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.

Consistent variations in personality traits and their potential for genetic improvement in biocontrol agents: Trichogramma evanescens as a case study.

Improvements in the biological control of agricultural pests require improvements in the phenotyping methods used by practitioners to select efficient biological control agent (BCA) populations in industrial rearing or field conditions. Consistent inter-individual variations in behaviour (i.e. animal personality) probably affect BCA efficiency, but have never been taken into account in the development of phenotyping methods, despite having characteristics useful for phenotyping: repeatable (by definition), often heritable, etc. We developed a video-tracking method targeting animal personality traits and evaluated the feasibility of its use for genetic improvement in the BCA Trichogramma evanescens, by phenotyping 1,049 individuals from 24 isogenic lines. We found consistent individual variations in boldness, activity and exploration. Personality differences between the 24 isogenic lines suggested a genetic origin of the variations in activity and exploration (broad-sense heritability estimates of 0.06 to 0.11) and revealed a trade-off between exploration and fecundity.

Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing.

BACKGROUND: The rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments. RESULTS: We obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables. CONCLUSIONS: The pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.

QDD: a user-friendly program to select microsatellite markers and design primers from large sequencing projects.

SUMMARY: QDD is an open access program providing a user-friendly tool for microsatellite detection and primer design from large sets of DNA sequences. The program is designed to deal with all steps of treatment of raw sequences obtained from pyrosequencing of enriched DNA libraries, but it is also applicable to data obtained through other sequencing methods, using FASTA files as input. The following tasks are completed by QDD: tag sorting, adapter/vector removal, elimination of redundant sequences, detection of possible genomic multicopies (duplicated loci or transposable elements), stringent selection of target microsatellites and customizable primer design. It can treat up to one million sequences of a few hundred base pairs in the tag-sorting step, and up to 50,000 sequences in a single input file for the steps involving estimation of sequence similarity. AVAILABILITY: QDD is freely available under the GPL licence for Windows and Linux from the following web site: http://www.univ-provence.fr/gsite/Local/egee/dir/meglecz/QDD.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Scanning the European corn borer (Ostrinia spp.) genome for adaptive divergence between host-affiliated sibling species.

It has recently been shown that the European corn borer, a major pest of maize crops, is actually composed of two genetically differentiated and reproductively isolated taxa, which are found in sympatry over a wide geographical range in Eurasia. Each taxon is adapted to specific host plants: Ostrinia nubilalis feeds mainly on maize, while O. scapulalis feeds mainly on hop or mugwort. Here, we present a genome scan approach as a first step towards an integrated molecular analysis of the adaptive genomic divergence between O. nubilalis and O. scapulalis. We analysed 609 AFLP marker loci in replicate samples of sympatric populations of Ostrinia spp. collected on maize, hop and mugwort, in France. Using two genome scan methods based on the analysis of population differentiation, we found a set of 28 outlier loci that departed from the neutral expectation in one or the other method (of which a subset of 14 loci were common to both methods), which showed a significantly increased differentiation between O. nubilalis and O. scapulalis, when compared to the rest of the genome. A subset of 12 outlier loci were sequenced, of which 7 were successfully re-amplified as target candidate loci. Three of these showed homology with annotated lepidopteran sequences from public nucleotide databases.

Parasitoids (Hymenoptera) of Mealybug Pests (Hemiptera: Pseudococcidae) from Southern Brazil: Molecular and Morphological Characterization.

Parasitoids of three mealybug pests (Hemiptera: Pseudococcidae), Planococcus ficus (Signoret), Pseudococcus sociabilis Hambleton, and Pseudococcus viburni (Signoret) have been identified for the first time in Brazil. Mealybugs were collected in fruit-growing areas along southern Brazil during 2013-2016. An integrative approach, combining morphological and molecular methods, was used to identify the Brazilian parasitoids to the species level. Fifteen species were recorded, including 14 primary parasitoids belonging to Encyrtidae and Platygastridae and a single secondary parasitoid species belonging to Signiphoridae. The encyrtid parasitoids Acerophagus flavidulus (Brèthes), Anagyrus calyxtoi Noyes and Zaplatycerus sp., and the signiphorid secondary parasitoid Chartocerus axillaris De Santis are reported for the first time in Brazil.

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.

Representativeness of microsatellite distributions in genomes, as revealed by 454 GS-FLX titanium pyrosequencing.

BACKGROUND: Microsatellites are markers of choice in population genetics and genomics, as they provide useful insight into patterns and processes as diverse as genome evolutionary dynamics and demographic processes. The acquisition of microsatellites through multiplex-enriched libraries and 454 GS-FLX Titanium pyrosequencing is a promising new tool for the isolation of new markers in unknown genomes. This approach can also be used to evaluate the extent to which microsatellite-enriched libraries are representative of the genome from which they were isolated. In this study, we deciphered potential discrepancies in microsatellite content recovery for two reference genomes (Apis mellifera and Danio rerio), selected on the basis of their extreme heterogeneity in terms of the proportions and distributions of microsatellites on chromosomes. RESULTS: The A. mellifera genome, in particular, was found to be highly heterogeneous, due to extremely high rates of recombination, with hotspots, but the only bias consistently introduced into pyrosequenced multiplex-enriched libraries concerned sequence length, with the overrepresentation of sequences 160 to 320 bp in length. Other deviations from expected proportions or distributions of motifs on chromosomes were observed, but the significance and intensity of these deviations was mostly limited. Furthermore, no consistent adverse competition between multiplexed probes was observed during the motif enrichment phase. CONCLUSIONS: This approach therefore appears to be a promising strategy for improving the development of microsatellites, as it introduces no major bias in terms of the proportions and distribution of microsatellites.

Rapid and Differential Evolution of the Venom Composition of a Parasitoid Wasp Depending on the Host Strain.

Parasitoid wasps rely primarily on venom to suppress the immune response and regulate the physiology of their host. Intraspecific variability of venom protein composition has been documented in some species, but its evolutionary potential is poorly understood. We performed an experimental evolution initiated with the crosses of two lines of Leptopilinaboulardi of different venom composition to generate variability and create new combinations of venom factors. The offspring were maintained for 10 generations on two strains of Drosophila melanogaster differing in resistance/susceptibility to the parental parasitoid lines. The venom composition of individuals was characterized by a semi-automatic analysis of 1D SDS-PAGE electrophoresis protein profiles whose accuracy was checked by Western blot analysis of well-characterized venom proteins. Results made evident a rapid and differential evolution of the venom composition on both hosts and showed that the proteins beneficial on one host can be costly on the other. Overall, we demonstrated the capacity of rapid evolution of the venom composition in parasitoid wasps, important regulators of arthropod populations, suggesting a potential for adaptation to new hosts. Our approach also proved relevant in identifying, among the diversity of venom proteins, those possibly involved in parasitism success and whose role deserves to be deepened.

Natural enemies of armored scales (Hemiptera: Diaspididae) and soft scales (Hemiptera: Coccidae) in Chile: Molecular and morphological identification.

Scale insects (Hemiptera: Sternorrhyncha: Coccomorpha) are key pests of agricultural crops and ornamental plants worldwide. Their populations are difficult to control, even with insecticides, due to their cryptic habits. Moreover, there is growing concern over the use of synthetic pesticides for their control, due to deleterious environmental effects and the emergence of resistant populations of target pests. In this context, biological control may be an effective and sustainable approach. Hymenoptera Chalcidoidea includes natural enemies of scale insects that have been successfully used in many biological control programs. However, the correct identification of pest scale species and their natural enemies is particularly challenging because these insects are very small and highly specialized. Integrative taxonomy, coupling DNA barcoding and morphological analysis, has been successfully used to characterize pests and natural enemy species. In this study, we performed a survey of parasitoids and predators of armored and soft scales in Chile, based on 28S and COI barcodes. Fifty-three populations of Diaspididae and 79 populations of Coccidae were sampled over the entire length of the country, from Arica (18°S) to Frutillar (41°S), between January 2015 and February 2016. The phylogenetic relationships obtained by Bayesian inference from multilocus haplotypes revealed 41 putative species of Chalcidoidea, five Coccinellidae and three Neuroptera. Species delimitation was confirmed using ABGD, GMYC and PTP model. In Chalcidoidea, 23 species were identified morphologically, resulting in new COI barcodes for 12 species and new 28S barcodes for 14 species. Two predator species (Rhyzobius lophantae and Coccidophilus transandinus) were identified morphologically, and two parasitoid species, Chartocerus niger and Signiphora bifasciata, were recorded for the first time in Chile.

Molecular differentiation at nuclear loci in French host races of the European corn borer (Ostrinia nubilalis).

French populations of the European corn borer consist of two sympatric and genetically differentiated host races. As such, they are well suited to study processes that could be involved in sympatric speciation, but the initial conditions of host-race divergence need to be elucidated. Gene genealogies can provide insight into the processes involved in speciation. We used DNA sequences of four nuclear genes to (1) document the genetic structure of the two French host races previously delineated with allozyme markers, (2) find genes directly or indirectly involved in reproductive isolation between host races, and (3) estimate the time since divergence of the two taxa and see whether this estimate is compatible with this divergence being the result of a host shift onto maize after its introduction into Europe approximately 500 years ago. Gene genealogies revealed extensive shared polymorphism, but confirmed the previously observed genetic differentiation between the two host races. Significant departures from the predictions of neutral molecular evolution models were detected at three loci but were apparently unrelated to reproductive isolation between host races. Estimates of time since divergence between French host races varied from approximately 75,000 to approximately 150,000 years, suggesting that the two taxa diverged recently but probably long before the introduction of maize into Europe.

Effects of inbreeding on a gregarious parasitoid wasp with complementary sex determination.

Inbreeding and inbreeding depression are processes in small populations of particular interest for a range of human activities such as animal breeding, species conservation, or pest management. In particular, biological control programs should benefit from a thorough understanding of the causes and consequences of inbreeding because natural enemies experience repetitive bottlenecks during importation, laboratory rearing, and introduction. Predicting the effect of inbreeding in hymenopteran parasitoid wasps, frequently used in biological control programs, is nonetheless a difficult endeavor. In haplodiploid parasitoids, the purge of deleterious alleles via haploid males should reduce genetic load, but if these species also have complementary sex determination (CSD), abnormal diploid males will be produced, which may jeopardize the success of biological control introductions. Mastrus ridens is such a parasitoid wasp with CSD, introduced to control the codling moth, Cydia pomonella (L.). We studied its life history traits in the laboratory under two conditions: inbred (full-sib) and outbred (nonsib) crosses, across five generations, to examine the consequences of inbreeding in this species. We found that in inbred lines, nonreproducing females live less, the number of daughters produced was lower, and sex ratio (proportion of males) and proportion of diploid males were higher. Diploid males were able to produce fertile daughters, but fewer than haploid males. Lineage survival was similar for inbred and outbred lines across the five generations. The most significant decrease in fitness was thus a consequence of the production of diploid males, but this effect was not as extreme as in most other species with CSD, due to the fertility of diploid males. This study highlights the importance of determining the type of sex determination in parasitoid wasps used for biological control, and the importance of maintaining genetic diversity in species with CSD when importation or augmentation is the goal.

Integrative taxonomy methods reveal high mealybug (Hemiptera: Pseudococcidae) diversity in southern Brazilian fruit crops.

The Serra Gaúcha region is the most important temperate fruit-producing area in southern Brazil. Despite mealybugs (Hemiptera: Pseudococcidae) infesting several host plants in the region, there is a lack of information about the composition of species damaging different crops. A survey of mealybug species associated with commercial fruit crops (apple, persimmon, strawberry and grapes) was performed in Serra Gaúcha between 2013 and 2015, using both morphology and DNA analyses for species identification. The most abundant species were Pseudococcus viburni (Signoret), found on all four host plant species, and Dysmicoccus brevipes (Cockerell), infesting persimmon, vines and weeds. The highest diversity of mealybug species was found on persimmon trees, hosting 20 different taxa, of which Anisococcus granarae Pacheco da Silva & Kaydan, D. brevipes, Pseudococcus sociabilis Hambleton and Ps. viburni were the most abundant. A total of nine species were recorded in vineyards. Planococcus ficus (Signoret) and Pseudococcus longispinus (Targioni Tozzetti) were observed causing damage to grapes for the first time. A single species, Ps. viburni, was found associated with apples, while both Ps. viburni and Ferrisia meridionalis Williams were found on strawberry. Four of the mealybug species found represent new records for Brazil.

No inbreeding depression in laboratory-reared individuals of the parasitoid wasp Allotropa burrelli.

Inbreeding depression is a major concern in almost all human activities relating to plant and animal breeding. The biological control of pests with natural enemies is no exception, because populations of biocontrol agents experience a series of bottlenecks during importation, rearing, and introduction. A classical biological control program for the Comstock mealybug Pseudococcus comstocki (Hemiptera: Pseudococcidae) was initiated in France in 2008, based on the introduction of an exotic parasitoid, Allotropa burrelli Mues. (Hymenoptera: Platygastridae), a haplodiploid parasitoid imported from Japan. We evaluated the sensitivity of A. burrelli to inbreeding, to optimize rearing and release strategies. We compared several morphological and life-history traits between the offspring of siblings and the offspring of unrelated parents. We took into account the low level of genetic variability due to the relatively small size of laboratory-reared populations by contrasting two types of pedigree: one for individuals from a strain founded from a single field population, and the other generated by hybridizing individuals from two strains founded from two highly differentiated populations. Despite this careful design, we obtained no evidence for a negative impact of inbreeding on laboratory-reared A. burrelli. We discussed the results in light of haplodiploid sex determination and parasitoid mating systems, and classical biological control practices.

Identifying obstacles and ranking common biological control research priorities for Europe to manage most economically important pests in arable, vegetable and perennial crops.

EU agriculture is currently in transition from conventional crop protection to integrated pest management (IPM). Because biocontrol is a key component of IPM, many European countries recently have intensified their national efforts on biocontrol research and innovation (R&I), although such initiatives are often fragmented. The operational outputs of national efforts would benefit from closer collaboration among stakeholders via transnationally coordinated approaches, as most economically important pests are similar across Europe. This paper proposes a common European framework on biocontrol R&I. It identifies generic R&I bottlenecks and needs as well as priorities for three crop types (arable, vegetable and perennial crops). The existing gap between the market offers of biocontrol solutions and the demand of growers, the lengthy and expensive registration process for biocontrol solutions and their varying effectiveness due to variable climatic conditions and site-specific factors across Europe are key obstacles hindering the development and adoption of biocontrol solutions in Europe. Considering arable, vegetable and perennial crops, a dozen common target pests are identified for each type of crop and ranked by order of importance at European level. Such a ranked list indicates numerous topics on which future joint transnational efforts would be justified. © 2016 Society of Chemical Industry.

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.

Three new species of mealybug (Hemiptera, Coccomorpha, Pseudococcidae) on persimmon fruit trees (Diospyros kaki) in southern Brazil.

Brazil has the greatest insect diversity in the world; however, little is known about its scale insect species (Hemiptera: Coccomorpha). Mealybugs (Pseudococcidae) have been found in at least 50% of persimmon orchards Diospyros kaki L. in the southern part of the country. In this study three new mealybug species on persimmon trees located in the Serra Gaúcha Region, RS, Brazil, namely, Anisococcus granarae Pacheco da Silva & Kaydan, sp. n., Ferrisia kaki Kaydan & Pacheco da Silva, sp. n. and Pseudococcus rosangelae Pacheco da Silva & Kaydan, sp. n. are described. In addition, an identification key for the genera occurring on fruit orchards and vineyards in Brazil is provided, together with illustrations and molecular data for the new species.

Diploid male production correlates with genetic diversity in the parasitoid wasp Venturia canescens: a genetic approach with new microsatellite markers.

Sex determination is ruled by haplodiploidy in Hymenoptera, with haploid males arising from unfertilized eggs and diploid females from fertilized eggs. However, diploid males with null fitness are produced under complementary sex determination (CSD), when individuals are homozygous for this locus. Diploid males are expected to be more frequent in genetically eroded populations (such as islands and captive populations), as genetic diversity at the csd locus should be low. However, only a few studies have focused on the relation between population size, genetic diversity, and the proportion of diploid males in the field. Here, we developed new microsatellite markers in order to assess and compare genetic diversity and diploid male proportion (DMP) in populations from three distinct habitat types - mainland, island, or captive -, in the parasitoid wasp Venturia canescens. Eroded genetic diversity and higher DMP were found in island and captive populations, and habitat type had large effect on genetic diversity. Therefore, DMP reflects the decreasing genetic diversity in small and isolated populations. Thus, Hymenopteran populations can be at high extinction risk due to habitat destruction or fragmentation.

Investigating Biological Control Agents for Controlling Invasive Populations of the Mealybug Pseudococcus comstocki in France.

Pseudococcus comstocki (Hemiptera: Pseudococcidae) is a mealybug species native to Eastern Asia and present as an invasive pest in northern Italy and southern France since the start of the century. It infests apple and pear trees, grapevines and some ornamental trees. Biocontrol programmes against this pest proved successful in central Asia and North America in the second half of the 20th century. In this study, we investigated possible biocontrol agents against P. comstocki, with the aim of developing a biocontrol programme in France. We carried out systematic DNA-barcoding at each step in the search for a specialist parasitoid. First we characterised the French target populations of P. comstocki. We then identified the parasitoids attacking P. comstocki in France. Finally, we searched for foreign mealybug populations identified a priori as P. comstocki and surveyed their hymenopteran parasitoids. Three mealybug species (P. comstocki, P. viburni and P. cryptus) were identified during the survey, together with at least 16 different parasitoid taxa. We selected candidate biological control agent populations for use against P. comstocki in France, from the species Allotropa burrelli (Hymenoptera: Platygastridae) and Acerophagus malinus (Hymenoptera: Encyrtidae). The coupling of molecular and morphological characterisation for both pests and natural enemies facilitated the programme development and the rejection of unsuitable or generalist parasitoids.

Mealybug species from Chilean agricultural landscapes and main factors influencing the genetic structure of Pseudococcus viburni.

The present study aimed to characterize the distribution of mealybug species along Chilean agro-ecosystems and to determine the relative impact of host plant, management strategy, geography and micro-environment on shaping the distribution and genetic structure of the obscure mealybug Pseudococcus viburni. An extensive survey was completed using DNA barcoding methods to identify Chilean mealybugs to the species level. Moreover, a fine-scale study of Ps. viburni genetic diversity and population structure was carried out, genotyping 529 Ps. viburni individuals with 21 microsatellite markers. Samples from 16 localities were analyzed using Bayesian and spatially-explicit methods and the genetic dataset was confronted to host-plant, management and environmental data. Chilean crops were found to be infested by Ps. viburni, Pseudococcus meridionalis, Pseudococcus longispinus and Planococcus citri, with Ps. viburni and Ps. meridionalis showing contrasting distribution and host-plant preference patterns. Ps. viburni samples presented low genetic diversity levels but high genetic differentiation. While no significant genetic variance could be assigned to host-plant or management strategy, climate and geography were found to correlate significantly with genetic differentiation levels. The genetic characterization of Ps. viburni within Chile will contribute to future studies tracing back the origin and improving the management of this worldwide invader.