Isolation and characterization of microsatellite loci in the entomopathogenic nematode Heterorhabditis bacteriophora

Twenty microsatellite loci were identified from genomic DNA enrichment and expressed sequence tags of entomopathogenic nematode Heterorhabditis bacteriophora. Eight loci were found to be polymorphic in a Northeast Ohio H. bacteriophora population. Levels of polymorphism were evaluated in 31-56 individuals and the number of alleles ranged from two to three. The values of observed and expected heterozygosities ranged from 0 to 0.536 and from 0.223 to 0.616, respectively. All eight loci showed heterozygote deficiencies, but three conformed to Hardy-Weinberg equilibrium at the subpopulation level. This is the first set of microsatellite markers in entomopathogenic nematodes.     

Heterorhabditis atacamensis n. sp. (Nematoda: Heterorhabditidae), a new entomopathogenic nematode from the Atacama Desert, Chile

A new Heterorhabditis species of entomopathogenic nematode was isolated from soil of the Atacama Desert in Chile. The new species is characterized by morphometrics of the infective juvenile (IJ) with length (L)?=?611 (578-666)?μm, head to excretory pore length (EP)?=?115 (101-126)?μm, tail?=?69 (62-79)?μm long, (EP/tail)?×?100 (E%)?=?165 (149-182) and L/maximum body diameter (ratio a)?=?28 (25-31). The male has spicules 45 (40-49)?μm long, gubernaculum 20 (17-22)?μm long and (spicule length/anal body diameter)?×?100 (SW%)?=?205 (179-249). The hermaphroditic adult has shallow cuticular folds immediately anterior and posterior to the vulva, a slight post-anal swelling and a finely rounded tail terminus. Morphologically, H. atacamensis n. sp. resembles H. safricana, H. marelatus, H. downesi and H. amazonensis, but can be distinguished by characters of adult and IJ stages. In particular, for adult males, H. atacamensis n. sp. differs from H. amazonensis by the number and orientation of the genital papillae and from H. downesi by the position of the excretory pore; by the shape of the female tail terminus from H. downesi and by the position of the IJ hemizonid from H. marelatus. Heterorhabditis atacamensis n. sp. is further characterized by internal transcribed spacer (ITS) and D2D3 rDNA sequences, the closest species, H. safricana, being separated by 13?bp across 730?bp of the ITS (incorporating ITS1 (partial sequence), 5.8S (complete sequence), ITS2 (complete sequence)) and 5?bp across 592?bp of the partial 28S (incorporating D2D3) sequence. The morphological and molecular data confirm that H. atacamensis n. sp. is a valid species.     

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.     

Steinernema siamkayai n. sp. (Rhabditida: Steinernematidae), an entomopathogenic nematode from Thailand

Steinernema siamkayai n. sp. (Rhabditida: Steinernematidae) is a new entomopathogenic nematode isolated in Lohmsak, Thailand. Morphology, hybridisation and molecular studies indicated the distinctness of S. siamkayai n. sp. from other Steinernema spp. Diagnostic characters include: the total body length (398–495 m) and tail length (31-41m) of the third-stage infective juvenile and lateral field pattern with 6-8 longitudinal ridges; the presence of a tail mucro in both first and second generation females and males; the size and shape of the spicules and gubernaculum, and the arrangement of the genital papillae of the first and second generation males; and the shape of the vulva and tail of the first generation female and second generation female     

Vitellin-binding proteins in the nematode Oscheius tipulae (Nematoda, Rhabditida)

We describe the first application of a non-radioactive ligand-blotting technique to the characterization of proteins interacting with nematode vitellins. Chromatographically purified vitellins from the free-living nematode Oscheius tipulae were labeled with fluorescein in vitro. Ligand-blotting assays with horseradish peroxidase-conjugated anti-fluorescein antibodies showed that labeled vitellins reacted specifically with a polypeptide of approximately 100 kDa, which we named P100. This polypeptide is a specific worm's vitellin-binding protein that is present only in adult worms. Blots containing purified O. tipulae vitellin preparations showed no detectable signal in the 100 kDa region, ruling out any possibility of yolk polypeptides self-assembling under the conditions used in our assay. Experiments done in the presence of alpha-methyl mannoside ruled out the possibility of vitellins binding to P100 through mannose residues. Triton X-114 fractionation of whole worm extracts showed that P100 is either a membrane protein or has highly hydrophobic regions.     

Molecular and morphological characterization of Pristionchus pacificus (Nematoda: Rhabditida: Neodiplogastridae), a new record of an entomophilic nematode from Iran

During a survey of entomopathogenic nematodes from the Mashhad region, in northeastern Iran, an entomophilic nematode was isolated using the Galleria trap method. Morphological characterization showed that this isolate belongs to the family Neodiplogastridae. Detailed morphological and morphometric characterization including SEM data fit with Pristionchus pacificus Sommer, Carta, Kim et Sternberg, 1996, representing the first record of this genus and species from Iran. Iranian specimens of P. pacificus FUM 5 are characterized by the body length (0.69–0.83 mm), the stoma (6–10 μm) bearing a dorsal tooth and a conical elongated tail (146–220 μm). Molecular analyses using 18S and ITS rDNA genes further support this identification. Measurements, illustrations, and SEM photographs of the Iranian isolate are provided and the phylogenetic position of the species is discussed.     

Molecular Detection and Identification of Spotted Fever Group Rickettsiae in Ticks Collected from the West Bank,Palestinian Territories

Background

Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) rickettsiae. Although Spotted Fever is prevalent in the Middle East, no reports for the presence of tick-borne pathogens are available or any studies on the epidemiology of this disease in the West Bank. We aimed to identify the circulating hard tick vectors and genetically characterize SFG Rickettsia species in ixodid ticks from the West Bank-Palestinian territories.

Methodology/Principal Findings

A total of 1,123 ixodid ticks belonging to eight species (Haemaphysalis parva, Haemaphysalis adleri, Rhipicephalus turanicus, Rhipicephalus sanguineus, Rhipicephalus bursa, Hyalomma dromedarii, Hyalomma aegyptium and Hyalomma impeltatum) were collected from goats, sheep, camels, dogs, a wolf, a horse and a tortoise in different localities throughout the West Bank during the period of January-April, 2014. A total of 867 ticks were screened for the presence of rickettsiae by PCR targeting a partial sequence of the ompA gene followed by sequence analysis. Two additional genes, 17 kDa and 16SrRNA were also targeted for further characterization of the detected Rickettsia species. Rickettsial DNA was detected in 148 out of the 867 (17%) tested ticks. The infection rates in Rh. turanicus, Rh. sanguineus, H. adleri, H. parva, H. dromedarii, and H. impeltatum ticks were 41.7, 11.6, 16.7, 16.2, 11.8 and 20%, respectively. None of the ticks, belonging to the species Rh. bursa and H. aegyptium, were infected. Four SFG rickettsiae were identified: Rickettsia massiliae, Rickettsia africae, Candidatus Rickettsia barbariae and Candidatus Rickettsia goldwasserii.

Significance

The results of this study demonstrate the geographic distribution of SFG rickettsiae and clearly indicate the presence of at least four of them in collected ticks. Palestinian clinicians should be aware of emerging tick-borne diseases in the West Bank, particularly infections due to R. massiliae and R. africae.     

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 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 ceratophorum n. sp. (Nematoda: Steinernematidae), a new entomopathogenic nematode from north-east China

Steinernema ceratophorum n. sp. was isolated from soil in Liaoning and Jining Provinces of China. Morphological, cross-breeding and DNA studies support the distinctiveness of S. ceratophorum n. sp. in comparison with four morphologically similar Steinernema species: S. affine, S. intermedium, S. riobrave and S. bicornutum. Diagnostic characters include: body length of infective juvenile, juvenile lip region with two horn-like structures and lateral field with six or eight longitudinal ridges; tail tip of first and second generation males lacking mucron; spicules of first generation male curved, dark yellow in colour and 71 µm long; tail tip of first generation female with small mucronate projection; and second generation female with fine mucronate process about 5.5 µm long. In addition, the restriction fragment length pattern (RFLP) of the ITS region of the ribosomal DNA repeat unit is different from other Steinernema spp., and S. ceratophorum n. sp. did not hybridise with S. riobrave, the species with the most similar RFLP.     

Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode,Steinernema riobrave (Rhabditida: Steinernematidae)

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.     

Structure of the pharynx in the adult nematode Anguillicoloides crassus (Nematoda: Rhabditida)

The structure of the pharynx of the adult female nematode Anguillicoloides crassus (Spirurina) has been studied for the first time using light and transmission electron microscopy. The cylindrical pharynx consists of a short anterior muscular corpus and an enlarged posterior glandular and muscular postcorpus. The main cellular components of the pharynx of A. crassus include the muscle cells, the marginal cells, the nerve cells, and 1 dorsal and 2 subventral glands. New observations for nematodes include: (1) the non-contractile regions of pharyngeal musculature in the corpus have specific appearance; (2) the ventrosublateral longitudinal nerve in the pharynx has an enlarged, enucleated anterior part, with a pronounced palmate projections; and (3) abundant lysosomelike membranous bodies consisting of myelinlike figures of varied size present in marginal cells and pharyngointestinal valve. The 2 subventral glands and, apparently, the single dorsal gland, have their openings at the same level, i.e., at the border between the corpus and postcorpus. The pharyngeal-intestinal valve joins the pharynx to the intestine. Knowledge of the ultrastructure of these complex characters may be useful in understanding of functional features, and for comparative morphology as well as evolutionary considerations within the Chromadorea.     

Steinernema sichuanense n. sp. (Rhabditida, Steinernematidae), a new species of entomopathogenic nematode from the province of Sichuan, east Tibetan Mts., China

Steinernema sichuanense n. sp. is characterized by male, female and IJ. For male, the spicules are robust with prominent rostrum; gubernaculum has blunt anterior end; cuneus is arrow-shaped, pointed posteriorly. Second-generation male has a prominent mucron. For female, tail usually has one to four papillae-like projections on tail tip; post anal swelling is absent. For IJ, body length is about 710 microm; lateral field has six ridges; the formula of lateral field is 2, 5, 6, 4, 2 with two prominent submarginal ridges; tail usually has a dorsal depression. In Steinernema affine/intermedium group, the IJ of S. sichuanense n. sp. differs from S. affine by its absence of the internal tail spine; differs from Steinernema beddingi by its six ridges in lateral field compared to 4 for S. beddingi. For male mucron is absent in both generations of S. affine, S. intermedium and S. beddingi, whereas it is present in the second-generation of S. sichuanense sp. n. Morphology and morphometrics of spicules and gubernacula of the four species in S. affine/intermedium group are quite different based on SEM photographs. For female, the postanal swelling is absent in the first-generation of S. sichuanense n. sp. whereas S. affine and S. intermedium have slight swelling and S. beddingi has conspicuous swelling. The new species is further recognized by characterization of sequences of ITS and D2/D3 regions of the ribosomal DNA. The symbiotic bacterium associated to S. sichuanense belongs to the species Xenorhabdus bovienii.     

Behavioural and physiological responses of infective juveniles of the entomopathogenic nematode Heterorhabditis to desiccation

Infective juveniles (IJs) of entomopathogenic nematodes (EPNs) are susceptible to a wide variety of environmental factors, including desiccation, which limit their usefulness as biocontrol agents. Although EPNs can be subjected to a gradual loss of water in their natural environment they are not full anhydrobiotes, being able to survive only moderate levels of desiccation at high relative humidities (rh). We investigated the desiccation tolerance of IJs of several Heterorhabditisspecies and strains when exposed to fast and slow desiccation regimes. We also investigated the behavioural and biochemical responses of Heterorhabditis IJs when exposed to 98% rh for 4 days. IJs of H. megidis UK211 (but not IJs of H. indica) aggregate into large clumps when desiccated at high rh, but unlike Steinernema spp., neither H. megidis nor H. indica IJs showed any tendency to coil. Preincubation of H. megidis UK211 IJs at high (98%) rh enhances their ability to survive for 150 min at 57% rh. We show that preincubation of H. megidis and H. indica at 98% rh induces the synthesis of glycerol but not of trehalose, whereas identical preincubation conditions do induce trehalose synthesis in Steinernema carpocapsae and Aphelenchus avenae. The biosynthesis of glycerol rather than trehalose by IJs of two species of Heterorhabditis in response to moderate levels of desiccation indicates that Heterorhabditis is unlikely to have the necessary metabolic responses to desiccation required to enable it to enter into a fully anhydrobiotic state.     

Phoresy of the entomopathogenic nematode Heterorhabditis marelatus by a non-host organism, the isopod Porcellio scaber

Entomopathogenic nematodes are widespread in nature and commonly used in the biological control of insect pests. However, we understand little about how these organisms disperse. We show in a laboratory setting that the entomopathogenic nematode Heterorhabditis marelatus is phoretically dispersed by a non-host organism, the isopod Porcellio scaber. These species both inhabit tunnels excavated in the roots and lower stems of bush lupine (Lupinus arboreus) by the nematodes' primary prey, larvae of the ghost moth Hepialus californicus. Phoretic dispersal via P. scaber may play a role in the metapopulation dynamics of this nematode.     

Occurrence and distribution of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis indica in Indonesia

Soil samples from 79 sites on five islands of Indonesia were baited with insects for the recovery of entomopathogenic nematodes. Heterorhabditis and Steinernema were equally prevalent, and were recovered from 11.7% of samples representing 20.3% of sites sampled. Both genera were recovered from coastal sites only. Entomopathogenic nematodes were more prevalent on the Moluccan islands of Ambon and Seram than on Java or Bali. They were not detected on Sulawesi, where non-coastal sites only were sampled. RFLP analysis was used in the identification of nematode isolates. Heterorhabditis indica was the only heterorhabditid identified. Two RFLP types of Steinernema were identified.     

Isolation and molecular characterization of five entomopathogenic nematode species and their bacterial symbionts from eastern Australia

BioControl - Entomopathogenic nematodes (EPNs) are used in biological control of pest insects but their potential may be limited by strain availability from different bioregions and effectiveness...     

Phylogenetic and cophylogenetic relationships of entomopathogenic nematodes (Heterorhabditis: Rhabditida) and their symbiotic bacteria (Photorhabdus: Enterobacteriaceae)

Mutualistic association between entomopathogenic Photorhabdus bacteria and Heterorhabditis nematodes represents one of the emerging model systems in symbiosis studies, yet little is known about this partnership from a coevolutionary perspective. Herein, we investigated phylogenetic and cophylogenetic relationships of Heterorhabditis and Photorhabdus strains using molecular markers Internal Transcribed Spacer and gyrase B gene sequences, respectively. The phylogenies presented consistent, well supported, monophyletic groups in the parsimonious and likelihood analyses for both the nematode and bacterial strains and supported the placement of currently recognized taxa, from which a potentially new Heterorhabditis species represented by a Thailand strain MP68 was identified. While the nematode strains with distant geographic distributions showed no detectable phylogenetic divergence within H. bacteriophora or H. georgiana monophyletic groups, their respective symbiotic bacteria speciated into two Photorhabdus species: P. luminescens and P. temperata, indicating the occurrence of duplication. Although such evolutionary process reduces the phylogenetic congruence between Heterorhabditis nematodes and Photorhabdus bacteria, global cophylogenetic tests using ParaFit detected a highly significant correlation between the two phylogenies (ParaFitGlobal = 0.001). Further, the associations between H. zealandica, H. indica and H. megidis strains and their symbiotic bacteria exhibited significant contribution to the overall cophylogenetic structure. Overall, this study reveals evidence of coevolution between Photorhabdus bacteria and Heterorhabditis nematodes and provides a framework for further examination of the evolution of these associations.