Steinernema apuliae, Triggiani, Oreste, Ek, Zdenek Mrá Č & Reid, Alex, 2004

Triggiani, Oreste, Ek, Zdenek Mrá Č & Reid, Alex, 2004, Steinernema apuliae sp. n. (Rhabditida: Steinernematidae): a new entomopathogenic nematode from southern Italy, Zootaxa 460, pp. 1-12 : 3-11

Steinernema apuliae sp. n. ( Figures 1–3)

Measurements: Morphometrics of first and second generation paratype males of S. apuliae sp. n. are given in Table 1, morphometrics of first and second generation paratype females in Table 2 and morphometrics of the holotype (third­stage infective juvenile) and of thirdstage paratype juveniles in Table 3 .

TABLE 1: Morphometrics of first­ and second­generation paratype males of Steinernema apuliae sp. n. (mean, standard deviation of the mean, range; all measurements in µm).

* spicule length divided by anal body width, ** gubernaculum length divided by spicule length.

* Hyaline tail length in % of total tail length.**Anterior end to excretory pore in % of pharynx length.

Morphology

First generation males: body curved ventrally posteriorly, tapering toward lip region, J­shaped in heat­relaxed specimens. Cuticle with very faint transverse striae, lateral fields absent. Head rounded, almost continuous with body. Six lips fused at base, each lip bearing a labial papilla. Outer circle of four cephalic papillae present. Amphids distinct, located laterally between labial and cephalic circle of papillae ( Fig. 1 B). Excretory pore ( Fig. 1 C) variable in position, rarely slightly anterior, mostly at, and sometimes posterior to nerve ring (ranging from 75% to 92% of the length from anterior body end to base of pharynx). Stoma short, shallow, cheilorhabdions distinct, sclerotized, followed by the sclerotized smaller, sometimes indistinct prorhabdions. Pharynx muscular with procorpus extending into slightly swollen nonvalvate metacorpus, a narrower isthmus region surrounded by a nerve ring, and a rounded basal bulb. Cardia rather long, intestine generally with a wide lumen. Distinct, papilliform deirids situated at or slightly behind pharyngeal bulb. Well developed and reflexed testis. Paired symmetrical, slightly curved spicules and light brown in colour, distally bluntly pointed ( Fig. 1 G). Spicule manubrium distinctly elongated, with length: width ratio of 1.2–1.7:1; manubrium less then one third of spicule length (about 29%); calomus distinct, lamina slightly arched, ventral rostrum absent; velum narrow, indistinct, parallelly arched with lamina, extending from calomus to distal part of lamina. Gubernaculum boat­shaped in lateral view, distally much attenuated, proximal part varying from straight and bluntly pointed to slightly bent­like ( Fig. 1 H). Tail bluntly conoid, usually concave at ventral side, mucron absent. There are 23 genital papillae comprising 11 pairs and a single ventral papilla located just in front of the cloacal opening. Of the paired papillae, 6 pairs are located precloacal. Of these 5 pairs situated subventrally and 1 pair laterally. Five pairs postcloacal. Of these 2 pairs located adcloacal and subventral, very close to each other. The last 3 pairs are consistently located near the tail tip ( Fig. 1 D).

Second generation males: similar to the first­generation males, but slightly smaller in the body length and other measurements (Table 1; Fig. 2A ). Greatest body width much smaller. Excretory pore located more anterior than in the first generation (Table 1; Fig. 2A ). Tail mucron absent ( Fig. 2 C).

First generation females: body long, usually C­shaped on heat relaxation ( Fig. 1A). Cuticle only faintly striated. Head bluntly rounded, slightly tapering anteriorly. Morphology of head region and pharynx similar to the males ( Fig. 3A ). Excretory pore located rarely anterior, mostly at, and sometimes posterior to the nerve ring. Genital tract amphidelphic with reflexed ovaries. Vulva asymmetrical and oblique slit to symmetrical and transverse slit, slightly (3 µm) to distinctly (35 µm) protruding from the body contour (depending on the age). Slant and short vagina, leading into paired uteri. Narrow rectum, distinct anal opening. Tail of mature females short and obtuse, with small postanal lobe, almost long as is wide; in young females a longer tail with conical­like tip, which is more prominent in older females with the obtuse tail. The tip bears one large and two or three minute protuberances or papillae protruding from the body contour ( Fig. 3 B, 3C and 3D). Pigmy forms rarely observed.

Second generation females: similar to first generation females in general morphology, but smaller in dimension ( Table 2 ). Excretory pore located more anterior than in the first generation females. Vulva distinctly asymmetrical and oblique slit in all observed specimens ( Fig. 3 E and 3F). Conical, pointed tail, with a short protuberance or papilla­like structures on its tip, but not mucronated; tail longer than body width at anus ( Fig. 3 G). Pygmy forms not observed.

Infective third­stage juveniles: body of heat­relaxed specimens slightly C­shaped to almost straight, slender, slightly tapering towards anterior and posterior ends. Morphometrics of infective­stage juveniles are reported on Table 3 . Specimens from Galleria cultures often enclosed in second­stage cuticle that is indistinctly annulated and 1.5 to 2 µm thick in midbody region. Lateral fields at midbody region with eight equally distributed ridges (i.e. nine lines); all ridges and lines distinct and at approximately even distances ( Fig. 3 H). Phasmids weekly developed, posterior to the anus, with their opening approximately 40% of tail length located at ventral edge of the lateral fields. Head portion finely rounded, mostly slightly off­set from body contour by a depression of 11–13 µm wide ( Fig. 1 E). Four cephalic papillae and amphids indistinct. Papilliform deirids situated slightly behind pharyngeal bulb. Closed stoma. Pharynx with slightly expanded procorpus, narrower isthmus and subpyriform basal bulb with weakly developed valve apparatus. Basal bulb usually slightly displaced towards dorsal side by the excretory gland. Lentil­like hemizonid, about 5 µm long, mostly varying in position from beginning of bulb to its midsection.

Excretory pore posterior to mid­pharynx region (on average about 66% of the length from the anterior end to the base of the pharynx), cuticularized, about 1 µm wide, excretory duct extending posteriorly to the cardia region ( Fig. 3 I). Intestine separated from pharynx by a long cardia, which is followed by a slender bacterial vesicle. Gut filled with numerous fat globules, lumen not visible. Straight rectum, almost as long as anal body diameter. Anus 4­ 5 µm wide, sickle­shaped. Tail relatively wide (low ratio C'), conoid, straight, with terminus finely pointed, tip devoid of refractive inclusion ( Fig. 1 F). Hyaline portion occupying on average 41–42% of tail length ( Fig. 3 L).

DNA analysis

The RFLP (restriction fragment length polymorphism) profiles yielded by digestion of the ITS region with 17 restriction enzymes for Steinernema apuliae sp. n. ( Fig. 4 A) were compared to a database containing over 50 different Steinernema isolates (including 22 described species). Examination of the RFLP profiles showed S. apuliae sp. n. to be unique. S. apuliae sp. n. can be differentiated from S. arenarium ( Fig. 4 B) by 9/17 restriction enzymes (Alu I, Dde I, Hae III, Hha I, Hind III, Hinf I, Pvu II, Rsa I and Sal I) and from S. glaseri ( Fig. 4 C) by 11/17 enzymes (Alu I, Dde I, Hae III, Hha I, Hind III, Hinf I, Hpa II, Pvu II, Rsa I, Sal I and Sau3A I).

Type of locality and host

The type population was collected by using Galleria mellonella (L.) larvae as bait from a soil sample picked up from a saltpan near Margherita di Savoia, in the district of Bari at an altitude of 30 m above sea level, in October 1996. The natural host is unknown.

Type of material

Holotype infective juvenile, paratype third­stage juveniles, males and females of first and second generations are deposited in the Laboratory of Insect Pathology, Institute of Entomology, Czech Academy of Sciences, Č eské Budĕjovice. Additional paratypes (thirdstage juveniles, males and females) are at the Department of Biology and Chemistry, Section of Entomology and Zoology, University of Bari; paratype third­stage juveniles are in the Nematode Collection of CABI Bioscience, Egham, U.K.

Differential diagnosis

Steinernema apuliae sp. n. is mainly characterized by the following morphological characteristics: males with light brown, slightly curved spicules with longitudinally shaped manubrium and absence of a tail mucron in both generations. Females possess a characteristically asymmetrical oblique slit vulva distinctly behind the mid­body. Position of excretory pore is mostly posterior in both sexes. One distinct and two or three minute protuberances or papillae are located on the first female tail tip. Third­stage juveniles have long body, on average over 1000 µm. Hyaline tail portion is medium long, slightly over 40% of the tail length. Excretory pore is near the posterior­pharynx region (58­77%). Lip region broad, continuous, and rounded. Lateral fields with equally developed eight ridges (equivalent to nine lines) at even distances.

Steinernema glaseri and S. arenarium are the only described Steinernema species found in Europe closely resembling S. apuliae sp. n. in morphometrics and other morphological characters ( Poinar, 1978; de Doucet & Gabarra, 1994; Mráček et al., 1999). However, females of S. apuliae sp. n. have an asymmetrical, oblique slit vulva, whereas S. glaseri and S. arenarium females have a more symmetrical and transverse slit vulva. Vulval opening is slightly posterior in S. apuliae females. Males of S. glaseri possess a hooked spicule tip that was never observed in S. apuliae and S. arenarium . Hyaline tail portion of IJs of S. apuliae seems to be slightly shorter. Pair of marginal ridges is slightly annulated in S. glaseri and smooth in S. arenarium and S. apuliae . Finally, PCR­RFLP analysis can be used to clearly differentiate these species.

More species, such as S. cubanum Mráček, Hernández & Boemare 1994 , S. puertoricense Roman & Figueroa 1994 , S. longicaudum Shen & Wang 1992 , S. karii Waturu, Hunt & Reid 1997 , S. loci Phan, Nguyen & Moens, 2001 and S. diaprepesi Nguyen & Duncan, 2002 , belong to the “glaseri­group”. There is an important zoogeographical barrier for torrid species of S. cubanum , S. puertoricense , S. karii , and S. loci . The infective juvenile ratio (D%) is very low for S. karii (56.6%) and S. loci (57%), whereas 66% for S. apuliae .

The ratio c´for S. longicaudum IJs is very high (4.22) due to the tail length over 90 µm tail length, as it is for S. puertoricense . S. diaprepesi has shorter IJs, about 1000 µm, and a ratio (D%) close to 50%.

The morphology of males is similar among these species; however, S. apuliae possesses a strongly asymmetrical vulva that differs from that with a double flapped epiptygma of S. puertoricense . S. cubanum is morphologically and morphometrically very similar to S. apuliae ; however, its vulva position is about 50% that of the body length whereas in S. apuliae it is significantly, behind the mid­body, varying mostly between 55% to 57% of the body length.

Life cycle and crossbreeding

Steinernema apuliae sp. n. is easily reared on G. mellonella larvae in the laboratory at 20 to 22°C. The Galleria larvae were killed after two days of exposition to IJs; first generation of adults appeared three days after host death occurred and new IJs started to emerge after one week. There were no positive interbreedings of S. apuliae with S. glaseri and S. arenarium .

Distribution and ecology

Steinernema apuliae sp. n. has been found exclusively in southern Italy in the Apulia and Basilicata regions ( Fig. 5 ) ( Triggiani & Tarasco, 2002). Isolates were only found in sandy soil with one exception in silty soil (Table 4). They prevailed on seacoast with scant vegetation and a pinewood forests where Thaumetopoea pityocampa species ( Lepidoptera , Thaumetopoeidae ) is common.

Figures

FIGURE 4. RFLP profiles for (A) Steinernema apuliae sp. n., (B) S. arenarium and (C) S. glaseri. M molecular weight markers (band sizes shown in base pairs); Sf, the ITS region from S. feltiae (UK site 76) digested with Alu I; lanes 1 to 17 the ITS region for each isolate digested with the following restriction enzymes; 1, Alu I; 2, BstO I; 3, Dde I; 4, EcoR I; 5, Hae III; 6, Hha I; 7, Hind III; 8, Hinf I; 9, Hpa II; 10, Kpn I; 11, Pst I; 12, Pvu II; 13, Rsa I; 14, Sal I; 15, Sau 3 A I; 16, Sau 96 I; 17, Xba I.

FIGURE 5. Map of collecting sites. Solid circles are the sites where S. apuliae was found.

FIGURE 2. A, SEM second generation male of S. apuliae sp. n, dorsal view of posterior region of male and 2 pairs of adcloacal papillae (a and b), 3 pairs of papillae near tail tip; B, second generation male, Nomarski LM, esophagus and excretory pore; C, second generation male, Nomarski LM, mail tale with spicules and gubernaculum.

FIGURE 3. A, SEM of first generation female of S. apuliae sp. n., head with 6 labial and 4 cephalic papillae; B, first generation female, Nomarski LM, tail with apical papillae or protuberance; C, SEM of first generation female, tail with apical papillae or protuberances; D, SEM of first generation female, lateral view of tail with conical­like tip and apical papilla or protuberance; E, second generation female, Nomarski LM, asymmetrical vulva; F, SEM of second generation female, asymmetrical vulva; G, SEM of second generation female, ventral view of tail with an apical protuberance; H, SEM of infective juvenile, lateral field with 8 identical ridges; I, infective juvenile, Nomarski LM, esophageal portion with excretory pore and hemizonid; L, infective juvenile, Nomarski LM, tail with hyaline portion.

Tables

TABLE 2. Morphometrics of first­ and second­generation paratype females of Steinernema apuliae sp. n. (mean, standard deviation of the mean, range; all measurements in µm).

Character	First generation (n = 15)	Second generation (n = 20)
Body length	5086 ± 1700 (2828 – 9474)	2362 ± 433 (1818 – 3192)
Greatest diameter	271 ± 40 (227 – 383)	144 ± 25 (105 – 196)
Stoma length	8.1 ± 1.1 (6.4 – 11.5)	5.5 ± 0.6 (4.7 – 6.2)
Stoma width	14 ± 1.2 (11 – 16)	9.3 ± 0.8 (7.9 – 10.6)
Anterior end to Excretory pore	184 ± 15 (141 – 199)	158 ± 18 (129 – 187)
Pharynx length	229 ± 11 (207 – 246)	225 ± 24 (196 – 266)
Tail length	62 ± 8.2 (47 – 74)	61 ± 4.4 (51 – 67)
Anal body width	69 ± 6.2 (54 – 78)	46 ± 9.7 (35 – 70)
A	19 ± 5.7 (11 – 33)	16 ± 1.6 (12 – 19)
B	22 ± 6.8 (13 – 39)	14 ± 2.1 (12 – 21)
C	82 ± 29 (52 – 173)	46 ± 5.6 (40 – 64)
C'	3 ± 0.3 (2.4 – 3.6)	1.3 ± 0.1 (1 – 1.5)
V	55 ± 1.8 (50 – 58)	57 ± 1.9 (55 – 61)
D %	80 ± 4.7 (68 – 87)	70 ± 5.1 (60 – 78)

TABLE 3: Morphometrics of infective­stage juveniles of Steinernema apuliae sp. n. (mean, standard deviation of the mean, range; all measurements in µm).

Character	Holotype	Paratypes (n = 19)	Laboratory populations (n = 20)
Body length	1192	1064 ± 66 (945 – 1212)	1063 ± 81 (869 – 1192)
Greatest diameter	38	37 ± 1.9 (34 – 41)	36 ± 1.8 (33 – 40)
Anterior end to Excretory pore	100	95 ± 4.2 (86 – 102)	93 ± 21 (41 – 57)
Anterior end to Nerve ring	119	111 ± 5.3 (103 – 104)	109 ± 6.7 (100 – 122)
Pharynx length	146	145 ± 6.4 (137 – 159)	147 ± 8.5 (134 – 164)
Tail length	73	71 ± 5.4 (63 – 80)	74 ± 6.6 (64 – 83)
Hyaline tail length	33	30 ± 2.7 (25 – 34)	31 ± 2.6 (27 – 35)
Anal body width	25	23 ± 1.4 (20 – 26)	23 ± 1.2 (21 – 25)
A	31	29 ± 1.4 (26 – 31)	29 ± 2 (26 – 30)
B	8.2	7.3 ± 0.3 (6.8 – 7.8)	7.2 ± 0.6 (6.5 – 7.3)
C	16.3	15 ± 0.7 (14 – 17)	14 ± 0.7 (14 – 16)
C'	2.9	3.0 ± 0.2 (2.7 – 3.3)	3.3 ± 0.3 (2.8 – 3.8)
E	1.4	1.3 ± 0.1 (1.2 – 1.5)	1.3 ± 0.1 (1.1 – 1.5)
H % *	46	42 ± 2.6 (38 – 47)	41 ± 2.5 (35 – 45)
D %**	68	66 ± 2.3 (63 – 70)	66 ± 0.05 (58 – 77)