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Keywords:

  • Anopheles aconitus;
  • An. fluviatilis;
  • An. minimus;
  • An. varuna;
  • filariasis vector;
  • malaria vector;
  • mitochondrial COII;
  • molecular systematics;
  • mosquito distribution;
  • mosquito morphology;
  • 28S ribosomal DNA;
  • sibling species;
  • SSCPs;
  • Hainan;
  • Yunnan;
  • China

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Abstract. Mosquitoes of the Anopheles minimus group (Diptera: Culicidae) from nine Provinces of southern China were identified morphologically and by molecular characterization, using single-strand conformation polymorphisms (SSCPs) and sequence data for the D3 region of the 28S ribosomal DNA and the mitochondrial COII locus. Species A and C (sensu Green et al., 1990) of the An. minimus complex were found to be sympatric in Yunnan Province. Species A occurs eastward from Yunnan through southern Guangxi, Hainan, Guangdong and Taiwan Provinces, whereas species C occurs northward to northern Guangxi, Guizhou and Sichuan Provinces. Morphological and molecular evidence (based on specimens from the field and four isofemale lines) shows that An. minimus forms A and B (sensu Yu & Li, 1984) are morphological variants of species A, which is accepted as An. minimus Theobald sensu stricto (type-locality: Pokfulam, Hong Kong). The so-called subspecies x of An. minimus (sensu Baba, 1950) is reinterpreted as An. aconitus Dönitz. The distribution and vector status of members of the An. minimus group are discussed in relation to the historical and current transmission of malaria and filariasis in China. Both An. minimus A and C have been implicated as widespread vectors of malaria, whereas only species A has been found in Hainan, where An. minimus s.l. was a vector of Bancroftian filariasis. The presence of An. aconitus in Hainan and Yunnan Provinces is confirmed, but the occurrence of An. varuna Iyengar and An. fluviatilis James, which were previously recorded in China, could not be verified.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mosquitoes of the Anopheles minimus group (of the Myzomyia Series of subgenus Cellia) recorded from China include An. aconitus, An. fluviatilis and An. varuna as well as the An. minimus complex (Lu et al., 1997). Anopheles minimus sensu lato is widespread in hilly areas throughout the Oriental Region, extending north to about 32°30′ N in China, west to Uttar Pradesh in India, south through peninsular Malaysia and east to Taiwan and the Ryukyu archipelago of Japan.

Anopheles minimus s.l. is regarded as an important vector of human malaria throughout its distribution, including southern China (Ho & Feng, 1958; Reid, 1968; Harrison et al., 1990). For example, in the Feibar District of Hainan Province, Wu et al. (1993) found An. minimus s.l. at 18 sites, constituting 52% of anophelines collected. The vectorial capacity of An. minimus s.l. populations was correlated with malaria infection rates among inhabitants living in three types of residential quarters with different socioeconomic conditions. Recent cases of malaria in mainland China were all caused by Plasmodium vivax, although P. falciparum and P. malariae have been reported in Hong Kong and Hainan (Li, 1981; Zhang et al., 1991; Zhu & Huang, 1995). Although lymphatic filariasis is now almost eliminated from China (Xu, 1997; WHO, 2002), An. minimus s.l. was a vector of Wuchereria bancrofti in Hainan (Lu et al., 1997).

During the first half of the 20th century, An. minimus was not perceived as taxonomically complex, until Baba (1950) reported two forms of eggs from An. minimus collected in Guangdong Province and designated one form as ‘subspecies x’, but this received scant recognition. Yu & Li (1984) described An. minimus forms A and B from Hainan Island, based on morphological characters of larvae, pupae and adults. Yu (1987) reported these forms in several Provinces of China (Fujian, Guangdong, Guangxi, Yunnan) and noted differences in their esterase electromorphs. Green et al. (1990) showed that An. minimus consists of two species in western Thailand, based on sympatric occurrence of homozygotes of two enzyme loci in the absence of heterozygotes, and informally designated them as species A and C. Sharpe et al. (1999, 2000) confirmed the presence of species A and C in western Thailand, and suggested the possible presence of a sympatric third species (represented by their specimen no. 157). Baimai (1989) reported that C. A. Green and colleagues had recognized a third genetic species D in sympatry with species A and C in western Thailand, based on electrophoretic data, but it remains unclear whether or not specimen no. 157 of Sharpe et al. (1999, 2000) belongs to that putative species D (they were collected at the same locality in Kanchanaburi Province). Recently, Somboon et al. (2001) provided morphological, cytogenetic, molecular and hybridization evidence for the recognition of another sibling species, designated E, of the An. minimus complex on Ishigaki Island of the Rykyus, Japan.

Anopheles minimus species A is the predominant species of the An. minimus complex in Thailand (Green et al., 1990) and has also been recorded in India, Laos, Cambodia and Vietnam (Subbarao, 1998; Van Bortel et al., 1999, 2000; Kengne et al., 2001). Anopheles minimus species C has been reported only from western and northern Thailand (Kanchanaburi, Tak and Chiang Mai Provinces) (Green et al., 1990; Sharpe et al., 1999) and Hoa Binh Province of northern Vietnam (Van Bortel et al., 1999, 2000; Kengne et al., 2001) where it occurs in sympatry with species A. Using the population genetic parameter theta (θ), Sharpe et al. (2000) estimated that the long-term effective population size of species C is approximately half that of species A, implying that species C is far more widely distributed than was known at that time.

Regarding the wider distributions of other members of the An. minimus group recorded from China, An. aconitus has been reported from Thailand, Vietnam, Laos and Cambodia, whereas An. varuna has been reported from India, Sri Lanka, Thailand and Vietnam (Harrison, 1980; Sharpe et al., 1999, 2000; Van Bortel et al., 2000; Kengne et al., 2001). Anopheles fluviatilis was thought to occur across southern Asia from Taiwan to Yemen until Harrison (1980) reinterpreted its distribution as restricted to areas west of Myanmar, i.e. the Indian subcontinent and the Middle East. Harrison (1980) considered fluviatilis-like specimens from Myanmar and more easterly countries to be variants of An. minimus s.l. It is now known from cytogenetic evidence that An. fluviatilis comprises at least three sibling species, designated species S, T and U (Subbarao et al., 1994).

Adults of An. minimus A and C, An. aconitus and An. varuna appear very similar to one another, with overlapping ranges of morphological character variations, often resulting in their misidentification (Harrison, 1980). To overcome this difficulty, various molecular methods have been developed, as summarized by Van Bortel et al. (2002), including single-strand conformation polymorphisms (Sharpe et al., 1999), restriction patterns of the ITS2 fragment of rDNA (Van Bortel et al., 2000) and random amplified polymorphic DNA markers (Kengne et al., 2001), to facilitate identification of these and other species of the Myzomyia Series (Harbach, 1994).

Unresolved taxonomic questions and the lack of reliable distribution data for members of the An. minimus group in southern China limit the usefulness of available information on their biology, ecology and vector status in relation to effective malaria control. To help overcome these deficiencies, therefore, we combine the evidence from morphological and molecular investigations to elucidate the systematics and distribution of all known members of the An. minimus group in southern China.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Specimen collection

Mosquitoes were collected at 37 localities in Chongqing, Sichuan, Yunnan, Guizhou, Guangxi, Hainan, Guangdong and Hong Kong Provinces (Table 1) during July–September 2000 and August–September 2001. Most localities visited were areas with relatively high malaria incidence and/or places where An. minimus s.l. had been collected previously by other researchers. Adults resting near or landing on humans and tethered bovines were captured with aspirators between 20.00 and 23.00 hours. Mosquitoes were killed by exposing them to ethyl acetate vapour, placed individually in Beem capsules (Agar Scientific Ltd, Stansted, U.K.) and stored in plastic bags with silica gel. Larvae and pupae were collected in streams and irrigation ditches at some sites and reared to adults. Progeny broods were raised from a number of wild-caught, blood-fed females to obtain adults with associated larval and pupal exuviae for morphological study. Geographical and ecological data were recorded for all collections. Information on malaria incidence was gathered from local public health offices.

Table 1.  Localities, dates and numbers of An. minimus A, An. minimus C and An. aconitus collected in southern China. *Samples from established colony; – indicates that the species was not encountered at the collection site, or its occurrence there is unknown in the case of colony material.
ProvinceCounty: TownLatitude/longitudeDate of collectionAn. minimus AAn. minimus CAn. aconitus
ChongqingBeibei: Tianshengqiao29°48′N/106°24′E13–15/7/00
Nanchuan: Chengjiao29°12′N/107°06′E16–17/7/00
GuangdongConghua: Jiangpu23°30′N/113°30′E30/8/00, 24/8/01
Huidong: Daling22°54′N/114°48′E2/9/00, 20–22/8/0145
Taishan: Nongwen22°N/112°42′E31/8/005
GuangxiFangcheng; Dalu21°54′N/108°06′E16/8/00
Lingzhan: Lingyun24°22′N/106°30′E 21
Longlin: Jiuzhou24°36′N/105°48′E12/8/007
Longsheng: Rixin25°48′N/110°E26–27/8/0145
Nanning: Fuwu22°54′N/108°18′E14/8/00
Pubei: Jiangcheng22°15′N/109°30′E3/8/01
Pubei: Liuyun22°27′N/109°48′E4/8/01
Shangsi: Zhaoan22°18′N/107°54′E15/8/00, 1–2/8/015
GuizhouGuanling: Duanqiao25°57′N/105°36′E6/8/0011
Huishui: Chengjiao26°12′N/106°42′E9/8/00
Luodian: Chengjiao25°30′N/105°36′E10/8/00
Puding: Pingshang26°18′N/105°42′E5/8/00
Wangmuo: Chengjiao25°12′N/106°06′E11/8/009
HainanBaisha: Yunbang19°24′N/109°06′E21/8/00
Baoting: Nanling18°21′N/109°36′E23/8/00, 12/8/0186
Changjiang: Shilu19°18′N/109°03′E22/8/00, 7–8/8/013115
Changjiang: Wangxia19°N/109°09′E9–10/8/01274
Danzhou: Bayi19°30′N/109°21′E11/8/01105
Ledong: Jianfenglin19°36′N/108°42′E24/8/00
Qiongzhong: Zhayun19°N/109°36′E20/8/00
Tunchang: Nankun19°21′N/109°54′E19/8/005
Hong KongDa O22°18′N/113°54′E7/9/00
Pokfulam22°16′N/114°8′E7–9/98
Yuen Long22°24′N/114°E5–6/9/00
SichuanJunlian: Shuangteng28°06′N/104°12′E20/7/008
Leibo: Chengjiao28°12′N/103°36′E22/7/00
Pingshan: Xinshi28°36′N/103°48′E21/7/00
Qianwei: Gongping29°12′N/104°E18–19/7/0011
TaiwanPingtung: Manchow22°39′N/120°27′E8/008
Taichung24°12′N/120°42′E8/009
YunnanDaguan: Jili27°42′N/103°54′E17–19/9/0032
Jingdong: Chengjiao24°24′N/100°48′E27/9/011313
*Lincang23°57′N/100°06′E 65
*Luxi: Wanding24°12′N/98°06′E19995
Menghai: Daluo21°48′N/100°06′E31/7/00
Mengla: Menglun21°54′N/101°12′E30/7/00, 21/9/01161121
Mengla: Xiangming21°23′N/101°35′E29/7/00142
Menglian: Mengma22°18′N/99°30′E1/8/001
Simao: Cuiyun22°42′N/100°54′E2/8/00, 9/9/0114135
*Simao: Nandaohe22°42′N/100°54′E 29
Yuanjiang: Dashuping23°30′N/102°E26/7/0054
*Yuanjiang: Ganzhuang23°30′N/102°E7/0010

In addition to recently captured mosquitoes, the following material was used in the study. (1) Specimens from four isofemale line laboratory colonies: Lingzhan (Guangxi Province), Yuanjiang and Simao (Yunnan Province) colonies maintained in the Guangxi Medical University; and Lincang (Yunnan Province) colony maintained in the Yunnan Institute of Malaria. (2) Samples from natural populations in Taichung and Pingtung, Taiwan provided by P. Somboon, and Luxi in Yunnan Province provided by X. Z. Wang. (3) Some 153 old, pinned specimens received from various insect collections in China.

Morphological identification

The keys of Harrison (1980) were used to identify adults and larvae. Results were used to target specimens for morphological and molecular analyses. Morphological studies focused on features of adult females that purportedly differentiate various species and forms of An. minimus, including: (1) the presence or absence of the humeral pale (HP) and presector pale (PSP) wing spots; (2) the separation or fusion of the accessory sector pale (ASP) and sector pale (SP) spots; and (3) the presence or absence of median pale scaling on vein M1 (= M1+2 of Harrison, 1980; Yu & Li, 1984; Yu, 1987).

Molecular identification

Mosquitoes were homogenized in buffer (0.01 m Tris, pH 7.8, 0.005 m EDTA, 0.5% SDS), and digested with 50 µL/mL proteinase K at 37°C overnight (Sambrook et al., 1989). DNA was then extracted with equal volumes of phenol/chloroform/isoamyl alcohol (25 : 24 : 1) twice and chloroform/isoamyl alcohol (24 : 1) once, followed by ethanol precipitation with 0.3 m sodium acetate and 2.0 volumes of 100% ethanol on ice for 60 min. After centrifugation at 13 000 g for 30 min and discarding the supernatant, the pellet was washed with 70% ethanol, dried and re-suspended in 20 µL TE buffer (10 mm Tris, 1 mm EDTA, pH 8.0) before storage at 4°C. A negative control was included with every set of extractions.

Two regions of DNA were amplified using PCR: 335–362 bp third domain (D3) of the 28S gene of ribosomal DNA (rDNA) using primers D3a (5′-GACCCGTCTTGAAACACGGA-3′, forward) and D3b (5′-TCGGAAGGAACCAGCTACTA-3′, reverse), and 685 bp of the mitochondrial cytochrome oxidase II gene using primers LEU (5′-TCTAATATGGCAGATTAGTGCA-3′, forward) and LYS (5′-ACTTGCTTTCAGTCATCTAATG-3′, reverse) (Sharpe et al., 2000). Amplifications were performed in 50 µL volumes overlaid with two drops of mineral oil on a HYBAID OmniGene cycler (Thermo Hybaid, Ashford, U.K.). Each PCR included 1/100 DNA of a whole mosquito, 5 µL 10 × ReddyMixTM buffer (ABgene, Epsom, U.K.), 200 µm dNTPs, 2 mm MgCl2, 600 µm of each primer and 1.3 units of Thermoprime Plus DNA Polymerase (ABgene). Reactions started with denaturation at 95°C for 5 min, followed by 35 cycles, each cycle consisting of denaturation for 40 s at 95°C, annealing for 40 s at 55°C and extension for 1 min at 72°C, with a final extension at 72°C for 6 min. PCR products were electrophoresed through ethidium bromide-stained 1% agarose gels in 1 × TBE and visualized under UV light to check for successful amplification.

The single-strand conformation polymorphisms (SSCPs) of Sharpe et al. (1999) were used for the identification of An. minimus A and C, An. aconitus and An. varuna. SSCPs developed for the identification of An. minimus E, An. pampanai Büttiker & Beales, An. fluviatilis, An. jeyporiensis James and An. culicifacies Giles (B. Chen, unpublished) were used to verify the absence of these species in the collections noted above. A 20 µL aliquot of each PCR product of D3 was precipitated with 20 µL isopropanol and 2 µL sodium acetate (3 m, pH 5.2), dried at room temperature and re-suspended in 10 µL of formamide loading buffer. Half of the sample was loaded onto SSCP gel cast in a Protean II system (Bio-Rad Laboratories Ltd, Hemel Hempstead, U.K.) and made of MDE gel solution (Flowgen, Ashby de la Zouch, U.K.), 0.6 × TBE, TEMED and 10% ammonium persulphate. Electrophoresis was carried out in 0.6 × TBE at 4°C under a constant voltage of 300 V for 18 h (Sharpe et al., 1999). The gel was then fixed in 10% glacial acetic acid for 30 min in a plastic tray with agitation, stained in a solution comprising 0.1% AgNO3 and 0.056% formaldehyde for 30 min, developed in a pre-chilled solution comprising 3% Na2CO3 (w/v), 0.056% formaldehyde and 0.002 mg/mL sodium thiosulphate twice until all bands became visible (about 10–15 min). The gel was finally washed in 10% glacial acetic acid for 2–3 min and dried on a Gel Dryer (Bio-Rad Laboratories Ltd) at 27°C for 2 h.

DNA sequencing

Whenever atypical SSCP banding patterns were observed, the D3 region of the sample was sequenced. The COII genes of up to 12 individuals per species per locality were sequenced to confirm the D3 locus identification. PCR products were cleaned using a spin column (Promega Wizard PCR Preps, Madison, Wisconsin, U.S.A.) and sequenced in both directions in an ABI 377 automated sequencer (PE Applied Biosystems, Warrington, U.K.). Sequences were edited manually and aligned using ClustalX (Thompson et al., 1997).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Morphological and molecular identification

Approximately 1000 of some 4000 Anopheles specimens collected in southern China were found to be species of the Myzomyia Series of subgenus Cellia (Harbach, 1994). DNA was successfully extracted from 686 of these individuals. We tried to extract DNA from one or two legs or the abdomen of a number of old, pinned specimens rather than destroy them entirely, but this did not work well; therefore, data for these specimens are not included here. PCR products of amplified D3 regions were run on SSCP gels. The D3 region was sequenced from 79 of these samples and the COII gene was sequenced from 480. Consequently, 203 wild-caught mosquitoes from Yunnan, Guangxi, Hainan, Guangdong and Taiwan Provinces were unambiguously identified as An. minimus A, 172 from Sichuan, Yunnan, Guizhou and Guangxi Provinces were identified as An. minimus C, and 62 from Yunnan and Hainan Provinces were identified as An. aconitus (Table 1; Fig. 1). The numbers of An. minimus A and C and An. aconitus comprised 29.6, 25.1 and 9.2%, respectively, of all specimens identified as members of the Myzomyia Series. No specimens of An. varuna or An. fluviatilis, two species previously recorded from various provinces in southern China, were found in the collections.

image

Figure 1. Outline map of southern China, showing localities sampled and geographical distribution of three species of the An. minimus group. The area below the dotted line indicates the distribution of An. minimus s.l. inferred from historical records and personal communications.

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D3 sequence for An. minimus A

The D3 sequence of rDNA serves as the standard for the identification of An. minimus A and C (Sharpe et al., 1999). We sequenced D3 from 32 samples of An. minimus A identified by SSCPs of D3 and confirmed by COII sequences (Chen, Harbach and Butlin, unpublished) that were collected from southern China, Thailand and Vietnam, including samples from Thailand and Vietnam previously analysed by Sharpe et al. (1999) and Somboon et al. (2001). We found the D3 sequences of all of these samples to be identical. However, they differed by one base pair from the D3 sequence for An. minimus A that Sharpe et al. (1999) entered into GenBank (AF114019). Because this is apparently an error that unwittingly occurred when the sequence was entered into the database, we entered the correct D3 sequence into GenBank (accession number AJ459420) to clarify GenBank entry AF114019 on behalf of Sharpe et al. D3 sequences obtained from 28 An. minimus C also identified by SSCPs of D3 and confirmed by COII sequences are homologous with the D3 sequence that Sharpe et al. (1999) entered into GenBank (AF114017) for this species.

Morphological variation

Wings of 150 An. minimus A, 144 An. minimus C and 34 An. aconitus from various localities in southern China were examined for the presence of HP and PSP spots, the separation of ASP and SP spots, and the presence of a median pale spot on vein M1. These characters were also examined in colony material of An. minimus A and C. The results are shown in Table 2. An HP spot was found to occur in low frequencies in wild-caught females of all three species (7.3–15.6%), but was not observed at all in females of An. minimus A and C from laboratory colonies. The most probable explanation for the latter observation is that the HP spot was absent in the females that were used to start the isoline colonies. A PSP spot was present much more frequently in wild-caught An. minimus A (88.4–100%) than in wild-caught specimens of An. minimus C (46.9–75.0%) or An. aconitus (20.0–28.6%). The frequency of occurrence of a PSP spot in colony specimens of An. minimus A (81.5–94.1%) fell within the range for wild-caught mosquitoes, whereas this spot was absent in colony samples of An. minimus C. A separate ASP spot was always absent in An. aconitus and occurred in roughly equal frequencies in An. minimus A and C. One or two median pale spots on vein M1 occurred much more frequently in wild-caught An. minimus A (68.1% of females, 100% of males) than in wild-caught An. minimus C (28.1% of females, 62.5% of males); however, there was no significant difference in the occurrence of these spots in colony samples of the two species (75–100%). Because of the variable occurrence of the four wing spot characters in both An. minimus A and C, they cannot be used with any degree of confidence to distinguish them.

Table 2.  Occurrence of selected spots (as percentage of specimens with spot present on one or both wings) among wild-caught mosquitoes and colony specimens of An. minimus A and C and An. aconitus from southern China. Numbers of specimens examined are indicated in parentheses.
SpeciesSexHP spot presentPSP spot presentSeparate ASP spotM1 without median spot
An. minimus A (wild-caught)M (138)7.388.417.468.1
F (12)010083.3100
An. minimus C (wild-caught)M (128)15.646.923.428.1
F (16)0755062.5
An. aconitus (wild-caught)M (20)1020050
F (14)14.328.60100
An. minimus A (Lincang colony)M (34)094.12.979.4
F (27)3.781.53.781.5
An. minimus C (Simao colony)M (15)0046.793.3
F (11)0063.6100
An. minimus C (Yuanjiang colony)M (4)007575
F (5)0010080
An. minimus C (Lingzhan colony)M (15)0080100

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

An. minimus forms A and B

Yu & Li (1984) and Yu (1987) defined An. minimus‘forms’ A and B (hereafter termed morphotypes A and B) and recorded their distribution in Hainan, Fujian, Yunnan, Guangdong and Guangxi Provinces. They described morphotype A as having one or two small median pale spots (rarely absent) on vein M1, the cibarial armature of females with cone filaments bifurcated apically, and the dorsomentum (their mental plate) of larvae with 7, rarely 9, teeth. They distinguished morphotype B by the absence of a median pale spot on vein M1, cone filaments of females without bifurcated tips, and the dorsomentum of larvae with 9 or 11 teeth. Jiang et al. (1987) reported differences in the electrophoretic banding patterns of non-specific esterases between these two morphotypes, but considered that the differences were far from indicative of species or subspecies status. Among 208 An. minimus s.l. collected in Yayun Village, Shilu Town, Changjiang County, Hainan Province (Yu et al., 1989), morphotype A comprised 9.2% of specimens captured on humans (1.0%) and bovines (8.2%), whereas morphotype B accounted for 90.9% of the specimens (1.0% from humans and 89.9% on bovines).

The number of dorsomental teeth of larvae does not provide complete separation of morphotypes A and B, and the cibarial armature of females is extremely small, requires meticulous dissection and its fine structure cannot be discerned with confidence under a conventional microscope. Therefore, we used the reportedly diagnostic character of vein M1 to distinguish specimens as either morphotype A or B. Specimens with pale scaling on the middle of vein M1 were recorded as morphotype A, and all of those with this vein entirely dark except at the base and apex were recorded as morphotype B. Based on these criteria, morphotypes A and B were found to be sympatric in 20 of 22 localities where An. minimus s.l. were collected during this study, including Yayun Village in Hainan Province where Yu & Li originally collected their ‘forms’ A and B (see above), and Sichuan and Guizhou Provinces. In Taiwan, Teng et al. (1998) found both morphotypes A and B in five counties where they studied seasonal fluctuations of An. minimus s.l. More importantly, these two morphotypes were found together in each of the four laboratory colonies studied here (Table 2). Wild populations of An. minimus species A had more morphotype B individuals (68.1% of females, 100% of males) than wild populations of An. minimus species C (28.1% of females, 62.5% of males) (Table 2). Thirty-one individuals of An. minimus s.l. collected from Yayun Village in Hainan Province were all identified as species A on the basis of DNA sequences, which explains why Yu et al. (1989) encountered such a high proportion of morphotype B at this locality. In the case of colony material, both An. minimus species A and C have a higher proportion (75–100%) of morphotype B. It is interesting to note that other species of the An. minimus group can also be divided into morphotypes A and B on the basis of this character: within An. aconitus 50% of females and 100% of males were identifiable as morphotype B (Table 2), and within An. jeyporiensis 53.3% of females and 83.3% of males were identifiable as this morphotype. Finally, no differences were observed in the sequences of D3 and COII between specimens identified as morphotypes A and B. Therefore, we conclude that the characters used by Yu & Li (1984), Yu (1987) and Yu et al. (1989) to define morphotypes A and B have no taxonomic significance, and these ‘forms’ are nothing more than morphological variants of the same biological species.

An. minimus subspecies x

Baba (1950) found two egg forms of An. minimus s.l. in an area that presently includes Guangdong and Hainan Provinces and the most southern part of Guangxi Province (specific locality not mentioned). The smaller one, with a smooth frill, he termed An. minimus minimus. The comparatively larger one, with an irregular frill, he designated as An. minimus subspecies x, but did not distinguish the corresponding adult and larval stages of these subspecies in his identification keys. The differences in egg morphology observed by Baba (1950) are not the same as those observed between species A and C from Thailand (Sharpe, 1997). Anopheles minimus species A was found in Guangdong, Hainan and southern Guangxi, and An. aconitus in Hainan (Table 1). Although we have not studied eggs of the An. minimus group in China, our interpretation of Baba's identification keys is that both of his so-called ‘subspecies’ conform with the morphological characters of An. aconitus (cf. Harrison, 1980), whereas his An. minimus refers to An. fluviatilis (see below). Baba did not include An. aconitus in his keys because his paper was published before this species was recognized as a member of the Chinese mosquito fauna. Anopheles aconitus normally has distal pale scaling on the proboscis (rarely absent), whereas in An. minimus the proboscis is usually entirely dark-scaled (infrequently with a small ventral pale patch distally) (Harrison, 1980). Baba used the presence of pale scaling on the proboscis to identify subspecies x and subspecies minimus together in one part of a couplet, and the absence of pale scaling as the alternative, leading to another couplet for separating An. fluviatilis from An. culicifacies. Larvae of subspecies x, subspecies minimus and An. fluviatilis are inseparable in Baba's key for the identification of this life stage.

Sibling species of the An. minimus complex

This research establishes for the first time the presence of two species of the An. minimus complex in China, and also delimits their distributions. It appears that only species A and C occur in southern China. We found no evidence from D3 sequences for the presence of other forms, i.e. species D (Baimai, 1989), specimen no. 157 (Sharpe et al., 1999) or species E (Somboon et al., 2001).

Green et al. (1990) found that a humeral pale (HP) wing spot was present more often in An. minimus C than in An. minimus A in western Thailand: 78% in the former as opposed to only 5% in the latter. Among mosquitoes collected in the same locality visited by Green et al., Sharpe (1997) found an HP spot in 63% of species C and 9% of species A. A similar percentage of species A females from southern China possess HP spots (7.3%), but surprisingly fewer species C females exhibit this character (15.6%) (Table 2). Except for a few males from the Lincang colony, HP spots were absent in colony specimens (males and females) of both species A and C (Table 2). From these findings, it is clear that the presence of HP spots cannot be used with any degree of confidence to differentiate species C from species A. In our samples, significantly more individuals of species A have a PSP spot on at least one wing (88.4% of females, 100% of males) than individuals of species C (46.9% of females, 75% of males). Whereas a similar proportion of species A from colonies had PSP spots (94.1% of females, 81.5% of males), no PSP spots were present in colony specimens of species C (Table 2). The PSP spot is also unreliable for species differentiation.

Distribution of An. minimus A and C

Lu et al. (1997) recorded An. minimus s.l. from 16 provinces in southern China: Sichuan, Chongqing, Hubei, Henan, Anhui, Zhejiang, Yunnan, Guizhou, Guangxi, Hainan, Hunan, Jiangxi, Guangdong, Fujian, Hong Kong and Taiwan. The taxon occurs throughout Yunnan Province (Dong, 1997). It has been reported from as far north as Nanjiang in Sichuan Province, but the western boundary of its distribution in this province is unknown (X. T. Lei, personal communication). The northernmost occurrence records for the taxon are Danjiangkou and Tongbai in Hubei and Henan Provinces, respectively (Wang & Liu, 1996). It has been recorded from 10 counties south of Nanling in Anhui Province (N. C. Zheng, personal communication), and in almost all counties south of Hangzhou in Zhejiang Province (M. H. Qiu, personal communication). In summary, the An. minimus complex occurs throughout southern China from Yunnan Province eastward and from Hainan Island northward to approximately 32.5° N latitude (Fig. 1).

Anopheles minimus A is known to occur in Thailand, India, Vietnam, Laos and Cambodia (Green et al., 1990; Subbarao, 1998; Van Bortel et al., 1999, 2000; Kengne et al., 2001). This study extends its distribution into five southern provinces of China, Yunnan, Guangxi, Hainan, Duangdong and Taiwan. Until now, An. minimus C was only known from localities in western and north-western Thailand (Green et al., 1990; Sharpe et al., 1999) and northern Vietnam (Van Bortel et al., 1999, 2000; Kengne et al., 2001). During the present study, species C was found in the south-central provinces of China (Yunnan, Guangxi, Guizhou and Sichuan), which confirms the prediction of Sharpe et al. (2000) that species C should be more widely distributed than the localities in Thailand. Available evidence suggests that species A and C are sympatric in Yunnan and Guangxi Provinces, and species C extends north-eastward into Guizhou and Sichuan Provinces, whereas species A extends eastward and southward into Guangdong, Hainan and Taiwan Provinces (Fig. 1). Further study is required to determine whether one or both species occur as far north as 32.5°, but the available distributional data for the two species, coupled with the fact that species C is more closely related to the more eastern species E (Somboon et al., 2001), suggest that species C is likely to be more widely distributed than species A. At this time, species A appears to occur no farther north than approximately 24.5° N latitude.

Nomenclature of An. minimus s.l.

Anopheles minimus was originally described by Theobald (1901) from a single female collected in Pokfulam, Hong Kong, but the specimen apparently no longer exists (Theobald, 1910; Yamada, 1925). Harrison (1980) stated: ‘If a neotype is ever needed, numerous adults with associated immature skins collected only 20–22 km from Pokfulam (Hong Kong Island) in Sai Kung District, New Territories, are deposited in the USNM’. Sharpe (1997) examined some of the latter specimens but was unable to determine whether they were representatives of species A or C. She attempted to sequence the D3 region of 28S rDNA from several specimens but the sequences turned out to be those of a yeast-like organism rather than a mosquito. Because species A was known to have a much wider distribution than species C, it has been suggested that the former is likely to be conspecific with the species originally described as An. minimus from Hong Kong. Because only species A has been found in Guangdong Province immediately north of Hong Kong, our data lend support to this idea. However, before the name of minimus can be unambiguously assigned to species A, a neotype unequivocally identified as species A from as near as practicable to the original type locality must be designated to fix the application of the name. In the case of sibling species, type specimens should be selected from individually reared progeny of wild-caught females. Progeny broods are invaluable for defining members of species complexes because they provide conspecific material that can be used for combined morphological and molecular characterization (Linton et al., 2001a,b).

One of us (B.C.) collected mosquitoes at the type locality in July–September 1998, but no specimens of the An. minimus complex were encountered. Collections were also made in Yuen Long, New Territories, where the samples in the USNM mentioned by Harrison (1980) were collected, and at Da O, Lantau Island in Hong Kong in 2000, but no members of the complex were found at either of these places. It is fairly certain that An. minimus has disappeared from the original type locality and adjacent areas as a result of environmental change. For this reason, we considered designating a neotype from samples collected in Guangdong Province, but we were unsuccessful in our attempts to obtain progeny broods of this taxon during our field trips. Unfortunately, the taxonomic identity of An. minimus s.s. remains unresolved and a neotype is needed before an available or new name can be applied to the other species.

Anopheles aconitus

Anopheles aconitus is broadly distributed from Sri Lanka, India and Nepal in the west to Hainan Island in the east, and south from southern China through South-east Asia into Indonesia as far east as Babar Island in the Lesser Sunda chain (Harrison, 1980). It is recorded from five provinces in southern China, Yunan, Guizhou, Guangxi, Hainan and Zhejiang (Lu et al., 1997), and may also occur in Guangdong Province (Baba, 1950). This study confirms its presence in Yunnan and Hainan Provinces (Table 1). Further investigation is needed to verify the presence of this species in other provinces.

Anopheles varuna

The distribution of An. varuna is uncertain because morphological identification of adult females is unreliable (Harrison, 1980; Green et al., 1990; Sharpe et al., 1999, 2000; Van Bortel et al., 2001). Whereas Covell (1944) and Rao (1961) listed An. varuna from southern China, Feng (1938) and Chow (1949) did not. Liu et al. (1959) referred to Chinese reports of An. varuna in Guizhou and Yunnan Provinces, and showed that these most certainly applied to dark-winged An. minimus. Based on published records and the examination of specimens, Harrison (1980) stated that An. varuna occurs in Bangladesh, Myanmar, India, Nepal, Sri Lanka, Thailand and possibly Vietnam, but not in China. Despite this, Lu et al. (1997) listed it among the mosquito fauna of Yunnan, Guangxi, Hainan, Fujian and Zhejiang Provinces. We did not find An. varuna in Yunnan, Guangxi or Hainan Provinces, or any of the other provinces where we collected members of the An. minimus group (Fig. 1). For this reason, we agree with Liu et al. (1959) and Harrison (1980) that this species does not occur in China. Recent application of molecular methods has confirmed the presence of An. varuna in Thailand and Vietnam (Van Bortel et al., 2000, 2001; Kengne et al., 2001).

Anopheles fluviatilis

Anopheles fluviatilis s.l. is a vector of malaria in India and Nepal. According to Lu et al. (1997), this taxon occurs in 10 Provinces of China: Sichuan, Yunnan, Guizhou, Guangxi, Hainan, Zhejiang, Anhui, Jiangxi, Fujiang and Taiwan. However, Harrison (1980) studied specimens from Taiwan, Hong Kong, Thailand and Myanmar, which were previously identified as An. fluviatilis, and found they were variants of An. minimus. We examined samples from Yunnan, Guizhou and Hainan Provinces in Chinese museums previously identified as An. fluviatilis on the basis of having a long preapical dark band on the maxillary palpi. In agreement with Harrison (1980), we found that palpal variations are common in Chinese specimens of An. minimus, i.e. the preapical dark band exhibits a range of variation from completely absent to slightly longer than the preapical pale band. We obtained D3 sequences from some of the fluviatilis-like museum samples and found that they correspond to either An. minimus A or C. Therefore, we agree with Harrison (1980) that specimens from China with fluviatilis-like maxillary palpi are merely variants of An. minimus s.l., and An. fluviatilis should be removed from the list of mosquito species occurring in China.

History and current status of malaria in China

Historically, malaria has been a major health problem in certain areas of China. Prior to 1949, approximately 30 million malaria cases occurred in the country each year, with some localities having a parasite incidence as high as 800 cases per 1000 population. Malaria cases and incidence decreased to 6.97 million and 122.90/0000, respectively, in 1954, but unfortunately rose to 24.115 million and 296.100/0000 in 1970 (Table 3). After 1970, both the malaria cases and parasite incidence rapidly decreased to 18 620 and 0.140/0000 in 2000. Economic development, especially in the last 20 years, has contributed significantly to the decline of malaria. Improved living conditions and mosquito prevention, especially in areas where An. sinensis Wiedemann occurs, have reduced mosquito–human contact. Environmental change has also contributed to the decline of malaria. Widespread use of agricultural insecticides, improved cultivation systems and the construction of water conservancy works have eliminated many of the habitats used by immature An. anthropophagus Xu & Feng, An. minimus and An. dirus Peyton & Harrison. This consequently has narrowed species distributions, reduced their densities and weakened their transmission capability. More importantly, since the nation-wide malaria control programme was launched in 1955, large-scale malaria control activities, including the establishment of control facilities, professional training and implementation of integrated control measures (Tang et al., 1991; Liu et al., 1995), have been conducted with significant success in all parts of mainland China.

Table 3.  Malaria cases and incidence per 10 000 population in China from 1954 to 2000, based on Liu et al. (1995), an unpublished report by the Chinese Ministry of Public Health for 1997 and published reports for 1998–2000 (Advisory Committee on Malaria, 1999, 2000; Gu & Zheng, 2001).
YearMalaria casesMalaria incidence
19546970000122.90
196010236800155.40
197024115000296.10
1980330030033.78
19901174001.06
1995497000.42
1996372000.31
1997346000.29
1998313190.25
1999290390.23
2000186200.14

In Taiwan, malaria was transmitted mainly by An. minimus s.l. in the hills, foothills and plains, whereas An. sinensis was the chief vector in areas of rice cultivation (Yip, 2000). Malaria eradication from Taiwan, mainly by means of DDT house-spraying, was achieved through the following phases: preparatory (1946–1951), attack (1952–1957), consolidation (1958–1964) and maintenance (1965 onwards). In November 1965, malaria eradication from Taiwan was certified by the World Health Organization (Taiwan, 1991), but the vectors remain endemic, so Taiwan is still receptive to possible resumption of transmission if malaria parasites are reintroduced.

During recent decades, Plasmodium vivax is the only species of malaria remaining endemic in the region north of 33° N (north of the Qinling Mountains and the Huaihe River), and is the predominant form of malaria throughout the area south of 33° N. In the early 1950s, malaria due to P. falciparum accounted for 30% of all cases in the region between 25 and 33° N (from the Nanling Mountains northward) and 10–20% in the region south of 25° N (from the Nanling Mountains southward). In the early 1960s, falciparum malaria accounted for 20–80% of all cases when a malaria outbreak occurred in southern Anhui, Jiangsu and northern Zhejiang Provinces (see Tang et al., 1991). After a brief resurgence in 1988–1989, falciparum malaria markedly declined in China. In 1990, indigenous cases of falciparum malaria were found only in 58 counties of four provinces. However, at the same time, non-indigenous falciparum cases were reported from 78 counties of eight provinces, which accounted for 8.95% of all parasitologically confirmed cases. Now falciparum malaria is only found in two provinces, Yunnan and Hainan. In 1999, some 3904 cases of falciparum malaria were recorded in Yunnan Province and 705 in Hainan Province. The following year, the number of cases attributable to P. falciparum in these two provinces fell to 1466 and 522, respectively (WHO, 2002). Most of the people affected belonged to ethnic minority groups living in remote and inaccessible mountains and forested areas. Malaria caused by P. malariae and P. ovale is seldom seen in China (Tang et al., 1991; Lu, 1999).

Although significant progress has been made, China still faces the threat of malaria. Eighty-two counties with 40.4 million people are at risk (malaria incidence = 1.1–100/0000), especially 36 counties (6 in Hainan, 27 in Yunnan, 1 in Sichuan, 1 in Henan and 1 in Jiangsu Province) with 8.5 million people where the incidence of malaria is currently greater than 10.10/0000 (unpublished report by the Chinese Ministry of Public Health on the status of malaria for 1997). Some 36.51 million people were still at risk in 2000 (WHO, 2002). Unfettered population movements that jeopardize malaria control efforts characterize the southern international border regions where it is estimated that malaria cases may be underreported by as much as 40% by county and township hospitals, and a greater percentage by village health workers and private health providers. In border areas, unreported cases have been found to be 8–10 times more numerous than reported cases. Although the confirmed numbers of malaria cases in 1999 and 2000 are, respectively, 29 039 and 18 620, taking missing records into account, the actual number of malaria cases was estimated to be at least 250 000–300 000 for each year (Advisory Committee on Malaria, 2000; Gu & Zheng, 2001). In the forested, hilly regions of Hainan Province, the chief vector (An. dirus) is exophilic, and, thus, residual spraying and the use of insecticide-treated bednets are not particularly effective for its control. Malaria cases in Guangdong Province have increased markedly, partly due to human population movements in the 1990s (Zhu & Huang, 1995).

Regions of malaria incidence

Ho & Feng (1958) and Ho (1965) divided China into four geographical regions of malaria incidence: the north-west region, a region north of 32° N, another between 25 and 32° N, and one south of 25° N. Liu et al. (1995) modified these divisions based on more recent data on the incidence of malaria. Information on malaria incidence gathered from local public health offices and recent publications during this study generally concurs with the epidemiological regions of Liu et al. (1995), which are shown in Fig. 2 with some modification. The Western Region is without malaria except for sporadic infections in Xingjiang Province transmitted by An. messeae Falleroni and An. sacharovi Favre. The Northern Region, where An. sinensis is the only vector, has sporadic transmission with an incidence of <10/0000 (malaria has been eliminated in the northern and western areas of this region). The Central Region, where An. anthropophagus, An. minimus, An. sinensis and An. kunmingensis Dong & Wang are the principal vectors, is predominantly hypo-endemic (incidence < 10/0000), with some areas having incidence greater than 10/0000. Finally, the Southern Region, where An. minimus and An. anthropophagus are the principal vectors, is a hypo- and meso-endemic region.

image

Figure 2. Regions of malaria endemicity in China, based on vector species, malaria prevalence and geographical criteria (modified from Liu et al., 1995).

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Malaria vector status of An. minimus s.l.

In addition to the An. minimus complex, the main malaria vectors in suitable habitats of China are An. anthropophagus, Anopheles dirus s.l., An. kunmingensis and An. sinensis. Indications of the infection risk and degree of transmission by these vectors are provided in Table 4. Anopheles minimus s.l. has long been regarded as a principal malaria vector throughout its distribution in China (Ho & Feng, 1958), coinciding with the Southern and Central Regions of malaria endemicity noted above (cf. Figs 1 and 2). Our collections were made across the Southern Region and in the western part of the Central Region, with emphasis on malarious areas. In Yunnan Province, south of 25° N latitude, An. minimus is regarded as the main vector below 1500 m (Dong, 1999), with an average sporozoite rate of 7.05% (Dong, 1997). Seasonal fluctuations of An. minimus populations and malaria cases coincide in this area of the province (Wang et al., 1999). Both An. minimus A and C were collected from this area, mostly in sympatry (Table 1). Anopheles minimus is the principal vector of malaria in Guangdong and Hainan Provinces (Wu et al., 1993; Zhu & Huang, 1995) where only An. minimus A has been found, implicating this sibling species as a vector. Anopheles minimus is also the main malaria vector in southern Guizhou Province (Wang et al., 1997) and northern Guangxi Province (J. R. Huang, personal communication). We only found An. minimus C in these areas, implying that this species is responsible for malaria transmission there. Anopheles anthropophagus and An. sinensis are thought to be the main vectors in north-eastern Yunnan and south-eastern Sichuan Provinces (Lei et al., 1997; W.B. Li, personal communication), but this needs to be carefully investigated in light of the fact that we collected a large number of An. minimus C in those areas (Table 1; Fig. 1).

Table 4. . Main vector species and their relationships to counties, their populations and malaria in China, in 1997, based on an unpublished report by the Chinese Ministry of Public Health for 1997. Incidence per 10 000 population.
Vector speciesCountiesPopulationMalaria casesMalaria incidence
An. dirus s.l.103009000343611.42
An. minimus s.l.21498568000132511.34
An. anthropophagus192113441000146261.30
An. kunmingensis3477880005440.70
An. sisnensis61439591900027320.07

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by a Wellcome Trust Travelling Research Fellowship 058569/Z/99/Z award to B.C. A number of people provided field assistance, mosquito samples and information used in the study. We are especially grateful to Hanbin Chen, Jianren Huang, Xintian Lei, Weiben Li, Zhigang Liao, Baolin Lu, Jianming Peng, Fengyi Qu, Rosie Sharpe, Jianrong Shi, Huanhuan Shi, Pradya Somboon, Cathy Walton, Xuezhong Wang, Yuan Yu, Peisheng Zhou and Taihua Zhu.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Advisory Committee on Malaria, Chinese Ministry of Health (1999) Malaria situation in the People's Republic of China in 1998. Chinese Journal of Parasitology and Parasitic Diseases, 17, 193195.
  • Advisory Committee on Malaria, Chinese Ministry of Health (2000) Malaria situation in the People's Republic of China in 1999. Chinese Journal of Parasitology and Parasitic Diseases, 18, 129131.
  • Baba, K. (1950) A study of anopheline mosquitoes in relation to the epidemiology of malaria in Canton Delta, South China. Transactions of the Kansai Entomological Society, 15, 111.
  • Baimai, V. (1989) Speciation and species complexes of the Anopheles malaria vectors in Thailand. Proceeding of the Third Conference on Malaria Research, Thailand Chiangmai, 18–20 October 1989, pp. 146162.
  • Chow, C.Y. (1949) The identification and distribution of Chinese anopheline mosquitoes. Journal of the National Malariology Society, 8, 121131.
  • Covell, G. (1944) Notes on the distribution, breeding places, adult habits and relation to malaria of the anopheline mosquitoes of India and the Far East. Journal of Malaria Institute of India, 5, 399434.
  • Dong, X.S. (1997) The advance of mosquito research in Yunnan. Annual Report on Malaria Control and Research for 1995–96, Yunnan Institute of Malaria Control, 1997, 7276.
  • Dong, X.S. (1999) The malaria vectors and their ecology in Yunnan Province. Annual Report on Malaria Control and Research for 1997–98, Yunnan Institute of Malaria Control, 1999, 167.
  • Feng, L.G. (1938) A critical review of literature regarding the records of mosquitoes in China. Part I. Subfamily Culicinae, tribe Anophelini. Peking Natural History Bulletin, 12, 169181.
  • Green, C.A., Gass, R.F., Munstermann, L.E. & Baimai, V. (1990) Population genetic evidence for two species in Anopheles minimus in Thailand. Medical and Veterinary Entomology, 4, 2534.
  • Gu, Z.C. & Zheng, X. (2001) Malaria situation in the People's Republic of China in 2000. Chinese Journal of Parasitology and Parasitic Diseases, 19, 257259.
  • Harbach, R.E. (1994) Review of the internal classification of the genus Anopheles (Diptera: Culicidae): the foundation for comparative systematics and phylogenetic research. Bulletin of Entomological Research, 84, 331342.
  • Harrison, B.A. (1980) Medical entomology studies – XIII. The Myzomyia Series of Anopheles (Cellia) in Thailand, with emphasis on intra-interspecific variations (Diptera: Culicidae). Contributions of the American Entomological Institute, 17, 1195.
  • Harrison, B.A., Rattanarithikul, R., Peyton, E.L. & Mongkolpanya, K. (1990) Taxonomic changes, revised occurrence records and notes on the Culicidae of Thailand and neighboring countries. Mosquito Systematics, 22, 196227.
  • Ho, C. (1965) Study on malaria in China. Science Bulletin, 5, 402.
  • Ho, C. & Feng, L.C. (1958) Studies on malaria in new China. Chinese Medical Journal, 77, 533551.
  • Jiang, C.S., Liu, Z.D., Yu, Y. & Peng, X.M. (1987) Studies on the patterns of nonspecific estarase isozymes of Anopheles (Cellia) minimus Theobald. Acta Entomologica Sinica, 30, 229230.
  • Kengne, P., Trung, H.D., Baimai, V., Coosemans, M. & Manguin, S. (2001) A multiplex PCR-based method derived from random amplified polymorphic DNA (RAPD) markers for the identification of species of the Anopheles minimus group in Southeast Asia. Insect Molecular Biology, 10, 427435.
  • Lei, X.T., Lai, Q., Zhang, S.W., Wei, H.Y. & Xiao, N. (1997) Malaria incidence and geographical distribution of Anopheles vectors in Sichuan. Journal of Applied Parasitic Diseases, 5, 6971.
  • Li, C.K. (1981) Epidemiology of malaria in the New Territories Region, Hong Kong (1975–80). Journal of the Society of Community Medicine, Hong Kong, 12, 2940.
  • Linton, Y.-M., Harbach, R.E., Chang, M.S., Anthony, T.G. & Matusop, A. (2001a) Morphological and molecular identity of Anopheles (Cellia) sundaicus (Rodenwaldt) (Diptera: Culicidae), the nominotypical member of a malaria vector species complex in Southeast Asia. Systematic Entomology, 26, 357366.
  • Linton, Y.-M., Samanidou-Voyadjoglou, A., Smith, L. & Harbach, R.E. (2001b) New occurrence records for Anopheles maculipennis and An. messeae in northern Greece based on DNA sequence data. European Mosquito Bulletin, 11, 3136.
  • Liu, L.C., Fang, C.C. & Hu, M.H. (1959) Study on the forms of Anopheles minimus Theobald, 1901. Acta Entomologica Sinica, 9, 154160.
  • Liu, C.F., Qian, H.L., Tang, L.H., Zheng, X., Gu, Z.C. & Zhu, W.D. (1995) Current malaria stratification in China. Chinese Journal of Parasitology and Parasitic Diseases, 13, 813.
  • Lu, B.L. (1999) Integrated Pest Management of Mosquitoes, 2nd edn. Science Press, Beijing.
  • Lu, B.L. et al.[sic] (1997) Fauna Sinica, Insecta, Vol. 9, Diptera. Culicidae II. Science Press, Beijing.
  • Rao, V.V. (1961) Vectors of Malaria in India, 2nd edn. National Society of India for Malaria and Mosquito-Borne Diseases, Delhi.
  • Reid, J.A. (1968) Anopheline mosquitoes of Malaya and Borneo. Studies from the Institute for Medical Research Malaya, 31, 1520.
  • Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold. Spring Harbor Press, New York.
  • Sharpe, R.G. (1997) The status of cryptic species within Anopheles minimus. PhD Thesis, University of Leeds, Leeds.
  • Sharpe, R.G., Harbach, R.E. & Butlin, R.K. (2000) Molecular variation and phylogeny of members of the Minimus Group of Anopheles subgenus Cellia (Diptera: Culicidae). Systematic Entomology, 25, 263272.
  • Sharpe, R.G., Hims, M.M., Harbach, R.E. & Butlin, R.K. (1999) PCR-based methods for identification of species of the Anopheles minimus group: allele-specific amplification and single-strand conformation polymorphism. Medical and Veterinary Entomology, 13, 265273.
  • Somboon, P., Walton, C., Sharpe, R.G., Higa, Y., Tuno, N., Tsuda, Y. & Takagi, M. (2001) Evidence for a new sibling species of Anopheles minimus from the Ryukyu Archipelago, Japan. Journal of the American Mosquito Control Association, 17, 98113.
  • Subbarao, S.K. (1998) Anopheles species complexes in South-east Asia. WHO Technology Publication, SEARO, 18, 182.
  • Subbarao, S.K., Nanda, N., Vasantha, K., Dua, V.K., Malhotra, M.S., Yadav, R.S. & Sharma, V.P. (1994) Cytogenetic evidence for three sibling species in Anopheles fluviatilis (Diptera: Culicidae). Annals of the Entomological Society of America, 87, 116121.
  • Taiwan (1991) Malaria Eradication in Taiwan. Monograph xxii+300 pp. Department of Health, The Executive Yuan, Taipei, Republic of China.
  • Tang, L.H., Qian, H.L. & Xu, S.H. (1991) Malaria and its control in the People's Republic of China. Southeast Asian Journal of Tropical Medicine and Public Health, 22, 467476.
  • Teng, H.J., Wu, Y.L., Wang, S.L. & Lin, C. (1998) Effect of environmental factors on abundance of Anopheles minimus (Diptera: Culicidae) larvae and their seasonal fluctuation in Taiwan. Environmental Entomology, 27, 324328.
  • Theobald, F.V. (1901) A Monograph of the Culicidae or Mosquitoes1. British Museum (Natural History), London.
  • Theobald, F.V. (1910) A Monograph of the Culicidae or Mosquitoes,5. British Museum (Natural History), London.
  • Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 24, 48764882.
  • Van Bortel, W., Harbach, R.E., Trung, H.D., Roelants, P., Backeljau, T. & Coosemans, M. (2001) Confirmation of Anopheles varuna in Vietnam, previously misidentified and mistargeted as the malaria vector An. minimus. American Journal of Tropical Medicine and Hygiene, 65, 729732.
  • Van Bortel, W., Tho Sochanta, Harbach, R.E., Duong Socheat, Roelants, P., Backeljau, T. & Coosemans, M. (2002) Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species. Medical and Veterinary Entomology, 16, 329334.
  • Van Bortel, W., Trung, H.D., Manh, N.D., Roelants, P., Verle, P. & Coosemans, M. (1999) Identification of two species within the Anopheles minimus complex in northern Vietnam and their behavioural divergences. Tropical Medicine and International Health, 4, 257265.
  • Van Bortel, W., Trung, H.D., Roelants, P., Harbach, R.E., Backeljau, T. & Coosemans, M. (2000) Molecular identification of Anopheles minimus s.l. beyond distinguishing the members of the species complex. Insect Molecular Biology, 9, 335340.
  • Wang, L.L. & Liu, Y.R. (1996) The known species of Anopheles mosquitoes and its relation to disease in Hubei Province. Chinese Journal of Vector Biology and Control, 7, 170173.
  • Wang, X.L., Zhou, N.C., Chen, Z.Y. & Zhou. X. (1997) Comparison of malaria incidence in different areas of Guizhou in 1980–95. Public Hygiene of China, 13, 33.
  • Wang, X.Z., Du, Z.W., Lu. Y.R., Zhu, G.J., Huang, R., Gu, Y., Takagi, M. & Tusda, Y. (1999) Bionomics of Anopheles minimus and its role in malaria transmission in south parts of Yunnan. Annual Report on Malaria Control and Research for 1997–98, Yunnan Institute of Malaria Control, 1999, 155.
  • WHO/WPRO (2002) Malaria, other vector-borne and parasitic diseases. http://www.malaria.wpro.who.int.
  • Wu, K.C., Chen, W.J., Wang, Z.G., Cai, X.Z., Deng, D., Hu, L.K., Liu, Z.Y., Zhu, G., Guan, D.H. & Jiang, W.K. (1993) Studies on distribution and behaviour of Anopheles minimus and its role of malaria transmission in Hainan Province at present. Chinese Journal of Parasitology and Parasitic Diseases, 11, 120123.
  • Xu, S.H. (1997) Control of filariasis in China. Chinese Journal of Parasitic Disease Control, 10, 246249.
  • Yamada, S. (1925) A review of the adult anopheline mosquitoes of Japan: systematic descriptions, their habits, and their relations to human diseases (Part II.). Scientific Reports to the Government Institute of Infections Disease (Tokyo), 4, 447493.
  • Yip, K. (2000) Malaria eradication: the Taiwan experience. Parassitologia, 42, 117126.
  • Yu, Y. (1987) Studies on the two forms of Anopheles (Cellia) minimus Theobald, 1901 in China (Diptera: Culicidae). Mosquito Systematics, 19, 143145.
  • Yu, Y., Fan, B.R., Peng, X.M. & Zeng, L.H. (1989) Observations on the house frequenting behaviour and host preference of two forms of Anopheles (Cellia) minimus Theobald in Hainan. Acta Entomologia Sinica, 32, 253254.
  • Yu, Y. & Li, M.X. (1984) Notes on the two forms of Anopheles (Cellia) minimus Theobald, 1901 in Hainan Island. Journal of Parasitology and Parasitic Diseases, 2, 9598.
  • Zhang, Z.X., Huang, R., He, G.J. & Xiao, Z.R. (1991) The effect of deltamethrin-impregnated mosquito nets for controlling Anopheles minimus and An. sinensis. Chinese Journal of Parasitic Disease Control, 4, 257.
  • Zhu, T.H. & Huang, Q.L. (1995) The current epidemiological characteristics of malaria in Guangdong Province. Chinese Journal of Parasitic Disease Control, 8, 13.