with a redescription of

International Journal of Nematology

Vol. 24, No. 2, pp. 141-148

December, 2014

Description of Diplolaimelloides rushikondai sp. n. (Monhysteridae: Nematoda) and first report of the males of Myolaimus indicus (Myolaimidae: Nematoda) from India Nadia Sufyan, Md. Mahamood*, Gaurav K. Singh and Irfan Ahmad Department of Zoology, Aligarh Muslim University, Aligarh 202002, India * Present address: Soil Ecology Group, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang - 110016, China Corresponding author’s e-mail: [email protected]

Abstract. During the screening of nematodes collected from across India, a new species belonging to the genus Diplolaimelloides and males of Myolaimus indicus were discovered. Diplolaimelloides rushikondai sp. n. is characterized by a well-defined two-chambered stoma, absence of ocelli, two pairs of caudal papillae, four pairs of bursal papillae arranged in (2+2) pattern, tubular gubernaculum and SL: GL ratio = 5.2-6.2. A dichotomous key to identification of species is provided. Males of Myolaimus indicus lack spicules and gubernaculum, have seven pairs of bursal papillae; four pairs precloacal, flask-shaped; and three pairs postcloacal smaller in size. Keywords. Diplolaimelloides rushikondai sp. n, India, males, Myolaimus indicus, new, taxonomy.

INTRODUCTION

species was found to be new to science. The species is being described herein as Diplolaimelloides rushikondai sp. n.

enus Diplolaimelloides, which Meyl (1954) proposed, includes all the species of the genus Diplolaimella with males and bursa with fine genital papillae. Earlier the most important taxonomic characters considered were the presence or absence of ocelli, distance of ocelli and amphidial fovea from anterior end, tail length and de Man’s ratios. However, characters such as bursal and caudal papillae, morphology of spicules and gubernaculum have helped in better diagnosis of species. Timm (1966) described the females of Diplolaimelloides meyli for the first time and considered Diplolaimella monhysteroides as species dubia. He synonymized Diplolaimella schneideri with D. ocellata and the genus Diplolaimita with Diplolaimella. Gerlach & Riemann (1973) published Bremerhaven-checklist which comprised of all aquatic species of the family Monhysteridae. Pastor de Ward & Lo Russo (2009) recognized ten species under the genus Diplolaimelloides. During the examination of soil samples collected from the edge of a drain near Rushikonda beach, Vishakapatnam, Andhra Pradesh, India, an interesting population of Diplolaimelloides was collected. On further examination, the

Myolaimus indicus was first described by Ali et al. (1971) from the banks of the river Devan-Nadi in Mysore, India, based only on females. M. indicus collected from the eastern bank of Loktak Lake in Manipur, revealed the presence of males of this species. It is being described herein with some additional information on stoma, excretory system, female reproductive system and males are being reported for the first time.

G

MATERIALS AND METHODS The nematodes were extracted from moist soil samples by the sieving and decantation and modified Baermann’s funnel techniques (Flegg, 1967). Extracted nematodes were fixed in FA (4:1) for 24h and then transferred to glycerinealcohol (5 parts glycerine, 95 parts 30% alcohol) for slow dehydration in a desiccator. Dehydrated specimens were mounted in anhydrous glycerine on glass slides using the wax ring method (de Maeseneer & d’Herde, 1963). All observations, drawing and photographs were made on an Olympus BX 50 DIC microscope.

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International Journal of Nematology Vol. 24, No.2, 2014

Figure 1. Diplolaimelloides rushikondai sp. n. A-J. A: Entire female. B: Entire male. C: Female anterior region (showing stoma). D: Pharyngeal region. E & G: Female posterior regions. F: Female reproductive system. H: Male posterior region. I: Spicules. J: Gubernaculum.

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Diplolaimelloides rushikondai sp. n.: Sufyan et al.

Table 1. Measurements (in μm) of Diplolaimelloides rushikondai sp. n. Holotype (male)

Paratypes females (n =9) Mean ± S.D. (Range)

Paratype males (n = 10) Mean ± S.D. (Range)

L

550

679±209 (620–748)

587±46 (545–683)

a b c

37 6.9 6

32±3.5 (37–26) 7±0.3 (6.8–8.0) 4.9±1.0 (4–6)

37.0 ±3.4 (32.1–42.7) 6.7± 0.5 (6.0–7.6) 6±1 (5.4–7.8)

cc

7

9±2 (7–11)

6.4±1.0 (4.7–8.8 )

-63.4 15 7 8 80 53 ---

58±3 (55–62) -21.5±2.0 (20–25) 8.7±1.0 (8–10) 7.8±0.3 (7–8) 99±8 (85–115) 64±9 (50–75) 267±27 (220–300) 20.5±1.0 (20–22)

56.5±5.5 (44–64) 56.5±5.3 (44- 64 ) 16 ±1 (15–17) 6.5±0.5 (6–7 ) 6.7±1.0 (6–8) 88.0±4.5 (80–95) 48.5±5.0 (40–55 ) ---

--

406±20 (357–443)

--

-13 25 95 300 47 8

133±32 (50–165) 15.5±0.5 (15–16) 13±1 (12-15) 140±23 (110–175 ) ----

-15.5±2.0 (12–18 ) 23 ±2 (20– 25) 98±8 (85–110 ) 333±50 (250–425 ) 45.5±2.0 (42–50) 8.3±0.3 (8– 9)

Characters

V T Maximum body width Lip width Length of stoma Pharynx Nerve ring from anterior end Anterior gonad VBD Vulva from anterior end Vulva-anus distance ABD Rectum/ Cloaca Tail Testis Spicules along axis Gubernaculum

outstretched, anteriorly blunt, present on right side of intestine. Oocytes arranged in single row in maturation zone and in multiple rows in germinal zone towards the anterior end. Uterus well developed, divisible into a proximal glandular part with a narrow lumen and distal tubular part. Uterine eggs usually one or two. Vagina anteriorly directed, supported by proximal and distal circular muscles. Vaginal lumen cuticularized. Vulva a small transverse slit at 55-62% of body length. Vulva-anus distance 8-11 times of anal body diameter. Rectum 0.8-1.0 anal body diameter long. Female tail long filiform, 7-11 times of anal body diameter. Three caudal glands arranged in tandem, opening into a spinneret chamber through a 5-6 μm long spinneret.

DESCRIPTIONS Diplolaimelloides rushikondai sp. n. (Fig. 1)

Measurements: See Table I. Females: Body 0.62-0.75 mm long, straight to slightly ventrally curved upon fixation, tapering towards the both ends. Outer cuticle smooth and inner cuticle striated. Lip region truncate. Labial sensilla small and setiform, six labial and four cephalic. Stoma sclerotized, 0.7-1.0 times lip diam. long. Cheilostom sub-cylindrical, leading through a tube-like extension into an umbrella-shaped posterior chamber without any denticles. Amphidial apertures spiral, 3-5 μm wide, located at 1.0-1.5 lip diam. from anterior end. Pharynx cylindrical, muscular, without a terminal bulb. Nerve ring located at 50-70% of pharyngeal length. Small excretory cell at the level of cardia. Cardia small conoid. Progaster connecting the intestine and pharynx. Intestine with prominent nucleated cells and lumen with longitudinal marking. Female reproductive system mono-prodelphic. Ovary

Males: Anterior end similar to that of females, body slightly smaller in length. Testis outstretched on right side of intestine. Tail divisible into two parts, anterior conical part 0.8-2.0 times of anal body diameters and filamentous posterior part 4.1-6.2 anal body diameters or 64-94% of tail length. Bursa 22-30 μm long, with four pairs of post-cloacal papillae arranged in (2+2) pattern. Two pairs of caudal papillae, one dorso-lateral pair just below bursa and a ventrolateral pair about three anal body diameters posterior to 143


cloaca. Spicules long and setose, slightly curved, with a welldefined capitulum. Gubernaculum small, wedge-shaped, proximal end raised to form a groove. SL (Spicules length): GL (Gubernaculum length) ratio 5.2-6.2. Three caudal glands, opening through 4-5 μm long spinneret into a spinneret chamber.

*Key to males of all known species of

Diplolaimelloides 1. With five papillae in bursa .…............…….…...….…... 2 With less than five papillae in bursa.……....……......…. 3 2. Bursal papillae pattern (1+1+1+1+1) …...….. D. palustris Bursal papillae pattern (1+1+2+1) ………...… D. altherri 3. With four papillae in bursa ……………...……………... 4 With three papillae in bursa in pattern (1+2)……..……. 6 4. Spicules longer than 45 μm ..……………....................... 5 Spicules shorter than 40 μm ………………....... D. brucei 5. Gubernaculum wedge shaped .......... D. rushikondai sp. n. Gubernaculum rod like ……...............…………. .D. deyli Gubernaculum tube-like ……….................. D. tehuelchus 6. With one male tail papilla ………...……………..…….. 7 With more than one tail papillae …...…...........…….….. 8 7. Spicules shorter than 30 μm ………........…. D. islandicus Spicules longer than 30 μm ………............. D. deconincki 8. With two male tail papillae (1+1) ….........…… D. oschei With three male tail papillae (1+1+1) ……........……… 9 9. Spicules angular and long gubernaculum (20 μm) ...................................................... D. warwicki Spicules arcuate and short gubernaculum (9-10 μm)……………........................………. D. meyli

Type habitat and locality: Moist soil collected from the edge of a drain, near Rushikonda beach, Vishakhapatnam, Andhra Pradesh, India. Type specimens: Holotype male on slide Diplolaimelloides rushikondai sp. n. /1; nine paratype females and ten paratype males on slides Diplolaimelloides rushikondai sp. n. / 2-12; deposited in the nematode collection of Department of Zoology, Aligarh Muslim University, Aligarh, India Diagnosis and relationship: Diplolaimelloides rushikondai sp. n. is characterized by well-defined two-chambered stoma, long, setose spicules, wedge shaped gubernaculum, four pairs of bursal papillae arranged in (2+2) pattern and two pairs of caudal papillae. The new species resembles D. deyli Andrássy, 1958, D. brucei Hopper 1970, D. tehuelchus Pastor de Ward & Lo Russo, 2009, in having four pairs of bursal papillae. However, it differs from D. deyli in the shape of stoma (distinctly two-chambered vs funnel-shaped anterior part and posterior chamber often indistinct), ocelli (absent vs present), shape of gubernaculum (wedge shaped vs rod-like, distally curved), transverse striation along spicule (absent vs present), SL: GL ratio (5.2-6.2 vs 3.7), length of bursa (22-30 μm vs 55-77 μm), smaller conical part of tail (15-16 μm vs 35-38 μm). It can be further differentiated from D. bruciei in SL: GL ratio (5.2-6.2 vs 1.3), ocelli (absent vs present), in the size and shape of gubernaculum (8-9 μm, wedge-shaped vs 23-28, tubular proximally divided into arms). D. rushikondai sp. n. further differs from D. tehuelchus in SL: GL ratio (5.26.2 vs 6.6-7.1), ocelli (absent vs present), and in the number of caudal papillae (only two pairs vs four pairs and seven unpaired caudal papillae). Etymology: The species is named after the place it was found. Bionomics: Previously described species of Diplolaimelloides were widely distributed. D. deyli Andrássy, 1958 has been reported from brackish water in Egypt, Africa. Of the Asian species, D. palustris Tsalolikhin, 1985 was reported from brackish water and D. meyli Timm, 1961 was reported from Bay of Bengal. Four species have been reported from America (D. altherri Meyl, 1954 from brackish water, D. brucei Hooper, 1970 from Barataria bay, D. warwicki Pastor de Ward and Lo Russo, 2009 and D. tehuelchus Pastor de Ward and Lo Russo, 2009 both from lagoon). Three species have been reported from Europe - D. deconincki (Gerlach, 1951) Meyl, 1954 from Kiel Bay, Germany, D. islandicus (De Coninck, 1943) Meyl, 1954 from Iceland and D. oschei Meyl, 1954 from brackish water, Germany. Diplolaimelloides rushikondai sp. n. is reported from brackish water near Rushikonda beach, Vishakapatnam, India.

*

Modified and amended from the key proposed by Pastor de Ward & Lo Russo (2009).

Myolaimus indicus Ali, Farooqui & Suryawanshi, 1971 (Fig. 2)

Measurements: See Table II. Female: Body slightly ventrally curved upon fixation, tapering towards the anterior and posterior ends. Cuticle loose, finely striated, outer cuticle showing wrinkles and attached to body on ventral side near excretory pore, vulva and behind the anus. Lateral fields with two, very closely placed ridges. Lip region continuous with body, lips fused; labial papillae well-developed. Labial sensilla small, six labial and four cephalic. Buccal cavity wide, triangular at base. Stoma with well-cuticularised cheilostom, gymnostom and stegostom. Gymnostom and stegostom fused. Dorsal stegostomal wall with a prominent tooth, subventral wall bulged into a ring-like structure. Amphidial apertures small, round, located less than one lip diameter from anterior end or at the level of middle of stoma. Pharynx divisible into muscular corpus, narrow isthmus and distinct basal bulb. Anterior pharynx cylindrical, 1.2-1.5 times of posterior pharynx. Basal bulb ellipsoidal with well-developed valve plates. Nerve ring just posterior to corpus, encircling isthmus. Excretory pore at the base of corpus, excretory gland and excretory duct distinct. Excretory cell at the level of nerve ring. 144


Figure 2. Myolaimus indicus A-M. A: Entire female. B. Entire male. C: Pharyngeal region. D: Female anterior region (showing dorsal tooth). E: Female anterior region (showing sub-ventral ring). F: Female anterior region (showing amphid). G: Lateral field. H: Pharyngeal region showing position of excretory pore and excretory cell. I: Female reproductive system. J & K: Female posterior regions. L & M: Male posterior regions (showing bursa and bursal papillae).

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Table 2. Measurements (in μm) of Myolaimus indicus Ali, Farooqui & Suryawanshi, 1971.

Character L a b c c’ V Lip width Lip height Stoma Corpus Isthmus Basal bulb Pharynx Nerve ring from anterior end Excretory pore from anterior end Cardia Basal bulb width Post uterine sac VBD Vulva - anus distance Rectum/ Cloaca Tail ABD Cuticular extension at vulva Gonad Maximum body diameter Nerve ring % Excretory pore % Testis

Females (n=10) Mean ± S.D. (Range)

Males (n=9) Mean ± S.D. (Range)

615±82 (479-727) 24±3 (17.1-27.2) 4.0±0.5 (4-5) 8.8±0.9 (7-10) 6.0±0.7 (5-7.5) 50±3 (47.5-57.5) 8.0±1.3 (7-10) 3.5±0.5 (3-5) 9.5±1.0 (8-10) 80.0±6.5 (68-87) 39± 4 (32-45) 18±3 (12-22) 136±12 (113-152 ) 86.5±7.0 (73-95) 69.5±6.5 (58-77) 4.5±0.5 (4-5) 14.0±1.5 (12-15) 18.5±1.5 (16-20) 26.0±1.5 (20-40) 237±47 (145-300) 25.5±2.0 (23-30) 73±7 (60-85 ) 12.0±1.5 (10-15) 13±2.0 (10-15) 184±29 (135-222) 26±5 (20-40) 61±9 (34.5-67.5) 52.0±5.5 (42.5-62.5) --

420±36 (360-478) 23.5±2.0 (20-26.1) 3.7±0.1 (3.4-3.9) 64±9 (46.8-79.6) 1.4±0.4 (1-2) -6.5±0.5 (6-7) 3.5±0.5 (3-7) 6.3±0.5 (6-7) 66.5±5.5 (60-76) 33.5±4.0 (24-38) 14.5±2.0 (11-17) 115±8 (99-128) 14.5±2.0 (11-17) 58±5 (50-66) 3.0±0.5 (3-4) 11.0±0.5 (10-12) ---16±2 (13-20) 6.5±1.0 (5-9) 5.0±1.5 (5-9) --18±1 (16-20) 64±2 (58.5-66.5 ) 50.5±3.5 (43-54) 241±29 (195-285)

Female reproductive system mono-prodelphic. Ovary long, reflexed extending beyond vulva. Oviduct joins the ovary anteriorly. Spermatheca small, continuous, usually containing sperms. Uterus can be differentiated into glandular and muscular part of equal length. Post-vulval sac 0.5-0.9 vulval body diameter long. Sperms present both in the uterus and the post-vulval sac. Vulval lips flap-like, enlarged. Cuticular extension at vulval region 10-15 μm from the inner body wall. Rectum 1.8-2.5 anal body diameters long, with three rectal glands. Tail sharp conoid, 58 anal body diameters long.

shaped forming a group, sub ventral in position. GP5 lateral, GP6 and GP7 subventral, present at tail tip. GP4 smaller than GP1-GP3, GP6 & GP7 are smallest of all. Phasmids present close to GP5. GP3 present near cloaca. Spicules and gubernaculum absent. Bursa peloderan. Tail short, conoid, 0.9-2 anal body diameters long.

Habitat and locality: Moist soil collected from the eastern bank of Loktak Lake, Manipur, India. Voucher specimens: Ten females and nine males on slides Myolaimus indicus LT/ 1-10 deposited in the nematode collection, Department of Zoology, Aligarh Muslim University, Aligarh, India.

Male: Body slightly smaller than females. Lateral fields undifferentiated. Anterior end similar to that of females. Reproductive system monorchic, testis reflexed, present on right side of intestine. Seven pairs of bursal papillae, dimorphic in size and arrangement; anterior four flask-

Remarks: Males of Myolaimus indicus can be characterized by the absence of spicules, gubernaculum and ridges in lateral fields, presence of peloderan bursa and seven 146


Figure. 3. A-E. Myolaimus indicus. F-I. Diplolaimelloides rushikondai sp. n. A: Anterior region showing dorsal tooth. B: Anterior end showing subventral wall. C: Lateral field. D & E: Male posterior regions showing bursal papillae and bursa. F: Anterior region. G. Anterior region showing amphid. H & I: Male posterior regions showing spicular apparatus, bursa and bursal papillae. Scale bars: A-D, F-G & I = 10 μm; E & H = 20 μm.

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pairs of bursal papillae; anterior four subventrally grouped together, GP5 lateral, GP6 and GP7 again subventral. Females are characterized by the presence of wide stoma, ring-like structure at subventral wall, circular amphids in the middle of stoma, pointed dorsal tooth, excretory cell at the level or anterior to nerve ring, lateral fields with two ridges, and flap-like cuticular extension at vulva. Descriptions and measurements of our specimens agree well with the original specimens but amphidial apertures are small pore-like as compared to slit-like in the type population and the stoma shows a ring-like bulge. Bärmann et al. (2009) commented that the position of the secretary cell could be unique in Myolaimus indicus if it occurred anterior to the nerve ring as depicted in the drawing (though not mentioned in the description of Ali et al., 1971). Study of our specimens confirms the unusual position of the excretory cell i.e. at the level of nerve ring but does not conform to the position as shown by Ali et al., (1971). Notwithstanding these minor differences (shape of amphidial opening and position of excretory cell), we are confident that our population is conspecific to that of Ali et al. (1971). M. indicus appears to be related to M. heterurus (Cobb, 1920), as shown by Ali et al., (1971) on the basis of female characters only. Now as the males have been discovered, it can be differentiated from the other species on the basis of shape of male tail, number and arrangement of bursal papillae. M. indicus now closely resembles to M. goodeyorum (Andrássy, 1984), M. heterurus (Cobb, 1920), M. hortulanus (Bärmann, 2009), and M. xylophilus (Bärmann, 2009), in number of bursal papillae. However, it differs from M. goodeyorum in male body length (0.36-0.48 vs 0.7-0.75), lateral fields in females (differentiated vs undifferentiated), and c’ value (2-3 vs 5.0-7.5). Further it can differentiated from M. heterurus in the arrangement of bursal papillae (GP3 & GP4 separated vs GP3 overlaps with GP4). From M. hortulanus it differs in shape of dorsal tooth (pointed vs rounded), shape of buccal cavity (barrel shaped vs funnel shaped). It differs from M. xylophilus in shape of dorsal tooth (pointed vs rounded), lateral field in females (differentiated vs undifferentiated).

Acknowledgements: This work was financially supported by the University Grants Commission through Maulana Azad National Fellowship, Ministry of Environment and Forests through All India Coordinated Project on Taxonomy and by the Department of Science and Technology (DST) through a Fast Track Project Scheme.

LITERATURE CITED Ali, S. M., M. N. Farooqui and M. V. Suryawanshi 1971. Myolaimus indicus n. sp. (Rhabditida: Myolaimidae) and Metadorylaimus coomansi n. sp. (Dorylaimida: Tylencholaimidae) from Marathwada, India. Nematologica 16, 577-583. Bärmann, E. V., A. Fürst von Lieven and W. Sudhaus 2009. Revision and phylogeny of Myolaimus Cobb, 1920 (Secernentea, nematode) with the description of four new species. Journal of Nematode Morphology and Systematics 12, 145-168. De Maeseneer J. and C. J. d'Herde 1963. Methodes utilisees pour l'etude des anguillules libres du sol. Revue Agriculture, Bruxelles 16, 441-447. Flegg, J. J. M. 1967. Extraction of Xiphinema and Longidorus species from soil by a modification of Cobb’s decanting and sieving technique. Annals of Applied Biology 60, 420-437. Giblin-Davis, R. M., N. Kanzaki, P. De Ley, D. S. Williams, E. Schierenberg, E. J. Ragsdale, Y. Zeng and B. J. Center 2010. Ultrastructure and life history of Myolaimus byersi n. sp.; a phoretic associate of the crane fly Limonia schwarzi (Tiplulidae) in Florida. Nematology 12, 519-542. Hopper, B. E. 1970. Diplolaimelloides brucei n. sp. (Monhysteridae: Nematoda), prevalent in marsh grass, Spartina alterniflora Loisel. Canadian Journal of Zoology 48, 573-575. Meyl, A. H. 1954. Die fadenwürmer (Nematoden) einiger Salztellen südöstlich von Braunscheweig. Abhandlungen der Braunschweigischen Wissenschaftlichen Gesellschaft 6, 84-106. Pastor de ward, C. T. and V. Lo Russo 2009. Distribution of Diplolaimella and Diplolaimelloides species from Patagonian lagoons and coastal waters (Nematoda: Monhysteridae), Chubut, and Santa Cruz Provinces (Argentina). Journal of the Marine Biological Association of the United Kingdom 89, 711-718. Timm, R. W. 1966. Some observations on the nematode Genera Diplolaimella and Dipllolaimeloides Proceedings of Pakistan Academy of Sciences 3, 52-125.

Bionomics: All of fifteen species of Myolaimus are widely distributed. Nine species are reported from Europe, three from North America and one each from South America, Africa and Asia respectively. These species mostly inhabit soil, moss, saprobic or, exceptionally aquatic habitats. However, M. byersi Giblin-Davis et al., 2010, shows phoretic association with the crane fly. Interestingly, earlier, M. indicus was described from a semi-aquatic habitat in peninsular India and it is reported here from a similar habitat of Loktak Lake situated in North-eastern part of India.

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