Induction of antibacterial activity in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 in the anterior and posterior body part of the ligated larvae.

1. Induction of antibacterial activity was investigated in the ligated fifth instar larvae of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 into the haemocoel in the anterior and in the posterior body part, followed by activity determination by inhibition zone assay of the haemolymph at 12 and 24 hr after immunization. 2. At 12 hr after immunization, high antibacterial activity, approximately 6.8-7.5 mm in the anterior body part and 4.5-6.4 mm in the posterior body part in diameter of a clear zone (2.0 mm for no activity) was detectable in day 3 larvae. This result was in good contrast to expression of lectin gene in the ligated flesh fly, Sarcophaga peregrena (Shiraishi and Natori, 1988, FEBS Lett. 232, 163-166), in which only the anterior part of insect responded to stimulus of injury. 3. Antibacterial activity at 24 hr after immunization in days 3 or 4 ligated larvae was lower than that at 12 hr; 4.0-4.5 mm of activity was observed in the anterior body part, and no activity was observed in day 3 ligated larvae in the posterior body part. 4. Acid polyacrylamide gel electrophoresis of the haemolymph of immunized insects followed by overlay assay showed that the size of antibacterial activity bands were similar between the haemolymph from 12 hr and from 24 hr, and between the anterior and the posterior body part. This result was contradictory to the observation of activity by inhibition zone assay. The activity bands were associated with peptides that were similar to cecropin-like peptides A and B in the silkworm.(ABSTRACT TRUNCATED AT 250 WORDS)     

家蚕血淋巴体外黑化观察及相关黑色素合成催化酶基因

观察不同生长期家蚕幼虫血淋巴在体外的黑化速度和对大肠杆菌生长的影响结果显示,随食桑生长幼虫血淋巴的黑化速度逐渐变快,对大肠杆菌生长的抑制作用逐渐增强。RT-PCR实验显示,黑色素合成催化酶Bm Tan、Bm Po-1、Bm Yellow-f和Bm Ddc等的基因在家蚕5 L 3 d血淋巴中表达量高,Bm Black、Bm Yellow和Bm Pah等的基因也有明显表达。q PCR分析显示,黑化病蚕中Bmtan、Bmddc、Bmyellow、Bmebony和Bmblack,尤其Bmddc表达发生了显著上调。与对照相比,Ddc酶的抑制剂能显著抑制脂多糖对血淋巴的诱导黑化作用。用大肠杆菌注射家蚕幼虫,血淋巴中多巴和多巴胺的含量明显上升。这些表明家蚕幼虫血淋巴黑化与防御免疫有关,Bmddc很可能在幼虫血淋巴的免疫黑化中发挥作用。     

柞蚕滞育蛹的体液防卫反应

本文对柞蚕Antheraea pernyi滞育蛹体液防卫的某些特性做了报道。结果表明：(1)经注射大肠杆菌诱导6h后,体液中出现了抗菌活性物质。诱导7d后,抗菌活力达到峰值,15d后活力消失。用生理盐水诱导的反应较快,4d后达到峰值,10d后活力消失。(2)经大肠杆菌诱导后的体液对苏芸金杆菌、大肠杆菌、枯草杆菌及金黄葡萄球菌均有杀菌效应。在最初5min内杀菌效率较高。注射生理盐水诱导的体液较大肠杆菌诱导的杀菌效果低。(3)用昆虫病原菌苏芸金杆菌及非病原菌大肠杆菌作不同的诱导源,诱导1d后的体液对大肠杆菌的抗菌活力差别不显著,但对柞蚕蛹的生活力及发育有明显影响;用苏芸金杆菌诱导的柞蚕滞育蛹3d后死亡,用大肠杆菌诱导的蛹能正常羽化。(4)在一定范围内用不同剂量的大肠杆菌进行诱导,其抗菌活力不受影响,但超过一定限度,既使是非病原菌也可以突破昆虫的防卫功能。     

大肠杆菌及聚肌胞核苷酸对柞蚕、家蚕蛹诱导产生溶菌酶、抗菌肽及凝集素的动力学

大肠杆菌D₃₁诱导柞蚕蛹产生抗菌多肽。同时,溶菌酶和凝集素活性都比诱导前有明显增高.其活力高峰、抗菌多肽在第7天左右、溶菌酶在第5天,而凝集素在第3天即达最高水平。不同品种的柞蚕蛹,经诱导产生的三种活性物质其活力差异不明显,但741、河四和小混品种中的抗菌多肽P_9A及P_9B组成比例较高。上述三种活性物质的诱导变化与性别有关,雄性高于雌性。比较了柞蚕蛹和家蚕经细菌诱导后上述三种活性物质的变化,家蚕凝集素活力很低,诱导后活力增高不明显。抗菌活力及溶菌酶活力的提高程度柞蚕也高于家蚕。聚肌胞核苷酸(Poly I:C)也能诱导两种蚕产生抗菌多肽及溶菌酶,但活力提高的显著程度都不及大肠杆菌诱导,凝集素活力变化也不显著。     

家蚕感染蛹虫草后的生理生化变化

蛹虫草分生孢子侵染5龄家蚕Bombyx mori后,家蚕血淋巴中总糖、海藻糖、蛋白质和甘油酯含量均有不同程度的下降,其中甘油酯含量下降最为明显。海藻糖酶活性在侵染初期也明显降低。接种后家蚕体内的保护酶超氧化物歧化酶、过氧化物酶和过氧化氢酶活性也有较大变化,其中超氧化物歧化酶活性上升最为明显,在4日内由441.841 U/mL升至601.255 U/mL。     

九香虫血淋巴及其纯化蛋白抑菌活性的研究

对九香虫AsporgopuschinensisDallas血淋巴及其血淋巴蛋白质分离物的抗菌活性进行了研究,抗菌活性检测指示菌为大肠杆菌Escherichiacoli和金黄色葡萄球菌Staphilocalliesacereus。测定结果表明,九香虫血淋巴及其离心上清液都具有明显的抗菌活性。用凝胶过滤法从血淋巴蛋白分离提纯获得一种小分子肽,SDS PAGE电泳为单一带,分子量约为1～1 4 4kD。该小分子蛋白对大肠杆菌和金黄色葡萄球菌都有抑菌作用,与血淋巴对2种细菌的抗菌性一致,表明其是九香虫血淋巴中具抗菌作用的主要物质之一。     

Quantitative determination of the juvenile hormones in the haemolymph of Locusta migratoria during normal development and after implantation of corpora allata

Simultaneous quantitative determination of the three naturally occurring juvenile hormones in insects (JH-I, JH-II and JH-III) was performed on haemolymph samples of both normally developing locusts and locusts implanted with active corpora allata, using capillary gas chromatography with electron capture detection.In fourth instar female larvae, 24–48 hr after the third ecdysis, as well as in adult females, 18 days after the imaginal ecdysis, only JH-III was detected. In fifth instar female larvae JH-III was present in very low concentrations, if at all.After implantation of four pairs of corpora allata taken from young fourth instar female larvae or one pair or corpora allata taken from adult females into fifth instar female larvae 0–24 hr after ecdysis, an elevation of the JH-III titre was observed. Neither JH-I nor JH-II could be detected. The amount of JH-III, already elevated 2 hr after implantation, remained high for several days in comparison to that of control insects. On the third day after the subsequent moult the JH-III level was comparable to that of normally developing fifth instar larvae. Factors involved in the achievement of the haemolymph JH-titre are discussed.     

Determination of Caffeic Acid in the Digestive Juice of Silkworm Larvae and Its Antibacterial Activity against the Pathogenic Streptococcus faecalis AD–4

Caffeic acid (CA) was identified as an antibacterial substance in the digestive juice of silkworm larvae. Concentrations of CA in the digestive juice decreased from 460 ppm to 3 ppm as the starvation period of the larvae was prolonged from 1 hr to 12 hr. The specimens of digestive juice (ca. pH 10.0) containing 460 ppm and 222 ppm of CA had antibacterial activity against Streptococcus faecalis AD–4, a pathogenic intestinal bacterium affecting silkworm larvae, and an alkaline medium (pH 10.0) containing 500 ppm of authentic CA also exhibited antibacterial activity.

These findings suggest that CA plays an important role in the defense mechanism of silkworm larvae against the bacterial infection.     

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.     

Induced immunity in Antheraea assama Ww larvae against flacherie causing Pseudomonas aeruginosa AC-3

This study reports for the first time the induction of immunity in Antheraea assama Ww larvae against bacterial flacherie. In silkworms group of disease caused by bacteria are collectively called "flacherie." This refers to the flaccid condition of the larvae due to the infections of bacterial strains pathogenic to muga silkworm. Antibacterial activity against pathogenic Pseudomonas aeruginosa AC-3 causing flacherie, was induced by injection of heat-killed cells of the same strain. Experiments on larval survivability and viable cell count revealed peak immune response on third day. Comparison of the amount of food ingested, excreta produced and larval weight of the saline-injected control, live bacteria-challenged larvae and heat-killed bacteria-injected larvae "(vaccinated)" confirmed the development of immunity against bacterial infection in the "vaccinated" set. The haemolymph of A. assama larvae was analyzed for proteins associated with bacterial infection. Out of the total 32 detected proteins, eleven (A1-2, A15-20, A22-23, and A29) were constitutively synthesized in both the control and live bacteria-injected larvae. Four inducible proteins A4, A9-10, and A21 were detected in the haemolymph of the live bacteria-injected larvae. Synthesis of rest of the proteins varied between the control and their live bacteria-injected counterparts. General protein profile of "vaccinated" larvae injected with live bacteria were found to be similar to that of the saline-injected control.     

Insect immunity. Purification and characterization of a family of novel inducible antibacterial proteins from immunized larvae of the dipteran Phormia terranovae and complete amino-acid sequence of the predominant member, diptericin A

Injury or injection of live bacteria into third instar larvae of the dipteran insect Phormia terranovae results in the appearance in the haemolymph of at least five groups of heat-stable, more or less basic peptides with antibacterial activity against Escherichia coli. Three of these peptides have been purified. The amino acid sequence has been completely established for one of these and partially (first 40 residues from the N-terminus) for the two others. The sequences show marked homologies indicating that the three peptides belong to a common family. They are not related to other known antibacterial peptides from insects [lysozymes, cecropins (including sarcotoxin I) and attacins]. We propose the name of diptericins for this new family of antibiotic molecules.     

Exoproteinases of the type A in pathogenesis of insect bacterial diseases

Immune inhibitors produced in infected larvae of Galleria mellonella by such entomopathogens as Pseudomonas aeruginosa, Serratia marcescens and Heterorhabditis bacteriophora effectively blocked in vitro bactericidal activity of insect haemolymph against Escherichia coli D31, both in Galleria mellonella and Pieris brassicae pupae previously vaccinated with Enterobacter cloacae. Even at a trace concentration, the extracellular proteinases, by proteolytic degradation, totally destroyed the activity of cecropin peptides from Galleria and cecropin-like and attacin-family proteins from Pieris, but no ability to destroy antibacterial activity was shown by extracts obtained from Galleria larvae killed by massive doses of bacterial saprophytes. It is suggested that by blocking antibacterial immune response of the host, the proteinases help the bacteria to multiply in the haemolymph, thus they could be considered an important factor in the pathogenesis of bacterial diseases of insects.     

中国家蚕抗菌肽人工合成类CMⅣ基因在甜菜夜蛾虫体中的表达

将人工合成的中国家蚕抗菌肽类CMⅣ基因与抗菌肽信号肽基因连接 ,经EcoRⅠ、HindⅢ双酶切后 ,克隆于pFASTBacⅠ的EcoRⅠ、HindⅢ酶切位点之间 ,得到重组转座载体pFASTBac ABP ,经测序证明阳性克隆正确。将重组转座载体转化HD1 0Bac大肠杆菌 ,得到重组Bacmid ABP。将重组Bacmid转染sf2 1细胞及感染甜菜夜蛾 (Laphygmaexigua)幼虫 ,在培养细胞上清及虫体血淋巴中均测到抗菌活性。经Northernblotting证明感染甜菜夜蛾幼虫中有类CMⅣmRNA的存在。且表达产物在酸性电泳中电泳行为与天然抗菌肽CMⅣ组分相似。为进一步利用昆虫细胞及虫体生产抗菌肽药物打下了基础。     

RH-5992--an ecdysone agonist on model system of the silkworm Bombyx mori

RH-5992 is a novel synthetic non-steroidal ecdysteroid agonist with a high selectivity towards Lepidopteran species. The effect of ecdysone agonist RH-5992 on larval period, larval weight, silk gland weight and haemolymph protein profile were examined in the model organism, the larvae of silkworm, Bombyx mori. The LD50 values were found to be 16.21 and 12.01 micrograms/larva for 72 and 96 hr respectively. In the present study, three sublethal concentrations of 1/5th, 1/10th and 1/20th of LD50 at 72 hr were selected and applied on the mid-dorsal line of the silkworm B. mori. The maximum mortality of 35% was observed in the group which received the highest (3.2 micrograms/larva) concentration of RH-5992. The mortality rate was found to be dose dependent as well as time dependent. Interesting results were observed in haemolymph profile of the RH-5992 treated larvae as staining intensity of 30 kDa protein decreased significantly whereas the effect was not marked on other major proteins like storage proteins and vitellogenin polypeptides. From the results, it is confirmed that RH-5992 causes changes in larval characters and protein profile of silkworm B. mori. It is proposed that RH-5992 may cause negative effect specifically on reproductive characters like development of ovary and egg production due to decrease in 30 kDa protein.     

家蚕天蚕素cDNA原核表达及抗菌活性检测

采用RT-PCR方法从家蚕Bombyx mori新疆品种新蚕三号组织中扩增天蚕素cDNA片段,回收并克隆至Pmd18-T载体,进行序列分析。基因序列分析结果与已发表的天蚕素B的序列同源性为98%,表明所克隆的新疆家蚕天蚕素cDNA为独特的cDNA片段。将天蚕素基因与Pgex-4T-1融合表达载体中的谷胱甘肽转移酶基因融合,在大肠杆菌中表达, 结果表明经IPTG 诱导30 min后,pGEX-4T-1/天蚕素转化后的大肠杆菌生长明显受到抑制;当诱导210 min 后,大肠杆菌数量又开始增加,逐渐恢复至正常水平。说明天蚕素与谷胱甘肽转移酶基因融合表达后,在IPTG存在的短时间内仍然对原核细胞有较强的抗菌抑杀作用。     

不同家蚕品系的耐热性特征

研究了家蚕Bombyx mori 3个品系(Pure Mysore,NB4D2和CSR2）5龄幼虫和蛹在不同温度（35,38和40℃）下的耐热性,采用Probit分析测定了它们在各温度下的LT50值和置信限。结果表明：多化性品系Pure Mysore在高温下的存活率高于两个二化性品系NB4D2和CSR2,而两个二化性品系中NB4D2表现出更好的耐热性。家蚕幼虫接触38℃高温6 h和40℃高温3 h后,其血淋巴中出现90,70和29 kDa的热激蛋白条带。在恢复过程中,NB4D2和CSR2的血淋巴中未见29 kDa蛋白条带,而Pure Mysore幼虫的血淋巴中29 kDa蛋白仍然表达。当幼虫置于高温下时,血淋巴中90和70 kDa蛋白表达,但是检测不到29 kDa蛋白。研究认为热激蛋白表达与热带家蚕不同品系的耐热性以及与同一品系不同发育阶段的耐热性具有相关性。     

Haemolymphal activation of protyrosinase and the site of synthesis of haemolymph protyrosinase in larvae of the fleshfly, Sarcophaga barbata

When whole blood from 5 day third instar larvae of the fleshfly, Sarcophaga barbata was incubated under nitrogen at 25°C for 16 hr in the presence of salivary glands there was an increase in its protyrosinase content, which amounted to 53% of that which occurs in vivo over the same period. The protyrosinase in ammonium sulphate fractions of haemolymph that were allowed to stand at 4°C for 24 hr following the incubation at 25°C was found to have autoactivated. Analysis of all these fractions revealed the presence of a protyrosinase activator in the 30% saturated ammonium sulphate fraction. When proenzyme and haemolymph activator were mixed there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme, but was independent of the activator concentration and was comparable to that obtained using the cuticle activator. The level of activator in the haemolymph increased as larvae aged from 4 to 7 days.The effect of several compounds on the catecholase activity of the activated haemolymph protyrosinase and on the cuticle enzyme is reported and the significance of haemolymphal activation of protyrosinase is discussed.     

蜚蠊灭菌肽的诱导及初步分离分析

昆虫经诱导盾产生灭菌肽的研究近年来已有很大进展,有关这方面的研究工作绝大多数都是以有翅亚纲内生翅类鳞翅目(主要是蚕类)昆虫和少数双翅目昆虫为材料.本文首次以有翅亚纲外生翅类蜚蠊目的美洲蜚蠊(Ptriplaneta americana L.)为实验昆虫,用Escherichia coil K₁₂ strain D₃₁作诱导源,对不同发育期、不同性别、不同成虫期的蜚蠊进行诱导后,采用含菌培养基平板测活方法,就存在个体数及能产生抗菌物质的个体数进行了初步的研究.发现成虫日龄在10天之内的雄性蜚蠊能够产生抗菌物质的个体百分比最高.抗菌物质出现的高峰期是在诱导后第三、四天.用滴滴涕和溴氰菊酯作诱导源对雄性蜚蠊的诱导实验表明,杀虫剂也能诱导蜚蠊产生抗菌物质,而且所诱导产生抗菌物质的活性强度(用抑菌圈直径表示)高于大肠杆菌所诱导的.滴滴涕和溴氰菊酯的重复诱导可提高蜚蠊产生抗菌物质个体百分比.蜚蠊经诱导后产生的抗菌物质具有广谱性,对苏云金杆菌、金黄色葡萄球菌、枯草杆菌及绿脓杆菌等有较强的抗菌活性,而对大肠杆菌D₃₁、大肠杆菌、粘质沙雷氏杆菌和溶壁微球菌等有较弱的抗菌活性.用肽类物质的指纹图谱法分离蜚蠊血淋巴抗菌物质,发现经诱导后血淋巴中确有新的肽类物质产生,该物质具抗菌活性,用DABITC法分析,其N-末端氨基酸为赖氨酸.     

Changes in haemolymph constituents of American bollworm, Helicoverpa armigera (Hübner), infected with nucleopolyhedrovirus

Six types of haemocytes viz., prohaemocytes, plasmatocytes (round, fusiform, vermiform and spindle shaped), granular cells, spherule cells, oenocytoids and adipohaemocytes were found in the haemolymph of larvae of American bollworm H. armigera. The total and differential haemocyte counts (THC and DHC) in H. armigera haemolymph were affected by nucleopolyhedrovirus (NPV) treatment. There was a general decrease in THC in response to NPV treatment in both young and old larvae. However the decrease was more apparent in 5 and 8 day old larvae than in 10 day old larvae. The differential haemocytes showed less of granular cells and more of spherule cells and prohaemocytes in the old larvae. Plasmatocytes and granular cells in 10 day old larvae initially phagocytosed polyhedra; however, disintegrated after 3 to 4 hr. The haemolymph of NPV treated larvae melanized slowly particularly in old larvae. Phenoloxidase (PO) activity decreased positively with granular cells and oenocytoids in 10 day old treated larvae. Cellular fraction had high level of PO activity, which was transferred to plasma in response to NPV infection in the older larvae. The role of NPV pathogenesis vis-à-vis immunity in insect is discussed.     

Identification of the juvenile hormone from the cricket, Teleogryllus commodus, and juvenile hormone titre changes

In the cricket, Teleogryllus commodus, eggs, haemolymph of 7th and 8th (last)-larval instars, and haemolymph of adults of both sexes contain only juvenile hormone III. While in the male the hormone titre is independent of previous mating experience, juvenile hormone concentration in haemolymph taken from females 36–38 hr after mating (an event which is followed by oviposition) is at a level 5 times higher than that of virgin females. Based on data gleaned from several research groups the identification of juvenile hormone III as the exclusive juvenile hormone in the Order Orthopteroidea is discussed.