Influences of host size and host species on theinfectivity and development of Heterorhabditismegidis (strain NLH-E87.3)

The infectivity, time to first emergence of infective juveniles (IJs), total number of IJs per insect and IJs body length of the entomopathogenic nematode Heterorhabditis megidis (strain NLH-E87.3) after development in larvae of two insect hosts, Galleria mellonella (greater wax moth) and Otiorhynchus sulcatus (vine weevil) was studied. At a dose of 30 IJs, larvae of G. mellonella show to be significantly more susceptible than O. sulcatus larvae. At a dose of one IJ, vine weevil larvae were more susceptible. The number of invading infective juveniles (IJs) increased with host size while the host mortality at a dose of one IJ decreased with the increase of host size. Time to first emergence was longer at a dose of one IJ per larva and increased with the increase of host size in both insect species. Reproduction of IJs differed between host species, host sizes and doses of nematodes. Generally, the IJs body size increased with an increasing host size. The longest infective juveniles were produced at the lowest IJ doses. Results are discussed in relation to the influence of different host species and their different sizes on the performance of H. megidis (strain NLH-E87.3) as a biological control agent.     

Orientation of Heterorhabditis megidis to insect hosts and plant roots in a Y-tube sand olfactometer

The host-searching behaviour of Heterorhabditis megidis strain NLH-E 87.3 in the presence of insect hosts and plant roots, offered individually and in combination, was studied using a newly developed Y-tube olfactometer filled with sand. Within a period of 24 hours infective juveniles (IJs) were significantly attracted to living G. mellonella larvae and caused 100% larval mortality. Otiorhynchus sulcatus larvae, however, did not elicit host-oriented movement of IJs and no larval mortality was observed. Roots of strawberry plants induced a negative response in IJs. The combination of strawberry roots and O. sulcatus larvae, however, strongly attracted IJs leading to 37% host mortality. It was shown that this type of Y-tube choice arena is a useful tool in studying the searching behaviour of entomopathogenic nematodes in a semi-natural habitat.     

Host influences on the pathogenicity of Heterorhabditis megidis

The infectivity of infective juveniles(IJs) of Heterorhabditis megidis (strain NLH-E87.3) produced on small, medium and large larvae ofGalleria mellonella, and on medium and largelarvae of Otiorhynchus sulcatus was tested underlaboratory conditions against G. mellonella andO. sulcatus larvae. Infective juvenilesoriginating from small G. mellonella exposed toan initial dose of one IJ were more infectious thanthose from small cadavers exposed to a dose of 30 IJs.Independent of the initial inoculum size, IJs fromsmall cadavers of G. mellonella were moreinfectious than those from medium and large cadavers.At a dose of one IJ per larva, IJs originating frommedium size O. sulcatus cadavers were moreinfective against G. mellonella than againstO. sulcatus larvae. Large G. mellonellalarvae were less susceptible to all IJ batches thanmedium and small sized larvae.     

Virulence of entomopathogenic nematodes to larvae of the guava weevil, Conotrachelus psidii (Coleoptera: Curculionidae), in laboratory and greenhouse experiments

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil and causes severe reduction in fruit quality. This weevil is difficult to control with insecticides because adults emerge over a long period, and larvae develop to the fourth-instar inside the fruit and move to the soil for pupation. We assessed the virulence of entomopathogenic nematodes to fourth-instar larvae in soil by comparing their susceptibility to nine species or strains: Heterorhabditis bacteriophora HP88, H. baujardi LPP7, and LPP1, H. indica Hom1, Steinernema carpocapsae All and Mexican, S. feltiae SN, S. glaseri NC, and S. riobrave 355. In petri dish assays with sterile sand at a concentration of 100 infective juveniles (IJs) of a given nematode species/strain, larval mortality ranged from 33.5 to 84.5%, with the heterorhabditids being the most virulent. In sand column assays with H. baujardi LPP7, H. indica Hom1, or S. riobrave 355 at concentrations of 100, 200, and 500 IJs, mortality was greater than the control only for H. baujardi (62.7%) and H. indica (68.3%) at the highest concentration. For H. baujardi LPP7 in a petri dish assay, the time required to kill 50 and 90% of the larvae (LT₅₀ and LT₉₀) for 100 IJs was 6.3 and 9.9 days, whereas the lethal concentration required to kill 50 and 90% of the larvae (LC₅₀ and LC₉₀) over 7 days was 52 and 122.2 IJs. In a greenhouse study with guava trees in 20-L pots, 10 weevil larvae per pot, and concentrations of 500, 1000 or 2000 IJs, H. baujardi LPP7 caused 30 and 58% mortality at the two highest concentrations. These results show that H. baujardi is virulent to fourth-instar larvae and has potential as a biological control agent in IPM programs.     

Sustainable control of black vine weevil larvae,Otiorhynchus sulcatus (Coleoptera: Curculionidae) with Heterorhabditis bacteriophora in strawberry

Efficacy, persistence and recovery of the nematode Heterorhabditis bacteriophora was tested in the laboratory after application of the nematode to strawberry roots by dipping. To mitigate nematode sedimentation and improve attachment to strawberry roots, carboxy-methyl-cellulose was added to the nematode solution. Mortality of black vine weevil Otiorhynchus sulcatus varied between 90 and 96% in the pot trials.     

The effect of the CpTi gene in strawberry against attack by vine weevil (Otiorhynchus sulcatus F. Coleoptera: Curculionidae)

The degree of protection against insect feeding conferred upon transgenic strawberry lines expressing the Cowpea trypsin inhibitor was evaluated under glasshouse conditions. Insect bioassays were carried out using vine weevil (Otiorhynchus sulcatus) in two experiments and in both experiments there was a highly significant reduction in damage by weevil larvae on the transgenic lines.     

Behavioural responses of the vine weevil, Otiorhynchus sulcatus, to semiochemicals from conspecifics, Otiorhynchus salicicola, and host plants

The vine weevil Otiorhynchus sulcatus is a parthenogenetic reproducing species which forages for suitable host plants at night, but is found congregated in dark places during the day. Frass of this weevil species is suspected to contain attractive compounds that are host‐plant related. Using a still‐air olfactometer, we tested adult vine weevils at night for their behavioural response to odours from conspecifics, feeding on a mixture of spindle tree (Euonymus fortunei) and yew (Taxus baccata), and to a sexually reproducing related species (Otiorhynchus salicicola), feeding on a mixture of ivy (Hedera helix) and cherry laurel (Prunus laurocerasus). Their attraction to conspecifics and O. salicicola appeared to be related to frass production. Freshly collected frass from O. sulcatus and from O. salicicola males and females was attractive. Prunus laurocerasus and H. helix have not been observed to be hosts of the vine weevil in the field. However, our tests showed that the vine weevil was attracted to mechanically damaged leaves of both plant species, whereas undamaged leaves were not attractive. Only undamaged young unfolding leaves of H. helix were also attractive. The attraction to odours from mechanically damaged host and non‐host plants suggested the involvement of compounds that are commonly found in many plant species. The involvement of plant compounds and/or aggregation pheromones in attraction to frass of the vine weevil and frass of the related weevil species O. salicicola is discussed.     

Reduction of black vine weevil larval growth by vesicular-arbuscular mycorrhizal infection

Larvae ofOtiorhynchus sulcatus (black vine weevil) were reared at three densities (zero, two or eight), on plants ofTaraxacum officinale (dandelion) with and without infection by the mycorrhizal fungusGlomus mosseae. On plants without the fungus, survival of larvae to late final instar was 84%, but this was only 43% on infected plants. The differential survival of larvae was evident in their effects on plant biomass. Significant interactions were found between larval density and infection, indicating that mycorrhizal presence mitigated the effects of herbivory at the low larval density. Infection byG. mosseae thus conferred some degree of resistance in roots to this insect and the consequences of this for horticultural and natural situations are discussed.     

Preference for plants damaged by conspecific larvae in ovipositing cabbage root flies: influence of stimuli from leaf surface and roots

In laboratory dual-choice assays females of the cabbage root fly, Delia radicum, prefer for oviposition plants with roots damaged by conspecific larvae to undamaged controls. Cauliflower and kale plants were inoculated with root fly eggs (25 per plant) and the hatching larvae were allowed to feed on the roots for various periods of time (1–17 days). After 4 (cauliflower) or 5 (kale) days of larval feeding the oviposition preference was most pronounced and flies laid between 64% and 68% of their eggs near plants with damaged roots. Later, with increasing damage but fewer surviving, and thus actively feeding, larvae, the magnitude of the preference declined. The preference for plants already damaged by conspecific larvae may contribute to the previously observed aggregated distribution of D. radicum eggs in Brassica crop fields.Further experiments revealed that the sensory cues inducing this oviposition preference originate from the complex consisting of the damaged roots, the surrounding substrate (soil) and associated microbes, rather than from the aerial plant parts. In choice assays using the root-substrate complex of damaged and control plants (aerial parts removed), the observed preference for damaged roots was similar to that found for the entire plant but was more pronounced. The damaged roots alone, compared to control roots, received up to 72% (cauliflower) and 75% (kale) of the eggs. By contrast, surrogate leaves sprayed with methanolic leaf surface extracts from the most preferred plants which had been damaged were not discriminated from surrogate leaved sprayed with extracts of the respective control plants. Analysis of glucosinolate levels in methanolic leaf surface extracts revealed that root damage resulted in enhanced concentrations of indole-glucosinolates on the leaf surface in kale but not in cauliflower. Although indole-glucosinolates are oviposition stimulants for the cabbage root fly, the induced changes were apparently too small to influence oviposition behaviour.     

Susceptibility of strawberry cultivars to the vine weevil Otiorhynchus sulcatus (Coleoptera: Curculionidae)

Abstract 1 The feeding preference of vine weevil, Otiorhynchus sulcatus (Fabricius), adults for foliage from 21 commercial cultivars of strawberries is investigated using binary choice tests with leaf disks, using ‘Honeoye’ as a standard against which all other cultivars are compared. ‘Delmarvel’, ‘Idea’, ‘Lester’, ‘Primetime’ and ‘Seneca’ are not preferred. 2 Variation in leaf nitrogen content is correlated with feeding preference, but does not explain all the variation, because outliers exist for both preferred and nonpreferred cultivars. 3 Removal of leaf hairs with adhesive tape permits their role in deterrence to be evaluated. Eight of the 21 cultivars have deterrent leaf hairs. Paradoxically, some highly preferred cultivars (e.g. ‘Latestar’ and ‘Tristar’) have deterrent leaf hairs, and four of the five nonpreferred cultivars lack significantly deterrent leaf hairs. 4 The 21 tested cultivars do not differ in their suitability for vine weevil larval development. 5 Differences in cultivar susceptibility to vine weevil in the field may involve interactions between the palatability and nutritional value of foliage, which influence fecundity and egg placement, and the tolerance of strawberry plants to larval feeding, determined by root biomass. The nonpreference traits found in the foliage of commercial cultivars of strawberry are present by chance. Accordingly, further selective breeding to enhance strawberry varietal tolerance to vine weevils may be possible.     

Influence of Insect Larvae and Plant Roots on the Host-finding Behaviour of Heterorhabditis megidis

We studied the host-finding and dispersion behaviour of Heterorhabditis megidis (strain NLHE 87.3) in the presence of Galleria mellonella or Otiorhynchus sulcatus larvae and strawberry roots. In large Petri dishes (19 cm diameter) filled with moist sand (8% w/w), and incubated at 15°C over 24 h, infective juveniles (IJs) responded positively to the presence of G. mellonella , to roots of a single strawberry plant and to O. sulcatus larvae in direct contact with roots of a single strawberry plant. A neutral or negative response was observed when IJs were presented with only O. sulcatus larvae or a combination of several strawberry plants with O. sulcatus larvae, either in contact or not in contact with the roots. IJs responded strongly to the combination of plant roots and feeding larvae indicating that the tritrophic interaction formed by IJs - O. sulcatus larvae - strawberry plants may be an infochemical-mediated interaction.     

Biological control of black vine weevil Otiorhynchus sulcatus in Ireland using Heterorhabditis megidis

Heterorhabditis megidis (UK211) was applied against black vine weevil (BVW, Otiorhynchus sulcatus) in potted plants in a polyethylene (2002) or glasshouse (2003) and in field planted strawberries (2003). Both potted and field strawberries were artificially infested with BVW larvae. In a 2002 pot planting in the polyethylene house, a single drench application of 25,000 H. megidis infective juveniles per plant in 50 ml of water in mid September, reduced the number of BVW larvae to 1.8/20 plants. A second application in early October gave a reduction of 0.2/20 plants and in the third application, the following March no live weevils were recovered, compared to the control which had 8.2 larvae/20 plants. In a 2003 pot planting in a glasshouse, similar treatments gave a reduction of 5.2, 5.4 and 0.8 larvae/20 plants, respectively, compared to the control where 26.2 larvae/20 plants were recovered. In an artificially, BVW infested field trial, similar treatments gave a reduction to 2.2 larvae/20 plants in the single September treatment, and 2 larvae/20 plants in the single October treatment. The double (September and October) application reduced BVW larvae further to 1.6/20 plants and the triple (September, October and April) application to 0.4 larvae/20 plants, compared to the control where four larvae corresponded to every 20 plants. There was, therefore, little difference between the single and double autumn treatments indoors or in the field, and it mattered little whether the single application in the field was made in September or October under the conditions of 2003. Early spring application gave a significant reduction in BVW in each of the three experiments.     

Investigations on the parasitism ofOtiorrhynchus salicicola andO. Sulcatus [Col.: Curculionidae] byHeterorhabditis sp. [Nematoda]

Experiments were conducted to study the efficacy of the insect parasitic nematodeHeterorhabditis sp. (HW79) as a biological control agent ofOtiorrhynchus salicicola. This weevil species is reported as a pest of ornamental plants in Switzerland and Italy. Dipping plastic boxes containing heavily infested cuttings of laurel (Prunus laurocerasus) in a nematode suspension resulted in approximately 100% parasitisation of full-grown larvae, pupae and non-emerged young adults. The average dose resulting from dipping varied between 56,000 and 62,000 nematodes per liter soil. This experiment was run under natural outdoor conditions. In a further outdoor experiment, pottedLigustrum plants were inoculated with eggs ofO. salicicola and later 20,000 infective juvenile nematodes per liter soil were added to the soil surface. The resulting weevil mortality in the treated pots was 78%. In seven greenhouse tests using the same nematode dose in pots with horticultural soil to which weevil larvae had been added, weevil mortality varied between 76% and 100%, the arithmetic average being 90%. These results indicate that Heterorhabditid nematodes may provide an effective means of controllingO. salicicola. In an other experiment usingO. sulcatus larvae, the influence of application time on nematode efficacy was investigated. When nematodes were added a few days before weevil larvae had hatched from the eggs, no parasitic effect was obtained. Nematode applications done shortly after larval hatching however, resulted in complete weevil control. These results are of significance in timing nematode applications in practice.      

Efficacy of entomopathogenic nematodes against neonate larvae of <Emphasis Type="Italic">Capnodis tenebrionis</Emphasis> (L.) (Coleoptera: Buprestidae) in laboratory trials

The efficacy of five entomopathogenic nematode strains of the families Steinernematidae and Heterorhabditidae was tested against the neonate larvae of Capnodis tenebrionis. The nematode strains screened included two of Steinernema carpocapsae (Exhibit and M137), and one each of S. feltiae (S6), S. arenarium (S2), and Heterorhanditis bacteriophora (P4). Exposure of neonate larvae of Capnodis to 10 and 150 infective juveniles (IJs) per larva (equivalent to 3 and 48 IJs/cm² respectively) in test tubes with sterile sand, resulted in mortality between 60–91% and 96–100%, respectively. At a concentration of 150 IJs/larva, all of the nematode strains were highly virulent. Both S. carpocapsae strains (Exhibit and M137) caused infection and mortality to larvae more quickly than the other strains. However, at a lower concentration assay (10 IJs/larva), S. arenarium was the most virulent strain. The penetration rate as an indicator of entomopathogenic nematode infection was also evaluated. The highest value was recorded for S. arenarium (36%), followed by H. bacteriophora (30.6%), S. feltiae (23.1%), and S. carpocapsae (20.7%).     

Olfactory responses of the vine weevil, Otiorhynchus sulcatus, to tree odours

Abstract A Y-tube olfactometer and a still-air olfactometer were developed to determine the attractiveness of several host plants for the vine weevil ( Otiorhynchus sulcatus (F.); Coleoptera: Curculionidae). Odours of weevil-damaged yew ( Taxus baccata ) and spindle trees ( Euonymus fortunei ) are attractive to the vine weevil, but Rhododendron and strawberry ( Fragaria  ×  ananassa ) are not. Undamaged Euonymus is attractive to the weevils in springtime but not in late summer. When clean air or undamaged Euonymus is the alternative, weevils strongly prefer weevil-damaged Euonymus foliage, and this preference is retained throughout the year. Hence, plant damage plays a role in attraction of the vine weevil. In contrast to the permanent attractiveness of weevil-damaged Euonymus , mechanically damaged plants gradually lose the attractiveness that they have early in the growing season. This suggests that emission of volatiles, produced by the plants in response to weevil damage, is important for attraction of the weevils because the weevils may use these plant odours to find suitable food plants throughout the season. Apart from weevil-damage-related plant volatiles, green leaf volatiles must also play a significant role, as indicated by the fact that weevils prefer: early season, undamaged Euonymus over clean air; early season, mechanically damaged Euonymus over undamaged Euonymus ; and, throughout the season, had no preference when mechanically damaged Euonymus is tested against weevil-damaged Euonymus . Thus, monitoring traps may be developed by the use of green leaf volatiles and/or herbivore-induced volatiles, as attractants.     

Effects of a novel microsporidium on the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae)

A newly discovered microsporidium infecting the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae), provisionally placed in the genus Canningia, was studied to determine its impact on O. sulcatus. O. sulcatus populations from several locations were sampled and evaluated for microsporidiosis. A very low prevalence of the disease was observed in all locations surveyed (<3.0%). Laboratory studies were conducted by orally exposing both larvae and adults of O. sulcatus to varying concentrations of Canningia sp. spores. Larval bioassays at a variety of dosages (0, 10, etc.) were performed to evaluate pathogen infectivity, larval survival and growth. Adult bioassays (dosages: 0, 10, etc.) were performed to evaluate longevity, fecundity and mechanisms of vertical pathogen transmission. Larvae and adults were infected in all spore treatments. Larval growth was significantly reduced at dosages above 10 spores/larva. Adults infected at all dosages experienced high levels of mortality and fecundity was reduced to zero. Greenhouse trials were performed to determine if larvae feeding in soil acquired infections when spores were topically applied as a drench application (0, 10⁵, 10⁶, 10⁷ spores/pot). Established larvae feeding on plant roots in pots developed infections when exposed to drench treatments of 10⁶ and 10⁷ spores/pot after 14-21 days. Canningia sp. is an acute pathogen of O. sulcatus infective to both larvae and adults. Topically applied spores also infected larvae feeding on roots in soilless potting media, suggesting the possibility of using this pathogen in a microbial control program.     

Effect of potting media on the efficacy and dispersal of entomopathogenic nematodes for the control of black vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae)

The effect of five commercial potting media, peat, bark, coir, and peat blended with 10% and 20% compost green waste (CGW) on the virulence of six commercially available entomopathogenic nematodes (EPN), Heterorhabditis bacteriophora UWS1, Heterorhabditis megidis, Heterorhabditis downesi, Steinernema feltiae, Steinernema carpocapsae, and Steinernema kraussei was tested against third-instar black vine weevil (BVW), Otiorhynchus sulcatus. Media type was shown to significantly affect EPN virulence. Heterorhabditis species caused 100% larval mortality in all media whereas Steinernema species caused 100% larval mortality only in the peat blended with 20% CGW. A later experiment investigated the effect of potting media on the virulence of EPN species against BVW by comparing the vertical dispersal of EPN in the presence and absence of BVW larva. Media type significantly influenced EPN dispersal. Dispersal of H. bacteriophora was higher than H. megidis, H. downesi, or S. kraussei in all media, whereas, S. feltiae and S. carpocapsae dispersal was much reduced and restricted to peat blended with 20% CGW and coir, respectively. In the absence of larvae, most of the EPN species remained in the same segment they were applied in, suggesting that the larvae responded to host volatile cues. Greenhouse trials were conducted to evaluate the efficacy of most virulent strain, H. bacteriophora in conditions more representative of those in the field, using 2.5 × 10⁹ infective juveniles/ha. The efficacy of H. bacteriophora UWS1 against third-instar BVW was 100% in peat, and peat blended with 10% and 20% CGW but only 70% in bark and coir, 2 weeks after application. These studies suggest that potting media significantly affects the efficacy and dispersal of EPN for BVW control.     

Use of a nematode, Heterorhabditis heliothidis, to control black vine weevil, Otiorhynchus sulcatus, in potted plants

Application of aqueous suspensions of infective juvenile Heterorhabditis heliothidis, isolate T327, to the soil resulted in up to 100% parasitisation of larvae of the black vine weevil, Otiorhynchus sulcatus, in potted yew, raspberries and grapes in nurseries, and over 87% parasitisation on potted cyclamens and strawberries. Pupae and newly emerged adults on grapevines were also parasitised. Another isolate, T310, produced 92.5 to 98.5% parasitism of O. sulcatus larvae on potted cyclamens in glasshouse, but was less effective on strawberries. Neoaplectana bibionis was found to be less effective than H. heliothidis T327 strain. The use of these nematodes provides an economical and effective method for controlling O. sulcatus on potted plants in glasshouses and nurseries.     

Host plant preference and performance of the vine weevil Otiorhynchus sulcatus

Abstract 1 The relationship between reproductive performance and preference for potential host plants of the vine weevil is investigated, as shown in tests on contact (or feeding) preference, presented herein, and tests on olfactory preference, published elsewhere. 2 Assessment of reproductive performance shows that the host‐plant range of the adult vine weevil Otiorhynchus sulcatus in Europe is limited to one gymnosperm genus (Taxus sp.) and a broad range of angiosperm plants in two subclasses of the Dicotyledonae, namely Dilleniidae and Rosidae. The successful reproduction on very distantly related plant taxa suggests that the original weevil‐ and plant‐habitat has mediated the current host‐plant range of the vine weevil. 3 Contact‐preference tests with equally suitable hosts, such as Aronia, Fragaria, Euonymus and Taxus, and one less suitable host, Humulus, indicate a mismatch between contact preference and performance and, as far as olfactory preferences are known, these match neither the contact preferences nor the performance. This mismatch may arise because (i) host plant species offered do not occur in weevil habitat in Europe (e.g. Aronia and the cultivated Fragaria come from North America) and (ii) predation (or disease) risks differ among host plants, thereby altering effective reproductive performance. 4 With respect to performance on novel hosts (Thuja, Prunus) and bad hosts (Rhododendron), some between‐individual variation is found within a single population, suggesting that local populations harbour (possibly genetic) variation for adaptation to new hosts. How this variation is maintained in the face of strong selection pressures on local populations of flightless and thelytokous weevils, is an important question for understanding the broad host plant range in the vine weevil.     

Evaluation of entomopathogenic nematodes against the olive fruit fly, Bactrocera oleae (Diptera: Tephritidae)

Infectivity of six entomopathogenic nematode (EPNs) species against Bactrocera oleae was compared. Similar infection levels were observed when third-instar larvae were exposed to infective juveniles (IJs) on a sand-potting soil substrate. When IJs were sprayed over naturally infested fallen olives, many larvae died within treated olives as well as in the soil; Steinernema feltiae caused the highest overall mortality of 67.9%. In addition, three laboratory experiments were conducted to optimize a time period for S. feltiae field application. (1) Abundance of fly larvae inside fallen olives was estimated over the 2006–2007 season with the highest number of susceptible larvae (3 mm and larger) per 100 olives being observed during December, 2006. (2) S. feltiae efficacy against fly larvae dropped to the soil post-IJ-application was determined. B. oleae added to the substrate before and after nematode application were infected at similar levels. (3) Effect of three temperature regimes (min–max: 10–27, 6–18, and 3–12 °C) corresponding to October through December in Davis, California on S. feltiae survival and infectivity was determined. After 8 weeks, the IJs at the 3–12 °C treatment showed the highest survival rate. However, the cold temperature significantly limited S. feltiae infectivity. Our results demonstrate that B. oleae mature larvae are susceptible to EPN infection both in the soil and within infested olives. Being the most effective species, S. feltiae may have the potential to suppress overwintering populations of B. oleae. We suggest that November is the optimal time for S. feltiae field application in Northern California.