Geographical Variation of the Oriental Fruit Fly, Bactrocera dorsalis, Occurring in Taiwan

오리엔탈과실파리 유전변이 - 대만 지역 집단변이

Abstract

This study analyzed genetic variation of the Oriental fruit fly (OFF), Bactrocera dorsalis, which is designated to be a quarantine insect pest in Korea. OFF samples endemic to Taiwan were collected at three different locations (Taipei, Taichung, and Kaohsiung) for three days from July 30 to August 1 in 2018 and assessed in their age and mitochondrial DNA sequence variations. In these places, 1,085 OFF males were collected using methyl eugenol lure while 30 males of Zeugodacus cucurbitae and one male of Bactrocera tau were collected using Cuelure. A protein diet lure attracted 6 flies including one OFF and 5 flies of Z. cucurbitae. Male heads of OFF contained pterin, which increased in contents with age from 32 to $59{\mu}g/head$. There was a local variation in pterin amounts in OFF heads, in which Kaohsiung population had lower amounts of pterin than Taipei and Taichung populations. Genetic distance among these three populations were measured by random amplified polymorphic DNA and showed that Taipei population was separated from Taichung/Kaohsiung cluster. Genetic variation was also analyzed in sequence variations in cytochrome oxidase I (CO-I) and NADH dehydrogenase I (ND-I). There was 7.8% variation in CO-I sequence (360 residues) and 6.6% variation in ND-I sequence (213 residues). These polymorphic sites are proposed to be used to develop SNP (single nucleotide polymorphism) markers characteristic to Taiwan OFF populations.

본 연구는 국내 금지급 과실파리인 오리엔탈과실파리(Bactrocera dorsalis)에 대한 유전적 변이를 분석하였다. 이를 위해 오리엔탈과실파리가 자생하는 대만 지역을 대상으로 2019년 동일한 시기(3일간: 7월 30일~8월 1일)에 서로 다른 세 지역(타이페이, 타이중, 카오슝)에서 과실파리류를 채집하여 나이 변이 및 미토콘드리아 서열 변이를 각각 비교하였다. 세 지역에서 채집된 오리엔탈과실파리는 1,085마리로서 메틸유제놀 유인제에 모두 유인되었으며, 큐루어 유인제에는 30마리의 오이과실파리(Zeugodacus cucurbitae) 및 1마리의 타우과실파리(Bactrocera tau)만 채집되었다. 단백질먹이 유인제에는 총 6마리가 포획되었으며 이 가운데 오리엔탈과실파리는 1마리가 포함되었으며 나머지는 오이과실파리였다. 오리엔탈과실파리 수컷의 머리에는 테린이 포함되었으며 나이가 증가함에 따라 각 머리에는 $32{\mu}g$에서 $59{\mu}g$까지 테린 함량이 증가하였다. 대만 세 지역의 수컷 집단들은 테린 양에 차이를 나타냈으며, 카오슝 집단이 타이페이와 타이중 집단에 비해 적은 테린 양을 보유하였다. 이들 세 지역 사이에 유전적 거리가 RAPD (random amplified polymorphic DNA)를 이용하여 분석되었으며 타이페이 집단이 타이중 및 카오슝 집단들과 차이를 나타내는 것으로 나타났다. 유전적 변이는 미토콘드리아의 cytochrome oxidase I (CO-I)과 NADH dehydrogenase I (ND-I)을 각각 비교하였다. CO-I 영역 가운데 360개 염기서열을 비교한 결과 7.8%의 염기서열 변이를 나타냈다. ND-I 영역을 비교한 결과 213개 염기서열 가운데 6.6%의 염기서열 변이를 보였다. 이들 변이 서열을 대만 지역에 발생하는 오리엔탈과실파리의 특이적 SNP (single nucleotide polymorphism) 마커로 개발하는 데 추천한다.

Fig. 1. B. dorsalis larvae infesting wax apple (Syzygium samarangense) in National Taiwan University. (A) A damaged fruit containing larvae (B) Anterior morphology showing mouth part (C) Posterior morphology showing spiracles (D) CO-I sequence and Blast search.

Fig. 2. Age variation of B. dorsalis male adults among different places of Taiwan. (A) Head pterin amounts in different ages of B. dorsalis adults reared at 25℃. (B) Pterin amounts in three different locations in Taiwan. In each locality, 30 male heads were analyzed. Different letters above standard error bars indicate significant different among at type I error = 0.05 (LSD test).

Fig. 3. Genetic distance analysis among three local populations of B. dorsalis using RAPD. Their hierarchical clustering using 8041 (A) and 8011 (B) RAPD markers.

Fig. 4. Phylogenetic analysis of three local populations of B. dorsalis using CO-I (A) and ND-I sequences. Three locations include 6 fly samples of Taipei (TP1-TP6), Taichung (TC1-TC6), and Kaohsiung (TK1-TK6). All sequences were aligned with BioEdit 7.2 program and trimmed off. The processed sequences were phylogenetically analyzed with Neighbor-joining method using MEGA 6.0 program (Tamura et al., 2013). Figures at tree nodes indicate bootstrap values obtained after 1,000 repetitions.

Fig. 5. Sequence alignments of CO-I (A) and ND-I (B) of B. dorsalis collected from three locations in Taiwan: Taipei (TP), Taichung (TC), and Kaohsiung (TK). Alignment was performed by two steps. At first step, 6 individual DNA samples were aligned to produce a conserved sequence using Clustal W program of DNAStar-SeqMag. In next step, three conserved sequences were aligned. Arrows indicate hot spots containing polymorphic sequences at least two locations.

Table 1. Statistic of fruit fly collection from July 30 to Aug 1 in Taiwan

Table 2. ANOVA of pterin amounts in B. dorsalis heads

Table 3. Polymorphic sites in 360 nucleotides of CO-I of B. dorsalis in three Taiwan populations: Taipei, Taichung, and Kaohsiung

Table 4. Polymorphic sites in 213 nucleotides of ND-I of B. dorsalis in three Taiwan populations: Taipei, Taichung, and Kaohsiung