Juvenile Hormone Analog Research Articles

Gene expression response of the non-target gastropod Physella acuta to Fenoxycarb, a juvenile hormone analog pesticide

Pesticides are an environmental problem. The search for new pest control methods has focused on compounds with low or no toxic effects in non-target organisms. Analogs of the juvenile hormone (JH) interfere endocrine system of arthropods. However, the lack of effect on non-target species requires confirmation. This article analyzes the impact of Fenoxycarb, an analog of JH, on Physella acuta, an aquatic gastropod. For 1 week, animals were exposed to 0.01, 1, and 100 μg/L and the RNA was isolated to analyze the gene expression by retrotranscription and Real-Time PCR. Forty genes related to the endocrine system, the DNA repair mechanisms, the detoxification mechanisms, oxidative stress, the stress response, the nervous system, hypoxia, energy metabolism, the immune system, and apoptosis were analyzed. Three of the genes, AchE, HSP17.9, and ApA, showed responses to the presence of Fenoxycarb at 1 μg/L, with no statistically significant responses in the rest of the genes and at the remaining concentrations. From the results, it can be concluded that Fenoxycarb shows a weak response at the molecular level in P. acuta in the tested time and concentrations. However, Aplysianin-A, a gene related to immunity, was altered so the long-term effect could be relevant. Therefore, additional research is required to confirm the safety of Fenoxycarb in non-arthropod species in the long term.

The retinoid metabolism of Gammarus fossarum is disrupted by exogenous all-trans retinoic acid, citral, and methoprene but not by the technical formulation of glyphosate

Over the last decade, fluctuations of retinoids (RETs), also known as vitamin A and derivatives, have proved to be useful biomarkers to assess the environmental chemical pressure on a wide variety of non-target vertebrates. This use of RET-based biomarkers is of particular interest in the non-target sentinel species Gammarus fossarum in which RETs were shown to influence crucial physiological functions. To study and probe this metabolism in this crustacean model, a UHPLC-MS/MS method was developed to 1) identify and 2) monitor several endogenous RETs in unexposed females throughout their reproductive cycle. Then, females were exposed in controlled conditions to exogenous all-trans retinoic acid (atRA) and citral (CIT), a RA synthesis inhibitor, to simulate an excess or deficiency in RA. Perturbation of vitamin A metabolism by pesticides was further studied in response to methoprene (MET), a juvenile hormone analog as well as glyphosate (GLY). The developed method allowed, for the first time in this model, the identification of RA metabolites (all-trans 4-oxo and 13-cis 4-oxo RA), RA isomers (all-trans and 13-cis RA) as well as retinaldehyde (RALD) isomers (all-trans, 11-cis, and 13-cis RALD) and showed two distinct phases in the reproductive cycle. Retinoic acid successfully increased the tissular concentration of both RA isomers and CIT proved to be efficient at perturbating the conversion from RALD to RA. Methoprene perturbed the ratios between RA isomers whereas GLY had no observed effects on the RET system of G. fossarum females. We were able to discriminate different dynamics of RET perturbations by morphogens (atRA or CIT) or MET which highlights the plausible mediation of RETs in MET-induced disorders. Ultimately, our study shows that RETs are influenced by exposure to MET and strengthen their potential to assess aquatic ecosystem chemical status.

Group V Chitin Deacetylases Influence the Structure and Composition of the Midgut of Beet Armyworm, Spodoptera exigua.

Chitin deacetylase (CDA) can accelerate the conversion of chitin to chitosan, influencing the mechanical properties and permeability of the cuticle structures and the peritrophic membrane (PM) in insects. Putative Group V CDAs SeCDA6/7/8/9 (SeCDAs) were identified and characterized from beet armyworm Spodoptera exigua larvae. The cDNAs of SeCDAs contained open reading frames of 1164 bp, 1137 bp, 1158 bp and 1152 bp, respectively. The deduced protein sequences showed that SeCDAs are synthesized as preproteins of 387, 378, 385 and 383 amino acid residues, respectively. It was revealed via spatiotemporal expression analysis that SeCDAs were more abundant in the anterior region of the midgut. The SeCDAs were down-regulated after treatment with 20-hydroxyecdysone (20E). After treatment with a juvenile hormone analog (JHA), the expression of SeCDA6 and SeCDA8 was down-regulated; in contrast, the expression of SeCDA7 and SeCDA9 was up-regulated. After silencing SeCDAV (the conserved sequences of Group V CDAs) via RNA interference (RNAi), the layer of intestinal wall cells in the midgut became more compact and more evenly distributed. The vesicles in the midgut were small and more fragmented or disappeared after SeCDAs were silenced. Additionally, the PM structure was scarce, and the chitin microfilament structure was loose and chaotic. It was indicated in all of the above results that Group V CDAs are essential for the growth and structuring of the intestinal wall cell layer in the midgut of S. exigua. Additionally, the midgut tissue and the PM structure and composition were affected by Group V CDAs.

Methoprene treatment and its effect on male reproductive organ size and female remating in a fruit fly.

Methoprene, a juvenile hormone analog, is used to accelerate sexual maturation in males of species of economic importance in support to the sterile insect technique (SIT). In the SIT, mass-reared sterile males are released into the field and need to survive until they reach sexual maturation, find a wild female, mate with her and then induce female sexual refractoriness, so she will not remate with a wild counterpart. The use of methoprene shortens the time between release and copulation. However, in South American fruit flies, Anastrepha fraterculus, the ability of methoprene-treated males to inhibit female remating has been shown to be lower than wild males, when methoprene was applied by pupal immersion or topical application. Here we evaluated the possibility of incorporating methoprene into the male diet at different doses and the ability of those males to inhibit female remating, as well as the effect of methoprene on male reproductive organ size, due to the possible correlation between male accessory gland size and their content, and the role of male accessory gland proteins in female inhibition. We found that A. fraterculus males fed with methoprene in the adult protein diet at doses as high as 1% were less likely to inhibit female remating, however, at all other lower doses males had the same ability as untreated males to inhibit female remating. Males fed with methoprene had bigger male accessory glands and testes compared to methoprene-deprived males. We demonstrate that the incorporation of methoprene in adult male diets is possible in this species and potentially useful as a post-teneral, pre-release supplement at doses as low as 0.01%. Even at higher doses, the percentage of females remating after 48 h from the first copulation is sufficiently low in this species so as not compromise the efficiency of the SIT.

Autophagy genes AMBRA1 and ATG8 play key roles in midgut remodeling of the yellow fever mosquito, Aedes aegypti.

The function of two autophagy genes, an activating molecule BECN1 regulated autophagy (AMBRA1) and autophagy-related gene 8 (ATG8) in the midgut remodeling of Aedes aegypti was investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of RNA samples collected from the last instar larvae and pupae showed that these two genes are predominantly expressed during the last 12h and first 24h of the last larval and pupal stages, respectively. Stable ecdysteroid analog induced and juvenile hormone (JH) analog suppressed these genes. RNA interference (RNAi) studies showed that the ecdysone-induced transcription factor E93 is required for the expression of these genes. JH-induced transcription factor krüppel homolog 1 (Kr-h1) suppressed the expression of these genes. RNAi-mediated silencing of AMBRA1 and ATG8 blocked midgut remodeling. Histological studies of midguts from insects at 48h after ecdysis to the final larval stage and 12h after ecdysis to the pupal stage showed that ATG gene knockdown blocked midgut remodeling. AMBRA1 and ATG8 double-stranded (dsRNA)-treated insects retained larval midgut cells and died during the pupal stage. Together, these results demonstrate that ecdysteroid induction of ATG genes initiates autophagy programmed cell death during midgut remodeling. JH inhibits midgut remodeling during metamorphosis by interfering with the expression of ATG genes.

Insect Growth Disruptors Cause Mouthpart Malformations, Inhibition of Feeding, and Mortality in the Neotropical Brown Stink Bug Euschistus heros (Hemiptera: Pentatomidae)

The Neotropical brown stink bug, Euschistus heros, is the most important sucking pest of soybean crops, quantitatively and qualitatively affecting grain and seed. This study aimed to determine mouthpart deformities, feeding inhibition, and E. heros nymphs’ mortality when in contact with insect growth disruptors (IGDs). The effects of two insecticides of the chitin biosynthesis inhibitor group (CBIs), lufenuron and diflubenzuron, and the juvenile hormone analog pyriproxyfen were tested in the laboratory. CBI application promoted mouthpart deformities, feeding inhibition, alteration of metamorphosis, and mortality of E. heros nymphs. Although pyriproxyfen did not cause mouthpart deformation, it caused certain mortality for nymphs, possibly due to physiological disorders. Lufenuron caused feed inhibition and mortality in less time when compared with diflubenzuron and pyriproxyfen. It was also more toxic to 4th instar nymphs. The insecticides, when used in higher concentrations, increased nymph’s mortality in less time. Nymphs with deformed mouthparts were unable to feed and died. All IGDs in the tested concentrations caused 100% mortality and prevented adult formation.

Sublethal effects of fenoxycarb on female reproduction in Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract The cotton bollworm, Helicoverpa armigera (Hünber) (Lepidoptera: Noctuidae), is a polyphagous pest that causes severe damage to economical crops in most parts of the world. The juvenile hormone analogues are a group of insect growth regulators that increase larval ages and disrupt larval development. Moreover, they affect reproductive system in adult insects. In this study, the effects of sublethal doses of fenoxycarb on fecundity, fertility, and longevity parameters, as well as on oocyte size, ecdysteroid titer in ovaries, and expression level of the ultraspiracle (USP), ecdysone receptor (EcR), hormone receptor 3 (HR3), and vitellogenin (Vg) genes in adult female H. armigera, were examined. Feeding on artificial diet containing fenoxycarb (LC10 and LC25) significantly decreased the percentage of larval survival rate, pupation rate, pupal weight, fecundity, fertility, and oviposition period. It also clearly shortened female longevity. Fenoxycarb treatment suppressed the expression of the USP, EcR, and Vg genes in H. armigera but had no effect on the HR3 gene expression level in either pupae or adults. Overall, our results show that using fenoxycarb as a juvenile hormone analogue can reduce reproduction in H. armigera and may work as an alternative to the use of insecticides for controlling this important agricultural pest.

Characterization of the RACK1 gene of Aips cerana cerana and its role in adverse environmental stresses.

Receptors for Activated C Kinase 1 (RACK1s) are a kind of multifunction scaffold protein that plays an important role in cell signal transductions and animal development. However, the function of RACK1 in the Chinese honeybee Apis cerana cerana is little known. Here, we isolated and identified a RACK1 gene from Apis cerana cerana, named AccRACK1. By bioinformatic analysis, we revealed a high nucleic acid homology between AccRACK1 and RACK1 of Apis cerana. RT-qPCR analyses demonstrated AccRACK1 was mostly expressed in 3rd instar larvae, darked-eyed pupae and adults (one and thirty days post-emergence), suggesting it might participate in the development of A. cerana cerana. Moreover, the expression of AccRACK1 was highest in the thorax, followed by the venom gland. Compared to the blank control group, AccRACK1 was induced by 24 and 44°C, HgCl2 and pesticides (paraquat, pyridaben and methomyl) but inhibited by 14°C, H2O2, UV light and cyhalothrin. Additionally, 0.05, 0.1, 1, 5 and 10mg/ml PPN (juvenile hormone analogue pyriproxyfen) could promote the expression of AccRACK1, with 1mg/ml showing the highest upregulation, suggesting it was regulated by hormones. Further study found that after knockdown of AccRACK1 by RNAi, the expression of the eukaryotic initiation factor 6 of A. cerana cerana (AcceIF6), an initiation factor regulating the initiation of translation, was inhibited, indicating AccRACK1 might affect cellular responses by translation. These findings, taken together, suggest AccRACK1 is involved in the development and responses to abiotic stresses of A. cerana cerana, and therefore, it may be of critical importance to the survival of A. cerana cerana.

Fenoxycarb exposure affects antiviral immunity and HaNPV infection in the cotton bollworm, Helicoverpa armigera.

Application of insect growth regulators (IGR) is a good option for insect pest management because of their fewer adverse effects on humans and domestic animals. These compounds are capable of interfering with normal growth and development by mimicking the actions of hormones such as juvenile hormone (JH) or ecdysone. The effect of JH and its analogs on some aspects of insect immunity has been determined, yet their possible effects on antiviral immunity response has not been investigated yet. Considering the importance of antiviral response in viral replication, in this study the effects of the JH analog (JHA), fenoxycarb on the antiviral immunity pathway core genes [i.e. micro (mi)RNA, small interfering (si)RNA and apoptosis] of Helicoverpa armigera (Hubner) larvae were investigated. The effect of fenoxycarb on the susceptibility of the larvae to H.armigera nuclear polyhedrosis virus (HaNPV) also was assessed. The results showed that the transcription level of miRNA (Dicer1, Ago1), siRNA (Dicer2, Ago2) and apoptosis (Caspase1, Caspase5) core genes in H.armigera larvae were decreased significantly after 24, 48 and 96 h feeding on a diet containing lethal and sublethal doses of fenoxycarb. Moreover, the mortality rate to HaNPV in the larvae treated with fenoxycarb increased compared to the control, leading to an increased replication of HaNPV. Together, our results suggest that the antiviral immune system could be modulated by JHA and facilitate HaNPV replication in the larvae, increasing the mortality rate of the insect larvae. Understanding the effect of JHA on antiviral immunity is an important step toward the process of exploiting JHAs and viral pathogens to control insect pests. © 2022 Society of Chemical Industry.

The ecdysis triggering hormone system is essential for reproductive success in Mythimna separata (Walker).

Ecdysis triggering hormone (ETH) was originally discovered as a key hormone that regulates insect moulting via binding to its receptor, ETH receptor (ETHR). However, the precise role of ETH in moth reproduction remains to be explored in detail. ETH function was verified in vivo using Mythimna separata (Walker), an important cereal crop pest. RT-qPCR analysis revealed that transcriptional expression profiles of MsepETH showed evident sexual dimorphism in the adult stage. MsepETH expression increased in the females on day 3 and persisted thereafter till day 7, consistent with female ovarian maturation, and was merely detectable in males. Meanwhile, MsepETH expression levels were significantly higher in the trachea than in other tissues. MsepETHR-A and MsepETHR-B were expressed in both sexes and were significantly higher in the antennae than in other tissues. MsepETH and MsepETHR knockdown in females by RNA interference significantly reduced the expression of MsepETH, MsepETHR-A, MsepETHR-B, MsepJHAMT, and MsepVG, which delayed egg-laying and significantly reduced egg production. RNAi 20-hydroxyecdysone (20E) receptor (EcR) decreased MsepETH expression whereas injecting 20E restored egg production that had been disrupted by MsepETH interference. Meanwhile, RNAi juvenile hormone (JH) methoprene tolerant protein (Met) also decreased MsepETH expression and smearing JH analog methoprene (Meth) restored egg production. In conclusion, the reproduction roles of ETH, JH, and 20E were investigated in M. separata. These findings will lay the foundation for future research to develop an antagonist that reduces female reproduction and control strategies for pest insects.

Risk assessment of bisphenol analogues towards mortality, heart rate and stress-mediated gene expression in cladocerans Moina micrura.

Bisphenol A (BPA) is a well-known endocrine-disrupting compound that causes several toxic effects on human and aquatic organisms. The restriction of BPA in several applications has increased the substituted toxic chemicals such as bisphenol F (BPF) and bisphenol S (BPS). A native tropical freshwater cladoceran, Moina micrura, was used as a bioindicator to assess the adverse effects of bisphenol analogues at molecular, organ, individual and population levels. Bisphenol analogues significantly upregulated the expressions of stress-related genes, which are the haemoglobin and glutathione S-transferase genes, but the sex determination genes such as doublesex and juvenile hormone analogue genes were not significantly different. The results show that bisphenol analogues affect the heart rate and mortality rate of M. micrura. The 48-h lethal concentration (LC50) values based on acute toxicity for BPA, BPF and BPS were 611.6µg L-1, 632.0µgL-1 and 819.1µgL-1, respectively. The order of toxicity based on the LC50 and predictive non-effect concentration values were as follows: BPA > BPF > BPS. Furthermore, the incorporated method combining the responses throughout the organisation levels can comprehensively interpret the toxic effects of bisphenol analogues, thus providing further understanding of the toxicity mechanisms. Moreover, the output of this study produces a comprehensive ecotoxicity assessment, which provides insights for the legislators regarding exposure management and mitigation of bisphenol analogues in riverine ecosystems.

Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae.

Feeding by pests is one of the most important reasons for reductions in agricultural crop yield. This study aimed to reveal how juvenile hormone (JH) participates in larval feeding regulation of the Asian corn borer Ostrinia furnacalis. Larvae of O.furnacalis exhibit a daily circadian feeding rhythm, with a peak at ZT18 and a trough at ZT6 under both photoperiod (LD) and constant dark (DD) conditions, which may be eliminated by application of fenoxycarb, a JH active analogue. JH negatively regulates larval feeding as a downstream factor of neuropeptide F (NPF), in which knocking down JH increases larval feeding amount along with body weight and length. The production of JH in the brain-corpora cardiaca-corpora allata (brain-CC-CA) is regulated by brain NPF rather than gut NPF, which was demonstrated in Drosophila larvae through GAL4/UAS genetic analysis. In addition, feeding regulation of JH is closely related to energy homeostasis in the fat body by inhibiting energy storage and promoting degradation. The JH analogue fenoxycarb is an effective pesticide against O.furnacalis, controlling feeding and metabolism. The brain NPF system regulates JH, with functions in food consumption, feeding rhythms, energy homeostasis and body size. This study provides an important basis for understanding the feeding mechanism and potential pest control of O.furnacalis. © 2022 Society of Chemical Industry.

Diallyl trisulfide reduced the reproductive capacity of male Sitotroga cerealella via the regulation of juvenile and ecdysone hormones

Environmental pollution and resistance in animals are major concerns for the application of synthetic pesticides. Diallyl trisulfide (DAT), an active compound in garlic essential oil, is a novel tool for active and safe control of agricultural insect pests. In this study, we analysed the effects of DAT (0.01 μL/L) on the protein content in male reproductive tissues (accessory glands, ejaculatory ducts, and testis), and juvenile hormone (JH) and ecdysone titres in a highly detrimental pest of stored products, Sitotroga cerealella. Evaluation of the expression profile of JH and ecdysone pathway-related genes in various tissues indicated that the accessory gland protein and ecdysone titres were markedly decreased after DAT fumigation, whereas the testis protein content and JH titre were increased. However, the protein content of the ejaculatory ducts remained unchanged between the treated and control groups. Further investigation revealed that DAT disrupted the mRNA expression of key enzymes involved in JH and ecdysone pathways. While increased mRNA levels of juvenile hormone acid O-methyltransferase (JHMAT) and Kruppel homologue 1 (Kr-h1) were observed after 4 and 7 h of DAT fumigation, the levels of juvenile hormone epoxide hydrolase (JHEH) were substantially reduced 3 h post-fumigation. mRNA levels of the ecdysone-responsive gene, FTZF1, and cytochrome P450 enzyme, CYP315A1, were notably decreased at 7 h and 4 h, respectively, post-fumigation, whereas CYP314A1 and CYP302A1 mRNA levels decreased after 3 h and 4 h, respectively. While DAT fumigation disrupted sperm number in the testis, ejaculatory ducts, and seminal vesicles, topical application of the 20-hydroxyecdysone (20E) analogue also lowered sperm number in the ejaculatory ducts. Topical application of methoprene, a JH analogue, increased the protein content in the testes, but not in the accessory glands or ejaculatory ducts. However, the survival rate was not affected by the topical application of methoprene or 20E. These data suggest that DAT regulates JH and ecdysone via its molecular pathway genes and modulates endocrine secretion during the male reproductive process.

CRISPR-Cas9 Genome Editing Uncovers the Mode of Action of Methoprene in the Yellow Fever Mosquito, Aedes aegypti.

Methoprene, a juvenile hormone (JH) analog, is widely used for insect control, but its mode of action is not known. To study methoprene action in the yellow fever mosquito, Aedes aegypti, the E93 (ecdysone-induced transcription factor) was knocked out using the CRISPR-Cas9 system. The E93 mutant pupae retained larval tissues similar to methoprene-treated insects. These insects completed pupal ecdysis and died as pupa. In addition, the expression of transcription factors, broad complex and Krüppel homolog 1 (Kr-h1), increased and that of programmed cell death (PCD) and autophagy genes decreased in E93 mutants. These data suggest that methoprene functions through JH receptor, methoprene-tolerant, and induces the expression of Kr-h1, which suppresses the expression of E93, resulting in a block in PCD and autophagy of larval tissues. Failure in the elimination of larval tissues and the formation of adult structures results in their death. These results answered long-standing questions on the mode of action of methoprene.

The endocrine disruptor, fenoxycarb modulates gut immunity and gut bacteria titer in the cotton bollworm, Helicoverpa armigera

The endocrine system modulates several physiological functions such as development and metamorphosis in insects. The normal growth and development of insects is interfered with insect growth regulators (IGRs), which act as mimics of insect hormones like juvenile hormone (JH) and ecdysone hormone. The effects of JH and its analogs on systemic immunity have been identified. However the effect of these compounds on local gut immunity is largely unknown. In this study, the effects of JH analog fenoxycarb on the local gut immunity of Helicoverpa armigera, gut bacteria population, and their role in the pathogenicity of Bacillus thuringiensis (Bt) were analyzed. The results showed that feeding fenoxycarb causes a decrease in the transcription level of IMD (Relish and PGPR-LC), ROS (DUOX and SOD) related genes and antimicrobial peptides (AMPs), followed by an overpopulation of gut bacteria. The fenoxycarb-treated larvae showed higher susceptibility to Bt compared to the control larvae. Overall, these findings collectively suggest that JH analog affects local gut immunity and gut bacteria titer.

Evaluation on the Activity and Efficacy of Omniprene™ WSP and XWSP Against the Southern House Mosquito Culex quinquefasciatus in Simulated Catch Basins.

Mosquitoes and mosquito-borne diseases remain one of the major public health burdens. In most cases, vector control is the main or the only intervention to mitigate these problems. We are facing the challenges of traditional, emerging, or resurging vectors and diseases, yet the availability and affordability of safe and effective mosquito control products are at a historical low. Development of new active ingredients (AI) and novel formulations based on currently available AI are demanded by mosquito control operations. This paper validated the bioactivity in the laboratory and evaluated the semi-field efficacy for 2 newly registered controlled-release products based on juvenile hormone analog S-methoprene: OmniPrene™ water-soluble pouch (WSP) and extended water-soluble pouch (XWSP). Along with technical S-methoprene, these 2 formulations showed high inhibition of adult emergence in laboratory bioassays against Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus that are commonly found in catch basins. High initial and long-term residual efficacy were demonstrated in simulated catch basins against Cx. quinquefasciatus, where the OmniPrene WSP consistently provided over 90% control for 15 wk (105 days) and OmniPrene XWSP did the same for 38 wk (266 days). Considering the need for reliable mosquito control products, combined with commonly encountered product performance issues in catch basins, OmniPrene WSP and XWSP can be viable tools to combat mosquito species of public health concern that are associated with urban drainage systems.

Identification of species-specific juvenile hormone response elements in the fall armyworm, Spodoptera frugiperda

Juvenile hormones (JH) regulate insect development and reproduction. The JH analogs (JHA) are used as insecticides. However, JHAs are rarely used in managing pests such as the fall armyworm, Spodoptera frugiperda that cause damage during larval stages. The insecticides that antagonize JH action and induce stoppage of feeding and precocious metamorphosis might work better to control these pests. Treating insects with JHA insecticides induces the expression of an early JH response gene, Krüppel homolog 1 (Kr-h1) by working through JH response elements (JHRE) present in its promoter. In this study, we identified JHREs present in the promoter of Kr-h1 gene of a global pest, S. frugiperda, and used them to develop a JHRE-reporter cell platform to screen for JH analogs. JHA, methoprene induced the expression of SfKr-h1 both in vitro and in vivo. JHRE present in the promoters of two SfKr-h1 isoforms, SfKr-h1α and SfKr-h1β were identified. In Sf9 cells, the knockout of isoform-specific JHRE affected JH response in an isoform-specific manner. We also found that S. frugiperda JHRE (SfJHRE) did not function in the mosquito Aedes aegypti Aag2 cells and Tribolium castaneum TcA cells. Similarly, Ae. aegypti AaJHRE and T. castaneum TcJHRE were only functional in cells derived from these insects. The nucleotide sequence at the 3'end to the conserved core JHRE E-box sequence seems to be responsible for the species specificity observed. Two stable cell lines expressing the luciferase and enhanced green fluorescent protein genes under the control of SfJHRE were established. These cell lines responded well to JHA; these two JHRE-reporter cell lines could be used in screening assays to identify insecticides to manage S. frugiperda and other major pests.

Myofilaments promote wing expansion and maintain genitalia morphology in the American cockroach, Periplaneta americana.

Insects are the most widely distributed and successful animals on the planet. A large number of insects are capable of flight with functional wings. Wing expansion is an important process for insects to achieve functional wings after eclosion and healthy genital morphology is crucial for adult reproduction. Myofilaments are functional units that constitute sarcomeres and trigger muscle contraction. Here, we identified four myofilament proteins, including Myosin, Paramyosin, Tropomyosin and Troponin T, from the wing pads of nymphs in the American cockroach, Periplaneta americana. RNAi-mediated knockdown of Myosin, Paramyosin, Tropomyosin and Troponin T in the early stage of final instar nymphs caused a severely curly wing phenotype in the imaginal moult, especially in the Paramyosin and Troponin T knockdown groups, indicating that these myofilament proteins are involved in controlling wing expansion behaviours during the nymph-adult transition. In addition, the knockdown resulted in abnormal external genitalia, caused ovulation failure, and affected male accessory gland development. Interestingly, the expression of myofilament genes was induced by methoprene, a juvenile hormone (JH) analogue, and decreased by the depletion of the JH receptor gene Met. Altogether, we have determined that myofilament genes play an important role in promoting wing expansion and maintaining adult genitalia morphology, and their expression is induced by JH signalling. Our data reveal a novel mechanism by which wing expansion is regulated by myofilaments and the functions of myofilaments are involved in maintaining genitalia morphology.

‘Insecticidal efficacy of a hormonal analogue on the post-embryonic development of a flesh fly, Sarcophaga ruficornis (Diptera: Sarcophagidae)’

ABSTRACT Sarcophaga ruficornis (Fabricius 1794) causes myiasis in humans and other vertebrates. It has a wide distribution across tropical countries. In the present investigation, the effect of a juvenile hormone analogue, fenoxycarb has been observed on the last instar larvae of S. ruficornis. The last instar larvae (0, 1 and 2-days old) of this dipteran fly were treated topically with sub-lethal doses (10, 20, 40, 60 and 80 µg/µl/larva) of fenoxycarb. Several effects were observed such as mortality, abnormal pupariation, reduction pupariation, adult emergence and eclosion failure. Pupal-adult intermediates and the emergence of abnormal adults were also observed. Log Inhibitory concentration (LogIC50) causing inhibition of normal pupariation was 1.68, 1.8 and 1.681 µg/µl in 0, 1 and 2-days of larval treatments respectively. Whereas LogIC50 values inhibiting the adult emergence were 1.297, 1.359 and 1.482 µg/µl in 0, 1, and 2-days of larval treatments respectively. Results manifest the potency of fenoxycarb in the control of larval-adult transformation in S. ruficornis.

Development and characterization of Spodoptera mauritia ovarian primary cell culture and evaluation of fenoxycarb toxicity.

The rice swarming caterpillar, Spodoptera mauritia (Biosduval, 1833) (Lepidoptera: Noctuidae), has been considered a severe pest and has caused the extreme loss of rice paddies in the nursery stages. The massive reproductive efficiency of pests results from balanced endocrine functioning. Insect treatments with exogenous either juvenile hormones (JH) or juvenile hormone analogues (JHAs) during low endogenous JH titers disrupt metamorphosis andovarian development. Hence, it was thought worthwhile to develop a primary ovarian cell culture of Spodoptera mauritia to study the biological efficiency of JHA, fenoxycarb, at the cellular level. The study envisioned the cell characteristics and growth properties and found that most cells were spherical. Spindle-shaped cells were also present during the initial stage of active cell division. The majority of the cells grew attached to the bottom of the culture plates, and a few grew in suspension. The cell doubling time was 144 ± 14h. The growth curve exhibited a sigmoid pattern of cell proliferation at first, followed by a declining phase. These properties indicate that the primary ovarian culture is a suitable candidate for developing a novel cell line of S. mauritia. The preliminary study investigated the cytotoxicity of the insect growth regulator fenoxycarb in ovarian, primary cell culture. Incubated cells showed noticeable morphological changes, including cell shrinkage and apoptotic body formation; inhibition of cellular proliferation; and apoptosis-inducing effects in ovarian, primary cell culture of S. mauritia. Evidently, from these results, it is clear that juvenile hormone mimics can react in a meaningful way to control the rice pest S. mauritia by inducing cellular apoptosis in ovarian cells, which may cause adult females to become sterile.