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Summary The entomopathogenic nematode Steinernema siamkayai has been a subject of several detailed studies but its morphological, molecular and ecological characterisation is still incomplete. In the present study we studied morphology of several strains of S. siamkayai using morphometrics and light and scanning electron microscopy (SEM) and we aimed to complete its molecular characterisation by obtaining the sequence of the D2-D3 region of the 28S rDNA in addition to ITS rDNA and mitochondrial (12S and COI) DNA studies. Furthermore, we studied the species life cycle and we attempted to assess its distribution using a meta-analysis of the GenBank records. Finally, insecticidal properties were evaluated against Helicoverpa armigera and Spodoptera litura. The SEM study revealed the presence of the adcloacal pair of genital papillae in males. Duration of the life cycle of the Indian strains did not differ from the previous records; however, we have observed production of the infective juveniles by the first-generation adults. For the first time in entomopathogenic nematodes, we have recorded intra-individual variability in the D2-D3 region of the 28S rDNA and we discuss potential implications of this phenomenon for the use of this marker in EPN systematics. Our analysis of distribution has shown that S. siamkayai is ubiquitous throughout the Indian subcontinent but is rarely found in South East Asia. The pathogenicity results showed that the present species effectively killed the tested pests under laboratory conditions, demonstrating its potential as a biocontrol agent.

A new species of entomopathogenic nematode, Steinernema riojaense n. sp., was isolated during a survey in La Rioja province of Spain. Morphological and morphometric traits place the new species in the glaseri-group, which is characterised by having the largest infective juvenile (IJ) with an average body length in excess of 1000 μm. The IJ of S. riojaense n. sp. possess a body length of 1043 (869-1172) μm, the lateral fields are formed by eight equally spaced and developed ridges (nine incisures) in the mid-body region, giving the formula 2, 6, 8, 4, the excretory pore is located posterior to the mid-pharynx region (D% = 58), and the hyaline region occupies ca half of the tail length. The male spicules are moderately curved with a sharp tip and are golden-brown in colour with the manubrium elongate and having a length to width ratio of 1.7:1. The first-generation male tail is non-mucronate, whereas a small papilla-like mucron is developed in the second generation. There are 11 pairs of genital papillae plus a single precloacal papilla. The first-generation female possesses a moderately protruding vulva and a slightly protruding postanal swelling. The second-generation female possesses a slightly protruding vulva and a postanal swelling. The new species is further characterised by sequences of the internal transcribed spacer (ITS) and partial 28S regions (D2-D3) of the ribosomal DNA (rDNA). Phylogenetic analyses confirm that S. riojaense n. sp. belongs to the glaseri-group and forms a monophyletic subclade with other European species.

Simple Summary Due to their potential entomopathogenicity, nematodes of the genus Oscheius have been in the spotlight of the scientific interest in recent years. Unfortunately, some of these species are poorly described and have inadequate or no molecular support. This fact complicates the systematics of the group, and a revision of these species is necessary to elucidate their taxonomic status. In the present study, we provide a detailed description based on the morphological, morphometrical, and molecular characteristics of Oscheius siddiqii from Uttar Pradesh, India, including the first scanning electron microscopy (SEM) studies of the species. Furthermore, based on morphological and molecular data, the status of some Oscheius species is discussed, and several synonymisations are proposed. Abstract An insect parasitic nematode belonging to the genus Oscheius was recovered from the agricultural soils from the Hapur district in western Uttar Pradesh, India. Morphological studies on this species exhibited its high resemblance with two Pakistani species: Oscheius siddiqii and O. niazii. No molecular data are available for these taxa but, morphologically, both species do not differ significantly from our strains and each other. Hence, these nematodes can be considered conspecific, and the correct name for this taxon is O. siddiqii, the first described species. The phylogenetic analyses of the ITS-, 18S-, and the 28S rDNA sequences showed that O. siddiqii is a sister taxon to the group formed by Oscheius microvilli, O. myriophilus, O. safricanus, and several unidentified Oscheius species. Additionally, our analyses show that based on molecular and morphological data, the species Oscheius rugaoensis and O. microvilli cannot be distinguished from O. chongmingensis and O. myriophilus, respectively, and are thus considered junior synonyms of these taxa. Furthermore, the available data are not sufficient to evaluate the status of Oscheius basothovii and O. safricanus, which are, in consequence, considered species inquirendae. These findings highlight the necessity of the proper morphological and molecular characterisation of the described Oscheius species.

Alloionema appendiculatum Schneider, 1859 (Rhabditida: Alloionematidae) is a common parasite of terrestrial molluscs capable of causing high snail mortality in heliculture. It has also been described as a parasite of the invasive slug Arion vulgaris Moquin-Tandon, 1855 (Pulmonata: Arionidae), a serious pest in central Europe. Nontheless, our knowledge about the ecology of this nematode is poor. Our study aims to provide new information about some as yet unknown aspects of the biology of this species, mainly its prevalence, attraction to slug associated cues, development, progeny production, and influence on the mortality and feeding activity of its host. The results of our study provide new information about the saprobic life cycle and natural prevalence of the species, but also show that, in standard conditions, A. appendiculatum has a very weak influence on the mortality and feeding activity of A. vulgaris. We concede that the effect of A. appendiculatum on the slug host can be strongly influenced by bacterial associates, even though the role of bacteria in nematode development is questionable and needs further research. Based on our results, A. appendiculatum does not seem to be a suitable agent for the biocontrol of A. vulgaris.

Summary The lateral field pattern of infective juveniles of the nematode family Steinernematidae is an important taxonomic character. Scanning electron microscopy (SEM) shows the number of ridges and lines or incisures clearly, but does not provide other details. In the present study, ten species from six clades of Steinernematidae have been studied for their lateral field morphology using SEM and high pressure freezing (HPF) with transmission electron microscopy (TEM). Both methods indicated the same number of ridges and lines, although HPF/TEM resulted in a more detailed morphology with differences between the species. The tips of the ridges are either finely rounded or pointed and the lines are V-shaped or have a broadened bottom. These characters represent an additional pattern that may be characteristic for some species within the phylogenetic clades. Further studies of the lateral field morphology of other species is needed to ascertain whether each pattern is clade specific and phylogenetically valuable.

Bulb mites of the genus Rhizoglyphus are cosmopolitan pests of onion, garlic and ornamental plants. Despite the growing awareness against the use of pesticides, growers continue to use insecticides as the main control method. Thus, the search for environmentally safe alternative control methods, such as effective biocontrol agents, is of paramount importance. In the present study, the bulb mites were exposed to the infected juveniles of 20 strains of Steinernema and Heterorhabditis species applied at a single dose of 300 IJs per mite, and the invasion rate and mite mortality were assessed. Furthermore, the effect of the culture supernatants of the selected symbiotic bacteria of the genus Xenorhabdus on bulb mite mortality was tested. Our data show that both Steinernema and Heterorhabditis nematodes are able to invade and kill bulb mites, but in general, the invasion and resulting mortality were relatively low. The highest invasion rate of ca 30 IJs per mite was observed in Heterorhabditis taysearae. The mortality reached the maximum of 30% in the most efficient species, namely Steinernema huense, H. bacteriophora and H. amazonensis. The effect of the culture supernatants was generally low, though there were considerable differences in the efficacy among Xenorhabdus species and strains. To conclude we demonstrate that some EPN species are able to invade and kill bulb mites but their effect is in general quite low. Based on present data, EPNs and the metabolites of their symbiotic bacteria do not seem to represent a viable option for bulb mite biocontrol as a standalone approach.

Terrestrial gastropod molluscs are widely distributed and are well known as pests of many types of plants that are notoriously difficult to control. Many species of nematodes are able to parasitize land snails and slugs, but few of them are lethal to their host. Species and/or populations of mollusc-parasitic nematodes (MPNs) that kill their hosts are promising for biological control purposes. The recent discovery of new nematode species of the genus Phasmarhabditis in Europe and the associations between Alloionema spp. and slugs are expanding the possibilities of using MPNs as control agents. However, very little is known about the distribution and ecology of these species. Using molecular techniques based on qPCR methods for quick identification and quantification of various species of MPN isolated directly from the soil or from infected hosts can assist in providing information on their presence and persistence, as well as the composition of natural assemblages. Here, we developed new primers and probes for five species of the genus Phasmarhabditis and one species of the genus Alloionema. We employed these novel molecular techniques and implemented a published molecular set to detect MPN presence in soil samples coming from natural and agricultural areas in Switzerland. We also developed a method that allows the detection and quantification of Phasmarhabditis hermaphrodita directly from the tissues of their slug host in a laboratory experiment. The new molecular approaches were optimized to a satisfactory limit of detection of the species, with only few cross-amplifications with closely related species in late cycles (>32). Using these tools, we detected MPNs in 7.5% of sampled sites, corresponding to forest areas (P. hermaphrodita and Alloionema appendiculatum) and wheat-oriented agricultural areas (Phasmarhabditis bohemica). Moreover, we confirmed that the method can be used to detect the presence of P. hermaphrodita inside slug hosts, with more detections in the susceptible slug Deroceras larvae compared to the resistant Arion vulgaris. These primers/probe sets provide a novel and quick tool to identify MPNs from soil samples and infected slugs without having to culture and retrieve all nematode life stages, as well as a new tool to unravel the ecology of nematode-slug complexes., This work was funded by the Swiss National Science Foundation: Grants NRP68 and SNSF Div. I-III, reference 166632. The Government of Spain supports RCH with a Ramón y Cajal contract award: RYC-2016-19939.

A population of a nematode species belonging to the genus Oscheius was isolated in western Uttar Pradesh, India. Morphological and morphometrical studies on this species showed its high similarity with six species described previously from Pakistan (Oscheius citri, O. cobbi, O. cynodonti, O. esculentus, O. punctatus and O. sacchari). The molecular analysis of the ITS1-5.8S-ITS2 rDNA sequences of the Indian population and the six species described from Pakistan showed that all the sequences are almost identical. Thus, based on morphological and molecular characteristics, all of the six above-mentioned Pakistani species and Indian strain do not differ from each other, hence can be considered synonyms. The correct name for this taxon is the first described species O. citri. Additionally, the phylogenetic analysis of the 18S rDNA and the 28S rDNA sequences showed that Oscheius citri is sister to the clade formed by O. chongmingensis and O. rugaoensis from China. The high similarity of morphological and morphometric characteristics of O. citri and other species, O. maqbooli, O. nadarajani, O. niazii, O. shamimi and O. siddiqii, suggest their conspecificity; however, lack of molecular data for these species does not allow this hypothesis to be tested.

Entomopathogenic nematodes and fungi are globally distributed soil organisms that are frequently used as bioagents in biological control and integrated pest management. Many studies have demonstrated that the combination of biocontrol agents can increase their efficacy against target hosts. In our study, we focused on another potential benefit of the synergy of two species of nematodes, Steinernema feltiae and Heterorhabditis bacteriophora, and the fungus Isaria fumosorosea. According to our hypothesis, these nematodes may be able to disseminate this fungus into the environment. To test this hypothesis, we studied fungal dispersal by the nematodes in different arenas, including potato dextrose agar (PDA) plates, sand heaps, sand barriers, and glass tubes filled with soil. The results of our study showed, for the first time, that the spreading of both conidia and blastospores of I. fumosorosea is significantly enhanced by the presence of entomopathogenic nematodes, but the efficacy of dissemination is negatively influenced by the heterogeneity of the testing arena. We also found that H. bacteriophora spread fungi more effectively than S. feltiae. This phenomenon could be explained by the differences in the presence and persistence of second-stage cuticles or by different foraging behavior. Finally, we observed that blastospores are disseminated more effectively than conidia, which might be due to the different adherence of these spores (conidia are hydrophobic, while blastospores are hydrophilic). The obtained results showed that entomopathogenic nematodes (EPNs) can enhance the efficiency of fungal dispersal.

During a survey in agricultural fields of the sub-humid region of Meerut district, India, two strains of entomopathogenic nematodes, labelled CS31 and CS32, were isolated using the Galleria baiting technique. Based on morphological and morphometric studies, and molecular data, the nematodes were identified as Steinernema pakistanense, making this finding the first report of this species from India. For the first time, we performed a molecular and biochemical characterization of the bacterial symbiont of S. pakistanense. Furthermore, a co-phylogenetic analysis of the bacteria from the monophyletic clade containing a symbiont of S. pakistanense, together with their nematode hosts, was conducted, to test the degree of nematode–bacteria co-speciation. Both isolates were also tested in a laboratory assay for pathogenicity against two major pests, Helicoverpa armigera and Spodoptera litura. The morphology of the Indian isolates corresponds mainly to the original description, with the only difference being the absence of a mucron in first-generation females and missing epiptygmata in the second generation. The sequences of bacterial recA and gyrB genes have shown that the symbiont of S. pakistanense is closely related to Xenorhabdus indica, which is associated with some other nematodes from the ‘bicornutum’ group. Co-phylogenetic analysis has shown a remarkable congruence between the nematode and bacterial phylogenies, suggesting that, in some lineages within the Steinernema / Xenorhabdus complex, the nematodes and bacteria have undergone co-speciation. In the virulence assay, both strains caused a 100% mortality of both tested insects after 48 h, even at the lowest doses of 25 infective juveniles per insect, suggesting that S. pakistanense could be considered for use in the biocontrol of these organisms in India.

Summary The mucron in male steinernematid nematodes may be missing or present either in the first or second generation, or missing in both generations, with variable shape. However, for many species, the information on mucron morphology is incomplete and its taxonomic significance and distribution remain unclear. The present study assessed mucron morphology in males of 26 species using LM and SEM microscopy. For other species we summarised the published data. Furthermore, ancestral character state reconstruction analysis was performed to assess the distribution of mucron morphology within steinernematid phylogeny. In most species, papilla/spine-like or filamentous mucrons occur at least in the second generation. The species gathered in single phylogenetic groups have a similar mucron morphology. Generally, species with a prominent filamentous mucron belong to the ‘kraussei/feltiae’ and ‘carpocapsae’ groups, whereas mostly non-mucronated species occur in ‘glaseri’ and ‘riobrave’ groups. For future descriptions a precise mucron characterisation in both generations of the male is recommended.

Simple Summary Colorado potato beetle (CPB) Leptinotarsa decemlineata is the potato plant’s most destructive pest. Recently, resistance to the traditional insecticides has appeared, thus new environmentally friendly control agents are highly needed. In our study, we searched for the most effective entomopathogenic agents that could be used to decrease the emergence of CPB adults from the soil. We selected two entomopathogenic nematodes (Steinernema carpocapsae and S. feltiae) and one strain of fungus (Beauveria bassiana). The suspension application was done on the leaves, plus by watering the pods and the field plots. All the treatments had an obvious effect, but in the field, only the fungal treatment showed a promising result. Further research is needed to develop the most effective application for field usage. Abstract Colorado potato beetle Leptinotarsa decemlineata is among the most destructive pests of potatoes quickly developing resistance to traditional insecticides. In the present study, we tested the effect of various species and strains of entomopathogenic nematodes on CPB adults, and subsequently, the most effective nematodes were applied alone and in combination with entomopathogenic fungus B. bassiana in pots with potato plants and in the field and their effect on the number of emerging adults was evaluated. In the experimental infections, both the nematode invasion and pathogenicity were variable, and, in several strains, the mortality reached 100%. In pot experiments, soil application of nematodes S. carpocapsae 1343 and S. feltiae Jakub and fungus significantly decreased numbers of emerging CPB adults, while, after the application on leaves, only fungal treatment was effective. The field application of fungus B. bassiana significantly decreased the number of emerging CPB adults in comparison to control sites by ca. 30% while the effect of nematodes and the nematodes–fungus combination was not significant. In conclusion, we demonstrate the necessity of thorough bioassays to select the most effective nematode strains. Entomopathogenic nematodes have the potential to effectively decrease the emergence of CPB adults, but further research is needed to improve the effectiveness in the field.

Phasmarhabditis bohemica n. sp. is described and illustrated from the body of Deroceras reticulatum from the village of Chelčice, the Czech Republic. Morphological and molecular data showed that the new isolate is close to other species of Phasmarhabditis, particularly P. californica and P. papillosa. Females are characterised by a body length of 2079 (1777-2222) μm and a long tapering tail with prominent papilliform phasmids located laterally in the mid-tail region. Males are 1683 (1515-1818) μm long. They have a peloderan bursa, with nine pairs of rays, 1/1/1/2/1/3, and a reflexed testis 495 (434-555) μm long. Dauer juveniles are thin, 553 (474-636) μm long, with prominent lateral fields consisting of two prominent ridges and three incisures. Small subunit (18S), ITS, and D2-D3 expansion segments of the large subunit of ribosomal DNA were used to analyse the phylogenetic relationships of sequenced species in Phasmarhabditis and other closely related species. Our preliminary observations suggest that the newly described species may be a facultative mollusc-parasitic nematode that is able to survive permanently in the saprobic phase on decaying organic matter. The ecology, morphology, and phylogenetic positions of P. bohemica n. sp. are discussed.

Steinernema beitlechemi n. sp. is a new species of entomopathogenic nematode isolated from soil samples collected in Bethlehem, Free State province, South Africa. The infective juveniles of S. beitlechemi n. sp. are characterised by a body length of 696 (606-768) μm, position of nerve ring of 96 (86-105) μm from the head and lateral fields with six ridges (i.e., seven lines) in mid-body. First-generation males of S. beitlechemi n. sp. can be distinguished by a spicule length of 93 (88-100) μm and 12 pairs of genital papillae. First-generation females have a moderately protruding vulva and a rounded tail tip bearing one spine-like projection. The new species is further characterised by sequences of the ITS and partial 28S regions of the ribosomal DNA. Phylogenetic analyses show S. beitlechemi n. sp. as a member of a strongly supported monophyletic clade with three other African species: S. sacchari, S. cameroonense and S. nyetense.

A new nematode species belonging to the family Alloionematidae was originally isolated by Dr Marie Anne Félix on 16 December 2007 from a rotting coconut at the boat boarding point in Estuary island resort, Poovar, Kerala, India. Both morphologically and molecularly, the new isolate resembles Neoalloionema tricaudatum but, unlike N. tricaudatum this species has no phasmidial ‘horns’ in the juvenile stage, even though prominent structures in the mid-tail of dauer juveniles were observed. Females are characterised by a body length of 947 (869-1030) μm, vulva in the mid-body region, long, and tapering tail with a minute post-anal swelling. Males are 678 (606-757) μm long with a long tapering tail that is very thick at base. They are further characterised by seven pairs of genital papillae and a single genital papilla. Dauer juveniles are only 391 (364-434) μm long, thin and almost straight when heat-killed. Large, prominent phasmids are located laterally at the mid-tail region and lateral fields consisting of a single ridge are present. Several morphological and molecular differences encouraged us to describe the nematode as N. indicum n. sp. The ecology of the newly described species is unknown, but it is probably a saprophytic bacteriophagous nematode preferring rotting organic material.

A new nematode species belonging to the family Rhabditidae, genus Phasmarhabditis, was isolated in the city of Bari (strain BAR) and near the village Gravina (strain GRA) in Apulia, Italy, and is described herein as Phasmarhabditis apuliae n. sp. The original hosts were Milax sowerbyi and M. gagates. Females are 2623 (2262-2848) μm long with a long tapering tail. Prominent papilla-like phasmids are present. Males are 2096 (1898-2363) μm long. They have a peloderan bursa with nine pairs of rays arranged as 1 + 1 + 1 + 2 + 1 + 3. Each ray bears a single papilla. A non-paired papilla is located near the ventral appendage anterior to the cloacal aperture. Prominent papilla-like phasmids are situated close to the tail tip. Dauer juveniles have lateral fields formed from two prominent wide ridges and have three incisures. Phasmarhabditis apuliae n. sp. seems to be a facultative mollusc-parasitic nematode that is capable, in a similar manner to P. hermaphrodita, of long-term survival in the saprophytic phase on decaying organic matter. Ecology, morphology and phylogenetic relationships of Phasmarhabditis nematodes are discussed.

In Brazil, the sugarcane billbug Sphenophorus levis (Coleoptera: Curculionidae) bores sugarcane rhizomes in its larval stage, causing high losses to the crop. Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis and Steinernema (Nematoda: Rhabditidae) could be applied in the sugarcane fields to control the larvae and pupae of S. levis inside rhizomes. However, it is not clear whether this insect leaves an opening in the rhizome that would allow EPNs to enter and reach the insects in their immature stages. In addition, there is still no record of the natural occurrence of EPNs in the immature stages of S. levis or on the losses this insect can cause to sugarcane crops. This work aimed to investigate aspects of S. levis behavior on sugarcane plants and to determine the cane damage and crop loss percentage it causes through observations and regular evaluations of plants in the field; as well as to assess the natural occurrence of EPNs in the insect population found inside the cane rhizomes. For every 1% increase in its damage to the rhizomes, S. levis caused a 1% loss in the cane crop due to reductions of 0.318% in the weight of the stems and of 0.68% in the number of shoots generated by the rhizomes. The insect leaves openings that link the tunnels inside the root to the outside; these openings are made by adults during oviposition or by last-instar larva to allow the adults to exit. Almost 90% of the openings were made below the ground, allowing EPNs to enter the tunnels to reach the insect life stages inside the root. Steinernema carpocapsae was found to cause natural infection in S. levis inside the root, providing up to 60% pupal mortality. This information may help to establish the level of economic damage caused by S. levis and highlights EPNs as a potential alternative for S. levis control; their application just after cane harvesting is suggested in order to reduce the S. levis population in the next plant generation.

Entomopathogenic nematodes (EPN) are excellent biological control agents to fight soil-dwelling insect pests. In a previous survey of agricultural soils of Switzerland, we found mixtures of free-living nematodes (FLN) in the genus Oscheius, which appeared to be in intense competition with EPN. As this may have important implications for the long-term persistence of EPN, we studied this intraguild competition in detail. We hypothesized that (i) Oscheius spp. isolates act as scavengers rather than entomopathogens, and (ii) cadavers with relatively small numbers of EPN are highly suitable resources for Oscheius spp. reproduction. To study this, we identified Oscheius spp. isolated from Swiss soils, quantified the outcome of EPN/Oscheius competition in laboratory experiments, developed species-specific primers and probe for quantitative real-time PCR, and evaluated their relative occurrence in the field in the context of the soil food web. Molecular analysis (ITS/D2D3) identified MG-67/MG-69 as Oscheius onirici and MG-68 as O. tipulae (Dolichura-group). Oscheius spp. indeed behaved as scavengers, reproducing in ∼64% of frozen-killed cadavers from controlled experiments. Mixed infection in the laboratory by Oscheius spp. with low (3 IJs) or high (20 IJs) initial EPN numbers revealed simultaneous reproduction in double-exposed cadavers which resulted in a substantial reduction in the number of EPN progeny from the cadaver. This effect depended on the number of EPN in the initial inoculum and differed by EPN species; Heterorhabditis megidis was better at overcoming competition. This study reveals Oscheius spp. as facultative kleptoparasites that compete with EPN for insect cadavers. Using real-time qPCR, we were able to accurately quantify this strong competition between FLN and EPN in cadavers that were recovered after soil baiting (∼86% cadavers with >50% FLN production). The severe competition within the host cadavers and the intense management of the soils in annual crops readily explain the low EPN numbers in Swiss field samples. The developed molecular tools can be used to elucidate the extent to which the competitive interactions affect EPN populations. This can help to develop strategies to achieve good persistence and natural EPN recycling, in particular in systems where native EPN levels are low, such as annual crops.

The internal transcribed spacers of rDNA are among the most important molecular markers used in the taxonomy and phylogeny of entomopathogenic nematodes. However, their ease of use and the results they provide can be confounded by the presence of intra-individual variability. In the present study we screened 40 strains of S. feltiae and S. kraussei and nine strains from the “glaseri” group. In two selected strains of S. feltiae, and one strain each of S. kraussei and S. glaseri, PCR products of four virgin females were cloned, and five clones per individual female were sequenced and analyzed. In S. feltiae, intra-individual variability was compared with intraspecific variability. We revealed a high frequency of intra-individual variability, which reached 65 % in S. feltiae, 30 % in S. kraussei, and almost 90 % in the “glaseri” group. Analysis of the cloned products showed intra-individual variability in each of the tested strains and individuals. Maximum within-individual variability was over 1 % in S. feltiae and S. kraussei, and in S. glaseri reached up to 4 %. In S. feltiae, intra-individual variability equaled the variability among S. feltiae strains from different parts of the world. Our data show that intra-individual variability in the ITS regions is quite frequent in entomopathogenic nematodes. The implications of this fact to the group taxonomy are discussed.

Background and Aims: As part of a research consortium that explores ways to improve soil health we study how entomopathogenic nematodes (EPNs) can be better exploited for the biological control of soil dwelling insect pests in annual crops. Methods: We evaluated how tillage might affect belowground interactions in two 30 year running Swiss field trials by combining traditional (insect bait) and molecular (novel real time qPCR protocols) methods. Soil samples (April and October 2013) were evaluated for the presence and activity of EPN soil food web assemblage comprising 13 EPN species six nematophagous fungi one ectoparasitic bacterium and the free living nematodes (FLN) of the Acrobeloides group. Results: Mortality of sentinel larvae as well as qPCR analyses (for which we provide seven new primers/probes sets) found only trace levels of six EPN species dominated by heterorhabditids species. Analysis of nematode progeny revealed that EPN compete intensely with FLN for insect cadavers. Overall it appears that temperate annual cropping systems provide poor environments for EPN and that tillage does not negatively affect the natural occurrence of EPN. Conclusions: Natural occurrence of EPN in Swiss tillage soils was very low and augmentation may be a promising strategy to improve the control of root pests of annual crops.

Alloionema appendiculatum is a common juvenile parasite of many terrestrial molluscs. Its third-stage juveniles (dauers) invade the foot muscle of snails and slugs and develop into fourth-stage juveniles, which then leave the host. Later they mature and reproduce in the soil. A population of A. appendiculatum was isolated from infected individuals of the invasive slug Arion vulgaris (= A. lusitanicus), collected in the city of České Budějovice, Czech Republic, and was designated as the AL strain. This nematode is also able to reproduce on pig kidney in laboratory culture. Our isolate was compared primarily with the description published by Mengert (1953) and in some aspects with the original description published by Schneider (1859). Insufficient morphology, morphometrics, phylogeny and an absence of pictorial material encouraged us to re-describe this frequent slug parasite and to add some new information on its life-cycle. The species is characterised by the absence, in adults, of ridges in the lateral fields. At this life stage the stoma is short, narrow and approximately twice as long as it is broad. Fourth-stage female juveniles produce a mucus-like substance from the phasmids. Males possess six pairs of papillae and a single inconspicuous papilla. The nematode has both parasitic and saprophytic life-cycles. Parasitic adults are bigger than the saprophytes and have a thick, digitate tail, whereas the smaller saprophytic generation has a filiform tail. The species is amphimictic, displaying a higher proportion of females, but males are frequent.

Surveying the biodiversity of entomopathogenic nematodes in Zulia and Falcón states, north-western Venezuela, two populations of a newSteinernemaspecies were isolated from soil samples. Morphological, morphometric and molecular data indicate that the new species belong to the ‘bicornutum-group’ ofSteinernema. The new species can be separated from the other species of the group due to the size and shape of their spicules, which are the shortest within the group, and the presence of prominent deirids in the male of both generations at basal bulb level. Lateral field with eight ridges at mid-body of which submarginal ridges are less distinct, but the third and sixth ridges eventually become wider posteriorly to form the last two ridges. Female possesses small but distinct double epiptygmata and anal swellings present in 50% of the specimens. Phylogenetic analyses of thebicornutum-group based on both ITS and D2-D3 regions showed a clear separation of the new species from all other species. We describe these two populations and designate them asSteinernema papillatumn. sp.

The effect of different growing substrates on the development of the slug parasite Phasmarhabditis hermaphrodita has been studied in a series of laboratory experiments. Wild, laboratory and Nemaslug strains of P. hermaphrodita and their monoxenic counterparts with Moraxella osloensis (CCM 5605T) were reared on homogenised pig kidney, Deroceras reticulatum, Arion lusitanicus, and Galleria mellonella, the faeces of D. reticulatum and A. lusitanicus, or leaf compost. Development time, yield, lipid reserves and the body length of nematodes were assessed. All P. hermaphrodita strains were able to grow and reproduce on all tested substrates; however, yields were markedly higher on invertebrate media. Lipid content and body size varied across the substrates and strains. A uniform response of all nematode strains and the most dramatic differences among substrates were recorded in yields, which could indicate that the quality of the substrate is expressed mainly in yield. Monoxenized strains had higher yield, leng...

A new entomopathogenic nematode, Steinernema huense sp. n., belonging to the carpocapsae group, was recovered in Bach Ma National Park (Thua Thien Hue province) Vietnam. Steinernema huense sp. n. infective juveniles are characterised by short body length of 527 (444-571) μm, distance from anterior end to excretory pore of 43 (38-46) μm, anterior end to end of pharynx of 116 (103-129) μm; tail length of 50 (43-56) μm, D% = 37 (34-39) and E% = 85 (79-93), H% = 44 (39-52) and in having six ridges (i.e., seven lines) in the lateral field. For first generation males, the diagnostic characters include the spicule length of 67 (60-72) μm; D% = 48 (41-61); SW% = 195 (168-238) and GS% = 76 (68-81). Both generations possess a minute filamentous mucron, which can be rudimentary in the first generation, and 23 genital papillae. Spicules are finely curved, brownish and slender with a prominent rostrum. First generation females have a non-protruding, symmetrical vulva, with double flapped epiptygma. The tail of mature females is obese with a short conical tip (peg) lacking minute protuberances, and post-anal swelling not developed. The new species is further characterised by sequences of ITS and D2-D3 regions of the ribosomal DNA. According to molecular data, the symbiotic bacterium of S. huense sp. n. is closely related to Xenorhabdus stockiae.

Several taxonomic studies of the entomopathogenic nematode family Steinernematidae have reported on the minute tail projections on the tail tips of females of the first generation; however, the occurrence and morphology of these traits in other steinernematid species and their taxonomic significance are still unclear. In the present study, the tail projections in females of seven steinernematid species belonging to four phylogenetic clades were examined. The projections are present in all of the tested species and their number and shape is consistent within species but significantly differs among species. It thus seems that these structures may have taxonomic significance at the species level. Based on these results, we suggest that every description of a new species in the family Steinernematidae should contain a precise morphology of the female tail, focused on these projections, using both light and scanning electron microscopy.

The efficacy of different concentrations of the commercial neem-based insecticide, Nimbicidine® and the ecdysone agonist compound Methoxyfenozide (RH-2485), was evaluated against larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). RH-2485 and Nimbicidine® significantly (p 0.05) difference was noticed on total number of eggs laid by the female when lower concentration 0.0001 ppm of RH-2485 was applied in the same stage as compared to the control. In the second part of this study, the invasion rate of Stei...

Entomopathogenic and molluscoparasitic nematodes are important parasites of many insects and molluscs, respectively. Due to their infectivity, the possibility of mass production by industrial techniques and the relative safety to nontarget organisms and environment, these organisms represent an attractive agent for biological control of many pests. This chapter summarises the current knowledge of the diversity of these organisms. In this chapter, we review the recent advances in production, storage, application techniques genetic improvement and safety of these organisms.

A new species of entomopathogenic nematode, Steinernema pwaniensis n. sp., belonging to the glaseri group, is described from Tanzania. The infective juveniles of S. pwaniensis n. sp. are characterized by a body length of 978 μm (808–1131), distance from anterior end to excretory pore of 86 μm (80–95) and a tail length of 87 μm (75–95). The ratios a, c and E% of S. pwaniensis n. sp. are substantially lower than those of all other African ‘glaseri’ group members. The first-generation males of S. pwaniensis n. sp. can be distinguished by a large spicule length of 92 μm (80–97) and by the absence of the caudal mucron, while second-generation males possess a short spine-like mucron. First-generation females have a peg-like tail tip bearing three spine-like projections. Second-generation females can be recognized by a slightly protruding vulva and well-developed post-anal swelling. The new species is further characterized by sequences of the internal transcribed spacer (ITS) and partial 28S regions of the ribosomal DNA. Phylogenetic analyses show that S. pwaniensis n. sp. forms a strongly supported monophyletic clade with two other East African species, S. ethiopiense and S. karii.

The effect of intraspecific competition on the development and reproduction of the slug parasitic nematode Phasmarhabditis hermaphrodita was studied in a series of laboratory experiments. Different inocula concentrations (1, 10, 25, 50, 100, 250, 500 and 1000) of P. hermaphrodita dauer juveniles (DJ) were applied to 0.02 g of homogenised slug Deroceras reticulatum on agar in multi-well plates and the development time, yield, lipid content, and body length of females and DJs were recorded. There was an inverse relationship between dose and recorded female length or lipid content. In newly emerged DJs the body length and lipid content were relatively invariable up to a dose of 250 DJs (ranging between 850 and 925 µm, 6.8 and 7.4, respectively), and decreased at higher doses (500 and 1000 DJs). The yield increased with increasing dosage to its optimum at a dose of 100 (200,000 DJs per 1 g of substrate) DJs and thereafter decreased. In the treatment with the highest dose (1000 DJs), only a minor port...

In the present study the response of Phasmarhabditis hermaphrodita to different cues of Deroceras reticulatum was examined on agar plates and in sand filled olfactometers. The entomopathogenic nematode (EPN) Steinernema feltiae and the cues of Galleria mellonella were used as a reference. The movement of P. hermaphrodita in both arenas was more intensive in comparison to S. feltiae. On the agar plates it was significantly attracted to D. reticulatum both as cadavers and in homogenized form. Slug feces, mucus and the feces of G. mellonella caused a high, but non-directional, reaction. In olfactometers the slug cues evoked a markedly higher reaction, albeit non-directional, in the case of living slugs and feces, and a negative reaction in the case of slug cadavers and homogenized slugs. We demonstrate that P. hermaphrodita is well able to detect host associated volatile cues and move in mineral substrate. S. feltiae was attracted to both insect and slug associated cues and its strong attraction to slug cadavers suggests that EPNs could also scavenge on carcasses other than those of insects.

A population of Oscheius chongmingensis was isolated from a soil sample collected from an alfalfa field in the vicinity of Hailar, Inner Mongolia, and was designated as the Tumian strain. This nematode has a facultative entomopathogenic activity and reproduces on the Galleria mellonella larvae. We compared our isolate with the original description of O. chongmingensis, checked several paratype slides of this species and compared it with cultures of the original Shanghai strain. Poor morphology, morphometrics and some errors in the original description of O. chongmingensis encouraged us to re-describe this species. The species is characterised by having five distinct ridges in the lateral field in adults.. The stoma of O. chongmingensis is long and narrow, in adults approximately 3.5-4.5 times as long as wide, whereas in dauer juveniles it may be 6-7 times longer than wide. Males possess a ventral pair of small bristle-like sensilla posterior to the cloacal opening. Both the Tumian and original strain are amphimictic with a higher proportion of females, but males are not rare (ca 2.5:1).

Three isolates (Dero-1, Dero-8 and Mosisa-1) of a new entomopathogenic nematode, S. ethiopiense sp. n., were isolated by baiting soil samples from the Mendi area, Western Wollega, Ethiopia, with last instar wax moth larvae Galleria mellonella. Infective juveniles of S. ethiopiense sp. n. have a body length of 898 (768-1010) μm, a maximum of eight identical ridges (i.e., nine lines) in the lateral field, excretory pore located at mid-pharynx, hyaline layer occupying approximately half of the tail and c′ = 3.2. First generation males lack a caudal mucron, whereas second generation males possess a short spine-like mucron. The spicules are slightly arcuate, golden-brown in colour and have an ellipsoid or oblong manubrium. First generation females lack a postanal swelling and have a minute protuberance on the tail tip whereas second generation females have a postanal swelling and protruding vulva. Based on the morphology, morphometrics and DNA analysis, the new species belongs to the glaseri group. The closest relative species is the afro-tropical S. karii recorded from Kenya. The BLAST analysis of the ITS region of the rDNA revealed a similarity of 93% with S. karii, supporting the validity of S. ethiopiense sp. n. as a new species. In the phylogenetic trees the new species groups together only with S. karii (bootstrap value of 100%), but is also separated from S. karii by a bootstrap value of 100% or 70%.

Various aspects of the coexistence of two sympatric entomopathogenic nematode species Steinernema affine and Steinernema kraussei were studied in the field and laboratory. The seasonal dynamics of both nematodes and local insects, and nematode vertical and horizontal distribution were observed in the field in 2007. A series of subsequent laboratory experiments studied the host range of both species and their competition in selected experimental and natural hosts. S. affine was dominant in the natural habitat reaching a mean density of 25,000 infective juveniles per square meter and being several times more abundant than the latter species. Both nematode species had a patchy distribution and occurred in all vertical strata of the tested upper 10 cm soil profile, however S. affine was more abundant in the top 4 cm whereas S. kraussei preferred lower soil layers (6–10 cm). No evident spatial relationship between the two species was found. In laboratory bioassays both species were able to attack and multiply in the same spectrum of local insects from various dipteran and coleopteran families that probably serve as natural hosts of entomopathogenic nematodes. No evidence suggesting any host differentiation between the two species was found. Competition experiments showed a strong dominance of S. affine in all tested insects and its superiority appears to be universal. Generally it seems that both species share an ecological niche and thus the avoidance of competition with the latter species seems to be a crucial factor for S. kraussei. Patchy distribution and implicit differences in horizontal distribution probably markedly contribute to the coexistence of both species.

In the past 70 years, EPN research in the Czech Republic has contributed to the field with a number of important achievements, especially in the taxonomy of entomopathogenic nematodes (EPNs). Leading persons involved have been insect pathologist Dr. Jaroslav Weiser and his pupil Dr. Zdeněk Mracek. In this short overview, we will focus on the most important advances in EPN research provided by investigations in the Czech Republic.

The genus Alloionema (Rhabditida: Alloionematidae) currently includes three nominal species—the type species, Alloionema appendiculatum, and two recently described species: A. similis and A. californicum. Comparison of morphological, molecular and ecological characters demonstrates that A. similis and A. californicum belong to the same species, providing support for their synonymization, as proposed in this publication.

Ptilodactyla crenatostriata Redtenbacher, 1868 (Coleoptera: Byrrhoidea: Ptilodactylidae) described from Rio de Janeiro, Brazil, is transferred to the genus Phytocerum Costa, Vanin, Lawrence & Ide, 2003 (Coleoptera: Elateroidea: Cerophytidae). Based on the examination of the holotype, deposited in the Naturhistorisches Museum Wien, Austria, Phytocerum crenatostriatum comb. nov. is redescribed and its main diagnostic characters are figured. It is similar to P. birai Costa, Vanin, Lawrence & Ide, 2003 and P. cayennense (Bonvouloir, 1870). An updated checklist of the extant and fossil Cerophytidae of the World is provided.

Entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae (Rhabditida, Nematoda) are obligate pathogens of insects and are associated with specific symbiotic bacteria of the genera Xenorhabdus and Photorhabdus , respectively. Due to their ability to infect various insects, the possibility of mass production by industrial techniques, and safety to non-target organisms and the environment, these nematode-bacteria complexes are attractive agents for the biological control of many insect pests. In this chapter, a brief characteristic of these organisms is given, together with information on their use in the biological control of insect pests.

We determined the complete mitochondrial genome of the facultative entomopathogenic nematode Oscheius chongmingensis. The mitogenome length was 15,413 bp and similar to other Rhabditids contains genes for 2 rRNAs, 22 tRNAs, and 12 proteins (ATPase subunit 8 is missing). Predicted tRNAs indicated the secondary structure typical for chromadorean nematodes. Gene order is similar to that observed in the genus Caenorhabditis. The control AT-rich region is considerably large (2061 bp, 84% of AT), positioned in between tRNAAla and tRNAPro and has several microsatellite-like (AT)n elements.

Phasmarhabditis bonaquaense n. sp. is described and illustrated from the body of Malacolimax tenellus , from the locality of Ceske Svýcary near the village of Dobra Voda, the Czech Republic. Females are characterized by a body length of 2349 (1878–2626) µm and a cupola shaped tail with a long hyaline hair-like tail tip. Extremely prominent papilla-like phasmids present. Males 1829 (1414–2121 ) µm long. Peloderan bursa with nine pairs of rays (papillae), 1/1/1/2/1/3. One non-paired apparent papilla-like structure located near the ventral appendage anterior to the cloaca. Prominent papilla-like phasmids located close to the tail tip. Small subunit (18S), ITS, and D2-D3 expansion segments of the large subunit of ribosomal DNA were used to analyze the phylogenetic relationships of sequenced species in the genus Phasmarhabditis and other closely related species. Phasmarhabditis bonaquaense n. sp. varied from other related nematodes both in morphological characterizations and phylogenetic analysis. The life cycle of the newly described species is not well known but it is probably a facultative, mollusc-parasitic nematode able to survive permanently in the saprobic phase on decaying organic matter.

The Colorado potato beetle, Leptinotarsa decemlineata, has developed resistance to most registered pesticides and has become one of the most difficult insect pests to control. Development of new biopesticides targeting this pest might solve the resistance problem and contribute to sustainable crop production. Laboratory experiments were conducted to assess the efficacy of Isaria fumosorosea (syn. Paecilomyces fumosoroseus) strain CCM 8367 against L. decemlineata when applied alone or combined with the entomopathogenic nematode Steinernema feltiae. The last-instar larvae of the Colorado potato beetle showed the highest susceptibility to I. fumosorosea followed by pre-pupae and pupae. The median lethal concentration (LC50) was estimated to be 1.03×106 blastospores/ml. The strain CCM 8367 was more virulent, causing 92.6% mortality of larvae (LT50 = 5.0 days) compared to the reference strain Apopka 97, which caused 54.5% mortality (LT50 = 7.0 days). The combined application of the fungus with the nematodes increased the mortality up to 98.0%. The best results were obtained when S. feltiae was applied simultaneously with I. fumosorosea (LT50 = 2.0 days); later application negatively affected both the penetration rate and the development of the nematodes. We can conclude that the strain CCM 8367 of I. fumosorosea is a prospective biocontrol agent against immature stages of L. decemlineata. For higher efficacy, application together with an entomopathogenic nematode is recommended.

Living and freeze-killed natural and laboratory hosts, with different susceptibility to entomopathogenic nematodes, were exposed to the larvae of Steinernema affine and Steinernema kraussei in two different experimental arenas (Eppendorf tubes, Petri dishes), and the success of the colonisation and eventual progeny production were observed. Both nematodes were able to colonise both living and dead larvae of Galleria mellonella (Lepidoptera) and adult Blatella germanica (Blattodea) even though the progeny production in dead hosts was lower on average. Living carabid beetles, Poecilus cupreus, and elaterid larvae (Coleoptera) were resistant to the infection, however, both nematodes were able to colonise and multiply in several dead P. cupreus and in a majority of dead elaterid larvae. By scavenging, EPNs can utilise cadavers of insects that are naturally resistant to EPN infection, and so broaden their host range.

The interactions of two sympatric entomopathogenic nematodes Steinernema affine and Steinernema. kraussei were studied in a series of laboratory experiments. Single species, simultaneous and sequential infections of Galleria mellonella were performed in Eppendorf tubes and the invasion rate, nematode progeny production and the number of hosts producing nematode progeny were observed. The invasion rate of S. affine was not affected by the mixed infection whereas the invasion of the latter species was strongly reduced. S. affine out-competed S. kraussei in all treatments and the progeny production of the latter species occurred only sporadically. In comparison to single species infections, per-host progeny production of S. affine was affected only in the treatments with a low dose of S. affine, where it was 30–50% lowered. Generally, in the presence of the latter species, S. affine was able to infect and multiply in a higher number of hosts in comparison to single species infection, especially at a low inoculation rate. S. affine invaded and multiplied also in hosts already infected and even killed by S. kraussei producing a normal amount of progeny. Generally the results suggest that the interactions between steinernematid species can be more complex, including a positive effect of one species on another.

During a random survey of entomopathogenic nematodes in the provinces of Sichuan and Gansu (eastern Tibet) in 2004, soil samples from several sites were collected and tested for the incidence of entomopathogenic nematodes. A new species was collected in this survey and it is described herein as Steinernema cholashanense n. sp. Steinernema cholashanense n. sp. is characterized by morphology and morphometry of the IJ and male. For the IJ, the new species can be recognized by the average body length 843 microm, esophagus length 125 microm, H%=39% and E%=81%. The lateral field pattern is 2, 5, 7, 4, 2. The male of the first generation is characterized by spicule shape and length and especially with prominent velum and the presence of a mucron on both generations. The average body length of the IJ of S. cholashanense n. sp. (843 microm) is shorter than that of S. oregonense (980 microm), S. kraussei (951 microm) and S. litorale (909 microm), similar to that of S. feltiae (849 microm), but longer than that of S. weiseri (740 microm), S. jollietti (711 microm) and S. hebeiense (658 microm). Esophagus length of the new species (125 microm) is closer to that of S. jollieti (123 microm) but longer than that of S. weiseri (113 microm) and shorter than that of S. oregonense (132 microm), S. kraussei (134 microm) and S. feltiae (136 microm). E% of the new species (81) is similar to that of S. kraussei (80), but smaller than that of S. jollieti (88), S. weiseri (95), S. oregonense (100) and S. feltiae (119). Spicule head length of the new species is almost the same as its width, this character is similar to that of S. kraussei but it is different from this species by its prominent velum. The new species can be recognized further by characteristics of sequences of ITS and D2D3 regions and cross hybridization with closely related species, S. feltiae, S. kraussei and S. oregonense.

The effect of dry conditions on the population dynamics of the entomopathogenic nematode (EPN) Steinernema affine was studied for one month in the exceptionally dry period in the summer of 2003 in the oak wood in the vicinity of Ceske Budejovice, Czech Republic. Soil moisture, soil temperature, and the abundance of suitable insect hosts were monitored. The abundance of infective juveniles (IJs) was correlated with soil moisture and both these values were gradually decreasing during the study period and finally rapidly increased at the end of the investigation. During this period there was a decline in the number of insects suitable as hosts for S. affine, but not in numbers of unsuitable insects. We hypothesise that the observed decrease in IJ numbers was probably caused by the persistence of IJs in host cadavers due to low ambient moisture.

The seasonal dynamics of entomopathogenic nematodes (EPNs) of the genus Steinernema and Heterorhabditis were studied during one season in meadow and oak wood habitats, in the vicinity of Ceske Budejovice, Czech Republic. The influences of soil temperature, moisture, and abundance of suitable hosts on EPN dynamics were investigated. The host range of these nematodes, in both habitats was also observed. A total of four EPN species were found in both habitats. Steinernema affine was the dominant species both in oak wood and in meadow. Additionally, the oak wood habitat was inhabited by S. kraussei and S. weiseri; the meadow habitat by Heterorhabditis bacteriophora. The mean abundance of total EPN community was 28,000ind./m(2) in oak wood and 11,000ind./m(2) in meadow. The seasonal dynamics of entomopathogenic nematodes in both habitats were characterized by high nematode densities in the beginning of the season, followed by a rapid decrease, and then stabilization. EPN abundances did not show any apparent correlation with soil temperature and moisture, but they were negatively correlated with the abundance of suitable insect hosts. Inter- and intraspecific competition for limited nutrients (hosts) probably played a major role in EPN seasonal dynamics. Broad host range of entomopathogenic nematodes in both habitats was predominantly represented by dipteran and coleopteran larvae. Most common hosts belonged to the families Asilidae, Bibionidae, and Empididae (Diptera), as well as Carabidae and Curculionidae (Coleoptera).

In der stabilisierten Kultur von PK-Zellen wurde eine 53 Std 45 min dauernde Amitose und in der Deckglaskultur eines embryonalen Mausskeletmuskels eine etwa 31 Std dauernde Kernknospung beobachtet. In beiden Fallen wurden die Zellen 3 Tage nach der beendeten Teilung beobachtet. Es kam nicht zu einer Teilung der Zellen, die keine Unterschiede gegenuber den ubrigen Zellen der Kultur aufwiesen. Insbesondere waren keine Degenerationszeichen auf ihnen zu beobachten. Einige Zellen derselben Kulturen teilten sich mitotisch.