Editor's evaluation: A transcriptomic atlas of Aedes aegypti reveals detailed functional organization of major body parts and gut regional specializations in sugar-fed and blood-fed adult females

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Mosquitoes transmit numerous pathogens, but large gaps remain in our understanding of their physiology. To facilitate explorations of mosquito biology, we have created Aegypti-Atlas (http://aegyptiatlas.buchonlab.com/), an online resource hosting RNAseq profiles of Ae. aegypti body parts (head, thorax, abdomen, gut, Malpighian tubules, ovaries), gut regions (crop, proventriculus, anterior and posterior midgut, hindgut), and a gut time course of blood meal digestion. Using Aegypti-Atlas, we provide insights into regionalization of gut function, blood feeding response, and immune defenses. We find that the anterior and posterior midgut possess digestive specializations which are preserved in the blood-fed state. Blood feeding initiates the sequential induction and repression/depletion of multiple cohorts of peptidases. With respect to defense, immune signaling components, but not recognition or effector molecules, show enrichment in ovaries. Basal expression of antimicrobial peptides is dominated by holotricin and gambicin, which are expressed in carcass and digestive tissues, respectively, in a mutually exclusive manner. In the midgut, gambicin and other effectors are almost exclusively expressed in the anterior regions, while the posterior midgut exhibits hallmarks of immune tolerance. Finally, in a cross-species comparison between Ae. aegypti and Anopheles gambiae midguts, we observe that regional digestive and immune specializations are conserved, indicating that our dataset may be broadly relevant to multiple mosquito species. We demonstrate that the expression of orthologous genes is highly correlated, with the exception of a 'species signature' comprising a few highly/disparately expressed genes. With this work, we show the potential of Aegypti-Atlas to unlock a more complete understanding of mosquito biology. Editor's evaluation Hixson et al. provide a large overview of gene expression level of the mosquito Aedes aegypti through the use of RNA-seq. They analyse gene expression changes in the digestive tract, as well as the 3 body regions and the ovaries in various conditions. These organ-specific transcriptomes fill a hole in our understanding of mosquito vector biology and will be an excellent starting point for many researchers to produce new projects. https://doi.org/10.7554/eLife.76132.sa0 Decision letter eLife's review process Introduction Hematophagous mosquito vectors contribute substantially to the world's disease burden through the transmission of Plasmodium parasites and arboviruses ("World Health Organisation, 2020a | Vector-borne diseases,"). With the rise of insecticide resistance (Moyes et al., 2017; Ranson et al., 2011; "WHO | Insecticide resistance," World Health Organisation, 2020b), and the expansion of mosquito species' ranges potentiated by climate change (Liu et al., 2020), new and more efficient means of vector control are needed, grounded in a solid understanding of mosquito physiology. The FlyAtlas project (Chintapalli et al., 2007; Leader et al., 2018) contributed to the field of insect physiology, not only by characterizing the functions associated with the major body parts and organs of Drosophila melanogaster, but by making it possible to quickly ascertain where in the body, and in what quantity, any gene of interest is transcribed. FlyAtlas also showed how the various contributions of different organs added up to constitute the whole fly transcriptome, providing an estimate of the relative transcriptional yield of the parts of the Drosophila melanogaster body. Similar anatomical datasets have been created for several species of Anopheles (Baker et al., 2011; Martínez-Barnetche et al., 2012; Sreenivasamurthy et al., 2017), but not for Aedes mosquitoes. While transcriptional profiles have been created for various Ae. aegypti body parts in diverse studies (e.g head (Ptitsyn et al., 2011), Malpighian tubules (Li et al., 2017), midgut (Angleró-Rodríguez et al., 2017; Cui and Franz, 2020; Dong et al., 2017; Hyde et al., 2020; Liu et al., 2016; Raquin et al., 2017; Xiao et al., 2017), ovaries (Akbari et al., 2013; Nag et al., 2021), carcass (Akbari et al., 2013; Choi et al., 2012), the lack of uniformity of strain and methodology between these studies makes direct comparison difficult. The mosquito gut is of special interest to vector control, as it is central to the hematophagous lifestyle and serves as the first interface between a mosquito and the pathogens it transmits. Insect guts are highly regionalized organs, with specialized functions differentially distributed along their length (Benguettat et al., 2018; Buchon and Osman, 2015; Buchon et al., 2013; Dutta et al., 2015; Lemaitre and Miguel-Aliaga, 2013). The mosquito gut is divided into five anatomically distinct regions: foregut (comprising pharynx, dorsal diverticula, and crop), proventriculus, anterior midgut, posterior midgut, and hindgut. The crop and dorsal diverticula are muscular cuticle-lined sacs which store imbibed sugar for gradual release into the midgut (Clements, 1992). In Anopheles gambiae, the proventriculus and anterior midgut were found to exhibit an enrichment of transcripts encoding antimicrobial peptides (AMPs) and anti-Plasmodium factors (Warr et al., 2007) suggesting that these regions serve a special defensive function against orally acquired pathogens. Within the midgut, digestive functions are believed to be divided between the anterior and posterior regions, with the former specializing in the digestion of nectar but playing no direct role in the digestion of the blood meal, which is processed in the posterior midgut (Hecker, 1977). The hindgut receives and eliminates excreta from the midgut and urine from the Malpighian tubules (Clements, 1992). Genome-wide transcriptomic profiles have the potential to reveal much more detailed information about the regionalization of function in the mosquito gut. In recent years, many groups have used microarrays and RNAseq to create transcriptomic profiles of mosquito midguts, mostly in the context of infection (Baker et al., 2011; Cui and Franz, 2020; Dong et al., 2017; Hyde et al., 2020; Raquin et al., 2017; Vedururu et al., 2019; Vlachou et al., 2005; Warr et al., 2007). However, to our knowledge, only a single microarray-based study has ever characterized the transcriptomes of individual mosquito midgut regions (Warr et al., 2007) and no transcriptomic profiles have been generated for either foregut or hindgut tissues in any mosquito species. The Ae. aegypti gut's response to the ingestion of a blood meal has been characterized as a biphasic process. In the first phase, mRNAs encoding a cohort of 'early' serine endopeptidases, transcribed in response to juvenile hormone (JH) secretion during previtellogenic maturation, are translated (Bian et al., 2008; Felix et al., 1991; Jiang et al., 1997; Noriega et al., 1996; Noriega et al., 1997; Noriega et al., 2001). In the second, ecdysone signaling mediates the transcription of a 'late' cohort of endo and exopeptidases, with the transcripts of key peptidases and overall proteolytic activity typically peaking around 24 hr post blood meal (pbm) (Barillas-Mury et al., 1991; Brackney et al., 2010; Gooding, 1966; Graf and Briegel, 1982; Graf et al., 1998; He et al., 2021; Isoe et al., 2009b; Noriega et al., 2002). The transcriptional timeline is, however, more complex than a simple early translation/late transcription paradigm might suggest. Several studies have, by RT-qPCR and microarray, documented de novo transcription of peptidases and other genes peaking much earlier than 24 hr pbm (Brackney et al., 2010; Isoe et al., 2009b; Sanders et al., 2003). However, we found no RNAseq data to allow us to compare gut or midgut transcriptomes genome-wide in sugar-fed conditions and across the time-course of bloodmeal digestion. Here, we aimed to complement existing datasets by, first, contextualizing the adult female gut's transcriptome with profiles of the whole body and other major body parts and organs (head, thorax, abdomen, Malpighian tubules, ovaries) with the dual goal of (a) creating an at-a-glance reference for the anatomical distribution of transcripts, genome-wide and (b) facilitating the identification of tissue-specific marker genes which may, in the future, be useful in the construction of tissue-specific expression systems. Second, we created profiles for the five anatomically distinct regions of the adult female gut (crop, proventriculus, anterior midgut, posterior midgut, hindgut) to facilitate explorations of the regionalization of gut function. Finally, we profiled transcriptional responses to blood feeding in the anterior and posterior midgut and at multiple timepoints (6, 24, and 48 hr) after blood meal ingestion. We opted to profile Aedes aegypti, as it is among the most important insect vectors (Powell, 2018), with an increasing worldwide range (Iwamura et al., 2020), and a recently updated genome assembly (Matthews et al., 2018). All the resulting transcriptional data can be accessed at the Aegypti-Atlas online database we constructed (http://aegyptiatlas.buchonlab.com/). With RNAseq profiles for body parts, gut regions, and blood-fed guts in hand, we launched a broad investigation into multiple aspects of mosquito biology. Our inquiries were guided by the following questions: What functions are associated with each body part? What are the functional specializations of the gut regions? How does the gut transcriptome change in the context of blood feeding, and how are those changes distributed in the midgut? What can our atlas tell us about the organization of mosquitoes' antimicrobial defenses? Finally, in a cross-species comparison between the midguts of Ae. aegypti and Anopheles gambiae (s.l.), we asked whether the gut's structure-function relationship is conserved between two hematophagous mosquito vector species. Results The transcriptional landscape of an adult mosquito reflects the embryonic origins and functional signatures of its major tissues In order to develop a transcriptomic atlas of some of the main body parts of Ae. aegypti, we generated RNAseq profiles from the whole bodies and dissected body parts (head, thorax, abdomen, gut, Malpighian tubules, and ovaries) of sucrose-fed adult female mosquitoes of a field-derived 'Thai' strain from a region where arbovirus transmission is endemic (League et al., 2019). For the dissected body parts (but not whole body), we omitted the anterior-most section of the thorax as well as the final segments of the abdomen to exclude transcripts from salivary glands and from sperm in the spermatheca (see Figure 1—figure supplement 1A for diagram). We first evaluated the relative variance between body part and whole-body profiles by Principal Component Analysis (PCA) (Figure 1A). All transcriptomes from the same body part clustered together closely, providing evidence for the replicability of our data. On PC1, body parts were grouped in a manner that accorded with their divergent embryonic origin (primarily mesoderm/ectoderm for head, thorax, and abdomen, endoderm for gut and Malpighian tubules) and whole-body transcriptomes were clustered in between. Ovaries, which contain the germline, strongly separated from the other, exclusively somatic, body parts on PC2. Altogether, PCA indicated that the transcriptomes of body parts are largely defined by their embryonic origin, and that germline tissues possess a highly distinctive transcriptional signature. Figure 1 with 3 supplements see all Download asset Open asset The transcriptomes of Aedes aegypti body parts reflect their embryonic origins and functional specializations. (A) Principal component analysis of the transcriptomes of whole body and body parts; 3–6 replicates per body part. Circled clusters contain body parts of dominantly endodermal (left) and mesodermal/ectodermal (right) derivation. (B) Expression of body-part-specific putative tissue markers (in TPM) (C) Calculated estimates of body parts' contributions to whole body transcriptome; Bubble plots: classic Fisher topGO gene ontology enrichment analysis of genes enriched 5 x (head, gut thorax, Malpighian tubules, abdomen) or 2 x (ovaries) relative to whole body (DESeq2, padj <0.05); Treemaps: 20 highest expressed genes per body part, grouped by category/function; percentages reflect the proportion of the transcriptome comprised by the displayed genes. Each body part we sequenced expressed a set of unique or nearly unique transcripts some of which, we hypothesize, may be exclusive tissue markers. We conducted a census of putative tissue markers (here defined as genes expressed at five or more transcripts per million (TPM) in one body part and enriched at least 50-fold compared to all other body parts). Figure 1B presents a selection of markers from each body part (full census in Supplementary file 1). The quantity of markers ranged from 8 in the abdomen to 391 in the ovaries, which expressed more markers than all other body parts combined; nearly one in fifty of all genes in the genome qualified as an ovary marker. We further noted that ovaries simultaneously expressed the greatest number of highly enriched transcripts relative to other dissected body parts (Figure 1—figure supplement 1B) and the smallest number of highly enriched transcripts relative to whole body (Figure 1—figure supplement 1C). This apparent paradox led us to hypothesize that the ovaries make a disproportionately large contribution to the transcriptome of the whole female mosquito. A gene that is uniquely expressed in the ovaries will not be highly enriched relative to the whole mosquito body if the ovarian transcriptome comprises a large proportion of the whole body's transcriptome. To validate our sequencing results, we performed RT-qPCR amplification of one putative marker gene from each body part. We selected a translation factor (AAEL013144) as a reference gene for our assay over more traditional candidates (Dzaki et al., 2017), on the basis of its robust expression and relatively small variation across samples Figure 1—figure supplement 2A. The results of our RT-qPCR confirmed the anatomical specificity of each selected marker (Figure 1—figure supplement 2B Fig). The existence of transcripts specific to each body part allowed us to calculate their relative contribution to the whole body's transcriptome (see the 'mosquito equation' in Materials and methods). Expressed as percentages, the body part contributions we obtained were: head (4.7%), thorax (19.4%), abdomen (26.2%), gut (14.0%), Malpighian tubules (1.7%), and ovaries (28.8%). It should be noted that the percentage calculated for the thorax is likely to slightly underestimate its actual contribution, as a portion of the thorax was removed (Figure 1—figure supplement 1A) to eliminate salivary glands. This removal likely accounts, at least in part, for the fact that the sum of the calculated scaling factors is 94.9%, rather than 100%. We validated our estimates by using them to predict the whole-body expression of every gene in the genome, then plotting those predictions against observed values from the whole-body transcriptome (Figure 1—figure supplement 3A). Calculated and observed values were highly correlated (slope of 1.00, R2 = 0.95) after removal of a single outlying gene (AAEL018689, a mitochondrial ribosomal RNA) which was underpredicted by our calculation. This analysis validates our hypothesis that the ovaries contribute a disproportionate number of transcripts to the whole-body transcriptome. Further, our data demonstrate that, due to the preponderance of ovarian contribution, multiple tissues have only a low representation in the body, severely limiting the ability to detect tissue-specific changes in whole-mosquito experiments. To examine the biological processes and molecular functions enriched in each body part, we performed a Gene Ontology Enrichment Analysis (GOEA) of genes 5 x (or, for ovaries, 2 x) enriched in each body part (in comparison to whole body, DESeq2 padj <0.05). An extended list of GO categories may be found in Supplementary file 2. We complemented this approach by identifying the twenty highest expressed genes in each body part (treemaps in Figure 1C), reasoning that the highest expressed genes in a transcriptome may also give insight into function. In brief, the head showed enrichment for sensory machinery and neuronal signaling, the thoracic carcass (which houses flight muscle) showed signs of enhanced metabolic activity, and the abdominal carcass (which contains mostly fat body) displayed enrichment for regulatory proteolytic enzymes (CLIP-domain serine endopeptidases), defense, and lipid transport. The gut was enriched with defense-related genes including immune-activating peptidoglycan recognition proteins (PGRPs), AMPs, and immune-modulating amidase PGRPs. It also showed enrichment for digestive enzymes, especially peptidases. Notably, its three most prevalent transcripts were the non-CLIP serine endopeptidases early trypsin (ET), female-specific chymotrypsin (CHYMO), and juvenile hormone-regulated chymotrypsin (JHA15) which, together, accounted for more than one third of its transcriptome. (Note: Vectorbase gene IDs for all genes named in this text are listed in Supplementary file 3). The Malpighian tubules, which serve a function analogous to mammalian kidneys, expressed large quantities of ion transporters, especially vacuolar ATPases, which may be required to create proton gradients for use in secondary active transport (Weng et al., 2003). Because the ovaries did not express many highly enriched genes, we adjusted our analysis to examine genes expressed 2 x higher than in the whole body and discovered categories related to cell division and germline maintenance. We also observed that a small cohort of mitochondrial genes (COX1, COX2, COX3, ATP6, and a 16 s gene) were among the highest expressed transcripts in several body parts, especially those containing tissues that carry out energy-intensive functions (head, thorax, Malpighian tubules). Overall, we concluded that the most prominent transcripts and enriched GO categories in each body part were consistent with expectations for the tissues they house. We take these results as a validation of our data set, and as confirmation that the segments of the mosquito carcass (head, thorax, abdomen) perform well as proxies for important tissues (brain/eyes, flight muscle, fat body). Gut region-specific transcriptomes reveal functional compartmentalization of the mosquito midgut To evaluate the regionalization of gut function, we generated RNAseq profiles for the five main regions of the mosquito digestive tract: crop and dorsal diverticula (hereafter referred to as 'crop' for brevity), proventriculus, anterior midgut, posterior midgut, and hindgut. A PCA of these profiles (Figure 2A) demonstrated clear segregation between regions, and close clustering of all replicates from the same region, indicating clearly distinct transcriptomes. The anterior and posterior midgut, which are from clustered together the crop and which are of The proventriculus, which is from was between these two clusters on transcriptomes clustered with posterior midgut suggesting that the posterior midgut more to the transcriptome of the whole gut than the other few genes in the posterior midgut showed enrichment compared to the whole gut (Figure supplement 1A). on genes enriched in each region (Figure complemented by an of the twenty highest expressed genes in each (Figure that the regions of the gut and hindgut) expressed genes in lipid ion transport and numerous serine The proventriculus was enriched for signaling and defensive genes and the highly expressed gambicin, In the anterior midgut, and proteins were the most enriched The highest expressed gene in this region was a of the of transcription factors of function in mosquitoes. the posterior midgut transcriptome that of the whole gut, only a of genes were enriched 5 x in this region relative to whole gut. We also noted that a large proportion of the genome was either or in the posterior midgut relative to the other gut regions (Figure supplement To genes enriched in the posterior midgut, we calculated enrichment against an of the other gut We found that the posterior midgut is highly enriched for peptidases non-CLIP serine and genes in enriched categories recognition transport and these categories a region that is to and large quantities of and to process and the resulting Overall, our data demonstrate that the five gut regions are highly specialized functional Figure 2 with 1 supplement see all Download asset Open asset gut transcriptomic and reveal digestive specializations. (A) Principal component analysis of the transcriptomes of whole gut and gut 3–6 Circled clusters contain body parts of endodermal (left) and (right) derivation. (B) Bubble plots: classic Fisher topGO gene ontology enrichment analysis of enriched genes proventriculus, and anterior 5 x enriched over DESeq2 padj for posterior enriched over all other gut regions, DESeq2 padj (C) Treemaps: 20 highest expressed genes per region, grouped by category/function; percentages reflect the proportion of the transcriptome comprised by the displayed genes. in categories of digestive enzymes and are as percentages of the region with the highest transcriptional in the by The region with the highest is as with its displayed in the of transcriptional by of of digestive by region by regional The central function of the gut is a highly sequential process. To how digestion and are distributed along the gut, we the regional expression of enzymes and in the digestion and of and We that the expression of a set of digestive of function reflects the of each region in that function of their transcriptome they to that We first of sugar transporters, and with a digestive function genes with function, peptidases or orthologous serine endopeptidases, components, For complete and see Supplementary file We the expression of the genes from each (in TPM) and calculated their relative expression by region (Figure The highest in different categories of were divided among several regions, with the greatest from crop and anterior midgut, from anterior midgut, and the of more expressed in the posterior midgut and hindgut. The crop was the greatest in lipid proteins and transporters, while the anterior midgut showed the greatest in and The highest in sugar and digestion were found in the anterior and posterior midgut, The hindgut was a disproportionate in transporters, suggesting that many of the of digestion from the midgut are These data demonstrate that distinct gut regions different of their transcriptomes in the digestion and of different at a of of should be used in the of expression as (in TPM) is distinct from the number of transcripts of a gene or in a The five regions not produce of transcripts their transcriptional be by their transcriptional yield to an estimate of their relative contributions to specific We the from each region (Figure and the expression values from Figure by yield to an estimate of the transcriptional of each region with respect to each digestive (Figure We found that the posterior midgut, by of its transcriptional is the not only of but of most categories of transcripts with digestive and However, the proventriculus and anterior midgut together are the of more than of all and and contribute a of all sugar These our that the anterior midgut in the digestion and of while the posterior midgut is for most and lipid digestion. Blood feeding initiates a of transcriptomic over the course of digestion The gut of the hematophagous mosquito changes blood To these changes at a we generated RNAseq profiles for sugar-fed guts as well as guts at hr (hereafter referred to as hr for brevity), 24 and 48 hr after feeding on a PCA (Figure that the transcriptome changes at least the first 24 hr to a by 48 hr suggesting that the transcriptional of digestion is a process. Figure 3 with 3 supplements see all Download asset Open asset The gut's transcriptome blood meal digestion. (A) Principal component analysis of the transcriptomes of sugar-fed and blood-fed 3–6 replicates per (B) Fisher topGO gene ontology enrichment analysis of blood-fed guts at each in comparison to (DESeq2, padj of 2 x (C) in categories of digestive enzymes and by are as percentages of the region with the highest transcriptional in the by The region with the highest is as with its displayed in the of whole in selected gene by Expression of selected as a percentage of The region with the highest expression (in TPM) is as with its displayed in the expression of selected peptidases at 24, and meal by reference 3 To evaluate the functional changes to the blood-fed gut's transcriptome, we performed a (Figure the gut at each to its at the at the final hr) were also compared to sugar-fed The analysis was to genes that were up or at least padj <0.05). We found that, at 24 hr after ecdysone their et al., activity was A of five genes were including the to and categories to and secretion were also by at hr a small number of genes associated with and secretion were while genes associated with of translation were However, by 24 hr all these changes were genes associated with transport and translation were 48 hr and translation were all Notably, translation