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.     

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.     

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。     

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。     

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。     

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.     

Storage proteins of the fall webworm,Hyphantria cunea drury

Two storage proteins, storage protein-1 (SP1) and storage protein-2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8-day-old 7th instar larvae). SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively. Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4-day-old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.     

Properties,synthesis, and accumulation of storage proteins in Pieris rapae L.

Two kinds of storage proteins (SP-1, SP-2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP-1 and SP-2 dropped after pupation as the proteins were being deposited in fat bodies. SP-2 is present in a larger amount than SP-1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body. SP-1 has a molecular weight of 500,000 and consists of one type of subunit (M_r 77,000), while SP-2 has a molecular weight of 460,000 and is composed of two types of subunits (M_r 80,000 and 69,000). The pl values of SP-1 and SP-2 were determined to be 6.97 and 7.06, respectively. Fat body cells from 1-day-old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken up in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage. Morphological changes in the fat body tissues were observed during the larval-pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues.     

伯氏致病杆菌IDP16 蛋白抑制大蜡螟的免疫反应

[目的]从伯氏致病杆菌(Xenorhabdus bovienii)胞外组分中分离纯化出能够抑制大蜡螟(Galleria mellonella)免疫反应的一种蛋白,研究其在昆虫病原线虫及其共生菌致病过程中的作用.[方法]采用硫酸铵沉淀和柱层析的方法对活性蛋白进行分离和纯化,通过体内注射并观察血淋巴黑化进行活性蛋白的筛选;采用荧光微球和琼脂糖小球评价活性蛋白对血细胞吞噬、包被作用的影响;采用双向电泳结合质谱分析对活性蛋白进行鉴定,设计引物用PCR的方法克隆其编码基因,利用pET 30a载体进行原核表达,以亲和层析纯化重组蛋白.[结果]纯化得到一个昆虫免疫抑制蛋白,命名为IDP16,该蛋白可显著抑制大蜡螟血淋巴中的多酚氧化酶活性,降低血细胞的吞噬和包被作用.克隆得到其编码基因并进行了原核表达,重组蛋白仍具有免疫抑制活性.[结论]伯氏致病杆菌产生的IDP16蛋白能够抑制昆虫的免疫反应,在共生菌和宿主昆虫互作过程中起着重要的作用.