The mechanism of compensation in juvenile hormone synthesis following unilateral allatectomy in Diploptera punctata

Following unilateral allatectomy in the viviparous cockroach, Diploptera punctata, the remaining corpus allatum (CA) can synthesize juvenile hormone (JH) at rates equal to that of a pair of control CA. A single CA undergoes changes in volume and cell number similar in magnitude to those occurring during the normal cycle of JH synthesis. There is no hyptertrophy of the CA associated with compensation in JH synthesis. Therefore, the rate of JH synthesis per cell or per unit volume of single CA following unilateral allatectomy is twice that of a single control CA.     

In vitro effects of juvenile hormone analog on wing disc morphogenesis under ecdysteroid treatment in the female-wingless bagworm moth Eumeta variegata (Insecta: Lepidoptera, Psychidae)

Female adults of the bagworm moth, Eumeta variegata, lack wings completely, whereas male adults of this species have functional wings. We previously found that ecdysteroid induces apoptotic events in the female wing rudiment of E. variegata in vitro, whereas the male wing discs cultured with 20-hydroxyecdysone (20E) underwent apolysis and then cell differentiation. To investigate whether juvenile hormone (JH) in involved in sex-specific cellular response to ecdysteroid during wing development between sexes of E. variegata, we tested the effects of juvenile hormone analog (JHA), methoprene, and 20E on wing disc morphogenesis between sexes in vitro. Using transmission electron microscopy (TEM), we found that both higher concentration of JHA (5 μg/ml) and 20E (1 μg/ml) addition induced cell death (apoptosis) in the male wing discs but not induced cell death in the female wing rudiments in vitro in E. variegata. These culture experiments clearly detected the differential responses of wing discs to JHA under ecdysteroid treatment between sexes. We propose two important hypotheses: (1) JH is not significantly involved in the suppression of the female wing rudiment morphogenesis under 20E treatment, (2) female wing rudiment has lost the ability for cell proliferation in response to the stimulus of 20E.     

Suppression of allatotropin simulates reproductive diapause in the mosquito Culex pipiens

The cessation of juvenile hormone (JH) production is a key endocrine event that halts ovarian development and hence initiates diapause in females of the mosquito, Culex pipiens. The shutdown in endocrine activity of the corpora allata (CA), the source of JH, was manifested in the smaller size of CA in females reared under short daylengths (diapause) compared to those reared under long daylengths (nondiapause), as well as in low expression of the mRNA encoding allatotropin, the neuropeptide that promotes JH biosynthesis in the CA. Genes encoding both allatotropin and allatostatin were identified in C. pipiens, but only expression levels of allatotropin differed in the two types of females. Knockdown of allatotropin mRNA using RNA interference in females programmed for nondiapause resulted in a cessation of ovarian development akin to diapause. This arrest in development could be reversed with an application of JH. Our results thus suggest that suppression of allatotropin is a critical link in regulating the shutdown of the CA during diapause.     

Ultrastructural studies on the fungus,Nomuraea rileyi,infecting the velvetbean caterpillar,Anticarsia gemmatalis

Combined scanning and transmission electron microscopy was used to study the fine structure of the developmental stages of Nomuraea rileyi infecting host larvae of Anticarsia gemmatalis. Larvae were dusted with large numbers of fungal conidia, which germinated and penetrated the cuticle within 6 hr post-treatment. Within 24 hr, penetration hyphae had reached the cuticular epidermis and, via a budding process, initiated the hyphal body stage in the hemocoel. The hyphal bodies, suspended in hemolymph, multiplied and spread throughout the host larvae. By 6–7 days post-treatment, the majority of larvae were mummified. Within 12 hr postmortem numerous conidiophores emerged producing a confluent mycelial mat over the entire cuticular surface. Numerous hydrophobic conidia were formed on phialides present on the aerial conidiophores.     

Ecdysis triggering hormone ensures proper timing of juvenile hormone biosynthesis in pharate adult mosquitoes

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in 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 (or pharate adult) becomes again “competent” to synthesize JH, which would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP₃R-operated mobilization of endoplasmic reticulum Ca²⁺ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.     

Juvenile hormone acid methyltransferase activity in imaginal discs of Manduca sexta prepupae

The occurrence of a peak of juvenile hormone (JH) during the prepupal period has been noted in several lepidopterans. In Manduca sexta and Hyalophora cecropia this peak is known to prevent the precocious onset of adult differentiation in imaginal tissues. However, it has previously been observed in our laboratory that corpora allata (CA) of this age are incapable of making JH owing to a lack of the terminal synthetic enzyme, juvenile hormone acid methyltransferase (JHAMT). Since the CA are required for normal pupation, it is likely that JH acid is the product released by the prepupal CA. Therefore, we analyzed whether JH acid treatment would prevent precocious adultoid differentiation in allatectomized M. sexta larvae. JH acid injections were found to be as effective as JH in normalizing pupation, and acted in a time- and dose-dependent manner. This finding led to a question of whether injected or endogenous JH acid could be methylated to JH. Homogenates of several tissues from prepupae were assayed for the presence of JHAMT. Of the tissues assayed, only imaginal discs possessed significant levels of the enzyme. These results support our previously proposed mechanism for production of the prepupal JH peak in M. sexta.     

Hormonal modulation of reproduction and fertility signaling in polistine wasps

In social insects, it has been suggested that reproduction and the production of particular fertility-linked cuticular hydrocarbons (CHC) may be under shared juvenile hormone (JH) control, and this could have been key in predisposing such cues to later evolve into full-fledged queen pheromone signals. However, to date, only few studies have experimentally tested this “hormonal pleiotropy” hypothesis. Here, we formally test this hypothesis using data from four species of Polistine wasps, Polistes dominula, Polistes satan, Mischocyttarus metathoracicus, and Mischocyttarus cassununga, and experimental treatments with JH using the JH analogue methoprene and the anti-JH precocene. In line with reproduction being under JH control, our results show that across these four species, precocene significantly decreased ovary development when compared with both the acetone solvent-only control and the methoprene treatment. Consistent with the hormonal pleiotropy hypothesis, these effects on reproduction were further matched by subtle shifts in the CHC profiles, with univariate analyses showing that in P. dominula and P. satan the abundance of particular linear alkanes and mono-methylated alkanes were affected by ovary development and our hormonal treatments. The results indicate that in primitively eusocial wasps, and particularly in Polistes, reproduction and the production of some CHC cues are under joint JH control. We suggest that pleiotropic links between reproduction and the production of such hydrocarbon cues have been key enablers for the origin of true fertility and queen signals in more derived, advanced eusocial insects.     

Juvenile hormone and photoperiodically controlled polymorphism in Aphis fabae: Postnatal effects on presumptive gynoparae

Long days (short nights) (LD 16:8) and high temperatures (> 15°C) have an apterizing effect on the short day (LD 12:12) induced, presumptive gynopara of Aphis fabae. Transfer of presumptive gynoparae to long days (15°C) or to 25°C (short days) at varying times during postnatal development demonstrate that the adult form is determined by the second day of the second instar, i.e. 5 days after birth at 15°C. Transfer on day 1 induces maximum apterization with the proportion of aphids affected decreasing with age at transfer.Apterization induced by long days immediately after birth can, to some extent, be cancelled by return to short days but only up to day 4. Thus long days are morphogenetically more potent than short days at the beginning of larval development. At temperatures above 15°C the proportion of aphids apterized increases almost linearly.Apterized insects can be distinguished from juvenilized insects in the fifth-instar. Topical application of juvenile hormone (JH) induces both apterization and juvenilization of presumptive gynoparae but at different times during larval development, JH treatment during the early-instars promotes apterization but induces little juvenilization, whereas maximum juvenilization, without apterization, is produced by middle-instar treatment. The apterizing effects of JH are, thus, not due to its neotenic action.The response profile of JH-induced apterization is similar to that observed with long days and 25°C. It is suggested that such conditions increase endogenous JH levels in A. fabae. The three naturally occurring JH's differ in activity in the order JH I > JH II > JH III. Both long-day and JH-apterized insects switch from the normal ovipara production of the adult gynopara to vivipara production.     

Relationships between frontal‐gland formation and mandibular modification during JH III‐induced presoldier differentiation in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

Termite soldiers engage in colony defense and they possess weapons in order to attack enemies. Defensive strategies vary among species depending on the morphology of the soldier's weapons. Both the frontal gland and elongated mandibles are formed during soldier differentiation of Reticulitermes speratus workers, which is associated with an increase in juvenile hormone (JH) titer. It was shown that the amount of JH applied and coexistence with soldiers affected mandibular elongation of the induced presoldiers. To determine the relationship between applied JH concentration and frontal‐gland development of R. speratus, mandibular modification and frontal‐gland formation of presoldiers induced by 20, 40 and 80 µg JH III in the presence or absence of soldiers were observed. Both presoldier differentiation rates and their mandibular lengths were affected by the presence of soldiers in treatments with 20 and 40 µg JH III. Especially in the treatment with 20 µg JH III, frontal‐pore malformation was observed in some presoldiers. All presoldiers with a malformed frontal pore had almost the same sized mandibles, compared with those of stationarily molted workers. Interestingly, sections of these presoldiers showed that frontal‐gland invagination occurred incompletely in comparison with presoldiers with relatively elongated mandibles. Consequently, frontal‐gland formation might be affected by JH titer (applied and endogenous JH), and individuals with a malformed frontal pore were probably differentiated by JH titers only slightly above the threshold for presoldier differentiation.     

Methoprene does not affect juvenile hormone titers in honey bee (Apis mellifera) workers

Methoprene, a juvenile hormone (JH) analog, is a widely used insecticide that also accelerates behavioral development in honey bees (Apis mellifera). JH regulates the transition from nursing to foraging in adult worker bees, and treatment with JH or methoprene have both been shown to induce precocious foraging. To determine how methoprene changes honey bee behavior, we compared JH titers of methoprene‐treated and untreated bees. Behavioral observations confirmed that methoprene treatment significantly increased the number of precocious foragers in 3 out of 4 colonies. In only 1 out of 4 colonies, however, was there a significant difference in JH titers between the methoprene‐treated and control bees. Further, in all 4 colonies, there was no significant differences in JH titers between precocious and normal‐aged foragers. These results suggest that methoprene did not directly affect the endogenous JH secreted by corpora allata. Because methoprene caused early foraging without changing workers’ JH titers, we conclude that methoprene most likely acts directly on the JH receptors as a substitute for JH.     

Monitoring the effects of juvenile hormones and 20-hydroxyecdysone on yolk polypeptide production of Drosophila melanogaster with enzyme immunossay

ABSTRACT. A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting and quantifying small amounts of yolk polypeptides (YP) in studies on the hormonal control of vitellogenesis in Drosophila melanogaster Meigen. Monoclonal antibodies were incorporated as primary antibodies in the ELISA procedure to ensure selectivity in YP detection. The fact that YP concentration increases immediately after adult eclosion presents some difficulties in designing hormonal regulation experiments. Female adults decapitated immediately after eclosion remain alive for several days and virtually no YP is detected in the haemolymph 24 h after decapitation. The surgical procedure does not interfere with the competence of the fat bodies to respond to exogenous source of hormones. The effect of juvenile hormone (JH) on vitellogenesis can be studied by topical application of test material to these decapitated adults. A juvenile hormone analogue. Methoprene applied at 0.2 μg/fly or greater, restores YP production. The relative potencies of JH I₂ II₃ III and ZR 515 are compared at the same dose of 0.25 μg/fly. Their ranking in terms of re-initiating vitellogenesis is ZR-515 < JH IIFat bodies which are left attached to the body wall, are successfully maintained in culture. With this in vitro system, synthetic hormone can be administered precisely to the organ culture. After a short incubation period, aliquots of medium are removed for the quantification of YP. Incubation of fat bodies with a physiological dose of the 20-hydroxyecdysone (20-HE) stimulates the production and release of YP into the medium. This represents the first direct experimental evidence for 20-HE stimulation of Drosophila fat bodies for YP production in the absence of other endogenous factors that might either promote or interfere with vitellogenesis     

Bioassays of compounds with potential juvenoid activity on Drosophila melanogaster: Juvenile hormone III, bisepoxide juvenile hormone III and methyl farnesoates

Metabolites of the 6,7,10,11 bisepoxide juvenile hormone III (JHB₃), and other potential juvenoids, were tested for juvenile hormone activity using early instar or early stage pupae of Drosophila melanogaster. Importantly, methyl farnesoates were tested as they might have JH-like activity on Dipteran juveniles. Larvae were exposed to compounds in medium, or the compounds were applied to white puparia. In the assays employed in the present study, there was no indication for JH activity associated with the metabolites of JHB₃. The activity of methyl farnesoate (MF) was higher than that of JH III and far greater than bisepoxide JH III. As opposed to the two endogenous juvenile hormones, methyl farnesoate has weak activity in the white puparial bioassay. When fluorinated forms of methyl farnesoate, which is unlikely to be converted to JH, were applied to Drosophila medium to which fly eggs were introduced, there was a high degree of larval mortality, but no evidence of subsequent mortality at the pupal stage. One possible explanation for the results is that methyl farnesoate is active as a hormone in larval stages, but has little activity at the pupal stage where only juvenile hormone has a major effect.     

Morphological and Structural Changes During the Yeast-to-Mold Conversion of Phialophora dermatitidis

The details of the morphological and structural events occurring during yeast-to-mold conversion of the human pathogenic fungus Phialophora dermatitidis as seen by phase-contrast microscopy and electron microscopy are described and illustrated. Budding yeasts growing exponentially were observed to have thin walls and a cytoplasm exhibiting the characteristics of rapidly growing cells including numerous mitochondria, abundant ribosomes, few vacuoles, and little accumulation of storage material. In contrast, thick-walled yeasts were characterized by less apparent or significantly fewer mitochondria and ribosomes and the presence of considerable amounts of storage materials. Microscope observations of yeast-to-mold conversion revealed that only thick-walled yeasts having prominent lipid bodies in their cytoplasm converted to hyphal forms. Typically, the thick-walled yeast formed two to a number of moniliform hyphal cells which in turn often produced true hyphae. The results indicated that yeasts of P. dermatitidis must acquire spore-like characteristics by becoming thick-walled and by accumulating considerable endogenous substrate reserves before they convert and produce hyphae.     

Regulation of vitellogenin synthesis and uptake in the boll weevil, Anthonomus grandis

Abstract. Hormonal factors influencing reproductive development were examined in adult boll weevils, Anthonomus grandis (Boheman) (Coleoptera: Curculionidae). Long-day, high-temperature rearing conditions promote reproduction whereas short-day, low-temperature conditions do not. Implants of corpora allata (CA), brains, or brains plus retrocerebral complexes taken from long-day donors, or hormone analogue treatments were used to examine onset of vitellogenin synthesis and uptake in decapitated bodies of adult weevils reared in short-day, low-temperature conditions. Weevils decapitated within 2 days after eclosion and reared in short-day, low-temperature conditions never initiated vitellogenin production or ovarian development. Females and males decapitated on day 2 showed haemolymph vitellogenin within 5 days following treatment with Juvenile Hormone (JH) analogue or implantation of CA, but not after implantation of brain alone or implantation of muscle (sham). Uptake of vitellogenin into the oocytes did not occur unless both JH analogue and brain were given as replacement therapy. These experiments indicated that JH is necessary and sufficient to stimulate vitellogenin synthesis in this species but that a brain factor must be present for vitellogenin uptake.     

The induction of normal and teratoid viviparae by a juvenile hormone and kinoprene in two species of aphids

ABSTRACT. Topical application of a juvenile hormone (JH1) and the JH mimic, kinoprene, to short-day, ovipara-producers of Megoura viciae usually leads to the production of oviparous/viviparous intermediate forms in the progeny sequence, in place of the expected sexual females (oviparae). The ovaries of these abnormal forms may contain embryos rather than the haploid yolky eggs of oviparae; 'mixed' ovaries containing both haploid eggs and embryos are also observed. The intermediates range in form from winged (alate) to wingless (apterous). The fully alate individuals usually contain only parthenogenetic ovaries but differ from the naturally occurring alate viviparae in that they are invariably infertile, have fewer antennal sense organs and often bear pheromone releasing glands on the metathoracic tibiae. The hormonally induced production of normal viviparae is difficult in this species but has been achieved by rearing short-day aphids on kinoprene-treated bean plants.
In Aphis fabae similar oviparous/viviparous teratomorphs have been reported and, in addition, single JH1 treatments were shown to induce normal viviparae at the end of the progeny sequence. Multiple applications, beginning prenatally and continuing through the postnatal development of the gynopara (winged ovipara-producer), showed that the numbers of viviparae born were related to the earliness of the treatment and to the dosage. The results are compared with the effect of a switch in photoperiod and discussed in relation to the endocrine control of aphid polymorphism.     

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.     

The all-or-none rule in morphogenetic action of juvenile hormone on insect epidermal cells

We have investigated ultrastructural changes in the integuments of larval–adult and larval–pupal intermediates produced by exogenous application of juvenile hormone (JH) analogues in Pyrrhocoris apterus (Hemiptera), and Galleria mellonella and Manduca sexta (Lepidoptera). Ultrastructural analysis of the epidermis of these intermediates always revealed the presence of only two types of epidermal cell, which produced morphologically perfect cuticles of the previous and future developmental stages. There were no intermediate cuticles at the level of individual cells. It has been determined that a single epidermal cell constitutes the lowest elementary unit in the perception and realization of the developmental messages conveyed by JH to its target tissues. Further investigations revealed that the responses of individual epidermal cells to JH were strictly autonomous and qualitative, i.e. they were executed according to the ''yes-or-no'' or ''all-or-none'' rule. The neighbouring epidermal cells could realize independently, side-by-side, the quite dissimilar +JH (somatic growth) or -JH (metamorphosis) developmental programmes, although each of them formed biochemically, functionally, and ontogenetically different structures. The qualitative on- and off- signal given by JH for induction of the stationary (+JH) developmental cycle was limited to relatively short, genetically determined, and stage-specific developmental periods of cellular susceptibility to JH. The mosaic mixtures of the heterochronic, larval–pupal or adult epidermal cells, which we found in different proportions on the bodies of the intermediates, revealed two variable, development-related factors: (i) the presence or absence of a minimum effective concentration of JH, and (ii) positive or negative sensitivity of a particular epidermal cell to JH.     

Altered juvenile hormone metabolism, reproduction and stress response in Drosophila adults with genetic ablation of the corpus allatum cells

Juvenile hormone (JH), which controls many developmental and physiological processes in Drosophila melanogaster, is synthesized de novo in the specialized endocrine glands, corpus allatum (CA). The present study concerns JH metabolism, reproduction and stress resistance in Drosophila with genetic ablation of a part of CA cells. The correlated regulation of JH biosynthesis and degradation in Drosophila adults has been found: ablation of CA cells led to (1) a dramatic decrease in activity of the key regulatory enzyme of JH biosynthesis, juvenile hormone acid methyl transferase and (2) a considerable increase in JH-hydrolyzing activity. It has been also shown that ablation of CA cells caused three significant physiological changes: (1) an increase in the intensity of response of JH degradation system to heat stress; (2) a disturbance of reproduction; (3) a decrease in stress resistance. Pharmacological rise of JH level rescued JH-hydrolyzing activity, fecundity and stress resistance in CA-ablated females. Pronouncedly, all the physiological effects caused by CA ablation were significant in females but not in males indicating a sexual dimorphism of JH physiological roles in Drosophila adults.