Compatibility between Forficula auricularia and entomopathogenic nematodes to be used in pome fruit pest management

Use of predators, parasitoids and entomopathogens as biocontrol agents in pome fruit production can lead to more efficient and sustainable pest management programmes. The European earwig (Forficula auricularia Linnaeus [Dermaptera: Forficulidae]) is a major predator of key pests in pome fruit orchards, and entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae are obligate parasites of a large number of insect species. Therefore, the interaction between earwigs and EPNs can play an important role in pest management programmes. Susceptibility of the European earwig to Steinernema carpocapsae, Steinernema feltiae (Steinernematidae) and Heterorhabditis bacteriophora (Heterorhabditidae) was evaluated. S. carpocapsae was the only tested EPN capable of killing the European earwig. However, the European earwig can detect the presence of S. carpocapsae and therefore avoid nematode‐treated shelters. An earwig deterrent activity in EPN‐killed codling moth larvae that reduces the foraging of European earwig on insect cadavers containing nematodes and allows nematodes to complete their life cycle was also assessed with the three species of nematodes. These findings suggest a positive compatibility between the European earwig and EPNs.     

Multifunctional weaponry: The chemical defenses of earwigs

Earwigs protect themselves against predators using pincer-like cerci and/or malodorous exudates secreted from abdominal glands. Little is known about the chemistry of these secretions and their potential functions. However, because earwigs live in aggregations and overwinter in soil, they are exposed to high microbial loads throughout their lifecycle, and we therefore hypothesized that the secretions are used not only to deter predators but also to combat pathogens and parasites in their environment. We analyzed the defensive secretions of the European earwig Forficula auricularia, the short-winged earwig Apterygida media and the woodland earwig Chelidurella guentheri by gas chromatography–mass spectrometry. The secretions of all three species contained 2-methyl-1,4-benzoquinone and 2-ethyl-1,4-benzoquinone, whereas A. media also produced 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone. The latter has not been identified in the exudates of insects before. The composition and/or quantity of these components were species-specific and partially sex-specific. All secretions showed antimicrobial activity against Gram-positive and Gram-negative bacteria as well as two entomopathogenic fungi. Furthermore, the secretion of F. auricularia displayed nematicidal activity against Caenorhabditis elegans. Our data support the hypothesis that earwig secretions are multifunctional, serving both to deter predators and sanitize the microenvironment.     

Abundance,interannual variation and potential pest predator role of two co‐occurring earwig species in citrus canopies

Earwigs are usually considered pest predators in orchards. Because of its worldwide distribution, most research on earwigs focuses on the European earwig Forficula auricularia Linnaeus (Insecta: Dermaptera: Forficulidae). However, very little is known of this species in Mediterranean citrus orchards. Earwigs and aphids were collected monthly during 5 years (2006–2010) from citrus canopies. Two species of earwigs were found: F. auricularia and Forficula pubescens Gené (=Guanchia pubescens), with the latter seldom cited in the literature. The goals of this study were (i) to document the abundance of these two earwig species in Mediterranean citrus canopies; (ii) to determine whether they are positively or negatively associated with each other, or randomly distributed; (iii) to measure the interannual variation of the abundance of both species during a 5‐year period and (iv) to evaluate the potential role of earwigs as pest predators in citrus canopies. As compared to colder regions, F. auricularia active period in citrus canopies in our study site lasted longer. Both species co‐occurred randomly in canopies. In 2006, both species showed approximately the same abundance, but in 2010, F. pubescens abundance in canopies was 28 times greater than that of F. auricularia. The potential role of earwigs as pest predators is higher in the Mediterranean than in other colder regions, because of the longer active period. F. auricularia is a sedentary generalist predator, already present in citrus canopies at the onset of most pest outbreaks, while F. pubescens arrived later to the canopies, but most likely was abundant enough to contribute in the control of citrus pests.     

Effect of Exsheathment on Motility and Pathogenicity of Two Entomopathogenic Nematode Species

The effect of sheath loss on motility and pathogenicity of the entomopathogenic nematodes, Heterorhabditis bacteriophora and Steinernema carpocapsae, was examined using both naturally and chemically exsheathed (desheathed) infective juveniles. Exsheathed S. carpocapsae showed increased motility on agar compared to sheathed nematodes. The presence of a host increased motility threefold in all S. carpocapsae treatments. These results suggest that activation of S. carpocapsae host finding may result from sheath loss in addition to host stimuli. Desheathed H. bacteriophora were significantly less motile than the sheathed or exsheathed groups. The decreased motility may be due to adverse effects of the chemical treatment for desheathment. Sheath loss did not affect the pathogenicity of either species.     

Ontogeny in the European earwig (Forficula auricularia) and grain crops interact to exacerbate feeding damage risk

The preference of herbivores for different host plants can be modulated by plant ontogeny. In agricultural pest management, this has implications for sowing dates and pest monitoring. In the last 20 years, the European earwig (Forficula auricularia), a cosmopolitan pest, has been increasingly implicated in damage to grain crops in Australia. Among these, rapeseed, Brassica napus, appears especially at risk, but little information on F. auricularia as a grain pest is available. We tested the susceptibility of seven grain crops commonly grown in Australia to infestation by F. auricularia using closed microcosm experiments, exposing plant seedlings at two early growth stages to four different life stages of F. auricularia. Lucerne and rapeseed were shown to be the most vulnerable crops, and younger seedlings experienced significantly more damage than older seedlings across all crop types. Fourth instar F. auricularia were found to cause greater feeding damage than younger or older earwigs, while adults collected in winter generally caused more damage than those collected in summer. Surprisingly, even second instar F. auricularia caused greater damage than summer adults. This variation could reflect the ontogenetically dynamic nutritional needs of earwigs. Recent studies of F. auricularia's life cycle in southern Australia indicate that these damaging life stages have some overlap with sowing dates of the crops tested here, exposing their vulnerable seedling stage to infestation. The phenology of F. auricularia in southern Australia therefore partly drives its ability to act as a pest. Future monitoring will likely need to track the distribution of F. auricularia life stages in order to effectively mitigate risks to vulnerable crops.     

Entomopathogenic Nematodes for Control of Codling Moth,Cydia pomonella(Lepidoptera: Tortricidae): Effect of Nematode Species, Concentration, Temperature, and Humidity

The susceptibility of codling moth diapausing larvae to three entomopathogenic nematode species was assessed in the laboratory using a bioassay system that employed cocooned larvae within cardboard strips. The LC₅₀values forSteinernema carpocapsae, S. riobrave,andHeterorhabditis bacteriophorawere 4.7, 4.8, and 6.0 infective juveniles/cm², respectively. When a discriminating concentration of 10 infective juveniles/cm²of each of the three nematode species was evaluated at 15, 20, 25, and 30°C,S. carpocapsaewas the most effective nematode with mortalities ranging from 66 to 90%. Mortalities produced byS. riobraveandH. bacteriophoraat the four temperatures were 2–94 and 25–69%, respectively. Studies were also conducted to test infectivity at 10, 35, and 40°C. No mortality was produced by any of the nematode species at 10°C.S. riobravewas the most infective nematode at 35°C producing 68% mortality which was more than twice that observed forS. carpocapsaeorH. bacteriophora.Codling moth larvae treated with 10 infective juveniles/cm²ofS. carpocapsaeand kept in 95+% RH at 25°C for 0–24 h followed by incubation at 25–35% RH indicated that more than 3 h in high humidity was needed to attain 50% mortality. Trials ofS. carpocapsae, S. riobrave,andH. bacteriophoraat 50 infective juveniles/cm²against cocooned larvae on pear and apple logs resulted in reductions of codling moth adult emergence of 83, 31, and 43%, respectively, relative to control emergence. Trials of the three entomopathogenic nematodes at 50 infective juveniles/cm²against cocooned larvae in leaf litter resulted in 99 (S. carpocapsae), 80 (S. riobrave), and 83% (H. bacteriophora) mortality, respectively. Our results indicate good potential of entomopathogenic nematodes, especiallyS. carpocapsae,for codling moth control under a variety of environmental conditions.     

Attraction of entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to the red palm weevil (Rhynchophorus ferrugineus)

The red palm weevil (RPW), Rhynchophorus ferrugineus, is a serious pest of date palms. Its larvae bore deep into the trunk disrupt the vascular tissues and kill the infested trees. Behavioral features of entomopathogenic nematodes (EPNs), reflected by attraction and distribution patterns, are fundamental aspect in determining their parasitic ability and potential management of RPW. We studied the attraction behavior of the EPNs Steinernema carpocapsae and Heterorhabditis bacteriophora to the RPW under simulated natural conditions in tubes to evaluate their infective potential. In all experiments, a certain proportion of infective juveniles (IJs) (16–20%) stayed near the inoculated site and a major proportion (38–48%) was attracted to the host end. Both H. bacteriophora and S. carpocapsae were efficient crawlers, climbing up and descending when locating their insect host. They were efficiently attracted to the various larval sizes and stages of the RPW life cycle. Host localization by ascending movement was more prominent in S. carpocapsae than in H. bacteriophora. In general, H. bacteriophora is classified as a cruiser forager and S. carpocapsae as an ambusher. However, in this study, we discovered a higher percentage of cruiser foragers among S. carpocapsae IJs. They dispersed much faster and their cruising behavior was prominent characteristic in controlling the cryptic RPW concealed in organic habitats.     

Effect of insect host age and diet on the fitness of the entomopathogenic nematode-bacteria mutualism

Insect host age and diet were evaluated as potential factors that could affect the fitness of the entomopathogenic nematode-bacterium mutualistic partnership. Two nematode species were considered: Steinernema carpocapsae and Heterorhabditis sonorensis, together with their symbionts Xenorhabdus nematophila and Photorhabdus luminescens, respectively. The tobacco hornworm, Manduca sexta, was used as the insect host. Insect developmental stage was a factor that impacted nematode virulence. Non-wandering 5th instar M. sexta were found to be more susceptible to nematode infection compared to wandering 5th instars. This was more noticeable for S. carpocapsae than for H. sonorensis. The nutritional status of the host also had an effect on the fitness of the two nematode species tested. In general, insects fed with the reduced diet content were less susceptible to nematode parasitism. The least observed mortality (0.5 %) was in those M. sexta larvae exposed to the low H. sonorensis dose. Host diet also had an effect on the production of IJ progeny in the insect cadavers. For both nematode species tested, the highest yield of emerging IJs was observed from those insect hosts fed with the low nutrient diet and exposed to the highest nematode inoculum. However, for both nematode species tested, the nutritional status of the host did not significantly affect time of emergence of IJ progeny or the reassociation with their bacterial symbionts (expressed as cfu/IJ). This is the first study on the effect of insect host physiology on both EPN and their symbiotic bacteria fitness.     

Biology and host range testing of Triarthria setipennis and Ocytata pallipes (Diptera: Tachinidae) for the control of the European earwig (Forficula auricularia)

The European earwig (Forficula auricularia) is an invasive species in the Falkland Islands, causing considerable problems for local horticulture, as well threatening indigenous ecosystems. To assess the potential of a classical biological control introduction two parasitoid fly species, Triarthria setipennis and Ocytata pallipes (Diptera: Tachinidae), were collected from sites in southern and northern England and then tested for their suitability as earwig biological controls at Egham, UK. Both species had previously been introduced into North America for earwig control however little is known of their long-term efficacy and host specificity. Host range tests including both target and non-target species were done. As there are no native Dermaptera on the Falkland Islands, tests were restricted to the field cricket Gryllus assimilis and the Dubia cockroach Blaptica dubia, as representatives of insect orders phylogenetically closely related to earwigs. A second cricket species (Gryllus bimaculatus) was included in an egg-depositing experiment for O. pallipes. Both tachinid species successfully parasitised and emerged from earwigs under laboratory conditions but no signs of parasitisation and development were observed in either the cricket or cockroach.     

Effect of Entomopathogenic Nematode Concentration on Survival during Cryopreservation in Liquid Nitrogen

Entomopathogenic nematodes are used for biological control of insect pests. A method for improved cryopreservation of infective juvenile stage nematodes has been developed using Steinernema carpocapsae and Heterorhabditis bacteriophora. Optimum survival for both species was achieved with 12,000 infective juveniles/ml in glycerol and 7,500/ml in Ringer''s solution. For S. carpocapsae, maximum survival also was observed with 60,000 infective juveniles/ml in glycerol and 25,000/ml in Ringer''s solution. These concentrations resulted in 100% post-cryopreservation survival of S. carpocapsae and 100% retention of original virulence to Galleria mellonella larvae. This is the first report of achieving 100% survival of an entomopathogenic nematode after preservation in liquid nitrogen. Maximum survival of H. bacteriophora following cryopreservation was 87%.     

Field evaluation of entomopathogenic nematodes for biological control of striped flea beetle, Phyllotreta striolata (Coleoptera: Chrysomelidae)

The striped flea beetle, Phyllotreta striolata (F.) (Coleoptera: Chrysomelidae) is a key pest of crucifer vegetables in Southern China. The use of entomopathogenic nematodes (EPNs) within an integrated pest management approach may offer an effective and environmentally safe strategy to suppress outbreaks of this pest. In the present study, the efficacy of Steinernema carpocapsae All and Heterorhabditis indica LN2 for the control of P. striolata in the field was evaluated, as well as the combined application of EPNs and azadirachtin against the pest. Both nematode species were capable of reducing populations of the soil-dwelling stages of P. striolata, thus leading to a reduction of the adult populations and the associated shot-hole damage on the leaves. Nematode treatments also increased cabbage yields as compared to the control and azadirachtin treatments alone. Azadirachtin alone was not effective to prevent damage by P. striolata, but it could enhance the control effect of S. carpocapsae shortly after application. Osmotically treated infective juveniles (IJs) of S. carpocapsae All performed as well as untreated IJs.     

Infectivity of Steinernema carpocapsae and S. feltiae to Larvae and Adults of the Hazelnut Weevil,Curculio nucum: Differential Virulence and Entry Routes

We investigated the existing susceptibility differences of the hazelnut weevil, Curculio nucum L. (Coleoptera:, Curculionidae) to entomopathogenic nematodes by assessing the main route of entry of the nematodes, Steinernema carpocapsae strain B14 and S. feltiae strain D114, into larvae and adult insects, as well as host immune response. Our results suggested that S. carpocapsae B14 and S. feltiae D114 primarily entered adult insects and larvae through the anus. Larvae were more susceptible to S. feltiae D114 than S. carpocapsae B14 and adults were highly susceptible to S. carpocapsae B14 but displayed low susceptibility to S. feltiae D114. Penetration rate correlated with nematode virulence. We observed little evidence that hazelnut weevils mounted any cellular immune response toward S. carpocapsae B14 or S. feltiae D114. We conclude the differential susceptibility of hazelnut weevil larvae and adults to S. carpocapsae B14 and S. feltiae D114 primarily reflected differences in the ability of these two nematodes to penetrate the host.     

Gold-conjugated Reagents for the Labeling of Carbohydrate-Recognition Domains and Glycoconjugates on Nematode Surfaces

Various fluorescent conjugated lectins have been used for the detection of glycoconjugates on nematode surfaces under light microscopy. Several problems have been experienced with these reagents including penetration of the cuticle by fluorescent lectins, non-glycoconjugate specificity, strong nematode autofluorescence at the emission wavelength of the fluorescent dye, and prevention of persistent visualization due to rapid quenching of the fluorescent components. Gold-conjugated reagents combined with silver enhancement alleviated these difficulties when working with three phytonematode species (Heterodera avenae, H. latipons, and Meloidogyne javanica) and two entomopathogenic species (Steinernema carpocapsae and S. glaseri) under light-microscopy visualization of binding by fluorescent lectins and neoglycoproteins. Moreover, gold-conjugated reagents resulted in stable bindings that enabled long-term observations.     

A mechanism of acquired resistance against an entomopathogenic nematode by Agrotis ipsilon feeding on perennial ryegrass harboring a fungal endophyte

Perennial ryegrass forms a symbiotic relationship with the fungus Neotyphodium lolii, which provides many benefits including resistance to herbivory through the production of alkaloids. The impact of endophytic grass on the third trophic level has received little attention. The black cutworm, Agrotis ipsilon, is less susceptible to the entomopathogenic nematode, Steinernema carpocapsae, when it consumes the endophytic grass. We examined the potential mechanisms of the resistance exhibited by A. ipsilon against S. carpocapsae. Although A. ipsilon larvae fed on endophytic grass had similar numbers of nematodes attached and that successfully developed into adults, they had significantly lower mortality than larvae fed on endophyte-free grass when exposed to nematodes for 1.5 h. We examined the effects of N. lolii produced ergot alkaloids, ergotamine tartrate, ergonovine maleate, ergocryptine, and erogcristine on nematode viability and infectivity. Ergonovine malate increased and ergocristine decreased the rates of nematode infectivity, whereas other treatments had no significant effect. We also investigated the effects of ergocristine on Xenorhabdus nematophila, the symbiotic bacterium of S. carpocapsae. Bacterial growth and pathogenicity were significantly reduced when the bacterium was grown in nutrient broth containing 200 μg/ml concentration of ergocristine. We conclude that herbivores capable of developing on endophytic grasses may acquire some level of resistance against S. carpocapsae due to the toxic effects of ergocristine on the bacterium, X. nematophila. Our results underscore the ability of N. lolii to affect trophic interactions through the production of alkaloids.     

Simple In Vivo Production and Storage Methods for Steinernema carpocapsae Infective Juveniles

Methods are described for standardized in vivo production, rapid harvest, and storage, in a concentrated form, of infective juveniles of the entomopathogenic nematode, Steinernema carpocapsae Mexican strain Kapow selection. Nematodes were stored in nematode wool configurations, consisting of mats of intertwined infective juveniles. Freshly harvested nematodes are readily available in adequate quantities for laboratory and small-scale field evaluations as well as cottage industry production.     

Influence of Soil pH and Oxygen on Persistence of Steinernema spp.

Survival of infective juveniles of Steinernema carpocapsae and Steinernema glaseri gradually declined during 16 weeks of observation as the tested soil pH decreased from pH 8 to pH 4. Survival of both species of Steinernema dropped sharply after 1 week at pH 10. Survival or S. carpocapsae and S. glaseri was similar at pH 4, 6, and 8 during the first 4 weeks, but S. carpocapsae survival was significantly greater than S. glaseri at pH 10 through 16 weeks. Steinernema carpocapsae and S. glaseri that had been stored at pH 4, 6, and 8 for 16 weeks, and at pH 10 for 1 or more weeks were not infective to Galleria mellonella larvae. Steinernema carpocapsae survival was significantly greater than that of S. glaseri at oxygen:nitrogen ratios of 1:99, 5:95, and 10:90 during the first 2 weeks, and survival of both nematode species declined sharply to less than 20% after 4 weeks. Survival of both nematode species significantly decreased after 8 weeks as the tested oxygen concentrations decreased from 20 to 1%, and no nematode survival was recorded after 16 weeks. Steinernema carpocapsae pathogenicity was significantly greater than that of S. glaseri during the first 2 weeks. No nematode pathogenicity was recorded at oxygen concentrations of 1, 5, and 10% after 2 weeks and at 20% after 16 weeks.     

Virulence and sex-dependent invasion efficiency of entomopathogenic nematodes on developmental stages of Phthorimaea operculella (Lep., Gelechidae)

Virulence and invasion efficiency of the three entomopathogenic nematodes, Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae against the potato tuber moth (PTM), Phthorimaea operculella was evaluated. Also evaluated were the sex ratio of Steinernema spp. and host stages to determine if 1) the developmental stage of the host affects sex ratio of nematodes; 2) infective juveniles (IJs) concentration affects sex ratio in host developmental stages and 3) the establishment of IJs is affected by developmental stages of host. The PTM pre-pupa and pupa were exposed to IJs in filter substrate petri dish bioassays. By increasing the IJs concentrations, the number of established Steinernema spp. in both PTM stages increased and only decreased at the highest concentration. No reduction in established nematode numbers at the highest concentration was observed for H. bacteriophora. Sex ratio of S. carpocapsae in pre-pupa was affected by IJ concentration. PTM was more susceptible to Steinernema spp. than H. bacteriophora. Pre-pupa were more susceptible to S. feltiae but S. carpocapsae recorded as the most virulent EPN on pupa. Invasion efficiencies were similar for Steinernema and considerably higher than for H. bacteriophora. Despite a higher invasion efficiency of Steinernema into pupae, mortality was lower compared to pre-pupa No correlation was recorded between the invasion efficiencies of the EPNs and mortalities of PTM. The results showed that the invasion efficiency is not appropriate criterion to reflect the virulence of studied EPNs. Compared to H. bacteriophora both tested Steinernema spp. were good candidates for further studies as biocontrol agents of PTM.     

Effects of Crop Residue on the Persistence of Steinernema carpocapsae

We determined the effects of crop residue on the persistence of an entomopathogenic nematode, Steinernema carpocapsae. During 2 consecutive years, nematodes were applied at rates of 2.5 × 10₄ and 1.0 × 10⁵ infective juveniles/m² to small field plots planted with corn. Nematode persistence was monitored by exposing Galleria mellonella larvae to soil samples from plots with and without crop residue (approximately 75% coverage of soybean stubble). Persistence of S. carpocapsae was significantly greater in crop residue plots than in plots without residue. In crop residue plots that received the higher rate of nematode application, larval mortality did not significantly decrease during the study period (3 to 5 days) and remained above 85%. In nematode-treated plots without crop residue, however, larval mortality fell from over 96% to below 11% and 35% in the first and second trials, respectively. The increased crop residue may have benefited nematode persistence through protection from desiccation or ultraviolet light. We conclude that increased ground cover in cropping systems (e.g., due to reduced tillage) may lead to increased insect pest suppression with entomopathogenic nematodes.     

Optimization of Inoculation for In Vivo Production of Entomopathogenic Nematodes

Entomopathogenic nematodes are potent biopesticides that can be mass-produced by in vitro or in vivo methods. For in vivo production, consistently high infection rates are critical to efficiency of the process. Our objective was to optimize in vivo inoculation of Steinernema carpocapsae and Heterorhabditis bacteriophora in Galleria mellonella and Tenebrio molitor by determining effects of inoculation method, nematode concentration, and host density. We found immersing hosts in a nematode suspension to be approximately four times more efficient in time than pipeting inoculum onto the hosts. The number of hosts exhibiting signs of nematode infection increased with nematode concentration and decreased with host density per unit area. This is the first report indicating an effect of host density on inoculation efficiency. We did not detect an effect of nematode inoculum concentration on nematode yield per host or per gram of host. Yield was affected by host density in one of the four nematode-host combinations (S. carpocapsae and T. molitor). We conclude that optimization of inoculation parameters is a necessary component of developing an in vivo production system for entomopathogenic nematodes.     

Predictive thresholds for forecasting the compatibility of <Emphasis Type="Italic">Forficula auricularia</Emphasis> and <Emphasis Type="Italic">Aphelinus mali</Emphasis> as biological control agents against woolly apple aphid in apple orchards

The woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae) is a well-known pest of apple orchards world-wide. Several studies have demonstrated variable control of WAA populations by the European earwig, Forficula auricularia (L.) (Dermaptera: Forficulidae) and the WAA parasitoid Aphelinus mali (Halderman) (Hymenoptera: Aphelinidae). We examine whether a beneficial interaction between F. auricularia and A. mali exists and calculate optimal numbers for each species to maintain WAA infestations below acceptable levels. We demonstrate that trees possessing >14 earwigs per trunk trap per week within the first seven weeks post-blossom contained WAA infestations well below acceptable levels. Where these earwig thresholds were not met, a first generation of A.mali greater than 0.5 wasps per tree was required. If these beneficial insect targets were not met, severe WAA infestations occurred. Our findings suggest that if F. auricularia and A. mali numbers exceed these thresholds chemical intervention may not be required.