Distribution of Anopheles in Vietnam, with particular attention to malaria vectors of the Anopheles minimus complex

Background The distribution of anopheline mosquitoes in Vietnam was examined, with a particular interest for the two sibling species of the Anopheles minimus complex (Cellia: Myzomyia), An. minimus and Anopheles harrisoni, respectively former species A and C. Because the morphological identification of both sibling species is difficult and may lead to misidentifications, accurate data on their respective distribution are missing. This is of fundamental importance since the two species seem to exhibit differential vectorial capacities for malaria transmission. Methods Large entomological surveys based on cattle collections and molecular identifications of An. minimus s.l. were carried out in 23 sites throughout northern, central and south-eastern regions of Vietnam. Results Based on previous molecular works and our data, the distribution of anopheline species and the relative densities of An. minimus and An. harrisoni were mapped. It is noteworthy that there was a high specific biodiversity at each study site. Anopheles minimus s.l. and Anopheles sinensis were the main anopheline species in the northern region, whereas Anopheles aconitus and Anopheles vagus were the most frequent ones in the central region. The southern limit of An. harrisoni was increased to the latitude of 11°N. Sympatry between both sibling species has been extended to new provinces. Conclusion Malaria transmission is still high in central Vietnam and along bordering countries. Therefore, it is important to know and map the precise distribution of the main and secondary malaria vectors in Vietnam for applying efficient vector control programmes. Moreover, these maps should be regularly updated and linked to environmental characteristics relative to disease epidemiology, and environmental and climatic changes occurring in southeast Asia.


Background
The main malaria vector Anopheles minimus s.l. (Myzomyia Series, Funestus Group) is composed of three sibling species, An. minimus (former species A), Anopheles harrisoni (former species C), and An. minimus species E [1]. Anopheles minimus and An. harrisoni can be sympatrically distributed over the southeast Asian mainland whereas species E is restricted to the Ishigaki Island in the Ryukyu Archipelago, Japan, a malaria free region [2,3]. Because the morphological identification of the two sympatric species is unreliable [4], accurate data on the distribution of An. minimus and An. harrisoni are missing [3]. Morphological misidentifications with the closely related sympatric species, such as Anopheles aconitus, Anopheles pampanai and Anopheles varuna, are common [5,6]. Recently, molecular identification assays have been developed which can be used to shed light on the specific distribution of each sibling species [7][8][9][10].
Anopheles minimus s.l. occurs in hilly, forested areas [5]. Northern Vietnam exhibits different types of landscapes ranging from the plains of the Red River to high mountains (3,143

meters). The most common larval habitats of
An. minimus s.l. are streams or canals, with slow-running water partially shaded by grassy margins [5]. In central and south-eastern region of Vietnam, both An. minimus s.l. and Anopheles dirus s.l. occur [3,11]. The latter is the main malaria vector associated with forests or rubber plantations. Even if malaria epidemics have been hardly reported in northern Vietnam for the past 10 years, central Vietnam is still a high malaria risk area [12,13].
To date, throughout Vietnam, An. minimus was molecularly reported in 17 provinces; and An. harrisoni confirmed in nine provinces ( Figure 1, Table 1).
Since these sibling species may exhibit different vectorial capacities [14,15], it is fundamental to know the precise distribution of each species and to infer potential risk zones. Moreover, the southeast Asian region is currently under strong environmental changes that may alter with the vector distribution and malaria epidemiology. Therefore, the overall aim of this work was (1) to define the anopheline biodiversity in Vietnam; (2) to review the literature on molecular records of An. minimus s.l.; (3) to compile a distribution map for the two members of the

Methods
A total of 23 sites in 15 provinces were selected for mosquito collections in different geographical areas of northern, central and south-eastern Vietnam ( Figure 1, red dots, Table 2). Adult mosquitoes were captured on cattle bait once, from October 2003 to November 2004, during a period ranging from 3 to 10 nights (Table 2). One to 5 cattle-sheds were sampled by two collectors from 21 h to midnight ( Table 2). Details on mosquito collections are given in Table 2.
Adult mosquitoes were morphologically identified in the field using the standardized key for the medically important anophelines of southeast Asia [16]. Specimens of An. minimus s.l. were DNA-extracted [17] and identified with an allele specific-PCR (AS-PCR) assay [18].

Results and Discussion
This work provides updated distribution of anopheline biodiversity in Vietnam ( Figure 2) and deepens and extends our knowledge on the distribution of An. minimus, An. harrisoni and associated anopheline species  Table 1. The close-up on the northern region is for a better reading.
line fauna throughout Vietnam and not to focus only on malaria vectors. In addition, studies have shown the zoophilic behaviour of known malaria vectors [15,19], which makes this collection method the most appropriate for our objective of mapping the anopheline biodiversity in Vietnam. The results illustrated and reinforced that oriental Anopheles are clearly opportunistic mosquitoes with trophic behaviour heterogeneities as previously found in Vietnam [15] and Thailand [20]. It is noteworthy that no individuals of An. pampanai and An. varuna were identified, although both species have already been recorded in central Vietnam (Khanh Hoa and Binh Thuan Provinces [7,8]). One assumption could be that An. pampanai and An. varuna, being so closely related to An. aconitus [5], may have been morphologically misidentified as An. aconitus [5]. The An. aconitus populations, identified on morphological characters only, need to be screened with the AS-PCR assay in order to avoid any potential misidentifications. If this assumption is confirmed, the density of An. aconitus would have been over-estimated. The results showed that An. minimus s.l., main malaria vector, and secondary malaria vectors such as An. aconitus, are present throughout Vietnam independently of the type of landscapes, except the costal fringe where other malaria vectors are present.
Based on morphological identifications, a total of 19 anopheline species were collected and identified throughout the northern to south-eastern regions of Vietnam (Table 3) out of the 38 taxa of the Indochina fauna [12].
A total of 706 An. minimus s.l. were DNA-extracted and identified as An. minimus or An. harrisoni for all the sites, except five sites with samples not available and three sites with no An. minimus s.l. populations (Table 3). Only two misidentifications in the An. minimus samples (two An. minimus s.l instead of two An. aconitus) were noticed for the VSLA site (northern region). No sites with An. harrisoni only were found neither for the northern region nor the central one ( Figure 3). Interestingly enough, sites where An. harrisoni was dominant, An. minimus was often rare. This is the case in two sites in the Son La Province (northern region). This may reflect a competition between these two sibling species which needs to be further investigated. Molecular species identifications were intentionally not carried out for the Dirus and Maculatus complexes, because the Minimus complex was targeted. Recently, Obsomer et al. [11] published an updated review and clarified the distribution of the Dirus complex in southeast Asia.

Northern Vietnam
Anopheles minimus s.l. and An. sinensis were the main species collected on cattle over the northern region, followed by An. aconitus and An. jeyporiensis (Figure 2). Two sites were negative for An. minimus s.l. (VNAA and VLCB) and one site (VTHB) presented a very low density (Table 3, Figure 2).
The VNAA village is surrounded by pine plantations. The overall number of anophelines collected was low (204 specimens during six nights) and An. minimus s.l. was not present during the time of our survey. The absence of An. minimus s.l. in the VLCB site is likely explained by the high altitude (1,256 m). Actually in Vietnam, An. minimus s.l. is commonly found at an altitude ranging from 200 to 800 meters and becomes quite rare at altitudes above 1,500 meters [5,21]. The low density of An. minimus s.l. in the VTHB site (only three specimens) may be due to very recent agricultural changes linked to modified irrigation systems that may have disturbed the mosquito larval habitats, phenomenon also observed in Thailand [22] and India [23]. The main species in this site were An. sinensis and Anopheles philippinensis, the latter species having much lower densities in all the other sites and is known to occur in agricultural areas [24]. Map of the relative densities for each anopheline taxon Figure 2 Map of the relative densities for each anopheline taxon. The close-up on northern region is for a better reading.
Regarding the distribution reported in previous studies in northern Vietnam (Figure 1), the results bring new insights on the distribution of the Minimus complex. For the first time, the anopheline composition of the extreme eastern area of the northern region was analysed and the sympatry of the two species has been reported in the three new study sites in Quanh Ninh (VQNA, VQNB) and Lang Son (VLSA) Provinces. More westwards, Phuc et al. [10] reported An. minimus only in their two sites in Lao Cai Province. However, one site (VLCA) was found positive for both sibling species in this province. Further south, the two sites VTHA and VNAB showed two different situations in the hilly areas of Thanh Hoa and Nghe An Provinces, respectively sympatric populations and An. minimus only.
In northern Vietnam, malaria is relatively well controlled but still occurs at the border of China and Laos [12]. However, the presence of main vectors, such as An. minimus and possibly An. harrisoni, represents a permanent threat for the resurgence of malaria in this region with the move-ment of infected human populations from endemic areas such as central Vietnam and bordering countries.

Central and south-eastern Vietnam
Anopheles aconitus and An. vagus were the most frequent species in these regions ( Table 3, Figure 2). Anopheles dirus s.l. was found in low densities in two sites only (VGLA and VPYA) (Table 3, Figure 2). It occurs typically in forests and forested fringes, either natural or man-made, such as rubber plantations where its contribution to malaria transmission is quite important in central Vietnam and throughout southeast Asia [11]. It is a highly anthropophilic mosquito [11] which explains the low densities collected on cattle during our surveys (Table 3).
One site (VKTA) was negative for An. minimus s.l. and four sites among the seven had low densities ranging between one and six specimens (Table 3, Figure 2). Anopheles harrisoni was recently found in Khanh Hoa Province [9] (site 24, Figure 1) and as recorded previously in three northern sites, the densities of this species are now more important Distribution of the two sibling species of the Minimus Complex based on records obtained from this study and from the liter-ature Figure 3 Distribution of the two sibling species of the Minimus Complex based on records obtained from this study and from the literature. Numbers refer to Table 1. The close-up on northern region is for a better reading.   than those of An. minimus which was reportedly the dominant species until 1999 [9]. This was the first record of this species in central Vietnam at a latitude of 12°N [9]. These results increased the southern limit of An. harrisoni to Binh Duong Province (VBGA), located in south-eastern Vietnam, at a latitude of 11°N, less than 100 km northwest of Ho Chi Minh City. This is a new site where An. minimus and An. harrisoni are sympatric.
Anopheles aconitus, the Maculatus complex and An. vagus were collected in high densities in central Vietnam (Table  3). Anopheles maculatus s.l. is thought to be one of the main vector in the Oriental region [25][26][27], and An. aconitus is considered a secondary vector in Bangladesh, India, Indonesia (Java) and Thailand [22,25,28,29]. Compiling distribution maps based on large sample collections is not an academic exercise. Knowing precise and accurate geographic distributions of vector species is an important prerequisite for: (1) an adequate choice of zones where vector control actions should focus, (2) a better selection of future study sites for entomologists working on secondary or local vector species, (3) studies on anopheline biodiversity relating to environmental and climatic changes, (4) analyses of landscape-species associations, and (5) modelling malaria risk maps or comparison of predictive ecological maps with field observations.

Conclusion
Since malaria transmission still occurs in central Vietnam and population movement from the centre to the north are important, vector control measures must be maintained. In addition, entomological, epidemiological and climatic data need to be integrated into a Geographic Information System to follow the future trends of the disease and to assess the malaria risk zones. These maps should be regularly updated and linked to environmental characteristics of disease epidemiology relating to environmental and climatic changes occurring in southeast Asia.