drugs currently
available, and as such, insecticides and source reduction are
common. Recent focus has shifted towards mosquito biomanipulation techniques such as paratransgenic techniques
(ex. Wolbachia) preventing viral transmission, and the sterile
insect technique, that prevents future progeny through the
release of sterile males4.

RESULTS & DISCUSSION

RNA Sequencing: Each library generated between 14
and 28 million 101 bp single end reads.
Read Processing: 83% of reads mapped to genome
(17% unmapped).

Choosing Candidate Genes: Three criteria were used to

generate a list of possible candidate genes: i) highly
differentially expressed, ii) highly differentially expressed and
immune related, and iii) marked as intriguing by literature
reviews.

Differential Expression: Since Cuffdiff and DESeq were

both extremely conservative, GFOLDs DE gene list was
used. The top DE expressed genes can be seen in Figure 3.
Genes were putatively classified as acting in either an 'antiviral (AV) or 'pro-viral (PV)' manner, based on their direction
of change in conditions of interest.

Anti-viral genes:
AAEL011539: Metalloproteinase
AAEL015458: Transferrin
AAEL005444: Pyrokinin
AAEL001878: Lipase
AAEL010195: Trypsin

AV

Pro-viral genes:
AAEL001650: MD2-Like Receptor
AAEL004386: Peroxidases
AAEL013338: Lethal (2)
AAEL002606: Odorant binding

PV

Figure 3 Heatmap of top DE genes, shown as belonging to the proviral (PV) or anti-viral (AV) group. Treatment groups represented as:
Cali-S bloodfed (Sb), Cali-S virus fed (Sv), Cali-R bloodfed (Rb) and
Cali-R virus fed (Rv), with associated time point

Figure 1 Schematic of Cali-R strain with dengue virus

Clustering: As seen in Figure 4, samples clustered into

either the 48 hour time group or the 24 and 36 hour time
group. Samples clustered by viral condition in the 48 hour
group, while clustered by time, strain and finally viral
condition in the 24 and 36 hour group.

Research Question:
Can we use RNA sequencing to determine what is
happening in the midgut of these resistant Cali Ae. aegypti
mosquitoes?

Future Directions: qPCR will be used to validate

RNAseq DE results from all candidate genes of interest.
Sequencing of regions of interest will be completed to validate
variant calling. Expression studies will be completed to
investigate possible phenotypic variant effects. Lastly, RNAi
knock-downs will be completed on selected pro-viral genes to
try and reverse phenotype (from S to R) via alternation of
gene expression.
Alternative Applications: We could apply similar
procedures to other emerging arboviruses such as
Chikungunya. If knock-downs are successful, gene editing
could enable virus control through creating and propagating
populations of resistant mosquitoes.
Climate Change Implications: Changes in travel,
urbanization, and climate have already indirectly expanded
disease range by extending ideal Ae. aegypti habitats. Future
and more drastic climatic changes will most likely exasperate
this issue10.

METHODS

REFERENCES

Library Preparation: Cali-R and Cali-S were fed blood or

blood + Dengue-2. Midguts were dissected for each of the 12
treatments (Table 1). Subsequently, RNA extraction, polyA
selection, cDNA synthesis, purification, PCR amplification,
and illumina sequencing were completed.
Strain
S or R

Time (hours post feeding)

24 or 36 or 48

Table 1 Outline of 12 experimental treatment groups

RNA Sequencing: Libraries were multiplexed using the

Illumina miSeq platform.
Read Processing: See Figure 2. (Genome obtained
from VectorBase6)

http://pixshark.com

CONCLUSION

Situation in Cali, Colombia: 70% of Cali, Colombian

field mosquitoes are susceptible (S), while 30% are resistant
(R) (Figure 1), and do not transmit dengue 5.

Condition
Blood or Blood + DENv2

Mairuhu, A. T. A., Wagenaar, J., Brandjes, D. P. M., and van Gorp, E. C. M. (2004).
Dengue: an arthropod-borne disease of global importance. Eur J Clin Microbiol Infect Dis. 23,
425 433.
2
Barnes, E. (2005). Diseases and Human Evolution. University of New Mexico Press.
Albuquerque, N.Y.
3
Chauhan, C., Behura, S. K., deBruyn, B., Lovin, D. D., Harker, B. W., Gomez-Machorro,
C., Mori, A., Romero-Severson, J., and Severson, D. W. (2012). Comparative Expression
Profiles of Midgut Genes in Dengue Virus Refractory and Susceptible Aedes aegypti across
Critical Period for Virus Infection. PLoS One. 7(10), e47350.
4
McGraw, E. A. and ONeill, S. L. (2013). Beyond insecticides: new thinking on an ancient
problem. Nat. Rev. Microbiol. 11, 181 193.
5
Ocampo, C. B., Caicedo, P. A., Jaramillo, G., Bedoya, R. U., Baron, O., Serrato, I. M.,
Cooper, D. M., and Lowenberger, C. (2013). Differential Expression of Apoptosis Related Genes
in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus. PLoS One.
8(4), e61187.
6
Megy, K. (2012). VectorBase: improvement to a bioinformatics resource for invertebrate
vector genomics. Nucleic Acids Research. 40, D729 734.
7
R Development Core Team (2008). R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0.
8
Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing.
arXiv preprint arXiv:1207.3907 [q-bio.GN] 2012
9
Cingolani, P., Platts, A., Wang, L. L., Coon, M., Nguyen, T., Wang, L., Ruden, D. M.
(2012). A program for annotating and predicting the effects of single nucleotide polymorphisms,
SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly, 6(2), 80
92. doi:10.4161/fly.19695.
10
Bhatt, S., Gething, P. W., Brady, O. J., Messina, J. P., Farlow, A. W., Moyes, C. L.,
Drake, J. M., Brownstein, J. S., Hoen, A. G., Sankoh, O., Myers, M. F., George, D. B., Jaenisch,
T., Wint, G. R. W., Simmons, C. P., Scott, T. W., Farrar, J. J., and Hay, S. I. (2013). The global
distribution and burden of dengue. Nature. 496, 504 507.
1

Figure 4 Hierarchical clustering of treatments based on

normalized gene expression values for all genes

Variant Calling: 258,303 unique variants found, 19,599

of these predicted to have a high effect, 2,041 falling within
exons (Figure 5).

ACKNOWLEDGEMENTS
CIDEIM Vector Biology and Control Team: Idalba Mildred
Serrato, Julieth Mina, Mabel Moreno, Anglica Aponte, Ana
Lucia Estrada Jaramillo
SFU: Timothy Loh, Alessandra Guarneri, Liliana Lopez,
Rachel Halipchuk, Carson Gill, Chris Combe, Jeffery Yung, Jim
Mattson, Fiona Brinkman, Geoff Winsor

Figure 2 Flowchart of Bioinformatic processes used to align, map,

and process treatment reads

Clustering: R7 was used to visualize expression patterns

by performing complete linkage hierarchical clustering on DE
data.
Variant Calling: Freebayes8 was used to call variants,
after which SnpEff9 was run in order to predict the effect of
each of the variants.

Figure 5 Venn diagram of all four groups denoting shared and

unshared exonic variants