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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.

We report the complete mitogenome of Sitona obsoletus, an agricultural pest in New Zealand and some European countries. Like other Sitona mitogenomes, the 6 tRNA gene box is ordered RNSAEF, support...

The introduced parasitoid Microctonus aethiopoides (Hymenoptera Braconidae) appears to be an effective biocontrol agent for clover root weevil Sitona lepidus Gyllenhal (Coleoptera Curculionidae) in New Zealand pastures As these pastures lack accessible nectar sources hemipteran honeydew may be the most readily available energy source for the parasitoid Experiments were undertaken to compare longevity of newly emerged M aethiopoides adults caged on white clover firstly with and without black legume aphid Aphis craccivora Kock (Hemiptera Aphididae) and then with additional water and sucrose solution treatments Parasitoids with access to aphid honeydew lived significantly longer (mean longevity 81 days) than those with water or no liquid (both 68 days) but not as long as those fed sucrose solution (109 days) Therefore the abundance of aphids commonly present on clovers may enhance M aethiopoides longevity and therefore efficacy in the field especially in mid spring when host availability is very low

Two strains of the hymenopteran parasitoid Microctonus aethiopoides have been released in New Zealand for the biological control of Sitona weevil species. One attacks Sitona discoideus, a pest of lucerne, and the other attacks Sitona lepidus, a pest of clover. Two other Microctonus species also attack weevils in pasture; M. hyperodae was released for the biological control of Listronotus bonariensis and the native M. zealandicus attacks Irenimus spp. These Microctonus species can attack non-target weevil hosts and the identification of the larvae of the different Microctonus species and the separation of adults of M. aethiopoides strains can only be achieved by molecular methods. This paper describes a simple polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method for distinguishing between the two M. aethiopoides strains, M. hyperodae and M. zealandicus. This PCR-RFLP method requires minimal molecular equipment and is cheaper and/or faster than other molecular methods.

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.

Insect pests that have a root-feeding larval stage often cause the most sustained damage to plants because their attrition remains largely unseen, preventing early diagnosis and treatment. Characterising movement and dispersal patterns of subterranean insects is inherently difficult due to the difficulty in observing their behaviour. Our understanding of dispersal and movement patterns of soil-dwelling insects is therefore limited compared to above ground insect pests and tends to focus on vertical movements within the soil profile or assessments of coarse movement patterns taken from soil core measurements in the field. The objective of this study was to assess how the dispersal behaviour of the clover root weevil (CRW), Sitona lepidus larvae was affected by differing proportions of host (clover) and non-host (grass) plants under different soil water contents (SWC). This was undertaken in experimental mini-swards that allowed us to control plant community structure and soil water content. CRW larval survival was not affected either by white clover content or planting pattern or SWC in either experiment; however, lower clover composition in the sward resulted in CRW larvae dispersing further from where they hatched. Because survival was the same regardless of clover density, the proportion of infested plants was highest in sward boxes with the fewest clover plants (i.e. the low host plant density). Thus, there is potential for clover plants over a larger area to be colonised when the clover content of the sward is low.

The effects of larval herbivory by clover root weevil (CRW, Sitona lepidus) on seven white clover (Trifolium repens) and two red clover (Trifolium pratense) progenies from plants showing tolerance of CRW in field trials were tested in two controlled glasshouse experiments. CRW larvae recovered from red clover plants were, on average, fewer (6.3 larvae/g root dry weight) and shorter (5.7 mm/g root dry weight) than those from white clover (16.7 larvae, 7.1 mm length).This confirmed previous findings that red clover is an inferior host for this pest. In the first experiment, progeny of four of the white clover selections for CRW tolerance showed high tolerance (shoot dry weights −2.9% to +3.1% different from their respective weevil-free controls) compared with a 13–24% shoot weight loss for two white clover cultivars. These results confirm field trial observations that there is variation for CRW tolerance in white clover and also that CRW tolerance is hereditable in at least some populations. The se...

Two experiments were carried out at Ruakura in soil taken from Whatawhata hill country pasture. The relative susceptibility of several annual clovers (Trifolium spp.) and perennial white clover (T. repens ) to slugs (Deroceras reticulatum), clover root weevil (Sitona lepidus) adults, native crickets (Nemobius sp.) or wheat bugs (Nysius huttoni) was tested by sowing seed of the clovers into separate rows in turf. Susceptibility of clovers to clover cyst nematode (Heterodera trifolii) was tested by sowing each clover variety into Whatawhata soil inoculated with the nematode. Plant growth was assessed in both experiments 4 weeks after sowing. White clover was more susceptible to pests than the annual clovers, with slugs and clover root weevil significantly reducing seedling survival, and clover cyst nematode significantly reducing plant growth. Subterranean clover (T. subterraneum) cv. Denmark was the least affected by pests, showing no significant reduction in survival in the presence of slugs, the most damaging pest, and no significant decrease in plant root and shoot dry weight when exposed to nematodes.

Clover root weevil (Sitona lepidus) (CRw), a white clover pest from the Northern Hemisphere, was first found in the North island in 1996. its 2006 detection in the south island coincided with the introduction from europe of an insect parasitoid for biocontrol of CRw. upon detecting CRw in the south island, we surveyed for suitable locations to release the biocontrol agent. our goals were to reduce clover damage and to minimise the number of releases required by allowing the parasitoid to spread with the pest. we released it in three large CRw infestations where it quickly became established. although the parasitoid’s natural spread rate appears similar to that of CRw, additional geographically isolated CRw infestations were detected which have probably arisen from accidental human assisted transportation, and the parasitoid will take several years to reach them. Three further releases of the biocontrol agent have been made, and more are planned.

Soil-dwelling insect herbivores are significant pests in many managed ecosystems. Because eggs and larvae are difficult to observe, mathematical models have been developed to predict life-cycle events occurring in the soil. To date, these models have incorporated very little empirical information about how soil and drought conditions interact to shape these processes. This study investigated how soil temperature (10, 15, 20 and 25 °C), water content (0.02 (air dried), 0.10 and 0.25 g g−1) and pH (5, 7 and 9) interactively affected egg hatching and early larval lifespan of the clover root weevil (Sitona lepidus Gyllenhal, Coleoptera: Curculionidae). Eggs developed over 3.5 times faster at 25 °C compared with 10 °C (hatching after 40.1 and 11.5 days, respectively). The effect of drought on S. lepidus eggs was investigated by exposing eggs to drought conditions before wetting the soil (2–12 days later) at four temperatures. No eggs hatched in dry soil, suggesting that S. lepidus eggs require water to remain viable. Eggs hatched significantly sooner in slightly acidic soil (pH 5) compared with soils with higher pH values. There was also a significant interaction between soil temperature, pH and soil water content. Egg viability was significantly reduced by exposure to drought. When exposed to 2–6 days of drought, egg viability was 80–100% at all temperatures but fell to 50% after 12 days exposure at 10 °C and did not hatch at all at 20 °C and above. Drought exposure also increased hatching time of viable eggs. The effects of soil conditions on unfed larvae were less influential, except for soil temperature which significantly reduced larval longevity by 57% when reared at 25 °C compared with 10 °C (4.1 and 9.7 days, respectively). The effects of soil conditions on S. lepidus eggs and larvae are discussed in the context of global climate change and how such empirically based information could be useful for refining existing mathematical models of these processes.

The egg, larval, pupal and adult abundance of the clover root weevilSitona lepidusGyllenhal (Coleoptera: Curculionidae) was monitored at three sites for the first ten years following the discovery of this exotic pest in the Waikato region of New Zealand. The species went through an initial boom and bust cycle at two sites, with populations reaching up to 1800 larvae m−2. Thereafter, winter larval populations were relatively stable, ranging between 450–750 m−2. Unlike in the Northern Hemisphere,S. lepiduswas found to have two generations a year in the Waikato region of New Zealand. Pasture white clover content at the time of peak adult numbers was positively related to the subsequent peak larval populations for each generation. The factors contributing to the emergence ofS. lepidusas one of the most important pasture pests in New Zealand are discussed.

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.

Most arthropod traps available for biosecurity surveillance are either generic and incur problems with large bycatches, or are targeted at one or a few species and have limited application. Traps designed to be of intermediate specificity were tested at three high risk sites in Christchurch. Traps with and without attractants targeting weevils (Curculionidae), and two trap types targeting cobweb spiders (Theridiidae) were evaluated. The weevil traps captured nine weevil species from six genera, including the first detection of Sitona lepidus in the South Island. Significantly more weevils were captured in the traps with attractants. The spider traps captured spiders in the families Theridiidae, Linyphiidae and Agelenidae, including the first detection of Nesticodes rufipes (Theridiidae) in the South Island. For all traps, bycatches were small and readily processed, and included a cricket, Gryllodes sigillatus, that is not established in New Zealand. Traps of intermediate specificity have potential for operational use in high risk site surveillance.

The effects of root herbivory at five densities of Sitona lepidus larvae (overall means between 4 and 333 larvae m‐2) were assessed over 2 years on newly established perennial ryegrass‐white clover swards in a small plot trial. Initial larval establishment in autumn 2003 was positively related to clover content in plots, and there was no significant impact on clover herbage yield in the first year. Nodule damage in winter 2003 increased with larval density, and results suggested an overcompensatory response in nodule production. A 34–35% reduction in clover yield between highest and lowest S. lepidus densities was recorded for both cultivars in the second year, with greatest losses in spring 2004. This coincided with reductions in clover root and stolon weights. Plant parasitic nematodes and grass grub larvae were most abundant in the plots with lowest weevil numbers. These results confirm field observations that S. lepidus is a major pest of pastures.

Four experimental releases of the parthenogenetic strain of Microctonus aethiopoides from Ireland were made in early 2006 in the Waikato Manawatu (two sites) and Hawkes Bay By early winter establishment was confirmed at all sites with parasitism levels exceeding 10 in the target host Sitona lepidus a serious pest of white clover in New Zealand Subsequent monitoring revealed considerable betweensite variation At the Hawkes Bay and Manawatu Feilding sites where overwintering weevil adults were still present in October over 30 parasitism was found in newly emerged weevils in December 2006 In contrast at the Waikato and Manawatu Bulls sites where there was no overlap of host generations parasitism was below detectable levels during spring and early summer but recovered subsequently The Irish M aethiopoides appears to have four generations a year and diapauses over winter as a first instar larva Following the initial success releases have commenced in other regions

Insect herbivores that have soil-dwelling larval stages usually lay eggs directly or indirectly into the soil. Following egg hatch, emergent larvae must locate host plant roots to avoid starvation and this represents the most vulnerable part of the life cycle. We present a model for this aspect of the life cycle, specifically modelling the egg development rate and survival time of the clover root weevil, Sitona lepidus. The model is based on a partial differential equation, developed from age-structure models that are widely used in ecology. The model incorporates stochastic random variation caused by environmental fluctuation and genetic variation in a population, and treats chronological time and biological age as two independent variables. The average developmental rate and the impact of randomness are described by a first-order and a second-order derivative term, respectively. The significance of this model is that it can combine two biological events (egg development and larval survival time) into a single functional event, a potentially important feature for soil-dwelling insects because their concealed habitat does not permit all biological events to be observed. The model was tested against experimental observations of egg development and larval survival time under different soil conditions, firstly by considering egg development and larval survival time as independent biological events and secondly by combining both into a single functional event. Model simulations and experimental observations were in close agreement in all cases. To further test whether the model could be applied to other insect taxa and incorporate more than two biological events, we compared model simulations with published experimental results for the cereal leaf beetle (Oulema duftschmidi). Simulations of egg hatching and the larval development through several instars compared favourably with all experimental observations, demonstrating that the model has multiple applications.

Clover root weevil ( Sitona lepidus, CrW) is a major New Zealand pasture pest. This study used computer simulation and decision support modelling to simulate CrW herbivory, estimate the long-term consequences on clover abundance, pasture production and quality, and financial implications to a typical sheep and beef farmer. Three farm scenarios were explored, the absence of C rW and the presence of C rW with and without additional nitrogen (N). For a hypothetical 325 ha Waikato sheep and beef farm, C rW decreased mean clover abundance from 21 to 13%, pasture production from 9200 to 7900 kg dM/ha/year, pasture quality from 10.5 to 10.2 MJM e/kg dM and N fixation from 60 to 42 kg N/ha/year. This resulted in a 16% reduction in the annual gross margin. however, assuming current prices and costs, and that an N response could be consistently achieved, urea could be used to replace the reduction in N fixation without affecting profits.

Clover root weevil (CRW) (Sitona lepidus) continues to have a severe impact on white clover in pastures in the North Island of New Zealand Trials were established in October 2005 as part of a continuing evaluation of the insectpathogenic fungus Beauveria bassiana F418 strain for management of this pest F418 was applied as a rice biopolymer formulation targeting CRW larvae in the clover root feeding zone (35 cm below the surface) Overall there was a reduction in CRW numbers at all of the test sites The number of larvae recovered was significantly lower in F418treated than control plots after 3 weeks and the number of pupae was similarly lower in F418 than control plots after 6 weeks Higher levels of fungal propagules were recovered from 06 cm in the soil profile than 610 cm

A European biotype of Microctonus aethiopoides was identified as the best candidate biocontrol agent for Sitona lepidus a serious New Zealand pasture pest A Moroccan biotype was already present throughout the country and hence there was no requirement to obtain Hazardous Substances and New Organisms (HSNO) Act approval to release new biotypes However as research had shown mating between the two biotypes produced hybrids with poor efficacy against target hosts and that the Moroccan biotype attacked several native weevil genera serious reservations were held about introducing the European biotype Concerns were overcome with the identification of a parthenogenetic strain of European M aethiopoides from Ireland which has little risk of hybridisation and a narrower host range than the Moroccan biotype Following regulation of M aethiopoides as a risk species this strain was considered to be a new organism Approval was sought and gained under the HSNO Act to release the strain

1. Few entomological studies include soil-dwelling insects in mainstream ecological theory, for example the preference–performance debate. The preference–performance hypothesis predicts that when insect herbivores have offspring with limited capacity to relocate in relation to a host plant, there is a strong selection pressure for the adult to oviposit on plants that will maximise offspring performance. 2. This paper discusses the proposition that insect herbivores that live above ground, but have soil-dwelling offspring, should be included in the preference–performance debate. Twelve relevant studies were reviewed to assess the potential for including soil insects in this framework, before presenting a preliminary case study using the clover root weevil (Sitona lepidus) and its host plant, white clover (Trifolium repens). 3. Maternal S. lepidus preferentially oviposited on T. repens plants that had rhizobial root nodules (which enhance offspring performance) rather than T. repens plants without nodules, despite plants having similar foliar nutritional quality. This suggests that adult behaviour above ground was influenced by below-ground host-plant quality. 4. A conceptual model is presented to describe how information about the suitability for offspring below ground could underpin oviposition behaviour of parental insects living above ground, via plant- and soil-mediated semiochemicals. These interactions between genetically related, but spatially separated, insect herbivores raise important evolutionary questions such as how induced plant responses above ground affect offspring living below ground and vice versa.

White clover ( Trifolium repens ) is an important pasture legume but is often difficult to sustain in a mixed sward because, among other things, of the damage to roots caused by the soil-dwelling larval stages of S. lepidus . Locating the root nodules on the white clover roots is crucial for the survival of the newly hatched larvae. This paper presents a numerical model to simulate the movement of newly hatched S. lepidus larvae towards the root nodules, guided by a chemical signal released by the nodules. The model is based on the diffusion–chemotaxis equation. Experimental observations showed that the average speed of the larvae remained approximately constant, so the diffusion–chemotaxis model was modified so that the larvae respond only to the gradient direction of the chemical signal but not its magnitude. An individual-based lattice Boltzmann method was used to simulate the movement of individual larvae, and the parameters required for the model were estimated from the measurement of larval movement towards nodules in soil scanned using X-ray microtomography. The model was used to investigate the effects of nodule density, the rate of release of chemical signal, the sensitivity of the larvae to the signal, and the random foraging of the larvae on the movement and subsequent survival of the larvae. The simulations showed that the most significant factors for larval survival were nodule density and the sensitivity of the larvae to the signal. The dependence of larval survival rate on nodule density was well fitted by the Michealis–Menten kinetics.

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.

Populations of adult clover root weevil (Sitona lepidus) can infl ict signifi cant numbers of their distinctive feeding notches on the leaves of white clover (Trifolium repens) plants. However, it is assumed that because white clover is bred for defoliation, the damage on established plants has little impact. Two experiments were undertaken to quantify the daily leaf consumption levels of adult weevils, and how this varied with weevil size and sex, clover cultivar and previous leaf damage. Overall, adults consumed between 0.75 and 1.2 mg dry matter/day depending on cultivar and sex. Sexual differences were observed between two contrasting clover cultivars, with females consuming more Grassland Prestige and males Grasslands Kopu. Clover dry matter loss through adult feeding was estimated at about 50 kg/ha from early summer until midwinter in the Waikato, the period of peak adult activity.

The rapid spread of clover root weevil (Sitona lepidus) (CRW) since its introduction in the early 1990s, threatens the competitive advantage of New Zealand's pastoral industry. When CRW was discovered, it had already spr ead too far for containment. The insect's distribution currently covers the North Island and there is no reason to prevent its spread ultimately throughout NZ. With no competing species, CRW is more damaging in NZ than in its native Europe. Clover root weevil affects white clover nitrogen (N) fixation while simultaneously reducing the clover content of pastures thereby lowering total forage quality. Legume germplasm was screened for resistance/tolerance and while no resistance was discovered, vigorous growing white c lover plants showed tolerance to CRW and gains from selection for tolerance were achieved. Eliminating remnant clover before pasture renovation or growing a crop between grass stages reduces the resident CRW population and improves clover re-establishment. However, CRW can reinvade and potentially return to its original density. Two candidate biological control agents are being pursued for release later in 2005. Clover root weevil's impact on pastoral farming varies, partially due to environmental variation, which dictates clover growth and CRW development. Keywords: clover root weevil, cultivar selection, life cycle analysis, pasture management, Sitona lepidus

This study investigated the ability of neonatal larvae of the root-feeding weevil, Sitona lepidus Gyllenhal, to locate white clover Trifolium repens L. (Fabaceae) roots growing in soil and to distinguish them from the roots of other species of clover and a co-occurring grass species. Choice experiments used a combination of invasive techniques and the novel technique of high resolution X-ray microtomography to non-invasively track larval movement in the soil towards plant roots. Burrowing distances towards roots of different plant species were also examined. Newly hatched S. lepidus recognized T. repens roots and moved preferentially towards them when given a choice of roots of subterranean clover, Trifolium subterraneum L. (Fabaceae), strawberry clover Trifolium fragiferum L. (Fabaceae), or perennial ryegrass Lolium perenneL. (Poaceae). Larvae recognized T. repens roots, whether released in groups of five or singly, when released 25 mm (meso-scale recognition) or 60 mm (macro-scale recognition) away from plant roots. There was no statistically significant difference in movement rates of larvae.

Laboratory experiments were undertaken to investigate attraction of first instar clover root weevil (Sitona lepidus) towards different clover nodules Two choice tests were used to assess larval preference between red and white clover nodules and nitrogen fixing and nonnitrogen fixing white clover nodules The assays were run over 24 h in individual Petri dishes No difference in attraction was found between red and white clover nodules but larvae showed a strong preference towards nitrogen fixing white clover nodules (P0001)

The fungus Beauveria bassiana is a virulent pathogen of the clover root weevil a major introduced pest of clover in New Zealand Trials to investigate establishment of fungal inoculum in pasture were conducted in the Waikato region of New Zealand Granular formulations of conidia based on biopolymer technology successfully allowed the established of fungus in pasture However it did not support survival of inoculum into the second year Conidia on rice and conidial emulsions resulted in more successful establishment These results have implications for development of a biopesticide for clover root weevil based on B bassiana

1. In contrast to above-ground insects, comparatively little is known about the behaviour of subterranean insects, due largely to the difficulty of studying them in situ. 2. The movement of newly hatched (neonate) clover root weevil (Sitona lepidus L. Coleoptera: Curculinidae) larvae was studied non-invasively using recently developed high resolution X-ray microtomography. 3. The movement and final position of S. lepidus larvae in the soil was reliably established using X-ray microtomography, when compared with larval positions that were determined by destructively sectioning the soil column. 4. Newly hatched S. lepidus larvae were seen to attack the root rhizobial nodules of their host plant, white clover (Trifolium repens L.). Sitona lepidus larvae travelled between 9 and 27 mm in 9 h at a mean speed of 1.8 mm h(-1). 5. Sitona lepidus larvae did not move through the soil in a linear manner, but changed trajectory in both the lateral and vertical planes.

Red clover (Trifolium pratense) has been observed to persist longer than white clover (T repens) during field screening of legumes for tolerance of clover root weevil (Sitona lepidus) It has been suggested that this is linked to adult feeding preferences A laboratory trial was undertaken to confirm the feeding preference for white clover over red and to determine whether adults became habituated to red clover over time Groups of clover root weevil adults were caged on either white clover cv Grasslands Huia or 3 lines of Grasslands Pawera red clover At weeks 2 4 6 and 8 subsamples of adults were removed from the cages and were subjected to a 24 h choice test It was found that although adults showed a strong preference for white clover those that had been continuously fed red clover were more likely to feed on red clover in the choice test than those that had previously been fed white clover

The parasitoid Microctonus aethiopoides Loan (Hymenoptera: Braconidae) was introduced to New Zealand in 1982 for control of Sitona weevil, Sitona discoideus Gyllenhal (Coleoptera: Circulionidae), a pest of lucerne. Unfortunately, New Zealand populations of M. aethiopoides do not parasitise clover root weevil, Sitona lepidus Gyllenhal, but European populations do. Several PCR-based methods have demonstrated the existence of genetic variation between and amongst European and New Zealand populations of M. aethiopoides. However, enzyme electrophoresis methods have the potential to provide less expensive population markers, and these could also prove more useful than PCRbased markers for studying gene flow between European and New Zealand M. aethiopoides populations. This paper reports a preliminary assessment of allozyme variation between European and New Zealand M. aethiopoides. Nine enzymes and general proteins were assayed, but the only variation was observed at esterase (EST) with two alleles present at each of two loci.

Clover root weevil, Sitona lepidus, remains a threat to New Zealand’s white-clover based pastoral production system. Unfortunately, the New Zealand-established strain of M. aethiopoides (originally from Morocco) active against Sitona discoideus, has been found to have no potential as a control agent against S. lepidus. It was therefore unexpected that a European strain of M. aethiopoides was found to be highly active against the weevil. This contribution examined the effect of Moroccan x European strain crosses on the value of M. aethiopoides as a control agent of S. discoideus and S. lepidus. It was found that such strain hybridisation led to significant reductions in the impact of this parasitoid species as a control agent against either S. lepidus or S. discoideus. The widespread implications of this finding for biological control are discussed.

Protease inhibitors (PIs) cause mortality in a range of insects and transgenic plants expressing PIs have been protected against pest attack The effects of feeding two purified PIs to larvae of Argentine stem weevil (Listronotus bonariensis) and clover root weevil (Sitona lepidus) were investigated in laboratory bioassays The serine PI aprotinin and the cysteine PI E64 were administered continuously in artificial diets to neonate or oneweekold larvae Aprotinin had no effect on clover root weevil larvae but significantly inhibited growth of Argentine stem weevil larvae E64 caused significant mortality in clover root weevil larvae and significant reductions in growth of Argentine stem weevil larvae

One of the most significant pests of white clover (Trifolium repens L.) is the clover root weevil (Sitona lepidus L. Coleoptera; Curculionidae). The adult of this species feeds on the leaves of the plant. However, root feeding by the larvae is more insidious. An experiment is described that was designed to test the hypothesis that root nodule herbivory by early instar larvae of S. lepidus reduces the assimilation of C by plants of white clover. White clover plants were grown (± larvae) in individual growth chambers and the net carbon (C) assimilation by each plant was estimated by monitoring CO2 flux in the chambers. White clover plants which had been infested with larvae had a significantly (P

Plots of two cultivars of white clover (Trifolium repens) Grasslands Prestige (smallleaved early flowering) and Grasslands Kopu (largeleaved later flowering) were established in coldframe beds Clover root weevil (Sitona lepidus) larval numbers were manipulated to obtain winter larval densities ranging from 0 to over 1000/m2 Increasing winter larval densities increased Prestige seed production (g/m2) when harvested on 28 December and 10 January and the trend was still evident on 24 January No such relationships were found in Kopu for the early and middle harvests but at the late harvest increasing winter larval densities had a negative impact on seed production The results indicate that the effect of clover root weevil larvae on second year clover seed crops may vary substantially between cultivars and may even be positive rather than negative

Several candidate biological control agents of Sitona lepidus have been identified since a search commenced in 1997. Interestingly, Microctonus aethiopoides from Europe is a much more effective parasitoid of S. lepidus than the M. aethiopoides ecotype already established in New Zealand. To assess further the suitability of the European M. aethiopoides for biological control of S. lepidus, 1599 infected S. lepidus were shipped to New Zealand quarantine during late 2000. These yielded 267 parasitoid pupae from which 204 adult parasitoids were reared. This material was obtained from a wide geographical range in Europe and has been used to establish cultures in New Zealand quarantine based on genetic material from France, England, Norway, Finland, Romania, Ireland, Scotland, Italy and Wales. This contribution presents an overview of the work associated with the importation of the parasitoids and the effort now being made to maintain genetic diversity. Planned research is also discussed.

The decimation of white clover in Waikato and Bay of Plenty pastures owing to the invasion clover root weevil (CRW) initiated an extensive screening of legume germplasm to identify plant material that could survive and remain productive under high CRW pressure. A total of 50 000 plants from 275 lines and six species, were planted on seven commercial farms and evaluated for tolerance/ resistance to CRW under conventional farming practices. Plant productivity and level of CRW damage were both highest on white clover. Significant CRW damage was also observed on red clover and caucasian clover, but not on birdsfoot trefoil, lotus major or lucerne. However, the productivity under stock grazing of the latter three species was not sufficient to consider them suitable alternatives to white clover. Within the white clover lines there was encouraging variability in the level of CRW damage, and in the ratio of damage to shoot production. While root damage is of greater importance than shoot damage, the latter reflects CRW activity and egg deposition and is a good indicator of likely larval development and root damage. Factors such as farm type (dairy farms versus dry stock farms) had some influence on shoot production, but very little influence on CRW damage. Climatic conditions appeared to have strong influences on clover shoot production and CRW damage, with dry conditions from late spring to early autumn having a particularly suppressive effect on CRW damage and CRW population growth. Top performing lines have been selected from this screening and are currently being evaluated in more detail under high CRW pressure. Keywords: clover, clover root weevil, plant screening, Sitona lepidus, tolerance, Trifolium repens

Clover root weevil, Sitona lepidus (syn. flavescens) (Coleoptera: Curculionidae), has recently arrived in New Zealand. It has emerged as a pest of white clover (Trifolium repens) in the central North Island and appears likely to spread throughout the country. This contribution describes efforts made in 1998 to source biological control agents of S. lepidus from Europe and North America. Six species of parasitoids (one nearctic, five palaearctic) have been recorded in the literature as attacking S. lepidus. Three species which warrant investigation as candidate biological control agents were identified in this study. These were the palaearctic species Microsoma exiguum (Diptera: Tachinidae), Microctonus aethiopoides (Hymen‐optera: Braconidae), and Pygostolus falcatus (Hymenoptera: Braconidae). Also, a preliminary description is provided of a disease which was observed to infect S. lepidus adults collected in California.

A slant board experiment investigated the effect of four white clover (Trifolium repens L) root types on tolerance of root feeding by larvae of clover root weevil (Sitona lepidus Gyllenhål) Root damage was determined by image analysis and larval weight gains were measured Differences in root type did not have a major impact on initial damage by clover root weevil However different root types were affected differently by the clover root weevil A clover genotype with long fine roots had more functional root length after larval feeding than genotypes with different root types

The efficacy of the entomopathogenic nematodes Heterorhabditis zealandica, H. bacteriophora, Steinernema carpocapsae and S. feltiae against clover root weevil (CRW) larvae and pupae was determined in petri dish and pot experiments. Field collected third instar (L3)‐pupal CRW immature stages were used, and experiments were conducted in a controlled environment room at 18 °C. Weevil survival and development was assessed 7 and 10 days after nematode inoculation for petri and pot experiments respectively. All nematodes significantly reduced CRW survival, and all except S. feltiae prevented adult development. Under the conditions used in this study, Heterorhabditis spp. were generally more effective than Steinernema spp. Results are discussed in terms of the potential use of entomopathogenic nematodes as biocontrol agents for this clover pest.

Microctonous aethiopoides Loan has been introduced into New Zealand to control the lucerne pest Sitona discoideus Gyllenhal (Coleoptera: Curculionidae). Sitona lepidus Gyllenhal (Coleoptera: Curculionidae) a pest of clover ( Trifolium spp.), has recently established in New Zealand. Laboratory experiments to test the potential of M. aethiopoides to parasitize S. lepidus has resulted in very low levels of parasitism. To investigate whether there were behavioural or physiological barriers to successful parasitism, two experiments were conducted using the insect pathogenic bacterium. Serratia marcescens Bizio as a marker for parasitoid ovipositor penetration. Firstly, M. aethiopoides 'treated' with S. marcescens were exposed to weevils and rapid weevil mortality was used to indicate ovipositor penetration. Up to 50% mortality of S. lepidus occurred, which was comparable with mortality observed in the permissive host Listronotus bonariensis . Dissection of S. lepidus exposed to parasitoids treated with distill...

A small plot trial was undertaken in the Waikato to determine the potential impact of Sitona lepidus adults on white clover seed production. Five densities of adult weevil were caged on 1 m2 plots from the time of peak adult emergence in November 1998 until harvest in late January 1999. In the laboratory, weevil adults were found to severely damage clover inflorescences when no leaves were present. Although feeding damage on leaves was highly visible in the field plots, with over 80% of the leaflets damaged at the highest density of 600 adults/m2, weevil adults were found to have no effect on flowering or seed production.

Three clover root weevil (Sitona lepidus) adults infected with the entomopathogenic fungus, Beauveria bassiana, were collected from pastures in the Waikato in 1997. Genotyping using rDNA comparisons showed the strain from weevils to be a cosmopolitan type. Application of fungal conidia topically and by contaminating leaf, soil and root surfaces showed that both B. bassiana isolated from S. lepidus in New Zealand and commercial preparations of this species were pathogenic to adult and larval clover root weevil, with up to 100% mortality within two to four weeks of inoculation. Isolates varied in their effectiveness against larvae and adults, with two isolates effective against adults found to be less pathogenic to larvae than a third isolate.

Measurements of feeding damage by adult clover root weevil (Sitona lepidus) on both seedlings and excised leaf material from thirteen pasture legume species were made in four laboratory experiments. Clover root weevil showed no feeding preferences in the choice and non-choice feeding experiments using seedlings. However, in both choice and non-choice experiments using excised leaf material from mature plants, food intake was higher on perennial Trifolium spp. compared to annual Trifolium spp. Feeding on leaf material from mature Medicago spp. and Lotus spp. was very limited in both choice and non-choice experiments.

In a year of spring–summer drought on a sandy volcanic ash soil, ryegrass/caucasian clover (RG/ CC) and RG/white clover (RG/WC) pastures had similar rapid increases in pasture growth rates during September. Growth rates peaked around 25 October (65–70 kg DM/ha/day) for RG/CC and around 14 October (50–60 kg DM/ha/day) for RG/ WC. As drought intensified the declines in pasture growth rates were similar, but delayed by up to 3 weeks for RG/CC. Both pastures reached minimum summer growth rates of

Laboratory experiments were carried out to determine whether either of the two braconid parasitoids Microctonus aethiopoides Loan or Microctonus hyperodae Loan, already established in New Zealand, would parasitise the recently introduced clover root weevil, Sitona lepidus Gyllenhal. No parasitism by either parasitoid was recorded in two no-choice experiments. In a third experiment, M. aethiopoides was exposed to S. lepidus and S. discoideus in either choice or no-choice cages. In this case, a very low level of parasitism of S. lepidus (1%) occurred in no-choice cages, but this was increased to about 6% in cages where both weevil species were present. It was concluded that neither parasitoid was likely to be a suitable biological control agent for

Clover root weevil (Sitona lepidus) is established in Auckland, Waikato and Bay of Plenty provinces. A pasture survey in 1997 estimated that the rate of its spread at about 35 km annually. Adults feed on the foliage and larvae on roots and nodules of white clover. Pasture populations appear to be 10 times higher in New Zealand than in the UK. New Zealand's temperate climate and high clover content in pastures may offer a very favourable environment for S. lepidus, with serious implications for New Zealand's pastoral farming. Keywords: clover root weevil, pasture pests, Sitona lepidus, Trifolium repens, white clover

Antennal olfactory receptor neurons (ORNs) for pheromone and plant volatile compounds were identified and characterized in male and female clover root weevil, Sitona lepidus (Gyllenhal), using the single sensillum recording technique with five pheromone-related compounds, and 40 host and non-host plant volatile compounds. Overall, seven different types of olfactory sensilla containing specialized ORNs were identified in each sex of S. lepidus. Among them, three different types of sensilla in the males and two types in the females housed ORNs specialized for pheromone-related compounds. The ORNs in males were specialized for 4-methyl-3,5-heptanedione or one or more of four stereoisomers of 5-hydroxy-4-methyl-3-heptanone. In contrast, female sensilla did not contain ORNs sensitive to 4-methyl-3,5-heptanedione while they contained ORNs sensitive to and specialized for the stereoisomers of (4S,5S)-5-hydroxy-4-methyl-3-heptanone. In addition to the pheromone-related ORNs, four types of olfactory sensilla contained ORNs responsive to plant volatile compounds in male S. lepidus, and five types in females. Most of the ORNs identified in S. lepidus showed a high degree of specificity to specific volatile compounds although some of the active compounds showed overlapping response spectra in the ORNs across different types of sensilla. The most active plant volatile compounds were the four green leaf volatile compounds, (E)-2-hexenol, (Z)-2-hexenol, (Z)-3-hexenol and (E)-2-hexenal, and isomers of two monoterpenols, (±)-linalool and (±)-α-terpineol, all eliciting strong responses from relatively large numbers of ORNs in male and female S. lepidus. Our study indicates that S. lepidus has a set of highly sensitive and selective ORNs for pheromone and plant volatile compounds. Further work is needed to elucidate the behavioral implications of these findings.