A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Soluble interleukin‐1 receptor,a potential negative regulator of orange‐spotted grouper Epinephelus coioides interleukin‐1 system

In this study, the cDNA sequence encoding interleukin‐1 (Il‐1) receptor‐like protein of orange‐spotted grouper Epinephelus coioides was obtained. The newly identified sequence was named soluble type I Il‐1 receptor (sIl‐1rI) owing to its structural composition, which had two Ig‐like domains, lack of transmembrane region and the Toll/interleukin‐1 receptor (TIR) domain, similar to the brown rat Rattus norvegicus soluble Il‐1rI. In addition, sequence comparison and phylogenetic analysis indicated that E. coioides sequence had a closer relationship with Il‐1rI than Il‐1rII. Real‐time PCR revealed that sil‐1rI mRNA expression presented a process of decrease, restoration and increase in Cryptocaryon irritans‐infected E. coioides. The negative correlation between Il‐1β and sil‐1rI mRNA in C. irritans‐infected head‐kidney implied the potential negative regulatory role of sil‐1rI in E. coioides Il‐1 system. The leucocytes incubated with lipopolysaccharide or polyriboinosinic polyribocytidylic acid exhibited different expression profiles of sil‐1rI. Recombinant Il‐1β (rIl‐1β) protein was capable of inducing sil‐1rI mRNA under the concentration of 100 ng ml^?1, suggesting that high dosage or excess Il‐1β would stimulate the expression of sil‐1rI to maintain the homoeostasis of E. coioides Il‐1 system. For the first time, the role of teleost Il‐1rI in parasite infection has been identified, and soluble Il‐1r was found in fish.     

滇西亚种树鼩体外寄生虫自然感染状况及分析

目的调查滇西亚种树鼩体外寄生虫的自然感染状况,为建立树鼩质量控制标准提供依据。方法对野生的滇西亚种树鼩和自繁F1代树鼩各60只分别用尸检法和活体法检测,随即采用拔毛或用透明胶带粘取腋下、耳根、腹股沟、肛门附近等处的毛发制片,置于体视镜和显微镜下观察虫体和虫卵。结果野生滇西亚种树鼩和自繁F1代树鼩体外寄生虫自然感染率分别为：虱子25%/6%、蚤5%/0、蜱1.6%/0、水蛭1.6%/0、虱子和蚤混合感染1.6%/0。结论野生树鼩体外寄生虫的自然感染多为虱子和蚤,其次还有蝉、水蛭,总感染率明显高于自繁F1代。使用灭虱灵对于驱除树鼩体外寄生虫具有较好的疗效。     

斜带石斑鱼TLR3基因的克隆及其在刺激隐核虫感染时的表达分析

TLR3(Toll like receptor 3)是Toll样受体家族的重要成员,通过识别病原相关分子模式,诱导宿主天然免疫应答。研究从斜带石斑鱼(Epinephelus coioides)中克隆得到TLR3 cDNA序列,全长为2937 bp,包括107bp的5′非编码区、100 bp的3′非编码区和编码909个氨基酸的2730 bp的开放阅读框。TLR3全长氨基酸序列包含1个信号肽、18个富含亮氨酸的重复序列(Leucine-rich Repeat LRR)、1个跨膜结构域和1个胞内TIR结构域(IL-R1 homologous region)。同源比对显示,斜带石斑鱼TLR3与其他已报道硬骨鱼类的TLR3具有较高的同源性(52%—67%)。组织表达分析显示,TLR3在健康斜带石斑鱼的组织中具有较广的表达分布,其中在前脑、体肾和脾脏中表达量较高。刺激隐核虫(Cryptocaryon irritans)感染斜带石斑鱼后:在皮肤中TLR3的表达量呈现先降低后升高的趋势,从感染后第7天开始上调,并在第10天达到高峰;而在脾脏中,TLR3的表达量在感染6h时就显著上调并达到峰值。结果表明斜带石斑鱼TLR3在抗刺激隐核虫的免疫应答过程中可能发挥重要作用。     

Conservation and versatility of a new set of primers for long‐PCR amplification of complete insect mitochondrial genomes based on Haematobia irritans mtDNA sequences

The amplification of complete mitochondrial genomes by long PCR (polymerase chain reaction) has been a major contribution to the large‐scale sequencing of arthropodan mitochondrial genomes. In this work, we designed six conserved long‐PCR primers to successfully recover the entire mitochondrial genome of the horn fly Haematobia irritans (Diptera: Muscidae) in two overlapping fragments. The conservation and versatility of these primers were tested for 17 other species from four major insect orders: Diptera (14), Coleoptera (1), Lepidoptera (1) and Hymenoptera (1). The amplification of complete mitochondrial genomes in orders other than Diptera suggested an even broader application of these primers, especially within the Hexapoda.     

卵形鲳鯵对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳鯵(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示:两种免疫方法都能让卵形鲳鯵的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。       

Use of electroporation as an option to transform the horn fly,Haematobia irritans: a species recalcitrant to microinjection

The horn fly, Haematobia irritans, is a serious pest of cattle in North America. The control of horn flies has primarily relied on insecticides. However, the heavy use of insecticides has led to the development of insecticide resistance in horn flies. Novel methods to control horn flies are greatly needed. Transgenic technology is an effective tool to genetically modify insects and may lead to novel methods of pest control based on genomic approaches. Here we report a piggyBac‐mediated transformation of the horn fly via electroporation. Transformation with a DsRed fluorescent marker protein coding region was verified by PCR analysis of individual fly bodies and pupal cases and sequencing of PCR products. However, Southern blot analysis failed to indicate the DsRed gene was integrated into the horn fly genome. Thus, the electroporation protocol may have caused the DsRed gene to be integrated into bacterial symbionts of the horn fly.     

Effects of intraspecific larval competition on the development of the African buffalo fly Haematobia thirouxi potans

The effects of larval crowding on development, survival and size of the African buffalo fly, Haematobia thirouxi potans (Bezzi), were examined in small (10 or 20 g) and 1 000 g masses of cattle dung in the laboratory at 25°C. Dung was infested with fly eggs at densities which ranged from 0.05 to 32.0 eggs per g dung; in most cases 90–100% of eggs hatched. The duration of larval developmentin 10 g dung masses was extended by larval crowding at densities above 0.8 eggs per g dung and the development period with 32 eggs per g dung (11 days) was double that observed in uncrowded dung masses. Both size and survival decreased with increasing larval density, but stunting was observed at lower densities than those which reduced survival and so size was the more sensitive index of the adequacy of the larval environment. The effects of larval crowding became evident at lower larval densities in the 1 000 g dung masses than in the 10 g dung masses. There was a minimum size below which third instar larvae failed to pupariate. The probability of survival was related to pupal size and a high proportion of the smaller individuals failed to eclose.

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Effets de la competition intraspécifique sur le développement des larves, la taille des pupes et la survie d'Haematobia thirouxi potans

Résumé Les effets du surpeuplement larvaire sur le développement, la survie et la taille d'Haematobia thirouxi potans, ont été examinés sur de petites quantités de bouse (10 et 20 g) et de plus importantes (1 000 g), au laboratoire à 25°C. Les bouses étaient contaminées avec des oeufs de la mouche à des densités de 0,05 à 32 oeufs par gramme de bouse. Dans la plupart des cas le taux d'éclosion était de 90–100%. La durée du dévelopement larvaire dans 10 g de bouse a été prolongée pour les densités supérieures à 0,8 oeuf per g, et la durée développement pour 32 oeufs per g (11 jours) était le double de celle obtenue grand il n'y avait pas surpeuplement. La taille et la survie diminuent toutes les deux avec la densité larvaire, mais la miniaturisation était observée à des densités où la survie n'était pas modifiée; ainsi la taille est un indice plus sensible de l'adéquation de l'habitat larvaire. Les effets du surpeuplement larvaires deviennent clairs à des densités plus faibles avec 1 000 g de la bouse qu'avec 10 g. Il y a eu une taille minimale endessous de laquelle les larves étaient incapables de former une pupe. La probabilité d'éclosion était liée à la taille de la pupe, et une plus forte proportion de petits individus ne sont pas parvenu à éclore.

     

Control of Muscidae on cattle by flucythrinate ear-tags, the behaviour of these flies on cattle and the effects on fly-dislodging behaviour

In two trials using diary heifers, in the south of England, ear-tags, containing flucythrinate, completely controlled Haematobia irritans (L). Musca autumnalis De Geer on the face were reduced by 41% using one tag and by 76% using two tags per heifer. A single ear-tag reduced numbers of Hydrotaea irritans (Fallén) on the body by 90% and on the teats by more than 55%. Other minor pestering flies were similarly controlled. Control was sustained over the entire 14 weeks duration of the trials which effectively encompassed the fly season of the three main pests.Ear-flicks by the heifers were related to dislodging M. autumnalis from the face, and tail flicks to dislodging Hy. irritans from the belly. These are the two commonest species of nuisance flies and are known to carry pathogenic bacteria.A management plan intended to maximise effective use of insecticidal ear-tags while minimising risks of insecticide resistance is described.

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Résumé Des boucles, contenant du flucythrinate, ont protégé totalement des génisses pour la production de lait contre Haematobia irritans L. lors de deux traitements expérimentaux dans le sud de l'Angleterre.Les effectifs de Musca autumnalis De Geer sur la face des génisses ont été réduits de 41% en utilisant un boucles et de 76% en utilisant deux. Un seul boucles réduit les effectifs d'Hydrotaea irritans Fallén de 90% sur le corps et de plus de 55% sur les pis. Les effectifs d'autres mouches nuisibles de moindre intérêt ont été réduits eux aussi. L'efficacité a été maintenue pendant les 14 semaines du traitement ce qui déborde efficacement le période de vol des 3 principales espèces de mouches nuisibles. Les battements d'oreille des génisses sont destinés à chasser M. autumnalis du visage, et les coups de queue à chasser H. irritans du ventre.Ce sont les deux principales espèces de mouches nuisibles connues comme vecteurs de bactéries pathogènes.Un programme est proposé pour optimaliser l'efficacité des boucles, tout en limitant les risques de résistance aux insecticides.

     

Mites of the familyMacrochelidae as predators of two species of dung-breeding pest flies

Nine species of macrochelid mites [Macrocheles “glaber” (Müller),M. aestivus Halliday,M. mammifer Berlese,M. peniculatus Berlese,M. robustulus (Berlese),M. merdarius (Berlese),M. peregrinus Krantz,M. eurygaster Krantz,Glyptholaspis confusa (Foà) were tested in the laboratory as predators of 2 species of dung-breeding pest flies (Musca vetustissima Walker,Haematobia irritans exigua de Meijere). The mites varied in their ability to attack the 2 fly species at both egg and larval stages. larger mites were generally more effective predators than the smaller species. The level of predation imposed byM. “glaber” onMusca vetustissima also varied with the developmental stage of both predator and prey.