Steinernema beddingi  Qiu, Hu, Zhou, Pang & Nguyen, 2005

 

 

 

Description

 

Male, first generation: (Fig. 1) Body C-shaped and curved posteriorly when killed by gentle heat. Cuticle smooth. Stoma shallow and wide. Pharynx extending nearly to mouth opening. Cheilorhabdions and prorhabdions sclerotised, small but distinct. Pharynx muscular with a cylindrical procorpus, metacorpus swollen, isthmus well defined, basal bulb enlarged. Nerve ring often surrounding anterior portion of basal bulb. Excretory pore opening located near posterior part of metacorpus. Lateral fields and phasmids not observed. Gonad single and reflexed. Spicules paired, colorless and moderately curved. Spicule length/width about 4.6. Spicule head shorter than wide with a length/width about 1:2 (Fig. 3). Lateral lobe prominent, beginning anteriorly at shaft and extending posteriorly to spicule tip. Ventral lobe enlarged anteriorly and ventrally to form a prominent rostrum and ending before, but close to, spicule tip (Fig. 1E; Fig. 4A and B). Dorsal lob starting from shaft and terminating at a level similar to ventral lob. Spicule terminus blunt. Velum not observed. Gubernaculum boat-shaped in lateral view. In ventral view, gubernaculum tapering anteriorly and evenly. Corpus fused and cuneus not seen (Fig. 4C to F). Tail long and concave ventrally. No mucron-like projection was observed at tip of tail. Twenty-three genital papillae were seen by SEM, including five pairs and one single preanal papillae, two pairs adanal, one pair lateral and three pair postanal (two subterminal and one subdorsal) papillae (Fig. 1B; Fig 3F).

Male, second generation: Morphologically similar to the first generation male, such as tail concaves ventrally and without mucron, but most of measurements are smaller and excretory pore is located more anterior than that in the first generation.

Female, first generation: (Fig. 2) Body C-shaped when killed by gentle heat. Cuticle smooth, head rounded and slightly truncated anteriorly. Six lips fused, each with a papilla. Four cephalic papillae occur further back on head. Amphidial aperture distinct and located behind lateral labial papillae. Morphology of anterior region of female similar to male. Lateral fields and phasmids not observed. Gonads didelphic and reflexed. Vulva a transverse slit, usually slightly protruding and symmetrical. Vagina short and muscular. Epiptygma not observed. Tail wide and with a wedge-like projection on tip. Postanal swelling distinct.

Female, second generation: Similar to the first generation females but much smaller. Vulva is slightly more protruding than the first generation. Postanal swelling and tail projection as prominent as those of the first generation.

Infective juveniles: Body slender, enclosed in a sheath. Anterior end rounded, labial region smooth, papillae not seen. Amphidian apertures slit¨Cshaped. Four prominent cephalic papillae. Mouth and anus closed. Pharynx long and narrow, isthmus distinct and surrounded by nerve ring. Excretory pore located anterior to nerve ring, near middle of pharynx. Cuticle with prominent transverse striations. Lateral field begining anteriorly with two slightly raised ridges at a position slightly anterior to excretory pore. Another two ridges appear between previous two at a position close to base of pharynx and become gradually more prominent at middle portion of body and then fade gradually after level of anus (Fig. 3D and E). Middle two ridges slightly smaller than other two and whole lateral field distinctly protrudes from body surface. Tail attenuate and tapering gradually with a hyaline portion occupying about 45% of tail length. No spine like structure was seen on tail tip. Phasmid inconspicuous. There was no substantial difference between IJ produced from the artificial medium and from Galleria larvae except that the former was slightly short than the later (Table 1).

 

 

Diagnostic characters

S. beddingi is characterised morphologically by the combination of the features of various developmental stages of the nematode. For IJ, body length averaging 743  (700-790) ¦Ìm; distance from anterior end to excretory pore 70 (64-75) ¦Ìm, to the base of pharynx 125 (113-130) ¦Ìm; tail length 77 (72-83) ¦Ìm; D and E value 57 (52-64) and 92 (84-103), respectively; lateral field at the mid-body position has only four ridges, which raise distinctly from body surface; tail attenuates and tapering gradually without a dorsal constrict or a spine on the tail tip. For males, tail has a concave in the ventral part and without mucron on tip; spicules and gubernacula are colorless; spicule head very short, dorsal and ventral lobs ending before the spicule tip while distal end of lateral lob suddenly narrowed both dorsally and ventrally to form a blunt tip; gubernaculum without cuneus. For females, vulva is a symmetric, transverse-slit without epiptygma, tail with a wedge-like structure on the tip and a conspicuous postanal swelling.

Molecular

 

The sequence length and composition of ITS regions (AY603397) of S. beddingi are 1069 bp  and 280A, 234G, 373T, 182C, GC% = 38.9, and partial 28S rDNA (AY603396) are 465 bp  and 123A, 138G, 127T, 77C, GC% = 46.2.

 

 

Relationships 

Both morphometrical (Tables 1) and molecular (Fig. 5-8) data show congruently that S. beddingi n. sp. is close to S. affine and S. intermedium. However, the new species can be separated from these two described species by the morphological characters of various developmental stages of the nematodes (Table 4). The morphometrics of S. beddingi IJ are very close to those of S. affine and S. intermedium except that the tail is longer, 77 (72-83) ¦Ìm compared to 64 (53-72) ¦Ìm for S. intermedium and 66 (64-74) ¦Ìm for S. affine. IJ of the new species can also be distinguished from the two described species mentioned above by morphology of tail and lateral field:  S. beddingi  IJ tail attenuates and tapering gradually with no spine on tail tip, while S. affine IJ usually has a spine like structure on tail tip, and tail of S. intermedium IJ usually has a constriction on the dorsal side. The middle portion of lateral field of the new species has four raised ridges while that of the other two species has 6 ridges. For the first generation males, the spicule and gubernaculum length of S. beddingi, 71 (63-78) ¦Ìm and 43 (38-48) ¦Ìm, respectively, is shorter than those of S. intermedium 91 (84-100) ¦Ìm and 64 (56-75) ¦Ìm, respectively. The morphology of spicule and gubernaculum also differed substantially from the two described species (Adams & Nguyen 2002; Fig. 4), for example, no velum was observed on spicules of S. beddingi n. sp while it was observed clearly on spicules of both S. intermedium and S. affine; no cuneus was observed on S. beddingi gubernacula while both S. affine and S. intermedium has a needle type cuneus. For the first generation females, the new species has a distinct postanal swelling but the other two species have not. S. beddingi  can also be distinguished from S. affine and S. intermedium genetically by either the ITS or 28S D3 domain rDNA sequence, and biologically by cross tests.

S. beddingi can be distinguished from other described Steinernema species that have a similar mean IJ body length (ranged from 650 ¦Ìm to 850 ¦Ìm), including S. pakistanense Shahina, Anis, Reid, Rowe and Maqbool 2001, S. ceratophorum Jian, Reid and Hunt 1997, S. bicornutum Tallosi, Peters, Ehlers 1995, S. feltiae (Filipjev, 1934) Wouts, Mracek, Gerdin and Bedding 1982, S. thanhi Phan, Nguyen and Moens 2001, S. sangi Phan, Nguyen and Moens 2001, S. weiseri Mracek, Sturhan and Reid 2003 and S. monticolum Stock, Choo and Kaya 1997 by morphological characters of various stages. For example, it can be distinguished from all nematodes mentioned above by the spicule and gubernaculum morphology of the first generation males (Fig. 4; Adams and Nguyen, 2002) as well as lateral field morphology and ridge number of IJ (the number of ridges at the middle portion of the above mentioned nematodes is six to eight while that of the new species is four and the whole lateral filed raised distinctly from body surface). The new species can also be separated from S. pakistanense, S. ceratophorum and S. bicornutum by IJ head structure (IJ of these three species possess a horn like structure on their head).

 

 

Type host and locality

 

The type host of this nematode in nature is unknown as it was recovered from soil using Galleria larvae as bait. The soil sample was collected from a cabbage field in An-fen-ying town (latitude N24.58, longitude E102.28, altitude 1850m, mean annual temperature 16oC, precipitation 1000 mm/year), about 60 km from Kunming, Yunnan province, People¡¯s Republic of China.

 

Type materials

 

Holotype male, allotype female, five paratype first generation males, five paratype females, fifteen paratype infective juveniles and other population slides deposited in the State Key Lab for Biocontrol, School of Life Sciences, Zhongshan University, Guangzhou 510275, China. One slide that contains about ten paratype IJ and two slides, each contains one paratype male and one female will be deposited in USDA Nematode Collection, Beltsville, Maryland, USA. Living infective juveniles is also preserved in liquid nitrogen in the nematode collection of SKLB, Zhongshan University.

 

Reproducibility and symbiotic bacteria

 

S. beddingi reproduced poorly in Galleria larvae, usually without second generation adults in the infected Galleria larval cadaver and only a few thousands IJ were obtained from a larvae averagely. However, it proliferated well in the artificial medium using the method described. IJ produced either from Galleria larvae or the artificial medium were highly pathogenic to Galleria larvae, with most of larvae dying within 24 hrs when 15 larvae were exposed to about 2000 IJ in a Petri dish lined with two layer of moist filter paper at 23 oC. It is unknown why S. beddingi n. sp. reproduced poorly in Galleria larvae. The symbiotic bacteria of S. beddingi n. sp. have been isolated and preserved in liquid nitrogen. It produced a yellow pigment on the artificial medium mentioned and possessed many characters of Xenorhabdus species, such as phase variation, negative to peroxide, etc. A phylogenetic tree based on the 16S rDNA sequence showed the symbiotic bacteria of S. beddingi n. sp. claded with but can be clearly distinguished from X. bovienii (Qiu et al., unpublished data).

 

Etymology

 

            The species was named after R. A Bedding from the Australian Center for International Agricultural Research (ACIAR).

 

 

 

Reference

 

Qiu, L,  Hu, X..,  Zhou, Y.,  Pang, Y. and  Nguyen K. B. 2005. Steinernema beddingi n. sp. (Nematoda:Steinernematidae), a new entomopathogenic nematodes fron Yunan, China. Nematology 7, 737-749.