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19Russian Journal of Nematology, 2012, 20 (2), 119-126
Addenum to the description of Steinernema jollieti Spiridonov, Krasomil-Osterfeld & Moens, 2004
Henryk Wojciech Skrzypek1, Waldemar Stanislaw Kazimierczak1, Anna Maria Kreft1 , Sergei E. Spiridonov2 and Zdenek Mracek3
1Department of Zoology and Ecology of Invertebrates, Institute of Environmental Protection, John Paul II Catholic
University of Lublin, Krasnicka Avenue 102, 20-718 Lublin, Poland 2Centre of Parasitology, A.N. Severtsov's Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii
prospect, 33, Moscow, Russian Federation, 119071 3Laboratory of Insect Pathology, Institute of Entomology, Biological Centre, Czech Academy of Sciences, Branisovska 31,
370 05 Ceske Budejovice, Czech Republic e-mail: mracek@entu.cas.cz
Accepted for publication 14 November 2012
Summary. Additonal morphological data are provided for Steinernema jollieti Spiridonov, Krasomil-Osterfeld, Moens, 2004. A light and scanning electron microscopy were used to particularize taxonomically important characters missing in the first decsription. The GS% and SW% indices are provided for males. The distribution of genital papillae on male tail is illustrated with SEM images. Structure of female tail and vulvar area are decsribed for females of the first and second generation. The relationships of S. jollieti with other steinernematid feltiae/kraussei group species are discussed. Key words: entomopathogenis nematodes, infective juveniles, morphology, Steinernematidae.
Steinernema jollieti Spiridonov, Krasomil-Osterfeld & Moens, 2004 was described from a soil sample collected close to a stream waterbed in the Bush-Augusta State Park region, Missouri valley near St. Louis (Spiridonov et al., 2004). This species belongs to the kraussei/feltiae group (Nguyen et al., 2007). The species description presented all important morphological, morphometric and genetic data; however, the microphotographic documentation of valuable characters are represented only by a scanning electron microscopy (SEM) of infective juvenile (IJ) lateral field. The main goal of our paper is to add SEM and DIC microphotography of adult and infective juvenile nematodes and particularize some taxonomically important characters according to suggestion published in Hominick et al. (1997).
MATERIALS AND METHODS
The S. jollieti living culture was recieved from the collection of Institute of Parasitology RAS, Moscow. The nematodes were reared on the late instar larvae of Galleria mellonella L.. For examination with SEM the first generation males, females and IJ were used. Larvae of G. mellonella, infected with 50-70 IJ per insect at 22.5±0,5°C,
were dissected in Ringer's solution (Woodring & Kaya, 1988) 4-5 days after the nematode and host established contact. Males, females and IJ thus obtained were collected in centrifuge tubes, heat relaxed (55°C, 3-5 min), fixed for 24-36 h in 2% glutaraldehyde solution (POCH) in 0.15 M cacodylate buffer (pH 7.2, POCH) at 4°C, rinsed in cacodylate buffer four times (10 min each change) and post-fixed for the next 12-24 h in 1% osmium tetroxide (Sigma Aldrich) with the same buffer and temperature. Fixed nematodes were rinsed in cacodylate buffer four times again (10 min intervals) and dehydrated in ethanol series (POCH) (10, 30, . . . , 90% at 10 min intervals, 100% at 20 min intervals with three changes). After exchanging the ethanol for acetone (POCH), specimens were critical point-dried (Polaron Range CPD 7501) in liquid CO2, mounted on SEM stubs, sputter-coated with 20 nm gold-palladium layer (Polaron Range SC 7620) and examined in a LEO 1430VP scanning electron microscope at an accelerating voltage of 15 kV in high vacuum mode.
For a light microscopy nematodes were fixed in 2% glutaraldehyde (25% glutaraldehyde diluted with Ringer's solution), processed to anhydrous glycerin according to Seinhorst method (Seinhorst, 1959) and observed using a Leica 5500B.
Fig. 1. Steinernema jollieti. A-F: Male. A: First generation head; B: Second generation head; C: First generation tail with genital papillae (laterally); D: Second generation tail with genital papillae (laterally) and mucron; E: First generation spicule; F: First generation gubernaculum (ventrally). EP - excretory pore, ES - oesophagus, NR - nerve ring, An- anus, S-P - single preanal papilla, Mu - mucron.
Fig. 2. Steinernema jollieti. A-D: Female. A: First generation head; B: First generation vulva; C: First generation tail; D: Second generation tail; E-F: Infective juvenile. E: Head with esophagus; F: Tail with hyaline layer. ES -oesopahgus, V - vulva, An - anus, NR - nerve ring, BP - bacterial pouch (receptacle), H - hyaline portion of tail.
Fig. 3. Steinernema jollieti. A-F: Male first generation. A: Head "en face view"B: head region; C: Tail region with genital papillae; D: Tail region around cloaca with a papilla-like structure at the cloaca base; E-F: Tail tip. EP -excretory pore, D - deirid, CP -cephalic papilla, LP - labial papilla, A - amphid opening, 1-11 - genital papillae, PD-postdeirid, S-P - single preanal papilla, Pl-S - papilla-like structure, G-C J - gubernaculum-cuticle junction, Mu -mucron, P - phasmid opening.
Fig. 4. Steinernema jollieti. A-D: Female first generation. A: Head region; B: Vulva; C: Hind body portion; D: Tail with a conical peg. EP - excretory pore, D - deirid, V - vulva, An - anus, PD - postdeirid, T - tail.
Fig. 5. Steinernema jollieti. A-D: Infective juvenile. A: Forebody portion; B: Head; C: Lateral field; D: Tail with anus and phasmids. EP- excretory pore, CP -cephalic papilla, A - amphid opening, LF - laterail fields, An - anus, P -phasmid opening.
Fig. 6. An arrangement of adcloacal genital papillae. A: Steinernema jollieti; B: S. feltiae. A - amphid, An - anus, BP - bacterial pouch, CP - cephalic papilla, EP - excretory pore, ES - esophagus, H - hyaline layer, LF - lateral field, LP - labial papilla, Mu - mucron, NR - nerve ring, P - phasmid, S-P - single preanal papilla, V - vulva, G-C J - gubernaculum-cuticule junction, D - deirid, PD - postdeirid, Pl-S - papilla-like structure, 1-11 -genital papillae.
RESULTS
Comparison of the description data and topotype strain. There are several discrepancies and missing data in the original description of S. jollieti. With respect to the published drawings (Spiridonov et al., 2004) it is evident that several characters must be corrected and added.
Males (Figs. 1, 3). Spicules slightly to moderately, not strongly, curved. Manubrium elongated, robust in the first generation with ratio length/width 1.5-2 : 1. Second generation manubrium slender with ratio at least 2:1. Gubernaculae very variable in their shape, often with a depressed dorsal side on the proximal portion forming narrow neck. Proximal end slightly bent, never hooked. Typical mucron absent in the first generation; however, papilla-like (mucron-like) structure visible in 50% of specimens under SEM (Fig. 3F).
Second generation males possess either conical in younger males (length 2-3 ^m) or filamentous mucron in older specimens, (length 5-8 ^m).
Important ratios in the first generation GS% 84 vs 79 (76-81) and SW% 145 ra 169 (154-183) were recalculated from the original description vs measured from specimens of our living culture, respectively. In the second generations the GS% 72 vs 79 (76-83) and SW% 187 vs 149 (128-164) were recalculated vs measured, respectively.
In the original description the arragement of genital papillae in S. jollieti was mentioned as specific due to postcloacal position of only three pairs of papillae, whereas other species of the feltiae/kraussei group possess five postcloacal pairs. However, in Nguyen et al. (2007) only three postcloacal pairs are mentioned, also for S. kraussei (Steiner, 1923) Travassos, 1927 and from five postcloacal pairs in S. feltiae (Filipjev, 1934) Wouts, Mracek, Gerdin & Bedding, 1982 two pairs are more adcloacal. Generally, the correct position of
genital papillae can be resolved by using SEM. The position of papilae that are close to the cloaca may be influenced by the bend of fixed male tail and two adcloacal pairs may vary slightly in their position. In our observation (Fig.6), the arrangement of three postcloacal papillae in S. jollieti was found to be relatively constant and different from S. kraussei and S. feltiae. However, more SEM studies of genital papillae in species of the feltiae/kraussei group are needed to resolve if the postcloacal papillae arrangement can be considered as a specific trait distiguishing S. jollieti from other species of the feltiae/kraussei group. The single-preanal papilla is bigger than that in S. feltiae and S. kraussei. Moreover, there is a papilla-like structure at the base of cloaca that is characteristic for some species of feltiae/kraussei group.
Infective juveniles (Figs 2 E-F, 4 and 5). Head rounded, not offset from the body contour. Important ratio c'4.5 and %E 88 were recalculated from the original description vs our measurements of living culture, 4.2 (3.9-4.3) and 92 (81-98), respectively. Ratio H 55% (46-60) ranks S. jollieti among species that have hyaline layer longer than 50% of tail length. Described mucron on the IJ tail tip was not found either in living or fixed specimes and this character cannot be considered taxonomically important. Tail was found slightly dorsally depressed.
Females (Figs. 2 A-D and 4). First generation possess the smooth conical tail with a peg on the tip bearing three minute indistinct papilla like structures, one on the tip and two situated laterally that may represent an inconspicuous phasmid openings. Postcloacal swelling not developed. Second generation females possess the conical tail extending to the filamentous mucron. Anal opening is surrounded by the moderately protruding preanal fold and postcloacal swelling.
character/species jollieti feltiae x krausseix x oregonense . . x weisen
head rounded rounded flattened flattened flattened
lateral ridges 6 8 7 7 8
c' 4.5 x 4.8 (4.5-5.1)y 3.8 4.7 3.7 (3.2-4.1)
% D 48 (46-50) 46 (44-50)y 47 50 (40-60) 51 (44-55)
% E 88 x 74 (67-81)y 80 100 (90-110) 95
% H 55 (46-60) 44 (37-51)y 38 (35-40) 47 36 (34-39)
Bold- character different from S. jollieti; x recalculated from the species description, y after Nguyen et al., (2007), c' - tail length divided by anal body width, %D - distance from anterior end to excretory pore divided by pharynx length x-100%, %E - distance from anterior end to excretory pore divided by tail length x-100%, %H - hyaline posrtion of tail divided by tail length x 100%.
Table 1. Infective juvenile characters distinguishing some Holarctic species of the feltiae/kraussei group of Steinernema.
Relationship. Steinernema kraussei, S. feltiae, S. oregonense Liu & Berry, 1996, and S. weiseri Mracek, Sturhan & Reid, 2003 represent feltiae/kraussei group species with a Holoarctic distribution. Of these S. kraussei and S. feltiae were found in both Palearctic and Neoarctic regions. Steinernema jollieti infective juvenile differs from these species by characters in Table 1. Of these characters we consider as the most important six ridges in the lateral field, whereas other mentioned species have seven or eight ridges, respectively. Rounded head of S. jollieti differs from those flattened in S. kraussei, S. oregonense and S. weiseri. Ratio c'4.5 is different from S. kraussei 3.8 and S. weiseri 3.7 (3.2-4.1), respectively. H% is much higher in S. jollieti 55 (46-60) when compared with S. feltiae 44 (37-51), S. kraussei 38 (35-40) and S. weiseri 36 (34-39), respectively. Second generation females possess characteristically formed anal surroundings. While a postanal swelling is common in many steinernematid species, preanal fold offset from body contour seem to be quite unique for S. jollieti.
The SW% value of the first generation male of S. jollieti is higher than that of S. oregonense and S. weiseri at 169 (154-183) vs 110 and 140, respectively, whilst the GS% value did not bring significant differences.
ACKNOWLEDGEMENT
The study was supported by the Czech Ministry of Education, Youth and Sport project KONTAKT
ME 10078. The studies of S.E. Spiridonov were partially supported by RFBR research project 11-04-00590a.
REFERENCES
Hominick W.M., Briscoe B.R., del Pino F.G., Heng J., Hunt D.J., Kozodoy E., Mracek 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 definition. Journal of Helminthology. 71: 271-298.
Nguyen K.B., Hunt, D.J. & Mracek Z. 2007. Steinernematidae: species description. In: Nguyen K.B. & Hunt, D.J (eds). Entomopathogenic nematodes: Systematics, Phylogeny and Bacterial symbionts.. Nematology Monographs and Persepctives (series editors Hunt, D.J. & Perry, R.N.). Brill, Leiden, The Netherlands, pp. 121-609. Spiridonov, S. E., Krasomil-Osterfeld, K. & Moens, M. 2004. Steinernema jollieti sp.n. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from the American Midwest. Russian Journal of Nematology 12: 85-95. Woodring, J. L. & Kaya, H. K. 1988. Steinernematid and heterorhabditid nematodes: a handbook of techniques. Southern Cooperative Sens Bulletin 331. Fayetteville, AK, USA, Arkansas Agricultural Experiment Station, 30 pp. Seinhorst, J. W. 1959. A rapid method for transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4: 67-69.
& W. Skrzypek, W. S. Kazimierczak, A. M. Kreft, S.E. Spiridonov, Z. Мгасек. Дополнения к описанию 8(етегпгта]оШей Spiridonov, Кга5отП-Оз1ег1еИ & Моеш, 2004 .
Резюме. Приводятся дополнительные данные по морфологии 81етегпета ]оШвИ Spiridonov, Кга8отП-О81ег1еШ & Moens, 2004, отсутствующие в первоописании. Для их получения была использована световая и сканирующая электронная микроскопия. Приводятся значения индексов GS% и SW% для самцов. Распределение генитальных папилл на хвостовом конце самцов иллюстрируется с помощью СЭМ. Приводятся сведения об организации хвостового конца и вульвы у самок первого и второго поколения. Обсуждаются филетические отношения X ]оШвИ с другими видами штейнернематид.
