Steinernema aciari sp. n. (Nematoda: Steinernematidae), a new entomopathogenic nematode from Guangdong, China

A new species of entomopathogenic nematode, Steinernema aciari sp. n. was described. It was recovered from a soil sample collected from Haimen town, Shantou district in the eastern coast of Guangdong province, the People's Republic of China during a survey for entomopathogenic nematodes. S. aciari sp. n. belongs to the Steinernema glaseri group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For male, the new species can be recognized by spicule length (86+/-6.3 microm); spicule tip blunt with a hook-like structure; gubernaculum with a short and Y-shaped cuneus and corpus well-separated posteriorly. For infective juvenile, the combination of the following characters: body length (1113+/-68 microm), distance from anterior end to excretory pore (95+/-3.7 microm), tail length (78+/-5.2 microm), and E % (123+/-7) can be used to differentiate the new species from other nematodes. For female, the tail (conoid with a long mamillate terminus and a distinct postanal swelling) and vulva (slightly protruding from body surface with conspicuous double flapped epiptygma) shapes can be used as diagnostic characters for the new species. The new species can also be distinguished from other Steinernema species by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA, and from the close related species S. glaseri, Steinernema longicaudum CWL05, and Steinernema guangdongense by cross-breeding test.     

Steinernema cholashanense n. sp. (Rhabditida, Steinernematidae) a new species of entomopathogenic nematode from the province of Sichuan, Chola Shan Mountains, China

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.     

Steinernema biddulphi n. sp., a New Entomopathogenic Nematode (Nematoda: Steinernematidae) from South Africa

A new species of entomopathogenic nematode (EPN), Steinernema biddulphi n. sp., was isolated from a maize field in Senekal, Free State Province of South Africa. Morphological and molecular studies indicated the distinctness of S. biddulphi n. sp. from other Steinernema species. Steinernema biddulphi n. sp. is characterized IJs with average body length of 663 μm (606–778 μm), lateral fields with six ridges in mid-body region forming the formula 2,6,2. Excretory pore located anterior to mid-pharynx (D% = 46). Hyaline layer occupies approximately half of tail length. Male spicules slightly to moderately curved, with a sharp tip and golden brown in color. The first generation of males lacking a mucron on the tail tip while the second generation males with a short filamentous mucron. Genital papillae with 11 pairs and one unpaired preanal papilla. The new species is further characterized by sequences of the internal transcribed spacer (ITS) and partial 28S regions (D2-D3) of the ribosomal DNA (rDNA). Phylogenetic data show that S. biddulphi n. sp. belongs to the “bicornutum” clade within the Steinernematidae family.     

Steinernema ichnusae sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from Sardinia Island (Italy)

A new Steinernema species was isolated from three different sandy soil samples along the Platamona Beach, in the north-west coast of Sardinia Island (Italy). This new species is characterized by the following morphological characters: infective third-stage juvenile with a body length of 866 ± 61 (767-969) μm, distance from head to excretory pore of 63 ± 2.7 (59-68) μm, tail length of 81 ± 3.2 (76-89) μm, ratio E (%) 77 ± 3.4 (68-83); male tail with a mucron only in the second generation, spicule length of 66 ± 1.4 (64-67) μm and gubernaculum length of 44 ± 1.4 (43-46) μm in the first generation male; female of first generation with a slight vulval protrusion and ratio D (%) of 53 ± 4.0 (47-63). The new species differs distinctly from the related species (S. feltiae, S. kraussei, S. litorale, S. oregonense and S. cholashanense) in some morphometric values such as percentage of hyaline portion, ratios of gubernaculum/spicule length, spicule head length/width. The DNA analyses of the internal transcribed spacers and D2D3 regions show that the studied nematode isolates are a new species. Cross hybridisation tests with S. feltiae, S. kraussei, S. litorale, S. weiseri and S. oregonense showed that these species were reproductively isolated.     

Screening of entomopathogenic nematodes for virulence against the invasive western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) in Europe

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

Steinernema brazilense n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Mato Grosso, Brazil

A new entomopathogenic nematode, Steinernema brazilense n. sp., was isolated from a single soil sample collected from a natural forest in Mato Grosso do Sul state, Brazil. S. brazilense n. sp. is characterized morphologically by features of infective juveniles (IJ), males and females. For the IJ, body length averaging 1157 (1023-1284) μm, distance from anterior end to excretory pore 95 (87-102) μm, from anterior end to end of esophagus 148 (139-153) μm, tail length 85 (80-104) μm, D% and E% values 63 (58-70) and 106 (95-118.0), respectively. Lateral field pattern variable; the formula for the arrangement of ridges from head to tail is: 2, 4, 6, 8, 6, 2. For the male, the diagnostic characters include spicule averaging 83 (75-89) μm; D% about 65; the ratio SW% about 192. The length of spicule head is greater than width. Lateral field with one narrow ridge. First generation females are characterized by the presence of a ventral postanal swelling. S. brazilense n. sp. is morphologically close to Steinernema diaprepesi. It can be differentiated from S.diaprepesi by its longer IJ body length (1157 vs 1002 μm), longer distance from anterior end to excretory pore (110 vs 75 μm), a longer tail length (103 vs 83 μm); males of the new species with longer spicule (83 vs 79 μm). The new species can be distinguished further from other members of Steinernema glaseri group by characteristics of rDNA of ITS and D2D3 regions.     

Steinernema diaprepesi n. sp. (Rhabditida: Steinernematidae), a Parasite of the Citrus Root Weevil Diaprepes abbreviatus (L) (Coleoptera: Curculionidae)

A nematode collected from Diaprepes abbreviatus is identified and described as a new species, Steinernema diaprepesi n. sp. The new species is closely related to S. feltiae, S. glaseri, and S. oregonense and can be distinguished from these species by the following characteristics: Males: Spicule averaging 79 (71-90) µm and spicule shape; D% (distance from anterior end to excretory pore/ esophagus length × 100) about 80; the ratio SW (spicule length/anal body width) about 1.8. Females: Vulva with short, double- flapped epiptygma; tail terminus usually with 5 papillae-like structures. Infective juveniles: Body averaging 1,002 (880-1,133) µm, EP (distance from anterior end to excretory pore) = 74 (66-83) µm; tail length = 83 (65-91) µm, and E% (EP/tail length × 100) = 89.6 (78-114). Lateral field pattern variable, the formula for the arrangement of ridges from head to tail is: 2, 6, 7, 8, 4, 2. The portion with eight ridges is the longest. This new species can be differentiated further from three closest species (S. feltiae, S. glaseri, and S. oregonense) by characteristic sequences of their ITS regions, including sequence lengths, ratios of similarity, composition, and differences in base characters in sequence alignment.     

Steinernema weiseri n. sp. (Rhabditida,Steinernematidae), a new entomopathogenic nematode from Europe

Steinernema weiseri n. sp. is described from a roadside with apple trees near Ceské Budejovice, Czech Republic. The species is also widely distributed in Germany and Slovakia, from where it had previously been reported as Steinernema spec. F. The British Steinernema sp. D1 is considered conspecific with S. weiseri n. sp. Males of the new species are mainly characterised by light brown, slightly curved spicules with a long manubrium and the presence of a short tail mucron in the second generation. Third-stage infective juveniles are characterised by a `medium size' body and tail length, short hyaline tail portion (mostly around 1/3 of tail length), the excretory pore situated in the mid-pharynx region, lip region slightly offset, angular and flattened, and the lateral field having nine equally developed lines separated by eight distinct ridges. S. weiseri n. sp. is most similar to S. feltiae, with which it did not hybridise. RFLP analysis of the ITS region of the rDNA repeat shows S. weiseri n. sp. to be distinct from 50 other Steinernema species and isolates. The new species was found in a wide range of habitats and is readily maintained on Galleria mellonella larvae.     

Directional movement of entomopathogenic nematodes in response to electrical field: effects of species, magnitude of voltage, and infective juvenile age

Entomopathogenic nematodes respond to a variety of stimuli when foraging. Previously, we reported a directional response to electrical fields for two entomopathogenic nematode species; specifically, when electrical fields were generated on agar plates Steinernema glaseri (a nematode that utilizes a cruiser-type foraging strategy) moved to a higher electric potential, whereas Steinernema carpocapsae, an ambush-type forager, moved to a lower potential. Thus, we hypothesized that entomopathogenic nematode directional response to electrical fields varies among species, and may be related to foraging strategy. In this study, we tested the hypothesis by comparing directional response among seven additional nematode species: Heterorhabditis bacteriophora, Heterorhabditis georgiana, Heterorhabditis indica, Heterorhabditis megidis, Steinernema feltiae, Steinernema riobrave, and Steinernema siamkayai. S. carpocapsae and S. glaseri were also included as positive controls. Heterorhabditids tend toward cruiser foraging approaches whereas S. siamkayai is an ambusher and S. feltiae and S. riobrave are intermediate. Additionally, we determined the lowest voltage that would elicit a directional response (tested in S. feltiae and S. carpocapsae), and we investigated the impact of nematode age on response to electrical field in S. carpocapsae. In the experiment measuring diversity of response among species, we did not detect any response to electrical fields among the heterorhabditids except for H. georgiana, which moved to a higher electrical potential; S. glaseri and S. riobrave also moved to a higher potential, whereas S. carpocapsae, S. feltiae, and S. siamkayai moved to a lower potential. Overall our hypothesis that foraging strategy can predict directional response was supported (in the nematodes that exhibited a response). The lowest electric potential that elicited a response was 0.1 V, which is comparable to electrical potential associated with some insects and plant roots. The level of response to electrical potential diminished with nematode age. These results expand our knowledge of electrical fields as cues that may be used by entomopathogenic nematodes for host-finding or other aspects of navigation in the soil.     

斯氏线虫属一新种(小杆目:斯氏线虫科)

本文描述了斯氏线虫属一新种:尖尾斯氏线虫,并根据形态特征与属内的已知种进行了比较。     

Interaction of microbial populations in Steinernema (Steinernematidae,Nematoda) infected Galleria mellonella larvae

Infection of Galleria mellonella larvae with the entomopathogenic nematodes Steinernema feltiae (A21 and R strains) and Steinernema glaseri (Dongrae) resulted in several species of bacteria, including the respective bacterial symbiont, Xenorhabdus spp., growing in the infected insect cadavers. These other bacteria were Enterococcus in all three nematode infections studied and Acinetobacter in the S. feltiae infections. The respective populations of these bacteria changed with time. Following infection of G. mellonella larvae with any one of the Steinernema sp., only Enterococcus bacteria were detected initially in the dead larvae. Between 30 and 50h post-infection Xenorhabdus bacteria were detected and concurrent with this Enterococcus population declined to zero. This was probably due to secondary metabolites with antibacterial properties that were produced by Xenorhabdus. In the S. feltiae (both R and A21 strains) infections a third bacterium, Acinetobacter, appeared at about 130h (in S. feltiae A21 infections) or 100h (in S. feltiae R infections) and increased in population size to approximately that of Xenorhabdus. It was demonstrated that Enterococcus, orginating from the G. mellonella digestive tract, was sensitive to the organically soluble antimicrobials produced by Xenorhabdus but Acinetobacter, which was carried by the nematode, was not.     

Effectiveness of different species of entomopathogenic nematodes for biocontrol of the Mediterranean flatheaded rootborer, Capnodis tenebrionis (Linné) (Coleoptera: Buprestidae) in potted peach tree

The susceptibility of larvae of the Mediterranean flatheaded rootborer (Capnodis tenebrionis) to 13 isolates of entomopathogenic nematodes was examined using GF-677 potted trees (peachxalmond hybrid) as the host plant. The nematode strains tested included nine Steinernema feltiae, one S. affine, one S. carpocapsae and two Heterorhabditis bacteriophora. Nematodes showed the ability to locate and kill larvae of C. tenebrionis just after they enter into the roots of the tree. S. feltiae strains provided an efficacy ranging from 79.68% to 88.24%. H. bacteriophora strains resulted in control of 71.66-76.47%. S. carpocapsae (B14) and S. affine (Gspe3) caused lower control of C. tenebrionis larvae (62.03% and 34.76%, respectively). The influence of foraging strategy and the use of autochthonous nematodes to control C. tenebrionis larvae inside the roots is discussed.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Spinitectus macrospinosus n. sp. (Nematoda: Cystidicolidae) from the channel catfish Ictalurus punctatus in southern Manitoba and its distribution in other Ictalurus spp

Spinitectus macrospinosus n. sp., a parasite of the channel catfish Ictalurus punctatus, from the Red and Assiniboine rivers in southern Manitoba, Canada, is characterized by spines reaching a maximum length of 21.8-26.8 microm (means for males and females, respectively) in the anterior esophageal region, arranged in 4 sectors in the anterior region of the body. Spines in this region of the body increase in size and decrease in number (from a maximum of 6 to 3 per sector). The combination of spine length, number, and arrangement and the position of the excretory pore (between spine rows 6 and 7) readily distinguishes it from all its North American congeners, including the species it is most similar to, S. carolini. It is different from S. gracilis and S. acipenseri in having a posteriorly directed vagina, an excretory pore between spine rows 6 and 7, a longer stoma, a right spicule with a terminal ventral barb, and a heart-shaped caudal mucron. Spinitectus macrospinosus is similar to other North American species of Spinitectus, e.g., S. carolini, S. micracanthus, S. mexicanus, S. osorioi, and S. humbertoi, in having the anterior rows of spines arranged in 4 sectors. It is also similar to S. micracanthus and S. carolini in possessing a relatively long stoma, a posteriorly directed vagina, a right spicule with a terminal ventral barb, and a heart-shaped caudal mucron. Furthermore, it is similar to S. carolini in possessing an "area rugosa" with 2 rows of precloacal cuticular cleats. In southern Manitoba, S. macrospinosus appears to mature only in the channel catfish. Reexamination of museum specimens revealed that the nematode also is found in I. furcatus in Kentucky Lake (Kentucky-Tennessee) and in I. lacustris in Lake Texoma (Oklahoma-Texas).     

<Emphasis Type="Italic">Steinernema costaricense</Emphasis> n. sp. and <Emphasis Type="Italic">S. puntauvense</Emphasis> 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.     

Identification of symbiotic bacteria (Photorhabdus and Xenorhabdus) from the entomopathogenic nematodes Heterorhabditis marelatus and Steinernema oregonense based on 16S rDNA sequence

Two species of entomopathogenic nematodes, Heterorhabditis marelatus and Steinernema oregonense, were described recently from the west coast of North America. It is not known whether the bacterial symbionts of these nematodes are also unique. Here we compared partial 16S rRNA sequences from the symbiotic bacteria of these two nematodes with sequence from previously described Photorhabdus and Xenorhabdus species. The 16S sequence from the new Xenorhabdus isolate appears very similar to, although not identical to, that of X. bovienii, the common symbiont of S. feltiae. The new Photorhabdus isolate appears to be very distinct from other known Photorhabdus species, although its closest affinities are with the P. temperata group. We also verified a monoxenic association between each isolate and its nematode by amplifying and sequencing bacterial 16S sequence from crushed adult and juvenile nematodes and from bacterial cultures isolated from infected hosts.     

Morphological and Molecular Characterization of Longidorus americanum n. sp. (Nematoda: Longidoridae), a Needle Nematode Parasitizing Pine in Georgia

We describe and illustrate a new needle nematode, Longidorus americanum n. sp., associated with patches of severely stunted and chlorotic loblolly pine, (Pinus taeda L.) seedlings in seedbeds at the Flint River Nursery (Byromville, GA). It is characterized by having females with a body length of 5.4-9.0 mm; lip region slightly swollen, anteriorly flattened, giving the anterior end a truncate appearance; long odontostyle (124-165 µm); vulva at 44%-52% of body length; and tail conoid, bluntly rounded to almost hemispherical. Males are rare but present, and in general shorter than females. The new species is morphologically similar to L. biformis, L. paravineacola, L. saginus, and L. tarjani but differs from these species either by the body, odontostyle and total stylet length, or by head and tail shape. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other Longidorus species. Phylogenetic relationships of Longidorus americanum n. sp. with other longidorids based on analysis of this DNA fragment are presented. Additional information regarding the distribution of this species within the region is required.     

A novel subspecies of entomopathogenic nematode Steinernema feltiae protense subsp. N.(Nematoda: steinernematidae) from Iakutiia

A new subspecies of entomopathogenic nematode, Steinernema feltiae protense subsp. n., isolated from Yakutia grasslands is described. These nematodes demonstrate a high invasion activity to insect-hosts at 6 degrees C, maximum infestation of insect-hosts was registered at 13-21 degrees C.