Steinernema taiwanensis, Tseng & Hou & Tang, 2018

Tseng, Ching-Tzu, Hou, Roger F. & Tang, Li-Cheng, 2018, Steinernema taiwanensis n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Taiwan, Zootaxa 4434 (3), pp. 466-480 : 468-475

Steinernema taiwanensis * n. sp.

( Figs. 1–8 )

*Specific epithet named after the isolated country.

Description of infective juvenile. The morphometrics of IJs are shown in Table 1. Body elongate and slender, straight or slightly curved when heat-killed. Cephalic region rounded and stoma closed. No horn-like structures presented on labial region. Four cephalic papillae prominent, labial papillae not seen and labial region smooth. Amphidial apertures not prominent ( Figs. 5A, 5B ). Cuticle marked with transverse striations. Lateral field pattern begins anteriorly with one line which goes for a distance posteriorly. Another two incisures appeared to form two ridges and the number of ridges are changing into six, seven, and eight. Nearing phasmid, only two ridges were observed. With above arrangement, lateral field formula from head to tail is 2, 6, 7, 8, and 2 ( Figs. 5C to 5H). Pharynx with thin corpus and metacorpus swollen slightly. Isthmus presented and surrounded by nerve ring. Excretory pore anterior to nerve ring ( Fig. 2A ). Basal bulb elongated with visible valve and cardia presented but inconspicuous ( Fig. 2B ). Phasmid presented and located next to lateral field near mid-tail ( Fig. 5G ). Hyaline portion comprises about 54 (53–55) % of tail length ( Fig. 1C ; Table 1). No dorsal constriction presented in tail region ( Fig. 2C, 2D ).

First-generation male. The morphometrics of males are shown in Table 1. Body ventrally curved, J-shaped when heat-killed ( Fig. 3A ). Cephalic extremity truncate with cephalic papillae and labial papillae presented. Cheilorhabdions distinct and well-sclerotized. Esophagus with cylindrical corpus and lightly swollen metacorpus. Narrow isthmus presented and surrounded by nerve ring just above basal bulb. Cardia prominent. Excretory pore is always located near metacorpus, above nerve ring and basal bulb ( Figs. 1D , 3B ). Excretory duct and excretory gland observed. Genital system monorchic and reflexed. 11 pairs of genital papillae plus 1 single papilla presented ( Figs. 6A, 6B ). Spicules paired, dark yellow to dark brown in color ( Fig. 3C ). Head (manubrium) of spicules has rounded terminus, shaft (calomus) short but could be easily seen, blade (lamina) tapering to the posterior and curved moderately, blade terminus sharp, velum presented but usually thin. Each spicule has two internal ribs ( Figs. 1G , 3E ). Gubernaculum boat-shaped in lateral view, anterior end curved ventrally, swollen slightly in the middle and tapering gradually till tip ( Figs. 1H, 1I , 3E ). Shape of gubernaculum head variable, round, hook or horn-liked. Tail conoid shorter than anal body diameter, mucron presented in a few observed individuals.

Second-generation male. Similar to the first generation, except their bodies are thinner and smaller (Table 1). Spicule and gubernaculum are also shorter while their shape are not different from those of first generation ( Fig. 3D ). Tail mucron presented only in a few individuals.

First-generation female. The morphometrics of females are shown in Table 1. Body giant and robust, spiral or curved strongly while alive, and C-shaped when heat-killed. Cuticle smooth, bit with some faint annuli. Head rounded and continued with body. Four cephalic papillae and six labial papillae presented ( Figs. 6C, 6D ). Cheilorhabdions small but well-sclerotized. Esophagus with cylindrical corpus and metacorpus swollen slightly. Isthmus presented and surrounded by nerve ring. Excretory pore located near metacorpus, just anterior to nerve ring. Excretory duct and excretory gland presented. Basal bulb elongated with visible valve and cardia presented ( Figs. 1J , 4A ). Vulva located near midway of the body in a ventro-median position, fully developed females have protruding vulval lips while there are only slightly protruding ones presented in young females, epiptygmata absent ( Figs. 1K , 4B , 6E ). Gonads amphidelphic and reflexed. Post-anal swelling is usually not well developed, sometimes even absent in young females ( Figs. 4C, 4D , 6F ). Most mature females have slightly developed postanal swelling. Young females possess tail tapering to a sharp end while some individuals have papilla-like or protuberance structures on tip ( Figs. 4C, 4D , 6G ). As for fully developed females, their tail is slightly shorter than anal body diameter (Table 1). The tail shape of most individuals is more conoidal than young ones with papilla-like or protuberance structures on tip, but a few individuals observed still remain a sharp-ended tail.

Second-generation female. Similar to the first generation female, except smaller and thinner in most morphometric measurements (Table 1). Tail significantly longer than anal body diameter (Table 1) and tapering to a sharp terminus ( Fig. 4E ).

Type host and locality. The natural insect host of the novel species is unknown, and the type isolate was isolated from grassland in southern part of Pingtung County, Taiwan (22°12'23"N, 120°53'25"E), where the soil type was yellow-brown loam.

Type material. Slides of holotype male (first generation) (NMNS-7814-001), 3 paratype first-generation males (NMNS-7814-002, NMNS-7814-003), 2 paratype females (NMNS-7814-004), and 3 paratype IJ (NMNS- 7814-005) have been deposited in National Museum of Natural Science , Taichung, Taiwan. Slides with other paratypes were deposited at Museum of Entomology and the Insect Microbial Control Laboratory , Department of Entomology , College of Agriculture and Natural Resources, National Chung Hsing University ( NCHU) , Taichung, Taiwan. Living IJs are kept in the Insect Microbial Control Laboratory , NCHU .

Diagnosis and relationships. Steinernema taiwanensis n. sp. is characterized by IJ body length of 1012 (983– 1045) µm and distance from anterior end to nerve ring of 124 (120–127) µm. The lateral field formula from head to tail is 2, 6, 7, 8, 2. Tail length is 90 (79–96) µm and has no dorsal constriction in tail region. Males have 23 genital papillae (11 pairs and 1 single papilla). The shape of spicule, spicule length 94 (89–99) µm, gubernaculum length 68 (65–70) µm, and the SW% value 213 (210–214) could be used as identification characteristics. Females of first generation have no epiptygmata and slightly developed post-anal swelling.

Steinernema taiwanensis n. sp. first generation males could be distinguished from S. longicaudum by a longer gubernaculum (68 (65–70) vs 60 (54–65) µm) ( Table 2) and different shapes of spicule. First-generation S. longicaudum males have spicules with distinct shaft and well-curved blade while S. taiwanensis n. sp. possesses spicules with short shaft and not strongly curved blade. Also, spicules of S. taiwanensis n. sp. males have smooth blade tip, whereas those from S. longicaudum narrowed suddenly to form a flattened terminus.

First-generation males of S. taiwanensis n. sp. could be distinguished from S. guangdongense by a smaller body length (1712 (1528–1920) vs 1809 (1627–2126) µm) ( Table 2). Both generations of S. guangdongense have small double-flapped epiptygmata while it is absent in S. taiwanensis n. sp. IJs of S. taiwanensis n. sp. have longer distance from head to nerve ring (124 (120–127) vs 102 (88–111) µm) and a longer pharynx (147 (143–150) vs 134 (123–144) µm) than S. guangdongense ( Table 3). Tail of S. taiwanensis n. sp. IJ has no dorsal constriction as shown in S. guangdongense . The lateral field formula of S. guangdongense (2, 7, 8, 7, 4, 2) is also different from that of S. taiwanensis n. sp. (2, 6, 7, 8, 2).

Males of S. taiwanensis n. sp. can be separated from S. pui by shorter distances from head to excretory pore (114 (97–127) vs 152 (130–180) µm) and to nerve ring (129 (124–132) vs 157 (143–188) µm), and by a shorter pharynx (162 (146–169) vs 196 (175–228) µm). Tail length of first generation males of S. taiwanensis n. sp. is shorter than that of S. pui (26 (25–28) vs 32 (29–38) µm). Both species could also be differentiated by the length of spicule (94 (89–99) vs 84 (78–88) µm), gubernaculum length (68 (65–70) vs 62 (58–65) µm), and the SW% value (213 (210–214) vs 152 (140–184)) ( Table 2). Also, S. taiwanensis n. sp. has a lower number of genital papillae than S. pui (23 vs 25). First generation females of S. pui exhibit a short double flapped epiptygmata while it is absent in S. taiwanensis n. sp. IJs of S. taiwanensis n. sp. can be distinguished from S. pui by longer tail (90 (79–96) vs 69 (60–80) µm), by a higher H% value (54 (53–55) vs 45 (40–50)), a lower E% value (86 (78–88) vs 125 (109–142)), and a lower c ratio (11 (9–12) vs 15 (13–16)) ( Table 3). The lateral field formula of S. pui (2, 8, 6, 5, 2) is also different from that of S. taiwanensis n. sp. (2, 6, 7, 8, 2).

The most significant difference between S. taiwanensis n. sp. and S. hermaphroditum is that the latter species has hermaphrodites presented in the first adult generation but not the former. Males of S. taiwanensis n. sp. could be different from S. hermaphroditum by a longer distance from anterior end to excretory pore (114 (97–127) vs 75 (65–80) µm), by longer spicule length (94 (89–99) vs 68 (65–70) µm) and gubernaculum length (68 (65–70) vs 48 (47–50) µm), higher D% value (70 (66–75) vs 48 (44–50)) and SW% value (213 (210–214) vs 150 (140–160)), but a shorter tail (26 (25–28) vs 34 (28–35) µm) ( Table 2). Males of S. hermaphroditum have rectangular manubrium, which could be differentiated from rounded terminus of S. taiwanensis n. sp. The number of their genital papillae is also different (23 from S. taiwanensis n. sp. vs 21 from S. hermaphroditum ). IJs of S. taiwanensis n. sp. have longer distances from anterior end to excretory pore than those of S. hermaphroditum (82 (80–84) vs 65 (63–68) µm) and to nerve ring (124 (120–127) vs 103 (95–113) µm) ( Table 3).

First-generation males of S. taiwanensis n. sp. can be distinguished from S. lamjungense by the length of gubernaculum (68 (65–70) vs 57 (50–66) µm) and the SW% value (213 (210–214) vs 173 (151–193)) ( Table 2). Velum presented in S. taiwanensis n. sp. but usually thin, while S. lamjungense possesses strong and distinct ones. Females of S. taiwanensis n. sp. have no epiptygmata while both generations of S. lamjungense have small epiptygmata. Endotokia matricida is rarely observed in S. lamjungense , but always existed in S. taiwanensis n. sp. IJs of S. taiwanensis n. sp. could be separated from S. lamjungense by body length (1012 (983–1045) vs 832 (690– 950) µm) and maximum body diameter (35 (33–37) vs 27 (23–31) µm), the distances from head to excretory pore (82 (80–84) vs 68 (61–73) µm) and to nerve ring (124 (120–127) vs 93 (82–100) µm), and a longer pharynx (147 (143–150) vs 127 (111–142) µm), but a lower H% value (54 (53–55) vs 61 (55–69)) ( Table 3). Tail of S. lamjungense IJs usually has slightly dorsal constriction and blunt terminus while that of S. taiwanensis n. sp. does not have dorsal constriction and sharp terminus. The lateral field formula of S. lamjungense (2, 6, 7, 8, 3, 2) is also different from that of S. taiwanensis n. sp. (2, 6, 7, 8, 2).

TABLE]. Morphometrics of Steinernema taiwanensis n. sp. All measurements are in µm anđ in the form: mean ± s.đ. (range). the form: mean (range); abbreviations as in Table 1; na, data not available.

Molecular characterization and phylogenetic analysis. Comparison with other known nematode species in GenBank database indicated that our isolate belongs to Steinernema . The sequence of ITS region from S. taiwanensis n. sp. can be separated from other species in Longicaudum -clade by 45–120 bp ( Table 4), whereas the D2D3 expansion segments of the 28S rRNA were separated by 13–38 bp ( Table 4).

Phylogenetic analysis of ITS and D2D3 regions supported that S. taiwanensis n. sp. belongs to the Longicaudum -clade ( Figs. 7 , 8 ). In the ITS tree, S. taiwanensis n. sp. comprises a monophyletic group with S. guangdongense and S. longicaudum , and formed a group with S. pui , S. lamjungense , and S. hermaphroditum ( Fig. 7 ). In the D2D3 tree, S. taiwanensis n. sp. forms a monophyletic group with S. guangdongense and S. longicaudum , and grouped with S. pui , S. hermaphroditum and S. lamjungense ( Fig. 8 ).

Symbiotic bacterium. The molecular and phylogenetic analysis of the symbiotic bacterium isolated from S. taiwanensis n. sp. revealed that it is close to Xenorhabdus griffiniae Tailliez et al. Further studies on the symbiotic bacterium are undergoing.

In conclusion, both morphological and phylogenetic analyses confirmed that the new isolate belongs to Longicaudum -clade, and is closely related to S. guangdongense and S. longicaudum but it is distinctly different from these two species according to its morphological characterizations of IJ, male and female. Therefore, the newly isolated nematode from Taiwan is described as a novel species, S. taiwanensis n. sp.

Figures

FIGURE 1. Steinernema taiwanensis n. sp. A–C) infective juvenile: A) anterior region with nerve ring and excretory pore; B) anterior region showing pharyngeal part; C) tail with anus and hyaline region. D–I) first generation male: D) anterior region of first generation with nerve ring, excretory pore and pharynx; E) tail region with anus; F) tail region showing spicule and gubernaculum; G) spicule; H–I) gubernaculum. J–L) young first generation female: J) anterior region with nerve ring, excretory pore and pharynx; K) vulva; L) tail region with anus, showing not well-developed post-anal swelling. (Scale bars: A, C, F–I = 20µm; B, E, L = 10µm; D = 30µm; J–K = 100µm)

FIGURE 2. Light micrographs of infective juvenile of Steinernema taiwanensis n. sp. A) anterior region showing excretory pore (arrow); B) anterior region (pharyngeal region); C) tail region showing anus (arrow); D) tail region showing no dorsal constriction. (Scale bars: A–D = 20µm).

FIGURE 3. Light micrographs of male of Steinernema taiwanensis n. sp. A) full view of a first-generation adult; B) anterior region (pharyngeal region) showing excretory pore (arrow) (first generation); C) tail region showing spicule and gubernaculum (first generation); D) tail region (second generation); E) spicules and gubernaculum (first generation). (Scale bars: A–B = 100µm; C–D = 50µm; E = 20µm)

FIGURE 4. Light micrographs of female of Steinernema taiwanensis n. sp. A) anterior region (pharyngeal region) showing excretory pore (arrow) (young first generation); B) vulva (young first generation); C,D) tail region (young first generation); E) tail region (second generation). (Scale bars: A–E = 20µm)

FIGURE 5. Scanning electron micrographs of infective juvenile of Steinernema taiwanensis n. sp. A) anterior region showing smooth cuticle, three of four cephalic papillae (CP), and one amphid (AM); B) anterior area showing the beginning of lateral field pattern with one incisure (arrow) maintaining a short distance; C,D) lateral field pattern of anterior area where several incisures appear to form ridges; E,F) from lateral field pattern, ridges extend rearward; G) two ridges and phasmid (PH) in tail region; H) tail region showing the lateral field and phasmid.

FIGURE 6. Scanning electron micrographs of first-generation adults of Steinernema taiwanensis n. sp. A,B) male, posterior region showing genital papillae and a single papilla (s). C–G) young female: C,D) anterior and lateral view of head region showing mouth, labial papillae, and cephalic papillae; E) vulva; F,G) tail region with anus, showing not well-developed postanal swelling and papilla-like structures on tip.

FIGURE 7. Phylogenetic relationships of Steinernema taiwanensis n. sp. with other species in Longicaudum-clade based on the sequences of rDNA ITS region. Steinernema khoisanae, S. jeffreyense, S. tophus, and S. innovationi as outgroups.

FIGURE 8. Phylogenetic relationships of Steinernema taiwanensis n. sp. with other species in Longicaudum-clade based on the sequences of D2D3 region of 28S rDNA. Steinernema khoisanae, S. tophus, and S. innovationi as outgroups.