家蝇抗菌肽Attacin-2基因的克隆、序列分析和诱导表达

攻击素(attacin)作为昆虫抗菌肽之一, 在昆虫的先天免疫中起着重要作用。本研究通过家蝇Musca domestica EST序列筛选并结合RACE技术克隆了家蝇的Attacin-2基因(Mdatta2) cDNA序列。其全长819 bp, 包含一个726 bp的完整开放阅读框(open reading frame, ORF), 以及42 bp的5′末端非翻译区(5′-UTR)和51 bp的3′末端非翻译区(3′-UTR), 编码241个氨基酸残基, 推导的多肽N端22个氨基酸残基为信号肽序列。同源性分析表明, 其氨基酸序列与嗜凤梨果蝇Drosophila ananassae的Attacin一致性最高(46%)。以邻接法(Neighbor-Joining, NJ)构建的系统关系表明, 家蝇的Attacin-2与其他双翅目昆虫的Attacin起源于共同的祖先, 属于Attain_C超家族。应用实时荧光定量PCR的方法研究家蝇幼虫在受到外源细菌刺激时Mdatta2基因的表达, 结果显示, 在大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus分别刺激后3 h和6 h, 家蝇幼虫Mdatta2表达量出现显著上调。Mdatta2基因在家蝇幼虫体内呈诱导型表达, 表达水平随诱导时间的不同而变化, 提示Mdatta2基因可能在家蝇免疫防御过程中起着重要作用。     

Developmental patterns in the antioxidant defenses of the housefly, Musca domestica

The activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione S-transferases, GSSG reductase, thiol transferases, gamma glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase, and the concentrations of H2O2 and reduced and oxidized glutathione were determined in the various developmental stages of houseflies. Housefly development was correlated with a progressive increase of cellular oxidizing equivalents and a loss of cellular reducing capacity. The loss of reducing equivalents appeared to result from a decrease in the activity of enzymes involved in glutathione and NADPH synthesis and a concomitant increase in glutathione-oxidizing enzymes. Relatively little change was observed in SOD activity during housefly development; however, the electrophoretic pattern of MnSOD varied in a manner specific to developmental stage. A striking increase in H2O2 concentration occurred prior to pupation possibly due to changes in substrate catabolism. These results support the hypothesis that the cellular environment becomes progressively more oxidizing during development.     

Diuresis in the housefly (Musca domestica) and its control by neuropeptides

Diuresis was studied in vivo by measuring the loss of tritiated water. The basal rate of water loss (5 nl/min) represents respiratory and cuticular losses, whereas higher rates reflect urine output, which reaches 20 nl/min after injection of 1 microl distilled water. This response to hypervolemia involves release of a diuretic hormone(s) into the hemolymph. However, housefly diuretic peptides increased urine output to a maximum of only 7 nl/min, and higher rates may require fluid reabsorption from the hindgut to be reduced. Diuresis is partially blocked by injected anti-muscakinin antibodies, providing evidence of a hormonal function for this insect myokinin.     

家蝇蛹凝集素的免疫调节作用

本实验研究了从家蝇蛹体内分离的一种凝集素,研究了其免疫调节的性质。首先将收集的家蝇蛹在缓冲液中研磨,得到粗提物,经过亲和吸附、竞争洗脱等步骤得到了纯品。电泳结果表明家蝇蛹凝集素分子量大约为55kD。通过检测巨噬细胞分泌的细胞因子IL-6、IL-12等实验,证明了家蝇蛹凝集素浓度在5μg/mL时对与巨噬细胞分泌IL-6有显著增强作用,家蝇蛹凝集素的浓度在10μg/mL时对巨噬细胞分泌IL-12有显著效果。通过小鼠脾淋巴细胞增殖试验结果可知家蝇蛹凝集素对于混合淋巴细胞增殖有促进作用。以上试验结果说明家蝇蛹凝集素免疫调节作用,为天然免疫增强剂的开发提供了一定的参考依据。     

The Optics of the Compound Eye of the Honeybee (Apis mellifera)

The optical system of the compound eye of the worker honeybee, as a representative of the closed-rhabdom type of eye, was investigated and its function analyzed. Measurements of refractive indices of the elements of the optical system were made with an interference microscope. With the use of the resulting measurements, the optical system was analyzed by means of a ray-tracing procedure implemented for the IBM 7094 digital computer, and by means of the Gaussian thick lens formulae. The more detailed results of the ray-tracing technique were used for further analyses. Direct visual confirmation of the focal point was obtained. The rhabdom and the surrounding zone of lower refractive index act together as a wave guide, as demonstrated by the presence of several wave guide modes in the rhabdom. An admittance function was defined as the percentage of the rays reaching the rhabdom with respect to those entering the ommatidium. Good agreement with experimental results was found. The characterization of the visual field of an ommatidium by means of an admittance function permits the analysis of the influence of different stimuli on the eye.     

家蝇幼虫体内一种抗菌蛋白的分离纯化

30%H2O2诱导家蝇幼虫90min,继续饲喂24h后利用硫酸铵分级盐析、Sephadex G-25 和Sephadex G-75两步凝胶过滤、CM-Sepharose Fast Flow弱阳离子交换的纯化方法,得到一种电泳纯的抗菌蛋白,经过VDS凝胶扫描得到其分子量为28kDa,命名为AP28。抑菌活性分析表明,AP28对实验中涉及的大多数革兰氏阳性菌和阴性菌都有明显的抑制作用。     

Barbs Facilitate the Helical Penetration of Honeybee (Apis mellifera ligustica) Stingers

The stinger is a very small and efficient device that allows honeybees to perform two main physiological activities: repelling enemies and laying eggs for reproduction. In this study, we explored the specific characteristics of stinger penetration, where we focused on its movements and the effects of it microstructure. The stingers of Italian honeybees (Apis mellifera ligustica) were grouped and fixed onto four types of cubic substrates, before pressing into different substrates. The morphological characteristics of the stinger cross-sections were analyzed before and after penetration by microscopy. Our findings suggest that the honeybee stinger undergoes helical and clockwise rotation during penetration. We also found that the helical penetration of the stinger is associated directly with the spiral distribution of the barbs, thereby confirming that stinger penetration involves an advanced microstructure rather than a simple needle-like apparatus. These results provide new insights into the mechanism of honeybee stinger penetration.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

Honeybee workers (Apis mellifera capensis) compete for producing queen-like pheromone signals

Physical fights are the usual means of establishing dominance hierarchies in animal societies. This form of dominance behaviour is most strongly expressed in honeybee queens who engage in fights to the death to establish themselves in the colony. Workers can also compete for reproductive dominance resulting in the establishment of stable hierarchies. They do not engage each other physically, but use pheromones that mimic those produced by queens. The dynamics of pheromone production in paired workers suggests that they engage in a pheromonal contest. Because queen pheromones suppress ovary activation, the contest results in the sterility of the loser.     

Circadian rhythm gene regulation in the housefly Musca domestica

The circadian mechanism appears remarkably conserved between Drosophila and mammals, with basic underlying negative and positive feedback loops, cycling gene products, and temporally regulated nuclear transport involving a few key proteins. One of these negative regulators is PERIOD, which in Drosophila shows very similar temporal and spatial regulation to TIMELESS. Surprisingly, we observe that in the housefly, Musca domestica, PER does not cycle in Western blots of head extracts, in contrast to the TIM protein. Furthermore, immunocytochemical (ICC) localization using enzymatic staining procedures reveals that PER is not localized to the nucleus of any neurons within the brain at any circadian time, as recently observed for several nondipteran insects. However, with confocal analysis, immunofluorescence reveals a very different picture and provides an initial comparison of PER/TIM-containing cells in Musca and Drosophila, which shows some significant differences, but many similarities. Thus, even in closely related Diptera, there is considerable evolutionary flexibility in the number and spatial organization of clock cells and, indeed, in the expression patterns of clock products in these cells, although the underlying framework is similar.     

Segmentation gene expression in the housefly Musca domestica.

Drosophila and Musca both belong to the group of higher dipteran flies and show morphologically a very similar early development. However, these two species are evolutionary separated by at least 100 million years. This presents the opportunity for a comparative analysis of segmentation gene expression across a large evolutionary distance in a very similar embryonic background. We have analysed in detail the early expression of the maternal gene bicoid, the gap genes hunchback, Krüppel, knirps and tailless, the pair-rule gene hairy, the segment-polarity gene engrailed and the homoeotic gene Ultrabithorax. We show that the primary expression domains of these genes are conserved, while some secondary expression aspects have diverged. Most notable is the finding of hunchback expression in 11-13 stripes shortly before gastrulation, as well as a delayed expression of terminal domains of various genes. We conclude that the early developmental gene hierarchy, as it has been defined in Drosophila, is evolutionary conserved in Musca domestica.     

Geographical patterns of mitochondrial DNA variation in Apis mellifera iberiensis (Hymenoptera: Apidae)

An extensive survey of mitochondrial haplotypes in honeybee colonies from the Iberian Peninsula has corroborated previous hypotheses about the existence of a joint clinal variation of African (A) and west European (M) evolutionary lineages. It has been found that the Iberian Peninsula is the European region with the highest haplotype diversity (12 haplotypes detected of the M lineage and 10 of the A lineage). The frequency of A haplotypes decreases in a SW-NE trend, while that of M haplotypes increases. These results are discussed in relation to hypotheses about the African origin of Apis mellifera and an early colonization of west Europe during intermediate Pleistocene glaciation events, followed by a regional differentiation. The extant pattern of haplotype frequency and distribution seems to be influenced at a regional scale by adaptation to local climatic conditions and the mobile beekeeping that has become a large-scale practice during the last decades. Other previous anthropogenic influences (Greek, Roman and Arab colonizations) are thought to be of minor importance in present day populations.     

Problems of housefly (Musca domestica) control due to multiresistance to insesticides.

The development of chemical control of Musca domestica on Danish farms 1945--72 is outlined. It has been strongly influenced by successive development of resistance and failure of control by one insecticide after another. The chlorinated hydrocarbons used as residual sprays failed 1947--51. Organophosphorus compounds (OPC) were widely used from 1953, first as strips impregnated with parathion and residual sprays with diazinon. Resistance to OPC was first found in 1955, diazinon was given up in 1957--59 and parathion strips failed in the early '60's. Trichlorfon paint-on baits were widelyused 1958--64 and serious resistance did not appear until 1967, induced by selective pressure of fenthion and dimethoate used as residual sprays. High resistance to the contact effect of trichlorfon now occurs everywhere in Denmark. However, trichlorfon baits are still able to kill many flies. Residual sprays with fenthion, ronnel and fenitrothion were used to some extent 1960--70, but increased resistance reducing the residual effect developed in 2--3 years. Dimethoate was used on the majority of farms 1965--72. It was very effective the first years and resistance increased slowly until 1971--72, when high to extreme dimethoate-resistance became general on Danish farms. This was associated with high resistance to other OPC for fly control, e.g. fenthion, fenitrothion, bromophos, and tetrachlorvinphos, and to carbamates, with the result that no generally effective residual sprays were available. In 1971--72 frequent treatments with synergized pyrethroids have been tried. However, the method is often expensive, and serious resistance problems have appeared on a few farms. In this situation preventive, sanitary measures to eliminate or reduce fly breeding in manure are becoming decisive again, but difficult to practise due to lack of farm labour. The extreme Danish situation is compared with those in other areas, and probable reasons for differences in resistance and control problems are discussed, as well as possibilities for strategies to reduce resistance development.