Two independent LAMP assays for rapid identification of the serpentine leafminer, Liriomyza huidobrensis (Blanchard, 1926) (Diptera: Agromyzidae) in Australia

Liriomyza huidobrensis is a leafminer fly and significant horticultural pest. It is a quarantine listed species in many countries and is now present as an established pest in Australia. Liriomyza huidobrensis uses a broad range of host plants and has potential for spread into various horticultural systems and regions of Australia. Rapid in-field identification of the pest is critically needed to assist efforts to manage this pest. Morphological identification of the pest is effectively limited to specialist examinations of adult males. Generally, molecular methods such as qPCR and DNA barcoding for identification of Liriomyza species require costly laboratory-based hardware. Herein, we developed two independent and rapid LAMP assays targeted to independently inherited mitochondrial and nuclear genes. Both assays are highly sensitive and specific to L. huidobrensis. Positive signals can be detected within 10 min on laboratory and portable real-time amplification fluorometers. Further, we adapted these assays for use with colorimetric master mixes, to allow fluorometer free in-field diagnostics of L. huidobrensis. Our LAMP assays can be used for stand-alone testing of query specimens and are likely to be essential tools used for rapid identification and monitoring of L. huidobrensis

Mud crab susceptibility to disease from white spot syndrome virus is species-dependent

<p>Abstract</p> <p>Background</p> <p>Based on a report for one species (<it>Scylla serrata</it>), it is widely believed that mud crabs are relatively resistant to disease caused by white spot syndrome virus (WSSV). We tested this hypothesis by determining the degree of susceptibility in two species of mud crabs, <it>Scylla olivacea </it>and <it>Scylla paramamosain</it>, both of which were identified by mitochondrial 16 S ribosomal gene analysis. We compared single-dose and serial-dose WSSV challenges on <it>S. olivacea </it>and <it>S. paramamosain</it>.</p> <p>Findings</p> <p>In a preliminary test using <it>S. olivacea </it>alone, a dose of 1 × 10⁶WSSV copies/g gave 100% mortality within 7 days. In a subsequent test, 17 <it>S. olivacea </it>and 13 <it>S. paramamosain </it>were divided into test and control groups for challenge with WSSV at 5 incremental, biweekly doses starting from 1 × 10⁴and ending at 5 × 10⁶copies/g. For 11 <it>S. olivacea </it>challenged, 3 specimens died at doses between 1 × 10⁵and 5 × 10⁵copies/g and none died for 2 weeks after the subsequent dose (1 × 10⁶copies/g) that was lethal within 7 days in the preliminary test. However, after the final challenge on day 56 (5 × 10⁶copies/g), the remaining 7 of 11 <it>S. olivacea </it>(63.64%) died within 2 weeks. There was no mortality in the buffer-injected control crabs. For 9 <it>S. paramamosain </it>challenged in the same way, 5 (55.56%) died after challenge doses between 1 × 10⁴and 5 × 10⁵copies/g, and none died for 2 weeks after the challenge dose of 1 × 10⁶copies/g. After the final challenge (5 × 10⁶copies/g) on day 56, no <it>S. paramamosain </it>died during 2 weeks after the challenge, and 2 of 9 WSSV-infected <it>S. paramamosain </it>(22.22%) remained alive together with the control crabs until the end of the test on day 106. Viral loads in these survivors were low when compared to those in the moribund crabs.</p> <p>Conclusions</p> <p><it>S. olivacea </it>and <it>S. paramamosain </it>show wide variation in response to challenge with WSSV. <it>S. olivacea </it>and <it>S. paramamosain </it>are susceptible to white spot disease, and <it>S. olivacea </it>is more susceptible than <it>S. paramamosain</it>. Based on our single-challenge and serial challenge results, and on previous published work showing that <it>S. serrata </it>is relatively unaffected by WSSV infection, we propose that susceptibility to white spot disease in the genus <it>Scylla </it>is species-dependent and may also be dose-history dependent. In practical terms for shrimp farmers, it means that <it>S. olivacea </it>and <it>S. paramamosain </it>may pose less threat as WSSV carriers than <it>S. serrata</it>. For crab farmers, our results suggest that rearing of <it>S. serrata </it>would be a better choice than <it>S. paramamosain </it>or <it>S. olivacea </it>in terms of avoiding losses from seasonal outbreaks of white spot disease.</p

Assessment of population genetic structure in the arbovirus vector midge, Culicoides brevitarsis (Diptera Ceratopogonidae), using multi-locus DNA microsatellites

Bluetongue virus (BTV) is a major pathogen of ruminants that is transmitted by biting midges (Culicoides spp.). Australian BTV serotypes have origins in Asia and are distributed across the continent into two distinct episystems, one in the north and another in the east. Culicoides brevitarsis is the major vector of BTV in Australia and is distributed across the entire geographic range of the virus. Here, we describe the isolation and use of DNA microsatellites and gauge their ability to determine population genetic connectivity of C. brevitarsis within Australia and with countries to the north. Eleven DNA microsatellite markers were isolated using a novel genomic enrichment method and identified as useful for genetic analyses of sampled populations in Australia, northern Papua New Guinea (PNG) and Timor-Leste. Significant (P < 0.05) population genetic subdivision was observed between all paired regions, though the highest levels of genetic sub-division involved pair-wise tests with PNG (PNG vs. Australia (F-ST = 0.120) and PNG vs. Timor-Leste (F-ST = 0.095)). Analysis of multi-locus allelic distributions using STRUCTURE identified a most probable two-cluster population model, which separated PNG specimens from a cluster containing specimens from Timor-Leste and Australia. The source of incursions of this species in Australia is more likely to be Timor-Leste than PNG. Future incursions of BTV positive C. brevitarsis into Australia may be genetically identified to their source populations using these microsatellite loci. The vector's panmictic genetic structure within Australia cannot explain the differential geographic distribution of BTV serotypes

Towards a global barcode library for Lymantria (Lepidoptera: Lymantriinae) tussock moths of biosecurity concern

Background: Detecting and controlling the movements of invasive species, such as insect pests, relies upon rapid and accurate species identification in order to initiate containment procedures by the appropriate authorities. Many species in the tussock moth genus Lymantria are significant forestry pests, including the gypsy moth Lymantria dispar L., and consequently have been a focus for the development of molecular diagnostic tools to assist in identifying species and source populations. In this study we expand the taxonomic and geographic coverage of the DNA barcode reference library, and further test the utility of this diagnostic method, both for species/subspecies assignment and for determination of geographic provenance of populations. Methodology/Principal Findings: Cytochrome oxidase I (COI) barcodes were obtained from 518 individuals and 36 species of Lymantria, including sequences assembled and generated from previous studies, vouchered material in public collections, and intercepted specimens obtained from surveillance programs in Canada. A maximum likelihood tree was constructed, revealing high bootstrap support for 90% of species clusters. Bayesian species assignment was also tested, and resulted in correct assignment to species and subspecies in all instances. The performance of barcoding was also compared against the commonly employed NB restriction digest system (also based on COI); while the latter is informative for discriminating gypsy moth subspecies, COI barcode sequences provide greater resolution and generality by encompassing a greater number of haplotypes across all Lymantria species, none shared between species. Conclusions/Significance: This study demonstrates the efficacy of DNA barcodes for diagnosing species of Lymantria and reinforces the view that the approach is an under-utilized resource with substantial potential for biosecurity and surveillance. Biomonitoring agencies currently employing the NB restriction digest system would gather more information by transitioning to the use of DNA barcoding, a change which could be made relatively seamlessly as the same gene region underlies both protocols

Regional patterns of genetic structure among Australian populations of the mud crab, Scylla serrata (Crustacea: Decapoda): evidence from mitochondrial DNA

The population genetic structure of the estuarine crab, Scylla serrata (Forsk嬬 1775), was examined among shelf-connected locations and across a historical bio-geographic barrier. Over 300 individuals were sampled from multiple locations within coastal regions (western, northern and eastern) of Australia and analysed for mutational differences at a mitochondrial coding gene (COI). Analysis of molecular variance indicated mitochondrial haplotypes to be structured regionally (P < 0.001), which contrasted with evidence of genetic panmixia within regions. Regional genetic structure broadly correlated with hydrological circulation, supporting the contention that release of propagules away from the estuary may allow genetic connectivity among widespread shelf-connected S. serrata populations. That similar patterns of maternal gene flow are absent among trans-oceanic populations may indicate that the spatial scale of effective dispersal for this species is generally limited to areas of coastal shelf. Two distinct clades of haplotypes were geographically separated either side of the Torres Strait, a narrow sea channel connecting the northern and eastern regions of coastal Australia. This pattern of historical genetic separation is concordant with a number of other marine species across northern Australia and may indicate a shared history of vicariance induced by eustasy. Alternatively, we suggest that sundering of S. serrata populations resulting in cladogenesis may have its origins outside of the northern Australian region.Griffith Sciences, Griffith School of EnvironmentNo Full Tex

Mitochondrial DNA evidence for rapid colonisation of the Indo- West Pacific by the mudcrab Scylla serrata

Scylla serrata (Forskål, 1775) is widely distributed throughout mangrove habitats of the Indo-West Pacific (IWP) coastal waters. This study investigated the phylogeographic distribution of S. serrata mitochondrial DNA haplotypes sampled throughout the species range. Adults were sampled from three west Indian Ocean locations (N = 21), five west Pacific sites (N = 28) and three sites from northern and eastern Australia (N = 76). Temperature-gradient gel-electrophoresis and sequencing of 549 base pairs of a mtDNA gene (cytochrome oxidase 1) identified 18 distinct haplotypes. Haplotypes cluster into two clades separated by ≃2% sequence-divergence. One clade is widespread throughout the IWP, the other is strictly confined to northern Australia. Genealogical assessment of sequenced haplotypes suggests that the historical spread of S. serrata throughout the IWP has occurred rapidly and recently (<1 million years before present) from a west Pacific origin. The fact that many locations contain a single unique haplotype suggests limited contemporary gene flow between trans-oceanic sites, and that recent historical episodes of population founding and retraction have both determined and affected the current distribution of S. serrata populations. Contrary to that reported for other widespread species of IWP taxa, there is no pattern of regional separation of Indian from Pacific Ocean populations. However, results do suggest a vicariant separation of northern Australian crabs prior to the IWP radiation. We speculate that this separation may have resulted in the formation of a new species of Scylla

Colonisation of the south-west Australian coastline by mud crabs: evidence for a recent range expansion or human-induced translocation?

Mud crabs (Portunidae; Scylla spp.) have become established recently in some south-west Australian estuaries - almost 1000 km south of their recorded distribution. Colonisation may have occurred by a natural range expansion from the north-west or by translocation from source(s) within the Indo-West Pacific. To identify the species and the potential source population(s), genetic analyses was used to compare south-west crabs (N = 32) to other populations. Levels of diversity at two independent genetic markers were also compared to obtain relative estimates of effective population size between colonist and suspected source population(s). Comparisons of mitochondrial DNA sequences (COI) indicated that all south-west crabs were Scylla serrata. Indeed, the sole haplotype found among colonists was identical to one prevalent but endemic to more diverse north-west Australian populations. In contrast, source and colonist populations had equally high levels of genetic diversity at two microsatellite loci. It is argued that the south-west region was colonised by large numbers of S. serrata from north-west Australia through a recruitment event enhanced by the strong 1999/2000 Leeuwin Current. Differences in diversity among nuclear and mitochondrial loci may reflect different responses to the colonisation process; it is predicted that such differences are prevalent among plankton-dispersed species.Griffith Sciences, Griffith School of EnvironmentNo Full Tex