Juvenile hormone (JH) esterase activity but not JH epoxide hydrolase activity is downregulated in larval Adoxophyes honmai following nucleopolyhedroviruses infection

Juvenile hormones (JHs) and ecdysteroids are critical insect developmental hormones. JH esterase (JHE) and JH epoxide hydrolase (JHEH) are JH-selective enzymes that metabolize JH and thus regulate the titer of JH. Baculoviruses are known to alter host endocrine regulation. The nucleopolyhedroviruses, AdhoNPV and AdorNPV, are known to have slow and fast killing activity against Adoxophyes honmai (Lepidoptera: Tortricidae), respectively. Here we found that when penultimate (4th) instar A. honmai are inoculated with AdhoNPV or AdorNPV, the mean survival time is 9.7 and 8.2 days, respectively. The larvae molted once but did not pupate. The AdhoNPV- or AdorNPV-infected larvae did not show a dramatic increase in JHE activity as was found in mock-infected larvae, instead they showed a marked decrease in JHE activity. In contrast, both viral infections had no effect on JHEH activity. In order to further characterize the JHE activity, the JHE-coding sequence of A. honmai (ahjhe) was cloned and confirmed to encode a biologically active JHE. Quantitative real-time PCR analysis of ahjhe expression in 4th and 5th instar A. honmai revealed that AdhoNPV and AdorNPV are able to reduce ahjhe expression levels.     

Depletion of juvenile hormone esterase extends larval growth in Bombyx mori

Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production.     

The role of exogenous JH I,JH III and Anti-JH (precocene II) on queen induction of 4.5-day-old worker honey bee larvae

Worker larvae at an age of 4½ days were fed one of several mixtures of reconstituted royal jelly adjusted to a refractive index of 1.3825 and supplemented with JH I, JH III or Anti-JH (precocene II). In addition, juvenile hormone was topically applied to larvae of the same age. It was readily apparent that caste induction is concentration-dependent and that 4?-day-old worker larvae can still develop into queens under laboratory conditions, providing that they have not stopped feeding or can be induced to commence feeding again. These findings are contrary to the general belief that queen induction is not possible after a socalled sensitive period of 3–3½ days. Queens resulted only from honey bee larvae exposed to royal jelly containing 1 μg of JH I. In addition, oral application at this concentration resulted in the only case in which the normal mean weights of worker honey bees were exceeded. All other concentrations of juvenile hormone were not sufficient to initiate queen induction, although its lower concentration may have influenced the production of intercastes.Precocene II did not play a role in queen induction and it also did not interfere with the growth of developing larvae or adults. In addition, the lack of malformations in honey bees treated with precocene II indicates that the use of such a compound as a control agent in insect populations will probably not be detrimental to honey bee larvae that are at least 4½ days old. However, large doses of precocene will quickly kill most 3½-day-old honey bee larvae.The evidence presented here clearly indicates that caste determination is regulated by the endocrine system in honey bee larvae. Food intake in honey bee larvae may well be regulated by the endocrine system. Thus, an apparently inhibited corpus allatum (C.A.) could be reactivated by food intake coupled with juvenile hormone. The food intake restriction that worker larvae normally encounter in the hive probably results in a cessation of C.A. activity. The increase in food intake by queen larvae, on the other hand, carries an increase in growth and accompanying morphological changes necessary for queen development. This concept may also explain the development of intercastes encountered in in vitro studies. Only those larvae that follow a normal food intake sequence, i.e. moderate during the first 3–4 days or so, will develop into queens. Conversely, those larvae that take in too much food during the early portion of development may achieve incomplete development of the neurosecretory system and, thus, develop into intercastes.     

Coordinated changes in JH biosynthesis and JH hemolymph titers in Aedes aegypti mosquitoes

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. A decrease in JH titer during the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa once again synthesizes JH, which plays an essential role in orchestrating reproductive maturation. In spite of the importance of Aedes aegypti as a vector, a detailed study of the changes of JH hemolymph titers during the gonotrophic cycle has never been performed. In the present studies, using a high performance liquid chromatography coupled to a fluorescent detector (HPLC–FD) method, we measured changes in JH levels in the hemolymph of female mosquitoes during the pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers during the gonotrophic cycle of female mosquito. Feeding high sugar diets resulted in an increase of JH titers, and mating also modified JH titers in hemolymph. In addition these studies confirmed that JH titer in mosquitoes is fundamentally determined by the rate of biosynthesis in the CA.     

Expression pattern of enzymes related to juvenile hormone metabolism in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis> L.

The physiological balance of juvenile hormone (JH) in insects depends on its biosynthesis and degradation pathway. Three key enzymes namely, juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol kinase (JHDK) are required for degradation in insects. Our present results showed that JHE and JHEH exhibited expression in almost all the tissues. This indicated that JHE and JHEH might degrade JH simultaneously. In addition, the highest levels of JHDK were observed in the midgut, with trace level being found in the malpighian tubule and haemocytes. Since the midgut is a digestive organ and not a JH target, it was hypothesized that both JHE and JHEH hydrolyzed JH to JH diol (JHd) which was then transported to midgut and hydrolyzed further by JHDK, to be finally excreted out of the body. Also the expression studies on JH degradation enzymes in different tissues and stages indicated that the activities of the three enzymes are specific and coincident with the JH functions in silkworm, Bombyx mori L.     

A whole genome screening and RNA interference identify a juvenile hormone esterase-like gene of the diamondback moth,Plutella xylostella

Juvenile hormone (JH) plays a crucial role in preventing precocious metamorphosis and stimulating reproduction. Thus, its hemolymph titer should be under a tight control. As a negative controller, juvenile hormone esterase (JHE) performs a rapid breakdown of residual JH in the hemolymph during last instar to induce a larval-to-pupal metamorphosis. A whole genome of the diamondback moth (DBM), Plutella xylostella, has been annotated and proposed 11 JHE candidates. Sequence analysis using conserved motifs commonly found in other JHEs proposed a putative JHE (Px004817). Px004817 (64.61 kDa, pI = 5.28) exhibited a characteristic JHE expression pattern by showing high peak at the early last instar, at which JHE enzyme activity was also at a maximal level. RNA interference of Px004817 reduced JHE activity and interrupted pupal development with a significant increase of larval period. This study identifies Px004817 as a JHE-like gene of P. xylostella.     

Role of juvenile hormone esterase and epoxide hydrolase in reproduction of the cotton bollworm, Helicoverpa zea

The role of juvenile hormone (JH) esterase (JHE) and epoxide hydrolase (EH) in reproduction of the cotton bollworm, Helicoverpa zea, was investigated. Peak emergence of male and female bollworm adults occurred early in the scotophase. Female adults were added to males in a 1:2 ratio, respectively, at the beginning of the first photophase after emergence (d0). The highest oviposition rates for mated females were noted on d 2-4. The in vitro JH III esterase and JH III EH activity was measured in whole body homogenates of virgin and mated females from d0 to d8 post-emergence. Maximal JHE activity for virgin females occurred on d2 (1.09+/-0.14(+/-1 SEM) nmol of JH III degraded/min/mg protein), which was approximately twice that of mated females on the same day. The same results were observed for EH where the activity peaked on d2 at 0.053+/-0.003 as compared to 0.033+/-0.003 nmol of JH III degraded/min/mg protein, respectively. By d4, both JHE and JH EH activities declined significantly in virgin and mated females and were the same through d7. The developmental changes and effects of mating on JH degradation were similar when measured per insect. The highest levels of JHE and JH EH activity/min/mg protein in d2 virgin and mated females was found in ovaries followed by the carcass and then haemolymph; no EH activity was found in haemolymph as expected. For ovary, the JHE and JH EH activity was highest in virgin compared to mated females. The role of both enzymes in the regulation of reproduction is discussed.     

Control of juvenile hormone esterase activity in Galleria mellonella larvae

The juvenile hormone esterase (JHE) activity in Galleria mellonella larvae was measured after exposure to different experimental conditions that affect larval-pupal transformation. The data show that stimulation of production of JHE is closely coupled with the developmental signals that intiate larval-pupal metamorphosis. Injury, which delays pupation, delays the appearance of JHE activity if the larvae are injured within 48 hr after the last larval moult. Chilling of day-0 larvae induces a supernumerary larval moult and inhibits the appearance of JHE. However, JHE activity increases in chilled larvae when their commitment for an extra larval moult is reversed by starvation. Starvation is effective in reversing the commitment for an extra larval moult if commenced within 48 hr after chilling, thereby suggesting a critical period for that commitment. These data suggest that the stimulus for JHE synthesis and/or release occurs approximately within 48 hr after the last larval ecdysis. A series of studies involving implantation of brain, suboesophageal ganglion and fat body into chilled, as well as chilled and ligated larvae suggest that a factor from the brain is involved in stimulation or production of JHE in Galleria larvae.JH, which suppresses JHE activity in day-3, -5 and early day-6 Galleria larvae, stimulates the production of JHE in late day-6 larvae, suggesting that reprogramming in larval fat body may occur on day 6 of the last larval stadium.     

Characterization and functional study of juvenile hormone diol kinase gene in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

The brown planthopper, Nilaparvata lugens (Stål) is an important pest in rice. It has been widely recognized that the juvenile hormone (JH) is regulated by its hydrolase, which includes juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol kinase (JHDK). In this paper, we cloned the gene of Jhdk and the gene expression at different stages of N. lugens was analysed, and the relationship with Jhe and Jheh was studied after silencing the jhdk gene of N. lugens (Nljhdk) through double-stranded RNA (dsRNA) feeding. We also explored the expression of the three JH hydrolase after indoxacarb treatments. RT-PCR was used to amplify the full length Jhdk cDNA, and the Nljhdk gene was expressed throughout all the development periods tested and showed the lowest level at the 4th instar and the highest in the 5th instar. The expression level of Nljhdk in male adults was higher than that of female adults. Through feeding, dsRNA against Nljhdk successfully knocked down the target gene, which had no significant effect on the expression of the jhe gene of N. lugens (Nljhe), while the expression of Nljheh was upregulated. Indoxacarb could inhibit N. lugens reproduction, and the expression level of Nljhe and Nljhdk increased with the increasing of indoxacarb concentration, but the expression of the jheh gene of N. lugens (Nljheh) was reduced. These studies provide a line of experimental evidence in N. lugens to support that Nljhdk encodes the functional protein involved in JH degradation and further showed the relationship of the three hydrolases and the mechanism of indoxacarb inhibition of the fecundity of N. lugens.     

Improved insecticidal efficacy of a recombinant baculovirus expressing mutated JH esterase from Manduca sexta

Juvenile hormone esterase (JHE), a member of the carboxylesterase family (EC 3.1.1.1), metabolizes JH that is found in juvenile insects. A highly conserved amphipathic alpha helix is found on the surface of known JHEs. This helix is implicated in receptor-mediated binding and endocytosis of JHE by the pericardial cells resulting in the clearance of JHE activity from the hemolymph. In this study, Lys-204 and Arg-208 of the amphipathic alpha helix of the JHE of Manduca sexta (MsJHE) were mutated to histidine residues generating MsJHE-HH. Pharmacokinetic studies following the injection of MsJHE-HH into the hemocoel of larval M. sexta, Heliothis virescens, and Agrotis ipsilon indicated that MsJHE-HH and wild type MsJHE are cleared at similar rates. The infectivity (lethal concentration and lethal time) of a recombinant baculovirus, AcMsJHE-HH, expressing MsJHE-HH was not significantly different than that of a recombinant baculovirus, AcMsJHE, expressing MsJHE in first instars of H. virescens and A. ipsilon. However, the mass of AcMsJHE-HH-infected larvae was 40–50% lower than that of larvae infected with AcMsJHE, and 70–90% lower than that of wild type AcMNPV-infected larvae.     

Suppression of JH biosynthesis by JH analog treatment: Mechanism of suppression and roles of allatostatins and nervous connections in the cockroach Diploptera punctata

Juvenile hormone analogs are known to inhibit the production of juvenile hormone (JH) by the corpora allata (CA). However, the mechanism of this inhibition remains undefined. We have used two JH mimics, fenoxycarb and pyriproxyfen, to examine the mechanism of suppression in the cockroach, Diploptera punctata. Denervation experiments demonstrated the importance of nervous connections between the brain and CA for the inhibition of JH biosynthesis by fenoxycarb. Fenoxycarb treatment alters the sensitivity of CA to allatostatin treatment in vitro. Suppression of JH biosynthesis by fenoxycarb following denervation of the CA showed that innervation was in part responsible for the inhibition. Similarly, maximal inhibition by Dippu-AST7 requires intact nervous connections between the brain and CA, particularly during rapid vitellogenesis. qPCR analysis of brain, CA, ovary and midgut extracts revealed that both allatostatin and its receptor Dippu-ASTR2 show increased levels of expression following topical fenoxycarb treatment, particularly in brain tissue on days 4 and 5 of the first gonadotrophic cycle and in CA on day 4. The correlation between inhibition of JH biosynthesis and increased expression of AST and ASTR2 in brains and CA, together with increased sensitivity of CA to allatostatin in vitro, suggests that allatostatin may be one of the effectors by which fenoxycarb inhibits JH biosynthesis.     

Expression of methyl farnesoate epoxidase (mfe) and juvenile hormone esterase (jhe) genes and their relation to social organization in the stingless bee Melipona interrupta (Hymenoptera: Apidae)

Social organization in highly eusocial bees relies upon two important processes: caste differentiation in female larvae, and age polyethism in adult workers. Juvenile Hormone (JH) is a key regulator of both processes. Here we investigated the expression of two genes involved in JH metabolism - mfe (biosynthesis) and jhe (degradation) - in the context of social organization in the stingless bee Melipona interrupta. We found evidence that the expression of mfe and jhe genes is related to changes in JH levels during late larval development, where caste determination occurs. Also, both mfe and jhe were upregulated when workers engage in intranidal tasks, but only jhe expression was downregulated at the transition from nursing to foraging activities. This relation is different than expected, considering recent reports of lower JH levels in foragers than nurses in the closely related species Melipona scutellaris. Our findings suggest that highly eusocial bees have different mechanisms to regulate JH and, thus, to maintain their level of social organization.     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.     

Larval juvenile hormone treatment affects pre-adult development,but not adult age at onset of foraging in worker honey bees (Apis mellifera)

Previous research has shown that juvenile hormone (JH) titers increase as adult worker honey bees age and treatments with JH, JH analogs and JH mimics induce precocious foraging. Larvae from genotypes exhibiting faster adult behavioral development had significantly higher levels of juvenile hormone during the 2nd and 3rd larval instar. It is known that highly increased JH during this period causes the totipotent female larvae to differentiate into a queen. We treated third instar larvae with JH to test the hypothesis that this time period may be a developmental critical period for organizational effects of JH on brain and behavior also in the worker caste, such that JH treatment at a lower level than required to produce queens will speed adult behavioral development in workers. Larval JH treatment did not influence adult worker behavioral development. However, it made pre-adult development more queen-like in two ways: treated larvae were capped sooner by adult bees, and emerged from pupation earlier. These results suggest that some aspects of honey bee behavioral development may be relatively insensitive to pre-adult perturbation. These results also suggest JH titer may be connected to cues perceived by the adult bees indicating larval readiness for pupation resulting in adult bee cell capping behavior.