Heterorhabditis floridensis, Nguyen & Gozel & Köppenhöfer & Adams, 2006

Nguyen, Khuong B., Gozel, Ugur, Köppenhöfer, Heather S. & Adams, Byron J., 2006, Heterorhabditis floridensis n. sp. (Rhabditida: Heterorhabditidae) from Florida, Zootaxa 1177, pp. 1-19 : 4-17

Heterorhabditis floridensis sp. n.

( Figs. 1–7)

Description

Male: Measurements are in Table 1. Body curved ventrally when heat killed. Head truncate, sometimes slightly swollen. Labial papillae 6, cephalic papillae not observed. Amphid prominent. Stoma with sclerotized rod­ or barrel­shaped cheilorhabdions; other rhabdions not distinguishable forming a funnel­shaped structure below cheilorhabdions. Pharynx with cylindrical corpus, metacorpus sometimes slightly enlarged. Nerve ring surrounding isthmus just anterior to basal bulb. Basal bulb with reduced valve. Cardia present, protruding into intestine. Excretory pore usually posterior to basal bulb. Testis monorchic, reflexed; distance from end of basal bulb to testis flexure variable. Vas deferens well developed. Spicules paired, separate, rostrum absent, ventral side usually flattened. Gubernaculum slightly curved ventrally. Bursa peloderan. The number of genital papillae from one to six (from anterior end) are unchanged and typical for Heterorhabditis spp. ( Nguyen et al. 2004). The distribution of these papillae is variable. From anterior to posterior, pair one is well anterior to the cloaca, its tips reach beyond the bursal rim; pairs two and three usually (90%) occur in a group ( Fig. 2F), sometimes (10%) separated ( Fig. 2E). The two papillae standing immediately anterior to cloaca also reach beyond the bursal rim. Pairs four, five and six usually (40%) form a group separately, or papillae four and five form a group, with pair six standing separately (20%). These papillae are situated just posterior to the cloaca, with pair 4 curved outward (laterally). Frequently (40%), papillae four, five, six, and seven form a group ( Fig. 3D). The number and distribution of papillae in the terminal group are also highly variable. The observation of papillae in the terminal group of ten bursa yield the following results: 4/10 (40%) with 2 pairs of papillae ( Fig. 3B); 3/10 (30%) with 3 papillae on one side and 2 papillae on the other side ( Fig. 3D); 2/ 10 (20%) with one pair of papillae Fig. 3A), and 1/10 (10%) with three pairs of papillae ( Fig. 3C). In general, the first papilla in the terminal group is prominent; the last two papillae, if present, are weaker, shorter, or appear as small sclerotized dots. Under SEM observation, only pairs seven was seen in the terminal group; in one male eight pair were observed ( Fig. 2D). In addition to bursal papillae, a pair of smaller papillae was observed on the posterior edge of the cloacal opening ( Fig. 2F). The tail is conoid, slightly curved ventrally.

Female: Hermaphroditic female C­shaped in fixative, body robust, always with many eggs in young females and many eggs and juveniles in mature females. Cuticle smooth under light microscope but finely annulated with SEM. Head region tapering anteriorly. Labial region with six prominent lips appearing protruding into stoma ( Fig. 4A,B), each lip with a labial papilla on top. Cephalic papillae not observed with SEM. In face view, mouth hexagonal in shape ( Fig. 4A). Amphid present, pore­like. Stoma with refractile cheilorhabdions. Posterior part of stoma funnel­shaped, enclosed by anterior part of pharynx. Pharynx with cylindrical corpus. Isthmus very short or undistinguishable. Nerve ring near anterior end of basal bulb. Basal bulb prominent with inconspicuous valve, but lumen of pharynx in basal bulb well sclerotized. In young female, anterior end of intestine with thick wall. Intestinal cells prominent, especially between basal bulb and flexure of anterior ovary, and from rectum to flexure of posterior ovary. Intestinal lumen well defined in these two regions. Cardia present. Gonads didelphic, amphidelphic. Vulva with a transverse slit, located on a slightly protruding area, anterior to mid­body (V=44–49%). In ventral view, vulva elliptical encircled by arch­shaped annules anteriorly and posteriorly ( Fig. 4D). Vagina short. Tail longer than anal body diameter, conoid with pointed terminus ( Fig. 4C,E,F). Sometimes, near the end, tail widens then narrows down to a pointed tip. Post­anal swelling present. Phasmid inconspicuous. Amphimictic female similar to hermaphroditic female but smaller. Vulva rarely protruding, usually (67%) covered with exudates or copulation plug after mating ( Fig. 5F). Post­anal swelling much smaller.

n = number of specimens measured.

EP = distance from anterior end to excretory pore.

NR = distance from anterior end to nerve ring.

ES = distance from anterior end to end of pharynx.

V = distance from anterior end to vulva/body length X 100.

Infective juvenile: Body elongate. Sheath (second­stage cuticle) present immediately after harvesting, but many infective juvenile (IJ) losing sheath in storage. Ensheathed infective juvenile with body length close to that of H. bacteriophora and H. indica . Labial region with six annules without longitudinal incisures ( Fig. 6B). Anterior part of body (about 3–5 body diameters from anterior end) with tessellate pattern. Posterior part with longitudinal ridges. Tail long, pointed. Exsheathed IJ body annulated, without longitudinal ridges. Labial region with a dorsal tooth, labial and cephalic papillae absent. Amphid prominent, pore­like ( Fig. 6B). Excretory pore posterior to nerve ring but just anterior to base of pharynx ( Fig. 6A). Excretory duct pronounced, well cuticularized except in anterior part, 3–5 µm from excretory pore. Hemizonid when observed, 3–4 annules anterior to excretory pore ( Fig. 6A). Pharynx typical for Heterorhabditis . Lateral field at the center of the body with 2 ridges. Phasmid not observed under light microscope. Tail elongate conoid with pointed terminus. Tail length without sheath is about 61% of tail with sheath.

Type host and locality

Natural host unknown. The nematode was collected by baited soil sample in Desoto county, from a wooded area next to the highway (SR 70), GPS coordinates: N 27.20855 and W 81.75035 .

Type material Holotype (male), and paratype (hermaphrodites, females, males, and infective juve­ niles in 3% formalin) isolated from haemocoel of G. mellonella deposited in the United States Department of Agriculture Nematode Collection ( USDANC), Beltsville , Maryland.

Slides of one male and one female of the second generation, and several infective juveniles deposited in the California Collection of Nematodes , University of California Davis Nematode Collection, Davis, California.

Several slides of hermaphrodites, males, females and all stages in 4% formalin are maintained in the Department of Entomology and Nematology, University of Florida , Gainesville, Florida, USA .

Diagnosis

Heterorhabditis floridensis n. sp. is characterized by unique male, female, and infective juvenile characters. For males, the number of papillae in the terminal group of bursa varies, either with 2 pairs of papillae (40%), 3 papillae on one side and 2 papillae on the other side (30%), one pair of papillae (20%), or with three pairs of papillae(10%). SW and GS values are 157 and 54, respectively. Females possess a typical vulva pattern ( Fig. 4D). For infective juveniles, EP=109 (101–122) µm, ES=135 (123–142) µm, tail length=103 (91–113) µm, and a=27.6 (25–32). This new species can be further characterized by molecular characteristics of ITS regions of ribosomal DNA.

Relationships

Heterorhabditis floridensis n. sp. can be distinguished from other species of Heterorhabditis by morphological characters of males, females, and infective juveniles. As shown in the phylogenetic tree ( Fig. 7) and morphological comparisons, the new species is most similar to H. mexicana , H. baujardi , and H. indica . Measurements of infective juveniles of the new species are different from these three species by bold­faced numbers in Table 3. For males of H. floridensis n. sp., the number of papillae in the terminal group is variable as stated in the diagnosis, and measurements of some characteristics differ from other taxa as presented in Table 2. For females, the vulval pattern of the new species ( Fig. 5A) is quite different from that of the other morphologically closely related species, H. mexicana ( Fig. 5B), H. bacteriophora ( Fig. 5C), H. indica ( Fig. 5D). Unfortunately, the vulval pattern of H. baujardi could not be obtained for comparison. The new species can be distinguished from H. megidis , H. zealandica , and H. marelatus by the presence of three pairs of bursal papillae in the terminal group ( Fig. 6, Nguyen et al. 2004), and the body length (more than 600 µm) of the infective juvenile of these three species.

DNA characterization

The ITS rDNA regions, flanked by primers 18S and 26S, of Heterorhabditis floridensis n. sp. are characterized by length (1012 base pairs [bp], ITS1= 393 bp, ITS2=214 bp), and nucleotide usage composition ( Table 4). Compared to sequences of eight other species in the genus Heterorhabditis , the sequence length of the new species is longer than that of seven other species, and shorter than that of only one species, H. bacteriophora (1021 bp). The ITS1 sequence length of the new species is longer than most of the other species (4– 17 bp longer) but is one bp shorter than H. mexicana (ITS1=394 bp), The sequence length of the ITS2 region differs (1–3 bp) from six species ( H. baujardi , H. downesi , H. indica , H. marelatus , H. mexicana , H. zealandica ), but is six bp shorter than H. megidis and 14 bp shorter than H. bacteriophora . The nucleotide composition of all nine species of Heterorhabditis is presented in Table 4. The reconstructed nucleotide character transformations ( Figure 7) show that the new species differs from its closest taxon H. mexicana at 16 aligned positions three of which are unambiguous, polarized autapomorphies. It is most divergent from Heterorhabditis zealandica (193 aligned positions). Genetic distances between the new species and others are presented in Table 5.

Phylogenetic Analysis

Parsimony, likelihood and distance based tree building approaches produced a single, concordant phylogenetic hypothesis for the genus Heterorhabditis . The topology of this tree is identical with that produced for H. mexicana ( Nguyen et al., 2004) except the branch for H. mexicana in the former is replaced by the new clade ( Fig. 7). The new species and its sister taxon H. mexicana form a clade with H. baujardi .

n = number of specimens measured.

EP = distance from anterior end to excretory pore.

NR = distance from anterior end to nerve ring.

ES = distance from anterior end to end of pharynx.

V = distance from anterior end to vulva/body length X 100.

n. sp. and other related species.

1) This sequence is not as complete as in other species.? Not available.