Multiplex TaqMan Real-Time PCR Assay for Sensitive Detection of Two Weevil Species (Coleoptera: Curculionidae).

Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus. This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/µl to 1 × 10-6 pg/µl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.

Out of the forest: past and present range expansion of a parthenogenetic weevil pest, or how to colonize the world successfully.

Previous research revealed complex diversification patterns in the parthenogenetic weevil Naupactus cervinus. To understand the origin of clonal diversity and successful spreading of this weevil, we investigated its geographic origin and possible dispersal routes and whether parthenogens can persist in habitats under unsuitable environmental conditions. This study is based on samples taken throughout a broad area of the species' range. We used both mitochondrial and nuclear markers and applied phylogenetic and network analyses to infer possible relationships between haplotypes. Bayesian phylogeographic analyses and ecological niche modeling were used to investigate the processes that shaped genetic diversity and enabled the colonization of new geographic areas. Southeastern Brazil emerges as the original distribution area of N. cervinus. We detected two range expansions, one along natural corridors during the Pleistocene and the other in countries outside South America during recent times. Isolation due to climate shifts during the early Pleistocene led to diversification in two divergent clades, which probably survived in different refugia of the Paranaense Forest and the Paraná River delta. The origin of the clonal diversity was probably a complex process including mutational diversification, hybridization, and secondary colonization. The establishment of N. cervinus in areas outside its native range may indicate adaptation to drier and cooler conditions. Parthenogenesis would be advantageous for the colonization of new environments by preventing the breakup of successful gene combinations. As in other insect pests, the present distribution of N. cervinus results from both its evolutionary history and its recent history related to human activities.

A PCR-Based Diagnostic System for Differentiating Two Weevil Species (Coleoptera: Curculionidae) of Economic Importance to the Chilean Citrus Industry.

A PCR-based method was developed to identify Naupactus cervinus (Boheman) and Naupactus xanthographus (Germar), two curculionids affecting the citrus industry in Chile. The quarantine status of these two species depends on the country to which fruits are exported. This identification method was developed because it is not possible to discriminate between these two species at the egg stage. The method is based on the species-specific amplification of sequences of internal transcribed spacers, for which we cloned and sequenced these genome fragments from each species. We designed an identification system based on two duplex-PCR reactions. Each one contains the species-specific primer set and a second generic primer set that amplify a short 18S region common to coleopterans, to avoid false negatives. The marker system is able to differentiate each Naupactus species at any life stage, and with a diagnostic sensitivity to 0.045 ng of genomic DNA. This PCR kit was validated by samples collected from different citrus production areas throughout Chile and showed 100% accuracy in differentiating the two Naupactus species.

Flutuação populacional de curculionídeos-das-raízes em citros / Population dynamics of root weevils in citrus

O objetivo neste trabalho foi estudar a flutuação populacional de curculionídeos-das-raízes em citros. Foram feitas amostragens quinzenais das fases de larva, de pupa e adulta, em laranjeiras de quatro fazendas, em Itapetininga, São Paulo, de março de 2000 a fevereiro de 2002. A emergência dos adultos foi quantificada em gaiolas sob a copa das plantas e a população de adultos, na copa das plantas, com pano-de-batida; as larvas e pupas em trincheiras abertas no solo, sob a copa das plantas. Com base na flutuação populacional das fases, é possível afirmar que os insetos são univoltinos. A maioria dos adultos emergiu de outubro a abril, com picos no início do período. Foram encontradas as espécies Parapantomorus fluctuosus (Boheman), Naupactus cervinus Boheman e Naupactus versatilis Hustache, sendo as duas últimas as mais freqüentes, embora com picos em épocas diferentes. Ocorreram larvas praticamente o ano inteiro, com picos populacionais no período de junho a janeiro. A população de curculionídeos foi menor no segundo ano de estudo, provavelmente em conseqüência da estiagem. Dentre as fases estudadas, os adultos e as larvas, amostrados com pano-de-batida e em trincheiras no solo, podem servir para detecção, quantificação e previsão da ocorrência dos curculionídeos-das-raízes dos citros.

The goal of this study was to evaluate the population dynamics of citrus root weevils. Samples of the larval, pupa and adult phases were taken every 15 days in four citrus farms in Itapetininga, State of São Paulo, Brazil, between March, 2000 and February, 2002. The emergence of adults was evaluated in cages under plant canopy and the adult population at plant canopy with a beating cloth; larvae and the pupae in ditches opened in the soil, under plant crowns. Based on the population dynamics, the insects are univoltine. Most adults emerged from October to April, with peaks at the beginning of the period. Species Parapantomorus fluctuosus (Boheman), Naupactus cervinus Boheman and Naupactus versatilis Hustache were more frequent in Itapetininga, SP, although the peaks and times were different. Larvae occurred almost during the whole year, with population peaks from June to January. The number of curculionids was lower in the second year of the study for all insect phases, probably due to drought. Adults and larvae sampled, with beating cloths and in cages, could be used to detect, quantify and predict the occurrence of citrus root weevils.