Low effective population sizes in Amblyomma variegatum, the tropical bont tick.

Effective population sizes have rarely been estimated in ticks despite the importance of this parameter for evaluating the evolutionary and adaptive potential of tick populations. The present study was aimed at evaluating the effective population sizes of Amblyomma variegatum, the tropical bont tick, in three villages in Burkina Faso. For this purpose, microsatellites markers were developed. Eight out of 19 assessed markers provided good amplification results with 4 to 24 alleles recorded per marker on 216 genotyped ticks. The within-samples polymorphism was congruent with Hardy-Weinberg expectations at four markers while sex linkage and/or null alleles were observed at the others. As sampling involved two tick generations, effective population sizes were independently estimated by two methods insensitive to heterozygosity: the first one is based on linkage disequilibrium analysis within a single cohort while the second uses the changes in allele frequencies across generations. Both methods estimated the number of reproducing ticks ranging from two to a few tens reproductive adults per village and cohort. Such small estimates are congruent with the rarity of records of acaricide resistance in A. variegatum.

Colonization of the Mediterranean basin by the vector biting midge species Culicoides imicola: an old story.

Understanding the demographic history and genetic make-up of colonizing species is critical for inferring population sources and colonization routes. This is of main interest for designing accurate control measures in areas newly colonized by vector species of economically important pathogens. The biting midge Culicoides imicola is a major vector of orbiviruses to livestock. Historically, the distribution of this species was limited to the Afrotropical region. Entomological surveys first revealed the presence of C. imicola in the south of the Mediterranean basin by the 1970s. Following recurrent reports of massive bluetongue outbreaks since the 1990s, the presence of the species was confirmed in northern areas. In this study, we addressed the chronology and processes of C. imicola colonization in the Mediterranean basin. We characterized the genetic structure of its populations across Mediterranean and African regions using both mitochondrial and nuclear markers, and combined phylogeographical analyses with population genetics and approximate Bayesian computation. We found a west/east genetic differentiation between populations, occurring both within Africa and within the Mediterranean basin. We demonstrated that three of these groups had experienced demographic expansions in the Pleistocene, probably because of climate changes during this period. Finally, we showed that C. imicola could have colonized the Mediterranean basin in the Late Pleistocene or Early Holocene through a single event of introduction; however, we cannot exclude the hypothesis involving two routes of colonization. Thus, the recent bluetongue outbreaks are not linked to C. imicola colonization event, but rather to biological changes in the vector or the virus.

Protozoan and bacterial pathogens in tick salivary glands in wild and domestic animal environments in South Africa.

A total of 7364 ticks belonging to 13 species was collected from 64 game animals (belonging to 11 species) and from 64 livestock animals (cattle and sheep) living in close vicinity at 6 localities in 3 South African Provinces (Free State, Mpumalanga, and Limpopo). The geographic distribution of all tick species was congruent with the literature except for Haemaphysalis silacea. From each infested host, a maximum of 10 males and 10 females of each tick species were dissected to isolate the salivary glands. Salivary glands were screened for tick-borne pathogens using polymerase chain reaction followed by reverse line blotting and sequencing. This approach allowed us to evaluate the exposure of wild and domestic hosts to tick-borne pathogens in their respective environments. Among the 2117 examined ticks, 329 (15.5%), belonging to 8 species, were infected and harboured 397 infections. Among those, 57.7% were identified to species level and were assigned to 23 pathogen species of the genera Babesia, Theileria, Anaplasma, and Ehrlichia. In 3 out of 6 localities, salivary glands from ticks infesting wild ruminants displayed significantly higher infection prevalence and pathogen mean density than salivary glands from ticks infesting livestock animals. Four piroplasm species [Theileria bicornis, Babesia sp. (sable), Theileria sp. (giraffe), and Theileria sp. (kudu)] were detected for the first time in ticks. The tick species Rhipicephalus evertsi evertsi, Rhipicephalus (Boophilus) decoloratus, Hyalomma rufipes, Rhipicephalus appendiculatus, and Amblyomma hebraeum were associated with a broader pathogen range than previously known, and thus new vector-pathogen combinations are described. In addition, previously unknown coinfection patterns in tick salivary glands are reported.

Tick-borne pathogens in the blood of wild and domestic ungulates in South Africa: interplay of game and livestock.

We screened for tick-borne pathogens blood samples from 181 wild and domestic ungulates belonging to 18 host species in 4 South African Provinces. Polymerase chain reaction followed by reverse line blotting and sequencing allowed detecting 16 tick-borne pathogen species belonging to the genera Babesia, Theileria, Anaplasma, and Ehrlichia. Ten pathogen species were involved in 29 new host-pathogen combinations. Most infections (77.9%) involved more than one pathogen species. Principal component analysis (PCA) assigned the 163 infections, identified to species level, to 4 groups. Three groups were associated with sheep, cattle, and horse and their respective wild counterparts. Each group was characterised by high homogeneity in pathogen assemblage and host phylogenetic status. These groups characterised the most privileged transmission routes between and among wild and domestic ungulates. The 4th group showed high heterogeneity in pathogen assemblage and host phylogenetic status. This group seems to indicate frequent spill over events in impala of pathogens that usually circulate among cattle- or sheep-related species. Within 6 localities, we sampled an equal number of wild and domestic animals (n=128). On this dataset once having controlled for the significant variation among localities, the infection prevalence and intensity of infection did not differ significantly between wild and domestic hosts. This suggests that both animal types, domestic and wild hosts, could act as evenly efficient sources of infection for themselves and for each other. Overall, this study shed new light on the pathogen circulation naturally achieved at the interplay between wild and domestic ungulates.

Circulation and suspended sediment transport in a coral reef lagoon: the south-west lagoon of New Caledonia.

The south-west lagoon of New Caledonia is a wide semi-open coral reef lagoon bounded by an intertidal barrier reef and bisected by numerous deep inlets. This paper synthesizes findings from the 2000-2008 French National Program EC2CO-PNEC relative to the circulation and the transport of suspended particles in this lagoon. Numerical model development (hydrodynamic, fine suspended sediment transport, wind-wave, small-scale atmospheric circulation) allowed the determination of circulation patterns in the lagoon and the charting of residence time, the later of which has been recently used in a series of ecological studies. Topical studies based on field measurements permitted the parameterisation of wave set-up induced by the swell breaking on the reef barrier and the validation of a wind-wave model in a fetch-limited environment. The analysis of spatial and temporal variability of suspended matter concentration over short and long time-scales, the measurement of grain size distribution and the density of suspended matter (1.27 kg l(-1)), and the estimation of erodibility of heterogeneous (sand/mud, terrigenous/biogenic) soft bottoms was also conducted. Aggregates were shown to be more abundant near or around reefs and a possible biological influence on this aggregation is discussed. Optical measurements enabled the quantification of suspended matter either in situ (monochromatic measurements) or remotely (surface spectral measurements and satellite observations) and provided indirect calibration and validation of a suspended sediment transport model. The processes that warrant further investigation in order to improve our knowledge of circulation and suspended sediment transport in the New Caledonia lagoon as well as in other coral reef areas are discussed, as are the relevance and reliability of the numerical models for this endeavour.

Swift sympatric adaptation of a species of cattle tick to a new deer host in New Caledonia.

The occurrence and frequency of sympatric speciation in natural systems continue to be hotly debated issues in evolutionary biology. This might reflect the timescale over which evolution occurs resulting in there being few compelling observations of the phenomenon (lake fishes, phytophagous insects and Island trees). Despite predictions, few examples of sympatric speciation have been recorded in animal parasites, at least widely accepted as such. Here we show that, in New Caledonia, the monophasic (exploiting one individual host per generation) cattle tick Rhipicephalus microplus has evolved in contact with two sympatric host species into two differentiated genetic pools: on the cattle, its original host and on rusa deer, a new host for this tick. This sympatric isolation has occurred over a relative short period of time (i.e. less than 244 tick generations) as a consequence of differential selection pressure imposed by hosts. It is most likely that this phenomenon has occurred in many other places across the globe where this tick has come in contact with different host species in sympatry with cattle.

Population genetic structure of wild and farmed rusa deer (Cervus timorensis russa) in New-Caledonia inferred from polymorphic microsatellite loci.

Historical records indicate that 12 rusa deer (Cervus timorensis russa) were introduced in New-Caledonia during the 1870s. We used eight polymorphic microsatellite DNA loci to assess the genetic differentiation and diversity of farmed and wild deer populations. Past genetic bottlenecks were detected in both sub-populations, although higher genetic diversity was maintained in farmed populations, probably due to the regular introduction of reproducers from wild populations and from other farms. The genetic structure of farmed and wild populations differed significantly. There was a significant isolation by distance for wild populations, whereas farmed populations were significantly differentiated between farms independently from their geographical proximity. Wild rusa deer consisted of small populations (with effective population sizes ranging between 7 and 19 individuals depending on the methods used), with a low parent-offspring dispersion range (0.20-2.02 km). Genetic tools and direct observations provided congruent estimates of dispersion and population sizes. We discuss the relevance of our results for management purposes.

Modelling the spatial configuration of refuges for a sustainable control of pests: a case study of Bt cotton.

The 'high-dose-refuge' (HDR) strategy is widely recommended by the biotechnology industry and regulatory authorities to delay pest adaptation to transgenic crops that produce Bacillus thuringiensis (Bt) toxins. This involves cultivating nontoxic plants (refuges) in close proximity to crops producing a high dose of Bt toxin. The principal cost associated with this strategy is due to yield losses suffered by farmers growing unprotected, refuge plants. Using a population genetic model of selection in a spatially heterogeneous environment, we show the existence of an optimal spatial configuration of refuges that could prevent the evolution of resistance whilst reducing the use of costly refuges. In particular, the sustainable control of pests is achievable with the use of more aggregated distributions of nontransgenic plants and transgenic plants producing lower doses of toxin. The HDR strategy is thus suboptimal within the context of sustainable agricultural development.

Mechanisms of resistance to organophosphates in Tetranychus urticae (Acari: Tetranychidae) from Greece.

We investigated the mechanisms conferring resistance to methyl-parathion (44-fold) and to methomyl (8-fold) in Tetranychus urticae from Greece by studying the effect of synergists on the resistance and the kinetic characteristics of various enzymes in a resistant strain (RLAB) and a susceptible reference strain (SAMB). It is shown that S,S,S-tributyl phosphorotrithioate, a synergist that inhibits esterases and glutathione S-transferases, and piperonyl butoxide, a synergist that inhibits cytochrome P450 mediated monooxygenases, did not affect the level of methyl-parathion or methomyl resistance in RLAB and that resistance ratios to both insecticides did not change significantly in the presence of either synergist. Isoelectric focusing of esterase allozymes on single mites revealed no differences in staining intensity and glutathione S-transferase activity was not significantly different in the two strains. The activity of two cytochrome P450 monooxygenase groups was compared. No significant difference of 7-ethoxyresorufin-O-diethylase activity was observed between strains that were two-fold higher in RLAB than in SAMB. The kinetic characteristics of acetylcholinesterase, the target enzyme of organophosphates and carbamates, revealed that acetylcholinesterase in RLAB was less sensitive to inhibition by paraoxon and methomyl in comparison with SAMB. I(50), the inhibitor concentration inducing 50% decrease of acetylcholinesterase activity was greater (119- and 50-fold with paraoxon and methomyl, respectively) and the bimolecular constant k(i) was lower (39- and 47-fold with paraoxon and methomyl, respectively) in RLAB compared to SAMB.

Dissecting the cost of insecticide resistance genes during the overwintering period of the mosquito Culex pipiens.

In several insects, there appears to be a high fitness cost associated with insecticide resistance genes during the overwintering period. In order to understand when and how this cost operates, all mosquitoes entering a natural cave for overwintering were regularly sampled, and their resistance genes at two loci (Ester and Ace.1) were individually identified. During the main period of entry (October and November), susceptible mosquitoes were first observed, followed by resistant ones, this trend being similar for both loci. This observation is best explained by a migration phenomenon, northern and more susceptible populations starting to overwinter first, followed by southern and more resistant ones. During the remaining part of the winter (December-March), mosquitoes entering the cave were still caught and they probably corresponded to individuals leaving a former overwintering site in search of a more suitable one. A lower overall frequency of resistant phenotypes was found in the second part of the winter at Ester, suggesting that a large fitness cost (42%) had operated. A decreasing frequency of resistant phenotypes was also found at Ace.1, indicating a large survival cost of resistant mosquitoes (7% for the homozygote Ace.1R) in their former overwintering site. These results are discussed in the light of the local evolution of these resistance genes in southern France.

Resistance to Bacillus sphaericus in Culex pipiens (Diptera: Culicidae): interaction between recessive mutants and evolution in southern France.

In southern France, failure to control Culex pipiens L. with Bacillus sphaericus Neide toxin (Bs) was first detected in 1994, at the extreme east of the Languedoc-Roussillon coast. This failure was due to a single recessive mutant, sp-1R. Two complementary strategies were used to test whether sp-1R had invaded the Bs-controlled area by 1998. First, a strain (BP) was selected from resistant larvae sampled in the western part of the Bs-controlled area. In BP strain, resistance involved a single recessive gene, sp-2R, distinct from sp-1R, that conferred a similarly high resistance in the homozygous state (approximately 6,000-fold). Combining one copy of sp-1R and one of sp-2R conferred a > 100-fold resistance. Second, Bs-resistance was monitored among the offspring of field females crossed to sp-1RR homozygous males. Females were sampled in 20 localities of southern France and three localities of the Llobregat delta (Barcelona, Spain) where C. pipiens control is also intensive. The 537 females in the study produced enough larvae to infer their genotype: 462 progenies were susceptible and the survival rate of 51 others was explained by the presence of sp-1R and/or sp-2R. The remaining 24 cases indicated that other factors could confer resistance when combined with sp-1R. The current data showed that, even when recessive, resistant mutants can rapidly increase in frequency, providing some interactions that protect them from disappearance. We discuss the consequences of this finding on the current strategies aimed to avoid or delay resistance in the pests controlled with B. sphaericus or B. thuringiensis Berliner toxins.

Recombination between two amplified esterase alleles in Culex pipiens.

Esterase gene amplification at the Ester superlocus provides organophosphate resistance in the mosquito Culex pipiens (L.). In this study we explored the possibility of recombination between two amplified esterase alleles, thus generating a composite amplified allele. To do that, females heterozygous for two distinct amplified alleles (Ester(2) and Ester(4)) were crossed with males homozygous for a third resistance allele (Ester(8)). Among analyzed offspring, one recombinant composite allele (Ester(2-4)) was detected, providing a rate of recombination of approximately 0.2%. This is the first report of a recombination between two distinct amplified esterase alleles. This phenomenon renders the predictability of allele evolution considerably more complex than was previously thought.

Insecticide resistance in the mosquito culex pipiens: what have we learned about adaptation?

Resistance to organophosphate (OP) insecticide in the mosquito Culex pipiens has been studied for ca. 30 years. This example of micro-evolution has been thoroughly investigated as an opportunity to assess precisely both the new adapted phenotypes and the associated genetic changes. A notable feature is that OP resistance is achieved with few genes, and these genes have generally large effects. The molecular events generating such resistance genes are complex (e.g., gene amplification, gene regulation) potentially explaining their low frequency of de novo occurrence. In contrast, migration is a frequent event, including passive transportation between distant populations. This generates a complex interaction between mutations and migration, and promotes competition among resistance alleles. When the precise physiological action of each gene product is rather well known, it is possible to understand the dominance level or the type of epistasis observed. It is however difficult to predict a priori how resistance genes will interact, and it is too early to state whether or not this will be ever possible. These resistance genes are costly, and the cost is variable among them. It is usually believed that the initial fitness cost would gradually decrease due to subsequent mutations with a modifier effect. In the present example, a particular modifier occurred (a gene duplication) at one resistance locus, whereas at the other one reduction of cost is driven by allele replacement and apparently not by selection of modifiers.

Resistance to xenobiotics and parasites: can we count the cost?

The nature and cost of single genes of major effect is one of the longest running controversies in biology. Resistance, whether to xenobiotics or to parasites, is often paraded as an obvious example of a single gene effect that must carry an associated fitness 'cost'. However, a review of the xenobiotic resistance literature shows that empirical evidence for this hypothesis is, in fact, scarce. We postulate that such fitness costs can only be fully interpreted in the light of the molecular mutations that might underlie them. We also derive a theoretical framework both to encompass our current understanding of xenobiotic resistance and to begin to dissect the probable cost of parasite resistance.

Interruption of chemical mosquito control and evolution of insecticide resistance genes in Culex pipiens (Diptera: Culicidae).

Within the Llobregat Delta (Barcelona, Spain), Culex pipiens L. has been the target of organophosphate insecticide (OP) control for 10 yr (1982-1992). Between 1991 and 1992, OPs were replaced by Bacillus-based toxins in all the mosquito control programs within > 150 km of this area. The distribution of several OP-resistance genes was surveyed within the Llobregat Delta and neighboring populations (< 25 km) during the 2 yr following this regional pesticide change to investigate how the change in selection pressure affected the dynamics of OP-resistance genes. The immigration failure of the A2-B2 resistant esterases and the observed difference in OP-resistance dynamics between isolated and nonisolated populations may indicate fitness disadvantages associated with OP-resistance genes, hence a tendency for a decrease in OP-resistance. In contrast, one OP-resistance gene further increased in frequency, whereas the frequencies of some others were maintained. These unexpected results question the importance of pesticides from sources other than mosquito control, and the variability of pleiotropic fitness costs among pesticide resistance genes.

Analytical aspects of population-specific DNA fingerprinting for individuals.

An emerging problem of some interest is whether we can determine the population membership of a single individual, using a population-specific "genetic fingerprint." The levels of accuracy and precision required are beyond the reach of allozyme analysis, and attention has shifted to DNA polymorphisms. There are different types of DNA markers available for population surveys: RFLPs, mini- and micro-satellites, and RAPDs, and each type has its own strengths and weaknesses. We present a generic analysis that relates gene pool separation to our ability to assign individuals, an analysis that does not depend on the type of marker. We then review strengths and weaknesses of different DNA markers, in the context of DNA fingerprinting. Codominant loci are best. It is possible to gain more information per marker with multiallelic loci, but diminishing returns set in rapidly, and it is better to add loci. A modest number of independent loci is best, each locus with a modest number of alleles and with each allele in modest frequency.

An overview of the evolution of overproduced esterases in the mosquito Culex pipiens.

Insecticide resistance genes have developed in a wide variety of insects in response to heavy chemical application. Few of these examples of adaptation in response to rapid environmental change have been studied both at the population level and at the gene level. One of these is the evolution of the overproduced esterases that are involved in resistance to organophosphate insecticides in the mosquito Culex pipiens. At the gene level, two genetic mechanisms are involved in esterase overproduction, namely gene amplification and gene regulation. At the population level, the co-occurrence of the same amplified allele in distinct geographic areas is best explained by the importance of passive transportation at the worldwide scale. The long-term monitoring of a population of mosquitoes in southern France has enabled a detailed study to be made of the evolution of resistance genes on a local scale, and has shown that a resistance gene with a lower cost has replaced a former resistance allele with a higher cost.

Pleiotropy of adaptive changes in populations: comparisons among insecticide resistance genes in Culex pipiens.

Resistance to toxicants is a convenient model for investigating whether adaptive changes are associated with pleiotropic fitness costs. Despite the voluminous literature devoted to this subject, intraspecific comparisons among toxicant resistance genes are rare. We report here results on the pleiotropic effect on adult survival of Culex pipiens mutants involved in the same adaptation: the resistance to organophosphorus insecticides. This field study was performed in southern France where four resistance genes sequentially appeared and increased in frequency in response to intense insecticide control. By repeated sampling of overwintering females through winter, we analysed the impact of each of three resistance genes on adult survival. We showed that (i) the most recent gene seems to be of no disadvantage during winter, (ii) the oldest affects survival in some environmental conditions, and (iii) the third induces a constant, severe and dominant survival cost. Such variability is discussed in relation to the physiological changes involved in resistance.