Short Report: Natural Hybrid Between Anopheles Kleini and Anopheles Sinensis

Short Report: Natural Hybrid between Anopheles kleini and Anopheles sinensis

Deepak Joshi , Wej Choochote , and Gi-Sik Min * Department of Biological Sciences, Inha University, Incheon, South Korea; Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

Abstract. While analyzing wild mosquitoes caught in Paju, South Korea, we identified one egg-laying hybrid female

between Anopheles kleini and Anopheles sinensis . Additional evidence was obtained by identifying several F 1 progeny and conducting self-crossing between them. Sequencing of mitochondrial cytochrome c oxidase subunit 1 sequence suggested that the maternal origin of the female should be An. sinensis . Additionally, observation of only two distinct

genotypes of F 1 progeny (double band, hybrid type, and single-band An. sinensis type) more closely resembled a situation of natural back-crossing between a hybrid female and male An. sinensis. Results of self-crossing between F1 progeny was impaired and yielded abnormally low larval hatchings (3.7%). Overall, the observation of 1 female from 658 caught-wild mosquitoes indicated possible natural hybridization among members of the Hyrcanus group from South Korea.

Members of the Hyrcanus group of mosquitoes recently eny were cloned and sequenced. Additionally, to trace mater- gained renewed attention in numerous entomologic and epi- nal lineage of the hybrid female, mitochondrial cytochrome c demiologic studies because some members have been asso- oxidase subunit 1 (CO1) sequence was determined according ciated with the recent re-emergence of malaria in eastern to the methods of Park and others.13 Self-crossing was carried 1–3 Asia, including South Korea and China. Although nearly out between F1 progeny using the method described by Park 30 anopheline species are members of the Hyrcanus group, and others. 13 A total of 18 randomly selected females were only six species are indigenous to South Korea.4 These include successfully mated with brother progeny and allocated into Anopheles sinensis , An. lesteri , An. pullus , An. sineroides , An. three batches (each batch contained six females). Fertility of 5–7 kleini , and An. belenrae . F 1 progeny was determined by determining the hatching rate For sexually reproducing organisms, there are barriers that of eggs in each batch. We also examined spermathecae of the prevent interbreeding of two or more closely related species females. For this examination, seven of 18 females from the that are actually or potentially sympatric, thereby render- three batches were randomly selected and dissected, and the ing the species reproductively isolated. However, evidence presence or absence of sperm was confirmed. regarding the existence of natural hybrids gives rise to the From 16 light-trap collections, we successfully identified opinion that these barriers could be comparatively weak 658 female anopheline mosquitoes. Among these mosquitoes, in closely related species. In rare cases, when these barriers there were 360 An. sinensis , 258 An. pullus , 20 An. belenrae , 15 are broken, hybrids are produced. In particular, there are An. kleini, 3 An. sineroides, 1 An. lesteri, and 1 hybrid female. a few reports that indicate the existence of natural hybrids The hybrid female, which showed two bands, was identified in sympatric populations of Anopheles mosquitoes. To date, by polymerase chain reaction (PCR) of the sample obtained such hybrids are believed to exist in the An. gambiae com- on September 18, 2008, from Samok-ri, Paju ( Figure 1 ). plex, especially between An. gambiae and An. arabiensis. 8–11 Sequence identities of the double bands were in complete In the past, hybrid mosquitoes were identified from the study agreement with ITS2 28S ribosomal DNA sequences of An. of polytene chromosomes and crossing experiments, 8,9 but in kleini and An. sinensis (Supplementary Figure 1, available recent years, molecular markers have detected rare hybrids.11 online at www.ajtmh.org ). Identity of the CO1 sequence from We describe a single gravid female with sufficient evidence to the hybrid matched that of An. sinensis, suggesting that it may suggest that it is a potential hybrid between An. sinensis and have been derived from natural crossing between a female An. kleini . An. sinensis and a male An. kleini (Supplementary Figure 2 , Light-trap collections were conducted during 2004–2008 available online at www.ajtmh.org ). Sequences used in the in Manu-ri, Majeong-ri, and Samok-ri in Paju County, South current study have been deposited in GenBank under acces- Korea. Collected females were allowed to lay eggs inside sion numbers GQ265915–GQ265919. screen-topped paper cups containing moist filter paper. Hybrid sterility and fertility have been investigated in the Preliminary identification of females was conducted by using An. gambiae complex, a group that is currently reported to the egg characteristics reported by Otshuru and Ohomri12 and have natural hybrids. It was reported that female and male multiplex markers developed by Li and others.7 Subsequently, produced from crosses between An. gambiae and An. arabi- females were identified by using an internal transcribed spacer ensis are fertile and sterile, respectively.8 In the present study, 2 (ITS2) 28S ribosomal DNA–based multiplex assay devel- the hybrid female was fertile because she produced viable and oped in our laboratory (Joshi D, unpublished data). abundant F1 progeny. Thus, her status resembled that of the DNA extractions were performed from a female that showed female hybrid obtained from An. gambiae and An. arabien- double bands and several of its progeny (larva and adults). sis crosses as described by Davidson. 8 During PCR identifi-

Double bands obtained from the hybrid female and her F 1 prog- cation of 16 randomly selected F1 progeny, 6 progeny showed double bands similar to their mother (matching to An. sinensis and An. kleini ). The remaining 10 progeny showed 1 band * Address correspondence to Gi-Sik Min, Department of Biological that matched only An. sinensis . However none of the progeny Sciences, Inha University, Incheon 402-751, South Korea. E-mail: were identified with 1 band that matched An. kleini ( Figure 2 ). [email protected] This result led us to assume that the mating partner of a hybrid 1020 HYBRID BETWEEN AN. KLEINI AND AN. SINENSIS 1021

T able 1

Results of self-crossing between F1 progeny of the parental female suspected as the natural hybrid of Anopheles sinensis and An. kleini Total no. of artificially induced females No. of eggs Egg batches No. hatched (%) 6 300 First 0 (0) 6 249 Second 0 (0) 6 292 Third 31 (10.6) 18 841 31 (3.7)

F igure 1. Polymerase chain reaction identification of wild-caught female mosquitoes on September 18, 2008, from Samok-ri, Paju, South ity and the F1 progeny she produced appeared sterile in self- Korea. Lane M, 1-kb DNA marker (Enzymonics, Seoul, South Korea); crossing experiments. Therefore, further study will be required lanes 2–10, wild-caught female mosquitoes. Bands of 1,077, 481, and to verify the fertility of F1 progenies from laboratory back 385 basepairs (bp) are matched to Anopheles sinensis , An. belenrae , crosses. Given that progeny of hybrids can survive and are fer- and An. kleini , respectively. Lane 4, female-produced double bands (1,077 bp and 385 bp). tile, hybridization might be an important mechanism for gene introgression from one species to another among the members of Hyrcanus group in nature. female should be a normal An. sinensis male. This is precisely the same situation as in the back cross between a hybrid female Received March 11, 2009. Accepted for publication August 18, 2009. and a normal male. Note: Supplementary Figures can be found online at www.ajtmh.org . The result of the crossing experiment between F prog- 1 Acknowledgment: We thank Park Mi-Hyun for valuable assistance in eny was analyzed in three steps. First, larval hatching was the collection of the mosquitoes and other experimental tasks. observed. In the first and second batches, hatching of larvae was not observed by 6–7 days post oviposition, and relatively Financial support: This work was supported by Korea Research Foundation Grant (KRF-2004-041-C00266). low larval emergence (10.6%) was seen in the remaining third batch ( Table 1 ). Second, the status of embryogenic Authors’ addresses: Deepak Joshi and Gi-Sik Min, Department of Biological Sciences, Inha University, 253 Yonghyun-dong, Nam-gu, development was confirmed, in which dissections of 310 eggs Incheon, 402-751, South Korea. Wej Choochote, Department of representing the first two batches were performed. There was Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, no sign of embryo development within any of the dissected 50200, Thailand. eggs (0% embryonation rate). Third, during spermathecae, Reprint requests: Gi-Sik Min, Department of Biological Sciences, Inha examinations of seven females showed that only one con- University, 253 Yonghyun-dong, Nam-gu, Incheon, 402-751, South tained sperm. Korea, E-mail: [email protected]. On the basis of data from PCR identification of F1 progenies ( Figure 2 ), our study showed self-crossing between F 1 REFERENCES progeny of the hybrid female that were produced from the natural back crossing between the hybrid female and male An. 1. World Health Organization, 2005. World malaria report–2005. Available at: http://rbm.who. int/wmr2005/profiles/republic sinensis . 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