BackgroundTaxonomy that utilizes morphological characteristics has been the gold standard method to identify mosquito species. However, morphological identification is challenging when the expertise is limited and external characters are damaged because of improper specimen handling. Therefore, we explored the applicability of mitochondrial cytochrome C oxidase subunit 1 (COI) gene-based DNA barcoding as an alternative tool to identify mosquito species. In the present study, we compared the morphological identification of mosquito specimens with their differentiation based on COI barcode, in order to establish a more reliable identification system for mosquito species found in Singapore.MethodsWe analysed 128 adult mosquito specimens, belonging to 45 species of 13 genera. Phylogenetic trees were constructed for Aedes, Anopheles, Culex and other genera of mosquitoes and the distinctive clustering of different species was compared with their taxonomic identity.ResultsThe COI-based DNA barcoding achieved a 100% success rate in identifying the mosquito species. We also report COI barcode sequences of 16 mosquito species which were not available previously in sequence databases.ConclusionsOur study utilised for the first time DNA barcoding to identify mosquito species in Singapore. COI-based DNA barcoding is a useful tool to complement taxonomy-based identification of mosquito species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-014-0569-4) contains supplementary material, which is available to authorized users.

Background Genetic markers are employed widely in molecular studies, and their utility depends on the degree of sequence variation, which dictates the type of application for which they are suited. Consequently, the suitability of a genetic marker for any specific application is complicated by its properties and usage across studies. To provide a yardstick for future users, in this study we assess the suitability of genetic markers for molecular systematics and species identification in helminths and provide an estimate of the cut-off genetic distances per taxonomic level. Methods We assessed four classes of genetic markers, namely nuclear ribosomal internal transcribed spacers, nuclear rRNA, mitochondrial rRNA and mitochondrial protein-coding genes, based on certain properties that are important for species identification and molecular systematics. For molecular identification, these properties are inter-species sequence variation; length of reference sequences; easy alignment of sequences; and easy to design universal primers. For molecular systematics, the properties are: average genetic distance from order/suborder to species level; the number of monophyletic clades at the order/suborder level; length of reference sequences; easy alignment of sequences; easy to design universal primers; and absence of nucleotide substitution saturation. Estimation of the cut-off genetic distances was performed using the ‘K-means’ clustering algorithm. Results The nuclear rRNA genes exhibited the lowest sequence variation, whereas the mitochondrial genes exhibited relatively higher variation across the three groups of helminths. Also, the nuclear and mitochondrial rRNA genes were the best possible genetic markers for helminth molecular systematics, whereas the mitochondrial protein-coding and rRNA genes were suitable for molecular identification. We also revealed that a general gauge of genetic distances might not be adequate, using evidence from the wide range of genetic distances among nematodes. Conclusion This study assessed the suitability of DNA genetic markers for application in molecular systematics and molecular identification of helminths. We provide a novel way of analyzing genetic distances to generate suitable cut-off values for each taxonomic level using the ‘K-means’ clustering algorithm. The estimated cut-off genetic distance values, together with the summary of the utility and limitations of each class of genetic markers, are useful information that can benefit researchers conducting molecular studies on helminths.

Background: Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics. Methods: To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results: The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions: This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

BackgroundThe mosquito Aedes aegypti (L.) is a major vector of viral diseases like dengue fever, Zika and chikungunya. Aedes aegypti exhibits high morphological and behavioral variation, some of which is thought to be of epidemiological significance. Globally distributed domestic Ae. aegypti have often been grouped into (i) the very pale variety queenslandensis and (ii) the type form. Because the two color forms co-occur across most of their range, there is interest in understanding how freely they interbreed. This knowledge is particularly important for control strategies that rely on mating compatibilities between the release and target mosquitoes, such as Wolbachia releases and SIT. To address this question, we analyzed nuclear and mitochondrial genome-wide variation in the co-occurring pale and type Ae. aegypti from northern Queensland (Australia) and Singapore.Methods/FindingsWe typed 74 individuals at a 1170 bp-long mitochondrial sequence and at 16,569 nuclear SNPs using a customized double-digest RAD sequencing. 11/29 genotyped individuals from Singapore and 11/45 from Queensland were identified as var. queenslandensis based on the diagnostic scaling patterns. We found 24 different mitochondrial haplotypes, seven of which were shared between the two forms. Multivariate genetic clustering based on nuclear SNPs corresponded to individuals’ geographic location, not their color. Several family groups consisted of both forms and three queenslandensis individuals were Wolbachia infected, indicating previous breeding with the type form which has been used to introduce Wolbachia into Ae. aegypti populations.ConclusionAedes aegypti queenslandensis are genomically indistinguishable from the type form, which points to these forms freely interbreeding at least in Australia and Singapore. Based on our findings, it is unlikely that the presence of very pale Ae. aegypti will affect the success of Aedes control programs based on Wolbachia-infected, sterile or RIDL mosquitoes.

Background Genetic markers like the nuclear ribosomal RNA (rRNA) genes, internal transcribed spacer regions, mitochondrial protein-coding genes, and genomes have been utilized for molecular identification of parasitic trematodes. However, challenges such as the design of broadly applicable primers for the vast number of species within Digenea and the genetic markers’ ability to provide sufficient species-level resolution limited their utility. This study presented novel and broadly applicable primers using the mitochondrial 12S and 16S rRNA genes for Digenea and aimed to show their suitability as alternative genetic markers for molecular identification of orders Plagiorchiida, Echinostomida, and Strigeida. Results Our results revealed that the mitochondrial 12S and 16S rRNA genes are suitable for trematode molecular identification, with sufficient resolution to discriminate closely related species and achieve accurate species identification through phylogenetic placements. Moreover, the robustness of our newly designed primers to amplify medically important parasitic trematodes encompassing three orders was demonstrated through successful amplification. The convenience and applicability of the newly designed primers and adequate genetic variation of the mitochondrial rRNA genes can be useful as complementary markers for trematode molecular-based studies. Conclusions We demonstrated that the mitochondrial rRNA genes could be alternative genetic markers robust for trematode molecular identification and potentially helpful for DNA barcoding where our primers can be widely applied across the major Digenea orders. Furthermore, the potential of the mitochondrial rRNA genes for molecular systematics can be explored, enhancing their appeal for trematode molecular-based studies. The novelty of utilizing the mitochondrial rRNA genes and the designed primers in this study can potentially open avenues for species identification, discovery, and systematics in the future.

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Background Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics. Methods To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.