Elucidating the complex taxonomic status of the major malaria vector taxa and characterising the individual species within each complex is important for understanding the complexity of the vector system in the south-east Asian region and will allow to estimate the impact of vector control measures. This applies to countries such as Laos, Cambodia and Vietnam that spend about 60% of their malaria control budget on implementing vector control activities. We used isozyme electrophoresis to clarify the Anopheles minimus s.l. species composition in northern Vietnam and identify behavioural divergences of individual species. Using different collection methods, adult mosquitoes were caught at monthly intervals from June to November 1995 in four villages. An. minimus s.l. could be distinguished from closely related species, An. aconitus and An. jeyporiensis, at the Octanol dehydrogenase (Odh) enzyme locus. Significant positive Fis values gave clear evidence of nonrandom mating within the An. minimus s.l. population. The highest heterozygote deficiency was observed at locus Odh, which was diagnostic for 2 sympatric An. minimus species in Vietnam similar to the An. minimus A and C species known from Thailand. We found no evidence for restricted gene flow between monthly samples, villages, or collection methods in either of the two An. minimus species. They occurred in sympatry, but in different proportions depending on the collection site, and had dissimilar resting and biting behaviours. Thus a vector control strategy will have a nonuniform effect on the various components of this diverse vector system.

The feasibility of identifying parasite DNA and specific mRNAs from wild caught Anopheles dirus mosquitoes was assessed using dried mosquito salivary glands preserved on filter paper. We were able to detect Plasmodium falciparum, Plasmodium vivax This study also shows that the preservation of mosquito salivary glands on filter paper, and the down-stream extraction of parasite DNA and RNA from those, offers a powerful resource for molecular epidemiological studies on malaria. Keywords: Salivary glands, Plasmodium knowlesi, PCR, RT-PCRMalaria transmission occurs in the forested areas in the southern and central provinces of Vietnam (Erhart et al., 2004) and constitutes a considerable public health problem in these areas. In order to fully understand the complexities of malaria transmission, it is important to identify and characterize malaria parasites in mosquito vectors. Evidence for the generation of genetic diversity during sexual proliferation in mosquito vectors has previously been accumulated using laboratory isolates of genetically diverse parasites, as well as with samples from field settings (Babiker et al., 1994; Menegon et al., 2000 knowlesi, were spotted onto chromatography-grade filter papers (ET31CHR;Whatman, Maidstone, UK). Each blood-spotted filter paper was immediately air-dried and stored in a sealed plastic bag at room temperature until RT-PCR or PCR analysis was performed. To examine the detection limit for mosquito salivary gland-extracted parasite mRNA, salivary glands from laboratory reared Anopheles stephensi were prepared in the same way as were the wild-caughtAn. dirus. Salivary glands were dissected from laboratory colony reared An.stephensi more than 1 week after blood feeding on either mouse or human blood and dried on E31CHR filter paper at room temperature. Gametocyte suspensions were obtained from cultured 3D7-9A by pyrimethamine treatment at 10 -6 M from day 7 after thawing until harvest on day 12. Ten times serially diluted cultured gametocyte suspension was added to the spot on the filter paper where the salivary glands were attached. Extraction of RNA and reverse transcription was carried out as previously described (Maeno et al., 2003). Briefly, dried spotted filter paper was cut into small pieces and total RNA and genomic DNA (gDNA) was extracted with ISOGEN (Nippon gene, Tokyo, Japan) according to the manufacturer's instructions. The extracted total RNA was transcribed to synthesize cDNA which was subsequently subjected to PCR using specific The detection limit of parasite mRNA by RT-PCR was one gametocyte per salivary gland for pfg377 mRNA and 10 gametocytes per gland for pfs16 mRNA (Fig. 1A). We extracted both parasite gDNA and mRNA from the salivary glands of wild caught mosquitoes. Region 3 of pfg377 mRNA was detected in all of the three dried salivary gland samples and both the mRNA and gDNA of pfg377 were detected in two samples (Fig. 1B). The PCR products showed the same molecular size as the RT-PCR products by electrophoresis. No PCR product was observed by ...

Anopheles sundaicus species A of the Southeast Asian A. sundaicus complex is formally named Anopheles epiroticus Linton & Harbach based on DNA sequence differentiation of the whole nuclear ITS2 region and a portion of both the cytochrome b and cytochrome c oxidase I mitochondrial genes. Detailed comparative morphological studies of the adult, larval and pupal stages did not reveal any differential or diagnostic differences that reliably distinguish A. epiroticus from A. sundaicus s.s. Information is provided on the bionomics and systematics of the new species.

The nomenclatural identity of species C of the Anopheles minimus complex is resolved by excluding the available junior synonyms of the nominotypical member of the complex and naming it An. harrisoni Harbach & Manguin, sp. n. Anopheles formosaensis I Tsuzuki, An. christophersi Theobald and An. christophersi var. alboapicalis Theobald are retained as junior synonyms of An. minimus Theobald based on the provenance of type specimens in geographical areas where An. harrisoni is not known to occur. A lectotype is designated for An. vincenti Laveran, which thus becomes the senior name of the specific entity known as An. jeyporiensis James. Molecular data that diagnose An. harrisoni are reviewed and the holotype female is contrasted with the neotype series of An. minimus. Available information on the bionomics and distribution of the new species is included.

BackgroundReduced artemisinin susceptibility and artemisinin-based combination therapy (ACT)-resistance against Plasmodium falciparum and chloroquine (CQ)-resistant P. vivax malaria has been reported in Vietnam. Two therapeutic efficacy studies were conducted in Thuan Bac District (Ninh Thuan Province, Vietnam) in 2015 and 2016 to determine the extent of reduced artemisinin susceptibility and ACT resistant falciparum malaria, and CQ-resistant vivax malaria were present.MethodsTwenty-seven patients with falciparum malaria were randomized to receive artesunate alone (AS ~ 4 mg/kg/day) for 4 days followed by dihydroartemisinin (DHA) (2.2 mg/kg)–piperaquine (PPQ) (18 mg/kg) daily for 3 days or artemether (AM) (1.7 mg/kg)–lumefantrine (LUM) (12 mg/kg) twice daily for 3 days. Sixteen subjects with vivax malaria received CQ (total 25 mg/kg over 3 days). The therapeutic efficacy study for treating falciparum malaria was complemented with molecular analysis for artemisinin and piperaquine resistance, and in vitro drug susceptibility testing. Patient’s drug exposure following both falciparum and vivax treatment studies was determined.ResultsTwenty-five of 27 patients treated with the artemisinin regimens completed the 42-day follow-up period. None had parasites present on day 3 after commencing treatment with no incidence of recrudescence (100% curative rate). One patient on AS + DHA–PPQ was lost to follow-up and one patient had Plasmodium falciparum and Plasmodium vivax infection on day 0 by PCR. Of the vivax patients, 15 of 16 completed CQ treatment and two had a recurrence of vivax malaria on day 28, a failure rate of 13.3% (2/15). No mutations in the Pfkelch-13 gene for artemisinin resistance or exo-E415G gene polymorphism and amplification in plasmepsins 2 and 3 for piperaquine resistance were observed. In vitro testing of patient’s falciparum parasites indicated susceptibility (low IC50 nM values) to dihydroartemisinin, lumefantrine, piperaquine and pyronaridine. Patient’s drug exposure to artesunate and lumefantrine was comparable to published data, however, blood CQ concentrations were lower.ConclusionsClinical findings, molecular analysis and in vitro testing revealed that the falciparum parasites at Phuoc Chien Commune were artemisinin susceptible. The clinical failure rate of the 15 vivax patients who completed CQ treatment was 13%. Further studies are required to determine whether CQ-resistant vivax malaria is present at the commune.Electronic supplementary materialThe online version of this article (10.1186/s12936-019-2640-2) contains supplementary material, which is available to authorized users.