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DNA identification of species of the Anopheles maculipennis complex and first record of An. daciae in Belgium.

ABSTRACT: The present study aimed at identifying the members of the Anopheles maculipennis complex (Diptera: Culicidae) occurring in Belgium. Therefore, the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) loci were sequenced in 175 and 111 specimens, respectively, collected between 2007 and 2019. In parallel, the suitability of two species-diagnostic PCR-RFLP assays was tested. The identified specimens included: An. maculipennis s.s. (N = 105), An. daciae (N = 62), An. atroparvus (N = 6) and An. messeae (N = 2). Each species was characterized by unique ITS2 haplotypes, whereas COI only supported the monophyly of An. atroparvus, a historical malaria vector in Belgium. Species identification results were further supported by unique PCR-RFLP banding patterns. We report for the first time An. daciae in Belgium, where it was found to co-occur with An. maculipennis s.s. The latter was the most prevalent in the collection studied (60%) and appears to have the widest distribution in Belgium. As in other studies, An. daciae and An. messeae appeared the most closely related species, up to the point that their species status remains debatable, while their ecological differences, including vector competences, need further study.

SUBMITTER: Smitz N

PROVIDER: S-EPMC8453948 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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DNA identification of species of the Anopheles maculipennis complex and first record of An. daciae in Belgium.

Smitz N N De Wolf K K Gheysen A A Deblauwe I I Vanslembrouck A A Meganck K K De Witte J J Schneider A A Verlé I I Dekoninck W W Gombeer S S Vanderheyden A A De Meyer M M Backeljau T T Müller R R Van Bortel W W

Medical and veterinary entomology 20210505 3

The present study aimed at identifying the members of the Anopheles maculipennis complex (Diptera: Culicidae) occurring in Belgium. Therefore, the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) loci were sequenced in 175 and 111 specimens, respectively, collected between 2007 and 2019. In parallel, the suitability of two species-diagnostic PCR-RFLP assays was tested. The identified specimens included: An. maculipennis s ...[more]

PMID: 33951205

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Project description:BackgroundDetermining the host feeding patterns of mosquitoes by identifying the origin of their blood-meals is an important part of understanding the role of vector species in current and future disease transmission cycles. Collecting large numbers of blood-fed mosquitoes from the field is difficult, therefore it is important to maximise the information obtained from each specimen. This study aimed to use mosquito genome sequence to identify the species within Anopheles maculipennis sensu lato (An. maculipennis s.l.), identify the vertebrate hosts of field-caught blood-fed An. maculipennis s.l. , and to test for the presence of myxoma virus (Poxviridae, genus Leporipoxvirus) in specimens found to have fed on the European rabbit (Oryctolagus cuniculus).MethodsBlood-fed An. maculipennis s.l. were collected from resting sites at Elmley Nature Reserve, Kent, between June and September 2013. Hosts that An. maculipennis s.l. had fed on were determined by a PCR-sequencing approach based on the partial amplification of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes were then identified to species by sequencing a region of the internal transcribed spacer-2. DNA extracts from all mosquitoes identified as having fed on rabbits were subsequently screened using PCR for the presence of myxoma virus.ResultsA total of 94 blood-fed Anopheles maculipennis s.l. were collected, of which 43 (46%) provided positive blood-meal identification results. Thirty-six of these specimens were identified as Anopheles atroparvus, which had fed on rabbit (n = 33, 92%) and cattle (n = 3, 8%). Seven mosquitoes were identified as Anopheles messeae, which had fed on cattle (n = 6, 86%) and dog (n = 1, 14%). Of the 33 An. atroparvus that contained rabbit blood, nine (27%) were positive for myxoma virus.ConclusionsResults demonstrate that a single DNA extract from a blood-fed mosquito can be successfully used for molecular identification of members of the An. maculipennis complex, blood-meal identification, and for the targeted detection of a myxoma virus. This study shows that An. atroparvus has a strong feeding preference for both healthy and myxoma-infected rabbits, providing evidence that this species may play a significant role in the transmission of myxomatosis among wild rabbit populations in the United Kingdom (UK).

Project description:BackgroundThe Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex.MethodsThe AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex.ResultsDespite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers.ConclusionsThe DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS.

Dataset Information

DNA identification of species of the Anopheles maculipennis complex and first record of An. daciae in Belgium.

Publications

DNA identification of species of the Anopheles maculipennis complex and first record of An. daciae in Belgium.

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