Hydrolytic degradation of juvenile hormones in haemolymph and corpora allata of Galleria mellonella (L).

Hydrolytic rates of juvenile hormones (JHs) I, II and III by the corpora cardiaca-corpora allata complex (CC-CA) and by the haemolymph of Galleria mellonella remain in the same order (III greater than I greater than II in CC-CA and I greater than III greater than II in haemolymph) throughout the last larval instar. Haemolymph hydrolytic activity shows peak at the end of feeding when 80 pmol JH I versus 15 pmol JH II is degraded per 1 microliter and minute; hydrolysis rapidly declines in the apolysing insects. Hydrolytic rates in CC-CA reach a maximum of 240 fmol/pair per min for JH III and 85 fmol/pair per min for JH II in pharate pupae. Brain implantations or chilling of freshly ecdysed last instar larvae, which are known to elevate JH titer and induce supernumerary larval molt, do not affect JH hydrolysis. The results indicate that the dominance of JH II in Galleria may be at least partly controlled by preferential hydrolysis of homologs I and III.     

Caste specific modulation of juvenile hormone titers in Apis mellifera

The titers of JH III were studied in the larval and pupal stages of the two female honey bee castes, the queen and the worker. Whereas the early larval stages, L3 and L4, had to be pooled, all the last instar larvae, pupae, and newly hatched adults, were titered individually. The queen stages produce two-fold higher JH III titers in comparison with the worker stages. Both have relatively high titers during the early larval instars, decreasing from an average of 450 pmol/g at L3 to about 20 pmol/g in the queen and 75 pmol/g at L3 to 5 pmol/g at L5 in the worker. Both castes build up another JH III peak at the end of their spinning phase when entering the pharate pupa stage, with about 200 pmol/g in the queen and 60 pmol/g in the worker. No JH III was found in the pupal stage; the queen only develops a new JH III titer in the late pupal stage.     

Degradation of juvenile hormone and methylation of juvenile hormone acid by corpora cardiaca–corpora allata of the cockroach,Nauphoeta cinerea: II. Physiological aspects

In vitellogenic females of Nauphoeta cinerea, injected (10R)-juvenile hormone (JH) III was degraded more rapidly than racemic JH III: we measured a half-life of 21 min (with or without coinjection of lipophorin) for the former and 24 min (with coinjection of lipophorin) and 43 min (without coinjection of lipophorin) for the latter. One to two hours after injection, JH III acid was the major metabolite observed; in addition, several highly polar products were found. The half-life of injected racemic JH III acid was 19 min with coinjection of lipophorin and 4 min without. The JH III acid titer in hemolymph was low (around 5–10 pmol/ml) in last instar larvae and previtellogenic and pregnant females and reached higher values (40–100 pmol/ml) in vitellogenic and ovulating females. Racemic JH III acid could be methylated in vitro to JH III by corpora cardiaca–corpora allata (CC-CA) from penultimate instar larvae and females at stages between adult ecdysis and ovulation and at the very end of pregnancy, but not by CC-CA from last instar larvae and adult females at earlier stages of pregnancy. This indicates that CC-CA are capable of methylating JH III acid only at stages when JH III is detectable in the hemolymph. In double-labelling experiments with CC-CA from vitellogenic females and L-[¹⁴C]methionine and [³H]JH III acid as precursors, we observed that only a small proportion (1–8%) of total biosynthesized JH III was derived from JH III acid when the latter was present at physiological concentration. This suggests that in vivo recycling of JH III acid by CC-CA plays only a minor role in the regulation of the titer of JH III and JH III acid.     

Influence of the braconid Glyptapanteles liparidis on the juvenile hormone titer of its larval host,the gypsy moth,Lymantria dispar

The increase in the juvenile hormone (JH) III titer in the hemolymph of Lymantria dispar larvae that were parasitized by the endoparasitoid braconid, Glyptapanteles liparidis, during the host's premolt to third instar, coincided with the molt of the parasitoid larvae to the second instar between day 5 and 7 of the fourth host instar. It reached a maximum mean value of 89 pmol/ml on day 7 of the fifth instar while it remained below 1 pmol/ml in unparasitized larvae. Only newly molted fifth instar hosts showed a low JH III titer similar to that of the unparasitized larvae. JH II, which is the predominant JH homologue in unparasitized gypsy moth larvae, also increased relative to controls in the last two samples (days 7 and 9) from parasitized fourth and fifth instars. Compared to unparasitized larvae, a generally reduced activity of JH esterase (JHE) was found in parasitized larvae throughout both larval stages. The reduction in enzyme activity at the beginning and at the end of each instar, when the JHE activity in unparasitized larvae was high, may be in part responsible for the increased JH II and JH III titers in parasitized larvae. Ester hydrolysis was the only pathway of JH metabolism in the hemolymph of unparasitized and parasitized gypsy moth larvae as detected by chromatographic assays. © 1996 Wiley-Liss, Inc.     

In vivo fluctuation of JH,JH acid,and ecdysteroid titer,and JH esterase activity,during development of fifth stadium Manduca sexta

Juvenile hormone (JH), JH acid, and ecdysteroid titer, and JH esterase activity, were measured in hemolymph from synchronous last stadium larvae of Manduca sexta. JH and JH acids were identified and quantified by GC-MS: JH I and II (and the corresponding acids) were the predominant JH homologs detected in males or females. Maximum levels of JHs and JH acids were observed just following ecdysis to the fifth (last) stadium (day 0, 0 hr) and at the prepupal stage (day 6–day 7). JH titer (≥ 1 ng JH I or II/ml) was higher than JH acid titer (∼0.7 ng JH I acid or JH II acid/ml) in very early fifth stadium larvae. However, this was reversed at the prepupal stage when higher titers of JH acids than JH were observed. JH acid titer began to rise prior to JH titer at the prepupal stage. JH esterase activity rose significantly only after JH or JH acid titers had begun to decline; maximum JH esterase activity was observed at day 3 and day 8. Ecdysteroid titer (measured by RIA) decreased during the last larval molt to a low level by day 0 (0 hr) and to undetectable levels at day 0 (12 hr) of the fifth stadium, by which time JH and JH acid levels had also declined substantially. Just prior to wandering, a small ecdysteroid peak was noted and a slightly elevated level of ecdysteroid was maintained for a further 2 days before a surge in ecdysteroid titer occurred at the prepupal stage, in synchrony with JH and JH acid titer maxima. There was no sexual dimorphism in timing or magnitude of JH, JH acid, and ecdysteroid titer or JH esterase activity.     

Juvenile hormone biosynthesis by corpus cardiacum-corpus allatum complexes of larval Lymantria dispar

• 1.1. A radiochemical assay was used to examine juvenile hormone (JH) synthesis and secretion in vitro by incubating two pairs of larval corpus cardiacum-corpus allatum complexes (CC-CA) from, Lymantria dispar, in 50 μl of osmotically balanced Grace's medium containing 1 μC1 [³H-methyl]-methionine for 6 hr.
• 2.2. For CC-CA of fourth instar female larvae, maximal incorporation of ³H-methyl was 0.15 pmol/pr/hr between days 2 and 3. High pressure liquid chromatographic (HPLC) analysis suggested that the biosynthetic products are mainly JH III with a little JH II at times.
• 3.3. For CC-CA of last instar female larvae, incorporation of ³H-methyl was 0.48 pmol/pr/hr at the beginning of the stadium and decreased to negligible levels by day 10. HPLC analysis suggested that CC-CA of last instar larvae produced only JH III. Volume increases in CA during the last instar were associated with declining activities of JH secretion.
• 4.4. Comparisons of maximal rates of ³ H-methyl incorporation by each unit volume of CA revealed that in the last instar each unit volume (μm³) of glandular tissue secreted 50% more JH than in the fourth instar.

     

Correlations between juvenile hormone esterase activity,ecdysone titre and cellular reprogramming in Galleria mellonella

Juvenile hormone esterase (JHE) activity, ecdysone titre, and developmental competence of the epidermis were determined in last instar larvae and pupae of Galleria mellonella. Haemolymph JHE activity reaches a peak before increases are observed in ecdysone titre both during larval-pupal and pupal-adult metamorphosis. JHE activity is low during the penultimate larval instar although general esterase activity is relatively high. In last instar larvae two ecdysone peaks are noted after the increase in JHE activity. Furthermore, epidermal cell reprogramming occurs just after the increase in haemolymph JHE activity and possibly before the first increase in ecdysone titre. This was tested by injection of high doses of β-ecdysone into last instar larvae of different ages resulting in rapid cuticle deposition. Reprogramming occurred if the resulting cuticle was of the pupal type. These correlative observations may increase our understanding of the relative importance of an ecdysone surge in the absence of JH in reprogramming of the insect epidermis.     

Parasitism-induced effects of Glyptapanteles liparidis (Hym., Braconidae) on the juvenile hormone titer of its host, Lymantria dispar: the role of the parasitoid larvae

Parasitization by the gregarious larval endoparasitoid Glyptapantles liparidis induces a dramatic increase in the hemolymph juvenile hormone (JH) titer (especially JH III) of its host larva, Lymantria dispar. Here, we investigated the role of the parasitoid larvae in JH synthesis and release by in vitro and in vivo experiments. GC-MS analyses confirmed that the rising hemolymph JH titer coincided with the time at which the parasitoids molt to the second larval instar. Peak values in host hemolymph titers were observed prior to parasitoid emergence, and titers dropped to negligible levels within 24 h after parasitoid emergence. Whole body extracts from excised second instar parasitoids yielded JH III and trace amounts of JH II. The in vitro secretory activity of the corpora allata (CA) of L. dispar larvae was not enhanced by parasitization. When the host's CA were separated by neck ligation, we found elevated JH III titers, but no JH II in the hemolymph of the posterior section, which contained the parasitoids. Parasitoids that were kept in in vitro culture produced and released only JH III. The parasitoids’ ability to secrete JH and to molt independently from their host's molting cycles indicates that at least second instar parasitoids are hormonally self-reliant.     

Titres of juvenile hormone I,II and III in Spodoptera littoralis (Noctuidae) from the egg to the pupal moult and their modification by the egg-larval parasitoid Chelonus inanitus (Braconidae)

Physico-chemical analysis of juvenile hormones (JHs) of Spodoptera littoralis revealed highest quantities in the second half of embryonic development and in newly hatched 1st instar larvae. At these stages, mostly JH II, JH I and little JH III were found, while in later stages only JH II and JH III were found. Titres fluctuated in a similar manner in all larval instars, being lowest during the moults. In last (=6th) instar larvae, JHs disappeared in the late feeding-digging stage and again increased in the early prepupal stage. Parasitisation with Chelonus inanitus, a solitary egg-larval parasitoid which induces in its host the precocious onset of metamorphosis in the 5th instar, did not alter JH homologue composition but led to a disappearance of JHs in the 5th instar. Implantation of a parasitoid larva into early 5th instar larvae containing polydnavirus/venom caused a drop in the JH titre which indicates that the parasitoid larva plays an important role in the manipulation of the host's JH titre. In the parasitoid larva, only JH III was found; titres were highest in the 2nd larval instar, a stage when the host is in the 5th instar and contains almost no JHs. Thus, JHs of the parasitoid and the host fluctuate in an independent manner.     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

A putative new juvenile peptide hormone in lepidopteran insects.

A growth-blocking peptide (GBP) with repressive activity against juvenile hormone (JH) esterase has been isolated from the last (6th) instar larval plasma of the armyworm Pseudaletia separata (Lepidoptera: Noctuidae) parasitized by the parasitoid wasp Apanteles kariyai (Hymenoptera: Braconidae) (1,2). This study demonstrates that GBP not only exists in the plasma of parasitized last instar larvae, but also in the plasma of unparasitized penultimate (5th) instar larvae, while the plasma of last instar larvae does not contain any detectable amount of GBP. The detection of GBP in unparasitized penultimate instar larvae, before the final larval molt, demonstrates that this factor is naturally occurring in the insect larva before the last larval instar and is seemingly coordinating, along with JH, the regulation of juvenile characteristics. This finding suggests the existence of a new type of juvenile peptide hormone in lepidopteran insects.     

Haemolymph juvenile hormone esterase during the life cycle of the tobacco hornworm,Manduca sexta (L.)

Juvenile hormone (JH) esterase activity was found in the plasma of larvae, pupae and adults of wild-type tobacco hornworms, Manduca sexta. There was a single peak of plasma JH esterase activity approx. 28 h prior to ecdysis in each instar from the second through the fourth instar and a peak of activity prior to both wandering and pupation in the fifth (last) instar. JH esterase activity was high in newly formed male and female pupae but declined to minimal levels by day 1 of the pupal stage. For the remainder of the pupal period, activity was at background levels. JH esterase activity increased again in newly emerged, virgin male and female adults but declined and remained at a low level 1 day after emergence through death. Gel filtration analysis of larval, pupal and adult plasma resolved a single peak of JH esterase activity with an apparent molecular weight of 66,000. However, isoelectric focusing revealed three forms with isoelectric points of 5.5, 5.8 and 6.1. These isoelectric forms were also found in black and white mutants of last instar M. sexta and in purified JH esterase from wild-type larvae. The plasma JH esterase activity metabolized JH I 2–3 times faster than JH III and was sensitive to inhibition by octylthio-1,1,1-trifluoro-2-propanone and insensitive to O,O-diisopropyl phosphorofluoridate. Gel filtration, isoelectric focusing, substrate specificity and developmental studies suggest that the same JH esterases are found in the plasma of larvae, pupae and adults and appear to be different from general (α-NA) esterase.     

Effects of ultralow doses of fenoxycarb on juvenile hormone–regulated physiological parameters in the silkworm,Bombyx mori L.

Effects of fenoxycarb at ultralow doses were investigated on juvenile hormone (JH)–regulated parameters in the silkworm, B. mori. Like JH, this non-terpenoid carbamate is able to induce permanent larvae in the last larval instar. However, whereas micrograms of JH are needed to produce this effect, only a few picograms of fenoxycarb are necessary to induce the same effect. The effects of fenoxycarb observed in this study were only visible from day 4 of the last larval instar—that is, when the JH titer has dropped to undetectable levels and JH-repressed physiological parameters would naturally be expressed. We observed that the permanent larvae induced with low doses of fenoxycarb (100 pg/larva) had no 20-hydroxyecdysone (20E) peak. Their prothoracic glands (Pgs) were completely inactive and very weakly sensitive to prothoracicotropic hormone (PTTH). Fenoxycarb at doses of 1 ng/larva also significantly inhibited silk gland growth and coloration, whereas carotenoid content of the hemolymph was maintained at high levels, which could reflect an inhibition of its uptake by the silk glands. Total hemolymph protein levels in last instar larvae were also depressed at these doses. So, it seems that low doses of fenoxycarb are sufficient to maintain in a juvenilized status the physiological parameters that are normally expressed when JH titer has declined. Moreover, from an endocrinological viewpoint, we demonstrated that the corpora allata (CA) are not necessary for fenoxycarb to induce those effects and discussed its possible mode of action. Arch. Insect Biochem. Physiol. 37:178–189, 1998. © 1998 Wiley-Liss, Inc.     

Haemolymph ecdysteroids in the solitary and gregarious larvae of Schistocerca gregaria

In the penultimate and last instar larvae of Schistocerca gregaria, 20-hydroxyecdysone (20E) makes up 74–84% of detected ecdysteroids in the females, and 63–74% in the males. Remaining ecdysteroids include ecdysone, a compound with HPLC and TLC retention times of makisterone A, and highly polar metabolites. Except for the last instar females, the contents of ecdysone and the unknown compound are higher in the solitary phase, while that of polar metabolites is higher in the gregarious phase. The phases also differ in that the molt-inducing ecdysteroid peaks last longer in the gregarious than in the solitary larvae. Peak concentrations reach 3.0–4.0 μg 20E equiv./ml in penultimate female instar, 2.5–3.0 μg/ml in penultimate male instar, and 1.5–2.0 μg/ml in the last larval instar of both sexes. © 1996 Wiley-Liss, Inc.     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

Juvenile hormone changes associated with diapause induction, maintenance, and termination in the beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae)

At 22°C and under a long-day photoperiod of L:D 16:8, all the last fifth instar Loxostege sticticalis larvae undergo prepupal stage and pupate without diapause. Under a short-day photoperiod of L:D 12:12, in contrast, they all enter diapause with approximately 36 days diapause maintenance and then terminate diapause spontaneously, although only 44% of the larvae terminated diapause successfully. Changes in hemolymph juvenile hormone (JH I) titers of diapause-destined larvae across diapause induction, maintenance and termination were examined using HPLC, and were compared with those of non-diapause-destined larvae from the fifth instar through pupation. JH I titer of the earliest fifth instar diapause-destined larvae remained at a high level with a peak of 220.4 ng/ml, though it decreased continuously to a minimum of 69.0 ng/ml on day 5 in the fifth instar when the larvae stopped feeding to enter diapause. During the diapause maintenance, JH I titer of the mature larvae increased significantly and maintained a high level until day 31 in prepupae. JH I titer declined and fluctuated at low level from 5 days before pupation. In contrast, JH I titer of both the fifth instar non-diapause-destined larvae and prepupae remained and fluctuated at low level consistently, as well as decreased before pupation. These results indicate that diapause induction and maintenance in this species might be a consequence of high JH, whereas diapause termination can be attributed to low JH titer, which was in agreement with the hormonal regulation observed in many other larval-diapausing insects.     

Endocrine alteration and precocious premetamorphic behaviors in the greater wax moth larvae,Galleria mellonella,parasitized by an endoparasitoid,Apanteles galleriae

Parasitization of Galleria mellonella (Lepidoptera: Pyralididae) larvae by a larval endoparasitoid Apanteles galleriae (Hymenoptera: Braconidae) leads to the precocious expression of premetamorphic behavior in the sixth (normally penultimate) instar host larvae prior to the parasitoid's emergence. We investigated the role of parasitization with A. galleriae on the alteration of development and/or behavior of its host. The ecdysteroid titer in the hemolymph of parasitized sixth instar larvae (the last instar of parasitized larvae) was higher than that of unparasitized ones, and the high ecdysteroid concentrations induced premetamorphic behaviors such as wandering and cocoon spinning. However, the epidermis of the parasitized larvae was not pupally committed through this stage. The activity of JH esterase in the parasitized larvae remained low, and application of a JH analogue to these larvae caused the production of a larval-type cocoon. These facts suggest that the parasitization by A. galleriae induces precocious premetamorphic behaviors of G. mellonella larvae by changing host endocrine conditions without causing the typical larval-pupal metamorphosis. Arch. Insect Biochem. Physiol. 34:257–273, 1997. © 1997 Wiley-Liss, Inc.