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

<jats:p>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.</jats:p>

section_content: 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 km 2, 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.
section_name: Introduction

section_content: 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.
section_name: Materials and Methods

section_content: 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 spe cies, three species belonging to the order Enoplida have been identified as new specie and described here as Belbolla octobulba sp. nov., Ironella gracilis sp. nov. and Oxystomina longiseta sp. nov.
section_name: Results

section_name: Description of Belbolla octobulba sp. nov. (Figures 2-4)

section_content: 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.
section_name: Type Material

section_content: The species epithet comes from the Latin words octo (eight) and bulba (bulbous), re ferring to the new species possessing eight pharyngeal bulbs.
section_name: Etymology

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

section_content: All measurement data are given in Table 1.
section_name: Measurements

section_content: 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. 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.
section_name: Results

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

section_content: 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).
section_name: Type Locality and Habitat

section_content: All measurement data are given in Table 1. 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 (Figures 2C and 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 (Figures 3B and 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.
section_name: Measurements

section_content: 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. [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. 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).
section_name: Differential Diagnosis and Discussion

section_content: The species is named after its relatively thin and slender body.
section_name: Etymology

section_content: 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).
section_name: Type Locality and Habitat

section_content: All measurement data are given in Table 2. 5A and 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 ).
section_name: Measurements

section_content: 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.
section_name: Differential Diagnosis and Discussion

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

section_content: 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).
section_name: Type Locality and Habitat

section_content: All measurement data are given in Table 3.
section_name: Measurements

section_content: 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).
section_name: Type Locality and Habitat

section_content: All measurement data are given in Table 3. 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 ).
section_name: Measurements

section_content: 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.
section_name: Differential Diagnosis and Discussion

section_content: 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.
section_name: Conclusions and Discussion

section_name: Institutional Review Board Statement: Not applicable.

section_content: Funding: This research was funded by the National Natural Science Foundation of China, grant number 41676146, and the Natural Science Foundation of Shandong Province, grant number ZR202103040101.

section_content: Informed Consent Statement: Not applicable.
section_name: Institutional Review Board Statement: Not applicable.

section_name: Acknowledgments:

section_content: Data Availability Statement: Data are contained within the article.

section_content: The authors thank Mengdi Chu and Ziwei Wang for their kind help in the sampling and are sincerely grateful to three anonymous referees for reviewing and improving on the manuscript.
section_name: Acknowledgments:

section_name: Conflicts of Interest:

section_content: The authors declare no conflict of interest.
section_name: Conflicts of Interest: