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23Russian Journal of Nematology, 2016, 24 (1), 17 - 31
Redescription of Steinernema thesami Gorgadze, 1988 (Rhabditida: Steinernematidae) from Georgia
12 1 Olegi A. Gorgadze , Elena S. Ivanova , Manana G. Lortkhipanidze and
Sergei E. Spiridonov2
'Institute of Zoology, Ilia State University, Kakutsa Cholokashvili Avenue 3/5, 0162, Tbilisi, Georgia 2Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences,
Leninskii Prospect 33, 119071, Moscow, Russia e-mail: [email protected]
Accepted for publication 20 April 2016
Summary. Redescription of Steinernema (Neoaplectana) thesami Gorgadze, 1988 was done based on the nematode strain isolated in Georgia in 1987 from an infected lepidopteran, Operophtera brumata, and since then maintained under laboratory conditions. Molecular analysis has shown the similarity of the ITS rDNA sequence of S. thesami with that of recently described S. tbilisiense Gorgadze, Lortkhipanidze, Ogier, Tailliez & Burjanadze, 2015 and an unidentified isolate from Krasnaya Polyana, near Sochi, Russian Federation. As molecular characteristics for both S. thesami and S. tbilisiense are identical, the latter is to be considered as the junior synonym for S. thesami. The phylogenetic relationships of S. thesami with other species of 'affine-intermedium' clade of the genus Steinernema are discussed. Key words: 'affine-intermedium' group, EPN, morphology, phylogeny.
The taxonomy of entomopathogenic nematodes (EPN) of the genus Steinernema is intensively studied as these nematodes are used in the control of insect pests and certain species have already been established as biocontrol agents (Bedding, 1998). The interest in EPN has resulted in a rapid increase in the number of Steinernema species described. However, there are a number of species described in the pre-molecular era, which are evidently in need of redescription according to modern standards with the use of all available methods. The species Neoaplectana (= Steinernema) thesami Gorgadze, 1988 is one such species, isolated in Mtskheta-Mtianeti Region of Georgia in 1987 from infested pupa of a winter moth Operophtera brumata Linnaeus, 1758 (Geometridae: Lepidoptera). To date, a total of five species of entomopathogenic nematodes of the family Steinernematidae have been described from Georgia (Kakulia & Veremtchuk, 1965; Gorgadze, 1988, 2001; Gorgadze & Lortkipanidze, 2006; Gorgadze et al., 2015), i.e., Neoaplectana (= Steinernema) georgica Kakulia & Veremtchuk, 1965; Neoaplectana (= Steinernema) thesami Gorgadze, 1988; Steinernema disparica Gorgadze, 2001; Steinernema gurgistana Gorgadze & Lortkipanidze, 2006 and S. tbilisiense (= S. tbilisiensis) Gorgadze, Lortkhipanidze, Ogier, Tailliez & Burjanadze, 2015. Steinernema georgica was considered as species inquirenda in Nguyen et
al. (2007), whilst S. thesami, S. disparica and S. gurgistana were not recognised, probably owing to their descriptions being published in Georgian. In this publication, we present the redescription of S. thesami and discuss its phylogenetic relationships within the 'affine-intermedium' clade of Steinernema.
MATERIALS AND METHODS
Nematode source. The isolate of S. thesami was maintained in the laboratory of enthomopathogenic nematodes of the Institute of Zoology (Ilia State University, Tbilisi, Georgia) by recycling through Bombyx mori instars or other insect larvae available since 1987. Infective juveniles were stored at 5°C.
Morphological studies. For morphological studies, ten larvae of G. mellonella were inoculated with infective juveniles (IJ) at a rate of ca 100 IJ larva-1 in Petri dishes lined with filter paper. First generation juvenile and adult stages were obtained by dissecting G. mellonella cadavers 5 days after inoculation; second generation males and females were recovered ten days after inoculation. Third-stage infective juveniles were collected after emergence from cadavers in White traps 10-14 days after inoculation, cleansed by passing the suspension through a milk filter and collected for morphological studies after 2 months storage.
Morphological studies were done in Tbilisi and Moscow on nematodes raised on wax moth of different provenance.
For light microscopy, nematodes were examined both live and preserved in 4% formaldehyde solution or triethanolamine formalin (TAF) (Courtney et al., 1955). Measurements and drawings were taken from formalin-fixed nematodes mounted on permanent slides after processing to glycerin according to Seinhorst (1959). All measurements are in micrometres and presented as the mean followed by the range in parentheses. Abbreviations: L, body length; W, maximum body width; ABD, anal body diameter; ES, pharynx length; NR, distance from anterior extremity to nerve ring; EP, distance from anterior extremity to excretory pore; T, length of tail; Sp, spicule length measured on the chord; SW, spicule width; Gub, gubernaculum length; GW, gubernaculum width; H, length of the hyaline region; V (%), distance from the anterior extremity to the vulva as a percent of body length; D (%) = EP/Ph * 100; E (%) = EP/T * 100; GS (%) = Gub/Sp * 100; H (%) = H/T * 100; SW (%) = Sp/ABD * 100; a, b, c, c', de Man indices.
Compound microscopes Motic-DMB1 and Nikon Eclipse E200 with drawing attachments were used. Photographs were taken using digital video camera Genius (G-Shot) DV 1110 and Leica microscope. Illustrations were finalised with WACOM Intuos A4 USB drawing tablet and Adobe Illustrator CS5. For scanning electron microscopy (SEM), the material was re-hydrated after formaldehyde, dehydrated in a graded ethanol series, critical point dried using a HCP-2 HITACHI dryer, mounted on aluminium stubs and coated with gold in a BIO-RAD SC502 sputter coater. Specimens were studied in Cam Scan and JCM-6380 LA SEM at Moscow State University.
Molecular characterisation. Suspensions of living juveniles and adults were washed several times with sterile water through centrifugation in 1.5 ml Eppendorf tubes. DNA was extracted using the Wizard® SV Genomic DNA Purification System (Promega) according to the manufacturer's protocol. For amplification of ITS rDNA, a pair of primers TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and AB28 (5' -ATATGCTTAAGTTCAGCGGGT-3') (Joyce et al., 1994) was used. The PCR cycling parameters included a primary denaturation step at 94°C for 5 min, followed by 34 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by a post-amplification extension step at 72°C for 5 min. PCR products were cleaned using Wizard® SV Gel and PCR Clean-Up System (Promega).
PCR products were sequenced with Genotech Ltd, Moscow. The sequences of other species and genotypes of Steinernema were downloaded from the GenBank and used to compare with the obtained one. The sequences were aligned using Clustal X with default values for gap opening and gap extension penalties. Different methods of alignment analysis: maximum parsimony (MP), neighbour joining (NJ) and maximum likelihood (ML) were performed with MEGA5 (Tamura et al., 2011).
DESCRIPTION
Steinernema thesami (Gorgadze, 1988) Gorgadze & Lortkipanidze, 2004
= Neoaplectana thesami (Gorgadze, 1988)
= S. tbilisiensis Gorgadze, Lortkhipanidze, Ogier, Tailliez & Burjanadze, 2015
Males 1st generation (Figs 1, 3 & 5; Table 1). Body plump, ventrally curved posteriorly, J-shaped when heat-relaxed, tapered to anterior end. Cuticle annulated. Lateral fields weakly developed and seen in SEM only as slight differentiation of cuticle. Deirids situated at level of pharyngeal bulb. Head continuous with body, truncate to rounded, six low, fused lips each bearing prominent labial papilla present (Figs 1B & 3A). Four slightly smaller cephalic papillae (2 subdorsal + 2 subventral) located posterior to labial papillae and a pair of slitlike amphids in lateral position at level of cephalic papillae. Mouth aperture subtriangular. Perioral disk absent. Stoma short, shallow 8-13 long and 7-10 wide, cheilostom broad, with thick rounded rhabdia; gymnostom short; stegostom funnel-shaped. Pharynx clavate, muscular, comprising corpus anteriorly reaching stoma base and slightly swollen posteriorly, short isthmus and pyriform basal, valvated bulb slightly wider than metacorpal expansion. Nerve ring surrounding isthmus. Excretory pore situated anterior to nerve ring, excretory duct weakly cuticularised at its distal, ca 10 long, part, totally ca 60 long, excretory cell located at bulb level. Cardia prominent, projecting into intestine. Intestine with wide lumen. Reproductive system monorchic, reflexed. Testis reflexion at ca 210-372 posterior to basal bulb, 590640 long. Spermatids located in vas deferens, large, 18-21 * 20-24, broadly elliptical in shape (Fig. 1I). Ejaculatory duct ca 170 long, with wide muscular walls. Immature sperm in ejaculatory duct ca 4-5 in dia. Spicules (Fig. 1J & L) paired, equal, colourless or yellowish, stout, with moderately defined, rounded manubria slightly longer than wide (ca 16-18 * 14-16 in size), shaft very short, ca 11-13 wide, blade moderately
Fig. 1. Steinernema thesami (Gorgadze, 1988). Males of the first generation: B, E, I-L; second generation males: C, F, G; first generation females: A, D, H, M-Q, V; second generation females: R-U, W; infective juveniles: X-ZZ. A-C, Head end; D, Anterior region; E, F, tail; G, tail extremity; H, sperm in uterus; I, spermatids in testis; J, spicules; K, gubernacula; L, spicules and gubernaculum; M-U, tail shape; V, W, vulva; X, anterior region; Y, head; Z, tail; ZZ, bacterial vesicle. Scale-bars in ^m. All in lateral position. Arrows: X, hemizonid; Z, phasmid..
Table 1. Morphometric characters of Steinernema thesami Gorgadze, 1988 (Tbilisi material). All measurements in ^m and in the form: mean ± standard deviation (range).
Males Females Infective
Character 1st generation 2nd generation 1st generation 2nd generation juveniles
Holotype Paratypes Paratypes Paratypes Paratypes Paratypes
n 20 20 20 20 50
L 2153 2030±239 1393±157 4763±569 2683±216 801±31
(1606-2332) (1201-1630) (3332-5795) (2356-3117) (714-880)
W 205 195±25 90±9 266±26 170±7 34±3
(122-235) (78-107) (214-321) (151-181) (29-39)
EP 132 130±10 119±9 143±16 128±11 72±3
(107-151) (107-137) (117-181) (112-151) (67-78)
NR 147 136±11 127±6 178±14 153±6 102±4
(117-156) (117-137) (147-220) (147-166) (93-107)
ES 200 191±10 174±8 265±12 212±7 140±5
(171-205) (161-186) (240-289) (200-225) (127-151)
ABW 78 72±8 48±4 94±9 58±5 19±2
(53-88) (39-58) (78-107) (49-68) (18-19)
TL 63 53±6 42±4 78±10 64±6 70±5
(44-68) (34-53) (63-98) (53-73) (63-78)
SL 68 68±6 (53-78) 55±3 (51-61) - - -
SW 14 15±2 (14-19) 13±0.9 (12-14) - - -
GL 53 49±2 (44-53) 37±3 (31-44) - - -
a 10.5 10.4±1.1 15.5±2.1 17.9±2 15.4±1.3 23±2
(9-13.2) (13-23.6) (14.1-23.2) (11.3-17.3) (20.4-27.4)
b 10.7 10.5±1.1 7.9±0.7 18±2 12.6±1.1 5.7±0.2
(8.7-12.6) (7-10.4) (13.8-21.5) (11.2-15) (5.4-6.2)
c 34 38±4.7 33.6±5.1 61±10 42.2±4.7 11.4±0.8
(30-47) (23.5-39) (42.7-81.4) (18-53.7) (10-13.3)
0.9 0.9±0.02 0.6±0.05 0.5±0.03 0.6±0.01 4.2±0.3
c (0.7-1.0) (0.6-0.7) (0.5-0.6) (0.5-0.6) (4.0-4.4)
HT - - - - - 31±3 (26-39)
H, % - - - - - 45±0.4 (37-54)
V, % - - - 55±3 (48.1-60.2) 56.5±2 (52.3-64.8) -
D, % 66 67±0.4 69±0.8 53±7 60±5 50±2
(57-75) (60-100) (42-72) (53-73) (47-53)
E, % 209 247±32 285±34 183±23 207±31 100±0.7
(186-300) (243-351) (153-253) (153-269) (87-123)
SW, % 87 94±1.4 (60-118) 114±1 (100-143) - - -
GS, % 77 72±1.4 (62-92) 66±0.6 (57-78) - - -
curved, 16-17 wide, rostrum absent, velum prominent, broad, not extending to spicule tip, distal tips bluntly rounded, 4 wide, sometimes appeared bifid (Fig. 5I & J). Blade with two lateral, prominent internal ribs. Gubernaculum ca 3/4 length of spicule and darker in colour than the latter, broad (8-10 wide), proximal end spoon-like, usually wide,
rounded but sometimes narrowed; distally, gubernaculum corpus open forming short wings curved upwards. Cuneus absent (Fig. 1K). Complement of 23 genital papillae arranged as typical for genus: single midventral precloacal papilla, five pairs of subventral papillae precloacal, two pairs adcloacal, one pair lateral postcloacal, two
Fig. 2. Steinernema thesami (Gorgadze, 1988). Infective juveniles. SEM images. A, Head end; B, Head; C, lateral field at anterior; D, lateral field at mid-body; E, body posterior showing lateral field; F, tail extremity. Scale-bars in цт. A-B, subapical view; C-F, lateral view. Abbreviations: a, amphid; cp, cephalic papilla; ph, phasmid.
pairs subterminal and one pair subdorsal. Phasmids indistinct. Tail short, ventrally curved, conoid, tip rounded, mucron absent (Figs 1E, 3C & D).
Males 2nd generation (Figs 1 & 3; Table 1). Similar to 1st generation but smaller (mean 2030 vs 1393). Lips more pronounced (Figs 1C & 3B). Stoma slightly longer. Spicules and gubernaculum shorter and thinner (Table 1). Tail mucron ca 4 (2-5) long present in 35% of specimens (Figs 1F, G, 3E &
F).
Females 1st generation (Figs 1, 4 & 5; Table 1).
Body C-shaped, obese, only slightly tapered to anterior end. Much larger in size than males. Cuticle
finely striated transversely. No lateral lines observed. Anterior end structure similar to that in males (Fig. 1B). Labial papillae prominent (Fig. 4A). Stoma 13 (12-15) long and 15 (12-18) wide (Fig. 1A). Corpus 141 (125-165) long and 32 (27-36) wide; isthmus 25 (20-36) long and 22 (20-28) long, bulb 54 (42-65) long and 51 (43-57) wide. Excretory duct up to 2-5 wide, its cuticularised distal end 12-18 long; total length of excretory duct 60-100. Reproductive system amphidelphic, ovaries reflexed. Spermia large, amoeboid, 7-9 * 11-13, forming short (2-5 cells) chains (Fig. 1H). Vagina straight, short, muscular, vulval lips small, inflated, symmetrical.
Fig. 3. Steinernema thesami (Gorgadze, 1988). Males of the first generation: A, C, D; second generation males: B, E, F. SEM images. A, B, head; C, E, F, tail region; D, tail extremity. Scale-bars in ^m. A-B, subapical view; C, dorsolateral view; D, subdorsal view; E-F, lateral view.
Vulva without epyptigmata, slit-like (Figs 1V & 4G), located just posterior to mid-body. Oviparous. Eggs ovoid, 49-54 * 45-47 in size, egg-shells thin, smooth. Anus an inverted crescent-shaped slit.
Slight postanal swelling usually present. Tail short (shorter than anal width), conoid. Tail tip shape
depending on obesity of females and varying from pointed in younger females to obtuse to knob-like in older and larger females (Figs 1M-Q & 4C-D). Sometimes, terminus bearing 2-3 minute warts (papilliform protruberances) seen mostly in SEM (Fig. 4C-D).
Table 2. Comparative morphometries of infective juveniles of Steinernema thesami Gorgadze, 1988 and the seven Steinernema strains of'ajfine-
intermedium' group. All measurements in |im and in the form: mean (range).
Character S. thesami * S. thesami ** S. beddingi S. sichuanense S. affine S. intermedium S. poinari S. arasbaranense
L 801 (714-880) 866 (713-948) 743 (700-790) 710 (606-787) 693 (608-880) 680 (608-800) 768 (687-848) 731 (693-795)
a 23 (20-27) 23 (20-28) 25 (23-27) 25 (23-28) 23 (21-28) 24 24 (21-28) 16(14-18)
b 5.7(5.4-6.2) 6.1 (5.2-6.7) 6.1 (5.8-6.6) 5.4 (4.9-6) 5.5(5.1-6) 5.6 5.5 (4.8-6.3) 6.5 (6-7)
c 11.4(10-13.3) 10.8(9.6-13) 9.6(8.7-10.7) 10(8.9-11) 10.5(9.5-12) 11 10(8.7-12) 8.7(7-10)
c 4.2 (4-4.4) 4.1 (3.5-4.8) - 4.3(3.8-5) 3.6 (3.3-4.2) 4 4.1 (3.3-4.6) 3.6 (3-5)
W 34 (29-39) 36 (34-41) - - 30 (28-34) 28 (25-35) 32 (29-37) 44 (41-50)
EP 72 (67-78) 72 (68-79) 70 (64-75) 64 (57-68) 62 (51-69) 65 (61-69) 64 (56-70) 56 (53-60)
NR 102 (93-107) 107 (98-112) 96 (80-113) 99(91-108) 95 (88-104) 92 (85-96) 98 (87-103) 84 (79-88)
ES 140(127-151) 140(132-151) 125 (113-130) 131 (121-142) 126 (115-134) 121 (110-131) 141 (119-151) 112(109-118)
TL 70 (63-78) 82 (70-93) 77 (72-83) 72 (64-76) 66 (64-74) 64 (53-72) 77 (68-87) 84 (79-101)
H 31 (26-39) 37 (31-48) - 31 (27-35) 25 (19-30) 32 (27-37) 38 (32-45) -
H% 45 (37-54) 45 (43-57) - 44 (45-51) 40 (36-45) 48 (45-51) 50 (43-56) -
ABW 19(18-19) 25 (17-36) - 17(14-19) 18(16-19) 16(13-18) 19(17-21) 23 (20-27)
D% 50 (47-53) 51 (47-55) 57 (52-64) 49 (46-53) 49 (43-53) 51 (48-58) 46 (40-55) 50 (45-54)
E% 100 (87-123) 90 (81-100) 92 (84-103) 86 (75-98) 94 (74-108) 96 (89-108) 84 (76-95) 67 (53-75)
Lateral field 4 4 4 6 6 6 6 -
References present study 1 2 3 4 4 5 6
* Original strain isolated in 1987, Tbilisi material.
** Isolated in 2011 and described as S. tbilisiense Gorgadze et al., 2015.
(1) Gorgadze etal., 2015; (2) Qiu etal., 2005; (3) Mracek etal., 2006; (4) Nguyen etal., 2007; (5) Mracek etal., 2014; (6)Nikdel etal., 2011.
Fig. 4. Steinernema thesami (Gorgadze, 1988). Females of the first generation: A, C, D, G; second generation females: B, E, F, H. SEM images. A, B, head; C, F, tail region; D, E, tail extremity; G, H, vulva. Scale-bars in ^m. A & B, sublateral view; C-E, lateral view; F-G, en face view; H, sublateral view.
D
Fig. 5. Steinernema thesami (Gorgadze, 1988). Light photographs. Females of the first generation: A, B, D, E; second generation female: C; first generation males: I, J; infective juveniles: F-H. A, anterior end; B, sperm in uterus; C, E, H, G, tail; D, sperm in extracted uterus; F, bacterial vesicle; G-H, tail with droplet insertion. Scale-bars: A, B, 50 ^m; C, 30 ^m; D, 12 ^m; E, 100 ^m; F, 30 ^m; G, H, 20 ^m; I, J, 15 ^m. Arrows: B, D, spermia; E, minute warts on tail tip; F, bacterial vesicle; G, H, droplet insertions; I, J, bifid appearance of spicule tip. All in lateral position.
Females 2nd generation (Figs 1 & 4; Table 1).
Smaller in size, more slender and with fewer eggs. Post-anal swelling expressed more strongly than in
1st generation. Short epiptigmata present or not (Figs 1W & 4H). Tail longer than wide, conoid, pointed or, rarely, mucronate (Figs 1R-U & 4E-F).
Infective juveniles (Figs 1, 2 & 5; Table 1).
Body C-shaped, tapering to both ends. Head end rounded, continuous with body contour, bearing four rather high cephalic papillae and small oval amphids (Figs 1Y & 2A-B). Cuticle annulated posterior to head region, annuli less than 1 wide. Lateral fields extending from ca 15 from anterior to tail terminus. Lateral field 18-20 wide at mid-body. Initially, it is expressed as three incisures, then at mid-body as a slightly elevated area where two prominent, marginal ridges positioned at short distance from field lateral margins and two lower ridges positioned in the centre; in tail region, central ridges disappearing (Fig. 2C-F). Stoma ca 4-7 long and 2 wide. Hemizonid situated opposite basal bulb (Fig. 1X). Pharynx with slender corpus slightly expanded at posterior, narrow isthmus and small elongated bulb with valves. Excretory pore at level of metacorpal expansion, cuticularised part of excretory duct short, entire duct extending to basal bulb level (Fig. 1X). Cardia distinct. Intestine containing numerous fat globules. Bacterial vesicle located in anterior part of intestine, oval or drop-
like, 28 (15-47) long and 15 (8-18) wide (measurements of Moscow culture) or 37-74 long and 21 wide (measurements of Tbilisi culture), usually with distinct, thin (ca 1 wide or less) wall, sometimes vesicle wall inconspicuous (Figs 1X, ZZ & 5F). Vesicle containing a number of rod-like bacterial cells (ca 5 long). Tail conical, slightly asymmetrical (sickle-shaped), with anterior part conical and posterior, hyaline region, somewhat cylindroid tapering to the point (Fig. 1Z). Anus crescent-shaped, inverted. Rectum ca 14 long. Phasmids present at border of hyaline portion. Hyaline part without spine but sometimes refractive droplet-like inclusions present (Fig. 5G-H).
Type habitat and locality. Georgia, Mtskheta-Mtianeti Region, Thesami village, alder thickets along Thesami river, 1105 m a.s.l.; coordinates: N 44°52'52", E 44°57'18", from infected pupa of Operophtera brumata Linnaeus, 1758 (Geometridae: Lepidoptera).
Other localities. Georgia, Tbilisi city, soil in a deciduous forest at the Kus Tba (Turtle Lake), 550 m a.s.l.; coordinates: N 41°42.645', E 44°45.708'.
100/96/100
-c
98/95/90
11 i Stei
99/100/100
99/87/100
-c
St in <.' r~n <*fn a at r'f? <* c cta & K.J^)5()29 1 Steifiernema t>icorn 1411*m KJ93X568
Steinernennt thili.sien.se KC287223 Steinernemcz thesami (typo isolate) Steinernemct sp. AY 1 7 1 29 1 (Russian Kcd. ) Steinernemi:z ,s/j. KJ696684 (Czech Rep.).
nc*rwc met intermedium A F 1 220 1 ft Steitiernemci proinari KH 1 45 ! 73 ernennt poind ri KK24 I 754 Stein&r-n&mci .siehndnen.se 1.5QSS4965 Steinernetnct up. AY 1 7 1 292 (Estonia) Si4-'i nurntvna bc-'tldinAY603397 Steinernema ar~<v.vficircjnt-'n^'cr FJS60039 Steinernem a affine AY 1 "7 1 29f> Steinemem a ajjine AY 17 1 289
Ci>tetn i Stein
Fig. 6. Phylogenetic relationships of Steinernema thesami (Gorgadze, 1988) with other species of 'affine-intermedium' group. Bootstrap 50% majority-rule consensus tree, based on 811 total characters with 219 parsimony informative. Gaps are treated as 'missing'. 1000 bootstrap replicates. Bootstrap values are presented near nodes as MP, NJ, ML.
Table 3. Comparative morphometries of 1st generation males of Steinernema thesami Gorgadze, 1988 (Tbilisi material) and seven Steinernema spp. strains of'affine-intermedium' group. All measurements in |im and in the form: mean (range).
Character S. thesami * S. thesami ** S. beddingi S. sichuanense S. affine S. intermedium S. poinari S. arasbaranense
T 2030 1652 2215 1852 1800 2400 1815 1265
l (1606-2332) (1911-2140) (1750-2700) (1555-2202) (1400-2100) (1600-3000) (1475-2363) (1057-1645)
a 10.4 (9-13.2) 13(10.1-17.3) - 15 (12-18) 15 14 12(9.4-14) 10(8-12)
b 10.5 (8.7-12.6) 9(7.5-11) 11 9.9(8.1-12) 12 13 10(8.5-12) 9(8-11)
c 38 (30-47) 33 (25.5-43) 43 40 (33-49) 35 44 38(31-46) 37 (29-47)
c' 0.9(0.7-1.0) 0.7(0.7-0.8) 0.8 0.9(0.8-0.9) 0.9 0.7 0.7 1 (0.7-1)
W 195 (122-235) 127(98-156) - 122 (91-141) 118 (95-164) 169(113-207) 158(118-219) 125 (98-143)
EP 130(107-151) 118(102-142) 114(95-138) 95 (88-105) 94 (82-114) 137(114-155) 99 (78-114) 65 (51-87)
NR 136(117-156) 134(117-156) 143 (125-165) - 105 (89-117) 146(120-168) - 106 (98-122)
ES 191 (171-205) 186 (166-200) 195 (175-218) 188(172-199) 153 (136-174) 190(155-209) 182(151-211) 133 (126-143)
TL 53 (44-68) 48 (39-59) 52 (41-63) 46 (43-54) 51 (45-56) 54 (45-59) 48 (43-59) 32 (31-36)
SL 68 (53-78) 67 (58-73) 71(63-78) 68 (65-72) 70 (67-86) 91 (84-100) 70 (62-78) 75 (70-82)
GL 49 (44-53) 44 (34-53) 43 (38-48) 7(40-51) 46 (37-56) 64 (56-75) 49 (45-57) 48 (42-53)
D% 67 (57-75) 62 (54-72) 58 (54-63) 51 (45-56) 61 67 (58-76) 54 (42-64) 50 (43-59)
GS% 72 (62-92) 66 (57-75) 61 (55-66) 80 (69-89) 66 69 (62-77) 70 (58-87) 63 (52-70)
SW% 94 (60-118) 115 (93-149) 108 (88-132) 130 (120-140) 117 124(103-139) 109 (98-123) 187(174-213)
E% 247(186-300) 235 (194-310) 219 207 180 253 207(167-246) 187(157-223)
Mucron absent absent absent absent present absent absent -
References present study 1 2 3 4 4 5 6
* Original strain isolated in 1987.
** Isolated in 2011 and described as S. tbilisiense Gorgadze etal., 2015.
(1) Gorgadze etal., 2015; (2, 4)Nguyen etal., 2007; (3) Mracek etal., 2006; (5)Mracekefa/.,2014; (6)Nikdel etal., 2011.
Molecular analysis and phylogenetic position
of S. thesami (Gorgadze, 1988). The length of the ITS rDNA sequence obtained for S. thesami was 797 bp. This sequence was used for BLAST-search in NCBI GenBank and revealed two most similar deposited sequences: Steinernema sp. AY 171291 from Russia and Steinernema sp. KC287223 from Georgia. The first sequence originates from Steinernema sp. isolated from the spruce wood soil. The soil sample was collected in 2000 near Krasnaya Polyana (43°42' N, 40°15' E; 900 m a.s.l.), in the mountains over Black Sea city-resort of Sochi and baited with wax moth larvae. The invasive ability of the strain decreased rapidly and it was lost after year long attempts to culture it on wax moth. The second sequence was obtained from the type culture of the recently described species S. tbilisiense Gorgadze et al., 2015. The newly obtained sequence of S. thesami was completely identical with that of Steinernema sp. AY171291 from Sochi. A 1 bp difference was reported between the sequences of S. thesami and S. tbilisiense. The level of the nucleotide differences in ITS rDNA of several Steinernema species of 'affine-intermedium' group are presented in Table 4. The interspecific differences for this group account for 17-23 bp minimum while intraspecific once range from 1 to 4 bp (as it is obvious for S. affine and S. poinari). Phylogenetic relationships of S. thesami with other species and strains of 'affine-intermedium' group are presented on Fig. 6. In all the obtained phylograms the clade with S. thesami and two conspecific strains was in basal position for entire group.
Type and voucher material. S. thesami
(isolated in 1987): holotype 1st generation male (slide no. D-412), paratype 1st and 2nd generation males (slide no. D-416-430), paratype 1st and 2nd generation females (slide no. D-431-445) and infective juveniles (slide no. D-446-457) are deposited in Tbilisi, in the collection stock of the museum of the Institute of Zoology of Ilia State University.
Steinernema thesami (isolated in 2011 and described as S. tbilisiense): holotype 1st generation male (slide no. D-337), paratype 1st and 2nd generation males (slide no. D-338-358), paratype 1st and 2nd generation females (slide no. D-359-374) and infective juveniles (slide no. D-375-391) are deposited in Tbilisi, in the collection stock of the museum of the Institute of Zoology of Ilia State University.
Voucher materials. 1st generation male no. 14255; 1st generation female no. 14256 and IJ no. 14257 are deposited in the Museum of the
Helminthological Collections, Centre of Parasitology, Severtsov Institute of Ecology and Evolution, Moscow.
Bionomics. Developing nematodes emit a characteristic scent. The infection of the Colorado beetle, Leptinotarsa decemlineata Say, by S. thesami was reported (Gorgadze & Lortkipanidze, 2004).The studies on the pathogenicity of S. thesami for G. mellonella and Tenebrio molitor had shown that it can be suitable for the biological control of certain pest species (Gorgadze, 2000; Gorgadze et al., 2013).
Comparison of Steinernema thesami with other species of 'affine-intermedium' group. The
strain of S. thesami kept in the laboratory of Iliya State University is the original EPN isolate from the type locality of the nematode. Since its isolation nearly 30 years ago, it was supported by the regular recycling through lepidopteran instars. It seems probable that the long laboratory life of the strain was facilitated by the most suitable choice of hosts (the natural host of S. thesami also being lepidopteran). The morphology of all stages of S. thesami was re-examined after its culturing on wax moth larvae as advised in Nguyen et al. (2007) using light and scanning microscopy. Sequences of ITS and D2-D3 regions of ribosomal DNA of the nematode were obtained for the first time. Molecular data for the nematode have confirmed the complete identity of S. thesami and S. tbilisiense. The comparative analysis of morphological data of these two strains has shown insignificant differences between them. They include the smaller body and tail length of IJ of S. thesami (mean 806 vs 866) and the larger value of E% (100 vs 90). At the same time, 1st generation males of S. thesami are larger than S. tbilisiense (2030 vs 1652) with longer gubernacula (49 vs 44, GS% 72 vs 66), and having the greater value of D% (67 vs 62) and E% (247 vs 235) with smaller SW% (94 vs 115). As molecular characteristics for both S. thesami and S. tbilisiense are identical, the latter is to be considered as the junior synonym for S. thesami. The morphometric differences between the isolates can originate from the different food source or population plasticity; having examined nematodes reared on wax moth in Moscow, we also found slight differences in the metric characteristics between Moscow and Tbilisi cultures. Thus, the infective juveniles were generally shorter (mean 720 vs 806) with smaller bacterial vesicles.
The chief metric characteristics of the members of the 'affine-intermedium' group are given in Tables 2 & 3. The similarity of S. tbilisiense (= S. thesami) to the other species of this group was discussed
Table 4. Pairwise nucleotide differences between Steinernema species and unidentified strains of 'affine-
intermedium' group of steinernematids.
Species 1 2 3 4 5 6 7 8 9 10 11 12 13
1 Steinernema thesami -
2 Steinernema tbilisiense KC287233 1 -
3 Steinernema sp. AY171291 0 1 -
4 Steinernema sp. KJ696684 30 29 30 -
5 Steinernema intermedium AF122016 42 41 42 35 -
6 Steinernema poinari KF145173 23 22 23 26 29 -
7 Steinernema poinari KF241754 23 22 23 26 39 1 -
8 Steinernema sichuanense DQ884965 46 45 46 47 59 40 40 -
9 Steinernema sp. AY171292 26 25 26 32 45 17 17 37 -
10 Steinernema arasbaranense FJ860039 37 36 37 40 50 31 31 45 23 -
11 Steinernema beddingi AY603397 50 49 50 57 64 43 43 63 38 54 -
12 Steinernema affine AY171296 35 38 39 41 49 32 32 43 27 28 51 -
13 Steinernema affine AY171289 39 38 39 41 49 32 32 43 27 28 55 4 -
in Gorgadze et al. (2015) and hereby is given with some additions. The 'affine-intermedium' group presently contains S. affine (Poinar, 1985), S. intermedium (Poinar, 1988), S. beddingi (Qiu et al., 2005), S. sichuanense (Mracek et al., 2006), S. arasbaranense (Nikdel et al., 2011) and S. poinari (Mracek et al., 2014). Morphologically, the members of 'affine-intermedium' group are characterised by 500-900 long IJ which are C-shaped in relaxed state and possessing lateral fields with two prominent central ridges; bacterial vesicles with thin wall (Spiridonov et al., 2004). Spermia in female gonads are amoeboid and form short chains. The phylogenetic analysis of the position of S. tbilisiense (Gorgadze et al., 2015) has placed it closest to S. intermedium. In the present study, the phylogenetic relationships between S. intermedium and S. thesami were observed only in maximum likelihood analysis, but had low level of bootstrap support.
Males of S. thesami are closest to S. intermedium by having similar body size and body proportions, similar colourless, broad, blunt, curved spicules with wide velum and hooked gubernaculum but differ in lacking a spicule rostrum and cuneus in the gubernaculum and in the smaller size of both structures. Bifid appearance of spicule terminus is common in both species. Also similarly, mucrons are absent in the 1st generation males in both species but if developed in the 2nd, they are reduced. Females are similar in the tail shape of both generations. Infective juveniles of both species are with a characteristic (dorsal) constriction of tail but can be distinguished by longer body size and 4 vs 6 ridges of the lateral field. Refractive, droplet-like
elements in IJ tail of S. thesami are sometimes present but were not reported for S. intermedium.
Steinernema thesami and S. affine are similar in having spicule shape and female tail shape. First generation males of S. thesami can be distinguished from S. affine by more posterior position of excretory pore (130 vs 94), longer pharynx (191 vs 153), the greater E% value (247 vs 180) and by the absence vs presence of a tail mucron. Infective juveniles are distinguished by longer IJ (806 vs 693), 4 vs 6 ridges in lateral field and absence of tail spine (only short refractive elements are present).
It is separated from S. beddingi, to which S. thesami is close, by similarly sized IJ with similar structure of lateral field, males with blunt-tipped spicules and lacking tail mucron and similar female tail; it is differentiated by smaller manubria, presence vs absence of velum, reduced vs developed postanal swelling and absence vs presence of tail spine in IJ.
Steinernema thesami is very close to S. sichuanense in general morphometrics of adult stages and similar spicule and gubernaculum shape (except for the absence of a rostrum and cuneus in S. thesami), absence of mucron and similar shape of female tail. Infective juveniles of S. sichuanense have a dorsal tail constriction as in S. thesami but are shorter (710 vs 806), with 6 vs 4 ridges in lateral field, indistinct vs distinct deirids and much longer bacterial vesicle (81-115 x 23-28 vs 15-74 x 8-21).
The first generation males of S. thesami differ from S. poinari in having blunt vs pointed spicules; no rostrum; IJ can be distinguished by body length (806 vs 768), D% value (67 vs 54), tail length 70 vs 77 and presence of 4 vs 6 ridges of lateral field.
First generation males and females of S. thesami differ from S. arasbaranense in the absence of mucron and females in the absence of epyptigmata. Infective juveniles mostly differ in the number of ridges of a lateral field (four in thesami vs eight in arasbarense) and body length (806 vs 732) and the presence of refractive elements in hyalin portion vs its absence.
DISCUSSION
Comparative analysis of ITS rDNA sequence of S. thesami has revealed its complete identity with the sequence of Steinernema sp. from mountainous area over Sochi. Also, the strain described recently as S. tbilisiense is obviously conspecific with S. thesami, as these differ only in 1 bp, which is lower than reported intraspecific variability of this region. Thus, up to now S. thesami was reported three times. All three findings were made on the southern slope of Caucasus Ridge. Further surveys can only answer if this ridge is serving as natural barrier for the EPN fauna.
ACKNOWLEDGEMENTS
The Russian Foundation for Basic Research supported this work (project no. 15-04-06148-a).
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Резюме. По результатам переисследования типового изолята, выделенного в Грузии из зараженных чешуекрылых Operophtera brumata, дано переописание вида Steinernema (Neoaplectana) thesami Gorgadze, 1988. Типовой изолят культивировался в лаборатории с момента выделения в 1987 году. Анализ нуклеотидных последовательностей ITS rDNA S. thesami выявил их идентичность последовательностям недавно описанных S. tbilisiense Gorgadze, Lortkhipanidze, Ogier, Tailliez & Burjanadze, 2015, а также штейнернематид не определенного изолята из Красной Поляны, Сочи, Российская Федерация. Steinernema tbilisiense рассматривается как младший синоним S. thesami. Предложен анализ филогенетических связей S. thesami с другими видами группы видов 'affine-intermedium' рода Steinernema.
