Three New Species of Free-Living Marine Nematodes of the Order Enoplida Filipjev, 1929 (Nematoda) from the Yellow Sea, China

Abstract

Three new nematode species belonging to the order Enoplida were discovered in the muddy and sandy sediment along the Shandong peninsula coast of the Yellow Sea during a biodiversity investigation of marine nematodes in the Yellow Sea. They are named Belbolla octobulba sp. nov., Ironella gracilis sp. nov. and Oxystomina longiseta sp. nov. Belbolla octobulba sp. nov. is characterized by having eight pharyngeal bulbs; spicules arcuate, broad and even; gubernaculum with a pair of dorso-caudal apophyses, about a third of the length of spicule; and two winged precloacal supplements. Ironella gracilis sp. nov. differs from known species by body slender, buccal cavity divided into conical vestibulum and cylindrical posterior portion, three sclerotized teeth located in the vestibulum; both circles of outer labial setae and cephalic setae 10 µm apart; elongated spicules weakly arcuate with central strip and cephalate proximal end; gubernaculum conical without apophysis; and a tubular precloacal supplement with a long seta at its anterior end. Oxystomina longiseta sp. nov. can be distinguished from other known species by the relatively long outer labial setae and cephalic setae; arcuate spicules expanded proximally with a median rib; gubernaculum small and oblong; and two unequal precloacal supplementary setae, anterior one short, posterior one very long. Updated keys to eighteen species of Belbolla and keys to four species of Ironella are given.

1. Introduction

Free-living marine nematodes are the most dominant and diverse meiofauna in marine benthic habitats. They interact in many ways with different organisms in many food webs, and they play a very important role in the recycling of organic matter and energy flow to the higher trophic levels and through the benthic ecosystem [1,2]. They can also be used for biomonitoring. Identifying nematode species is therefore very important. Globally, more than 9860 species of marine nematodes have been recorded to date [2,3,4], but only about 500 species have been identified in China, and there are still many unknown species.

The Yellow Sea is located on the edge of the western Pacific Ocean, between the Chinese mainland and the Korean Peninsula. It is a semi-enclosed inland shallow sea basin. The area is 38 × 104 km², with an average depth of 44 m and a maximum depth of 140 m. Biodiversity surveys and taxonomical studies on nematodes in the Yellow Sea have been carried out in recent years. The abundance is 505 ± 238–2538 ± 676 individuals per 10 cm⁻² and the Shannon–Wiener diversity index is 1.859–3.79. More than 340 species were identified, and they belong to 2 classes, 13 orders, 45 families and 141 genera [2,3]. In abundance and occurrence, Daptonema [5] is the most dominant genus from the intertidal to subtidal zone up to 89 m deep. However, the study on nematodes in the intertidal zone of the Shandong peninsula is still not comprehensive. The total number of nematodes is unknown, and new species are always found in each sampling.

Currently, the classification of nematodes is usually based on morphological, molecular and developmental evidence. Molecular phylogenetic analyses have proven to be a valuable tool to clarify relationships among marine nematode taxa, which are impossible to determine based on morphology alone [6,7]. In the present paper, only morphological descriptions are carried out for three new species; no molecular phylogenetic analyses are conducted on them, due to their clear phylogenetic status.

The genus Belbolla [8] belonging to the family Enchelidiidae [9] was first proposed as Bolbella by Cobb in 1920 with the description of Bolbella tenuidens [5]. Currently, eighteen valid species have been recognized within the genus globally (electronic database: http://nemys.ugent.be (accessed on 1 October 2023)). Five of them, namely B. huanghaiensis [10]; B. sinica [4]; B. stenocephalum [10]; B. warwicki [10]; and B. zhangi [11], were originally described from the Chinese sea area. The Belbolla species have a high occurrence rate in the subtidal zone of the Yellow Sea, but they are rare in the intertidal zone, and only B. sinica and this new species have been found in this survey area.

The genus Ironella belonging to the family Ironidae [12] was erected by Cobb in 1920 with I. prismatolaima as the type species [5]. To date, only three species have been recorded around the world (electronic database: http://nemys.ugent.be (accessed on 1 October 2023)). Unfortunately, I. cobbi [13] was described only from females found at the Atlantic Shore of the USA by Timm. I. prismatolaima [5] was described only from males found at the Atlantic Shore of the USA by Cobb. I. riemanni [14] was found by Riemann in 1966 [15] in the Elbe estuary and in Kandalaksha Bay (the White Sea) by Platonova and Mokievsky in 1994 [14]. The new species is the first recorded species within Ironella in the Chinese sea area.

The genus Oxystomina [16] belonging to the family Oxystominidae [17] was established by Filipjev in 1921, and assigned O. elongata [16,18] as the type species. At present, there have been 56 nominal species of the genus worldwide (electronic database: http://nemys.ugent.be (accessed on 1 October 2023)). However, only 41 species were confirmed as valid after several revisions by nematologists [19,20,21,22]. Shimada et al. provided two dichotomous keys to 36 species of Oxystomina in 2020 [22]. The specimens of O. elegans [23] and O. elongata [16,18] were often found in intertidal to subtidal sediments of the Yellow Sea. O. longiseta sp. nov. is the first new species of the genus found in the survey area.

2. Materials and Methods

Sediment samples were collected at twenty sampling points using a 2.9 cm diameter sawn-off syringe from the top sediment layer (0–8 cm deep) in the intertidal zone along the coast of Shandong Peninsula in February and August 2022 (Figure 1). The samples were fixed with an equal amount of 10% formalin solution. At the sorting, the samples were poured through two sieves (500 and 42 µm mesh sizes), and they were washed with tap water to remove silt and separate macrofauna from meiofauna [24,25]. Meiofauna was sorted under a stereoscopic microscope. Nematodes were processed in a solution of glycerol and ethanol [26]. Finally, they were mounted in glycerin on permanent slides. Observation and measurement were carried out using a differential interference contrast microscope (Leica DM 2500) (CMS Gmbh, Wetzlar, Germany and assembled in Shanghai, China) and Leica software of LAS X version 3.3.3. Type specimens were deposited in the Marine Biological Museum of the Chinese Academy of Sciences, Qingdao.

Abbreviations used in the table and keys are as follows: a = body length/max. body diameter; abd = cloacal or anal body diameter; b = body length / pharynx length; c = body length/tail length; c′ = tail length/body diameter at cloaca or anus; V% = position of vulva as % of body length from anterior end.

3. Results

The mean abundance of nematodes along the coast of Shandong peninsula is 1663 ± 502 individuals 10 cm⁻². More than 200 species have been discovered. Among these species, three species belonging to the order Enoplida have been identified as new species and described here as Belbolla octobulba sp. nov., Ironella gracilis sp. nov. and Oxystomina longiseta sp. nov.

3.1. Description of Belbolla octobulba sp. nov. (Figure 2, Figure 3 and Figure 4)

3.1.1. Type Material

One male and two females were obtained and measured. Holotype male, paratype 1 (female) and paratype 2 (female) are all on slide 22YMDNQ3-2-1.

3.1.2. Etymology

The species epithet comes from the Latin words octo (eight) and bulba (bulbous), referring to the new species possessing eight pharyngeal bulbs.

3.1.3. Type Locality and Habitat

The holotype and paratypes were all collected from intertidal muddy sediments at the coast of Yangma Island, Yantai (37°26′1′′ N, 121°36′37′′ E) (Figure 1 Red 5).

3.1.4. Measurements

All measurement data are given in

Table 1. Individual measurements of Belbolla octobulba sp. nov. (in µm).

Characters	Holotype	Paratype
		♀1	♀2
Body length	3185	4076	3853
Maximum body diameter	96	139	134
Head diameter	14	18	11
Length of cephalic setae	10	10	9
Length of buccal cavity	21	21	17
Width of buccal cavity	10	11	11
Nerve ring from anterior end	335	368	341
Length of pharynx	717	760	740
Body diameter at pharyngeal base	80	100	91
Number of pharyngeal bulbs	8	8	8
Tail length	274	273	248
Body diameter at cloaca or anus	64	62	62
Spicule length along arc	124	-	-
Length of gubernacular apophysis	38	-	-
Length of anterior supplement	53	-	-
Length of posterior supplement	42	-	-
Posterior supplement from cloaca	251	-	-
Distance between both supplements	185	-	-
Vulva from anterior end	-	1986	1853
Body diameter at vulva	-	138	120
V%	-	49	48
a	33.0	29.3	28.8
b	4.4	5.4	5.2
c	11.6	14.9	15.5
c′	4.3	4.4	4.0

.

3.1.5. Description

Male. Body slender with thin and elongated neck. The front end of the body tapers to a very small diameter. Cuticle smooth with some somatic setae and mainly distributing in cervical and caudal region. Inner labial sensilla papilliform. Six outer labial setae and four cephalic setae situated in the same circle, located approximately one head diameter from the front of the body, each seta 10 μm long. Amphidial fovea not observed. Buccal cavity barrel-shaped, divided into two parts by a sclerotized transverse ring accompanying some denticles (Figure 2C and Figure 3A). A large right ventro-lateral tooth and two fewer prominent teeth (dorsal and left ventro-lateral in position) present. The anterior ten cervical setae (20–25 µm long) arranged in one circle (about 40 µm from head end), while the remaining setae arranged irregularly. The pharynx expanded gradually and modified into eight bulbs (Figure 3B and Figure 4C). The nerve ring situated at 47% pharyngeal length from front end of body. Excretory system not seen. Tail 4.3 cloacal body diameters long, conico-cylindrical with the distal one-fourth cylindrical and some short caudal setae (Figure 3D). Tail tip slightly swollen with a terminal spinneret. Three caudal glands within the tail.

Reproductive system containing two opposed outstretched testes. Paired spicules equal in length, arcuate, broad and even, 124 µm (1.9 cloacal body diameters) long as arc, 118 µm long as chord. Gubernaculum with a pair of dorso-caudal apophyses, approximately one third of spicule in length. Two well-developed winged precloacal supplements situated close together relatively far from the cloaca (Figure 3C). The previous one slightly larger than the latter one. The latter supplement situated 251 µm in front of the cloaca and distance between both supplements is 185 µm. A total of 1–2 paired short setae just anterior to cloaca.

Females. Body size slightly larger than male, lacking cervical and caudal setae. Tail equipped with two subterminal setae. Reproductive system didelphic, two ovaries opposed, reflexed. Vulva slightly raised (Figure 4D), located at 48–49% of body length from front end of body. Vagina sphincter well developed.

3.1.6. Differential Diagnosis and Discussion

Belbolla octobulba sp. nov. is characterized by having eight pharyngeal bulbs, spicules arcuate, broad and even, gubernaculum with a pair of dorso-caudal apophyses, about a third of the length of spicule, two typical winged precloacal supplements lying close together comparatively far from the cloacal opening, the previous supplement slightly larger than the latter one. At present, seven species in the genus, Belbolla gallanachmorae [8,27]; B. longispiculata [28]; B. teissieri [8,29]; B. tenuidens [8,9]; B. stenocephalum [10]; B. sundoensis [8,30]; and B. zhangi [11], have been found to have eight pharyngeal bulbs.

The new species is different from B. gallanachmorae by the distance from the posterior supplement to the proximal end of spicule almost equal to the spicule length vs. the distance longer than spicule length and gubernacular apophysis one third of spicule length vs. one fifth to one sixth of spicule length. The new species differs from B. longispiculata in having shorter spicules (1.9 vs. 10.8 cloacal body diameters) and different precloacal supplements (two winged vs. five papilliform). The new species differs from B. teissieri by longer body length (3.2 mm vs. 1.9 mm), longer spicules (115 µm vs. 39 µm) and shorter tail (4.3 cloacal body diameters with one-fourth posterior cylindrical part vs. 5.7 cloacal body diameters with half posterior cylindrical part). The new species differs from B. tenuidens by shorter length of supplements (0.7–0.8 vs. 1.4–1.5 cloacal body diameters long). The new species differs from B. stenocephalum in larger body size (3.1–4.1 mm in length and 96–139 µm in maximum width vs. 2.2–2.7 mm and 62–72 µm, respectively) and different spicules (stout with tapered distal tip vs. slender with swollen distal tip). The new species differs from B. sundoensis by distance between posterior supplement and the proximal end of spicule almost equal to the spicule length vs. the distance longer than the spicule length and gubernacular apophysis one third of spicule length vs. one fifth to one sixth of spicule length. The new species differs from B. zhangi by longer body length (3.1–4.1 mm vs. 2.2–2.8 mm) and length of gubernacular apophysis shorter than half spicule length vs. length of gubernacular apophysis longer than half spicule length. In addition, the new species differs from B. sinica [4] by a different number of pharyngeal bulbs (eight vs. seven), spicules arcuate and uniform vs. straight with proximal and distal ends tapered and middle part thickened, and gubernaculum with apophysis vs. without apophysis.

The distinction between B. octobulba sp. nov. and other known species is specified in the key below.

Figure 2. Drawing of Belbolla octobulba sp. nov. (A) Pharyngeal region of male, showing nerve ring and eight pharyngeal bulbs; (B) lateral view of male posterior end, showing two precloacal supplements, spicule, gubernaculum, gland cells and tail; (C) anterior end of male, showing buccal cavity with teeth, outer labial and cephalic setae, and cervical setae; (D) lateral view of entire female; (E) spicule and gubernaculum. Scale bar: (A,B) = 100 µm; (C) = 20 µm; (D) = 200 µm; (E) = 50 µm.

Figure 2. Drawing of Belbolla octobulba sp. nov. (A) Pharyngeal region of male, showing nerve ring and eight pharyngeal bulbs; (B) lateral view of male posterior end, showing two precloacal supplements, spicule, gubernaculum, gland cells and tail; (C) anterior end of male, showing buccal cavity with teeth, outer labial and cephalic setae, and cervical setae; (D) lateral view of entire female; (E) spicule and gubernaculum. Scale bar: (A,B) = 100 µm; (C) = 20 µm; (D) = 200 µm; (E) = 50 µm.

Figure 3. Micrograph of Belbolla octobulba sp. nov. (A) Anterior end of male, showing buccal cavity with teeth, outer labial and cephalic setae, and cervical setae; (B) pharyngeal region, showing nerve ring (arrow 1), bulbs and cardia (arrow 2); (C) lateral view of male cloacal region, showing two precloacal supplements, spicules and gubernaculum; (D) lateral view of male posterior end, showing spicule, gubernaculum and tail. Scale bar: (A) = 20 μm; (B–D) = 50 μm.

Figure 3. Micrograph of Belbolla octobulba sp. nov. (A) Anterior end of male, showing buccal cavity with teeth, outer labial and cephalic setae, and cervical setae; (B) pharyngeal region, showing nerve ring (arrow 1), bulbs and cardia (arrow 2); (C) lateral view of male cloacal region, showing two precloacal supplements, spicules and gubernaculum; (D) lateral view of male posterior end, showing spicule, gubernaculum and tail. Scale bar: (A) = 20 μm; (B–D) = 50 μm.

Figure 4. Micrograph of Belbolla octobulba sp. nov. (A,B) Anterior end of female, showing buccal cavity with teeth and cephalic setae; (C) pharyngeal region of female, showing bulbs; (D) lateral view of female middle region, showing vulva (arrow 1) and ovary (arrows); (E) lateral view of female posterior end, showing tail and anus. Scale bar: (A,B) = 20 μm; (C–E) = 50 μm.

Figure 4. Micrograph of Belbolla octobulba sp. nov. (A,B) Anterior end of female, showing buccal cavity with teeth and cephalic setae; (C) pharyngeal region of female, showing bulbs; (D) lateral view of female middle region, showing vulva (arrow 1) and ovary (arrows); (E) lateral view of female posterior end, showing tail and anus. Scale bar: (A,B) = 20 μm; (C–E) = 50 μm.

3.1.7. Updated Key to Species of the Genus Belbolla (Based on Huang and Zhang, 2005) [10]

• Absence of precloacal supplements…B. asupplementata (Juario, 1974) Andrássy, 1973 [8,31]

  -

  Presence of precloacal supplements …2

• Pharynx with four or seven bulbs…3

  -

  Pharynx with eight, nine or ten bulbs …7

• Four pharyngeal bulbs; male with two papilliform and two winged precloacal supple ments…B. vietnamica Gagarin & Nguyen Dinh Tu, 2016 [32]

  -

  Seven pharyngeal bulbs; male with two precloacal supplements…4

• Precloacal supplements winged; gubernacular apophysis absent …5

  -

  Precloacal supplements not winged; gubernacular apophysis present…6

• Spicules slender with handle-shaped proximal end, gubernaculum absent…B. heptabulba (Timm, 1961) Andrássy, 1973 [8,33]

  -

  Spicules broad with tapered proximal end, gubernaculum present…B. sinica Wang, Guo & Wang, 2022 [4]

• Tail conico-cylindrical, spicules broad, 1.3–1.5 abd…B. warwicki Huang & Zhang, 2005 [10]

  -

  Tail long, filiform, spicules slender, 2.5 abd…B. gracilis Gagarin & Thanh, 2016 [34]

• Pharynx with eight bulbs…8

  -

  Pharynx with nine or ten bulbs…15

• Spicules 10.8 abd long with knob-like proximal end; five papilliform supplements…B. longispiculata Nasira, Shahina & Shamim, 2014 [28]

  -

  Spicules shorter than 2 abd; two precloacal supplements…9

• Gubernacular apophysis longer than half spicule length …10

  -

  Gubernacular apophysis shorter than half spicule length …11

• Precloacal supplements 1.4–1.5 abd long…B. tenuidens (Cobb, 1920) Andrássy, 1973 [5,8]

  -

  Precloacal supplements 0.5–0.9 abd long, eight or nine pharyngeal bulbs…B. zhangi Guo & Warwick, 2001 [11]

• Spicule with swollen distal tip …B. stenocephalum Huang & Zhang, 2005 [10]

  -

  Spicule with tapered distal tip …12

• Distance between posterior supplement and proximal end of spicule equal or shorter than spicule length; gubernacular apophysis about one third of spicule length…13

  -

  Distance between posterior supplement and proximal end of spicule longer than spicule length; gubernacular apophysis one fifth to one sixth of spicule length…14

• Body length 1.9 mm, spicules 39 µm, tail 5.7 abd with half posterior cylindrical part…B. teissieri (Luc. et de Conink, 1959) Andrássy, 1973 [8,29]

  -

  Body length 3.2 mm, spicules 115 µm, tail 4.3 abd with one-fourth posterior cylindrical part…B. octobulba sp. nov.

• The wings of supplement almost equal to the diameter of its central part…B. sundoensis (Micoletzky, 1930) Andrássy, 1973 [8,30]

  -

  The wings of supplement twice the diameter of its central part …B. gallanachmorae (Inglis, 1961) Andrássy, 1973 [8,27]

• Spicule with a hook at the distal end…B. intarma Belogurov & Belogurova, 1980 [35]

  -

  Spicule without hook at the distal end…16

• Gubernacular apophysis shorter than 25 µm…17

  -

  Gubernacular apophysis longer than 40 µm…18

• Female having ten pharyngeal bulbs…B. californica (Allgén, 1951) Andrássy, 1973 [8,36]

  -

  Female having nine pharyngeal bulbs…B. wonkimi Rho, Lee, Lee & Min, 2020 [37]

• Male having ten pharyngeal bulbs, a = 19.8…B. insula Belogurov, Fadeeva & Belogurova, 1983 [38]

  -

  Male having nine pharyngeal bulbs, a = 34.5–41.5…B. huanghaiensis Huang & Zhang, 2005 [10]

3.2. Description of Ironella gracilis sp. nov. (Figure 5, Figure 6 and Figure 7)

3.2.1. Type Material

Four males and two females were acquired. Holotype male is on slide JM24(4-8). Paratypes male 2, male 3, female 1 and female 2 are successively on slides JM252(4-8), JM149(4-8), JM6(4-8) and JM665(4-8).

3.2.2. Etymology

The species is named after its relatively thin and slender body.

3.2.3. Type Locality and Habitat

Holotype and paratypes were all collected from intertidal fine sandy sediments at the Jimo coast of Qingdao along the Yellow Sea (120°41′31″ E, 36°22′32″ N) (Figure 1 Red 10).

3.2.4. Measurements

All measurement data are given in

Table 2. Individual measurements of Ironella gracilis sp. nov. (in µm).

Characters	Holotype	Paratypes
		♂1	♂2	♀1	♀2
Total body length	1469	1357	1244	1623	1513
Maximum body diameter	19	18	19	27	26
Head diameter	14	14	14	18	18
Length of inner labial setae	3	3	3	2.5	2.5
Length of outer labial setae	9	9	9	7	8
Length of cephalic setae	9	9	10	9	9
Distance between outer labial setae and cephalic seta	7	8	6	6	6
Depth of buccal cavity	26	29	26	34	33
Width of buccal cavity	4	5	4	7	7
Nerve ring from anterior end	106	103	93	113	117
Length of pharynx	316	304	284	326	329
Body diameter at pharyngeal base	17	17	19	25	25
Length of cardia	7	7	7	10	10
Spicule length along arc	61	57	57	-	-
Length of gubernaculum	7	7	7	-	-
Vulva from anterior end	-	-	-	825	797
V%	-	-	-	50.8	52.7
Body diameter at cloaca or anus	16	15	15	17	16
Tail length	99	96	85	110	122
a	77.3	75.4	65.5	60.1	58.2
b	4.6	4.5	4.4	5	4.6
c	14.8	14.1	14.6	14.8	12.4
c′	6.2	6.4	5.7	6.5	7.6

.

3.2.5. Description

Males. Body slender. Cuticle smooth. No somatic setae. Anterior end slightly widened. Buccal cavity large, divided into conical vestibulum and cylindrical posterior portion, 26–29 µm in depth and 4–5 µm in width (Figure 5A and Figure 6A). Three sclerotized teeth situated at the vestibulum. Anterior sensilla in three circles. Six inner labial setae 3 µm long; all the outer labial setae and cephalic setae 9 µm long (i.e., 64% head diameter). Both circles of outer labial setae and cephalic setae 10 µm apart. Amphid not seen. Pharynx expanded around buccal cavity and forming an anterior bulb, then narrow after the buccal cavity, cylindrical and slightly swelling at its base, not forming posterior bulb. Cardia small, conical. Excretory pore situated at the level of outer labial setae. Tail clavate, 5.7–6.4 cloacal body diameters in length, with half proximal conical and half distal cylindrical. Tail tip slightly swelling without terminal seta. Caudal glands precloacal.

Testis difficult to identify. Spicules 57–61 µm long (about 3.8 cloacal diameters), weakly arched with central strip, proximal end cephalate and distal end tapered (Figure 6C). Gubernaculum conical, 7 µm long, surrounding spicules distally, without apophyses. A tubular precloacal supplement 8 µm long, 9 µm in front of cloaca, with a bended seta at its anterior end, 8 µm in length (Figure 6D).

Females closely resemble males in most characteristics, except the body and the buccal cavity slightly large. Reproductive system didelphic, two ovaries reflexed and opposed; the anterior ovary somewhat shorter than the posterior one. Both anterior and posterior ovaries located to the left side of the intestine. Vulva raised, located in the mid-body (Figure 7C).

3.2.6. Differential Diagnosis and Discussion

Ironella gracilis sp. nov. is characterized by body slender, buccal cavity divided into conical vestibulum and cylindrical posterior portion with three sclerotized teeth in the vestibulum, both the circles of outer labial setae and cephalic setae 10 µm apart, outer labial setae and cephalic setae 9 µm long, spicules elongated, weakly arched, with central strip and cephalate proximally, gubernaculum conical without apophysis, a tubular precloacal supplement just in front of cloaca and with a long thick seta at its front end.

I. gracilis sp. nov. is most similar to I. riemanni [14] in body size. However, it differs from the latter species by body slender (demanian value a = 65.5–77.3 vs. 34.7–50.7 in males and 58.2–60.1 vs. 32.5–35 in females), vestibulum separated and with sclerotized walls (vs. vestibulum not separated and not sclerotized), tubular precloacal supplement large and close to cloaca (vs. small and far from cloaca, 20 µm in front of cloaca). The new species is similar to I. cobbi [13] in having vestibulum separated and with sclerotized walls, but differs from I. cobbi by slender body (a = 58.2–60.1 vs. 34 in females), both circles of outer labial setae and cephalic setae far apart (distance between the two circles of setae equal to l/3 of the corresponding diameter vs. l/6 of the corresponding diameter in the latter species). From the identification key below, the distinctions between I. gracilis sp. nov. and its congeners can be deduced.

3.2.7. Identification Key to Species of Ironella (Updated from Platonova & Mokievsky [14])

• Lips twisted at anterior ends into horizontal tubes, microodontia present…I. prismato laima Cobb, 1920 [5]

  -

  Lips not twisted, microodontia absent…2

• Distance between the circles of outer labial setae and cephalic setae equal to l/6 cbd… I. cobbi Timm, 1954 [13]

  -

  Distance between the circles of cephalic setae equal to 1/3 cbd…3

• Vestibulum separated and with sclerotized walls…I. gracilis sp. nov.

  -

  Vestibulum not separated and not sclerotized…. I. riemanni Platonova & Mokievsky, 1994 [14]

Figure 5. Drawing of Ironella gracilis sp. nov. (A) Anterior end of male, showing inner labial setae (1), outer labial setae (2), cephalic setae (3), buccal cavity, teeth and anterior bulb (arrow); (B) entire female; (C) entire male; (D) cloacal region of male, showing spicule, gubernaculum (arrow) and precloacal supplement; (E) posterior end of male, showing tail. Scale bar: (A,D,E) = 20 μm; (B,C) = 50 μm.

Figure 5. Drawing of Ironella gracilis sp. nov. (A) Anterior end of male, showing inner labial setae (1), outer labial setae (2), cephalic setae (3), buccal cavity, teeth and anterior bulb (arrow); (B) entire female; (C) entire male; (D) cloacal region of male, showing spicule, gubernaculum (arrow) and precloacal supplement; (E) posterior end of male, showing tail. Scale bar: (A,D,E) = 20 μm; (B,C) = 50 μm.

Figure 6. Microscopic images of Ironella gracilis sp. nov. (A) Anterior end of male, showing outer labial setae (arrow 1), cephalic setae (arrow 2) and buccal cavity; (B) anterior end of male, showing buccal cavity teeth; (C) lateral view of male posterior end, showing spicules and tail (broken); (D) lateral view of male posterior end, showing spicules, gubernaculum (arrow 1) and precloacal supplement with seta (arrow 2). Scale bar: (A–D) = 10 μm.

Figure 6. Microscopic images of Ironella gracilis sp. nov. (A) Anterior end of male, showing outer labial setae (arrow 1), cephalic setae (arrow 2) and buccal cavity; (B) anterior end of male, showing buccal cavity teeth; (C) lateral view of male posterior end, showing spicules and tail (broken); (D) lateral view of male posterior end, showing spicules, gubernaculum (arrow 1) and precloacal supplement with seta (arrow 2). Scale bar: (A–D) = 10 μm.

Figure 7. Microscopic images of Ironella gracilis sp. nov. (A) Anterior end of female, showing buccal cavity and anterior setae; (B) anterior end of female, showing buccal cavity, tooth and anterior bulb; (C) lateral view of female middle region, showing vulva (arrow), ovary and eggs; (D) posterior end of female, showing anus (arrow) and tail. Scale bars: (A,B) = 10 μm; (C) = 30 μm; (D) = 20 μm.

Figure 7. Microscopic images of Ironella gracilis sp. nov. (A) Anterior end of female, showing buccal cavity and anterior setae; (B) anterior end of female, showing buccal cavity, tooth and anterior bulb; (C) lateral view of female middle region, showing vulva (arrow), ovary and eggs; (D) posterior end of female, showing anus (arrow) and tail. Scale bars: (A,B) = 10 μm; (C) = 30 μm; (D) = 20 μm.

3.3. Description of Oxystomina longiseta sp. nov. (Figure 8, Figure 9 and Figure 10)

3.3.1. Type Material

Three males and three females were acquired. Holotype male, paratype 1 (male) and paratype 3 (female) are on slide 22WHRLW2-8-12; paratype 2 (male) and paratype 4 (female) are on slide 22WHRLW2-8-13; and paratype 5 (female) is on slide 22WHRLW2-8-11.

Figure 8. Drawing of Oxystomina longiseta sp. nov. (A) Anterior end of male, showing outer labial setae, cephalic setae, amphid (arrow 1) and epidermal gland cells (arrow 2); (B) anterior end of female, (C) entire male; (D) posterior end of male, showing short and long precloacal seta, spicule and epidermal gland cells (arrow); (E) magnifying spicule and gubernaculum; (F) entire female. Scale bar: (A,B,E) = 10 μm; (C,F) = 50 μm; (D) = 20 μm.

Figure 8. Drawing of Oxystomina longiseta sp. nov. (A) Anterior end of male, showing outer labial setae, cephalic setae, amphid (arrow 1) and epidermal gland cells (arrow 2); (B) anterior end of female, (C) entire male; (D) posterior end of male, showing short and long precloacal seta, spicule and epidermal gland cells (arrow); (E) magnifying spicule and gubernaculum; (F) entire female. Scale bar: (A,B,E) = 10 μm; (C,F) = 50 μm; (D) = 20 μm.

Figure 9. Micrograph of Oxystomina longiseta sp. nov. (A–C) Lateral view of male anterior end, showing outer labial setae (1), cephalic setae (2) and amphid; (D) lateral view of male posterior end, showing long precloacal seta, spicule, gubernaculum (arrow 1) and tail; (E) lateral view of male cloacal region, showing spicules, longer (arrow 1) and shorter (arrow 2) precloacal setae; (F) magnifying spicule. Scale bar: (A–F) = 10 μm.

Figure 9. Micrograph of Oxystomina longiseta sp. nov. (A–C) Lateral view of male anterior end, showing outer labial setae (1), cephalic setae (2) and amphid; (D) lateral view of male posterior end, showing long precloacal seta, spicule, gubernaculum (arrow 1) and tail; (E) lateral view of male cloacal region, showing spicules, longer (arrow 1) and shorter (arrow 2) precloacal setae; (F) magnifying spicule. Scale bar: (A–F) = 10 μm.

Figure 10. Micrograph of Oxystomina longiseta sp. nov. (A) Lateral view of female anterior end, showing outer labial setae, cephalic setae and amphid (arrow); (B) lateral view of female middle region, showing vulva and vagina (arrow), ovary (arrow 1) and degraded anterior uterus (arrow 2). Scale bar: (A) = 10 μm; (B) = 20 μm.

Figure 10. Micrograph of Oxystomina longiseta sp. nov. (A) Lateral view of female anterior end, showing outer labial setae, cephalic setae and amphid (arrow); (B) lateral view of female middle region, showing vulva and vagina (arrow), ovary (arrow 1) and degraded anterior uterus (arrow 2). Scale bar: (A) = 10 μm; (B) = 20 μm.

3.3.2. Etymology

The species epithet is derived from the Latin words longus (long) and seta (seta), referring to the new species possessing very long precloacal seta.

3.3.3. Type Locality and Habitat

Holotype and paratypes were all collected from intertidal fine sandy sediments at the Riluowan Bay of Weihai (37°31′11′′ N, 121°1′8′′ E) (Figure 1 Blue 7).

3.3.4. Measurements

All measurement data are given in

Table 3. Individual measurements of Oxystomina longiseta sp. nov. (in µm).

Characters	Holotype	Paratypes
		♂1	♂1	♀1	♀2	♀3
Total body length	2387	2221	2205	2019	2349	2540
Maximum body diameter	20	21	21	19	23	23
Head diameter	4	4	4	4	4	4
Length of outer labial setae	13	16	17	13	14	14
Length of cephalic setae	14	15	14	14	13	13
Distance between two circles of setae	11	11	11	10	11	11
Length of amphidial fovea	6	6	8	8	8	9
Width of amphidial fovea	5	4	4	4.5	4	4
Amphid from anterior end	30	29	32	38	32	31
Body diameter at amphid level	9	8	8	9	9	8
Length of pharynx	486	532	460	476	503	562
Body diameter at pharyngeal base	19	19	20	18	20	19
Spicule length along arc	26	27	26	-	-	-
Length of precloacal setae	35/8	41/11	38/8	-	-	-
From precloacal setae to cloaca	19	18	18	-	-	-
Body diameter at cloaca or anus	19	19	18	18	18	17
Tail length	90	86	85	80	85	82
Vulva from anterior end	-	-	-	1019	1200	1305
Body diameter at vulva	-	-	-	20	24	24
V%	-	-	-	50.5	51.1	51.4
a	119	106	105	106	102	111
b	4.9	4.2	4.8	4.2	4.7	4.5
c	26.5	25.8	25.9	25.2	27.6	31.0
c′	4.7	4.5	4.7	4.4	4.7	4.8

.

3.3.5. Description

Males. Body slender, elongated and very thin at the anterior end. Cuticle smooth. Epidermal gland cells scattering throughout the body. Inner labial sensilla papilliform and difficult to observe. Six outer labial setae 13–17 μm long. Four cephalic setae 14–15 μm long, located at 11 μm (about three times of head diameter) from front end of body. Amphidial fovea longitudinal oval (Figure 8A,B and Figure 9A–C), 5–6 μm long and 4–5 μm wide, located at 29–31 μm from front end of body. Buccal cavity minute. Pharynx slender with an expanded base. Nerve ring difficult to distinguish. No excretory pore and ventral cell observed. Cardia conical. Tail conico-cylindrical with a clavate distal part, 4.5–4.7 cloacal body diameters long, without terminal setae. Caudal glands extend into the precloacal region.

Reproductive system monorchic with an anterior outstretched testis. Spicules arcuate, 26–27 μm long, enlarged proximal end with a median rib, distal end tapered. Gubernaculum small, oblong, 4 μm long. One short and one long precloacal supplementary setae in front of cloaca; the anterior one thin and short, 8–11 μm long; the posterior one thick and very long, 35–41 μm long; located at the level of proximal end of spicule, or 18–19 μm in front of cloaca opening.

Females. Similar to males in most respects, but the body slightly thick. Reproductive system monodelphic, single posterior reflexed ovary. Vulva raised, situated at mid-body. Vagina straight, trumpeted, encircled by sphincter muscle. Uterus a wide and short tube. The degraded anterior uterus present (Figure 10B).

3.3.6. Differential Diagnosis and Discussion

Oxystomina longiseta sp. nov. can be easily identified by longer outer labial and cephalic setae within the genus; spicules with enlarged proximal end and a median rib; gubernaculum small and oblong; two unequal precloacal supplementary setae, anterior one thin and short, posterior one thick and very long.

The new species resembles O. alpatovi [19,39], O. affinis [40] and O. elongate [16,18] in having two unequal precloacal setae, but it differs from O. alpatovi by longer anterior sensory setae (longer than three head diameters vs. shorter than one head diameter) and much longer precloacal setae (the longer one 35–41 μm vs. 18 μm long), proximal end of spicule without median rib in the latter species. The new species differs from O. affinis in having very slender body with shorter tail (a = 102–111 vs. 56 in the latter species; c = 25.2–31 vs. 14); the latter species possessing very short anterior sensory setae and precloacal setae and proximal end of spicules not enlarged. The new species distinguishes from O. elongate by very slender body (a = 102.1–119.4 vs. 47–56), longer outer labial and cephalic setae (vs. 2.5 μm long), and much longer precloacal setae (the longer one 35–41 μm vs. about 10 μm long); spicule enlarged proximally with a median rib and tapered distally (vs. spicule with a slight bump ventrally at the proximal tip without median rib and rounded distally), gubernaculum oblong (vs. curved enclosing spicule tips). Relatively long anterior sensory setae and very long precloacal seta can be used to distinguish the new species from its similar congeners.

4. Conclusions and Discussion

Three new species, Belbolla octobulba sp. nov., Ironella gracilis sp. nov. and Oxystomina longiseta sp. nov., were discovered in the intertidal zone along the Shandong peninsula coast of the Yellow Sea. The genus Belbolla is common in the Yellow Sea, and five species within the genus were found, namely, B. huanghaiensis [10]; B. sinica [4]; B. stenocephalum [11]; B. warwicki [10]; and B. zhangi [11]. B. octobulba sp. nov. is the second new species after B. sinica found in the intertidal zone of the Yellow Sea. The new species can be easily identified from its congeners by eight pharyngeal bulbs, arcuate spicules broad and even, gubernacular apophyses one third of the length of spicule and two winged precloacal supplements. Even though only one male specimen was found for the identification, it was still considered as a new species due to its distinctive characteristic. Ironella is a rare genus, containing only three known species worldwide. Ironella gracilis sp. nov. is the first species found in the Chinese sea area. Oxystomina is a common genus in the Yellow Sea. The specimens of O. elegans [23] and O. elongata [16,18] were often found. O. longiseta sp. nov. is the first new species of the genus found in the Yellow Sea.