Barcoding pest species in a biodiversity hot-spot: the South African polyphagous broad-nosed weevils (Coleoptera, Curculionidae, Entiminae).

Polyphagous broad nosed weevils (Curculionidae: Entiminae) constitute a large and taxonomically challenging subfamily that contains economically significant agricultural pests worldwide. South Africa is a hot-spot for biodiversity and several species of indigenous and endemic genera of Entiminae have shifted on to cultivated plants, with some being phytosanitary pests. The sporadic pest status of many species (where the species has an occasional economic impact on the agricultural industry, but is not encountered often enough that is is readily recognisable by researchers and agricultural extension workers) and the presence of pest complexes and cryptic species represent an identification challenge to non-specialists. Furthermore, no comprehensive identification tools exist to identify immature stages that may be found in crops/soil. In this paper, a curated barcoding database with 70 COI sequences from 41 species (39 Entiminae, 2 Cyclominae) is initiated, to assist with the complexity of identification of species in this group.

Diversification, selective sweep, and body size in the invasive Palearctic alfalfa weevil infected with Wolbachia.

The alfalfa weevil Hypera postica, native to the Western Palearctic, is an invasive legume pest with two divergent mitochondrial clades in its invading regions, the Western clade and the Eastern/Egyptian clade. However, knowledge regarding the native populations is limited. The Western clade is infected with the endosymbiotic bacteria Wolbachia that cause cytoplasmic incompatibility in host weevils. Our aim was to elucidate the spatial genetic structure of this insect and the effect of Wolbachia on its population diversity. We analyzed two mitochondrial and two nuclear genes of the weevil from its native ranges. The Western clade was distributed in western/central Europe, whereas the Eastern/Egyptian clade was distributed from the Mediterranean basin to central Asia. Intermediate mitotypes were found from the Balkans to central Asia. Most Western clade individuals in western Europe were infected with an identical Wolbachia strain. Mitochondrial genetic diversity of the infected individuals was minimal. The infected clades demonstrated a higher nonsynonymous/synonymous substitution rate ratio than the uninfected clades, suggesting a higher fixation of nonsynonymous mutations due to a selective sweep by Wolbachia. Trans-Mediterranean and within-European dispersal routes were supported. We suggest that the ancestral populations diversified by geographic isolation due to glaciations and that the diversity was reduced in the west by a recent Wolbachia-driven sweep(s). The intermediate clade exhibited a body size and host plant that differed from the other clades. Pros and cons of the possible use of infected-clade males to control uninfected populations are discussed.

DNA barcoding for bio-surveillance of emerging pests and species identification in Afrotropical Prioninae (Coleoptera, Cerambycidae).

DNA barcoding has been succesfully used for bio-surveillance of forest and agricultural pests in temperate areas, but has few applications in the tropics and particulary in Africa. Cacosceles newmannii (Coleoptera: Cerambycidae) is a Prioninae species that is locally causing extensive damage in commercially-grown sugarcane in the KwaZulu-Natal Province in South Africa. Due to the risk of spread of this species to the rest of southern Africa and to other sugarcane growing regions, clear and easy identification of this pest is critical for monitoring and for phytosanitary services. The genus Cacosceles Newman, 1838 includes four species, most being very similar in morphology. The damaging stage of the species is the larva, which is inherently difficult to distinguish morphologically from other Cerambycidae species. A tool for rapid and reliable identification of this species was needed by plant protection and quarantine agencies to monitor its potential abundance and spread. Here, we provide newly-generated barcodes for C. newmannii that can be used to reliably identify any life stage, even by non-trained taxonomists. In addition, we compiled a curated DNA barcoding reference library for 70 specimens of 20 named species of Afrotropical Prioninae to evaluate DNA barcoding as a valid tool to identify them. We also assessed the level of deeply conspecific mitochondrial lineages. Sequences were assigned to 42 different Barcode Index Numbers (BINs), 28 of which were new to BOLD. Out of the 20 named species barcoded, 11 (52.4%) had their own unique Barcode Index Number (BIN). Eight species (38.1%) showed multiple BINs with no morphological differentiation. Amongst them, C. newmannii showed two highly divergent genetic clusters which co-occur sympatrically, but further investigation is required to test whether they could represent new cryptic species.

DNA Metabarcoding as a Tool for Disentangling Food Webs in Agroecosystems.

Better knowledge of food webs and related ecological processes is fundamental to understanding the functional role of biodiversity in ecosystems. This is particularly true for pest regulation by natural enemies in agroecosystems. However, it is generally difficult to decipher the impact of predators, as they often leave no direct evidence of their activity. Metabarcoding via high-throughput sequencing (HTS) offers new opportunities for unraveling trophic linkages between generalist predators and their prey, and ultimately identifying key ecological drivers of natural pest regulation. Here, this approach proved effective in deciphering the diet composition of key predatory arthropods (nine species.; 27 prey taxa), insectivorous birds (one species, 13 prey taxa) and bats (one species; 103 prey taxa) sampled in a millet-based agroecosystem in Senegal. Such information makes it possible to identify the diet breadth and preferences of predators (e.g., mainly moths for bats), to design a qualitative trophic network, and to identify patterns of intraguild predation across arthropod predators, insectivorous vertebrates and parasitoids. Appropriateness and limitations of the proposed molecular-based approach for assessing the diet of crop pest predators and trophic linkages are discussed.

Multilocus phylogeography of the world populations of Elaeidobius kamerunicus (Coleoptera, Curculionidae), pollinator of the palm Elaeis guineensis.

Elaeidobius kamerunicus Faust (Coleoptera, Curculionidae) is one of the specific pollinators on inflorescences of the African oil palm Elaeis guineensis Jacquin. This derelomine weevil is native to tropical Africa. During the late 20th century, it was introduced into all tropical regions where E. guineensis is grown, in order to improve its pollination and fruit set. Despite an overall success, a decline in pollination efficiency has been documented in several regions. In this study, we reconstructed a multilocus phylogeography of the world populations of E. kamerunicus, in order to explore its genetic diversity in its native and introduced ranges. Our results showed that African populations of E. kamerunicus are forming two differentiated mitochondrial clusters in West and central Africa, forming a contact zone along the Cameroon Volcanic Line. The existence of this sharp contact zone along this weak altitudinal barrier suggests that other parameters, such as climate, may be driving the distribution of populations. A differential genetic structure between mitochondrial and nuclear genes, and the strong level of genetic structure of the mitochondrial gene, also suggest sex-biased dispersal in this species, with males dispersing more than females. The genetic structure inferred from Asian and South American populations suggests that they originate from populations of both western and central tropical Africa and that a bottleneck has probably been experienced by these populations.

Deciphering host-parasitoid interactions and parasitism rates of crop pests using DNA metabarcoding.

An accurate estimation of parasitism rates and diversity of parasitoids of crop insect pests is a prerequisite for exploring processes leading to efficient natural biocontrol. Traditional methods such as rearing have been often limited by taxonomic identification, insect mortality and intensive work, but the advent of high-throughput sequencing (HTS) techniques, such as DNA metabarcoding, is increasingly seen as a reliable and powerful alternative approach. Little has been done to explore the benefits of such an approach for estimating parasitism rates and parasitoid diversity in an agricultural context. In this study, we compared the composition of parasitoid species and parasitism rates between rearing and DNA metabarcoding of host eggs and larvae of the millet head miner, Heliocheilus albipunctella De Joannis (Lepidoptera, Noctuidae), collected from millet fields in Senegal. We first assessed the detection threshold for the main ten endoparasitoids, by sequencing PCR products obtained from artificial dilution gradients of the parasitoid DNAs in the host moth. We then assessed the potential of DNA metabarcoding for diagnosing parasitism rates in samples collected from the field. Under controlled conditions, our results showed that relatively small quantities of parasitoid DNA (0.07 ng) were successfully detected within an eight-fold larger quantity of host DNA. Parasitoid diversity and parasitism rate estimates were always higher for DNA metabarcoding than for host rearing. Furthermore, metabarcoding detected multi-parasitism, cryptic parasitoid species and differences in parasitism rates between two different sampling sites. Metabarcoding shows promise for gaining a clearer understanding of the importance and complexity of host-parasitoid interactions in agro-ecosystems, with a view to improving pest biocontrol strategies.

A review of the genus Smicronyx Schoenherr (Coleoptera, Curculionidae, Curculioninae) in tropical Africa.

The genus Smicronyx Schoenherr (Curculionidae, Curculioninae, Smicronychini) contains several species considered as potential biocontrol agents of parasitic and hemi-parasitic plants of crops (Striga spp. (Orobanchaceae) and Cuscuta spp. (Convolvulaceae)). However, the diversity of species occurring in Africa has not been explored in detail. This study reviews the species of Smicronyx occurring in continental tropical Africa. In total, 16 species are recorded for this region: 6 species previously described (S. bisignatus Hoffmann, S. gossypii Marshall, S. guineanus Voss, S. kiboanus Voss, S. pauperculus Wollaston and S. remaudierei Hoffmann), 2 newly recorded for the area (S. albosquamosus Wollaston, S. syriacus Faust), and 8 species are described as new (S. adjamati sp. n., S. crassithorax sp. n., S. kenyanus sp. n., S. namibicus sp. n., S. rufus sp. n., S. buchnerae sp. n., S. zambianus sp. n., S. zonatus sp. n.). A key to the species and photographs of the habitus and male genitalia are provided.

Multi-scale and multi-site resampling of a study area in spatial genetics: implications for flying insect species.

The use of multiple sampling areas in landscape genetic analysis has been recognized as a useful way of generalizing the patterns of environmental effects on organism gene flow. It reduces the variability in inference which can be substantially affected by the scale of the study area and its geographic location. However, empirical landscape genetic studies rarely consider multiple sampling areas due to the sampling effort required. In this study, we explored the effects of environmental features on the gene flow of a flying long-horned beetle (Monochamus galloprovincialis) using a landscape genetics approach. To account for the unknown scale of gene flow and the multiple local confounding effects of evolutionary history and landscape changes on inference, we developed a way of resampling study areas on multiple scales and in multiple locations (sliding windows) in a single large-scale sampling design. Landscape analyses were conducted in 3*104 study areas ranging in scale from 220 to 1,000 km and spread over 132 locations on the Iberian Peninsula. The resampling approach made it possible to identify the features affecting the gene flow of this species but also showed high variability in inference among the scales and the locations tested, independent of the variation in environmental features. This method provides an opportunity to explore the effects of environmental features on organism gene flow on the whole and reach conclusions about general landscape effects on their dispersal, while limiting the sampling effort to a reasonable level.

A review of Smicronyx Schoenherr (Coleoptera, Curculionidae) of Israel, with description of two new species.

The species of the genus Smicronyx sensu lato (Curculionidae, Curculioninae) occurring in Israel are reviewed. A total of seven species is recorded. Two species are newly recorded for the country (S. pauperculus Wollaston, 1864 and S. albosquamosus Wollaston, 1854), and two new species are described: S. jordanicus Haran and S. longitarsis Haran. A key to species, diagnosis of the new species, new biological and distribution data together with line illustrations of some characters, including male genitalia, and colour photographs of the habitus are provided.

From east to west across the Palearctic: Phylogeography of the invasive lime leaf miner Phyllonorycter issikii (Lepidoptera: Gracillariidae) and discovery of a putative new cryptic species in East Asia.

Knowing the phylogeographic structure of invasive species is important for understanding the underlying processes of invasion. The micromoth Phyllonorycter issikii, whose larvae damage leaves of lime trees Tilia spp., was only known from East Asia. In the last three decades, it has been recorded in most of Europe, Western Russia and Siberia. We used the mitochondrial cytochrome c oxidase subunit I (COI) gene region to compare the genetic variability of P. issikii populations between these different regions. Additionally, we sequenced two nuclear genes (28S rRNA and Histone 3) and run morphometric analysis of male genitalia to probe for the existence of cryptic species. The analysis of COI data of 377 insect specimens collected in 16 countries across the Palearctic revealed the presence of two different lineages: P. issikii and a putative new cryptic Phyllonorycter species distributed in the Russian Far East and Japan. In P. issikii, we identified 31 haplotypes among which 23 were detected in the invaded area (Europe) and 10 were found in its putative native range in East Asia (Russian Far East, Japan, South Korea and China), with only two common haplotypes. The high number of haplotypes found in the invaded area suggest a possible scenario of multiple introductions. One haplotype H1 was dominant (119 individuals, 67.2%), not only throughout its expanding range in Europe and Siberia but, intriguingly, also in 96% of individuals originating from Japan. We detected eight unique haplotypes of P. issikii in East Asia. Five of them were exclusively found in the Russian Far East representing 95% of individuals from that area. The putative new cryptic Phyllonorycter species showed differences from P. issikii for the three studied genes. However, both species are morphologically undistinguishable. They occur in sympatry on the same host plants in Japan (Sendai) and the Russian Far East (Primorsky krai) without evidence of admixture.

Revision of the genus Afrosmicronyx Hustache (Coleoptera, Curculionidae, Curculioninae).

The species of the Afrotropical genus Afrosmicronyx Hustache (Curculionidae, Curculioninae, Smicronychini), biocontrol agents of parasitic plants in crops (Striga, Orobanchaceae), are revised. Ten species are recognized, two of them described as new (A. mirei Haran, sp. n. and A. madagascariensis Haran, sp. n.). A key to the species with photographs of the habitus and male genitalia of type specimens is provided.

Family-Level Sampling of Mitochondrial Genomes in Coleoptera: Compositional Heterogeneity and Phylogenetics.

Mitochondrial genomes are readily sequenced with recent technology and thus evolutionary lineages can be densely sampled. This permits better phylogenetic estimates and assessment of potential biases resulting from heterogeneity in nucleotide composition and rate of change. We gathered 245 mitochondrial sequences for the Coleoptera representing all 4 suborders, 15 superfamilies of Polyphaga, and altogether 97 families, including 159 newly sequenced full or partial mitogenomes. Compositional heterogeneity greatly affected 3rd codon positions, and to a lesser extent the 1st and 2nd positions, even after RY coding. Heterogeneity also affected the encoded protein sequence, in particular in the nad2, nad4, nad5, and nad6 genes. Credible tree topologies were obtained with the nhPhyML ("nonhomogeneous") algorithm implementing a model for branch-specific equilibrium frequencies. Likelihood searches using RAxML were improved by data partitioning by gene and codon position. Finally, the PhyloBayes software, which allows different substitution processes for amino acid replacement at various sites, produced a tree that best matched known higher level taxa and defined basal relationships in Coleoptera. After rooting with Neuropterida outgroups, suborder relationships were resolved as (Polyphaga (Myxophaga (Archostemata + Adephaga))). The infraorder relationships in Polyphaga were (Scirtiformia (Elateriformia ((Staphyliniformia + Scarabaeiformia) (Bostrichiformia (Cucujiformia))))). Polyphagan superfamilies were recovered as monophyla except Staphylinoidea (paraphyletic for Scarabaeiformia) and Cucujoidea, which can no longer be considered a valid taxon. The study shows that, although compositional heterogeneity is not universal, it cannot be eliminated for some mitochondrial genes, but dense taxon sampling and the use of appropriate Bayesian analyses can still produce robust phylogenetic trees.

Altitudinal Barrier to the Spread of an Invasive Species: Could the Pyrenean Chain Slow the Natural Spread of the Pinewood Nematode?

Mountain ranges may delimit the distribution of native species as well as constitute potential barriers to the spread of invasive species. The invasive pinewood nematode, Bursaphelenchus xylophilus, is a severe forest pest inducing pine wilt disease. It is vectored in Europe by a native long-horned beetle, Monochamus galloprovincialis. This study explored the potential of the Pyrenean chain to slow or prevent the natural spread of nematode-infested beetles from the Iberian Peninsula, where the nematode is established and is expanding its range, towards France and the rest of Europe. An analysis of the genetic structure and migration patterns of the beetle populations throughout the Pyrenean mountain range was combined with a spread model simulating the potential movements of nematode-infested beetles across it. The central part of the Pyrenees, which corresponds to the highest elevation zone, was shown to prevent gene flow between the French and Spanish populations of M. galloprovincialis on each side of the mountains. Conversely, strong admixture was detected between populations located on both sides of low elevation hills, and especially at the east and west extremities of the mountain range. Simulations of the spread of nematode-infested beetles under various thresholds of beetle survival and pine wilt disease expression gave results consistent with the variation in genetic make-up, suggesting that western and eastern hillsides may represent corridors favoring natural spread of the nematode from the Iberian Peninsula to France. Simulations also showed that temperature rise due to climate change may significantly reduce the extent of the barrier formed by highest elevations. Our results support the hypothesis that the Pyrenean chain represents a partial barrier to the natural spread of nematode-infested beetles. These results, which have to be considered together with potential human-assisted long-distance spread of the nematode, highlight priority zones for future pest monitoring and management programs. More generally, such an integrated approach could be used to assess the role of mountain chains in the potential spread of other invasive pests.

Detection of the Acetylcholinesterase Insecticide Resistance Mutation (G328A) in Natural Populations of Ceratitis capitata.

Wild Mediterranean fruit fly specimens collected from various regions worldwide were screened for the glycine to alanine (Gly->Ala) point mutation (G328A) in the acetylcholinesterase enzyme, presumably causing resistance to organophosphates. We found that the single nucleotide polymorphism (SNP) responsible for this amino acid change is located at the beginning of exon 6 of the Ccace2 gene. The identification of the exact location of the SNP permitted PCR primer design around this site and direct sequencing of the corresponding genomic region. We detected the resistance allele in natural Mediterranean fruit fly populations from Brazil and Spain, but not from other sites in four continents. The known treatment history of sites suggests that the resistance buildup is linked to organophosphate application in the field. The PCR-based detection provides a screening method useful for monitoring Mediterranean fruit fly insecticide resistance in local populations and improving pest management strategies accordingly.

Mitogenome sequences stabilize the phylogenetics of weevils (Curculionoidea) and establish the monophyly of larval ectophagy.

The weevils and their relatives (superfamily Curculionoidea) constitute a huge radiation of beetles, but basal relationships in this group remain controversial, in particular within the largest family, Curculionidae. We used next-generation sequencing to generate mitochondrial genome data comprising 12 protein coding genes for 27 taxa. Together with two published ingroup sequences, (sub)family relationships were assessed with Bayesian and ML searches under various models and partitioning schemes. Forward and reverse strands of the mitochondrial genome differed in nucleotide skew. Consequently synonymous codon usage differed substantially on either strand for each amino acid, whereby codons ending in AT and GC were favored on the forward and reverse strands, respectively. Data partitioning by forward/reverse strand and codon position greatly improved likelihood scores and nodal support, whereas the tree topology was largely stable. The analysis generally supports the basal position of several 'orthocerus' lineages with straight antennae and male genitalia of an ancestral type; a paraphyletic mixed group of Heteromorphi exhibiting mixed ancestral and derived antennal and genitalic characters; and the derived 'gonathocerous' lineages with kinked antennae corresponding to the strongly supported Curculionidae. The latter did not include the wood boring Platypodinae that was recovered as sister to Dryophthoridae, while the Scolytinae and Cossoninae formed two independent lineages of wood borers within Curculionidae. A basal split in Curculionidae placed the Entiminae and Hyperinae as sister to all other subfamilies with high support, which provides a new ecological concept for structuring the Curculionidae according to the ectophagous larval life style in the former versus endophagous larvae in all others. This basal split is also supported by gene order rearrangements in a tRNA cluster. Recent studies supporting the monophyly of wood boring weevils may be attributable to long-branch attraction, as molecular rates in their mitochondrial genomes were found to be higher than in other lineages, but this did not confound tree searches under combined analysis of mitochondrial protein coding genes.