CC BY
24Russian Journal of Nematology, 2014, 22 (2), 109-120
Addendum to the description of Steinernema carpocapsae (Weiser, 1955) Wouts, Mracek, Gerdin & Bedding, 1982
Zdenek Mracek, Vladimir Puza and Jiri Nermut'
Laboratory of Entomopathogenic Nematodes, Institute of Entomology, Biology Centre, Czech Academy of Sciences, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic
e-mail: vpuza@seznam.cz
Accepted for publication 2 September 2014
Summary. In the light of current morphological knowledge, the Steinernema carpocapsae morphology is not satisfactorily described, some important characters of this species are missing and no thorough molecular characterisation of this species has been performed. Steinernema carpocapsae represents a type of the 'carpocapsae' group and thus a main objective of this study was to add characters such as, infective juvenile hyaline layer with %H, tail morphometry with ratio c', shape and protuberances on the F1 tail etc., to the general knowledge about this species. We further aimed to sequence ITS region and D2-D3 regions of the ribosomal DNA, and compare them with the sequences for S. carpocapsae available in the GenBank NCBI database. Four S. carpocapsae isolates (strains) were studied (USA - DD136 and All, Russian - Agriotos and Swiss - DOK83). Morphology was identical for all isolates and morphometry close each to other. Infective juveniles possess rather narrow, continuous and hemispherically rounded head, lateral field with six ridges (seven lines) with the less prominent central pair, hyaline layer occupies about 40-44% of the tail length, ratio c' varies from 4.0 (3.8-4.1) to 4.2 (3.9-4.4) and ratio %E from 71 (59-82) to 79 (74-83). First generation females possess a characteristic blunt tail with a terminal, conical sharply ended peg and no other minute protuberances and rounded vulva slightly protruding from the body contour. All male generations possess the only minute mucron and there was little size difference between the first and second (other) generations. Both studied genetic markers effectively characterize S. carpocapsae as a species, showing very low interspecific variation. However, the status of the closely related species S. anatoliense and S. websteri seems to be doubtful. Key words: entomopathogenic nematodes, Steinernematidae, morphology.
Recently, the number of steinernematid species has increased rapidly. Even though significant differences in morphological characters between these species exist, all are classified in the genus Steinernema Travassos, 1927. To enable easier species characterisation, five groups of morphologically similar species were established (e.g., Hominick et al, 1997). Later, molecular data supported these groups as phylogenetic groups (e.g., Stock et al., 2001; Spiridonov et al., 2004).
The steinernematid species forming the 'carpocapsae' group are characterised by a short body length, finely rounded head, six (eight) ridges in the lateral field and anterior position of excretory pore of infective juveniles (IJ). Males possess slender, finely arcuate spicules with a prominent rostrum. The first described species of this group, S. carpocapsae (Weiser, 1955) Wouts, Mracek, Gerdin
& Bedding, 1982 was redescribed by Poinar (1967). However, in the light of current morphological knowledge, the S. carpocapsae morphology is not satisfactorily described and some important characters of this species are missing. Therefore, S. carpocapsae represents a type of this group so we added characters, such as, IJ hyaline layer with %H, tail morphometry with ratio c', shape and protuberances on the F1 tail etc., to the general knowledge about this species and complete Weiser's (1955) description and the redescriptions by Poinar (1967) and Nguyen et al. (2007), and emphasise those characters that should be compared with other species belonging to the 'carpocapsae' group.
A large number of ITS and D2-D3 of S. carpocapsae and related unidentified isolates are available in the GenBank NCBI database. Some sequences attributed to this species are doubtful, and
Fig. 1. Steinernema carpocapsae (light microscopy). A & B first generation female: A - tail with conical peg, B -vulva; C & D second generation female: C - tail, D - vulva; E & F male: E - first generation, F - second generation; G & H infective juvenile: G - tail, H - anterior portion. (Scale bars: A-B = 300 ^m; C-D = 87 ^m; E-F = 60 ^m; G-H = 27 ^m.). Abbreviations: bv - bacterial vesicle, ep - excretory pore, h - hemizonid, hy - hyaline layer beginning.
Fig. 2. Steinernema carpocapsae (scanning electron microscopy). A-H infective juvenile: A-C - head portion; D -lateral field in anterior portion; E - lateral field in the mid body; F & H - lateral field in the tail portion; G - tail ventrally. Abbreviations: ep - excretory pore, ph - phasmid.
some sequences attributed to other species show very high similarity to S. carpocapsae. Thus, we aimed to revise relevant sequences in the GenBank NCBI database.
MATERIALS AND METHODS
Steinernema carpocapsae has a Holarctic distribution (Hominick, 2002); however, its recovery from fields is rare (Mrácek et al., 2005). The original topotype culture of Weiser (1955) is lost. Therefore, we studied the following cultures: S. carpocapsae strain Agriotos, which came from the original culture of Dr Veremtchuk (1969) and it has been kept for 30 years in our living stock, US strains DD-136 and All, received from the collection of Dr Ralf Ehlers, Christian Albrechts University Kiel, Germany, and a Swiss strain DOK83 from the collection of Dr Raquel Campos-Herrera, University of Neuchatel, Switzerland. All these strains were propagated at the Laboratory of Entomopathogenic Nematodes, Institute of Entomology, Biology Centre ASCR in Ceské Budejovice.
Light microscopy (LM) and morphometry. IJ and adult nematodes were studied either heat-killed on glass slides in a drop of water or fixed in a hot TAF (Courtney et al., 1955) and transferred to glycerin by a slow evaporation method (Seinhorst, 1959). The measurements and examination of morphology were performed with an AMPLIVAL light microscope, Carl Zeiss Jena, and a Leitz DIAPLAN with Nomarski optics.
Scanning electron microscopy (SEM). Nematodes were fixed in 4% formalin buffered with 0.1 M sodium cacodylate at pH 7.2 for 24 h at 4-6°C and post-fixed with 2% osmium tetroxide solution for 12 h at 25°C. After dehydratation in a graded ethanol series, critical point drying with liquid COB2B, mounting on SEM stubs, and coating with gold (Nguyen & Smart, 1995) the nematodes were observed under SEM JEOL JSM 7401F.
Molecular characterisation. DNA was extracted from single females. Each female was transferred into a sterile Eppendorf tube (500 ^l) with 20 ^l of extraction buffer (17.7 ^l of ddH2O, 2 ^l of 10 x PCR buffer, 0.2 ^l of 1% tween and 0.1 ^l of Proteinase K). Buffer and nematode were frozen at -20°C for 20 min and then immediately incubated at 65°C for 1 h, followed by 10 min at 95°C. The lysates were cooled on ice, and then centrifuged (2 min, 9000 g) and 2 ^l of supernatant was used for PCR. A fragment of rDNA containing the internal transcribed spacer regions (ITS1, 5.8S, ITS2) was amplified using primers 18S: 5'-TTGATTACGTCCCTGCCCTTT-3' (forward), and
28S: 5'-TTTCACTCGCCGTTACTAAGG-3' (reverse) (Vrain et al., 1992). The other fragment containing D2-D3 regions of 28S rDNA was amplified using primers D2F: 5'-CCTTAGTAACGGCGAGTGAAA-3' (forward) and 536: 5'-CAGCTATCCTGAGGAAAC-3' (reverse) (Nguyen, 2007). The PCR master mix consisted of ddH2O 7.25 ^l, 10 x PCR buffer 1.25 ^l, dNTPs 1 ^l, 0.75 of each forward and reverse primers, polymerase 0.1 ^l and 1 ^l of DNA-extract. The PCR profiles were used as follows for ITS: 1 cycle of 94°C for 7 min followed by 35 cycles of 94°C for 60 s, 50°C for 60 s, 72°C for 60 s and a final extension at 72 °C for 7 min (Nguyen, 2007) and for 28S rDNA: 1 cycle of 94°C for 7 min followed by 35 cycles of 94°C for 60 s, 55°C for 60 s, 72°C for 60 s and a final extension at 72°C for 10 min (Mracek et al, 2006). PCR was followed by electrophoresis (45 min, 120 V) of 2 ^l of PCR product in a 1% TAE buffered agarose gel stained with ethidium bromide (20 ^l ETB per 100 ml of gel).
PCR-products were sequenced and the sequences were edited and deposited in the GenBank NCBI database under accession numbers KJ950291 (ITS sequence, strain NCR), KJ950292 (D2-D3 sequence, strain NCR) and KJ950293 (D2-D3 sequence, strain Dok83). ITS sequence of the strain Dok83 (KJ818295) and ITS and D2D3 sequences of the strains All (AY230164, AF331900) and DD136 (HM140694, HM140688) were downloaded from the GenBank NCBI database. An alignment of our samples together with sequences of the species of the 'carpocapsae' group, including other isolates of S. carpocapsae present in GenBank NCBI database, was produced for each amplified DNA-region using default ClustalW parameters in MEGA 5.05 (Tamura et al., 2011). Alignments were optimised manually in BioEdit (Hall, 1999) and dubious sequences (e.g., those with marked differences only at the edges) were removed. Pairwise distances between species of the 'carpocapsae' group were computed using MEGA (Tamura et al., 2011). Codon positions included were 1st + 2nd + 3rd + Noncoding.
The phylogenetic trees were obtained by the Minimum Evolution method (Rzhetsky & Nei, 1992) in MEGA 5.05 (Tamura et al, 2011). Representatives of other Steinernematid groups S. riobrave, S. abbasi, S. glaseri, S. feltiae, S. texanum, S. litorale, S. affine, S. intermedium and S. sichuanense were added to illustrate the position of the 'carpocapsae' group within the family and Caenorhabditis elegans was used as the outgroup taxon. The Minimum Evolution trees were searched
using the Close-Neighbour-Interchange (CNI) algorithm (Nei & Kumar, 2000). The Neighbour-joining algorithm (Saitou & Nei, 1987) was used to generate the initial trees. The evolutionary distances were computed using the p-distance method (Nei & Kumar, 2000) and are expressed as the number of base differences per site.
RESULTS
IJ head and neck region (Figs 1H, 2A-C). The
shape of head varies in its lip region in the steinernematid species. The fore part can be finely rounded to flattened. In the neck region, the head is continuous or slightly offset from the body contour. However, a detailed description of the S. carpocapsae head has not yet been published. Stanuszek (1974) reported the head of S. carpocapsae (erroneously identified as S. feltiae) was narrow, elongated, separated with lateral and ventral constriction. Sturhan (pers. comm.) described it as rather narrow, continuous and hemispherically rounded. This description seems to be characteristic for all species of the 'carpocapsae' group. Lens-like hemizonid is between the nerve ring and terminal bulb beginning.
IJ lateral fields (Figs 2D-H). Lateral field represents an important morphological character. Procedures of IJ preparation for a scanning electron microscopy may result in some cuticular artifacts and therefore the structure of the lateral field must be studied carefully. In our observation, all strains possessed six ridges (seven lines) with the less prominent central pair, the same number as reported by Poinar (1967). The lateral fields begin in the neck region as a single depression shortly splitting in three depressions. From these depressions the first pair of ridges raises with a wide flat space between them. In the flat space the remaining ridges increase gradually up to six. In the tail portion the two central pairs merge before the anus, whereas the prominent lateral pair leads to its tip.
IJ hyaline layer and %H (Fig. 1G). Valuable taxonomical characters recommended by Dr Sturhan (pers. comm.) that, unfortunately, are often forgotten in recent species descriptions. In S. carpocapsae, the hyaline portion is not well seen in a LM. In exsheathed IJ the length of layer (measured from the tip of hypodermal lining) varied between 19.1 and 28.7 ^m depending on the body length. Longer IJ had longer hyaline layer, but the ratio %H, which varied from 40 to 44, was not influenced.
IJ tail, ratio c' and %E. Ratio c' (tail length/tail width) is an often overlooked and little used character that may separate species quite precisely,
even within one group. For example, for 'carpocapsae' group species, it varies from 3.1 to 4.6 (Phan et al., 2014). In our measurements and calculations, the tail length varied from 48 to 57 ^m and when divided by the tail width, the average ratio c' of our four isolates varied from 4.0 (3.8-4.1) in the Swiss strain to 4.2 (3.9-4.4) in the Agriotos strain. In Nguyen et al. (2007), the ratio %E is reported to be 60 (54-66). By contrast, our results varied from 71 (59-82) in the DD 136 strain to 79 (74-83) in the Swiss strain.
Female tail and vulvar structure (Figs 1A-D, 3C & D). Female tail morphology provides two characters valuable in steinernematid taxonomy. Small papilla-like protuberances (mucrons) in the first generation females vary in number (from 0 to 4) and arrangement. Postanal swelling may absent or form slight, moderate or prominent protrusion. In S. carpocapsae the blunt tail possesses characteristic terminal conical sharply ended peg and no other minute protuberances. Postanal swelling not or very slightly developed. Second generation females possess conical tail without postanal swelling and with characteristic depression (All). In both generations, the rounded vulva is slightly protruding from the body contour.
Male mucron (Figs 1E & F, 3E-H). Male tail with a terminal mucron, either filamentous or spinelike, belongs to the important taxonomical characters. Generally in steinernematids, the mucron can be missing or present. Usually, if the mucron is developed, the first generation possesses a shorter mucron compared with the second generation, which is much longer. Stanuszek (1974) reported similar average length of the S. carpocapsae (erroneously identified as S. feltiae) mucron when compared first and second generations, 2.3 vs 2.4 ^m, respectively. In our measurements, in all generations only the minute mucron was developed and there was a little size difference between the first and second (other) generations, 2.2 vs 2.6 ^m, respectively. Ratios % GS and % SW were comparable to previously published data.
Molecular characterisation. ITS region. For the ITS region, the variability among different S. carpocapsae strains ranged from 0-1.1% with the exception of S. carpocapsae C101 (EU914605), which differed from other strains by 1.2-2.4% (Table 2). Such intraspecific variation in the ITS region is comparable to other steinernematid species (Spiridonov et al., 2004). The distance between S. carpocapsae strains and the closest species S. sasonense was between 4.3-6% (Table 2). All S. carpocapsae strains formed a highly supported monophyletic group (Fig. 4). The general topology
Fig. 3. Steinernema carpocapsae (scanning electron microscopy). A-D first generation female: A & B - head portion with labial and cephalic papillae, C - tail with conical peg, D - vulval portion; E-H males; E & F lateral view, G - ventrolateral view, H - ventral view. Abbreviation: m - mucron.
Table 1. Comparative morphometries of different Steinernema carpocapsae strains (IJ - infective juvenile and Ml -
first generation male). Data are in the form: mean (range).
Strain IJ BL IJc' IJ%D IJ%E IJ%H LF ridges M 1s %GS M 1%SW
DD 136 525 4.1 33 71 37 6 82 155
(404-606) (3.8-4.6) (28-35) (59-82) (33-42) (79-88) (148-166)
All 594 4.1 32 73 38 6 79 Yy
(545-626) (3.9-4.5) (29-34) (69-78) (34-44) (79-79) (143-156)
Agriotos 566 (485-626) 4.2 (3.9-4.4) 33 (29-36) 77 (69-84) 42 (40-44) 6 82 (77-86) 150 (144-177)
Dok83 626 4.0 35 79 39 6 77 161
(545-667) (3.8-4.1) (32-37) (74-83) (36-41) (71-80) (152-168)
Pieridarum x 556 (465-631) 3.9 38 81 60 173
carpocapsae y 558 (438-650)y 4.6z 26 (23-28)y 60 (54-66)y 42z 6y 72y 151y
s spicules measured along the chord; x recalculated, according to Stanuszek (1974); y from Nguyen et al. (2007); z from Phan et al. (2014). BL = body length, c' = tail length divided by tail width, %D = length to excretory pore divided by pharynx length, %GS = gubernaculum length divided by spicule length, %E = length to excretory pore divided by tail length, %H = hyaline divided by tail length, LF - lateral field, %SW = spicule length divided by tail width.
Gu3ss6)3 s. sp. Cotîi
ГТ CiUÉ>471 ЪЬ 5. .....МРЮ
"Î_AY171280S. rarrti from Wet «am
Ч—
ч
6U3956325.si. YNdeO <3U3SS620 S. sp. *Nd278 liOf Aï 170339 3. sc. GCC313 AY487920 S. ctrmgarense -AY487921 i.
dFJ418C45i 10 SG»b2 HQ317503S. sp SG*b5 GU39563üi sp. YNdS
----90042i iulMtltW UGOM 104191
- AY4S7918 S. trcrtoriense
-JX135S47 S. sp. Pah.B.S.eS
-GU130132 3L s p. TS1 HQ190044S. neppiense UGW! 104194 AY487915 S. SffSCPKPUe , GCL42 5- çarpo&ipiaç йсл 14 KJ9S0291 5. corpocopsoe NCR I— EU9148H S. C.Oj
1 ¡-''1132 J çorpocopsoc From l'-J.'.-. GQ421607 S. carpocapsae * GQ421615 S. Г'"." -С Т'Г? ; EUS98239 5. carpocapsae IRAIS KJS18295 S. carpocapsae Dekttä JF920967Î. rerpotppsôeAolrÈ г EU345421 5. sp. 1 Р5-2003 [ AF331913 i. carpocopsoe AY171283S. carpocopsoe ,AY330364 S. carpocapsae AW 1на4ГК729 5. fofpûcopifle rVRAII F N04 1кС621916 5. MDU5I15
НМ14069Д S. carpocapsae 00136 G U 39562 H. мчмещкое A24 KCS71260 5. carpocapsae SiS7 Kt 571262 5. carpocapsae SISll I EU2D0353 5. carpocapsae Bydr-1 ' EU2W352S.«JrfrortrpipeAI-BdJ*i-Andi GQ42L6ÛS S. carpocapsae CatuOl AFI21049 5. сarpoeppspe
AY17Q334 5. carpocapsae SGIB KCS7126S5. corpocapsoe IS34
. AY2 30183 5. scaptertsci -^¡d. AF12 2020 £ impterj'scp AF33191S S scepterisci
-AY171286 S. gloseri
-AY23015B 5. ûbÎMij"
-DQB35613 Я riobrovt
г-EF15256S S. tenirлит
______I-Afl21050S-fr/ti'ire
'— A&243441S. htaroie
- AF122016 S. intermedium
— AY230159S affine
GQ25S639Î. siamkayai SSRJC10 GQ2 53642 $ sftm*o>o<SSRKB KC62191S5-siomitoj4ii TH5H4 GÛ2SS654 S- sfomkxryaf SSRK26 GQ25B646 S. siamkayoi 55RK18 GdJSe643S.jfimkeye' SSBK14 GqîMSSllsitrmibiyiii $5Re23 GQ2S3660Î. Siomkavai S5RK33 r— JN571095Î. siaivkayai A PAK.P.S.EO 1 JX11044SS. sp. Pak.P.S.150 GQÎ58652S siamkoyoi SSBK24
tlNlS7772i. slomkoyoi RAK.S.P.79 Ш355565. sp. Pak.S.5.139 JX13S5S7S. sp. Pak.S.S.136 г AY1712315. SB. 1 SS-2003 72 Ш104453. Sp. Pak.P.5.161 I— JX1355S5 S. sp. Pat S 3-137 FJ46393S 5. siamkayoi SSRK2 FU63937J ЯиЛЮцИ" 5S8K4 I FJ4&3940 ■ siamkayoi RK 7
JQ2774605 sp. citiUJ J02 774 S9 5 sp. 50117 г- EF21Ë371S. sp. 5Gdl2 L HQ317502 5. sp. SStn2 EF216&70 S. sp. SGdll GU3S4215S.se. SGijl9 JN17 7514 5. sp. 5Gmtrl6 JN25I021 3. sp G U130134 S sp. G93 JN177S1S S. sp. SGmirl? GU13018? S sp. МЗЭ rtC625525J sp- ГШ S 5 86(1)6 L JÛ2 67993 5. siamkayoi PKMS1 GQ377416S. siamkayoi CM2 ■ JQ2Ü7992 S Siamkayoi CBES1
I 10267994 S. siamkayoi PKMS2 4 I ÜC62S529S. sp. PAK.e.S.95(A) f G037J415 S. siamkvyci CM "—HQ317505Î. sp. SGcjlE JM177S13 S Sp. 5Gm( SU 130185 S. sp KS QUI 30183 i. sp G6 -OU395629 S. sp. YNC230 bU 130186 S. sp M 24 GU354212 S sp. SGmlfll AF331917 S. siamkayai JF8&2S44 5. siamkayai Psk.S.S.10 -~ Jf392 545 i. SB. Pat. S.S.73
Fig. 4. Phylogenetic relationships of the species of the 'carpocapsae' group and other selected steinernematid species based on analysis of ITS rDNA regions. Steinernema affine and S. intermedium were used as outgroup taxa. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10000 replicates) are shown next to the branches. Branch lengths indicate evolutionary distances and are in the units of the number of base differences per site.
Tntile 1, Pairwise distances of ITS region between selected strains of Steimm2ma carpocapscte and other related species (percentage distances) (Sc = S. carpocapsae, Ssa iflsoHenie, Ssc = S. scapterisci).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
1 EU200353 Sc Badr-1
2 EU200352 Sc Al-Balka-Arida-1 0.0
3 AF121049 Sc 0.2 0.2
4 AF331913 Sc 0.4 0.4 0.1
5 6 AY170334 Sc SGIB AY 171282 Sc Russia 0.8 0.7 0.8 0.7 0.6 0.4 0.5 0.5 1.1
7 8 HM170694 Sc DDI36 AY230164 Sc All 0.3 0.7 0.3 0.7 0.0 0.5 0.1 0.1 0.7 1.1 0.4 0.4 0.0
9 KJ818295 Sc DOK83 0.6 0.6 0.4 0.5 1.0 0.0 0.4 0.4
10 KJ950291 Sc NCR 0.7 0.7 0.5 0.7 1.0 0.1 0.5 1.0 0.1
11 EU345421 S. sp. 1 PS-2008 0.5 0.5 0.3 0.1 0.9 0.4 0.0 0.3 0.6 0.8
12 EU598239 Sc IRA 18 0.7 0.7 0.4 0.5 1.0 0.0 0.4 0.4 0.0 0.1 0.4
13 HQ406729 HB All EN04 0.7 0.7 0.5 0.1 1.1 0.4 0.0 0.0 0.4 1.0 0.3 0.4
14 EU914854 Sc C101 2.0 2.0 1.7 1.7 2.4 1.2 1.6 1.7 1.3 1.2 1.7 1.3 1.7
15 GQ421605 Sc Caba02 0.2 0.2 0.0 0.1 0.6 0.4 0.0 0.5 0.4 0.5 0.3 0.4 0.5 1.7
16 GQ421606 Sc B cnl4 0.7 0.7 0.5 0.7 1.0 0.1 0.5 1.0 0.1 0.0 0.8 0.1 1.0 1.2 0.5
17 GQ421607 Sc Az20 0.6 0.6 0.3 0.5 0.9 0.0 0.4 0.9 0.0 0.1 0.6 0.0 0.9 1.3 0.3 0.1
18 GQ421615 R1 0.6 0.6 0.3 0.5 0.9 0.0 0.4 0.9 0.0 0.1 0.6 0.0 0.9 1.3 0.3 0.1 0.0
19 GU395621 Sc A24 0.5 0.5 0.2 0.4 0.8 0.7 0.3 0.7 0.6 0.7 0.5 0.7 0.7 2.0 0.2 0.7 0.6 0.6
20 JF920967 Sc Arak6 0.9 0.9 0.7 0.9 1.1 0.4 0.8 1.3 0.4 0.5 1.0 0.4 1.3 1.6 0.7 0.5 0.4 0.4 0.9
21 KC571260 Sc SiS7 0.2 0.2 0.0 0.1 0.6 0.4 0.0 0.5 0.4 0.5 0.3 0.4 0.5 1.7 0.0 0.5 0.3 0.3 0.1 0.7
22 KC571262 Sc SiSll 0.2 0.2 0.0 0.1 0.6 0.4 0.0 0.5 0.4 0.5 0.3 0.4 0.5 1.7 0.0 0.5 0.3 0.3 0.1 0.7 0.0
23 KC571265 Sc IS34 0.2 0.2 0.0 0.1 0.6 0.4 0.0 0.5 0.4 0.5 0.3 0.4 0.5 1.7 0.0 0.5 0.3 0.3 0.2 0.7 0.0 0.0
24 KC621916 Sc MDUStl5 0.8 0.8 0.5 0.1 1.1 0.4 0.0 0.0 0.4 1.0 0.3 0.4 0.0 1.7 0.5 1.0 0.9 0.9 0.8 1.3 0.5 0.5 0.5
25 AY487919,S'm 4.5 4.5 4.3 4.5 5.0 4.7 4.3 4.4 4.7 4.9 4.3 4.7 4.4 6.0 4.3 4.9 4.7 4.7 4.6 4.8 4.3 4.3 4.3 4.4
26 AF122020 Ssc 11 11 11 12 11 12 12 12 12 11 12 12 12 13 11 11 11 11 11 11 11 11 11 12 12
Tiible 3, Pairwise distances of D2D3 region between selected strains of Stsmernema carpocapsae and other related species (percentage distances) =S. websteri, Sc = S. carpocapsae, Sa = S. anatoliense, Sh = S. huense, Ssu = S. surkhetense, Sn = S. nepalense, Ssc = S. scapterisci).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
1 JF503100 .Sto AS1
2 GU56P058^w Alcazar 0.1
3 EF217323 .Sto JCL006 0.0 0.1
4 EF217324 ^H? JCL015 0.0 0.1 0.0
5 EF217325 S» JCL030 0.0 0.1 0.0 0.0
6 EF217326 ^h? JC1032 0.0 0.1 0.0 0.0 0.0
7 AY841762 ¿"w 0.0 0.1 0.0 0.0 0.0 0.0
8 KJ950292 Sc NCR 0.0 0.1 0.0 0.0 0.0 0.0 0.0
9 KJ950293&DOK83 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.0
10 HM140688 Sc DD136 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.0 0.0
11 AY169552 ScBW 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
12 GU3 95640 icBW 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2
13 GU3 95641 Sc A24 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.0
14 EU200356 Sa 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.4 0.4 0.4 0.2 0.4 0.4
15 GU5 69043 Sa 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.4 0.4 0.2 0.4 0.4 0.0
16 AY841761 Sa 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.4 0.4 0.4 0.2 0.4 0.4 0.0 0.0
17 HQ 190043 Ssu 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.8 0.7 0.7 1.1 0.7 0.7 1.3 1.0 1.3
18 HQ 190045 Sn 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.5 1.4 1.4 1.8 1.4 1.4 2.0 1.7 2.0 0.7
19 KF857582 1.5 1.3 1.4 1.4 1.4 1.4 1.4 1.4 1.6 1.6 1.8 1.4 1.4 2.1 1.7 2.1 1.1 1.5
20 GU3 95646 Ssc 5.5 5.6 5.5 5.5 5.5 5.5 5.5 5.6 5.5 5.5 7.0 5.6 5.6 5.7 5.7 5.7 4.8 5.2 5.6
21 AF331900& All* 34 33 33 33 33 33 33 33 34 34 37 34 34 33 33 33 30 30 34 34
22 EU598241 Sc IRA 18* 34 34 34 34 34 34 34 34 34 34 37 34 34 34 34 34 31 32 35 34 0.0
23 DQ145660 Sc* 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 38 37 0.4 0.4
41 sequence belongs to Heterorhabditis
Fig. 5. Phylogenetic relationships of the species of the 'carpocapsae' group and other selected steinernematid species based on analysis of D2D3 regions of the 28S rDNA. Caenorhabditis elegans was used as outgroup taxon. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10000 replicates) are shown next to the branches. Branch lengths indicate evolutionary distances and are in the units of the number of base differences per site.
with two well supported main clades and S. scapterisci occupying a basal position corresponds to the results of Phan et al. (2014). Monophyly of all the species represented by more than one population suggests that ITS region accurately characterises the species within the 'carpocapsae' group.
D2-D3 expansion of the 28S rDNA. The variability among different S. carpocapsae strains ranged from 0-0.2%. Surprisingly, the D2-D3 sequences attributed to S. anatoliense and especially S. websteri showed a minimum distance from the sequences of S. carpocapsae strains (Table 3). The other closest species S. surkhetense was separated by 0.7-1.3%.
In the phylogenetic analysis based on this region (Fig. 5), S. websteri and S. anatoliense fell within the well supported group with S. carpocapsae strains. It thus seems that these nematodes do not represent independent evolutionary lines, and are conspecific with S. carpocapsae. Unfortunately, sequences of the ITS region or other markers are not
available for these nematodes. Thus, the status of S. websteri and S. anatoliense seems to be doubtful and should be further examined.
Both the pairwise comparisons and phylogenetic tree further showed that D2-D3 sequences with accession numbers AF331900 (S. carpocapsae, strain All), EU598241 (S. carpocapsae, strain IRA18) and DQ145660 S. carpocapsae, strain All) belong in fact to the nematodes of the genus Heterorhabditis. Unfortunately, these sequences were used in some phylogenetic studies of the Steinernematidae family, causing placement of the 'carpocapsae' group to the basal position. Studies based on the ITS region sequence usually support the basal position of 'affine' group (e.g., Spiridonov et al., 2004). To prevent inappropriate usage, these sequences should be removed from the database or adequately renamed.
In any new species description of steinernematid nematodes, the sequences of both ITS and D2-D3 regions should be included.
ACKNOWLEDGEMENTS
The study was supported by the Czech Ministry
of Education, Youth and Sport in the programme
KONTAKT II, project LH 12105.
REFERENCES
Courtney, W.D., Polley, D. & Miller, V.I. 1955. TAF, an improved fixative in nematode technique. Plant Disease Reporter 39: 570-571.
Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98.
Hominick, W.M. 2002. Biogeography. In: Entomopathogenic Nematology (R. Gaugler Ed.). pp. 115-143. Wallingford, UK, CABI Publishing.
Hominick, W.M., Briscoe, B.R., Del Pino, F.G., Heng, J., Hunt, D.J., Kozodoy, E., Mräcek, Z., Nguyen, K.B., Reid, A.P., Spiridonov, S., Stock, P., Sturhan, D., Waturu, C. & Yoshida, M. 1997. Biosystematics of entomopathogenic nematodes: current status, protocols and definitions. Journal of Helminthology 71: 271-298.
Mräcek, Z., Becvär, S., Kindlmann, P. & Jersäkovä, J. 2005. Habitat preference for entomopathogenic nematodes, their insect hosts and new faunistic records for the Czech Republic. Biological Control 34: 27-37.
Mräcek, Z., Nguyen, K.B., Tailliez, P., Boemare, N. & Chen, S. 2006. Steinernema sichuanense sp. n. (Rhabditida, Steinernematidae) a new species of entomopathogenic nematode from the province of Sichuan, east Tibetan Mts., China. Journal of Invertebrate Pathology 93: 157-169.
Nei, M. & Kumar, S. 2000. Molecular Evolution and Phylogenetics. USA, Oxford University Press. 333 pp.
Nguyen, K.B. 2007. Methodology, morphology and identification. In: Entomopathogenic Nematodes: Systematics, Phylogeny and Bacterial Symbionts. Nematology Monographs and Perspectives, V. V (K.B. Nguyen & D.J. Hunt Eds.). pp. 59-119. Leiden, The Netherlands, Brill.
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. & Mräcek, Z. 2007. Steinernematidae: species descriptions. In: Entomopathogenic Nematodes: Systematics, Phylogeny and Bacterial Symbionts. Nematology Monographs and Perspectives, V. V (K.B. Nguyen & D.J. Hunt Eds.). pp. 121-609. Leiden, The Netherlands, Brill.
Phan, K.L., Mrâcek, Z., Pûza, V., Nermut, J. & Jarosovâ, A. 2014. Steinernema huense sp. n. a new entomopathogenic nematode (Nematoda: Steinernematidae) from Vietnam. Nematology (in press).
Poinar, G.O. Jr. 1967. Description and taxonomic position of the DD-136 nematode (Steinernematidae, Rhabditoidea) and its relationship to Neoaplectana carpocapsae Weiser. Proceedings of the Helminthological Society of Washington 34: 199-209.
Rzhetsky, A. & Nei, M. 1992. A simple method for estimating and testing minimum evolution trees. Molecular Biology and Evolution 9: 945-967.
Saitou, N. & Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406-425.
Seinhorst, J.W. 1959. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4: 67-69.
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.
Stanuszek, S. 1974. [Neoaplectana feltiae pieridarum, n. ecotype (Nematoda: Rhabditoidea, Steinernematidae) - a parasite of Pieris brassicae and Manestra brassicae L., morphology and biology]. Zeszyty Problemowe Postepow Nauk Rolniczych 154: 361-393 (in Czech).
STOCK, S.P., Campbell, J.F. & Nadler, S.A. 2001. Phylogeny of Steinernema Travassos, 1927 (Cephalobina: Steinernematidae) inferred from ribosomal DNA sequences and morphological characters. Journal of Parasitology 87: 877-889.
Tamura, K., Peterson, D., Peterson, n., Stecher, G., Nei, M. & Kumar, S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731-2739.
Veremtshuk, G.V. 1969. New species of entomopathogenic nematode from the genus Neoaplectana (Rhabditida: Steinernematidae). Parasitologia 3: 249-252.
Vrain, T.C., Wakarchuk, D.A., Levesque, A.C. & Hamilton, R.I. 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology 15: 563-573.
Weiser, J. 1955. [Neoaplectana carpocapsae n. sp, (Anguillulata, Steinernematidae), new parasite of codling moth caterpillars Carpocapsa pomonella L.]. VësMk Ceské Spolecnosti Zoologické 19: 44-52 (in Czech).
Z. Mräcek, V. Puza and J. Nermut'. Дополнения к описанию Steinernema carpocapsae (Weiser, 1955) Wouts, Mracek, Gerdin & Bedding, 1982.
Резюме. Морфология энтомопатогенных нематод Steinernema carpocapsae описана недостаточно полно, и некоторые важные особенности этого вида остаются неизученными. Недостает и подробного молекулярно-таксономического исследования. Steinernema carpocapsae представляет собой типовую форму группы «carpocapsae», поэтому дополнение первоописания данными о гиалиновой части и других морфометрических особенностях хвостового конца личинок, наличии выступов на оконечности хвостового конца самок первого поколения представляются необходимыми для понимания взаимоотношений в этой группе штейнернематид. Были получены последовательности ITS и D2-D3 участков рибосомальной ДНК и проведено их сравнение с другими последовательностями S. carpocapsae, имеющимися в ГенБанке (NCBI). Было исследовано 4 изолята S. carpocapsae (из США - DD136 и All, России - Agriotos, а также Швейцарии - DOK83). Морфологические особенности и морфометрия оказались сходными у всех изолятов. Инвазионные личинки характеризуются зауженным и полусферическим головным концом и контурами, продолжающими контуры тела; латеральными полями с 6-ю ребрами (семь линий), с менее различимой центральной парой. Гиалиновая часть хвостового конца составляет 40-44% от его общей длины, индекс с' варьирует от 4.0 (3.8-4.1) до 4.2 (3.9-4.4), а индекс %E от 71 (59-82) до 79 (74-83). Самки первого поколения характеризуются тупой оконечностью хвостового конца с терминальным конически завершающимся отростком (без дополнительных мелких выступов). Округлые губы вульвы слабо выступают над поверхностью тела. У самцов всех поколений имеется небольшой мукрон, лишь незначительно различающийся по длине между 1-м и 2-м поколениями. Оба генетических меркера показывают видовую самостоятельность и целостность S. carpocapsae как вида, выявляя лишь низкую внутривидовую нуклеотидную изменчивость. Сомнительным, правда, остается видовой статус близких видов S. anatoliense и S. websteri.
