Conservation Biological Control of Codling Moth (Cydia pomonella): Effects of Two Aromatic Plants, Basil (Ocimum basilicum) and French Marigolds (Tagetes patula).

The addition of flowering companion plants within or around crop fields is a promising strategy to strengthen pest regulation by their natural enemies. Aromatic plants are frequently used as companion plants, but their effects on natural enemies remain unclear under field conditions. Here, we evaluated the effects of two aromatic plant species on the parasitism of the codling moth (Cydia pomonella) and the recruitment of predatory arthropods (spiders, earwigs) in a factorial field experiment. Apple trees were intercropped with basil (Ocimum basilicum), French marigolds (Tagetes patula), or ryegrass (Lolium perenne). The association between apple trees and O. basilicum increases codling moth parasitism, but does not affect arthropod predator abundances. Furthermore, we find a general negative effect of T. patula on arthropod diversities and abundances, including the pest and its natural enemies. Finally, changes in the parasitism rate and arthropod community structure due to the aromatic plants do not reduce codling moth density or associated apple damage. Further experiments are needed to determine the mechanisms involved in aromatic plant effects on pest repellence and on natural enemy recruitment (volatile organic compound composition, floral resource supply, or pest density dependence).

Population Genetic Structure of Codling Moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), in Different Localities and Host Plants in Chile.

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is a major pest introduced to almost all main pome fruit production regions worldwide. This species was detected in Chile during the last decade of the 19th century, and now has a widespread distribution in all major apple-growing regions. We performed an analysis of the genetic variability and structure of codling moth populations in Chile using five microsatellite markers. We sampled the codling moth along the main distribution area in Chile on all its main host-plant species. Low genetic differentiation among the population samples (FST = 0.03) was found, with only slight isolation by distance. According to a Bayesian assignment test (TESS), a group of localities in the coastal mountain range from the Bío-Bío Region formed a distinct genetic cluster. Our results also suggest that the codling moth that invaded the southernmost locality (Aysén Region) had two origins from central Chile and another unknown source. We did not find significant genetic differentiation between codling moth samples from different host-plant species. Our results indicate high genetic exchange among codling moth populations between the different Chilean regions and host plants.

A global synthesis reveals biodiversity-mediated benefits for crop production.

Human land use threatens global biodiversity and compromises multiple ecosystem functions critical to food production. Whether crop yield-related ecosystem services can be maintained by a few dominant species or rely on high richness remains unclear. Using a global database from 89 studies (with 1475 locations), we partition the relative importance of species richness, abundance, and dominance for pollination; biological pest control; and final yields in the context of ongoing land-use change. Pollinator and enemy richness directly supported ecosystem services in addition to and independent of abundance and dominance. Up to 50% of the negative effects of landscape simplification on ecosystem services was due to richness losses of service-providing organisms, with negative consequences for crop yields. Maintaining the biodiversity of ecosystem service providers is therefore vital to sustain the flow of key agroecosystem benefits to society.

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

Limited genetic structure and demographic expansion of the Brassicogethes aeneus populations in France and in Europe.

BACKGROUND: The pollen beetle, Brassicogethes aeneus (Fabricius, 1775), is one of the most significant pests of oilseed rape. To shed light on past and current pollen beetle demography (dispersal, population size), 12 microsatellite markers were developed, and population genetic diversity and structure were analysed at different spatial scales in France and in Europe from 433 individuals collected in 18 winter oilseed rape fields. RESULTS: Genetic differentiation among the population samples was low but was significant between the Estonian sample and the rest of Europe. Isolation by distance was significant only at the European scale. Genetic variability was similar among the 18 population samples. Demographic inferences suggested a recent expansion of B. aeneus population size over Europe, possibly corresponding to an increase in oilseed rape crop area during past decades. CONCLUSION: Current population size and dispersal are not straightforward to estimate from the distribution of genetic variability in B. aeneus over Europe because of the complexity of the demographic history of this pest. Nevertheless, because gene flow was important enough to prevent strong genetic differentiation at large geographical scales, the management of pollen beetle populations should likely be thought of at a continental Europe level. © 2018 Society of Chemical Industry.

Molecular Tools for the Detection and the Identification of Hymenoptera Parasitoids in Tortricid Fruit Pests.

Biological control requires specific tools for the accurate detection and identification of natural enemies in order to estimate variations in their abundance and their impact according to changes in environmental conditions or agricultural practices. Here, we developed two molecular methods of detection based on PCR-RFLP with universal primers and on PCR with specific primers to identify commonly occurring larval parasitoids of the tortricid fruit pests and to estimate parasitism in the codling moth. Both methods were designed based on DNA sequences of the COI mitochondrial gene for a range of parasitoids that emerged from Cydia pomonella and Grapholitamolesta caterpillars (102 parasitoids; nine species) and a range of potential tortricid hosts (40 moths; five species) damaging fruits. The PCR-RFLP method (digestion by AluI of a 482 bp COI fragment) was very powerful to identify parasitoid adults and their hosts, but failed to detect parasitoid larvae within eggs or within young C. pomonella caterpillars. The PCR method based on specific primers amplified COI fragments of different lengths (131 to 463 bp) for Ascogaster quadridentata (Braconidae); Pristomerusvulnerator (Ichneumonidae); Trichomma enecator (Ichneumonidae); and Perilampus tristis (Perilampidae), and demonstrated a higher level of sensibility than the PCR-RFLP method. Molecular estimations of parasitism levels in a natural C. pomonella population with the specific primers did not differ from traditional estimations based on caterpillar rearing (about 60% parasitism in a non-treated apple orchard). These PCR-based techniques provide information about within-host parasitoid assemblage in the codling moth and preliminary results on the larval parasitism of major tortricid fruit pests.

Characterization of the pollen beetle, Brassicogethes aeneus, dispersal from woodlands to winter oilseed rape fields.

Many crop pests rely on resources out of crop fields; understanding how they colonize the fields is an important factor to develop integrated pest management. In particular, the time of crop colonization and damage severity might be determined by pest movements between fields and non-crop areas. Notably, the pollen beetle, Brassicogethes aeneus, previously named Meligethes aeneus, one of the most important pests of winter oilseed rape, overwinters in woodlands. As a result, its abundance increases in oilseed rape fields near wooded areas. Here, we assessed the spatio-temporal patterns of the dispersal from woodlands to oilseed rape fields in diversified landscapes of a same region. We observed on four dates the abundance of pollen beetles in 24 fields spread in the Eure department, France. We modeled the abundance as a result of the dispersal from the neighboring woodlands. We compared the modalities of dispersal corresponding to different hypotheses on the dispersal origin, kernel shape and sources of variability. Within oilseed rape the distance to the edges of woodlands is not the main determinant of pollen beetle abundance. On the contrary, the variability of the abundance between fields is largely explained by the dispersal from neighboring woodlands but there is considerable variability between dates, sites and, to a lesser extent, between fields. The two dispersal kernels received similar support from the data and lead to similar conclusions. The mean dispersal distance is 1.2 km but seems to increase from a few hundred meters the first week to more than two kilometers the fourth, allowing the pollen beetles to reach more distant OSR fields. These results suggest that early varieties away from woodlands and late varieties close to the woodlands may limit attacks at the time when oilseed rape is the most sensitive.

Impact of Agroecological Infrastructures on the Dynamics of Dysaphis plantaginea (Hemiptera: Aphididae) and Its Natural Enemies in Apple Orchards in Northwestern France.

Apple orchard production is facing new environmental and societal challenges, resulting, in particular, in strong pressure to reduce pesticide use. Cider-apple production, for which the perfect visual aspect of fruits is not a marketability imperative, offers good opportunities to study production systems that are developing new agronomic strategies, which could be subsequently extended to all apple-production types. Agroecological infrastructures play an important role in providing shelter, food resources, or reproduction habitats to many arthropods. Consequently, setting-up agroecological infrastructures in the vicinity of or within orchards could increase natural enemy presence and thus improve the biological control of pests. In this study, we focused on Dysaphis plantaginea (Passerini), one of the major pests in apple orchards in Europe, which causes important economic production losses. During two years (2014 and 2015), we monitored the population dynamics of D. plantaginea, its natural enemies, and mutualistic ants in commercial production cider-apple orchards. The influences of the cider-apple cultivar, insecticide use, and distance to agroecological infrastructures (hedgerows and flower strips) were assessed. Our results suggest that flower strips favor an increase in natural enemy abundance in the vicinity of the orchards and could thus play an important role in the production system by improving the biological control of D. plantaginea.

Measuring local genetic variability in populations of codling moth (Lepidoptera: Tortricidae) across an unmanaged and commercial orchard interface.

The genetic structure of adult codling moth, Cydia pomonella (L.), populations was characterized both inside a managed apple, Malus domestica Borkdhausen, orchard and in surrounding unmanaged hosts and nonhost trees in central Chile during 2006-2007. Adult males were collected using an array of sex pheromone-baited traps. Five microsatellite genetic markers were used to study the population genetic structure across both spatial (1-100 ha) and temporal (generations within a season) gradients. Analysis of molecular variance (AMOVA) found a significant, but weak, association in both the spatial and temporal genetic structures. Discriminant analysis also found significant differentiation between the first and second generation for traps located either inside or outside the managed orchard. The Bayesian assignment test detected three genetic clusters during each of the two generations, which corresponded to different areas within the unmanaged and managed apple orchard interface. The lack of a strong spatial structure at a local scale was hypothesized to be because of active adult movement between the managed and unmanaged hosts and the asymmetry in the insecticide selection pressure inside and outside the managed habitats. These data highlight the importance of developing area-wide management programs that incorporate management tactics effective at the landscape level for successful codling moth control.

Multiple origins of the sodium channel kdr mutations in codling moth populations.

Resistance to insecticides is one interesting example of a rapid current evolutionary change. DNA variability in the voltage-gated sodium channel gene (trans-membrane segments 5 and 6 in domain II) was investigated in order to estimate resistance evolution to pyrethroid in codling moth populations at the World level. DNA variation among 38 sequences revealed a unique kdr mutation (L1014F) involved in pyrethroid resistance in this gene region, which likely resulted from several convergent substitutions. The analysis of codling moth samples from 52 apple orchards in 19 countries using a simple PCR-RFLP confirmed that this kdr mutation is almost worldwide distributed. The proportions of kdr mutation were negatively correlated with the annual temperatures in the sampled regions. Homozygous kdr genotypes in the French apple orchards showed lower P450 cytochrome oxidase activities than other genotypes. The most plausible interpretation of the geographic distribution of kdr in codling moth populations is that it has both multiple independent origins and a spreading limited by low temperature and negative interaction with the presence of alternative resistance mechanisms to pyrethroid in the populations.

Estimating gene flow between refuges and crops: a case study of the biological control of Eriosoma lanigerum by Aphelinus mali in apple orchards.

Parasitoid disturbance populations in agroecosystems can be maintained through the provision of habitat refuges with host resources. However, specialized herbivores that feed on different host plants have been shown to form host-specialized races. Parasitoids may subsequently specialize on these herbivore host races and therefore prefer parasitizing insects from the refuge, avoiding foraging on the crop. Evidence is therefore required that parasitoids are able to move between the refuge and the crop and that the refuge is a source of parasitoids, without being an important source of herbivore pests. A North-South transect trough the Chilean Central Valley was sampled, including apple orchards and surrounding Pyracantha coccinea (M. Roem) (Rosales: Rosacea) hedges that were host of Eriosoma lanigerum (Hemiptera: Aphididae), a globally important aphid pest of cultivated apples. At each orchard, aphid colonies were collected and taken back to the laboratory to sample the emerging hymenopteran parasitoid Aphelinus mali (Hymenoptera: Aphelinidae). Aphid and parasitoid individuals were genotyped using species-specific microsatellite loci and genetic variability was assessed. By studying genetic variation, natural geographic barriers of the aphid pest became evident and some evidence for incipient host-plant specialization was found. However, this had no effect on the population-genetic features of its most important parasitoid. In conclusion, the lack of genetic differentiation among the parasitoids suggests the existence of a single large and panmictic population, which could parasite aphids on apple orchards and on P. coccinea hedges. The latter could thus comprise a suitable and putative refuge for parasitoids, which could be used to increase the effectiveness of biological control. Moreover, the strong geographical differentiation of the aphid suggests local reinfestations occur mainly from other apple orchards with only low reinfestation from P. cocinnea hedges. Finally, we propose that the putative refuge could act as a source of parasitoids without being a major source of aphids.

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.

Population genetic structure of two primary parasitoids of Spodoptera frugiperda (Lepidoptera), Chelonus insularis and Campoletis sonorensis (Hymenoptera): to what extent is the host plant important?

Plant chemistry can strongly influence interactions between herbivores and their natural enemies, either by providing volatile compounds that serve as foraging cues for parasitoids or predators, or by affecting the quality of herbivores as hosts or prey. Through these effects plants may influence parasitoid population genetic structure. We tested for a possible specialization on specific crop plants in Chelonus insularis and Campoletis sonorensis, two primary parasitoids of the fall armyworm, Spodoptera frugiperda. Throughout Mexico, S. frugiperda larvae were collected from their main host plants, maize and sorghum and parasitoids that emerged from the larvae were used for subsequent comparison by molecular analysis. Genetic variation at eight and 11 microsatellites were respectively assayed for C. insularis and C. sonorensis to examine isolation by distance, host plant and regional effects. Kinship analyses were also performed to assess female migration among host-plants. The analyses showed considerable within population variation and revealed a significant regional effect. No effect of host plant on population structure of either of the two parasitoid species was found. Isolation by distance was observed at the individual level, but not at the population level. Kinship analyses revealed significantly more genetically related--or kin--individuals on the same plant species than on different plant species, suggesting that locally, mothers preferentially stay on the same plant species. Although the standard population genetics parameters showed no effect of plant species on population structure, the kinship analyses revealed that mothers exhibit plant species fidelity, which may speed up divergence if adaptation were to occur.

Crosses prior to parthenogenesis explain the current genetic diversity of tropical plant-parasitic Meloidogyne species (Nematoda: Tylenchida).

The tropical and subtropical parthenogenetic plant-parasitic nematodes Meloidogyne are polyphagous major agricultural pests. Implementing proper pest management approaches requires a good understanding of mechanisms, population structure, evolutionary patterns and species identification. A comparative analysis of the mitochondrial vs nuclear diversity was conducted on a selected set of Meloidogyne lines from various geographic origins. Mitochondrial co2-16S sequences and AFLP markers of total DNA were applied because of their ability to evidence discrete genetic variation between closely related isolates. Several distinct maternal lineages were present, now associated with different genetic backgrounds. Relative discordances were found when comparing mitochondrial and nuclear diversity patterns. These patterns are most likely related to crosses within one ancestral genetic pool, followed by the establishment of parthenogenesis. In this case, they mirror the genetic backgrounds of the original individuals. Another aspect could be that species emergence was recent or on process from this original genetic pool and that the relatively short time elapsed since then and before parthenogenesis settlement did not allow for lineage sorting. This could also be compatible with the hypothesis of hybrids between closely related species. This genetic pool would correspond to a species as defined by the species interbreeding concept, but also including the grey area of species boundaries. This complex process has implications on the way genotypic and phenotypic diversity should be addressed. The phenotype of parthenogenetic lines is at least for part determined by the ancestral amphimictic genetic background. A direct consequence is, therefore, in terms of risk management, the limited confidence one can have on the direct association of an agronomic threat to a simple typing or species delineation. Risk management strategies and tools must thus consider this complexity when designing quarantine implementation, resistance breeding programmes or molecular diagnostic.

Isolation and characterization of polymorphic microsatellite loci in two primary parasitoids of the noctuid Spodoptera frugiperda: Chelonus insularis and Campoletis sonorensis (Hymenoptera).

Fifteen and 13 microsatellite loci were isolated, respectively, from Campoletis sonorensis Cameron and from Chelonus insularis Cresson. These two parasitic Hymenoptera are primary parasitoids of Lepidoptera in North, Central and South America, including the important agricultural pest Spodoptera frugiperda. Allelic diversity and heterozygosity were quantified in samples from Mexico. Each locus was polymorphic, with the number of alleles ranging from two to 16 in C. sonorensis and from four to 18 in C. insularis. Heterozygosity ranged from 0.088 to 0.403 in C. sonorensis and from 0.106 to 0.458 in C. insularis.

Diversity of insecticide resistance mechanisms and spectrum in European populations of the codling moth, Cydia pomonella.

Only a few of the registered insecticides against Cydia pomonella L. are still effective in areas where insecticide resistance has emerged in this pest. Resistance mechanisms are multiple, and their lone or cumulative effects in a single population are not completely understood. A detailed estimation of resistance spectrum is still required to define the suitable insecticides to use against a given population. The efficacy of ten insecticides was therefore investigated together with the resistance mechanisms expressed in four laboratory strains and 47 field populations of C. pomonella from five countries. Bioassays were performed using topical applications of diagnostic concentrations on diapausing larvae, and resistance mechanisms were analysed on adults emerging from control insects. All populations exhibited a reduced susceptibility to at least one insecticide when compared with the susceptible laboratory strain. Cross-resistances were observed between azinphos-methyl or phosalone and more recent compounds such as spinosad and thiacloprid. Resistances to azinphos-methyl, diflubenzuron, spinosad, tebufenozide and thiacloprid were significantly correlated with mixed-function oxidase activity, while increased glutathione-S-transferase and reduced non-specific esterase activities were correlated with resistance to azinphos-methyl and emamectin, respectively. Conversely, resistances to azinphos-methyl, tebufenozide and thiacloprid were negatively correlated with increased esterase activity. None of the observed mechanisms explained the loss of susceptibility of populations to chlorpyrifos-ethyl, and no significant correlation was detected between resistance to deltamethrin and the presence of the kdr mutation. The suitability of such non-target instars to monitor insecticide resistance in field populations is discussed.

Insecticide resistance may enhance the response to a host-plant volatile kairomone for the codling moth, Cydia pomonella (L.).

The behavioral and electroantennographic responses of Cydia pomonella (L.) to the ripe pear volatile ethyl (2E,4Z)-2,4-decadienoate (Et-E,Z-DD), were compared in insecticide-susceptible and -resistant populations originating from southern France. A dose-response relationship to this kairomonal attractant was established for antennal activity and did not reveal differences between susceptible and resistant strains. Conversely, males of the laboratory strains expressing metabolic [cytochrome P450-dependent mixed-function oxidases (mfo)] or physiological (kdr-type mutation of the sodium-channel gene) resistance mechanisms exhibited a significantly higher response to Et-E,Z-DD than those of the susceptible strain in a wind tunnel experiment. No response of the females to this kairomone could be obtained in our wind-tunnel conditions. In apple orchards, mfo-resistant male moths were captured at significantly higher rates in kairomone-baited traps than in traps baited with the sex pheromone of C. pomonella. Such a differential phenomenon was not verified for the kdr-resistant insects, which exhibited a similar response to both the sex pheromone and the kairomonal attractant in apple orchards. Considering the widespread distribution of metabolic resistance in European populations of C. pomonella and the enhanced behavioral response to Et-E,Z-DD in resistant moths, the development of control measures based on this kairomonal compound would be of great interest for the management of insecticide resistance in this species.

The Africanization of honeybees (Apis mellifera L.) of the Yucatan: a study of a massive hybridization event across time.

Until recently, African and European subspecies of the honeybee (Apis mellifera L.) had been geographically separated for around 10,000 years. However, human-assisted introductions have caused the mixing of large populations of African and European subspecies in South and Central America, permitting an unprecedented opportunity to study a large-scale hybridization event using molecular analyses. We obtained reference populations from Europe, Africa, and South America and used these to provide baseline information for a microsatellite and mitochondrial analysis of the process of Africanization of the bees of the Yucatan Peninsula, Mexico. The genetic structure of the Yucatecan population has changed dramatically over time. The pre-Africanized Yucatecan population (1985) comprised bees that were most similar to samples from southeastern Europe and northern and western Europe. Three years after the arrival of Africanized bees (1989), substantial paternal gene flow had occurred from feral Africanized drones into the resident European population, but maternal gene flow from the invading Africanized population into the local population was negligible. However by 1998, there was a radical shift with both African nuclear alleles (65%) and African-derived mitochondria (61%) dominating the genomes of domestic colonies. We suggest that although European mitochondria may eventually be driven to extinction in the feral population, stable introgression of European nuclear alleles has occurred.

THE ORIGIN OF WEST EUROPEAN SUBSPECIES OF HONEYBEES (APIS MELLIFERA): NEW INSIGHTS FROM MICROSATELLITE AND MITOCHONDRIAL DATA.

Apis mellifera is composed of three evolutionary branches including mainly African (branch A), western and northern European (branch M), and southeastern European (branch C) populations. The existence of morphological clines extending from the equator to the Polar Circle through Morocco and Spain raised the hypothesis that the branch M originated in Africa. Mitochondrial DNA analysis revealed that branches A and M were characterized by highly diverged lineages implying very remote links between both branches. It also revealed that mtDNA haplotypes from lineages A coexisted with haplotypes M in the Iberian Peninsula and formed a south-north frequency cline, suggesting that this area could be a secondary contact zone between the two branches. By analyzing 11 populations sampled along a France-Spain/Portugal-Morocco-Guinea transect at 8 microsatellite loci and the DraI RFLP of the COI-COII mtDNA marker, we show that Iberian populations do not present any trace of "africanization" and are very similar to French populations when considering microsatellite markers. Therefore, the Iberian Peninsula is not a transition area. The higher haplotype A variability observed in Spanish and Portuguese samples compared to that found in Africa is explained by a higher mutation rate and multiple and recent introductions. Selection appears to be the best explanation to the morphological and allozymic clines and to the diffusion and maintenance of African haplotypes in Spain and Portugal.