Applying fenoxycarb at the penultimate instar triggers an additional ecdysteroid surge and induces perfect extra larval molting in the silkworm

When the juvenile hormone analog fenoxycarb was topically applied to the silkworm Bombyx mori at the beginning of the 3rd or 4th (penultimate) instar, an extra larval molt was induced. The 5th instar period was shortened to about 5 days and the extra 6th instar ranged from 8 to more than 20 days, depending on the dose applied. Starvation before fenoxycarb treatment strongly enhanced the incidence of extra molting up to 100%. When 1 ng was applied in the 4th instar after a 2-day starvation, most larvae underwent an extra molt, metamorphosed to pupae, then to fertile adults. Combining starvation and fenoxycarb application thus induces a perfect extra molt efficiently. In perfect extra molting larvae, profiles of total ecdysteroid titer during the 4th and 5th instars were similar to that during the 4th instar in the control, and the ecdysteroid profile during the extra 6th instar was similar to that during the control 5th (last) instar. At ecdysteroid peaks, 20-hydroxyecdysone (20E) and ecdysone (E), generally regarded as the active molting hormone and its precursor, had similar titers in the 6th instar, whereas E was much less than 20E in the 4th and 5th instars in the extra molting larvae. E was also abundant only in the last larval instar in the control. These results suggest that both 20E and E contents are important for regulation of larval molt and metamorphosis in silkworms and that fenoxycarb triggers the extra molt by inducing an additional larval molt type of ecdysteroid surge before the last larval instar.     

Endocrine changes associated with the starvation-induced premature metamorphosis in the yellow-spotted longicorn beetle, Psacothea hilaris

Under 25 degrees C and a long day photoperiod, about half of the fourth instar Psacothea hilaris larvae molt to the fifth instar on day 13 and pupate about 18 days later; the rest pupate without a further larval molt with a mean fourth instar period of 24 days. However, starvation of fourth instar larvae exceeding a threshold weight induces premature pupation, resulting in the formation of small but morphologically normal adults. To clarify the endocrine basis for this premature pupation, hemolymph juvenile hormone (JH) and ecdysteroid titers were quantified during the fed and the starved periods. Normally fed fourth instar larvae exhibited two populations with regard to JH and ecdysteroid titers, one having JH titers ranging from 1.2 to 2.1 ng/ml through to day 13, the other, similarly high titers in the early part of the instar but low titers reaching 0.1 ng/ml on day 13. One population had ecdysteroid titers with a peak of 43 ng/ml on day 10, coinciding well with the period when some larvae normally molt to the fifth instar (day 13), the other, a small peak of 14 ng/ml on day 14 and a large peak of 70 ng/ml on day 17 coinciding well with the period just before the prepupa stage. When fourth instar larvae were starved after 4 days of feeding, JH titers decreased sharply in the next 24h and never recovered, and a small but significant increase (to 21 ng/ml) in ecdysteroid titers was observed on day 6, followed by a large peak of 63 ng/ml on day 11. Altogether, these results suggest that starvation induces a rapid decline in the JH titer, and this cues the early occurrence of a small ecdysteroid peak that commits larvae to early metamorphosis.     

Growth and development of Aedes aegypti larvae at limiting food concentrations

Mosquitoes have a complex life-cycle with dramatic changes in shape, function, and habitat. Aedes aegypti was studied by growing individual larvae at different concentrations of a defined rich food source. At higher food concentrations, rate of larval growth was faster, but the time required for 4th instar larvae to molt into the pupal stage was unexpectedly extended. These opposite tendencies resulted in constant times from hatching to pupation and up to adult eclosion at permissive food concentrations. The results demonstrate that nutritional conditions of 4th instar larvae impact initiation of the first metamorphic molt.     

Larval morphology of Atherigona orientalis (Schiner) (Diptera: Muscidae) —A species of sanitary and forensic importance

Larval morphology is documented using both light and scanning electron microscopy for all three instars of the muscid fly Atherigona orientalis (Schiner), which is a species of known sanitary and forensic importance found in tropical and subtropical areas of all biogeographic regions. The unpaired sclerite in a form of a spicule is reported herein in the second and the third instar larvae. Occurrence of this sclerite was hitherto unknown in the second instar larvae of Muscidae and was only known from the third instar of several species, however not in a form of a spicule. Our study is the first report of the occurrence of the “sensory organ X” in all three larval instars of a species representing the family Muscidae. The bubble membrane, previously known only from third instar cyclorrhaphan larvae, is reported herein for the first time in the second instar. Characters allowing for discrimination of A. orientalis larvae from other forensically important Muscidae are summarised.     

Some effects of the mole-inhibiting hormone and 20-hydroxyecdysone upon molting in the grass shrimp, Palaemonetes pugio.

The ligation studies by which critical periods for prothoracicotropic hormone (PTTH), juvenile hormone (JH) and ecdysone were determined for the last larval molt in Manduca sexta are reviewed in relation to recent experimental observations on Manduca larvae. Our two findings which challenged the accepted interpretation of these critical period determinations were: (1) Larval molting in vitro of the crochet epidermis from fourth instar larvae required ecdysone but not JH. (2) The hemolymph titer of JH was high and relatively constant before and during the period of PTTH secretion in the fourth instar. We present further experimental results bearing on the endocrine requirements for larval molting. Uninjected fourth instar larvae neck-ligatured prior to the supposed PTTH critical period initiated metamorphosis precociously. Immediate injection of ecdysone into such neck-or abdomen-ligatured larvae resulted in a larval molt. When the ecdysone injection was delayed, the proportion showing a larval response declined. Juvenile hormone injections (JHI) at the time of neck ligation prevented this loss of response to injected ecdysone. Delayed injections of subthreshold amounts of ecdysone into neck-ligatured larvae caused a delay in the onset of metamorphosis relative to uninjected controls. Additional ligation studies throughout the initiation of larval molting suggested that the prothoracic glands were increasing secretory activity during this period. On the basis of this evidence we have reinterpreted the ligation data for Manduca larvae as follows: the supposed PTTH critical period corresponds to attainment of subthreshold secretory activity of the prothoracic glands; the supposed JH critical period identifies onset of full secretory activity and delimits the physiological PTTH critical period for larval molting.     

Developmental changes in the response of the fat body of Manduca sexta to injections of Corpora cardiaca extracts

The Corpora cardiaca of larval and adult Manduca sexta contain glycogen phosphorylase-activating and adipokinetic activity. Depending on the age of the injected animal, injection of Corpora cardiaca extracts induces different responses. In larvae of the 1st and 2nd day of the Vth instar, phosphorylase is activated strongly by injections of Corpora cardiaca extracts, whereas in older animals it is only slightly activated. Mobilization of lipids in response to Corpora cardiaca extract injections is found only in animals injected after the imaginal molt; in younger animals this response is absent. It is argued that this difference in responsiveness may be due to a change in the properties of the target organ fat body.     

Changes in ecdysteroid and juvenile hormone titers in the hemolymph of Galleria mellonella larvae and pupae

The variations in circulating ecdysteroids and juvenile hormones (JH) in Galleria, from the end of the antepenultimate larval stage until emergence of adults, have been determined. The two hormonal families were extracted separately from the same hemolymph sample and quantified by two radioimmunoassays. Juvenile hormone RIA activity was about 35 nM in larvae of the antepenultimate and penultimate stages. It dropped before each molt and increased thereafter. Moreover, it gradually decreased during the last larval instar. In pupae, it was generally low, but it rose drastically during the late pupal development and in young adults. This rise was very much higher in females than in males. Three different RIA-active compounds were found; they were assumed to be JH-I, JH-II, and JH-III according to their retention times in HPLC. The three compounds were present in almost equal concentration in larvae of the penultimate stage: JH-I predominated, however, during the last larval instar. In late pupae, the main hormone was JH-III both in males and in females. There is no clear relationship between ecdysteroid and juvenile hormone changes, except for a female-specific ecdysteroid rise which coincides with the juvenile hormone release in late pupae. This double hormonal stimulation can be involved in the regulation of vitellogenin synthesis and deposition in oocytes.     

Allatinhibin, a neurohormonal inhibitor of juvenile hormone biosynthesis in Manduca sexta

The corpora allata (CA) of Manduca sexta larvae become incapable of synthesizing juvenile hormone (JH) early in the wandering stage of the last larval stadium. They then switch to the synthesis and release of JH acids. This change in CA activity is induced by an inhibitory factor--allatinhibin (AI)--from the brain. AI activity is present in the fifth (Vth) instar hemolymph from about Day 4 (day of wandering) until Day 7 (early prepupa). CA of early fifth instar larvae (uninhibited) incubated in vitro with brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes or brain alone from wandering larvae are inhibited as demonstrated by bioassay. On the basis of these observations, an in vitro-in vivo assay for AI was developed. Br-CC-CA or Br alone were first incubated in tissue culture medium overnight. Day 0 (0d) Vth instar CA incubated for 16 hr in such medium will lose the ability to induce a larval molt in allatectomized 0d IVth instar larvae if the medium contained AI activity. The highest AI activity was exhibited by the medium obtained from incubations of brain from wandering larvae whereas the medium from incubation of 0d Vth and 0d pupal brains showed no AI activity. Dose-response data show that AI is active at 0.03 brain equivalents/200 microliters medium. CA must be exposed to AI for 12-16 hr for manifestation of inhibition. AI causes a stable inhibition of CA. AI is heat-labile, protease sensitive, has a molecular size between 1.0 and 3.5 kDa, and is clearly distinct from the allatostatins described by others.     

Hot temperatures can force delayed mosquito outbreaks via sequential changes in Aedes aegypti demographic parameters in autocorrelated environments

Aedes aegypti L. (Diptera: Culicidae) is a common pantropical urban mosquito, vector of dengue, Yellow Fever and chikungunya viruses. Studies have shown Ae. aegypti abundance to be associated with environmental fluctuations, revealing patterns such as the occurrence of delayed mosquito outbreaks, i.e., sudden extraordinary increases in mosquito abundance following transient extreme high temperatures. Here, we use a two-stage (larvae and adults) matrix model to propose a mechanism for environmental signal canalization into demographic parameters of Ae. aegypti that could explain delayed high temperature induced mosquito outbreaks. We performed model simulations using parameters estimated from a weekly time series from Thailand, assuming either independent or autocorrelated environments. For autocorrelated environments, we found that long delays in the association between the onset of “hot” environments and mosquito outbreaks (10 weeks, as observed in Thailand) can be generated when “hot” environments sequentially trigger a larval survival decrease and over-compensatory fecundity increase, which lasts for the whole “hot” period, in conjunction with a larval survival increase followed by a fecundity decrease when the environment returns to “normal”. This result was not observed for independent environments. Finally, we discuss our results implications for prospective entomological research and vector management under changing environments.     

Ecdysteroids during the third larval instar in 1(3)ecd-1ts, a temperature-sensitive mutant of Drosophila melanogaster

The temperature-sensitive 1(3)ecd-1ts mutation (A. Garen, L. Kauvar, and J.A. Lepesant (1977). Proc. Natl. Acad. Sci USA 74, 5099-5103.) has been used in several laboratories to obtain Drosophila larvae deprived of moulting hormone. The development of mutants and controls during the third larval instar at permissive (20 degrees C) and restrictive temperatures (29 degrees C) was compared. Pupariation was inhibited when larvae were shifted to the restrictive temperature immediately at the second moult. The permanent larvae obtained remained active, did not leave the food, and reached a maximum weight superior to the weight of controls. Ecdysteroids were studied during the third larval instar by HPLC analysis and radioimmunoassays. A careful synchronization of the larvae at the second moult enabled the confirmation that at least one ecdysteroid peak occurs during the third larval instar, prior to the wandering stage in controls (20 or 29 degrees C). Ecdysone was then the predominant moulting hormone, whereas 20-hydroxyecdysone was the main ecdysteroid at the time of pupariation. Low levels of ecdysteroid were measured in mutant larvae shifted to 29 degrees C immediately at the second moult but larvae completely deprived of immunoreactive material were never observed. Nearly normal levels of ecdysteroids appeared at 27.5 degrees C. Feeding ecd-1 larvae maintained at restrictive temperature on 20-hydroxyecdysone-yeast mixture for 16 hr triggered abortive pupariation. Ecdysteroid levels were measured after the return of the larvae to the standard medium; normal levels were restored 24 hr later. The mutant ecd-1 appears to present interesting opportunities for the detailed study of the hormonal induction of a developmental process during the third larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)     

8种植物提取物对2种不同品系淡色库蚊的敏感性研究（英文）

目的研究开发有效的天然植物杀虫剂。方法按照WHO推荐的蚊幼虫敏感性测定方法,用8种植物的叶、茎、果和皮等提取物,对野生和敏感2个品系淡色库蚊进行敏感性测定。结果银杏外果皮提取物对淡色库蚊2个品系杀虫效果最强,其次为三尖杉和水杉,其他5种植物（重阳木、乌桕、香椿、杜仲和山茱萸）杀虫效果较弱。随后用银杏外果皮提取物对淡色库蚊卵、幼虫、蛹进行毒力测定,结果表明,Ⅰ龄幼虫最敏感,其次为Ⅱ龄、Ⅲ龄和Ⅳ龄幼虫。银杏外果皮提取物对敏感品系Ⅰ龄、Ⅱ龄、Ⅲ龄和Ⅳ龄的LC50分别为2.52、8.62、3.3和18.6mg/L;对野生品系Ⅰ龄、Ⅱ龄、Ⅲ龄和Ⅳ龄的LC50分别为7.4、15.2、19.2和25.0mg/L,明显高于敏感品系。银杏外果皮提取物在5～100mg/L浓度范围内不影响卵的孵化,在100mg/L以上时,影响蛹的羽化。结论8种植物提取物对淡色库蚊的敏感性各不相同,银杏外果皮提取物有明显的杀幼效果,值得进一步研究和开发。     

Ecdysone-inducible functions of larval fat bodies in Drosophila

Late in the third instar larval stage of Drosophila melanogaster, the titer of the steroid hormone ecdysone increases sharply. This increase is blocked in the temperature-sensitive mutant ecd(1) after a temperature shift from 20 degrees C to 29 degrees C. The mutant was used to prepare three samples of late third instar larvae with different titers of ecdysone; the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysone-supplemented food, and also high in a third sample that was kept at 20 degrees C, providing a control for normal development. The effect of the high titer of ecdysone on proteins of the larval fat bodies was examined by comparing two-dimensional gel electrophoresis patterns of total proteins in stained gels. There were proteins at five positions in the gels for the high-ecdysone samples that were not detected at the corresponding positions in the gel for the low-ecdysone sample. The effect of ecdysone on these proteins was further studied by injecting [(35)S]methionine into the larvae at both early and late third instar stages, in order to label proteins synthesized before and after the increase in ecdysone titer. The results indicate that ecdysone induces two major responses in the fat bodies; certain proteins that were synthesized earlier in the fat bodies and secreted into the hemolymph are incorporated back into the fat bodies, and other proteins are newly synthesized. Attempts to induce prematurely the synthesis of the new proteins by exposing early third instar larvae to exogenous ecdysone were unsuccessful, suggesting that development must proceed further before the fat bodies can respond to ecdysone.By in vitro translation of RNA isolated from fat bodies of low-and high-ecdysone samples of larvae, it was shown that ecdysone greatly increases the amount of translatable messenger RNA for one of the newly synthesized proteins. A clone of DNA complementary to the induced messenger RNA has been isolated from a population of lambda bacteriophage carrying segments of the Drosophila genome. Using the cloned DNA to measure amounts of complementary poly(A)-RNA in the fat bodies by DNA.RNA hybridization, we detected about 50 times more complementary poly(A)-RNA in the high-ecdysone sample of larvae than in the low-ecdysone sample. This finding provides direct evidence that ecdysone induces an increase in the amount of the messenger RNA. The ecdysone-induced appearance of a major messenger RNA in late third instar larval fat bodies represents a developmental response to ecdysone that appears to be gene-specific, tissue-specific, and stage-specific, and it has exceptionally favorable features for further molecular studies of the control of gene expression by a steroid hormone.     

Plasma cortisol and thyroid hormone concentrations in pre-weaning Australian fur seal pups

The hormonal factors that influence development from birth to weaning in otariid seals is still largely unknown. In the present study, a suite of thyroid hormones and cortisol were measured in Australian fur seal pups in order to determine baseline concentrations as well as to describe their endocrinology over this critical developmental period. A cross-section of newborn pups from a breeding colony located on Kanowna Island, Australia were sampled at six different times over the course of the 10 month lactation period. Sample times were designed to correspond to periods of heightened physiological change during pre-weaning development: post-natal, pre-molt, the initiation of molt, mid-molt, period of peak milk intake and weaning. Results indicate that the greatest hormonal changes were associated with the post-natal stage and molt, with molt showing the greatest changes, as has been reported for several species of pinnipeds. Two forms of thyroid hormones analyzed (Total T₄, and Free T₃), increased with the initiation of the molt, and Free T₃ exhibited a second increase that was associated with the period of peak milk intake. The T₃:T₄ ratio was significantly lower during the initiation of molt than either pre- or mid-molt. The study was able to describe physiological change during the first year of life in Australian fur seals as well as document basal concentrations of thyroid hormones and cortisol in pups of this species.     

Juvenile hormone inhibits ecdysone secretion and responsiveness to prothoracicotropic hormone in prothoracic glands of Bombyx mori

The role of juvenile hormone (JH) in the regulation of prothoracic gland activity was investigated during the early days of the last (fifth) larval instar of Bombyx mori. Allatectomy on the day of larval ecdysis into the fifth instar or 1 day before ecdysis shortened the time between larval ecdysis and gut purge. Prothoracic glands of the freshly ecdysed fifth instar larvae were inactive and did not respond to the prothoracicotropic hormone (PTTH), whereas those larvae that were allatectomized 1 day before ecdysis exhibited secretory activity in vitro and were capable of responding to PTTH. When corpora allata were removed from freshly ecdysed fifth instar larvae, the prothoracic glands became competent to respond to PTTH in 6 hr and exhibited secretory activity in vitro 9 hr after the allatectomy. Treatment of allatectomized larvae with a JH analog resulted in the recovery of the normal inactive state of the glands. These data suggest that JH acts during the early stages of the instar to suppress both the secretory activity of prothoracic glands and also the acquisition of competence to respond to PTTH.     

Induction of DNA synthesis by 20-hydroxyecdysone in the prothoracic gland cells of the silkworm Bombyx mori during the last larval instar.

DNA synthesis in the prothoracic gland cells of the silkworm Bombyx mori was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU), and its developmental changes during the last larval instar were examined. During the first 3 days of the last larval instar, no DNA-synthesizing cells were detected. On day 4, the number of DNA-synthesizing cells increased and peaked on day 7. When larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves throughout the last larval instar, the number of DNA-synthesizing cells dramatically increased 2 days after 20-hydroxyecdysone treatment and reached a high level on day 5. The mechanism by which 20-hydroxyecdysone treatment induces DNA synthesis of prothoracic gland cells was further examined by using continuous in vitro BrdU labeling for a 2-day incubation. We conclude that 20-hydroxyecdysone may exert its growth-promoting action indirectly.     

Effects of a juvenile hormone analogue on the morphology and biology of Aedes scutellaris malayensis Colless (Diptera: Culicidae)

Effects of 2-methyl-9(4-isopropylphenyl)-2,6-dimethyl nonane, or MV-678, a juvenile hormone analogue, on Aedes scutellaris malayensis Colless were investigated under laboratory conditions (29 degrees +/- 2 degrees C and 86 +/- 3% RH). The MV-678 was tested against the first, second, third and fourth instar larvae. The concentrations used were 0.0032, 0.016, 0.08, 0.4 and 2.0 mg/l. The morphogenetic aberrations were determined and divided into 8 groups, among which they included (1) death larvae, (2) late fourth instar larvae before pupation, (3) larvae with pupae partly emerged, (4) white pupae, (5) brown pupae, (6) elephantoid pupae of which pupae with adults visible inside, (7) pupae with apparently adults partly emerged and (8) death adults. The percentage mortality rates were found to be relatively high in pupal and larval stages when they were treated with 2.0 mg/l. The LC50 values were 0.26, 0.175, 0.06 and 0.032 mg/l for the first, second, third and fourth instar larvae respectively. The effectiveness of MV-678 at 2.0 mg/l was about 11 days under the open air conditions (28 degrees +/- 2 degrees C and 72 +/- 3% RH). When the fourth instar larvae were treated with 0.4 and 2.0 mg/l of MV-678, the LC50 values were 3.1 and 7.1 days respectively.     

Caste and metamorphosis: hemolymph titers of juvenile hormone and ecdysteroids in last instar honeybee larvae

Juvenile hormone (JH) and ecdysteroid titers are critical factors for caste development and metamorphosis in the last larval instar of the honeybee, Apis mellifera. Two highly sensitive radioimmunoassays were used for the determination of these hormones in the hemolymph. For juvenile hormone, which is of prime importance for the control of caste development in honeybees, our data show a caste-specific peak in queen larvae of the early fifth instar. A second peak appears in prepupae of both castes which probably is responsible for the regulation of the pupal moult. A single peak of ecdysteroids was observed in prepupae of both castes. In queens, however, the titer increases distinctly earlier than in worker larvae. The ecdysteroid composition of this prepupal peak was determined by high-performance liquid chromatography separation followed by radioimmunoassay. Makisterone A proved to be the main ecdysteroid compound, but 20-hydroxyecdysone was also found in significant amounts.     

Essential roles for four cytoplasmic intermediate filament proteins in Caenorhabditis elegans development

The structural proteins of the cytoplasmic intermediate filaments (IFs) arise in the nematode Caenorhabditis elegans from eight reported genes and an additional three genes now identified in the complete genome. With the use of double-stranded RNA interference (RNAi) for all 11 C. elegans genes encoding cytoplasmic IF proteins, we observe phenotypes for the five genes A1, A2, A3, B1, and C2. These range from embryonic lethality (B1) and embryonic/larval lethality (A3) to larval lethality (A1 and A2) and a mild dumpy phenotype of adults (C2). Phenotypes A2 and A3 involve displaced body muscles and paralysis. They probably arise by reduction of hypodermal IFs that participate in the transmission of force from the muscle cells to the cuticle. The B1 phenotype has multiple morphogenetic defects, and the A1 phenotype is arrested at the L1 stage. Thus, at least four IF genes are essential for C. elegans development. Their RNAi phenotypes are lethal defects due to silencing of single IF genes. In contrast to C. elegans, no IF genes have been identified in the complete Drosophila genome, posing the question of how Drosophila can compensate for the lack of these proteins, which are essential in mammals and C. elegans. We speculate that the lack of IF proteins in Drosophila can be viewed as cytoskeletal alteration in which, for instance, stable microtubules, often arranged as bundles, substitute for cytoplasmic IFs.     

Regulation of prothoracicotropic hormone release by juvenile hormone in the penultimate and last instar larvae of Mamestra brassicae

The penultimate instar larvae of Mamestra brassicae have one ecdysteroid peak 2 days after ecdysis which induces larval ecdysis. Allatectomy deleted this peak and caused a larger peak 7 days later just before precocious pupation, in a pattern similar to the ecdysteroid titer of the last instar. Application of juvenile hormone (JH) immediately after allatectomy restored the normal larval pattern and larval ecdysis. The brain of the normal Day 1 or 2 penultimate instar larva had higher prothoracicotropic hormone (PTTH) activity than that of the allatectomized larva. Therefore, JH in the penultimate instar activates the brain to secrete and/or produce PTTH. Allatectomy of last instar larvae prolonged the period from gut purge to pupation by causing a delay of the ecdysteroid peak necessary for pupation. Application of JH restored the normal timing of ecdysteroid release. Thus, JH is important for the occurrence of the ecdysteroid surge on Day 7, since its absence delayed this surge by a day.