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.     

Molecular identification of some forensically important blowflies of southern Africa and Australia

One major aspect of research in forensic entomology is the investigation of molecular techniques for the accurate identification of insects. Studies to date have addressed the corpse fauna of many geographical regions, but generally neglected the southern African calliphorid species. In this study, forensically significant calliphorids from South Africa, Swaziland, Botswana and Zimbabwe and Australia were sequenced over an 1167 base pair region of the COI gene. Phylogenetic analysis was performed to examine the ability of the region to resolve species identities and taxonomic relationships between species. Analyses by neighbour-joining, maximum parsimony and maximum likelihood methods all showed the potential of this region to provide the necessary species-level identifications for application to post-mortem interval (PMI) estimation; however, higher level taxonomic relationships did vary according to method of analysis. Intraspecific variation was also considered in relation to determining suitable maximum levels of variation to be expected during analysis. Individuals of some species in the study represented populations from both South Africa and the east coast of Australia, yet maximum intraspecific variation over this gene region was calculated at 0.8%, with minimum interspecific variation at 3%, indicating distinct ranges of variation to be expected at intra- and interspecific levels. This region therefore appears to provide southern African forensic entomologists with a new technique for providing accurate identification for application to estimation of PMI.     

Annual and spatial activity of dung flies and carrion in a Mediterranean holm-oak pasture ecosystem

The annual activity and spatial distribution of Muscidae and Calliphoridae were investigated in a holm-oak ('dehesa') ecosystem in western Spain over two years in pasture and woodland habitats, using wind-orientated traps baited with a mixture of fresh cattle faeces, liver and sodium sulphide solution. Lucilia sericata (Meigen) was always the dominant species and, with Chrysomya albiceps (Weidemann), Hydrotaea ignava (Harris), Muscina levida (Harris) and Muscina prolapsa (Harris), was more abundant during the second than the first year. By contrast, Calliphora vicina Robineau-Desvoidy, Calliphora vomitoria (L.), Hydrotaea armipes (Fallén), Hydrotaea penicillata (Rondani) and Hydrotaea dentipes (Fabricius) were more numerous during the first than the second year of the study. In summer, the Diptera sampled were significantly more abundant in a wooded than a pasture area. However, in autumn, while H. penicillata remained significantly more abundant in woodland, L. sericata became more abundant in the pasture, whereas C. vicina was captured in open and wooded areas in similar proportions. During winter and spring the populations sampled were relatively small. The changing patterns of abundance are discussed in relation to differences in climate within and between years.     

Development, survival and fecundity of the urine fly, Scatella (Teichomyza) fusca and predation by the black dumpfly, Hydrotaea aenescens

Development, survival and fecundity for Scatella (Teichomyza) fusca Maquart (Diptera: Ephydridae) were studied at 20 ± 1 °C and 85 ± 10% r.h. Mean (± S.E.) developmental times of eggs, larvae and pupae were 4.0 ± 0.06, 14.9 ± 0.19 and 14.6 ± 0.11 days, respectively, the mean (± S.E.) survival of the original egg cohort to the start of larval, pupal and adult stage being 77.2 ± 3.2%, 54.5 ± 2.6% and 47.9 ± 3.5%, respectively. Females and males displayed approximately straight survivorship curves during adult life, implying constant mortality rates. Mean (± S.E.) adult longevity was 41.6 ± 2.98 days for females and 51.2 ± 3.91 days for males. Assuming a stable age distribution the population consisted of 56% eggs, 31% larvae, 6% pupae and 7% adults. Oviposition peaked when females were 25 days old, and the highest reproductive values (RV_x) (mean ± S.E.) ranged from 129.1 ± 7.57 to 138.5 ± 6.83 for individuals 17–27 days old. A female deposited a mean (± S.E.) of 614.7 ± 35.9 female eggs over a maximum life span of 93 days. The basic reproductive rate (R ₀) (mean ± S.E.) was 173.0 ± 14.2 female offspring per female and the intrinsic rate of natural increase of female individuals (r) (mean ± S.E.) was 0.088 ± 0.001 day^-1. The mean (± S.E.) generation time (T) was 57.8 ± 0.78 days. In cultures with equal numbers of first instar S. fusca larvae and predacious third instar larvae of Hydrotaea aenescens (synonymous Ophyra aenescens) Wiedemann, mean (± S.E.) survival to the adult stage of S. fusca (16.7 ± 8.8%) was significantly lower than in controls with S. fusca alone (58.3 ± 7.4%). The potential significance of predation by H. aenescens on S. fusca in pig farms is discussed.     

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.