Background
The susceptibility of
Anopheles mosquitoes to
Plasmodium infection is defined by the ability of a mosquito vector to support parasite development from gamete fertilization through to sporozoite production. This susceptibility ranges from complete receptiveness, where all individuals support infection, to the opposite end of the spectrum, total refractoriness, where no individuals support infection. The majority of mosquito vectors are positioned somewhere between the two extremes depending on geographical origin of both the parasite and the mosquito [
1‐
10]. In essence, there is a degree of compatibility that exists between parasite and mosquito and the extent of this compatibility determines the successful transmission of infection.
Theoretical co-evolutionary models propose that parasites are locally adapted to their hosts and that parasite fitness decreases as geographical distance from the host increases [
11]. Thus,
Plasmodium falciparum parasites would be expected to transmit more successfully through a local, indigenous, mosquito species rather than a non-local species. Local adaptation in
Plasmodium was investigated previously during the era of malaria eradication, when experiments largely focused on the susceptibility of
Anopheles mosquitoes from non-malarious areas to tropical
P. falciparum parasites. In particular, the re-introduction of malaria to Europe was examined, leading to the discovery that indigenous European mosquitoes are almost completely refractory to tropical
P. falciparum infection [
1,
3‐
5]. In contrast, work carried out in North America demonstrated that the indigenous vector,
Anopheles freeborni, is highly susceptible to many species of
Plasmodium from widely separated geographical areas (reviewed [
12]).
Contemporary patterns of human migration provide numerous occasions for allopatric parasite populations to interbreed allowing the transference of novel phenotypes, such as drug resistance, to new parasite populations both within and between continents. A better understanding of the limitations imposed by mosquito susceptibility would be advantageous in predicting the potential spread of these undesirable phenotypes and allow cost-effective vector control measures to be implemented. This paper aims to build on previous investigations and examine the compatibility between parasite and host by examining the transmission of tropical P. falciparum parasites through non-indigenous tropical vectors. Indeed an extensive survey of the literature reveals that this is the first published report describing the attempted challenge of the African vector Anopheles gambiae with Thai and Papua New Guinean (PNG) P. falciparum parasites.
Results
1,197 mosquitoes were dissected and examined; 403 (33.7%) were infected with
P. falciparum. Infection rate was 33% (230 of 696) for
An. stephensi and 34.5% (173 of 501) for
An. gambiae. Ten
P. falciparum isolates from three geographical origins were used to evaluate the differential susceptibility of
An. stephensi and
An. gambiae to tropical parasites (Table
1). Variation in the infection rate between
P. falciparum isolates was clearly observed with a number of parasite isolates failing to infect one or both mosquito species. In particular, the isolates PNG12.2 and PNG51.1 from PNG appeared to be non-infectious producing no oocysts in either mosquito species. Similarly, PNG37.2, 1776.3.7, and M24 produced a very limited number of mosquito infections suggesting that they are only minimally infective. The remaining five parasite isolates appeared to be highly infective and produced some surprising results. The African parasite K39, and 3D7, infected both
An. gambiae and
An. stephensi equally (
P > 0.01) although the actual infection prevalence demonstrated by the two isolates was markedly different, with K39 infection levels three times higher than 3D7.
Table 1
Infection prevalence of different Plasmodium falciparum isolates in two mosquito species: Anopheles stephensi and Anopheles gambiae
?
| 3D7 | 32/136 | 24 | 19/108 | 18 | 0.257 |
Africa
| K39 | 89/119 | 75 | 117/136 | 86 | 0.023 |
| M24 | 0/78 | 0 | 2/32 | 6 | 0.083 |
Thailand
| P018 | 32/42 | 76 | 14/38 | 37 | <0.001* |
| P019 | 60/78 | 77 | 14/66 | 21 | <0.001* |
| P020 | 11/24 | 46 | 6/41 | 15 | <0.001* |
Papua New Guinea
| PNG12.2 | 0/117 | 0 | - | - | N/A |
| PNG37.2 | 2/31 | 7 | 0/21 | 0 | 0.35 |
| PNG51.1 | 0/31 | 0 | 0/41 | 0 | N/A |
| 1776.3.7 | 4/40 | 10 | 1/18 | 6 | 0.577 |
The Thailand parasites provided the most interesting results with all three Thai isolates demonstrating significant differences (P < 0.001) in infection prevalence between the two mosquito species. P018 and P019 infected An. stephensi at a comparable level to K39 but unlike K39, this prevalence diminished sharply in An. gambiae. Similarly, P020 infectivity was significantly reduced in An. gambiae but it also displayed a lower prevalence in An. stephensi, than P018 and P019.
Amongst infected mosquitoes, the intensity of infection was variable both across different parasite isolates and between mosquito species (Table
2). Median oocyst number per infected midgut varied from 18 (K39) to 1 (1776.3.7) with similar variations in 25–75 percentiles ranging from 1–46 (K39) to 1-1 (1776.3.7). As with prevalence, K39 was shown to produce the highest infection intensities but neither K39 nor 3D7 displayed any difference in oocyst numbers between mosquito species. Unlike infection prevalence, only one Thai isolate demonstrated a statistically significant difference in infection intensity; P019 produced significantly more oocysts per infected
An. stephensi mosquito than
An. gambiae (
P < 0.001). Neither of the other Thai isolates displayed this trend but this appears to be due to a lower intensity of infection in
An. stephensi rather than an increase of infection intensity in
An. gambiae.
Table 2
Intensity of infection of different Plasmodium falciparum isolates in paired feeds to Anopheles stephensi and Anopheles gambiae
3D7 | 1.5 (1–2) | 2 (1–3) | 0.251 |
K39 | 12 (1–46) | 18 (6–46) | 0.343 |
M24 | N/A: no infection | 1 (N/A: too few infections) | - |
P018 | 3 (1–9) | 3 (1–4) | 0.297 |
P019 | 10 (2–41) | 2 (1–2) | < 0.001* |
P020 | 2 (2–4) | 2 (1–2) | 0.711 |
PNG12.2 | N/A: no infection | - | - |
PNG37.2 | N/A: too few infections | N/A: no infection | - |
PNG51.1 | N/A: no infection | N/A: no infection | - |
1776.3.7 | 1 (1-1) | 1 (N/A: too few infections) | 0.8 |
Discussion
Population-based data on microsatellites [
17], mitochondrial DNA haplotypes [
18], and chromosome-wide SNP haplotypes [
19] reveal that
P. falciparum has a well-differentiated population structure demonstrating strong clustering according to geographical origin. Whether this differentiation is solely due to geographical separation or indicative of a biological barrier remains an open question. Geographical separation is assumed, but in an era when modern transportation has led to a global community, it seems improbable that parasite populations stay differentiated by geography alone. The data presented in this paper clearly demonstrate significant differences in transmission success of parasites according to geographical origin of the mosquito and it seems apposite to propose a role for the vector in facilitating population separation.
The infectivity of 3D7 in this study was visibly lower than previous studies [
14], but crucially, the reduction in infectivity was observed in both mosquito species and thus judged insignificant to the overall conclusions. Without knowing the geographical origins of 3D7, it is impossible to determine which mosquito species would be the indigenous transmission vector and indeed 3D7 demonstrates no significant differences, either in prevalence or infection intensity, between the two mosquito vectors. The African isolate, K39, also demonstrates the ability to infect both mosquito species equally successfully, and it can be hypothesized that this may be due to maintenance of ancestral genetic diversity or, as recently suggested by Sinden and colleagues, that a longer period of insect-parasite co-evolution may lead to an increase in parasite strategies against the insect immune system such that ancestral strains could be expected to be transmitted by a broader range of mosquito species [
20].
In all the Thai parasites examined, infection prevalence was significantly reduced in
An. gambiae. This trend was not, however, borne out in infection intensity with only P019 demonstrating a significant reduction in oocyst numbers in infected
An. gambiae mosquitoes. While these observations are interesting and provide an insight into parasite-vector transmission dynamics, it is important to note that no parasite-mosquito combination produced total refractoriness as observed previously in other compatibility studies [
1,
3‐
5]. Unlike some studies, these experiments used laboratory maintained colonies of mosquitoes rather than wild-caught populations. Laboratory-maintained mosquito colonies are highly inbred and consequently can demonstrate markedly reduced microsatellite DNA polymorphism and heterozygosity [
21], which may make them genetically dissimilar to the originally sampled population but whether they are representative of wild-caught populations in regards to transmission potential remains to be determined. Natural mosquito populations have been shown to demonstrate marked differences to
P. falciparum infection with resistance to infection occurring at relatively high frequencies [
22], thus, wider ranges in infection prevalence may be observed in the more diverse field populations. These preliminary experiments do demonstrate the potential for important differences in transmission potential of Thai compared to African parasites within
An. gambiae, however, repetition of these results using wild-caught F1 reared progeny would be valuable.
PNG infection results ranged from zero to low infection among the four gametocyte-producing isolates tested in both mosquito species; PNG37.2 and 1776.3.7 produced successful infections. Poor exflagellation (1–2 exflagellation events in 20 fields) was observed in all the PNG isolates except 1776.3.7. Although this is far from an assured method of determining successful infection outcome, it does serve as an indication of the culture infectivity. Exflagellation of 1776.3.7 was excellent (>5 exflagellation events per field), comparable to K39, yet only 10% of
An. stephensi mosquitoes were infected. Similarly, gametocyte production in these parasite isolates was comparable to the Thai and African parasites and there were no discernable microscopic distinctions regarding gametocyte health. This study represents the first published observations regarding PNG parasite isolates infecting
An. stephensi or
An. gambiae and it may be that parasites from this region do not transmit successfully through these non-indigenous mosquito species. Paired feedings of PNG parasites to
An. stephensi/
An. gambiae concurrent with the native PNG mosquito vector,
An. farauti, are essential to determine true parasite-mosquito incompatibilities and determine the validity of these results. Interestingly, recent investigations have shown that
An. farauti is only barely susceptible to
P. falciparum strains from South America and Africa, with the respective authors urging the need to study co-indigenous parasite-mosquito combinations in this region [
8,
10].
A high level of parasite-mosquito compatibility in An. farauti may explain the low infection success of PNG parasite isolates in these studies, but the same can not be said for M24. Only two mosquitoes were positive for this isolate and both had a single oocyst. It appears that this parasite isolate displays very limited infectivity which is unrelated to geographical origin of the mosquito.
Studying the local adaptation and compatibility of
P. falciparum isolates to different mosquito species is of use regarding gene transfer between allopatric parasite populations. Drug resistance is a burgeoning problem in malaria and global transportation links allow parasites to be rapidly transported between malarious areas. People within areas of intense malaria transmission habitually contain multiple parasite clones that are able to cross and recombine within the mosquito, leading to novel genotypes. In this manner, drug resistance genes can spread through populations. The spread of chloroquine resistance is well documented and is thought to have arisen independently in four different geographical areas – Southeast Asia, twice in South America and later in PNG [
23]. Reports from Thailand suggest that resistance first occurred in the late 1950s but the subsequent spread into neighbouring areas, such as India, did not occur for 10–15 years. It appears that the spread of resistance within the limits of
Anopheles balabacensis s.l. was fairly rapid but outside the confines of this permissive vector complex, progress was markedly slower. This could simply be due to the efficiency of the
An. balabacensis complex but, as Wernsdorfer suggested [
24], the possibility of a parasite-vector incompatibility reducing transmission success cannot be discounted.
Authors' contributions
JCCH and KPD conceived and designed the study. JCCH conducted the experimental work with assistance from MT and LRC and supervision from KPD. JCCH drafted the manuscript with KPD. All authors read and approved the final manuscript.