Species-level identification of the blowfly Chrysomya megacephala and other Diptera in China by DNA barcoding.

The blowfly Chrysomya megacephala, or oriental latrine fly, is the most common human-associated fly of the oriental and Australasian regions. Chrysomya megacephala is of particular interest for its use in forensic entomology and because it is a disease vector. The larvae are economically important as feed for livestock and in traditional Chinese medicine. Identification of adults is straightforward, but larvae and fragments of adults are difficult to identify. We collected C. megacephala, its allies Chrysomya pinguis and Protophormia terraenovae, as well as flies from 11 other species from 52 locations around China, then sequenced 658 base pairs of the COI barcode region from 645 flies of all 14 species, including 208 C. megacephala, as the basis of a COI barcode library for flies in China. While C. megacephala and its closest relative C. pinguis are closely related (mean K2P divergence of 0.022), these species are completely non-overlapping in their barcode divergences, thus demonstrating the utility of the COI barcode region for the identification of C. megacephala. We combined the 208 C. megacephala sequences from China with 98 others from public databases and show that worldwide COI barcode diversity is low, with 70% of all individuals belonging to one of three haplotypes that differ by one or two substitutions from each other, reflecting recent anthropogenic dispersal from its native range in Eurasia.

Rapid and specific detection of porcine parvovirus using real-time PCR and high resolution melting (HRM) analysis.

BACKGROUND: Porcine parvovirus (PPV) is the important causative agent for infectious infertility, which is a fairly tough virus that multiplies normally in the intestine of pigs without causing clinical signs in the world. RESULTS: We developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the detection of PPV. Primers targeting the VP gene were highly specific, as evidenced by the negative amplification of closely related viruses, such as porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The performance of unlabeled real time PCR was compared to TaqMan real time PCR, and the detection limits of the two methods were nearly equal. Moreover, there was good correlation between Cp and diluted genomic DNA when tested with the two methods. The assay has the accuracy of 100% in reference to labeled real time PCR, when it was tested on 45 clinical samples. CONCLUSIONS: The present study demonstrated that the established assay integrating real-time PCR and HRM is relatively cost-effective and more stable, which provides an alternative tool for rapid, simple, specific and sensitive detection of PPV.

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis.

Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini.

Initial analysis of the hemocyanin subunit type 1 (Hc1 gene) from Locusta migratoria manilensis.

Hemocyanins are copper-containing (Cu(+)) proteins that transport oxygen in many arthropods hemolymph. We characterized Hc1 gene from the grasshopper species Locusta migratoria manilensis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression at different developmental stages. The cDNA of Hc1 gene (GenBank accession no.:HQ213937) is 2271 bp in length and the open reading frame is 2016 bp, which encodes a 672 amino acids protein with a calculated molecular mass of 77.9 kD and the isoelectric point of 6.06. Sequence alignment analysis result showed that this gene shares 94.7% identity with Schistocerca americana EHP. In addition, analysis of quantitative RT-PCR indicated that, LmiHc1 was expressed in the embyro (24, 39, 62, 86, 144, and 193 h after hatch), nymphs (1st instar, 2nd instar, 3rd instar, 4th instar and 5th instar) and in adult. These results showed that Hc1 plays an important role in grasshopper, which may be related to an enhanced oxygen supply. Phylogenetic analysis of insecta based on Hc1 are basically consistent with the morphology.

Two new species of Episymploce Bey-Bienko, 1950 (Blattodea, Ectobiidae, Blattellinae) from China.

Two new species of Episymploce Bey-Bienko from China are described. Nine individuals of E. sichuanensis sp. nov. were collected from Sichuan Province and four individuals of E. maxima, sp. nov. were collected from Guangxi Province. Morphology, especially the wings, specialized abdominal tergum and genitalia of adults, are described and illustrated in detail. Episymploce sichuanensis sp. nov. is similar to E. kunmingi (Bey-Bienko, 1969), but can be easily distinguished by the reduced wings, bifurcated two processes at the hind margin of the supra-anal plate, and the unspecialized first abdominal tergum (T1). Episymploce maxima sp. nov. is similar to E. taiheizana Asahina, 1979 but is distinguished by its large size, the lateromedial margins of the subgenital plate without processes, and the unspecialized T1. A key to the recorded Episymploce species from China is provided in this paper.

The genomic and functional landscapes of developmental plasticity in the American cockroach.

Many cockroach species have adapted to urban environments, and some have been serious pests of public health in the tropics and subtropics. Here, we present the 3.38-Gb genome and a consensus gene set of the American cockroach, Periplaneta americana. We report insights from both genomic and functional investigations into the underlying basis of its adaptation to urban environments and developmental plasticity. In comparison with other insects, expansions of gene families in P. americana exist for most core gene families likely associated with environmental adaptation, such as chemoreception and detoxification. Multiple pathways regulating metamorphic development are well conserved, and RNAi experiments inform on key roles of 20-hydroxyecdysone, juvenile hormone, insulin, and decapentaplegic signals in regulating plasticity. Our analyses reveal a high level of sequence identity in genes between the American cockroach and two termite species, advancing it as a valuable model to study the evolutionary relationships between cockroaches and termites.

The complete mitochondria genome of Chrysomya phaonis (Seguy, 1928) (Diptera: Calliphoridae).

Chrysomya phaonis (Seguy, 1928) is one of the blowflies of great medical and forensic importance. In this paper, we report that the entire genome of mitochondrial DNA of C. phaonis is 15,831 bp in length, which consists of 39 genes including13 protein-coding genes, 24 tRNA genes, 2 mitochondrial ribosomal RNA genes, and a 992 bp non-coding A + T-rich region. The overall base compositions of A, G, C, and T are 38.79%, 9.75%, 14.15%, and 37.31%, respectively. We provide the first complete mitochondrial genome of C. phaonis, and should provide useful information for phylogenetic and species identification for C. phaonis.

Mitochondrial genomes suggest that hexapods and crustaceans are mutually paraphyletic.

For over a century the relationships between the four major groups of the phylum Arthropoda (Chelicerata, Crustacea, Hexapoda and Myriapoda) have been debated. Recent molecular evidence has confirmed a close relationship between the Crustacea and the Hexapoda, and has included the suggestion of a paraphyletic Hexapoda. To test this hypothesis we have sequenced the complete or near-complete mitochondrial genomes of three crustaceans (Parhyale hawaiensis, Squilla mantis and Triops longicaudatus), two collembolans (Onychiurus orientalis and Podura aquatica) and the insect Thermobia domestica. We observed rearrangement of transfer RNA genes only in O. orientalis, P. aquatica and P. hawaiensis. Of these, only the rearrangement in O. orientalis, an apparent autapomorphy for the collembolan family Onychiuridae, was phylogenetically informative.We aligned the nuclear and amino acid sequences from the mitochondrial protein-encoding genes of these taxa with their homologues from other arthropod taxa for phylogenetic analysis. Our dataset contains many more Crustacea than previous molecular phylogenetic analyses of the arthropods. Neighbour-joining, maximum-likelihood and Bayesian posterior probabilities all suggest that crustaceans and hexapods are mutually paraphyletic. A crustacean clade of Malacostraca and Branchiopoda emerges as sister to the Insecta sensu stricto and the Collembola group with the maxillopod crustaceans. Some, but not all, analyses strongly support this mutual paraphyly but statistical tests do not reject the null hypotheses of a monophyletic Hexapoda or a monophyletic Crustacea. The dual monophyly of the Hexapoda and Crustacea has rarely been questioned in recent years but the idea of both groups' paraphyly dates back to the nineteenth century. We suggest that the mutual paraphyly of both groups should seriously be considered.

One new species in the cockroach genus Jacobsonina Hebard 1929 (Blattodea, Ectobiidae, Blattellinae) from Mainland China.

One new species of Jacobsonina Hebard from China is described and illustrated: Jacobsonina erebis sp. nov.. A key to all known species in this genus, except for J. lugubris (Brunner von Wattenwyl, 1893), based on males, is provided. 

A formal re-description of the cockroach Hebardina concinna anchored on DNA Barcodes confirms wing polymorphism and identifies morphological characters for field identification.

BACKGROUND: Hebardina concinna is a domestic pest and potential vector of pathogens throughout East and Southeast Asia, yet identification of this species has been difficult due to a lack of diagnostic morphological characters, and to uncertainty in the relationship between macroptyrous (long-winged) and brachypterous (small-winged) morphotypes. In insects male genital structures are typically species-specific and are frequently used to identify species. However, male genital structures in H. concinna had not previously been described, in part due to difficulty in identifying conspecifics. METHODS/PRINCIPAL FINDINGS: We collected 15 putative H. concinna individuals, from Chinese populations, of both wing morphotypes and both sexes and then generated mitochondrial COI (the standard barcode region) and COII sequences from five of these individuals. These confirmed that both morphotypes of both sexes are the same species. We then dissected male genitalia and compared genital structures from macropterous and brachypterous individuals, which we showed to be identical, and present here for the first time a detailed description of H. concinna male genital structures. We also present a complete re-description of the morphological characters of this species, including both wing morphs. CONCLUSIONS/SIGNIFICANCE: This work describes a practical application of DNA barcoding to confirm that putatively polymorphic insects are conspecific and then to identify species-specific characters that can be used in the field to identify individuals and to obviate the delay and cost of returning samples to a laboratory for DNA sequencing.

Time-lapse imaging of chick cardiac precursor cells.

The chick embryo is easily accessible and has therefore been widely used in developmental biology studies. In particular, the early embryo can be removed from the egg and cultured, which allows real-time observations and imaging. Here, we describe ex vivo electroporation followed by long-term time-lapse microscopy, image capture, and processing. We have applied this approach to characterise the migration route of cardiac progenitor cells (CPCs) in live embryos. The heart is the first organ to function during vertebrate development and it is essential for the continued growth and survival of the embryo. In the chick, cardiac progenitors have been mapped to the anterior and mid-primitive streak at Hamburger-Hamilton stage 3. However, until recently it was not possible to observe cell migration trajectories directly. Furthermore, we used grafting of beads or cell pellets or electroporation of expression plasmids to show that Wnt3a acts as a repulsive signal to guide the movement of cardiac progenitors.

Cardiac progenitor migration and specification: The dual function of Wnts.

The heart is the first organ to function during vertebrate development and cardiac progenitors, are among the first cell lineages to be established from mesoderm cells emerging from the primitive streak during gastrulation. Cardiac progenitors have been mapped in the epiblast of pre-streak embryos. In the early chick gastrula they are located in the mid-primitive streak, from which they enter the mesoderm bilaterally. However, migration routes of cardiac progenitors have never been directly observed within the embryo and the factor(s) controlling their movement are not known. Furthermore, it is not understood how signals controlling cell movement are integrated with those that determine cell fate. Long-term video microscopy combined with GFP labelling and image processing enabled us to observe the movement patterns of prospective cardiac cells in whole embryos in real time. Embryo manipulations and the analysis of explants suggest that Wnt3a plays a crucial role in guiding these cells through a RhoA dependent mechanism involving negative chemotaxis. Wnt3a is expressed at high levels in the amniote primitive streak and ectopic signalling activity caused wider movement trajectories resulting in cardia bifida, which was rescued by dominant-negative Wnt3a. Our studies revealed Wnt3a-RhoA mediated chemo-repulsion as a novel mechanism guiding cardiac progenitors. This activity can act at long-range and does not interfere with cardiac cell fate specification.

Wnt3a-mediated chemorepulsion controls movement patterns of cardiac progenitors and requires RhoA function.

The heart is the first organ to function during vertebrate development and cardiac progenitors are among the first cell lineages to be established. In the chick, cardiac progenitors have been mapped in the epiblast of pre-streak embryos, and in the early gastrula they are located in the mid-primitive streak, from which they enter the mesoderm bilaterally. Signals controlling the specification of cardiac cells have been well documented; however, migration routes of cardiac progenitors have not been directly observed within the embryo and the factor(s) controlling their movement are not known. In addition, it is not clear how cell movement is coordinated with cell specification in the early embryo. Here we use live imaging to show that cardiac progenitors migrate in highly directed trajectories, which can be controlled by Wnt3a. Ectopic Wnt3a altered movement trajectories and caused cardia bifida. This was rescued by electroporation of dominant-negative DN-Wnt3a into prospective cardiac cells. Explant essays and mutant analysis showed that cellular guidance involved repulsion in response to Wnt3a and required RhoA function. It has been shown that Wnt3a inhibits cardiogenic cell specification through a beta-catenin-dependent pathway. On the basis of our results, we propose that Wnt3a concomitantly guides the movement of cardiac progenitors by a novel mechanism involving RhoA-dependent chemorepulsion.

Ribosomal DNA gene and phylogenetic relationships of Diplura and lower hexapods.

The monophyly of Diplura and its phylogenetic relationship with other hexapods are important for understanding the phylogeny of Hexapoda. The complete 18SrRNAgene and partial 28SrRNA gene (D3-D5 region) from 2 dipluran species (Campodeidae and Japygidae), 2 proturan species, 3 collembolan species, and 1 locust species were sequenced. Combining related sequences in GenBank, phylogenetic trees of Hexapoda were constructed by MP method using a crustacean Artemia salina as an outgroup. The results indicated that: (i) the integrated data of 18SrDNA and 28SrDNA could provide better phylogenetic information, which well supported the monophyly of Diplura; (ii) Diplura had a close phylogenetic relationship to Protura with high bootstrap support.