Changes of haemolymph protein profile in the larva of Pericallia ricini (Fabricius) parasitised by the Braconid Wasp, Apanteles taragamae Viereck (Hymenoptera: Braconidae)

Parasitism by the braconid wasp, A. taragamae caused alterations in the haemolymph polypeptides of woolly bear larvae of P. ricini. Analysis of haemolymph proteins by SDS-PAGE and densitometry showed that the quantities of haemolymph proteins were reduced dramatically in the parasitised larvae. Simultaneously, parasitism induced large amount of 95 kDa polypeptides in the haemolymph of the parasitised larvae. Also, a remarkable induction of 43 and 45 kDa polypeptides which are not detectable in non-parasitised larvae appeared in the parasitised larvae.     

Changes taking place in the electrophoretic haemolymph protein pattern during metamorphosis of Polytela gloriosae (Lepidoptera)

The haemolymph proteins of the larva, pupa and adult of Polytela gloriosae have been fractioned by Polyacrylamide gel disc electrophoresis. In the haemolymph of the fifth instar larval stage a total of ten protein fractions have been detected. The concentration of the protein fractions 2, 3, 4, 9 and 10 shows oscillations in their concentration in the early fifth instar, middle fifth instar and late fifth instar larval stage. In all 11 protein fractionswere detected in the haemolymph of different stages of the pupa. The protein bands 1, 7 and 10 of the pupa appear newly in the haemolymph as these bands were not found in the haemolymph of the larvae. The protein fraction 9 of larva was not found in the pupa. In the haemolymph of adult insect sexual difference was observed in the haemolymph protein pattern. In the haemolymph of adult female a total of 10 protein fractions were detected while from the male haemolymph a total of 8 protein fractions were detected. The pupal band 7 was not found in the adults of both the sexes. In the haemolymph of larva and adult one pigmented protein fraction was observed. No pigmented protein fraction was found in the haemolymph of pupa. Iron - containing protein fraction and the acid mucopolysaccharides were not found in the haemolymph. The protein fractions 3, 4, 5, 6 and 7 of adult haemolymph were darkly stained by the Schiff reagent and, thus, they are the fractions of glycoprotein. One protein fraction of lipoprotein was also found in the haemolymph.     

Inhibition of the formation of protein storage granules by glutaurine in the larval fat body cells of Mamestra brassicae (Insecta, Lepidoptera)

Correlative changes in the protein contents of haemolymph and fat body and the accumulation of protein storage granules in the fat body cells of Mamestra brassicae were investigated during the last larval stage in normally developing larvae and following administration of glutaurine (1 X 10(-4) mg/g body weight). The protein content of the haemolymph of untreated larvae increased up to the 4th day of the stage, declined during days 5 and 6, and increased again before pupation. In the glutaurine-treated larvae the amount of proteins in the haemolymph was as high as in the controls during the first four days but continued to rise up to the end of the stage. The protein content of the fat body started to increase from the 3rd day and heavy accumulation of protein storage granules in the cells of fat body was observed on the 5th and following days. The protein content of the fat body of glutaurine-treated larvae remained at a low level and the protein storage granules were absent in the cells. The inhibition of the selective uptake of haemolymphatic storage proteins by fat body following glutaurine treatment is suggested.     

The influence of oral secretions from larvae on levels of proteinases in colony members of Solenopsis invicta Buren (Hymenoptera: Formicidae)

The role of larvae in the digestion of protein in colonies of the red imported fire ant, Solenopsis invicta Buren was examined. Proteinase activity was significantly higher in the midguts of workers tending larvae compared to workers maintained without larvae. Food type (honey, egg yolk or soybean oil) influenced proteolytic activity in isolated workers but not in workers maintained with larvae. Proteinase activity in the labial glands and midguts of larvae maintained with or without workers was not significantly different. The total protein concentration in larval oral secretions was approximately 60–70% of that in the haemolymph. However, the percentage of free amino acids in the oral secretions was 2 to 3 times greater than that in the haemolymph.     

Cathepsin L-like cysteine proteinase (DcCathL) from Delia coarctata (wheat bulb fly): Basis of insecticidal activity

A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly (Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.5. DcCathL was insecticidal to lepidopteran larvae when injected into haemolymph, causing mortality that was accompanied by systemic melanisation, suggesting that DcCathL was affecting the immune-related proteolytic activation cascade leading to production of active phenoloxidase. This process is normally negatively regulated by serpins in the haemolymph. Recombinant serpins from cabbage moth (Mamestra brassicae) did not inhibit DcCathL, and were susceptible to degradation by the enzyme in vitro in buffer and extracted haemolymph. When M. brassicae larvae were co-injected with a lethal dose of DcCathL and exogenous recombinant serpins, no mortality or systemic melanisation was observed, suggesting that the insecticidal effects of DcCathL in vivo result from degradation of endogenous serpins.     

A sex-influenced protein in Chironomus (Diptera)

A protein, detected electrophoretically in Chironomus, was found to be sex-influenced since it is present in large amounts in females and absent in males (although on native gels, but not SDS gels, an electrophoretically equivalent band is present). This protein was found to be composed of two subunits of similar molecular weight (approx. 17,800 Da) and has the characteristics of both a haemoglobin as well as a vitellogenin. Its presence in the female haemolymph was correlated with the stages of development and in none of the species tested did it appear until the fourth instar; however, it persists throughout the subsequent stages. Tissue distribution studies indicate that this sex-influenced protein is found not only in the haemolymph of the female larvae but also in the pupal ovaries and adult eggs. An antiserum raised against the protein was used to confirm the presence of this protein in these tissues in a number of species, and in isolating the gene. The results suggest that this protein may be required for respiration as well as egg formation.     

Changes in haemolymph proteins of the fall armyworm, Spodoptera frugiperda (J. E. Smith), associated with parasitism by the braconid parasitoid Cotesia marginiventris (Cresson)

Changes in haemolymph proteins of the fall armyworm, Spodoptera frugiperda, associated with parasitism by the parasitoid Cotesia (= Apanteles) marginiventris were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis. As early as hour 4 after parasitization treatment, several electrophoretically slow-migrating, high-molecular-weight proteins were detected in the host's haemolymph. These proteins were detected earlier in haemolymph from parasitized larvae than in haemolymph from control larvae, and their concentrations were higher in heavily parasitized host larvae (≥ 3 eggs/host) than in lightly parasitized larvae (1 egg/host). Additionally, unique proteins that migrated electrophoretically with bovine serum albumin appeared in the haemolymph of parasitized larvae at hour 8 after parasitization treatment and were evident in haemolymph collected through to hour 64.     

Effects of methoprene,a juvenile hormone analogue,on the larval and pupal stages of the yellow fever mosquito, Aedes aegypti

Exposure of early fourth-instar larvae of Aedes aegypti to the juvenile hormone analogue Altosid ZR15^® (methoprene) significantly increased the concentration of carbohydrates in the haemolymph of late fourth-instar larvae and reduced the haemolymph carbohydrate concentration of 24-h-old pupae relative to controls. Such treatment also effected a decline in haemolymph amino nitrogen levels of the pupal stage and a depletion of haemolymph proteins in late fourth-instar larvae as well as pupae. Two of nine protein fractions in the haemolymph of larvae were significantly depleted following methoprene treatment. Fourteen soluble protein fractions were present in the haemolymph of control pupae; two of these were missing from the pupae which were treated as larvae with methoprene. A further protein fraction, common to the haemolymph of both treated and control pupae, was significantly reduced in concentration as a consequence of exposure to methoprene. The juvenile hormone analogue impaired the capacity of the fat bodies of late fourth-instar larvae and pupae to synthesise proteins, resulting in a lowered concentration of fat body proteins. Glycogen levels in the fat bodies of treated larvae were significantly lower than in controls and glycogenolysis was suppressed due to an overall depletion of glycogen phosphorylase and, in pupae, a lowered ratio of active: inactive enzyme. The data are consistent with the proposition that the juvenile hormone analogue elicits neuroendocrinological changes in the target insect.     

冰核活性细菌(INAB)对菜青虫血淋巴免疫系统影响的研究

实验采用浸泡和喂食2种方法,研究了冰核活性细菌(INAB)在不同处理时间对菜青虫Pierisrapae血淋巴免疫系统的影响。结果表明,采用水浴法可使菜青虫血淋巴免疫力被破坏,而喂食法效果则不明显;同时结果还表明冰核活性细菌主要是在低温条件下通过体表侵入菜青虫血淋巴而影响其免疫力。     

菜青虫感染玫烟色拟青霉后血淋巴蛋白质含量及几种保护酶活力的变化

对玫烟色拟青霉Paecilomyces fumosoroseus侵染3、4龄菜青虫Pieris rapae后,其血淋巴中蛋白质含量、体内保护酶及谷胱甘肽S-转移酶活力进行了研究。结果表明,感病的3、4龄菜青虫血淋巴中蛋白质含量明显低于同期未感染的幼虫;感病虫体内超氧化物歧化酶、过氧化氢酶和过氧化物酶的活力发生了不同程度的改变,其中对3龄菜青虫体内酶活力的影响比4龄幼虫大。此外被侵染的3、4龄菜青虫体内谷胱甘肽S-转移酶活力在感病前期显著高于同期未感染的菜青虫,而在感病后期明显低于同期未感染的菜青虫。     

Freeze–thawing method: a bleeding method from lepidopteran larvae utilizing a spontaneous insect body contraction after a freezing-thawing treatment

Insect haemolymph contains growth promotor(s) for cultured insect cells and is frequently used as an additive to the culture media. Insect haemolymph serves as a pool of a protein product produced by a virus vector–insect host system. Haemolymph collection is an essential step in the above process, which should limit the scale and cost of their performance. In the present study, a simple procedure for bleeding from lepidopteran larvae, Bombyx mori , has been developed which utilized a spontaneous contraction of the insect body after a freezing–thawing treatment. In the case of fifth-instar B. mori , 60 to 80% of the total haemolymph was collected by this method. The authors applied the method to a haemolymph collection from frozen larvae stored at −80°C for longer than 1 month. Preservability of the frozen larvae enabled the development of a system dealing with a huge bulk of insects. The bleeding method was effective under cooled condition at 0°C or 4°C, which was desired for protein handling. Development of a large system would result in a cost reduction for the insect haemolymph products such as insect cell-culture additive. Furthermore, the above bleeding method was applied to the nuclear polyhedrosis virus-infected B. mori larvae and up to 80% of the total haemolymph was collected from the virus-infected larvae. It suggests the bleeding method as an effective means of haemolymph collection in the protein productive system using a virus vector and its insect host.     

Proteins of the fat body of non-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella: Effect of juvenile hormone

The proteins of the fat body of non-diapausing, pre-diapausing, and newly-diapaused larvae of the southwestern corn borer, Diatraea grandiosella, were examined. Since a low titre of juvenile hormone (JH) is present in the haemolymph throughout the final instar of non-diapausing larvae, the hormone does not appear to stimulate the pre-metamorphic synthesis of proteins. In contrast, the high titre of JH in the haemolymph during the final instar of pre-diapausing larvae appears to stimulate the synthesis of selected proteins. For example, pre-diapausing larvae store in their fat body a low molecular weight protein which has been named the ‘diapause-associated protein’. When non-diapausing larvae were treated topically with C₁₇-JH or a JH mimic, from 50 to 70% entered a diapause-like state as fully grown larvae. These hormone-treated larvae accumulated the diapause-associated protein and a high molecular weight protein in their fat bodies. Both of these proteins were shown to be released from the fat body of newly-diapaused larvae in vitro, and may function in the haemolymph during diapause. The high molecular weight protein, isolated from the haemolymph, was shown to contain neutral and polar lipids, including biochromes. Its storage in the fat body and release into the haemolymph may be essential for the transport of lipids during diapause. The fat body proteins of newly-diapaused larvae of the southern cornstalk borer, Diatraea crambidiodes, were also examined electrophoretically. They were found to contain a similar protein pattern to that of D. grandiosella, including the presence of a diapause-associated protein.     

柞蚕感染微孢子虫后血淋巴免疫应答蛋白质的分离与鉴定

了解柞蚕Antheraea pernyi感染微孢子虫初期血淋巴内免疫系统及刺激应答相关蛋白质种类, 本研究以柞蚕5龄雌幼虫的起蚕（结束4眠, 刚完成蜕皮的幼虫）添食柞蚕微孢子虫Nosema pernyi为材料, 对感染后血淋巴利用SDS-PAGE进行分离后, 利用LC-MS/MS质谱技术和蛋白质组学分析对差异蛋白质条带进行鉴定。结果显示： 感染微孢子虫144 h后, 血淋巴中分子量约为44 kD （AP44）和28 kD （AP28）的蛋白质条带表达量增高。质谱分析AP28和AP44蛋白质条带样品, 共鉴定117个不重复蛋白质, 其中2个样品共有蛋白质12个, AP28独有蛋白质52个, AP44独有蛋白质53个。对质谱数据利用COG数据库进行搜寻鉴定, 显示AP28和AP44的鉴定蛋白质中涉及柞蚕免疫系统及刺激应答生物过程的蛋白质共有29个, 其中AP28中包括热激蛋白、 泛素样蛋白、 泛素结合酶E2、 保幼激素环氧水解酶、 微管结合蛋白、 溶菌酶、 ADP-核糖基化因子、 防御蛋白、 肽聚糖识别蛋白等15个, AP44中包括DRK、 酚氧化酶原、 类免疫球蛋白等10个; 二者共有热激蛋白hsp21.4、 酚氧化酶原、 抗菌肽等4个。本研究结果可以为今后研究柞蚕对微孢子虫的免疫应答及防御机制提供参考。     

Effect of dietary cholesterol deficiency upon its distribution in the larvae of the housefly, Musca domestica

The free sterol, total phospholipids and protein content of the various tissues and haemolymph lipoproteins obtained from the larvae of Musca domestica, reared on the diets containing 0.56 μmole cholesterol/g wet weight of diet (normal) and 0.05 μmole cholesterol/g wet weight of diet (deficient) have been determined. The cholesterol in the diet was found to be taken up by the larvae and distributed between all the tissues examined. About 60% of the free sterol in the larvae was recovered from the composite gut fraction and muscle. Cholesterol deficiency reduced both the growth of larvae and the free sterol content of the various tissues and haemolymph when compared to that of normal larvae. Cholesterol deficiency resulted in a slightly higher proportion of sterol and protein of the larval haemolymph being associated with the lipoproteins having slower electrophoretic mobility. Most of the different tissues from the cholesterol deficient larvae contained a much smaller proportion of their normal free sterol content than of their phospholipid or protein; the brain tissue however contained a higher percentage of free sterol and the haemolymph a much lower percentage than would be expected from the lowering of phospholipid and protein content as a result of the deficiency. When the sterol content was expressed relative to the protein, the ratio was higher in the brain tissue of both the normal and deficient larvae than the ratio present in the remaining tissues, apart from the composite gut fraction of the normal larvae. The results suggest that a disproportionate amount of available cholesterol was being concentrated into the nervous system of the cholesterol deficient insect.A rather higher proportion of the total sterol fraction recovered from the various tissues and haemolymph lipoproteins of cholesterol deficient larvae behaved as ‘polar metabolites’ of cholesterol when compared with that of normal larvae.     

The biochemical effects of potassium chloride on the silkworm, （ Bombyx mori L.）

The supplementation with 50, 100 and 150μg/mL potassium chloride to the fifth instar larvae of the silkworm Bombyx mori on fat body glycogen, protein, total lipids and haemolymph protein and trehalose were analyzed. The fat body glycogen and protein and haemolymph protein were increased significantly in all the treated groups; whereas fat body total lipids increased only in 100 and 150μg/mL and haemolymph trehalose increased only in 150μg/mL potassium chloride-treated groups when compared with those of the corresponding parameters of the carrier controls.     

Analysis of endoparasitoid-released proteins and their effects on host development in the system Chelonus inanitus (Braconidae)-Spodoptera littoralis (Noctuidae)

Having shown earlier that the larva of C. inanitus is essential in inducing the precocious onset of metamorphosis in polydnavirus/venom containing S. littoralis, we here analysed release of proteins by parasitoid larvae and their effects on host development. Parasitoid larvae released proteins in vivo and in vitro in a stage dependent manner. An approximately 212 kD protein was released from the mid 1st instar onwards and additional smaller proteins could be associated mainly with the 2nd and 3rd instar. When parasitoids were implanted into S. littoralis larvae, parasitoid-released proteins were seen 6 hr later. When parasitoids were removed from hosts, parasitoid-released proteins persisted in the host haemolymph for some time. Injection of antiserum against parasitoid-released proteins after removal of the parasitoid larva accelerated the disappearance of the 212 kD protein and reduced the number of larvae entering metamorphosis precociously. Repeated injections of concentrated parasitoid medium into polydnavirus/venom containing larvae caused a reduction of the head capsule width and formation of miniature 6th instar larvae; this effect was not seen in the absence of polydnavirus/venom. These observations suggest that proteins released by the parasitoid might play a role in modifying host metamorphosis in the presence of polydnavirus/venom, and the temporal appearance of the 212 kD protein makes it the most interesting candidate for being involved in such an effect.     

Parasitism of Lacanobia oleracea (Lepidoptera) by the ectoparasitoid, Eulophus pennicornis, is associated with a reduction in host haemolymph phenoloxidase activity

When haemolymph from fifth instar Lacanobia oleracea was incubated in vitro, rapid melanization occurred. Similar levels of melanization occurred in haemolymph from larvae that had been experimentally injected with venom from the ectoparasitic wasp, Eulophus pennicornis. In contrast, haemolymph from larvae parasitized by this wasp melanized more slowly and less extensively. Phenoloxidase assays indicated that enzyme activity was present in haemocyte lysate supernatants, serum and plasma from L. oleracea and that on day 5 post-parasitization, fractions prepared from parasitized larvae had significantly less phenoloxidase activity than similar fractions from untreated or experimentally envenomated larvae. In addition, no PO activity was detectable in wasp venom, and the venom had no effect on L. oleracea plasma phenoloxidase activity in vitro. These results indicate that parasitism of L. oleracea by E. pennicornis suppresses host haemolymph phenoloxidase activity and that this suppression is not induced by adult wasp venom. The results are discussed with reference to the survival advantages of suppressing the activity of this host enzyme, and to the possible source(s) of putative suppressive factors.     

中华甲虫蒲螨寄生双条杉天牛幼虫血淋巴变化

为了解中华甲虫蒲螨Pyemotes zhonghuajia Yu and Zhang对双条杉天牛Semanotus bifasciatus(Motschulsky)的致死过程,将已发育成熟的中华甲虫蒲螨膨腹体移入放有双条杉天牛2龄幼虫的玻璃指形管中,在蒲螨的一个生活史60h内,观察其取食行为,并在移入后每隔12h用考马斯亮蓝法分别测定天牛幼虫和蒲螨血淋巴中总蛋白质含量。结果显示,蒲螨蛋白含量总体呈现上升的趋势,在36～48h上升幅度最大,达61.78%;而天牛幼虫血淋巴总蛋白质含量则呈总体下降趋势,与对照相比,在36～48h之间下降最为明显;蒲螨总蛋白质含量在24h后处理组与对照组差异均为极显著,天牛幼虫总蛋白质含量在48h后处理组与对照组差异显著,这表明蒲螨在24h已搜寻到寄主开始取食,且取食效果明显,而天牛幼虫在48h后表现为麻痹致死,36～48h蒲螨取食对天牛致死起关键作用。12h时处理组的双条杉天牛幼虫总蛋白质含量比对照组略高,推测是由于幼虫对外寄生物蒲螨具有一定的免疫防御反应。     

Immunological analysis of lipophorin in the haemolymph,ovaries, and testes of the fall webworm,Hyphantria cunea (Drury)

Lipophorin (LP) was purified from haemolymph in last instar larvae of Hyphantria cunea (Drury) by KBr density gradient ultracentrifugation and gel filtration. LP is composed of Apo-LP I and Apo-LP II with molecular weights of 230 kDa and 80 kDa, respectively. The level of haemolymph LP in early pupae was somewhat greater than in last instar larvae. In males, this LP concentration is maintained throughout pupal development, whereas the level of haemolymph LP decreases in female pupae beginning at day 7, coincident with the onset of vitellogenesis in the fall webworm. In both male and female adults, haemolymph LP concentrations were dramatically increased in comparison to their pre-adult levels. Actually, LP was found in the ovary by immunodiffusion, tandem-crossed immunoelectrophoresis, and Western blotting. Location of LP in the ovary was also traced by immunogold labelling. Also, LP appeared in small amounts in protein yolk bodies of the ovary at an early stage of vitellogenesis, when nurse cells are bigger than the oocyte, but in greater amounts at those stages when the oocyte is larger than nurse cells—that is, when vitellogenesis is actively taking place. This fact clearly reveals that LP is synthesized by fat body and released into the haemolymph, and then taken up by the growing ovary during vitellogenesis. Also, LP was detected in testes by immunological analysis. Western blotting showed that LP was present in testicular fluid but not in the peritoneal sheath and cysts. To test whether LP is also synthesized in testes, testes and fat body tissues were cultured in vitro, indicating that fat body synthsizes LP but testes do not. The result showed that the haemolymph LP itself is taken up into the testes. © 1994 Wiley-Liss, Inc.     

大斑芫菁滞育幼虫在滞育不同阶段体内糖类和醇类含量的变化

在以卵滞育的昆虫中昆虫滞育时的生理代谢特点已经得到了大量研究。本文对以末龄幼虫（5龄）滞育的大斑芫菁Mylabris phalerate（Pallas）在不同滞育阶段体内糖类和醇类代谢的特征进行了研究。结果表明： 滞育个体血淋巴中的海藻糖含量高于非滞育个体,且随滞育时间的加大逐渐升高,滞育5个月时达到最大值,为5.61 μmol/mL。糖原的含量随滞育的进程逐渐减少,滞育初期（0.5个月）为0.72 mg/mL,到滞育末期（5个月）时仅为0.1 mg/mL。滞育个体脂肪体中的海藻糖含量都高于非滞育个体,滞育1个月时为非滞育个体的3倍,至滞育末期时达非滞育个体的5倍,为2.5 μmol/g脂肪体。糖原含量总体变化趋势是随滞育时间的加大逐渐减少,滞育早期和中期都高于非滞育个体。在滞育过程中血淋巴积累的小分子多元醇主要为甘油,其次是山梨醇;而在脂肪体中主要为甘油,其次是甘露醇,少量积累山梨醇：表明大斑芫菁滞育幼虫主要积累的是海藻糖和一些小分子多元醇。滞育幼虫在准备滞育时储备了大量糖原,这些糖原可能为滞育期间海藻糖、山梨醇和甘油的代谢提供了原料。