Drosophila suzukii is a pest of global concern due to its great impact on several crops. The entomopathogenic nematode Steinernema carpocapsae was highly virulent to the larvae of the fly although some immune mechanisms were triggered along the infection course. Thus, to understand the gene activation profile we performed a comparative transcriptome of D. suzukii larvae infected with S. carpocapsae and Xenorhabdus nematophila to map the differentially expressed genes involved in the defence response. The analysis exposed the induction of genes involved in the humoral response such as the antimicrobial peptides and pattern-recognition receptors while there was a suppression of the cellular defence. Besides, genes involved in melanisation, and clot formation were downregulated hindering the encapsulation response and wound healing. After the infection, larvae were in a stress condition with an enrichment of metabolic and transport functionalities. Concerning the stress response, we observed variations of the heat-shock proteins, detoxification, and peroxidase enzymes. These findings set a genetical comprehensive knowledge of the host-pathogen relation of D. suzukii challenged with S. carpocapsae which could support further comparative studies with entomopathogenic nematodes.

Entomopathogenic nematodes (EPNs) are closely associated with Popillia japonica and potentially used as their biological control agents, although field results proved inconsistent and evoked a continual pursuit of native EPNs more adapted to the environment. Therefore, we surveyed the Azorean Archipelago to isolate new strains of Heterorhabditis bacteriophora and to evaluate their virulence against the model organism Galleria mellonella under laboratory conditions. Six strains were obtained from pasture and coastal environments and both nematode and symbiont bacteria were molecularly identified. The bioassays revealed that Az172, Az186, and Az171 presented high virulence across the determination of a lethal dose (LD50) and short exposure time experiments with a comparable performance to Az29. After 72 hours, these virulent strains presented a mean determination of a lethal dose of 11 infective juveniles cm-2, a lethal time (LT50) of 34 hours, and achieved 40% mortality after an initial exposure time of only 60 minutes. Az170 exhibited an intermediate performance, whereas Az179 and Az180 were classified as low virulent strains. However, both strains presented the highest reproductive potential with means of 1700 infective juveniles/mg of larvae. The bioassays of the native EPNs obtained revealed that these strains hold the potential to be used in biological control initiatives targeting P. japonica because of their high virulence and locally adapted to environmental conditions.

A new species of the genus Oscheius, Oscheius cyrus n. sp., collected in the moist soils taken from forest heights in the north of Iran, is recorded. A comprehensive description, comprising molecular (internal transcribed spacer (ITS), 18S, and 28S rDNA genes) information, morphometrics data, light microscope and scanning electron microscope images, is supplied. The species resembles Oscheius myriophilus. However, the highest ranges for female body length, female tail, infective juvenile tail length, median bulb, absence of epiptygma and lateral field incisures number vary. The new species was distinguished from Oscheius insectivorus by the general lip region. The male was not found. Molecular analysis showed that the new species has the most similarity to O. myriophilus both in the ITS and 18S regions. Morphological and molecular data confirmed its belonging to the Insectivora-group. Furthermore, the species of Ochrobactrum pseudogrignonense was reported as a dominant associated bacterium of the new Oscheius species. Finally, the mortality of the host after seven days varied from 20% to 82.5%, depending on nematodes’ concentration.

The use of native entomopathogenic nematodes as biocontrol agents is a strategy to decrease the environmental impact of insecticides and achieve sustainable agriculture crops. In this study, the effect of the surface culture of Steinernema sp. JAP1 over two solid media at 23–27°C on infective juvenile (IJ) production and pathogenicity against Galleria mellonella larvae were investigated. First, the bacterial lawn on the surface of the media with egg yolk (P2) or chicken liver (Cl) were incubated in darkness at 30°C for 48 and 72 h, and 100 surface-sterilized IJs were added. Four harvests were conducted within the next 35 days and the mean accumulated production was superior on Cl (210 × 103 IJs) than on P2 (135 × 103 IJs), but the productivity decreased up to 10% when the incubation time of the bacterial lawn was of 72 h. The mean pathogenicity of in vitro- and in vivo-produced IJs were of 47–64% and 31%, respectively. It is worth noting that none of the two solid media had a statistically significant difference in IJ pathogenicity. Considering that the maximum multiplication factor of IJs on solid media was 2108 and that the pathogenicity against G. mellonella was outstanding, Steinernema sp. has a good potential for in vitro mass production.

Liquid culture is the most scalable technology for the industrial production of entomopathogenic nematodes. Variability of the recovery after inoculation into cultures of Photorhabdus luminescens remains a persistent problem in the mass production of Heterorhabditis sp. In order to enhance infective juvenile (IJ) recovery and improve nematode population management, we analysed the correlation between the nematode Heterorhabditis megidis (strain KV – 136) development in liquid cultures, the density of bacteria of P. luminescens and the culture agitation speed. Analyses focused on the impact of different agitation speeds (160 rpm and 200 rpm) on the dynamics of population growth of H. megidis in liquid cultures at constant biotic and abiotic parameters (initial dose of nematodes introduced to the culture 2300 IJs/ml, temperature 25°C, the number of bacterial colonies 0.3 × 107/ml). The performed experiments showed that the agitation speed of 200 rpm favourably affected the density of bacteria of P. luminescens (24.14 × 107/ml). High density of bacteria at this agitation speed resulted in an earlier (on the fifth day of the culture) maximum increase in the number of hermaphroditic individuals (1239.6 H/ml) than in the culture at an agitation speed of 160 rpm.

Entomopathogenic nematodes (EPNs) Steinernema and Heterorhabditis and their symbiotic bacteria, Xenorhabdus and Photorhabdus, have been successfully used for the control of insect pests. The objectives of this study were to survey the EPNs and symbiotic bacteria in the agricultural areas of the Phitsanulok province, Thailand, and to study the association between the soil parameters and presence of EPNs. We collected 200 soil samples from 40 soil sites in agricultural areas (field crops, horticulture crops and forest). The prevalence of EPNs was 8.0% (16/200). Fifteen of the EPN isolates were molecularly identified (based on 28S ribosomal DNA and internal transcribed spacer regions) as Steinernema siamkayai. Seven isolates of Xenorhabdus stockiae were identified using recombinase A sequencing. Phylogenetic analysis revealed that all the Steinernema and Xenorhabdus isolates were closely related to S. siamkayai (Indian strain) and X. stockiae (Thai strain), respectively. Significantly more EPNs were recovered from loam than from clay. Although the association between soil parameters (pH, temperature and moisture) and the presence of EPNs was not statistically significant, the elevation levels of the soil sites with and without EPNs were found to be different. Moreover, statistical comparisons between the agricultural areas revealed no significant differences. Therefore, we concluded that S. siamkayai is associated with X. stockiae in agricultural areas and that there is no association between the soil parameters of agricultural areas and presence of EPNs, except for soil texture and the elevation. Steinernema siamkayai may be applied as a biocontrol agent in agricultural areas.

The potato tuber moth, Phthorimaea operculella (Zeller), is a serious pest of potato and other commercial crops belonging to the Solanaceae family. In recent years, it has become an emerging problem in potato-growing regions of the Nilgiri hills of southern India. It is responsible for the reduced quality and quantity of marketable potatoes. In this regard, the development of an eco-friendly control method for the management of the potato tuber moth is urgently required. Therefore, in the present study, the virulence of Steinernema cholashanense CPRSUS01 originally isolated from the potato rhizosphere was tested on fourth-instar larvae and pupae of P. operculella. Steinernema cholashanense caused the greatest mortality in the fourth-instar larval stage (100%) than the pupae (30%). In addition to this, penetration and reproduction of this nematode was also studied in fourth-instar larvae of P. operculella and this is the first report of penetration and reproduction of any entomopathogenic nematode species on potato tuber moth larvae. The reproduction capacity of S. cholashanense on P. operculella is higher (702 infective juveniles mg−1 body weight). Our results indicated that S. cholashanense has good potential as an alternative tool for the management of P. operculella. But before including S. cholashanense in the integrated pest management program of P. operculella, its efficacy should be tested under field conditions.

In laboratory experiments, we investigated how media with varying ratio of peat:sand and two levels of compaction influence dispersal success of entomopathogenic nematode (EPN) species with different foraging strategies: Steinernema carpocapsae (ambusher), Heterorhabditis downesi (cruiser) and Steinernema feltiae (intermediate). Success was measured by the numbers of nematodes moving through a 4 cm column and invading a wax moth larva. We found that both compaction and increasing peat content generally decreased EPN infective juvenile (IJ) success for all three species. Of the three species, H. downesi was the least affected by peat content, and S. carpocapsae was the most adversely influenced by compaction. In addition, sex ratios of the invading IJs of the two Steinernema species were differentially influenced by peat content, and in the case of S. feltiae, sex ratio was also affected by compaction. This indicates that dispersal of male and female IJs is differentially affected by soil parameters and that this differentiation is species-specific. In conclusion, our study shows that organic matter: sand ratio and soil compaction have a marked influence on EPN foraging behaviour with implications for harnessing them as biological pest control agents.

A survey on the occurrence of entomopathogenic nematodes (EPNs) was conducted in selected banana fields from three regions in Tanzania, namely Mbeya (Southern Highlands), Morogoro (Lowland) and Pwani (Coast). The main objective of this study was to isolate EPNs naturally occurring in banana fields in Tanzania and to test their effect on banana weevil (Cosmopolites sordidus Germar 1824). We report for the first time the presence of EPNs in Tanzania where four (4.4%) out of 90 samples contained nematodes in the genera Steinernema and Heterorhabditis. EPNs were only isolated in the coastal region in soils with a high sand content. The virulence of nine EPN isolates was tested against larvae and adults of C. sordidus. All isolates caused mortality of the larval stages, whereas the adults appeared resistant to nematode infection. Larval mortality was found to increase significantly with increasing nematode dose. It was also shown that nematodes were able to penetrate and establish in the banana weevil larvae in increasing numbers with increasing nematode dose. The study indicates the potential for including EPNs in management strategies of banana weevil.

The entire ribosomal DNA repeat unit of a steinernematid species (Nashes isolate) was cloned as three separate EcoR I fragments in the plasmid pUC18. An equimolar cocktail of these three clones was used to identify Steinernema species on Southern blots as each species displays its own unique restriction fragment length polymorphisms. The clones also identified two new species isolated in a soil survey of coastal regions of Britain. One of the clones (pSn4.0) can detect length heterogeneities in the rDNA repeat unit of various isolates of some of the species, particularly the most common in the United Kingdom, S. feltiae. These differences in the rDNA repeat unit length remained constant over several years for one isolate of S. feltiae, but were different for each of the geographical isolates studied to date.

The presence of entomopathogenic nematodes in soil from 15 sites on 10 sampling occasions over a period of 28 months was documented. Sites were chosen to represent habitats classified as heathland, hedgerow, roadside verge, pasture or woodland. Bioassays utilized Galleria larvae and most of the nematodes obtained were identified asSteinernema (= Neoaplectana) bibionis (Bovien). A sibling species which fails to interbreed with S. bibionis was also found at 2 of the sites. Bioassays were at 15 and 20°C and there was no significant difference in the results at these temperatures. There was no evidence for seasonality in the presence of the nematodes and at 2 sites they were found on every sampling occasion. Otherwise, sites converted unpredictably from positive to negative or negative to positive over the 28-month sampling period.

We combined polymerase chain reaction (PCR) amplification of DNA sequences and important morphological characters as a technique to differentiate nematode isolates in the genus Steinernema. Five decamer oligonucleotide primers were used to generate random amplified polymorphic DNA (RAPD) fragments from 11 nematode isolates. The primers generated 8–12 fragments, ranging from 220 to 1300 bp in size. Reproducible amplified DNA fragments of 11 isolates showed obviously inter- or intra-specific polymorphisms, enabling us to differentiate easily the nematode species and isolates. Combining RAPD–PCR fragments with the examination of morphological characters of infective juveniles and 1st-generation males, we identified isolate OH1S, collected from Newport, Oregon, as S.feltiae; isolate OS21, collected from Grants Pass, Oregon, belonged to a previously undescribed species. Our study may provide a rapid and reliable method for the identification of Steinernema nematodes.

Steinernema carpocapsae (Rhabditida: Steinernematidae) host recognition behaviour was assessed and compared with 2 measures of host suitability. Previous research showed that S. carpocapsae infective juveniles respond to host cues in a hierarchical order, with attraction to Galleria mellonella volatiles being stimulated by contact with G. mellonella cuticle. We measured host recognition behaviour by calculating the percentage response of S. carpocapsae infective juveniles to volatiles produced by G. mellonella last instars after the nematodes were exposed to the cuticle of 11 candidate arthropod hosts and 2 control surfaces. Host suitability was measured by nematode-induced mortality to candidate hosts at 2 nematode doses and the level of reproduction supported by each host. The highest recognition response was scored for Agrotis ipsilon (Lepidoptera: Noctuidae). This insect also incurred nearly 100% mortality due to nematode infection and supported the highest level of reproduction. Non-insect arthropods tested (Chilopoda and Isopoda) stimulated no behavioural response and were not susceptible to nematode infection. Other insect species elicited intermediate levels of the recognition response. There were significant correlations between behavioural response and nematode-induced mortality at the lower dose. The level of reproduction supported by the candidate hosts was also correlated with S. carpocapsae behavioural response.

Foraging strategies of eight species of entomopathogenic nematodes were predicted from their response to host volatile cues and dispersal behaviour on 2-dimensional substrates. Positive directional response to chemical cues and similar distances travelled on smooth (agar) or nictation substrates (agar overlaid with sand grains) by Heterorhabditis bacterio-phora, Heterorhabditis megidis, Steinernema anomali, and Steinernema glaseri suggest their cruising approach to finding hosts. The absence of directional response and less distance travelled on nictation substrate, than on smooth agar by Steinernema carpocapsae and Steinernema scapterisci suggest their ambushing mode of foraging. Steinernema feltiae and Steinernema sp. responded directionally to host volatiles, but travelled less distance on the nictation substrate than on smooth agar; the two species also did not nictate. The cruiser species located hosts more effectively in the sand columns, whereas the ambushers were more effective at finding hosts on filter paper. Steinernema feltiae and Steinernema sp. performed equally on filter paper and in the sand column. We conclude that H. bacteriophora, H. megidis, S. anomali and S. glaseri cruise to find hosts, whereas S. carpocapsae and S. scapterisci ambush hosts. Steinernema feltiae and Steinernema sp. are intermediary in the search continuum sharing some characteristics of both ambush and cruise foragers.

Studies of foraging strategies are often complicated by competing goals of the forager. In contrast, non-feeding infective juvenile entomopathogenic nematodes forage exclusively for a single host. Two questions were posed: (1) what is the relationship between metabolic rate, energy reserves and foraging strategy and (2) when a foraging strategy fails, will an infective-stage parasite switch strategies? Three species of entomopathogenic nematodes were stored in water and changes in their behaviour, metabolic rate, energy reserves, and infectivity were measured throughout the storage period. Steinernema carpocapsae ambushes insect hosts, whereas S. glaseri and Heterorhabditis bacteriophora cruise forage. Steinernema carpocapsae was least active and had the lowest metabolic rate. Heterorhabditis bacteriophora was more active and had the highest metabolic rate. Steinernema glaseri was most active and had an intermediate metabolic rate. Neither cruising species changed foraging strategy. Steinernema carpocapsae decreased nictation (a behaviour associated with ambushing only) and increased their locomotory rate. Any change in searching strategy occurred without assessment of the profitability or distribution of potential hosts, but the advantage this confers is unknown.

Search behaviour of two entomopathogenic nematode species with different foraging strategies was compared by measuring parameters of unrewarded search after contact with host cues. Steinernema glaseri cruises in search of hosts. Steinernema carpocapsae ambushes hosts. Nematodes should respond to contact with relevant host cues by shifting their search from ranging to localized after contact with them. We predicted that cruising foragers rely on chemical cues more heavily than ambushers. These species were also tested for host affinities. Nematodes were tracked by image analysis after exposure to faeces, cuticle or food of either Popillia japonica or Spodoptera exiqua. Steinernema glaseri responded to selected host cues by shifting from ranging to localized search, characterized by decreased locomotory rate, distance travelled, search area and the proportion of the test period spent moving. Steinernema carpocapsae did not respond to host cues. Steinernema glaseri responds to selected chemical host cues for host location, whereas S. carpocapsae does not.

Genomic DNA extracted from entomopathogenic nematodes isolated from 89 soil samples taken throughout the United Kingdom was hybridized with the ribosomal DNA clone from Caenorhabditis elegans (pCe7). When the DNA was digested with EcoR I and Hind III in a double digest, 5 distinct RFLP (restriction fragment length polymorphism) types were observed. While the prevalence of the 5 types was not equal, no correlation with geographical location, soil type or habitat could be detected. Subsequent hybridizations of total genomic DNA from the various RFLP types divided them into 2 groups. The most prevalent group, identified as Steinernema feltiae ( = bibionis), contained 2 of the RFLP types (Al and A2). The other group contained the remaining 3 RFLP types (B1, B2 and B3). Although similar to S. feltiae ( = bibionis), the members of the B-types can be distinguished from this species on morphological grounds and preliminary crossbreeding experiments have demonstrated that the 2 groups are reproductively isolated.

Soil from 403 hedgerow, roadside verge, woodland, heathland or field sites was assessed for presence of entomopathogenic rhabditid nematodes with aGalleria larva baiting technique. Steinernematids were recovered from 48 % of the sites but only one site yielded Heterorhabditis sp. The species recovered most frequently was Steinernema bibionis (Bovien). Another unidentified Steinernema sp. was also isolated. Prevalence of steinernematids in the different types of habitat formed continuum so that roadside verges harboured them most often while heathland sites yielded them least often. Mean soil temperatures at 5 cm were about 15 °C in fields and verges, 13 °C in hedgerows and heathland and 12 °C in woodland. Prevalence of the nematodes varied in different parts of Britain and was influenced by soil type. They were associated with calcareous soils, especially those with a calcareous subsoil horizon. Soils which are periodically or seasonally water-logged were suitable. Soils enriched by humus and hence high in organic matter frequently harboured the nematodes and some suitable soils had significant clay contents or subsoils high in clay. It appears that the two steinernematids are relatively unspecialized species, occurring in diverse habitats and soil types and are adapted to cooler temperatures.

Entomopathogenic nematode species available in Europe were screened for their efficacy against both the root-feeding larvae and silk-feeding adults of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Laboratory screening tests were aimed at the selection of candidate biological control agents for the management of this invasive alien pest in Europe. Steinernema glaseri, S. arenarium, S. abassi, S. bicornutum, S. feltiae, S. kraussei, S. carpocapsae and Heterorhabditis bacteriophora were studied to determine their virulence against third instar larvae and adults of D. v. virgifera in small-volume arenas (using nematode concentrations of 0.5, 0.8, 7.9 and 15.9 infective juveniles cm–2). All nematode species were able to invade and propagate in D. v. virgifera larvae, but adults were rarely infected. At concentrations of 7.9 and 15.9 cm–2, S. glaseri, S. arenarium, S. abassi and H. bacteriophora caused the highest larval mortality of up to 77%. Steinernema bicornutum, S. abassi, S. carpocapsae and H. bacteriophora appeared to have a high propagation level, producing 5970±779, 5595±811, 5341±1177 and 4039±1025 infective juveniles per larva, respectively. Steinernema glaseri, S. arenarium, S. feltiae, S. kraussei and H. bacteriophora were further screened at a concentration of 16.7 nematodes cm–2 against third instar larvae in medium-volume arenas (sand-filled trays with maize plants). Heterorhabditis bacteriophora, S. arenarium and S. feltiae caused the highest larval mortality with 77±16.6%, 67±3.5%, and 57±17.1%, respectively. In a next step, criteria for rating the entomopathogenic nematode species were applied based on results obtained for virulence and propagation, and for current production costs and availability in Europe. These criteria were then rated to determine the potential of the nematodes for further field testing. Results showed the highest potential in H. bacteriophora, followed by S. arenarium and S. feltiae, for further testing as candidate biological control agents.

Entomopathogenic nematode infective juveniles are likely to encounter both uninfected and infected insects and host quality depends on the stage of the infection. We hypothesized that nematode response to infected hosts will change over the course of an infection. Here, we tested this hypothesis by focusing on the influence of host infection status on long-range attraction to host volatile cues. The attraction response of 3 nematode species (Steinernema carpocapsae, S. glaseri and S. riobrave) with different foraging strategies to infected and uninfected insects (Galleria mellonella and Tenebrio molitor) was tested at 24 h intervals from start of infection to emergence of infective juveniles from depleted host. As expected, based on their foraging strategies, S. carpocapsae was not very responsive to hosts, S. glaseri was highly responsive and S. riobrave was intermediate. Generally, the level of attraction did not change with time after infection and was similar between infected and uninfected hosts. An exception was S. glaseri infected T. molitor, which tended to be less attractive to S. glaseri than uninfected hosts. These results suggest that any influence of host infection status on infection behaviour is occurring at subsequent steps in the host-infection process than host attraction, or involves non-volatile cues.