Oscheius onirici, Torrini, Giulia, Mazza, Giuseppe, Carletti, Beatrice, Benvenuti, Claudia, Roversi, Pio Federico, Fanelli, Elena, Luca, Francesca De, Troccoli, Alberto & Tarasco, Eustachio, 2015

Torrini, Giulia, Mazza, Giuseppe, Carletti, Beatrice, Benvenuti, Claudia, Roversi, Pio Federico, Fanelli, Elena, Luca, Francesca De, Troccoli, Alberto & Tarasco, Eustachio, 2015, Oscheius onirici sp. n. (Nematoda: Rhabditidae): a new entomopathogenic nematode from an Italian cave, Zootaxa 3937 (3), pp. 533-548 : 535-542

Oscheius onirici sp. n.

( Figs. 1–3 , Table 1 )

Female: Body almost straight to slightly bent on ventral side when relaxed by gentle heat. Cuticle finely annulated. Lateral fields with basically three longitudinal ridges, delimited by four lines. In few specimens, an additional short running line could be seen in the middle part of body ( Fig. 2 H). Six lips, separate, continuous with body contour; six bristle-like labial sensilla, one on each lip. Amphidial apertures elliptical ( Fig. 3 E), located on lateral lips. Stoma rhabditoid type, ca four times longer than diam. Cheilostom not sclerotized, gymnostom culticularized, stegostom (pharyngeal collar) conspicuous, enveloping ca 50% of stoma length. Glottoid apparatus well developed, isomorphic. Pharyngeal corpus cylindrical, devoid of valvate median bulb, ending in a round basal bulb. Nerve ring localized at ca 64–67% of pharyngeal length. Excretory pore conspicuous, ventrally located at level ranging 73–100% of pharyngeal length. Hemizonid not clearly observed. Reproductive system amphidelphic. Ovaries reflexed anteriorly. Vulva in form of a transverse slit. Vagina ca 16% of corresponding body diameter. Rectum long, ranging 2.4–2.8 anal body diam. long, sometimes expanded proximally. Spermatheca filled with sperm. Tail conical, gradually tapering to a fine point, ca 4 anal body diam. long. Phasmids pore-like, located behind anus at 14 to 25 % of tail length.

Male: Rare (only twelve, were found). Body straight to open C when heat-killed. General morphology similar to female except for smaller size and reproductive system. Testis single, anterior end reflexed ventrally. Spicules paired, of equal length, separate, with a small, rounded manubrium; lamina expanded in its proximal part, with a more or less evident velum and ending in a rounded tip. Gubernaculum slender and slightly curved, ca 46% of spicule length. Bursa peloderan, not open; nine pairs of papillae of different lengths, arranged in a 1+1+1/3+3 pattern. Pairs 4–6 and 7–9 more closely spaced than pairs 1–3. Pairs 5 and 8 curved dorsally and not reaching rim of bursa. Tail ca 1.1 anal body diam. long. In three males, the tail protruded slightly outside the bursa ( Fig. 1 I)

Dauer juveniles: Body straight when heat-killed. Stoma and pharynx morphology similar to adult. Lateral fields with two longitudinal ridges. Nerve ring localized at ca 67% of pharyngeal length. Excretory pore located at ca 83% of pharyngeal length. Tail elongate, conical tapering to a hyaline part 11.5 ± 1.6 (9.5–16) Μm long.

Type host and locality. Natural host unknown, since material was recovered by soil samples inside the karst cave “Grotta del Lago” in the Monti Pisani area: 10°28’24.8’’E, 43°44’17.7’’N, municipality of San Giuliano Terme, Pisa Province, Tuscany Region, Central Italy ( Mazza et al. 2014).

Type material. Holotype male, ten paratype females and ten dauer juveniles are mounted on glass slides and deposited at the laboratory of Nematology of the CRA-ABP of Florence ( Italy). Two glass slides with two paratype males, two females and five dauer juveniles were deposited in the laboratory of Department of Soil, Plant and Food Sciences, Section of Entomology and Zoology, University of Bari “A. Moro”, Bari ( Italy) and in the nematode collection at the Institute for Sustainable Plant Protection ( IPSP), CNR, Bari, Italy. Additional paratypes were distributed to the United States Department of Agriculture Nematode Collection, Beltsville, MD, University of California Riverside Nematode Collection and WaNeCo, Plant Protection Service, The Netherlands, Wageningen (one glass slide with two males and two females for each Collection).

Etymology. The name of the species refers to the dream [from greek őνειρον, -ου, τò (oneiron)] because one of the authors had a premonitory dream on the discovery of this new species with the indication on the precise locality of the collection.

Nematode entomopathogenicity. Oscheius onirici sp. n. infected 46% of Galleria larvae and 58% of Tenebrio larvae, while S. carpocapsae killed 100% of these insects in one week (LT50: 6.8 ( Galleria ) and 5.2 ( Tenebrio ) days for O. onirici , sp. n. and 1.4 days for S. carpocapsae ). IJs of both nematodes emerged from all cadavers within 72 h after host death, and from all the dissected dead larvae nematodes of different developmental stages were recovered. Galleria and Tenebrio larvae death was sufficiently rapid with a 33% of insect larvae death within 120 hours.

Diagnosis and relationships. Oscheius onirici sp. n. is characterized by a small size (mean body length of the female is 671 Μm, of the male 510 Μm and of the dauer juvenile 334 Μm), cuticle finely annulated, stoma rhabditoid type, ca three times longer than its diameter and a lateral field marked by three ridges (four longitudinal lines) in adults. Female have an amphidelphic reproductive system, a long rectum (ca 2.4 anal body diam. long), a spermatheca with sperm, and a conical tail, gradually tapering to a finely pointed terminus (ca 4 anal body diam. long). Males are characterized by a short, rounded tail, peloderan bursa with nine pairs of papillae of different lengths, arranged in a 1+1+1/3+3 pattern, spicules small and slender.

The peloderan bursa, expansile rectum and the not crochet-needle-shaped spicules place O. onirici sp. n. in the Dolichura -group of Oscheius ( Sudhaus & Hooper 1994, Sudhaus & Fitch 2001). Species comparisons in the Dolichura -group are presented in Table 2 .

12.4‾17.1 7.7‾11.2 9.4 ± 0.7 6.1 9.7 ± 0.9 7.4‾8.3 ‾ 7.0 (8.2‾11.2) (5.4‾7.0) (8.6‾11.8) (6.2‾8.5) ‾ ‾ 7.0±0.7 7.7 4.3 ± 0.4 ‾ ‾ 5.2 (5.9‾8.2) (6.9‾9.6) (3.5‾5.0) (4.2‾6.4) 48‾52 48.7‾55 54±1.7 53 51.0 ± 1.3 45.5‾49.1 50.6* 48.6 (51‾57) (50‾56) (47.3‾52.6) (46.4‾51.7)

Max. bοdy diam. 57‾96 54‾74 ‾ 47 36 ± 5.3 46‾79 ‾ 33 (38‾61) (30‾51) (29‾42)

Pharynx 197‾234 130‾197 255 ± 10.5 143 137 ± 6.2 ‾ 128 140

(231‾273) (126‾162) (126‾146) (129‾151)

Excretοry pοre ‾ ‾ 207 ± 25 ‾ 108 ± 13.2 ‾ ‾ 105

(178‾225) (92‾146) (84‾120)

Nerve ring ‾ ‾ ‾ ‾ 90 ± 5.0 ‾ ‾ 100

(80‾98) (81‾109)

Τail length 83‾117 73‾116 144 ± 13.5 145 69 ± 5.5 ‾ 120 83 (121‾172) (116‾172) (63‾81) (70‾95) ......continued on the next page Reference Sudhaus (1974) volk (1950) Andersοn & Sudhaus & present study Korner (1954) Pοtts (1910) Sudhaus (1993)

Sudhaus (1984) Hοοper (1994)

Calculated

After οriginal drawing (Pοtts, 1910) and repοrted by Sudhaus & Hοοper (1994) Oscheius onirici sp. n. is most closely related to O. tipulae ( Sudhaus 1993) , for similar body size, but can be separated by shorter body length (510 vs 676) and shorter spicules in males and shorter tail length in both adult stages. Females also differ in the value of ratio c (9.7 vs 7.0 in O. tipulae ). Body length of the adult of the new species is similar to O. sechellensis , but it is clearly different for the presence of four lateral lines vs two.

Oscheius onirici sp. n. can be distinguished from O. pseudodolichura ( Körner 1954) by smaller size of both adult stages. They also differ in the value of ratio b (female: 4.4–6.0 vs 6.6–8.0; male: 3.8–4.6 vs 6.4) and females differ in the value of ratio c (8.6–11.8 vs 7.4–8.3). Oscheius onirici sp. n. differs from O. bengalensis ( Sudhaus 1974) , O. dolichuroides by smaller size of both adult stages and also for shorter pharynx, shorter male spicules and gubernaculum and shorter female tail (see Table 2 ). Based on measurements by Völk (1950), O. onirici sp. n. differs from O. dolichura ( Schneider 1866) in having a shorter pharynx in males (126–146 vs 130–197) shorter tail in both adult stages, shorter male spicules and gubernaculum and four vs two lines in lateral fields.

Finally, O. onirici sp. n. can be separated from O. guentheri by the shorter adults body size, by the shorter tail and by bursa arrangement, though in O. guentheri is uncertain. Adults of O. onirici sp. n. also differ in the value of ratio c (female: 8.6–11.8 vs 5.4–7.0; male: 21.2–30.4 vs 18.4–20.8).

Since different standard were used to assess classification of EPNs, according to Dillman et al. (2012) O. onirici sp. n. is an entomopathogenic nematode, because of its ability to reproduce inside the body of the insects and because Galleria and Tenebrio larvae death was sufficiently rapid that it can be distinguished from phoretic, necromenic, and other parasitic associations. This new species is the first EPN of Dolichura -group.

Figures

FIGURE 1. Oscheius onirici sp. n. A – C: Entire body of female (A), male (B) and dauer juvenile (C); D: Female pharyngeal region; E: Stoma; F: Male pharyngeal region; G: Female tail region; H, I: Male tail in ventral and lateral view, respectively; J: Morphology of male spicule and gubernaculum; K: Female vulval region.

FIGURE 2. Light micrographs of Oscheius onirici sp. n. A: Female pharyngeal region; B: Female anterior part of intestine, showing bacteria (bracket); C: Female tail; D: Vulval region, with embryonated eggs in the uterus; E: Ventral view of male tail; F: Lateral view of male tail; G: Male tail showing bursal rays arrangement; H: Female lateral field at midbody; arrowed is a discontinuous fifth line, occasionally visible in some of the specimens; I: Female anterior genital tract showing the spermatheca (bracket). (Scale bars = 10 Μm).

FIGURE 3. Scanning electron micrographs of Oscheius onirici sp. n. (A, B, C: Juvenile; D, E: Female): A: Entire body; B: Tail; C: Lateral field at mid-body; D, E: Lip region, showing lips with their labial sensilla (D) and amphidial aperture (E, arrowed). (Scale bars: A = 50 Μm; B, C = 5 Μm; D, E = 2 Μm).

Tables

TABLE 1. Morphometrics of Oscheius onirici sp. n. All measurements are in Μm: mean ± s. d. (range).

Character	Male	Female	Infective juveniles
n L	Holotype Paratypes – 10 648 510±35.2 (443–553)	Paratypes 20 671±65.5 (584–801)	Paratypes 20 334±19.6 (304–370)
a b c	17.5 16.1±1.2 (14.6–18.2) 4.3 4.1±0.3 (3.8–4.6) 26 24.2±3.1 (21.2–30.4)	19±2.0 (15.8–22.5) 4.9±0.5 (4.4–6.0) 9.7±0.9 (8.6–11.8)	18.8±2.6 (14.9-23.9) 3.6±0.1 (3.4-3.8) 6.9±0.3 (6.3-7.6)
c'	1.3 1.2±0.2 (0.9–1.5)	4.3±0.4 (3.5–5.0)	32.6±2.7 (27.9-36.7)

TABLE 2. Cοmparisοn οf species in the Dolichura - grοup οf Oscheius. All measurements are in μm ∶ mean ± s. d. (range). Impοrtant characters are indicated in bοld.

	O. bengalensis	O. dolicura	O. dolicuroides	O. guentheri	O. onirici sp. n.	O. pseudodolicura	O. sechellensis	O. tipulae
Τype lοcality	Bengal	‾	Kenia	vietnam	Italy	‾	Seychelles	Germany
Τype hοst⁄habitat	Algae ( Siphonocladus )	‾	Scarabaeidae larva	Rice plants ( Oryza sativa )	Sοil cave	‾	Mοss	Tipula paludosa larvae
Lateral lines	4	2	4	4	4	4	‾	4
Female L a b	1160‾1643 17.0‾23.4 5.5‾7.5	686‾1110 14‾19.8 5.3‾8.6	1355 ± 129 (167‾1580) 27 ± 2.7 (22‾31) 5.4 ± 0.5 (4.6‾6.2)	863 (733‾1071) 18.3 (15.8‾20.7) 6.1 (5.6‾6.6)	671 ± 65.5 (584‾801) 19 ± 2.0 (15.8‾22.5) 4.9 ± 0.5 (4.4‾6.0)	915‾1216 15.4‾19.9 6.6‾8.0	680 ‾ ‾	603 (505‾691) 18.4 (16.3‾19.9) 4.3 (3.9‾4.9)