Effect of lighting conditions on endocrine events in Galleria mellonella

Galleria mellonella larvae reared in a light:dark (LD) 12:12 regime terminate feeding and pupate 1 day earlier than insects kept in constant darkness. Rearing conditions have no influence on the body weight attained at pupation. In both rearing conditions body weights attained by females were greater on the average by 50% than the body weights of males.In LD 12:12 all distinctive changes in the juvenile hormone titre and juvenile hormone esterase activity occurred 1 day earlier than in constant darkness. The peak value of juvenile hormone esterase activity was by 22% higher in insects reared under light conditions as compared with animals kept in constant darkness.Last-instar G. mellonella larvae reared in constant darkness were more sensitive to cooling and juvenile hormone analogue application. The chilling-induced elevation of juvenile hormone titre was also higher in constant darkness. The chilling-induced elevation of the brain allatotropic activity was not influenced by rearing conditions (brain activity measured as the number of extra-larval moults produced by hosts implanted with chilled brains). However, rearing in an LD 12:12 regime decreases the host sensitivity to implanted brains.     

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.     

Effects of anti-juvenile hormone “ETB” on the development and metamorphosis of the silkworm, Bombyx mori

As in the tobacco hornworm Manduca sexta, the synthetic juvenile hormone analogue ETB (ethyl 4-[2-(tert-buthylcarbonyloxy)butoxy]benzoate) showed both juvenile hormone-like and anti-juvenile hormone activities in the silkworm, Bombyx mori. When ETB was topically applied to allatectomized 4th-instar larvae, the compound counteracted the effects of allatectomy, such as induction of precocious metamorphosis and black pigmentation in the larval markings. Therefore, ETB had juvenile hormone activity, but it could neither induce brown pigmentation in the markings nor induce an extra-larval moult as can juvenile hormone.When intact 3rd-instar larvae were treated with the compound, the majority underwent precocious metamorphosis in the 4th-instar, and later formed fertile miniature adults. Some moulted into larval-pupal intermediates or 5th-instar larvae with darkened larval markings and/or with abnormality of specific regions of the silk-gland. The optimal dose for such anti-juvenile effects was about 1–10 μg/larva, and higher doses showed less activity. Such anti-juvenile hormone effects of ETB were counteracted by administration of the juvenile hormone analogue, methoprene, before a certain critical time in the 4th-instar. The corpora allata of treated larvae appeared cytologically normal, and the corpora allata from ETB-induced miniature moths secreted juvenile hormone when implanted into allatectomized 4th-instar larvae.     

Lepidopteran anti-juvenile hormones: Effects on development of Apanteles congregatus in Manduca sexta

Parasitism by the braconid wasp Apanteles congregatus decreases the effectiveness of the anti-juvenile hormone agents ETB (ethyl 4-[2-{ittert-butyl carbonyloxy}bytoxy]benzoate) and fluoromevalonolactone (FMev) in inducing precocious metamorphosis of Manduca sexta larvae. Topical application of 1–200 μg ETB to parasitized third-instar larvae had no effect on either host or parasite development, whereas doses of 50μg or more ETB applied to unparasitized third-instar larvae caused formation of larval-pupal intermediates after the fourth instar. Parasitism also decreased the effectiveness of 100–200 μg FMev in causing metamorphosis at the moult following its application. In contrast to ETB, FMev disrupted development of the parasitoids. No wasps emerged when preterminal stage hosts were treated with FMev and the hosts formed larval-pupal intermediates. After treatment of terminal stage hosts with FMev, the number of emerging parasitoids was reduced by one-third. Precocene II (100 μg per larvae) had no effect on development of either M. sexta or A. congregatus.     

Factors influencing the developmental capacity of Galleria larval epidermis

The nature of the cuticle secreted by integument from a day-1 penultimate instar larval Galleria when cultured in vivo in the abdomen of a last instar larva varied with the age of the host. When placed in a day-5 last instar larva, the implanted integument secreted a pupal cuticle at the time the host metamorphosed and became a pupa. However, when placed in a day-7 last instar larva the implant, from the same stage donor, secreted a larval cuticle at the time the host pupated. Experimental studies involving implantation of the integument for a 24 hr period, into various developmental stages of normal and ligated last instar larvae, pupae, and pharate adults, prior to placing it in a day-7 last instar larva suggest that a non-hormonal factor present in day-4 and -5 last instar larvae is important to initiate pupal syntheses.     

Annual Changes in the Greater Wax Moth (Galleria Mellonella L.) Development and Sensitivity to Juvenilizing Treatments

The greater wax moth (Galleria mellonella L.) larvae reared in constant conditions showed endogenous annual changes in the sensitivity to juvenilizing treatments, i.e. cooling and JHA administration. Also control, untreated larvae showed annual changes in normal development. The number of spontaneously appearing extra-larval molts, the number of animals entering the state of permanent larva, as well as the sex-ratio in Galleria population changed with respect to the season of the year. The possible mechanisms involved in these phenomena are discussed.     

Disruptive developmental effects of benzyl-1,3-benzodioxole derivatives on unparasitized and parasitized Manduca sexta larvae

Treatment of tobacco hornworm larvae with the benzyl-1,3-benzodioxole derivative J-2710 immediately after ecdysis to the fourth instar disrupted development either during the moult to the fifth instar or shortly thereafter. Larvae given topical applications of 100 μg J-2710 in 1 μl acetone suffered 100% mortality, often after secreting moulting fluid in large pockets between the epidermis and the cuticle later in the fourth instar. Larvae that successfully ecdysed had abnormalities of the mouthparts and cervix that interfered with normal feeding, inhibiting growth in the fifth instar. Larvae of the gregarious endoparasitic wasp Cotesia congregata (=Apanteles congregatus) frequently failed to emerge from host Manduca sexta larvae treated with high doses of J-2710, particularly when the host failed to feed normally. Less potent disruptive effects on Manduca and Cotesia were seen after treatment of larvae with the derivatives J-3370 and J-2581.No anti-juvenile hormone action of J-2710 was observed. J-2710-treated M. sexta larvae showed no precocious metamorphosis and the developmental effects of J-2710 were not prevented by co-application of the juvenile hormone analogue methoprene in doses ranging from 1 to 100 μg/larva. Moreover, J-2710 had no effect on the action of methoprene in the black larval assay for juvenile hormone-like activity, unlike results reported to occur using the Galleria wax wound assay.     

Instars ofMicroplitis rufiventris [Hym.: Braconidae] and their relative developmental speed under different photoperiods

The internal parasiteMicroplitis rufiventris Kok. passes through 3 instars but moults 3 times within its host. The last moult occuring just at emergence time. The morphology of the egg and larval stages of the parasite are discussed. At 27°C and a photoperiod of 6 h (6L:18D) the endo-developmental cycle of the parasite can summarize as follows: Egg 18–24 h; instar 1,4 days (fighting phase 48 h; feeding phase 30–48 h); instar 2, 12–18 h and instar 3,3 days. The effect of different photoperiods on the relative speeds of the endo-developmental stages of the parasite at each of 30, 25, 20°C were carefully studied. At the first 2 temperatures, the short photoperiod (6L:18D) accelerated the development of larval instars, while both of 18L:6D or 0L:24D slowed down the development. Under the latter photoperiods some larvae failed to moult and had emergence problems. The influence of photoperiod is significantly noticeable at 20°C. The incubation period of the egg-stage was prolonged significantly at 18L:6D and the development of larval instars was significantly faster and refined at 6L:18D. The factor(s) inhibiting the development of the egg-stage perhaps differ from those affecting the larval development. The ventral area of the host mid-gut among malpighian tubes seems to be where the surplus parasite larvae are eliminated by physical attack. A physiologically suppressed parasite larva is able to attack its developed competitor of the same age. Teratocytes cells perhaps play a part in eliminating the surplus parasite larvae by physiological suppression.     

Chilled Galleria mellonella larvae: mechanism of supernumerary moulting

ABSTRACT. Supernumerary larval instars were produced when Galleria mellonella L. (Lepidoptera) larvae were chilled at 0°C. Although sensitivity to cooling stress of the last instar and younger larvae were generally the same, only penultimate and the last instar larvae showed a significant correlation between their age and the number of additional larval moults. Chilling stress induced a rapid and persistent increase in the JH titre of the last instar larvae. Severing the ventral nerve cord resulted in a predictable loss of the ability to produce supernumerary moults in chilled last instar larvae. The data suggest that sensory input stimulates allatotropic hormone secretion by the brain of chilled larvae. The possible mechanism controlling supernumerary moulting is discussed.     

Stage dependent influences of polydnaviruses and the parasitoid larva on host ecdysteroids

In the solitary egg-larval parasitoid Chelonus inanitus (Braconidae) both polydnavirus and the parasitoid larva manipulate host development. Parasitization leads to a premature drop in juvenile hormone titre and a precocious onset of metamorphosis in the 5th larval instar. The C. inanitus bracovirus (CiBV) alone causes a reduction in host ecdysteroid titres at the pupal cell formation stage and prevents pupation. Here we report three new findings. (1) We show that parasitization causes a reduction in haemolymph ecdysteroid titre immediately after the moult to the 5th instar; similarly low values were seen in nonparasitized larvae after the moult to the 6th instar. These data along with parasitoid removal experiments indicate that the low ecdysteroid titre after the moult is a very early sign of the upcoming metamorphosis. (2) In vitro experiments with prothoracic glands and brain extracts showed that CiBV affects both prothoracic glands and prothoracicotropic hormone after the stage of pupal cell formation. (3) In the haemolymph of parasitized larvae the ecdysteroid titre increased in the late cell formation stage, i.e. immediately before egression of the parasitoid. In vitro experiments showed that late 2nd instar parasitoids release ecdysteroids and are thus very likely responsible for the rise in host ecdysteroids.     

Neuroendocrine regulation of corpus allatum activity in Manduca sexta: The endocrine basis for starvation-induced supernumerary larval moult

When newly-ecdysed 5th instar larvae of Manduca sexta were starved for 3 days and thereafter fed on standard diet the majority (90%) of the surviving larvae moulted into 6th instars. Allatectomy prior to starvation abolished the supernumerary moult, while denervation of the corpora allata (CA) had no effect.Cautery of medial neurosecretory cells, but not of the lateral cells, prevented supernumerary moulting and pupation ensued. Transplantation of brains from young 5th instar donors into larvae, whose medial neurosecretory cells were cauterized prior to starvation, restored the extra larval moult. Neither CA nor corpora cardiaca (CC) could be substituted for the medial neurosecretory cells.For induction of the supernumerary moult the medial neurosecretory cells are required only until day 1 after refeeding whereas the CA are required until day 3 after refeeding. Allatectomy on day 3 after refeeding resulted in the production of black 6th instar larvae.We conclude that starvation-induced supernumerary moulting is due to activation of the CA by allatotropin produced by medial neurosecretory cells in the brain. The anteromedial cells (group II) appear to be the source of allatotropin.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

The occurrence of the second moult of Ascaris lumbricoides and Ascaris suum

Maung M. 1978. The occurrence of the second moult of Ascaris lumbricoides and Ascaris suum. International Journal for Parasitology 8: 371–378. Eggs of Ascaris lumbricoides and A. suum were cultured at 28°C and observed daily. Larvae were released by pressure, by artificial hatching with CO₂, and by natural hatching after infection of laboratory mice. The early stages of development in the egg were observed to comprise two moults, one occurring immediately after the other. Both moults were initiated within the egg, but the time of completion of the second moult varied considerably, and in some instances was not completed until the larvae reached the liver of experimentally infected animals.     

Control of prothoracic gland activity in larvae of Galleria mellonella

Prothoracic glands of last instar wax moth larvae maintain spontaneous secretory activity both in decapitated larvae and in isolated abdomens into which they have been transplanted, as judged by their ability to induce secretion of a new cuticle. Their activity is hormonally stimulated by the brain and inhibited by the prothoracic and mesothoracic ganglia. The subesophageal ganglion seems to suppress the inhibitory influence of the thoracic ganglia. The prothoracic glands of larvae decapitated at different times during the last instar all respond to brain implantation, and this response does not change when brains are implanted at increasing intervals after decapitation. The prothoracotropic activity of the isolated brain is highest in brains of pupae and adults but is relatively and consistently low in brains of last instar larvae. The results demonstrate that the control of prothoracic glands is a complex process governed by the nervous integration of various stimuli.     

The effects of cholesterol on the development of Heliothis zea

Heliothis zea was reared on an artificial diet, which lacked supplementation with plant materials, in order to determine the effects of cholesterol on the development of this insect. A number of parameters of larval development were found to be dependent upon the concentration of dietary sterol including: the number of moults which the larvae completed within a particular time interval, the ability of the larvae to pupate and the survival of the larvae. The number of moults which a larva completed prior to pupation, though, was independent of the concentration of sterol.     

Factors affecting change in sensitivity of prophoracic glands to juvenile hormone in Mamestra brassicae

The prothoracic glands of the early last-instar larva of Mamestra brassicae (day 0–3) were found previously to be insensitive to stimulation by juvenile hormone, whereas those later in the instar (from day 4 on) were activated by this hormone. When neck-ligatured young larvae (day-1, day-2 and day-3) were given juvenile hormone 5–10 days after ligation, pupation was induced. Similarly, juvenile hormone induced pupation of isolated abdomens which contained prothoracic glands taken from neck-ligatured day-3 larvae 5 days after ligation. If the glands were exposed to prothoracicotropic hormone (PTTH) from implanted brains before they were transplanted to isolated abdomens, their sensitivity to juvenile hormone activation was enhanced. Ecdysone but not 20-hydroxyecdysone given every 3 hr for 12 hr also slightly enhanced sensitivity. These results suggest that prothoracic glands from either day-1, day-2 or day-3 larvae can slowly acquire a sensitivity to juvenile hormone activation by prolonged incubation in the absence of factors from the head. The acquisition of sensitivity occurs more rapidly in the presence of both a factor from the brain, presumably PTTH, and ecdysone released from the prothoracic glands themselves.     

Factors influencing juvenile hormone esterase activity in the wax moth, Galleria mellonella

The effects of juvenile hormone, antiallatotropins, selected surgical procedures and starvation on the juvenile hormone esterase levels in Galleria larvae and pupae were investigated. JH reduced JH esterase activity in larvae but induced the enzyme in 1-day-old pupae. In vitro studies confirmed that the peak of synthesis and/or release of JH esterase from the fat body of last instar larvae occurred 4 days after ecdysis. These studies also showed that fat body from JH-treated larvae released much less enzyme than controls. Antiallatotropins, precocene 2 and ZR 2646 also reduced JH esterase levels in larvae, but ZR 2646 induced JH esterase in pupae. In starved larvae, JH esterase did not increase during the first five days. A minimum of 36 hr of feeding was necessary for the larval esterase activity to increase on schedule on day 4 of the last larval stadium. When day-l larvae were ligated behind the head or the prothorax, they had lower JH esterase levels and yet showed a slight increase in the enzyme when the larvae reached the age of 4 days. The significance of these results is discussed in relation to the possible control of esterase activity during metamorphosis.     

Some aspects of activity regulation of Galleria mellonella PTTH cells

Using the Galleria prothoracicotropic bioassay, five small neurosecretory cells occurring in each dorsolateral part of protocerebrum of Galleria mellonella brain were identified as prothoracicotropic hormone (PTTH) cells. It was found that the critical period for the release of PTTH from a brain implanted in neck-ligated larva lasts up to the third day after implantation. The content of paraldehyde-fuchsin positive neurosecretory material (NSM) in PTTH cells was determined during the penultimate and last larval instar, during pupal instar, and in starved or poststarvation fed or space-deprived last instar larvae. Two peaks of NSM in PTTH cells were found in the penultimate instar (in freshly molted, and 76-h-old larvae), four peaks in the last instar larvae (in freshly molted, and in 67-, 132-, and 174-h-old larvae), and one peak in the pupal instar (in 56-76-h-old pupae). It was also observed that upon starvation NSM accumulated in PTTH cells, while after 3 h of poststarvation feeding it was released. In permanent space-deprived last instar larvae no NSM occurred in PTTH cells. In all investigated larval instars a rapid release of NSM from PTTH cells was found a few hours after molt associated with the beginning of the feeding period. The significance of the NSM content in PTTH cells is discussed in relation to ecdysteroid titer.     

In vitro bioassay for the brain allatotropic hormone of Galleria mellonella (Lepidoptera).

An in vitro sensitive bioassay for the Galleria mellonella brain allatotropic hormone (ATTH) was developed. This assay measures the rate of juvenile hormone (JH) synthesis in corpora cardiacacorpora allata complex (CC-CA) stimulated in vitro by ATTH released from the brain during short-term in vitro incubation, or by ATTH extracted from the tissue with methanol. CC-CA of the late VIth instar (VI3) larvae were used for assessment of ATTH. The maximum activation of test CC-CA by ATTH occurred at a concentration of 2 brain equivalents (per 100 ul medium). The highest ATTH activity was exhibited by the brains of chilled VII1 larvae: ATTH extracted from freshly dissected brains, or ATTH released from these brains during 6 h in vitro incubation, activated JH synthesis in the CC-CA nearly five or four times, respectively. The brain of VII1 hydroprenetreated larvae were ATTH inactive.     

Dependence of the parasitoid Gonia cinerascens on the hormones of its lepidopterous hosts

Development of first instar larvae of Gonia cinerascens, which rest in the muscles of host caterpillars, is triggered by the release of the host's ecdysteroids when the juvenile hormone is absent. Ecdysteroids act on the parasitoid directly and at the same time induce physiological and biochemical changes in the host, which are indispensable for the parasitoid's development. These changes do not occur when metamorphosis of the host is suppressed with the juvenile hormone. Normally the parasitoids initiate development at the larval-pupal transformation of the host, but under experimental conditions, they do so whenever a high ecdysteroid titre is coupled with the proper internal environment in the host, that is in decapitated caterpillars, isolated host abdomens, and when implanted into host pupae. Activated parasitoids moult into the second instar and migrate to the exuvial space of the host; this migratory behaviour is also triggered by ecdysteroids and may be induced experimentally in the first instar parasitoids. Unknown clues direct the migrating parasitoids under the wings and appendages of the host pharate pupal stage. The second instar parasitoids, which anchor to the integument of the host pupae, apparently develop independently of the host's hormones: they can produce third instar larvae, pupae, and adult flies when cultured in vitro.