Genetic population structure and introgression in Anopheles dirus mosquitoes in South-east Asia

Genetic structure and species relationships were studied in three closely related mosquito species, Anopheles dirus A, C and D in Thailand using 11 microsatellite loci and compared with previous mitochondrial DNA (mtDNA) data on the same populations. All three species were well differentiated from each other at the microsatellite loci. Given the almost complete absence of mtDNA differentiation between An. dirus A and D, this endorses the previous suggestion of mtDNA introgression between these species. The high degree of differentiation between the northern and southern population of An. dirus C (RST = 0.401), in agreement with mtDNA data, is suggestive of incipient species. The lack of genetic structure indicated by microsatellites in four populations of An. dirus A across northern Thailand also concurs with mtDNA data. However, in An. dirus D a limited but significant level of structure was detected by microsatellites over ~400 km in northern Thailand, whereas the mtDNA detected no population differentiation over a much larger area (>1200 km). There is prior evidence for population expansion in the mtDNA. If this is due to a selective sweep originating in An. dirus D, the microsatellite data may indicate greater barriers to gene flow within An. dirus D than in species A. Alternatively, there may have been historical introgression of mtDNA and subsequent demographic expansion which occurred first in An. dirus D so enabling it to accumulate some population differentiation. In the latter case the lack of migration-drift equilibrium precludes the inference of absolute or relative values of gene flow in An. dirus A and D.     

Complex population history of two Anopheles dirus mosquito species in Southeast Asia suggests the influence of Pleistocene climate change rather than human-mediated effects

Anopheles dirus and Anopheles baimaii are closely related species which feed on primates, particularly humans, and transmit malaria in the tropical forests of mainland Southeast Asia. Here, we report an in-depth phylogeographic picture based on 269 individuals from 21 populations from mainland Southeast Asia. Analysis of 1537 bp of mtDNA sequence revealed that the population history of A. baimaii is far more complex than previously thought. An old expansion (pre-300 kyr BP) was inferred in northern India/Bangladesh with a wave of south-eastwards expansion arriving at the Thai border (ca 135-173 kyr BP) followed by leptokurtic dispersal very recently (ca 16 kyr BP) into peninsular Thailand. The long and complex population history of these anthropophilic species suggests their expansions are not in response to the relatively recent (ca 40 kyr BP) human expansions in mainland Southeast Asia but, rather, fit well with our understanding of Pleistocene climatic change there.     

SCAR markers and multiplex PCR-based identification of isomorphic species in the Anopheles dirus complex in Southeast Asia

The Anopheles dirus Peyton & Harrison complex of mosquitoes (Diptera: Culicidae) comprises seven known species, including important malaria vectors in Southeast Asia. Specific identification of each species of the complex, which cannot be distinguished using morphological characters, is crucial for understanding vector ecology and implementing effective control measures. Derived from individual random amplified polymorphic DNA (RAPD) markers, sequence characterized amplified regions (SCAR) were analysed for the design of specific paired-primers. Combination of six SCAR primers resulted in the development of a simple, robust, single multiplex PCR able to identify three important malaria vectors among the four most common species (A, B, C, D) of the complex: species A from several Southeast Asian countries, species B from Perlis, Malaysia, and species C and D from Thailand.     

Population genetic structure of Anopheles arabiensis mosquitoes in Ethiopia and Eritrea

This study examined the population genetic structure of the major malaria vector, Anopheles arabiensis mosquitoes, in Ethiopia and Eritrea. Ethiopia and Eritrea have great geographical diversity, with high mountains, rugged plateaus, deep gorges, and rolling plains. The plateau is bisected diagonally by the Great Rift Valley into the Northwestern Highlands and the Southeastern Highlands. Five A. arabiensis populations from the Northwestern Highlands region and two populations from high-altitude sites in the Great Rift Valley were genotyped using six microsatellite markers to estimate the genetic diversity and population genetic structure of A. arabiensis. We found that A. arabiensis populations from the Northwestern Highlands and the Great Rift Valley region showed a similar level of genetic diversity. The genetic differentiation (F(ST)) of the five mosquito populations within the Northwestern Highlands region was 0.038 (P <.001), while the two populations within the Great Rift Valley showed little genetic differentiation (F(ST) = 0.007, P <.01). The degree of genetic differentiation between the Northwestern Highlands region and the Great Rift Valley region was small but statistically significant (F(ST) = 0.017, P <.001). The population genetic structure of A. arabiensis in the study area did not follow the isolation-by-distance model (r(2) = 0.014, P >.05). The low F(ST) estimates for A. arabiensis populations in Ethiopia and Eritrea are consistent with the general population genetic structure of this species in East Africa based on other molecular markers.     

Dental perspectives on the population history of Southeast Asia

This article uses metric and nonmetric dental data to test the "two-layer" or immigration hypothesis whereby Southeast Asia was initially occupied by an "Australo-Melanesian" population that later underwent substantial genetic admixture with East Asian immigrants associated with the spread of agriculture from the Neolithic period onwards. We examined teeth from 4,002 individuals comprising 42 prehistoric and historic samples from East Asia, Southeast Asia, Australia, and Melanesia. For the odontometric analysis, dental size proportions were compared using factor analysis and Q-mode correlation coefficients, and overall tooth size was also compared between population samples. Nonmetric population affinities were estimated by Smith's distances, using the frequencies of 16 tooth traits. The results of both the metric and nonmetric analyses demonstrate close affinities between recent Australo-Melanesian samples and samples representing early Southeast Asia, such as the Early to Middle Holocene series from Vietnam, Malaysia, and Flores. In contrast, the dental characteristics of most modern Southeast Asians exhibit a mixture of traits associated with East Asians and Australo-Melanesians, suggesting that these populations were genetically influenced by immigrants from East Asia. East Asian metric and/or nonmetric traits are also found in some prehistoric samples from Southeast Asia such as Ban Kao (Thailand), implying that immigration probably began in the early Neolithic. Much clearer influence of East Asian immigration was found in Early Metal Age Vietnamese and Sulawesi samples. Although the results of this study are consistent with the immigration hypothesis, analysis of additional Neolithic samples is needed to determine the exact timing of population dispersals into Southeast Asia.     

Human population history at the crossroads of East and Southeast Asia since 11,000 years ago

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Evidence for late Pleistocene population expansion of the malarial mosquitoes, Anopheles arabiensis and Anopheles gambiae in Nigeria

Anopheles gambiae Giles s.s. and Anopheles arabiensis Patton (Diptera: Culicidae) are major vectors of malaria in Nigeria. We used 1115 bp of the mitochondrial COI gene to assess their population genetic structures based on samples from across Nigeria (n = 199). The mtDNA neighbour-joining tree, based on F(ST) estimates, separated An. gambiae M and S forms, except that samples of An. gambiae M from Calabar clustered with all the An. gambiae S form. Anopheles arabiensis and An. gambiae could be combined into a single star-shaped, parsimonious haplotype network, and shared three haplotypes. Haplotype diversity values were high in An. arabiensis and An. gambiae S, and intermediate in An. gambiae M; all nucleotide diversities were relatively low. Taken together, patterns of haplotype diversity, the star-like genealogy of haplotypes, five of seven significant neutrality tests, and the violation of the isolation-by-distance model indicate population expansion in An. arabiensis and An. gambiae S, but the signal was weak in An. gambiae M. Selection is supported as an important factor shaping genetic structure in An. gambiae in Nigeria. There were two geographical subdivisions in An. arabiensis: one included all southern localities and all but two central localities; the other included all northern and two central localities. Re-analysing an earlier microsatellite dataset of An. arabiensis using a Bayesian method determined that there were two distinctive clusters, northern and southern, that were fairly congruent with the mtDNA subdivisions. There was a trend towards decreasing genetic diversity in An. arabiensis from the northern savannah to the southern rainforest that corroborated previous data from microsatellites and polytene chromosomes.     

Transmission of the OS strain of Plasmodium inui to Saimiri sciureus boliviensis and Aotus azarae boliviensis monkeys by Anopheles dirus mosquitoes

Eight Saimiri and 7 Aotus monkeys were exposed to infection with the OS strain of Plasmodium inui via the bites of from 2 to 7 Anopheles dirus mosquitoes. All Saimiri monkeys developed high-level infections of from 152,000 to 500,000/mm3 after prepatent periods of from 14 to 17 days. Only 1 Aotus monkey developed a patent infection after a period of 28 days. Feeding on these animals failed to result in infection of An. dirus mosquitoes.     

Rodent Plasmodium: population dynamics of early sporogony within Anopheles stephensi mosquitoes

Early sporogony of Plasmodium parasites involves 2 major developmental transitions within the insect vector, i.e., gametocyte-to-ookinete and ookinete-to-oocyst. This study compared the population dynamics of early sporogony among murine rodent Plasmodium (Plasmodium berghei, Plasmodium chabaudi, Plasmodium vinckei, and Plasmodium yoelii) developing within Anopheles stephensi mosquitoes. Estimates of absolute densities were determined for gametocytes, ookinetes, and oocysts for 108 experimental infections. Total losses throughout early sporogony were greatest in P. vinckei (ca. 250,000-fold loss), followed by P. yoelii (ca. 70,000-fold loss), P. berghei (ca. 45,000-fold loss), and P. chabaudi (ca. 15,000-fold loss). The gametocyte-to-ookinete transition represented the most severe population bottleneck. Numerical losses during this transition (ca. 3,000- to 30,000-fold, depending on species) were orders of magnitude greater than losses incurred during the ookinete-to-oocyst transition (3- to 14-fold). There were no significant correlations between gametocyte and ookinete densities. Significant correlations between ookinete and oocyst densities existed for P. berghei, P. chabaudi, and P. yoelii (but not for P. vinckei), and were best described by nonlinear functions (P. berghei = sigmoid, P. chabaudi = hyperbolic, P. yoelii = sigmoid), indicating that conversion of ookinetes to oocysts in these species is density dependent. The upper theoretical limit for oocyst density on the mosquito midgut for P. chabaudi and P. yoelii (ca. 300 oocysts per midgut) was higher than for P. berghei (ca. 30 oocysts per midgut). This study provides basic information about population processes that occur during the early sporogonic development of some common laboratory model systems of malaria.     

Predicted Distribution of Major Malaria Vectors Belonging to the Anopheles dirus Complex in Asia: Ecological Niche and Environmental Influences

Methods derived from ecological niche modeling allow to define species distribution based on presence-only data. This is particularly useful to develop models from literature records such as available for the Anopheles dirus complex, a major group of malaria mosquito vectors in Asia. This research defines an innovative modeling design based on presence-only model and hierarchical framework to define the distribution of the complex and attempt to delineate sibling species distribution and environmental preferences. At coarse resolution, the potential distribution was defined using slow changing abiotic factors such as topography and climate representative for the timescale covered by literature records of the species. The distribution area was then refined in a second step using a mask of current suitable land cover. Distribution area and ecological niche were compared between species and environmental factors tested for relevance. Alternatively, extreme values at occurrence points were used to delimit environmental envelopes. The spatial distribution for the complex was broadly consistent with its known distribution and influencing factors included temperature and rainfall. If maps developed from environmental envelopes gave similar results to modeling when the number of sites was high, the results were less similar for species with low number of recorded presences. Using presence-only models and hierarchical framework this study not only predicts the distribution of a major malaria vector, but also improved ecological modeling analysis design and proposed final products better adapted to malaria control decision makers. The resulting maps can help prioritizing areas which need further investigation and help simulate distribution under changing conditions such as climate change or reforestation. The hierarchical framework results in two products one abiotic based model describes the potential maximal distribution and remains valid for decades and the other including a biotic mask easy to update with frequently available information gives current species distribution.     

Life history consequences of larval foraging depth differ between two competing Anopheles mosquitoes

1. Anopheline larvae are surface feeders and allocate most of their time to search for food at the water surface. However, species of the Anopheles gambiae Giles complex may also show bottom feeding. The consequences of this foraging tactic for life history are unknown, yet may be relevant to understand inter‐specific competition patterns. 2. The diving ability and activity of larvae of the main African malaria vectors, An. coluzzii and An. gambiae, at two different water depths (14 and 30 cm) were assessed. We further explored the biological relevance of diving for food harvesting by monitoring key life history traits in two species treatments (single or mixed species) and two food treatments (surface or bottom feeding). 3. Overall, An. coluzzii larvae showed more diving activity than An. gambiae. When feeding at the bottom both species, and especially An. gambiae, showed a delayed emergence and a reduced emergence rate. Moreover, An. gambiae also suffered a reduced wing length. 4. Mixed‐species rearing had a detrimental effect on the life history traits of An. gambiae but not on An. coluzzii, suggesting a competitive advantage for the latter in our experimental conditions. 5. The present results confirm that anopheline larvae are able to forage for food at the bottom of their breeding site and that An. coluzzii shows a superior diving activity than An. gambiae and this at a lower cost. These behavioural differences probably reflect specific adaptations to different aquatic habitats, and may be important in shaping species distributions and the population biology of these important vector mosquitoes.     

High Resolution Population Distribution Maps for Southeast Asia in 2010 and 2015

Spatially accurate, contemporary data on human population distributions are vitally important to many applied and theoretical researchers. The Southeast Asia region has undergone rapid urbanization and population growth over the past decade, yet existing spatial population distribution datasets covering the region are based principally on population count data from censuses circa 2000, with often insufficient spatial resolution or input data to map settlements precisely. Here we outline approaches to construct a database of GIS-linked circa 2010 census data and methods used to construct fine-scale (∼100 meters spatial resolution) population distribution datasets for each country in the Southeast Asia region. Landsat-derived settlement maps and land cover information were combined with ancillary datasets on infrastructure to model population distributions for 2010 and 2015. These products were compared with those from two other methods used to construct commonly used global population datasets. Results indicate mapping accuracies are consistently higher when incorporating land cover and settlement information into the AsiaPop modelling process. Using existing data, it is possible to produce detailed, contemporary and easily updatable population distribution datasets for Southeast Asia. The 2010 and 2015 datasets produced are freely available as a product of the AsiaPop Project and can be downloaded from: www.asiapop.org.     

The Anopheles dirus complex: spatial distribution and environmental drivers

Background

The Anopheles dirus complex includes efficient malaria vectors of the Asian forested zone. Studies suggest ecological and biological differences between the species of the complex but variations within species suggest possible environmental influences. Behavioural variation might determine vector capacity and adaptation to changing environment. It is thus necessary to clarify the species distributions and the influences of environment on behavioural heterogeneity.

Methods

A literature review highlights variation between species, influences of environmental drivers, and consequences on vector status and control. The localisation of collection sites from the literature and from a recent project (MALVECASIA) produces detailed species distributions maps. These facilitate species identification and analysis of environmental influences.

Results

The maps give a good overview of species distributions. If species status partly explains behavioural heterogeneity, occurrence and vectorial status, some environmental drivers have at least the same importance. Those include rainfall, temperature, humidity, shade, soil type, water chemistry and moon phase. Most factors are probably constantly favourable in forest. Biological specificities, behaviour and high human-vector contact in the forest can explain the association of this complex with high malaria prevalence, multi-drug resistant Plasmodium falciparum and partial control failure of forest malaria in Southeast Asia.

Conclusion

Environmental and human factors seem better than species specificities at explaining behavioural heterogeneity. Although forest seems essential for mosquito survival, adaptations to orchards and wells have been recorded. Understanding the relationship between landscape components and mosquito population is a priority in foreseeing the influence of land-cover changes on malaria occurrence and in shaping control strategies for the future.     

Malaria and the Anopheles mosquitoes of Tajikistan

Surveys of Anopheles mosquitoes were conducted in urban, rural, and natural areas of Tajikistan to obtain updated information on their distributions, especially in southern districts of the country where malaria is a prevalent disease. Nine species of Anopheles are found in Tajikistan. Anopheles superpictus, An. claviger, An. hyrcanus, and An. pulcherrimus are the most widespread and abundant species. Investigations in northern Tajikistan confirmed the presence of An. artemievi and the absence of An. martinius, both members of the An. maculipennis complex of malaria vectors. Anopheles barianensis, An. lindesayi, and An. marteri sogdianus, species previously recorded in the country, were not encountered during our surveys. The history of Anopheles and malaria research in Tajikistan is reviewed and bionomical and distributional information is provided for each of the nine species.     

Genetic Admixture in the Culturally Unique Peranakan Chinese Population in Southeast Asia

The Peranakan Chinese are culturally unique descendants of immigrants from China who settled in the Malay Archipelago ∼300–500 years ago. Today, among large communities in Southeast Asia, the Peranakans have preserved Chinese traditions with strong influence from the local indigenous Malays. Yet, whether or to what extent genetic admixture co-occurred with the cultural mixture has been a topic of ongoing debate. We performed whole-genome sequencing (WGS) on 177 Singapore (SG) Peranakans and analyzed the data jointly with WGS data of Asian and European populations. We estimated that Peranakan Chinese inherited ∼5.62% (95% confidence interval [CI]: 4.76–6.49%) Malay ancestry, much higher than that in SG Chinese (1.08%, 0.65–1.51%), southern Chinese (0.86%, 0.50–1.23%), and northern Chinese (0.25%, 0.18–0.32%). A sex-biased admixture history, in which the Malay ancestry was contributed primarily by females, was supported by X chromosomal variants, and mitochondrial (MT) and Y haplogroups. Finally, we identified an ancient admixture event shared by Peranakan Chinese and SG Chinese ∼1,612 (95% CI: 1,345–1,923) years ago, coinciding with the settlement history of Han Chinese in southern China, apart from the recent admixture event with Malays unique to Peranakan Chinese ∼190 (159–213) years ago. These findings greatly advance our understanding of the dispersal history of Chinese and their interaction with indigenous populations in Southeast Asia.     

Selection of Anopheles dirus for refractoriness and susceptibility to Plasmodium yoelii nigeriensis

Two lines of the Oriental malaria vector mosquito Anopheles dirus species A (Diptera: Culicidae), one fully refractory and one fully susceptible to Plasmodium yoelii nigeriensis (an African rodent malaria parasite), were established after 17 generations of mass selection, followed by single female selection for one or two generations. Prior to selection, the stock colony of An. dirus was 17% refractory. Both lines of An. dirus produced abundant ookinetes that started to invade the midgut within 24h post-infection, as seen in histological sections. In most of the refractory mosquitoes, oocysts stopped development <12 h post-invasion, indicating a rapid defence mechanism. Dead P. y. nigeriensis parasites were apparently localized as small melanized spots (2-5 microm) seen in wet preparations of mosquito midguts dissected 5-7 days post infective bloodmeal. In some refractory An. dirus females, apart from the spots, a small number of totally encapsulated oocysts (c. 10 microm) were also present. These larger melanized parasites predominated in a few females: they appeared 2-3 days post-infection as a secondary delayed defence mechanism. The progeny of reciprocal matings between susceptible and refractory lines had approximately 50% susceptibility. Backcrosses of F1 hybrids with susceptible or refractory lines increased or decreased the susceptibility of backcross progeny accordingly. Overall, these results suggest polygenic control of susceptibility to P. y. nigeriensis infection. The refractory line of An. dirus showed normal susceptibility to natural infections of the human malarias P. falciparum and P. vivax from local patients.     

Evolutionary history of Helicobacter pylori sequences reflect past human migrations in Southeast Asia

The human population history in Southeast Asia was shaped by numerous migrations and population expansions. Their reconstruction based on archaeological, linguistic or human genetic data is often hampered by the limited number of informative polymorphisms in classical human genetic markers, such as the hypervariable regions of the mitochondrial DNA. Here, we analyse housekeeping gene sequences of the human stomach bacterium Helicobacter pylori from various countries in Southeast Asia and we provide evidence that H. pylori accompanied at least three ancient human migrations into this area: i) a migration from India introducing hpEurope bacteria into Thailand, Cambodia and Malaysia; ii) a migration of the ancestors of Austro-Asiatic speaking people into Vietnam and Cambodia carrying hspEAsia bacteria; and iii) a migration of the ancestors of the Thai people from Southern China into Thailand carrying H. pylori of population hpAsia2. Moreover, the H. pylori sequences reflect iv) the migrations of Chinese to Thailand and Malaysia within the last 200 years spreading hspEasia strains, and v) migrations of Indians to Malaysia within the last 200 years distributing both hpAsia2 and hpEurope bacteria. The distribution of the bacterial populations seems to strongly influence the incidence of gastric cancer as countries with predominantly hspEAsia isolates exhibit a high incidence of gastric cancer while the incidence is low in countries with a high proportion of hpAsia2 or hpEurope strains. In the future, the host range expansion of hpEurope strains among Asian populations, combined with human motility, may have a significant impact on gastric cancer incidence in Asia.