Insect Science

Ruvbl1 (also known as TIP49, Pontin) encodes an ATPase of the AAA+ protein superfamily involved in several cellular functions, including chromatin remodeling, control of transcription, and cellular development (motility, growth, and proliferation). Here, we used an in-vivo RNA interference (RNAi) approach to evaluate the effect of Ruvbl1 silencing on the physiology of the corn planthopper, Peregrinus maidis. Silencing of P. maidis Ruvbl1 (PmRuvbl1) was correlated with visible morphology changes in female individuals with significant increases in body mass observed at 8 and 12 days after double strand RNA (dsRNA) injection. Ovary morphology was significantly affected in adult females with PmRuvbl1 silenced, with no mature oocytes observed at 8 and 12 days after gene silencing. Whereas no significant difference in egg laying was observed 4 days after dsRNA injection, significantly fewer eggs were laid in plants at 8 and 12 days after dsRNA treatment. Furthermore, dramatic reductions in egg hatching were observed at all time points after PmRuvbl1 silencing, compared to dsGFP-injected controls. These results extend PmRuvbl1 functions as a putative regulator of P. maidis reproduction and demonstrate the potential of Ruvbl1 to be further exploited as a target for RNAi-mediated insect control.

Trichogramma, a genus of egg parasitoid wasps, are widely used as biological control agents and serve as model organisms in parasitoid research. Despite their significance, the understanding of RNA interference (RNAi) in Trichogramma remains very limited. In this study, we investigated RNAi-associated genes by bioinformatic approaches and experimentally assessed the feasibility of RNAi and the susceptibility of environmental RNAi in Trichogramma. We found that Trichogramma genomes contain a complete set of genes in the RNAi pathway and exhibit extensive gene expansion of dsRNase, which may influence RNAi efficiency by degrading dsRNA. We demonstrated successful RNAi through pupal microinjection in T. dendrolimi Matsumura, providing a technical approach for future gene functional studies. In addition, we observed no evidence of susceptibility to environmental RNAi in either T. dendrolimi adults or larvae, which might be attributed to the extensive expansion of dsRNase. This low environmental RNAi sensitivity in Trichogramma could suggest a reduced risk of RNAi-based pest management strategies affecting nontarget Trichogramma populations. Overall, this study presents a technical approach for conducting gene functional studies in Trichogramma and provides a foundation for evaluating the nontarget effects of RNAi-based pest control strategies on Trichogramma.

Selecting an appropriate target gene is critical to the success of feeding RNA interference (f-RNAi)-based pest control. Gene targets have been chosen based on their ability to induce lethality. However, lethality induction by f-RNAi is slow-acting and crop damage can progress during this time. Here, we show that f-RNAi of death-associated inhibitor of apoptosis protein 1 (diap1), but not two conventional targets vacuolar ATPase subunit A and E, induces acute feeding cessation in the solanaceous pest, Henosepilachna vigintioctopunctata during 24-48 hours. We also found that the feeding cessation by diap1 f-RNAi has species-specificity and occurs with only 1.6 ng dsRNA. Our results suggest that diap1 is an appropriate target in the context of rapid reduction of crop damage. We propose that acute feeding disorder should be assessed as a novel criterion for selecting appropriate target genes for RNAi-based pest control in addition to the conventional criterion based on lethality.

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.

The sex determination mechanism for hemipteran species remains poorly understood. During the sex determination of the brown planthopper (BPH), Nilaparvata lugens, one species of Hemiptera, the functions of doublesex (Nldsx) and NlTra-2 (NlTra-2) genes were identified in our previous studies. Here, we identify an upstream gene for Nldsx in the sex determination cascade, NlFmd, which acts as female determinant gene for N. lugens. The sex-specific transcript of NlFmd (NlFmd-F) encodes an arginine/serine-, and proline-rich protein that is essential for female development. The knockdown of NlFmd resulted in the development of pseudomales, with sex-specific alternative Nldsx processing, and maternal RNA interference (RNAi) against NlFmd generates male-only progeny. Moreover, homologous genes for NlFmd have also been identified in two rice planthopper species, the white-backed planthopper (WBPH, Sogotalla furcifera) and the small brown planthopper (SBPH, Laodelphax striatellus), and these genes appear to be involved in the sex determination cascades for these species. Our data suggest that the sex determination cascade in Delphacidae is conserved.

Dalbulus maidis [(DeLong & Wolcott), corn leafhopper], a phloem-feeding insect, is the most efficient vector of maize stunting pathogens (Spiroplasma kunkelii, Maize bushy stunt phytoplasma, and Maize rayado fino virus) in the Americas. Studies involving gene editing in insects are rapidly providing information that can potentially be used for insect vector and plant disease control. RNA interference (RNAi), a sequence-specific gene silencing method, is one of the most widely used molecular tools in functional genomics studies. RNAi uses exogenous double-stranded RNA (dsRNA) or small interfering RNA (siRNA) to prevent the production of proteins by inhibiting the expression of their corresponding messenger RNA (mRNA). In this study, we measured the efficacy of gene silencing, and its effects on D. maidis mortality as proof of concept that RNAi is a viable tool for use in genetic pest control of phloem-feeding insects. Oral delivery of dsRNA using an artificial diet was used to silence two key insect genes, vacuolar ATP synthase subunit B, and subunit D (V-ATPase B and V-ATPase D). Our results showed reduced gene expression of V-ATPase B and V-ATPase D after ingestion of dsRNA, and significantly higher mortality, and wing deformation, associated with reduced gene expression, compared to control insects that were not orally fed dsRNA. These results reveal RNAi as a viable tool for use in genetic pest control of phloem-feeding insects, and a way for further functional genomic studies, such as identification of potential target genes for either population suppression or population replacement of this vector of maize diseases.

Intraspecific brood parasitism (IBP), where a parasitic female lays eggs in the nest of another female of the same species, occurs in insects and birds. Also, quasi-parasitism (QP), where a parasitic female copulates with a host male at his nest and lays eggs that are fertilized by the male, has been documented in a few monogamous birds, but QP has not been observed in any insects. Burying beetles, genus Nicrophorus, use small vertebrate carcasses for reproducing and providing biparental care for their offspring. IBP has been observed in one burying beetle by laboratory experiments, but has not been well reported under natural conditions. IBP and QP may occur under natural conditions in burying beetles. Here we focused on a burying beetle, Nicrophorus quadripunctatus. Ten broods, consisting of larvae and their parental female and male, were collected from a deciduous forest. To investigate the kin relationship between parents and larvae, eight microsatellite DNA loci were used. We detected three types of parasitic larvae: 1) larva not related to either its parental female or male, 2) larva not related to its parental female, but unknown regarding its parental male, and 3) larva not related to its parental female, but related to its parental male. These results suggested that IBP and QP can occur with certain frequencies in the reproduction of N. quadripunctatus under natural conditions. QP is thought to have a benefit for a parental male to enhance his paternity within one brood in this species.

Exportin 1 (XPO1) is the major karyopherin-{beta} nuclear receptor mediating the nuclear export of hundreds of proteins and some classes of RNA and regulates several critical processes in the cell, including but not limited to, cell-cycle progression, transcription, translation, oncogenesis and longevity. Viruses have co-opted XPO1 to promote nucleocytoplasmic transport of viral proteins and RNA. Maize mosaic virus (MMV) is an Alphanucleorhabdovirus transmitted in a circulative propagative manner by the corn planthopper, Peregrinus maidis. MMV replicates in the nucleus of plant and insect hosts, and it remains unknown whether MMV co-opts P. maidis XPO1 (PmXPO1) to complete its life cycle. Because XPO1 plays multiple regulatory roles in cell functions and virus infection, we hypothesized that RNAi-mediated silencing of XPO1 would simultaneously and negatively affect MMV accumulation and insect physiology. Although PmXPO1 expression was not modulated during MMV infection, PmXPO1 knockdown negatively affected MMV accumulation in P. maidis at 12 and 15 days after microinjection. Likewise, PmXPO1 knockdown negatively affected P. maidis survival and reproduction. PmXPO1 exhibited tissue specific expression patterns with higher expression in the ovaries compared to the guts of adult females. Survival rate was significantly lower for PmXPO1 knockdown females, compared to controls, but no effect was observed for males. Adult females with PmXPO1 knockdown were heavier and had a larger abdomen compared to controls at 4, 8 and 12 days after dsRNA microinjection. Consistent with an increase in weight, glyceride content specifically and significantly increased in PmXPO1 knockdown female planthoppers. Ovary development was significantly inhibited, and mature eggs were not observed in adult females with PmXPO1 knockdown. Consistent with a major role of PmXPO1 in ovary function and egg production, oviposition and egg hatch in plants was dramatically reduced in dsRNA PmXPO1 treated insects compared with control. Altogether, these results suggest that PmXPO1 is a positive regulator of P. maidis reproduction and that it plays a proviral role in the insect vector supporting MMV infection.

Genetic correlations among behavioural traits are often controlled by pleiotropic genes. Many studies suggest the existence of genetic correlations among behavioural traits based on artificial selection experiments in the laboratory. However, few studies have examined whether behavioural correlations in the laboratory are maintained in the field, where natural selection works. Artificial selection experiments showed a behavioural correlation among death feigning, walking movement, and locomotor activity in the red flour beetle (Tribolium castaneum). This study investigated whether this behavioural correlation is observed in wild T. castaneum populations. We also collected beetles from various regions in Japan and investigated the geographic variation in these traits. There was geographic variation in the three behavioural traits. However, these behavioural traits were not correlated. The results suggest that the genetic correlations among behavioural traits are not maintained in the field. Therefore, the results derived from laboratory experiments may be overestimated. The same correlation between traits was not believed to arise in the field, as the indoor results may have been caused by unrealistic selection pressures. Further laboratory and field investigations are both needed.

In the era of global warming, much attention has been paid to the possibility that high temperature may influence diverse insects not only directly but also indirectly via effects on their symbiotic microorganisms. The stinkbug Plautia stali develops a midgut symbiotic organ that harbors a specific bacterial symbiont indispensable for its growth and survival. Being maintainable in laboratory and tractable experimentally, P. stali is recently highlighted as a model system to investigate the mechanisms underpinning insect-microbe symbiotic interactions. In this study, we reared newly-emerged adult insects of P. stali under different temperature conditions for 8 days and monitored how their symbiotic organs and symbiotic bacteria are affected. While all insects survived at temperatures from 25{degrees}C to 37{degrees}C, some insects died at 38{degrees}C, 39{degrees}C and 40{degrees}C, wherein mortality rates increased as temperature elevated. While the symbiotic organs of the normal insects exhibited vivid yellow color, the symbiotic organs of the insects reared at 35{degrees}C or higher frequently exhibited abnormal colors such as pale yellow, yellowish white, or white, the extent of which became more severe as temperature elevated. Symbiont quantification revealed that, while the symbiont titers were almost constant for 8 days at 25{degrees}C and 30{degrees}C, the symbiont titers on the 8th day drastically declined to 1/100 at 35 and 1/10000 at 37{degrees}C and 39{degrees}C. Based on these results, we propose that rearing at 37{degrees}C for a week is a recommended treatment regime by which the symbiont is effectively suppressed with minimal damage to the host insect.

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

In insects, the tyramine receptor 1 (TAR1) has been shown to control several physiological functions, including olfaction. We investigated the molecular and functional profile of the Halyomorpha halys type 1 tyramine receptor gene (HhTAR1) and its role in olfactory functions of this pest. Molecular and pharmacological analyses confirmed that the HhTAR1 gene codes for a true TAR1. The RT-qPCR analysis revealed that HhTAR1 is expressed mostly in adult brain and antennae as well as in early development stages (eggs, 1st and 2nd instar nymphs). In particular, among the antennomeres that compose a typical H. halys antenna, HhTAR1 was more expressed in flagellomeres. Scanning electron microscopy (SEM) investigation revealed the type and distribution of sensilla on adult H. halys antennae: both flagellomeres appear rich in trichoid and grooved sensilla, known to be associated with olfactory functions. Through a RNAi approach, topically delivered HhTAR1 dsRNA induced a 50 % gene downregulation after 24 h in H. halys 2nd instar nymphs. An innovative behavioral assay revealed that HhTAR1 RNAi-silenced 2nd instar nymphs were less susceptible to the alarm pheromone component (E)-2 decenal as compared to control. These results provide critical information concerning the TAR1 role in olfaction regulation, especially alarm pheromone reception, in H. halys. Furthermore, considering the emerging role of TAR1 as target of biopesticides, this work paves the way for further investigation on innovative methods for controlling H. halys.

Chagas disease is estimated to affect 8 million people worldwide and is responsible for circa 10,000 deaths in Latin America every year. Vector control has been developed as the main strategy to control disease spreading. The identification of new targets is essential, and genes containing small open reading frames (smORFs - < 100 amino acids) are present as hundreds of putative new candidate targets in insect genomes. Here, we show that the prototypic smORF containing gene mille-pattes/polished-rice/tarsalless (mlpt/pri/tal) is essential for post-embryonic development of the kissing bug Rhodnius prolixus and for the metacyclogenesis of the Trypanosoma cruzi parasite during nymphal stages. Injection of double-stranded RNA against mlpt (Rp-dsmlpt) during nymphal stages leads to a plethora of phenotypes which impairs post-embryonic development. First, nymphs of fourth or fifth stage injected with Rp-dsmlpt do not moult. Second, vector digestive physiology is largely modified, and haemozoin is significantly increased in the posterior midgut of Rp-dsmlpt nymphs. Third, Rp-mlpt knockdown inhibits the metacyclogenesis of Trypanosoma cruzi, the etiologic agent of Chagas disease. Thus, our study provides the first evidence of a smORF-containing gene regulating vector physiology, parasitic cycle and disease transmission.

The emission of artificial light at night (ALAN) is rapidly increasing worldwide. Yet, evidence for its detrimental effects on various species is accumulating. While the effects of ALAN on phenotypic traits have been widely investigated, effects on the molecular level are less well understood. Here we aimed to integrate the effects of ALAN at the transcriptomic and the phenotypic level. We tested these effects on Chironomus riparius, a multivoltine, holometabolous midge with high ecological relevance for which genomic resources are available. We performed life-cycle experiments in which we exposed midges to constant light and control conditions for one generation. We observed higher EmT50 and reduced fertility under ALAN. From the observed decline in population size due to the reduced fertility, we predicted the population size to decline to 1% after 200 days. The transcriptomic analysis revealed expression changes of genes related to circadian rhythmicity, moulting, catabolism and oxidative stress. From the transcriptomic analysis we hypothesised that under ALAN, oxidative stress is increased, and that moulting begins earlier. We were able to confirm both hypotheses in two posthoc experiments, showing that transcriptomics are a powerful tool in predicting physiological outcomes before they are even observable.

The Japanese Archipelago, comprising a series of isolated yet interconnected islands, had been geographically separated from the Eurasian continent. The linear topography presents a unique biogeographic context for dispersing organisms from the continent. In this study, we utilized mitochondrial DNA (mtDNA) and single nucleotide polymorphism (SNP) variation to elucidate the dispersal history of the Japanese clouded butterfly Parnassius glacialis across the Japanese Archipelago, including North Island (Hokkaido), Main Island (Honshu) and Shikoku Island. Our analysis of mtDNA (COI, COII) at 1192 base pairs (bps) revealed 49 haplotypes and identified three distinct haplotype groups in the network. These groups correspond geographically to East Japan, West Japan, and Chugoku-Shikoku. The Chugoku-Shikoku group is the most ancient lineage. Interestingly, the Chugoku-Shikoku lineage showed a closer network connection to the East Japan lineage than the geographically proximate West Japan lineage. Divergence time estimates suggest that the Chugoku-Shikoku lineage diverged from the continental P. glacialis approximately 3.05 million years ago (MYA). Subsequently, from the Chugoku-Shikoku lineage, the East Japan and West Japan lineages diverged around 1.05 MYA, with the subsequent divergence between the East and West Japan lineages occurring at approximately 0.62 MYA. Based on the 3067 SNP genotypes, population structure analysis revealed five distinct genetic structures within the Japanese Archipelago, indicating geographical differentiation. From the analyses by mtDNA and SNP variations, four primary genetic barriers were identified: between Hokkaido and Honshu, between East and Central Japan, within the Kansai region, and within the Chugoku region. The former three lines corresponded to the Blakiston Line, the Itoigawa-Shizuoka Tectonic Line, and Lake Biwa, respectively. These findings suggest that P. glacialis diverged from the continental P. glacialis and expanded its range across the Japanese Archipelago via the North and South routes, establishing its current distribution.

The major plant pest fall armyworm (FAW), Spodoptera frugiperda, is native to the Americas and has colonized African and Asian countries in the Eastern hemisphere since 2016, causing severe damage to multiple agricultural crop species. However, the genetic origin of these invasive populations require more in-depth exploration. We analyzed genetic variation across FAW genomes of 153 newly sequenced individuals from Eastern hemisphere and 127 individuals mostly originating from the Americas. The global genetic structure of FAW shows that the FAW in American has experienced deep differentiation, largely consistent with the Z-chromosomal Tpi haplotypes commonly used to differentiate "corn-strain" and "rice-strain" populations. Results indicate that the invasive Eastern hemisphere populations are different from the American ones and have relatively homogeneous population structure, consistent with the common origin and recent spreading from Africa to Asia. Our analyses suggest that north-and central American "corn-strain" FAW are the most likely sources of the invasion into the Eastern hemisphere. Furthermore, evidence based on genomic, transcriptomic and mitochondrial haplotype network analysis suggest that there has been an earlier independent introduction of FAW into Africa that introgressed into the recent invasive population.

Akt (also known as protein kinase B) belongs to the multifunctional serine/threonine kinase family and is an important component of the insulin signaling pathway that plays a key role in many biological processes such as cell growth, proliferation, and survival. However, few studies have reported the effect of Akt on reproduction in Hemiptera. In this study, we cloned and characterized the Akt gene from Coridius chinensis (CcAkt). The open reading frame of CcAkt has a length of 1,563 bp and encodes 520 amino acids. It has a conserved pleckstrin homology domain, a serine/threonine protein kinase catalytic domain, and a serine/threonine protein kinase domain. Phylogenetic analysis showed that CcAkt and HhAkt of Halyomorpha halys had the highest similarity. Analysis of temporal and spatial expression patterns revealed that CcAkt is expressed throughout development and in various tissues of C. chinensis adults. CcAkt expression was high in female adult and the fourth instar nymph stage of C. chinensis. In C. chinensis adult, CcAkt expression was highest in the testis and ovary. Injection of bovine insulin induced CcAkt expression, whereas that of 20-hydroxyecdysone significantly reduced CcAkt expression. Both hormones, however, induced the expression of vitellogenin (Vg) and vitellogenin receptor (VgR). In unmated females, CcAkt knockout resulted in decreased expression of CcVg and CcVgR, stunted the development of the ovarioles, decreased the number of eggs and hatching rate. These findings suggest that CcAkt may be involved in regulating the reproduction of C. chinensis.

Insects living in temperate regions often accumulate a large amount of glycerol during winter to avoid freezing. This seasonal accrual of glycerol is generally produced from glycogen reserves through the pentose phosphate pathway. An alternative pathway to produce glycerol is through glycolysis, normally used for pyruvate production for eventual ATP synthesis. Aside from seasonal accumulation, some insects will also rapidly increase glycerol production as a short-term response to a sudden cold event, thereby increasing cold hardiness when necessary. In the eastern spruce budworm Choristoneura fumiferana, this plasticity in cold hardiness is locally adapted, where northern populations produce more glycerol upon cold shock. Here we investigate how glycerol is produced during the rapid plastic response to fluctuating cold conditions, and whether this pathway could be a target of local adaptation. After a period of repeated cold exposure, we found evidence of increased enzyme activity and increased mRNA abundance of several proteins associated with glycolysis, and a downregulation in expression of glucose-6-phosphate dehydrogenase, associated with pentose phosphate. Pyruvate production is prevented through downregulation of glyceraldehyde-3-phosphate dehydrogenase. We found higher overall enzyme activity and glycerol accumulation in a northern population from Alberta, although there was no evidence of an interaction effect between population and cold shock treatment. This is one the first studies to show a mechanistic basis of such plasticity in cold hardiness.

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.

Strains of Euschistus heros (Hemiptera: Pentatomidae) with resistance to thiamethoxam (NEO) and lambda-cyhalothrin (PYR), generated by selection with these insecticides in the laboratory, have been recently reported in Brazil. However, the mechanisms conferring resistance to these insecticides in E. heros remain unresolved. We utilized comparative transcriptome profiling and single nucleotide polymorphism (SNP) variant calling of susceptible and laboratory-selected resistant strains of E. heros to investigate the molecular mechanism(s) underlying resistance. The E. heros transcriptome was assembled using approximately 190.1 million paired-end reads, which generated 91,673 transcripts with a mean length of 720 bp and N50 of 1795 bp. Approximately, 54.8% of the assembled transcripts ware functionally annotated against the NCBI nr database, with most sequences (43%) being similar to the pentatomids Halyomorpha halys (43%) and Nezara viridula (29%). Comparative gene expression analysis between the susceptible (SUS) and NEO strains identified 215 significantly differentially expressed (DE) transcripts. DE transcripts associated with the metabolism of xenobiotics were all up-regulated in the NEO strain. The comparative analysis of the SUS and PYR strains identified 204 DE transcripts, including an esterase (esterase FE4), a glutathione-S-transferase, an ABC transporter (ABCC1), and aquaporins that were up-regulated in the PYR strain. We identified 9,588 and 15,043 non-synonymous SNPs in the PYR and NEO strains respectively in comparisons with the SUS strain. One of the variants (D70N) detected in the NEO strain occurs in a subunit (5) of the nicotinic acetylcholine receptor, the target-site of neonicotinoid insecticides. Nevertheless, the position of this residue was found very variable among 5 from insect species. In conclusion, neonicotinoid and pyrethroid resistance in laboratory-selected strains of E. heros is associated with a potential metabolic resistance mechanism mediated by the overexpression of several proteins commonly involved in the three phases of xenobiotic metabolism. Together these findings provide insight into the potential basis of resistance in E. heros and will inform the development and implementation of resistance management strategies against this important pest. HighlightsO_LI419 DE genes were observed in E. heros insecticide-resistant strains C_LIO_LI24,631 SNPs were identified in E. heros insecticide-resistant strains C_LIO_LIE. heros insecticide-resistant strains overexpress metabolic resistance genes C_LIO_LILambda-cyhalothrin-resistant E. heros overexpresses cuticular proteins C_LIO_LIThiamethoxam-resistant E. heros carries the target-site mutation D70N in nAChRalpha5 C_LI