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45Russian Journal of Nematology, 2011, 19 (1), 53 - 74
Two new species of Steinernema Travassos, 1927 with short infective juveniles from Nepal
Hari Bahadur Khatri-Chhetri 15, Lieven Waeyenberge 2, Sergei Spiridonov 3, Hira Kaji Manandhar 4, Maurice Moens 2' 5
1 District Agriculture Development Office, Dailekh, Department of Agriculture, Nepal e-mail: [email protected] 2 Institute of Agriculture and Fisheries Research, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium 3Centre of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninskii
prospect, 33, Moscow, 119071, Russia 4Plant Pathology Division, Nepal Agricultural Research Council, Khumaltar, Lalitpur, Nepal 5Laboratory of Agrozoology, Department of Crop Protection, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
Accepted for publication 28 November 2010
Summary. During a survey conducted in 2007 in Nepal, two new species of entomopathogenic nematodes, Steinernema nepalense sp. n. and Steinernema surkhetense sp. n. were recovered from soil samples collected in Itahari, Sunsari and Birendranagar, Surkhet, respectively. The infective juveniles of S. nepalense sp. n. are recognised by body length 479 (424-513) pm, pharynx length 38 (32-46) pm, tail length 53 (48-61) pm, H% = 63 (52-71) and E% = 71 (58-87). The lateral field pattern is 2, 7, 8, 6, 5, 4, 2. The males have slender, well curved yellowish-brown spicules (71 (56-78) pm) and 25 genital papillae. The infective juveniles of S. surkhetense sp. n. can be recognised by the body length averaging 415 (393450) pm, pharynx length 32 (28-34) pm, H% = 57 (44-73) and E% = 72 (54-84). The lateral field pattern is
2, 8, 7, 5, 2. The males have well curved brownish spicules (70 (58-78) pm) with thin velum and 23/25
genital papillae. The analysis of ITS-rDNA sequences places both species in the 'carpocapsae-scapterisci-
tami' group.
Key words: ITS-rDNA, molecular phylogeny, morphology, SEM, steinernematid taxonomy.
Entomopathogenic nematodes (EPN) of the genus Steinernema Travassos, 1927 are naturally occurring, obligate and lethal pathogens of different insect hosts. The third-stage infective juveniles (IJ) of this genus are symbiotically associated with bacteria of the genus Xenorhabdus Thomas & Poinar, 1979. Once the bacteria are released in the insect haemolymph, they proliferate and provide the favourable environment for nematode growth and development. The potential as a biological control agent of insect pests of different crops has been explored in both laboratory bioassays and in field releases (Shapiro-Ilan et al., 2002). They exhibit great variation in their host specificity, infectivity, survival and thus in their biological control potentialities. Because of their local adaption, indigenous nematodes are considered as suitable for the control of local pests (Bedding, 1990). Exploration of indigenous EPN is therefore getting attention around the world.
Hunt (2007) documented 55 species of Steinernema, 11 species of Heterorhabditis and one
species of Neosteinernema as valid species. Since then, more than 10 species of Steinernema and at least four species of Heterorhabditis have been described.
A survey conducted in Nepal yielded four known species of Steinernema (Steinernema abbasi Elawad, Ahmad & Reid, 1997; S. siamkayai Stock, Somsook & Reid, 1998; S. feltiae (Filipjev, 1934) Wouts, Mracek, Gerdin & Bedding, 1982 and S. cholashanense Nguyen, Puza & Mracek, 2008) along with Heterorhabditis indica Poinar, Karunakar & David, 1992 (Khatri-Chhetri et al., 2010). Two new species of Steinernema were described recently from material collected during the survey (Khatri-Chhetri et al., 2011a, b). Two other Nepalese isolates are described herein as Steinernema nepalense sp. n. and Steinernema surkhetense sp. n.
MATERIAL AND METHODS
Nematode isolation. Soil samples (approximately 1000 g) were composites of 5-7 random sub-samples taken at a depth of 0-15 cm in
an area of approximately 25 m2. The samples were placed in polyethylene bags, transported to the laboratory and kept in a cool place until processing. When processing the samples, a plastic container (ca 500 ml) was filled with a representative volume of soil (ca 250-450 g). Entomopathogenic nematodes were isolated using the insect-baiting method described by Bedding & Akhurst (1975). Five to seven last instar Gallería mellonella (L.) larvae (Lepidoptera, Pyralidae) were used for this purpose. Suspected nematode-killed cadavers were placed in modified White traps (White, 1927) to allow the infective stages to come out. Emerging nematodes were pooled from each sample and used to infect fresh G. mellonella larvae. Recovered nematodes were multiplied ín vívo with last instar larvae of G. mellonella at 22 ± 3°C, cleaned, cultured in aerated water and stored in an incubator at 12-15°C (Woodring & Kaya, 1988).
Light microscopy. First and second generation males and females were collected randomly from different cadavers of Gallería at 5 and 9 days postinoculation, respectively. Gallería cadavers were dissected in Ringer's solution (Woodring & Kaya, 1988). IJ were pooled from White traps during the week after their first emergence from Gallería cadavers. Nematodes were killed by heat, fixed (Seinhorst, 1959 as modified by De Grisse, 1969) and mounted on permanent slides. Examination of morphology was done with an Olympus BX51 microscope equipped with differential interference contrast optics. Morphometrics were taken using digital image software (CellD Soft Imaging System, Olympus Company, Japan).
Scanning electron microscopy. For scanning electronic microscopy (SEM), fixed nematodes were transferred to a drop of 4% formalin. The specimens were washed three times with 4% glutaraldehyde (buffered with cacodylate, pH 7.2) and cleaned again by ultrasonic treatment for 8 min. The nematodes were then dehydrated by passing them through a gradual ethanol concentration gradient of 30, 50, 75, 95% (45 min each) and 100% (overnight) at 25°C and then critical point dried with liquid CO2, mounted on SEM stubs, coated with gold and studied using a JEOL LSM-840 scanning electronic microscope. Spicules and gubernacula were prepared as described by Nguyen & Smart (1995).
Cross hybridization test. Reproductive compatibility of S. nepalense sp. n. and S. sur-khetense sp. n. with S. carpocapsae (All strain), S. siamkayai (CD1, Nepalese strain; Khatri-Chhetri et al., 2010) and with each other was tested with the method reported by Nguyen & Duncan (2002) using
Galleria mellonella haemolymph. No other related species were available for this test.
Molecular characterisation. DNA was extracted from three IJ, which were hand-picked, placed in a 40 ^l drop of double distilled water on a glass slide and cut into two or more pieces under a dissecting microscope. Nematode fragments were transferred in 10 ^l water to a sterile Eppendorf tube containing 8 ^l lysis buffer (final concentration: 50 mM KCl, 10 mM Tris-Cl pH 8.3, 1.5 mM MgCl2, 1 mM DTT, 0.45% Tween 20). Two ^l of proteinase K (600 ^g ml-1) was added and nematode lysates were frozen at -70°C for a minimum of 15 min. The samples were then removed from the freezer and immediately incubated at 65°C for 1 h, followed by 10 min at 95°C. After centrifugation (1 min, 16.000 g), 5 ^l of the crude DNA extract was used for PCR. The remainder of the crude DNA-extract was stored at -20°C for future use. An rDNA fragment containing the internal transcribed spacer regions ITS1 and ITS2, and the 5.8S rRNA gene was amplified by PCR using the forward primer TW81 5'-GTT TCC GTA GGT GAA CCT GC-3' and the reverse primer AB28 5'-ATA TGC TTA AGT TCA GCG GGT-3' (Joyce et al., 1994). A second rDNA fragment containing the D2D3 expansion segment of the 28S rRNA gene was amplified using the forward primer D2A 5'-ACA AGT ACC GTG AGG GAA AGT TG-3' and the reverse primer D3B 5'-TCG GAA GGA ACC AGC TAC TA-3' (De Ley et al., 1999). The PCR reaction mixture contained 5 ^l 10 x PCR reaction buffer, 2 mM MgCl2, 200 ^M of each dNTP, 1 ^M forward and reverse primer, 2 U Taq Polymerase (Invitrogen, Merelbeke, Belgium), 5 ^l crude DNA-extract and sterile water up to a volume of 50 ^l. The PCR-programme settings for the ITS and D2D3 segment were as described by Joyce et al. (1994) and De Ley et al. (1999), respectively. After electrophoresis of 5 ^l PCR product in a 1.5% TAE buffered agarose gel (1 h, 100 V), the gel was stained in an ethidium bromide bath (1mg l-1) for 10 min and photographed under UV-light. When there was a positive result, the remainder of the PCR product was purified after electrophoresis in a 1% TAE buffered agarose gel (1 h, 100 V) following the instructions included in the Wizard SV Gel and PCR Clean-Up System kit (Promega Benelux, Leiden, The Netherlands). Subsequently, the concentrations of the purified PCR products were measured using a UV spectrophotometer (Nanodrop ND-1000, Isogen Life Sciences, Sint-Pieters-Leeuw, Belgium). The purified PCR products were sequenced (Macrogen Inc., Seoul, Korea) in both directions to obtain overlapping sequences of the forward and reverse
DNA strand. Finally, the sequences were visualised, edited and analysed using software packages Chromas 2.00 (Technelysium Pty, Helensvale, Australia) and BioEdit 7.0.4.1 (Hall, 1999), and deposited in GenBank (http://www.ncbi.nlm.nih.gov/). The ITS-sequence was compared with sequences of Steinernema species available in Genbank (NCBI). The sequences of those nematodes belonging to the same group (carpocapsae-scapterisci-tami) with addition of one representative of the other EPN-groups (S. bicornutum, S. feltiae and S. glaseri) were used for phylogenetic analysis. An alignment was produced using Clustal X 1.61 (Thompson et al., 1997) and subsequently Maximum Parsimony (MP) and Minimum Evolution (ME) trees were calculated by PAUP*4.0b10 (Swofford, 1998). The MP and ME tree were calculated using heuristic search option with 100 replicates (adding of sequences randomly and gaps treated as missing data). Bootstrap values were calculated with 100 replicates. Pairwise taxa divergence was computed by PAUP as absolute distance values; the mean character differences were adjusted for missing data and percentage of total domain length. The GTR+I+G model was selected (Hierarchical Likelihood Ratio Tests, -lnL = 15827.9619) for maximum likelihood analysis using ModelTest 3.5 (Posada & Crandall, 1998). The programme MtGui by Nuin (2005) was also used as an interface to prepare Modeltest results for ML analysis in PAUP* 4.0b10. In agreement with Nguyen et al. (2010), Caenorhabditis elegans Maupas, 1900 (EU131007) was used as outgroup.
DESCRIPTIONS
Steinernema nepalense sp. n. (Figs 1- 4 & 10, 11)
Measurements: See Table 1.
Infective third-stage juveniles. Heat relaxed specimens have a short (around 479 ^m) slightly curved sometimes straight, slender body; slightly tapering towards anterior and posterior ends (Fig. 2A). Cuticular striation vague under light microscope, but distinct with SEM, 1-1.25 ^m thick in mid-body region. Lateral field (Fig. 2B-I) beginning anteriorly with one line at the fourth or fifth annule. Two additional lines start after some distance forming two ridges. After 12 annules after the start of two ridges, each ridge divides into three. Both ridges share the middle single ridge and thus the number of ridges increases to seven, where two marginal ridges are more prominent than others. Five annules behind, the middle ridge further divides into two and number of ridges reaches eight. Two submarginal ridges are thinner than others,
which become invisible after some distance and the number of ridges reduces to six. These six ridges are prominent and uniform for a long distance. Before the anus, the widths of the ridges are irregular and not all the ridges are visible. Here, the number of ridges reduces to five for short distance, with one submarginal line being more prominent than others. At the level of anus, one more ridge disappears, four ridges remain up to the level of phasmid. Thereafter, only two prominent marginal lines remain in the tail. With the above arrangement, the formula of the lateral field is 2, 7, 8, 6, 5, 4, 2. Deirid not observed. Phasmid (Fig. 2I) pore-like, posterior to the anus, in anterior half of the tail. Head region rounded, not annulated. Stoma slightly opened. Labial papillae not observed; amphids crescent-like, prominent in SEM (Fig. 2B); four cephalic papillae distinct, posterior to amphids. Pharynx with uniform or slightly expanded corpus, narrower isthmus and subpyriform basal bulb with very weakly developed valve. Hemizonid distinct, lens-like, located near the level of nerve ring. Excretory pore in mid-pharynx region, cuticularised. Cardia present. Bacterial vesicle oval to elongated, size around 20 x 11 ^m. Intestine filled with numerous fat globules, lumen not visible. Genital primordia sometimes prominent. Rectum straight. Hyaline layer occupies slightly more than one half (63%) of tail length, terminus finely pointed.
First generation male. Body mostly C-shaped, sometimes J-shaped in heat relaxed specimens. Cuticle with faint transverse striae, lateral differentiation of cuticle in the shape of single round or flattened ridge (Fig. 3B). Head rounded, mostly offset from the rest of body. Six lips, each lip bearing a labial papilla. Four cephalic papillae present. Amphids distinct, crescent shaped, located laterally between labial and cephalic circle of papillae. Excretory pore mostly at the base of metacorpus and always anterior to nerve ring. Stoma short, shallow, cheilorhabdions distinct. Pharynx muscular with procorpus extending into a slightly swollen metacorpus, a narrower isthmus region surrounded by a nerve ring, and a large, valvated basal bulb. Cardia prominent, intestine generally with a wide lumen. Well developed reflexed testis. Testis reflection ventral in position and less than half of testis length. Paired, symmetrical, moderately curved yellowish-brown slender spicules (Fig. 3F-G). Spicule manubrium shape variable, usually elongated with length longer than width; manubrium almost one fourth of spicule length; long calomus separating manubrium from lamina. Lamina well curved anteriorly and dorsally with two
Fig. 1. Steinernema nepalense sp. n. A, B: Infective juvenile. A: Head and pharynx region; B: Tail region. C, D: First generation male. C: Head region; D: Tail region. E, F: First generation female. E: Vulva region; F: Tail region. G: Second generation male, tail region. H, I: Second generation female. H: Vulva region; I: Tail region. (Scale bar: A, B, D, G, H, I: 20 pm; C: 50 pm; E, F: 100 pm).
Fig. 2. Steinernema nepalense sp. n. SEM and LM pictures of infective juvenile. A: Entire view; B: Head region showing amphid (a) and cephalic papillae(c); C, D, E, F, G, H: Lateral field showing 7, 8, 6, 5 ridges; I: Lateral field showing 2 ridges and phasmid (p); J: Tail region showing anus; K: Head region. (Scales: A: 100 pm; B, C, D, E, F, G, H, I: 1 pm; J: 10 pm, K: 20 pm).
thin internal ribs. Rostrum not well developed, velum thin and distinct. Spicule terminus round. Gubernaculum slender (Fig. 3H-I), boat-shaped in lateral view, with anterior part curved up ventrally, posterior part usually tapering. In ventral view, cuneus long, Y-shaped, corpus with two wings. Tail bluntly conoid, short mucron present (2.8 ± 0.93 ^m). The 25 genital papillae comprise 12 pairs and a single mid-ventral papilla located just anterior to cloacal opening. Of these, seven pairs are located precloacal (six pairs subventral and one pair lateral) and five pairs of postcloacal papillae (two pairs adcloacal, two pairs subterminal, and one pair subdorsal (Fig. 3C-E)). The most posterior two pairs are consistently located near the tail tip. Next to these genital papillae, one pair of sensory/papillae-like structures protruding from the edges of cloaca are also present (Fig. 3C-E).
Second generation male. Second-generation male similar to that of the first generation except body, spicule and gubernaculum shorter and thinner (Table 1).
First generation female. Body variable in length, usually C-shaped sometimes coiled (Fig. 4A) on heat relaxation. Head rounded, sometimes slightly offset from body. Morphology of head region and pharynx similar to males. Excretory pore anterior to nerve ring. Genital tract amphidelphic with reflexed ovaries. Vulva transverse slit-like, slightly swollen or sometimes well swollen equal lips, double flapped epiptygma present (Fig. 4D, Fig. 1 E). Vulva opening at 58% of the-body length. Well cuticularised short vagina leading into paired uteri. Narrow rectum, distinct anal opening. Postanal swelling mostly present, sometimes not well developed or slightly developed (Fig. 4F, G). Tail shape variable, mostly dome shaped with small mucron.
Fig. 3. Steinernema nepalense sp. n. SEM pictures of first generation male. A: En-face view; B: Lateral field; C, D: Tail region showing arrangement of genital papilla; E: Close up of tail end; F, G: Spicule; H, I: Gubernaculum. H: Ventral view; I: Lateral view. (Scales: 10 pm).
Fig. 4. Steinernema nepalense sp. n. LM pictures of female. A, B, D, F, G: First generation. A: Entire view; B: Head region; D: Vulva region; F, G: Tail region; C, E, H: Second generation. C: Head region, E: Vulva region; H: Tail region. (Scales: A: 1000 pm; B, D, F, G : 100 pm; C, H, E: 20 pm).
Pigmy forms not observed. Endotokia matricida rarely observed.
Second generation female. Similar to first generation females in general morphology (Fig. 4,
C), but smaller (Table 1). Body slightly curved on heat relaxation. Gymnostom with thick, well sclerotized walls. Vulval opening behind mid-body. Tail conical with mucron. Uniformly swollen vulval
Table 1. Morphometries of Steinernema nepalense sp. n. (SUI1). All measurements in pm and in the form: mean ± standard deviation (range).
Characters Male Male (paratypes) Female (paratypes) Infective juvenile (paratypes)
Holotype First generation Second generation First generation Second generation
n 1 20 20 20 20 20
Body length (L) 1555 1295 ±155 769 ± 46 4763±901 1050 ±141 479 ± 21
(1050-1556) (707-861) (2846-6308) (839-1440) (424-513)
Body width (W) 135 105 ± 15.0 59 ± 4.1 192 ± 31 74 ± 12 24 ± 1.7
(76-135) (51-66) (148-267) (52-95) (21-27)
Excretory pore (EP) 72 62 ± 7.9 54 ± 5.2 77 ± 14 61 ± 7.3 38 ± 4.4
(43-75) (46-69) (41-96) (53-86) (32-46)
Nerve ring (NR) 119 107 ± 8.0 97 ± 5.6 143 ± 17 111 ± 14 72 ± 3.3
(94-130) (87-106) (117-168) (98-165) (66-80)
Pharynx length (ES) 143 135 ± 10 127 ± 5.9 198 ± 24 146 ± 5.4 100 ± 5.6
(120-163) (118-136) (157-229) (135-157) (91-115)
Tail length (T) 20 21 ± 2.2 16 ± 1.3 27 ± 9.4 41 ± 4.5 53 ± 3.5
(16-25) (14-18) (11-45) (28-49) (48-61)
Anal body diameter (ABD) 32 33 ± 3.3 26 ± 1.8 69 ± 17 32 ± 12 14 ± 1.1
(28-41) (24-30) (37-107) (25-84) (13-17)
Anterior end to testis curvature 415 369 ± 76 273 ± 37 - - -
(224-535) (210-337)
Spicule length (SP) 73 71 ± 5.7 63 ± 4.6 - - -
(56-78) (55-69)
Gubernaculum length (G) 60 56 ± 3.2 42 ± 3.7 - - -
(47-60) (36-50)
Distance to vulva (V) - - - 2508±561 606 ± 94 -
(1567-3618) (420-834)
Hyaline tail length (HT) - - - - - 33 ± 3.7
(28-40)
a (L / W) 11.51 12 ± 0.9 13 ± 1.0 25 ± 2.8 14 ± 2.2 20 ± 1.5
(11-14) (11-15) (19-28) (10-19) (17-23)
b (L / ES) 10.87 9.6 ± 0.9 6.0 ± 0.4 24 ± 3.2 7.2 ± 0.8 4.8 ± 0.3
(8-11) (5.3-6.9) (17-30) (6.1-9.2) (4.4-5.4)
c (L / T) 77.75 63 ± 8.3 49 ± 4.9 196 ± 80 26 ± 3.9 9.1 ± 0.8
(49-79) (39-58) (77-440) (21-35) (7-10)
D% 50.34 46 ± 5.2 43 ± 5.1 39 ± 7.1 41 ± 4.3 38 ± 5.6
(EP / ES x 100) (34-54) (37-57) (18-49) (35-55) (31-51)
E% (EP / T x 100) 360 297 ± 35 346 ± 48 310±107 149 ± 23 71 ± 7.9
(243-365) (283-460) (136-609) (120-210) (58-87)
SW% 228 218 ± 25 240 ± 21 - - -
(SP / ABD x 100) (165-260) (207-283)
GS% 82.19 79 ± 5.9 67 ± 4.2 - - -
(G /S P x 100 (70-93) (58-74)
V% (V / L x 100) - - - 52 ± 3.2 58 ± 5.8 -
(46-58) (41-70)
H% - - - - - 63 ± 6.0
(HT / T x 100) (52-71)
lips with small epiptygma (Fig. 4, E). Postanal swelling mostly developed (Fig. 4, H).
Type specimens and deposition of sequences.
Holotype male (UGMD 104194), nine male paratypes (UGMD 104194) and six female paratypes (UGMD 104195) of S. nepalense sp. n. are deposited in the nematode eolleetion of the Institute of Zoology, Ghent University, K.L. Ledeganekstraat 35, Ghent, Belgium. Eight male paratypes (N 1104) and five female paratypes (N 1105) of S. nepalense sp. are also deposited in the nematode collection of Centre of Parasitology of A.N. Severtsov Institute of Eeology and Evolution
of Russian Academy of Sciences, Leninskii prospect, 33, Moscow, 119071, Russia. Sequences of the ITS region and the D2D3 segment of S. nepalense sp. n. were deposited in NCBI Genbank under accession numbers HQ190044 and HQ190045, respectively.
Type locality. Steinernema nepalense sp. n. was recovered from a soil sample collected from subtropical low altitude area, at Itahari (87°16'E 26°39'N, 96 m a. s. l.), Sunsari district of eastern Nepal. The soil sample was taken from a field cultivated to lentil in December, 2007. Soil was silt loam, acidic (pH: 3.6) with moderate organic matter content.
Cross hybridization test. Cross-hybridisation assays between S. nepalense sp. n., S. carpocapsae (All strain), and S. siamkayai (CD1, Nepalese strain) yielded no progeny. In about 10% of the cases, S. nepalense sp. n. produced progeny in first crossbreeding experiment with these two species. However, these hybrids did not produce a following generation. In the majority of experiments no progeny production was observed. In the controls (conspecific crossbreedings), offspring was produced. No signs of hermaphroditism were observed in both species when single female was observed.
Ethymology. The specific epithet is derived from the country where the type locality is situated.
Diagnosis and relationship. Steinernema nepalense sp. n. is characterised by morphology and morphometrics of IJ and adults. For IJ, the new species can be recognized by the short body length 479 (424-513) pm, pharynx length 38 (32-46) pm, tail length 53 (48-61) pm, H% = 63 (52-71) and E% = 71 (58-87). The lateral field pattern is 2, 7, 8, 6, 5, 4, 2. The males have slender, well curved yellowish-brown spicules with prominent but thin velum. Gubernaculum slender, boat-shaped in lateral view, cuneus Y-shaped, corpus with two wings. Spicule and gubernaculum length of the first generation males is 71 (56-78) pm and 56 (47-60) pm. Male have two thin ridges in lateral field and tail mucron present in both generations. There are 25 genital papillae, comprising 12 pairs and a single mid-ventral papilla located just anterior to cloacal opening. Of these, seven pairs are located precloacal (six pairs subventral and one pair lateral) and five pairs postcloacal (two pairs adcloacal, two pairs subterminal, and one pair subdorsal). The most posterior two pairs are consistently located near the tail tip. Besides these genital papillae, one pair of sensory/papillae-like structures protruding from the edges of cloaca is also present. Vulva mostly is protruding with equal lips and double flapped epiptygma is present. Postanal swelling slightly developed in first but well developed in second generation females.
The body length, 479 (424-513) pm of IJ of S. nepalense sp. n. is longer than S. cumgarense Phan, Spiridonov, Subbotin & Moens, 2006 (402 (384432) pm), S. eapokense Phan, Spiridonov, Subbotin & Moens, 2006 (402 (370-434) pm), S. surkhetense. sp. n. (present study) (415 (393-450) pm), S. backanense Phan, Spiridonov, Subbotin & Moens, 2006 (438 (351-473) pm) and S. siamkayai Stock, Somsock & Reid, 1998 (446 (398-495) pm), but shorter than S. sasonense Phan, Spiridonov, Subbotin & Moens, 2006 (486 (456-516) pm), S.
tami Luc, Nguyen, Reid & Spiridinov, (2000; 530 (400-600) pm), S. carpocapsae (Weiser, 1955) Wouts, Mrácek, Gerdin & Bedding, 1982 (558 (438650) pm) and S. colombiense López-Núñez, Plichta, Góngora-Botero & Stock, 2008 (636 (549-732) pm). The lateral fields with maximum of eight ridges and the lateral line formula (2, 7, 8, 6, 5, 4, 2) seem similar to that of S. backanense, S. cumgarense, S. eapokense and S. sasonense but due to lack of SEM pictures, this formula is not clearly depicted in the original description of these species. This applies to other species of the group too (Table 4). The spicules of S. nepalense sp. n. (71 (66-78) pm) are longer than those of S. backanense (58 (53-62) pm) and S. carpocapsae (65 (66-72) pm), S. eapokense (69 (63-74) pm), S. surkhetense sp. n. (70 (58-78) pm), equal to S. sasonense (71 (66-78) pm) and S. colombiense (71 (64-77) pm), but shorter than those of S. cumgarense (76 (68-81) pm), S. tami (77 (7184) pm) and S. siamkayai (78 (75-80) pm). Steinernema nepalense sp. n. has an additional one pair of genital papillae, the number being 24+1 compared with 22+1 in all other species of the group except S. surkhetense sp. n. (22/24 + 1). The manubrium of the spicules of S. nepalense sp. n. is mostly elongated, and differs from S. carpocapsae having wider than long, S. siamkayai and S. colombiense having rhomboid and S. cumgarense and S. eapokense having bifurcation of outline of velum. The slender boat-shaped gubernaculum of new species is different from S. carpocapsae having plump gubernaculum, S. backanense, S. cumgarense, S. eapokense and S. sasonense all having straight gubernaculum with ventrally curved proximal end.
Steinernema surkhetense sp. n. (Figs 5-9, 10,11 )
Measurements: See Table 2.
Infective third-stage juveniles. Heat relaxed specimens have a short slender, slightly curved or straight in fixation body, slightly tapering towards anterior and posterior ends (Fig. 6A). Cuticular annulation vague under light microscope, but distinct under SEM, 1 pm wide in mid-body region. Lateral field (Fig. 6B-F) beginning anteriorly with a single ridge at second annule. At annule 14 or 15, two additional lines appear forming two ridges. At the level of excretory pore, each ridge divides into four, the number of ridges increasing from two to eight; two submarginal ridges are not prominent. This remains for the anterior half of the body. In the posterior half, one submarginal ridge of one side disappears, the number of ridges becoming seven, another ridge of that side widens. One marginal and
one submarginal ridge gradually disappear, thus five ridges until the level of phasmid. Behind phasmid level, central three ridges disappear and number of ridges becomes two in tail. Lateral field formula 2, 8, 7, 5, 2. Deirid not observed. Phasmid pore-like (Fig. 6F), posterior to anus, approximately 30% distance of tail from anus. Head region slightly truncate and usually slightly offset from the body contour, not annulated (Fig. 6B). Stoma slightly open. Labial papillae not observed; amphids slitlike, prominent in SEM (Fig. 6B); four cephalic papillae distinct, posterior to amphids. Pharynx with slightly expanded procorpus, narrower isthmus and subpyriform basal bulb with very weakly developed valve. Hemizonid distinct, lens-shaped, located at
the beginning of basal bulb. Excretory pore in anterior third of pharynx region (Fig. 6B), cuticularised, about 1 pm wide. Cardia present. Bacterial vesicle oval or round, size around 8 x 5 pm (Fig. 6G). Intestine filled with numerous fat globules, lumen not visible. Genital primordia prominent. Rectum straight, anus sickle-shaped. Hyaline layer occupies more than one-half of tail length. Tail attenuate with pointed terminus.
First generation male. Body curved ventrally posteriorly, tapering toward anterior end, mostly C-shaped, rarely J-shaped in heat relaxed specimens. Cuticle with very faint transverse striae, lateral field absent. Head rounded, slightly offset from the rest of body. Six lips bearing labial papilla. Four cephalic
Table 2. Morphometrics of Steinernema surkhetense sp. n. (SUI1). All measurements in pm and in the form: mean ± standard deviation (range).
Characters Male Male (paratypes) Female (paratypes) Infective juvenile (paratypes)
Holotype First generation Second generation First generation Second generation
n 1 20 20 20 20 20
Body length (L) 1194 1224 ±194 824 ± 89 2970 ± 922 1072 ± 101 415 ± 15
(887-1576) (621-945) (1894-5692) (875-1290) (393-450)
Body width (W) 115 105 ± 19 73 ± 13 154 ± 34 80 ± 10 21 ± 1.8
(75-132) (56-96) (102-236) (64-97) (18-25)
Excretory pore (EP) 44 55 ± 10 44 ± 4 53 ± 13 137 ± 21 32 ± 1.5
(43-78) (35-50) (33-86) (96-195) (28-34)
Nerve ring (NR) 86 92 ±19 90 ± 10 93 ± 17 159 ± 13 63 ± 3.1
(59-140) (73-109) (65-114) (142-198) (57-70)
Pharynx length (ES) 110 115 ± 15 122 ± 11 132 ± 31 193 ± 16 92 ± 4.1
(86-146) (106-141) (52-190) (173-236) (84-101)
Tail length (T) 18 19 ± 2.1 19 ± 3.6 19 ± 5.0 103 ± 15 45 ± 3.2
(16-23) (15-27) (12-30) (65-135) (38-53)
Anal body diameter (ABD) 28 31 ± 3.8 27 ± 2.3 47 ± 10 28 ± 3.2 12 ± 0.8
(23-38) (23-32) (25-68) (21-34) (11-14)
Anterior end to testis 390 291 ±102 207 ± 67 - - -
curvature (150-499) (122-335)
Spicule length (SP) 74 70 ± 5.2 64 ± 5.4 - - -
(58-78) (55-76)
Gubernaculum length (G) 55 52 ± 5.1 44 ± 5.6 - - -
(42-63) (35-53)
Distance to vulva (V) - - - 1585±458 535 ± 42 -
(1110-3051) (453-605)
Hyaline tail length (HT) - - - - - 26 ± 3.9
(20-32)
a (L / W) 10.38 12 ± 1.3 12 ± 1.9 19 ± 2.7 13 ± 1.4 19 ± 1.8
(10-14) (8.8-15) (13-25) (11-16) (16-24)
b (L / ES) 10.85 11 ± 1.6 6.8 ± 0.8 24 ± 8.8 5.6 ± 0.5 5 ± 0.2
(8.0-15) (5.2-8.0) (14-49) (4.6-6.7) (4.3-4.8)
c (L / T) 66.33 65 ± 12 44 ± 6.9 161 ± 54 11 ± 1.6 9 ± 0.5
(43-85) (31-59) (91-309) (8.7-15) (8.3-10)
D% (EP / ES x100) 40 48 ± 8.7 36 ± 3.3 44 ± 20 71 ± 10 35 ± 2.2
(37-64) (31-42) (28-117) (50-87) (31-40)
E% (EP / T x 100) 244 295 ± 66 237 ± 45 292 ± 84 137 ± 28 72 ± 6.4
(205-429) (167-313) (156-453) (85-189) (54-84)
SW% 264 230 ± 38 239 ± 31 - - -
(SP / ABD x 100) (168-330) (191-292)
GS%(G / SP x 100) 74.32 75 ± 5.0 68 ± 6.8 - - -
(66-84) (57-79)
V% (V / L x 100) - - - 54 ± 3.4 50 ± 2.7 v
45-59 (42-54)
H% (HT / T x 100) - - - - - 57 ± 7.4
(44-73)
Fig. 5. Steinernema surkhetense sp. n. A, B: Infective juvenile. A: Head and pharynx region; B: Tail region. C, D First generation male. C: Close-up of tail region, D: Tail region. E, F, G: First generation female. E: Head region; F Vulva region; G: Tail region. H: Second generation male, tail region. I, J: Second generation female. I: Tail region; J Vulva region. (Scale bar: A, B, C, H, I, J: 20pm; D, E: 50 pm; F, G: 100 pm).
Fig. 6. Steinernema surkhetense sp. n. SEM and LM pictures of infective juvenile. A: Entire view. B: Anterior region showing mouth, amphid (a), cephalic papillae(c), excretory pore (EP), one lateral line divided into two and two into eight ridges; C: Lateral field with 8 ridges; D: Lateral field with 7 ridges; E: Lateral field showing 7 ridges and reducing to 5; F: Tail region showing lateral field with 2 ridges, anus (a) and phasmid (p); G: Showing bacterial vesicle (bv). (Scales: A: 100 pm; B, C, D, E, F: 10pm; G: 20 pm).
papillae present (Fig. 7F). Amphids distinct, slitlike, located between labial and cephalic circle of papillae. Excretory pore always anterior to nerve ring, mostly located at metacorpus level. Stoma short, shallow, funnel-shaped gymnostom with thick, well sclerotised walls. Pharynx muscular with procorpus extending into a slightly swollen metacorpus, a narrower isthmus region surrounded by a nerve ring, and a large, valvated basal bulb. Cardia prominent, intestine generally with a wide lumen. Well developed and reflexed testis. Testis reflection ventral in position and about half of testis length. Paired, symmetrical, moderately curved, spicules (Fig. 8A) of brownish colour. Spicule manubrium
elongated oval (Fig. 8A), tapering anteriorly; manubrium about one fourth of spicule length; calomus separating manubrium from lamina. Lamina well curved anteriorly and dorsally, with two internal ribs less prominent, slender and almost straight posteriorly. Rostrum not developed; thin velum, velum margin parallel to spicule edge not covering the distal part of lamina. Spicule terminus blunt. In lateral view, gubernaculum boat-shaped (Fig. 8B), anterior part curved up. In ventral view, cuneus: Y-shaped, post-forked, corpus with two wings opened (Fig. 8BDE). In dorsal view, gubernaculum with two marginal ridges (Fig. 8C). Tail bluntly conoid, short (2.78 ± 0.66 pm) mucron
(Fig. 8C, D). Genital papillae 23/25, comprising 11/12 pairs and a single mid-ventral papilla. Specimens with 23 (Fig. 7B,C) genital papillae have seven precloacal pairs (six pairs subventral and one pair lateral) and four post cloacal pairs (one pair subventral, two pairs subterminal and one pair subdorsal). The most posterior two pairs are consistently located near the tail tip. The specimens with 25 genital papillae (Fig. 7A) have one extra pair precloacal papillae closely located with second anterior pair. Besides these genital papillae, one pair of sensory/papillae-like structures protruding from the edges of cloaca is also present (Fig. 7, C-E).
Second generation male. Second-generation males similar to the first generation except length and width of body, spicule and gubernaculum (Table 2).
First generation female. Body variable in length, usually C-shaped sometimes coiled on heat relaxation (Fig. 9A). Cuticle faintly striated. Lateral field not observed. Head truncated and slightly offset. Morphology of stoma and pharynx similar to the males. Cheilorhabdions prominent. Cardia prominent. Excretory pore located anterior to nerve ring, duct well cuticularized. Genital tract amphidelphic with reflexed ovaries. Vulva transverse slit-like, lips protruding, mostly equal lips, small epiptygma present (Fig. 9C). Vulva opening around mid-body. Short vagina leading into paired uteri. Postanal swelling usually not present, if present usually not well developed. Tail shape variable, mostly dome-shaped. Tail
Fig. 7. Steinernema surkhetense sp. n. SEM pictures of first generation male. A, B, C, D: Tail regions showing arrangement of genital papillae. A: Specimen with 27; B: Specimen with 25; C: Close-up of B; D: Close-up of A. E: Close-up of cloacal papilla. F: Face view. (Scales: A, B, C, D, F: 10 pm; E: 1 pm).
Fig. 8. Steinernema surkhetense sp. n. SEM pictures of spicules and gubernaculum of first generation male. A: Pair of spicules. B, C, D, E: Gubernaculum. B, D: Ventro-lateral view; C: Dorsal view; E: Ventral view. (Scale: 10 pm).
Fig. 9. Steinernema surkhetense sp. n. LM pictures of female. A, B, C, D: First generation. A: Entire view; B: Head region; C: Vulva region; D: Tail region. E, F, G: Second generation. E: Entire view; F: Vulva region; G: Tail region. (Scales: A, E: 500 pm; B, C, D, F, G: 50 pm).
terminus with short mucron (Fig. 9D). Endotokia matricida rarely observed.
Second generation females. Similar to first generation females in general morphology, but smaller (Table 2). Body straight or slightly curved on heat relaxation. Vulval opening at mid-body. Tail long, tapering to pointed end. Slightly protruding vulva mostly with very small epiptygma (Fig. 9 F). Postanal swelling slightly or not developed (Fig. 11G).
Type specimens and deposition of sequences. Holotype male (UGMD 104191), five male paratypes (three in UGMD 104191 and two in UGMD 104192) and 12 infective juvenile paratypes (UGMD 104193) of S. surkhetense sp. n. are deposited in the nematode collection of the Institute of Zoology, Ghent University, K.L. Ledeganckstraat 35, Ghent, Belgium. Eight male paratypes (N 1105) and 14 infective juvenile paratypes (N 1106) of S. surkhetense sp. n. are also deposited in the nematode collection of Centre of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninskii prospect, 33, Moscow, 119071, Russia. Sequences of the ITS region and the D2D3 segment of S. surkhetense sp. n. were also deposited in NCBI Genbank under accession numbers HQ190042 and HQ190043, respectively.
Type locality. Steinernema surkhetense sp. n. was recovered from the soil sample collected from Birendranagar (81°38E 28°35'N, 674 m a. s. l.), Surkhet district of western Nepal. Soil samples were collected from agricultural field cultivated with black gram (Vigna mungo (L.) Hepper). Soil was loamy acidic (pH: 5.2) with low organic matter content (1.14 %).
Distribution. Another isolate of the same species was recovered from a sample taken in neighbourhood of the type locality (81°37'E 28°35'N, 690 m) in a sissoo (Dalbergia sissoo Roxb.) forest with ground cover of different broad-and narrow-leaved weeds.
Cross hybridization test. Cross-hybridisation between S. surketense sp. n. and S. carpocapsae (strain All), S. siamkayai (CD1, Nepalese strain) yielded no progeny. In about 10% of the cases, S. surkhetense sp. n. produced progeny in first crossbreeding experiments; however, these hybrids did not produce a progeny in the following generation. In the majority of experiments no progeny production was observed. In the controls (conspecific crossbreeding), offspring was produced. No signs of hermaphroditism were observed in both species when single female was observed.
Ethymology. The specific epithet is derived from the district Surkhet where the type population was found.
Diagnosis and relationship. Steinernema surkhetense sp. n. is characterised by morphology and morphometrics of IJ and adults. Infective juveniles of this species can be recognized by the body length averaging 415 (393-450) pm, pharynx length 32 (28-34) pm, H% = 57 (44-73) and E% = 72 (54-84). The lateral field pattern is 2, 8, 7, 5, 2. The males have well curved brownish spicules with thin velum. Spicule terminus is blunt. In lateral view, gubernaculum is boat-shaped, cuneus Y-shaped, post-forked, corpus with two wings opened posteriorly. Spicule length of the first generation males is 70 (58-78) pm and gubernaculum length is 52 (42-63) pm. Tail mucron present in both generation males. Genital papillae 23/25, comprising 11/12 pairs and a single mid-ventral papilla. Besides these genital papillae, one pair of sensory/papillae-like structures protruding from the edges of cloaca is also present. Specimens with 23 genital papillae have seven precloacal pairs (six pairs subventral and one pair lateral) and four post cloacal pairs (one pair subventral, two pairs subterminal and one pair subdorsal). The most posterior two pairs are consistently located near the tail tip. The specimens with 25 genital papillae have one additional pair precloacal papillae closely located with second anterior pair. Vulva is slightly protruding, mostly with equal lips and a presence of small epiptygma. Postanal swelling usually is not present, if present usually not well developed in both generation females.
The body length of IJ of Steinernema surkhetense sp. n. (415 (393-450) pm) is longer than S. cumgarense (402 (384-432) pm), S. eapokense (402 (370-434) pm) but shorter than S. backanense (438 (351-473) pm), S. siamkayai (446 (398-495) pm), S. nepalense sp. n. (479 (424-513) pm), S. sasonense (486 (456-516) pm), S. tami (530 (400600) pm), S. carpocapsae (558 (438-650) and S. colombiense (636(549-732) pm). The spicule length of S. surkhetense sp. n. (70 (58-78) pm) is longer than S. backanense (58 (53-62) pm) and S. carpocapsae (65 (66-72) pm) and shorter than rest of the species of the group. The lateral line formula (2, 8, 7, 5, 2) is different from all other species within the group.
DISCUSSION
The sequence length of the S. nepalense sp. n. ITS-region is 814 base pairs (bp), ITS1 = 277 bp, ITS2 = 302 bp; the D2D3-segment is 614 bp long. The sequence length of the ITS-region of S. surkhetense sp. n. is 817 base pairs (bp), ITS1 = 277 bp, ITS2 = 306 bp; the D2D3-region is 614 bp long.
The pairwise distance comparison of ITS region of these species to other Steinernema species is presented in Table 5.
Maximum parsimony analysis showed that the alignment resulted in 1111 characters of which 401 are excluded, 203 are constant, 252 variable characters are parsimony uninformative and 255 characters are parsimony informative. The phylogenetic relationship of14 species of Steinernema based on maximum parsimony (Fig. 10, tree length = 980, CI = 0.7673, HI = 0.2327, CI excluding uninformative characters = 0.6514, HI excluding uninformative characters = 0.3486, RI 0.5614, RC = 0.4328) and minimum evolution (Fig. 11, ME score = 1.25122) are presented. In both MP and ME trees, S. nepalense sp. n. and S. surkhetense sp. n. are grouped together and form a subgroup within the 'carpocapsae' group. Bootstrap support for this clade is 60 in ME trees.
The combination of molecular and morphological features demonstrate that S. nepalense sp. n. and S. surkhetense sp. n. are members of 'carpocapsae-scapterisci-tami' group sensu Spiridonov et al. (2004). In molecular and phylogenetic studies, they clustered with the members of this group. These new species can be separated from each other and other species
belonging to the group by morphological and morphometrical characters of IJ and adults, as well as by molecular characters. The comparison of the morphometric data (Table 3) and morphological features (Table 4) are presented.
Based on molecular information, S. nepalense sp. n. and S. surkhetense sp. n. are closer to one another than to any other species of the group, thus clustering in the same clade in phylogenetic trees. However, both new species can be distinguished from each other by morphological and morphometric characters. Infective juveniles of S. nepalense sp. n. can be distinguished from each other by slightly longer body size (479 (424-513) pm) vs (415 (393-450) pm), longer EP distance (38 (32-46) pm) vs (32 (28-34) pm), slightly longer tail length (53(48-61) pm) vs (45 (38-53) pm) and lateral line formula 2, 7, 8, 6, 5, 4, 2 vs 2, 8, 7, 5, 2. First generation males of both species can be distinguished from each other by lateral field (presence of single flattened ridge on lateral field of S. nepalense sp. n. vs absence in S. surkhetense sp. n.), lower SW% value (218 (165-260) vs 230 (168330)), longer gubernaculum (56 (47-60) vs 52 (4263) pm) and slender in comparison.
c
EU131007 Caenorhabditis elegans HQ190044 Steinernema nepalense sp. n. HQ190042 Steinernema surkhetense sp. n. 'AY487920 Steinernema cumgarense 'AY171280 Steinernema tami 'AF331917 Steinernema siamkayai 'AY487921 Steinernema eapokense 'AY487918 Steinernema backanense 'AY171282 Steinernema carpocapsae EU345421 Steinernema colombiense 'AY487919 Steinernema sasonense 'AY230183 Steinernema scapterisci 'DQ310469 Steinernema feltiae 'AY171288 Steinernema glaseri ■AY171279 Steinernema bicornutum
Fig. 10. Phylogenetic relationships (Maximum Parsimony) of Steinernema nepalense sp. n. SUI1, Steinernema surkhetense sp. n. SKB8 with Steinernema spp. of 'carpocapsae group' based on ITS-rDNA sequences. Caenorhabditis elegans (EU131007) was used as outgroup. Bootstrap values are indicated.
90
99
88
65
Table 3. Comparative morphometries of third stage juveniles and first generation males of Steinernema nepalense sp. n., Steinernema surkhetense sp. n. and related species of the "carpocapsae-scaptirisci-tami" group. All measurements are in um and in the form: mean (range).
na: information not available. Abbreviations as in Table 1.
ON
vo
Species* Juveniles Male I References
L EP T D% E% SP GL D% SW% GS%
S. backanense 438 31 42 34 73 58 43 41 219 74 Phan et al., 2006
(351-473) (28-33) (39-46) (30-37) (64-83) (53-62) (36-48) (30-48) (184-256) (66-79)
S. carpocapsae 558 38 53 26 60 65 47 41 172 71 Poinar, 1967
(438-650) (30-56) (46-61) (23-28) (54-66) (66-72) (39-56) (27-55) (140-200) (59-88) Nguyen et al., 2007
S. colombiense 636 35 41 29 205 71 50 40 200 150 Lopez-Nunez et al.,
(549-732) (31-40) (32-53) (25-33) (138-284) (64-77) 47-63 (30-50) (150-270) (110-180) 2008
S. cumgarense 402 35 36 41 99 76 58 45 277 76 Phan et al., 2006
(384-432) (33-38) (34-38) (38-43) (87-103) (68-81) (48-66) (35-52) (250-313) (71-83)
S. eapokense 402 29 39 34 73 69 50 43 246 73 Phan et al., 2006
(370-434) (26-31) (35-45) (32-37) (66-80) (63-74) (47-56) (37-48) (221-282) (69-80)
S. nepalense sp. n. 479 38 53 38 71 71 56 46 218 79 Present study
(424-513) (32-46) (48-61) (31-51) (58-87) (56-78) (47-60) (34-54) (165-260) (70-93)
S. sasonense 486 32 47 32 69 71 53 40 250 75 Phan et al., 2006
(456-516) (30-34) (42-52) (30-34) (61-76) (66-78) (50-56) (37-45) (219-306) (69-82)
S. siamkayai 446 35 36 37 96 78 54 42 170 70 S rock et al., 1998
(398-495) (29-38) (31-41) (31-43) (95-112) (75-80) (47-65) (35-49) (140-220) (60-80)
S. surkhetense sp. n. 415 32 45 35 72 70 52 48 230 75 Present study
(393-450) (28-34) (38-53) (31-40) (54-84) (58-78) (42-63) (37-64) (168-330) (66-84)
S. tami 530 36 50 31 73 77 48 44 200 62 Luc et al., 2000
(400-600) (34-41) (42-57) (28-34) (67-86) (71-84) (38-55) (30-60) (140-300) (na)
o
o
l-h Co
TO
T3
SL
Table 4. Comparative morphology of Steinernema nepalense sp. n., Steinernema surkhetense sp. n. and related species
of the "carpocapsae-scaptirisci-tami" group.
Species* IJ Male 1" generation Female 1" generation
Genital
Lateral field Spicule Gubernaculum papillae Mucron Vulva Tail
S. backanense Four ridges in centre, 2 Elongated funnel-shaped manubrim, Straight with ventrally 22+1 P Protuberance, unequal Subconical, spike
pronounced marginal; velum present curved proximal end lips, like mucron
submarginal poorly epiptygma present
discernible in anterior
half of the body
S. carpocapsae Six ridges Head wider than long, bluntly pointed Cuneus arrowhead or Y, 22+1 P Protuberance vulva, Bluntly conical to
terminus, velum present post forked, corpus with epiptygma absent dome shaped, with
two wings or without spine
S. coïombiense Six ridges in mid body Ochre-brown, manubrium rhomboidal Manubrium of 24+1 A Vulva protruding, Tail blunt, conoid,
with a rounded 'knob-like' protrusion, gubernaculum curved asymmetric lips lacking mucro.
lamina with rostrum, velum present, ventrally and bifurcate (posterior larger than
narrow, blade terminus blunt anterior)
S. cumgarense As in S. backanense Elongated funnel shape manubrim, velum Straight with ventrally 22+1 P Equitorial protuding, Dome shaped with
present, bifurcation of outerline of velum curved proximal end epiptygma present peg like mucron
and ventral rib of spicule near rostrum
S. eapokense As in S. backanense Elongated funnel shape manubrim, velum Straight with ventrally 22+1 P Equitorial with Dome shaped with
present, bifurcation of outerline of velum curved proximal end petal like protuding peg like mucron
and ventral rib of spicule near rostrum structure
S. nepalense sp. n. 2,7,8,6,5,4,2 Brownish, shape variable mostly elongated Long, slender, boat shaped 24+1 P Protuberance vulva, Dome shaped with
funnel-shaped manubrim, shaft long, laterally, cuneus Y, corpus double flap epiptygma mucron
velum thin, finely round terminus with two wings
S. sasonense As in S. backanense Elongated funnel-shaped manubrim, Straight with ventrally 22+1 P Equitorial with Dome shaped with
velum present curved proximal end petal like structure spike like mucron
protuding
S. siamkayai 6 or 8 at mid body Manubrim rhomboid, velum present, Long, slender, distally 22+1 P protuberant, double Conoid and
orange-brown forked in ventral or dorsal flapped epiptygma, mucronate
S. surkhetense s p. n. 2,8,7,5,2 Brownish, manubrium elongated oval, Gubernaculum boat-shaped, 22/24+1 P Vulva protuding mostly Mostly dome-
tapering anteriorly; thin velum, spicule cuneus: Y, corpus with two equal lips, small shaped, terminus
terminus blunt wings, opened, dorsally epiptygma present with short mucron
with two marginal ridges
S. îami 8 ridges in mid body Head slightly elongated, velum thin, Boat shaped, cuneus: Y 22+1 P Not protruding, no conoid with peg like
terminus bluntly pointed, yellow-brown epiptygma mucron
P: present, A: absent, na: not available
* Descriptions from references cited in Table 3, except some characteristics of S. siamakayai from Nguyen et al., 2007.
Table 5. Pairwise distances (ITS regions) of Steinernema species of the carpocapsae group. Below diagonal: total character differences, above diagonal: mean
character differences.
SN Species ITS region
1 2 3 4 5 6 7 8 9 10 11 12
1 S. nepalense sp. n. - 0.05429 0.04977 0.04491 0.08536 0.05239 0.06153 0.12264 0.05089 0.08549 0.07044 0.32535
2 S. surkhetense sp. n. 36 - 0.05731 0.05838 0.09696 0.06787 0.07492 0.13364 0.06636 0.10166 0.08675 0.33012
3 S. backanense 33 38 - 0.04341 0.08828 0.04826 0.05828 0.11635 0.04374 0.08536 0.07938 0.32692
4 S. sasonense 30 39 29 - 0.08472 0.04191 0.05954 0.11388 0.03742 0.08030 0.07142 0.32324
5 S. cumgarense 56 64 58 56 - 0.07926 0.04885 0.14218 0.07774 0.05151 0.03647 0.34782
6 S. carpocapsae 35 45 32 28 52 - 0.06000 0.11006 0.00449 0.08702 0.07197 0.32695
7 S. eapokense 40 49 38 39 32 39 - 0.12440 0.05846 0.04580 0.03522 0.33495
8 S. scapterisci 78 85 74 73 90 70 78 - 0.11006 0.13924 0.12836 0.33818
9 S. colombiense 34 44 29 25 51 3 38 70 - 0.08244 0.07044 0.32695
10 S. tami 56 67 67 53 34 57 30 88 54 - 0.03647 0.34354
11 S. siamkayai 46 57 57 47 24 47 23 81 46 24 - 0.33441
12 S. glaseri 204 206 204 203 216 205 206 209 205 213 207 -
■EU131007 Caenorhabditis eiegans ■HQ190044 Steinernema nepalense sp. n. ■HQ190042 Stelnernema suikhelense sp. n.
■AY487918 Steinernema backanense
■AY487919 Steinernema sasonense
AY171282 Steinernema carpocapsae
■EU345421 Steinernema colombiense
■AY487920 Steinernema cumgarense
AY171280 Steinernema tami
■ AF331917 Steinernema siamkayai ■AY487921 Steinernema eapokense
■ AY230183 Steinernema scapterisci
■ AY171279 Steinernema bicornutum -DQ310469 Steinernema feltiae
■ AY171288 Steinernema glaseri
Fig. 11. Phylogenetic relationships (Minimum Evolution) of Steinernema nepalense sp. n. SUI1, Steinernema surkhetense sp. n. SKB8 with Steinernema spp. of 'carpocapsae group' based on ITS-rDNA sequences. Caenorhabditis elegans (EU131007) was used as outgroup. Bootstrap values are indicated.
60
9/
85
/5
Both S. nepalense sp. n. and S. surkhetense sp. n. reproduced well on G. mellonella larvae at 22 ± 3°C. Nematode-infected G. mellonella larvae died after one or two days. First generation adults appeared after three days. Second generation adults appeared seven days after inoculation. After 12 days the first cohort of infective juveniles migrated into a water trap. Besides G. mellonella, both species reproduced well on rice moth, Corcyra cephalonica Stainton, lesser wax moth larvae, Achroia grisella Fabricius and silk worm Bombyx mori L.
ACKNOWLEDGEMENT
We appreciate the technical assistance of Ms Nancy de Sutter and Shrinkhala Manandhar in laboratory at ILVO, Merelbeke, Belgium and at NARC, Khumaltar, Nepal, respectively. We thank Mr Rudra Paudel for his assistance at the soil sample collection in Surkhet area. We thank Ms Marjolein Couvreur, Institute of Zoology, Ghent University, Belgium for assistance at the scanning electron microscope. We also thank Prof. Ralf-Udo Ehlers, Christian-Albrechts-University Kiel, Germany for providing S. carpocapsae for cross hybridisation. The participation of Dr S.E. Spiridonov was partially supported by RFBR grant
11-04-00590a. The Vlaamse Interuniversitaire Raad-University Development Co-operation (VLIR-UOS), Belgium is gratefully acknowledged for providing a PhD scholarship to the first author.
REFERENCES
BEDDING, R.A. 1990. Logistics and strategies for introducing entomopathogenic nematode technology into developing countries. In: Entomopathogenic Nematodes in Biological Control (R. Gaugler & H.K. Kaya. Eds.). pp. 233-246. CRC Press, Boca Raton, Florida.
BEDDING, R.A., Akhurst, R.J. 1975. A simple technique for the determination of insect parasitic Rhabditid nematodes in soil. Nematologica 21: 109-110. De Grisse, A.T. 1969. Redescription ou modifications de quelques techniques utilisées dans L'etude de nématodes phyotparasitaires. Mededelingen Rijksfaculteit Landbouwwetenschappen Gent 34: 315-359. De Ley, P., Felix, M.A., Frisse, L.M., Nadler, S.A., STERNBERG, P.W. & THOMAS, W.K. 1999. Molecular and morphological characterisation of two reproductively species with mirror-image anatomy (Nematoda: Cephalobidae). hematology 2: 591-612. HALL, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for
Windows95/98/NT. Nucleic Acids Symposium Series 41: 95-98.
HUNT, D.J. 2007. Overview of taxonomy and systematics. In: Entomopathogenic Nematodes: Systematics, Phylogeny and Bacterial Symbionts. Nematology Monographs and Perspectives 5 (K.B. Nguyen & D.J. Hunt. Eds.). pp. 27-57. Brill, LeidenBoston.
Joyce, S.A., Reid, A., Driver, F. & Curran, J. 1994. Application of polymerase chain reaction (PCR) methods to the identification of entomopathogenic nematodes. In: COST 812 Biotechnology: Genetics of entomopathogenic nematodes bacterium complexes (A.M. Burnell, R.-U. Ehlers & J.-P. Masson. Eds.). pp. 178-187. Proceedings of symposium and workshop, St Patrick's College, Maynooth, County Kildare, Ireland. Luxembourg, European Commission, DGXII.
Khatri-Chhetri, H.B., Waeyenberge, L, Manandhar, H. K. & MOENS, M. 2010. Natural occurrence and distribution of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Nepal. Journal of Invertebrate Pathology 103: 7478.
Khatri-Chhetri, H.B., Waeyenberge, L., Spiridonov, S.E., Manandhar, H. K. & Moens, M. 2011a. Steinernema everestense n. sp. (Rhabditida: Steinernematidae), a new species of entomopathogenic nematode from Pakhribas, Dhankuta, Nepal. Nematology 13: 443-462.
Khatri-Chhetri, H.B., Waeyenberge, L., Spiridonov, S.E., Manandhar, H. K. & Moens, M. 2011b. Steinernema lamjungense n. sp. (Rhabditida: Steinernematidae), a new species of entomopathogenic nematode from Lamjung district, Nepal. Nematology 13: 589-605.
Lopez-Nunez, J.C., Plichta, K., Gongora-Botero, C.E. & SToCK, S.P. 2008. A new entomopathogenic nematode, Steinernema colombiense n. sp. (Nematoda: Steinernematidae), from Colombia. Nematology 10: 561-574.
Luc, P.V., Nguyen, K.B., Reid, A.P. & Spiridonov, S.E. 2000. Steinernema tami sp. n. (Rhabditida: Steinernematidae) from Cat Tien Forest, Vietnam. Russian Journal of Nematology 8: 33-43.
NGUYEN, K.B., SMART, G. C. 1995. Scanning electron microscope studies of Steinernema glaseri (Nematoda: Steinernematidae). Nematologica 41: 183-190.
Nguyen, K.B., Hunt, D.J. & MrAcek, Z. 2007. Steinernematidae: species descriptions. In: Entomopathogenic Nematodes: Systematics, Phyllogeny and Bacterial Symbionts. Nematology Monographs and Perspectives 5 (K.B. Nguyen & D.J. Hunt. Eds.). pp 121-609. Brill, Leiden-Boston.
NGUYEN, K.B. & DUNCAN, L.W. 2002. Steinernema diaprepesi n. sp. (Rhabditida: Steinernematidae), a parasite of the citrus weevil Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae). Journal of Nematology 34: 159-170. Phan, K.L., Spiridonov, S. E., Subbotin, S.A. & MoENS, M. 2006. Four new species of, Steinernema Travasos, 1928 with short infective juveniles from Vietnam. Russian Journal of Nematology 14: 11-29.
POINAR, G.O. 1967. Description and Taxonomic position of the DD-36 nematode (Steinernematidae: Rhabditoidea) and its relationship to Neoplectana carpocapsae Weiser. Proceeding of Helminthological Society of Washington 34: 199-209.
SEINHORST, J.W. 1959. A rapid method for transfer of nematodes from fixative to anhydrous glycerine. Nematologica 4: 67-69. Shapiro-Ilan, D.I., Gouge, D.H. & Koppenhoffer, A.M. 2002. Factors affecting commercial success: case studies in cotton, turf and citrus. In: Entomopathogenic Nematology (R. GAUGLER. Ed.). pp. 333-356. CABI publishing, Wallingford, UK.
SPIRIDONOV, S.E., REID, A.P., PODRUCKA, K., SUBBOTIN, S.A. & Moens, M. 2004. Phylogenetic relationships within the genus Steinernema (Nematoda: Rhabditida) as inferred from analyses of sequences of the ITS1-5.8S-ITS2 region of rDNA and morphological features. Nematology 6: 547-566.
STOCK, S.P., SOMSOOK, V., Reid, A.P. 1998. Steinernema siamkayai n. sp. (Rhabditida: Steinernematidae), an entomopathoganic nematodes from Thailand. Systematic Parasitology 41: 105-113.
SWOFFORD, D.L. 1998. PAUP*, phylogenetic analysis using parsimony and other methods. Version 4. Sunderland, Massachusetts, Sinauer Associates. 128 pp.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & HIGGINS, D.G. 1997. The ClustalX windows inference: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Research 24: 4876-4882.
WEISER, J. 1955. Neoplectana carpocapsae n. sp. (Angullulinata: Steinernematidae) novy, cizopasnic housenik obalece jableeneho, Carpocapsa pomonella L. Vestnik Ceskoslovenske Spolecnosti Zoologicke 19: 44-52.
WHITE, G.F. 1927. A method of obtaining infective nematodes larvae from cultures. Science 66: 302-303.
WOODRING, J.L., KAYA, H.K. 1988. Steinernematid and Heterorhabditid Nematodes. A handbook of techniques. Southern Cooperative Series Bulletin 331, Arkansas Agricultural Experiment Station, Fayetteville, Arkansas. 30 pp.
Hari Bahadur Khatri-Chhetri, L. Waeyenberge, S.E. Spiridonov, Hira Kaji Manandhar, M. Moens.
Два новых вида Steinernema Travassos, 1927 с инвазионными личинками малой длины из Непала. Резюме. Во время обследования почв Непала в 2007 году из почвенных проб, собранных близ Итахари (округ Сансари) и Бирендранагар (округ Сурхет), были выявлены два новых вида штейнернематид - Steinernema nepalense sp. n. и Steinernema surkhetense sp. n. Инвазионные личинки S. nepalense sp. n. характеризуются длиной тела 479 (424-513) мкм, длиной пищевода 38 (32-46) мкм, длиной хвостового отдела 53 (48-61) мкм, H% = 63 (52-71) и E% = 71 (58-87). Формула латерального поля 2, 7, 8, 6, 5, 4, 2. У самцов желтовато-коричневые спикулы, тонкие, явно изогнутые, длиной 71 (56-78) мкм и имеется 25 генитальных папилл. Инвазионные личинки S. surkhetense sp. n. отличаются длиной тела 415 (393-450) мкм, длиной пищевода 32 (28-34) мкм, H% = 57 (44-73) и E% = 72 (54-84). Формула латерального поля 2, 8, 7, 5, 2. Спикулы у этого вида коричневые, длиной 70 (58-78) мкм с тонким велюмом. Насчитывается 23 или 25 генитальных папилл. Филогенетический анализ нуклеотидных последовательностей ITS-rDNA указывает на принадлежность этих двух видов к группе 'carpocapsae-scapterisci-tami'.
