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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.

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.

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.

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.

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.

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.

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...

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%.