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New agricultural pest species attacking introduced crops may evolve from pre-existing local herbivores by ecological speciation, thereby becoming a species by becoming a pest. We compare the evolutionary pathways by which two maize pests (the Asian and the European corn borers, ACB and ECB) in the genus Ostrinia (Lepidoptera, Crambidae) probably diverged from an ancestral species close to the current Adzuki bean borer (ABB). We typed larval Ostrinia populations collected on maize and dicotyledons across China and eastern Siberia, at microsatellite and mitochondrial loci. We found only two clusters: one on maize (as expected) and a single one on dicotyledons despite differences in male mid-tibia morphology, suggesting that all individuals from dicotyledons belonged to the ABB. We found evidence for migrants and hybrids on both host plant types. Hybrids suggest that field reproductive isolation is incomplete between ACB and ABB. Interestingly, a few individuals with an 'ABB-like' microsatellite profile collected on dicotyledons had 'ACB' mtDNA rather than 'ABB-like' mtDNA, whereas the reverse was never found on maize. This suggests asymmetrical gene flow directed from the ACB towards the ABB. Hybrids and backcrosses in all directions were obtained in no-choice tests. In laboratory conditions, they survived as well as parental strain individuals. In Xinjiang, we found ACB and ECB in sympatry, but no hybrids. Altogether, our results suggest that reproductive isolation between ACB and ABB is incomplete and mostly prezygotic. This points to ecological speciation as a possible evolutionary scenario, as previously found for ECB and ABB in Europe.

Defining the taxonomic identity of organisms is a prerequisite for their study, and in the case of economically important species, misidentification may lead to the application of inappropriate prevention and control strategies. Flat mites of the Brevipalpus genus include several crop pests and the systematics of these mites represents a challenge for acarologists. Many of the most economically important Brevipalpus species have repeatedly been inaccurately identified. Such problematic classification has been attributed to the likely occurrence of cryptic species in the genus. In this study, we used an integrative approach that combined molecular analyses, including sequence-based species delimitation, with detailed morphological identification using traits that have recently showed to be taxonomically informative. Sequences of mitochondrial cytochrome c oxidase subunit I (COI) were obtained from individuals collected from host plants belonging to 14 genera and 13 families across 29 locations in the Americas (Brazil, Chile, USA). The phylogenetic analyses included previously published Brevipalpus sequences from GenBank, and the final data set was classified into 44 haplotypes. Six putative species were recognised by COI-based species delimitation analysis, and morphological evidence supported each of these species. The integrative approach revealed the occurrence of cryptic species in the Brevipalpus genus and contributed to the clarification of previously noted incongruences. The results presented here allow for the evaluation of taxonomic characteristics in a phylogenetic context and indicate new characters for the differentiation of Brevipalpus species. In addition, Brevipalpus incognitus n. sp. Ferragut & Navia, a cryptic species detected in this study, is described based on morphological and molecular traits. Implications of the advances in Brevipalpus systematics presented herein with respect to pest management are briefly discussed., We sincerely thank Dalva L. de Queiroz Santana, Embrapa Florestas, Colombo, Parana; Aloyseia Noronha, Eduardo Chumbinho de Andrade, Juliana Freitas-Astua and Francisco F. L. Barbosa, Embrapa Mandioca e Fruticultura, Cruz das Almas, Bahia; Alberto Luiz Marsaro Jr, Embrapa Roraima, Boa Vista, Roraima; Ricardo Adaime da Silva, Embrapa Amapa, Macapa, Amapa; Patricia Maria Drumond, Embrapa Acre, Rio Branco, Acre; Manoel Guedes C. Gondim Jr and Aleuny C. Reis, Universidade Federal Rural de Pernambuco, Recife, Pernambuco; Jose. C. M. Poderoso, Universidade Federal de Sergipe, Sao Cristovao, Sergipe, all from Brazil; and Jose Carlos V. Rodrigues, University of Puerto Rico, San Juan, Puerto Rico, for their help with sampling; Tatiane G. C. M. Galasso, Brazil, for help with extensive laboratory work. We also thank Maries Pages and Fabien Condamine, INRA, UMR CBGP, France, for their help concerning the Pons analysis. This study was funded by Brazil - National Council for Scientific and Technological Development (CNPq) (call CNPq/MAPA/SDA No 064/2008, grant 578353/2008-3), Embrapa Macroprograma 3 and Fundacao de Apoio a Pesquisa do Distrito Federal (FAP-DF). Authors DN and RSM are also grateful to CNPq for the research and pos-doc fellowships, respectively.

Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when silenced, constitute promising targets for the development of more specific and safer control means., Author Summary Plant-parasitic nematodes are annually responsible for more than $100 billion crop yield loss worldwide and those considered as causing most of the damages are root-knot nematodes. These nematodes used to be controlled by chemicals that are now banned from use because of their poor specificity and high toxicity for the environment and human health. In the absence of sustainable alternative solutions, new control means, more specifically targeted against these nematodes and safe for the environment are needed. We searched in root-knot nematode genomes, genes conserved in various plant-damaging species while otherwise absent from the genomes of non target species such as those of chordates, plants, annelids, insect pollinators and mollusks. These genes are probably important for plant parasitism and their absence from non-target species make them interesting candidates for the development of more specific and safer control means. Further bioinformatics pruning of this set of genes yielded 16 novel candidates that could be biologically tested. Using RNA interference, we knocked down each of these 16 genes in a root-knot nematode and tested the effect on plant parasitism efficiency. Out of the 16 tested genes, 12 showed a significant and reproducible diminution of infestation when silenced and are thus particularly promising.

Expanding genomic data on plant pathogens open new perspectives for the development of specific and environment friendly pest management strategies based on the inhibition of parasitism genes that are essential for the success of infection. Identifying such genes relies on accurate reverse genetics tools and the screening of pathogen knock-down phenotypes. Root-knot nematodes are major cosmopolitan crop pests that feed on a wide range of host plants. Small interfering RNAs (siRNAs) would provide a powerful tool for reverse genetics of nematode parasitism genes provided that they could (1) target genes expressed in inner tissues of infective nematodes and (2) target genes expressed during parasitism. In this study, we show that siRNAs can access inner tissues of the infective juveniles during soaking and accumulate in the esophagus, amphidial pouches and related neurons of the nematode. We provide evidence that siRNAs can trigger knock-down of the parasitism gene Mi-CRT, a calreticulin gene expressed in the esophageal glands of Meloidogyne incognita. Mi-CRT knock-down in infective juveniles affected nematode virulence. However, Mi-CRT knock-down was not persistent after plant infection, indicating that siRNA-mediated RNAi is best suited for functional analysis of genes involved in pre-parasitic stages or in the early steps of infection.