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.

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.

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.

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.

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.

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.

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.

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.

Microsatellite and mitochondrial data provide evidence for a single major introduction for the Neartic leafhopper Scaphoideus titanus in Europe.

Scaphoideus titanus, a leafhopper native to North America and invasive in Europe, is the vector of the Flavescence dorée phytoplasma, the causal agent of the most important form of grapevine yellows in European vineyards. We studied 10 polymorphic microsatellite loci and a 623 bp fragment of the mitochondrial cytochrome oxidase II gene in native S. titanus from north-eastern America and introduced European populations, to elucidate the colonization scenario. Consistent with their recent history, invasive European populations were less genetically diverse than American populations for both types of markers, suggesting a recent bottleneck. Significant isolation by distance was detected between American populations but not between European populations. None of the European mitochondrial haplotypes was found in the American vineyards, from which they are assumed to have originated. The precise source of the invasive S. titanus populations therefore remains unclear. Nevertheless, the high heterozygosity of North-East American populations (which contained 92% of the observed alleles) suggests that this region is part of the native range of S. titanus. Clustering population genetics analyses with microsatellite and mitochondrial data suggested that European populations originated from a single introduction event. Most of the introduced populations clustered with populations from Long Island, the Atlantic Coast winegrowing region in which Vitis aestivalis occurs.

Estimation of demo-genetic model probabilities with Approximate Bayesian Computation using linear discriminant analysis on summary statistics.

Comparison of demo-genetic models using Approximate Bayesian Computation (ABC) is an active research field. Although large numbers of populations and models (i.e. scenarios) can be analysed with ABC using molecular data obtained from various marker types, methodological and computational issues arise when these numbers become too large. Moreover, Robert et al. (Proceedings of the National Academy of Sciences of the United States of America, 2011, 108, 15112) have shown that the conclusions drawn on ABC model comparison cannot be trusted per se and required additional simulation analyses. Monte Carlo inferential techniques to empirically evaluate confidence in scenario choice are very time-consuming, however, when the numbers of summary statistics (Ss) and scenarios are large. We here describe a methodological innovation to process efficient ABC scenario probability computation using linear discriminant analysis (LDA) on Ss before computing logistic regression. We used simulated pseudo-observed data sets (pods) to assess the main features of the method (precision and computation time) in comparison with traditional probability estimation using raw (i.e. not LDA transformed) Ss. We also illustrate the method on real microsatellite data sets produced to make inferences about the invasion routes of the coccinelid Harmonia axyridis. We found that scenario probabilities computed from LDA-transformed and raw Ss were strongly correlated. Type I and II errors were similar for both methods. The faster probability computation that we observed (speed gain around a factor of 100 for LDA-transformed Ss) substantially increases the ability of ABC practitioners to analyse large numbers of pods and hence provides a manageable way to empirically evaluate the power available to discriminate among a large set of complex scenarios.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2011-30 September 2011.

This article documents the addition of 299 microsatellite marker loci and nine pairs of single-nucleotide polymorphism (SNP) EPIC primers to the Molecular Ecology Resources (MER) Database. Loci were developed for the following species: Alosa pseudoharengus, Alosa aestivalis, Aphis spiraecola, Argopecten purpuratus, Coreoleuciscus splendidus, Garra gotyla, Hippodamia convergens, Linnaea borealis, Menippe mercenaria, Menippe adina, Parus major, Pinus densiflora, Portunus trituberculatus, Procontarinia mangiferae, Rhynchophorus ferrugineus, Schizothorax richardsonii, Scophthalmus rhombus, Tetraponera aethiops, Thaumetopoea pityocampa, Tuta absoluta and Ugni molinae. These loci were cross-tested on the following species: Barilius bendelisis, Chiromantes haematocheir, Eriocheir sinensis, Eucalyptus camaldulensis, Eucalyptus cladocalix, Eucalyptus globulus, Garra litaninsis vishwanath, Garra para lissorhynchus, Guindilla trinervis, Hemigrapsus sanguineus, Luma chequen. Guayaba, Myrceugenia colchagüensis, Myrceugenia correifolia, Myrceugenia exsucca, Parasesarma plicatum, Parus major, Portunus pelagicus, Psidium guayaba, Schizothorax richardsonii, Scophthalmus maximus, Tetraponera latifrons, Thaumetopoea bonjeani, Thaumetopoea ispartensis, Thaumetopoea libanotica, Thaumetopoea pinivora, Thaumetopoea pityocampa ena clade, Thaumetopoea solitaria, Thaumetopoea wilkinsoni and Tor putitora. This article also documents the addition of nine EPIC primer pairs for Euphaea decorata, Euphaea formosa, Euphaea ornata and Euphaea yayeyamana.

Weak spatial and temporal population genetic structure in the rosy apple aphid, Dysaphis plantaginea, in French apple orchards.

We used eight microsatellite loci and a set of 20 aphid samples to investigate the spatial and temporal genetic structure of rosy apple aphid populations from 13 apple orchards situated in four different regions in France. Genetic variability was very similar between orchard populations and between winged populations collected before sexual reproduction in the fall and populations collected from colonies in the spring. A very small proportion of individuals (∼2%) had identical multilocus genotypes. Genetic differentiation between orchards was low (F(ST)<0.026), with significant differentiation observed only between orchards from different regions, but no isolation by distance was detected. These results are consistent with high levels of genetic mixing in holocyclic Dysaphis plantaginae populations (host alternation through migration and sexual reproduction). These findings concerning the adaptation of the rosy apple aphid have potential consequences for pest management.

High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries.

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.

Biological invasions in agricultural settings: insights from evolutionary biology and population genetics.

Invasion biology and agriculture are intimately related for several reasons and in particular because many agricultural pest species are recent invaders. In this article we suggest that the reconstruction of invasion routes with population genetics-based methods can address fundamental questions in ecology and practical aspects of the management of biological invasions in agricultural settings. We provide a brief description of the methods used to reconstruct invasion routes and describe their main characteristics. In particular, we focus on a scenario--the bridgehead invasion scenario --which had been overlooked until recently. We show that this scenario, in which an invasive population is the source of other invasive populations, is evolutionarily parsimonious and may have played a crucial role in shaping the distribution of many recent agricultural pests.

Inbreeding depression is purged in the invasive insect Harmonia axyridis.

Bottlenecks in population size reduce genetic diversity and increase inbreeding, which can lead to inbreeding depression. It is thus puzzling how introduced species, which typically pass through bottlenecks, become such successful invaders. However, under certain theoretical conditions, bottlenecks of intermediate size can actually purge the alleles that cause inbreeding depression. Although this process has been confirmed in model laboratory systems, it has yet to be observed in natural invasive populations. We evaluate whether such purging could facilitate biological invasions by using the world-wide invasion of the ladybird (or ladybug) Harmonia axyridis. We first show that invasive populations endured a bottleneck of intermediate intensity. We then demonstrate that replicate introduced populations experience almost none of the inbreeding depression suffered by native populations. Thus, rather than posing a barrier to invasion as often assumed, bottlenecks, by purging deleterious alleles, can enable the evolution of invaders that maintain high fitness even when inbred.

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.