Using axenic and gnotobiotic insects to examine the role of different microbes on the development and reproduction of the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae).

Kissing bugs (Hempitera: Reduviidae) are obligately and exclusively blood feeding insects. Vertebrate blood is thought to provide insufficient B vitamins to insects, which rely on symbiotic relationships with bacteria that provision these nutrients. Kissing bugs harbour environmentally acquired bacteria in their gut lumen, without which they are unable to develop to adulthood. Rhodococcus rhodnii was initially identified as the sole symbiont of Rhodnius prolixus, but modern studies of the kissing bug microbiome suggest that R. rhodnii is not always present or abundant in wild-caught individuals. We asked whether R. rhodnii or other bacteria alone could function as symbionts of R. prolixus. We produced insects with no bacteria (axenic) or with known microbiomes (gnotobiotic). Gnotobiotic insects harbouring R. rhodnii alone developed faster, had higher survival, and laid more eggs than those harbouring other bacterial monocultures, including other described symbionts of kissing bugs. R. rhodnii grew to high titre in the guts of R. prolixus while other tested species were found at much lower abundance. Rhodococcus species tested had nearly identical B vitamin biosynthesis genes, and dietary supplementation of B vitamins had a relatively minor effect on development and survival of gnotobiotic R. prolixus. Our results indicate that R. prolixus have a higher fitness when harbouring R. rhodnii than other bacteria tested, that this may be due to R. rhodnii existing at higher titres and providing more B vitamins to the host, and that symbiont B vitamin synthesis is probably a necessary but not sufficient function of gut bacteria in kissing bugs.

Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito Aedes aegypti.

Gut microbes positively affect the physiology of many animals, but the molecular mechanisms underlying these benefits remain poorly understood. We recently reported that bacteria-induced gut hypoxia functions as a signal for growth and molting of the mosquito Aedes aegypti In this study, we tested the hypothesis that transduction of a gut hypoxia signal requires hypoxia-induced transcription factors (HIFs). Expression studies showed that HIF-α was stabilized in larvae containing bacteria that induce gut hypoxia but was destabilized in larvae that exhibit normoxia. However, we could rescue growth of larvae exhibiting gut normoxia by treating them with a prolyl hydroxylase inhibitor, FG-4592, that stabilized HIF-α, and inhibit growth of larvae exhibiting gut hypoxia by treating them with an inhibitor, PX-478, that destabilized HIF-α. Using these tools, we determined that HIF signaling activated the insulin/insulin growth factor pathway plus select mitogen-activated kinases and inhibited the adenosine monophosphate-activated protein kinase pathway. HIF signaling was also required for growth of the larval midgut and storage of neutral lipids by the fat body. Altogether, our results indicate that gut hypoxia and HIF signaling activate multiple processes in A. aegypti larvae, with conserved functions in growth and metabolism.

Bacteria-mediated hypoxia functions as a signal for mosquito development.

Mosquitoes host communities of microbes in their digestive tract that consist primarily of bacteria. We previously reported that several mosquito species, including Aedes aegypti, do not develop beyond the first instar when fed a nutritionally complete diet in the absence of a gut microbiota. In contrast, several species of bacteria, including Escherichia coli, rescue development of axenic larvae into adults. The molecular mechanisms underlying bacteria-dependent growth are unknown. Here, we designed a genetic screen around E. coli that identified high-affinity cytochrome bd oxidase as an essential bacterial gene product for mosquito growth. Bioassays showed that bacteria in nonsterile larvae and gnotobiotic larvae inoculated with wild-type E. coli reduced midgut oxygen levels below 5%, whereas larvae inoculated with E. coli mutants defective for cytochrome bd oxidase did not. Experiments further supported that hypoxia leads to growth and ecdysone-induced molting. Altogether, our results identify aerobic respiration by bacteria as a previously unknown but essential process for mosquito development.

Evolution of DNA Methylation across Insects.

DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects.

Ovary ecdysteroidogenic hormone requires a receptor tyrosine kinase to activate egg formation in the mosquito Aedes aegypti.

Mosquitoes are major disease vectors because most species must feed on blood from a vertebrate host to produce eggs. Blood feeding by the vector mosquito Aedes aegypti triggers the release of two neurohormones, ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs), which activate multiple processes required for egg formation. ILPs function by binding to the insulin receptor, which activates downstream components in the canonical insulin signaling pathway. OEH in contrast belongs to a neuropeptide family called neuroparsins, whose receptor is unknown. Here we demonstrate that a previously orphanized receptor tyrosine kinase (RTK) from A. aegypti encoded by the gene AAEL001915 is an OEH receptor. Phylogenetic studies indicated that the protein encoded by this gene, designated AAEL001915, belongs to a clade of RTKs related to the insulin receptor, which are distinguished by an extracellular Venus flytrap module. Knockdown of AAEL001915 by RNAi disabled OEH-mediated egg formation in A. aegypti. AAEL001915 was primarily detected in the mosquito ovary in association with follicular epithelial cells. Both monomeric and dimeric AAEL001915 were detected in mosquito ovaries and transfected Drosophila S2 cells. Functional assays further indicated that OEH bound to dimeric AAEL001915, which resulted in downstream phosphorylation of Ak strain transforming factor (Akt). We hypothesize that orthologs of AAEL001915 in other insects are neuroparsin receptors.

Mosquitoes rely on their gut microbiota for development.

Field studies indicate adult mosquitoes (Culicidae) host low diversity communities of bacteria that vary greatly among individuals and species. In contrast, it remains unclear how adult mosquitoes acquire their microbiome, what influences community structure, and whether the microbiome is important for survival. Here, we used pyrosequencing of 16S rRNA to characterize the bacterial communities of three mosquito species reared under identical conditions. Two of these species, Aedes aegypti and Anopheles gambiae, are anautogenous and must blood-feed to produce eggs, while one, Georgecraigius atropalpus, is autogenous and produces eggs without blood feeding. Each mosquito species contained a low diversity community comprised primarily of aerobic bacteria acquired from the aquatic habitat in which larvae developed. Our results suggested that the communities in Ae. aegypti and An. gambiae larvae share more similarities with one another than with G. atropalpus. Studies with Ae. aegypti also strongly suggested that adults transstadially acquired several members of the larval bacterial community, but only four genera of bacteria present in blood fed females were detected on eggs. Functional assays showed that axenic larvae of each species failed to develop beyond the first instar. Experiments with Ae. aegypti indicated several members of the microbial community and Escherichia coli successfully colonized axenic larvae and rescued development. Overall, our results provide new insights about the acquisition and structure of bacterial communities in mosquitoes. They also indicate that three mosquito species spanning the breadth of the Culicidae depend on their gut microbiome for development.

RNAi-mediated knockdown of two orphan G protein-coupled receptors reduces fecundity in the yellow fever mosquito Aedes aegypti.

G protein-coupled receptors (GPCRs) control numerous physiological processes in insects, including reproduction. While many GPCRs have known ligands, orphan GPCRs do not have identified ligands in which they bind. Advances in genomic sequencing and phylogenetics provide the ability to compare orphan receptor protein sequences to sequences of characterized GPCRs, and thus gain a better understanding of the potential functions of orphan GPCRs. Our study sought to investigate the functions of two orphan GPCRs, AAEL003647 and AAEL019988, in the yellow fever mosquito, Aedes aegypti. From our phylogenetic investigation, we found that AAEL003647 is orthologous to the SIFamide-2/SMYamide receptor. We also found that AAEL019988 is orthologous to the Trapped in endoderm (Tre1) receptor of Drosophila melanogaster. Next, we conducted a tissue-specific expression analysis and found that both receptors had highest expression in the ovaries, suggesting they may be important for reproduction. We then used RNA interference (RNAi) to knock down both genes and found a significant reduction in the number of eggs laid per individual female mosquito, suggesting both receptors are important for Ae. aegypti reproduction.

Microbiomes of Blood-Feeding Triatomines in the Context of Their Predatory Relatives and the Environment.

The importance of gut microbiomes has become generally recognized in vector biology. This study addresses microbiome signatures in North American Triatoma species of public health significance (vectors of Trypanosoma cruzi) linked to their blood-feeding strategy and the natural habitat. To place the Triatoma-associated microbiomes within a complex evolutionary and ecological context, we sampled sympatric Triatoma populations, related predatory reduviids, unrelated ticks, and environmental material from vertebrate nests where these arthropods reside. Along with five Triatoma species, we have characterized microbiomes of five reduviids (Stenolemoides arizonensis, Ploiaria hirticornis, Zelus longipes, and two Reduvius species), a single soft tick species, Ornithodoros turicata, and environmental microbiomes from selected sites in Arizona, Texas, Florida, and Georgia. The microbiomes of predatory reduviids lack a shared core microbiota. As in triatomines, microbiome dissimilarities among species correlate with dominance of a single bacterial taxon. These include Rickettsia, Lactobacillus, "Candidatus Midichloria," and Zymobacter, which are often accompanied by known symbiotic genera, i.e., Wolbachia, "Candidatus Lariskella," Asaia, Gilliamella, and Burkholderia. We have further identified a compositional convergence of the analyzed microbiomes in regard to the host phylogenetic distance in both blood-feeding and predatory reduviids. While the microbiomes of the two reduviid species from the Emesinae family reflect their close relationship, the microbiomes of all Triatoma species repeatedly form a distinct monophyletic cluster highlighting their phylosymbiosis. Furthermore, based on environmental microbiome profiles and blood meal analysis, we propose three epidemiologically relevant and mutually interrelated bacterial sources for Triatoma microbiomes, i.e., host abiotic environment, host skin microbiome, and pathogens circulating in host blood. IMPORTANCE This study places microbiomes of blood-feeding North American Triatoma vectors (Reduviidae) into a broader evolutionary and ecological context provided by related predatory assassin bugs (Reduviidae), another unrelated vector species (soft tick Ornithodoros turicata), and the environment these arthropods coinhabit. For both vectors, microbiome analyses suggest three interrelated sources of bacteria, i.e., the microbiome of vertebrate nests as their natural habitat, the vertebrate skin microbiome, and the pathobiome circulating in vertebrate blood. Despite an apparent influx of environment-associated bacteria into the arthropod microbiomes, Triatoma microbiomes retain their specificity, forming a distinct cluster that significantly differs from both predatory relatives and ecologically comparable ticks. Similarly, within the related predatory Reduviidae, we found the host phylogenetic distance to underlie microbiome similarities.

The Peptide Hormone CNMa Influences Egg Production in the Mosquito Aedes aegypti.

Mosquito reproduction is regulated by a suite of hormones, many acting through membrane-bound receptor proteins. The Aedes aegypti G protein-coupled receptors AAEL024199 (AeCNMaR-1a) and AAEL018316 (AeCNMaR-1b) were identified as orthologs of the Drosophila melanogaster CNMa receptor (DmCNMaR). The receptor was duplicated early in the evolution of insects, and subsequently in Culicidae, into what we refer to as CNMaR-1a and CNMaR-1b. AeCNMaR-1a is only detected in male mosquito antennae while AeCNMaR-1b is expressed at high levels in mosquito ovaries. Using a heterologous cell assay, we determined that AeCNMa activates AeCNMaR-1a with a ~10-fold lower concentration than it does AeCNMaR-1b, though both receptors displayed half maximal effective concentrations of AeCNMa in the low nanomolar range. Finally, we show that injections of AeCNMa into blood-fed mated female Ae. aegypti resulted in fewer eggs laid.

Sources of variation in dietary requirements in an obligate nutritional symbiosis.

The nutritional symbiosis between aphids and their obligate symbiont, Buchnera aphidicola, is often characterized as a highly functional partnership in which the symbiont provides the host with essential nutrients. Despite this, some aphid lineages exhibit dietary requirements for nutrients typically synthesized by Buchnera, suggesting that some aspect of the symbiosis is disrupted. To examine this phenomenon in the pea aphid, Acyrthosiphon pisum, populations were assayed using defined artificial diet to determine dietary requirements for essential amino acids (EAAs). Six clones exhibiting dependence on EAAs in their diet were investigated further. In one aphid clone, a mutation in a Buchnera amino acid biosynthesis gene could account for the clone's requirement for dietary arginine. Analysis of aphid F1 hybrids allowed separation of effects of the host and symbiont genomes, and revealed that both affect the requirement for dietary EAAs in the clones tested. Amino acid requirements were minimally affected by secondary symbiont infection. Our results indicate that variation among pea aphids in dependence on dietary amino acids can result from Buchnera mutation as well as variation in the host genotype.

Transcriptome Sequencing Reveals Large-Scale Changes in Axenic Aedes aegypti Larvae.

Mosquitoes host communities of microbes in their digestive tract that consist primarily of bacteria. We previously reported that Aedes aegypti larvae colonized by a native community of bacteria and gnotobiotic larvae colonized by only Escherichia coli develop very similarly into adults, whereas axenic larvae never molt and die as first instars. In this study, we extended these findings by first comparing the growth and abundance of bacteria in conventional, gnotobiotic, and axenic larvae during the first instar. Results showed that conventional and gnotobiotic larvae exhibited no differences in growth, timing of molting, or number of bacteria in their digestive tract. Axenic larvae in contrast grew minimally and never achieved the critical size associated with molting by conventional and gnotobiotic larvae. In the second part of the study we compared patterns of gene expression in conventional, gnotobiotic and axenic larvae by conducting an RNAseq analysis of gut and nongut tissues (carcass) at 22 h post-hatching. Approximately 12% of Ae. aegypti transcripts were differentially expressed in axenic versus conventional or gnotobiotic larvae. However, this profile consisted primarily of transcripts in seven categories that included the down-regulation of select peptidases in the gut and up-regulation of several genes in the gut and carcass with roles in amino acid transport, hormonal signaling, and metabolism. Overall, our results indicate that axenic larvae exhibit alterations in gene expression consistent with defects in acquisition and assimilation of nutrients required for growth.

Functional and evolutionary analysis of the genome of an obligate fungal symbiont.

Nutritional symbionts of insects include some of the most bizarre genomes studied to date, with extremely reduced size, biased base composition, and limited metabolic abilities. A monophyletic group of aphids within the subfamily Cerataphidinae have lost the bacterial symbiont common to all other Aphididae (Buchnera aphidicola), which have been replaced by a eukaryotic one, the yeast-like symbiont (YLS). As symbionts are expected to experience reduced effective population size (Ne) and largely clonal life cycles, we used this system as a model to test the hypothesis that chronically high levels of genetic drift will result in an increase in size of a eukaryotic genome. We sequenced the genome of the YLS of the aphid Cerataphis brasiliensis and observed elevated rates of protein sequence evolution and intron proliferation in YLS orthologs relative to those of its closest-sequenced relative, consistent with predictions. A moderate amount of repetitive DNA was found along with evidence of directed mutation to prevent proliferation of repetitive elements. Despite increased intron numbers, the overall genome structure appears not to have undergone massive expansion and is around 25 Mb in size. Compared with Buchnera, the YLS appears to have a much broader metabolic repertoire, though many gene families have been reduced in the YLS relative to related fungi. The patterns observed in the YLS genome suggest that its symbiotic lifestyle is permissive to intron proliferation and accelerated sequence evolution, though other factors appear to limit its overall genome expansion.

Phylogenetic investigation of Peptide hormone and growth factor receptors in five dipteran genomes.

Peptide hormones and growth factors bind to membrane receptors and regulate a myriad of processes in insects and other metazoans. The evolutionary relationships among characterized and uncharacterized ("orphan") receptors can provide insights into receptor-ligand biology and narrow target choices in deorphanization studies. However, the large number and low sequence conservation of these receptors make evolutionary analysis difficult. Here, we characterized the G-protein-coupled receptors (GPCRs), receptor guanylyl cyclases (RGCs), and protein kinase receptors (PKRs) of mosquitoes and select other flies by interrogating the genomes of Aedes aegypti, Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster, and D. mojavensis. Sequences were grouped by receptor type, clustered using the program CLANS, aligned using HMMR, and phylogenetic trees built using PhyML. Our results indicated that PKRs had relatively few orphan clades whereas GPCRs and RGCs had several. In addition, more than half of the Class B secretin-like GPCRs and RGCs remained uncharacterized. Additional studies revealed that Class B GPCRs exhibited more gain and loss events than other receptor types. Finally, using the neuropeptide F family of insect receptors and the neuropeptide Y family of vertebrate receptors, we also show that functional sites considered critical for ligand binding are conserved among distinct family members and between distantly related taxa. Overall, our results provide the first comprehensive analysis of peptide hormone and growth factor receptors for a major insect group.

Ovary ecdysteroidogenic hormone functions independently of the insulin receptor in the yellow fever mosquito, Aedes aegypti.

Most mosquito species must feed on the blood of a vertebrate host to produce eggs. In the yellow fever mosquito, Aedes aegypti, blood feeding triggers medial neurosecretory cells in the brain to release insulin-like peptides (ILPs) and ovary ecdysteroidogenic hormone (OEH). Theses hormones thereafter directly induce the ovaries to produce ecdysteroid hormone (ECD), which activates the synthesis of yolk proteins in the fat body for uptake by oocytes. ILP3 stimulates ECD production by binding to the mosquito insulin receptor (MIR). In contrast, little is known about the mode of action of OEH, which is a member of a neuropeptide family called neuroparsin. Here we report that OEH is the only neuroparsin family member present in the Ae. aegypti genome and that other mosquitoes also encode only one neuroparsin gene. Immunoblotting experiments suggested that the full-length form of the peptide, which we call long OEH (lOEH), is processed into short OEH (sOEH). The importance of processing, however, remained unclear because a recombinant form of lOEH (rlOEH) and synthetic sOEH exhibited very similar biological activity. A series of experiments indicated that neither rlOEH nor sOEH bound to ILP3 or the MIR. Signaling studies further showed that ILP3 activated the MIR but rlOEH did not, yet both neuropeptides activated Akt, which is a marker for insulin pathway signaling. Our results also indicated that activation of TOR signaling in the ovaries required co-stimulation by amino acids and either ILP3 or rlOEH. Overall, we conclude that OEH activates the insulin signaling pathway independently of the MIR, and that insulin and TOR signaling in the ovaries is coupled.

Effect of Host Genotype on Symbiont Titer in the Aphid-Buchnera Symbiosis.

Obligate nutritional symbioses require balance between the energetic needs of the host and the symbiont. The resident symbiont population size within a host may have major impacts on host fitness, as both host and symbiont consume and supply metabolites in a shared metabolite pool. Given the massive genome degradation that is a hallmark of bacterial endosymbionts of insects, it is unclear at what level these populations are regulated, and how regulation varies among hosts within natural populations. We measured the titer of the endosymbiont Buchnera aphidicola from different clones of the pea aphid, Acyrthosiphon pisum, and found significant variation in titer, measured as Buchnera genomes per aphid genome, among aphid clones. Additionally, we found that titer can change with the age of the host, and that the number of bacteriocytes within an aphid is one factor likely controlling Buchnera titer. Buchnera titer measurements in clones from a sexual cross indicate that the symbiont genotype is not responsible for variation in titer and that this phenotype is likely non-heritable across sexual reproduction. Symbiont titer is more variable among lab-produced F1 aphid clones than among field-collected ones, suggesting that intermediate titer is favored in natural populations. Potentially, a low heritability of titer during the sexual phase may generate clones with extreme and maladaptive titers each season.