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.     

Neurohormonal control of ecdysone production: Comparison of insects and crustaceans

Summary

Ecdysteroid synthesis is regulated in insects by prothoracicotropic hormone (PTTH) and in crustaceans by molt-inhibiting hormone (MIH). These neurohormones exert opposite effects on their respective target tissues, PTTH stimulating the prothoracic glands and MIH inhibiting the Y-organs. The present work reviews recent progress in the neurohormonal regulation of prothoracic gland and Y-organ function. The steroid products of these glands are briefly discussed, as is current information on the structures of PTTH and MIH. Focus is placed on the mechanism of action of these hormones at the cellular level, as well as developmental changes in cellular sensitivity to PTTH. Though exerting different effects on ecdysteroid secretion, both PTTH and MIH increase cyclic nucleotide second messengers, are influenced by alterations in cellular calcium, and are likely to activate protein kinases. The contrasting steroidogenic effects of PTTH and MIH probably arise from differences in the cellular kinase substrates. In insects, such substrates enhance ecdysteroid secretion, possibly by increasing the translation of glandular proteins. In crustaceans, MIH-stimulated changes lead to the inhibition of both protein synthesis and steroidogenesis.     

Endocrine changes during diapause development in the cabbage army moth Mamestra brassicae

Many insects undergo diapause to survive adverse seasons. Although the mechanism of diapause induction is the subject of extensive study, that of diapause termination remains poorly understood. In the present study, we show the endocrine processes leading to the termination of pupal diapause in Mamestra brassicae. Diapause of this insect is terminated if the pupae are exposed to a low temperature for several weeks. During this period, the prothoracic glands (PGs) of pupae acquire the potential to secrete sufficient ecdysteroids necessary for inducing adult development. The main endocrine changes observed under the low temperature conditions are: (i) the increase in activity of the PGs in two steps; (ii) the increase in responsiveness of the glands to prothoracicotropic hormone (PTTH); and (iii) two‐step increase in PTTH gene expression in the brain. The timing of the first and second increases in PG activity roughly coincides with that of the two steps of increase in PTTH gene expression, and the timing of the increase in the responsiveness of the PGs to PTTH coincides with the second, larger increase in PTTH gene expression. The ablation of the PGs prior to cooling pupae does not affect the increase in PTTH gene expression, whereas brain removal results in a failure to increase PG activity, strongly suggesting that PTTH is the master regulator of diapause development and termination.     

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain-corpora cardiaca-corpora allata complexes were removed from intact fifth-instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20-hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.     

Lysozyme as Pathogen-Recognition Protein in the Hemolymph of Galleria mellonella

ABSTRACT Recognition of invading micro-organisms into hemolymph is a pivotal event for triggering diverse immune mechanisms in insects. It has been known that this recognition was mediated by the binding of hemolymph proteins to pattern-molecules on the cell surface of microbes. Recently, I found that the lysozyme in the G. mellonella hemolymph has binding affinity to cell-walls of Gram (-), (±) bacteria and fungus ( Candida albicans ). After the hemolymph was incubated with heat-killed microbes and treated with acidic buffer containing high concentration of NaCl, several plasma proteins detached from microbes were detected by reverse phase HPLC and SDS-PAGE analyses. Of binding proteins, it was assumed that the major one might be a lysozyme, which was previously characterized in the G. mellonella hemolymph. Furthermore immunoblot analysis performed with antiserum to G. mellonella lysozyme revealed that it was a lysozyme.     

Prothoracicotropic hormone induces tyrosine phosphorylation in prothoracic glands of the silkworm,Bombyx mori

In the present study, we investigated the tyrosine phosphorylation of Bombyx mori prothoracic glands using phosphotyrosine‐specific antibodies and Western blot analysis. Results showed that prothoracicotropic hormone (PTTH) stimulates a rapid increase in tyrosine phosphorylation of at least 2 proteins in prothoracic glands, one of which was identified as extracellular signal‐regulated kinase (ERK). The phosphorylation of another 120‐kDa protein showed dose‐ and time‐dependent stimulation by PTTH in vitro. In vitro activation of tyrosine phosphorylation was also verified by in vivo experiments: injection of PTTH into day‐6 last‐instar larvae greatly increased tyrosine phosphorylation. Treatment of prothoracic glands with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, also resulted in tyrosine phosphorylation of several proteins and increased ecdysteroidogenesis. The PTTH‐stimulated phosphorylation of the 120‐kDa protein was markedly attenuated by genistein, a broad‐spectrum tyrosine kinase inhibitor, but not by HNMPA‐(AM)₃, a specific inhibitor of insulin receptor tyrosine kinase. PP2, a more‐selective inhibitor of the Src‐family tyrosine kinases, partially inhibited PTTH‐stimulated tyrosine phosphorylation, but not ecdysteroidogenesis. This result implies the possibility that in addition to ERK, the phosphorylation of the 120‐kDa protein, which is not Src‐family tyrosine kinase, is likely also involved in PTTH‐stimulated ecdysteroidogenesis in B. mori. © 2010 Wiley Periodicals, Inc.     

大蜡螟幼虫的体色遗传规律

对大蜡螟Galleria mellonella幼虫不同颜色品系的普通遗传学分析表明,大蜡螟幼虫的体色遗传是常染色体遗传且符合复等位基因遗传规律。深黄色基因（AA）对灰黑色基因(BB)和灰色基因(CC)为显性,深黄色基因(AA)对白黄色基因(DD)、灰黑色基因(BB)对白黄色基因(DD)和灰色基因(CC)、灰色基因(CC)对白黄色基因(DD)为不完全显性。基因型为AD、BD、CD的个体,其表现型均为黄色;基因型为AA、BC的个体,其表现型均为深黄色。     

Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae

Candida albicans is a dimorphic human pathogen in which the yeast to hyphal switch may be an important factor in virulence in mammals. This pathogen has recently been shown to also kill insects such as the Greater Wax Moth Galleria mellonella when injected into the haemocoel of the insect larvae. We have investigated the effect of previously characterised C. albicans mutations that influence the yeast to hyphal transition on virulence in G. mellonella larvae. There is a good correlation between the virulence of these mutants in the insect host and the virulence measured through systemic infection of mice. Although the predominant cellular species detected in G. mellonella infections is the yeast form of C. albicans, mutations that influence the hyphal transition also reduce pathogenicity in the insect. The correlation with virulence measured in the mouse infection system suggests that Galleria may provide a convenient and inexpensive model for the in vivo screening of mutants of C. albicans.     

Metamorphic shortening of interganglionic connectives of Galleria mellonella (Lepidoptera) in vitro: stimulation by ecdysone analogues

In the absence of other organ systems, β-ecdysone (0·05 to 0·10 μg/ml culture medium) stimulates the shortening of interganglionic connectives of Galleria mellonella that occurs during metamorphosis. There is a direct relations-ship between the amount of β-ecdysone in the medium and the fraction of the sample shortening to at least half the initial length. β-Ecdysone is ～ 140 × more active than α-ecdysone in eliciting the response. When β-ecdysone and other ecdysone analogues are assayed on this system at uniform dosages (10 μg/ml tissue culture medium), the order of effectiveness (percentage sample shortening to at least half the initial length) is: cyasterone > ponasterone-A = β-ecdysone > inokosterone > α-ecdysone. 22-Iso-α-ecdysone is ineffective in stimulating shortening.     

Electron microscopical-immunocytochemical evidence of ecdysteroids in the prothoracic gland of Galleria mellonella

Summary Fixation of prothoracic glands of Galleria mellonella with a solution containing saponin permits immunocytochemical staining of the entire gland. By this means ecdysteroids were demonstrated electron microscopically to be present in the hyaloplasm and microtubules.Supported by Sächsische Akademie der Wissenschaften zu Leipzig     

青蒿琥酯抗蜡螟烟曲霉感染自噬机制初步研究

目的 探究青蒿琥酯在蜡螟感染烟曲霉后,对蜡螟的自噬相关蛋白的表达影响。方法 用一定量的烟曲霉的活化孢子感染蜡螟,经过1 h,用青蒿琥酯注射一组蜡螟,两性霉素B注射一组蜡螟,剩余的作为感染组。12 h后,取各组蜡螟进行病理切片染色,观察各组的病理情况;取各组的蜡螟的淋巴液,收集各组的孢子,用真菌活性检测试剂盒检测孢子活性并将淋巴细胞分离、裂解,离心取上清,用Western-blot法检测上清液中的Dectin-1、ROS、LC3Ⅱ的表达水平。结果 青蒿琥酯注射组的蜡螟病理切片中的孢子比感染组数量少且聚集在一起,未长出菌丝,而体外分离的真菌孢子,青蒿琥酯组的活性明显受到抑制。经Western-blot显示青蒿琥酯能增强淋巴细胞Dectin-1、ROS、LC3Ⅱ的表达。结论 青蒿琥酯可通过抑制烟曲霉的活性和增强蜡螟淋巴细胞的自噬水平,对抗蜡螟烟曲霉感染。     

Inhibition of Beauveria bassiana Proteases and Fungal Development by Inducible Protease Inhibitors in the Haemolymph of Galleria mellonella Larvae

Injection of zymosan or dead yeast cells enhanced the inhibitory activity against exocellular Beauveria bassiana proteases in the cell - free haemolymph of Galleria mellonella larvae . Pre - injected larvae exhibited no decreased mortality after subsequent injection with living B. bassiana blastospores but survived for a prolonged time before death . Increased levels of protease inhibitors in the haemolymph were also observed after injection of B. bassiana proteases . In contrast , no enhanced inhibitory activity against B. bassiana proteases was detected in infected larvae when mycosis was initiated with conidia which enabled the fungus to invade host larvae through the integument in a natural manner . B. bassiana proteases were not completely inhibited by the addition of cell - free haemolymph . Protease inhibitors obtained after heat and trichloroacetic acid precipitation of cell - free haemolymph were added to the protein medium of B. bassiana to study the effect on its growth in vitro. Enriched fractions from pre - injected larvae delayed fungal growth in comparison with fractions from untreated larvae , suggesting that delayed mortality of immunized G. mellonella larvae infected with B. bassiana is due to enhanced levels of protease inhibitors . A non - virulent form of the same strain exhibited reduced capacity to release proteases in vitro. The results strongly suggest that the capacity of insects to release inhibitors against fungal proteases influences their susceptibility against entomopathogenic fungi .     

Cytoplasmic free calcium in single cells of centric diatoms. The use of fura-2

Summary Cytoplasmic free calcium concentration has been measured in centric diatoms using Fura-2. Cells hydrolysed Fura-2 acetoxymethyl ester and accumulated free Fura-2 in the cytoplasm. The [Ca²⁺] distribution in two species (Coccinodiscus concinnus andGuinardia flaccida) has been mapped using digital image analysis.Abbreviations [Ca _cyt ²⁺ ] Cytoplasmic free calcium concentration - Quin-2/AM Quin-2 acetoxymethyl ester - Fura-2/AM Fura-2 acetoxymethyl ester DMSO dimethyl sulphoxide - CCTV closed circuit television     

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

Toxicity of mycotoxins of Fusarium sambucinum for feeding in Galleria mellonella

Fusarium sambucinum Fuckel showed antifeedant activity towards larvae of Galleria mellonella L. when incorporated into insect diet. The activity appeared mostly due to the concentration of trichothecenes present in the fungal extracts. Diacetoxyscirpenol and neosolaniol showed similar levels of activity and were significant potent antifeedants against larvae at 50 and 100 ppm. On the contrary, enniatin B showed no activity up to 100 ppm.     

Tris stimulates ecdysteroid secretion via Ca2+ messenger system in the prothoracic glands of Locusta migratoria

Abstract.  Secretion of ecdysteroids by the prothoracic glands of the migratory locust, Locusta migratoria L., is enhanced in vitro by tris(hydroxymethyl)aminomethane (tris) in a dose-dependent manner. Glands from larvae on the second day of the penultimate stadium are most sensitive. This action of tris depends on the uptake of calcium; increased production of ecdysteroid does not occur in the presence of cadmium, verapamil or TMB-8, or in calcium-free media. The concentration of unbound Ca²⁺, [Ca²⁺]_i, in the cytoplasm is measured with the aid of FURA 2/AM. Tris causes a rise of [Ca²⁺]_i that is fully suppressed by lanthanum and partially by nitrendipine. Two antagonists of IP₃ receptors elicit mutually opposite effects: heparin blocks, whereas 2-APB accelerates, the rise in [Ca²⁺]_i. Ryanodine exerts only a slight effect. It is proposed that tris activates locust prothoracic glands in a Ca²⁺-dependent manner that exhibits many similarities to the transduction pathway of prothoracicotropic hormone.     

大蜡螟性肽受体基因GmelSPR克隆及表达分析

交配是昆虫繁衍的重要方式,雌性交配后会显现出一系列保护后代而产生的行为,SPR则是重要的交配行为调控受体基因。本研究根据前期转录组SPR基因数据信息,利用RT-PCR扩增出大蜡螟SPR基因的完整CDS序列,命名为GmelSPR（GenBank登录号：OR347698）。生物信息学分析显示该基因编码区全长1 263 bp,编码420个氨基酸,预测分子量48.70718 kDa,理论等电点8.70,有7个跨膜螺旋区,具有典型G蛋白偶联受体蛋白特征。系统进化树比对结果显示大蜡螟GmelSPR与螟蛾科昆虫脐橙螟Amyelois transitella、印度谷螟Plodia interpunctella置信度较高。大蜡螟不同状态、不同组织中表达分析表明,未交尾雌蛾、雄蛾和已交尾雌蛾、雄蛾GmelSPR均在头部较高表达,且在未交尾雌蛾、雄蛾中表达量均高于已交尾雌蛾、雄蛾。本研究克隆了大蜡螟SPR基因,克隆结果表明GmelSPR与螟蛾科昆虫亲缘较近;表达谱分析则显示GmelSPR可能参与大蜡螟交配行为,为进一步研究大蜡螟中SPR基因功能奠定基础。     

Fungal infection causes changes in the number,morphology and spreading ability of Galleria mellonella haemocytes

The most effective and important strategy in the insect immune response is based on cellular reactions incorporating haemocytes. The present study uses Galleria mellonella (Lepidoptera: Pyralidae) as a host to study the pathogenesis caused by the entomopthoralean fungus Conidiobolus coronatus (Entomophthorales). Five types of haemocytes with different morphologies and behaviour are observed in the haemolymph of G. mellonella: granulocytes (GRs), plasmatocytes (PLs), spherulocytes (SPs), oenocytes (OEs) and prohaemocytes (PRs). During in vitro cultivation, three morphological subtypes of PLs are distinguished: flattened PLs, sun‐like PLs and oval PLs. In fresh smears of haemolymph observed under phase‐contrast microscopy, only flattened PLs are identified. No morphological changes are observed between fresh smears and in vitro cultures for GR, OE, SP and PR. Haemocytes cultured in vitro form a cellular network composed of PLs and GRs. Changes in the numbers, morphology and behaviour of haemocytes induced by fungal infection are compared with those observed in normally‐developing untreated larvae. Infection results in a significant drop in the number of haemocyte types. Fresh smears of haemocytes from mycosed larvae reveal malformed OEs, vacuolized PLs and GRs, as well as PLs with apoptotic blebs. Haemocytes from mycosed larvae incubated in vitro look similar, with degranulated GRs and vacuolized PLs forming microaggregations, as well as deformed OEs; only the SPs remain unharmed. Fungal infection impairs the ability of haemocytes to attach and spread on the culture dish. The actin cytoskeleton of haemocytes from mycosed larvae appear disorganized.     

Humoral stimulation of the larval and adult prothoracic glands in Schistocerca gregaria

Fluctuations in ecdysteroid production by explanted prothoracic glands (PG) during the penultimate and last larval instars parallel changes in ecdysteroid titer in the hemolymph. The in vitro output of ecdysteroids increases up to 30-fold when PG are co-cultured with the brain. Maximal amounts of ecdysteroids are produced when both PG and brain are taken from larvae at the time of the molt-inducing ecdysteroid peaks (days 2–3 in the penultimate and days 5–6 in the last instar), and also from day 3 last instar larvae that exhibit a small rise of hemolymph ecdysteroids. Detailed investigations on penultimate instar larvae revealed that their PG become sensitive to the stimulation on day 1 (about 24 h after ecdysis), but the stimulatory brain potential is restricted to days 2 and 3. Both the stimulatory capacity of the brain and the sensitivity of PG are lost on days 4 and 5, i.e., after the ecdysteroid surge on day 3. PG explanted from young adults do not secrete appreciable amounts of ecdysteroids but can be stimulated to ecdysteroid production with active larval brains. Arch. Insect Biochem. Physiol. 36:85–93, 1997. © 1997 Wiley-Liss, Inc.     

JNK MAP kinase is involved in the humoral immune response of the greater wax moth larvae Galleria mellonella

We investigated the participation of MAP kinases in the response of Galleria mellonella larvae to immune challenge. JNK MAP kinase was activated in fat body 10-15 min after LPS injection in vivo. The level of JNK activation was time- and LPS dosage-dependent. JNK MAP kinase isolated from cell-free extract of fat bodies dissected from immune stimulated larvae phosphorylated c-Jun protein in vitro. The activity of Gm JNK kinase was abolished in the presence of the JNK specific inhibitor SP600125. Our data indicate a correlation between JNK phosphorylation and induction of antimicrobial activity in the insect hemolymph after immune stimulation. Hemolymph from larvae pre-treated with JNK specific inhibitor SP600125 showed a reduced level of antibacterial activity after LPS injection. JNK inhibition by SP600125 abolished antibacterial activity of the in vitro culture of G. mellonella fat body. Finally, we also show a correlation between JNK-dependent immune response of G. mellonella larvae and elevated temperature.