Journal of Asia-Pacific Entomology 20 (2017) 935–941 Contents lists available at ScienceDirect Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape Genetic diversity and spread of Lissorhoptrus oryzophilus (Coleoptera: MARK Curculionidae) in China, based on amplified fragment length polymorphism ⁎ Shang-Wei Lia, Fang-Chao Weia, Juan Dua, Mao-Fa Yanga,b, a Provincial Key Laboratory for Agricultural Pest Management of Mountainous Region, Institute of Entomology, Guizhou University, Guiyang, Guizhou 550025, China b College of Tobacco Science, Guizhou University, Guiyang, Guizhou 550025, China ARTICLE INFO ABSTRACT Keywords: The rice water weevil (RWW), Lissorhoptrus oryzophilus Kuschel, is one of the most destructive pests causing AFLP heavy rice yield loss worldwide. In the present study, amplified fragment length polymorphism (AFLP) was used Genetic differentiation to analyze the genetic differentiation and transmission routes of 26 RWW geographical populations from Lissorhoptrus oryzophilus Guizhou and 6 other provinces in China. The genetic distance between the 26 populations ranged from 0 to Rice pest 0.693; the genetic distance between Guizhou and 6 other provinces populations varied from 0.043 to 0.693, with Spread pathway an average of 0.382. There was higher genetic variation among the populations from Guizhou and 6 other provinces compared with populations within Guizhou. The Mantel test showed no correlation between the ge- netic and geographical distances of the 26 populations. According to genetic distance, clustering, and time that RWW was found in these regions, we deduced that the pest entered Guizhou and spread in this province most likely via three routes: (1) from Hebei to Zunyi and southward to the south-central regions; (2) from Sichuan to Bijie and eastward to the central regions; and (3) from Yunnan through Anshun to the central regions and Qiandongnan prefecture. RWW spread in a natural manner, including flight, crawling, swimming, and by human-associated mechanisms, comprising dissemination through paddies and hay and hitchhiking on human transportations. The rice water weevil (RWW), Lissorhoptrus oryzophilus Kuschel identified as parthenogenic L. oryzophilus (Liu, 2010). In 2013, RWW (Coleoptera: Curculionidae), native to the Mississippi River Basin in the infested 24,140 ha of paddies in 971 villages, 125 towns, 26 counties United States, is now one of the most destructive rice insect pests in (districts, county-level cities), and 7 prefecture cities in Guizhou, China and is thus considered as a quarantine pest. The weevil was in- causing significant rice yield losses. L. oryziophlus is spread through itially recognized as a rice pest in Georgia in the early 1880s (Chen parthenogenesis, flight, swimming, and hitchhiking on human trans- et al., 2005; Isely and Schwardt, 1934). In the late 1950s, partheno- portations (Chen et al., 2005). genic RWW was observed in Northern California (Bowling, 1964; Lange L. oryzophilus is a semi-aquatic insect that undergoes complete and Grigarick, 1959). In 1976, parthenogenic RWW was detected in a metamorphosis, passing through four distinct developmental stages, paddy field in Aichi prefecture, Japan, and this pest was assumed to i.e., egg, larva, pupa, and adult. Adult weevils are omnivorous pests hitchhike with hay from California into Asia (Japan Plant Protection affecting many host plants, including 64 species in 10 families in China, Association, 1986; Saito et al., 2005). Subsequently, RWW spread to the and the larvae feed on 15 species in 5 families (Chen et al., 2005; Sun Korean peninsula and mainland China in 1988 and to Taiwan in 1990 et al., 1996). However, these weevils primarily feed on Gramineae and (Hirao, 1988; Shih, 1991; Sun et al., 1996). RWW was first detected in Cyperaceae, particularly rice plants (Lupi et al., 2009). Rice plants are Tanghai county, Hebei province, in May 1988 (Nagata, 1990; Sun et al., infected with both larval and adult RWW. Adult weevils chew the leaf 1996). Since then, RWW has expanded to most parts of China, such as epidermis, leaving skeletonized longitudinal scars on the upper surface the provinces of Liaoning, Shandong, Zhejiang, Fujian, Anhui, Hunan, of the rice leaves. Most economic damage occurs through the larvae, as Jiangxi, and Guizhou. It is likely that the weevil is currently distributed these organisms feed in or on rice roots, resulting in root pruning. through mainland China, except for Xinjiang and Tibet. In May 2010, Larval feeding and root pruning decrease nutrient absorption, tillering, the Pingba county farmers in Guizhou province reported weevil in- and grain number and weight. Infested plants often show yellowing, festations to the Agriculture Committee; subsequently, the weevils were stunting, and thinning and are more susceptible to lodging, which ⁎ Corresponding author at: Guizhou University, Huaxi District, Guiyang 550025, Guizhou, China. E-mail address: [email protected] (M.-F. Yang). http://dx.doi.org/10.1016/j.aspen.2017.06.012 Received 13 February 2017; Received in revised form 6 June 2017; Accepted 22 June 2017 Available online 24 June 2017 1226-8615/ © 2017 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved. S.-W. Li et al. Journal of Asia-Pacific Entomology 20 (2017) 935–941 delays maturity and reduces yield. Yield losses are higher in flooded Table 1 rice paddies because these rice plants are more prone to oviposition and Collection sites of rice water weevils, including geographical coordinates. infestation (Stout et al., 2002). Generally, RWW causes rice yield losses – Sampling time Locations (County/District/City, Longitudes Latitudes of approximately 15% 20%, a severe reduction in yield of more than Province) 50%, or even total crop failure (Ding, 2002; Shang and Zhang, 2004; Yu et al., 2006). RWW primarily reproduces sexually in the USA, whereas 5/2011 Xifeng, Guizhou 106.69E 27.09N in China, only females are present and reproduce by parthenogenesis. 5/2011 Huaxi, Guizhou 106.53E 26.43N 5/2011 Pingba, Guizhou 106.32E 26.34N Two generations of RWW are commonly observed per year in the USA, 5/2011 Jinyang, Guizhou 106.56E 26.64N while in China, the number of generations varies among different rice- 5/2011 Baiyun, Guizhou 106.62E 26.71N growing regions. It is difficult to prevent and control the occurrence of 5/2011 Xiuwen, Guizhou 106.63E 26.86N RWW due to the rapid spread, wide range of survival, high fecundity, 5/2011 Puding, Huizhou 105.86E 26.31N 5/2011 Xixiu, Guizhou 105.92E 26.30N and parthenogenesis of these pests. Currently the RWW population is 5/2011 Liuzhi, Guizhou 105.67E 26.15N controlled using the integrated approaches involving plant quarantine, 5/2011 Changshun, Guizhou 106.27E 26.22N insecticides, water bodies management, and light and bait trapping; 5/2011 Nanming, Guizhou 106.81E 26.50N however, the application of insecticides remains the primary method 5/2011 Longli, Guizhou 106.86E 26.51N for controlling the weevils in China (Zhang et al., 2006). Therefore, it is 5/2011 Kaiyang,Guizhou 106.93E 27.03N 5/2011 Qingzhen, Guizhou 106.44E 26.55N imperative to utilize biological control and cultivate rice varieties with 5/2011 Guiding, Guizhou 107.16E 26.48N high resistance to RWW. 5/2011 Fuquan, Guizhou 107.41E 26.58N Amplified fragment length polymorphism (AFLP) is a DNA finger- 5/2011 Zhijing, Guizhou 105.91E 26.54N printing technique that facilitates the scanning of multiple loci across 5/2011 Qianxi, Guizhou 105.79E 26.78N the entire genome to identify polymorphism-derived point mutations, 6/2012 Suiyang, Guizhou 107.04E 27.85N 6/2012 Tongzi, Guizhou 106.85E 28.30N insertions, deletions, and rearrangements (Vos et al., 1995). This 5/2011 Hengshan, Hunan 112.58E 27.38N technique involves three major steps: preparing the genomic DNA as 5/2011 Longchang, Sichuan 105.22E 29.39N the AFLP template; amplifying the restriction fragments; and analyzing 6/2012 Songming, Yunnan 103.12E 25.31N the gel for the amplified fragments (Vuylsteke et al., 2007). AFLP 7/2011 Tanghai, Hebei 118.46E 39.27N 6/2008 Xiangshan, Zhejiang 121.87E 29.46N combines the advantages of both restriction fragment length poly- 4/2011 Leping, Jiangxi 117.17E 28.86N morphism (RFLP) and random amplified polymorphic DNA (RAPD) with rapidness, rich polymorphisms, good reproducibility, and reliable results. In addition, AFLP technology does not require any previous NanoDrop 2000 spectrophotometer (ThermoScientific, Wilmington, knowledge of the genome sequence (Chial, 2008; Vuylsteke et al., DE) and agarose gel electrophoresis. 2007). AFLP analysis is widely used for genetic map construction, gene mapping, germplasm resource identification, molecular phylogeny, and population genetics. This technique has also been broadly applied for AFLP protocol entomological research (Kneeland et al., 2013; Lall et al., 2010; Watanabe et al., 2014). Yang (2008) examined the genetic poly- AFLP was performed according to Vos et al. (1995), with some morphisms of various geographical RWW populations using AFLP modifications. Restriction digestion and ligation steps were performed analysis, and the results showed that the Italian population was more in a 20-μL reaction containing 200 ng of genomic DNA, 2 μLof10× closely related to the Asian population than to the American popula- reaction buffer, 3 μL of 10 mM ATP, 4 U each of two restriction en- tion, suggesting that the RWW in Italy might have originated from Asia. zymes (Hind III and Mse I), 1 μL of T4 DNA ligase (Dingguo Bio- In the present study, the spread pathway of RWW in China was traced technology Co., Beijing, China), 1 μLofHind III-adapter (Hind III-F and through AFLP analysis, with an emphasis on the routes of transmission -R), 1 μLofMse I-adapter (Mse I-F and -R), and 9 μL of sterile distilled in Guizhou. water. This mixture was incubated for 5 h at 37 °C for adequate di- gestion and overnight at 4 °C for sufficient ligation.