在藏羚羊上发现的中国第四种皮蝇

对于从可可西里藏羚羊上采集到的藏羚羊皮蝇Hypoderma sp.3龄幼虫进行了形态学观察,发现其在伪头、第10腹节上的刺、气门板形态上与中国现有记录的3种皮蝇（牛皮蝇、纹皮蝇和中华皮蝇）有着明显的区别。对藏羚羊皮蝇的线粒体COI 基因种属特异性序列研究表明,其与牛皮蝇、纹皮蝇和中华皮蝇对应序列的相似性分别为88.1％,87.8％和88.8％。据此认为采自藏羚羊的此种皮蝇可能为中国境内又一皮蝇新种或新记录种,为中国第四种皮蝇。     

Utility of mitochondrial and ribosomal genes for differentiation and phylogenesis of species of gastrointestinal bot flies

Larvae of Gasterophilus spp. (Diptera: Oestridae) cause gastrointestinal myiasis of equids. However, their identification may be problematic due to morphological similarities between species infesting identical regions of the digestive tract. In this study, genes encoding for mitochondrial cytochrome oxidase I (COI) and for the 16S and 28S ribosomal subunits of the most commonly encountered Gasterophilinae subfamily species [i.e., Gasterophilus haemorrhoidalis (L.), Gasterophilus inermis (Brauer), Gasterophilus intestinalis (De Geer), Gasterophilus nasalis (L.), and Gasterophilus pecorum (F.)] were studied, together with Gyrostigma pavesii (Corti), a rhinoceros parasite, and Hypoderma lineatum (De Villers), as outgroup taxa. Analysis identified interspecific differences that allowed their unequivocal identification. The high genetic homology among the sequences of G. haemorrhoidalis and G. intestinalis (i.e., 100, 99.86, and 99.46% in the 28S, COI, and 16S genes, respectively) strongly support the hypothesis that they are morphotypes of the same species. Phylogenetic analyses (maximum-likelihood and parsimony) were performed using PAUP; all analyses supported monophyly of subfamily Gasterophilinae. This study confirms the utility of the COI and 16S and 28S rRNA genes to address diagnostic and phylogenetic questions in Gasterophilus species.     

A third species of Hypoderma (Diptera: Oestridae) affecting cattle and yaks in China: molecular and morphological evidence

Cattle and yak hypodermosis in China is caused by Hypoderma bovis and H. lineatum, with a prevalence reaching up to 98-100% of the animals and maximum intensities exceeding 400 warbles for each animal. A third species, H. sinense, is also considered by Chinese researchers to affect livestock. The molecular characterization of the most variable region of the mitochondrial cytochrome oxidase I gene and of the ribosomal 28S gene has been performed for the third-stage larvae collected from cattle and yaks in China and identified (on the basis of the spinulation on the ventral side of the 10th segment) as H. bovis, H. lineatum, and H. sinense. Amplicons were digested with the HinfI and BfaI restriction enzymes, which provided diagnostic profiles to simultaneously differentiate the 3 Hypoderma species. Third-stage larvae of H. sinense were also examined by scanning electron microscopy, which revealed proper morphological characteristics different from those of H. bovis and H. lineatum. The molecular and morphological evidence herein reported support the existence of a third species of Hypoderma affecting cattle and yaks in China, and the results provide new tools for unequivocal identification of this species and present key components for the evaluation of its endogenous cycle and pathogenicity in animals and humans.     

Morphological variability and genetic identity in Rhinoestrus spp. causing horse nasal myiasis

Larvae of Rhinoestrus purpureus (Brauer) and Rhinoestrus usbekistanicus Gan (Diptera: Oestridae) cause nasal myiases of equids. During a recent epidemiological survey in southern Italy some morphological and taxonomical doubts arose concerning the identification of Rhinoestrus third stage larvae on the basis of the features of the posterior spiracles and the distribution of dorsal spines on the third segment. Four different morphotypes were retrieved: R. usbekistanicus-like, R. purpureus-like and two morphotypes with shared features. The genes encoding for the mitochondrial cytochrome oxidase I (COI) and for the ribosomal subunits 16S and 28S of the four morphotypes of Rhinoestrus were investigated to determine whether they belonged to a single taxon or they displayed genetic differences indicative of more than one species. The three genes showed a very low level of sequence variation (COI 0-0.43%, 16S 0-1.45%, 28S 0-0.23%) falling within the intraspecific ranges previously described for Oestridae species. Finally, the peritreme features and the spinulation of the third segment of the four morphotypes examined could not be used to differentiate the two species.     

Myiasis caused by Oestridae: serological and molecular diagnosis

Myiasis-causing Oestridae (bot flies) infect several animal species world-wide, from palaearctic to subtropical/tropical areas. Oestrids affect livestock production causing abortion, reduced milk production, losses in weight and fertility, poor hide quality and an impairment of the host's immune system. In the last few years much research has been carried out on the immunology of these infestations, in order to acquire efficient and reliable diagnostic serological tools; the genome of the different species of Oestridae has been studied to further their molecular identification, taxonomy and phylogenesis. The immunodiagnostic methods for many myiasis causing Oestrids have proven to be a viable alternative to the clinical parasitological examination or the post-mortem examination. Numerous serological tests have been developed for the diagnosis of bovine hypodermosis caused by Hypoderma bovis and Hypoderma lineatum, and ELISAs using larval hypodermin C as the antigen are currently used on serum, individual and pooled milk samples to detect the presence of circulating anti-Hypoderma antibodies. In Italy the best period to sample the animals is November-January, since it is in this period that the antibody kinetics of the animals reaches a peak. Recently the efficacy of the ELISA test on pasteurized milk samples has been demonstrated, allowing the diagnosis of bovine hypodermosis also in areas where there is no information on the presence of the disease and the sampling of the animals is laborious. The cross-reactivity between Przhevalskiana silenus antigens and anti-Hypoderma antibodies led to assessing the usefulness of a simple and cost-effective ELISA for the diagnosis of goat warble fly infection. In particular, it has been demonstrated that infected goats display an antibody peak in November-December in blood and milk, thus making this period suitable for sampling. Although no extensive data is available on the immunology of sheep and goat oestrosis caused by Oestrus ovis, the efficacy of ELISA has been demonstrated by correlating serological results with clinical post-mortem examinations. No immunological techniques are currently used to diagnose gasterophilosis of equids and only one study reports the efficacy of ELISA for detecting anti-Gasterophilus antibodies in infected equids. Several studies have been conducted into the molecular characterization of the mitochondrial DNA (mtDNA)--in particular of the gene encoding for the cytochrome oxidase I (COI)--for many free-living and parasitic arthropods for diagnostic, taxonomic and phylogenetic purposes. As regards Oestridae causing myiasis, the first study features a PCR-RFLP assay of the most common Italian species (i.e. H. bovis, H. lineatum, Gasterophilus intestinalis, P. silenus, O. ovis), which showed clear genetic differences among the genera examined, but no inter-specific variation between the two species of Hypoderma considered. The molecular characterization of the most variable region of the COI gene (encoding for the region from E4 to the terminal COOH) was able to clearly differentiate H. bovis and H. lineatum. The E4-COOH region of the COI gene has been characterized for 18 oestrid species and from a taxonomical point of view, molecular data confirm the morphological classification, with the examined species divided into four subfamilies. New insights have also been gained on the molecular differentiation of the most common species of Hypoderma (i.e. H. bovis, H. lineatum, Hypoderma actaeon, Hypoderma diana and Hypoderma tarandi) and, in particular, the restriction enzyme BfaI, provides a diagnostic profile that can be used to simultaneously differentiate all the species examined. The characterization of the E4-COOH COI gene and the hypervariable region of the gene encoding for the ribosomal Isu revealed the identity of Hypoderma sinense as a new species, infecting cattle and yaks in China. Finally, the molecular analysis of the same mitochondrial and ribosomal regions showed that P. silenus, Przhevalskiana aegagri and Przhevalskiana crossii are morphotypes of the same species.     

Analysis of sequence variation in Gnathostoma spinigerum mitochondrial DNA by single-strand conformation polymorphism analysis and DNA sequence

Morphological variations were observed in the advance third stage larvae of Gnathostoma spinigerum collected from swamp eel (Fluta alba), the second intermediate host. Larvae with typical and three atypical types were chosen for partial cytochrome c oxidase subunit I (COI) gene sequence analysis. A 450 bp polymerase chain reaction product of the COI gene was amplified from mitochondrial DNA. The variations were analyzed by single-strand conformation polymorphism and DNA sequencing. The nucleotide variations of the COI gene in the four types of larvae indicated the presence of an intra-specific variation of mitochondrial DNA in the G. spinigerum population.     

Molecular identification of two species of myiasis-causing Cuterebra by multiplex PCR and RFLP

The myiasis-causing flies Cuterebra grisea (Coquillet) and Cuterebra fontinella (Clark) (Diptera: Oestridae) are normally parasites of mice, predominantly of the genus Peromyscus. The morphological similarities of these species and the existence of intermediate morphotypes bearing characters of both species make the identification of adults problematic; furthermore the identification of larvae is apparently not possible. This study presents two molecular approaches to discriminate between these species using specific band patterns: (i) species-specific primers designed in the cytochrome oxidase II (COII) region used in multiplex polymerase chain reaction (PCR) and (ii) restriction fragment length polymorphism (RFLP) on amplified segments of cytochrome oxidase I (COI) gene. Both methods were tested on Cuterebra larvae and on adult museum specimens. The two techniques showed a clear difference between C. grisea and C. fontinella, although species-specific primers were more successful than RFLP for degraded DNA. No intraspecific variation in RFLP and species-specific amplifications were detected for the two species of Cuterebra. The results exhibit discrepancies between molecular and morphological identification, suggesting that some of the adults were misidentified.     

Two new species of Brueelia Kéler, 1936 (Ischnocera, Philopteridae) parasitic on Neotropical trogons (Aves, Trogoniformes)

Two new species of Brueelia are described and illustrated. These new species and their type hosts are: Brueelia sueta ex Pharomachrus pavoninus (Spix, 1824), the Pavonine Quetzal and Brueelia cicchinoi ex Trogon viridis Linnaeus, the White-tailed Trogon. Both new species differ from the only Brueelia described on Trogon mexicanus by many morphological features, including those present in the male genitalia and female vulvar margin. Partial sequences of the mitochondrial cytochrome oxidase I (COI) gene for these two new species differ from one another by 13.6% uncorrected p-distance. Whereas Brueelia cicchinoi is only 0.3% divergent from previously published COI sequences identified as Brueelia sp. from the Mexican Trogon melanocephalus Gould, 1936 and Trogon massena Gould, 1938. We also found Brueelia cicchinoi on Trogon melanurus, Trogon collaris and Pharomachrus pavoninus. Thus Brueelia cicchinoi is found on multiple trogoniform hosts across an extremely large geographic distribution and has one of the largest number of host associations among Brueelia species.     

Discrimination of Cricotopus species (Diptera: Chironomidae) by DNA barcoding

Chironomids (Diptera) typically comprise the most abundant group of macroinvertebrates collected in water quality surveys. Species in the genus Cricotopus display a wide range of tolerance for manmade pollutants, making them excellent bioindicators. Unfortunately, the usefulness of Cricotopus is overshadowed by the difficulty of accurately identifying larvae using current morphological keys. Molecular approaches are now being used for identification and taxonomic resolution in many animal taxa. In this study, a sequence-based approach for the mitochondrial gene, cytochrome oxidase I (COI), was developed to facilitate identification of Cricotopus species collected from Baltimore area streams. Using unique COI sequence variations, we developed profiles for seven described Cricotopus sp., four described Orthocladius sp., one described Paratrichocladius sp. and one putative species of Cricotopus. In addition to providing an accurate method for identification of Cricotopus, this method will make a useful contribution to the development of keys for Nearctic Cricotopus.     

Association and description of males, females and larvae of two New Caledonian Xanthochorema species (Trichoptera: Hydrobiosidae) based on mitochondrial 16S and COI sequences

A method for associating larvae, females and males of Trichoptera is demonstrated for New Caledonian Hydrobiosidae species of the genus Xanthochorema, using cytochrome oxidase subunit I (COI) and 16S mitochondrial gene sequences. Two species, X. caledon Kimmins, 1953 and X. celadon Schmid, 1989 , previously with unknown larvae and undescribed females, were associated, and males, females and larvae of both species are described. Mitochondrial COI and 16S gene fragments are demonstrated to be useful for association of sexes and life stages of the two species, and distance measures show that the method is likely to also be useful for other species within the genus. The associations are well supported by high bootstrap and jackknife values.     

Use of nuclear and mitochondrial DNA PCR and sequencing for molecular identification of Diphyllobothrium isolates potentially infective for humans

Tapeworms of the genus Diphyllobothrium (Cobold, 1858) are widely distributed all around the world and some of them are agents of human diphyllobothriasis. Approximately 50 species have been described within the Diphyllobothrium genus but only 13 are human pathogens. Species identification by using morphological criteria is very difficult. We determined the value of 18S ribosomal RNA gene, internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 gene (COI) sequences to differentiate between Diphyllobothrium isolates. Sequences from 18 isolates (larvae or adults) of D. latum, D. nihonkaiense, D. ditremum, D. dentriticum and D. stemmacephalum species were obtained. COI region sequences analysis was clearly more discriminative than those of the ITS1 and 18S rRNA and was a useful tool for identifying specimens.     

A DNA barcode library for Korean Chironomidae (Insecta: Diptera) and indexes for defining barcode gap

Non-biting midges (Diptera: Chironomidae) are a diverse population that commonly causes respiratory allergies in humans. Chironomid larvae can be used to indicate freshwater pollution, but accurate identification on the basis of morphological characteristics is difficult. In this study, we constructed a mitochondrial cytochrome c oxidase subunit I (COI)-based DNA barcode library for Korean chironomids. This library consists of 211 specimens from 49 species, including adults and unidentified larvae. The interspecies and intraspecies COI sequence variations were analyzed. Sophisticated indexes were developed in order to properly evaluate indistinct barcode gaps that are created by insufficient sampling on both the interspecies and intraspecies levels and by variable mutation rates across taxa. In a variety of insect datasets, these indexes were useful for re-evaluating large barcode datasets and for defining COI barcode gaps. The COI-based DNA barcode library will provide a rapid and reliable tool for the molecular identification of Korean chironomid species. Furthermore, this reverse-taxonomic approach will be improved by the continuous addition of other speceis’ sequences to the library.     

COI barcoding of true bugs (Insecta, Heteroptera)

Several recent studies have proposed that partial DNA sequences of the cytochrome c oxidase I (COI) mitochondrial gene might serve as DNA barcodes for identifying and differentiating between animal species, such as birds, fish and insects. In this study, we tested the effectiveness of a COI barcode to identify true bugs from 139 species collected from Korea and adjacent regions (Japan, Northeastern China and Fareast Russia). All the species had a unique COI barcode sequence except for the genus Apolygus (Miridae), and the average interspecific genetic distance between closely related species was about 16 times higher than the average intraspecific genetic distance. DNA barcoding identified one probable new species of true bug and revealed identical or very recently divergent species that were clearly distinguished by morphological characteristics. Therefore, our results suggest that COI barcodes can reveal new cryptic true bug species and are able to contribute for the exact identification of the true bugs.     

Evolutionary and structural analysis of the cytochrome c oxidase subunit I (COI) gene from Haematobia irritans, Stomoxys calcitrans and Musca domestica (Diptera: Muscidae) mitochondrial DNA.

This work describes the molecular characterization of the cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA from three species of great medical and veterinary importance: the horn fly, Haematobia irritans, the stable fly, Stomoxys calcitrans and the house fly, Musca domestica (Diptera: Muscidae) (Linnaeus). The nucleotide sequence in all species was 1536 bp in size and coded for a 512 amino acid peptide. The nucleotide bias for an A+T-rich sequence is linked to three features: a high A+T content throughout the entire gene, a high A+T content in the third codon position, and a predominance of A+T-rich codons. An anomalous TCG (serine) start codon was identified. Comparative analysis among members of the Muscidae, Scatophagidae, Calliphoridae and Drosophilidae showed high levels of nucleotide sequence conservation. Analysis of the divergent amino acids and COI protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COI protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species.     

Genetic variation tracks ecological segregation in Pacific island black flies

Geographic isolation is widely viewed as a key component of insular radiations on islands. However, strong ecological affinities may also reinforce isolation and promote genetic divergence. The black fly fauna in the Society Islands French Polynesia is notable for the number of closely related endemic species (31), and the morphological and habitat diversity of the larvae. Here, we measure ecological and morphological differences within and between two closely related species, Simulium oviceps and Simulium dussertorum and relate these differences to genetic distance. Phylogenetic analyses of a 920 bp fragment of the cytochrome oxidase I (COI) gene revealed a well-supported, ecologically divergent S. oviceps clade (larvae found in rivers instead of cascades) that shows little morphological differentiation. For both S. oviceps and S. dussertorum, genetic distance among populations is related to larval habitat, with cascade populations showing greater isolation from each other than river populations. Our data support the hypothesis that larval ecological shifts have played a role in the radiation of this black fly fauna.     

Molecular identification of dipteran pests (Diptera: Sciaroidea) from shiitake mushroom

On shiitake farms, mycophagous maggots can cause serious damage by preventing formation of the fruiting body. Recently, these pests have significantly reduced shiitake production in Korea. However, larvae and female adults cannot be identified due to their lack of morphological characteristics. Therefore, farmers and applied entomologists are unable to determine which species is the primary cause of the shiitake damage. In this study, mycophagous flies (colonized larvae) were collected from damaged shiitake farms and subsequently identified by matching identified males with the cytochrome c oxidase subunit I (COI) sequences from the larvae. Divergences of the COI sequences among the species discriminated the clusters clearly, and the mycophagous pests were identified as Camptomyia corticalis and C. heterobia. Interestingly, these two species coexisted under the bark of shiitake oak bed logs.     

Species composition and seasonal occurrence of Phyllophaga (Coleoptera: Scarabaeidae) infesting intensely managed Bermudagrass in Oklahoma

Larvae of Phyllophaga spp. (Coleoptera: Scarabaeidae) are important turfgrass pests in many regions of the United States. However, not all of the species associated with turfgrass are known, including species most likely to be of economic concern in Oklahoma turfgrasses, especially Bermuda grass. This study documented the species composition and seasonal occurrence of Phyllophaga associated with high maintenance Bermuda grass turf in Oklahoma over a 2-yr period. In 2005 and 2006, adult Phyllophaga spp. were collected with blacklight traps from selected golf courses throughout Oklahoma Phyllophaga larvae were obtained from Bermuda grass stands at selected sod production facilities adjacent to or near the light traps. We collected 20 species of Phyllophaga beetles in light traps, and nine species of Phyllophaga larvae from turfgrass. Peak flight periods for most species occurred in May and June, but some were captured as early as mid-April and others as late as September. The cytochrome c oxidase I (COI) gene from adults and larvae was amplified using polymerase chain reaction, sequenced, and then used to compare larval DNA against DNA from identified adults. These results confirmed the validity of using COI sequences to identify species of some Phyllophaga larvae. The identifications will aid in optimizing the timing of insecticide applications against Phyllophaga white grubs as discussed.     

Discrimination of two Japanese water pennies,Eubrianax granicollis Lewis and E. ramicornis Kiesenwetter (Coleoptera: Psephenidae), based on laboratory rearing and molecular taxonomy

Two psephenid beetles, Eubrianax granicollis Lewis and E. ramicornis Kiesenwetter, are common species on the main islands of Japan (i.e. Honshu, Shikoku and Kyushu), but diagnostic characters for larval identification are unknown. Two types of field‐collected Eubrianax larvae from Honshu and Kyushu were discriminated based on the distributions of granules on the dorsal surface. These larvae were assigned to E. granicollis and E. ramicornis by comparing them with larvae of the two species obtained via laboratory rearing. The two species were also identified unambiguously on the basis of their mitochondrial cytochrome oxidase subunit I (COI) gene sequences. Larval and pupal morphology are described based on laboratory‐reared specimens.     

Using COI gene sequence to barcode two morphologically alike species: the cotton bollworm and the oriental tobacco budworm (Lepidoptera: Noctuidae)

Due to limited morphological difference, the two closely related sister species, the cotton bollworm, Helicoverpa armigera (Hübner) and the oriental tobacco budworm, H. assulta (Guenée) (Lepidoptera: Noctuidae), are very difficult to distinguish, especially at the larvae stage. Recently, DNA sequence has been widely used as a bio-barcode for species identification. In this study, we attempted to distinguish H. armigera and H. assulta using the mitochondrial cytochrome C oxidase subunit I gene (COI) gene sequence as the barcode. We determined a 658 bp segment of the COI gene for 28 individuals of H. armigera, 8 individuals of H. assulta, and 10 individuals of Mamestra brassicae (as the outgroup) in Yunnan Province, southwest of P. R. China, together with one H. assulta and two H. armigera reported sequences from GenBank. Twenty-three haplotypes were identified in all 49 samples. As expected, network analysis of the haplotypes of the three species presented a clustering pattern consistent with the respective species status. Haplotypes of the same species differed from each other by no more than three nucleotide substitutions. However, each haplotype of H. armigera differed from that of H. assulta by at least 22 nucleotide substitutions. Both species differed from M. brassicae by more than 50 nucleotide substitutions. 17 unique diagnostic nucleotides were also used to discriminate the two species. The finding of large COI sequence differences between H. armigera and H. assulta suggested that it could be used to distinguish the two morphologically alike species and be employed for quick species identification during pest control.