Genomic Shifts, Phenotypic Clines, and Fitness Costs Associated With Cold Tolerance in the Asian Tiger Mosquito.

Climatic variation is a key driver of genetic differentiation and phenotypic traits evolution, and local adaptation to temperature is expected in widespread species. We investigated phenotypic and genomic changes in the native range of the Asian tiger mosquito, Aedes albopictus. We first refine the phylogeographic structure based on genome-wide regions (1,901 double-digest restriction-site associated DNA single nucleotide polymophisms [ddRAD SNPs]) from 41 populations. We then explore the patterns of cold adaptation using phenotypic traits measured in common garden (wing size and cold tolerance) and genotype-temperature associations at targeted candidate regions (51,706 exon-capture SNPs) from nine populations. We confirm the existence of three evolutionary lineages including clades A (Malaysia, Thailand, Cambodia, and Laos), B (China and Okinawa), and C (South Korea and Japan). We identified temperature-associated differentiation in 15 out of 221 candidate regions but none in ddRAD regions, supporting the role of directional selection in detected genes. These include genes involved in lipid metabolism and a circadian clock gene. Most outlier SNPs are differently fixed between clades A and C, whereas clade B has an intermediate pattern. Females are larger at higher latitude yet produce no more eggs, which might favor the storage of energetic reserves in colder climate. Nondiapausing eggs from temperate populations survive better to cold exposure than those from tropical populations, suggesting they are protected from freezing damages but this cold tolerance has a fitness cost in terms of egg viability. Altogether, our results provide strong evidence for the thermal adaptation of A. albopictus across its wide temperature range.

Demographic inferences and climatic niche modelling shed light on the evolutionary history of the emblematic cold-adapted Apollo butterfly at regional scale.

Cold-adapted species escape climate warming by latitudinal and/or altitudinal range shifts, and currently occur in Southern Europe in isolated mountain ranges within "sky islands". Here, we studied the genetic structure of the Apollo butterfly in five such sky islands (above 1,000 m) in France, and infer its demographic history since the last interglacial, using single nucleotide polymorphisms (ddRADseq SNPs). The Auvergne and Alps populations show strong genetic differentiation but not alpine massifs, although separated by deep valleys. Combining three complementary demographic inference methods and species distribution models (SDMs) we show that the LIG period was highly unfavourable for Apollo that probably survived in small population in the highest summits of Auvergne. The population shifted downslope and expanded eastward between LIG and LGM throughout the large climatically suitable Rhône valley between the glaciated summits of Auvergne and Alps. The Auvergne and Alps populations started diverging before the LGM but remained largely connected till the mid-Holocene. Population decline in Auvergne was more gradual but started before (~7 kya vs. 800 ya), and was much stronger with current population size ten times lower than in the Alps. In the Alps, the low genetic structure and limited evidence for isolation by distance suggest a nonequilibrium metapopulation functioning. The core Apollo population experienced cycles of contraction-expansion with climate fluctuations with largely interconnected populations overtime according to a "metapopulation-pulsar" functioning. This study demonstrates the power of combining demographic inferences and SDMs to determine past and future evolutionary trajectories of an endangered species at a regional scale.

Long-term persistence and recycling of Bacillus thuringiensis israelensis spores in wetlands sprayed for mosquito control.

Bacillus thuringiensis subsp. israelensis (Bti) is the main larvicide used to control mosquitoes worldwide. Although there is accumulating evidence of Bti having environmental effects on non-target fauna, relatively few field studies have documented the fate of Bti spores in the environment. Spore density was quantified over a 6-yr period (2012-2017) in Mediterranean marshes sprayed with Vectobac 12AS (32 ITU/ha) since 2006 to reduce the nuisance caused by Aedes caspius. Bti spores were naturally found in all habitat types. Spore density expressed as colony-forming units per gram of soil (CFU g-1) increased significantly at treated sites by a factor of 22 to 500 times relative to control sites, with mean values of 7730 CFU g-1 in halophilous scrubs, 38,000 in reed beds, 49,000 in bulrush beds and 50 000 in rush beds. Spore density varied little in the first months after the spraying season (April-October), but increased sharply in spring, just before the annual launch of mosquito control. Considering that Bti is an insect pathogen that cannot proliferate without a suitable insect host, this unexpected recrudescence in spring could be related to the warming of water that triggers activity and development of benthic organisms such as chironomids, which may contribute to Bti proliferation by ingesting accumulated spores at the surface of sediments. While spore density tends to decrease over time, presumably during the summer period as a result of increased UV exposure, three to four years were necessary for spore density to return to normal levels after mosquito-control interruption. This study is important because it demonstrates that environmental effects of mosquito-control using Bti can far exceed the short period of Bti efficacy against lentic mosquitoes. Considering that Bti is a microbial agent, these long-term effects should be addressed at multiple levels of ecosystem organization from a one-health perspective.

Landscape does matter: Disentangling founder effects from natural and human-aided post-introduction dispersal during an ongoing biological invasion.

Environmental features impacting the spread of invasive species after introduction can be assessed using population genetic structure as a quantitative estimation of effective dispersal at the landscape scale. However, in the case of an ongoing biological invasion, deciphering whether genetic structure represents landscape connectivity or founder effects is particularly challenging. We examined the modes of dispersal (natural and human-aided) and the factors (landscape or founders history) shaping genetic structure in range edge invasive populations of the Asian tiger mosquito, Aedes albopictus, in the region of Grenoble (Southeast France). Based on detailed occupancy-detection data and environmental variables (climatic, topographic and land-cover), we modelled A. albopictus potential suitable area and its expansion history since first introduction. The relative role of dispersal modes was estimated using biological dispersal capabilities and landscape genetics approaches using genome-wide SNP dataset. We demonstrate that both natural and human-aided dispersal have promoted the expansion of populations. Populations in diffuse urban areas, representing highly suitable habitat for A. albopictus, tend to disperse less, while roads facilitate long-distance dispersal. Yet, demographic bottlenecks during introduction played a major role in shaping the genetic variability of these range edge populations. The present study is one of the few investigating the role of founder effects and ongoing expansion processes in shaping spatial patterns of genetic variation in an invasive species at the landscape scale. The combination of several dispersal modes and large proportions of continuous suitable habitats for A. albopictus promoted range filling of almost its entire potential distribution in the region of Grenoble only few years after introduction.

One, two or more species? Mitonuclear discordance and species delimitation.

Delimiting species boundaries is central to understand ecological and evolutionary processes, and to monitor biodiversity patterns over time and space. Yet, most of our current knowledge on animal diversity and phylogeny relies on morphological and mitochondrial (mt) DNA variation, a popular molecular marker also used as a barcode to assign samples to species. For morphologically undistinguishable sympatric species (cryptic species), the congruence of several independent markers is necessary to define separate species. Nuclear markers are becoming more accessible, and have confirmed that cryptic species are widespread in all animal phyla (Fiser, Robinson, & Malard, 2018). However, striking differences between the mitochondrial and nuclear variation patterns are also commonly found within single species. Mitonuclear discordance can result from incomplete lineage sorting, sex-biased dispersal, asymmetrical introgression, natural selection or Wolbachia-mediated genetic sweeps. But more generally, the distinct mode of transmission of these two types of markers (maternal vs. biparental) is sufficient to explain their distinct sensitivity to purely demographic events such as spatial range and population size fluctuations over time. In a From the Cover manuscript in this issue of Molecular Ecology, Hijonosa et al. (2019) show that highly divergent mtDNA lineages coexist in a widespread European butterfly (Figure 1). None of the hundreds of nuclear markers analyzed was associated with mt lineages, nor was Wolbachia variation. These findings rule out the presence of cryptic species but shed light on complex demographic history of lineage divergence/fusion during the Pleistocene climatic fluctuations, and pave the way to a better integration of both mt and nuclear information in demographic models.

Unravelling the invasion history of the Asian tiger mosquito in Europe.

Multiple introductions are key features for the establishment and persistence of introduced species. However, little is known about the contribution of genetic admixture to the invasive potential of populations. To address this issue, we studied the recent invasion of the Asian tiger mosquito (Aedes albopictus) in Europe. Combining genome-wide single nucleotide polymorphisms and historical knowledge using an approximate Bayesian computation framework, we reconstruct the colonization routes and establish the demographic dynamics of invasion. The colonization of Europe involved at least three independent introductions in Albania, North Italy and Central Italy that subsequently acted as dispersal centres throughout Europe. We show that the topology of human transportation networks shaped demographic histories with North Italy and Central Italy being the main dispersal centres in Europe. Introduction modalities conditioned the levels of genetic diversity in invading populations, and genetically diverse and admixed populations promoted more secondary introductions and have spread farther than single-source invasions. This genomic study provides further crucial insights into a general understanding of the role of genetic diversity promoted by modern trade in driving biological invasions.

Alkaline phosphatases are involved in the response of Aedes aegypti larvae to intoxication with Bacillus thuringiensis subsp. israelensis†Cry toxins.

Bacillus thuringiensis subsp. israelensis (Bti) is a natural pathogen of dipterans widely used as a biological insecticide for mosquito control. To characterize the response of mosquitoes to intoxication with Bti, the transcriptome profile of Bti-exposed susceptible Aedes aegypti larvae was analysed using Illumina RNA-seq. Gene expression of 11 alkaline phosphatases (ALPs) was further investigated by real time quantitative polymerase chain reaction and ALP activity was measured in the susceptible strain and in four strains resistant to a single Bti Cry toxin or to Bti. These strains were unexposed or exposed to their toxin of selection. Although all resistant strains constitutively exhibited a higher level of transcription of ALP genes than the susceptible strain, they showed a lower total ALP activity. The intoxication with different individual Cry toxins triggered a global pattern of ALP gene under-transcription in all the one-toxin-resistant strains but involving different specific sets of ALPs in each resistant phenotype. Most of the ALPs involved are not known Cry-binding proteins. RNA interference experiment demonstrated that reducing ALP expression conferred increased the survival of larvae exposed to Cry4Aa, confirming the involvement of ALP in Cry4Aa toxicity.

Hybridization promotes speciation in Coenonympha butterflies.

Hybridization has become a central element in theories of animal evolution during the last decade. New methods in population genomics and statistical model testing now allow the disentangling of the complexity that hybridization brings into key evolutionary processes such as local adaptation, colonization of new environments, species diversification and extinction. We evaluated the consequences of hybridization in a complex of three alpine butterflies in the genus Coenonympha, by combining morphological, genetic and ecological analyses. A series of approximate Bayesian computation procedures based on a large SNP data set strongly suggest that the Darwin's Heath (Coenonympha darwiniana) originated through hybridization between the Pearly Heath (Coenonympha arcania) and the Alpine Heath (Coenonympha gardetta) with different parental contributions. As a result of hybridization, the Darwin's Heath presents an intermediate morphology between the parental species, while its climatic niche seems more similar to the Alpine Heath. Our results also reveal a substantial genetic and morphologic differentiation between the two geographically disjoint Darwin's Heath lineages leading us to propose the splitting of this taxon into two different species.

Gene expression patterns and sequence polymorphisms associated with mosquito resistance to Bacillus thuringiensis israelensis toxins.

BACKGROUND: Despite the intensive use of Bacillus thuringiensis israelensis (Bti) toxins for mosquito control, little is known about the long term effect of exposure to this cocktail of toxins on target mosquito populations. In contrast to the many cases of resistance to Bacillus thuringiensis Cry toxins observed in other insects, there is no evidence so far for Bti resistance evolution in field mosquito populations. High fitness costs measured in a Bti selected mosquito laboratory strain suggest that evolving resistance to Bti is costly. The aim of the present study was to identify transcription level and polymorphism variations associated with resistance to Bti toxins in the dengue vector Aedes aegypti. We used RNA sequencing (RNA-seq) for comparing a laboratory-selected strain showing elevated resistance to Bti toxins and its parental non-selected susceptible strain. As the resistant strain displayed two marked larval development phenotypes (slow and normal), each phenotype was analyzed separately in order to evidence potential links between resistance mechanisms and mosquito life-history traits. RESULTS: A total of 12,458 genes were detected of which 844 were differentially transcribed between the resistant and susceptible strains. Polymorphism analysis revealed a total of 68,541 SNPs of which 12,571 SNPs exhibited more than 40% frequency difference between the resistant and susceptible strains, affecting 2,953 genes. Bti resistance is associated with changes in the transcription level of enzymes involved in detoxification and chitin metabolism. Among previously described Bti-toxin receptors, four alkaline phosphatases (ALPs) were differentially transcribed between resistant and susceptible larvae, and non-synonymous changes affected the protein sequence of one cadherin, six aminopeptidases (APNs) and four α-amylases. Other putative Cry receptors located in lipid rafts, such as flotillin and glycoside hydrolases, were under-transcribed and/or contained non-synonymous substitutions. Finally, immunity-related genes showed contrasted transcription and polymorphisms patterns between the two developmental resistant phenotypes, suggesting the existence of trade-offs between Bti-resistance, life-history traits and immunity. CONCLUSIONS: The present study is the first to analyze the whole transcriptome of Bti-resistant mosquitoes by RNA-seq, shedding light on the importance of studying both transcription levels and sequence polymorphism variations to get a comprehensive view of insecticide resistance.

Chemical and biological insecticides select distinct gene expression patterns in Aedes aegypti mosquito.

Worldwide evolution of mosquito resistance to chemical insecticides represents a major challenge for public health, and the future of vector control largely relies on the development of biological insecticides that can be used in combination with chemicals (integrated management), with the expectation that populations already resistant to chemicals will not become readily resistant to biological insecticides. However, little is known about the metabolic pathways affected by selection with chemical or biological insecticides. Here we show that Aedes aegypti, a laboratory mosquito strain selected with a biological insecticide (Bacillus thuringiensis israelensis, Bti) evolved increased transcription of many genes coding for endopeptidases while most genes coding for detoxification enzymes were under-expressed. By contrast, in strains selected with chemicals, genes encoding detoxification enzymes were mostly over-expressed. In all the resistant strains, genes involved in immune response were under-transcribed, suggesting that basal immunity might be a general adjustment variable to compensate metabolic costs caused by insecticide selection. Bioassays generally showed no evidence for an increased susceptibility of selected strains towards the other insecticide type, and all chemical-resistant strains were as susceptible to Bti as the unselected parent strain, which is a good premise for sustainable integrated management of mosquito populations resistant to chemicals.

Persistence and recycling of bioinsecticidal Bacillus thuringiensis subsp. israelensis spores in contrasting environments: evidence from field monitoring and laboratory experiments.

Sprays of commercial preparations of the bacterium Bacillus thuringiensis subsp. israelensis are widely used for the control of mosquito larvae. Despite an abundant literature on B. thuringiensis subsp. israelensis field efficiency on mosquito control, few studies have evaluated the fate of spores in the environment after treatments. In the present article, two complementary experiments were conducted to study the effect of different parameters on B. thuringiensis subsp. israelensis persistence and recycling, in field conditions and in the laboratory. First, we monitored B. thuringiensis subsp. israelensis persistence in the field in two contrasting regions in France: the Rhône-Alpes region, where mosquito breeding sites are temporary ponds under forest cover with large amounts of decaying leaf matter on the ground and the Mediterranean region characterized by open breeding sites such as brackish marshes. Viable B. thuringiensis subsp. israelensis spores can persist for months after a treatment, and their quantity is explained both by the vegetation type and by the number of local treatments. We found no evidence of B. thuringiensis subsp. israelensis recycling in the field. Then, we tested the effect of water level, substrate type, salinity and presence of mosquito larvae on the persistence/recycling of B. thuringiensis subsp. israelensis spores in controlled laboratory conditions (microcosms). We found no effect of change in water level or salinity on B. thuringiensis subsp. israelensis persistence over time (75 days). B. thuringiensis subsp. israelensis spores tended to persist longer in substrates containing organic matter compared to sand-only substrates. B. thuringiensis subsp. israelensis recycling only occurred in presence of mosquito larvae but was unrelated to the presence of organic matter.

Pre-selecting resistance against individual Bti Cry toxins facilitates the development of resistance to the Bti toxins cocktail.

The bioinsecticide Bacillus thuringiensis subsp. israelensis is a larvicide used worldwide for mosquito control, which contains three Cry toxins and one Cyt toxin. We investigated for the first time in Aedes aegypti (1) the evolution of resistance and cross-resistance of strains selected with each Cry toxin, and (2) the effect of pre-selection with Cry toxin on the evolution of resistance to a mix of Bti toxins. Cross resistance was higher between Cry4Ba and Cry11Aa than between Cry4Aa and either Cry4Ba or Cry11Aa, suggesting both common and specific mechanisms of resistance. Pre-selecting resistance to each Cry toxins facilitated the development of resistance to the full Bti toxins cocktail.

Chromosome-Level Assembly and Annotation of the Pearly Heath Coenonympha arcania Butterfly Genome.

We present the first chromosome-level genome assembly and annotation of the pearly heath Coenonympha arcania, generated with a PacBio HiFi sequencing approach and complemented with Hi-C data. We additionally compare synteny, gene, and repeat content between C. arcania and other Lepidopteran genomes. This reference genome will enable future population genomics studies with Coenonympha butterflies, a species-rich genus that encompasses some of the most highly endangered butterfly taxa in Europe.

Increase in larval gut proteolytic activities and Bti resistance in the Dengue fever mosquito.

The bioinsecticide Bacillus thuringiensis var. israelensis (Bti) is increasingly used worldwide for mosquito control. Although no established resistance to Bti has been described in the field so far, a resistant Aedes aegypti strain (LiTOX strain) was selected in the laboratory using field-collected leaf litter containing Bti toxins. This selected strain exhibits a moderate resistance level to Bti, but a high resistance level to individual Cry toxins. As Bti contains four different toxins, generalist resistance mechanisms affecting mosquito tolerance to different toxins were expected in the resistant strain. In the present work, we show that the resistant strain exhibits an increase of various gut proteolytic activities including trypsins, leucine-aminopeptidases, and carboxypeptidase A activities. These elevated proteolytic activities resulted in a faster activation of Cry4Aa protoxins while Cry4Ba or Cry11Aa were not affected. These results suggest that changes in proteolytic activities may contribute to Bti resistance in mosquitoes together with other mechanisms.

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately.

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches.

BACKGROUND: Bacillus thuringiensis var. israelensis (Bti) is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis). RESULTS: Several Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti-receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut. CONCLUSIONS: By combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti-resistance mechanisms in mosquitoes.

Fate of Bacillus thuringiensis subsp. israelensis in the field: evidence for spore recycling and differential persistence of toxins in leaf litter.

Bacillus thuringiensis subsp. israelensis is a bioinsecticide increasingly used worldwide for mosquito control. Despite its apparent low level of persistence in the field due to the rapid loss of its insecticidal activity, an increasing number of studies suggested that the recycling of B. thuringiensis subsp. israelensis can occur under specific, unknown conditions. Decaying leaf litters sampled in mosquito breeding sites in the French Rhône-Alpes region several months after a treatment were shown to exhibit a high level of larval toxicity and contained large amounts of spores. In the present article, we show that the high concentration of toxins found in these litters is consistent with spore recycling in the field, which gave rise to the production of new crystal toxins. Furthermore, in these toxic leaf litter samples, Cry4Aa and Cry4Ba toxins became the major toxins instead of Cyt1Aa in the commercial mixture. In a microcosm experiment performed in the laboratory, we also demonstrated that the toxins, when added in their crystal form to nontoxic leaf litter, exhibited patterns of differential persistence consistent with the proportions of toxins observed in the field-collected toxic leaf litter samples (Cry4 > Cry11 > Cyt). These results give strong evidence that B. thuringiensis subsp. israelensis recycled in specific breeding sites containing leaf litters, and one would be justified in asking whether mosquitoes can become resistant when exposed to field-persistent B. thuringiensis subsp. israelensis for several generations.

Decreased toxicity of Bacillus thuringiensis subsp. israelensis to mosquito larvae after contact with leaf litter.

Bacillus thuringiensis subsp. israelensis is a bacterium producing crystals containing Cry and Cyt proteins, which are toxic for mosquito larvae. Nothing is known about the interaction between crystal toxins and decaying leaf litter, which is a major component of several mosquito breeding sites and represents an important food source. In the present work, we investigated the behavior of B. thuringiensis subsp. israelensis toxic crystals sprayed on leaf litter. In the presence of leaf litter, a 60% decrease in the amount of Cyt toxin detectable by immunology (enzyme-linked immunosorbent assays [ELISAs]) was observed, while the respective proportions of Cry toxins were not affected. The toxicity of Cry toxins toward Aedes aegypti larvae was not affected by leaf litter, while the synergistic effect of Cyt toxins on all B. thuringiensis subsp. israelensis Cry toxins was decreased by about 20% when mixed with leaf litter. The toxicity of two commercial B. thuringiensis subsp. israelensis strains (VectoBac WG and VectoBac 12AS) and a laboratory-produced B. thuringiensis subsp. israelensis strain decreased by about 70% when mixed with leaf litter. Taken together, these results suggest that Cyt toxins interact with leaf litter, resulting in a decreased toxicity of B. thuringiensis subsp. israelensis in litter-rich environments and thereby dramatically reducing the efficiency of mosquitocidal treatments.

Identifying insecticide resistance genes in mosquito by combining AFLP genome scans and 454 pyrosequencing.

AFLP-based genome scans are widely used to study the genetics of adaptation and to identify genomic regions potentially under selection. However, this approach usually fails to detect the actual genes or mutations targeted by selection owing to the difficulties of obtaining DNA sequences from AFLP fragments. Here, we combine classical AFLP outlier detection with 454 sequencing of AFLP fragments to obtain sequences from outlier loci. We applied this approach to the study of resistance to Bacillus thuringiensis israelensis (Bti) toxins in the dengue vector Aedes aegypti. A genome scan of Bti-resistant and Bti-susceptible A. aegypti laboratory strains was performed based on 432 AFLP markers. Fourteen outliers were detected using two different population genetic algorithms. Out of these, 11 were successfully sequenced. Three contained transposable elements (TEs) sequences, and the 10 outliers that could be mapped at a unique location in the reference genome were located on different supercontigs. One outlier was in the vicinity of a gene coding for an aminopeptidase potentially involved in Bti toxin-binding. Patterns of sequence variability of this gene showed significant deviation from neutrality in the resistant strain but not in the susceptible strain, even after taking into account the known demographic history of the selected strain. This gene is a promising candidate for future functional analysis.

Specialized nursery pollination mutualisms as evolutionary traps stabilized by antagonistic traits.

We examine the conditions for the transition from antagonism to mutualism between plants and their specialists nursery pollinators in a reference case which is the Trollius europaeus-Chiastocheta interaction. The mechanistic model we developed shows that a specialization of T. europaeus on Chiastocheta could be the result of an attempt to escape over-exploitation by closing its flower. The pressure for such an escape increases with the parasite's frequency and its pollination efficiency but decreases in the presence of alternative pollinators. The resulting specialization is a priori an unstable one, leading either to strong evolutionary oscillations, or to evolutionary suicide due to over-exploitation of the plants. It becomes stable if the plants develop a defense mechanism to regulate their parasite's population size and limit seed-exploitation. The development of a counter-measure by the latter can destabilize the mutualism depending on the costs linked to such a trait. On the other hand, we find that a specialization on a purely mutualistic basis would require a preexisting high diversity of flower-opening within the population.