Insect Biochemistry and Molecular Biology

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.

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.

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.

Mating often changes behavior and physiology of female insects. In many species, these changes have been attributed to receipt of seminal fluid molecules (SFMs). SFMs influence phenotypes including feeding, egg production, and response to male courtship and insemination attempts. These same phenotypes are potential targets for management of insect pests. Aedes aegypti mosquitoes are the primary vector of several pathogens including dengue, Zika, and chikungunya. Within an hour after an initial insemination, female Ae. aegypti are generally refractory to subsequent inseminations, a response attributed to SFMs. However, the specific molecules involved in inducing long-term insemination refractoriness have not been identified. In a previous study, we identified adipokinetic hormone (AKH) precursor protein as an SFM in Ae. albopictus. AKH is a well-studied insect neuropeptide that impacts phenotypes including those related to metabolism, locomotion, and reproduction. In this study, we investigated whether AKH is an SFM in Ae. aegypti and whether it impacts female re-insemination patterns. We first established that AKH is produced in the male reproductive tract and transferred to females during mating, and is, therefore, an SFM. We then created an AKH-null line which allowed us to demonstrate that seminal fluid AKH contributes to long-term insemination refractoriness of females. Together, these results have established a novel expression pattern for AKH and identified AKH as a contributor to Ae. aegypti insemination refractoriness, laying the groundwork for understanding the evolution and mode of action of novel seminal fluid proteins as well as for investigating novel pathways or approaches for mosquito control.

Across diverse insect taxa, the behavior and physiology of females dramatically changes after mating - processes largely triggered by the transfer of seminal proteins from their mates. In the vinegar fly Drosophila melanogaster, the seminal protein sex peptide (SP) decreases the likelihood of female flies remating and causes additional behavioral and physiological changes that promote fertility including increasing egg production. Although SP is only found in the Drosophila genus, its receptor, sex peptide receptor (SPR), is the widely-conserved myoinhibitory peptide (MIP) receptor. To test the functional role of SPR in mediating post-mating responses in a non-Drosophila dipteran, we generated two independent Spr-knockout alleles in the yellow fever mosquito Aedes aegypti. Although SPR is needed for post-mating responses in Drosophila and the cotton bollworm Helicoverpa armigera, Spr mutant Ae. aegypti show completely normal post-mating decreases in remating propensity and increases in egg laying. In addition, injection of synthetic SP or accessory gland homogenate from D. melanogaster into virgin female mosquitoes did not elicit these post-mating responses. Our results indicate that Spr is not required for these canonical post-mating responses in Ae. aegypti, indicating that unknown signaling pathways are likely responsible for these behavioral switches in this disease vector.

Ommochromes are widespread pigments in invertebrates utilized for screening pigments in compound eyes and for reddish coloration in epidermis and wings. Ommochromes are derived from 3-hydroxykynurenine (3OHK), which is incorporated into cells from hemolymph or synthesized from tryptophan within cells. While the synthetic pathway from tryptophan to 3OHK has been well characterized, the gene responsible for cellular uptake of 3OHK has been poorly understood. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of ommochrome pigments. By using positional cloning method, we found that a novel monocarboxylate transporter, 3-hydroxykynurenine transporter (3OHKT), is responsible for the recessive mutant maternal brown of Tsujita (b-t) of B. mori. In b-t mutant, the color of the eggs is light brown, whereas the color of the compound eyes is normal, and we identified a 2-kb deletion in 3OHKT gene. TALEN-mediated knockout of 3OHKT gene produced the same coloration phenotype as b-t mutant, and the complementation test between b-t mutant and 3OHKT knockout strain proved that 3OHKT is responsible for b-t phenotype. 3OHKT protein was localized in the cellular membrane, and LC-MS analysis indicated that the uptake of 3OHK from hemolymph into the ovary was suppressed in the b-t mutant. Moreover, we confirmed that 3OHKT gene is specifically expressed at the reddish region and the time of pigmentation in the pupal wing of nymphalid butterflies. RNA interference of 3OHKT prevented reddish pigmentation in wings, highlighting its general involvement in ommochrome-based pigmentation other than compound eyes. SignificanceOmmochromes are widely distributed pigments in invertebrates and are synthesized from intracellular tryptophan or 3-hydroxykynurenine (3OHK). Ommochrome-based red markings on butterfly wings are often used for sexual selection, warning colors and mimicry. Most genes involved in the ommochrome synthesis pathway have been elucidated from analyses of eye color mutants in Drosophila. However, this study reveals that the ommochrome synthesis pathway has a different genetic repertoire depending on the tissues, and that the novel monocarboxylate transporter identified in this study has a major role in ommochrome pigmentation other than in compound eyes. In particular, our results suggest that classical ommochrome-related genes are rarely involved in the wing pigmentation of the nymphalid butterflies.

The color pattern of insects is one of the most diverse adaptive evolutionary phenotypes. However, the molecular regulation of this color pattern is not fully understood. In this study, we found that the transcription factor Bm-mamo is responsible for black dilute (bd) allele mutations in the silkworm. Bm-mamo belongs to the BTB zinc finger family and is orthologous to mamo in Drosophila melanogaster. This gene has a conserved function in gamete production in Drosophila and silkworms and has evolved a pleiotropic function in the regulation of color patterns in caterpillars. Using RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) technology, we showed that Bm-mamo is a repressor or has dark melanin patterns in the larval epidermis. Using in vitro binding assays and gene expression profiling in wild-type and mutant larvae, we also showed that Bm-mamo likely regulates the expression of related pigment synthesis and cuticular protein genes in a coordinated manner to mediate its role in color pattern formation. This mechanism is consistent with the dual role of this transcription factor in regulating both the structure and shape of the cuticle and the pigments that are embedded within it. This study provides new insight into the regulation of color patterns as well as into the construction of more complex epidermis features in some insects.

Herbivorous insects can identify their host plants by sensing plant secondary metabolites as chemical cues. We previously reported the two-factor host acceptance system of the silkworm Bombyx mori larvae. The chemosensory neurons in the maxillary palp (MP) of the larvae detect mulberry secondary metabolites, chlorogenic acid (CGA), and isoquercitrin (ISQ), with ultrahigh sensitivity, for host plant recognition and feeding initiation. Nevertheless, the molecular basis for the ultrasensitive sensing of these compounds remains unknown. In this study, we demonstrated that two gustatory receptors (Grs), BmGr6 and BmGr9, are responsible for sensing the mulberry compounds with attomolar sensitivity for host plant recognition by silkworm larvae. Calcium imaging assay using cultured cells expressing the silkworm putative sugar receptors (BmGr4-10) revealed that BmGr6 and BmGr9 serve as receptors for CGA and ISQ with attomolar sensitivity in human embryonic kidney 293T cells. CRISPR/Cas9-mediated knockout (KO) of BmGr6 and BmGr9 resulted in a low probability of making a test bite of the mulberry leaves, suggesting that they lost the ability to recognize host leaves specifically. Electrophysiological recordings showed that the loss of host recognition ability in the Gr-KO strains was due to a drastic decrease in MP sensitivity toward ISQ in BmGr6-KO larvae and toward CGA and ISQ in BmGr9-KO larvae. Our findings unraveled that the two Grs, which have been regarded as sugar receptors, are molecules responsible for detecting plant phenolics in host plant recognition.

Neuroparsin is a common insect neurohormone produced in large neuroendocrine cells in the brain and is important in mosquito reproduction. Although it is present in many flies including many Drosophila species, it was lost from D. melanogaster and a few closely related species. Three different lines of transgenic D. virilis were produced: One that expresses the yeast transcription factor gal4 under the control of the neuroparsin promoter (NP-gal4), while others codes for proteins under the control of the gal4 promoter, either enhanced green fluorescent protein (UAS-eGFP) or the D. virilis ortholog of reaper, an apoptosis inducing protein (UAS-rpr). Crosses between UAS-eGFP and NP-gal4 revealed that expression of NP-gal4 was correct. Crosses between UAS-rpr and NP-gal4 completely eliminated the neuroparsin neuroendocrine cells, but were without effect on reproduction.

Heme is a prosthetic group of proteins involved in vital physiological processes in aerobic organisms. It participates in redox reactions crucial for cell metabolism due to the variable oxidation state of its central iron atom. However, excessive heme can be cytotoxic due to its prooxidant properties. Therefore, the control of intracellular heme levels ensures the survival of organisms, especially those that deal with high concentrations of heme during their lives, such as hematophagous insects. The feline leukemia virus C receptor (FLVCR) is a membrane protein responsible for heme transport in mammalian cells. In our study, we found that RpFLVCR serves as a heme exporter in the midgut of the hematophagous insect Rhodnius prolixus, a vector for Chagas disease. Silencing RpFLVCR decreased hemolymphatic heme levels and increased the levels of intracellular dicysteinyl-biliverdin, a product of R. prolixus heme degradation, indicating heme retention inside midgut cells. FLVCR silencing led to increased expression of heme oxygenase (HO), ferritin, and mitoferrin mRNAs while downregulating the iron importers Malvolio 1 and 2. In contrast, HO gene silencing increased FLVCR and Malvolio expression and downregulated ferritin, revealing crosstalk between heme degradation/export and iron transport/storage pathways. Furthermore, RpFLVCR silencing strongly increased oxidant production and lipid peroxidation, reduced cytochrome c oxidase activity and activated mitochondrial biogenesis, effects not observed in RpHO-silenced insects. These data support FLVCR function as a heme exporter, playing a pivotal role in heme/iron metabolism and maintenance of redox balance, especially in an organism adapted to face extremely high concentrations of heme.

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

Insects have various strategies like mimicry or camouflage to avoid predation. Swallowtail butterfly larvae switch from a black and white pattern mimicking bird droppings to a green camouflage pattern in the fifth (final) instar. This larval pattern switch is regulated during the juvenile hormone (JH)-sensitive period, when JH titer declines rapidly, and clawless (cll), abdominal-A (abd-A), and Abdominal-B (Abd-B) function during this period. However, the molecular mechanism behind the background green color, a crucial aspect of the camouflage pattern, remains poorly understood. Here, we used Papilio memnon, which switches to the camouflage pattern in the fifth instar but is greenish from the third instar, to investigate the mechanism of camouflage pattern formation, particularly the larval green coloration. Through RNA sequencing, we found that BBPs forming a gene cluster are upregulated in the green regions of P. memnon larvae during the fourth instar, whereas P. xuthus larvae, which have not yet turned green, showed minimal BBPs expression. When BBP1 and BBP2, which were particularly highly expressed, were knocked down by RNAi, there was a phenotypic change in green to yellow in both fourth and fifth instar larvae. Expression analysis and knockdown experiments were conducted also for JHBP, which had been previously reported, and confirmed that it is involved in the synthesis of yellow pigment. Furthermore, knockdown of Ubx resulted in no phenotypic change in fourth instar larvae, but in fifth instar larvae, the eyespots pattern characteristic of the camouflage pattern almost entirely disappeared, suggesting that Ubx is also functional only during JH-sensitive period. Our results indicate that the switch from mimetic to camouflage patterns resulted from the function of cll, abd-A, Abd-B, and Ubx prepatterning genes during the JH-sensitive period. And the increased expression of BBPs and JHBPs, independent of the JH-sensitive period, contributed to the development of green coloration.

Ecdysteroids are major hormones in insects and control moulting, growth, reproduction, physiology, and behaviour. The biosynthesis of ecdysteroids such as 20-hydroxyecdysone (20E) from dietary sterols is well characterised, but ecdysteroid catabolism is poorly understood. Ecdysteroid kinases (EcKs) mediate the reversible phosphorylation of ecdysteroids, which has been implicated in ecdysteroid recycling during embryogenesis and reproduction in various insects. However, to date only two EcK-encoding genes have been identified, in the silkworm Bombyx mori and the mosquito Anopheles gambiae. Previously, we identified two ecdysteroid kinase-like (EcKL) genes--Wallflower (Wall) and Pinkman (pkm)--in the model fruit fly Drosophila melanogaster that are orthologs of the ecdysteroid 22-kinase gene BmEc22K. Here, using gene knockdown, knockout and misexpression, we explore Wall and pkms possible functions and genetically test the hypothesis that they encode EcKs. Wall and pkm null mutants are viable and fertile, suggesting they are not essential for development or reproduction, whereas phenotypes arising from RNAi and somatic CRISPR appear to derive from off-target effects or other artefacts. However, misexpression of Wall results in dramatic phenotypes, including developmental arrest, and defects in trachea, cuticle and pigmentation. Wall misexpression fails to phenocopy irreversible ecdysteroid catabolism through misexpression of Cyp18a1, suggesting Wall does not directly inactivate 20E. Additionally, Wall misexpression phenotypes are not attenuated in Cyp18a1 mutants, strongly suggesting Wall is not an ecdysteroid 26-kinase. We hypothesise that the substrate of Wall in this misexpression experiment and possibly generally is an unknown, atypical ecdysteroid that plays essential roles in Drosophila development, and may highlight aspects of insect endocrinology that are as-yet uncharacterised. We also provide preliminary evidence that CG5644 encodes an ecdysteroid 22-kinase conserved across Diptera.

UDP-glycosyltransferases (UGTs) constitute a superfamily of enzymes that play a vital role in the biotransformation of diverse hydrophobic substrates into more hydrophilic products, thereby facilitating their excretion from the cell through transporters. The significance of UGTs in conferring insecticide resistance has been emphasized in various insect species. In this study, we characterised Anopheles funestus UGT genes genome-wide and explored their evolution and association with pyrethroid resistance. We combined genome-wide association of pooled-template sequencing (GWAS-PoolSeq) with the transcriptomic profile of pyrethroid-resistant An. funestus populations, and deep targeted sequencing of UGTs from 80 individual mosquitoes collected in Malawi, Uganda, Cameroon and the two laboratory colonies (FANG and FUMOZ) to investigate the role of UGTs in pyrethroid resistance. We identified common overexpression of UGT310B2 (AFUN000679) in the resistant laboratory colony (FUMOZ) and resistant field populations from Malawi, Cameroon and Uganda. Significant gene-wise Fst differentiation between the resistant and putatively susceptible populations was observed for UGT301C2 and UGT302A3 in Malawi, as well as UGT306C2 in Uganda. Furthermore, the gene-wise Tajimas D density curves of the sequenced regions provided insights into genome-wide processes elucidating population structures within An. funestus populations from these three countries, supporting previous observations. Additionally, we identified significantly differentiated nonsynonymous mutations within UGT genes, which may potentially contribute to pyrethroid resistance. The identified role of An. funestus UGT genes in pyrethroid resistance has direct implications for current vector control strategies, management approaches, and the prediction of potential cross-resistance to other insecticides that can be directly detoxified by UGTs.

Among insect chemosensory receptors, the broadly conserved Ionotropic Receptors (IRs) remain some of the least investigated. Several studies have documented IR activation by recording insects neuronal activity in situ, some demonstrated their activation when expressed in oocytes from Xenopus, others made use of the Drosophila "ionotropic receptor decoders" to functionally mis-express IRs from the same species or from the closely related D. sechellia. Here we demonstrated that both substituting tuning IRs of D. melanogaster and expressing heterologous IRs from other insects alongside the Drosophila native ones result in functional heteromeric complexes. By these methods, we functionally characterized the IR41a1 subunit of the codling moth Cydia pomonella, which demonstrated binding to polyamines with different pharmacological characteristics and the IR75d subunit of the spotted wing drosophila Drosophila suzukii, which binds hexanoic acid. Then we expressed the D. suzukii acid sensor IR64a into the D. melanogaster "ionotropic receptor decoder" neuron, which resulted in the inhibition to the main activators of other neurons housed in the same sensillum of D. melanogaster, as an evidence of its possible functional expression, but it did not show response to acids. In situ hybridization on the antennae of D. suzukii unveiled a wide expression of this subunit in neurons proximal to the sacculus. Structural analysis did not explain absence of IR64a binding to acids, but it identified key amino acid features that may justify possible hexanoic acid binding for IR75d. While our findings add to the derophanization efforts conducted on the chemosensors of the aforementioned pests, they demonstrated potential for the use of neurons of transgenic Drosophila as a tool to functionally characterize IRs from different insect species.

Drosophila melanogaster has been at the forefront of genetic studies and biochemical modelling for over a century. Yet, the functions of many genes are still unknown mainly because no phenotypic data are available. Herein, we present first evidence data regarding the particular molecular and other quantifiable phenotypes, such as viability and anatomical anomalies, induced by a novel P{lacW} insertional mutant allele of CG18135 gene. So far, the CG18135 functions have only been theorized based on electronic annotation and presumptive associations inferred upon high-throughput proteomics or RNA sequencing experiments. The descendants of individuals harboring the CG18135P{lacW}CG18135 allele were scored in order to assess mutant embryos, larvae and pupae viability versus Canton Special. Our results revealed that the homozygous CG18135P{lacW}CG18135/CG18135P{lacW}CG18135 genotype determines significant lethality both at the inception of larval stage and during pupal development. Few imago escapers that breach the puparium and even more rarely fully exit from it exhibit specific eye depigmentation, wing abnormal unfolding and strong locomotor impairment with apparent spasmodic legs movements. Their maximum lifespan is shorter than two days. When using the quantitative Real-Time PCR (qRT-PCR) method to confirm that CG18135 is indeed upregulated in males compared to females an unexpected gene upregulation was also detected in heterozygous mutants comparative to wild-type flies, probably because of regulatory perturbations induced by P{lacW} transposon. Our work provides the first phenotypic evidence for the essential role of CG18135, a scenario in accordance with the putative role of this gene in the carbohydrate binding processes.

The Lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily (LU super family) of proteins are involved in diverse biological processes. In Drosophila melanogaster, members of the LU superfamily have undergone lineage-specific gene duplication and acquired specialized functions in distinct tissues. A glycosylphosphatidylinositol (GPI)-anchored LU family protein Belly roll (Bero) has recently been shown to regulate larval escape behavior; however, its cellular expression profile and potential roles remain incompletely understood. In this study, we generated a bero-GAL4T2A transgenic line to delineate endogenous bero expression. This analysis revealed that bero is expressed in the peptidergic neurons in the central nervous system (CNS) that had not been documented in previous studies, as well as in the peripheral nervous system (PNS) and non-neuronal tissues, such as the anal pad and epidermis. Reanalysis of publicly available single-cell RNA sequencing (scRNA-seq) datasets demonstrated that bero is expressed in several peptidergic neurons. These findings suggest that Bero is specifically expressed in diverse peptidergic neurons and may play important roles in coordinating hormonal and neural regulation in D. melanogaster.

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.

Many insect species rely on diverse terpenoids for their development and interorganismal interactions. However, little is known about terpenoid biosynthesis in insects. The monoterpenoid sex pheromones of mealybugs and scale insects (Coccoidea) are particularly enigmatic, with several species producing unique structures presumed to result from the irregular coupling activity of unidentified isopentenyl diphosphate synthases (IDSs). Enzymes capable of similar transformations have previously only been described from a few plant, bacterial and archaeal species. To investigate if insect irregular monoterpenes can be biosynthesised by similar enzymes, we performed a comprehensive search for IDS coding sequences in the genome of Planococcus citri, a widespread agricultural pest. We complemented the available P. citri genome data with newly generated short- and long-read transcriptome data. The identified candidate genes had homology to both short- and long-chain IDSs and some appeared to be paralogous, indicating gene duplications and consequent IDS gene family expansion in P. citri. We tested the activity of eleven candidate gene products, confirming in vitro regular activity for five enzymes, one of which (transIDS5) also produced the irregular prenyl diphosphates, maconelliyl and lavandulyl diphosphate. Targeted mutagenesis of selected aspartates and a lysine in the active site of transIDS5 uncovered their importance for chain-length preference and irregular coupling. This work provides an important foundation for deciphering terpenoid biosynthesis in mealybugs, as well as a potential source of enzymes for the biotechnological production of sustainable insect pest management products.

Diapause in insects is a developmental arrest that serves as an adaptation to severe environmental conditions. In the silkworm Bombyx mori, embryonic diapause in the next generation is maternally induced by the diapause hormone (DH). However, the downstream factors responsible for initiating diapause have remained unidentified. The pnd and pnd-2 mutants produce pigmented and non-diapausing eggs despite intact DH signaling, indicating that the pnd and pnd-2 genes act downstream as potential initiators of diapause. Here, we analyzed their expression dynamics and functions. Expression analyses showed that pnd was strongly upregulated in diapause-destined eggs in a DH-signal-dependent manner and was suppressed by HCl treatment that prevents diapause initiation. In contrast, pnd-2 exhibited a transient expression peak independent of DH-signal and was unaffected by HCl treatment. RNAi-mediated knockdown of either gene inhibited the diapause initiation, and injection of pnd mRNA into non-diapause-destined eggs induced a diapause-like state in embryos. These findings demonstrate that pnd and pnd-2 are required downstream of the DH-signal to initiate diapause, providing key insights into the molecular mechanism underlying embryonic diapause induction in B. mori. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/667111v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@7e9516org.highwire.dtl.DTLVardef@6dddc2org.highwire.dtl.DTLVardef@fa11baorg.highwire.dtl.DTLVardef@1a2054e_HPS_FORMAT_FIGEXP M_FIG C_FIG