Insights into the whole genome sequence of Bacillus thuringiensis NBAIR BtPl, a strain toxic to the melon fruit fly, Zeugodacus cucurbitae.

Bacillus thuringiensis is the most widely used biopesticide, targets a diversity of insect pests belonging to several orders. However, information regarding the B. thuringiensis strains and toxins targeting Zeugodacus cucurbitae is very limited. Therefore, in the present study, we isolated and identified five indigenous B. thuringiensisstrains toxic to larvae of Z. cucurbitae. However, of five strains NBAIR BtPl displayed the highest mortality (LC50 = 37.3 µg/mL) than reference strain B. thuringiensis var. israelensis (4Q1) (LC50 = 45.41 µg/mL). Therefore, the NBAIR BtPl was considered for whole genome sequencing to identify the cry genes present in it. Whole genome sequencing of our strain revealed genome size of 6.87 Mb with 34.95% GC content. Homology search through the BLAST algorithm revealed that NBAIR BtPl is 99.8% similar to B. thuringiensis serovar tolworthi, and gene prediction through Prokka revealed 7406 genes, 7168 proteins, 5 rRNAs, and 66 tRNAs. BtToxin_Digger analysis of NBAIR BtPl genome revealed four cry gene families: cry1, cry2, cry8Aa1, and cry70Aa1. When tested for the presence of these four cry genes in other indigenous strains, results showed that cry70Aa1 was absent. Thus, the study provided a basis for predicting cry70Aa1 be the possible reason for toxicity. In this study apart from novel genes, we also identified other virulent genes encoding zwittermicin, chitinase, fengycin, and bacillibactin. Thus, the current study aids in predicting potential toxin-encoding genes responsible for toxicity to Z. cucurbitae and thus paves the way for the development of B. thuringiensis-based formulations and transgenic crops for management of dipteran pests.

Immunological function of Bombyx Toll9-2 in the silkworm (Bombyx mori) larval midgut: Activation by Escherichia coli/lipopolysaccharide and regulation of growth.

Toll receptors are important regulators of insects' innate immune system which, upon binding of pathogen molecules, activate a conserved signal transduction cascade known as the Toll pathway. RNA interference (RNAi) is a powerful tool to study the function of genes via reverse genetics. However, due to the reported refractory of RNAi efficiency in lepidopteran insects, successful reports of silencing of Toll receptors in the silkworm Bombyx mori have not been reported yet. In this study, a Toll receptor of the silkworm Bombyx Toll9-2 (BmToll9-2) was cloned and its expression and function were analyzed. The results showed that BmToll9-2 contains an ectodomain (ECD) with a signal peptide and nine leucine-rich repeats, a transmembrane helix, and a cytoplasmic region with a Toll/interleukin-1 domain. Phylogenetic analysis indicates that BmToll9-2 clusters with other insect Toll9 receptors and mammalian Toll-like receptor 4. Oral infection of exogenous pathogens showed that the Gram-negative bacterium Escherichia coli and its main cell wall component lipopolysaccharide (LPS), as well as the Gram-positive bacterium Staphylococcus aureus and its main cell wall component peptidoglycan, significantly induce BmToll9-2 expression in vivo. LPS also induced the expression of BmToll9-2 in BmN4 cells in vitro. These observations indicate its role as a sensor in the innate immunity to exogenous pathogens and as a pathogen-associated receptor that is responsive to LPS. RNAi of BmToll9-2 was effective in the midgut and epidermis. RNAi-mediated knock-down of BmToll9-2 reduced the weight and growth of the silkworm. Bacterial challenge following RNAi upregulated the expression of BmToll9-2 and rescued the weight differences of the silkworm, which may be related to its participation in the immune response and the regulation of the microbiota in the midgut lumen of the silkworm larvae.

Combinatorial regulatory states define cell fate diversity during embryogenesis.

Cell fate specification occurs along invariant species-specific trajectories that define the animal body plan. This process is controlled by gene regulatory networks that regulate the expression of the limited set of transcription factors encoded in animal genomes. Here we globally assess the spatial expression of ~90% of expressed transcription factors during sea urchin development from embryo to larva to determine the activity of gene regulatory networks and their regulatory states during cell fate specification. We show that >200 embryonically expressed transcription factors together define >70 cell fates that recapitulate the morphological and functional organization of this organism. Most cell fate-specific regulatory states consist of ~15-40 transcription factors with similarity particularly among functionally related cell types regardless of developmental origin. Temporally, regulatory states change continuously during development, indicating that progressive changes in regulatory circuit activity determine cell fate specification. We conclude that the combinatorial expression of transcription factors provides molecular definitions that suffice for the unique specification of cell states in time and space during embryogenesis.

The transcriptional landscape underlying larval development and metamorphosis in the Malabar grouper (Epinephelus malabaricus).

Most teleost fishes exhibit a biphasic life history with a larval oceanic phase that is transformed into morphologically and physiologically different demersal, benthic, or pelagic juveniles. This process of transformation is characterized by a myriad of hormone-induced changes, during the often abrupt transition between larval and juvenile phases called metamorphosis. Thyroid hormones (TH) are known to be instrumental in triggering and coordinating this transformation but other hormonal systems such as corticoids, might be also involved as it is the case in amphibians. In order to investigate the potential involvement of these two hormonal pathways in marine fish post-embryonic development, we used the Malabar grouper (Epinephelus malabaricus) as a model system. We assembled a chromosome-scale genome sequence and conducted a transcriptomic analysis of nine larval developmental stages. We studied the expression patterns of genes involved in TH and corticoid pathways, as well as four biological processes known to be regulated by TH in other teleost species: ossification, pigmentation, visual perception, and metabolism. Surprisingly, we observed an activation of many of the same pathways involved in metamorphosis also at an early stage of the larval development, suggesting an additional implication of these pathways in the formation of early larval features. Overall, our data brings new evidence to the controversial interplay between corticoids and thyroid hormones during metamorphosis as well as, surprisingly, during the early larval development. Further experiments will be needed to investigate the precise role of both pathways during these two distinct periods and whether an early activation of both corticoid and TH pathways occurs in other teleost species.

Functional expression and characterization of CAPA receptor in the digestive tract and life stages of Drosophila suzukii, and differential activities with insect PRXamide peptides.

Spotted-wing drosophila, Drosophila suzukii (Matsumura), is an invasive vinegar fly that is a major threat to the small fruits industries globally. Insect capa genes encode multiple neuropeptides, including CAPA-periviscerokinin (CAPA-PVK) peptides, that are specifically known to cause diuresis or anti-diuresis in various organisms. Here we identified and characterized a corresponding G protein-coupled receptor (GPCR) of the D. suzukii CAPA-PVK peptides: CAPA receptor (CAPA-R). To better characterize the behavior of D. suzukii CAPA-R, we used insect cell-based functional expression assays to evaluate responses of CAPA-R against D. suzukii CAPA-PVKs, CAPA-PVKs from five species in Insecta, one species from Mollusca, modified CAPA-PVK peptides, and some PRXamide family peptides: pyrokinin (PK), diapause hormone (DH), and ecdysis-triggering hormone (ETH). Functional studies revealed that the D. suzukii CAPA-R is strongly activated by both of its own natural D. suzukii CAPA-PVKs, and interestingly, it was strongly activated by other CAPA-PVK peptides from Frankliniella occidentallis (Thysanoptera), Solenopsis invicta (Hymenoptera), Helicoverpa zea (Lepidoptera) and Plutella xylostella (Lepidoptera). However, D. suzukii CAPA-R was not activated by Mollusca CAPA-PVK or the other PRXamide peptides. Gene expression analyses showed that the CAPA-R was highly expressed in the Malpighian tubules and moderately in hindgut compared to other digestive organs or the rest of body, supporting diuretic/antidiuretic functionality. When compared across life stages of D. suzukii, expression of CAPA-R was approximately 1.5x greater in the third instar than the other stages and minimally detected in the eggs, 4-day old pupae and 3-day old adults. Our results functionally characterized the D. suzukii CAPA-R and a few short peptides were identified as potential biological targets to exploit the CAPA-R for D. suzukii management.

Death-Associated LIM-Only Protein Reduces Cry1Ac Toxicity by Sequestration of Cry1Ac Protoxin and Activated Toxin in Helicoverpa armigera.

The extensive use of Bacillus thuringiensis (Bt) in pest management has driven the evolution of pest resistance to Bt toxins, particularly Cry1Ac. Effective management of Bt resistance necessitates a good understanding of which pest proteins interact with Bt toxins. In this study, we screened a Helicoverpa armigera larval midgut cDNA library and captured 208 potential Cry1Ac-interacting proteins. Among these, we further examined the interaction between Cry1Ac and a previously unknown Cry1Ac-interacting protein, HaDALP (H. armigera death-associated LIM-only protein), as well as its role in toxicology. The results revealed that HaDALP specifically binds to both the Cry1Ac protoxin and activated toxin, significantly enhancing cell and larval tolerance to Cry1Ac. Additionally, HaDALP was overexpressed in a Cry1Ac-resistant H. armigera strain. These findings reveal a greater number of Cry1Ac-interacting proteins than previously known and demonstrate, for the first time, that HaDALP reduces Cry1Ac toxicity by sequestering both the protoxin and activated toxin.

Fitness costs, realized heritability, and mechanism of resistance to tenvermectin B in Plutella xylostella.

Tenvermectin B (TVM-B) and five TVM-B analogs were produced by fermentation of a genetically engineered strain Streptomyces avermitilis HU02, and TVM-B is being developed as a new insecticide. Through 11 generations of resistance selection against TVM-B in the diamondback moth, Plutella xylostella, the median lethal concentration (LC50) was increased from 14.84 to 1213.73 mg L-1. The resistance to TVM-B in P. xylostella developed fast and its realized heritability was high (h2 = 0.2901 (F7), h2 = 0.4070 (F11)). However, the relative fitness was 0.6916 suggesting a fitness cost in the resistant strains. The fitness cost was partially explained by the upregulation of the detoxification enzyme activity by 2.15 folds in carboxylate esterase (CarE) and the gene expressions of ATP-binding cassette transporter gene (ABCC2) and the alpha subunit of the glutamate-gated chloride channel (GluCl) by 1.70- and 2.32 folds, respectively. The resistance was also explained by two points of mutations at the alpha subunit of the glutamate-gated chloride channel in the P. xylostella (PxGluClα) subunit in F11. However, there was little change in the binding affinity. These results provided helpful information for the mechanism study of TVM-B resistance and will be conducive to designing rational resistance management strategies in P. xylostella.

Insect cuticular protein; gene expression, genomic structure, transcriptional regulation, speculated cuticular structure, clarified through the genomic analysis of Bombyx mori.

JH and ecdysone signaling regulate insect metamorphosis through the master transcription factors, Krüppel homolog 1 (kr-h1), Broad-Complex (BR-C), and E93. Ecdysone signaling activates successively expressed ecdysone responsive transcription factors (ERTFs), and the interaction between ERTFs determines the expression profiles of ERTFs themselves. Through the construction of expressed sequence tag (EST) database of Bombyx mori from many tissues, the existence of a large number of cuticular protein (CP) genes was identified in wing disc cDNA library of the 3 days after the start of wandering (W3). From the genomic analysis, 12 types of CP clusters of CP genes were identified. DNA sequences of CP genes revealed the duplication of CP genes, which suggests to reflect the insect evolution. These CP genes responded to ecdysone and ecdysone pulse; therefore, CP genes were applied for the analysis of transcriptional regulation by ERTF. The binding sites of ERTF have been reported to exist upstream of CP genes in several insects, and the activation of CP genes occurred by the binding of ERTFs. Through the analysis, the following were speculated; the successive appearance of ERTFs and the activation of target genes resulted in the successively produced CPs and cuticular layer. The sequence of the ERTF and CP gene expression was the same at larval to pupal and pupal to adult transformation. The involvement of several ERTFs in one CP gene expression was also clarified; BmorCPG12 belongs to group showing expression peak at W3 and was regulated by two ERTFs; BHR3 and ßFTZ-F1, BmorCPH2 belongs to group showing expression peak at P0 and was regulated by two ERTFs; ßFTZ-F1 and E74A. The involvement of BHR39 as a negative regulator of CP gene expression was found. Larval, pupal, and adult cuticular layers were supposed to be constructed by the combination of different and similar types of CPs, through the expressed timing of CP genes.

The role of heterochronic gene expression and regulatory architecture in early developmental divergence.

New developmental programs can evolve through adaptive changes to gene expression. The annelid Streblospio benedicti has a developmental dimorphism, which provides a unique intraspecific framework for understanding the earliest genetic changes that take place during developmental divergence. Using comparative RNAseq through ontogeny, we find that only a small proportion of genes are differentially expressed at any time, despite major differences in larval development and life history. These genes shift expression profiles across morphs by either turning off any expression in one morph or changing the timing or amount of gene expression. We directly connect the contributions of these mechanisms to differences in developmental processes. We examine F1 offspring - using reciprocal crosses - to determine maternal mRNA inheritance and the regulatory architecture of gene expression. These results highlight the importance of both novel gene expression and heterochronic shifts in developmental evolution, as well as the trans-acting regulatory factors in initiating divergence.

SoxC and MmpReg promote blastema formation in whole-body regeneration of fragmenting potworms Enchytraeus japonensis.

Regeneration in many animals involves the formation of a blastema, which differentiates and organizes into the appropriate missing body parts. Although the mechanisms underlying blastema formation are often fundamental to regeneration biology, information on the cellular and molecular basis of blastema formation remains limited. Here, we focus on a fragmenting potworm (Enchytraeus japonensis), which can regenerate its whole body from small fragments. We find soxC and mmpReg as upregulated genes in the blastema. RNAi of soxC and mmpReg reduce the number of blastema cells, indicating that soxC and mmpReg promote blastema formation. Expression analyses show that soxC-expressing cells appear to gradually accumulate in blastema and constitute a large part of the blastema. Additionally, similar expression dynamics of SoxC orthologue genes in frog (Xenopus laevis) are found in the regeneration blastema of tadpole tail. Our findings provide insights into the cellular and molecular mechanisms underlying blastema formation across species.

The ALS-associated KIF5A P986L variant is not pathogenic for Drosophila motoneurons.

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by the death of motoneurons. Several mutations in the KIF5A gene have been identified in patients with ALS. Some mutations affect the splicing sites of exon 27 leading to its deletion (Δ27 mutation). KIF5A Δ27 is aggregation-prone and pathogenic for motoneurons due to a toxic gain of function. Another mutation found to be enriched in ALS patients is a proline/leucine substitution at position 986 (P986L mutation). Bioinformatic analyses strongly suggest that this variant is benign. Our study aims to conduct functional studies in Drosophila to classify the KIF5A P986L variant. When expressed in motoneurons, KIF5A P986L does not modify the morphology of larval NMJ or the synaptic transmission. In addition, KIF5A P986L is uniformly distributed in axons and does not disturb mitochondria distribution. Locomotion at larval and adult stages is not affected by KIF5A P986L. Finally, both KIF5A WT and P986L expression in adult motoneurons extend median lifespan compared to control flies. Altogether, our data show that the KIF5A P986L variant is not pathogenic for motoneurons and may represent a hypomorphic allele, although it is not causative for ALS.

Schlank orchestrates insect developmental transition by switching H3K27 acetylation to trimethylation in the prothoracic gland.

Insect developmental transitions are precisely coordinated by ecdysone and juvenile hormone (JH). We previously revealed that accumulated H3K27 trimethylation (H3K27me3) at the locus encoding JH signal transducer Hairy is involved in the larval-pupal transition in insects, but the underlying mechanism remains to be fully defined. Here, we show in Drosophila and Bombyx that Rpd3-mediated H3K27 deacetylation in the prothoracic gland during the last larval instar promotes ecdysone biosynthesis and the larval-pupal transition by enabling H3K27me3 accumulation at the Hairy locus to induce its transcriptional repression. Importantly, we find that the homeodomain transcription factor Schlank acts to switch active H3K27 acetylation (H3K27ac) to repressive H3K27me3 at the Hairy locus by directly binding to the Hairy promoter and then recruiting the histone deacetylase Rpd3 and the histone methyltransferase PRC2 component Su(z)12 through physical interactions. Moreover, Schlank inhibits Hairy transcription to facilitate the larval-pupal transition, and the Schlank signaling cascade is suppressed by JH but regulated in a positive feedback manner by ecdysone. Together, our data uncover that Schlank mediates epigenetic reprogramming of H3K27 modifications in hormone actions during insect developmental transition.

Expression and Transcriptional Targets of TGFß-RII in Paracentrotus lividus Larval Skeletogenesis.

Organisms from the five kingdoms of life use minerals to harden their tissues and make teeth, shells and skeletons, in the process of biomineralization. The sea urchin larval skeleton is an excellent system to study the biological regulation of biomineralization and its evolution. The gene regulatory network (GRN) that controls sea urchin skeletogenesis is known in great details and shows similarity to the GRN that controls vertebrates' vascularization while it is quite distinct from the GRN that drives vertebrates' bone formation. Yet, transforming growth factor beta (TGF-ß) signaling regulates both sea urchin and vertebrates' skeletogenesis. Here, we study the upstream regulation and identify transcriptional targets of TGF-ß in the Mediterranean Sea urchin species, Paracentrotus lividus. TGF-ßRII is transiently active in the skeletogenic cells downstream of vascular endothelial growth factor (VEGF) signaling, in P. lividus. Continuous perturbation of TGF-ßRII activity significantly impairs skeletal elongation and the expression of key skeletogenic genes. Perturbation of TGF-ßRII after skeletal initiation leads to a delay in skeletal elongation and minor changes in gene expression. TGF-ß targets are distinct from its transcriptional targets during vertebrates' bone formation, suggesting that the role of TGF-ß in biomineralization in these two phyla results from convergent evolution.

High expression of serine protease, Brachyurin in the posterior midgut of black soldier fly (Hermetia illucens) during horse dropping processing.

OBJECTIVE: Livestock droppings cause some environmental problems, but they have the potential to be used as effective biomass resources. The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is suitable for efficiently processing such resources. By using BSF larvae for the disposal of livestock droppings, we can obtain two valuable products: protein resources and organic fertilizer. However, there is insufficient research on the digestive enzymes suitable for processing this waste. Here, we aimed to construct an efficient BSF processing system using livestock droppings, and we explored the digestive enzymes involved in this process. RESULTS: First, we investigated the characteristics of transcripts expressed in the midgut of BSF larvae and found that immune response-related genes were expressed in the midgut. Then, we investigated digestive enzymes and identified a novel serine protease, HiBrachyurin, whose mRNA was highly expressed in the posterior midgut when BSF larvae fed on horse droppings. Despite the low protein content of horse droppings, larvae that fed on horse droppings accumulated more protein than those in the other groups. Therefore, HiBrachyurin may contribute to digestibility in the early stage of protein degradation in BSF larvae fed on horse droppings.

Exploring the contribution of the salivary gland and midgut to digestion in the swede midge (Contarinia nasturtii) through a genomics-guided approach.

The larvae of Contarinia nasturtii (Kieffer) (Diptera: Cecidomyiidae), the swede midge, targets the meristem of brassica crops where they induce the formation of galls and disrupt seed and vegetable production. Previously, we examined the salivary gland transcriptome of newly-hatched first instar larvae as they penetrated the host and initiated gall formation. Here we examine the salivary gland and midgut transcriptome of third instar larvae and provide evidence for cooperative nutrient acquisition beginning with secretion of enzymes and feeding facilitators followed by gastrointestinal digestion. Sucrose, presumably obtained from the phloem, appeared to be a major nutrient source as several α-glucosidases (sucrases, maltases) and ß-fructofuranosidases (invertases) were identified. Genes encoding ß-fructofuranosidases/invertases were among the most highly expressed in both tissues and represented two distinct gene families that may have originated via horizontal gene transfer from bacteria. The importance of the phloem as a nutrient source is underscored by the expression of genes encoding regucalcin and ARMET (arginine-rich mutated in early stages of tumor) which interfere with calcium signalling and prevent sieve tube occlusion. Lipids, proteins, and starch appear to serve as a secondary nutrient sources. Genes encoding enzymes involved in the detoxification of glucosinolates (myrosinases, arylsulfatases, and glutathione-S-transferases) were expressed indicative of Brassicaceae host specialization. The midgut expressed simple peritrophins and mucins typical of those found in Type II peritrophic matrices, the first such description for a gall midge.

A Malpighian Tubule-Specific P450 Gene SlCYP9A75a Contributes to Xenobiotic Tolerance in Spodoptera litura.

Phytophagous insects are more predisposed to evolve insecticide resistance than other insect species due to the "preadaptation hypothesis". Cytochrome P450 monooxygenases have been strongly implicated in insecticide and phytochemical detoxification in insects. In this study, RNA-seq results reveal that P450s of Spodoptera litura, especially the CYP3 clan, are dominant in cyantraniliprole, nicotine, and gossypol detoxification. The expression of a Malpighian tubule-specific P450 gene, SlCYP9A75a, is significantly upregulated in xenobiotic treatments except α-cypermethrin. The gain-of-function and loss-of-function analyses indicate that SlCYP9A75a contributes to cyantraniliprole, nicotine, and α-cypermethrin tolerance, and SlCYP9A75a is capable of binding to these xenobiotics. This study indicates the roles of inducible SlCYP9A75a in detoxifying man-made insecticides and phytochemicals and may provide an insight into the development of cross-tolerance in omnivorous insects.

Preliminary monitoring of knockdown resistance (kdr) mutation in Anopheles stephensi: insights from a malarious area in Southeastern Iran.

BACKGROUND: Anopheles stephensi is recognized as the main malaria vector in Iran. In recent years, resistance to several insecticide classes, including organochlorine, pyrethroids, and carbamate compounds, has been reported for this medically important malaria vector. The main objective of the present study was to evaluate the insecticide susceptibility status of An. stephensi collected from the southern part of Iran, and to clarify the mechanism of resistance, using bioassay tests and molecular methods comparing the sequence of susceptible and resistant mosquitoes. METHODS: Mosquito larvae were collected from various larval habitats across six different districts (Gabrik, Sardasht, Tidar, Dehbarez, Kishi and Bandar Abbas) in Hormozgan Provine, located in the southern part of Iran. From each district standing water areas with the highest densities of Anopheles larvae were selected for sampling, and adult mosquitoes were reared from them. Finally, the collected mosquito species were identified using valid keys. Insecticide susceptibility of An. stephensi was tested using permethrin 0.75%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, and DDT 4%, following the World Health Organization (WHO) test procedures for insecticide resistance monitoring. Additionally, knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene was sequenced and analysed among resistant populations to detect possible molecular mechanisms of observed resistance phenotypes. RESULTS: The susceptibility status of An. stephensi revealed that resistance to DDT and permethrin was found in all districts. Furthermore, resistance to all tested insecticides in An. stephensi was detected in Gabrik, Sardasht, Tidar, and Dehbarez. Analysis of knockdown resistance (kdr) mutations at the vgsc did not show evidence for the presence of this mutation in An. stephensi. CONCLUSION: Based on the results of the current study, it appears that in An. stephensi from Hormozgan Province (Iran), other resistance mechanisms such as biochemical resistance due to detoxification enzymes may be involved due to the absence of the kdr mutation or non-target site resistance. Further investigation is warranted in the future to identify the exact resistance mechanisms in this main malaria vector across the country.

Rearing condition influences gene expression in postlarval American lobster (Homarus americanus).

The American lobster (Homarus americanus) is an economically important species in the western Atlantic and its climate-driven range shift northward along the east coast of the United States is well documented. The thermal tolerance of lab-reared postlarvae of this species has been extensively investigated to better understand settlement and recruitment dynamics. However, there have been few studies focused on wild-caught postlarvae, and even fewer that have analyzed lab-rearing conditions in context of diet. In this study, we investigated gene transcriptional changes in postlarvae caught in the wild, as well as postlarvae reared in the laboratory on a brine shrimp diet or a wild-sourced zooplankton diet. We found between wild-caught and brine shrimp-reared larvae 3,682 differentially expressed genes, and between wild and zooplankton-reared postlarvae 3,939 differentially expressed genes. Between the two lab-reared groups fed different diets 2,603 genes were differentially expressed. We also exposed individuals in all rearing groups to chronic temperature treatments of 8°C and 26°C and found that both temperature extremes elicit 68-95% fewer transcriptional changes in wild postlarvae compared to either lab-reared group. In wild postlarvae, we identified differential expression of transcripts within the FoxO signaling pathway, a signaling pathway with a central role in cellular physiology, as potential molecular markers for cold tolerance in the American lobster. These findings contextualize the current literature on lab-reared postlarvae as containing conservative estimates for in situ organisms and can be used to inform population distribution modeling efforts. They also provide evidence for the need to adjust lab-rearing techniques or source wild larval crustaceans to augment studies of larval biology.

A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.

Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.

Molecular insights into the functional analysis of P450 CYP321A7 gene in the involvement of detoxification of lambda-cyhalothrin in Spodoptera frugiperda.

Fall armyworm, Spodoptera frugiperda (J. E. Smith), is a widely recognized global agricultural pest that has significantly reduced crop yields all over the world. S. frugiperda has developed resistance to various insecticides. Insect cytochrome P450 monooxygenases (CYPs or P450s) play an important role in detoxifying insecticides, leading to increased resistance in insect populations. However, the function of the specific P450 gene for lambda-cyhalothrin resistance in S. frugiperda was unclear. Herein, the expression patterns of 40 P450 genes in the susceptible and lambda-cyhalothrin-resistant populations were analyzed. Among them, CYP321A7 was found to be overexpressed in the resistant population, specifically LRS (resistance ratio = 25.38-fold) derived from a lambda-cyhalothrin-susceptible (SS) population and FLRS (a population caught from a field, resistance ratio = 63.80-fold). Elevated enzyme activity of cytochrome P450 monooxygenases (P450s) was observed for LRS (2.76-fold) and the FLRS (4.88-fold) as compared to SS, while no significant differences were observed in the activities of glutathione S-transferases and esterases. Furthermore, the knockdown of CYP321A7 gene by RNA interference significantly increased the susceptibility to lambda-cyhalothrin. Remarkably, the knockdown of CYP321A7 reduced the enzymatic activity of P450 by 43.7%, 31.9%, and 22.5% in SS, LRS, and FLRS populations, respectively. Interestingly, fourth-instar larvae treated with lambda-cyhalothrin at the LC30 dosage had a greater mortality rate due to RNA interference-induced suppression of CYP321A7 (with increases of 61.1%, 50.0%, and 45.6% for SS, LRS, and FLRS populations, respectively). These findings suggest a link between lambda-cyhalothrin resistance and continual overexpression of CYP321A7 in S. frugiperda larvae, emphasizing the possible importance of CYP321A7 in lambda-cyhalothrin detoxification in S. frugiperda.