Genetics and evolution of triatomines: from phylogeny to vector control.

Triatomines are hemipteran bugs acting as vectors of the protozoan parasite Trypanosoma cruzi. This parasite causes Chagas disease, one of the major parasitic diseases in the Americas. Studies of triatomine genetics and evolution have been particularly useful in the design of rational vector control strategies, and are reviewed here. The phylogeography of several triatomine species is now slowly emerging, and the struggle to reconcile the phenotypic, phylogenetic, ecological and epidemiological species concepts makes for a very dynamic field. Population genetic studies using different markers indicate a wide range of population structures, depending on the triatomine species, ranging from highly fragmented to mobile, interbreeding populations. Triatomines transmit T. cruzi in the context of complex interactions between the insect vectors, their bacterial symbionts and the parasites; however, an integrated view of the significance of these interactions in triatomine biology, evolution and in disease transmission is still lacking. The development of novel genetic markers, together with the ongoing sequencing of the Rhodnius prolixus genome and more integrative studies, will provide key tools to expanding our understanding of these important insect vectors and allow the design of improved vector control strategies.

Morphometric and molecular differentiation of Phlebotomus (Phlebotomus) sandflies.

The closely related sandfly species of the subgenus Phlebotomus namely, Phlebotomus papatasi (Scopoli, 1786), Phlebotomus duboscqi Neveu-Lemair, 1906 and Phlebotomus bergeroti Parrot, 1934 (Diptera: Psychodidae), are major vectors of Leishmania major (Kinetoplastida: Trypanosomatidae), the causative agent of cutaneous leishmaniasis in the Old World. Although allopatric in most of their distribution, the three species exist sympatrically in many places in central and eastern Sudan. Males of the three species can be distinguished using morphological characters; however, females are much harder to identify, thus complicating epidemiological studies. We carried out a morphometric and a molecular study to determine reliable morphological features and develop a polymerase chain reaction (PCR) assay for distinguishing females of these species. Males and females from each species were collected from sites in Sudan, East Africa and from one site in Mali, West Africa. Males were analysed morphologically and 20 characters and 10 character ratios were used in a stepwise discriminant analysis. This led to the identification of four characters with high discriminant loading scores sufficient for accurate male species identification. Male DNA was then used for the development of a PCR-based species diagnostic based on the second internal transcribed spacer (ITS2) of the ribosomal DNA. A set of four primers was developed to generate fragment sizes that are specific to each species and can reliably identify females as well as hybrid DNA. Both the morphometric and the molecular findings of this study have important applications for studies of the epidemiology of cutaneous leishmaniasis.

Male accessory gland proteins induce female monogamy in anopheline mosquitoes.

The role of male accessory gland (MAG) secretions in inducing refractoriness to further mating in mosquitoes (Diptera: Culicidae) was established in the late 1960s. In a set of simple experiments, MAG extract was injected intra-thoraxically into the hemocoel of virgin Aedes aegypti (L.), Culex pipiens pipiens (L.) and Anopheles quadrimaculatus Say females. This subsequently caused most females to remain unmated when exposed to males. For anophelines these findings were later challenged by a study involving intra-abdominal injections of MAG extracts into Anopheles gambiae Giles s.l. and Anopheles albimanus Wiedmann females, which failed to induce refractoriness to further mating. These findings led to controversy about the respective role of sperm and accessory gland peptides in inducing female monogamy in Anopheles and are at odds with our current understanding of the mating process in Drosophila spp. (Diptera: Drosophillidae) and other dipterans. Here we confirm the function of MAG secretions in anophelines experimentally by showing that intra-thoracic injections in Anopheles stephensi Liston and in the M and S molecular forms of An. gambiae s.s. result in the expected female monogamy. Cross-injections of MAG extracts between the M and S molecular forms of An. gambiae, two cryptic taxa within An. gambiae s.s. which are thought to be undergoing incipient speciation, also elicited effective refractoriness, suggesting that the two sub-taxa have not diverged with regard to sex peptides responsible for female monogamy. Importantly, this also suggests that the rare cases of re-mating following cross-mating observed in this species may not be a form of reproductive barrier between molecular forms.

Ecological immunology of mosquito-malaria interactions: Of non-natural versus natural model systems and their inferences.

There has been a recent shift in the literature on mosquito/Plasmodium interactions with an increasingly large number of theoretical and experimental studies focusing on their population biology and evolutionary processes. Ecological immunology of mosquito-malaria interactions - the study of the mechanisms and function of mosquito immune responses to Plasmodium in their ecological and evolutionary context - is particularly important for our understanding of malaria transmission and how to control it. Indeed, describing the processes that create and maintain variation in mosquito immune responses and parasite virulence in natural populations may be as important to this endeavor as describing the immune responses themselves. For historical reasons, Ecological Immunology still largely relies on studies based on non-natural model systems. There are many reasons why current research should favour studies conducted closer to the field and more realistic experimental systems whenever possible. As a result, a number of researchers have raised concerns over the use of artificial host-parasite associations to generate inferences about population-level processes. Here I discuss and review several lines of evidence that, I believe, best illustrate and summarize the limitations of inferences generated using non-natural model systems.

Experimental infection studies of UK Culicoides species midges with bluetongue virus serotypes 8 and 9.

This paper describes a rapid, standardised method for testing the susceptibility to bluetongue virus (BTV) of northern Palaearctic Culicoides species midges that can be used to assess the competence of both field-caught and laboratory-infected midges. The method has been used to show that Culicoides scoticus can replicate btv serotype 8 and BTV serotype 9 strains to more than 3 log(10) TCID50/midge, the first evidence of the potential of this species to transmit BTV.

A set of broadly applicable microsatellite markers for analyzing the structure of Culex pipiens (Diptera: Culicidae) populations.

Microsatellite markers were isolated and developed from Culex pipiens quinquefasciatus Say (Diptera: Culicidae) sampled in Johannesburg, South Africa, to identify those that are broadly useful for analyzing Cx. pipiens complex populations between continents. Suitable loci should be 1) inherited in a codominant Mendelian manner, 2) polymorphic, 3) selectively neutral, 4) randomly associated, 5) without null alleles, and 6) applicable across broad regions and between diverse biotypes. Loci in Cx. p. quinquefasciatus from Johannesburg ranged from two to 17 alleles per locus and expected heterozygosities (H(e)) were 0.02-0.87. Loci in Cx. p. pipiens L. from Johannesburg had five to 19 alleles per locus and H(e) values ranging from 0.57 to 0.93, whereas those from George, South Africa, had five to 17 alleles per locus and H(e) values ranging from 0.54 to 0.88. Loci in North American mosquitoes were more variable. Cx. p. quinquefasciatus from South Carolina had five to 19 alleles per locus and H(e) values ranging from 0.64 to 0.90, whereas Cx. p. pipiens from Massachusetts had six to 28 alleles per locus and with H(e) values ranging from 0.65 to 0.94. All loci were associated randomly. Overall, four of nine of these new loci satisfied all six criteria for broad utility for analyzing the genetic structure of Cx. pipiens populations.

Evidence for subdivision within the M molecular form of Anopheles gambiae.

The principal vector of malaria in sub-Saharan Africa, Anopheles gambiae is subdivided into two molecular forms M and S. Additionally, several chromosomal forms, characterized by the presence of various inversion polymorphisms, have been described. The molecular forms M and S each contain several chromosomal forms, including the Savanna, Mopti and Forest forms. The M and S molecular forms are now considered to be the reproductive units within A. gambiae and it has recently been argued that a low recombination rate in the centromeric region of the X chromosome has facilitated isolation between these forms. The status of the chromosomal forms remains unclear however. Therefore, we studied genetic differentiation between Savanna S, Forest S, Forest M and Mopti M populations using microsatellites. Genetic differentiation between Savanna S and Forest S populations is very low (F(ST) = 0.0053 +/- 0.0049), even across large distances. In comparison, the Mopti M and Forest M populations show a relatively high degree of genetic differentiation (F(ST) = 0.0406 +/- 0.0054) indicating that the M molecular form may not be a single entity, but could be subdivided into at least two distinct chromosomal forms. Previously it was proposed that inversions have played a role in the origin of species within the A. gambiae complex. We argue that a possible subdivision within the M molecular form could be understood through this process, with the acquisition of inversions leading to the expansion of the M molecular form into new habitat, dividing it into two distinct chromosomal forms.

An unusual distribution of the kdr gene among populations of Anopheles gambiae on the island of Bioko, Equatorial Guinea.

In West Africa, Anopheles gambiae exists in discrete subpopulations known as the M and S molecular forms. Although these forms occur in sympatry, pyrethroid knock-down resistance (kdr) is strongly associated with the S molecular form. On the island of Bioko, Equatorial Guinea we found high frequencies of the kdr mutation in M form individuals (55.8%) and a complete absence of kdr in the S form. We also report the absence of the kdr allele in M and S specimens from the harbour town of Tiko in Cameroon, representing the nearest continental population to Bioko. The kdr allele had previously been reported as absent in populations of An. gambiae on Bioko. Contrary to earlier reports, sequencing of intron-1 of this sodium channel gene revealed no fixed differences between M form resistant and susceptible individuals. The mutation may have recently arisen independently in the M form on Bioko due to recent and intensive pyrethroid application.

Effect of seminal fluids in mating between M and S forms of Anopheles gambiae.

Previous studies have shown that sympatric populations of M and S molecular forms of Anopheles gambiae sensu stricto exhibit strong assortative mating. In the few documented cases of cross-mating between M and S forms, females that mated with amale of the alternative form were often also mated with a male of their own form. A potential explanation for the association between cross-mating and double mating could be that male accessory gland or sperm proteins that are responsible for inducing refractoriness to further mating by females have diverged between the M and S forms. This mechanism of postmating reproductive isolation would have important implications for our understanding of the speciation processes in the An. gambiae complex. We tested for this mechanism, by comparing the likelihood of mating, feeding, and laying eggs, as well as the fertility of females presented with males of their own form or the alternate form in the laboratory. We also compared the likelihood of remating in cross-mated and assortatively-mated females, and we analyzed their progeny to unravel patterns of sperm precedence. We found that cross-mated females differed from assortatively-mated females only in terms of egg-hatching rate and larval survival but that these effects could be attributed to hybrid vigor rather than differential response to seminal products. Cross-mating between forms was not associated with remating behavior. These results indicate that the sex proteins responsible for inhibiting further insemination and triggering the gonotrophic cycle in females have not diverged between these M and S populations. We discuss alternative explanations for the patterns of cross-mating and multiple mating observed in the field.

DNA analysis of transferred sperm reveals significant levels of gene flow between molecular forms of Anopheles gambiae.

Anopheles gambiae populations in west Africa are complex, being composed of multiple, sympatric subpopulations. Recent studies have failed to reveal significant genetic differences among subpopulations, stimulating a debate regarding the levels of gene flow among them. The observed homogeneity may be the consequence of substantial contemporary gene flow or it may be that reproductive isolation is complete, but too recent for the accumulation of significant levels of genic divergence. Here, we report the results of a study estimating contemporary levels of gene flow between An. gambiae subpopulations by analysing females and transferred sperm removed from their reproductive systems. A total of 251 female and associated sperm extracts was analysed from a single site in Mali. Two molecular forms of An. gambiae, the M- and S-forms, occurred in sympatry at this site. Overall, we found very strong positive assortative mating within forms, however, we did observe significant hybridization between forms. In the M subpopulation 2/195 females (1.03%) contained sperm from S-form males and in 55 S-form females we found one female containing M-form sperm (1.82%). We also identified a mated M xS hybrid adult female. From mating frequencies, we estimate the Nem between the M- and S-form at 16.8, and from the adult hybrid frequency at 5.6. These values are consistent with our earlier estimate, based on FST for 21 microsatellite loci in which Nem = 5.8. We conclude that the general lack of genetic divergence between the M and S subpopulations of An. gambiae can be explained entirely by contemporary gene flow.

The coevolutionary potential of a 'generalist' parasite, the hen flea Ceratophyllus gallinae.

Hosts exert selection pressures on their parasites and it is often assumed that host-parasite coevolution with each host is less intense in a generalist parasite than for a parasite with a narrow host range. Selection pressure on the parasite, however, is rather determined by host specificity, i.e. the relative importance of each host, than simply by the range of hosts. The determination of host specificity requires an assessment of the prevalence and intensity of parasite infestation within each host's nests, as well as the local abundance of each host species. Since the hen flea, Ceratophyllus gallinae, is a rather generalist parasite of birds it could be concluded that there has been weak coevolution with each of its hosts. By reviewing the literature on the prevalence and intensity of hen flea infestations in bird nests we estimated the number of individuals produced in the nest of each host species. The comparative analysis shows (1) that the prevalence of infestation is highest in hole-nesting avian families, (2) that prevalence and intensity of infestation among bird families are highly correlated, and (3) that hole-nesting Paridae have the highest intensities of infestation and harbour the majority of the flea population. These results underline the fleas' potential for coevolution with Paridae despite their extensive host range.