嗜线虫致病杆菌血腔毒素Tp40对大蜡螟幼虫体内酶活和中肠组织的影响

嗜线虫致病杆菌Xenorhabdus nematophila在侵入到寄主昆虫血腔后能够成功地逃避或抑制寄主昆虫的免疫反应并快速杀死昆虫。为深入了解嗜线虫致病杆菌的杀虫机理,明确关键的致病因子,作者应用盐析和制备型非变性凝胶电泳等方法,从嗜线虫致病杆菌HB310菌株的细胞内分离纯化了一种新的杀虫蛋白——Tp40,该蛋白对大蜡螟Galleria mellonella具有高血腔注射活性,对大蜡螟5龄幼虫的LD₅₀为68.54 ng/头。本文检测了该毒素对大蜡螟幼虫的致病特性,注射Tp40毒素后,大蜡螟幼虫表现出兴奋和痉挛等症状,当以不低于（70±0.02）ng/头的剂量注射Tp40,大蜡螟幼虫均在20 min内死亡,但试虫的体色 、血淋巴的颜色以及血细胞的形态没有发生明显的变化。对大蜡螟体内酶活性的测定结果显示,在注射LD₅₀剂量的Tp40蛋白后,试虫体内羧酸酯酶和乙酰胆碱酯酶活力都明显的高于对照（P<0.05）,而酚氧化酶活力显著低于对照（P<0.05）。对大蜡螟幼虫中肠的组织病理学研究显示：这种42 kDa蛋白能够破坏试虫的中肠组织,导致其肠壁细胞出现排列紊乱、脱落和围食膜消失。据此推测,Tp40与嗜线虫致病杆菌对寄主昆虫的免疫抑制有关,寄主中肠组织可能是其作用靶标之一。     

Pathology of Spiroplasma floricola in Galleria mellonella larvae

Spiroplasma floricola strain 23-6, originally isolated from tulip tree flowers, was injected into larvae of the greater wax moth. Histopathology and cytopathology of disease larvae were studied by histochemical, fluorescent antibody, and electron microscopical methods. The gut was empty, polysaccharides in fat and muscle tissue were reduced, the fat body was broken down, and phospholipids were depleted in larvae 4 days after injection. Fluorescein conjugated S. floricola antibody was adsorbed onto hemocytes, sarcolemma, gut epithelial membrane, and the cortex of the ventral ganglia. By electron microscopy, spiroplasmas were found in hemocoel, hemocytes, pericardial cells, connective tissues, basement membranes, epidermal cells of the cuticle, the neural lamella, and the peripheral glial cells of the ventral nerve cord, and on midgut and epidermal membranes. It is postulated that the cytopathological effects induced in the body of the insect released nutritional elements that allow extensive reproduction of S. floricola.     

Anti-Listeria activities of Galleria mellonella hemolymph proteins

We report the use of antimicrobial hemolymph proteins from the model host Galleria mellonella as an inhibitor for various Listeria strains, providing a novel source for antilisterial therapeutics. We also have shown that specific virulence-associated genes known to mediate antimicrobial resistance of Listeria in mammalian models indicated a similar function in Galleria.     

Detoxification of destruxins in Galleria mellonella L. larvae

1. Destruxins applied by intrahaemocoelic injection into Galleria mellonella larvae quickly accumulate in hindgut and are extensively metabolized by opening their cyclic structure.2. The process of detoxification corresponds to physiological state of larvae from early symptoms of paralysis to complete recovery within several hours.     

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.     

Apolipophorin-III affects the activity of the haemocytes of Galleria mellonella larvae

Apolipophorin-III (apoLp-III) impaired the adhesion of plasmatocytes and a granular cell-subpopulation of larval Galleria mellonella to glass slides. The protein bound to haemocytes, limited the responses of the plasmatocytes to Bacillus subtilis and increased the percentage of a subgroup of granular cells with adhering bacteria. The total number of bacteria adhering to all the haemocytes on the slides declined. Injections of apoLp-III slowed bacterial removal from the haemolymph without affecting total haemocyte counts and impaired haemocyte attachment to glass slides. Purified apoLp-III bound to B. subtilis. ApoLp-III in serum bound to bacteria within 5 min, peaked at 15 min and was either shed or dissociated by 60 min. ApoLp-III bound to B. subtilis lowered the adhesion of the bacteria to the haemocytes and slowed the removal of the bacteria from the haemolymph.     

影响大蜡螟幼虫体色的环境因素初探

大蜡螟幼虫体色各式各样 ,通过对比实验分析了温度、湿度、密度、光照和食物等环境因素对大蜡螟幼虫体色的影响。实验结果表明 :不同温度、湿度、密度、光照条件下大蜡螟幼虫的体色均未变化。在研究食物对大蜡螟幼虫体色的影响实验中食蜂巢的白黄色幼虫身体内部发黑 ,这可能是黑色的蜂巢透射出的颜色。实验为进一步研究大蜡螟幼虫体色的遗传机制打下了基础。     

Linkage between diet humidity,metabolic water production and heat dissipation in the larvae of Galleria mellonella

Last instar Galleria larvae respond to a decrease of dietary water content from 12 to 1% by reducing their energetic net growth efficiency from 42.5 to 36.3%. This reduction is linked to increased production of metabolic water, the proportion of which in total water requirement increases from 57 to 97%. Fluctuations in metabolic water output are controlled by changes in oxidative catabolism of the same type of substrates (respiratory quotient is identical under different water regimes). Elevated generation of metabolic water is associated with increased metabolic flow and with enhanced uncoupling of mitochondrial respiration from oxidative phosphorylation. The uncoupling allows for large and variable production of metabolic water in insects adapted to dry environments: the relative degree of uncoupling is 43.3% in Cydia larvae, which live in excess water, but 52.3% in the larvae of Ephestia, which live on a dry substrate; fluctuations between 54.4 and 59.6% in the larvae of Galleria depend on dietary moisture and larval age.     

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。     

Hemagglutinating properties of apolipophorin III from the hemolymph of Galleria mellonella larvae

In search for factors that cause encapsulation of foreign bodies in insect hemolymph we discovered that larval hemolymph of Galleria mellonella caused aggregation of mammalian erythrocytes. The hemagglutinating agent was identified as an 18-kDa protein that did not react with lectins. The sequence of 81 amino acids in three protein fragments and the properties of the protein revealed that it was Galleria homologue of apolipophorin III (apoLp-III*). ApoLp-III was found in high amounts in the hemolymph of Galleria larvae, pupae, and adults, as well as in the molting fluid. The hemagglutinating action of the whole hemolymph or the purified apoLp-III was independent of the presence of sugars in the medium. This indicated that it was not mediated by carbohydrates on the erythrocyte surface. The hemagglutination was inhibited at low pH (3.0), in the absence of calcium ions, and in the presence of certain bacterial lipopolysaccharides or their essential component, the 2-keto-3-deoxyoctonate-3-deoxyoctulosonic acid (KDO). It is suggested that interaction of apoLp-III with lipopolysaccharides in bacterial cell walls may play a role in insect immune reactions. Arch. Insect Biochem. Physiol. 38:119–125, 1998. © 1998 Wiley-Liss, Inc.     

The encapsulation of foreign tissue implants in Galleria mellonella larvae.

When implanted into the haemocoel of the wax moth, Galleria mellonella, fragments of nerve cord from the locust Schistocerca gregaria were shown to be encapsulated by host haemocytes, in a two phase system. The initial reaction to the implant was observed within 5 min of implantation and involved the contact and lysis of the granular haemocytes on the nerve cord, and the release of granular material around it which resulted in localised clot formation.The second phase involved plasmatocyte adhesion to the implant ca. 30 min after its introduction into the haemocoel, but only at sites of granular cell lysis. These cells continued to be added to the capsule until 60 to 72 hr after implantation, and during this time granular release continued, although no further granular cell lysis occurred. The complete capsule (72 hr) could be divided into three distinct regions of cells showing various modifications from free plasmatocytes.It is suggested that encapsulation in this species involves some chemotactic element which originates from the granular cells and acts specifically on the plasmatocytes.     

Involvement of apolipophorin III in antibacterial defense of Galleria mellonella larvae

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. We investigated involvement of apoLp-III in the antibacterial response in Galleria mellonella larvae. Immune challenge with Gram-negative (Escherichia coli, Klebsiella pneumoniae) and Gram-positive (Micrococcus luteus) bacteria led to an increase in the level of apoLp-III in G. mellonella hemolymph, 0.5-2h and 8h after treatment, respectively. ApoLp-III purified from larval hemolymph as well as that present in hemolymph extracts adsorbed on the surface of different bacteria. The adsorption capacity of apoLp-III on bacterial cells prompted us to investigate the effect of this phenomenon on bacterial growth. Our results demonstrate antibacterial activity of apoLp-III against selected Gram-positive and Gram-negative bacteria in vitro. Among bacteria tested, Salmonella typhimurium and K. pneumoniae were the most sensitive to apoLp-III. LIVE/DEAD staining of bacteria incubated with purified apoLp-III revealed their growth inhibition; however, neither morphological changes in the cell shape nor formation of cell aggregates was noticed. The results suggest that apoLp-III is a multifunctional protein in G. mellonella hemolymph.     

Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae)

Bacillus thuringiensis is one of the most widely used sources of biorational pesticides, as well as a key source of genes for transgenic expression to provide pest resistance in plants. In this study the effect of Bacillus thuringiensis ssp. galleriae (Bt) infection on the activity of superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), concentrations of oxidated and reduced thiols (RSSR/RSH) and malondialdehyde (MDA) was tested in the midgut of Galleria mellonella larvae. We found that Bt infection resulted in increased activities of SOD, GST, malondialdehyde and RSSR/RSH ratio the first day after inoculation. However, catalase activity decreased on the first and following days after bacterial infection by Bt. Our results confirm the hypothesis that Bt infection increases the level of oxidative stress in the larval midgut. In light of this study, it seems possible that oxidative damage contributes to cell death in the midgut during bacteriosis.