Behavioural response of Heterorhabditis megidis towards plant roots and insect larvae

The behavioural response of infective juveniles (IJs) of Heterorhabditis megidis (strain NLH-E87.3) to cues from roots of strawberry (Fragaria x ananassa Duch.), thuja (Thuja occidentalis L.) and to larvae of the black vine weevil, Otiorhynchus sulcatus, was studied. Choice assays were conducted in an Y-tube olfactometer filled with moist sand. Infective juveniles were activated by the presence of intact roots of both strawberry and thuja plants. Some nematodes aggregated in the compartments with roots but most moved away from the roots to the opposite side. Given a choice, IJs showed a preference for strawberry roots above O. sulcatus larvae. No difference in preference was observed between thuja roots and O. sulcatus larvae. The combination of strawberry roots with vine weevil larvae was preferred above roots alone. In the assays with thuja roots and larvae versus thuja roots alone, however, IJs were stimulated to move but showed preference for the opposite compartment away from the arms with roots and larvae. Nematodes responded differently to mechanically damaged roots as opposed to roots damaged by vine weevil larvae. In assays with damaged thuja roots, IJs were most attracted by the roots damaged by larvae, whereas in the strawberry assays IJs showed a clear preference for the mechanically damaged roots. When challenged with a choice between strawberry and thuja roots, IJs moved preferentially to strawberry than to thuja roots. A preference for the combination of strawberry roots plus larvae over the thuja roots plus larvae was also observed.     

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.     

Effects of Density, Age and Host Cues on the Dispersal of Heterorhabditis megidis

Agar plate assays were used to assess the effect of density, incubation time and age of nematodes and the presence of insect hosts on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis (strain NLH-E87.3). IJs dispersed faster and further at high densities than at low densities. Dispersal was also influenced by the age of the IJs. Individuals stored for a period of 1.5 and 4.5 weeks showed to be more active than those stored for 2.5 and 3.5 weeks. The presence of a host insect enhanced the dispersion of nematodes. The increasing in the incubation period showed that IJs responded positively to host cues from Galleria mellonella but poorly to cues from Otiorhynchus sulcatus larvae.     

Entomopathogenic nematodes to control black vine weevil (Coleoptera: Curculionidae) on strawberry.

We investigated the potential of heterorhabditid nematodes to control larvae of the black vine weevil, Otiorhynchus sulcatus (F.), in 2 field experiments in commercial strawberry plantings. In both experiments, nematodes were applied directly onto the straw mulch, or onto the soil after temporary removal of the mulch. Heterorhabditis marelatus Lui & Berry (Rhabditida: Heterorhabditidae) reduced numbers of weevil larvae and the percentage of plants infested in both experiments, irrespective of straw removal. In the 1st field experiment, a sponge-packed H. marelatus formulation produced lower numbers of O. sulcatus larvae per strawberry plant (mean O. sulcatus larvae per plant = 0.7) and proportion of infested plants (42%) compared with a vermiculite formulation (mean O. sulcatus larvae per plant = 1.8, proportion infested plants 67%) and an untreated control (mean O. sulcatus larvae per plant = 1.9, proportion infested plants 75%). In the first 2 wk after application, more H. marelatus were found in soil samples collected from plots treated with sponge-packed nematodes, than from plots treated with vermiculite-formulated nematodes. In the 2nd field experiment, sponge-packed formulations of H. bacteriophora Poinar (Rhabditida: Heterorhabditidae) and H. marelatus were tested. H. marelatus caused a reduction in both numbers of weevil larvae (mean O. sulcatus larvae per plant = 0.1) and proportion of infested plants (9%) but H. bacteriophora did not (mean O. sulcatus larvae per plant = 0.45, proportion infested plants 34%). More H. bacteriophora were recovered from soil samples than H. marelatus during the first 7 d of this experiment. However, laboratory studies revealed no difference in the persistence of these 2 nematodes in sand.     

Aggregation of entomopathogenic nematodes, heterorhabditis spp. and steinernema spp., among host insects at 9 and 20 degrees C and effects on efficacy

The negative binomial distribution (NBD) with density-dependent k was used to test and describe the distribution of nematodes in Otiorhynchus sulcatus, Galleria mellonella, and Spodoptera exigua at 9 and 20 degrees C. Aggregation was greater in O. sulcatus than in the other two hosts. In case of G. mellonella exposed to the heterorhabditid HF85, the distribution of nematodes in the insects was random. There was only a small effect of low temperature on aggregation; this effect was stronger for O. sulcatus than for the other two hosts. Aggregation of nematodes among insects in a population increases the infection chance of already infected insects and decreases the infection chance of uninfected insects. Consequently, more nematodes will have to penetrate O. sulcatus than G. mellonella or S. exigua to cause the same proportion of infection in a population. The relevance of aggregation of nematodes for control O. sulcatus is discussed. Copyright 1999 Academic Press.     

Plants protect their roots by alerting the enemies of grubs

Plant roots in the soil are under attack from many soil organisms. Although many ecologists are aware of the presence and importance of natural enemies in the soil that protect the plants from herbivores, the existence and nature of tritrophic interactions are poorly understood. So far, attention has focused on how plants protect their above-ground parts against herbivorous arthropods, either directly or indirectly (i.e. by getting help from the herbivore's enemies). This article is the first in showing that indirect plant defences also operate underground. We show that the roots of a coniferous plant ( Thuja occidentalis ) release chemicals upon attack by weevil larvae ( Otiorhynchus sulcatus ) and that these chemicals thereby attract parasitic nematodes ( Heterorhabditis megidis ).     

Rapid age-related changes in infection behavior of entomopathogenic nematodes

Nonfeeding infective juvenile (IJ) entomopathogenic nematodes (EPNs) are used as biological agents to control soil-dwelling insects, but poor storage stability remains an obstacle to their widespread acceptance by distributors and growers as well as a frustration to researchers. Age is one factor contributing to variability in EPN efficacy. We hypothesized that age effects on the infectiousness of IJs would be evident within the length of time necessary for IJs to infect a host. The penetration behavior of "young" (<1-wk-old) and "old" (2- to 4-wk-old) Heterorhabditis bacteriophora (GPS 11 strain), Steinernema carpocapsae (All strain), and Steinernema feltiae (UK strain) IJs was evaluated during 5 "exposure periods" to the larvae of the wax moth, Galleria mellonella. Individual larvae were exposed to nematode-infested soil for exposure periods of 4, 8, 16, 32, and 64 hr. Cadavers were dissected after 72 hr, and the IJs that penetrated the larvae were counted. Larval mortality did not differ significantly between 72- and 144-hr "observation periods," or points at which larval mortality was noted, for any age class or species. However, age and species effects were noted in G. mellonella mortality and nematode penetration during shorter time periods. Initial mortality caused by S. carpocapsae and H. bacteriophora IJs declined with nematode age but increased with S. feltiae IJ age. Young S. carpocapsae IJs penetrated G. mellonella larvae at higher rates than old members of the species (27-45% vs. 1-4%). Conversely, old S. feltiae IJs had higher penetration rates than young IJs (approximately 8 to 57% vs. 4 to approximately 31%), whereas H. bacteriophora IJs had very low penetration rates regardless of age (3-5.6%). Our results show that the effect of age on IJ infectiousness can be detected in IJs aged only 2 wk by a 4-hr exposure period to G. mellonella. These results have important implications for storage and application of EPNs and suggest the possibility of shortening the time required to detect nematodes in the soil.     

Biological control of the black vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae) with entomopathogenic nematodes (Nematoda: Rhabditida)

Trials conducted under glasshouse conditions showed that control of Otiorhynchus sulcatus larvae in strawberry plants can be effective using Steinernema carpocapsae and Heterorhabditis megidis, given that temperature and moisture extremes are avoided. In field experiments, the double line T-Tape® drip irrigation system performed better than the single line T-Tape® system, effectively distributing the nematodes along and across strawberry raised beds, and placing them close to the root zone where O. sulcatus larvae feed. As soil temperatures are satisfactory for nematode infectivity from late spring to early autumn, nematode applications were aimed at late instar larvae during spring, and early instar larvae during summer. Late summer field treatment with S. carpocapsae induced 49.5% reduction of the early instar larvae, and field application of the same nematode species in late spring resulted in 65% control of late instar larvae. In the same trial, spring application of H. megidis caused 26% mortality of late instar larvae of O. sulcatus.     

拟双角斯氏线虫D43品系鞘蛋白对大蜡螟幼虫的免疫抑制作用

侵染期的拟双角斯氏线虫Steinernema ceratophorum D43品系体外都包裹着一个透明的体鞘。为探明体鞘对线虫侵染力的影响, 了解鞘蛋白（sheath proteins, SPs）对大蜡螟Galleria mellonella 幼虫的免疫抑制作用, 本研究通过化学方法使拟双角斯氏线虫D43脱鞘, 以对寄主的致死率和侵入点数量为指标, 与包鞘线虫比较对大蜡螟幼虫的侵染力; 采用乙醇提取的方法获得线虫鞘蛋白, 利用双向电泳和质谱技术对鞘蛋白进行鉴定分析; 从血细胞数量和酚氧化酶活力两个方面评价鞘蛋白对大蜡螟幼虫免疫反应的抑制作用。结果表明： 0.5%次氯酸钠处理20 min可以保证95%以上的线虫存活和脱鞘。与包鞘线虫相比, 脱鞘线虫对大蜡螟幼虫的致死率显著降低, 致死时间延后, 节间膜侵入点数量显著减少。以35%乙醇提取的鞘蛋白提取物可鉴定出6种鞘蛋白, 其中一个被鉴定为丝氨酸蛋白酶。此外, 血腔注射鞘蛋白提取物可导致试虫血细胞数量明显降低, 酚氧化酶活力受到显著抑制。由此说明, 体鞘对拟双角斯氏线虫D43的侵染力具有显著影响, 鞘蛋白在抑制寄主昆虫免疫反应中发挥重要作用。     

Factors influencing the infectivity of a Canadian isolate of Steinernema kraussei (Nematoda: Steinernematidae) at low temperature

The effectiveness of Canadian isolate 76 of Steinernema kraussei, at 10 degrees C, in penetrating Galleria mellonella larvae (percentage parasitism and number of IJs developed to adult nematodes) was measured at different host densities (differing number of larvae and size of experimental arena) and for different durations of exposure. The greater the size of the inoculum of infective juvenile nematodes per unit area and the longer the duration of exposure, the greater the number of larvae that were killed and the larger the number of mature nematodes in the larval host. The infection rate (alpha) and the adjusted infection rate (beta) were determined using the modified Anderson model. This model successfully described the behavior of the S. kraussei-G. mellonella interaction.     

Infectivity, distribution, and persistence of the entomopathogenic nematode Steinernema carpocapsae all strain (Rhabditida: Steinernematidae) applied by sprinklers or boom sprayer to dry-pick cranberries.

We evaluated infectivity, distribution, and persistence of commercially produced Steinernema carpocapsae (Weiser) All strain applied through solid set sprinkler irrigation or boom sprayer to 2 dry-pick cranberry farms on peat soil in British Columbia in 1993. Most infectivity assays used Galleria mellonella (L.) larvae. When possible, larvae of the target pest, Otiorynchus sulcatas (F.) were used as assay organisms. Nematodes in almost all samples of nematode suspensions diluted from shipping containers, from spray tanks, or collected in cups after passage through application equipment were infective to G. mellonella larvae. When O. sulcatus larvae were used as assay organisms, 93% (n = 14) of assays from the spray tank and 67% (n = 12) of assays after application showed infectivity. In the spring, sprinklers delivered nematodes to only 15 of 20 sample points on the 0.2-ha plot; delivery by the boom sprayer was better but 2 of 20 points on the 0.2-ha plot received approximately twice as many nematodes as the other points. In the fall, nematode delivery by both systems was more even. However, the average number of nematodes per milliliter of sprayed water collected from the 20 samples on each farm after each application did not correspond to the rates of nematodes applied. Persistence of nematodes in the soil was encouraging, but percentage of infectivity was lower than expected. After application in the spring, assays using G. mellonella larvae showed the presence of infective nematodes in soil samples (0-5 and 5-10 cm deep) on each sampling day (0, 3, 7, and 25) after application by boom sprayer, and on days 0, 3, and 7 after application through sprinklers. In the fall, G. mellonella assays showed infective nematodes in soil samples on each sampling day (0, 3, 7, and 25) after application by boom sprayer, and on days 0, 3, 7, 35, 60, 135, and 250 after application through sprinklers. In the spring, when assays lasted 4 d, percentage of infectivity rose to a maximum of 45% on the 3rd d after application by boom sprayer and declined thereafter. In the fall, when assays lasted 10 d, percentage of infectivity rose to a maximum of 58% on the 7th d after application through sprinklers and remained between 20 and 58% until day 135, declining thereafter; infectivity after boom application remained between 37 and 45% on days 3 and 7, and began to decline on day 25. Nematode infectivity was not compromised in peat soil, muck, or silty clay loam, but infectivity in loam (that may have contained nematicide residues) was very low. We suggest that the inconsistent control of O. sulcatus by S. carpocapsae on British Columbia cranberry farms may be partially explained by problems associated with application and factors related to nematode entry into the soil.     

Gnotobiological study of infective juveniles and symbionts of Steinernema scapterisci: A model to clarify the concept of the natural occurrence of monoxenic associations in entomopathogenic nematodes.

Gnotobiology of Steinernema scapterisci and bacteriological study of its symbiont confirmed that this nematode harbors a symbiotic species of Xenorhabdus, as do other Steinermena species. Based on phenotypic and 16S rDNA data, this Xenorhabdus strain UY61 could be distinguished from other Xenorhabdus species. Bacteria reported previously as being associated with this nematode and belonging to several other genera were probably contaminating bacteria located in the intercuticular space of the infective juveniles (IJs). These bacteria were detrimental to nematode reproduction in Galleria mellonella. Axenic S. scapterisci and its symbiont Xenorhabdus strain UY61 alone were not pathogenic to G. mellonella. The combination of both partners reestablished the pathogenicity of the complex toward G. mellonella. This combination also gave the best yields of IJs when produced in this insect and in vitro production on artificial diet.     

Improved Control of Otiorhynchus sulcatus at 9°C by Cold-stored Heterorhabditis megidis UK211

The effect of storage temperature (9 and 20°C) on North West European Heterorhabditis megidis isolate UK211 for control of Otiorhynchus sulcatus larvae at 9°C is assessed. O. sulcatus mortality increased from -5.3% (corrected mortality) using freshly produced nematodes, to 27.1% using nematodes that had been cold-stored for 12 weeks. The number of nematodes invading the insect larvae increased almost 27-fold. Nematode storage at 9°C for 11 to 12 weeks weeks resulted in significantly higher O. sulcatus mortality (41%) than storage at 20°C for 2 to 3 weeks (12%). Thus, cold storage does enhance nematode infectivity for O. sulcatus larvae.     

Host plant recognition by the root feeding clover weevil, Sitona lepidus (Coleoptera: Curculionidae)

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.     

The development of Steinernema feltiae (Nematoda Steinernematidae) in the sciarid fly Bradysia paupera (Diptera: Sciaridae)

There was only one generation of Steinernema feltiae in Bradysia paupera. Infection occurred after 3 h, adults developed 27 h after invasion and new infective juveniles (IJ) were produced after 48 h. Stunted females were produced in B. paupera larvae and in other small hosts and these stunted females produced small IJs. The small IJs were capable of infecting hosts and normal sized Us were produced in succeeding generations in Galleria mellonella.     

THE BIOLOGY AND CONTROL OF OTIORRHYNCHUS CLAVIPES BONSD. (RHYN. COLEOP.), A PEST OF STRAWBERRIES

Otiorrkynchus clavipes Bonsd. is a serious pest of strawberries around Cheddar, Somerset. The weevils nibble the foliage, the larvae eat the roots of the plants, and burrow into the rootstock.
The beetles appear in two waves, pupating in the autumn and emerging en masse in the spring, or pupating in the late spring and summer and emerging in succession between mid-June and the end of August. Both sexes are found and can be distinguished by the posterior thoracic sterna, which are concave in males and convex in females. Mating is alternated with egg-laying and the beetles lay between 100 and 300 eggs per female. On emergence the beetles eat voraciously, and roughly one egg is laid for every 10 sq.mm. of leaf consumed. Both parthenogenetic and fertile eggs are laid and hatch in between 17 and 24 days.
The larvae burrow rapidly when hatched and can only penetrate soil spaces which will admit their head capsules. The distribution of head capsule widths in mixed populations of larvae of all ages shows there are five instars. While feeding, the larvae are clustered within the root range of the plants and find their way to their food from plant to plant, guided by the exudates from the strawberry roots. As the larvae grow older they descend in the soil and turn to white soft pupae in smooth earthen cells 6–8 in. below the plants.
Adult damage to foliage is unimportant but larval damage to roots is serious and has been largely responsible for restricting the effective life of the strawberry beds to at most 2 years. Although weevil attack can be avoided by growing the crop in a 1-year rotation, control is difficult owing to the intensive cropping, which creates the most suitable conditions for the spread of the infestations. The adults are killed with 10% DDT dust; and the larvae by DDT and BHC preparations poured on to the soil, at a pint per plant, or by DD injection. The limitations and possibilities of various methods of control are discussed.     

Comparative study of the entomopathogenic nematode, Steinernema carpocapsae, reared on mutant and wild-type Xenorhabdus nematophila

The symbiotic interaction between Steinernema carpocapsae and Xenorhabdus nematophila was investigated by comparing the reproduction, morphology, longevity, behavior, and efficacy of the infective juvenile (IJ) from nematodes reared on mutant or wild-type bacterium. Nematodes reared on the mutant X. nematophila HGB151, in which an insertion of the bacterial gene, rpoS, eliminates the retention of the bacterium in the intestinal vesicle of the nematode, produced IJs without their symbiotic bacterium. Nematodes reared on the wild-type bacterium (HGB007) produced IJs with their symbiotic bacterium. One or the other bacterial strain injected into Galleria mellonella larvae followed by exposing the larvae to IJs that were initially symbiotic bacterium free produced progeny IJs with or without their Xenorhabdus-symbiotic bacterium. The two bacterial strains were not significantly different in their effect on IJ production, sex ratio, or IJ morphology. IJ longevity in storage was not influenced by the presence or absence of the bacterial symbiont at 5 and 15 °C, but IJs without their bacterium had greater longevity than IJs with their bacterium at 25 and 30 °C, suggesting that there was a negative cost to the nematode for maintaining the bacterial symbiont at these temperatures. IJs with or without their symbiotic bacterium were equally infectious to Spodoptera exigua larvae in laboratory and greenhouse and across a range of soil moistures, but the absence of the bacterial symbiont inhibited nematodes from producing IJ progeny within the host cadavers. In some situations, such as where no establishment of an alien entomopathogenic nematode is desired in the environment, the use of S. carpocapsae IJs without their symbiotic bacterium may be used to control some soil insect pests.     

Effects of Pratylenchus penetrans and Rhizoctonia fragariae on Vigor and Yield of Strawberry

Microplot and small field-plot experiments were conducted to determine the effects of Pratylenchus penetrans on strawberry yield over several seasons and to evaluate the effects of nematode control on strawberry vigor and yield. Pratylenchus penetrans alone or in combination with the black root rot pathogen, Rhizoctonia fragariae, reduced strawberry yield in microplots over time. There were no differences in effects on yield among R. fragariae anastomosis groups A, G, or I. The interaction of the two pathogens appeared to be additive rather than synergistic. In field plots infested with P. penetrans alone, plant vigor and yield were increased by the application of carbofuran and fenamiphos nematicides. Nematode control was transitory, as P. penetrans populations were initially suppressed but were not different in samples taken 10 months after treatment. These data highlight the error in associating causality between plant damage and nematode populations based on a correlation of root disease with nematode diagnostic assays from severely diseased plants. These findings may help to explain how nematode numbers can sometimes be higher in healthy plants than in severely diseased plants that lack sufficient roots to maintain nematode populations. Because nematode populations from up to a year before harvest are better correlated with berry yield, preplant nematode diagnostic assays taken a year in advance of harvest may be superior in predicting damage to perennial strawberry yield.     

Interactions of two idiobiont parasitoids (Hymenoptera: Ichneumonidae) of codling moth (Lepidoptera: Tortricidae) with the entomopathogenic nematode Steinernema carpocapsae (Rhabditida: Steinernematidae)

Simultaneous use of parasitoids and entomopathogenic nematodes for codling moth (CM) control could produce an antagonistic interaction between the two groups resulting in death of the parasitoid larvae. Two ectoparasitic ichneumonid species, Mastrus ridibundus and Liotryphon caudatus, imported for classical biological control of cocooned CM larvae were studied regarding their interactions with Steinernema carpocapsae. Exposure of M. ridibundus and L. caudatus developing larvae to infective juveniles (IJs) of S. carpocapsae (10 IJs/cm2; approximately LC(80-90) for CM larvae) within CM cocoons resulted in 70.7 and 85.2% mortality, respectively. However, diapausing full grown parasitoid larvae were almost completely protected from nematode penetration within their own tightly woven cocoons. M. ridibundus and L. caudatus females were able to detect and avoid ovipositing on nematode-infected cocooned CM moth larvae as early as 12h after treatment of the host with IJs. When given the choice between cardboard substrates containing untreated cocooned CM larvae and those treated with an approximate LC95 of S. carpocapsae IJs (25 IJs/cm2) 12, 24, or 48h earlier, ovipositing parasitoids demonstrated a significant preference for untreated larvae. The ability of these parasitoids to avoid nematode-treated larvae and to seek out and kill cocooned CM larvae that survive nematode treatments enhances the complementarity of entomopathogenic nematodes and M. ridibundus and L. caudatus.     

Field attraction of the vine weevil Otiorhynchus sulcatus to kairomones

Root weevils in the genus Otiorhynchus are cited as one of the most important pests in the major nursery and small fruit production areas throughout the United States, western Canada, and northern Europe. A major problem in combating weevil attack is monitoring and timing of control measures. Because of the night-activity of the adult weevils growers do not observe the emerging weevils in a timely manner and oviposition often starts before effective control measures are taken. Several vine weevil electroantennogram-active plant volatiles were identified from a preferred host plant, Euonymus fortunei. Main compounds evoking antennal responses on the weevils' antennae were (Z)-2-pentenol, (E)-2-hexenol, (Z)-3-hexenol, methyl benzoate, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene, methyl eugenol, and (E, E)-alpha-farnesene. Several of these compounds were tested alone and in mixtures on attractiveness for the vine weevil Otiorhynchus sulcatus (F.) in field-grown strawberry in Oregon. O. sulcatus were attracted to (Z)-2-pentenol (approximately 3 x more than control) and a 1:1 ratio mixture of (Z)-2-pentenol and methyl eugenol (4.5 x more than control). This is the first report of field-active attractants for O. sulcatus which holds promise for the development of new monitoring strategies for growers in the near future.