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.     

Traumatic myiasis in dogs caused by Wohlfahrtia magnifica and its importance in the epidemiology of wohlfahrtiosis of livestock

Abstract.  In the province of Al Hoceima, northern Morocco, and on two farms in Hungary, dogs were inspected for the presence of traumatic myiasis. Nine and four infested dogs were found in Morocco and Hungary, respectively. All the larvae and adults reared from them in the laboratory were identified as Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae). To our knowledge, these are the first cases of wohlfahrtiosis in dogs to be reported in these countries. All infested animals lived close to livestock, where wohlfahrtiosis was endemic. Infested body sites included limbs (six cases), external genitalia (two), ears (three), nose (one) and neck (one). Developing larvae caused severe welfare problems and tissue destruction in most cases. Although the number of cases reported here is small, wohlfahrtiosis in dogs may be very important from an epidemiological perspective because farm and stray dogs can act as both reservoirs and carriers of this parasitic fly species. Therefore, education of dog owners concerning the risk factors in endemic regions is recommended in order to reduce the prevalence of wohlfahrtiosis in dogs and thereby in livestock. Both owners and veterinarians should pay regular attention to any wounds and to the natural orifices of dogs, especially during the fly seasons.     

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。     

Gonotrophic and embryonic development of the Palaearctic screwworm fly Wohlfahrtia magnifies

Abstract. Gonotrophic and embryonic development of Wohlfahrtia magnifica were studied on pupae and adults reared in the laboratory. Gonotrophic development in screwworm begins 8 days after pupation in yellow eye pharate adults. The adult female emerges with its oocytes at stage 3 and requires only 2 days for complete oogenesis and 5 days for complete embryogenesis. Mating occurs 2–5 days after adult emergence, starting the embryonic development. Each female produces an average of 76.8 active larvae which can produce a traumatic lesion in their hosts. When food sources were regularly supplied, synchronization of gonotrophic stages can be observed. These stages together with the embryonic ones are described.     

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.     

First report of the exotic blue land planarian, Caenoplana coerulea (Platyhelminthes, Geoplanidae), on Menorca (Balearic Islands, Spain)

In April 2009 two specimens of a terrestrial flatworm were collected from under a rock in an orchard at Ciutadella de Menorca on the easternmost Balearic island of Menorca (Spain). Their external morphology suggested that both specimens belonged to the invasive blue planarian Caenoplana coerulea, a species which is native to eastern Australia. Sequence data of a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) and of the entire 18S ribosomal RNA confirm its identification. This is one of the first records of the species in Europe where it has only been found in one locality in the United Kingdom, France and NE Spain.     

Molecular markers to discriminate among four pest species of Helicoverpa (Lepidoptera: Noctuidae)

The four significant pest species in the Helicoverpa genus (H. armigera, H. assulta, H. punctigera and H. zea) are morphologically similar and can only be reliably distinguished through dissection of adult genitalia. Two partial regions of the mitochondrial DNA (mtDNA), the cytochrome oxidase subunit I (COI) and the cytochrome b (Cyt b) genes were amplified by PCR and digested with restriction endonucleases. The restriction patterns, generated by the endonucleases BstZ17I and HphI, demonstrated reliable differentiation of the four Helicoverpa pest species. This technique is fast, reliable and effective at distinguishing specimens irrespective of their life stages and offers support to conventional taxonomic differentiation based on morphological characters.     

A sensitive and reliable restriction enzyme assay to distinguish between the mosquitoes Culex torrentium and Culex pipiens

Culex pipiens pipiens Linnaeus and Culex torrentium Martini (Diptera: Culicidae) are closely related vector species that exist sympatrically in Europe. The two species are morphologically almost identical and can only be distinguished with certainty by characters of the male genitalia. Hence, correct species identification and conclusions on distribution and vector status are very difficult and often neglected. Therefore, we developed a reliable and simple mitochondrial cytochrome c oxidase subunit I (COI) gene restriction enzyme assay to discriminate between Cx. pipiens and Cx. torrentium, based on the analysis of morphologically identified male specimens. We sequenced approximately 830 bp in the 3′ region of the mitochondrial COI gene of 18 morphologically identified males of Cx. pipiens and Cx. torrentium. Two restriction enzymes (FspBI and SspI) that could distinguish between the two species according to species‐specific differences in these sequences were chosen. The restriction enzymes were tested on 227 samples from Sweden and verified by sequencing 44 of them. The enzyme FspBI correctly identified all investigated samples; the enzyme SspI identified all samples except one Cx. torrentium. We hope the method and the findings presented here will help to shed light on the true distribution and relative proportions of the two species in Europe.     

Polymorphism hides cryptic species in Oerstedia dorsalis (Nemertea, Hoplonemertea)

The marine hoplonemertean Oerstedia dorsalis is considered to be a highly polymorphic species with extensive geographic distribution. We show, based on mitochondrial cytochrome c oxidase subunit I, 16S and nuclear internal transcribed spacer sequences, that there are genetic subsets withing this species. Seventy-one specimens of various colours from different geographic localities (in Europe) were sequenced and analysed using statistical parsimony and Bayesian analysis. Both analyses supported nine major clades. We conclude that O. dorsalis hides different species with geographic resolution. These species, however, appear to be polymorphic as well, and we find no diagnostic features in pigmentation or external characters to separate species within this complex.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 556–567.     

Grey flesh fly (Wohlfahrtia vigil) parasitism of a Preble's meadow jumping mouse (Zapus hudsonius preblei)

A Preble's meadow jumping mouse (Zapus hudsonius preblei) parasitized by five grey flesh fly (Wohlfahrtia vigil) larvae was discovered at the United States Air Force Academy in Colorado Springs, Colorado (USA), in June 1998. This is the first documented case of grey flesh fly parasitism of jumping mice (Family Dipodidae). The lesion was approximately 6 mm wide and was partially hidden under a mat of wet fur. Myiasis was found in one (0.7%) of 146 jumping mice captured at the Academy in 1998. The Preble's meadow jumping mouse is considered a threatened subspecies by the United States Fish and Wildlife Service. Although grey flesh fly myiasis can be fatal, it is unknown whether it is affecting populations of Preble's meadow jumping mouse. This is the first report of grey flesh fly myiasis in free-ranging wildlife in Colorado.     

Phylogenetics of the millipede genus Brachycybe Wood, 1864 (Diplopoda: Platydesmida: Andrognathidae): patterns of deep evolutionary history and recent speciation

The genus Brachycybe Wood is a little known group of millipedes comprising eight nominal species distributed throughout North America, Japan, South Korea, Taiwan, and China. The group's species are relatively morphologically homogenous and have been described primarily on the basis of differences in somatic morphology largely ignoring the often-used characters in millipede taxonomy and systematics - male genitalia (the gonopods). The objectives of this study were to survey male gonopods with the aim of evaluating inter-specific variation, assess existing species boundaries and phylogeny using molecular characters, examine the historical biogeography of the genus, and estimate the timing of lineage divergence using a molecular clock. We surveyed two mitochondrial genes (cytochrome c oxidase I and cytochrome b) and one nuclear protein-coding gene (glutamyl and prolyl-tRNA synthetase) each comprising 952, 746, and 555 aligned base pairs respectively. Phylogenetic inference coupled with an analysis of species delimitation using a generalized mixed Yule coalescent model recovered eight species, two of which were considered to be cryptic. Molecular dating analyses coupled with ancestral range reconstructions indicate that the group is quite ancient (age>50 million years) and its origins are likely traced back geographically to the mountains of California's Sierra Nevada and Coastal Ranges. The genus Brachycybe appears to have expanded its range at least twice out of present day California into eastern North America and at least once into Asia. This study highlights the need for integrative approaches to describe biodiversity and furthers the evidence for cryptic diversity even in groups where genitalia are generally thought to be rapidly evolving, diagnostic features.     

Slow genital and genetic but rapid non-genital and ecological differentiation in a pair of spider species (Araneae,Pholcidae)

Like most animals with internal fertilization, spiders tend to have species-specific genitalia, allowing closely related species to be identified by their reproductive morphology more easily than by non-genital characters. This implies that genitalia evolve on average more rapidly than non-genital morphological traits. Here we describe two putative species of Pholcidae from Taiwan (Pholcus pingtung, n. sp.; Pholcus chengpoi, n. sp.) that differ conspicuously in their microhabitat (rocks vs. leaves), coloration, color pattern, and body proportions, but have almost indistinguishable genitalia and cytochrome oxidase I (co1) sequences. The two species have identical yet highly unusual male cheliceral modifications, strongly arguing for sister species. Despite the almost identical genitalia and co1 sequences, we treat the two ‘morphs’ as species for three reasons: (1) they are easily distinguished by several characters; no intermediate specimens were found; (2) subtle yet consistent differences in genital (uncus) shape support the idea of reproductively isolated entities beyond the more conspicuous non-genital differences; (3) each locality provided both types of microhabitat but only one of the two species, arguing against environmental plasticity or polymorphism. We conclude that probably a very recent expansion into a novel microhabitat has led to speciation and rapid ecological and non-genital differentiation, with insufficient time to accumulate significant genital and genetic differences.     

Genetic and morphometric diversity of the goldcrest (Regulus regulus) populations in the Azores

We studied the genetic diversity and phylogeography of the goldcrest Regulus regulus from the archipelago of the Azores (North Atlantic Ocean) based on sequences of two mitochondrial genes (cytochrome b and the NADH dehydrogenase subunit 2) and one nuclear gene in the Z-chromosome (intron 9 of the aconitase 1) from 69 individuals, and 41 birds from the Canary Islands and continental Europe for outgroup comparison. To understand the level of concordance between the genetic data and possible morphometric variability, 197 adult living birds from the seven Azorean islands where the species breeds were analysed in terms of eight morphometric characters. Our results are in accordance with previous studies, indicating a recent expansion of goldcrests throughout the archipelago and a low divergence in relation to continental Europe. Within the Azores, there is evidence of historical and/or recent gene flow among the island's populations, revealing a lack of current genetic structure within the archipelago. Only goldcrests from Flores Island seem to be genetically distinct and showed significantly larger body mass and tarsus length than birds on the other islands.     

Speciation in the globeflower fly Chiastocheta spp. (Diptera: Anthomyiidae) in relation to host plant species, biogeography, and morphology

Phylogenetic relationships among the Chiastocheta species (Diptera, Anthomyiidae) were investigated using a 1320-bp mitochondrial DNA fragment including parts of cytochrome oxidase subunits I and II. Larvae of the Chiastocheta genus feed exclusively on Trollius (Ranunculaceae) seeds. Six Chiastocheta species (and two vicariant taxa) coexist on Trollius europaeus in Europe, where they were shown to be the sole pollinator of their host plant. In Asia, several Trollius species are parasitized by Chiastocheta spp., and several other Trollius species are free of Chiastocheta. The plant-insect association is thus obligate in Europe but facultative in Asia. This system therefore provides a unique opportunity to study the evolution of an obligate mutualism between a plant and its seed parasite pollinators and its consequences on insect diversification. Most parsimonious, distance, and maximum likelihood analyses of 16 haplotypes from Europe and 14 from Asia show that European species do not form a monophyletic group. The species relationship indicated by egg morphology is only partly supported by molecular data. Moreover, a strong discrepancy between nucleotide variation and male genitalia morphology variation was found, particularly within and among European species coexisting on a single host plant. Molecular divergence across species ranges from 0 to 4.6%, suggesting a recent origin of the genus and several events of diversification, involving both host shifts and within-host-plant radiation. We discuss alternative evolutionary scenarios that are consistent with molecular data.     

Molecular phylogeny of the freshwater fish family Cobitidae (Cypriniformes: Teleostei): delimitation of genera, mitochondrial introgression and evolution of sexual dimorphism

The family Cobitidae represents a characteristic element of the Eurasian ichthyofauna. Despite diverse features of sexual dimorphism, comparably few morphological characters have been utilized for taxonomic studies resulting in many unresolved puzzles. Here we present the phylogenetic relationships of Cobitidae as inferred from the mitochondrial cytochrome b gene and the nuclear gene RAG-1. Analyses of both markers show a group of eight nominal genera, which all occur in Europe and eastern, northern and western Asia, forming a monophyletic lineage (northern clade) while all other clades inhabit South and Southeast Asia (southern lineages). While all eight southern lineages correspond to genera as defined by morphological studies, only four lineages were reliably recovered within the northern clade, and of these only one (Sabanejewia) corresponds to a formerly considered genus. The genera Cobitis, Iksookimia and Niwa?lla were polyphyletic. A comparison of the two markers shows several incongruities within the northern clade and mitochondrial introgression at least in the genus Misgurnus. Mapping the characters of sexual dimorphism on our cladogram, we identified five character states that are diagnostic for certain lineages. Estimations of the divergence times dated the separation of the northern clade from the southern lineages to the middle Eocene (46 MYA) and the origin of "Cobitis"misgurnoides, the basal taxon of the northern clade, during early Oligocene (30-35 MYA). The geographic distribution of the major clades supports recently developed hypotheses about the river history of East Asia and further suggests that a range expansion of the northern clade in late Miocene (15 MYA) led to the colonisation of Europe by three already distinct genera.     

A revision of the lygaeid genus Pseudopachybrachius (Hemiptera)

Abstract. The rhyparochromine lygaeid genus Pseudopachybrachius is revised. Four species are recognized in the perplexing vinctus-gvoup (viz capicola, reductus, vinctus and guttus) based on the genitalia details and external features. One new species, P.concepcioni sp.nov., is described from the West Indies and South America. New distributional records are given for all species. Pamera dubia Reuter 1882 is synonymized with Pseudopachybrachius reductus (Walker, 1872) and a neotype is designated for Rhyparochromus inconspicuus Dallas, 1852. A key and diagnostic characters of seven species are given. Phylogenetic and zoogeographic relationships are discussed. Illustrations of the genitalia, a cladogram and diagnostic external features are included.     

Evidence for lock-and-key mechanisms in the internal genitalia of the Apamea moths (Lepidoptera, Noctuidae)

Abstract. Fifty of the fifty-six species of the genus Apamea known from North America and three Palaearctic species were analysed for lock-and-key characters in their internal genitalia, mainly in the male vesica and the female bursa copulatrix. There were an average of 4.5 such characters per species, structurally corresponding in the two sexes. Anatomically they form a postcopulatory but prezygotic isolation mechanism. In some closely related species, the internal genitalia are very similar, but these species have a precopulatory isolation mechanism in the presence or absence of male abdominal coremata. Closely related species did not have more lock-and-key characters than unrelated species, which is taken to indicate absence of character displacement. The anatomical distribution of the lock-and-key characters was examined and the organs of eight species are illustrated.
The lock-and-key hypothesis has been abandoned by several earlier authors but mainly on consideration of external genitalia. In Apamea the invariable functional correspondence between sexes in the sperm transferring organs, and the overall species-specifity of characters but non-existence of interspecific differences under a precopulatory mechanism indicate that (a) lock-and-keys are functioning and (b) they act as isolation mechanisms. Alternative hypotheses of genitalic evolution are reviewed.     

So close and so different: comparative phylogeography of two small mammal species, the yellow-necked fieldmouse (Apodemus flavicollis) and the woodmouse (Apodemus sylvaticus) in the Western Palearctic region

In Europe, concordant geographical distribution among genetic lineages within different species is rare, which suggests distinct reactions to Quaternary ice ages. This study aims to determine whether such a discrepancy also affects a pair of sympatric species, which are morphologically and taxonomically closely related but which have slight differences in their ecological habits. The phylogeographic structures of two European rodents, the Yellow-necked fieldmouse (A. flavicollis) and the woodmouse (Apodemus sylvaticus) were, therefore, compared on the basis of mitochondrial DNA cytochrome b (mtDNA cyt b) sequences (965 base pairs) from 196 specimens collected from 59 European localities spread throughout the species distributions. The results indicate that the two species survived in different ways through the Quaternary glaciations. A. sylvaticus survived in the Iberian Peninsula from where it recolonized almost all Europe at the end of the last glaciation. Conversely, the refuge from which A. flavicollis recolonized Europe, including northern Spain, during the Holocene corresponds to the Italo-Balkan area, where A. sylvaticus suffered a serious genetic bottleneck. This study confirms that even closely related species may have highly different phylogeographic histories and shows the importance of ecological plasticity of the species for their survival through climate change. Finally, it suggests that phylogeographic distinctiveness may be a general feature of European species.     

Morphological and molecular species delimitation within the Holarctic Ilybius angustior complex with a focus on Beringia (Coleoptera: Dytiscidae)

A fourth species of the Holarctic Ilybius angustior complex, Ilybius minakawai n. sp., is described from the Island of Sakhalin in the Russian far east. Male genitalia are diagnostic within this species complex, although from body size and shape the new species cannot be separated from I. churchillensis Wallis from north Alaska and the Canadian low arctic. The small and narrow body characteristic of these two species represents an extreme of the cold climate form of the widespread I. angustior (Gyllenhal). Low variation within the studied large ribosomal and cytochrome c oxidase subunit I mitochondrial genes suggest that speciation events within the I. angustior-complex are fairly recent. In combination with known geographical ranges, the low genetic variation within this species complex suggest speciation within Pleistocene refugia including Beringia.