Regulation of soldier caste differentiation by microRNAs in Formosan subterranean termite (Coptotermes formosanus Shiraki).

The soldier caste is one of the most distinguished castes inside the termite colony. The mechanism of soldier caste differentiation has mainly been studied at the transcriptional level, but the function of microRNAs (miRNAs) in soldier caste differentiation is seldom studied. In this study, the workers of Coptotermes formosanus Shiraki were treated with methoprene, a juvenile hormone analog which can induce workers to transform into soldiers. The miRNomes of the methoprene-treated workers and the controls were sequenced. Then, the differentially expressed miRNAs (DEmiRs) were corrected with the differentially expressed genes DEGs to construct the DEmiR-DEG regulatory network. Afterwards, the DEmiR-regulated DEGs were subjected to GO enrichment and KEGG enrichment analysis. A total of 1,324 miRNAs were identified, among which 116 miRNAs were screened as DEmiRs between the methoprene-treated group and the control group. A total of 4,433 DEmiR-DEG pairs were obtained. No GO term was recognized as significant in the cellular component, molecular function, or biological process categories. The KEGG enrichment analysis of the DEmiR-regulated DEGs showed that the ribosome biogenesis in eukaryotes and circadian rhythm-fly pathways were enriched. This study demonstrates that DEmiRs and DEGs form a complex network regulating soldier caste differentiation in termites.

Interactive Effects of Salinity and Mosquito Larvicides Toxicity to Larvae of Aedes Taeniorhynchus.

Understanding the influence of salinity on the efficacy of mosquito larvicides in brackish water habitats is crucial for effective salt-marsh Aedes taeniorhynchus control. This study investigated the interactive effects of salinity on the toxicity of 3 commonly used mosquito larvicides: Bacillus thuringiensis israelensis (VectoBac® 12AS), spinosad (Natular® SC), and S-methoprene (Altosid® 12AS) against Ae. taeniorhynchus larvae. Four salinity levels (0 ppt [parts per thousand], 8 ppt, 16 ppt, and 32 ppt) were tested in laboratory bioassays. The results revealed distinct responses of these larvicides to varying salinity levels. VectoBac 12AS displayed consistent efficacy across all salinity levels, indicating its suitability for brackish water habitats. In contrast, Natular 2EC exhibited increased effectiveness with higher salinity, making it a preferable choice for saline environments. Altosid 12AS showed its highest efficacy in freshwater, with reduced effectiveness as salinity increased. These findings underscore the need to consider salinity levels when selecting and applying mosquito larvicides in diverse aquatic habitats. Understanding the complex interplay between salinity and larvicide performance is essential for optimizing mosquito control strategies and mitigating mosquito-borne diseases in various environments.

Accelerating sexual maturation of male Anastrepha ludens (Diptera: Tephritidae) fruit flies by adding two juvenile hormone analogues.

BACKGROUND: Improving the mating competitiveness and survival of sterile males are direct means to increase the effectiveness of the sterile insect technique (SIT). Some insecticide growth regulators, such as the juvenile hormone analogue (JHA) methoprene, have been used to improve the mating competitiveness of male tephritid flies by reducing their sexual maturation period. However, the application of methoprene reduces fly resistance to stress and decreases survival. Here, we compared the effects of methoprene and pyriproxyfen (PPF), another JHA, in Anastrepha ludens males. PPF is an insect growth regulator that exhibits higher negative effects on the larval molting process than methoprene or natural juvenile hormone. Both compounds were administered at two doses (0.05% and 0.10%) via the male diet immediately after emergence. RESULTS: Our results show that both PPF and methoprene reduced male sexual maturation. However, PPF-treated males exhibited a shorter maturation period and obtained more matings at a given age than methoprene-treated males. No significant differences were observed between the two PPF doses tested (0.05% and 0.10%). Male survival was equally reduced by the two compounds. CONCLUSION: Our results demonstrate that PPF accelerated sexual development without reducing the mating propensity of sterile male flies and can be used as a suitable alternative for methoprene. © 2023 Society of Chemical Industry.

Comparative Activity And Efficacy of Sumilarv 0.5G and Altosid Xr Briquet Against Culex Quinquefasciatus and Aedes Aegypti in Simulated Catch Basins.

Mosquito control plays a crucial role in the mitigation of mosquito-borne diseases. Larviciding that targets one of the aquatic stages is among the routine practices in mosquito control operations. One of the most extensive and challenging mosquito production sources in urban environments is underground storm drain systems. Along with the research and development of biorational larvicides in recent decades, numerous products based on microbial and insect growth regulators have become available. However, the performance of these products often varies because of product design and challenges associated with urban storm drain systems. This paper validates the comparative bioactivity and semifield efficacy of 2 control release products based on pyriproxyfen and S-methoprene. In laboratory bioassays, pyriproxyfen was significantly more active than S-methoprene against the test species, Culex quinquefasciatus Say and Aedes aegypti (L.). Culex quinquefasciatus was less susceptible than Ae. aegypti to both test materials. During a 26-wk-long semifield evaluation using the cast concrete simulated catch basins, the inhibition of emergence pretreatment and posttreatment in untreated control was negligible. The Sumilarv 0.5G applied at 75 g per catch basin provided 100% IE, whereas the Altosid XR briquet applied at 1 per catch basin yielded only partial control fluctuating from 12.7% to 82.7% (average 40.7%) of Cx. quinquefasciatus and 8.0% to 78.8% (average 37.4%) of Ae. aegypti. The Altosid XR briquet had an average residual weight of 59.9% at the end of semifield evaluation. Results are discussed in relation to field mosquito control operations in urban storm drain systems.

Investigating the role of the ROS/CncC signaling pathway in the response to xenobiotics in Spodoptera frugiperda using Sf9 cells.

Spodoptera frugiperda (fall armyworm, FAW) is an invasive polyphagous lepidopteran pest that has developed sophisticated resistance mechanisms involving detoxification enzymes to eliminate toxic compounds it encounters in its diet including insecticides. Although its inventory of detoxification enzymes is known, the mechanisms that enable an adapted response depending on the toxic compound remain largely unexplored. Sf9 cells were used to investigate the role of the transcription factors, Cap n' collar isoform C (CncC) and musculoaponeurotic fibrosarcoma (Maf) in the regulation of the detoxification response. We overexpressed CncC, Maf or both genes, and knocked out (KO) CncC or its repressor Kelch-like ECH associated protein 1 (Keap1). Joint overexpression of CncC and Maf is required to confer increased tolerance to indole 3-carbinol (I3C), a plant secondary metabolite, and to methoprene, an insecticide. Both molecules induce reactive oxygen species (ROS) pulses in the different cell lines. The use of an antioxidant reversed ROS pulses and restored the tolerance to I3C and methoprene. The activity of detoxification enzymes varied according to the cell line. Suppression of Keap1 significantly increased the activity of cytochrome P450s, carboxylesterases and glutathione S-transferases. RNAseq experiments showed that CncC mainly regulates the expression of detoxification genes but is also at the crossroads of several signaling pathways (reproduction and immunity) maintaining homeostasis. We present new data in Sf9 cell lines suggesting that the CncC:Maf pathway plays a central role in FAW response to natural and synthetic xenobiotics. This knowledge helps to better understand detoxification gene expression and may help to design next-generation pest insect control measures.

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.

Efficacy of stored grain insecticides against 2 pest beetles in rough rice.

Insecticidal grain protectants can provide control of pest insects during grain storage, but studies which directly compare the efficacy of insecticides in rough rice are few. We examined methoprene, deltamethrin, commercial formulations of methoprene + deltamethrin, ß-cyfluthrin, and diatomaceous earth applied to rough rice over a 6-month period for control of lesser grain borers, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), and rice weevils, Sitophilus oryzae (L.) (Coleoptera: Curculionidae). We found that methoprene, deltamethrin, or a combination of the 2 were the most effective controls of both beetles in 22.7-liter metal containers. Damage mass and beetle mass in methoprene-treated rice were reduced by 38% and 55%, respectively, when compared to the nontreated control. Similarly, the mixture of deltamethrin and methoprene reduced damage mass by 32% and total beetle mass by 45% below that of the nontreated control. Deltamethrin reduced total beetle mass by 38% relative to the nontreated rice. In vial assays, only the combination of deltamethrin and methoprene provided control of R. dominica. Diatomaceous earth had the greatest impact upon S. oryzae, but it did not affect R. dominica. Based on these results, a commercial formulation of deltamethrin and methoprene is likely to provide the best overall control of stored grain beetles, though diatomaceous earth may be effective when use of chemical insecticides is undesirable or when S. oryzae is the sole pest present.

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.

Krüppel-homologue 1 regulates the development of Tuta absoluta and its cascade regulation pattern in the juvenile hormone signalling pathway.

Tomato leaf miner, Tuta absoluta (Meyrick), is one of the most destructive quarantine pests globally. It has been confirmed that Krüppel-homologue 1 (kr-h1) plays a key role in the regulation of juvenile hormone (JH). However, it is unclear how kr-h1 regulates the synthesis of JH and its cascade regulation pattern in tomato leaf miner. Here, we obtained the six JH signalling genes (kr-h1, Methoprene-tolerant, Forkhead box O, Juvenile acid methyltransferase, Juvenile hormone esterase and Fatty acid synthase 2), and applied RNA interference to explore the role of kr-h1 and the seven genes (plus Vitellogenin) regulation relationship in T. absoluta. Bioinformatics analysis revealed the structural characteristics of kr-h1 protein and JH receptor Met, which contained eight C2H2 zinc finger structures and three typical domains of the bHLH-PAS family, respectively. The expression levels of Met and Vg were upregulated after RNAi of kr-h1 gene, while the gene levels of JHAMT and FAS2 were downregulated. Furthermore, topical application of JH analogue to second instar larvae could induce the expression of kr-h1 and inhibit the expression of Met. Our study reveals the mechanism by which kr-h1 regulates JH pathway genes, which could be applied to control the growth of tomato leaf miners.

Delivery of Methoprene-Tolerant dsRNA to Improve RNAi Efficiency by Modified Liposomes for Pest Control.

The application of RNA interference (RNAi) technology for pest control is environmentally friendly and accurate. However, the efficiency of RNAi is often inconsistent and unreliable, and finding a suitable carrier element is considered critical to success in overcoming biotic and abiotic barriers to reach the target site. The fall armyworm, Spodoptera frugiperda (FAW), which is one of most important global agricultural pests, has recently spread rapidly to other parts of the world. In this study, a method to improve the stability and RNAi efficiency of the dsRNA carrier complex was reported. Methoprene-tolerant gene (Met) was selected as a target, a gene which is critical to the growth and development of FAW. Biomaterials nanoliposomes (LNPs) were modified with polyethylenimine (PEI) to deliver the dsRNA of Met. The synthesized Met3@PEI@LNPs reached a size of 385 nm and were found to load dsRNA effectively. Through stability and protection assays, it was found that LNPs provided reliable protection. In addition, the release curve also demonstrated that LNPs were able to prevent premature release under alkaline condition of the insect midgut but accelerate the release after entering the acidic environment of the target cells. The cell transfection efficiency of the prepared LNPs reached 96.4%. Toxicity tests showed that the use of LNPs could significantly improve the interference efficiency, with 91.7% interference efficiency achieved when the concentration of dsRNA in LNPs was only 25% of that of the control. Successful interference of Met demonstrated it could significantly shorten the larval period and make the larvae pupate earlier, thus achieving the purpose of control. In this study, we have demonstrated the use of nanotechnology to provide a novel RNAi delivery method for pest control.

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.

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.

Residual efficacy of selected larvicides against Culex pipiens pipiens (Diptera: Culicidae) under laboratory and semi-field conditions.

Mosquitoes are a threat worldwide since they are vectors of important pathogens and parasites such as malaria, dengue, yellow fever, and West Nile. The residual toxicity of several commercial mosquito larvicides was evaluated for the control of Culex pipiens pipiens under controlled laboratory and semi-field conditions during late spring and summer of 2013. The evaluation included six different active ingredient formulations, i.e., diflubenzuron Du-Dim), Bacillus thuringiensis var. israelensis (Bti) (Vectobac), spinosad (Mozkill), S-methoprene (Biopren), temephos (Abate), and polydimethylsiloxane (PDMS) (Aquatain), that are currently registered of and had been registered in the past for mosquito control. Under controlled laboratory conditions, the residual activity ranged from 1 week (S-methoprene) up to 2 months (spinosad, PDMS). Exposure of larvicides under semi-field conditions resulted in noticeable differences regarding their efficacy as compared to the laboratory bioassays. Exposure of S-methoprene, Bti, and spinosad, for up to 3 days, resulted in similar adult emergence to the controls. On the other hand, the residual efficacy of diflubenzuron, temephos, and PDMS ranged from 14 to 28 days, depending on the season of exposure. Longevity and fecundity of adults that had emerged from surviving larvae, in most of the cases tested, did not differ significantly from that of the controls. The results of the present study demonstrate the necessity of both field and laboratory studies to draw safe conclusions regarding the efficacy of larvicides against mosquitoes and the selection of the proper formulation for each application scenario. In addition, defining the seasonal variation in the residual toxicity of the tested formulations could be useful for improving mosquitos' management programs.

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.

Effect of juvenile hormone on phenoloxidase and hemocyte number: The role of age, sex, and immune challenge.

Hormones are key factors in determining the response of organisms to their environment. For example, the juvenile hormone (JH) coordinates the insects' development, reproduction, and survival. However, it is still unclear how the impact of juvenile hormone on insect immunity varies depending on the sex and reproductive state of the individual, as well as the type of the immune challenge (i.e., Gram-positive or Gram-negative bacteria). We used Tenebrio molitor and methoprene, a JH analog (JHa) to explore these relationships. We tested the effect of methoprene on phenoloxidase activity (PO), an important component of humoral immunity in insects, and hemocyte number. Lyophilized Gram-positive Staphylococcus aureus or Gram-negative Escherichia coli were injected for the immune challenge. The results suggest that JH did not affect the proPO, PO activity, or hemocyte number of larvae. JH and immune challenge affected the immune response and consequently, affected adult developmental stage and sex. We propose that the influence of JH on the immune response depends on age, sex, the immune response parameter, and the immune challenge, which may explain the contrasting results about the role of JH in the insect immune response.

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.

Juvenile hormone acts on male accessory gland function via regulating l-asparaginase expression and triacylglycerol mobilization in Aedes aegypti.

Hormones control the reproductive development of Aedes aegypti mosquitoes. The adult male reproductive process and mating behavior require adequate nutrients and energy. Understanding the molecular mechanism linking hormones, energy metabolism, and reproduction in male mosquitoes is important. In this study, we found that the size of the male accessory gland, an essential part of the male reproductive system, gradually increased after eclosion. However, it was significantly reduced in male mosquitoes deficient in methoprene-tolerant (Met), the receptor of juvenile hormone. Likewise, egg hatchability of females that mated with Met-depleted males showed the same downward trend. The mRNA level of the gene encoding accessory gland protein, l-asparaginase (ASNase), was reduced in Met dsRNA-treated males. Electrophoretic mobility shift assay and quantitative reverse transcription-PCR results revealed that Met was capable of binding directly to the promoter of ASNase and activated its transcription. RNA interference of ASNase in males resulted in the reduction of egg hatchability of the females with which they mated. These results showed that Met influenced the fecundity of male mosquitoes by directly upregulating the expression of the ASNase gene. Moreover, the levels of triacylglycerol and the sizes of lipid droplets were decreased by 72-78 h after eclosion in the fat body cells, whereas both of them increased in Met-depleted male mosquitoes, indicating that Met knockdown reduced lipid catabolism. These data demonstrate that Met might influence the egg hatchability of females by regulating lipid metabolism and the development of the male accessory gland in male mosquitoes.

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.