Juvenile hormone receptor Methoprene tolerant: Functions and applications.

During the past 15years, after confirming Methoprene tolerant (Met) as a juvenile hormone (JH) receptor, tremendous progress has been made in understanding the function of Met in supporting JH signal transduction. Met role in JH regulation of development, including metamorphosis, reproduction, diapause, cast differentiation, behavior, im`munity, sleep and epigenetic modifications, have been elucidated. Met's Heterodimeric partners involved in performing some of these functions were discovered. The availability of JH response elements (JHRE) and JH receptor allowed the development of screening assays in cell lines and yeast. These screening assays facilitated the identification of new chemicals that function as JH agonists and antagonists. These new chemicals and others that will likely be discovered in the near future by using JH receptor and JHRE will lead to highly effective species-specific environmentally friendly insecticides for controlling pests and disease vectors.

Comparative bioactivity of S-methoprene and novel S-methobutene against mosquitoes (Diptera: Culicidae).

Mosquitoes and mosquito-borne illnesses significantly impact public health and human well-being. To address this concern, environmentally compatible larvicides have become a critical component of integrated mosquito management. However, the number of available larvicides is at a historical low. Currently, larvicides that harness microbials and insect growth regulators account for most products. Screening of new active ingredients (AIs) or improvement of existing AIs is thus necessary to augment the capacity for mosquito control. S-methoprene possesses a similar molecular structure and identical function to mosquito juvenile hormone and has been one of the main targets for research and development. The efficacy and safety of S-methoprene have been well documented since the late 1960s, and numerous products have been commercialized to combat pests of economic importance. However, S-methoprene is vulnerable to environmental factors that lead to its degradation, which has created challenges in formulation development, particularly where extended efficacy is desired. A derivative of S-methoprene, namely S-methobutene, with molecular modification has become available. This derivative has demonstrated an enhanced activity of inhibition of emergence (IE) against species across the Aedes, Anopheles, and Culex genera at IE10, IE50, and IE90. Furthermore, S-methobutene consistently outperformed S-methoprene during a 120-day aging process against the southern house mosquito Cx. quinquefasciatus, where the IE% in S-methobutene was significantly higher than that in S-methoprene on most aging intervals. The former had significantly longer residual activity than the latter. The potential of S-methobutene for further development and application is discussed in consideration of its enhanced activity and stability.

Disruption of oogenesis and molting by methoprene and glyphosate in Gammarus fossarum: involvement of retinoic acid?

In the last decade, the freshwater amphipod Gammarus fossarum proved to be a promising sentinel species in active biomonitoring programs to assess the effects of environmental contamination on non-target organisms. Given that the highly conserved retinoid (RETs) metabolism supports many biological functions and is perturbed by xenobiotics and used as biomarker for vertebrates, we explored the RETs functions in the crustacean model Gammarus fossarum. More specifically, we studied the implication of all -trans retinoic acid (atRA) in the reproduction (embryo, oocyte, and juvenile production) and development (success and delay of molting) by exposing G. fossarum females to atRA and citral (CIT), a known inhibitor of RA synthesis. In parallel, we exposed gammarids to methoprene (MET) and glyphosate (GLY), two pesticides suspected to interfere with atRA metabolism and signaling and frequently found in water systems. After 14 days of exposure, atRA, CIT, and MET reduced the number of oocytes, whereas only MET caused a reduced number of embryos. After 44 days, MET and GLY showed a tendency to decrease juvenile production. The duration of the molting cycle increased following the exposures to atRA and MET, while the treatment with CIT caused a typical endocrine disruptive inverted U-shaped curve. The exposure to GLY led to increased duration of the molting cycle at the lowest concentrations and lowered molting success at the highest concentration tested. This study highlights for the first time the implication of RA in the oogenesis and molting of G. fossarum and suggests that it may be a potential mediator of MET-induced effects on these processes. This study adds to the comprehension of the reproductive and developmental control in G. fossarum and opens new research avenues to study the effects of xenobiotics on the RET system in this sentinel species. Ultimately, our study will drive the development of RET-based biomarkers for non-target aquatic invertebrates exposed to xenobiotics.

Non-target effects of methoprene and larvicidal surface films on invertebrate predators of mosquito larvae.

Mosquito larvicides are used across a variety of aquatic habitats, although when applied they likely affect other aquatic organisms. The removal or impairment of top insect predators via larvicides could be beneficial to mosquitoes by allowing their populations to rebound once pesticide levels dissipate. Our goal was to determine if two larvicide types, growth regulators (IGRs) and surface films (SFs), harm non-target aquatic insect communities, and if these chemicals influence the ability of predatory aquatic insects to regulate mosquitoes. We surveyed aquatic sites before and after IGR and SF-application, then compared changes in insect community structure. Evenness was lower in SF treated habitats, and when analyzing prey/controphic taxa only, evenness and diversity changed in untreated reference areas, suggesting that differences measured were due to other environmental factors, not larvicide presence. A field experiment was then conducted by exposing specific predatory aquatic insects to varying doses of IGRs and SFs and then placing them in mesocosms containing mosquito larvae. Surface films were directly lethal to adult dytiscids at recommended and high concentrations. Although we found no significant differences in mosquito emergence among all treatment levels, there was a trend of negative controls (no predator mesocosms) and SF-treated predators allowing the most mosquitoes to emerge compared to positive controls (predators not exposed to larvicides) and IGR-treated predators. Thus, these larvicides may have minimal effects on mosquito larvae predators, but the direct effects of surface films on insects that interact with the water's surface require further investigation.

Methoprene-tolerant and Krüppel homolog 1 are actors of juvenile hormone-signaling controlling the development of male sexual behavior in the moth Agrotis ipsilon.

In insects, juvenile hormone (JH) is critical for the orchestration of male reproductive maturation. For instance, in the male moth, Agrotis ipsilon, the behavioral response and the neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs), to the female-emitted sex pheromone increase with fertility during adulthood and the coordination between these events is governed by JH. However, the molecular basis of JH action in the development of sexual behavior remains largely unknown. Here, we show that the expression of the paralogous JH receptors, Methoprene-tolerant 1 and 2 (Met1, Met2) and of the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) within ALs raised from the third day of adult life and this dynamic is correlated with increased behavioral responsiveness to sex pheromone. Met1-, Met2- and Kr-h1-depleted sexually mature males exhibited altered sex pheromone-guided orientation flight. Moreover, injection of JH-II into young males enhanced the behavioral response to sex pheromone with increased AL Met1, Met2 and Kr-h1 mRNA levels. By contrast, JH deficiency suppressed the behavioral response to sex pheromone coupled with reduced AL Met1, Met2 and Kr-h1 mRNA levels in allatectomized old males and these inhibitions were compensated by an injection of JH-II in operated males. Our results demonstrated that JH acts through Met-Kr-h1 signaling pathway operating in ALs, to promote the pheromone information processing and consequently the display of sexual behavior in synchronization with fertility to optimize male reproductive fitness. Thus, this study provides insights into the molecular mechanisms underlying the hormonal regulation of reproductive behavior in insects.

Ligand-dependent protein interactions of the juvenile hormone receptor captured in real time.

Juvenile hormone (JH) signalling provides vital regulatory functions during insect development via transcriptional regulation of genes critical for the progression of metamorphosis and oogenesis. Despite the importance of JH signalling, the underlying molecular mechanisms remain largely unknown. Our current understanding of the pathway depends on static end-point information and suffers from the lack of time-resolved data. Here, we have addressed the dynamic aspect of JH signalling by monitoring in real time the interactions of insect JH receptor proteins. Use of two tags that reconstitute a functional luciferase when in proximity enabled us to follow the rapid assembly of a JH receptor heterodimer from basic helix-loop-helix/Per-Arnt-SIM (bHLH-PAS) proteins, methoprene-tolerant (Met) and taiman (Tai), upon specific JH binding to Met. On a similar timescale (minutes), the dissociation of Met-Met complexes occurred, again strictly dependent on Met interaction with specific agonist ligands. To resolve questions regarding the regulatory role of the chaperone Hsp90/83 in the JHR complex formation, we used the same technique to demonstrate that the Met-Hsp83 complex persisted in the agonist absence but readily dissociated upon specific binding of JH to Met. Preincubation with the Hsp90 inhibitor geldanamycin showed that the chaperone interaction protected Met from degradation and was critical for Met to produce the active signalling dimer with Tai. Thus, the JH receptor functions appear to be governed by principles similar to those regulating the aryl hydrocarbon receptor, the closest vertebrate homologue of the arthropod JH receptor.

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.

MicroRNA miR-2765-3p regulates reproductive diapause by targeting FoxO in Galeruca daurica.

The forkhead box O (FoxO), as a conserved transcription factor, plays an indispensable role in regulating insect diapause. However, how FoxO is regulated to control diapause in insects remains unknown. In this study, we discovered functional binding sites for miR-2765-3p in the 3' untranslated region of FoxO in Galeruca daurica. The luciferase reporter assay showed that miR-2765-3p targeted FoxO and suppressed its expression. The expression profiles of miR-2765-3p and FoxO displayed opposite patterns during the female developmental process. Overexpression of miR-2765-3p by the injection of the miR-2765-3p agomir into adult females reduced FoxO expression, leading to the suppression of lipid accumulation, promotion of ovarian development, and inhibition of reproductive diapause. This is similar to the phenotype that results from the depletion of FoxO by injecting dsFoxO into adult females. In addition, the repression of miR-2765-3p by injecting the miR-2765-3p antagomir increased the FoxO transcript level, leading to the stimulation of lipid accumulation, depression of ovarian development, and induction of reproductive diapause. A hormone injection assay showed that the juvenile hormone (JH) agonist (methoprene) upregulated miR-2765-3p and downregulated FoxO. Notably, injecting methoprene rescued ovarian development defects associated with miR-2765-3p inhibition. These findings indicate that the JH/miR-2765-3p/FoxO axis plays a vital role in the regulation of reproductive diapause in G. daurica.

Involvement of Methoprene-tolerant and Krüppel homolog 1 in juvenile hormone-mediated vitellogenesis of female Liposcelis entomophila (End.) (Psocoptera: Liposcelididae).

Methoprene-tolerant (Met) as an intracellular receptor of juvenile hormone (JH) and the Krüppel-homolog 1 (Kr-h1) as a JH-inducible transcription factor had been proved to contribute to insect reproduction. Their functions vary in different insect orders, however, they are not clear in Psocoptera. In this study, LeMet and LeKr-h1 were identified and their roles in vitellogenesis and ovarian development were investigated in Liposcelis entomophila (Enderlein). Treatment with exogenous JH III significantly induced the expression of LeKr-h1, LeVg, and LeVgR. Furthermore, silencing LeMet and LeKr-h1 remarkably reduced the transcription of LeVg and LeVgR, disrupted the production of Vg in fat body and the uptake of Vg by oocytes, and ultimately led to a decline in fecundity. The results indicated that the JH signaling pathway was essential to the reproductive process of this species. Interestingly, knockdown of LeMet or LeKr-h1 also resulted in fluctuations in the expression of FoxO, indicating the complex regulatory interactions between different hormone factors. Besides, knockdown of both LeMet and LeKr-h1 significantly increased L. entomophila mortality. Our study provides initial insight into the roles of JH signaling in the female reproduction of psocids and provided evidence that RNAi-mediated knockdown of Met or Kr-h1 is a potential pest control strategy.

Juvenile hormone membrane signaling phosphorylates USP and thus potentiates 20-hydroxyecdysone action in Drosophila.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant (Met) and germ-cell expressed (Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C (PKC) which phosphorylated ultraspiracle (USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. The uspS35A mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20E signaling that delayed developmental timing. The uspS35A mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.

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-h1ß 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.

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 impact of insect growth regulators on adult emergence inhibition and the fitness of Aedes aegypti field populations in Thailand.

BACKGROUND: The frequent use of insecticides in vector control causes the development of insecticide resistance. Insect growth regulators (IGRs), which effect insect development, are used as a promising alternative to control resistant insect vectors. This study aimed to develop novel effective tools for Aedes aegypti control by evaluating the efficacy of different IGRs on larval development, blood feeding capacity, fecundity, and fertility in females and sperm productivity in males across geographical regions of Thailand. METHODS: The efficacy of 16 technical grade IGRs were evaluated against laboratory strain Ae. aegypti larvae in order to determine their emergence inhibition (EI) at 50% and 95% under laboratory conditions. Six IGRs were selected for fecundity, fertility, and sperm productivity studies using feed-through treatments at EI95 concentration levels against adult Ae. aegypti field strains. RESULTS: The results from larval bioassay tests indicate that juvenile hormone mimics (EI50 = 0.010-0.229 ppb; EI95 = 0.066-1.118 ppb) and chitin synthesis inhibitors affecting CHS1 (EI50 = 0.240-2.412 ppb; EI95 = 0.444-4.040 ppb) groups effectively inhibited adult Ae. aegypti emergence. Methoprene and fenoxycarb significantly reduced blood feeding capacity. Egg production was comparable among strains while methoprene, pyriproxyfen and diflubenzuron induced egg production. Egg retention was detected in females fed on diflubenzuron. Methoprene, fenoxycarb, diflubenzuron, and teflubenzuron reduced egg hatching rates in mosquito field strains compared to laboratory strain. Male mosquitoes fed on fenoxycarb showed significantly lower sperm production compared to other treatments. CONCLUSION: Juvenile hormone analogues and chitin synthesis inhibitors affecting CHS1 groups showed excellent results in adult emergence inhibition in this study. They also disrupted reproductive systems in both adult males and females. This study suggested that they can be used as an alternative larvicide in mosquito control programs.

Helicoverpa armigera miR-2055 regulates lipid metabolism via fatty acid synthase expression.

Insect hormones and microRNAs regulate lipid metabolism, but the mechanisms are not fully elucidated. Here, we found that cotton bollworm larvae feeding on Arabidopsis thaliana (AT) leaves had a lower triacylglycerol (TAG) level and more delayed development than individuals feeding on artificial diet (AD). Association analysis of small RNA and mRNA revealed that the level of miR-2055, a microRNA related to lipid metabolism, was significantly higher in larvae feeding on AT. Dual-luciferase reporter assays demonstrated miR-2055 binding to 3' UTR of fatty acid synthase (FAS) mRNA to suppress its expression. Elevating the level of miR-2055 in larvae by agomir injection decreased FAS mRNA and protein levels, which resulted in reduction of free fatty acid (FFA) and TAG in fat body. Interestingly, in vitro assays illustrated that juvenile hormone (JH) increased miR-2055 accumulation in a dosage-dependent manner, whereas knockdown of Methoprene tolerant (Met) or Kruppel homologue 1 (Kr-h1) decreased the miR-2055 level. This implied that JH induces the expression of miR-2055 via a Met-Kr-h1 signal. These findings demonstrate that JH and miRNA cooperate to modulate lipid synthesis, which provides new insights into the regulatory mechanisms of metabolism in insects.

Molecular Identification and Functional Characterization of Methoprene-Tolerant (Met) and Krüppel-Homolog 1 (Kr-h1) in Harmonia axyridis (Coleoptera: Coccinellidae).

Juvenile hormone (JH) plays a key role in regulating insect reproductive processes. Methoprene-tolerant (Met), as a putative JH receptor, transduces JH signals by activating the transcription factor krüppel homolog 1 (Kr-h1). To understand the effects of Met and Kr-h1 genes on female reproduction of natural enemy insects, the Met and Kr-h1 were identified and analyzed from Harmonia axyridis Pallas (HmMet and HmKr-h1). The HmMet protein belonged to the bHLH-PAS family with bHLH domain, PAS domains, and PAC domain. HmMet mRNA was detected in all developmental stages, and the highest expression was found in the ovaries of female adults. The HmKr-h1 protein had eight C2H2-type zinc finger domains. HmKr-h1 mRNA was highly expressed from day 7 to day 9 of female adults. The tissue expression showed that HmKr-h1 was highly expressed in its wing, leg, and fat body. Knockdown of HmMet and HmKr-h1 substantially reduced the transcription of HmVg1 and HmVg2, inhibited yolk protein deposition, and reduced fecundity using RNA interference. In addition, the preoviposition period was significantly prolonged after dsMet-injection, but there was no significant difference after dsKr-h1-silencing. However, the effect on hatchability results was the opposite. Therefore, we infer that both HmMet and HmKr-h1 are involved in female reproduction of H. axyridis, and their specific functions are different in certain physiological processes. In several continents, H. axyridis are not only beneficial insects, but also invasive pests. This report will provide basis for applying or controlling the H. axyridis.

MicroRNA let-7-5p targets the juvenile hormone primary response gene Krüppel homolog 1 and regulates reproductive diapause in Galeruca daurica.

MicroRNAs (miRNAs) regulate various biological processes in insects. However, their roles in the regulation of insect diapause remain unknown. In this study, we address the biological function of a conserved miRNA, let-7-5p in the regulation of a juvenile hormone primary response gene, Krüppel homolog 1 (Kr-h1), which modulates reproductive diapause in Galeruca daurica. The dual luciferase reporter assay showed that let-7-5p depressed the expression of Kr-h1. The expression profiles of let-7-5p and Kr-h1 displayed opposite patterns in the adult developmental stage. Injection of let-7-5p agomir in pre-diapause adult females inhibited the expression of Kr-h1, which consequently led to delay ovarian development, increase lipid accumulation, expand fat body, and induce reproductive diapause just as depleting Kr-h1 did. Conversely, injection of let-7-5p antagomir resulted in opposite effects by reducing fat storage and stimulating reproduction. Moreover, JH receptor agonist methoprene reduced the expression of let-7-5p, and rescued the ovarian development defects associated with let-7-5p overexpression. These results indicate that let-7-5p plays an important role in the regulation of reproductive diapause and development of G. daurica adults through its target gene Kr-h1.

Statewide Efficacy Assessment of Insect Growth Regulators Against Aedes albopictus (Diptera: Culicidae) in Sabah, Malaysia: An Alternative Control Strategy?

The efficacy of three groups of insect growth regulators, namely juvenile hormone mimics (methoprene and pyriproxyfen), chitin synthesis inhibitors (diflubenzuron and novaluron), and molting disruptor (cyromazine) was evaluated for the first time, against Aedes albopictus Skuse (Diptera: Culicidae) larvae from 14 districts in Sabah, Malaysia. The results showed that all field populations of Ae. albopictus were susceptible towards methoprene, pyriproxyfen, diflubenzuron, novaluron, and cyromazine, with resistance ratio values ranging from 0.50-0.90, 0.60-1.00, 0.67-1.17, 0.71-1.29, and 0.74-1.07, respectively. Overall, the efficacy assessment of insect growth regulators in this study showed promising outcomes and they could be further explored as an alternative to conventional insecticides.

Detection of juvenile hormone agonists by a new reporter gene assay using yeast expressing Drosophila methoprene-tolerant.

Juvenile hormones (JHs) are sesquiterpenoids that play important roles in the regulation of growth, metamorphosis, and reproduction in insects. Synthetic JH agonists (JHAs) have been used as insecticides and are categorized as a class of insect growth regulators (IGRs). Natural JHs and synthetic JHAs bind to the JH receptor methoprene-tolerant (Met), which forms a functional JH-receptor complex with steroid receptor coactivators, such as Drosophila melanogaster Taiman (Tai). The ligand-bound Met-Tai complex induces the transcription of JH response genes by binding to specific DNA elements referred to as JH response elements (JHREs). In the present study, we established a reporter gene assay (RGA) for detecting natural JHs and synthetic JHAs in a yeast strain expressing D. melanogaster Met and Tai. The yeast RGA system detected various juvenoid ligands in a dose-dependent manner. The rank order of the ligand potencies of the juvenoids examined in the yeast RGA linearly correlated with those of RGAs for Met-Tai established in mammalian and insect cells. Our new yeast RGA is rapid, easy to handle, cost-effective, and valuable for screening novel JHAs.

Behavioral flexibility in Wasmannia auropunctata (Hymenoptera: Formicidae).

Worker division of labor is a defining trait in social insects. Many species are characterized by having behavioral flexibility where workers perform non-typical tasks for their age depending on the colony's needs. Worker division of labor and behavioral flexibility were examined in the little fire ant Wasmannia auropunctata (Roger, 1863), for which age-related division of labor has been found. Young workers perform nursing duties which include tending of brood and queens, and colony defense, while older workers forage. When nurses were experimentally removed from the colony, foragers were observed carrying out nursing and colony defense duties, yet when foragers were removed nurses did not forage precociously. We also administered juvenile hormone analog, methoprene, to workers. When methoprene was applied, foragers increased their nursing and defense activities while nurses became mainly idle. The behavioral flexibility of foragers of the little fire ant may be evidence of an expansion of worker's repertoires as they age; older workers can perform tasks they have already done in their life while young individuals are not capable of performing tasks ahead of time. This may be an important adaptation associated with the success of this ant as an invasive species.

Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica.

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.