Steinernema khuongi n. sp. (Panagrolaimomorpha, Steinernematidae), a new entomopathogenic nematode species from Florida, USA.

In this study, molecular (ribosomal sequence data), morphological and cross-hybridization properties were used to identify a new Steinernema sp. from Florida, USA. Molecular and morphological data provided evidence for placing the novel species into Clade V, or the 'glaseri-group' of Steinernema spp. Within this clade, analysis of sequence data of the rDNA genes, 28S and internal transcribed spacer (ITS), depicted the novel species as a distinctive entity and closely related to S. glaseri and S. cubanum. Additionally, cross-hybridization assays showed that the new species is unable to interbreed with either of the latter two species, reinforcing its uniqueness from a biological species concept standpoint. Key morphological diagnostic characters for S. khuongi n. sp. include the mean morphometric features of the third-stage infective juveniles: total body length (average: 1066 µm), tail length (average: 65 µm), location of the excretory pore (average: 80.5 µm) and the values of c (average: 16.4), D% (average: 60.5), E% (average: 126) and H% (average: 46.6). Additionally, males can be differentiated from S. glaseri and S. cubanum by the values of several ratios: D% (average: 68), E% (average: 323) and SW% (average: 120). The natural distribution of this species in Florida encompasses both natural areas and citrus groves, primarily in shallow groundwater ecoregions designated as 'flatwoods'. The morphological, molecular, phylogenetic and ecological data associated with this nematode support its identity as a new species in the S. glaseri-group.

Steinernema innovationi n. sp. (Panagrolaimomorpha: Steinernematidae), a new entomopathogenic nematode species from South Africa.

Morphological and molecular sequence data were combined with cross-hybridization studies and used to identify a new Steinernema sp. from Free State, South Africa. Molecular and morphological data indicate that the new species belongs to the 'glaseri-group' of Steinernema spp. Key morphological diagnostic characters for S. innovationi n. sp. include the morphometric features of the third-stage infective juveniles: total body length = 1054 (1000-1103) µm, tail length = 108 (97-117) µm, location of the excretory pore = 88 (82-91) µm, and D% = 58 (54-63), E% = 115 (104-137) and H% = 43 (37-46). Additionally, the morphology of the spicules and gubernaculum of the first-generation males are considered key diagnostic traits. Steinernema innovationi n. sp. was also characterized by analysis of both rDNA and mitochondrial gene sequence data, which further indicate the uniqueness of this Steinernema species.

Interactions between the entomopathogenic nematode Heterorhabditis sonorensis (Nematoda: Heterorhabditidae) and the saprobic fungus Fusarium oxysporum (Ascomycota: Hypocreales).

In this study, we assessed the effect of the saprobic fungus, Fusarium oxysporum (Ascomycota: Hypocreales) on the fitness of the entomopathogenic nematode Heterorhabditis sonorensis (Caborca strain). Sand column assays were considered to evaluate the effect of fungal mycelia on infective juvenile (IJ) movement and host access. Additionally, we investigated the effect of fungal spores on the nematodes' ability to search for a host, its virulence, penetration efficiency and reproduction. Three application timings were considered to assess interactions between the fungus and the nematodes. In vitro assays were also considered to determine the effect of fungal extracts on the nematode's symbiotic bacteria. Our observations indicate that presence and age of fungal mycelia significantly affect IJ movement in the sand columns and their ability to establish in the host. These results were also reflected in a reduced insect mortality. In particular, treatments with the 15 days old mycelia showed a significant reduction in insect mortality and penetration efficiency. Presence of fungal spores also impacted nematode virulence and reproduction. In particular, two of the application timings tested (simultaneous [EPN and fungal spores applied at the same time] and alternate I [EPN applied first, fungus applied 24h later]) resulted in antagonistic interactions. Moreover, IJ progeny was reduced to half in the simultaneous application. In vitro assays revealed that fungal extracts at the highest concentration tested (10mg/ml) inhibited the growth of the symbiotic bacteria. Overall, these results suggest that saprobic fungi may play an important role in regulating. EPN populations in the soil, and that they may be one of the factors that impact nematode survival in the soil and their access to insect hosts.

Heterorhabditis gerrardi n. sp. (Nematoda: Heterorhabditidae): the hidden host of Photorhabdus asymbiotica (Enterobacteriaceae: gamma-Proteobacteria).

A new entomopathogenic nematode species from Australia, Heterorhabditis gerrardi n. sp. (Nematoda: Heterorhabditidae) is described. Morphological and molecular studies together with cross-hybridization tests indicated that this nematode represents a new undescribed species, closely related to members in the 'indica-group'. However, the new species can be distinguished from other species in this genus by a combination of several qualitative and quantitative morphological traits. Key diagnostic features include: body size and excretory pore position of the third-stage infective juveniles; male bursa with a reduction of bursal rays, usually affecting the terminal set of papillae, with symmetrical or asymmetrical loss of one or two pairs; vulva of hermaphrodites more anteriorly located than in other species in the indica-group (V% average: 43), with non-protruding or slightly protruding lips, and longer tail length (average: 106 mum). The new species can be further characterized by molecular traits of sequence data from the internal transcribed spacer (ITS) region of ribosomal DNA. Additionally, the bacterial symbiont of this new species, Photorhabdus asymbiotica Kingscliff strain, was phenotypically characterized and compared with other P. asymbiotica strains. The Kingscliff strain revealed many characters not present in other strains of this species. We hypothesize that the newly found traits may contribute to the maintenance of this mutualistic association of the bacterium with its nematode host.

Tropical nematode diversity: vertical stratification of nematode communities in a Costa Rican humid lowland rainforest.

Comparisons of nematode communities among ecosystems have indicated that, unlike many organisms, nematode communities have less diversity in the tropics than in temperate ecosystems. There are, however, few studies of tropical nematode diversity on which to base conclusions of global patterns of diversity. This study reports an attempt to estimate nematode diversity in the lowland tropical rainforest of La Selva Biological Research Station in Costa Rica. We suggest one reason that previous estimates of tropical nematode diversity were low is because habitats above the mineral soil are seldom sampled. As much as 62% of the overall genetic diversity, measured by an 18S ribosomal barcode, existed in litter and understorey habitats and not in soil. A maximum-likelihood tree of barcodes from 360 individual nematodes indicated most major terrestrial nematode lineages were represented in the samples. Estimated 'species' richness ranged from 464 to 502 within the four 40 x 40 m plots. Directed sampling of insects and their associated nematodes produced a second set of barcodes that were not recovered by habitat sampling, yet may constitute a major class of tropical nematode diversity. While the generation of novel nematode barcodes proved relatively easy, their identity remains obscure due to deficiencies in existing taxonomic databases. Specimens of Criconematina, a monophyletic group of soil-dwelling plant-parasitic nematodes were examined in detail to assess the steps necessary for associating barcodes with nominal species. Our results highlight the difficulties associated with studying poorly understood organisms in an understudied ecosystem using a destructive (i.e. barcode) sampling method.

Evolution of host search strategies in entomopathogenic nematodes.

There is interspecific variation in infective juvenile behavior within the entomopathogenic nematode genus Steinernema. This variation is consistent with use of different foraging strategies along a continuum between ambush and cruise foraging. To address questions about the evolution of foraging strategy, behavioral and morphological characters were mapped onto a phylogeny of Steinernema. Three species, all in the same clade, were classified as ambushers based on standing bout duration and host-finding ability. One clade of six species were all cruisers based on both host-finding and lack of standing behavior. All species in the ambusher clade had a high rate of jumping, all species in the cruiser clade had no jumping, and most intermediate foragers exhibited some level of jumping. Response to volatile and contact host cues was variable, even within a foraging strategy. Infective juveniles in the ambusher clade were all in the smallest size category, species in the cruiser clade were in the largest size categories, and intermediate foragers tended to be more intermediate in size. We hypothesize that the ancestral Steinernema species was an intermediate forager and that ambush and cruise foraging both evolved at least once in the genus.

Phylogeny of Steinernema travassos, 1927 (Cephalobina: Steinernematidae) inferred from ribosomal DNA sequences and morphological characters.

Entomopathogenic nematodes in Steinernema, together with their symbiont bacteria Xenorhabdus, are obligate and lethal parasites of insects that can provide effective biological control of some important lepidopteran, dipteran, and coleopteran pests of commercial crops. Phylogenetic relationships among 21 Steinernema species were estimated using 28S ribosomal DNA (rDNA) sequences and morphological characters. Sequences of the rDNA internal transcribed spacers were obtained to provide additional molecular characters to resolve relationships among Steinernema carpocapsae, Steinernema scapterisci, Steinernema siamkavai, and Steinernema monticolum. Four equally parsimonious trees resulted from combined analysis of 28S sequences and 22 morphological characters. Clades inferred from analyses of molecular sequences and combined datasets were primarily reliably supported as assessed by bootstrap resampling, whereas those inferred from morphological data alone were not. Although partially consistent with some traditional expectations and previous phylogenetic studies, the hypotheses inferred from molecular evidence, and those from combined analysis of morphological and molecular data, provide a new and comprehensive framework for evaluating character evolution of steinernematids. Interpretation of morphological character evolution on 6 trees inferred from sequence data and combined evidence suggests that many structural features of these nematodes are highly homoplastic, and that some structures previously used to hypothesize relationships represent ancestral character states.

Developmental temperature effects on five geographic isolates of the entomopathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae).

The development of five geographic isolates of Steinernema feltiae at 5, 8, 10, 15, 20, 25, and 28 degrees C in wax moth, Galleria mellonella, larvae was examined. The isolates were from Mediterranean (Sinop from Turkey, SN from France, and Monterey from California), subtropical (Rafaela from Argentina), and tropical (MG-14 from Hawaii) regions. All isolates caused 100% mortality of wax moth larvae and developed and produced progeny between 8 and 25 degrees C. At 28 degrees C, mortality was 100%, but no progeny was observed. The highest infective juvenile production was observed at 15 degrees C for all isolates. In general, the tropical isolate, MG-14, had the lowest production of infective juveniles. The time of emergence of the infective juveniles from the host cadaver showed some differences among isolates, with the Sinop isolate having the earliest emergence time from cadavers at 15 degrees C (10 days) and 20 degrees C (8 days). At 25 degrees C, the infective juveniles of the Sinop, SN, and Rafaela isolates emerged from the cadavers from 5 to 7 days. Time of host death by all isolates showed no differences at 8, 10, 15, 20, and 28 degrees C. At 25 degrees C for all isolates (except the MG-14), shorter times to host death were observed. Host death occurred at 12 days at 8 degrees C, 9 to 11 days at 10 degrees C, 4 to 5 days at 15 degrees C, 3 days at 20 degrees C, and 2 days at 25 and 28 degrees C. For penetration efficiency, the Sinop, SN, and Rafaela isolates penetrated their hosts at 5, 8, and 10 degrees C. Penetration of the infective juveniles was consistently high for all isolates at 15, 20, 25, and 28 degrees C, but it was significantly lower for the MG-14 isolate at 15, 25, and 28 degrees C. No progeny production occurred at 28 degrees C, but nematode penetration did occur with the MG-14 isolate having significantly lower penetration than the other isolates. When nematodes were produced at 8, 15, and 23 degrees C in wax moth larvae, all isolates had infective juveniles with longer body lengths at 8 degrees C followed by 15 and 23 degrees C. To further verify body length at the different temperatures, beet armyworm, Spodoptera exigua, larvae and dog-food agar medium were used, respectively, for in vivo and in vitro culture of the Sinop isolate. Infective juvenile body length showed the same trends, with the longest being at 8 degrees C and decreasing in length from 15 to 23 degrees C. The data suggest that quality of food for the nematode and temperature (that is, developmental time) influence the body length of the infective juvenile.

Redescription of Steinernema longicaudum Shen & Wang (Nematoda: Steinernematidae); geographic distribution and phenotypic variation between allopatric populations.

Steinernema longicaudum Shen & Wang is redescribed based on a comparative morphological study of specimens from the type isolate from China, and two other isolates recovered from Korea and the USA. For the first and second generation female, the location of the vulva, shape of the vulval lips, and shape and length of the tail were newly observed diagnostic characters. A more detailed description of the morphology of the male spicules and gubernaculum, and the arrangement of the genital papillae is included. A description, based on scanning electron microscopy observations, of the lateral field pattern of the third-stage infective juveniles is also provided. Additionally, restriction fragment length polymorphism profiles based on the internal transcribed spacer region, and cross-breeding tests supplement the description of this species.

Revised list of type specimens on deposit in the university of california davis nematode collection.

The list of deposited type specimens is updated for the University of California Davis Nematode Collection, as recommended by the International Code of Zoological Nomenclature. The type collection includes 1,001 species and more than 11,000 individual specimens mounted on microscope slides. This list can be used as a reference to locate specimens but is not meant to clarify ambiguities that may exist concerning the type status of particular specimens.

A multivariate analysis of morphometric characters of Heterorhabditis species (Nemata: Heterorhabditidae) and the role of morphometrics in the taxonomy of species of the genus.

A multivariate analysis on the morphometrics of 7 Heterorhabditis species was conducted to review the status of the traditional morphometric characterization methods for differentiating species of these nematodes. Results from this study showed consistency in the selection of morphometric characters for discriminating among males and infective juveniles of Heterorhabditis species. For the males, testis reflexion (TREF) and total length (LENGTH) were the variables that contributed most in the discrimination among the different species, and for the infective juveniles, tail length (TAILL) and total length (LENGTH) were the variables that contributed most. Therefore, we consider that these morphometric characters are useful and reliable, and that they should be used for the identification of Heterorhabditis species/isolates.

A new species of Heterorhabditis from the Hawaiian Islands.

A new species of nematode of the genus Heterorhabditis (Nemata: Heterorhabditidae) was found during a survey of the soil entomopathogenic nematode fauna of the Hawaiian Islands. Heterorhabditis hawaiiensis sp. n. can be separated from all other species of Heterorhabditis by the length of the infective juvenile and the morphological characters of the spicules, gubernaculum, and bursa. Random amplified polymorphic DNA (RAPD) fragment analysis showed that this species also has a distinct genetic pattern in RAPD bands relative to the other 6 species or isolates of Heterorhabditis that were compared.