Background
Under the present scenario of human-driven environmental changes, global climate change is one the most relevant concerns. Climatic predictions point to a significant increase of summer droughts in south-western European regions over the next 60 years, but there is also an increased risk for more frequent flash floods during the same period [
1]. Since the life cycles and distribution of many insect vector species are directly influenced by climatological conditions, climate change has the potential to affect the incidence, seasonal transmission and geographic range of several vector-borne diseases [
2]. It is still not clear, however, if the impact of climate change will be beneficial or adverse. Mosquito populations may tend to expand with warming and changes in rainfall patterns, which will tend to increase disease transmission. On the other hand, mosquito reproduction and survival could be impaired by altered rainfall and increased aridity leading to a reduction in transmission [
2]. Nonetheless, the overall effect of anthropogenic climate change on vector-borne diseases remains debated, and the outcome may vary regionally [
3].
Malaria is the vector-borne disease with the highest impact in the World's human population. In 2008, there were
ca. 243 million cases, and an estimated 863,000 deaths attributed to malaria [
4]. Although at present malaria endemic areas are mainly restricted to tropical and subtropical regions, several models project a geographical expansion of potential malaria transmission in the next few decades, and more substantial changes later this century [
2].
Malaria was endemic in Europe until the mid 20
th century [
5]. The eradication of malaria in the European region was largely due to a combination of changes in farming and husbandry, improvement in house construction and vector control. However, in recent years, the disease re-emerged in residual foci in Eastern Europe (Azerbaijan, Georgia, Kyrgyzstan, Tajikistan, Turkey and Uzbekistan), resulting in more than 30,000 malaria cases in the year 2000 [
4]. Since then, intensive control activities have been re-implemented throughout the affected region, and the number of reported cases has been reduced substantially to 660 in 2008 [
4].
Although the risk of malaria re-emergence is uncertain for Western/Southern European countries, the present climate change situation gave rise to some concern. One of the reasons was a predicted increase in mosquito vectorial capacity, especially in the southern countries of Europe and the Mediterranean [
6]. This in conjunction with the increasing intercontinental human movement may favour the re-establishment of autochthonous malaria transmission.
The former European malaria vectors were mainly members of the
Anopheles maculipennis complex that are still widely distributed throughout the continent [
7]. This complex comprises 13 Palearctic sibling species, of which
Anopheles atroparvus,
Anopheles labranchiae and
Anopheles sacharovi were the main malaria vectors in the European region. In Europe, the distribution of
An. atroparvus ranges from Britain to Russia (north Caucasus). It is absent in some Mediterranean regions, such as southern Italy, Greece and Turkey [
8].
Because its importance as a disease vector has declined, research on the biology of An. atroparvus and its sibling species has decreased in the last decades. However, the concern with malaria re-emergence has resulted in a revival of interest in European Anopheles mosquitoes. In this context, knowledge about the population structure and levels of gene flow in this species is of major importance to infer the potential for the re-establishment and spreading of malaria transmission under a scenario of local introduction of parasites. Furthermore, if necessary, it will be a critical tool for the design of vector control plans. In this study, genetic and phenotypic variation was analysed, using microsatellites and geometric morphometrics, in order to determine patterns of population structure of An. atroparvus in Southern Europe.
Discussion
Microsatellite analysis of eight European samples of
An. atroparvus indicates levels of genetic diversity similar to those described for other anopheline species of tropical regions, particularly from sub-Saharan Africa. The estimates of mean expected heterozygosity (0.61≤
H
e
≤0.73) are within the range of those obtained for the Afrotropical primary malaria vectors
Anopheles gambiae s.s. (0.57≤
H
e
≤0.71; [
31]),
Anopheles arabiensis (0.65 ≤
H
e
≤0.78; [
32]) and
Anopheles funestus (0.64≤
H
e
≤0.78; [
33]). In temperate climates, anopheline populations display marked seasonal variations in abundance, reaching high densities only during the summer months [
7,
34]. The high levels of genetic diversity suggest that
An. atroparvus populations are able to maintain large effective population sizes in spite of the marked seasonality imposed by the winter cold temperatures. A similar scenario is also met by Afrotropical vector populations in dry savanna/sahelian regions. In
An. arabiensis, the strong seasonal fluctuations in abundance do not seem to affect the overall genetic diversity and current effective population size in dry areas of Sudan and Senegal, where rains last for less than five months [
35,
36].
Estimates of genetic differentiation among An. atroparvus samples spanning over 3,000 km suggest a shallow population structure weakly correlated with geographic distance. This was evident when the most distant sample of Romania was excluded from the isolation-by-distance analysis. In addition, there was no particular pattern of population subdivision that could be attributable to the presence of two potential barriers to gene flow, the Pyrenees and the Alps. These mountain chains physically isolate the populations from the Iberian and Italian Peninsulas, respectively. For example, F
ST
estimates between France and Portugal (0.010-0.016) were considerably lower than those between Portugal and Spain (0.048-0.056).
The shallow patterns of population structure here reported for
An. atroparvus are consistent with those observed in most primary malaria vector species from tropical climates (reviewed in [
37]). Among the possible reasons for these patterns are historical demographic perturbations, particularly population expansions. These events may mask current levels of population structure and gene flow by disrupting the balance between migration and drift [
37]. Evidence for recent population expansions have been documented for several malaria vectors such as
An. gambiae s.s. and
An. arabiensis[
38],
Anopheles dirus A and D [
39] and
Anopheles minimus[
40]. In the two later examples, the signatures of population expansion have been associated with Pleistocene climate changes. This scenario can also be hypothesized for
An. atroparvus as these populations were most likely affected by the Last Glacial Maximum,
ca. 18,000 years ago. In addition, population perturbations could also derive from the vector control actions implemented by the European malaria eradication programmes of the 1950's. Therefore, it is possible that the observed patterns of differentiation reflect differences in demographic history rather than contemporary gene flow among populations.
In contrast with within species comparisons, microsatellites revealed high differentiation between
An. atroparvus samples and the only
An. maculipennis s.s. sample analysed, a result consistent with their sibling species status. This was particularly evident by the FCA analysis in which there was a complete cluster separation between the two species. Values of
F
ST
between 0.20 and 0.30 are similar those described between other anopheline sibling species (
e.g. An. gambiae/An. arabiensis: 0.25, [
41];
An. dirus complex: 0.21-0.39, [
42]).
Phenotypic differentiation between
An. atroparvus and
An. maculipennis s.s. was not so evident. There was a partial overlapping between the clusters of the two species in the PCA. Furthermore, most
An. atroparvus specimens of France shared the same dimensional space with
An. maculipennis s.s. With the exception of this comparison, all the remaining
An. atroparvus samples had significantly lower wing centroid sizes. These results concur with the notion of a relatively recent divergence time among the Palearctic members of the
An. maculipennis complex not sufficient for the accumulation of phenotypic differences, in contrast to that of the Nearctic members of the complex [
43,
44]. A similar pattern was also observed by multivariate morphometric analysis between the recently separated
An. gambiae s.s. and
An. arabiensis in which the later species displayed significantly larger measures but still with overlapping distributions [
45].
Within
An. atroparvus, low to moderate levels of phenotypic differentiation were detected between samples by pairwise estimates of
D
M
. However, there was no correlation between phenotypic differentiation and geographic or genetic distances. In mosquito populations, phenotypic variation is influenced by an assortment of environmental factors that include temperature, altitude, nutritional factors at the immature stages and host population distribution [
45‐
47]. The levels of phenotypic variation in our samples are more likely to reflect the local environmental pressures to which these populations are subjected.
Conclusions
The genetic and phenotypic variation among populations of the former European malaria vector
An. atroparvus were analysed for the first time over a range of more than 3,000 km.The low levels of differentiation observed were not correlated with geographic distance or with potential physical barriers separating these populations. While these results may suggest considerable levels of gene flow, other explanations such as the effect of historical population perturbations can be hypothesized. Further genetic studies involving the analysis of temporal samples of
An. atroparvus will help clarifying the recent demographic history of this species. In addition, analysis should also be extended to northern European locations, where
An. atroparvus populations are also established and sometimes display biological differences [
7]. Such analysis would provide new insights on the effect of temperature clines in the genetic structure of this vector. This will be essential to more precisely determine the degree of contemporary gene flow and hence the potential for mosquito-mediated spread of malaria parasites in the event of a focal re-establishment of malaria transmission. Likewise, it remains to be ascertained which local factors are governing phenotypic variation among these populations and how these may impact mosquito physiological and bio-ecological traits influencing vectorial capacity.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JLV, CAS, EF, JML, CT, HB, MDL, LT, RA, MDB, SM-C, DB, RR, GN and DF carried out mosquito surveys and morphological identification of An. maculipennis s.l. samples. Molecular analyses were done by JLV, CAS, RA, PS and TLS. JP, PS and JLV carried out the statistical analysis of the genetic data. Geometric morphometrics was performed by BA, SSC, BD and NO. The study was conceived and design by JP, BA, DF, VER, MDB, RR and GN. JP, JLV, PS, BA and BD drafted the manuscript with the contributions of TLS, VER and RA. All authors read and approved the manuscript.