The Lectin from the Crustacean Liocarcinus depurator Recognizes O-acetylsialic Acids

A lectin that recognized sialic acids and agglutinated mouse erythrocytes was purified from hemolymph of the crab Liocarcinus depurator. It consisted of 38-kDa subunits and had a pI about 6.0. The specificity of the lectin was assayed by hemagglutination inhibition. N-acetylneuraminic acid (Neu5Ac) was a good inhibitor and its N-acetyl group at C-5 was critical for lectin-ligand interaction. Substitution of the C-9 hydroxyl on Neu5Ac with an O-acetyl group (9-O-Ac-Neu5Ac) increased the inhibitory potency of this molecule. Furthermore, O-acetyl substitution of all the hydroxyl groups yielded even better inhibitors (2,4,7,8,9-O-Ac-Neu5Ac and its 1-O-methyl ester). Removal of the hydroxyl or O-acetyl group connected to C-2 reduced the potency of these inhibitors. The lectin agglutinated and stimulated human but not mouse lymphocytes. It was also inhibited by Escherichia coli (O111:B4) lipopolysaccharide and agglutinated specific gram-negative bacteria. In vitro labeling with [³⁵S]methionine indicated that the lectin was synthesized in hepatopangreas of L. depurator. Immunofluorescence showed that among hemocytes it localized mainly in the large-granule population.     

Hemocytes of Biomphalaria glabrata: Factors affecting variability

Variability in the hemocyte number of two geographic strains of Biomphalaria glabrata was studied. In each strain a logarithmic increase in hemocyte number associated with increasing shell size was observed. A two fold increase in circulating hemocytes occurred 2 hr following the exposure of a susceptible strain of B. glabrata to miracidia of Schistosoma mansoni. The hemocyte number was dependent on the temperatures at which the snails were maintained.     

Infection of the European chafer,Amphimallon majalis by Bacillus popilliae: Light and electron microscope observations

Following ingestion of spores of Bacillus popilliae by grubs of the European chafer, Amphimallon majalis, vegetative rods were observed within phagocytic vacuoles of midgut columnar cells prior to establishing primary infection foci in regenerative nidi areas. This resulted in increased activity of regenerative nidi and extrusion of degenerating epithelial cells frequently containing vegetative rods of B. popilliae. Circulating hemocytes adhered to the hemocoelic surface of the basement membrane and formed inflammatory capsules immediately adjacent to the areas of bacterial proliferation. Bacilli in various stages of degradation were observed in membrane-limited vacuoles of both mesenteric epithelial cells and capsular hemocytes. Despite these host defense reactions, some vegetative cells resisted degradation and were successful in traversing the epithelial, basal lamina, and capsular barriers to enter the hemolymph.     

Cytopathology of spotted cucumber beetle hemocytes containing virus-like particles

Spherical, virus-like particles with an average diameter of 70 nm were consistently found in the hemocytes of field-collected, spotted cucumber beetles, Diabrotica undecimpunctata. The particles and associated inclusion bodies occurred both in the cytoplasm and nucleus. Hemocytes containing the particles exhibited structural features which were indicative of virus infection. These were (1) the appearance of fibril-containing membranous vesicles, 70 to 80 nm in diameter, in the perinuclear space and in the cisternae of rough endoplasmic reticulum, (2) accumulation of vesicle-containing cisternae in certain areas of cytoplasm, (3) appearance and condensation of granular material in viroplasm-like structures, and (4) appearance of virus-like particles. The results suggest that the particles are viral in nature and occur widely in leaf-feeding beetles.     

In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system

Extracellular serine protease cascades mediate immune signaling and responses in insects. In the tobacco hornworm Manduca sexta, nearly 30 serine proteases (SPs) and their homologs (SPHs) are cloned from hemocytes and fat body. Some of them participate in prophenoloxidase (proPO) activation and proSpätzle processing. Here we report the cDNA cloning of hemolymph protease-1b (HP1b), which is 90% identical and 95% similar to HP1a (formerly HP1). The HP1a and HP1b mRNA levels in hemocytes was down- and up-regulated after an immune challenge, respectively. Quantitative real-time polymerase chain reactions revealed their tissue-specific and development-dependent expression, mostly in hemocytes of the feeding larvae. We isolated HP1 precursor (proHP1) from larval hemolymph and observed micro-heterogeneity caused by N-linked glycosylation. Supplementation of the purified proHP1 to plasma samples from naïve larvae or induced ones injected with bacteria caused a small PO activity increase, much lower than those elicited by recombinant proHP1a/b, but no proteolytic cleavage was detected in the zymogens. Incubation of proHP1a/b or their catalytic domains with a cationic detergent, cetylpyridinium chloride, induced an amidase activity that hydrolyzed LDLH-p-nitroanilide. Since LDLH corresponds to the P4–P1 region before the proteolytic activation site of proHP6, we propose that the active but uncleaved proHP1 may cut proHP6 to generate HP6 that in turn activates proPAP1 and proHP8. The catalytic domain of HP1a/b, which by itself does not activate purified proHP6 or hydrolyze LDLH-p-nitroanilide, somehow generated active HP6, HP8, PAP1 and PO in plasma. Together, these results indicate that proHP1 participates in the proPO activation system, although detailed mechanism needs further exploration.     

Chemotactic attraction between hemocytes of the oyster,Crassostrea virginica,and bacteria

Experiments were conducted to ascertain whether there is chemotactic attraction by Bacillus megaterium and Micrococcus varians, both Gram-positive species, and Escherichia coli and Vibrio parahaemolyticus, both Gram-negative species, for hemocytes of the American oyster, Crassostrea virginica. It was ascertained quantitatively that oyster hemocytes are attracted to live E. coli, B. megaterium, and M. varians but not to heat-killed bacteria. Furthermore, oyster cells are not attracted to either live or heat-killed V. parahaemolyticus. It is concluded that the chemoattractant is some molecule emitted by living vegetative cells of certain Gram-positive as well as Gramnegative bacteria.     

一种体外分析亚洲玉米螟幼虫血细胞包囊的改进方法

包囊反应是昆虫清除侵入体内的外源物如病原物和寄生生物的一种非常重要的细胞免疫反应。由于受到不便于观察、操作复杂等问题的限制,很多包囊分析实验无法或难于在昆虫体内完成。体外包囊方法在一定程度上解决了这些问题。目前常用的体外包囊是在96孔板中加入昆虫血细胞和外源物,如凝胶珠进行培养观察,但这种方法存在着一些明显的缺陷。本文以亚洲玉米螟(Ostrinia furnacalis)幼虫血细胞为研究对象,使用0.2mL的离心管(Eppendorf tube)代替96孔板,并将其固定在匀速旋转的载体上培养,极大程度的模拟了反应物在昆虫体内的状态。结果表明,改进方法后昆虫血细胞的体外包囊效果得到明显提高,且血细胞的状态也得到明显改善;而添加抗凝剂会减弱血细胞对外源物的包囊能力。     

红褐斑腿蝗血细胞的形态与分类

利用光学显微镜和显微数码拍照系统,对红褐斑腿蝗Catantops pinguis(Stal)血细胞的形态进行观察和分类。结果在红褐斑腿蝗血淋巴中观察到5种血细胞,分别是原血胞、浆血胞、粒血胞、珠血胞和囊血胞。原血胞为小型圆形细胞,边缘圆滑、清晰,核质比例很大。粒血胞多为中型,形状不规则,边缘凹突不平,内含较大的异质性溶酶体颗粒。浆血胞多为中型,刚离体时形状较规则,常呈圆形、卵圆形。浆血胞内缺乏大的颗粒,细胞核大而圆形,细胞质内具许多小型颗粒状物质。浆血胞离体后形状变化较多,常发展出伪足,呈丝状、短芒状、钩状或片状伪足。珠血胞多为大、中型,外形大体呈圆形,但边缘由于大小不等的珠形内含物突出,呈花瓣状。囊血胞多为中型,圆形或椭圆形,细胞质内具有大小不一的带有折光性的颗粒或块状物,细胞边缘比较光滑。     

Breeding patterns in the pre-desert oniscid isopod Porcellio buddelundi of Matmata (Gabès,Tunisia)

Porcellio buddelundi inhabits arid areas in Tunisia. The reproductive pattern of a population at Oued El Jir, Matmata (Tunisia) was studied from July 2005 to June 2006. Monthly samples were taken during the study period. The overall sex ratio was biased toward females. Males, females, and newborns all had greater body-mass in autumn than in spring and their lowest mean body-mass was in June and July. Ovigerous females greater than 41.1 mg in body-mass, were collected from March through May and from September through October, suggesting seasonal reproduction with two breeding seasons: the longer one in spring (3 months) and another in the fall (2 months). Fecundity, which was positively correlated with the body-mass of females, varied between breeding seasons with a large number in spring and a small number in autumn. Seasonal variation of fecundity could be explained by the growth rate of ovigerous females affecting the fecundity more in spring than in autumn. The onset of breeding, in P. buddelundi, takes place when the day-length exceeds 12 h and the soil moisture decreases.     

粘虫和棉大卷叶螟幼虫体内肺结构的存在与功能验证

加拿大昆虫学家Locke提出鳞翅目幼虫体内有一个适应血细胞进行气体交换的肺结构。本文以Locke的研究为依据, 运用电镜及其他化学方法, 观察和研究了粘虫Leucania separata及棉大卷叶螟Sylepta derogata幼虫的肺结构和功能。结果表明: 肺存在于第8腹节的气管处, 该气管分支细短而丰富, 形成气管簇。亚甲基蓝标记血液发现, 血流方向为由前向侧后方向, 流经第8腹节和臀腔, 最后流回心脏。第8腹节气管簇的管壁比其他各节都薄, 且管壁内膜具有更多适合运动的细管, 这有利于气体的通透。正常情况下, 气管簇处有各种各样的血细胞聚集, 当缺氧胁迫时有大量的血细胞从组织中释放, 通过变形而紧贴气管簇, 以利于气体的交换。这些特征都与高等动物的肺功能相似, 因而判断此结构存在肺的功能。