Steinernema Spp Research Articles

Exploration of Entomopathogenic Nematodes in Organic Rice Field in Sleman Regency

Entomopathogenic nematodes (EPNs), consisting of genera Steinernema and Heterorhabditis are biological agents proven effective in killing insect pests. Organic rice fields and sandy soil textures are potential areas for exploring the presence of EPNs. This research aims to determine the diversity of genera and populations of entomopathogenic nematodes in organic rice fields in Sleman Regency. The study was conducted on organic rice fields and the UPN "Veteran" Yogyakarta Plant Protection Laboratory from July – August 2023. EPNs were obtained from Prambanan Sleman and Ngemplak Sleman, with six plots as sample units in every location. EPNs were isolated from soil samples by using Tenebri molitor larvae. The Whitehead tray method was used to isolate EPNs from the dead larvae, and the population of EPNs was counted. EPNs were identified based on the symptoms of color changes on the cuticle and morphological characters. The result showed that the cuticles of T. molitor larvae that died were blackish brown in Prambanan and Ngaliyan isolates, referred to Steinernema genera. EPNs were found in Prambanan_6, Ngaliyan_1, and Ngaliyan_2. The highest population of EPNs was on Ngaliyan_1 (38,00 EPNs/ml), and the lowest was on Prambanan_6 (2,13 EPNs/ml). Based on the morphological characters of EPNs, on the Prambanan_4, Ngaliyan_1, and Ngaliyan_2, EPNs were identified as Steinernema spp. The characteristics of Steinernema in the infective juvenile stage found are: slender body, anterior end slightly rounded and fused with the body, cylindrical stoma, no second cuticular sheath, not annulated, and conoid tail.

Isolation, Identification and Antimicrobial Activities of Four Bacteria Associated with Entomopathogenic Nematodes, Steinernema spp.

Recently, the search for novel products derived from natural sources has become necessary due to the decreasing effectiveness of current antibiotics in treating bacteria that are antibiotic-resistant. In this context, it is well known that entomopathogenic bacteria (EPBs) produce a broad range of secondary metabolites with antibacterial activities. Therefore, an in-vitro trial was conducted to isolate and identify non-symbiotic bacteria associated with entomopathogenic nematodes, Steinernema spp. and evaluate the antibacterial activity against four antibiotic-resistant bacteria. Four bacterial isolates, i.e. Pseudomonas alcaligenes ST-1, Paenibacillus barcinonensis ST-2, Bacillus mojavensis ST-3, and Bacillus megaterium ST-4 were non-symbiotically isolated from the heamolymph of dead Steinernema-infected Galleria mellonella larvae and molecularly characterized. The bacterial cells and filtrates from P. alcaligenes ST-1 strongly inhibited the growth of Staphylococcus aureus through disk diffusion (43 mm), minimum inhibitory concentration (2.5 µL/mL), and minimal bactericidal concentration (5 µL/mL) assay. Conclusively, the direct application of endogenous Steinernema-associated EPB as an antibacterial agent for antibiotic-resistant bacteria looked promising.

Biocontrol Potential of Four Indigenous Entomopathogenic Nematodes From Kenya

Globally, vegetable production including tomato is constrained by pests and diseases. The tomato leafminer, Tuta absoluta (Meyrick.), is a major pest of tomato, mainly managed using chemical pesticides. There is a need for integrated pest management (IPM), approaches that are human and environmentally friendly. The study aimed at molecular identification and virulence evaluation of four entomopathogenic nematodes (EPNs) against T. absoluta larvae. The DNA was extracted, sequenced, and phylogeny analyzed. The EPNs virulence was evaluated using the insect baiting technique at different nematode concentrations (0-Control, 100, 150, 200, and 250 infective juveniles/ml of distilled water). Larval mortality data were collected for five days. Molecular identification resulted in nucleotide sequence lengths of 877-895 base pairs (bp). All the isolates were found to be Steinernema species of EPNs. The % similarity of the isolates and their close relatives ranged between 82-100%. The isolates closely matched Steinernema spp. AY230184 (Sri Lanka); AY230186 (Kenya), JN651414 (Ethiopia), and MW151701 (Kenya). The sequences were deposited to Genbank as Steinernema sp. Isolate Kalro 75 (MW150871), Steinernema sp. Isolate Kalro S86 (MW150872), Steinernema sp. isolate Kalro97 (MW150873) and Steinernema sp. Isolate KalroR52 (MW150874). All the EPN isolates were virulent against T. absoluta. Isolate Kalro R52 and Kalro S86 recorded the highest mortality of 76±7.5%; 72±15.0% respectively in the 48 h. There was a significant difference (P < 0.001), between all the isolates and the Control. The four EPNs were virulent and have bio-control potential against, T. absoluta. Research on their virulence against T. absoluta in the field is recommended.

Efficacy of Entomopathogenic Nematodes Against Western Flower Thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), Under Laboratory Conditions

Western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is an important field and greenhouse pest of many crops worldwide. To control F. occidentalis is extremely challenging, because of its cryptic behaviour, short life cycle, and resistance to many insecticides. The life history and success rate of WFT on chrysanthemum (Dendranthema grandiflora) leaflets and green bean pods (Phaseolus vulgaris) showed that more first instar larvae hatched, and developed faster, with a higher survival rate, on the former than on the latter, making chrysanthemum a more attractive and suitable host than were the green beans. A total of 11 local South African entomopathogenic nematode species and the exotic Steinernema feltiae, were tested, under laboratory conditions, for pathogenicity against WFT. Generally, Heterorhabditis spp. were more virulent than were the Steinernema spp. The study showed that locally isolated Steinernema yirgalemense (66 %), Heterorhabditis baujardi (67 %), and Heterorhabditis bacteriophora (60 %) had potential for the control of F. occidentalis, in terms of targeting its soil-dwelling stages. Results from the temporal development study showed that both S. yirgalemense and H. baujardi were able to complete their life cycles in the host, and to produce a new cohort of infective juveniles.

MASS PRODUCTION OF ENTOMOPATHOGENIC NEMATODES OF LOCAL ISOLATES AS BIOLOGICAL CONTROL AGENTS OF COFFEE BERRY BORER (Hypothenemus hampei Ferr.)

Entomopathogenic Nematodes (EpNs) that serve as biological control agents include Steinernema spp and Heterorhabditis spp. EPNs Heterorhabditis indicus (Bromo Isolate) has a high toxicity against larvae Lepidoptera and Coleoptera and was successfully developed for mass production in Biological Control Laboratory of Jember University. These nematodes will be formulated as solid and liquid biopesticides. This research aims: to find EPNs local isolates from smallholder coffee plantations; to identify local and in vivo and in vitro cultured EPNs isolates; to determine the pathogenicity of EpNs local isolate against Coffee Berry Borrer (CBB) Hypothenemus hampei Ferr and to formulate EpNs in vitro culture as a liquid and solid formula. These formula were tested for the patogenicity and viability of EpNs to the larvae of H. hampei, Tenebrio molitor and Galleria melonella. The results showed that the pathogenecity of EpNs isolate to the larvae of CBB in Silo was 30% after 24 hours and 90% after 48 hours of in vivo inoculation. However, the mortality of CBB larvae was only 10% by liquid spraying on the coffee berry. The viability was 524 IJ (Infective Juvenile) on liquid formula packed on polyurethane sponge, and this was higher than that on solid formula (330 IJ).

Study of Steinernema hermaphroditum (Nematoda, Rhabditida), from the West Uttar Pradesh, India.

The entomopathogenic nematodes have been reported from all continents (except Antarctica) and almost all regions of the world. Surveys of EPNs in India has resulted in the recovery of several isolates of Steinernema. Among one of them, isolate CS34 was identified as S. hermaphroditum Stock, Griffin & Chaerani, 2004. We investigated the identification and the pathogenicity of S. hermaphroditum in District Meerut of Western Uttar Pradesh, India. The Steinernema was examined for its pathogenicity and accurate identification by the mean of morphological and molecular technique and its geographical distribution was mapped based on meta-analysis of the ITS GenBank records. The surveys of agricultural soils of district Meerut, India, resulted in the isolation of one strain from entomopathogenic nematode labelled CS34 through Galleria baiting technique. Morphological characters and morphometrical analysis indicated that the strain CS34 was closely related to the "glaseri" group of Steinernema spp. The Nblast results indicated that ITS rDNA sequence had no nucleotide differences in comparison with the S. hermaphroditum (JQ687355). However, one variation in the D2-D3 segment of 28S rDNA was observed in comparison with the AY598358. The phylogenetic analysis using ITS and 28S rDNA indicated that the Indian S. hermaphroditum could be placed together with other S. hermaphroditum, with strong posterior probability. Besides, the PCA analysis demonstrated some variability within the test populations. The distribution of S. hermaphroditum based on meta-analysis of the GenBank records showed its presence in the three Asian countries-India, Thailand and Indonesia. The Indian strain of S. hermaphroditum also tested positively for its virulence against three major pests, namely, Galleria mellonella, Helicoverpa armigera, and Spodoptera litura, with resultant which showed good efficacy on the mortalities. In conclusion, the economy of India is agriculture-based, but there are huge losses due to different insect pests infesting different crops. Steinernema hermaphroditum CS34 is an indigenous species to Indian subcontinent and efforts should be made to evaluate its virulence and pathogenicity against the other agricultural pests hampering productivity throughout the country. This may lead to incorporate S. hermaphroditum strain CS34 as a regular biological control agent against important lepidopteran pest in integrated pest management programs in the future.

Entomopathogenic Nematodes (EPN): Diversity in Indian Tropical Sugarcane Ecosystem and Its Biocontrol Potential Against White Grub Holotrichia serrata F. on Sugarcane

The root-feeding larvae of white grub Holotrichia serrata F. (Coleoptera: Scarabaeidae) causes serious damage in sugarcane fields in India. Generally, the damage caused by white grubs is in patches, but during severe outbreaks the entire crop in a field may be dried up. Entomopathogenic nematodes (EPN) have proven to be the safest and most effective way of controlling white grubs as both organisms spends a large portion of their life cycle in the soil. To ascertain the diversity of EPN, a survey was conducted in the white grub endemic areas of sugarcane ecosystem in the tropical India. Isolation, molecular identification of EPN and laboratory bioassays was conducted at Nematology Laboratory, ICAR-Sugarcane Breeding Institute, Coimbatore, India. Twenty-four EPN were isolated out of 243 samples, and frequency of occurrence of EPN was 9.87%. Among the 24 positive samples, eight EPN (three Heterorhabditis and five Steinernema) were isolated from dead larvae of naturally infested third instar white grub H. serrata. Out of 24 positive samples, 14 (58.3%) were heterorhabditis and 10 were (41.6%) sterinernematids. Sequencing and characterisation of the internal transcribed spacer region was used to identify all nematode isolates to species level. Out of 14 Heterorhabditis isolates, eight have grouped with H. indica in the indica-group and remaining six were unknown species. Out of 10 Steinernema isolates, two were grouped with Steinernema siamkayai and two isolates grouped close to S. backanense, while the remaining six unknown isolates clustered closely with S. affine, but these isolates form a separate cluster which is genetically different from other group of Steinernema spp. Bioassay studies were conducted under laboratory conditions against first, second and third instar larvae of white grub H. serrata. Among the five H. indica isolates, H. indica (DSM78) recorded maximum mortality of the grubs and low median lethal dose (LD50) value for all the three instars of white grub. Two field trials were conducted during June 2011 to evaluate the efficacy of H. indica (DSM78) against naturally infested white grub H. serrata sugarcane fields. In both the trials, reduction in number of grubs observed in the EPN treated plots compared to the control. The reduction in grub population due to EPN treatment ranged between 43.5 and 77% at field trial I and 30–78% at field trial II. The results of the present study clearly indicated that EPN, H. indica isolate DSM78 was found to be effective and a potential EPN isolate against all the three instars of white grub larvae under laboratory bioassays as well as in the field conditions.

Comparison of the methods applicable for the pathogenicity assessment of entomopathogenic nematodes

Single infective juveniles of Heterorhabditis bacteriophora, H. megidis (Nematoda: Heterorhabditidae), Steinernema arenarium, S. carpocapsae and S. feltiae (Nematoda: Steinernematidae) were used to infect single Galleria mellonella (Lepidoptera: Pyralidae) larvae. Four parameters of entomopathogenic nematodes pathogenicity were assessed: the mortality of insects, infectivity of nematodes, number of nematodes established per single G. mellonella, and degree of infective juveniles colonization (percent of infective juveniles which intestine was colonized by symbiotic bacteria). The accuracy, repeatability, and versatility for different species of EPNs in bioassay arenas were compared. Our modifications of the original methods yielded ~ 50% higher efficiency of infective juveniles in cell culture plates and > 20% higher efficiency in centrifuge test tubes. The efficiency of nematodes in cell culture plates (39–77%) was relatively low, especially in the case of Heterorhabditis spp. In the bioassay arena, infective juveniles migrated between cells. The results of our studies indicate that the pathogenicity of EPNs should be assessed in centrifuge test tubes. In these arenas, the infectivity of single IJs was ~ 90% for Heterorhabditis spp. and ~ 95% for Steinernema spp. The degree of colonization of the EPN isolates by symbiotic bacteria was in the range of 96–98%.

Susceptibility of cockroaches (Gromphadorhina portentosa, Nauphoeta cinerea and Blaptica dubia) exposed to entomopathogenic nematodes

ABSTRACTCockroaches are major pests, vectors of pathogenic bacteria and induce allergies. Current control methods use chemical pesticides, but they can be ineffective and costly and there are reports of resistance in the field; hence new control methods are needed. There are conflicting reports about the susceptibility of cockroaches to entomopathogenic nematodes (EPNs); so we investigated if EPNs could kill several diverse cockroach species, including the Madagascan hissing roach (Gromphadorhina portentosa), the Lobster roach (Nauphoeta cinerea) and Blaptica dubia. Female adult cockroaches were exposed to either commercial products containing Steinernema kraussei or a combination of Heterorhabditis spp. and Steinernema spp. at 50 and 150 nematodes per cm2 for 21 days. We also monitored feeding and the numbers of infective juveniles that were produced from each cockroach corpse. We found that S. kraussei were harmless to all cockroach species (at both doses) but when exposed to a mixture of Heterorhabditis spp. and Steinernema spp. B. dubia died after 6 days and its feeding was strongly inhibited. We also found that the mixture of Heterorhabditis spp. and Steinernema spp. could proliferate in the cadavers of B. dubia whilst S. kraussei could only reproduce in G. portentosa and B. dubia but not N. cinerea. In conclusion, S. kraussei was harmless to all three cockroach species but B. dubia was killed when exposed to Heterorhabditis spp. and Steinernema spp., highlighting the differences in the host range of EPNs.

Pathogenicity of Selected Kenyan Entomopathogenic Nematodes of Genus Steinernema against Banana Weevil (Cosmopolites Sordidus)

Banana weevil is a pest with a great impact on banana production in the world. The objective of this study was to evaluate the pathogenicity of the Kenyan entomopathogenic nematodes (EPNs) of genus Steinernema against the weevil Cosmopolites sordidus (Germar) under laboratory conditions. Adult weevils were trapped from banana fields in Maragua, Central Kenya and larvae extracted from the infested banana rhizomes. The adults were treated with three Kenyan EPNs Steinernema weiseri, Steinernema yirgalemense and the new Steinernema spp and with Steinernema carpocapsae ALL strain as a standard and plain distilled water as a control in five replicates. The treatments were 500ijs, 750ijs and 1000ijs per adult on petri dishes and 1000ijs, 3000ijs and 5000ijs per adult on pseudo-stems. The treatments for larvae in petri dishes were 300ijs, 400ijs and 500ijs. The adults were not susceptible to all the nematodes at all doses and preparations while larvae were highly susceptible to the four test EPNs at all concentrations. There was a significant difference at P<0.05 in the mortality of larvae between nematode Steinernema carpocapsae and Steinernema weiseri. A significant difference was observed between nematode Steinernema carpocapsae and Steinernema yirgalemense. There was also a significant difference between the new Steinernema spp and Steinernema weiseri and finally in the two nematodes the new Steinernema spp and Steinernema yirgalemense. All the test nematodes caused over 90 % larval mortality within 48 hours. The mean percent mortality of larva increased with nematode concentration for all the test nematodes. The results indicated that the four test EPNs significantly (P<0.05) caused high mortality within 24-48 hrs. The findings indicate that the banana weevil larvae are susceptible to the local entomopathogenic nematodes and potentially useful in the management of the banana weevil. It is however recommended that more research be conducted on the formulation and application technology to enhance their effectiveness in the field.

Interference competition in entomopathogenic nematodes: male Steinernema kill members of their own and other species

There is evidence of competition within and between helminth species, but the mechanisms involved are not well described. In interference competition, organisms prevent each other from using the contested resource through direct negative interactions, either chemical or physical. Steinernema spp. are entomopathogenic nematodes; they enter a living insect host which they kill and consume with the aid of symbiotic bacteria. Several studies have demonstrated intra- and interspecific competition in Steinernema, mediated by a scramble for resources and by incompatibility of the bacterial symbiont. Here we describe a mechanism by which male Steinernema may compete directly for resources, both food (host) and females, by physically injuring or killing members of another species as well as males of their own species. A series of experiments was conducted in hanging drops of insect haemolymph. Males of each of four species (Steinernemalongicaudum, Steinernemacarpocapsae, Steinernemakraussei and Steinernemafeltiae), representing three of the five phylogenetic clades of the genus, killed each other. Within 48h, up to 86% of pairs included at least one dead male, compared with negligible mortality in single male controls. There was evidence of intraspecific difference: one strain of S. feltiae (4CFMO) killed while another (UK76) did not. Males also killed both females and males of other Steinernema spp. There was evidence of a hierarchy of killing, with highest mortality due to S. longicaudum followed by S. carpocapsae, S. kraussei and S. feltiae. Wax moth larvae were co-infected with members of two Steinernema spp. to confirm that killing also takes place in the natural environment of an insect cadaver. When insects were co-infected with one infective juvenile of each species, S. longicaudum males killed both S. feltiae UK76 and Steinernema hermaphroditum. Wax moths co-infected with larger, equal numbers of S. longicaudum and S. feltiae UK76 produced mainly S. longicaudum progeny, as expected based on hanging drop experiments.

Steinernema tophus sp. n. (Nematoda: Steinernematidae), a new entomopathogenic nematode from South Africa.

A new entomopathogenic nematode, Steinernema tophus n. sp. is described from South Africa. Morphological, molecular (ribosomal gene sequence data) together with cross-hybridization studies were used for diagnostics and identification purposes. Both molecular and morphological data indicate the new species belongs to the 'glaseri-group' of Steinernema spp. Key morphological diagnostic traits for S. tophus n. sp. include the morphology of the spicules and gubernaculum. Morphometric traits of third-stage infective juveniles, including total body length (average 1,046 µm), tail length (average 70 µm), location of the excretory pore (average 92 µm), D% (average 63), E% (average 132) and H% (average 32) values are definitive. In addition to these morphological characters, analysis of rDNA (28S and ITS) gene sequences depict this Steinernema species as a distinct and unique entity.

Effects of Soil Chemical Characteristics on the Occurrence of Entomopathogenic Nematodes

Aims: This study was conducted to determine the effect of selected soil chemical characteristics on the occurrence of entomopathogenic nematodes (EPNs) under different land uses in Embu and Taita districts in Kenya. Study Design: The sampling points were systematically marked in a grid-mesh construction using GPS marking. Place and Duration of Study: Soil sampling was done between January 2008 and May 2008 in Embu district in the highlands of Central and Taita-Taveta district in the Coastal Original Research Article British Journal of Applied Science & Technology, 4(16): 2333-2343, 2014 2334 highlands of Kenya. Methodology: EPNs were baited from soil using Galleria mellonella larvae and infective juveniles identified using morphologicalbiometric characteristics. The nematode occurrence was evaluated through relative abundance and recovery frequency expressed as percentage from the soils. Results: EPNs were detected in 43.3% of the samples with Steinernema spp being the dominant species. The occurrence of EPNs is affected by selected soil chemical properties, land use systems and heavy metals. Conclusion: Soil fertility management practices and heavy metals influence the occurrence of EPNs and should be considered for their effective use as biological control agents.

Steinernema vulcanicum n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode species from Sicily (Italy)

AbstractA new species of entomopathogenic nematode, Steinernema vulcanicum n. sp., is described from the slopes of Mount Etna (Sicily, Italy). It was recovered from soil in a chestnut wood. Molecular and morphological data indicate that the new species belongs to the glaseri group of Steinernema spp. The new species can be distinguished from the other described species of the group by morphological characteristics, crossbreeding tests and by the DNA sequences of ITS1 rDNA, 12S and cox1 mitochondrial loci. Steinernema vulcanicum n. sp. is characterised by the long and slender male spicules, with manubrium wider than long, velum prominent, rounded tip with ventral concavity and tail without mucro. The female has double epiptygmata in both generations. The third-stage infective juveniles have an average body length of 1200 μm, excretory pore generally located level with posterior part of the pharyngeal corpus (D% = 47-68), lateral field pattern of variable formula and hyaline part of tail forming ca 50% of its length. The DNA sequences, life cycle and the endosymbiotic bacterium of S. vulcanicum n. sp., Xenorhabdus kozodoii, indicate the similarity of the new species with S. arenarium, S. apuliae and S. boemarei, from which it can be distinguished by both morphological and molecular characters.

Steinernema boemarei n. sp. (Nematoda: Steinernematidae), a new entomopathogenic nematode from southern France

A new entomopathogenic nematode, Steinernema boemarei n. sp., is described from southern France. Morphological, molecular (28S and ITS rDNA sequence data) and cross-hybridisation studies were used for diagnostics and identification purposes. Both molecular and morphological data indicate that the new species belongs to the 'glaseri-group' of Steinernema spp. Key morphological diagnostic traits for S. boemarei n. sp. include the presence of prominent deirids (cervical papillae) on adult males, the morphology of the spicules and gubernaculum, and the arrangement of the 23 genital papillae of the first generation males. Additionally, morphometric traits of the third-stage infective juvenile, including total body length (mean 1,103 micron), tail length (mean 86 micron), location of the excretory pore (mean 91 micron), and D% (mean 63), E% (mean 106) and H% (mean 41) values are definitive. In addition to these morphological characters, analysis of both 28S and ITS rDNA sequences depict this Steinernema species as a distinct and unique entity.

A new entomopathogenic nematode, Steinernema colombiense n. sp. (Nematoda: Steinernematidae), from Colombia

Abstract A new entomopathogenic nematode, Steinernema colombiense n. sp., is described from Colombia. Morphological, molecular (28S and ITS rDNA sequence data) and cross-hybridisation studies were used for diagnostics and identification purposes. In addition, 28S and ITS rDNA sequence data were used to assess evolutionary relationships of the new species with other Steinernema spp. Morphological diagnostic features for S. colombiense n. sp. include morphometric features of the third-stage infective juvenile, including body length of 636 (549-732) μm, narrow body diam. (31 (22-36) μm), position of the excretory pore (35 (31-40) μm), tail length (41 (32-53) μm), D% = 29 (25-33) and E% = 205 (138-284). In addition, males of first and second generations are characterised by the morphology of the spicules and gubernaculum, the number and arrangement of the genital papillae and the excretory pore position (at 67 (56-76) and 54 (46-63) μm, for first and second generations, respectively). In addition to these traits, 28S and ITS rDNA sequences analyses both showed this species to be a distinct and unique entity.

Biochemical and molecular profiling of indigenous Xenorhabdus isolates associated with Steinernema spp.

Bacterial symbionts were obtained from entomopathogenic nematodes Steinernema carpocapsae, S, riobrave, S. feltiae and S. tami, and identified as belonging to Xenorhabdus species by subjecting to biochemical characterization. The Xenorhabdus isolates were further characterized for their interspecific variation by protein profiling and RFLP analysis of 16S rDNA. The protein profiles recorded discernible differences with protein distribution ranging from 97Kda to 14Kda, but most of the fragments were common to all isolates. RFLP analysis of 16S rDNA of the isolates using eight restriction enzymes showed the distinctness of isolates and based on the restriction enzyme patterns, the isolates were grouped into two clusters. The study showed that a combination of biochemical and molecular techniques could be used for the identification and characterization of Xenorhabdus isolates.

Steinernema costaricense n. sp. and S. puntauvense n. sp. (Rhabditida: Steinernematidae), two new entomopathogenic nematodes from Costa Rica

Steinernema costaricense n. sp. and S. puntauvense n. sp. were recovered during a survey for native entomopathogenic nematodes in Costa Rica. Morphological data, molecular (28S rDNA sequence data) studies and cross-hybridisation tests were used for diagnostic and identification purposes. Additionally, 28S rDNA sequence data were used to assess the evolutionary relationships of the new species with other Steinernema spp. Morphological diagnostic features for S. costaricense n. sp. include: the body size of the infective juvenile (av. 1,696); the presence of protruding 'horn-like' cephalic papillae; the position of the excretory pore in the infective juvenile (av. 77 microm) and the first generation male (av. 117 microm); the D% value of the infective juvenile (av. 53) and the first generation male (av. 56); the E% value of the infective juvenile (av. 85); and the morphology of the spicules and gubernaculum of the first generation male. Diagnostic traits for S. puntauvense n. sp. are: the position of the excretory pore in the infective juveniles (av. 25 microm); the shape and size of the spicules and gubernaculum of the first generation male; and the shape of the tail of the first generation female. In addition to these traits, 28S rDNA sequences analysis and hybridisation tests showed that both new Steinernema species are distinct and unique entities.

Intraspecific variations and phylogenetic relationships of steinernematids isolated from Japan based on the sequences of the ITS region of the nuclear rRNA gene and the partial mitochondrial COI gene

DNA sequences of the internal transcribed spacer (ITS) region of the nuclear rRNA gene and mitochondrial cytochrome oxidase I (COI) gene were used to analyze intraspecific variations and infer phylogenetic relationships of steinernematids isolated from Japan. DNA fragment lengths of the amplified ITS region obtained from 19 Japanese steinernematid isolates ranged from 914-1,067 bp and varied considerably among the species. A few intraspecific variations were detected in Steinernema litorale, Steinernema spp. MY6 and MY8, and no variation was observed in S. monticolum and Steinernema sp. MY5 in the sequences of the ITS region. The fragment length of the amplified partial COI gene was 397 bp for all species. The intraspecific variations of the sequences ranged from 0.3%-7.8% (0-42 nucleotide differences) for S. monticolum, S. litorale, Steinernema spp. MY5, MY6, and MY8. Sequence comparisons and phylogenetic analyses reveal that the sequence of the COI region can distinguish the isolates within a species but is not as effective as the ITS region in elucidating the relationship among species.

A new entomopathogenic nematode, Steinernema robustispiculum n. sp. (Rhabditida: Steinernematidae), from Chumomray National Park in Vietnam

Steinernema robustispiculum n. sp. (Rhabditida: Steinernematidae) was isolated from woodland in Chumomray National Park, Sason, Sathay, Kontum, Vietnam. Its morphology, morphometrics, cross-hybridisation and the ITS-rDNA sequence analysis revealed that S. robustispiculum clearly differs from other known Steinernema spp. As in the cases of S. intermedium (Poinar, 1985), S. robustispiculum has very robust spicules, but it can be distinguished by the longer tail of the infective juvenile, lower E%, shorter spicules, the shape of the spicules, the number of genital papillae in the caudal region and the presence of a mucron on the male tail. S. robustispiculum has a lateral field resembling that of S. sangi Phan, Nguyen & Moens, 2001, but can be distinguished by a higher E%, higher D%, smaller length to width ratio of the spicules and the morphology of both the spicule head (manubrium) and the dorsal lobe of the spicule. The morphometrics of infective juveniles of S. robustispiculum are similar to those of S. monticolum Stock, Choo & Kaya, 1997; these species can be distingusihed by the position of the excretory pore, the smaller length to width ratio of the spicules, and the length and morphology of the spicule head (manubrium). The phylogenetic relationships within Steinernema Travassos, 1927, including the newly sequenced Vietnamese species S. robustispiculum n. sp., S. loci Phan, Nguyen & Moens, 2001, S. thanhi Phan, Nguyen & Moens, 2001 and S. sangi, are presented based on analyses of the ITS-rDNA. The ITS RFLP profiles obtained from 17 different restriction enzymes are also presented.