Insect Biochemistry and Molecular Biology

Olfaction and circadian rhythm gate different behaviors in mosquitoes that are important for their capacity to transmit disease. However, the mechanisms of odor detection, and the circadian-guided changes in olfactory sensitivity across different mosquito species, remain largely unexplored. To this end, we performed a circadian-dependent RNA-sequencing study of the peripheral olfactory- and brain tissues of female Anopheles culicifacies and Aedes aegypti mosquitoes. Data analysis revealed a significant upregulation of genes encoding: (a) odorant binding proteins (OBPs), required for transportation of odorant molecules towards the olfactory receptors, and (b) xenobiotic-metabolizing enzymes (XMEs) during the day time in Aedes aegypti and during the dusk-transition phase in Anopheles culicifacies. While XMEs primarily function in the elimination of toxic xenobiotics, concurrent elevation of XMEs and OBPs are hypothesized to act cumulatively to regulate perireceptor events and odorant sensitivity. Electroantennographic analysis with both Anopheles gambiae and Aedes aegypti against diverse behaviorally relevant odorants, combined with XMEs inhibitors and RNA interference, establish the proof-of-concept that XMEs function in perireceptor events during odorant detection and influence the odorant sensitivity in mosquitoes. Additionally, the RNA-sequencing and RNAi-mediated knockdown data revealed that daily temporal modulation of neuronal serine proteases may facilitate the consolidation of the brain function, and influence the odor detection process in both diurnal and nocturnal mosquitoes. These findings provide the impetus to further explore the species-specific rhythmic expression pattern of the neuro-olfactory encoded molecular factors, which could pave the way to develop and implement successful mosquito control methods. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/563057v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@181b845org.highwire.dtl.DTLVardef@168f22dorg.highwire.dtl.DTLVardef@f3994aorg.highwire.dtl.DTLVardef@d4fc06_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICircadian oscillation of perireceptor proteins possibly influences time-of-day dependent change olfactory sensitivity in diurnal and nocturnal mosquitoes C_LIO_LIDiurnal and nocturnal mosquitoes depict distinct dynamic change in perireceptor proteins C_LIO_LIInhibition of cytochrome P450 gene minimizes antennal response to different odorants C_LIO_LINeuronal serine protease may consolidate brain function and odor detection C_LI

Mosquitoes transmit diverse pathogens through repeated blood feeding, a process that subjects the midgut to significant mechanical stress and cellular damage. While hemocyte association with the midgut is known to occur following injury, the mechanisms promoting their recruitment remain poorly defined. Given the conserved roles of eicosanoid signaling in injury responses, we hypothesized that thromboxane A2 (TXA2) mediates hemocyte recruitment to the damaged midgut. Here, we demonstrate that chemically induced midgut damage significantly increases both the number of hemocytes attached to the midgut and those in circulation. Pharmacological inhibition of cyclooxygenase suppresses hemocyte recruitment whereas supplementation with a stable TXA2 analog restores the response, indicating that TXA2 signaling is required for this process. To identify candidate enzymes involved in TXA2 biosynthesis, we performed in silico docking analyses and identified two cytochrome P450 (CYP) candidates. Among these, CYP6D3 was shown to be strongly upregulated in hemocyte populations following midgut damage. RNA interference-mediated knockdown of CYP6D3 significantly reduced both hemocyte recruitment to the midgut and systemic TXB2 levels, supporting its role in the eicosanoid-mediated immune response. Together, our findings demonstrate that TXA2 signaling drives hemocyte recruitment to the damaged mosquito midgut and suggest a conserved lipid-mediated mechanism underlying insect tissue-associated immune responses that may influence vector competence.

The cytochrome P450 enzymes of the CYP4G subfamily are some of the most enigmatic insect P450s. The dipterans with sequenced genomes have two CYP4G paralogs. In Drosophila melanogaster, CYP4G1 is highly expressed in the oenocytes and catalyzes the last enzymatic step in the biosynthesis of cuticular hydrocarbons. In contrast, CYP4G15 is expressed in the brain glial cells, but its function is unknown. The Aedes aegypti genome encodes two CYP4Gs: CYP4G36 (ortholog of DmCYP4G1) and CYP4G35 (ortholog of DmCYP4G15). Here, we show that CYP4G35 is highly expressed in mosquito antennae, and the RNAi knockdown of CYP4G35 results in delayed host-seeking. Ae. aegypti CYP4G knockout lines confirmed delayed host-seeking behavior in CYP4G35 knockout females. Proteomics analysis of CYP4G35 KO females also corroborates the physiological findings and shows upregulation of proteins related to olfaction and other CYP4Gs to compensate for the lack of CYP4G35. Immunohistochemistry and in situ hybridization were used to localize CYP4G35 and demonstrated its expression in the sensilla lymph of the antennae and the tip of the proboscis. CYP4G35 and CYP4G36 fusion proteins with cytochrome P450 reductase demonstrated that, unlike CYP4G36, CYP4G35 lacks an oxidative decarbonylase function. Together, our data support a novel function of CYP4G35 in modulating olfactory response.

Larvae of the black soldier fly (Hermetia illucens, BSFL) are rich in valuable nutrients and offer a promising alternative protein source for animal feeds. Nonetheless, there is a pressing need to improve both the productivity and quality of BSFL proteins to ensure their viability, facilitating the industrial production. To fulfil the needs of different animals, it is necessary to adjust the profile of essential amino acids (AAs) in BSFL. Insects excrete surplus nutrients to maintain homeostasis; AAs are excreted by nutrient AA transporters (NATs) in the Malpighian tubules. We aimed to modify the composition of essential AAs by silencing the NAT in Malpighian tubules of BSFL (HiNATt). Silencing HiNATt resulted in a 56.2% decrease in body weight but a 77.3% increase in the total AA content. Notably, the contents of some valuable essential AAs were strongly increased (histidine, 156.8%; valine, 98.1%). These results suggest that inhibiting the function of HiNATt could modify the composition of accumulated AAs. This finding opens a new avenue for producing of BSFL with increased nutritional value as an alternative protein source.

The Colorado potato beetle (CPB) is a prominent insect pest of potatoes, tomatoes and eggplants all over the world, however, the management of CPB remains a challenging task for more than one hundred years. We have successfully developed bacteria-expressed dsRNA-mediated feeding RNA interference (RNAi) approach in our previous study. A critical step towards field management of CPB via feeding RNAi is to identify effective and environmentally safe target genes. NADPH-Cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The full length Leptinotarsa decemlineata CPR (LdCPR) cDNA was isolated from an imidacloprid resistant population. The LdCPR gene was ubiquitously expressed in all stages tested but showed an increase in expression during the early stage of embryonic development. The bacteria-expressed dsRNA-mediated feeding RNAi of LdCPR in adults caused systemic knock down expression of the gene coding for LdCPR in both adults and their eggs. Suppression of LdCPR expression increased susceptibility of imidacloprid in resistant beetles, as well as a significant decrease of fecundity in female beetles (29% less eggs/day) and the hatching rate (47%) of their eggs. These data suggest that LdCPR plays important roles in insecticide detoxification and biosynthetic pathways of endogenous compounds and may serve as an essential target to control CPB. HIGHLIGHTSO_LIHigh expression of LdCPR was observed in the egg stage. C_LIO_LISilencing of LdCPR reduced the CPR enzymatic activities. C_LIO_LILdCPR knockdown increased imidacloprid susceptibility. C_LIO_LILdCPR knockdown decreased the fecundity and enhanced embryonic lethality. C_LI

Nicotine is a plant-derived pyridine alkaloid with potent neurotoxic properties. A major pathway for detoxification of nicotine in mammals is via glucuronidation to produce nicotine N-glucuronide, but this process in insects remains poorly understood. Using mass spectrometry, we demonstrate that Drosophila melanogaster detoxifies nicotine through glycosylation, producing nicotine N-glycoside. Given that many new agrochemicals contain pyridine rings, we also investigated the metabolism of flonicamid and imidacloprid. We detected glycosylation of flonicamid, but not imidacloprid. A targeted RNAi screen across 21 UDP-glycosyltransferases (Ugts) identified Ugt35B1 as important for survival of nicotine exposure. CRISPR-based knockout of Ugt35B1 increases sensitivity to nicotine and flonicamid, but not to imidacloprid, nor to a structurally distinct neonicotinoid (thiamethoxam). Mass spectrometry of knockout and control flies confirms that Ugt35B1 glycosylates nicotine, its metabolite cotinine, and flonicamid. Together these findings establish Ugt35B1 as the principal UGT mediating nicotine detoxification in D. melanogaster, revealing a previously uncharacterized insect glycosylation pathway with potential implications for herbivory, insecticide detoxification and toxicology. Highlights- Drosophila detoxifies nicotine by glycosylation into nicotine N-glycoside. - A targeted RNAi screen identifies Ugt35B1 as critical for nicotine survival. - Ugt35B1 knockout sensitizes flies to nicotine and flonicamid, but not to imidacloprid or thiamethoxam. - First demonstration of an insect UGT mediating in vivo glycosylation of nicotine and cotinine.

The polyphagous pest Apolygus lucorum has become the dominant insect in Bacillus thuringiensis (Bt) cotton fields. The hormone 20-hydroxyecdysone (20E) regulates multiple events in insect development and physiology. 20E responses are controlled by pathways triggered by phospholipase C (PLC)-associated proteins. However, 20E-modulated genes whose expression is affected by PLC remain unknown. Here, isobaric tag for relative and absolute quantitation (iTRAQ) and immunoblot were carried out for comparing differentially expressed proteins (DEPs) in A. lucorum in response to 20E and the PLC inhibitor U73122, respectively. Totally 1624 DEPs were, respectively, found in the 20E/control, U73122/control, and 20E+U73122/control groups. Venn diagram analysis further revealed 8 DEPs that were shared among the three groups. Immunoblot validated these findings, which corroborated and highlighted the reliability of proteomics. KEGG enrichment analysis showed that the DEPs were included in diverse signaling pathways. The largest portion of DEPs among the three groups were categorized in metabolic pathways. In addition, DEPs among the three groups were also found to regulate the Ras-MAPK and PI3K-AKT pathways. This is the first time that iTRAQ was carried out to assess proteome alteration in A. lucorum nymphs in response to 20E and a PLC inhibitor. These findings provide novel insights into protein expression in A. lucorum in response to 20E, and a more comprehensive understanding of the function of PLC in 20E signal transduction.

Nobo is a glutathione transferase (GST) crucially contributing to ecdysteroid biosynthesis in insects of the orders Diptera and Lepidoptera. Ecdysone is a vital steroid hormone in insects, which governs larval molting and metamorphosis, and suppression of its synthesis has potential as a novel approach to insect growth regulation and combatting vectors of disease. In general, GSTs catalyze detoxication, whereas the specific function of Nobo in ecdysteroidogenesis is unknown. We report that Nobo from the malaria-spreading mosquito Anopheles gambiae is a highly efficient ketosteroid isomerase catalyzing double-bond isomerization in the steroids 5-androsten-3,17-dione and 5-pregnen-3,20-dione. These mammalian ketosteroids are unknown in mosquitoes, but the discovered prominent catalytic activity with these compounds suggests that the unknown Nobo substrate in insects has a ketosteroid functionality. Nobo Asp111 is essential for activity with the steroids, but not for conventional GST substrates. Further characterization of Nobo may guide the development of new insecticides to prevent malaria.

Most insects enter diapause, a state of physiological dormancy crucial for enduring harsh seasons, with photoperiod serving as the primary cue for its induction, ensuring proper seasonal timing of the process. Although the involvement of the circadian clock in the photoperiodic time measurement has been demonstrated through knockdown or knockout of clock genes, the precise molecular mechanisms in this context remain unclear. In bivoltine strains of the silkworm, Bombyx mori, embryonic diapause is maternally controlled and affected by environmental conditions experienced by mother moths during embryonic and larval stages. Previous research highlighted the role of core clock genes, including period (per), timeless (tim), Clock (Clk) and cycle (cyc), in photoperiodic diapause induction in B. mori. In this study, we focused on another clock gene, cryptochrome 1 (cry1), which functions as a photoreceptor implicated in photoentrainment of the circadian clock across various insect species. Phylogenetic analysis and conserved domain identification confirmed the presence of both Drosophila-type cry (cry1) and mammalian-type cry (cry2) genes in the B. mori genome, akin to other lepidopterans. Temporal expression analysis revealed higher cry1 gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per, tim, Clk and cyc) disrupting this temporal expression pattern. Using CRISPR/Cas9-mediated genome editing, we established a cry1 knockout strain in p50T, a bivoltine strain exhibiting clear photoperiodism during both embryonic and larval stages. Although the wild-type strain displayed circadian rhythm in eclosion under continuous darkness, the cry1 knockout strain exhibited arrhythmic eclosion, implicating B. mori cry1 in the circadian clock feedback loop governing behavior rhythms. Females of the cry1 knockout strain failed to induce photoperiodic diapause during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating that B. mori CRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in a cry1/tim double-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence of cry1 involvement in insect photoperiodism, specifically in diapause induction. HighlightsO_LIKnockouts of core clock genes disrupted the rhythmic expression of cryptochrome 1 (cry1). C_LIO_LIA cry1 knockout strain was established using CRISPR/Cas9. C_LIO_LIThe cry1 knockout strain lost its eclosion rhythm. C_LIO_LIKnockout of cry1 disrupted photoperiodic diapause induction. C_LIO_LIFemales of a cry1/tim double knockout strain produced only non-diapause eggs regardless of larval photoperiod. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/593801v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a2b363org.highwire.dtl.DTLVardef@af3adcorg.highwire.dtl.DTLVardef@41490corg.highwire.dtl.DTLVardef@22bc30_HPS_FORMAT_FIGEXP M_FIG C_FIG

Heliconius butterflies are highly specialized in Passiflora, laying eggs and feeding as larvae only on these plants. Interestingly, Heliconius butterflies and Passiflora plants both contain cyanogenic glucosides (CNglcs). While feeding on specific Passiflora species, Heliconius melpomene larvae are able to sequester simple cyclopentenyl CNglcs, the most common CNglcs in this plant genus. Yet, aromatic, aliphatic, and modified CNglcs have been reported in Passiflora species and they were never tested for sequestration by heliconiine larvae. As other cyanogenic lepidopterans, H. melpomene also biosynthesize the aliphatic CNglcs linamarin and lotaustralin, and their toxicity does not rely exclusively on sequestration. Although the genes encoding the enzymes in the CNglc biosynthesis have not yet been fully biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 are hypothesized to be involved in this pathway in H. melpomene. In this study, we determine how the CNglc composition and expression of the putative P450s involved in the biosynthesis of these compounds vary at different development stages of Heliconius butterflies. We also established which kind of CNglcs H. melpomene larvae can sequestered from Passiflora. By analysing the chemical composition of the haemolymph from larvae fed with different Passiflora diets, we observed that H. melpomene is able to sequestered prunasin, an aromatic CNglcs, from P. platyloba. They were also able to sequester amygdalin, gynocardin, [C13/C14]linamarin and [C13/C14]lotaustralin painted on the plant leaves. The CNglc tetraphyllin B-sulphate from P. caerulea was not detected in the larval haemolymph, suggesting that such modified CNglcs cannot be sequestered by Heliconius. Although pupae and virgin adults contain dihydrogynocardin resulting from larval sequestration, this compound was metabolized during adulthood, and not used as nuptial gift or transferred to the offspring. Thus, we speculate that dihydrogynocardin was catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship and calling. Mature adults had a higher concentration of CNglcs than any other developmental stages due to intense de novo biosynthesis of linamarin and lotaustralin. All CYP405As were expressed in adults, whereas larvae mostly expressed CYP405A4. Our results shed light on the importance of CNglcs in Heliconius biology and for their coevolution with Passiflora.

Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by Ae. aegypti. This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and other clinical aspects such as acute febrile illness and neurologic complications, for example, Guillain-Barre syndrome. One of the actual most promising strategies to mitigate arbovirus transmission involves releasing Ae. aegypti mosquitoes carrying the maternally inherited endosymbiont bacteria Wolbachia pipientis. The presence of Wolbachia is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits as fecundity and fertility. However, the mechanisms by which Wolbachia influences metabolic pathways leading to differences in egg production remains poorly known. To investigate the impact of co-infections on the reproductive tract of the mosquito, we applied an isobaric labeling-based quantitative proteomic strategy to investigate the influence of Wolbachia wMel and ZIKV infection in Ae. aegypti ovaries. To the best of our knowledge, this is the most complete proteome of Ae. aegypti ovaries reported so far, with a total of 3,913 proteins identified, also,were able to quantify a total of 1,044 Wolbachia proteins in complex sample tissue of Ae. aegypti ovary.. Furthermore, we discuss proteins and pathways altered in Ae. aegypti during ZIKV infections, Wolbachia infections, co-infection Wolbachia/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of Ae. aegypti. The modified aspects were mostly related to the immune priming enhancement by Wolbachia presence and the modulation of the Juvenile Hormone pathway caused by both microorganisms infection. HighlightsO_LIProteome changes in Ae. aegypti, Wolbachia, and ZIKV interactions C_LIO_LIA great diversity of Wolbachia proteins were quantified in Ae. aegypti ovary C_LIO_LIJuvenile Hormone pathway is modulated by both infections C_LIO_LIWolbachia enhances Ae. aegypti immune priming mechanism C_LIO_LIZIKV unsettles host immune response by reducing antimicrobial peptides production C_LIO_LICoinfection triggers oxidative stress and a lack of vitellogenin precursors C_LI

CRISPR/Cas9-mediated gene editing provides an effective method for deciphering the molecular mechanisms underlying mosquito development and mosquito-borne disease transmission, as well as for exploring genetic control strategies. However, delivering the Cas9 ribonucleoprotein complex by embryo injection to produce genetic modifications is challenging, is mostly confined to model mosquitoes and specialized laboratories, and has low editing efficiency. Here, we established an effective Receptor-Mediated Ovary Transduction of Cargo (ReMOT) control method, enabling the introduction of heritable mutations into Anopheles sinensis, the major malaria vector in China and Southeast Asia, via the injection of female adult mosquitoes. Injection of a mixture of P2C-DsRed and saponin resulted in red fluorescence in the ovaries, with a 100% success rate. Using this system, we knocked-out the pigment synthesis genes, Aswhite and Asyellow, using injected wild-type (WT) females mated with WT males, resulting in the highest efficiency of gene editing among mosquitoes under the same mating conditions. Furthermore, the gene-editing efficiency was increased by at least 2.1-fold using injected WT females mated with mutant males. This improved ReMOT control method exhibits high editing efficiency, with important benefits in terms of functional genomics research and genetic control strategies in An. sinensis. Moreover, this represents a convenient method for gene manipulation in laboratories that are unable to perform embryo injection or that lack embryo-injection equipment.

AaCPR100A is a structural protein, found in the soft cuticle of Ae. Aegypti. RNAi of AaCPR100A resulted in high mortality in Ae. Aegypti and abnormal egg development in the surviving mosquitoes. Over thirty proteins that could interact with AaCPR100A were screened out by yeast two-hybrid assay, and subsequently, further verification by hybrid and GST pull-down assays identified that G12-like had the strongest interaction with AaCPR100A. RNAi of G12-like suggested it may be related to larva development. Interestingly, the adults in which the G12-like gene was knocked down were sensitive to low temperature, and their egg shell formation, production, and hatching were affected. G12-like has the opposite effect in the upstream expression of AaCPR100A, promoting AaCPR100A function in the larval stage and inhibiting AaCPR100A in the adult stage. In all, functional studies of AaCPR100A and its interaction protein G12-like provide insight into its involvement in cuticle development and formation and egg shell formation.

Insects rely on their highly efficient and precise olfactory systems to find suitable mates, host plants and oviposition sites, and adapt to the changing environment. The odorant receptors (ORs) including pheromone receptors (PRs) play a vital role in this process. While extensive studies have been focusing on deorphanization of lepidopteran PR genes, the information on the ligand profiles of general ORs is still sparse. In the present study, we identified a repertoire of 61 ORs including the co-receptor Orco from antennal and ovipositor transcriptomes of the turnip moth Agrotis segetum, which clustered in all the major lepidopteran OR clades. We characterized the function of eight female-biased expressed ORs in Xenopus oocytes and found three ORs differentially tuned to plant volatile compounds that might be repulsive or attractive to the moths. AsegOR13 was broadly tuned to a number of herbivore-induced plant volatiles (HIPVs) while AsegOR20 was specific to citral; AsegOR17 was narrowly tuned to the alcohols, isoamyl alcohol, pentanol and benzyl alcohol, that are potentially attractive to moths. The orthologues of the three ORs in other moth species seem to share the conserved function. Our results support the hypothesis that insects recognize their host plants mostly by detecting the mixture of ubiquitous compounds, instead of taxonomically characteristic host compounds. The combination of narrowly and broadly tuned ORs will ensure both the accuracy of the most important odor signals and the plasticity of the olfactory system to the changes in the environment.

Some herbivorous insects have evolved sequestration mechanisms, the ability to take up and accumulate plant secondary metabolites for their own benefit. The domestic silkworm, Bombyx mori, and its wild ancestor, B. mandarina, take up flavonoids from mulberry leaves and accumulate the molecules as their glucosides in their tissues and cocoon shell. This sequestration enhances the cocoons protective property against ultraviolet or bacterial proliferation. Here, we show that an ATP-binding cassette transporter subfamily C (ABCC) gene, BmABCC4, plays a crucial role in the flavonoid sequestration in the silkworms. BmABCC4 is located at Green c, a cocoon color-associated locus predicted in 1941 and whose detailed position we previously identified. This transporter is expressed in the midgut and silk glands, and the expression is upregulated in the midgut in late period of final instar larva. Knockout of BmABCC4 significantly reduced the total flavonoid content in the tissues and cocoon shell. Our results suggest that BmABCC4 transports flavonoid glucosides from the midgut cells to hemolymph and from the silk gland cells to the silk gland lumen.

The genetic basis of multi-functional traits shaped by both natural and sexual selection remains poorly understood. In insects, cuticular hydrocarbons (CHCs) are an excellent example of such traits, providing protection against different micro-climatic conditions while simultaneously encoding cues predominantly used in sexual signaling. The fatty acid synthase (fas) gene family has been implied as an important cornerstone in initiating and maintaining CHC functionality, whereas their exact biosynthetic and regulatory mechanisms have remained poorly understood. Here, we characterize a single fatty acid synthase gene (fas3) impacting the main CHC functions in the parasitoid wasp model organism Nasonia vitripennis. Knockdown of fas3 significantly decreases wasp survival under desiccation stress while also completely depleting sexual attractiveness of female wasps, where this trait naturally functions as sex pheromone. Transcriptomic analyses revealed that fas3 regulates other fas and CHC-associated genes, as well as key biosynthetic pathway hubs. We also identified striking sex-specific expression differences in fas3 across individual developmental stages, suggesting divergent functional roles of this gene in males and females. These findings largely advance our knowledge on the multi-functionality of fas genes in governing survival and sexual signaling and underscore their relevance for future studies on metabolomics, ecological adaptation, and sexual communication. Author SummaryTraits that serve both adaptive and reproductive functions--such as those involved in survival and mating success--are often complex, and their genetic foundations remain poorly understood. In insects, chemical compounds on the outer cuticle represent a prime example: they help prevent desiccation and act as key sexual signals. In this study, we investigate the gene fas3, a member of the fatty acid synthase family, in the parasitoid wasp Nasonia vitripennis, whose knockdown drastically reduces these surface chemicals. Consequently, both of their natural functionalities, namely enhancement of survival in dry conditions and female sexual attractiveness, are severely impaired. Transcriptomic analysis further revealed that fas3 also regulates multiple other genes, including those in further major biosynthetic pathways. Our findings shed light on how a single gene can coordinate the expression of multifunctional traits, contributing to both ecological adaptation and sexual communication.

Bacillus thuringiensis is widely employed for biological control. It can effectively suppress populations of various mosquito species, including Aedes aegypti. However, the precise mechanism underlying the action of cry toxin secreted by Bacillus thuringiensis on Ae. aegypti remains elusive. In this study, we investigated one of the binding receptors of cry toxin, aminopeptidase N. Through comprehensive bioinformatics analysis involving whole-genome screening, genetic mapping, structural characterization, phylogenetic analysis, and spatiotemporal expression profiling, we identified twenty-nine homologs of Ae. aegypti aminopeptidase N. Further, we successfully expressed GST-APN3 protein in E. coli and demonstrated through ligand blot and ELISA assays that APN3 exhibits high affinity binding to Cry4Ba toxin (Kd = 20.53 nM). To elucidate the functional role of APN3 as a receptor mediating Cry4Ba activity in Ae. aegypti midgut cells (some of which express this gene at high levels), CRISPR/Cas9 technology was employed to knock out APN3. Our bioassay results revealed that APN3 knockout mosquito larvae had 2.9 to 4.1-fold higher resistance against Cry4Ba, indicating its crucial involvement as an active receptor mediating Cry4Ba activity. Overall, this study provides a foundation for elucidating the specific larvicidal mechanisms of Bt against mosquito populations.

During blood feeding, mosquitoes introduce substantial quantities of salivary proteins into their hosts to facilitate feeding. Arylalkylamine N-acetyltransferase-1 (aaNAT1) is a salivary protein prominently produced in the salivary glands of Aedes aegypti. Previous research has demonstrated that mosquitoes with AaaaNAT1 mutations fail to efficiently inhibit host blood coagulation; however, the precise regulatory mechanisms remain unknown. This study identified the primary metabolites and proteins interacting with AaaaNAT1 through integrated transcriptomic, proteomic, and metabolomic analyses. Combined with isothermal titration calorimetry (ITC), immunohistochemistry, ELISA, platelet function assays, and coagulation tests, we further established that AaaaNAT1 binds norepinephrine, reducing host platelet aggregation and thereby limiting activation of the host coagulation cascade and fibrinolytic system. Additionally, behavioral investigations showed that AaaaNAT1 knockdown in Aedes aegypti significantly prolonged the time required for female mosquitoes to locate a host and successfully obtain blood, while also markedly reducing blood consumption. This effect may stem from the inability of aaNAT1-depleted mosquitoes to effectively suppress host blood aggregation during feeding, as well as the downregulation of octopamine following AaaaNAT1 knockdown. Therefore, this study reveals a novel role for aaNAT1 distinct from its known functions in pigmentation and immunity, advancing our understanding of aaNAT1s role in insects.

Mosquitoes such as Aedes aegypti must consume a blood meal for the nutrients necessary for egg production. Several transcriptome and proteome changes occur post blood meal that likely corresponds with codon usage alterations. Transfer RNA (tRNA) is the adapter molecule that reads messenger RNA (mRNA) codons to add the appropriate amino acid during protein synthesis. Chemical modifications to tRNA enhance codons decoding, improving the accuracy and efficiency of protein synthesis. Here, we examined tRNA modifications and transcripts associated with the blood meal and subsequent periods of vitellogenesis in A. aegypti. More specifically, we assessed tRNA transcript abundance and modification levels in the fat body at critical times post blood-feeding. Based on a combination of alternative codon usage and identification of particular modifications, we identified that increased transcription of tyrosine tRNAs is likely critical during the synthesis of egg yolk proteins in the fat body following a blood meal. Altogether, changes in both the abundance and modification of tRNA are essential factors in the process of vitellogenin production after blood-feeding in mosquitoes.

Neuropeptide Y-related signaling, consisting of various neuropeptides and their receptors including, for example, the mammalian neuropeptide Y (NPY), pancreatic polypeptide (PP), and peptide YY (PYY) families, along with neuropeptide Y-like peptides in insects, is evolutionarily conserved across vertebrates and invertebrate organisms. Vertebrate NPY is known as an important regulator of energy homeostasis and feeding behaviour, while studies on one NPY-associated signaling system in arthropods, known as RYamide, have mainly focused on functions regulating feeding-related behaviours. The current study aimed to begin exploring an additional functional role of RYamide signaling related to excretory physiology in Aedes aegypti given that a candidate RYamide receptor, RYaR2, is enriched in the hindgut of adult mosquitoes based on publicly available RNA-seq databases. Herein, we report that the RYamide transcript is expressed in all post-embryonic stages with greatest abundance in adult male mosquitoes. Specifically, the central nervous system and the ventral nerve cord were demonstrated as the major sources of RYamide, as supported by RT-qPCR and intense RYamide immunoreactivity observed in the protocerebral posterior dorsomedial neurons and protocerebral anterolateral neurons in the mushroom body region, along with an intensively stained pair of neurons in the terminal abdominal ganglion (TAG). The pair of immunoreactive neurons in the TAG contain elaborate processes extending onto the hindgut, with a branch extending posteriorly over the rectum where fine processes are in close proximity to the rectal papillae (pads) while an anterior branch extends over the ileum terminating at the midgut-hindgut junction forming a fine circular plexus over the pyloric valve. Importantly, two orphan GPCRs, previously referred to as NPYLRs, were functionally deorphanized as bona fide RYamide receptors. Their responses to diverse peptidergic ligands were assessed, revealing that RYamides elicited the strongest receptor activation, with half maximal effective concentrations in the pico- to nanomolar range. Notably, our phylogenetic analysis revealed that while only a single RYaR is commonly found across most arthropods, culicine mosquito species including A. aegypti possess two RYaRs. The characterization of RYamide signaling with respect to excretory physiology involving the hindgut, particularly in species having two receptor isoforms such as culicine mosquitos, may provide valuable insights for development of novel, species-specific pest control strategies.