A New Species of Free-Living Nematode (Enoplida: Enchelidiidae) from the Mangrove Wetlands of China

: A new species, Belbolla mangrove sp. nov., isolated from the mangrove wetlands of Fujian and Zhejiang Provinces in China, is described and illustrated. Belbolla mangrove sp. nov. is characterized by a pharynx with four bulbs, a small gubernaculum with a short dorsocaudal apophysis, four weakly developed precloacal supplements, a conico-cylindrical tail with a terminal spinneret, and the absence of terminal setae. This new species differs from B. vietnamica by the absence of ocelli and the blunt and rounded proximal ends of the spicules. The 18S rDNA GenBank accession numbers of B. mangrove sp. nov. are provided.


Introduction
Free-living marine nematodes are the most abundant meiofaunal animals in the marine environment. These organisms have short life cycles, and their distribution is closely related to the benthic environment. They are sensitive to environmental changes and pollution, particularly at small spatial and short time scales. Therefore, they have been considered one of the best indicator species of the marine environment [1]. However, there are still obstacles and challenges to their use as bioindicators on a global scale, mainly due to the limited available information, particularly regarding their identification. The taxonomic study of marine nematodes is an important basic work in current biodiversity research; however, a significant portion of marine nematode species remains unidentified.
Free-living marine nematodes are key players in coastal wetlands, especially in bioturbation [2]. The genus Belbolla was renamed by Andrássy to replace the previously used name Bolbella Cobb, 1920, because Bolbella was a homonym of Bolbella Giglio-Toss, 1915 (Orthoptera-Mantidae) [3]. The common characteristic features of the genus Belbolla include the presence or absence of ocelli, a large and narrow buccal cavity divided into two parts, the presence of three teeth, with the largest tooth being the ventro-sublateral one, and the presence of a series of bulb-like muscular pharynx swellings (ranging from 4 to 10) in the posterior section of the pharynx [4]. So far, 20 valid species of the genus Belbolla have been recorded from 11 countries, including Germany, the USA, Britain, Vietnam, Bangladesh, China, Korea, Russia, Pakistan, Indonesia, and France [5,6]. The main distribution habitat of the genus is marine, and five species have been previously described in China. One species, B. zhangi Guo & Warwick, 2001 is from the Bohia Sea [7], while the other four species, namely B. huanghaiensis Huang and Zhang, 2005, B. stenocephalum Huang and Zhang, 2005, B. warwicki Huang and Zhang, 2005, and B. sinica Guo and Wang, 2022, are from the Yellow Sea [5]. On the other hand, only three species have been reported worldwide in estuarine and mangrove habitats. These include B. longispiculata Nasira, Shahina and Shamim, 2014 from the Indus River Estuary in Sindh, Pakistan; B. gracilis Gagarin and Thanh, 2016 from the Yen River Estuary mangrove in Vietnam; and B. vietnamica Gagarin and Nguyen Dinh Tu, 2016 from the Yen River Estuary mangrove in Vietnam. However, the genus Belbolla has not been studied and reported from mangrove wetlands in China. The classification and identification of Belbolla species are poorly known, and molecular identification based on DNA sequencing of the genus Belbolla has never been reported.
In China, the northernmost boundary of the natural distribution of mangrove wetlands is Fuding City in the Fujian Province, while the northern boundary of artificial introduction is Yueqing City in Zhejiang Province [8]. In recent years, we have investigated the biodiversity and community structure of free-living marine nematodes in mangrove wetlands in China, aiming to understand their spatial and temporal variation patterns and provide references for environmental monitoring. More than 50 new species of free-living nematodes have been discovered. Among them, a new species of the genus Belbolla, which has never been reported before, was discovered for the first time in the mangrove wetlands at the mouth of the Jiulong River and Ximen Island. The present study aims to provide a comprehensive description of this new species of Belbolla, enhancing our knowledge of the biodiversity of free-living nematodes in mangrove wetlands. Additionally, we report the 18S rDNA sequence of the new species B. mangrove sp. nov., which is essential to fill the DNA sequence gap of the genus Belbolla.

Sample Collection
Undisturbed sediment samples were taken from the Ximen Island mangrove wetland in Yueqin City, Zhejiang Province (28.34 • N, 121.17 • E) and the Jiulong Estuary mangrove wetland in Zhangzhou City (23.24 • N, 117.55 • E) of Fujian Province, China in December 2018 and May 2021, respectively. The samples were collected by inserting a syringe (2.9 cm inner diameter) into the sediment to a depth of 5 cm. The collected samples were fixed with 5% formalin and transported back to the laboratory. They were then stained with 0.1% rose Bengal for over 24 h [4]. A portion of the fresh samples was stored in a refrigerator at −20 • C for DNA extraction.

Meiofauna Extraction and Nematode Identification
The samples were rinsed with tap water to eliminate any remaining formalin and then filter through two sieves (with mesh size of 500 and 42 µm, respectively) until the water passing through the sieve appeared clean. Meiofauna was extracted using the flotation method (using Ludox™ (Sigma-Aldrich, Merck, Rahway, NJ, USA) with a specific gravity of 1.15) as described by Jonge et al. [9]. Afterwards, it was stained with 1% Rose Bengal for over 48 h to aid the detection of animals during sorting [10]. The meiofauna was sorted using a stereoscopic microscope (Nikon SMZ800 (Nikon, Tokyo, Japan). The nematodes were transferred to a cavity block filled with a solution consisting of a mixture of glycerol, pure ethanol, and distilled water at a ratio of 1:1:18. This allowed for the gradual evaporation of ethanol and some of the water before mounting specimens on slides [11]. Drawings were made with the aid of a camera lucida attached to the Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). Type specimens were deposited at Jimei University in Xiamen, China. Permanent slides were prepared as described by Shih et al. [12]. The holotype and paratypes were also deposited at Jimei University.

DNA Extraction and PCR Amplification
Total genomic DNA extraction was performed on three individuals of each newly discovered species of Belbolla mentioned in the previous section. The TIANamp Marine Animals DNA Kit Dp324-03 (TIANGEN BIOTECH, CO., LTD., Beijing, China) was used for this purpose, following the manufacturer's instructions provided in the manual, with minor adjustments made to the volume of buffer solution used at each step. The volumes were modified as follows: 20 µL of buffer GA; 5 µL of proteinase K (20 mg/mL); 20 µL of buffer GB; 20 µL of 99% ethanol; 50 µL of buffer GD; 60 µL of buffer PW; and 30 µL of buffer TE. The genomic DNA was stored in a −20 • C refrigerator for further use. The primers utilized for the 18S rDNA were as follows: MN18F: 5 -CGCGAATRGCTCATTACAACAGC-3 and Nem_18S_R: 5 -GGGCGGTATCTGATCGCC-3 [13,14]. The volumes for each reaction were set at 25 µL, comprising 6 µL of DNA template, 12.5 µL of 2X Pro Taq Master Mix, 0.5 µL for each forward and reverse primer, and 5.5 µL of double distillation H 2 O (ddH 2 O). The thermal cycling conditions for PCR consisted of an initial denaturation step at 94 • C for 2 min, followed by 40 cycles of 94 • C for 35 s, 50 • C to 55 • C for 35 s, and 72 • C for 60 s. The final extension step was performed at 72 • C for 10 min. Following the PCR assay, 5 µL of each PCR product was subjected to electrophoresis on a 1% agarose gel. The gel was then strained with 0.5 µg/mL ethidium bromide and visualized using an ultraviolet trans-illumination system. All positive PCR amplicons were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).

Sequencing and Analyses
A total of three sequences were obtained from individual nematodes of H. mangrove sp. nov. in this study. All sequences were deposited in GenBank ( Table 1). The 18S rDNA sequences of Enchelidiidae were retrieved from the GenBank and the sequences were selected for phylogenetic analysis. Oncholaimus sp. was used as an outgroup in the 18S rDNA sequence analysis. Sequences were aligned in the BioEdit version 7.0.9.0 sequence editor. A neighbor-joining (NJ) tree was constructed in MEGA 7 using the gamma-corrected Kimura 2-parameter distance method. The NJ tree was subsequently validated with bootstrap analysis using 1000 replicates [13]. Moreover, intergeneric thresholds based on gene sequences was calculated using Kimura 2-parameter distances (K2P) via MEGA 7.0 software. The parameter model was reconstructed with 1000 bootstrap replicates [12].

Terminology and Abbreviations
The following measurements were conducted on nematodes, all in µm, with the following abbreviations: a, body length divided by maximum body diameter; b, body length divided by pharynx length; c, body length divided by tail length; c', tail length divided by anal body diameter; a.b.d., anal body diameter; c.b.d., corresponding body diameter; v.b.d., vulval body diameter; V, vulva distance from the anterior body end; V%, position of vulva as a percentage of body length from the anterior body end [4].

Terminology and Abbreviations
The following measurements were conducted on nematodes, all in µm, with the following abbreviations: a, body length divided by maximum body diameter; b, body length divided by pharynx length; c, body length divided by tail length; c', tail length divided by anal body diameter; a.b.d., anal body diameter; c.b.d., corresponding body diameter; v.b.d., vulval body diameter; V, vulva distance from the anterior body end; V%, position of vulva as a percentage of body length from the anterior body end [4].

Description of Belbolla mangrove sp. nov.
Class Enoplea Inglis, 1983Order Enoplida Filipjev, 1929Family Enchelidiidae Filipjev, 1918 Genus Belbolla Andrássy, 1973 Belbolla mangrove sp. nov. (Figures 1 and 2; Tables 2 and 3)   A new species of the genus Belbolla was discovered in the mangrove sediments of China, adding to the existing 20 valid species documented in the literature (summarized in Table 2). A new species of the genus Belbolla was discovered in the mangrove sediments of China, adding to the existing 20 valid species documented in the literature (summarized in Table 2).

Type Locality and Habitat
Intertidal muddy sediment was collected from the mangrove wetlands of Ximen Island, (28.34 • N, 121.17 • E). Specimens were found in the surface layer of the sediment at a depth of 0-5 cm.

Etymology
The species was named after the mangrove habitat.

Measurements
The measurements of the new species are summarized in Table 3.

Morphological Description
The body is slender and cylindrical, tapering towards both ends, with a length ranging from 1454.0-1825.0 µm and a maximum body diameter ranging from 52.0-77.0 µm, approximately 4.9-7.7 times the width of the cephalic region, which is located around the level of posterior esophagus end. The cuticle is smooth without lateral differentiation. The head diameter is 9.2-10.6 µm and distinctly narrowed. The anterior part of the head is slightly separated through a constriction at the level of cephalic setae. There are six inner labial setae, each approximately 1.0-1.3 µm long. There are six outer labial setae and four cephalic setae that form a circle, with each seta measuring about 4.0-5.7 µm in length. Numerous cervical setae were scattered over the anterior region of the pharynx, reaching lengths of up to 5.0-6.0 µm. The buccal cavity has a thick wall, is large and wide, and is divided into two chambers by a cuticular ring: (i) a spacious anterior chamber, and (ii) a narrower and longer posterior chamber. The total length of the buccal cavity is 9.6-12.5 µm, with a width of 4.2-6.1 µm. There was a large right subventral tooth and two less prominent teeth (dorsal and left subventral). The amphideal fovea was pocket-like and located at the level of the posterior buccal chambers. Ocelli are absent. The pharynx measures 327.0-405.0 µm in length, gradually expanding posteriorly and modified into four pharyngeal bulbs with a corresponding diameter of 52.0-70.0 µm at the end of the pharynx. The nerve ring was located at 46.4-55.7% of the pharynx length, with a corresponding body diameter of 47.0-54.0 µm. The cardia was short and conoid. The tail was conico-cylindrical, tapering with the distal third being cylindrical. In males, the tail length was 5.3-5.8 times the anal body diameter, and in females, it was 5.4-6.3 times the anal body diameter. The tip of the tail was slightly swollen and had a terminal spinneret, without any terminal setae. The caudal glands were poorly visible.
Male: Diorchic, with the anterior testis on the right side of the intestine, while the posterior testis was located at the same position as the vas deferens. The spicules were equal in length, curved with rounded and blunt proximal ends, and tapered distally. They measured 53.0-57.9 µm as an arc (1.4-1.7 times the anal body diameter), and 42.0-45.8 µm as a chord ( Table 3). The gubernaculum was small, measuring 4.7-8.0 µm in length, 1.4-1.7 times the anal body diameter. There was a precloacal seta located close to the cloaca, measuring 2.0-2.3 µm in length. Four precloacal supplements were weakly developed, appearing pore-like on the cuticle and resembling vacuoles internally. The size of the vacuoles increased as they were positioned further away from the anus. The arrangement of the supplements is as follows: The first supplement was located 21.0-22.4 µm from the cloacal opening, the second supplement 50.0-60.5 µm, the third supplement 95.6-106.2 µm, and the fourth, which was the most anterior, was positioned 167.4-180.3 µm from the cloacal opening.
Females: The general appearance was similar to that of the male. The ovaries were paired and reflexed, both located to the right of the intestine. The vulva was transversely positioned at 49.9-57.4% of the body length (Figures 1 and 2, Table 3).

Differential Diagnosis
Belbolla mangrove sp. nov. is characterized by a total body length of 1454.0-1825.0 µm. The cuticle is smooth, and the buccal cavity is divided into two chambers by a cuticular ring. The amphideal fovea is pocket-like, and there are four pharyngeal bulbs. The species possesses a small gubernaculum with a dorsocaudal apophysis and weakly developed precloacal supplements. The tip of the species is slightly swollen and has a terminal spinneret, without any terminal setae. Currently, B. vietnamica is the only known species in the Belbolla genus with four pharyngeal bulbs.
Belbolla mangrove sp. nov. differs from 19 valid species of the genus Belbolla in having four pharyngeal bulbs, the last one (the 20th) also having four bulbs, and it also differs from all other species in the shape of the precloacal supplements. The new species, Belbolla mangrove sp. nov., closely resembles B. vietnamica as they both possess four pharyngeal bulbs. However, the newly discovered species in this study differs from B. vietnamica in five aspects: (1)  The new species shared weakly developed precloacal supplements with B. longispiculata; however, B. longispiculata differs from the new species in its eight pharyngeal bulbs, long spicules, and gubernaculum.

DNA Sequences Results
A total of three sequences of the 18S rDNA gene were obtained from three male individuals of B. mangrove sp. nov. All sequences were deposited in GenBank. The detailed information of individual specimens includes collection date, site, habitat, longitude, latitude, GenBank number, and the length of the amplified fragment (Table 1). Intergeneric thresholds of K2P distance divergence are as follows: 1.6-8.3% for 18S rDNA. Table 4. Comparison of the major morphological features of Belbolla mangrove sp. nov. discovered in this study and other species in the genus Belbolla reported in the literature ("-" not documented in the literature).

Discussion
So far, the genus Belbolla has been found in various localities of intertidal and shallow subtidal habitats worldwide, including the Atlantic Ocean, Indian Ocean, and Pacific Ocean [6]. The genus Belbolla is characterized by multiple pharyngeal bulbs, which serve as an important feature for classification [23]. Belbolla has been subdivided into three groups based on the number of pharyngeal bulbs. The first group is characterized by having seven or eight pharyngeal bulbs in males, and it comprises fifteen species. The second group is characterized by having nine or ten pharyngeal bulbs, and it comprises five species. The third group is characterized by having four pharyngeal bulbs in males, and only includes the species B. vietnamica. Huang et al. [26] provided a binary key for identifying the 12 species of the genus Belbolla based on the number of pharyngeal bulbs, number, shape, and size of precloacal supplements, the length and shape of the spicules, and the presence or absence of the gubernaculum and apophysis. Interestingly, the number of pharyngeal bulbs can be a variable feature within the same species. For B. zhangi, the most anterior bulb sometimes appears unclear, while for B. californica, the number of pharynx bulbs ranges from seven to nine [7,21]. Additionally, the presence or absence of ocelli was also an important feature within this genus. Among the total of 21 species (including the species newly described here), B. heptabulba, B. vietnamica, and B. sundaensis were reported to have ocelli [24,27,28]. One species was explicitly described as lacking ocelli, while no information regarding this feature was available for the remaining species. The major morphological features of the 21 species of the genus Belbolla were summarized in Table 4.
Whether the number of pharyngeal bulbs, the shape of the supplements, or the shape of the spicules and gubernaculum constitutes an important feature for distinguishing different species within the genus Belbolla requires further evidence. Marine nematodes are morphologically diverse, and traditional identification methods are morphologically based and therefore limited, while the development of DNA barcoding technology has provided new technical support for species identification. Currently, molecular markers such as the nuclear small subunit ribosomal RNA (18S rDNA), large subunit ribosomal RNA (28S rRNA), and the mitochondrial cytochrome oxidase subunit I (COI) are commonly used for taxonomic identification of marine nematodes [29]. Taxonomic identification of nematodes using various DNA fragments has been a common approach in the past decades [30]. Previous studies have indicated that 18S rDNA serves as a suitable molecular marker for nematode identification [31][32][33][34]. However, no species of the genus Belbolla has ever been sequenced. The GenBank database was searched, and a total of 27 sequences belonging to 18S rDNA were found in the family Enchelidiidae, which is the taxonomic family level of the genus Belbolla. However, most of the sequences have not been assigned exact species names, indicating that the molecular sequence data in the database have significant limitations in providing taxonomic evidence.
Therefore, in this study to better complement and enhance the database, we first provided 18S rDNA sequences of B. mangrove sp. nov. and compared them with the genera Bathyeurystomina, Calyptronema, Eurystomina, Pareurystomina, and Polygastrophora. Subsequently, other genera were analyzed, and a taxonomic phylogenetic tree was constructed using the sequence data ( Figure 3). The results show that B. mangrove sp. nov. is a separate species and exhibits good differentiation from species of other genera. In addition, due to no other species of the genus Belbolla being sequenced, the reference sequences were limited, and therefore we established intergeneric thresholds of K2P distance divergence as follows: 1.6-8.3% for 18S rDNA.

Conclusions
A new species of the genus Belbolla has been discovered from surface muddy intertidal sediments in two mangrove wetlands of China. This newly discovered species was named B. mangrove sp. nov. in recognition of its association with the mangrove habitat, marking the first report of the genus Belbolla in Chinese mangroves. The taxonomic and distinct morphological features of this species were described and its DNA sequence was obtained to provide fundamental data for molecular identification. The main morphological characteristics of all species within the genus were also summarized to facilitate species identification. These findings contribute to our understanding of the biodiversity of free-living nematodes in mangrove sediments, providing a foundation for further biodiversity research, which is crucial for the conservation of coastal wetlands.

Conclusions
A new species of the genus Belbolla has been discovered from surface muddy intertidal sediments in two mangrove wetlands of China. This newly discovered species was named B. mangrove sp. nov. in recognition of its association with the mangrove habitat, marking the first report of the genus Belbolla in Chinese mangroves. The taxonomic and distinct morphological features of this species were described and its DNA sequence was obtained to provide fundamental data for molecular identification. The main morphological characteristics of all species within the genus were also summarized to facilitate species identification. These findings contribute to our understanding of the biodiversity of free-living nematodes in mangrove sediments, providing a foundation for further biodiversity research, which is crucial for the conservation of coastal wetlands.