Up-regulation of apoptotic- and cell survival-related gene pathways following exposures of western corn rootworm to B. thuringiensis crystalline pesticidal proteins in transgenic maize roots.

BACKGROUND: Resistance of pest insect species to insecticides, including B. thuringiensis (Bt) pesticidal proteins expressed by transgenic plants, is a threat to global food security. Despite the western corn rootworm, Diabrotica virgifera virgifera, being a major pest of maize and having populations showing increasing levels of resistance to hybrids expressing Bt pesticidal proteins, the cell mechanisms leading to mortality are not fully understood. RESULTS: Twenty unique RNA-seq libraries from the Bt susceptible D. v. virgifera inbred line Ped12, representing all growth stages and a range of different adult and larval exposures, were assembled into a reference transcriptome. Ten-day exposures of Ped12 larvae to transgenic Bt Cry3Bb1 and Gpp34/Tpp35Ab1 maize roots showed significant differential expression of 1055 and 1374 transcripts, respectively, compared to cohorts on non-Bt maize. Among these, 696 were differentially expressed in both Cry3Bb1 and Gpp34/Tpp35Ab1 maize exposures. Differentially-expressed transcripts encoded protein domains putatively involved in detoxification, metabolism, binding, and transport, were, in part, shared among transcripts that changed significantly following exposures to the entomopathogens Heterorhabditis bacteriophora and Metarhizium anisopliae. Differentially expressed transcripts in common between Bt and entomopathogen treatments encode proteins in general stress response pathways, including putative Bt binding receptors from the ATP binding cassette transporter superfamily. Putative caspases, pro- and anti-apoptotic factors, as well as endoplasmic reticulum (ER) stress-response factors were identified among transcripts uniquely up-regulated following exposure to either Bt protein. CONCLUSIONS: Our study suggests that the up-regulation of genes involved in ER stress management and apoptotic progression may be important in determining cell fate following exposure of susceptible D. v. virgifera larvae to Bt maize roots. This study provides novel insights into insect response to Bt intoxication, and a possible framework for future investigations of resistance mechanisms.

Biases of STRUCTURE software when exploring introduction routes of invasive species.

Population genetic methods are widely used to retrace the introduction routes of invasive species. The unsupervised Bayesian clustering algorithm implemented in STRUCTURE is amongst the most frequently used of these methods, but its ability to provide reliable information about introduction routes has never been assessed. We simulated microsatellite datasets to evaluate the extent to which the results provided by STRUCTURE were misleading for the inference of introduction routes. We focused on an invasion scenario involving one native and two independently introduced populations, because it is the sole scenario that can be rejected when obtaining a particular clustering with a STRUCTURE analysis at K = 2 (two clusters). Results were classified as "misleading" or "non-misleading". We investigated the influence of effective size, bottleneck severity and number of loci on the type and frequency of misleading results. We showed that misleading STRUCTURE results were obtained for 10% of all simulated datasets. Our results highlighted two categories of misleading output. The first occurs when the native population has a low level of diversity. In this case, the two introduced populations may be very similar, despite their independent introduction histories. The second category results from convergence issues in STRUCTURE for K = 2, with strong bottleneck severity and/or large numbers of loci resulting in high levels of differentiation between the three populations. Overall, the risk of being misled by STRUCTURE in the context of introduction routes inferences is moderate, but it is important to remain cautious when low genetic diversity or genuine multimodality between runs are involved.

Cannibalism in invasive, native and biocontrol populations of the harlequin ladybird.

BACKGROUND: Cannibalism is widespread in both vertebrates and invertebrates but its extent is variable between and within species. Cannibalism depends on population density and nutritional conditions, and could be beneficial during colonisation of new environments. Empirical studies are needed to determine whether this trait might facilitate invasion of a new area in natural systems. We investigated whether the propensity for cannibalism in H. axyridis differs both between native and invasive populations and between invasive populations from the core and from the front of the invasive area in Western Europe. We also compared the propensity for cannibalism of these natural populations with that of laboratory-reared biocontrol populations. We measured the cannibalism rates of eggs by first instar larvae and adult females at two different individual densities of ladybirds from three types of population (invasive, native and biocontrol), in laboratory-controlled conditions. RESULTS: Cannibalism was significantly greater in larvae from invasive populations compared to native or biocontrol populations, but there was no difference in cannibalism rates between populations from the core or front of the invaded range. Cannibalism was significantly lower in larvae from biocontrol populations compared to wild (invasive and native) populations. No differences in cannibalism rates of adult females were found between any populations. While high population density significantly increased cannibalism in both larvae and adults, the norm of reaction of cannibalism to individual density did not change significantly during the invasion and/or laboratory rearing processes. CONCLUSION: This study is the first to provide evidence for a higher propensity for cannibalism in invasive populations compared to native ones. Our experiments also shed light on the difference in cannibalism evolution with respect to life stages. However, we are still at an early stage in understanding the underlying mechanisms and several different research perspectives are needed to determine whether the higher propensity for cannibalism is a general feature of the invasion process.

Complementarity of statistical treatments to reconstruct worldwide routes of invasion: the case of the Asian ladybird Harmonia axyridis.

Inferences about introduction histories of invasive species remain challenging because of the stochastic demographic processes involved. Approximate Bayesian computation (ABC) can help to overcome these problems, but such method requires a prior understanding of population structure over the study area, necessitating the use of alternative methods and an intense sampling design. In this study, we made inferences about the worldwide invasion history of the ladybird Harmonia axyridis by various population genetics statistical methods, using a large set of sampling sites distributed over most of the species' native and invaded areas. We evaluated the complementarity of the statistical methods and the consequences of using different sets of site samples for ABC inferences. We found that the H. axyridis invasion has involved two bridgehead invasive populations in North America, which have served as the source populations for at least six independent introductions into other continents. We also identified several situations of genetic admixture between differentiated sources. Our results highlight the importance of coupling ABC methods with more traditional statistical approaches. We found that the choice of site samples could affect the conclusions of ABC analyses comparing possible scenarios. Approaches involving independent ABC analyses on several sample sets constitute a sensible solution, complementary to standard quality controls based on the analysis of pseudo-observed data sets, to minimize erroneous conclusions. This study provides biologists without expertise in this area with detailed methodological and conceptual guidelines for making inferences about invasion routes when dealing with a large number of sampling sites and complex population genetic structures.

Estimation of the dispersal of a major pest of maize by cline analysis of a temporary contact zone between two invasive outbreaks.

Dispersal is a key factor in invasion and in the persistence and evolution of species. Despite the importance of estimates of dispersal distance, dispersal measurement remains a real methodological challenge. In this study, we characterized dispersal by exploiting a specific case of biological invasion, in which multiple introductions in disconnected areas lead to secondary contact between two differentiated expanding outbreaks. By applying cline theory to this ecological setting, we estimated σ, the standard deviation of the parent-offspring distance distribution, of the western corn rootworm, Diabrotica virgifera virgifera, one of the most destructive pests of maize. This species is currently invading Europe, and the two largest invasive outbreaks, in northern Italy and Central Europe, have recently formed a secondary contact zone in northern Italy. We identified vanishing clines at 12 microsatellite loci throughout the contact zone. By analysing both the rate of change of cline slope and the spatial variation of linkage disequilibrium at these markers, we obtained two σ estimates of about 20 km/generation(1/2). Simulations indicated that these estimates were robust to changes in dispersal kernels and differences in population density between the two outbreaks, despite a systematic weak bias. These estimates are consistent with the results of direct methods for measuring dispersal applied to the same species. We conclude that secondary contact resulting from multiple introductions is very useful for the inference of dispersal parameters and should be more widely used in other species.

Genome-wide survey and analysis of microsatellites in nematodes, with a focus on the plant-parasitic species Meloidogyne incognita.

BACKGROUND: Microsatellites are the most popular source of molecular markers for studying population genetic variation in eukaryotes. However, few data are currently available about their genomic distribution and abundance across the phylum Nematoda. The recent completion of the genomes of several nematode species, including Meloidogyne incognita, a major agricultural pest worldwide, now opens the way for a comparative survey and analysis of microsatellites in these organisms. RESULTS: Using MsatFinder, the total numbers of 1-6 bp perfect microsatellites detected in the complete genomes of five nematode species (Brugia malayi, Caenorhabditis elegans, M. hapla, M. incognita, Pristionchus pacificus) ranged from 2,842 to 61,547, and covered from 0.09 to 1.20% of the nematode genomes. Under our search criteria, the most common repeat motifs for each length class varied according to the different nematode species considered, with no obvious relation to the AT-richness of their genomes. Overall, (AT)n, (AG)n and (CT)n were the three most frequent dinucleotide microsatellite motifs found in the five genomes considered. Except for two motifs in P. pacificus, all the most frequent trinucleotide motifs were AT-rich, with (AAT)n and (ATT)n being the only common to the five nematode species. A particular attention was paid to the microsatellite content of the plant-parasitic species M. incognita. In this species, a repertoire of 4,880 microsatellite loci was identified, from which 2,183 appeared suitable to design markers for population genetic studies. Interestingly, 1,094 microsatellites were identified in 801 predicted protein-coding regions, 99% of them being trinucleotides. When compared against the InterPro domain database, 497 of these CDS were successfully annotated, and further assigned to Gene Ontology terms. CONCLUSIONS: Contrasted patterns of microsatellite abundance and diversity were characterized in five nematode genomes, even in the case of two closely related Meloidogyne species. 2,245 di- to hexanucleotide loci were identified in the genome of M. incognita, providing adequate material for the future development of a wide range of microsatellite markers in this major plant parasite.

Reconstructing routes of invasion using genetic data: why, how and so what?

Detailed knowledge about the geographical pathways followed by propagules from their source to the invading populations--referred to here as routes of invasion-provides information about the history of the invasion process and the origin and genetic composition of the invading populations. The reconstruction of invasion routes is required for defining and testing different hypotheses concerning the environmental and evolutionary factors responsible for biological invasions. In practical terms, it facilitates the design of strategies for controlling or preventing invasions. Most of our knowledge about the introduction routes of invasive species is derived from historical and observational data, which are often sparse, incomplete and, sometimes, misleading. In this context, population genetics has proved a useful approach for reconstructing routes of introduction, highlighting the complexity and the often counterintuitive nature of the true story. This approach has proved particularly useful since the recent development of new model-based methods, such as approximate Bayesian computation, making it possible to make quantitative inferences in the complex evolutionary scenarios typically encountered in invasive species. In this review, we summarize some of the fundamental aspects of routes of invasion, explain why the reconstruction of these routes is useful for addressing both practical and theoretical questions, and comment on the various reconstruction methods available. Finally, we consider the main insights obtained to date from studies of invasion routes.

Inferring population history with DIY ABC: a user-friendly approach to approximate Bayesian computation.

UNLABELLED: Genetic data obtained on population samples convey information about their evolutionary history. Inference methods can extract part of this information but they require sophisticated statistical techniques that have been made available to the biologist community (through computer programs) only for simple and standard situations typically involving a small number of samples. We propose here a computer program (DIY ABC) for inference based on approximate Bayesian computation (ABC), in which scenarios can be customized by the user to fit many complex situations involving any number of populations and samples. Such scenarios involve any combination of population divergences, admixtures and population size changes. DIY ABC can be used to compare competing scenarios, estimate parameters for one or more scenarios and compute bias and precision measures for a given scenario and known values of parameters (the current version applies to unlinked microsatellite data). This article describes key methods used in the program and provides its main features. The analysis of one simulated and one real dataset, both with complex evolutionary scenarios, illustrates the main possibilities of DIY ABC. AVAILABILITY: The software DIY ABC is freely available at http://www.montpellier.inra.fr/CBGP/diyabc.

Microsatellite-based parentage analysis reveals non-ideal free distribution in a parasitoid population.

Habitat selection by dispersers is the focus of much theoretical models, most of which are based on the assumption of negative density dependence. The archetype of these models is the ideal free distribution, characterized by an evolutionary stable state where more competitors aggregate in better habitats, so that the fitness benefit of resource abundance is equally offset by the cost of competition in all habitats. In this study, we used parentage analysis on microsatellite genotypes to test the ideal free distribution in a natural population of aphid parasitoids. Parentage analysis was conducted on parasitoids emerging from aphid colonies. We inferred the number of foundress females which had reproduced in each colony, as well as the number of offspring for each foundress. As predicted by the ideal free distribution, the number of offspring per foundress per colony did not depend on the number of hosts per colony. However, contrary to ideal free distribution predictions, it was affected by the number of foundresses per colony. In surprising contrast with the basic assumption of negative density dependence, individual fitness increased with the number of foundresses. Moreover, parentage analysis revealed a very low number of offspring per foundress per colony (mean = 1.8). This observed distribution questions the validity of classical models of habitat choice based on competition. Indeed, our results provide a new illustration reinforcing a growing body of theory and data on positive density dependence. Our results also suggest that the avoidance of hyperparasitism and predation, although generally neglected, may shape the distribution of parasitoids in the field.

A core set of microsatellite markers for western corn rootworm (Coleoptera: Chrysomelidae) population genetics studies.

Interest in the ecological and population genetics of the western corn rootworm, Diabrotica virgifera virgifera LeConte, has grown rapidly in the last few years in North America and Europe. This interest is a result of a number of converging issues related to the increasing difficulty in managing this pest and the need to characterize and understand gene flow in the context of insect resistance management. One of the key components needed for successful population genetics studies is the availability of suitable molecular markers. Using a standard group of microsatellite markers enables researchers from different laboratories to directly compare and share their data, reducing duplication of effort and facilitating collaborative work among laboratories. We screened 22 candidate microsatellite loci against five criteria to create a core set of microsatellite markers for D. v. virgifera population genetics studies. The criteria for inclusion were moderate to high polymorphism, unambiguous readability and repeatability, no evidence of null alleles, apparent selective neutrality, and no linkage between loci. Based on our results, we recommend six microsatellite markers to be included as a core set in future population genetics studies of D. v. virgifera along with any other microsatellite or genetic markers. As more microsatellites are developed, those meeting the criteria can be added to the core set. We encourage other groups of researchers with common interests in a particular insect species to develop their own core sets of markers for population genetics applications.

Within-species variability of the response to 20-hydroxyecdysone in peach-potato aphid (Myzus persicae Sulzer).

Phytoecdysteroids have been proposed as new tools for controlling crop pests because of their endocrine disruption and deterrent effects on insects and nematodes. There is increasing evidence of variability between taxa in sensitivity to phytoecdysteroids, but the genetic variability of this sensitivity within species is unknown. However, knowledge about this intraspecies variability is required for predicting evolution of the pest's response to new control methods. We assessed the variability of the response of the aphid Myzus persicae Sulzer, a major agricultural pest, to 20-hydroxyecdysone (20E). We determined the number of nymphs produced by six clones of M. persicae exposed to various concentrations of 20E and the capacity of these clones to detect 20E in choice experiments. High concentrations of 20E significantly decreased the number of nymphs produced for two clones and both increases and decreases in the number of offspring were detected at low concentrations. Two clones significantly avoided food with 20E, while one significantly preferred it, suggesting that 20E does not always act as a deterrent in this species. We conclude that genetic variability in the response to 20E exists in natural populations of M. persicae. The consequences of this finding on the sustainability of control methods using 20E are discussed.

Conflicts of Interest in GM Bt Crop Efficacy and Durability Studies.

Public confidence in genetically modified (GM) crop studies is tenuous at best in many countries, including those of the European Union in particular. A lack of information about the effects of ties between academic research and industry might stretch this confidence to the breaking point. We therefore performed an analysis on a large set of research articles (n = 672) focusing on the efficacy or durability of GM Bt crops and ties between the researchers carrying out these studies and the GM crop industry. We found that ties between researchers and the GM crop industry were common, with 40% of the articles considered displaying conflicts of interest (COI). In particular, we found that, compared to the absence of COI, the presence of a COI was associated with a 50% higher frequency of outcomes favorable to the interests of the GM crop company. Using our large dataset, we were able to propose possible direct and indirect mechanisms behind this statistical association. They might notably include changes of authorship or funding statements after the results of a study have been obtained and a choice in the topics studied driven by industrial priorities.

Asymmetry in host and parasitoid diffuse coevolution: when the red queen has to keep a finger in more than one pie.

BACKGROUND: Coevolution between pairs of antagonistic species is generally considered an endless "arms race" between attack and defense traits to counteract the adaptive responses of the other species. PRESENTATION OF THE HYPOTHESIS: When more than two species are involved, diffuse coevolution of hosts and parasitoids could be asymmetric because consumers can choose their prey whereas preys do not choose their predator. This asymmetry may lead to differences in the rate of evolution of the antagonistic species in response to selection. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of the hypothesis is that the consumer trophic level is more likely to win the coevolution race. TESTING THE HYPOTHESIS: We propose testing the asymmetry hypothesis by focusing on the tritrophic system plant/aphid/aphid parasitoid. The analysis of the genetic variability in the virulence of several parasitoid populations and in the defenses of several aphid species or several clones of the same aphid species could be compared. Moreover, the analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation may complement the detection of differences between host and parasitoid trophic specialization. IMPLICATIONS OF THE HYPOTHESIS: Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots. Thus this hypothesis profoundly changes our understanding of coevolution and may have important implications in terms of pest management.

Development of 23 polymorphic microsatellite loci in invasive silver wattle, Acacia dealbata (Fabaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for silver wattle, Acacia dealbata (Fabaceae), which is both an ornamental and an invasive weed species. It is native to southeastern Australia and invasive in Europe, Africa, Asia, and the Americas. METHODS AND RESULTS: The pyrosequencing of a microsatellite-enriched genomic DNA library of A. dealbata produced 33,290 sequences and allowed the isolation of 201 loci with a minimum of seven repeats of microsatellite motifs. Amplification tests led to the setup of two multiplex PCR mixes allowing the amplification of 21 loci. The polymorphism of these markers was evaluated on a sample of 32 individuals collected in southeastern Australia. The number of alleles and the expected heterozygosity varied between two and 11, and between 0.11 and 0.88, respectively. CONCLUSIONS: The level of polymorphism of this set of 23 microsatellites is large enough to provide valuable information on the genetic structure and the invasion history of A. dealbata.

The tomato borer, Tuta absoluta, invading the Mediterranean Basin, originates from a single introduction from Central Chile.

The Lepidopteran pest of tomato, Tuta absoluta, is native to South America and is invasive in the Mediterranean basin. The species' routes of invasion were investigated. The genetic variability of samples collected in South America, Europe, Africa and Middle East was analyzed using microsatellite markers to infer precisely the source of the invasive populations and to test the hypothesis of a single versus multiple introductions into the old world continents. This analysis provides strong evidence that the origin of the invading populations was unique and was close to or in Chile, and probably in Central Chile near the town of Talca in the district of Maule.

Fitness costs of insecticide resistance in natural breeding sites of the mosquito Culex pipiens.

Genetic changes conferring adaptation to a new environment may induce a fitness cost in the previous environment. Although this prediction has been verified in laboratory conditions, few studies have tried to document this cost directly in natural populations. Here, we evaluated the pleiotropic effects of insecticide resistance on putative fitness components of the mosquito Culex pipiens. Experiments using different larval densities were performed during the summer in two natural breeding sites. Two loci that possess alleles conferring organophosphate (OP) resistance were considered: ace-1 coding for an acetylcholinesterase (AChE1, the OP target) and Ester, a ''super locus" including two closely linked loci coding for esterases A and B. Resistance ace-1 alleles coding for a modified AChE1 were associated with a longer development time and shorter wing length. The pleiotropic effects of two resistance alleles Ester1 and Ester4 coding for the overproduced esterases A1 and A4-B4, respectively, were more variable. Both A1 and A4-B4 reduced wing length, although only A1 was associated with a longer preimaginal stage. The fluctuating asymmetry (FA) of the wing did not respond to the presence or to the interaction of resistance alleles at the two loci at any of the density levels tested. Conversely, the FA of one wing section decreased when larval density increased. This may be the consequence of selection against less developmentally stable individuals. The results are discussed in relation to the local evolution of insecticide resistance genes.

Genetic study of Coris julis (Osteichthyes, Perciformes, Labridae) evolutionary history and dispersal abilities.

Microsatellite markers have been used to study the genetic variability of rainbow wrasse (Coris julis) Mediterranean and Atlantic populations. Differentiation tests failed to reveal any significant genetic differentiation among samples from continental Portugal and the Azores, despite more than 1800 km of geographical separation. Preliminary results tended to indicate a significant genetic differentiation among Atlantic and Mediterranean samples. It also supported the specific status of Cape Verde populations (Coris atlantica). We compare these results with previous mtDNA analyses and propose a biogeographic scenario that could explain our results.

No clear effect of admixture between two European invading outbreaks of Diabrotica virgifera virgifera in natura.

In this study, we challenged the hypothesis that admixture may have had a positive impact in the context of the European invasion of the western corn rootworm (WCR), Diabrotica virgifera virgifera, LeConte. This beetle was introduced in Europe from the USA several times since the 1980's. The multiple introductions of this major pest of cultivated corn led to the formation of two major outbreaks in North Western (NW) Italy and in Central and South Eastern (CSE) Europe that eventually merged into a secondary contact zone where insects from both outbreaks interbreed. We collected about 600 insects from this contact zone and genotyped them using 13 microsatellite markers. Three types of information were obtained from the collected individuals: (i) their survival under starvation; (ii) their admixed status, determined through a Bayesian method of genetic clustering and (iii) their mating probability, studied via the detection, isolation and genotyping of sperm in female spermathecae. Twenty six % and 12% of the individuals were assigned to the NW Italy or the CSE Europe parental types, respectively, and 23% and 39% to the F1 and backcross hybrid types, respectively. Globally, our results do not reveal any significant impact of the admixed status on the mating probability and on the choice of mating partners. However the admixed status had a sex- and sampling site-dependent effect on survival in adults under starvation. In addition sex had an effect on survival, with mortality hazard about 3 times larger in males than in females. The consequences of these findings for the evolution of the admixture zone of northern Italy are discussed.

First insights into the genetic diversity of the pinewood nematode in its native area using new polymorphic microsatellite loci.

The pinewood nematode, Bursaphelenchus xylophilus, native to North America, is the causative agent of pine wilt disease and among the most important invasive forest pests in the East-Asian countries, such as Japan and China. Since 1999, it has been found in Europe in the Iberian Peninsula, where it also causes significant damage. In a previous study, 94 pairs of microsatellite primers have been identified in silico in the pinewood nematode genome. In the present study, specific PCR amplifications and polymorphism tests to validate these loci were performed and 17 microsatellite loci that were suitable for routine analysis of B. xylophilus genetic diversity were selected. The polymorphism of these markers was evaluated on nematodes from four field origins and one laboratory collection strain, all originate from the native area. The number of alleles and the expected heterozygosity varied between 2 and 11 and between 0.039 and 0.777, respectively. First insights into the population genetic structure of B. xylophilus were obtained using clustering and multivariate methods on the genotypes obtained from the field samples. The results showed that the pinewood nematode genetic diversity is spatially structured at the scale of the pine tree and probably at larger scales. The role of dispersal by the insect vector versus human activities in shaping this structure is discussed.

Secondary contact and admixture between independently invading populations of the western corn rootworm, Diabrotica virgifera virgifera in Europe.

The western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is one of the most destructive pests of corn in North America and is currently invading Europe. The two major invasive outbreaks of rootworm in Europe have occurred, in North-West Italy and in Central and South-Eastern Europe. These two outbreaks originated from independent introductions from North America. Secondary contact probably occurred in North Italy between these two outbreaks, in 2008. We used 13 microsatellite markers to conduct a population genetics study, to demonstrate that this geographic contact resulted in a zone of admixture in the Italian region of Veneto. We show that i) genetic variation is greater in the contact zone than in the parental outbreaks; ii) several signs of admixture were detected in some Venetian samples, in a bayesian analysis of the population structure and in an approximate bayesian computation analysis of historical scenarios and, finally, iii) allelic frequency clines were observed at microsatellite loci. The contact between the invasive outbreaks in North-West Italy and Central and South-Eastern Europe resulted in a zone of admixture, with particular characteristics. The evolutionary implications of the existence of a zone of admixture in Northern Italy and their possible impact on the invasion success of the western corn rootworm are discussed.