A Novel Peptidomimetic Insecticide: Dippu-AstR-Based Rational Design and Biological Activity of Allatostatin Analogs.

Insect neuropeptides play an essential role in regulating growth, development, reproduction, nerve conduction, metabolism, and behavior in insects; therefore, G protein-coupled receptors of neuropeptides are considered important targets for designing green insecticides. Cockroach-type allatostatins (ASTs) (FGLamides allatostatins) are important insect neuropeptides in Diploptera punctata that inhibit juvenile hormone (JH) synthesis in the corpora allata and affect growth, development, and reproduction of insects. Therefore, the pursuit of novel insecticides targeting the allatostatin receptor (AstR) holds significant importance. Previously, we identified an AST analogue, H17, as a promising candidate for pest control. Herein, we first modeled the 3D structure of AstR in D. punctata (Dippu-AstR) and predicted the binding mode of H17 with Dippu-AstR to study the critical interactions and residues favorable to its bioactivity. Based on this binding mode, we designed and synthesized a series of H17 derivatives and assessed their insecticidal activity against D. punctata. Among them, compound Q6 showed higher insecticidal activity than H17 against D. punctata by inhibiting JH biosynthesis, indicating that Q6 is a potential candidate for a novel insect growth regulator (IGR)-based insecticide. Moreover, Q6 exhibited insecticidal activity against Plutella xylostella, indicating that these AST analogs may have a wider insecticidal spectrum. The underlying mechanisms and molecular conformations mediating the interactions of Q6 with Dippu-AstR were explored to understand its effects on the bioactivity. The present work clarifies how a target-based strategy facilitates the discovery of new peptide mimics with better bioactivity, enabling improved IGR-based insecticide potency in sustainable agriculture.

Cyp6g2 is the major P450 epoxidase responsible for juvenile hormone biosynthesis in Drosophila melanogaster.

BACKGROUND: Juvenile hormones (JH) play crucial role in regulating development and reproduction in insects. The most common form of JH is JH III, derived from MF through epoxidation by CYP15 enzymes. However, in the higher dipterans, such as the fruitfly, Drosophila melanogaster, a bis-epoxide form of JHB3, accounted most of the JH detected. Moreover, these higher dipterans have lost the CYP15 gene from their genomes. As a result, the identity of the P450 epoxidase in the JH biosynthesis pathway in higher dipterans remains unknown. RESULTS: In this study, we show that Cyp6g2 serves as the major JH epoxidase responsible for the biosynthesis of JHB3 and JH III in D. melanogaster. The Cyp6g2 is predominantly expressed in the corpus allatum (CA), concurring with the expression pattern of jhamt, another well-studied gene that is crucial in the last steps of JH biosynthesis. Mutation in Cyp6g2 leads to severe disruptions in larval-pupal metamorphosis and exhibits reproductive deficiencies, exceeding those seen in jhamt mutants. Notably, Cyp6g2-/-::jhamt2 double mutants all died at the pupal stage but could be rescued through the topical application of JH analogs. JH titer analyses revealed that both Cyp6g2-/- mutant and jhamt2 mutant lacking JHB3 and JH III, while overexpression of Cyp6g2 or jhamt caused a significant increase in JHB3 and JH III titer. CONCLUSIONS: These findings collectively established that Cyp6g2 as the major JH epoxidase in the higher dipterans and laid the groundwork for the further understanding of JH biosynthesis. Moreover, these findings pave the way for developing specific Cyp6g2 inhibitors as insect growth regulators or insecticides.

Modeling insect growth regulators for pest management.

Insect growth regulators (IGRs) have been developed as effective control measures against harmful insect pests to disrupt their normal development. This study is to propose a mathematical model to evaluate the cost-effectiveness of IGRs for pest management. The key features of the model include the temperature-dependent growth of insects and realistic impulsive IGRs releasing strategies. The impulsive releases are carefully modeled by counting the number of implements during an insect's temperature-dependent development duration, which introduces a surviving probability determined by a product of terms corresponding to each release. Dynamical behavior of the model is illustrated through dynamical system analysis and a threshold-type result is established in terms of the net reproduction number. Further numerical simulations are performed to quantitatively evaluate the effectiveness of IGRs to control populations of harmful insect pests. It is interesting to observe that the time-changing environment plays an important role in determining an optimal pest control scheme with appropriate release frequencies and time instants.

The embryonic role of juvenile hormone in the firebrat, Thermobia domestica, reveals its function before its involvement in metamorphosis.

To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

Evaluation of Insect Growth Regulators (IGRs) as biological pesticides for control of Aedes aegypti mosquitoes.

BACKGROUND OBJECTIVES: Insect growth regulators (IGRs) are biological hormone analogue or mimics used as pesticides to inhibit the growth of larva during their molting and skin shedding. This study aimed to test the effect of IGRs on the eggs hatching and post-hatching inhibition of Aedes mosquitoes and understanding its effect in the mosquito breeding habitats for reduction in adult emergence. METHODS: Experiments on the evaluation of three insect growth regulators (IGRs) for the control of different stages of Aedes aegypti was carried out during 2020-21. Each experiment consisted of four treatments viz., Pyriproxyfen, Novaluron, and Larvicol at 1.0 ppm and distilled water as a control. All experiments were carried out in completely randomized design (CRD) except eggs which were carried out in factorial design each with three replications. RESULTS: All tested IGRs performed better in affecting eggs, larval and pupal stages of Ae. aegypti. Highest eggs hatching inhibition (80%) of fresh eggs occurred in Pyriproxyfen followed by Novaluron (66%) and lowest in Larvicol (62%). Eggs hatch inhibition of embryonated eggs was lower than fresh eggs. Pyriproxyfen caused 69%, Novaluron 59% and Larvicol 39% eggs hatch inhibition of embryonated eggs. Both Pyriproxyfen and Novaluron performed better in causing 98-100% larval mortality followed by Larvicol (39%). Larval development to pupal stage was completely prevented by both Pyriproxyfen and Novaluron. Although Larvicol resulted in lowest eggs hatch and larval inhibition but prevented pupae to emerge as adults. Results further showed 70-89% mortality of 3rd instar larvae of Ae. aegypti when exposed to Pyriproxyfen and Novaluron solutions after 30 days storage at lab. temperature (27±2°C), RH 70±5. INTERPRETATION CONCLUSION: None of the IGRs was more effective at the pupal stage but showed carry-on activity of growth inhibition and mortality of the successive stages of development when used against eggs stages. Therefore, we recommend early application of IGRs at mosquito habitats during the beginning and onset of the season when very early stages of mosquitoes are available in the field.

The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster.

Intestinal stem cells (ISCs) of the fruit fly, Drosophila melanogaster, offer an excellent genetic model to explore homeostatic roles of ISCs in animal physiology. Among available genetic tools, the escargot (esg)-GAL4 driver, expressing the yeast transcription factor gene, GAL4, under control of the esg gene promoter, has contributed significantly to ISC studies. This driver facilitates activation of genes of interest in proximity to a GAL4-binding element, Upstream Activating Sequence, in ISCs and progenitor enteroblasts (EBs). While esg-GAL4 has been considered an ISC/EB-specific driver, recent studies have shown that esg-GAL4 is also active in other tissues, such as neurons and ovaries. Therefore, the ISC/EB specificity of esg-GAL4 is questionable. In this study, we reveal esg-GAL4 expression in the corpus allatum (CA), responsible for juvenile hormone (JH) production. When driving the oncogenic gene, RasV12, esg-GAL4 induces overgrowth in ISCs/EBs as reported, but also increases CA cell number and size. Consistent with this observation, animals alter expression of JH-response genes. Our data show that esg-GAL4-driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of esg-GAL4 as a "specific" ISC/EB driver to examine ISC/EB-mediated animal physiology.

A Potential Multitarget Insect Growth Regulator Candidate: Design, Synthesis, and Biological Activity of Novel Acetamido Derivatives Containing Hexacyclic Pyrazole Carboxamides.

Insect growth regulators (IGRs) are important green insecticides that disrupt normal growth and development in insects to reduce the harm caused by pests to crops. The ecdysone receptor (EcR) and three chitinases OfChtI, OfChtII, and OfChi-h are closely associated with the molting stage of insects. Thus, they are considered promising targets for the development of novel insecticides such as IGRs. Our previous work identified a dual-target compound 6j, which could act simultaneously on both EcR and OfChtI. In the present study, 6j was first found to have inhibitory activities against OfChtII and OfChi-h, too. Subsequently, taking 6j as a lead compound, 19 novel acetamido derivatives were rationally designed and synthesized by introducing an acetamido moiety into the amide bridge based on the flexibility of the binding cavities of 6j with EcR and three chitinases. Then, their insecticidal activities against Plutella xylostella (P. xylostella), Ostrinia furnacalis (O. furnacalis), and Spodoptera frugiperda (S. frugiperda) were carried out. The bioassay results revealed that most of these acetamido derivatives possessed moderate to good larvicidal activities against three lepidopteran pests. Especially, compound I-17 displayed excellent insecticidal activities against P. xylostella (LC50, 93.32 mg/L), O. furnacalis (LC50, 114.79 mg/L), and S. frugiperda (86.1% mortality at 500 mg/L), significantly better than that of 6j. In addition, further protein validation and molecular docking demonstrated that I-17 could act simultaneously on EcR (17.7% binding activity at 8 mg/L), OfChtI (69.2% inhibitory rate at 50 µM), OfChtII (71.5% inhibitory rate at 50 µM), and OfChi-h (73.9% inhibitory rate at 50 µM), indicating that I-17 is a potential lead candidate for novel multitarget IGRs. This work provides a promising starting point for the development of novel types of IGRs as pest management agents.

In vitro insecticide resistance patterns in field strains of the sheep blowfly, Lucilia cuprina.

The control of the sheep blowfly relies on the use of insecticides. There have been several reports of in vitro and in vivo resistance to the most widely-used flystrike control chemical, dicyclanil. A recent report also described in vitro resistance to imidacloprid in a strain collected from a single property over three consecutive seasons that also showed resistance to dicyclanil. The present study aimed to use in vitro assays to examine five field-collected blowfly strains to determine if this co-occurrence of resistance to dicyclanil and imidacloprid was present more widely in field strains and to also measure resistance patterns to the other currently-used flystrike control chemicals. Each of the strains showed significant levels of resistance to both dicyclanil and imidacloprid: resistance factors at the IC50 of 9.1-23.8 for dicyclanil, and 8.7-14.1 for imidacloprid. Resistance factors at the IC95 ranged from 16.5 to 53.7, and 14.6-24.3 for dicyclanil and imidacloprid, respectively. Resistance factors were up to 8.5 for cyromazine at the IC95. Resistance to dicyclanil and imidacloprid was suppressed by co-treatment with the cytochrome P450 inhibitor, aminobenzotriazole, implicating this enzyme system in the observed resistances. We discuss the implications of the co-occurrence of resistance to dicyclanil and imidacloprid on insecticide rotation strategies for blowfly control. We also discuss the roles of insecticide resistance, environmental factors (e.g. rainfall), operational factors (e.g. insecticide application technique) and other animal health issues (e.g. scouring / diarrhoea) that together will impact on the likelihood of flystrike occurring at an earlier time point than expected after insecticide application.

Endocrine factors modulating vitellogenesis and oogenesis in insects: An update.

The endocrine system plays a pivotal role in shaping the mechanisms that ensure successful reproduction. With over a million known insect species, understanding the endocrine control of reproduction has become increasingly complex. Some of the key players include the classic insect lipid hormones juvenile hormone (JH) and ecdysteroids, and neuropeptides such as insulin-like peptides (ILPs). Individual endocrine factors not only modulate their own target tissue but also play crucial roles in crosstalk among themselves, ensuring successful vitellogenesis and oogenesis. Recent advances in omics, gene silencing, and genome editing approaches have accelerated research, offering both fundamental insights and practical applications for studying in-depth endocrine signaling pathways. This review provides an updated and integrated view of endocrine factors modulating vitellogenesis and oogenesis in insect females.

Chronic Effects of an Insect Growth Regulator (teflubenzuron) on the Life Cycle and Population Growth Rate of Folsomia candida.

Current standard toxicity tests on nontarget soil invertebrates mainly focus on the endpoints survival and reproduction. Such results are likely insufficient to predict effects at higher organizational levels, for example, the population level. We assessed the effects of exposure to the pesticide teflubenzuron on the collembolan Folsomia candida, by performing a full life-cycle experiment exposing single individuals via contaminated food (uncontaminated control and 0.2, 0.32, 0.48, 0.72, 1.08, and 1.6 mg/kg dry yeast). Several life-history traits were considered by following the growth and development of newly hatched individuals over a period of 65 days. We assessed survival, body length, time to first oviposition, cumulative egg production, and hatchability of eggs. A two-stage model was applied to calculate the population growth rate (λ) combined with elasticity analysis to reveal the relative sensitivity of λ to the effects of teflubenzuron on each life-history parameter. Body length was the least sensitive life-history parameter (median effective concentration = 1.10 mg teflubenzuron/kg dry yeast) followed by time to first oviposition (0.96 mg/kg), survival (median lethal concentration = 0.87 mg/kg), cumulative egg production (0.32 mg/kg), and egg hatchability (0.27 mg/kg). Population growth decreased with increasing concentrations of teflubenzuron (λ = 1.162/day in control to 1.005/day in 0.72 mg/kg dry yeast, with populations going extinct at 1.08 and 1.6 mg/kg dry yeast). Elasticity analysis showed that changes in juvenile survival had a greater impact on the population growth rate compared with the other life-history traits. Our study provides a comprehensive overview of individual-level effects of long-term exposure to teflubenzuron and integrates these effects to assess the potential risk to collembolan populations. Environ Toxicol Chem 2024;43:1173-1183. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Sublethal acetamiprid exposure induces immunity, suppresses pathways linked to juvenile hormone synthesis in queens and affects cycle-related signaling in emerging bees.

Acetamiprid is the only neonicotinoid registered in the European Union because the risks of neonicotinoids to honey bees and other pollinators are strictly regulated. Herein, we orally exposed honey bee colonies to sublethal concentrations of acetamiprid (20 µg/L) under isolated conditions. After one month of continuous exposure, the emerging bees and queens were collected and analyzed via high-throughput label-free quantitative proteomics using a data-independent acquisition strategy. Six and 34 significantly differentially expressed proteins (DEPs) were identified in the emerging bees and queens, respectively. Mrjp3 was the only DEP found in both sample types/castes, and its opposite regulation illustrated a differential response. The DEPs in the emerging bees (H/ACA RNP, Rap1GAP, Mrjp3, and JHE) suggested that sublethal exposure to acetamiprid affected cell cycle-related signaling, which may affect the life history of workers in the colony. The DEPs with increased levels in queens, such as Mrjps 1-4 and 6-7, hymenoptaecin, and apidaecin 22, indicated an activated immune response. Additionally, the level of farnesyl pyrophosphate synthase (FPPS), which is essential for the mevalonate pathway and juvenile hormone biosynthesis, was significantly decreased in queens. The impaired utilization of juvenile hormone in queens supported the identification of additional DEPs. Furthermore, the proteome changes suggested the existence of increased neonicotinoid detoxification by UDP-glucuronosyltransferase and increased amino acid metabolism. The results suggest that the continuous exposure of bee colonies to acetamiprid at low doses (nanograms per gram in feed) may pose a threat to the colonies. The different exposure routes and durations for the emerging bees and queens in our experiment must be considered, i.e., the emerging bees were exposed as larvae via feeding royal jelly and beebread provided by workers (nurse bees), whereas the queens were fed royal jelly throughout the experiment. The biological consequences of the proteomic changes resulting from sublethal/chronic exposure require future determination.

Structural Characterization and Functional Analysis of Mevalonate Kinase from Tribolium castaneum (Red Flour Beetle).

Mevalonate kinase (MevK) is an important enzyme in the mevalonate pathway that catalyzes the phosphorylation of mevalonate into phosphomevalonate and is involved in juvenile hormone biosynthesis. Herein, we present a structure model of MevK from the red flour beetle Tribolium castaneum (TcMevK), which adopts a compact α/ß conformation that can be divided into two parts: an N-terminal domain and a C-terminal domain. A narrow, deep cavity accommodating the substrate and cofactor was observed at the junction between the two domains of TcMevK. Computational simulation combined with site-directed mutagenesis and biochemical analyses allowed us to define the binding mode of TcMevK to cofactors and substrates. Moreover, TcMevK showed optimal enzyme activity at pH 8.0 and an optimal temperature of 40 °C for mevalonate as the substrate. The expression profiles and RNA interference of TcMevK indicated its critical role in controlling juvenile hormone biosynthesis, as well as its participation in the production of other terpenoids in T. castaneum. These findings improve our understanding of the structural and biochemical features of insect Mevk and provide a structural basis for the design of MevK inhibitors.

The BTB transcription factor, Abrupt, acts cooperatively with Chronologically inappropriate morphogenesis (Chinmo) to repress metamorphosis and promotes leg regeneration.

Many insects undergo the process of metamorphosis when larval precursor cells begin to differentiate to create the adult body. The larval precursor cells retain stem cell-like properties and contribute to the regenerative ability of larval appendages. Here we demonstrate that two Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (BTB) domain transcription factors, Chronologically inappropriate morphogenesis (Chinmo) and Abrupt (Ab), act cooperatively to repress metamorphosis in the flour beetle, Tribolium castaneum. Knockdown of chinmo led to precocious development of pupal legs and antennae. We show that although topical application of juvenile hormone (JH) prevents the decrease in chinmo expression in the final instar, chinmo and JH act in distinct pathways. Another gene encoding the BTB domain transcription factor, Ab, was also necessary for the suppression of broad (br) expression in T. castaneum in a chinmo RNAi background, and simultaneous knockdown of ab and chinmo led to the precocious onset of metamorphosis. Furthermore, knockdown of ab led to the loss of regenerative potential of larval legs independently of br. In contrast, chinmo knockdown larvae exhibited pupal leg regeneration when a larval leg was ablated. Taken together, our results show that both ab and chinmo are necessary for the maintenance of the larval tissue identity and, apart from its role in repressing br, ab acts as a crucial regulator of larval leg regeneration. Our findings indicate that BTB domain proteins interact in a complex manner to regulate larval and pupal tissue homeostasis.

Germ cell migration: Unexpected role of juvenile hormone before juvenile stages.

Juvenile hormone is best known for its role in maintaining juvenile-stage insects in their immature states during postembryonic development. A new study finds an unexpected role for this signaling lipid in guiding primordial germ cell migration during embryogenesis - possibly an ancestral function of isoprenoid signaling molecules.

Farnesol, a component of plant-derived honeybee-collected resins, shows JH-like effects in Apis mellifera workers.

Farnesol, a sesquiterpene found in all eukaryotes, precursor of juvenile hormone (JH) in insects, is involved in signalling, communication, and antimicrobial defence. Farnesol is a compound of floral volatiles, suggesting its importance in pollination and foraging behaviour. Farnesol is found in the resin of Baccharis dracunculifolia, from which honeybees elaborate the most worldwide marketable propolis. Bees use propolis to seal cracks in the walls, reinforce the wax combs, and as protection against bacteria and fungi. The introduction within a honeybee hive of a compound with potential hormonal activity can be a challenge to the colony survival, mainly because the transition from within-hive to outside activities of workers is controlled by JH. Here, we tested the hypothesis that exogenous farnesol alters the pacing of developing workers. The first assays showed that low doses of the JH precursor (0.1 and 0.01 µg) accelerate pharate-adult development, with high doses being toxic. The second assay was conducted in adult workers and demonstrated bees that received 0.2 µg farnesol showed more agitated behaviour than the control bees. If farnesol was used by corpora allata (CA) cells as a precursor of JH and this hormone was responsible for the observed behavioural alterations, these glands were expected to be larger after the treatment. Our results on CA measurements after 72 h of treatment showed bees that received farnesol had glands doubled in size compared to the control bees (p < 0.05). Additionally, we expected the expression of JH synthesis, JH degradation, and JH-response genes would be upregulated in the treated bees. Our results showed that indeed, the mean transcript levels of these genes were higher in the treated bees (significant for methyl farnesoate epoxidase and juvenile hormone esterase, p < 0.05). These results suggest farnesol is used in honeybees as a precursor of JH, leading to increasing JH titres, and thus modulating the pacing of workers development. This finding has behavioural and ecological implications, since alterations in the dynamics of the physiological changes associated to aging in young honeybees may significantly impact colony balance in nature.

Developmental exposure to hormone-mimicking insect growth disruptors alters expression of endocrine-related genes in worker honey bee (Hymenoptera: Apidae) brains and hypopharyngeal glands.

Division of labor within a honey bee colony creates a codependence between bees performing different tasks. The most obvious example of this is between the reproductive queen and worker bees. Queen bees lay 1,000 or more eggs a day, while young worker bees tend and feed queens. Young workers and queens can be exposed to pesticides when foragers return to the hive with contaminated resources. Previous research has found negative effects of larval exposure to insect-growth disruptors (IGD) methoxyfenozide and pyriproxyfen, on adult responsiveness to artificial queen pheromone. The present work investigates potential physiological and molecular mechanisms underpinning this behavioral change by examining the development of hypopharyngeal glands and ovaries as well as the expression of genes related to reproduction and worker endocrine signaling in the brain and hypopharyngeal gland tissues. Though hypopharyngeal gland and ovary development were not altered by developmental exposure to IGDs, gene expression differed. Specifically, in the brain tissue, ilp1 was downregulated in bees exposed to pyriproxyfen during development, and Kr-h1 was downregulated in both methoxyfenozide- and pyriproxyfen-exposed bees. In the hypopharyngeal glands, Kr-h1, EcR-A, EcR-B, and E75 were upregulated in honey bees exposed to methoxyfenozide compared to those in the pyriproxyfen or control treatments. Here we discuss these results and their potential implications for the health and performance of honey bee colonies.

Juvenile hormones direct primordial germ cell migration to the embryonic gonad.

Germ cells are essential to sexual reproduction. Across the animal kingdom, extracellular signaling isoprenoids, such as retinoic acids (RAs) in vertebrates and juvenile hormones (JHs) in invertebrates, facilitate multiple processes in reproduction. Here we investigated the role of these potent signaling molecules in embryonic germ cell development, using JHs in Drosophila melanogaster as a model system. In contrast to their established endocrine roles during larval and adult germline development, we found that JH signaling acts locally during embryonic development. Using an in vivo biosensor, we observed active JH signaling first within and near primordial germ cells (PGCs) as they migrate to the developing gonad. Through in vivo and in vitro assays, we determined that JHs are both necessary and sufficient for PGC migration. Analysis into the mechanisms of this newly uncovered paracrine JH function revealed that PGC migration was compromised when JHs were decreased or increased, suggesting that specific titers or spatiotemporal JH dynamics are required for robust PGC colonization of the gonad. Compromised PGC migration can impair fertility and cause germ cell tumors in many species, including humans. In mammals, retinoids have many roles in development and reproduction. We found that like JHs in Drosophila, RA was sufficient to impact mouse PGC migration in vitro. Together, our study reveals a previously unanticipated role of isoprenoids as local effectors of pre-gonadal PGC development and suggests a broadly shared mechanism in PGC migration.

E93 gene in the swimming crab, Portunus trituberculatus: Responsiveness to 20-hydroxyecdysone and methyl farnesoate and role on regulating ecdysteroid synthesis.

Ecdysone-induced protein 93 (E93) is a metamorphic determinant involved in crosstalk between 20-hydroxyecdysone (20E) and juvenile hormone (JH) during the insect molting process. The present study identified the E93 gene from the swimming crab, P. trituberculatus, and found it was widely distributed in adult tissues. PtE93 mRNA levels in Y-organ and epidermis fluctuated during the molt cycle, suggesting its involvement in juvenile molting. In vitro and in vivo treatments with 20E led to an induction of PtE93 expression in Y-organ and epidermis, while we found the opposite effect for methyl farnesoate (MF) treatments, a crustacean equivalent of insect JH. We also observed that two genes for ecdysteroid biosynthesis, Spook (Spo) and Shadow (Sad), were suppressed by 20E and induced by MF, showing a negative correlation between PtE93 and ecdysteroid biosynthesis. PtE93 RNA interference (RNAi) induced Spo and Sad expression levels, elevated ecdysteroid content in culture medium, and relieved the 20E inhibitory effect on ecdysteroid synthesis, indicating an inhibitory role of PtE93 on ecdysteroid synthesis. Overall, our results suggest that E93 may be involved in the crosstalk between 20E and MF during crustacean molting, and its presence in Y-organ is closely related to ecdysteroid synthesis.

Efficacy of the insect growth regulator novaluron in the control of dengue vector mosquitoes Aedes aegypti and Ae. albopictus.

Insect Growth Regulator (IGR) novaluron is an alternative to synthetic neuro-inhibitory insecticides. Present study was designed to assess appropriate dosages of novaluron for dengue vector control. Larvae of Aedes aegypti and Ae. albopictus were exposed to a concentration series of novaluron (Rimon EC10) for two fixed exposure periods of 7-days and 14-days to determined LC50 and LC99 values. Inhibition of adult emergence (IE50 and IE99) was determined by a 14-day exposure. Semi-field experiments were conducted by exposing cohorts of Ae. aegypti larvae to IE99, 2 × IE99 and 10 × IE99 novaluron concentrations in water storage buckets (10 L) and plastic barrels (200 L). For the 7-day exposure, LC50 values were 0.047-0.049 ppm and LC99 were 0.144-0.151 ppm. For 14-day exposure, these values were 0.002-0.005 ppm and 0.006-0.01 ppm respectively. For both species, IE99 was 0.001 ppb under semi-field conditions, and was effective for nearly 2 months. Novaluron concentration 0.01 ppb was effective up to 3 months, with an IE of 89-95%. Authorities should critically review a reduction of the presently recommended field dosage of 200 ppm novaluron by × 100 or more. This would provide the same efficacy but mitigate environmental pollution, development of vector resistance, and financial losses.

Differential gene expression and microRNA profile in corpora allata-corpora cardiaca of Aedes aegypti mosquitoes with weak juvenile hormone signalling.

The corpora allata-corpora cardiaca (CA-CC) is an endocrine gland complex that regulates mosquito development and reproduction through the synthesis of juvenile hormone (JH). Epoxidase (Epox) is a key enzyme in the production of JH. We recently utilized CRISPR/Cas9 to establish an epoxidase-deficient (epox-/-) Aedes aegypti line. The CA from epox-/- mutants do not synthesize epoxidated JH III but methyl farneosate (MF), a weak agonist of the JH receptor, and therefore have reduced JH signalling. Illumina sequencing was used to examine the differences in gene expression between the CA-CC from wild type (WT) and epox-/- adult female mosquitoes. From 18,034 identified genes, 317 were significantly differentially expressed. These genes are involved in many biological processes, including the regulation of cell proliferation and apoptosis, energy metabolism, and nutritional uptake. In addition, the same CA-CC samples were also used to examine the microRNA (miRNA) profiles of epox-/- and WT mosquitoes. A total of 197 miRNAs were detected, 24 of which were differentially regulated in epox-/- mutants. miRNA binding sites for these particular miRNAs were identified using an in silico approach; they target a total of 101 differentially expressed genes. Our results suggest that a lack of epoxidase, besides affecting JH synthesis, results in the diminishing of JH signalling that have significant effects on Ae. aegypti CA-CC transcriptome profiles, as well as its miRNA repertoire.