Non-model Insects Research Articles

A Synthetic Biology Approach to Transgene Expression in Insects.

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most nonmodel and pest insect species, empirical evidence for predictable modulation of gene expression levels is lacking. This knowledge gap is critical for genetic control systems, particularly in mosquitoes, where transgenic methods offer novel routes for pest control. Commonly, the choice of RNA polymerase II promoter (Pol II) is the primary method for controlling gene expression, but the options are limited. To address this, we developed a systematic approach to characterize modifications in translation initiation sequences (TIS) and 3' untranslated regions (UTR) of transgenes, enabling the creation of a toolbox for gene expression modulation in mosquitoes and potentially other insects. The approach demonstrated highly predictable gene expression changes across various cell lines and 5' regulatory sequences, representing a significant advancement in mosquito synthetic biology gene expression tools.

Efficient CRISPR/Cas9-mediated genome editing in the European corn borer, Ostrinia nubilalis.

The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work in O. nubilalis has identified genes associated with differences in life cycle, reproduction, and resistance to Bt toxins, the general lack of a robust gene-editing protocol for O. nubilalis has been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis in O. nubilalis using the CRISPR/Cas9 genome editing system. Precise loss-of-function (LOF) mutations were generated at two circadian clock genes, period (per) and pigment-dispersing factor receptor (pdfr), and a developmental gene, prothoracicotropic hormone (ptth). Precluding the need for a visible genetic marker, gene-editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F1 offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene-specific phenotypic differences in behaviour and development were identified in F0 mutants. Specifically, F0 gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F0 mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function in O. nubilalis and facilitate the development of similar screens in other Lepidopteran and non-model insects.

CRISPR/Cas12a ribonucleoprotein mediated editing of tryptophan 2,3-dioxygenase of Spodoptera frugiperda.

In insect genome editing CRISPR/Cas9 is predominantly employed, while the potential of several classes of Cas enzymes such as Cas12a largely remain untested. As opposed to Cas9 which requires a GC-rich protospacer adjacent motif (PAM), Cas12a requires a T-rich PAM and causes staggered cleavage in the target DNA, opening possibilities for multiplexing. In this regard, the utility of Cas12a has been shown in only a few insect species such as fruit flies and the silkworm, but not in non-model insects such as the fall armyworm, Spodoptera frugiperda, a globally important invasive pest that defies most of the current management methods. In this regard, a more recent genetic biocontrol method known as the precision-guided sterile insect technique (pgSIT) has shown successful implementation in Drosophila melanogaster, with certain thematic adaptations required for application in agricultural pests. However, before the development of a controllable gene drive for a non-model species, it is important to validate the activity of Cas12a in that species. In the current study we have, for the first time, demonstrated the potential of Cas12a by editing an eye color gene, tryptophan 2,3-dioxygenase (TO) of S. frugiperda by microinjecting ribonucleoprotein complex into pre-blastoderm (G0) eggs. Analysis of G0 mutants revealed that all five mutants (two male and three female) exhibited distinct edits consisting of both deletion and insertion events. All five edits were further validated through in silico modeling to understand the changes at the protein level and further corroborate with the range of eye-color phenotypes observed in the present study.

Skimming the skaters: genome skimming improves phylogenetic resolution of Halobatinae (Hemiptera: Gerridae)

Abstract Gerromorpha Popov, 1971 is a fascinating and diverse insect lineage that evolved about 200 Mya to spend their entire life cycle on the air–water interface and have since colonized all types of aquatic habitats. The subfamily Halobatinae Bianchi, 1896 is particularly interesting because some species have adapted to life on the open ocean—a habitat where insects are very rarely found. Several attempts have been made to reconstruct the phylogenetic hypotheses of this subfamily, but the use of a few partial gene sequences recovered only a handful of well-supported relationships, thus limiting evolutionary inferences. Fortunately, the emergence of high-throughput sequencing technologies has enabled the recovery of more genetic markers for phylogenetic inference. We applied genome skimming to obtain mitochondrial and nuclear genes from low-coverage whole-genome sequencing of 85 specimens for reconstructing a well-supported phylogeny, with particular emphasis on Halobatinae. Our study confirmed that Metrocorini Matsuda, 1960, is paraphyletic, whereas Esakia Lundblad, 1933, and Ventidius Distant, 1910, are more closely related to Halobatini Bianchi, 1896, than Metrocoris Mayr, 1865, and Eurymetra Esaki, 1926. We also found that Ventidius is paraphyletic and in need of a taxonomic revision. Ancestral state reconstruction suggests that Halobatinae evolved progressively from limnic to coastal habitats, eventually attaining a marine lifestyle, especially in the genus Halobates Eschscholtz, 1822, where the oceanic lifestyle evolved thrice. Our results demonstrate that genome skimming is a powerful and straightforward approach to recover genetic loci for robust phylogenetic analysis in non-model insects.

Genome-wide identification of yellow gene family in Hermetia illucens and functional analysis of yellow-y by CRISPR/Cas9.

The yellow gene family plays a crucial role in insect pigmentation. It has potential for use as a visible marker gene in genetic manipulation and transgenic engineering in several model and non-model insects. Sadly, yellow genes have rarely been identified in Stratiomyidae species and the functions of yellow genes are relatively unknown. In the present study, we first manually annotated and curated 10 yellow genes in the black soldier fly (BSF), Hermetia illucens (Stratiomyidae). Then, the conserved amino acids in the major royal jelly proteins (MRJPs) domain, structural architecture and phylogenetic relationship of yellow genes in BSF were analyzed. We found that the BSF yellow-y, yellow-c and yellow-f genes are expressed at all developmental stages, especially in the prepupal stage. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) system, we successfully disrupted yellow-y, yellow-c and yellow-f in the BSF. Consequently, the mutation of yellow-y clearly resulted in a pale-yellow body color in prepupae, pupae and adults, instead of the typical black body color of the wild type. However, the mutation of yellow-c or yellow-f genes did not result in any change in color of the insects, when compared with the wild type. Our study indicates that the BSF yellow-y gene plays a role in body pigmentation, providing an optimal marker gene for the genetic manipulation of BSF.

Enhancement of an entomopathogenic fungal virulence against the seedcorn maggot, Delia platura, by suppressing immune responses with a bacterial culture broth of Photorhabdus temperata subsp. temperata.

In Korea, there are two maggot species in the Delia genus that commonly infest the roots and stems of the Welsh onion, thus causing serious economic damage on the crop at the seedling stage. In this study, the seedcorn maggot (Delia platura) was detected in onion fields in two different localities in Korea. After overwintering, maggot infestations occurred throughout the entire growing seasons from transplantation to harvest, but their specific patterns of occurrence varied in the two localities examined. Entomopathogenic fungi induced significant virulence against the maggot larvae, in which a strain of Beauveria bassiana was effective, though it exhibited limited mortality in its insecticidal activity. To enhance this insecticidal activity, a culture broth from an entomopathogenic bacterium, Photorhabdus temperata temperata (Ptt), was added to B. bassiana treatment. The addition of Ptt broth significantly increased the insecticidal activity of B. bassiana in a dose-dependent manner. To elucidate this enhancement in insecticidal activity, the immunosuppressive activity of Ptt broth was assessed by identifying the immune responses of the seedcorn maggots. The seedcorn maggots possessed at least three different hemocytes with plasmatocytes, crystal cells, and lamellocytes. These hemocytes exhibited nodule formation in response to the fungal infection. In addition to the cellular immunity, the maggots exhibited inducible expressions of antimicrobial peptide (AMP) genes such as cecropin and defensin. The addition of Ptt broth suppressed the nodule formation and the AMP expressions in response to the fungal infection. Altogether, this study demonstrated the innate immune responses in a non-model insect, D. platura, along with the application of immunosuppression to develop a highly efficient biological control by enhancing the virulence of B. bassiana.

Regulation of feeding dynamics by the circadian clock, light and sex in an adult nocturnal insect.

Animals invest crucial resources in foraging to support development, sustenance, and reproduction. Foraging and feeding behaviors are rhythmically expressed by most insects. Rhythmic behaviors are modified by exogenous factors like temperature and photoperiod, and internal factors such as the physiological status of the individual. However, the interactions between these factors and the circadian clock to pattern feeding behavior remains elusive. As Drosophila, a standard insect model, spends nearly all its life on food, we rather chose to focus on the adults of a non-model insect, Agrotis ipsilon, a nocturnal cosmopolitan crop pest moth having structured feeding activity. Our study aimed to explore the impact of environmental cues on directly measured feeding behavior rhythms. We took advantage of a new experimental set-up, mimicking an artificial flower, allowing us to specifically monitor feeding behavior in a naturalistic setting, e.g., the need to enter a flower to get food. We show that the frequency of flower visits is under the control of the circadian clock in males and females. Feeding behavior occurs only during the scotophase, informed by internal clock status and external photic input, and females start to visit flowers earlier than males. Shorter duration visits predominate as the night progresses. Importantly, food availability reorganizes the microstructure of feeding behavior, revealing its plasticity. Interestingly, males show a constant number of daily visits during the 5days of adult life whereas females decrease visitations after the third day of adult life. Taken together, our results provide evidence that the rhythmicity of feeding behavior is sexually dimorphic and controlled by photoperiodic conditions through circadian clock-dependent and independent pathways. In addition, the use of the new experimental set-up provides future opportunities to examine the regulatory mechanisms of feeding behavior paving the way to investigate complex relationships between feeding, mating, and sleep-wake rhythms in insects.

Identification and Interpretation of A-to-I RNA Editing Events in Insect Transcriptomes.

Adenosine-to-inosine (A-to-I) RNA editing is the most prevalent RNA modification in the nervous systems of metazoans. To study the biological significance of RNA editing, we first have to accurately identify these editing events from the transcriptome. The genome-wide identification of RNA editing sites remains a challenging task. In this review, we will first introduce the occurrence, regulation, and importance of A-to-I RNA editing and then describe the established bioinformatic procedures and difficulties in the accurate identification of these sit esespecially in small sized non-model insects. In brief, (1) to obtain an accurate profile of RNA editing sites, a transcriptome coupled with the DNA resequencing of a matched sample is favorable; (2) the single-cell sequencing technique is ready to be applied to RNA editing studies, but there are a few limitations to overcome; (3) during mapping and variant calling steps, various issues, like mapping and base quality, soft-clipping, and the positions of mismatches on reads, should be carefully considered; (4) Sanger sequencing of both RNA and the matched DNA is the best verification of RNA editing sites, but other auxiliary evidence, like the nonsynonymous-to-synonymous ratio or the linkage information, is also helpful for judging the reliability of editing sites. We have systematically reviewed the understanding of the biological significance of RNA editing and summarized the methodology for identifying such editing events. We also raised several promising aspects and challenges in this field. With insightful perspectives on both scientific and technical issues, our review will benefit the researchers in the broader RNA editing community.

Overexpression of the F116V allele of CYP9A186 in transgenic Helicoverpa armigera confers high-level resistance to emamectin benzoate

Insect cytochrome P450s play important roles in the detoxification of xenobiotics and the metabolic resistance to insecticides. However, the approach for in vivo validation of the contribution of specific candidate P450s to resistance is still limited in most non-model insect species. Previous studies with heterologous expression and in vitro functional assays have confirmed that a natural substitution (F116V) in the substrate recognition site 1 (SRS1) of the CYP9A186 of Spodoptera exigua is a gain-of-function mutation, which results in detoxification capability of and thus high-level resistance to both emamectin benzoate (EB) and abamectin. In this study, we established an effective piggyBac-based transformation system in the serious agricultural pest Helicoverpa armigera and overexpressed in vivo a resistance P450 allele, CYP9A186-F116V, from another lepidopteran pest Spodoptera exigua. Bioassays showed that transgenic H. armigera larvae expressing CYP9A186-F116V obtained 358-fold and 38.6-fold resistance to EB and abamectin, respectively. In contrast, a transgenic line of Drosophila melanogaster overexpressing this P450 variant only confers ∼20-fold resistance to the two insecticides. This bias towards the resistance level revealed that closely related species might provide a more appropriate cellular environment for gene expression and subsequent toxicokinetics of insecticides. These results not only present an alternative method for in vivo functional characterization of P450s in H. armigera and other phylogenetically close species but also provide a valuable genetic engineering toolkit for the genetic manipulation of H. armigera.

Integrated analysis of miRNA profiles and gut bacterial changes in Altica viridicyanea following antibiotic treatment.

The gut bacteria involves in insect homeostasis by playing essential roles in host physiology, metabolism, innate immunity, and so forth. microRNAs (miRNAs) are endogenous small noncoding RNAs that posttranscriptionally regulate gene expression to affect immune or metabolic processes in insects. For several non-model insects, the available knowledge on the relationship between changes in the gut bacteria and miRNA profiles is limited. In this study, we investigated the gut bacterial diversity, composition, and function from Altica viridicyanea feeding on normal- and antibiotic-treated host plants using 16S rRNA amplicon sequencing; antibiotics have been shown to affect the body weight and development time in A. viridicyanea, suggesting that the gut bacteria of the normal sample were more diverse and abundant than those of the antibiotic-fed group, and most of them were involved in various physical functions by enrichment analysis. Furthermore, we executed small RNA transcriptome sequencing using the two experimental groups to obtain numerous sRNAs, such as piRNAs, siRNAs, and known and novel miRNAs, by data mapping and quality control, and furthermore, a total of 224 miRNAs were identified as significantly differentially expressed miRNAs, of which some DEMs and their target genes participated in immune- and metabolism-related pathways based on GO and KEGG annotation. Besides, regarding the regulatory roles of miRNA and target genes, a interaction network of DEM-target gene pairs from eight immune- or metabolism-related signaling pathways were constructed. Finally, we discovered that DEMs from above pathways were significantly positively or negatively correlated with gut bacterial alterations following antibiotic treatment. Collectively, the observations of this study expand our understanding of how the disturbance of gut bacteria affects miRNA profiles in A. viridicyanea and provide new valuable resources from extreme ranges for future studies on the adaptive evolution in insects.

Immune responses of the Asian onion moth, Acrolepiopsis sapporensis, and their genetic factors from RNA-Seq analysis.

A nonmodel insect, Acrolepiopsis sapporensis, has been analyzed in immune responses. The total hemocytes in the fifth instar larvae were 2.33 × 106 cells/mL. These hemocytes comprised at least five different types and different relative ratios: 47% granulocytes, 26% plasmatocytes, 11% oenocytoid, 8% prohemocytes, and 5% spherulocytes. Upon bacterial challenge, some of the hemocytes exhibited typical hemocyte-spreading behaviors, such as focal adhesion, and filopodial and lamellipodial cytoplasmic extensions. The hemocyte behaviors induced cellular immune responses demonstrated by nodule formation. In addition, the plasma collected from the immune-challenged larvae exhibited humoral immune responses by bacterial growth inhibition along with enhanced phenoloxidase enzyme activity. These cellular and humoral immune responses were further analyzed by determining the immune-associated genes from a transcriptome generated by RNA-Seq. A total of about 12 Gb sequences led to about 218,116 contigs, which were predicted to encode about 46,808 genes. Comparative expression analysis showed 8392 uniquely expressed genes in the immune-challenged larvae. Differentially expressed gene (DEG) analysis among the commonly expressed genes indicated that 782 genes were upregulated and 548 genes were downregulated in the expressions after bacterial challenge. These immune-associated genes included pattern recognition receptors, immune mediation/signaling genes, and various immune effectors. Specifically, the genetic components of the Toll, IMD, and JAK/STAT immune signaling pathways were included in the DEG database. These results demonstrate the immune responses of A. sapporensis larvae and suggest the genes associated with the immune responses in this nonmodel insect.

Sea level rise-induced habitat loss does not alter effective migration rate for the salt marsh insect Tumidagena minuta due to large genetic effective population size

IntroductionAs anthropogenic change alters and fragments habitats, it is apparent that evolutionary change can co-occur with ecological change, though the scale and consequences of this contemporary evolution remain unclear. In coastal salt marshes of eastern North America, the flood tolerant low elevation marsh grass (Spartina alterniflora), is displacing Spartina patens, the flood intolerant high elevation marsh grass. Rising seas restrict S. patens, once occupying large areas of many hectares, to increasingly small patches, some as small as a few square meters. MethodsUsing nine microsatellite loci, we examined the genetic diversity and population structure of Tumidagena minuta, a minute, flightless planthopper and specialist herbivore of S. patens. We sampled T. minuta from S. patens habitat patches of varying radius (3–82 meters) and distances (54–1,100 meters) to test how landscape variation affects population genetic parameters associated with microevolutionary processes. We sampled and genotyped 142 T. minuta individuals across six S. patens patches in a single marsh in New Jersey, USA. ResultsWe observed high polymorphism, observing between 7 and 28 alleles per locus and an average of 13.3 alleles per locus. We observed no genetic differentiation among sampled patches (RST = −0.0109). The contemporary genetic effective population size (Ne) was estimated at approximately 360 (95% confidence interval: 208–1325) based on two-locus linkage disequilibrium. Based on an estimate of Nem = 32.4 in the finite island model, the estimated gene flow rate among these patches was 0.09 migrants per generation. DiscussionThese estimates, which are rarely produced for non-model insects, suggest that, despite rapid and precipitous decreases in habitat size and connectivity, T. minuta populations have remained large and have experienced little genetic differentiation due to drift. Ecological changes in patch size and isolation at this scale have not influenced population genetic processes like effective migration rate for T. minuta, consistent with our expectations for an insect with a large population size.

Pupal RNA interference methods for analyzing adult development in stag beetles

AbstractHolometabolous insects dramatically change their morphology via molt, both from larva to pupa and from pupa to adult. In nonmodel insects, RNA interference (RNAi) is a strong tool for analyzing gene function during postembryonic development. In many cases, larval RNAi is effective for analyzing genes involved in morphogenesis via metamorphosis. However, RNAi of genes involved in development sometimes results in lethality before animals metamorphose to pupae and/or adults, making it impossible to analyze their function during the pupal period. In this study, we establish a pupal RNAi system in the stag beetle Dorcus rectus. We selected the genes white and scarlet for RNAi knockdown to investigate appropriate injection timing and position. Both genes are known to be involved in eye pigmentation. By using these candidate genes, we demonstrate the potential of pupal RNAi in this experimental system. This method will be useful for analyzing pupal‐specific morphogenesis including fine‐shaping of the enlarged male mandible in this species.

A rapidly evolving single copy histone H1 variant is associated with male fertility in a parasitoid wasp.

The linker histone H1 binds to the nucleosome core particle at the site where DNA enters and exits, and facilitates folding of the nucleosomes into a higher-order chromatin structure in eukaryotes. Additionally, some variant H1s promote specialized chromatin functions in cellular processes. Germline-specific H1 variants have been reported in some model species with diverse roles in chromatin structure changes during gametogenesis. In insects, the current understanding of germline-specific H1 variants comes mainly from the studies in Drosophila melanogaster, and the information on this set of genes in other non-model insects remains largely unknown. Here, we identify two H1 variants (PpH1V1 and PpH1V2) that are specifically predominantly expressed in the testis of the parasitoid wasp Pteromalus puparum. Evolutionary analyses suggest that these H1 variant genes evolve rapidly, and are generally maintained as a single copy in Hymenoptera. Disruption of PpH1V1 function in the late larval stage male by RNA interference experiments has no phenotype on spermatogenesis in the pupal testis, but results in abnormal chromatin structure and low sperm fertility in the adult seminal vesicle. In addition, PpH1V2 knockdown has no detectable effect on spermatogenesis or male fertility. Collectively, our discovery indicates distinct functions of male germline-enriched H1 variants between parasitoid wasp Pteromalus and Drosophila, providing new insights into the role of insect H1 variants in gametogenesis. This study also highlights the functional complexity of germline-specific H1s in animals.

Characterization of five pigmentation genes as transgenic markers in Spodoptera frugiperda (Lepidoptera: Noctuidae)

The fall armyworm, Spodoptera frugiperda (J. E. Smith), has become one of the most damaging pests worldwide since its invasion of Africa, Asia and Oceania from 2016, threatening plants in 76 families including important crops. Genetics-based methods have proved to be an efficient way to control pests, especially invasive species, but many difficulties must be overcome to develop a transgenic insect strain, especially for a non-model species. Here we thus sought to identify a visible marker that would facilitate the distinction between genetically modified (GM) and non-transgenic insects, thereby simplifying mutation identification and facilitating the broader application of genome editing tools in non-model insects. Five genes (sfyellow-y, sfebony, sflaccase2, sfscarlet, and sfok) that are orthologs of well-studied genes in pigment metabolism were knocked out using the CRISPR/Cas9 system to identify candidate gene markers. Two genes, Sfebony and Sfscarlet, were identified responsible for body and compound eye coloration, respectively, in S. frugiperda, and could be potential visual markers for genetics-based pest management strategies.

Triple electroantennography captures the range and spatial arrangement of olfactory sensory neuron response on an insect antenna

BackgroundElectroantennography (EAG) is a basic neuroscientific tool that is widely used to measure olfactory responses in insects. It is particularly adapted to probing the olfactory systems of non-model insect species in chemical ecology and evolutionary biology. As currently practiced, EAG measures have varying degrees of correlation with olfactory responses, especially for insects whose olfactory sensory neurons (OSNs) are arranged in zones on the antennae. This limitation was shown to be partly due to the fact that only a single antennal position was recorded. New methodsWe describe a setup using triple electroantennography (EAG3), whereby three antennal positions are recorded simultaneously. The spatial arrangement of the electrodes ensures the mechanical stability of the assembly. The EAG3 detector was coupled to a gas chromatograph (GC-EAD3), customized using a Dean’s switch to improve the EAG signals by chopper modulation. EAG3 signals were analysed through a current point model to estimate olfactory responses across the antenna. ResultsRecordings were performed on Tephritidae and Drosophila species, which have antennae of different shapes and sizes. We confirmed that the spatio-temporal pattern of antennal activation was stimulus dependent and allowed us to quantify the antennal olfactory response. Comparisons with existing methodCompared to typical single-probe EAG, we show that EAG3 improves response quantification and increases the range of compounds for which a sensory response is detected. ConclusionsOur EAG3 setup is an original low-cost and easy-to-use method. It offers a useful bridge between comprehensive neurophysiological investigations and the broader themes explored in chemical ecology.

Antibody development to identify components of IIS and mTOR signaling pathways in lepidopteran species, a set of non-model insects.

Nutritional stress impacts many insect species that have differing reproductive strategies and life histories, yet it is unclear how nutrient-sensing signaling pathways mediate tissue-specific responses to changes in dietary input. In Drosophila melanogaster , insulin/insulin-like growth factor (IIS) and mTOR-mediated signaling within adipocytes regulates oogenesis. To facilitate comparative study of nutrient-sensing pathway activity in the fat body, we developed antibodies to assess IIS (anti-FOXO) and mTOR signaling (anti-TOR) across three nymphalid species (Lepidoptera). By optimizing whole-mount fat body immunostaining, we find FOXO nuclear enrichment in adult adipocytes, like that observed in Drosophila . Additionally, we show a previously uncharacterized TOR localization pattern in the fat body.

Mining Lygus hesperus (western tarnished plant bug) transcriptomic data for transient receptor potential channels: Expression profiling and functional characterization of a Painless homolog

The transient receptor potential (TRP) family of cation channels are evolutionarily conserved proteins with critical roles in sensory physiology. Despite extensive studies in model species, knowledge of TRP channel functional diversity and physiological impact remains limited in many non-model insect species. To assess the TRP channel repertoire in a non-model agriculture pest species (Lygus hesperus), publicly available transcriptomic datasets were mined for potential homologs. Among the transcripts identified, 30 are predicted to encompass complete open reading frames that encode proteins representing each of the seven TRP channel subfamilies. Although no homologs were identified for the Pyrexia and Brivido channels, the TRP complement in L. hesperus exceeded the 13–16 channels reported in most insects. This diversity appears to be driven by a combination of alternative splicing, which impacted members of six subfamilies, and gene expansion of the TRPP subfamily. To validate the in silico data and provide more detailed analyses of L. hesperus TRP functionality, the putative Painless homolog was selected for more in depth analysis and its functional role in thermosensation examined in vitro. RT-PCR expression profiling revealed near ubiquitous expression of the Painless transcript throughout nymphal and adult development. Electrophysiological data generated using a Xenopus oocyte recombinant expression system indicated activation parameters for L. hesperus Painless homolog that are consistent with a role in noxious heat (40°–45 °C) thermosensation.

RNA interference knockdown of insulin receptor inhibits ovarian development in Chilo suppressalis.

The nutritional signaling pathway regulates an insect's size, development, and lifespan, as well as playing a vital role in reproduction. The insulin/insulin-like growth factor signaling (IIS) pathway plays a key role in the nutrition signaling pathway. As an integral component of the IIS pathway, insulin receptor (InR), a receptor tyrosine kinase, plays a role in the insulin pathway by controlling reproduction in many insect species. However, the precise molecular function of InR in non-model insect reproduction is poorly understood. In our study, Chilo suppressalis, a well-known rice pest, was used as a molecular system to determine the role of InR in insect reproduction. Sequencing the InR gene of C. suppressalis, comparing the amino acid sequence-specific structure, and constructing a phylogenetic tree revealed that this gene has four main domains: ligand binding L domain, Furin-like region, fibronectin type III domains, and Tyrosine kinase catalytic domain, which were all highly conserved in insects. By characterizing the spatiotemporal expression profile of InR in different developmental stages and tissues, we found that InR gene expression was highest on the 3-day old in female pupae, 6th instar larvae, and fat body on the 6-day old in female pupae. InR gene expression may promote the molting and pupation of larvae and play a role in reproduction in the fat body. Furthermore, the RNA interference knockdown of InR dramatically reduced yolk deposition and blocked oocyte maturation. After suppression of InR, the expression of several other genes fluctuated to varying degrees. In conclusion, InR is vital to reproduction and is expected to become a new target for pest management.

Insights Into Chemosensory Proteins From Non-Model Insects: Advances and Perspectives in the Context of Pest Management.

Nowadays, insect chemosensation represents a key aspect of integrated pest management in the Anthropocene epoch. Olfaction-related proteins have been the focus of studies due to their function in vital processes, such ashost finding and reproduction behavior. Hence, most research has been based on the study of model insects, namely Drosophila melanogaster, Bombyx mori or Tribolium castaneum. Over the passage of time and the advance of new molecular techniques, insects considered non-models have been studied, contributing greatly to the knowledge of insect olfactory systems and enhanced pest control methods. In this review, a reference point for non-model insects is proposed and the concept of model and non-model insects is discussed. Likewise, it summarizes and discusses the progress and contribution in the olfaction field of both model and non-model insects considered pests in agriculture.