Effective mosquito and arbovirus surveillance using metabarcoding.

Effective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next-generation sequencing (NGS) is a high-throughput technology that has the potential to modernize vector surveillance. When combined with DNA barcoding, it is termed 'metabarcoding.' The aim of our study was to establish a metabarcoding protocol to characterize pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269-bp section of the mitochondrial cytochrome c oxidase subunit I (COI) locus. Additionally, a 67-bp region of the RRV E2 gene was amplified from synthesized cDNA to screen for RRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using a DNA barcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species and RRV in every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species and RRV from a single mosquito. The primers used for amplification, number of PCR cycles and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches.

DNA-based identifications reveal multiple introductions of the vegetable leafminer Liriomyza sativae (Diptera: Agromyzidae) into the Torres Strait Islands and Papua New Guinea.

Leafmining flies (Diptera: Agromyzidae) can be serious economic pests of horticultural crops. Some genera such as Liriomyza are particularly problematic with numerous species, some of which are highly polyphagous (wide host range), which can only be confidently identified morphologically from adult males. In our study, DNA barcoding was employed to establish new locality records of the vegetable leafminer fly, Liriomyza sativae, from the islands of Torres Strait (Queensland, Australia) and the central highlands of Papua New Guinea (PNG). These records represent significant range extensions of this highly invasive plant pest. Specimens of immature leafminers (from leaf mines) were collected over a 5-year period during routine plant health surveys in ethanol or on FTA® filter paper cards, both methods proved effective at preserving and transporting insect DNA under tropical conditions, with FTA cards possessing some additional logistical benefits. Specimens were identified through sequencing two sections of the cytochrome oxidase I gene and the utility of each was assessed for the identification of species and intra-specific genetic lineages. Our study indicates that multiple haplotypes of L. sativae occur in PNG, while a different haplotype is present in the Torres Strait, with genetic regionalization between these areas apart from a single possible instance - one haplotype 'S.7' appears to be common between these two regions - interestingly this has also been the most common haplotype detected in previous studies of invasive L. sativae populations. The DNA barcoding methods employed here not only identified multiple introductions of L. sativae, but also appear generally applicable to the identification of other agromyzid leafminers (Phytomyzinae and Agromyzinae) and should decrease the likelihood of potentially co-amplifying internal hymenopteran parasitoids. Currently, L. sativae is still not recorded from the Australian mainland; however, further sampling of leafminer flies from Northern Australia and surrounding areas is required, as surveillance for possible Liriomyza incursions, as well as to characterize endemic species with which Liriomyza species might be confused.

Review and revision of Australian Germalus Stål, with new genera and further new species of Australian Geocorinae (Hemiptera: Heteroptera: Geocoridae).

During a review of Australian Germalus Stål, types of all species recorded from Australia, as well as of the type species G. kinbergi Stål, originally described from Mauritius Island, were examined and illustrated to redefine and redescribe the genus as well as all the included species. As a result of this, the following synonymies have become necessary: G. humeralis Distant as junior synonym of G. victoriae Bergroth; G. roseobistriatus Kirkaldy as junior synonym of G. lineolosus Distant. Lectotype females have been designated for Germalus victoriae Bergroth, and Germalus lineolosus Distant.        The following new taxa have been discovered in the material available for this study: Germalus australis Malipatil sp. nov., Germalus fuscovittatus Malipatil sp. nov., Germalus littoralis Malipatil sp. nov., Capitostylus kurandae Malipatil gen. et sp. nov., Unicageocoris griseus Malipatil gen. et sp. nov., and Ausogeocoris westraliensis Malipatil gen. et sp. nov. Keys to the six genera of Geocorinae, and to the six species of Germalus, now recognised in Australia, are provided.

A proline repeat polymorphism of the Frost gene of Drosophila melanogaster showing clinal variation but not associated with cold resistance.

Genetic polymorphisms underlying adaptive shifts in thermal responses are poorly known even though studies are providing a detailed understanding of these responses at the cellular and physiological levels. The Frost gene of Drosophila melanogaster is a prime candidate for thermal adaptation; it is up-regulated under cold stress and knockdown of this gene influences cold resistance. Here we describe an amino-acid INDEL polymorphism in proline repeat number in the structural component of this gene. The two main repeats, accounting for more than 90% of alleles in eastern Australia, show a strong clinal pattern; the 6P allele was at a high frequency in tropical locations, and the 10P allele was common in temperate populations. However, the frequency of these alleles was not associated with three different assays of cold resistance. Adult transcription level of Frost was also unrelated to cold resistance as measured through post chill coma mobility. The functional significance of the proline repeat polymorphism therefore remains unclear despite its clinal pattern. The data also demonstrate the feasibility of using Roche/454 sequencing for establishing clinal patterns.

Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence.

Directly labelling locus-specific primers for microsatellite analysis is expensive and a common limitation to small-budget molecular ecology projects. More cost-effective end-labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus-specific primers with 5' universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co-amplifying large numbers of size overlapping loci and without requiring locus-specific PCR conditions to be modified. In this study, we report a suite of four high-performance universal primers that can be employed in a three primer PCR approach for efficient and cost-effective fluorescent end-labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co-amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.

A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila.

Body size often shows adaptive clines in many ectotherms across altitude and latitude, but little is known about the genetic basis of these adaptive clines. Here we identify a polymorphism in the Dca (Drosophila cold acclimation) gene in Drosophila melanogaster that influences wing size, affects wing:thorax allometry and also controls a substantial proportion of the clinal wing-size variation. A polymorphism in the promoter region of Dca had two common alleles showing strong reciprocal clinal variation in frequency with latitude along the east coast of Australia. The Dca-237 allele increased towards the tropics where wing size is smaller. A within-population association study highlighted that an increase in the frequency of this allele decreased wing size but did not influence thorax size. A manipulated increase in the level of expression of Dca achieved through UAS-GAL4 was associated with a decrease in wing size but had no effect on thorax size. This was consistent with higher Dca expression levels in family lines with higher frequency of the Dca-237 allele. Genetic variation in the promoter region of the Dca gene appears to influence adaptive size variation in the eastern Australian cline of Drosophila melanogaster and accounts for more than 10% of the genetic variation in size within and between populations.

Lack of genetic structure among ecologically adapted populations of an Australian rainforest Drosophila species as indicated by microsatellite markers and mitochondrial DNA sequences.

Although fragmented rainforest environments represent hotspots for invertebrate biodiversity, few genetic studies have been conducted on rainforest invertebrates. Thus, it is not known if invertebrate species in rainforests are highly genetically fragmented, with the potential for populations to show divergent selection responses, or if there are low levels of gene flow sufficient to maintain genetic homogeneity among fragmented populations. Here we use microsatellite markers and DNA sequences from the mitochondrial ND5 locus to investigate genetic differences among Drosophila birchii populations from tropical rainforests in Queensland, Australia. As found in a previous study, mitochondrial DNA diversity was low with no evidence for population differentiation among rainforest fragments. The pattern of mitochondrial haplotype variation was consistent with D. birchii having undergone substantial past population growth. Levels of nuclear genetic variation were high in all populations while F(ST) values were very low, even for flies from geographically isolated areas of rainforest. No significant differentiation was observed between populations on either side of the Burdekin Gap (a long-term dry corridor), although there was evidence for higher gene diversity in low-latitude populations. Spatial autocorrelation coefficients were low and did not differ significantly from random, except for one locus which revealed a clinal-like pattern. Comparisons of microsatellite differentiation contrasted with previously established clinal patterns in quantitative traits in D. birchii, and indicate that the patterns in quantitative traits are likely to be due to selection. These results suggest moderate gene flow in D. birchii over large distances. Limited population structure in this species appears to be due to recent range expansions or cycles of local extinctions followed by recolonizations/expansions. Nevertheless, patterns of local adaptation have developed in D. birchii that may result in populations showing different selection responses when faced with environmental change.

Systematic relationships within the dasyurid marsupial tribe Sminthopsini--a multigene approach.

We report analyses of complete DNA sequences of the mitochondrial cytochrome b (1146 bp), 12S rRNA (974 bp), partial control region (371 bp) loci, and the nuclear protamine P1 (616 bp) gene from all but one species (Sminthopsis butleri) of the dasyurid marsupial tribe Sminthopsini, as well as several outgroups. Parsimony analyses of combined nuclear and mitochondrial data suggest that Antechinomys is sister to a clade consisting of Sminthopsis and Ningaui. Parsimony, maximum-likelihood, and mixed-model distance analyses consistently resolve several species groups within Sminthopsis. The Macroura group includes S. macroura, S. virginiae, S. douglasi, and S. bindi; S. butleri is also included here on the basis of partial 12S rRNA sequences. S. crassicaudata is resolved as sister to the Macroura clade. The Murina group includes S. murina, S. leucopus, S. gilberti, S. dolichura, and S. archeri. S. griseoventer and S. aitkeni are resolved as a clade, and there is moderate support for a group consisting of the genetically divergent species S. psammophila, S. hirtipes, S. youngsoni, and S. ooldea (possibly along with S. longicaudata and S. granulipes). Compositions of species groups are partially congruent with clades previously proposed on the basis of morphological data. Radiations within Sminthopsini appear to be coincident with major environmental changes since the mid-Miocene.