Juvenile Hormone Analog Research Articles

Identification and Functions of JHE 6 Specifically Expressed in Bombyx mori Silk Gland.

Juvenile hormone esterase (JHE) is the specific enzyme that degrades juvenile hormone (JH) and regulates the JH titer in insects. JH also regulates the development of the silk gland and the synthesis and secretion of silk proteins in Bombyx mori. Here, we identified nine possible JHE family members, Bmjhe1-9. Notably, Bmjhe6 is specifically expressed in the silk gland. Using semi-quantitative, quantitative real-time RT-PCR and Western blot, it was confirmed that Bmjhe6 was specifically expressed in the middle silk gland (MSG) with high levels in the anterior region of the MSG (A-MSG). The immunofluorescence localization analysis revealed that Bmjhe6 is produced within cells, secreted into the gland lumen, and co-transported with silk proteins into the anterior silk gland (ASG). In vitro hormone induction experiments demonstrated that Bmjhe6 responds to a JH analog, increasing its expression after 12-24 h, whereas 20-hydroxyecdysone inhibited it. In addition, Bmjhe6 knockdown using dsBmjhe6 injections accelerated larval development, resulting in increased larval body and silk gland weight. This induced disordered sericin genes (Ser2, Ser3) expression, and key genes in the JH synthesis pathway (BmKr-h1 and BmMet1) were significantly upregulated along with the transcription factors (SGF-1 and Sage). These results indicate that Bmjhe6 plays an important role in silk gland growth and silk protein synthesis by modulating JH signal.

Methoprene, a Juvenile Hormone Analog, Causes Winter Diapause Elimination in Univoltine Bee Species Osmia bicornis L.

Osmia bicornis syn. O. rufa is a univoltine bee species in which adults fly in spring and the offspring overwinter as cocooned imagoes. The flight period of solitary bees is short, so methods of control for development and emergence time are needed to synchronize the activity of managed pollinators with blooming. In our study, we tested the effectiveness of a juvenile hormone analog for the prevention of winter diapause. Bees developed in settled nests outdoors or in the laboratory (22 °C) until the end of the pre-pupa stage, then cocoons were removed from the nest cells and treated with a JH analog-methoprene-during the pupa and young imago stages. Then, bees were activated at 25 °C until the adults left the cocoons. Topical application of methoprene to the cocoon at the pupa or imago stage induced the emergence of some adult bees in the pre-diapause period, while no adults emerged when the bees were not treated with methoprene. Most adults emerged (about 50%) when treated with methoprene on 3-week-old cocooned imagoes. Bees treated in the pupal stage had a lower emergence rate (20-30%), but adult bees emerged earlier. The emergence time of adults for the laboratory group was, on average, from 70 to 91 days, and that for outdoor groups was from 57 to 72 days.

Juvenile hormone regulates silk gene expression by m6A RNA methylation.

Juvenile hormone (JH) is an indispensable insect hormone that is critical in regulating insect development and physiology. N6-methyladenosine (m6A) is the most abundant modification of RNA that regulates RNA fate in eukaryotic organisms. However, the relationship between m6A and JH remains largely unknown. Here, we found that the application of a Juvenile hormone analog (JHA) extended the larval period of Bombyxmori and increased the weight and thickness of the cocoon. Interestingly, global transcriptional patterns revealed that m6A-related genes are specifically regulated by JHA in the posterior silk gland (PSG) that synthesizes the major component of cocoon silk. By transcriptome and m6A sequencing data conjointly, we discovered that JHA significantly regulated the m6A modification in the PSG of B. mori and many m6A-containing genes are related to nucleic acid binding, nucleus, and nucleobase-containing compound metabolism. Notably, 547 genes were significantly regulated by JHA at both the m6A modification and expression levels, especially 16 silk-associated genes, including sericin2, seroin1, Serine protease inhibitors 4 (BmSPI4), Serine protease inhibitors 5 (BmSPI5), and LIM domain-binding protein 2 (Ldb). Among them, 11 silk associated genes were significantly affected by METTL3 knockdown, validating that these genes are targets of m6A modification. Furthermore, we confirm that JHA directly regulates the expression of BmSPI4 and BmSPI5 through m6A modification of CDS regions. These results demonstrate the essential role of m6A methylation regulated by JH in PSG, and elucidate a novel mechanism by which JH affects silk gland development via m6A methylation. This study uncoversthatm6A modification is a critical factor mediating the effect of JH in insects.

Influence of juvenile hormone analog on behavior in the red imported fire ant, Solenopsis invicta

Division of labor is a hallmark characteristic of social insect colonies. While it is understood that worker differentiation is regulated through either the queen or her brood, the understanding of the physiology behind task regulation varies within social species. Studies in eusocial insects have shown that juvenile hormone (JH) is associated with division of labor and the onset of foraging tasks. Although, outside of a few key species, this interaction has yet to be elucidated in the red imported fire ant, Solenopsis invicta. In this study, we evaluated the role of a JH analog, S-hydroprene in worker task transition in Solenopsis invicta. S-hydroprene was applied to nurses to observe behavioral changes. S-hyroprene application to nurses did not affect phototaxis, but there was a shift in behavior from internal, nest-based behaviors to external, foraging-based behaviors. These results show that JH may be implicated in worker task transition in S. invicta and may function similarly as it does in other eusocial insects.

Investigating the Regulatory Mechanism of the Sesquiterpenol Nerolidol from a Plant on Juvenile Hormone-Related Genes in the Insect Spodoptera exigua.

Various plant species contain terpene secondary metabolites, which disrupt insect growth and development by affecting the activity of juvenile hormone-degrading enzymes, and the juvenile hormone (JH) titers maintained in insects. Nerolidol, a natural sesquiterpenol belonging to the terpenoid group, exhibits structural similarities to insect JHs. However, the impact of nerolidol on insect growth and development, as well as its underlying molecular mechanism, remains unclear. Here, the effects of nerolidol on Spodoptera exigua were investigated under treatment at various sub-lethal doses (4.0 mg/mL, 1.0 mg/mL, 0.25 mg/mL). We found that a higher dose (4.0 mg/mL) of nerolidol significantly impaired the normal growth, development, and population reproduction of S. exigua, although a relatively lower dose (0.25 mg/mL) of nerolidol had no significant effect on this growth and development. Combined transcriptome sequencing and gene family analysis further revealed that four juvenile hormone esterase (JHE)-family genes that are involved in juvenile hormone degradation were significantly altered in S. exigua larvae after nerolidol treatment (4.0 mg/mL). Interestingly, the juvenile hormone esterase-like (JHEL) gene Sexi006721, a critical element responsive to nerolidol stress, was closely linked with the significant augmentation of JHE activity and JH titer in S. exigua (R2 = 0.94, p < 0.01). Taken together, we speculate that nerolidol can function as an analog of JH by modulating the expression of the enzyme genes responsible for degrading JH, resulting in JH disorders and ultimately disrupting the development of insect larvae. This study ultimately provides a theoretical basis for the sustainable control of S. exigua in the field whilst proposing a new perspective for the development of novel biological pesticides.

The promoting effects of pyriproxyfen on autophagy and apoptosis in silk glands of non-target insect silkworm, Bombyx mori

Pyriproxyfen is a juvenile hormone analogue. The physiological effects of its low-concentration drift during the process of controlling agricultural and forestry pests on non-target organisms in the ecological environment are unpredictable, especially the effects on organs that play a key role in biological function are worthy of attention. The silk gland is an important organ for silk-secreting insects. Herein, we studied the effects of trace pyriproxyfen on autophagy and apoptosis of the silk gland in the lepidopteran model insect, Bombyx mori (silkworm). After treating fifth instar silkworm larvae with pyriproxyfen for 24 h, we found significant shrinkage, vacuolization, and fragmentation in the posterior silk gland (PSG). In addition, the results of autophagy-related genes of ATG8 and TUNEL assay also demonstrated that autophagy and apoptosis in the PSG of the silkworm was induced by pyriproxyfen. RNA-Seq results showed that pyriproxyfen treatment resulted in the activation of juvenile hormone signaling pathway genes and inhibition of 20-hydroxyecdysone (20E) signaling pathway genes. Among the 1808 significantly differentially expressed genes, 796 were upregulated and 1012 were downregulated. Among them, 30 genes were identified for autophagy-related signaling pathways, such as NOD-like receptor signaling pathway and mTOR signaling pathway, and 30 genes were identified for apoptosis-related signaling pathways, such as P53 signaling pathway and TNF signaling pathway. Further qRT-PCR and in vitro gland culture studies showed that the autophagy-related genes Atg5, Atg6, Atg12, Atg16 and the apoptosis-related genes Aif, Dronc, Dredd, and Caspase1 were responsive to the treatment of pyriproxyfen, with transcription levels up-regulated from 24 to 72 h. In addition, ATG5, ATG6, and Dronc genes had a more direct response to pyriproxyfen treatment. These results suggested that pyriproxyfen treatment could disrupt the hormone regulation in silkworms, promoting autophagy and apoptosis in the PSG. This study provides more evidence for the research on the damage of juvenile hormone analogues to non-target organisms or organs in the environment, and provides reference information for the scientific and rational use of juvenile hormone pesticides.

Uncovering the role of juvenile hormone in ovary development and egg laying in bumble bees

Juvenile hormone (JH) regulates developmental and physiological processes in insects. In bumble bees, the hormone acts as a gonadotropin that mediates ovary development, but the exact physiological pathways involved in ovary activation and subsequent egg laying are poorly understood. In this study, we examine how queen hibernation state, caste, and species impact the gonadotropic effect of JH in bumble bee queens through methoprene (JH analogue) application. We extend previous research by assessing queen egg laying and colony initiation, alongside ovary development. Furthermore, we compared sensitivity of workers of both species to the juvenile hormone's gonadotropic effect. In both bumble bee species, the ovaries of hibernated queens were developed five to six days after breaking diapause, regardless of methoprene treatment. By contrast, methoprene did have a stimulatory effect on ovary development in non-hibernated queens. The dose needed to obtain this effect was higher in B. impatiens. Methoprene did not have gonadotropic effects in callow workers of both species. These results indicate that the physiological effect of exogenous methoprene application varies according to species, caste and hibernation status. Interestingly, despite gonadotropic effects in non-hibernated queens, oviposition was not accelerated by JH. This suggests that JH alone is insufficient to induce egg laying and that an additional stimulus, which is naturally present in hibernated queens, is required. Consequently, our findings indicate that other physiological processes, beyond a rise in JH alone, are required for oviposition and colony initiation.

Effects of different hormones as dietary supplements on biological characteristics of Coccinella septempunctata L.

AbstractWhen Coccinella septempunctata L. was fed on an artificial diet, development was prolonged and survival and reproduction were decreased, thus negatively affecting population growth. Juvenile hormone and moulting hormones are important factors in insect reproduction and development. This study aims to screen out the optimal hormones that can solve the problems such as slow population growth of the C. septempunctata during mass rearing, thus improving their effectiveness as a biological control agent. The effects of juvenile hormone analogues and moulting hormones on C. septempunctata larval growth and adult reproduction were investigated by supplementing an artificial diet with different doses of methoprene, kinoprene, juvenile hormone Ш, fenoxycarb and 20‐hydroxyecdysone. Multiple parameters were measured for C. septempunctata including development, survival, pupation, emergence, pre‐oviposition, fecundity and hatching rates. The addition of juvenile hormone analogues to the diet significantly lengthened larval development times and reduced pupation and emergence. Conversely, juvenile hormone analogues significantly shortened pre‐oviposition period, reduced survival and increased fecundity. The shortest pre‐oviposition period was 9.6 days, and the highest oviposition number was 578 eggs in response to 5 μL kinoprene. The highest hatching rate was 66% in 1.7 mg 20‐hydroxyecdysone treatment. The results of this study provide a theoretical basis for hormone selection and dosage in C. septempunctata artificial diets, thus enhancing the survival, pupation and emergence of larvae and improving the fecundity and hatching rate of ladybird adults.

Overexpression of BmJHBPd2 Repressed Silk Synthesis by Inhibiting the JH/Kr-h1 Signaling Pathway in Bombyx mori.

The efficient production of silkworm silk is crucial to the silk industry. Silk protein synthesis is regulated by the juvenile hormone (JH) and 20-Hydroxyecdysone (20E). Therefore, the genetic regulation of silk production is a priority. JH binding protein (JHBP) transports JH from the hemolymph to target organs and cells and protects it. In a previous study, we identified 41 genes containing a JHBP domain in the Bombyx mori genome. Only one JHBP gene, BmJHBPd2, is highly expressed in the posterior silk gland (PSG), and its function remains unknown. In the present study, we investigated the expression levels of BmJHBPd2 and the major silk protein genes in the high-silk-producing practical strain 872 (S872) and the low-silk-producing local strain Dazao. We found that BmJHBPd2 was more highly expressed in S872 than in the Dazao strain, which is consistent with the expression pattern of fibroin genes. A subcellular localization assay indicated that BmJHBPd2 is located in the cytoplasm. In vitro hormone induction experiments showed that BmJHBPd2 was upregulated by juvenile hormone analogue (JHA) treatment. BmKr-h1 upregulation was significantly inhibited by the overexpression of BmJHBPd2 (BmJHBPd2OE) at the cell level when induced by JHA. However, overexpression of BmJHBPd2 in the PSG by transgenic methods led to the inhibition of silk fibroin gene expression, resulting in a reduction in silk yield. Further investigation showed that in the transgenic BmJHBPd2OE silkworm, the key transcription factor of the JH signaling pathway, Krüppel homolog 1 (Kr-h1), was inhibited, and 20E signaling pathway genes, such as broad complex (Brc), E74A, and ultraspiracle protein (USP), were upregulated. Our results indicate that BmJHBPd2 plays an important role in the JH signaling pathway and is important for silk protein synthesis. Furthermore, our findings help to elucidate the mechanisms by which JH regulates silk protein synthesis.

Efficacy of Contact Insecticides for the Control of the Larger Grain Borer, Prostephanus truncatus (Horn), on Stored Maize

One of the most destructive insect species for stored maize is the larger grain borer, Prostephanus truncatus. Its control is challenging, as it seems to have a natural tolerance to active ingredients that are effective for other stored-product insect species that cause infestations in maize. The objective of the present study was to comparatively evaluate a wide range of insecticides that are currently in use in stored product protection for the control of P. truncatus. Specifically, three inert dusts—namely, a kaolin, a zeolite and a diatomaceous earth formulation—and three residual insecticides—i.e., the pyrethroid deltamethrin, the bacterial insecticide spinosad and the juvenile hormone analogue S-Methoprene—were evaluated against adults of P. truncatus. Adult mortality was assessed after 7, 14, 21 and 28 d of exposure, whereas progeny production was measured after an additional interval of 28 d for inert dusts and 65 d for the contact insecticides. Moreover, the number and weight of infested and uninfested kernels per vial was measured. Low mortality levels were recorded for the three inert dusts even for the highest application rate and after 28 d of exposure. Moreover, the inert dusts tested failed to suppress the progeny production of P. truncatus. In contrast, high mortality levels were recorded for deltamethrin and spinosad that exceeded 95% already after 7 d of exposure at the lowest application rate (0.5 ppm). The application of S-Methoprene did not result in high adult mortality rates, irrespective of the application rate and the evaluation interval. Deltamethrin, spinosad and S-Methoprene significantly suppressed progeny production of the species at the doses tested.

The females' response to male attractiveness: Mate choice, larvae production and differential brain protein expression

AbstractSexual Selection has been studied predominantly from a functional perspective with relatively limited investigation of the mechanistic basis of female choice. In this study, we evaluated female choice and larvae production in response to attractive or less attractive males and concurrently examined the protein profile in the brains of females, using the mealworm beetle Tenebrio molitor as a model system. Females were found to prefer males with high levels of pheromones, which requires the secretion of juvenile hormone (JH). Male attractiveness was enhanced using the JH analog methoprene, resulting in less female rejection and higher mating frequency and offspring production. Importantly, reproductive success was not due to differences in testis size or sperm production, or viability. Out of the 1833 proteins detected in the brain, only 32 were differentially expressed in females mating with attractive versus less attractive males. These proteins involved memory, metabolism, olfactory detection, reproduction, and movement. Although some brain proteins have been linked to mate choice in vertebrates, most of the differentially expressed molecules found in this study have not been previously reported concerning mate choice.

Hexamerin and allergen are required for female reproduction in the American cockroach, Periplaneta americana.

Reproduction is of great importance for the continuation of the species. In insects, the fat body is the major tissue for nutrient storage and involved in vitellogenesis, which is essential for female reproduction. Here, 2 proteins, hexamerin and allergen, were separated from the fat bodies of adult female American cockroaches (Periplaneta americana) and identified as storage proteins, encoding for 733 amino acids with molecular weight of 87.88kDa and 686 amino acids with molecular weight of 82.18kDa, respectively. The encoding genes of these 2 storage proteins are mainly expressed in the fat body. RNA interference-mediated knockdown of Hexamerin and Allergen in the early stage of the first reproductive cycle in females suppressed vitellogenesis and ovarian maturation, indicating that these storage proteins are involved in controlling reproduction. Importantly, the expression of Hexamerin and Allergen was repressed by knockdown of the juvenile hormone (JH) receptor gene Met and the primary response gene Kr-h1, and was induced by methoprene, a JH analog, in both in vivo and in vitro experiments. Altogether, we have determined that hexamerin and allergen are identified as storage proteins and play an important role in promoting female reproduction in the American cockroach. The expression of their encoding genes is induced by JH signaling. Our data reveal a novel mechanism by which hexamerin and allergen are necessary for JH-stimulated female reproduction.

Juvenile Hormone-Induced Abnormality of &lt;i&gt;Periplaneta americana&lt;/i&gt; (L.)

Application of juvenile hormone III (JH III) and a JH analogue, methoprene to last instars of Periplaneta americana suppressed adult emergence and caused morphological abnormalities such as twisted wings and asymmetry of appendages resulting in formation of either nymphoids or giant supernumerary nymphs and adultoids. The effects of JH III and methoprene were concentration dependent in inducing aberrations in morphogenetic moult as well as in significantly lowering the body mass (BM) and lipid reserves (LR) in P. americana.

Effects of adult-diet modifications on sexual maturation rate, body weight, and quantity of sperm transfer by male Mexican fruit fly (Diptera: Tephritidae).

Mass-reared sterile Mexican fruit flies (Anastrepha ludens [Loew]) are released along the US-Mexico border to control fruit fly damage and spread. It is more cost effective for the mass rearing program if males mature at a younger age because they can be held for a shorter time at the facility before release. In this study, adult male Mexican fruit fly diets were tested to determine if the current diet used at the mass rearing facility promotes younger mating and optimal sperm transfer compared to other diet formulations. Hydrolyzed yeast presentation methods were examined: an agar-free dry mixture of yeast and sugar (Y+S), yeast embedded in the diet during the agar boiling process (current method used) and sifted dry on the ager gel surface. Methoprene, a juvenile hormone analog was also tested as a supplement to agar gel diets with and without yeast. A greater proportion of males fed Y+S mated 1 day earlier than males fed other diets. Male mating age and diet did not significantly affect the quantity of sperm transferred; however, a non-significantly higher percentage of males fed diets with embedded yeast transferred enough sperm to fill all 3 spermathecae. The results suggest that the current diet used is optimal for mass-rearing the flies and that yeast presentation method has a significant effect on A. ludens male mating age but not on the quantity of sperm transferred to females.

Growth, Reproduction, and Transgenerational Effects of Kinoprene on Moina macrocopa.

Juvenile hormone analogues (JHAs) are a kind of effective insecticide. These compounds have relatively low toxicity for fish, birds, and mammals, so they are increasingly used in insect pest control. However, JHAs may cause various adverse effects in crustaceans as in insects, because they have a close evolutionary relationship and possess similar juvenile hormone systems. To date, the chronic toxicities of JHAs over generations lacked intensive investigation. The present study evaluated the acute, chronic, and transgenerational effects of a terpenoid JHA, kinoprene, using the water flea Moina macrocopa. The result of acute exposure shows that kinoprene exhibited high toxicity to M. macrocopa. The chronic results indicate that kinoprene inhibited the survivorship, development, and reproduction of the organism. Moreover, the adverse effects induced by kinoprene continued in the F2 generation with no direct exposure but recovered in the F3 generation.

De Novo Transcriptomic and Life-History Responses of Moina Micrura Under Stress Environment Conditions.

Moina micrura represents a promising model species for ecological and ecotoxicological investigations in tropical freshwater ecosystems. Illumina NovaSeq™ 6000 sequencing was employed in this study to analyze M. micrura across three distinct developmental stages: juvenile, adult, and male. Current study successfully annotated 51,547 unigenes (73.11%) derived from seven (7) different databases. A total of 554 genes were found to be significantly upregulated, while 452 genes showed significant downregulation between juvenile and male. Moreover, 1001 genes were upregulated, whereas 830 genes exhibited downregulation between the adult and male. Analysis of differentially expressed genes revealed upregulation of chitin, cuticle, myosin (MYO), mitogen-activated protein kinases (MAPK), fibrillin (FBN), cytochrome (CYP), glutathione s-transferase (GST), vitellogenin (VTG), acetylcholinesterase (AChE), and transforming growth factor beta (TGFB) under unfavorable environmental conditions (male), as compared to favorable environmental conditions (juveniles and adults). These alterations in gene expression significantly impact the phenological and life-history traits of M. micrura. Furthermore, the upregulation of hemoglobin (HMB), doublesex (DSX), juvenile hormone analogs (JHA), heat shock protein (HSP), and methyltransferase (METT) genes in males initiates the sex-switching effects observed in M. micrura. These findings hold substantial value for researchers interested in determining M. micrura sequences for future investigations of gene expression and comparative reproductive genome analysis within the Moina genus and cladoceran families.

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.

Accelerated abdominal lipid depletion from pesticide treatment alters honey bee pollen foraging strategy, but not onset, in worker honey bees.

Honey bee abdominal lipids decline with age, a change thought to be associated with the onset of foraging behavior. Stressors, such as pesticides, may accelerate this decline by mobilizing internal lipid to facilitate the stress response. Whether bees with stressor-induced accelerated lipid loss vary from controls in both the onset of foraging and nutritional quality of collected pollen is not fully understood. We asked whether stressors affect foraging behavior through the depletion of abdominal lipid, and whether stress-induced lipid depletion causes bees to forage earlier and for fattier pollen. We tested this by treating newly emerged bees with one of two pesticides, pyriproxyfen (a juvenile hormone analog) and spirodiclofen (a fatty acid synthesis disruptor), that may affect energy homeostasis in non-target insects. Bees fed these pesticides were returned to hives to observe the onset of foraging behavior. We also sampled foraging bees to assay both abdominal lipids and dietary lipid content of their corbicular pollen. Initially, spirodiclofen-treated bees had significantly more abdominal lipids, but these declined faster compared with controls. These bees also collected less, yet more lipid-rich, pollen. Our results suggest that bees with accelerated lipid decline rely on dietary lipid content and must collect fattier pollen to compensate. Pyriproxyfen treatment reduced the age at first forage but did not affect abdominal or collected pollen lipid levels, suggesting that accelerated fat body depletion is not a prerequisite for precocious foraging.

Determining optimal time to harvest yellow mealworms following treatment with pyriproxyfen, a juvenile hormone analogue, to maximise protein yield and reduce fat yield

Determining optimal time to harvest yellow mealworms following treatment with pyriproxyfen, a juvenile hormone analogue, to maximise protein yield and reduce fat yield

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.