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And Semi-Landmark-Based Geometric Morphometric to Identify Four JOURNAL OF ADVANCED VETERINARY AND ANIMAL RESEARCH ISSN 2311-7710 (Electronic) http://doi.org/10.5455/javar.2019.f345 September 2019 A periodical of the Network for the Veterinarians of Bangladesh (BDvetNET) VOL 6, NO. 3, PAGES 278–283 ORIGINAL ARTICLE Effectiveness of landmark- and semi-landmark-based geometric morphometric to identify four species of Culex mosquitoes in Thailand Tanawat Chaiphongpachara1, Sedthapong Laojun2 1Department of Public Health and Health Promotion, College of Allied Health Science, Suan Sunandha Rajabhat University, Bangkok, Thailand 2Bachelor of Public Health, College of Allied Health Sciences, Suan Sunandha Rajabhat University, Samut Songkhram, Thailand ABSTRACT ARTICLE HISTORY Objective: This research aims to study the effectiveness of landmark- and semi-landmark-based Received January 11, 2019 geometric morphometric (LMSL) in the identification of four Culex vectors, namely, C. quinquefas- Revised January 30, 2019 ciatus, C. visnui, C. sitiens, and C. whitmorei in Thailand and also compared the potential between Accepted February16, 2019 LMSL and the landmark-based geometric morphometric method (LM). Published June 17, 2019 Materials and Methods: The overall size of the wing sample was estimated by the centroid KEYWORDS size. Shape variables were computed as principal components of the “partial warp” calculated after generalized procrustes analysis of raw coordinates. Discriminant analysis of the canonical Geometric morphometrics, Culex variables performed to explore the shape dissimilarity between Culex species has been shown quinquefasciatus, Culex visnui, Culex sitiens, Culex whitmorei as a factor map and to calculate the Mahalanobis distance. Size and shape differences based on pairwise Mahalanobis distances were tested using non-parametric methods (1,000 cycles) with Bonferroni correction at a p-value of <0.05. Results: A total of 120 individuals were used that were divided into 30 individuals per Culex species. The mean CS of C. sitiens had the largest wings followed by C. visnui, C. quinquefasciatus, and C. This is an Open Access article whitmorei in LM and LMSM. The patterns of statistical difference in CS of both methods were distributed under the terms of the similar and wing shapes among Culex species were different based on a comparison of pairwise Creative Commons Attribution 4.0 Mahalanobis distances (p < 0.05) in both methods. For the cross-validated reclassification test, LM Licence (http://creativecommons.org/ provided Culex species separation ranging from 54% to 84% and 51% to 93% for LMSM. licenses/by/4.0) Conclusion: Thus, LMSM is another option to use for the identification in mosquito vectors that have a curved line on the wing specific to the species. Introduction billion people are at risk of infection, while for lymphatic Mosquitoes are small insects belonging to the order Diptera, which are capable of transmitting pathogens to remain at risk of infection [4]. humans, such as Zika virus, chikungunya virus, yellow filariasis,Thailand 856 is millionone of thepeople countries in 52 facingcountries the problemworldwide of Culex-borne diseases, including Japanese encephalitis and nematode [1]. Culex mosquitoes are one of the medically - importantfever, dengue genera, fever, which Japanese have encephalitis members most virus, widespread and filarial to-borne diseases is to reduce the mosquito population in throughout the world [2]. Japanese encephalitis and lym- thelymphatic area filariasis.. There areOne many effective ways way for to mosquito control mosquicontrol, which must be suitably selected with the target species of have Culex species as vectors [3]. Twenty-four countries [5] phatic filariasis are important tropical diseases that often they have similar morphological characteristics in some endemic areas for Japanese encephalitis, and more than 3 species.vectors. It is often difficult to identify mosquito species as in the South-East Asia and Western Pacific regions are Correspondence Tanawat Chaiphongpachara [email protected] Department of Public Health and Health Promotion, Col- lege of Allied Health Science, Suan Sunandha Rajabhat University, Bangkok, Thailand. How to cite: Chaiphongpachara T, Laojun S. Effectiveness of landmark- and semi-landmark-based geometric morphometric to identify four species of Culex mosquitoes in Thailand. J Adv Vet Anim Res 2019; 6(3):278–283. http://bdvets.org/javar/ Chaiphongpachara and Laojun./ J. Adv. Vet. Anim. Res., 6(3): 278–283, September 2019 278 Geometric morphometrics (GM) is a powerful tool Wing preparation arthropods Thirty-three mosquitoes were selected randomly per Culex 7] species for analyses (a total of 120 individuals, including betweento use for species species to eggsidentification, of Triatominae especially , trombiculid 30 individuals of C. quinquefasciatus, 30 individuals of C. mites[6, . Previous[9] research[10] has, mosquitoes applied GM [11, for12] identification. Currently, [8] visnui, 30 individuals of C. sitiens, and 30 individuals of C. whitmorei). The right wing of each adult Culex mosquito particularly, tsetse a landmark-based flies method because their wing was removed from the thorax and mounted on a micro- ismosquito the perfect wing organ is considered for GM analysisto help identification 7]. Although by GMthe landmark-based GM method (LM) is an effective technique of identifying some mosquito species,[6, it is not perfect stereomicroscopescope slide under a(Nikon 0.08–0.12 Corp., mm Tokyo, coverslip Japan) using under Hoyer’s 40× medium. A digital camera connected to a Nikon SMZ745T The landmark- andbecause semi-landmark-based it is difficult to analyze GM method the curve, (LMSL) which is onemay GM be magnificationLandmark-based was method used to (LM) photograph and Landmark- wing images. and semi- techniquethe specific that location was developed of the target to improve species. the weaknesses landmark-based method (LMSL) of LM in curve position analysis. LMSL is a combination of landmark and semi-landmark for analysis [10]. However, In this study, we used two different technical GM methods, the researches that have applied LMSL are relatively few, namely, LM and LMSL, to compare the effectiveness of both which may affect the actual use in other areas. methods. Both methods are similar for analysis and calcu- Thus, this research aims to study the effectiveness of lation but different at anatomical points. For LM, 11 land- Culex vectors in Thailand, namely, C. quinquefasciatus, C. visnui, C. sitiens, and C. whit- LMSL, 11 landmarks (same positions landmarks in LM) moreiLMSL .in Culex the identification quinquefasciatus of four has been considered as the combinedmarks in Fig. with 1A 12 were semi-landmarks digitized in at each the curvedwing image. line of Forthe C. wing vein between landmarks 9 and 10 (Fig. 1B) were dig- visnui and C. sitiens are Japanese encephalitis virus vectors, index was calculated for whileJapanese C. whitmorei encephalitis virus and filarial nematode It also vector, com - evaluating the accuracy of landmarks and semi-landmarks pared the potential between LMSL and LM as a guideline ofitized. both After methods that, bythe comparison repeatability of repeat plots [14]. for further application. is a filarial nematode vector. the CS, which is the square root of the sum of the squared Materials and Methods distancesThe overall from sizethe centroid of the wing to each sample landmark was estimated , and CSby variations of Culex species were shown by quantile boxes. Sample collection Differences of mean CS among species of both[15] methods were compared using non-parametric methods (1,000 In this study, four species of female Culex mosquitoes, as important vectors in Thailand, were used, namely, C. quinquefasciatus, C. visnui, C. sitiens, and C. whitmorei. We collected three species, including C. quinquefascia- tus, C. visnui, and C. whitmorei from Nam Nak village (13°22 34.9E) in Ratchaburi Province, Thailand and Cx. sitiens from the coastal community area of Samut36.0 SongkhramN, 99°16 Province, Thailand (13°24’34.3”N, . Independent mosquito traps (Woodstream Corporation, 100°00’52.9”E) during August 2015 once a week takenUSA) werefrom usedtraps for and adult sent female to the collectionCollege of atAllied night Health (6.00 Sciences,PM–6.00 AM).Suan Every Sunandha morning Rajabhat (6.00 AM), University, mosquitoes Samut were Songkhram Provincial Education Center for species iden- morphologic characters using the illustrated keys to the mosquitoestification by in stereomicroscopicThailand [13]. observation based on Figure 1. Positions of landmarks and semi-landmarks. (A) 11 landmarks for LM analysis and (B) 11 landmarks combined with 12 semi-landmarks for LMSL analysis.. http://bdvets.org/javar/ Chaiphongpachara and Laojun./ J. Adv. Vet. Anim. Res., 6(3): 278–283, September 2019 279 - cance level was set at p cycles)Shape and variables relevant were Bonferroni computed correction. as principal The composignifi- < 0.05. procrustes analysis of raw coordinates. Discriminant anal- ysisnents of of the the canonical “partial variableswarp” calculated performed after to generalizedexplore the shape dissimilarity between Culex species has been shown as a factor map and to calculate the Mahalanobis distance. Shape differences based on pairwise Mahalanobis dis- tances were tested using non-parametric methods (1,000 cycles) with Bonferroni correction at a p accuracy of Culex -value of <0.05. Each wing individual was then reclassified to test the distance scores. Pheneticspecies
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