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Juvenile hormone (JH) represses precocious metamorphosis of larval to pupal and adult transitions in holometabolous insects. The early JH-inducible gene Krüppel homolog 1 (Kr-h1) plays a key role in the repression of metamorphosis as a mediator of JH action. Previous studies demonstrated that Kr-h1 inhibits precocious larval–pupal transition in immature larva via direct transcriptional repression of the pupal specifier Broad-Complex (BR-C). JH was recently reported to repress the adult specifier gene Ecdysone-induced protein 93F (E93); however, its mechanism of action remains unclear. Here, we found that JH suppressed ecdysone-inducible E93 expression in the epidermis of the silkworm Bombyx mori and in a B. mori cell line. Reporter assays in the cell line revealed that the JH-dependent suppression was mediated by Kr-h1. Genome-wide ChIP-seq analysis identified a consensus Kr-h1 binding site (KBS, 14 bp) located in the E93 promoter region, and EMSA confirmed that Kr-h1 directly binds to the KBS. Moreover, we identified a C-terminal conserved domain in Kr-h1 essential for the transcriptional repression of E93. Based on these results, we propose a mechanism in which JH-inducible Kr-h1 directly binds to the KBS site upstream of the E93 locus to repress its transcription in a cell-autonomous manner, thereby preventing larva from bypassing the pupal stage and progressing to precocious adult development. These findings help to elucidate the molecular mechanisms regulating the metamorphic genetic network, including the functional significance of Kr-h1, BR-C, and E93 in holometabolous insect metamorphosis.

S-methoprene, an insect growth regulator, and Lysinibacillus sphaericus ( Ls ), an entomopathogenic bacterium, are important larvicides used to control Culex pipiens [L.] mosquitoes, the primary vector of West Nile virus, in the Chicago, IL USA region. Resistance to both agents has been documented globally including a report of resistance ratios greater than 100 to S-methoprene in the Chicago region. Laboratory studies have suggested the potential for unidirectional cross-resistance between S-methoprene and Ls , despite differing modes of action. Among wild populations of Cx. pipiens in the Chicago area, this study aimed 1) to assess resistance status to Ls , 2) confirm the presence of S-methoprene resistance ratios >100, 3) determine if higher S-methoprene resistance ratios are associated with higher Ls resistance ratios, or whether Ls resistance arises solely from Ls exposure, and (4) determine the relationship between Ls treatment history and resistance levels of that active ingredient. We assessed susceptibility to both S-methoprene and Ls in 32 Cx. pipiens populations: 19 with S-methoprene exposure but no Ls history, and 13 with multi-year exposure to both larvicide active ingredients. Ls susceptibility was evaluated using dose-response bioassays to estimate LC 50 , LC 90 , and resistance ratios. Susceptibility to S-methoprene was tested using diagnostic doses corresponding to resistance ratios of 10 and 100 at the LC 50 . Resistance ratios to S-methoprene exceeding 10 were detected in 30 of 32 sampled populations. Among the 13 sites with prior Ls exposure, 11 were observed with resistance ratios > 5. In contrast, none of the 19 populations without Ls exposure exhibited Ls resistance, despite exhibiting higher S-methoprene resistance ratios. This lack of overlap supports the conclusion that S-methoprene resistance does not confer cross-resistance to Ls in the studied region. Logistic regression revealed a strong association between Ls treatment history and resistance development. The probability of Ls resistance exceeded 80% after 10 Ls applications within an eight-year period. These findings emphasize the need to develop improved resistance management strategies for larvicidal insecticides.

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.

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. Graphical Abstract

The conserved role of juvenile hormone (JH) signals in preventing larvae from precocious metamorphosis has been confirmed in insects. Crustaceans have different metamorphosis types from insects; we previously proved that methyl farnesoate (MF) can prohibit larvae metamorphosis in mud crabs, but the molecular signal of this process still needs to be elucidated. In this study, methoprene-tolerant (Met) of Scylla paramamosain was obtained and characterized, which we named Sp-Met. Sp-Met contains a 3360 bp ORF that encodes 1119 amino acids; the predicted protein sequences of Sp-Met include one bHLH, two PAS domains, one PAC domain, and several long unusual Gln repeats at the C-terminal. AlphaFold2 was used to predict the 3D structure of Sp-Met and the JH binding domain of Met. Furthermore, the binding properties between Sp-Met and MF were analyzed using CD-DOCK2, revealing a putative high affinity between the receptor and ligand. In silico site-directed mutagenesis suggested that insect Mets may have evolved to exhibit a higher affinity for both MF or JH III compared to the Mets of crustaceans. In addition, we found that the expression of Sp-Met was significantly higher in female reproductive tissues than in males but lower in most of the other examined tissues. During larval development, the expression variation in Sp-Met and Sp-Kr-h1 was consistent with the immersion effect of MF. The most interesting finding is that knockdown of Sp-Met blocked the inhibitory effect of MF on metamorphosis in the fifth zoea stage and induced pre-metamorphosis phenotypes in the fourth zoea stage. The knockdown of Sp-Met significantly reduced the expression of Sp-Kr-h1 and two ecdysone signaling genes, Sp-EcR and Sp-E93. However, only the reduction in Sp-Kr-h1 could be rescued by MF treatment. In summary, this study provides the first evidence that MF inhibits crustacean larval metamorphosis through Met and that the MF-Met→Kr-h1 signal pathway is conserved in mud crabs. Additionally, the crosstalk between MF and ecdysteroid signaling may have evolved differently in mud crabs compared to insects.

Insect growth regulators, like S-methoprene, are heavily relied upon worldwide for larval mosquito chemical control due to their target specificity and long-lasting effects. In this study, susceptibility to S-methoprene was evaluated in Culex pipiens , a globally important vector species. Populations from 14 sites throughout the Chicago area with a long history of S-methoprene use and two sites with minimal use in Wisconsin were examined. Using a bioassay methodology and probit analyses, LC 50 and LC 90 values were calculated and compared to a susceptible laboratory strain to develop resistance ratios, then categorized for resistance intensity. The resistance ratios observed required the addition of another category, termed ‘extreme’ resistance, indicating resistance ratios greater than 100. ‘Low’ to ‘extreme’ levels of resistance to S-methoprene were detected throughout Illinois populations, with resistance ratios ranging from 2.33 to 1010.52. Resistance was not detected in populations where S-methoprene pressure has been very limited. These 'extreme’ resistance ratios observed have never been documented in a wild vector species mosquito population. The relationships between historical S-methoprene use, resistance detected with laboratory bioassays, and the potential for field product failure remain unclear. However, the profound resistance detected here demonstrates a potential critical threat to protecting public health from mosquito-borne diseases.

Mosquito larval control by biorational larvicides plays a crucial role in mosquito and mosquito-borne disease management. However, the availability of larvicides that meet the criteria of efficacy, safety, and quality is limited and conventional pesticides are no longer preferred for larval control. Although efforts are made to research new active ingredients (AIs), it is equally important to innovate new formulations based on currently available AIs such as microbial agents and insect growth regulators. Studies were therefore conducted to compare the laboratory activity and semi-field efficacy of OmniPrene® G and Altosid® Pellets with DR-tech, both containing 4.25% S-methoprene, at 2.8 kg/ha and 11.2 kg/ha against the yellow fever mosquito Aedes aegypti (L.) in outdoor microcosms. Both products performed equally in bioassays against the test species with comparable inhibition of emergence activities. In the semi-field study, the lower dose of Altosid Pellets at 2.8 kg/ha, showed lower efficacy than OmniPrene G during the initial 6 weeks; this difference became negligible on week 7, followed by higher efficacy in Altosid Pellets on weeks 8 and 9. More uniform efficacy was observed at the higher dose of 11.2 kg/ha. Equal performance was revealed during weeks 2 to 6, with the OmniPrene G outperforming the Altosid Pellets in week 1, but the opposite during weeks 7 to 9. Mortality patterns were similar in both products, i.e., majority of mortality occurred before emergence, although more incomplete emergence was noted in lower doses, particularly in Altosid Pellets. Overall, newly available OmniPrene G provided comparable activity and efficacy with Altosid Pellets against the test species, with the advantages of fast initial AI release and even coverage, particularly when applied at low doses.

Insects, as the most diverse and numerous group in the animal kingdom, are at least partly dependent on the reproduction process, which is strictly regulated by the ‘classic’ insect hormones: juvenile hormone (JH), and 20-hydroxyecdysone (20E). However, the regulatory mechanism governing the reproduction of JH and 20E in Spodoptera frugiperda remains unclear. In this study, ovarian development and ovulation in female S. frugiperda were assessed through dissection of the ovaries following treatment with JH analog (JHA) and 20E. Moreover, the expression patterns of the JH-signal and 20E-signal-related genes were determined by quantitative PCR (qPCR), and RNA interference (RNAi) was used to investigate the role of JH and 20E-induced genes. Ovarian development was observed by microdissection, and JH and 20E titers were determined by ELISA. Kr-h1, Vg, and USP expression were determined by qPCR. Dissection and qPCR results showed that JHA and 20E promoted ovarian development, egg maturation, and egg laying by upregulating Methoprene-Tolerant (Met) and Ecdysone Receptor (EcR)expression. Additionally, the RNAi results showed that the injection of dsMet and dsEcR markedly delayed ovarian development, inhibited egg maturation, and halted egg production. Knockdown of Met and EcR significantly reduced JH and 20E content and inhibited the transcription of Kr-h1 and USP. These results indicate that JH and 20E facilitate adult reproduction through the methoprene-tolerant gene and ecdysone receptor gene in female S. frugiperda.

The juvenile hormone analog S-methoprene is the only synthetic biopesticide that is registered with the United States Environmental Protection Agency to control arthropods of economic importance in public health, livestock, pets, urban, and stored products. The high activity, relative target specificity, and benign environmental profile of S-methoprene have been well documented. While the risk of resistance in mosquitoes to S-methoprene is generally low, there is a lack of information regarding cross resistance in S-methoprene-resistant mosquitoes to other pesticides. In this paper, a population of the southern house mosquito Culex quinquefasciatus Say from southern California acquired low levels of resistance to S-methoprene in the field, where the resistance ratios ranged 7.0- to 8.8-fold as compared with a laboratory reference colony. After 30 generations of laboratory selections by S-methoprene when resistance was elevated to 57.4- to 168.3-fold relative to an unselected population, various levels of cross resistance to other commonly used pesticides were revealed in the selected population. Cross resistance to the microbial mosquito larvicide Lysinibacillus sphaericus (Meyer & Neide) (Bacillales: Bacillaceae) was the most profound, amounting to 77.50- to 220.50-fold. The mechanism and potential management tactics toward cross resistance are discussed to preserve the unique value of this synthetic biopesticide.

Juvenile hormone III (JH) plays a key role in regulating the reproduction of female mosquitoes. Microarray time-course analysis revealed dynamic changes in gene expression during posteclosion (PE) development in the fat body of female Aedes aegypti . Hierarchical clustering identified three major gene clusters: 1,843 early-PE (EPE) genes maximally expressed at 6 h PE, 457 mid-PE (MPE) genes at 24 h PE, and 1,815 late-PE (LPE) genes at 66 h PE. The RNAi microarray screen for the JH receptor Methoprene-tolerant (Met) showed that 27% of EPE and 40% of MPE genes were up-regulated whereas 36% of LPE genes were down-regulated in the absence of this receptor. Met repression of EPE and MPE and activation of LPE genes were validated by an in vitro fat-body culture experiment using Met RNAi. Sequence motif analysis revealed the consensus for a 9-mer Met-binding motif, CACG C/ TG ᴬ/ Gᵀ/ AG. Met-binding motif variants were overrepresented within the first 300 bases of the promoters of Met RNAi–down-regulated (LPE) genes but not in Met RNAi–up-regulated (EPE) genes. EMSAs using a combination of mutational and anti-Met antibody supershift analyses confirmed the binding properties of the Met consensus motif variants. There was a striking temporal separation of expression profiles among major functional gene groups, with carbohydrate, lipid, and xenobiotics metabolism belonging to the EPE and MPE clusters and transcription and translation to the LPE cluster. This study represents a significant advancement in the understanding of the regulation of gene expression by JH and its receptor Met during female mosquito reproduction.