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

DNA barcoding and surveillance sampling strategies for Culicoides biting midges (Diptera: Ceratopogonidae) in southern India

Background: Culicoides spp. biting midges transmit bluetongue virus (BTV), the aetiological agent of bluetongue (BT), an economically important disease of ruminants. In southern India, hyperendemic outbreaks of BT exert high cost to subsistence farmers in the region, impacting on sheep production. Effective Culicoides spp. monitoring methods coupled with accurate species identification can accelerate responses for minimising BT outbreaks. Here, we assessed the utility of sampling methods and DNA barcoding for detection and identification of Culicoides spp. in southern India, in order to provide an informed basis for future monitoring of their populations in the region. Methods: Culicoides spp. collected from Tamil Nadu and Karnataka were used to construct a framework for future morphological identification in surveillance, based on sequence comparison of the DNA barcode region of the mitochondrial cytochrome c oxidase I (COI) gene and achieving quality standards defined by the Barcode of Life initiative. Pairwise catches of Culicoides spp. were compared in diversity and abundance between green (570 nm) and ultraviolet (UV) (390 nm) light emitting diode (LED) suction traps at a single site in Chennai, Tamil Nadu over 20 nights of sampling in November 2013. Results: DNA barcode sequences of Culicoides spp. were mostly congruent both with existing DNA barcode data from other countries and with morphological identification of major vector species. However, sequence differences symptomatic of cryptic species diversity were present in some groups which require further investigation. While the diversity of species collected by the UV LED Center for Disease Control (CDC) trap did not significantly vary from that collected by the green LED CDC trap, the UV CDC significantly outperformed the green LED CDC trap with regard to the number of Culicoides individuals collected. Conclusions: Morphological identification of the majority of potential vector species of Culicoides spp. samples within southern India appears relatively robust; however, potential cryptic species diversity was present in some groups requiring further investigation. The UV LED CDC trap is recommended for surveillance of Culicoides in southern India

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

Complete chloroplast genome of Serrated Tussock, Nassella trichotoma (Poaceae: Stipeae)

Nassella trichotoma is one of the most serious weed species in Australia. It is often confused with other Nassella and stipoid species, especially at the young seedling stage, adding another layer of complexity in effective weed management. We report here the complete chloroplast genome of N. trichotoma (137,568 bp, GenBank accession number KX792500.2) sequenced using Next Generation Sequencing technology (Illumina). The N. trichotoma was grouped closely with other Nassella species and separated from other Stipeae species in the phylogenetic tree constructed based on the complete chloroplast genome sequences. The sequence information could be used for further identification of novel DNA barcodes for correct weed identification and subsequently improve management of this invasive grass

ML tree from PhyML analysis of data set 2 (1,553 sequences).

<p>Some clusters collapsed. Numbers in parentheses following names are number of sequences within that group. Numbers on branches are RAxML bootstrap values followed by SH-like support values from PhyML expressed as a percentage, both shown only if ≥ 50. Asterisk indicates species not recovered as monophyletic.</p

Subtree for <i>Australothis</i> and <i>Helicoverpa</i> clade, from PhyML analysis of data set 2 (1,553 sequences).

<p>Species clusters collapsed. Numbers in parentheses following names are number of sequences within that group. Numbers on branches are RAxML bootstrap values followed by SH-like support values from PhyML expressed as a percentage, both shown only if ≥ 50. Asterisk indicates species incongruence due to misidentifications (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160895#sec008" target="_blank">discussion</a>).</p

DNA Barcoding the Heliothinae (Lepidoptera: Noctuidae) of Australia and Utility of DNA Barcodes for Pest Identification in Helicoverpa and Relatives

<div><p><i>Helicoverpa</i> and <i>Heliothis</i> species include some of the world’s most significant crop pests, causing billions of dollars of losses globally. As such, a number are regulated quarantine species. For quarantine agencies, the most crucial issue is distinguishing native species from exotics, yet even this task is often not feasible because of poorly known local faunas and the difficulties of identifying closely related species, especially the immature stages. DNA barcoding is a scalable molecular diagnostic method that could provide the solution to this problem, however there has been no large-scale test of the efficacy of DNA barcodes for identifying the Heliothinae of any region of the world to date. This study fills that gap by DNA barcoding the entire heliothine moth fauna of Australia, bar one rare species, and comparing results with existing public domain resources. We find that DNA barcodes provide robust discrimination of all of the major pest species sampled, but poor discrimination of Australian <i>Heliocheilus</i> species, and we discuss ways to improve the use of DNA barcodes for identification of pests.</p></div