Immunity of the greater wax moth Galleria mellonella

Investigation of insect immune mechanisms provides important information concerning innate immunity, which in many aspects is conserved in animals. This is one of the reasons why insects serve as model organisms to study virulence mechanisms of human pathogens. From the evolutionary point of view, we also learn a lot about host–pathogen interaction and adaptation of organisms to conditions of life. Additionally, insect‐derived antibacterial and antifungal peptides and proteins are considered for their potential to be applied as alternatives to antibiotics. While Drosophila melanogaster is used to study the genetic aspect of insect immunity, Galleria mellonella serves as a good model for biochemical research. Given the size of the insect, it is possible to obtain easily hemolymph and other tissues as a source of many immune‐relevant polypeptides. This review article summarizes our knowledge concerning G. mellonella immunity. The best‐characterized immune‐related proteins and peptides are recalled and their short characteristic is given. Some other proteins identified at the mRNA level are also mentioned. The infectious routes used by Galleria natural pathogens such as Bacillus thuringiensis and Beauveria bassiana are also described in the context of host–pathogen interaction. Finally, the plasticity of G. mellonella immune response influenced by abiotic and biotic factors is described.     

Apolipophorin-III in the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae).

The level of apolipophorin-III reached a maximum in the haemolymph of Galleria mellonella at the end of the feeding phase of the seventh larval instar and declined to a plateau value in the pupal and the adult stages. Apolipophorin-III was detected immunologically in fat body tissue, haemocyte lysates, and plasma. In its native state, apolipophorin-III may be associated with another protein with an apparent molecular mass of 77 kDa, possibly apolipophorin-II. Injections of octopamine did not cause lipid loading of high density lipophorin.     

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.     

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.     

大蜡螟气味受体基因Gmel/Orco的克隆与序列分析

为进一步研究大蜡螟嗅觉通讯分子机制和寻求新的大蜡螟防治技术,本研究克隆了大蜡螟Galleria mellonella L.的气味受体基因Gmel/Orco,并对其序列进行生物信息学分析。根据GenBank中已发表的鳞翅目昆虫非典型气味受体家族基因的氨基酸保守序列设计简并引物,采用RT-PCR方法扩增目的基因,将其克隆至T载体并测序。克隆获得大蜡螟气味受体Orco的cDNA序列,命名为Gmel/Orco(GenBank登录号:KT020861),序列分析结果显示,Gmel/Orco开放阅读框长1425 bp,编码474个氨基酸,分子量为53.36 k D,等电点为8.44,序列中有7个跨膜区,N-端在细胞膜内,C-端在细胞膜外。通过在Gen Bank中进行序列的同源性比较,该基因与已公布的鳞翅目螟蛾科、夜蛾科昆虫的非典型气味受体基因序列有较高的同源性。克隆所获得的基因属于非典型气味受体家族基因。     

大蜡螟作为试验昆虫资源的利用现状

随着对资源昆虫的不断认识,人们的目光开始逐渐转到对大蜡螟Galleria mellonella L.的开发利用方面,而不再仅仅局限于对它的防治方面。近些年来,大蜡螟逐渐被作为试验昆虫用于一些生物的研究。文章主要介绍大蜡螟被用于昆虫病原线虫、寄生蜂、新型隐球菌Cryptococcus neoformans、抗菌肽、抗菌免疫机制等方面的研究。     

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.     

Effects of food quality on trade‐offs among growth,immunity and survival in the greater wax moth Galleria mellonella

The resources available to an individual in any given environment are finite, and variation in life history traits reflect differential allocation of these resources to competing life functions. Nutritional quality of food is of particular importance in these life history decisions. In this study, we tested trade‐offs among growth, immunity and survival in 3 groups of greater wax moth (Galleria mellonella) larvae fed on diets of high and average nutritional quality. We found rapid growth and weak immunity (as measured by encapsulation response) in the larvae of the high‐energy food group. It took longer to develop on food of average nutritional quality. However, encapsulation response was stronger in this group. The larvae grew longer in the low‐energy food group, and had the strongest encapsulation response. We observed the highest survival rates in larvae of the low‐energy food group, while the highest mortality rates were observed in the high‐energy food group. A significant negative correlation between body mass and the strength of encapsulation response was found only in the high‐energy food group revealing significant competition between growth and immunity only at the highest rates of growth. The results of this study help to establish relationships between types of food, its nutritional value and life history traits of G. mellonella larvae.     

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.     

Novel staining procedures for whole mount preparations of greater wax moth (Galleria mellonella) embryos: toluidine blue-rhodamine B,plus calcofluor white

Although hematoxylin and eosin (H & E) staining of sectioned embryonic insect material is widely used, it is time-consuming and may not provide sufficient information. We evaluated new staining procedures for embryonic whole mounts of the greater wax moth, Galleria mellonella. We compared a combination of toluidine blue and rhodamine B (TB-RB) to H & E; we also investigated calcofluor white (CFW) staining. TB-RB staining produced staining similar to H & E. TB-RB staining was less time-consuming and improved visualization of the blastoderm and its differentiation into the germ disk and serosa membrane. CFW enhanced details of mitosis in nuclei post-fertilization and stained the primary serosal membrane. Staining of whole mounts with TB-RB and CFW enabled embryonic staging that was more rapid, convenient and effective than the routine approach using H & E and fluorescent probes.     

Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

大蜡螟成虫3个醛氧化酶基因的鉴定、序列特征与组织表达模式

醛氧化酶(AOXs)在昆虫的嗅觉生理代谢过程中起重要作用.本研究从大蜡螟Galleria mellonella成虫中鉴定了3个AOXs基因,命名为GmelAOX1、GmelAOX2和GmelAOX3.这3个基因均含有完整的开放阅读框,所编码的蛋白质均具有醛氧化酶的典型特征,如具有铁硫氧化还原中心、黄素腺嘌呤二核苷酸结合...     

Purification and characterization of a male-specific protein in the hemolymph of the wax moth,Galleria mellonella L.

A male-specific protein (MSP) present only in males was identified from the hemolymph of the wax moth, Galleria mellonella L., by polyacrylamide gel electrophoresis (PAGE) and purified by anion-exchange chromatography. MSP has a native molecular mass of 55 kDa and consists of two 27-kDa subunits. An isoelectric point of MSP was measured to be approximately 5.8. MSP is a glycoprotein that contains 1.7% carbohydrate. The compositional analysis of carbohydrate component indicated a predominance of fructose and glucose. MSP also contains large amounts of asparagine, aspartic acid, glutamine, glutamic acid, and lysine but small amounts of tyrosine, methionine, and tryptophan. Western blot analysis of the hemolymph of each developmental stage indicated that MSP is present in the hemolymph of 8-day-old pupa and adult. Also, results from Western blotting indicated that MSP is not present in the tissues of larvae and of female adults but appears in the fat body of male pupae and adult and testis of adult. The fat body and testis of male pupae and adult were cultured in vitro to trace the place and time of MSP synthesis. The fat body began to synthesize MSP in late pupae and showed active synthesis during the adult stage. The distribution of MSP in the testis was observed by electron microscopic immunogold labeling, using the antibody against MSP. MSP is present between the germinal cysts and is taken up through the basal surface of the seminiferous tubular epithelium. Arch. Insect Biochem. Physiol. 37:257–268, 1998. © 1998 Wiley-Liss, Inc.     

Passive vectoring of entomopathogenic fungus Beauveria bassiana among the wax moth Galleria mellonella larvae by the ectoparasitoid Habrobracon hebetor females

Females of the ectoparasitoid Habrobracon hebetor attack and envenomate numerous host individuals during oviposition. The vectoring of the entomopathogenic fungus Beauveria bassiana during the adhesion stage by ectoparasitoid females among the wax moth larvae Galleria mellonella was explored under laboratory conditions. Vectoring occurred both from infected parasitoids to wax moth larvae and from infected to healthy wax moth larvae by parasitoids. The efficacy of vectoring in both cases was dose dependent. Parasitoid females were unable to recognize infected larvae in a labyrinth test. In addition, the presence of H. hebetor females significantly (1.5–13 fold) increased the mycoses level in clusters of G. mellonella, with 40% of the larvae infected with fungal conidia. Envenomation by H. hebetor increased conidia germination on the cuticles of the wax moth larvae by 4.4 fold. An enhanced germination rate (2 fold) was registered in the n‐hexane epicuticular extract of envenomated larvae compared to that of healthy larvae. Both envenomation and mycoses enhanced the phenoloxidase (PO) activity in the integument of G. mellonella and, in contrast, decreased the encapsulation rate in hemolymphs. We hypothesize that changes in the integument property and inhibition of cellular immunity provide the highest infection efficacy of entomopathogenic fungi with H. hebetor.     

Wax moth,Galleria mellonella fat body receptor for high-density lipophorin (HDLp)

To identify and characterize the HDLp (high-density lipophorin) receptor from Galleria mellonella (LpRGm), we used techniques of ligand blotting. This method was, to our knowledge, first used to characterize the lipophorin receptor (LpR) in insects. LpRGm had an approximate molecular weight of 97 kDa under non-reducing conditions and bound the HDLp specifically. The time-course of lipophorin binding to their receptor protein was rapid. The binding of lipophorins to their receptors was saturable with a Kd of 34.33+/-4.67 microg/ml. Although Ca2+ was essentially required in the binding of HDLp to their receptors, interestingly increasing concentration of Ca2+ has shown to have a slight inhibitory effect. EDTA was used here as Ca2+ chelating reagent, because Mg2+ in the binding buffer did not affect the binding of HDLp to their receptors, and inhibited the binding of HDLp and LpRGm absolutely. Suramin (polysulfated polycyclic hydrocarbon), known to inhibit the binding of lipoproteins to their receptors, effectively abolished the binding of HDLp to their receptors. LpRGm showed the stage specific binding activity especially in day 1-3 last instar larval, prepupal, and day 1-3 adult stages.     

Upregulation of hemolymph and tissue ferritin by dietary HgCl2 in the wax moth Galleria mellonella

The effect of Hg treatment on hemolymph and tissue ferritin in the wax moth Galleria mellonella was examined by western blotting. At 48 h after feeding HgCl₂, the level of hemolymph ferritin increased approximately 1.8‐fold over that of control insects that were not fed HgCl₂, while there was a small increase in tissue ferritin. Time series experiments showed that tissue ferritin had a typically saturated pattern, with a maximum level from 24 to 72 h, although it decreased 12 h following HgCl₂ feeding, while hemolymph ferritin first decreased but subsequently increased. Tissue ferritin in the fat body, gut and Malpighian tubules, the main tissues of ferritin expression, was upregulated over time following treatment with Hg, and in particular, tissue ferritin in the gut increased by a large amount at 12–48 h. The results suggest that in G. mellonella, the ferritin‐inducible mechanisms following treatment with HgCl₂ are different for hemolymph and tissue ferritin, as are their biochemical properties.     

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.