Mitochondrial DNA barcoding of mosquito species (Diptera: Culicidae) in Thailand

Abstract

The correct identification of mosquito species is important for effective mosquito vector control. However, the standard morphological identification of mosquito species based on the available keys is not easy with specimens in the field due to missing or damaged morphological features during mosquito collections, often leading to the misidentification of morphologically indistinguishable. To resolve this problem, we collected mosquito species across Thailand to gather genetic information, and evaluated the DNA barcoding efficacy for mosquito species identification in Thailand. A total of 310 mosquito samples, representing 73 mosquito species, were amplified using mitochondrial cytochrome c oxidase subunit I (COI) primers. The average maximum intraspecific genetic variation of the 73 mosquito species was 1% ranged from 0–5.7%. While, average minimum interspecific genetic variation (the distance to the nearest neighbour) of the 73 mosquito species was 7% ranged from 0.3–12.9%. The identification of success rates based on the “Best Match,” “Best Close Match,” and “All Species Barcodes” methods were 97.7%, 91.6%, and 81%, respectively. Phylogenetic analyses of Anopheles COI sequences demonstrated a clear separation between almost all species (except for those between An. baimaii and An. dirus), with high bootstrap support values (97%–99%). Furthermore, phylogenetic analyses revealed potential sibling species of An. annularis, An. tessellatus, and An. subpictus in Thailand. Our results indicated that DNA barcoding is an effective molecular approach for the accurate identification of mosquitoes in Thailand.

Evaluation of Modern Techniques for Species Identification of Lutzia Mosquitoes (Diptera: Culicidae) in Thailand: Geometric Morphometrics and DNA Barcoding

“…Due to their low interspecific differences, it is impossible to distinguish between Lt. fuscana and Lt. halifaxi . The barcoding gap is a hiatus of difference between the greatest intraspecific genetic distance and the smallest interspecific distance, which is very important in determining the success of DNA barcoding [ 3 ]. Our assessment of genetic divergences revealed that this gap was not present between Lt. fuscana and Lt. halifaxii , indicating that the DNA barcoding method could not discriminate between the two species.…”

“…The total genomic DNA of the mosquitoes was extracted from 4 to 6 legs of each adult Lutzia specimen, using FavorPrep™ Mini Kits (Favorgen Biotech, Ping-Tung, Taiwan), following the manufacturer’s guidelines. Both universal barcode primers, including forward (5′-GGA TTT GGA AAT TGA TTA GTT CCT T-3′) and reverse (5′-AAA AAT TTT AAT TCC AGT TGG AAC AGC-3′) primers [ 38 ], were used to amplify a 709-bp fragment of COI ; the polymerase chain reaction (PCR) reaction mixture and PCR conditions were as described in a previous study [ 3 ]. Negative (water without DNA) and positive controls (DNA of Lutzia mosquitoes) were included in each PCR.…”

“…This genus consists of nine formally recognized species divided into three subgenera, including Insulalutzia (one species: Lt. shinonagai ), Lutzia (two species: Lt. allostigma and Lt. bigoti ), and Metalutzia (six species: Lt. agranensis , Lt. chiangmaiensi , Lt. fuscana , Lt. halifaxii , Lt. tigripes , and Lt. vorax ) [ 1 ]. Recently, phylogenetic analyses based on DNA sequence data on the first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) and the mitochondrial cytochrome c oxidase I (mtDNA- COI ) revealed that the Lutzia clade is classified as a species of the subgenera of Culex [ 2 , 3 ]. However, Lutzia is currently classified as a distinct genus from the genus Culex based on its fundamentally distinctive morphological and biological identity [ 1 ].…”

“…DNA barcoding is a recognized effective choice in supporting and confirming the results of GM [ 31 , 32 , 33 ]. In addition, DNA barcoding sequences of several mosquito species were recently collected across Thailand, which could be used to better confirm the results of GM [ 3 ].…”

Species Identification of the Major Japanese Encephalitis Vectors within the Culex vishnui Subgroup (Diptera: Culicidae) in Thailand Using Geometric Morphometrics and DNA Barcoding

“…However, this method is limited by the need for complete morphological specimens and practitioner expertise [ 10 ]. Currently, effective alternative methods for species identification include geometric morphometrics and DNA barcoding; both may support the standard method to accurately identify mosquito samples that are morphologically similar [ 16 , 17 , 18 , 19 , 20 , 21 , 22 ].…”