Building pyramids against the evolutionary emergence of pathogens.

Mutations allowing pathogens to escape host immunity promote the spread of infectious diseases in heterogeneous host populations and can lead to major epidemics. Understanding the conditions that slow down this evolution is key for the development of durable control strategies against pathogens. Here, we use theory and experiments to compare the efficacy of three strategies for the deployment of resistance: (i) a mixing strategy where the host population contains two single-resistant genotypes, (ii) a pyramiding strategy where the host carries a double-resistant genotype, (iii) a combining strategy where the host population is a mix of a single-resistant genotype and a double-resistant genotype. First, we use evolutionary epidemiology theory to clarify the interplay between demographic stochasticity and evolutionary dynamics to show that the pyramiding strategy always yields lower probability of evolutionary emergence. Second, we test experimentally these predictions with the introduction of bacteriophages into bacterial populations where we manipulated the diversity and the depth of immunity using a Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated (CRISPR-Cas) system. These biological assays confirm that pyramiding multiple defences into the same host genotype and avoiding combination with single-defence genotypes is a robust way to reduce pathogen evolutionary emergence. The experimental validation of these theoretical recommendations has practical implications in various areas, including for the optimal deployment of resistance varieties in agriculture and for the design of durable vaccination strategies.

Competition and coevolution drive the evolution and the diversification of CRISPR immunity.

The diversity of resistance challenges the ability of pathogens to spread and to exploit host populations. Yet, how this host diversity evolves over time remains unclear because it depends on the interplay between intraspecific competition among host genotypes and coevolution with pathogens. Here we study experimentally the effect of coevolving phage populations on the diversification of bacterial CRISPR immunity across space and time. We demonstrate that the negative-frequency-dependent selection generated by coevolution is a powerful force that maintains host resistance diversity and selects for new resistance mutations in the host. We also find that host evolution is driven by asymmetries in competitive abilities among different host genotypes. Even if the fittest host genotypes are targeted preferentially by the evolving phages, they often escape extinctions through the acquisition of new CRISPR immunity. Together, these fluctuating selective pressures maintain diversity, but not by preserving the pre-existing host composition. Instead, we repeatedly observe the introduction of new resistance genotypes stemming from the fittest hosts in each population. These results highlight the importance of competition on the transient dynamics of host-pathogen coevolution.

Chemosystematics Using Cuticular Compounds: A Powerful Tool to Separate Species in Mediterranean Dung Beetles (Coleoptera: Geotrupidae).

The use of chemical characters to infer a phylogeny is known to be promising to ascertain phylogenetic relationships in controversial groups. Dung beetle classifications containing the Geotrupidae family, based on morphological characters and genes, are debated with respect to the subfamilies, such as the Bolboceratids. In our study, we used different approaches to generate and compare the Geotrupidae phylogenies based on genetics and chemotaxonomy. Cuticular compounds were analyzed for 12 species of Mediterranean dung beetles to build a chemical phylogeny. In addition, mitochondrial and nuclear marker concatenation have been used to elaborate the molecular phylogeny. Using the cuticular compound continuous data, our results showed that each species was associated with a specific chemical pattern and that all individuals belonging to the same species displayed a similar chemical blend. The most distant species was Bolbelasmus gallicus, with an evident distinction from the other species due to several compounds. The maximum parsimony tree showed that all genera belonging to a Geotrupidae subfamily were grouped in the same clade, with B. gallicus species isolated in another clade, similar to the chemotaxonomy grouping. A strong positive correlation between chemotaxonomy and genetic phylogeny has been demonstrated, underlying a genetic basis for cuticular hydrocarbon variations. Our results are congruent with previous studies using morphological or genetic data. Our results also showed that only 10 compounds can be used to distinguish at least six species of dung beetle and that chemotaxonomy could become a useful and affordable tool to determine phylogenetic relationships in insects.

Avian malaria alters the dynamics of blood feeding in Culex pipiens mosquitoes.

BACKGROUND: Some Plasmodium species have the ability to modify the behaviour of their mosquito vectors. This is thought to be an adaptive strategy that maximizes the parasite's transmission. METHODS: The effect of Plasmodium relictum infections on the blood feeding behaviour of Culex pipiens quinquefasciatus mosquitoes was monitored. RESULTS: Plasmodium infections did not alter the proportion of blood fed mosquitoes but they did affect the dynamics and the size of the blood meal. Sporozoite-infected mosquitoes completed their blood meal 1.3 times later than uninfected mosquitoes and ended up with smaller blood meals. CONCLUSION: The potential adaptive nature of this manipulation of mosquito behaviour is discussed in the light of previous studies on other malaria models.

Variability in the durability of CRISPR-Cas immunity.

The durability of host resistance is challenged by the ability of pathogens to escape the defence of their hosts. Understanding the variability in the durability of host resistance is of paramount importance for designing more effective control strategies against infectious diseases. Here, we study the durability of various clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) alleles of the bacteria Streptococcus thermophilus against lytic phages. We found substantial variability in durability among different resistant bacteria. Since the escape of the phage is driven by a mutation in the phage sequence targeted by CRISPR-Cas, we explored the fitness costs associated with these escape mutations. We found that, on average, escape mutations decrease the fitness of the phage. Yet, the magnitude of this fitness cost does not predict the durability of CRISPR-Cas immunity. We contend that this variability in the durability of resistance may be because of variations in phage mutation rate or in the proportion of lethal mutations across the phage genome. These results have important implications on the coevolutionary dynamics between bacteria and phages and for the optimal deployment of resistance strategies against pathogens and pests. Understanding the durability of CRISPR-Cas immunity may also help develop more effective gene-drive strategies based on CRISPR-Cas9 technology. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

Timing malaria transmission with mosquito fluctuations.

Temporal variations in the activity of arthropod vectors can dramatically affect the epidemiology and evolution of vector-borne pathogens. Here, we explore the "Hawking hypothesis", which states that these pathogens may evolve the ability to time investment in transmission to match the activity of their vectors. First, we use a theoretical model to identify the conditions promoting the evolution of time-varying transmission strategies in pathogens. Second, we experimentally test the "Hawking hypothesis" by monitoring the within-host dynamics of Plasmodium relictum throughout the acute and the chronic phases of the bird infection. We detect a periodic increase of parasitemia and mosquito infection in the late afternoon that coincides with an increase in the biting activity of its natural vector. We also detect a positive effect of mosquito bites on Plasmodium replication in the birds both in the acute and in the chronic phases of the infection. This study highlights that Plasmodium parasites use two different strategies to increase the match between transmission potential and vector availability. We discuss the adaptive nature of these unconditional and plastic transmission strategies with respect to the time scale and the predictability of the fluctuations in the activity of the vector.

Evolutionary emergence of infectious diseases in heterogeneous host populations.

The emergence and re-emergence of pathogens remains a major public health concern. Unfortunately, when and where pathogens will (re-)emerge is notoriously difficult to predict, as the erratic nature of those events is reinforced by the stochastic nature of pathogen evolution during the early phase of an epidemic. For instance, mutations allowing pathogens to escape host resistance may boost pathogen spread and promote emergence. Yet, the ecological factors that govern such evolutionary emergence remain elusive because of the lack of ecological realism of current theoretical frameworks and the difficulty of experimentally testing their predictions. Here, we develop a theoretical model to explore the effects of the heterogeneity of the host population on the probability of pathogen emergence, with or without pathogen evolution. We show that evolutionary emergence and the spread of escape mutations in the pathogen population is more likely to occur when the host population contains an intermediate proportion of resistant hosts. We also show that the probability of pathogen emergence rapidly declines with the diversity of resistance in the host population. Experimental tests using lytic bacteriophages infecting their bacterial hosts containing Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR-Cas) immune defenses confirm these theoretical predictions. These results suggest effective strategies for cross-species spillover and for the management of emerging infectious diseases.

Complete avian malaria parasite genomes reveal features associated with lineage-specific evolution in birds and mammals.

Avian malaria parasites are prevalent around the world and infect a wide diversity of bird species. Here, we report the sequencing and analysis of high-quality draft genome sequences for two avian malaria species, Plasmodium relictum and Plasmodium gallinaceum We identify 50 genes that are specific to avian malaria, located in an otherwise conserved core of the genome that shares gene synteny with all other sequenced malaria genomes. Phylogenetic analysis suggests that the avian malaria species form an outgroup to the mammalian Plasmodium species, and using amino acid divergence between species, we estimate the avian- and mammalian-infective lineages diverged in the order of 10 million years ago. Consistent with their phylogenetic position, we identify orthologs of genes that had previously appeared to be restricted to the clades of parasites containing Plasmodium falciparum and Plasmodium vivax, the species with the greatest impact on human health. From these orthologs, we explore differential diversifying selection across the genus and show that the avian lineage is remarkable in the extent to which invasion-related genes are evolving. The subtelomeres of the P. relictum and P. gallinaceum genomes contain several novel gene families, including an expanded surf multigene family. We also identify an expansion of reticulocyte binding protein homologs in P. relictum, and within these proteins, we detect distinct regions that are specific to nonhuman primate, humans, rodent, and avian hosts. For the first time in the Plasmodium lineage, we find evidence of transposable elements, including several hundred fragments of LTR-retrotransposons in both species and an apparently complete LTR-retrotransposon in the genome of P. gallinaceum.

Mosquito age and avian malaria infection.

BACKGROUND: The immune system of many insects wanes dramatically with age, leading to the general prediction that older insects should be more susceptible to infection than their younger counterparts. This prediction is however challenged by numerous studies showing that older insects are more resistant to a range of pathogens. The effect of age on susceptibility to infections is particularly relevant for mosquitoes given their role as vectors of malaria and other diseases. Despite this, the effect of mosquito age on Plasmodium susceptibility has been rarely explored, either experimentally or theoretically. METHODS: Experiments were carried out using the avian malaria parasite Plasmodium relictum and its natural vector in the field, the mosquito Culex pipiens. Both innate immune responses (number and type of circulating haemocytes) and Plasmodium susceptibility (prevalence and burden) were quantified in seven- and 17-day old females. Whether immunity or Plasmodium susceptibility are modulated by the previous blood feeding history of the mosquito was also investigated. To ensure repeatability, two different experimental blocks were carried out several weeks apart. RESULTS: Haemocyte numbers decrease drastically as the mosquitoes age. Despite this, older mosquitoes are significantly more resistant to a Plasmodium infection than their younger counterparts. Crucially, however, the age effect is entirely reversed when old mosquitoes have taken one previous non-infected blood meal. CONCLUSIONS: The results agree with previous studies showing that older insects are often more resistant to infections than younger ones. These results suggest that structural and functional alterations in mosquito physiology with age may be more important than immunity in determining the probability of a Plasmodium infection in old mosquitoes. Possible explanations for why the effect is reversed in blood-fed mosquitoes are discussed. The reversal of the age effect in blood fed mosquitoes implies that age is unlikely to have a significant impact on mosquito susceptibility in the field.

Avian malaria: a new lease of life for an old experimental model to study the evolutionary ecology of Plasmodium.

Avian malaria has historically played an important role as a model in the study of human malaria, being a stimulus for the development of medical parasitology. Avian malaria has recently come back to the research scene as a unique animal model to understand the ecology and evolution of the disease, both in the field and in the laboratory. Avian malaria is highly prevalent in birds and mosquitoes around the world and is amenable to laboratory experimentation at each stage of the parasite's life cycle. Here, we take stock of 5 years of experimental laboratory research carried out using Plasmodium relictum SGS1, the most prevalent avian malaria lineage in Europe, and its natural vector, the mosquito Culex pipiens. For this purpose, we compile and analyse data obtained in our laboratory in 14 different experiments. We provide statistical relationships between different infection-related parameters, including parasitaemia, gametocytaemia, host morbidity (anaemia) and transmission rates to mosquitoes. This analysis provides a wide-ranging picture of the within-host and between-host parameters that may bear on malaria transmission and epidemiology.

Evolution of plastic transmission strategies in avian malaria.

Malaria parasites have been shown to adjust their life history traits to changing environmental conditions. Parasite relapses and recrudescences--marked increases in blood parasite numbers following a period when the parasite was either absent or present at very low levels in the blood, respectively--are expected to be part of such adaptive plastic strategies. Here, we first present a theoretical model that analyses the evolution of transmission strategies in fluctuating seasonal environments and we show that relapses may be adaptive if they are concomitant with the presence of mosquitoes in the vicinity of the host. We then experimentally test the hypothesis that Plasmodium parasites can respond to the presence of vectors. For this purpose, we repeatedly exposed birds infected by the avian malaria parasite Plasmodium relictum to the bites of uninfected females of its natural vector, the mosquito Culex pipiens, at three different stages of the infection: acute (∼ 34 days post infection), early chronic (∼ 122 dpi) and late chronic (∼ 291 dpi). We show that: (i) mosquito-exposed birds have significantly higher blood parasitaemia than control unexposed birds during the chronic stages of the infection and that (ii) this translates into significantly higher infection prevalence in the mosquito. Our results demonstrate the ability of Plasmodium relictum to maximize their transmission by adopting plastic life history strategies in response to the availability of insect vectors.

Dynamics of prevalence and diversity of avian malaria infections in wild Culex pipiens mosquitoes: the effects of Wolbachia, filarial nematodes and insecticide resistance.

BACKGROUND: Identifying the parasites transmitted by a particular vector and the factors that render this vector susceptible to the parasite are key steps to understanding disease transmission. Although avian malaria has become a model system for the investigation of the ecological and evolutionary dynamics of Plasmodium parasites, little is still known about the field prevalence, diversity and distribution of avian Plasmodium species within the vectors, or about the extrinsic factors affecting Plasmodium population dynamics in the wild. METHODS: We examined changes in avian malaria prevalence and Plasmodium lineage composition in female Culex pipiens caught throughout one field season in 2006, across four sampling sites in southern France. Using site occupancy models, we correct the naive estimates of Plasmodium prevalence to account for PCR-based imperfect detection. To establish the importance of different factors that may bear on the prevalence and diversity of avian Plasmodium in field mosquitoes, we focus on Wolbachia and filarial parasite co-infections, as well as on the insecticide resistance status of the mosquito. RESULTS: Plasmodium prevalence in Cx. pipiens increased from February (0%) to October (15.8%) and did not vary significantly among the four sampling sites. The application of site occupancy models leads to a 4% increase in this initial (naive) estimate of prevalence. The parasite community was composed of 15 different haemosporidian lineages, 13 of which belonged to the Plasmodium genus, and 2 to the Haemoproteus genus. Neither the presence of different Wolbachia types and of filarial parasites co-infecting the mosquitoes, nor their insecticide resistance status were found to affect the Plasmodium prevalence and diversity. CONCLUSION: We found that haemosporidian parasites are common and diverse in wild-caught Cx. pipiens mosquitoes in Southern France. The prevalence of the infection in mosquitoes is unaffected by Wolbachia and filarial co-infections as well as the insecticide resistant status of the vector. These factors may thus have a negligible impact on the transmission of avian malaria. In contrast, the steady increase in prevalence from February to October indicates that the dynamics of avian malaria is driven by seasonality and supports that infected birds are the reservoir of a diverse community of lineages in southern France.

The impact of insecticide resistance on Culex pipiens immunity.

Because of their role as vectors of diseases, the evolution of insecticide resistance in mosquitoes has been intensively investigated. Insecticide resistance is associated to a wide range of pleiotropic effects on several key life-history traits of mosquitoes such as longevity and behavior. However, despite its potential implications in pathogen transmission, the effects of insecticide resistance on mosquito immunity have received little, if any, attention. Here, we investigate the impact of insecticide resistance in Culex pipiens, an epidemiologically important vector of a wide array of pathogens. Using both isogenic laboratory strains and field-caught mosquitoes, we investigate the impact of two main insecticide resistance mechanisms (metabolic detoxification and target site modification) on the relative transcription of several genes involved in the immune response to pathogens, at both their constitutive and inducible levels. Our results show a discrepancy between the isogenic laboratory lines and field-collected mosquitoes: While in the isogenic strains, insecticide-resistant mosquitoes show a drastic increase in immune gene expression, no such effect appears in the field. We speculate on the different mechanisms that may underlie this discrepancy and discuss the risks of making inferences on the pleiotropic effects of insecticide-resistant genes by using laboratory-selected insecticide-resistant lines.

Both infected and uninfected mosquitoes are attracted toward malaria infected birds.

BACKGROUND: The biting behaviour of mosquitoes is crucial for the transmission of malaria parasites. This study focuses on the feeding behaviour of Culex pipiens mosquitoes with regard to the infection status by the avian malaria parasite Plasmodium relictum (lineage SGS1). METHODS: Uninfected and sporozoite-infected mosquitoes were provided with a choice between an uninfected bird and a bird undergoing a chronic P. relictum infection. Mosquito choice is assessed by microsatellite typing of the ingested blood. RESULTS: Chronically infected birds are more attractive to mosquitoes. This choice is not altered by the infection status of the mosquitoes: both infected and uninfected mosquitoes have similar host choice behaviours and are more attracted towards infected birds. CONCLUSIONS: These results support some, but not all predictions derived from the hypothesis that malaria parasites can manipulate the behaviour of their mosquito vectors to enhance their transmission. The possible mechanisms driving this manipulation, the evolutionary dynamics leading to the modification of the biting behaviour of mosquitoes by Plasmodium sp. as well as the implications for malaria epidemiology are discussed.

Malaria infection increases bird attractiveness to uninfected mosquitoes.

The epidemiology of vector-borne pathogens is largely determined by the host-choice behaviour of their vectors. Here, we investigate whether a Plasmodium infection renders the host more attractive to host-seeking mosquitoes. For this purpose, we work on a novel experimental system: the avian malaria parasite Plasmodium relictum, and its natural vector, the mosquito Culex pipiens. We provide uninfected mosquitoes with a choice between an uninfected bird and a bird undergoing either an acute or a chronic Plasmodium infection. Mosquito choice is assessed by microsatellite typing of the ingested blood. We show that chronically infected birds attract significantly more vectors than either uninfected or acutely infected birds. Our results suggest that malaria parasites manipulate the behaviour of uninfected vectors to increase their transmission. We discuss the underlying mechanisms driving this behavioural manipulation, as well as the broader implications of these effects for the epidemiology of malaria.

Insecticide resistance and malaria transmission: infection rate and oocyst burden in Culex pipiens mosquitoes infected with Plasmodium relictum.

BACKGROUND: The control of most vectors of malaria is threatened by the spread of insecticide resistance. One factor that has been hitherto largely overlooked is the potential effects of insecticide resistance on the ability of mosquitoes to transmit malaria: are insecticide-resistant mosquitoes as good vectors of Plasmodium as susceptible ones? The drastic physiological changes that accompany the evolution of insecticide resistance may indeed alter the ability of vectors to transmit diseases, a possibility that, if confirmed, could have major epidemiological consequences. METHODS: Using a novel experimental system consisting of the avian malaria parasite (Plasmodium relictum) and its natural vector (the mosquito Culex pipiens), two of the most common mechanisms of insecticide resistance (esterase overproduction and acetylcholinesterase modification) were investigated for their effect on mosquito infection rate and parasite burden. For this purpose two types of experiments were carried out using (i) insecticide-resistant and susceptible laboratory isogenic lines of Cx. pipiens and (ii) wild Cx. pipiens collected from a population where insecticide resistant and susceptible mosquitoes coexist in sympatry. RESULTS: The isogenic line and wild-caught mosquito experiments were highly consistent in showing no effect of either esterase overproduction or of acetylcholinesterase modification on either the infection rate or on the oocyst burden of mosquitoes. The only determinant of these traits was blood meal size, which was similar across the different insecticide resistant categories in both experiments. CONCLUSIONS: Insecticide resistance was found to have no effect on Plasmodium development within the mosquito. This is the first time this question has been addressed using a natural mosquito-Plasmodium combination, while taking care to standardize the genetic background against which the insecticide resistance genes operate. Infection rate and oocyst burden are but two of the factors that determine the vectorial capacity of mosquitoes. Other key determinants of parasite transmission, such as mosquito longevity and behaviour, or the parasite's incubation time, need to be investigated before concluding on whether insecticide resistance influences the ability of mosquitoes to transmit malaria.

Development of 10 microsatellite loci in the pulmonate snail Biomphalaria kuhniana (Mollusca, Gastropoda).

We characterized 10 variable microsatellite loci in the freshwater snail Biomphalaria kuhniana, as well as conditions for multiplexing and co-loading sets of loci. Two to five alleles were detected per locus over the two studied populations in Venezuela. High inbreeding coefficients suggest high selfing rates. Cross-species amplification provided some variability at eight and three loci in the other species belonging to the Biomphalaria straminea complex (B. straminea and B. intermedia), but was unsuccessful in more divergent species.

Development of eight microsatellite markers in the parsley frog (Pelodytes punctatus).

We characterized eight microsatellite loci to study spatial and temporal population structure of Pelodytes punctatus, a European anuran that has a peculiar breeding pattern among amphibians. The eight loci proved to be highly polymorphic with the number of alleles per locus ranging from two to 15 within two populations. Cross-amplification indicates that those markers may be also useful for closely related species from the same family.

Development of polymorphic microsatellite loci in the hermaphroditic freshwater snails Drepanotrema surinamense and Drepanotrema depressissimum.

We characterized new variable microsatellites in two congeneric species of hermaphroditic freshwater snails (Drepanotrema depressissimum and D. surinamense), as well as conditions for multiplexing and simultaneously genotyping sets of loci. D. depressissimum had high mean gene diversity (> 0.8) and large number of alleles (= 10.9) per population. Most loci and populations were at Hardy-Weinberg equilibrium. The F(ST) estimates were low among lesser Antilles populations and larger with a Venezuelan population. Far less diversity was found in D. surinamense with mean number of alleles and gene diversity per population of 2.8 and 0.34, respectively. Very few heterozygous individuals were observed. The most likely explanation is a high selfing rate (> 0.825) in this species. Unsurprisingly, the F(ST) estimates among populations were much higher than in D. depressissimum. Cross-species amplification in three congeneric species was on the whole unsuccessful.

The different sources of variation in inbreeding depression, heterosis and outbreeding depression in a metapopulation of Physa acuta.

Understanding how parental distance affects offspring fitness, i.e., the effects of inbreeding and outbreeding in natural populations, is a major goal in evolutionary biology. While inbreeding is often associated with fitness reduction (inbreeding depression), interpopulation outcrossing may have either positive (heterosis) or negative (outbreeding depression) effects. Within a metapopulation, all phenomena may occur with various intensities depending on the focal population (especially its effective size) and the trait studied. However, little is known about interpopulation variation at this scale. We here examine variation in inbreeding depression, heterosis, and outbreeding depression on life-history traits across a full-life cycle, within a metapopulation of the hermaphroditic snail Physa acuta. We show that all three phenomena can co-occur at this scale, although they are not always expressed on the same traits. A large variation in inbreeding depression, heterosis, and outbreeding depression is observed among local populations. We provide evidence that, as expected from theory, small and isolated populations enjoy higher heterosis upon outcrossing than do large, open populations. These results emphasize the need for an integrated theory accounting for the effects of both deleterious mutations and genetic incompatibilities within metapopulations and to take into account the variability of the focal population to understand the genetic consequences of inbreeding and outbreeding at this scale.