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Some insects are infected with maternally inherited bacterial endosymbionts that protect them against pathogens or parasitoids. The weevil Sitona obsoletus (=Sitona lepidus) is invasive in New Zealand, and suspected to contain such defensive symbionts, because it is particularly resistant to a Moroccan strain of the parasitoid Microctonus aethiopoides (which successfully attacks many other weevil species), and shows geographic variation in susceptibility to an Irish strain of the same parasitoid. Using 454 pyrosequencing, we investigated the bacterial community associated with S. obsoletus, two other exotic weevils (Sitona discoideus and Listronotus bonariensis) and two endemic New Zealand weevils (Irenimus aequalis and Steriphus variabilis). We found that S. obsoletus was infected by one strain of Wolbachia and two strains of Rickettsia, none of which were found in any other weevil species examined. Using diagnostic PCR, we found that S. obsoletus in the Northland region, where parasitism is highly variable, were primarily infected with Wolbachia and Rickettsia strain 2, indicating that these two symbionts should be investigated for potential defensive properties. In comparison, S. discoideus lacked any apparent maternally inherited bacterial endosymbionts. In the other weevil species, we found a different strain of Wolbachia and two different strains of Spiroplasma. Two weevil species (St. variabilis and L. bonariensis) were infected with distinct strains of Nardonella, the ancestral endosymbiont of weevils, whereas three weevil species (S. obsoletus, S. discoideus, and I. aequalis) lacked evidence for Nardonella infection. However, I. aequalis was consistently infected with a novel Enterobacteriaceae strain, suggesting that a symbiont replacement may have taken place, similar to that described for other weevil clades.

The clover root weevil, Sitona lepidus Gyll. is a pest of clovers (Trifolium spp.), particularly white clover (T. repens). Larva feeding severely impairs the capacity of white clover to fix atmospheric nitrogen, by attacking the nitrogen-fixing root nodules. Development of the egg stage was studied at six different constant temperatures ranging from 9 to 30°C to improve the basis for phenological forecasts. Development occurred over the entire range of temperatures, although hatching percentage at 30°C was only 24.4% compared to 90.1% at 28.5°C. Developmental time decreased with increase in temperature from 9 to 28.5°C but increased markedly at 30°C. At 28.5°C, the embryo development period was the shortest (10.35 days); whereas at 9°C, this parameter was the longest (55.08 days). Using linear regression, the lower threshold temperature was calculated 4.38°C and the thermal constant was 236.45 degree-days. The relationship between constant temperature and developmental rate was evaluated using nine models. The suitability of the models was evaluated based on eight indicators (R 2, RSS, R adj 2 , AIC, BIC, AICC, W j , and Z i ). Of the nonlinear models, the Logan-6, Sharpe and DeMichele, and Lactin models were the most accurate at calculating t opt of 27, 27.50, and 28°C, respectively. The results suggest that of the three models (Lactin, Briere, and Hilbert and Logan), that estimated critical temperatures, (t min, t opt, and t max), Lactin gave the most suitable fit of data. This response of S. lepidus to temperature can be used for developing phonological models to predict the timing of egg hatch which are important for management programs targeting the weevil.

The clover root weevil Sitona lepidus Gyllenhal (Coleoptera: Curculionidae) became an economically important pasture pest in New Zealand shortly after it was discovered in the Waikato region in 1996. A classical biological programme was initiated and an Irish biotype of Microctonus aethiopoides Loan (Hymenoptera: Braconidae) was released at four sites in the North Island in late summer 2006. These sites in Waikato, Hawke’s Bay and Manawatu (two sites) regions were monitored monthly and parasitoid establishment confirmed at all sites within four months. In the winter of 2007, parasitism exceeded 70%. A widespread North Island drought in summer 2008 had a severe impact on S. lepidus populations at the Waikato and the two Manawatu release sites, resulting in parasitism below detection levels in the following summer. However, populations recovered by autumn. Within three years at the Hawke’s Bay site, M. aethiopoides appears to be suppressing S. lepidus populations and has dispersed naturally over 60 km.

Predicted increases in atmospheric carbon dioxide (CO(2)) concentrations are expected to increase primary productivity in many terrestrial ecosystems, which could lead to plants becoming N limited. Studies suggest that legumes may partially overcome this by increasing biological nitrogen fixation. However, these studies have not yet considered how these changes may be affected by the altered dynamics of insect herbivores feeding on the plant. This study investigated how elevated CO(2) (700 microl l(-1)) affected the clover root weevil (Sitona lepidus), a significant pest of white clover (Trifolium repens). Adults feed on leaves aboveground where they lay eggs; soil-dwelling larvae initially feed on root nodules that house N(2)-fixing bacteria. Foliar C:N ratios rose by 9% at elevated CO(2), but the biggest responses were observed belowground, with increases in root mass (85% greater) and nodule abundance (220% more abundant). Root C:N ratios increased significantly from 10.95 to 11.60 under elevated CO(2), which increased even further to 13.13 when nodules were attacked by larval S. lepidus. Adult S. lepidus consumed significantly more leaf tissue at elevated CO(2) (0.47 cm(2) day(-1)) compared with ambient CO(2) (0.35 cm(2) day(-1)), suggesting compensatory feeding, but laid 23% fewer eggs at elevated CO(2). Even though fewer eggs were laid at elevated CO(2), 38% more larvae were recovered suggesting that larval survival was much better under elevated CO(2). Increased larval abundance and performance at elevated CO(2) were positively correlated with the number of nodules available. In conclusion, reduced foliar quality at elevated CO(2) was generally disadvantageous for adult S. lepidus living aboveground, but extremely beneficial for S. lepidus larvae living belowground, due to the enhanced nodulation. Climate change may, therefore, enhance biological nitrogen fixation by T. repens, but potential benefits (e.g. provision of N without chemical fertilizers) may be undermined by larger populations of S. lepidus larvae belowground.

The clover root weevil, Sitona lepidus, frequently feeds on N2 fixing rhizobial root nodules of white clover (Trifolium repens), which may contain isoflavonoids with defensive and plant regulatory properties. This study investigated the isoflavonoids present in N2 fixing (active) root nodules, root nodules that were not fixing N2 (inactive), and roots without nodules, and tested the behavioral responses of neonatal S. lepidus larvae to aglycones of the identified compounds. Formononetin concentrations were higher in the active nodules compared with inactive nodules and roots alone. Moreover, there was a statistically significant attraction to formononetin by S. lepidus in arena experiments, whereas the other isoflavonoids were unattractive. It is suggested that S. lepidus may have become tolerant to the toxic effects of formononetin with repeated exposure, and that it may play a role in root nodule location. Such coevolutionary relationships are widely reported for aboveground insects and plants, but the present study suggests they may also occur belowground.

Bacillus thuringiensis subsp. tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the δ-endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.