RNA-Seq analysis of blood meal induced gene-expression changes in Aedes aegypti ovaries

- RNA-Seq analysis of blood meal induced gene-expression changes in Aedes aegypti ovaries.
- Each cycle of egg maturation is tightly linked with the intake of a fresh blood meal for most species.
- Large-scale gene-expression changes occur following each blood meal in various tissues, including ovaries.
- Here we analyzed mosquito ovary transcriptome following a blood meal at three different time points to investigate blood-meal induced changes in gene expression in mosquito ovaries..
- Results: We collected ovaries from Aedes aegypti that received a sugar meal or a blood meal on days 3, 10 and 20 post blood meal for transcriptome analysis.
- Over 4000 genes responded differentially following ingestion of a blood meal on day 3, and 660 and 780 genes on days 10 and 20, respectively.
- Although most of the DEGs returned to the nonsignificant level compared to the sugar-fed mosquito ovaries following oviposition on days 10 and 20, there remained differences in the gene expression pattern in sugar-fed and blood-fed mosquitoes..
- Conclusions: Enrichment of OBPs following blood meal ingestion suggests that these genes may have other functions besides being part of the olfactory system.
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- Full list of author information is available at the end of the article.
- Mosquito-borne pathogens are responsible for some of the widespread infectious diseases worldwide, such as malaria, encephalitis, filariasis, dengue fever, and yellow fever [1, 2].
- The availability of the mosquito genome sequence provides an excellent opportunity to identify host gene targets to control pathogen transmission..
- After each gonotrophic cycle, mosquitoes return to their host-seeking stage for another blood meal.
- Mosquitoes that acquire pathogens during the first blood meal may transmit the pathogen to an uninfected host during these subsequent blood meals.
- In Aedes aegypti, an anautogenous mosquito, the pre- blood meal period in the first gonotrophic cycle also includes the post-eclosion development period, which persists from 72 h to until the uptake of the first blood meal.
- Oogenesis in the mosquito ovary begins post- eclosion, but the oocyte growth is attenuated at a resting stage until the ingestion of a blood meal after which egg development continues until oviposition (i.e., egg- laying).
- In the post blood-meal (PBM) period, mosqui- toes use about 20% of the blood nutrients to produce egg components within 48 h and another fraction to carry out intense biosynthetic activities, then excrete the rest [13, 14].
- Protein-rich blood meal is required for oocyte development and vitellogene- sis, during which yolk constituents (both protein and lipid) generated in the fat body are taken up by oocytes for storage and later use during embryogenesis.
- Vitello- genesis and oogenesis require a high level of coordin- ation of molecular events in the fat body and ovary [3]..
- A blood meal triggers the release of ecdysone by the ovaries.
- Clearly, a complex series of physiological events occurs in multiple tissues (e.g., midgut, fat body, and ovary) fol- lowing blood meal ingestion.
- RNA-Seq analysis provides a useful tool to analyze changes in gene expression in the whole organism as well as in pertinent tissues [19, 20].
- Comparing gene expression patterns at various time points between sugar-fed and blood fed mosquitoes and tissues, one can identify the organism’s or tissue-specific responses to the blood meal.
- Here, we used RNA-Seq to analyze differential gene ex- pression following a blood meal at three time points (Days 3, 10, and 20) in A.
- 96 h (i.e., the duration of the gonotrophic cycle) and also during embryonic development.
- In these studies, gene expression at late time points in the gonotrophic cycle was monitored in gravid ovaries.
- Mosquitoes are expected to return to the pre-blood meal stage following each gonotrophic cycle.
- Our results indicated that al- though gene expression patterns following the gono- trophic cycle at late time points do not completely match with that of the non-blood fed (i.e., sugar fed) control mosquito ovaries, most differentially expressed genes (DEGs), however, return to the sugar-fed control level.
- In addition, several detoxification and defense- specific genes are also expressed at the early time point, suggesting that ovaries become prepared to avoid the ill effects of the blood meal derived toxic metabolites or to effectively deal with the pathogens that may accompany the blood meal..
- aegypti ovary transcriptome We carried out experiments to determine the ovaries’ re- sponse to blood meal ingestion by RNA-Seq analysis..
- Conse- quently, by day 10, most mosquitoes in the blood-fed group had laid their eggs and returned to the non- gonotrophic stage, similar to SF females.
- However, in several mosquitoes there were one or few unlaid eggs in the ovaries.
- More than 77% of the reads mapped to the host genome, with about 94% mapping to the gene regions and 6% to the intergenic regions (Suppl.
- We carried out a principal component analysis (PCA) of SF and BF libraries to examine the clustering of data based on ingestion of a sugar meal or a blood meal.
- We have compared our RNA-Seq results with those of the previously reported transcriptome ana- lyses of A.
- Nature of DEGs in mosquito ovaries at different time points following blood meal ingestion.
- Most of the DEGs are not characterized.
- The name derives from the expression of many family mem- bers in the olfactory system of insects.
- Additionally, there were differences in the time (72 vs 96 h) of sample collection.
- It has been shown that significant changes in gene expression patterns occur in Aedes strains depending on the place of origin, number of gen- erations in the laboratory, and susceptibility to dengue infection [44]..
- 1 Principal component analysis of the ovary RNA-Seq data.
- Since we are studying the expression pattern in ovaries, these results suggest that ovaries may take part in the oviposition site selection or they may perform totally different functions..
- Some of the induced OBPs are known to be involved in sensitive detection of oviposition attractants.
- aegypti) not only binds to the mosquito oviposition pheromone, but is also involved in the recep- tion of some oviposition attractants [45].
- expressed in the male reproductive tissues and trans- ferred to the spermathecas of females [47].
- Several members of cytochrome P450 (CYP) family de- toxification genes had altered expression patterns in the BF samples.
- Four glutathione transferase genes exhibited 2–4 fold overex- pression in the BF samples.
- Defensin genes, GNBP genes, and Cecropin genes were over expressed following blood meal ingestion.
- In addition, expression of various defense- associated genes was induced following the blood meal..
- Gene expression in ovaries occurs in waves following a blood meal [21, 25].
- Genes that are up- or down-regulated early in the gonotrophic cycle are not the same that occur later during egg development.
- It is possible that changes in expression at the whole-body level may con- ceal the tissue-specific changes [22], or the defense re- lated genes are induced later in the gonotrophic cycle.
- It is likely that slightly overexpressed genes on day 3 could be leftover RNAs from high levels of overexpression early in the gonotrophic cycle.
- The expression of immunity genes in ovaries PBM may be one of the reasons that ovary infections occur late.
- It would be interesting to see the ovary’s response to an infectious blood meal..
- On days 10 and 20 PBM, most of the genes that had an altered expression pattern on day 3 in BF samples exhibited no significantly different expres- sion patterns compared to SF samples.
- The numbers in the overlapping areas indicate genes that were common to both or to all three different time points.
- overexpression in the day 10 sample (Table 1)..
- All DEGs were subject to gene ontology analysis using Blast2GO plug-in tool of the CLC workbench.
- These results suggest that mosquitoes are ready for another blood meal..
- Fold changes in the expression level ranged from 660-fold increase to an 83-fold decrease on day 3 PBM..
- aegypti mosquitoes following inges- tion of a Zika virus containing infectious blood meal [41].
- Among the genes tested, although the level of expression change does not match with that of the RNA-Seq analysis, the overall trend (over or under- expression) remained the same in both RT-qPCR and RNA-Seq analyses.
- Among the 10 genes tested, we in- cluded OBP46, an odorant-binding protein, to confirm that OBPs were differentially expressed following a blood meal in ovaries.
- Most of the DEGs on day 3 returned to the non- significant level at later time points.
- These results were expected since most of the mosquitoes have laid their eggs by day 10 and are ready for a second blood meal..
- Table 3 Differentially expressed defense-specific genes post blood meal.
- The result of our gene-ontology analysis is also similar to that of the gene expression studies by EST analysis in autogenous Georgecraigius.
- The data presented here represent ovary tran- scriptomes in mosquitoes that do not take a second blood meal during a gonotrophic cycle.
- Further studies with additional blood feeding in the same gonotrophic cycle are necessary to address this issue..
- A large number of genes were differentially expressed following ingestion of a blood meal in mosquito ovaries when compared to SF ovaries.
- Differential expression is highest on day 3 PBM and most of the DEGs return to the non-significant level by day 10, although expression was not identical to the SF samples.
- Since GO terms for both days 10 and 20 are similar, they are not shown in the figure.
- Following library concentration and quality control, final libraries were pooled to 10 nM and the concentration of the pool was determined using the Kapa Biosystems Universal qPCR kit.
- Compressed FASTQ files were extracted, trimmed, and filtered using the Table 4 Quantitative PCR validation of the RNA-Seq ressults.
- default parameters of the modified Mott trimming algo- rithm as implemented in CLC.
- More spe- cifically, reads were mapped to the VectorBase reference genome (Aedes aegypti L 5.0) with mismatch, insertion, and deletion costs parameters set at 2, 3, and 3, respect- ively, with a length fraction of 0.8 in mapping parameter, where 80% of the nucleotides in a read must be aligned to the reference genome.
- Functional annotation and gene ontology analysis were carried out using the Blast2go Plug-in tool of the CLC workbench.
- PBM: Post blood meal;.
- The funding bodies had no role in the design of the study, and collection, analysis and interpretation of the data and writing of the manuscript..
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