Lipophorin uptake by the larval fat body and adult ovary in the wax moth Galleria mellonella

Lipophorin (Lp) acts in the circulation of insects to selectively deliver lipids to target tissues. In the present study, we wanted to show that Lp is taken up into larval fat body cells and the adult ovary in Galleria mellonella. Larval fat body and adult ovary tissues were incubated at room temperature for 30 min with fluorescein isothiocyanate (FITC)‐labeled Lp. Fluorescence microscopy and sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) revealed that fat body and ovary tissues internalize fluorescence‐labeled Lp. The results suggest that both lipids and proteins are taken up by fat body cells and the ovary and also that large amounts of proteins and lipids taken up can serve as building blocks and as a source of energy. Immunological relationships with other insects were investigated using western blotting. The data showed that the Lp of Galleria mellonella is related to that of Hyphantria cunea.     

Receptor-mediated endocytosis of lipid and lipophorin by the larval fat body, adult ovary and testis in the wax moth Galleria mellonella

Lipophorin (Lp) in the hemolymph of insects is known to selectively deliver lipids from sites of absorption or synthesis to sites of storage and utilization, such as the fat body, ovary and testis; however, no study regarding this has been reported in Galleria mellonella. In the present study, we examined the process by which Lp is taken up into the larval fat body, adult ovary and adult testis, and the transfer of lipid by Lp to these tissues in Galleria mellonella. To investigate the involvement of a receptor in Lp endocytosis, the larval fat body, adult ovary and adult testis were incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)‐Lp, FITC‐Lp plus unlabeled Lp, and FITC‐Lp plus suramin, a receptor endocytic inhibitor. The amounts of FITC‐Lp in the three tissues were significantly decreased in the presence of unlabeled Lp and suramin, indicating that endocytosis of Lp by the tissues is mediated by a receptor. To examine the transfer of lipid by Lp, the tissues were incubated for 1 h at room temperature with 1,1′‐dilinoleyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI)‐Lp, DiI‐Lp plus unlabeled Lp, and DiI‐Lp plus suramin. The transfer of lipid by Lp was inhibited in the presence of unlabeled Lp and suramin, which is consistent with a receptor‐mediated process. Our results show that the transfer process of lipid by Lp and uptake of Lp itself is by receptor‐mediated endocytosis.     

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。     

Effects of Palpita unionalis and Galleria mellonella larval densities on functional response,egg dispersion and progeny sex ratio of Habrobracon hebetor

To assess the potential of the hymenopteran ectoparasitoid, Habrobracon hebetor Say as a biological control agent, we evaluated its response to different larval densities of two pyralid hosts, Palpita unionalis Hbn. and Galleria mellonella L. The former host is a serious pest of olive trees, whereas the latter is used as a factitious host in parasitoid mass rearing. In order to study the functional response of the parasitoid, five host densities (1, 2, 3, 5, 7) of either late instar larva per Petri dish were used. The shape of the functional response curve was determined using logistic regression and could be described as a type II response for both hosts, characterised by a monotonic decelerating increase in the number of hosts attacked with increasing host density. Female parasitoids allocated more eggs to the first larva attacked than all the remaining larvae. Aggregated dispersion patterns for parasitoid egg distribution at different host densities were estimated using the Green index. Multiple visits and ovipositions by females did not significantly affect the total number of progeny produced or their sex ratio. This study has generated novel information on egg laying, egg distribution and sex ratio of H. hebetor when reared on G. mellonella and has the potential to be used in the development of sustainable biological control programmes aimed at P. unionalis in olive orchards.     

Efficacy of Heterorhabditis baujardi LPP7 (Nematoda: Rhabditida) applied in Galleria mellonella (Lepidoptera: Pyralidae) insect cadavers to Conotrachelus psidii, (Coleoptera: Curculionidae) larvae

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil causing severe reduction in fruit quality. We assessed its susceptibility to Heterhorhabditis baujardi LPP7 infective juveniles (IJs) in the greenhouse and under field conditions applying the nematodes in cadavers of seventh instar Galleria mellonella larvae. Field persistence of these nematodes in the soil was evaluated through G. mellonella-baiting. Insect cadaver concentrations of 2, 4 and 6 applied in pots in the greenhouse experiment caused significant mortality compared to the control. Significance differences were observed in the field between control and treatments only when six cadavers per 0.25 m² were applied. Infective juveniles from the cadavers persisted 6 weeks after application in the field, but decreased greatly thereafter. Our work demonstrates that H. baujardi LPP7 IJs emerging from G. mellonella cadavers can be efficacious against guava weevil fourth instar larvae. Also, we demonstrated the long-term persistence of IJs in the soil.     

Comparison of the relative efficacy of an insect baiting method and selective media for diversity studies of Metarhizium species in the soil

Metarhizium are a commonly occurring group of entomopathogenic fungi normally found in soil. The most common methods to assess the diversity of Metarhizium species in soil are (i) the use of selective media and (ii) insect baiting using Galleria mellonella larvae. We compared the recovery efficiency from soil of four common species of Metarhizium (Metarhizium anisopliae, Metarhizium pingshaense, Metarhizium brunneum and Metarhizium robertsii) using these two methods. Firstly, we compared the number of colony forming units (CFU) produced in vitro when grown on two selective media, one containing chloramphenicol, thiabendazole and cycloheximidethe (CTC) and one based on the fungicide dodine (n-dodecylguanidine acetate) (DOD). Secondly, we artificially inoculated natural/non-sterile soil with the four fungal species at a rate of 2×10² and 2×10³ conidia g^?1of soil, baited with G. mellonella, and processed for evaluation using the selective media. The in vitro results showed that the greatest number of CFUs were recorded for M. brunneum. In contrast, when inoculated into soil, more G. mellonella larvae became infected by M. anisopliae. Finally, when using selective media, most CFUs recovered were for M. robertsii. The importance of our results in selecting a method to study the natural occurrence of Metarhizium in soil are discussed.     

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。     

温度对大蜡螟生长发育和繁殖的影响

为明确不同温度对大蜡螟(Galleria mellonella L.)生长发育和繁殖的影响,在24℃、28℃和31℃三个温度条件下,采用人工饲料饲养观察了大蜡螟的生长发育和繁殖状况。结果表明,温度对大蜡螟发育历期、生长发育速率以及繁殖具有显著影响(P0. 05)。在24～31℃范围内,随着温度升高,各虫态发育历期缩短,幼虫体长增长加快,发育速率加快。在24℃下世代历期最长(47. 96 d),31℃下世代历期最短(33. 68 d)。在同一温度下,雄蛾的寿命明显较雌蛾的寿命长,但成虫寿命、产卵前期和产卵期均随着温度升高而缩短。在24℃下产卵量最高为2 781. 50粒/雌,而在31℃下产卵量最低为1 943. 17粒/雌。     

The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme

A prophenoloxidase (PPO) was purified from the hemolymph of the larvae of Galleria mellonella. A 135-fold purification of the proenzyme with 25% yield was achieved by a combination of different chromatographic methods. An alternative micropreparation of pure PPO by a novel method for native electrophoresis in polyacrylamide gel is also described. The molecular mass of the native PPO was estimated to be 300 kDa by the pore-limit gradient electrophoresis in polyacrylamide gel. In the presence of sodium dodecyl sulphate, two closely migrating subunits of 80 and 83 kDa were detected under non-reducing conditions. The PPO was shown to be a glycoprotein and its isoelectric point was 6.2. The amino-acid composition of the purified protein was similar to the PPO from Bombyx mori. The monospecific antibody raised against the purified PPO crossreacted with the (pro)phenoloxidase in hemolymph of Manduca sexta. The activation of the PPO with chymotrypsin was investigated and two proteins of 67 and 50 kDa were found to be products of the proteolytic cleavage. The N-terminus of the G. mellonella PPO was blocked, but eleven partial internal sequences were determined after fragmentation of the purified PPO with trypsin. Three of these peptides exhibited significant homology with highly conserved sequences found in arthopod hemocyanins and insect storage proteins, which indicates that the PPO belongs to this family.     

Molecular Cloning and Characterization of Transferrin from Wax Moth, Galleria mellonella

Complete mRNA sequence of transferrin from Galleria mellonella was obtained, and compared with those of other species. Until now, two types of insect transferrin were reported. Transferrins in cockroach and termite have two iron binding sites while those in most other insect groups, studied for the protein, have only one. It was suggested that the presence of two types of transferrin was related with transferrin evolution, because vertebrate transferrins have two iron binding sites, called N and C terminal lobe. It was shown that G. mellonella transferrin also has only one iron binding site (N terminal lobe), and the deduced amino acid sequence was most similar to those of Manduca sexta and Bombyx mori.     

大蜡螟医学真菌感染模型的研究现况

在研究真菌感染中建立合适的真菌感染动物模型非常重要,大蜡螟幼虫作为昆虫动物模型之一,相比于其他的动物模型具有多种技术优势,目前已被广泛用于新型隐球菌、小孢子菌、红色毛癣菌、念珠菌属、暗色真菌、马尔尼菲青霉菌、黄曲霉和烟曲霉等多种致病菌的毒力、发病机制、免疫学改变、抗菌药物的开发以及系统性真菌感染的治疗等各个研究领域。研究表明大蜡螟感染模型研究结果与哺乳动物的结果相似,因此可以用大蜡螟来替代哺乳动物进行相关研究,从而减少了实验对哺乳动物的依赖性。     

Cytotoxic effects of parasitism and application of venom from the endoparasitoid Pimpla turionellae on hemocytes of the host Galleria mellonella

In parasitoid species devoid of polydnaviruses and virus‐like particles, venom appears to play a major role in suppression of host immunity. Venom from the pupal endoparasitoid Pimpla turionellae L. (Hymenoptera: Ichneumonidae) has previously been shown to contain a mixture of biologically active components, which display potent paralytic, cytotoxic, and cytolytic effects toward lepidopteran and dipteran hosts. The current study was undertaken to investigate if parasitism and/or envenomation by P. turionellae affects the frequency of apoptotic and necrotic hemocytes, hemocyte viability and mitotic indices in Galleria mellonella L. (Lepidoptera: Pyralidae) pupae and larvae. Our study indicates that parasitism and experimental envenomation of G. mellonella by P. turionellae resulted in markedly different effects on the ratio of apoptotic hemocytes circulating in hemolymph depending on the host developmental stages. The ratio of early and late apoptotic hemocytes increased in G. mellonella pupae and larvae upon parasitization and at high doses of venom when compared to untreated, null and Phosphate Buffered Saline (PBS) injected controls. In contrast, an increase in necrotic hemocytes was only observed in parasitized pupae at 24 h and no difference was observed in larvae. The lowest hemocyte viability values were observed with pupae as 69.87%, 69.80%, and 72.47% at 4, 8, and 24 h post‐parasitism. The ratio of mitotic hemocytes also decreased in pupae and larvae upon parasitization and at high doses of venom. Staining of hemocytes with annexin V‐FITC revealed green fluorescent ‘halos’ along the plasma membranes of venom treated cells within 15 min following exposure to venom. By 1 h post‐venom – treatment, the majority of hemocytes displayed binding of this probe, indicative of early stage apoptosis. These same hemocytes also displayed a loss of plasma membrane integrity at the same time points as evidenced by accumulation of propidium iodide in nuclei.