Orientation of agents of wound myiasis to hosts and artificial stimuli in Hungary

Abstract. The orientation of adults of the myiasis species Wohlfahrtia magnified and Lucilia sericata to artificial visual stimuli (cloth targets) and olfactory stimuli ('swormlure-4', a potent screwworm attractant), was studied in sheep pastures near to Sarbogard in Hungary. Experiments with odour-baited cloth targets, enclosed in electrocuting grids, demonstrated that colour was an important factor influencing catches on targets: a black target caught most flies of both species, with other colours in the following order of effectiveness, blue > white > yellow. Wohlfahrtia magnified did not respond to swormlure-4 in the strong manner that L. sericata did. The sex ratios of W. magnifica caught on targets (67.2% males) and hand-netted from fence posts (68.8% males) were similar and biased towards males, whereas that of L. sericata on targets was strongly biased towards females (15.6% males), indicating a fundamental difference in the response of these two myiasis species towards the swormlure-baited targets. The orientation of these two species towards hosts was also recorded together with that of a third species, Phormia regina . Electric nets placed beside infested sheep caught significantly more flies of all three species than those placed beside uninfested sheep or in the absence of sheep. The sex ratio of W. magnified caught around infested sheep was the reverse of that on targets, with 68.5% females. Wohlfahrtia magnifica responded more strongly to healthy, uninfested sheep than did L. sericata and P. regina . The potential for use of targets both for population monitoring and control is discussed.     

大胡蜂蜂毒中多肽和蛋白质结构和功能的多样性

为证明大胡蜂Vespa magnifica(Smith)蜂毒具有较大的药用开发价值,本研究采用超高效液相色谱-质谱和电泳技术对其多肽和蛋白质的分布进行分析,发现其蛋白质的相对分子质量主要分布在17~45kDa范围内。蜂毒多肽类物质的相对分子质量呈"单峰"式分布,61%在500~3000Da范围内,为大胡蜂蜂毒中多肽含量最为丰富的部分。通过牛津杯法对蜂毒的抑菌活性进行研究,且以HepG2人肝癌细胞及B16黑色素瘤细胞为研究对象,用MTT法检测蜂毒的细胞毒性活性,证明其具有良好的抑菌作用和细胞毒活性,其结果与已报道的其他蜂类既有相似性又存在具体差异,展示了大胡蜂蜂毒的分子多样性,为后续该毒素的物质基础研究及药用价值开发提供参考。     

A Novel Arginine Kinase with Substrate Specificity Towards <Emphasis Type="SmallCaps">d</Emphasis>-arginine

We determined the cDNA-derived amino acid sequences of two arginine kinases (AK1, AK2) from the annelid Sabellastarte indica, cloned the cDNAs into pMAL plasmid and expressed them in E. coli. The phylogenetic analyses suggested that Sabellastarte AKs have evolved from a CK-related gene, not from the usual AK gene. The recombinant Sabellastarte AK1 showed a broad specificity towards various guanidine compounds, while the Sabellastarte AK2 mainly showed stronger activity for both d- and l-arginine, a very unique substrate specificity not seen before in usual AKs. We isolated guanidino compounds from the body wall musculature of Sabellastarte, and found that the major compound is d-arginine with a concentration of 4.85 ± 0.51 mmol/kg. From these results, we suggest strongly that in Sabellastarte, d-arginine is the major phosphagen substrate and that the AK2 with substrate specificity towards d-arginine, catalyzes the phosphorylation of d-arginine.     

In vitro rearing of the screwworm fly Wohlfahrtia magnifica

Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae) is the main agent of traumatic myiasis in many European, African and Asian countries. Although it can be reared in vivo without technical difficulty, such rearing presents ethical problems. Studies were therefore made of in vitro rearing to facilitate development of laboratory colonies that could be used in a wide range of biological, physiological and applied studies of W. magnifica, particularly in the long period of the year when natural populations of the fly are unavailable for study. Parental colonies of W. magnifica were established from larvae collected from natural infestations of sheep and cattle in central Hungary. First stage larvae were harvested from gravid females and were reared in groups of 5-20 on one of six artificial diets. The diets were based on various combinations of five to seven of eight ingredients: water, agar, blood (heparinized or dried), ground meat, egg yolk, low-fat milk powder, yeast and 10% formol. The larvae were incubated on the diets at 37 degrees C. There was no mortality of first stage larvae, which appeared to feed together in foci, in a natural manner. However, during the second stage, and especially after renewal of diet associated with disturbance of the larvae, many larvae began to disperse, crawling over the surface of the media and feeding less intensively. Mortality of larvae during all larval stadia was 64-98%, compared to 33% in batches of third stage larvae collected from natural infestations. The mean weights of puparia from artificial diets ranged from 38.7 to 59.3 mg, compared to 92.2 mg of puparia from larvae collected from natural infestations. There was a high mortality in the pupal stage, from 61 to 100%. Only a maximum of 6% of first stage larvae were successfully reared to the adult stage. Further studies are needed to identify factors present or absent in the diets that contributed to the present poor development of W. magnifica in vitro.     

Geographic differentiation of monoterpenes from Abies procera and Abies magnifica

Cortical oleoresins from 352 Abies magnifica and A. procera trees, collected in 35 localities, were analysed for composition of their monoterpene fractions. The monoterpenes were composed primarily of -pinene, β-pinene, limonene and β-phellandrene, with camphene and 3-carene appearing only sporadically in larger amounts. Limonene, β-phellandrene, -pinene and 3-cerene were used to study the transitional populations in northern California and southern and central Oregon and the transitional-like populations in southern Sierra Nevada. The populations segregated latitudinally into three related clusters—above 44° (A.procera), between 44° and 40° (transitional), and below 40° (A.magnifica). A.magnifica from southern Sierra Nevada and A.magnifica from Mtn. Shasta differed in a number of parameters, with southern Sierra Nevada populations being chemically much closer to typical A. magnifica from central and northern Sierra Nevada. While monoterpene and morphological data did not allow a clear Interpretation of the status of the transitional and southern populations, the paleobotanical evidence favored the recent evolution over introgression. The desirability of additional studies was indicated.     

The role of volatile semiochemicals in mediating host location and selection by nuisance and disease-transmitting cattle flies

The role of volatile semiochemicals in mediating the location and selection within herds of Holstein-Friesian heifers by nuisance and disease-transmitting cattle flies was investigated using coupled gas chromatography-electrophysiology (GC-EAG), coupled gas chromatography-mass spectrometry (GC-MS), electrophysiology (EAG), laboratory behaviour and field studies. Using volatile extracts collected by air entrainment from heifers in the Netherlands, a number of active peaks were located by coupled GC-EAG for Musca autumnalis (de Geer) (Diptera: Muscidae) and Haematobia irritans (L.) (Diptera: Muscidae). Volatile samples were also collected from two heifers in Denmark shown in previous counting experiments to differ significantly in their fly loads. Coupled GC-EAG using Ha. irritans antennae revealed differences in the EAG response to the samples, with additional EAG activity in the sample collected from the heifer with the lower fly load. To identify more EAG active compounds, volatiles were also collected from 48-h-old urine by air entrainment. In total, 23 compounds were located and identified by coupled GC-EAG and GC-MS. Further electrophysiological testing of these compounds with five fly species [M. autumnalis, Ha. irritans, Hydrotaea irritans (L.) (Diptera: Muscidae), Stomoxys calcitrans (L.) (Diptera: Musicidae) and Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae)] showed that only some of the compounds were physiologically active across the range of flies tested. These included 1-octen-3-ol, 6-methyl-5-hepten-2-one, (Z)-3-hexen-1-ol, naphthalene, and all EAG active compounds identified from urine. Compounds showing significant EAG activity were tested for behavioural activity using a wind-tunnel designed for measuring upwind flight behaviour. At certain concentrations, 1-octen-3-ol, 6-methyl-5-hepten-2-one and 3-octanol increased upwind flight, whereas naphthalene, propyl butanoate and linalool reduced upwind flight. In field studies using small herds of heifers ranked according to their fly load, individual slow-release formulations of 1-octen-3-ol and 6-methyl-5-hepten-2-one, when applied to low and high fly loading heifers, reduced fly loads on these individuals. This study provides evidence for the hypothesis that the natural differential attractiveness within herds of Holstein-Freisian heifers, i.e. a single host species, for cattle flies is partly due to differences in volatile semiochemicals emitted from the host. It is suggested that this phenomenon applies to other vertebrate host species and their associated insect pests.     

The mastoparanogen from wasp

Mastoparans are a family of small peptides identified from the venom of hymenopteroid insects. Although they have been characterized as early as 1979, and so far are recognized as a leading biomolecule in potential drug therapy, their precursors, mastoparanogen, have still not been determined. In this paper, several mastoparans from the venom of the wasp Vespa magnifica (Smith) are reported. The cDNA of mastoparanogen is 236 base pairs in length, and encodes 40 amino acid residues, including a N-terminal acidic fragment and a C-terminal mature basic mastoparan, which contain multiple acidic amino acid residues and a tetradecapeptide with three lysines, INLKAIAALAKKLLG, respectively. The glycine at the tetradecapeptide end is the donator of –NH₄ for the amidation of the leucine at the C-terminal. As far as we know, this is the first report of the precursor of animal mastoparan.     

橙黄后窗网蛾的生物学初报

橙黄后窗网蛾Dysodia magnificaWhalley是油桐(Vernicia fordii(Hemsl.)Airy-Shaw)的食叶害虫,在浙江省丽水地区油桐上普遍发生。该虫1年发生3代,以蛹在土茧内越冬。越冬代成虫于次年4月中旬开始羽化,第1、2代成虫分别于6月下旬和8月下旬开始羽化。饲养中育出幼虫-蛹期寄蝇—蚕饰腹寄蝇,是橙黄后窗网蛾的一种有效天敌。     

黑须污蝇不同发育阶段转录组及嗅觉相关基因分析

【目的】建立黑须污蝇Wohlfahrtia magnifica转录组数据库,挖掘黑须污蝇基因数据,为更深入地研究双峰驼阴道蝇蛆病提供理论依据。【方法】采用高通量测序平台Illumina HiseqTM4000对黑须污蝇幼虫、蛹和成虫进行转录组测序,并进行生物信息学分析。利用黑须污蝇幼虫、蛹和成虫转录组数据,通过基因差异表达分析以及GO功能显著性富集分析的方法筛选出嗅觉相关基因,并通过荧光定量PCR技术对黑须污蝇幼虫、蛹和成虫中OBP99b,OBP56a和OBP99a 3个嗅觉相关基因表达水平进行验证。【结果】结果显示,每个黑须污蝇样本的转录组数据量在4.96 Gb以上,G+C含量在35.35%以上,Q20含量在97%以上。与NCBInr, GO, KEGG, Pfam, Swiss-Prot和eggNOG数据库进行对比,共注释到73 303条unigenes。其中eggNOG数据库注释到的unigenes最多,为35 066条,分布在23个蛋白功能中,其中的翻译修饰、蛋白质周转的unigenes所占比例较大;GO数据库注释到的unigenes数为29 193条,功能包括分子功能、细胞组分、生物学过程三大类50个分支,其中参与生物学过程和氧化还原过程的unigenes比例较大;KEGG数据库注释到的unigenes数为15 068条,其中参与信号转导过程的unigenes比例较大。上述数据表明该转录组测序结果较好,为后续研究奠定了基础。依据黑须污蝇幼虫、蛹、成虫转录组数据筛选到30个嗅觉相关基因,其中包含9个气味结合蛋白(OBP)基因,进一步基因注释发现OBP99b,OBP56a和OBP99a基因在黑须污蝇不同发育阶段表达差异显著(|log2FC|>1,P<0.05),这在荧光定量PCR分析结果中得到了验证。【结论】本研究获得了黑须污蝇幼虫、蛹和成虫转录组数据,筛选出黑须污蝇各发育阶段嗅觉相关基因,并验证了OBP99b,OBP56a和OBP99a基因在黑须污蝇幼虫、蛹和成虫3个发育阶段差异表达,为防治双峰驼阴道蝇蛆病提供了新思路。