Oscheius carolinensis n. sp. (Nematoda: Rhabditidae), a potential entomopathogenic nematode from vermicompost

Nematology, 2010, Vol. 12(1), 121-135

Oscheius carolinensis n. sp. (Nematoda: Rhabditidae), a potentialentomopathogenic nematode from vermicompost

Weimin YE 1,∗, Andrea TORRES-BARRAGAN 2 and Yasmin J. CARDOZA 2

1 Nematode Assay Section, Agronomic Division, North Carolina Department of Agriculture & Consumer Services,4300 Reedy Creek Road, Raleigh, NC 27607, USA

2 Department of Entomology, North Carolina State University, Campus Box 7613, Raleigh, NC 27695-7613, USA

Received: 11 January 2009; revised: 13 May 2009Accepted for publication: 13 May 2009

Summary – Oscheius carolinensis n. sp. (Rhabditidae) was recovered through the Galleria bait method from vermicompost producedin Raleigh, NC, USA. Morphological studies with light microscopy and scanning electron microscopy, as well as molecular analysesof the near-full-length small subunit rDNA gene (SSU), D2/D3 expansion segments of the large subunit rDNA gene (LSU) and internaltranscribed spacer (ITS), revealed this as a new species, described herein as Oscheius carolinensis n. sp. The new species is characterisedby a combination of characters including its unique DNA sequences, amphimictic reproduction, six separate lips with bristle-likesensilla, lateral field with four lines, leptoderan and open male bursa, arrangement of bursal papillae 1 + 1 + 1/3 + 3 + ph, evenlyspaced first, second and third papillae, and separate spicules that are distally shaped like a crochet needle. Oscheius carolinensis n.sp. belongs to the Insectivorus-group and is closest to O. colombianus, O. chongmingensis n. comb., O. insectivorus and O. lucianii.A Bacillus-like bacterium appears to be associated with this nematode, based on our microscopic and SEM observations. ExposedGalleria larvae were killed within 5 days and numerous nematodes were recovered from the cadavers within 48 h. Preliminary testsrevealed that this nematode is capable of infecting at least two other insect species (Pieris rapae and Tenebrio molitor) under laboratoryconditions and therefore has potential as a biological control agent. The status of Heterorhabditidoides chongmingensis is discussed,the genus is proposed as a junior synonym of Oscheius, and O. chongmingensis n. comb. is proposed.

Keywords – description, Heterorhabditidoides n. syn., Insectivorus-group, molecular, morphology, Oscheius chongmingensis n. comb.,phylogeny, SEM, taxonomy.

Entomopathogenic nematodes (EPN) from the familiesSteinernematidae and Heterorhabditidae (Rhabditina) arelethal parasites of insects that are widely distributed insoils throughout the world (Poinar & Thomas, 1984;Kaya & Gaugler, 1993; Gaugler, 2002; Nguyen & Smart,2004). Their ability actively to locate their insect hosts,specific association with highly virulent bacteria, highreproductive potential, the possibility of mass productionand harmless impact on vertebrates and plants makethese nematodes highly suitable for the development ofenvironmentally friendly alternatives for the control ofinsect pests (Kaya & Gaugler, 1993; Burnell & Stock,2000; Boemare, 2002; Gaugler, 2002).

Vermicompost, made by feeding various organic wastesto earthworms, has been found to show increased micro-bial activity and nutritional content, factors which promoteplant growth and health (Peyvast et al., 2007). Recent data

∗ Corresponding author, e-mail: [email protected]

have shown that vermicomposts are also consistently ca-pable of conferring plant resistance to many pathogensand arthropod pests in numerous species of economicallyimportant plants (Arancon et al., 2005). The mechanismsleading to vermicompost-mediated plant defences againstinsect pests have not been deciphered, although some au-thors suggest that pest inhibition is correlated with thelarge number of antagonistic microbes found in vermi-compost (Hu et al., 2003).

Another possible factor involved in vermicompost-mediated resistance in plants could be the presence ofinsect-parasitic organisms in this substrate. Therefore, thepresence of entomopathogenic organisms, including ne-matodes, was assessed using the Galleria bait method.Galleria cadavers recovered from this study were foundto contain a large number of rhabditid nematodes whichmost closely resemble the Insectivorus-group of Oscheius

© Koninklijke Brill NV, Leiden, 2010 DOI:10.1163/156854109X458464Also available online - www.brill.nl/nemy 121

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based on morphological and rDNA sequence data. The ne-matode is described herein as Oscheius carolinensis n. sp.

Materials and methods

ISOLATION OF EPN FROM VERMICOMPOST

Raleigh vermicompost (Red Hen Enterprises, Raleigh,NC, USA) was used to evaluate the presence of EPNbecause of its availability and proven negative effectson insect pests in our laboratory. The nematodes wereisolated using the Galleria bait method (Bedding &Akhurst, 1975; Meyling, 2007). Fifth instar larvae ofG. mellonella were heat-treated to prevent webbing insoil according to Meyling (2007). Five larvae were placedin containers with 100 cm3 vermicompost for a total ofsix replicates. The containers were incubated in darknessat 26◦C. After 3 days, the containers were turned overto facilitate larval exposure to the microorganisms in thesoil. The killed larvae were selected and surface-sterilisedfor 30 s with 1% NaClO prior to incubation in a moistchamber at 26◦C in darkness. Bait cadavers were observedevery 24 h to confirm EPN infection.

MORPHOLOGICAL OBSERVATIONS

Nematodes were heat-killed and fixed in 4% formalinand processed to glycerin by a modification of a glycerin-ethanol series of Seinhorst’s (1959) rapid method thenpermanently mounted on 25 × 75 mm microscope slides.Specimens were examined with a Leica DM2500 com-pound microscope with interference contrast at up to1000× magnification. Stoma terminology by De Ley etal. (1995) and Sudhaus and Fitch (2001) was employed.Drawings and measurements were made using a drawingtube. Spicules were measured from distal to proximal tipin a straight line. Live nematodes were heat-killed in tem-porary water mounts for all measurements and micropho-tographs to assure quality and accuracy. Morphometricdata were processed using Excel software (Ye, 1996).

SCANNING ELECTRON MICROSCOPY

Nematodes in distilled water were transferred by pipetteto modified BEEM capsules whose ends were cut openand fitted with 20 μm nylon mesh to hold the speci-mens through the entire preparation procedure (Bozzola& Russell, 1999). The capped capsules were then trans-ferred to a jar containing 3% glutaraldehyde in 0.1 Msodium cacodylate buffer, pH 7.2, at 4◦C. Samples were

washed in three changes of the same buffer, post-fixed in2% osmium tetroxide in 0.1 M sodium cacodylate buffer,pH 7.2, for 16 h at 4◦C in the dark in a refrigerator,washed in three more changes of the same buffer andthen dehydrated through a graded ethanol series to 100%,30 min per change, all on ice. Samples were allowedto come to room temperature and given two changes of100% ethanol. Capsules containing the samples were thencritical-point dried in liquid CO2 (Samdri-795, TousimisResearch, Rockville, MD, USA), mounted on aluminiumstubs with double-sided tape, sputter-coated with 50 Å ofgold-palladium (Hummer 6.2, Anatech USA, Hayward,CA, USA) and viewed using a Jeol JSM-5900LV (JEOLUSA, Peabody, MA, USA) at 10 kV at the Center for Elec-tron Microscopy, North Carolina State University.

MOLECULAR ANALYSES

Six isolates of nematodes collected from different in-sect larvae were used for molecular study. Extraction,PCR and sequencing of DNA were done separately foreach isolate to account for DNA polymorphism amongnematodes originating from different insects. A single ne-matode was picked into distilled water and its morpholo-gical identity was confirmed with light microscopy beforebeing placed into 50 μl of worm lysis buffer containingProteinase K for DNA extraction (Willams et al., 1992)and then crushed with a pipette tip. DNA samples werestored at −20◦C until used as a PCR template. Primersfor DNA amplification, PCR and DNA sequencing are thesame as described in Pedram et al. (2008).

The sequences were deposited into the GenBank data-base. DNA sequences were aligned by ClustalW(http://workbench.sdsc.edu, Bioinformatics and Compu-tational Biology group, Department of Bioengineering,UC San Diego, CA). The molecular sequences of O. caro-linensis n. sp. were compared with those of the other ne-matode species available at the GenBank sequence data-base using the BLAST homology search program.

The model of base substitution was evaluated usingMODELTEST (Posada & Crandall, 1998; Huelsenbeck& Ronquist, 2001). The Akaike-supported model, thebase frequencies, the proportion of invariable sites andthe gamma distribution shape parameters and substitu-tion rates were used in phylogenetic analyses. Bayesiananalysis was performed to confirm the tree topology foreach gene separately using MrBayes 3.1.0 (Huelsenbeck& Ronquist, 2001) running the chain for 1 × 106 genera-tions and setting the ‘burnin’ at 1000. We used the MarkovChain Monte Carlo (MCMC) method within a Bayesian

122 Nematology

https://www.researchgate.net/publication/5441630_Heterorhabditidoides_chongmingensis_gen_nov_sp_nov_Rhabditida_Rhabditidae_a_novel_member_of_the_entomopathogenic_nematodes?el=1_x_8&enrichId=rgreq-0ce0e068-6646-45de-8040-73913adf7547&enrichSource=Y292ZXJQYWdlOzIzMzUyNjM4NTtBUzoxMDMwNTYwODM1MjE1NTBAMTQwMTU4MTg5MzY3NA==

Oscheius carolinensis n. sp. from vermicompost

framework to estimate the posterior probabilities of thephylogenetic trees (Larget & Simon, 1999) using 50%majority rule.

NEMATODE ENTOMOPATHOGENICITY

Nematode entomopathogenicity was evaluated usingthree different insects, viz., fifth instar larvae of Galle-ria mellonella (Lepidoptera: Galleridae), sixth instar lar-vae of Tenebrio molitor (Coleoptera: Tenebrionidae), andthird instar larvae of Pieris rapae (Lepidoptera: Pieridae).A 10 cm diam. Petri dish lined with two layers of filterpaper (Whatman No. 1) and containing five larvae of thetested insect was used as experimental unit. Two thousandinfective juveniles were inoculated in to each Petri dish(i.e., 400 nematodes/larva). Distilled water was used asthe control. Petri dishes were stored at 25◦C in darknessin a completely randomised design with four replicates.Mortality of larvae was evaluated every 24 h. Dead lar-vae were transferred to White traps (Nguyen, 2005) to de-termine nematode emergence. Experiments were repeatedfour times.

Oscheius carolinensis* n. sp.(Figs 1-3)

MEASUREMENTS

See Table 1.

DESCRIPTION

Male

Body ventrally arcuate when heat-killed. Cuticlesmooth, finely annulated, annules ca 1 μm wide at mid-body. Lateral fields with three longitudinal ridges (fourlines) and about four fine, interrupted, longitudinal stri-ations next to both sides of ridges under SEM; lateralfields ca one-fifteenth body diam., extending from corpusto level of tail, but inconspicuous under light microscope.Some specimens without clear ridges, but with tessellatedlines in lateral field. Six lips, separate, continuous withbody contour; six bristle-like labial sensilla, one on eachlip; four bristle-like cephalic sensilla, one on each sub-lateral lip. Amphidial apertures elliptical, located on lat-eral lips just posterior to labial sensilla. Stoma opening

* Specific epithet named after the state of North Carolina, thetype locality.

triangular, each side with two lips. Stoma rhabditoid, cafour times as long as broad. Cheilostom not sclerotised (caone-fifth stoma length), gymnostom a sclerotised cylinder(ca one-third stoma length), stegostom about half of totalstoma length, pro/mesostegostom a sclerotised cylinder(ca one-third stoma length); glottoid apparatus well de-veloped, isomorphic; metastegostomatal flaps difficult toobserve under light microscope, but apparently hinged onrefractile dots separating metastegostom and telostegos-tom appearing as a stomatal floor. Pharyngeal collar sur-rounding ca 50% of buccal capsule. Procorpus cylindrical;metacorpus not clearly differentiated. Basal bulb rounded,with duplex haustrulum posterior to valve plates. Nervering usually surrounding mid-part of isthmus. Excretorypore conspicuous, usually ventrally located at level ofbasal bulb. Hemizonid not clearly observed. Testis single,anterior end reflexed ventrally; spermatocytes arranged inmultiple rows. Spicules paired, separate, in lateral viewslightly ventrally arcuate, distally shaped like a crochetneedle. Gubernaculum conspicuous, 44 ± 7.03% (29.4-50.0%) of spicule length. Bursa leptoderan, open; ninepairs of papillae of different lengths (1+1+1/3+3+ph),three precloacal papillae evenly spaced, of six postcloacalpapillae, fifth and eighth opening dorsally on bursa velum;phasmids short, next to ninth papillae. Tail ca 1.5 analbody diam. long, terminus filiform.

Female

Body slightly curved ventrally when relaxed by gentleheat. General morphology similar to male. Reproductivesystem didelphic, amphidelphic. Ovaries reflexed anteri-orly. Up to 60 embryos within uteri 34 ± 13.5 (11-60).Vulva a median transverse slit with lateral vulval flaps.Rectum 2.5 ± 0.3 (2.0-3.0) anal body diam. long. Tailelongate, slender, tapering gradually, ca 4.5 anal bodydiam. long. Phasmids conspicuous under SEM, pore-like,protuberant, located at one-third of tail region posterior toanus.

Juveniles

Body straight when heat-killed. Stoma and pharynxmorphology similar to adult. Amphidial apertures moreposterior to lip region than in adults. Tail elongate,conical.

TYPE HOST AND LOCALITY

Natural host unknown as material was recovered bybaiting vermicompost produced in Raleigh, NC, USA, in

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Fig. 1. Oscheius carolinensis n. sp. A: Pharyngeal region of female; B: Schematic representation of en-face view of lip region; C:Stoma; D: Lateral view of female tail region showing phasmid and rectum; E: Lateral view of vulva region and lateral field; F: Lateralview of bursa and spicules; G: Ventral view of bursa and spicules. (In F and G, rays 5 and 8, infilled in black, open dorsally to thebursa.)

2008 with Galleria mellonella larvae. The food source formaking vermicompost is local waste food.

TYPE MATERIAL

Holotype male, ten paratype males, ten paratype fe-males and 4% formalin-fixed voucher specimens de-posited in the USDA Nematode Collection, Beltsville,MD, USA; ten paratype males and ten paratype females

deposited at the University of California, Riverside Col-lection, Riverside, CA, USA; ten paratype males andten paratype females deposited at the University of Cal-ifornia, Davis Collection, Davis, CA, USA; ten paratypemales and ten paratype females deposited in the HaroldW. Manter Laboratory of Parasitology, University of Ne-braska State Museum, Lincoln, NE, USA; and ten pa-ratype males and ten paratype females deposited in theCanadian National Collection of Nematodes, Ottawa, ON,Canada.

124 Nematology


Fig. 2. Light micrographs of Oscheius carolinensis n. sp. A: Pharyngeal region of female; B: Tail region of female; C: Vulva region; D:Spicules and gubernaculum; E: Lateral view of spicules showing separated and distally shaped like a crochet needle; F: Ventral viewof bursa under phase contrast showing phasmid next to ninth papilla. (Scale bar = 20 μm.)

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126 Nematology


Table 1. Morphometrics of Oscheius carolinensis n. sp. All measurements are in μm and in the format: mean ± s.d. (range).

Character Male Female

Holotype Paratypes Paratypes

n – 20 20L 1370 1499 ± 283 (1000-2000) 1728 ± 265 (1360-2420)a 20.8 20.4 ± 2.5 (15.8-24.3) 18.2 ± 2.4 (14.9-23.2)b 6.8 7.0 ± 1.2 (4.9-8.9) 7.0 ± 1.0 (5.4-9.5)c 29.8 32.3 ± 8.4 (18.9-50) 11.0 ± 2.6 (8.4-17.8)c′ 1.6 1.4 ± 0.3 (1.0-2.3) 4.4 ± 0.9 (3.2-6.1)V – – 50.3 ± 1.8 (47.6-55.6)Pharynx 202 214 ± 17.2 (165-245) 247 ± 8.9 (228-264)Mid-body diam. 66 73 ± 9.8 (52-89) 95.7 ± 13.6 (67-123)Tail length 46 48 ± 8.6 (32-64) 160 ± 26.6 (108-206)Anal/cloacal body diam. 28 34 ± 4.6 (25-40) 36 ± 3.6 (28-43)Stoma length 21 20 ± 1.5 (18-23) 23 ± 2.5 (18-27)Stoma diam. 4.5 5.5 ± 0.8 (4.5-7) 5.4 ± 1.1 (4-7)Corpus length 140 133 ± 6.1 (116-144) 149 ± 25.0 (54-177)Isthmus length 42 47 ± 6.9 (33-58) 49 ± 6.3 (38-65)Basal bulb length 32 39 ± 5.6 (32-52) 45 ± 6.2 (35-62)Basal bulb diam. 26 33 ± 4.7 (26-44) 35 ± 2.0 (33-40)Anterior end to excretory pore 212 229 ± 31.6 (177-295) 231 ± 12.7 (205-262)Testis reflexion 206 209 ± 74 (108-352) –Rectum 98 83.9 ± 9.7 (70-100) –Spicule length 69 65 ± 8.9 (50-81) –Spicule width 7 8.2 ± 1.2 (6-10) –Gubernaculum length 33 29.4 ± 4.8 (20-35) –Stoma length/stoma diam. 4.7 3.8 ± 0.7 (2.9-5.1) 4.5 ± 1.2 (2.6-6.8)

OTHER MATERIAL

Besides the type material, 4% formalin-fixed specimensand glycerin-mounted permanent slides were deposited atthe Nematode Assay Section, Agronomic Division, NorthCarolina Department of Agriculture & Consumer Ser-vices, Raleigh, NC, USA. Live nematodes cultures in Gal-leria are available from the Department of Entomology,North Carolina State University, Raleigh, NC, USA. Livenematode cultures in NGM agar (3 g NaCl, 17 g Bacto-agar, 2.5 g Bacto-peptone, 1 ml cholesterol, 1 ml 1 MCaCl2, 1 ml 1 M MgSO4 and 25 ml 1 M KPO4 (pH 6) and975 ml water) plates deposited at the Caenorhabditis Ge-

netics Center (CGC) at the University of Minnesota, TwinCities, MN, USA, and in Dr David H.A. Fitch’s laboratoryat the Department of Biology, New York University, NewYork, NY, USA, under the strain name YEW1.

DIAGNOSIS AND RELATIONSHIPS

Oscheius carolinensis n. sp. is characterised by itsunique ribosomal DNA 18S, 28S D2/D3 and ITS se-quences, amphimictic reproduction, six separate lipswhich are continuous with the body contour, bristle-likelabial sensillum on each lip, bristle-like cephalic sen-silla (four in all) on each sublateral lip; lateral field with

Fig. 3. Scanning electron microscope photographs of Oscheius carolinensis n. sp. (A-C: male; D: Juvenile; E-I: female). A: Lip region,en-face view, showing two amphidial apertures (A), six labial sensilla (L) and four cephalic sensilla (C); B: Tail, left lateral view,showing leptoderan bursa ribs (r1-r9); C: Tail ventral view showing open bursa, ribs (r1-r9), cloaca opening and bacteria; D: Lipregion, lateral view, showing amphidial aperture (arrow); E: Tail, ventral view, showing anus and phasmids (arrows); F: Excretorypore; G: Phasmid and its opening; H: Lateral field at vulva region showing three ridges (four lateral lines), fine longitudinal striae andbacteria; I: Vulva region, ventral view.

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four lines; male bursa open, leptoderan, bursal papillaearranged 1 + 1 + 1/3 + 3 + ph with first, second andthird papillae spaced evenly, spicules separate and distallyshaped like a crochet needle.

The leptoderan bursa and crochet-needle-shapedspicules place O. carolinensis n. sp. in the Insectivorus-group of Oscheius (Sudhaus & Hooper, 1994; Sudhaus& Fitch, 2001). Species comparisons in the Insectivorus-group are compiled in Table 2 updated from Stock et al.(2005). This new species, on the basis of the amphimic-tic reproduction and four lateral lines, appears morpho-logically most similar to O. colombianus Stock, Caicedo& Calatayud, 2005, O. chongmingensis (Zhang, Liu, Xu,Sun, Yang, An, Gao, Lin, Lai, He, Wu & Zhang, 2008)n. comb. (ex Heterorhabditoides chongmingensis), O. in-sectivorus (Körner, 1954) Andrássy, 1976 and O. lucianiiMaupas, 1919 (= O. wohlgemuthi Völk, 1950).

Oscheius carolinensis n. sp. can be distinguished fromO. colombianus by larger body size, longer spicules andgubernaculum, presence of more embryos in the genitalsystem (up to 60 vs up to 18) and by the significantdivergence in ribosomal DNA 18S sequences between thetwo species. Oscheius carolinensis n. sp. can be separatedfrom O. chongmingensis n. comb. by disparities in theirribosomal DNA 18S, 28S D2/D3 and ITS sequences, thearrangement of the genital papillae with the first, secondand third papillae evenly spaced, leptoderan bursa, longerstoma, longer female tail vs the second and third papillaecloser than the first and second papillae, presence of apair of setiform caudal sensilla posterior to the cloacalopening (= cloacal sensory papillae of Sudhaus and Fitch,2001) and peloderan bursa. Oscheius carolinensis n. sp.can be separated from O. insectivorus by rDNA 18Sand 28S D2/D3 sequences, smaller body, stoma longerand also much longer than wide vs as long as width,shorter spicules and gubernaculum and fewer embryos infemale. Oscheius carolinensis n. sp. can be separated fromO. lucianii by the longer stoma. Oscheius carolinensis n.sp. is clearly different from O. shamimi by four vs sixlateral lines and six lips not forming vs forming threedoublets.

Oscheius carolinensis n. sp. differs distinctly by repro-ductive mode from five hermaphroditic species, namely:O. andrassyi Tabassum & Shahina, 2008; O. caulleryiMaupas, 1919; O. maqbooli Tabassum & Shahina, 2002;O. myriophilus Poinar, 1986; and O. necromenus Sudhaus& Schulte, 1989. Oscheius carolinensis n. sp. is clearlydifferent from O. andrassyi and O. maqbooli by four lat-eral lines vs six. Furthermore, it differs from O. andrassyi Ta

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128 Nematology


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Vol. 12(1), 2010 129

W. Ye et al.

by bursal papillal arrangement of 1 + 1 + 1/3 + 3 + phvs 1 + 2/3 + 3. Oscheius carolinensis n. sp. can be dis-tinguished from O. caulleryi by the arrangement of thegenital papillae, the first, second and third papillae be-ing evenly spaced vs second and third being closer thanthe first and second papillae, and by the longer spicules.Oscheius carolinensis n. sp. can be distinguished fromO. myriophilus by ribosomal DNA 18S and 28S D2/D3sequences, the arrangement of the genital papillae withthe first, second and third papillae evenly spaced vs thesecond and third closer than the first and second papil-lae, longer female and male and longer spicules. Oscheiusnecromenus differs from O. carolinensis n. sp. by thelonger testis reflexion (43-56% of gonad length), smallerfemales and males and shorter spicules.

MOLECULAR PHYLOGENETIC RELATIONSHIPS

For molecular analysis, the nearly full-length 18SrDNA, the D2/D3 expansion segment of 28S rDNA andthe ITS region were sequenced. No DNA polymorphismwas found from six nematode individuals from six dif-ferent Galleria larvae. The DNA sequences of O. caroli-nensis n. sp. are available in the GenBank database underthe accession numbers FJ547240 (18S), FJ547239 (28S)and FJ547241 (ITS). The rhabditid species from GenBankwith the highest matches after a Blastn search were se-lected for phylogenetic analysis. No DNA sequences areavailable in GenBank for O. andrassyi, O. caulleryi, O. lu-cianii, O. maqbooli, O. necromenus and O. shamimi in theInsectivorus-group.

ITS is a highly variable DNA fragment, and a Blastnsearch of the 1176 bp ITS of O. carolinensis n. sp. failedto result in good matches for any closely related ne-matode species. Moreover, very few rhabditid ITS se-quences were available, so further phylogenetic analysisusing these sequences was not pursued. To date, the high-est match in ITS from GenBank was O. chongmingensisn. comb. (GenBank accession number EF503690, 1007bp). Alignment of ITS sequences from these two speciesyielded 1158 total characters with 712 identical nu-cleotides (61.49%) and 169 insertions/deletions (14.59%).When these two species were compared for the 18S re-gion, they shared 1472 identical nucleotides (95.09%) andthree insertions/deletions (0%) over 1548 total charac-ters, as well as 431 identical nucleotides (88.32%) andseven insertions/deletions (0.01%) in the 28S region ofover 488 total characters. These results indicated, as ex-pected, the high divergence in the ITS region comparedwith the more conserved functional 18S and 28S regions.

The ITS sequence O. carolinensis n. sp. was comparedwith all sequenced rhabditids from the Fitch laboratory inNYU (Kiontke & Fitch, pers. comm.). The result revealedthat O. carolinensis n. sp. was unique, but closest to twounidentified Oscheius species from Costa Rica and Peru.

A Blastn search of O. carolinensis n. sp. on the 1680 bp18S region revealed the highest match as O. colombianus(GenBank accession number AY751546, 1670 bp). Align-ment of these two sequences yielded 1675 total characterswith 1614 identical nucleotides (96.36%) and seven inser-tions/deletions (0%). The 632 bp 28S D2/D3 sequence ofO. carolinensis n. sp. was similar to that of O. insectivorus(GenBank accession number EU195968, 3340 bp). Align-ment of these two sequences yielded 637 total characterswith 580 identical nucleotides (91.05%) and five inser-tions/deletions (0%). The large sequence disparity (>3%)in the three loci supported O. carolinensis n. sp. as a dis-tinct species when compared with all available Rhabditi-dae species in GenBank.

Figure 4 presents a phylogenetic tree based on thenearly full-length 18S rDNA from a multiple alignmentof 1703 total characters. This dataset has 1094 constantcharacters (64.2%). The average nucleotide compositionwas as follows: 26.46% A, 20.62% C, 26.02% G and26.90% T. Using Heterorhabditis as an outgroup taxon,this tree inferred many highly supported monophyleticgroups. All species of Oscheius are in a clade with 100%support, clearly separated from all other genera. Under thegenus Oscheius, two groups were well defined, i.e., theInsectivorus-group and the Dolichurus-group, each with100% support. The Insectivorus-group comprises O. car-olinensis n. sp., O. myriophilus, O. colombianus, O. insec-tivorus, O. chongmingensis n. comb. and an undescribednew species (GenBank accession number EU273597).Oscheius carolinensis n. sp. was inferred as the sisterto O. colombianus. The Dolichurus-group comprises O.dolichurus, O. dolichuroides, O. guentheri and O. tipu-lae. This is consistent with the morphological classifica-tion by Sudhaus and Hooper (1994) and molecular phy-logeny (Sudhaus & Fitch, 2001; Kiontke & Fitch, 2005;Kiontke et al., 2007).

Figure 5 presents a phylogenetic tree based on rDNA28S D2/D3 sequences from a multiple alignment of 664total characters. This dataset has 362 constant characters(54.5%). The average nucleotide composition was as fol-lows: 24.64% A, 20.45% C, 30.27% G and 24.63% T.Rooted by Heterorhabditis, the genus Oscheius was ina monophyletic clade but with only 53% support. How-ever, the Insectivorus-group and Dolichurus-group were

130 Nematology


Fig. 4. The 10 001st Bayesian likelihood tree inferred from 18S under GTR + I + G model (lnL = 9697.1895; freqA = 0.2646;freqC = 0.2062; freqG = 0.2602; freqT = 0.269; R(a) = 0.9399; R(b) = 3.4936; R(c) = 2.4954; R(d) = 0.5528; R(e) = 5.2698;R(f) = 1; Pinva = 0.4389; Shape = 0.7862). Posterior probability values exceeding 50% are given on appropriate clades.

inferred as monophyletic clades, each with 100% sup-port, thereby corroborating the 18S tree inferred from oursequences. Therefore, based on molecular data, the In-

sectivorus-group and Dolichurus-group were well definednatural groups within Oscheius. This result also revealedthat although the 28S D2/D3 sequences are not sufficient

Vol. 12(1), 2010 131

W. Ye et al.

Fig. 5. The 10 001st Bayesian likelihood tree inferred from 28S D2/D3 under GTR + I + G model (lnL = 4814.3682;freqA = 0.2464; freqC = 0.2045; freqG = 0.3027; freqT = 0.2463; R(a) = 0.5402; R(b) = 2.8212; R(c) = 1.5601; R(d) = 0.5081;R(e) = 4.7203; R(f) = 1; Pinva = 0.4237; Shape = 1.0757). Posterior probability values exceeding 50% are given on appropriateclades.

to resolve high level relationships, they are proficient in al-locating Oscheius species to their corresponding groups.Oscheius carolinensis n. sp. was inferred to be within

the Insectivorus-group and close to O. chongmingensis n.comb. and O. insectivorus. The O. colombianus 28S se-quence was not available for comparison. An unidentified,

132 Nematology


so called Heterorhabditis (GenBank accession numberAY177182) was embedded in the Insectivorus-group farfrom the other known Heterorhabditis species at the rootof the tree. This species had an identical DNA sequencewith another unidentified species in Rhabditidae (Gen-Bank accession number EF417154). A re-examination ofthese two strains will likely show that they belong to Os-cheius.

NEMATODE ENTOMOPATHOGENICITY

All healthy fifth instar Galleria larvae infested withnematode suspensions died. The nematodes have beensuccessfully maintained in Galleria culture using Whitetraps. Nematodes were able to kill T. molitor (Coleoptera:Tenebrionidae) and P. rapae (Lepidoptera: Pieridae) underlaboratory conditions.

Nematodes infecting the fifth instar Galleria larvaecaused 58.3% larval mortality 5 days after inoculation,with juveniles emerging from cadavers within 24 h afterhost death. When inoculated on T. molitor larvae, nema-todes killed 78.8% of the insects within 72 h. Nematodesemerged from the cadavers within 24 h. The fastest mor-tality rate observed was for the third instar P. rapae where100% mortality was achieved in less than 48 h after inoc-ulation. In this case, nematodes emerged from the cadaverwithin 2 h after death.

Further experiments are underway to determine the lifecycle, host range, host stage preference and characteri-sation of the bacteria associated with this nematode asshown in SEM (Fig. 3C, H).

DISCUSSION ON THE GENERIC STATUS OF

HETERORHABDITIDOIDES

Heterorhabditidoides Zhang, Liu, Xu, Sun, Yang, An,Gao, Lin, Lai, He, Wu & Zhang, 2008 was proposed asa new genus in the family Rhabditidae to contain a sin-gle species, H. chongmingensis (Zhang et al., 2008). Thisspecies was collected from a soil sample from Shang-hai, China, using the same Galleria bait method in ourstudy. This genus was characterised by six separate lipswith minute sensilla, pore-like amphidial apertures, tubu-lar stoma, longer than wide buccal tube, glottoid appa-ratus, no median bulb, valvate basal bulb, peloderan andopen male bursa arrangement of bursal papillae 1 + 1 +1/3 + 3 + ph, fifth and eighth papillae opening dorsallyon the bursal velum (Fig. 1B-F in Zhang et al., 2008),spicules separate and distally shaped like a crochet nee-dle (Fig. 1D in Zhang et al., 2008 and our observation on

paratypes), median female vulva, amphidelphic, didelphicovary, long rectum and conical tail. These characters com-pletely agree with the Insectivorus-group in Oscheius asclassified by Sudhaus and Hooper (1994) with the excep-tion of the peloderan bursa. Figures 1C and 2E of Zhanget al. (2008) showed a short tail tip beyond the bursa,thereby appearing pseudopeloderan. Only two male typespecimens from the USDA Nematode Collection wereavailable for study. Both had a peloderan bursa. Basedupon our observations, the filiform tail tip is very frag-ile and often disappears after dehydration to glycerin inpermanent slides. Thus, the genus Heterorhabditidoideswas not justified on morphological grounds because of alack of apomorphic characters. Furthermore, Zhang et al.(2008) considered H. chongmingensis as the only ‘ento-mopathogenic’ nematode species in the Rhabditidae andnot belonging to Heterorhabditidae or Steinernematidae.However, the fact is that many species in Rhabditidaeare necromenic or potentially insectivorous, particularlyOscheius (Poinar & Thomas, 1984; Sudhaus & Schulte,1989; Schulte, 1991; Richter, 1993; Sudhaus, 1993; Smart& Nguyen, 1994; Tabassum & Shahina, 2002; Nguyen &Smart, 2004; Carta & Osbrink, 2005; Stock et al., 2005;Tabassum & Shahina, 2008). This also appears to be thecase for O. carolinensis n. sp., the new species describedherein. An issue still to be resolved is whether these ne-matodes have casual or strict associations with their as-sociated bacteria in a true symbiotic mutualism. More in-formation is needed to confirm the ‘entomopathogenic’behaviour of both O. chongmingensis n. comb. and O.carolinensis n. sp. within the well defined clade of theInsectivorus-group within the genus Oscheius.

Zhang et al. (2008) used phylogenetic analysis of the18S rDNA and ITS rDNA sequence data to infer therelationships of Heterorhabditidoides with other rhabdi-tids, but erroneously regarded it as a monophyletic rela-tive to Oscheius, Rhabditis and Pellioditis. In fact, theirown figure (Zhang et al., 2008) showed that H. chong-mingensis was in a monophyletic clade with 100% sup-port together with O. colombianus, O. insectivorus and O.myriophilus as examined by 18S data. Based on our studyand the alignment between H. chongmingensis and Pel-lioditis typica (GenBank accession number AF036946),the closest species in Figure 7 of Zhang et al. (2008), ITSsequences were too divergent for a robust homologousmultiple alignment and thus were not suited for examin-ing high level phylogenetic relationships across genera inthis group.

Vol. 12(1), 2010 133

W. Ye et al.

Phylogenetic analysis on the 18S (Fig. 4) and 28SD2/D3 (Fig. 5) rDNA in our study revealed that H. chong-mingensis was in a highly supported Insectivorus cladewithin a highly supported Oscheius genus. Thus, Hetero-rhabditidoides is proposed as a new synonym of Os-cheius, and H. chongmingensis is transferred to the Insec-tivorus-group of this genus. Although O. chongmingensisn. comb. was not compared with any species of Oscheiusin the original species description, it is a unique species bya combination of characters including its amphimictic na-ture; open, pseudoleptoderan bursa; nine pairs of papillaeof different lengths (1 + 1 + 1/3 + 3), the second and thirdpapillae being closer than the first and second, a pair ofbristle-like papillae posterior to the cloacal opening, andthe rDNA 18S, 28S D2/D3 and ITS sequence data. It isworth noting that a species in the genus Oscheius fromnorthern China was listed as a new species in GenBank(EU273597-EU273599), but has not yet been described.This species was 1 bp different in the ITS region, but hadidentical 18S and 28S D2/D3 with O. chongmingensis n.comb. (Figs 4, 5), suggesting conspecificity.

REMARK ON THE SENSILLA PATTERN IN LIP REGION

Our observations by SEM revealed six separate lips inO. carolinensis n. sp. with a bristle-like labial sensillumon each lip and four cephalic sensilla, one on eachsublateral lip (Fig. 3A). This 6 + 4 sensilla pattern isprobably typical in Rhabditis and Oscheius and agreeswith other species descriptions, including O. myriophilus(Poinar, 1986) and O. chongmingensis n. comb. (Zhanget al., 2008). In other studies, two sensilla per lip werereported in O. colombianus (Stock et al., 2005) and O.guentheri (Sudhaus & Hooper, 1994), whilst two or threefine sensilla were reported for each lip in O. necromenus(Sudhaus & Schulte, 1989). This intrageneric divergenceneeds to be further verified by studying many morespecies and specimens by SEM.

Acknowledgements

The authors thank Dr Robin M. Giblin-Davis at the FortLauderdale Research and Education Center, University ofFlorida, for critical review of the manuscript, Dr ValerieM. Knowlton at Center for Electron Microscopy, NorthCarolina State University, for assisting with scanningelectron microscopy, Janet Griffiths and Michael Maherat the Department of Entomology, North Carolina StateUniversity, for technical support, Dr David H.A. Fitch

and Dr Karin Kiontke at the Department of Biology, NewYork University, for searching DNA sequences in theirdatabase, and Dr Zafar Handoo at USDA NematologyLaboratory for loaning type specimens. This study wassupported by a NCARS project award to Y.J.C. (No.NC02199).

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Oscheius carolinensis n. sp. (Nematoda: Rhabditidae), a potential entomopathogenic nematode from vermicompost

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