A New Species of Free-Living Nematode (Enoplida: Enchelidiidae) from the Mangrove Wetlands of China
by
, ,
Huilan Zhu
1,2,3,
Yuqing Guo
1,4,*,
Haichao Zhou
2,5,
Yi-Jia Shih
1,
Fenglan Li
2,3,6,
Fred Wang Fat Lee
6,
Steven Jingliang Xu
6 and
Nora Fung Yee Tam
3,6,7,* 1
Fisheries College, Jimei University, Xiamen 361021, China
2
Greater Bay Area Coastal Mangrove Wetland Research & Development Centre, Guangdong Neilingding Futian National Nature Reserve, Shenzhen 518040, China
3
Shenzhen Research Institute, City University of Hong Kong, Shenzhen 518060, China
4
Fujian Provincal Key Laboratory of Marine Fishery Resources and Eco-Environment, Jimei University, Xiamen 361021, China
5
College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China
6
School of Science and Technology, Hong Kong Metropolitan University, Hong Kong 999077, China
7
State Key Laboratory of Marine Pollution, City University of Hong Kong, Hong Kong 999077, China
*
Authors to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2023, 11(7), 1412; https://doi.org/10.3390/jmse11071412
Submission received: 15 May 2023
/
Revised: 5 July 2023
/
Accepted: 12 July 2023
/
Published: 14 July 2023
(This article belongs to the Special Issue Coastal Environments: Recent Advances in Conservation and Sustainable Development)
Abstract
: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.
1. 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.
2. Materials and Methods
2.1. 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.
2.2. 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.
2.3. 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 H2O (ddH2O). 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).
2.4. 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].
2.5. 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].
3. Results
3.1. Description of Belbolla mangrove sp. nov.
Class Enoplea Inglis, 1983
Order Enoplida Filipjev, 1929
Family Enchelidiidae Filipjev, 1918
Genus Belbolla Andrássy, 1973
Belbolla mangrove sp. nov.
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).
Table 2.
The valid species of the genus Belbolla reported in literature.
| Valid Species | Habitat |
|---|---|
| B. asupplementata Juario, 1974 | Marine |
| B. californica (Allgén, 1951) Andrássy, 1973 | Marine |
| B. gallanachmorae (Inglis, 1961) Andrássy, 1973 | Marine |
| B. gracilis Gagarin & Thanh, 2016 | Estuary mangrove |
| B. heptabulba (Timm, 1961) Andrássy, 1973 | Marine |
| B. huanghaiensis Huang & Zhang, 2005 | Marine |
| B. hoonsooi Rho, Lee, Lee & Choi, 2021 | Marine |
| B. insula Belogurov, Fadeeva & Belogurova, 1983 | Marine |
| B. intarma Belogurov & Belogurova, 1980 | Marine |
| B. koreensis Rho, Lee, Lee & Choi, 2021 | Marine |
| B. longispiculata Nasira, Shahina & Shamim, 2014 | Estuary |
| B. sinica Wang, Guo & Wang, 2022 | Marine |
| B. stenocephalum Huang & Zhang, 2005 | Marine |
| B. sundaensis (Micoletzky, 1930) Andrássy, 1973 | Marine |
| B. teissieri (Luc & De Coninck, 1959) Andrássy, 1973 | Marine |
| B. tenuidens (Cobb, 1920) Andrassy, 1973 | Marine |
| B. vietnamica Gagarin & Nguyen Dinh Tu, 2016 | Estuary mangrove |
| B. warwicki Huang & Zhang, 2005 | Marine |
| B. wonkimi Rho, Lee, Lee & Min, 2020 | Marine |
| B. zhangi Guo & Warwick, 2001 | Marine |
Table 3.
Measurements of individuals of the new species, Belbolla mangrove sp. nov. discovered in this study (-: not applicable).
Table 3.
Measurements of individuals of the new species, Belbolla mangrove sp. nov. discovered in this study (-: not applicable).
| Characters | Holotype | Paratypes | ||||||
|---|---|---|---|---|---|---|---|---|
| ♂1 | ♂2 | ♂3 | ♂4 | ♀1 | ♀2 | ♀3 | ♀4 | |
| Body length (μm) | 1454 | 1608 | 1482 | 1465 | 1825 | 1649 | 1790 | 1632 |
| Head diameter (μm) | 10.6 | 9.4 | 10.0 | 9.2 | 10.2 | 9.8 | 10.0 | 9.5 |
| Length of cephalic setae (μm) | 4.0–5.4 | 4.8–5.7 | 4.0–5.1 | 4.0–5.2 | 5.0–6.0 | 4.0–4.7 | 4.0–5.1 | 4.0–5.1 |
| Buccal cavity length (μm) | 11.2 | 11.3 | 11.2 | 9.6 | 12.0 | 12.0 | 12.5 | 11.6 |
| Buccal cavity diamtere (μm) | 5.2 | 5.5 | 3.7 | 4.2 | 6.1 | 5.0 | 4.5 | 4.6 |
| Amphids from the anterior end (μm) | 4.7 | 5.7 | 5.6 | 5.9 | 5.1 | 4.0 | 4.3 | 5.9 |
| Amphid c.b.d. (μm) | 11.2 | 10.2 | 9.7 | 12.0 | 11.3 | 9.6 | 10.0 | 9.9 |
| Nerve ring from the anterior end (μm) | 180 | 195 | 177 | 159 | 210 | 190 | 197 | 179 |
| Nerve ring c.b.d. (μm) | 47 | 52 | 52 | 47 | 45 | 50 | 54 | 47 |
| Pharynx length (μm) | 327 | 350 | 352 | 343 | 390 | 386.5 | 405 | 379.5 |
| Pharynx c.b.d. (μm) | 52 | 60 | 54 | 52 | 59 | 60 | 70 | 55 |
| Maximum body diameter (μm) | 52 | 61 | 54 | 55 | 66 | 62 | 77 | 58 |
| a.b.d. (μm) | 34.6 | 37.3 | 36.0 | 33.4 | 34.2 | 34.5 | 39.0 | 33.0 |
| Tail length (μm) | 182 | 207 | 197 | 195 | 190 | 217 | 212 | 206 |
| Spicule length along chord (μm) | 45.8 | 42.0 | 42.6 | 42.7 | - | - | - | - |
| Spicule length long arc (μm) | 57.9 | 54.0 | 53.0 | 53.8 | - | - | - | - |
| Gubernaculum length (μm) | 6.5 | 4.7 | 7.2 | 8.0 | - | - | - | - |
| Apophysis length (μm) | 6.8 | 6.3 | 6 | 5.7 | - | - | - | - |
| Supplements 1 distance from cloacal opening (μm) | 21.5 | 22.4 | 22.0 | 21.0 | - | - | - | - |
| Supplements 2 distance from cloacal opening (μm) | 60.5 | 58.9 | 53.0 | 50.0 | - | - | - | - |
| Supplements 3 distance from cloacal opening (μm) | 106.2 | 104.3 | 103 | 95.6 | - | - | - | - |
| Supplements 4 distance from cloacal opening (μm) | 167.4 | 180.3 | 173 | 168 | - | - | - | - |
| V (μm) | - | - | - | - | 1048 | 899 | 909 | 814 |
| Precloacal seta length (μm) | 2.3 | 2.0 | 2.0 | 2.1 | - | - | - | - |
| v.b.d. (μm) | - | - | - | - | 62.0 | 54.5 | 67.6 | 54.5 |
| V% | - | - | - | - | 57.4 | 54.5 | 50.8 | 49.9 |
| a | 28.0 | 26.4 | 27.4 | 26.6 | 27.7 | 26.6 | 23.2 | 28.1 |
| b | 4.4 | 4.6 | 4.2 | 4.3 | 4.7 | 4.3 | 4.4 | 4.3 |
| c | 8.0 | 7.8 | 7.5 | 7.5 | 9.6 | 7.6 | 8.4 | 7.9 |
| cʹ | 5.3 | 5.5 | 5.5 | 5.8 | 5.6 | 6.3 | 5.4 | 6.2 |
3.1.1. Type Material
Holotype: one male (♂1: WZ2018120212107); paratypes: three males (♂2: WZ2018120211113, ♂3: ZZ20210509114, ♂4: ZZ20210509107) and four females (♀1: WZ2018120212115, ♀2: ZZ20210509112, ♀3: ZZ20210509112, ♀4: ZZ20210509111). Among them, WZ2018120212107 and WZ2018120211113 were obtained from Ximen Island mangrove wetland in Wenzhou City of Zhejiang Province; while ZZ20210509114, ZZ20210509107, ZZ20210509112, ZZ20210509112, and ZZ20210509111 were from Jiulong Estuary mangrove wetland in Zhangzhou City of Fujian Province.
3.1.2. 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.
3.1.3. Etymology
The species was named after the mangrove habitat.
3.1.4. Measurements
The measurements of the new species are summarized in Table 3.
3.1.5. 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.
3.2. 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 presence or absence of ocelli (absence in the new species versus presence in B. vietnamica); (2) the shape of proximal spicule ends (blunt and round in the new species versus strongly bent in B. vietnamica), (3) the shorter length of the gubernacular dorsocaudal apophysis (6.0–6.8 μm in the new species versus 16.0–19.0 μm in B. vietnamica); (4) the development of the precloacal supplements (weakly developed in the new species versus well-developed in B. vietnamica); and (5) the presence or absence of terminal setae (without terminal setae in the new species versus with three terminal setae in B. vietnamica). 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.
3.3. 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).
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).
| Taxa | Number of Pharynx Bulb | Supplements | Spicule Length(μm) | AmphidsFovea | Ocelli | a (μm) | b (μm) | c (μm) | References |
|---|---|---|---|---|---|---|---|---|---|
| B. asupplementata Juario, 1974 | 7 | absent | 38.9 | pocket-like | not described | 42 | 3.7 | 12.3 | [15] |
| B. californica (Allgén, 1951) Andrássy, 1973 | 7-9 | 2 horseshoe-like | - | not described | not described | 20.78 | 3.73 | 14.9 | [16] |
| B. gallanachmorae (Inglis, 1961) Andrássy, 1973 | 8 | 2 winged | 69 | pocket-like | not described | 34.9 | 4 | 12.9 | [17] |
| B. gracilis Gagarin & Thanh, 2016 | 7 | 2 complex structure | 50 | pocket-like | not described | 31 | 9 | 6.7 | [18] |
| B. heptabulba (Timm, 1961) Andrássy, 1973 | 7 | 2 winged | 30-34 | not described | present | 25-29 | 3.4-3.7 | 11.8-14.7 | [19] |
| B. hoonsooi Rho, Lee, Lee, & Choi, 2021 | 8 | 2 winged | 127 | not described | absent | 30 | 5 | 13 | [20] |
| B. huanghaiensis Huang & Zhang, 2005 | 9 | 2 winged | 137 | not described | not described | 38.5 | 4.5 | 14.8 | [21] |
| B. insula Belogurov, Fadeeva, & Belogurova, 1983 | 10 | 2 winged | 182 | pocket-like | - | 19.8 | 3.5 | 13.4 | [22] |
| B. intarma Belogurov & Belogurova, 1980 | 9 | 2 | 144 | - | - | - | - | - | [23] |
| B. koreensis Rho, Lee, Lee, & Choi, 2021 | 8 | 2 winged | 119 | not described | absent | 34.0 | 5.0 | 12.0 | [20] |
| B. longispiculata Nasira, Shahina, & Shamim, 2014 | 8 | 5 papillae | 216 | not described | not described | 32.97 | 4.7 | 12.9 | [24] |
| B. Sinica wang, Guo & Wang, 2022 | 7 | 2 wined | 36 | not observed | absent | 36.0 | 3.9 | 13.9 | [5] |
| B. stenocephalum Huang & Zhang, 2005 | 8 | 2 winged | 98 | not described | not described | 36.3 | 3.7 | 12.3 | [21] |
| B. sundaensis (Micoletzky, 1930) Andrássy, 1973 | 8 | 2 | - | crescent | present | 28 | 4.16 | 13.6 | [25] |
| B. teissieri (Luc & De Coninck, 1959) Andrássy, 1973 | 8 | 3 papillae | 39 | oval | not described | 36.5 | 3.7 | 11.7 | [26] |
| B. tenuidens(Cobb, 1920) Andrassy, 1973 | 8 | 2 winged | - | - | not described | - | - | - | [27] |
| B. vietnamica Gagarin & Nguyen Dinh Tu, 2016 | 4 | 4 papillae | 55 | not described | present | 22 | 3.8 | 7.5 | [28] |
| B. warwicki Huang & Zhang, 2005 | 7 | 2 long pocket-shaped | 37 | not described | not described | 40.8 | 3.4 | 12.7 | [21] |
| B. wonkimi Rho, Lee, Lee, & Min, 2020 | 9 | 2 winged | - | oval | absence | - | 4 | 11 | [6] |
| B. zhangi Guo & Warwick, 2001 | 8-9 | 2 winged | 56 | not obvious | not described | 34.7 | 4.6 | 13.5 | [7] |
| B. mangrove sp. nov. | 4 | 4 | 57.9 | pocket-like | absent | 28 | 4.4 | 8 | This study |
4. 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.
5. 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.
Author Contributions
Conceptualization, data curation, writing—original draft preparation, and writing—review and editing, H.Z. (Huilan Zhu), Y.G., Y.-J.S., F.L., and N.F.Y.T.; methodology, H.Z. (Huilan Zhu); investigation, H.Z. (Huilan Zhu); funding acquisition, Y.G., H.Z. (Haichao Zhou), F.W.F.L., and S.J.X. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the National Science Foundation of China (31772416, 41976161); the Natural Science Foundation of Fujian Province (2022J01324); Science, Technology and Innovation Commission of Shenzhen Municipality (JCYJ20200109140605948), and also supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (UGC/IDS(R)16/19).
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Not applicable.
Acknowledgments
The authors would like to thank Peipei Yang, Rengui Zhou, Chao Pan, and Zhen Xiao for sample collection. We also thank the reviewers for their constructive criticism and improvement of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
- Ridall, A.; Ingels, J. Suitability of free-living marine nematodes as bioindicators: Status and future considerations. Front. Mar. Sci. 2021, 8, 685327. [Google Scholar] [CrossRef]
- Du, Y.F.; Gao, S.; Warwick, R.M.; Hua, E. Ecological functioning of free-living marine nematodes in coastal wetlands: An overview. Chin Sci. Bull. 2014, 59, 4692–4704. [Google Scholar] [CrossRef]
- Andrássy, I. Über vier homonyme nematodengattungen. Nematologica 1973, 19, 403–404. [Google Scholar]
- Platt, H.M.; Warwick, R.M. Free-living Marine Nematodes, Part I, British Enoplids. In Synopses of the British Fauna (New Series); The Linnean Society of London: London, UK, 1998; p. 248. [Google Scholar]
- Wang, M.; Guo, W.; Wang, C. Two new species, Parabathylaimus gracilis sp. nov. and Belbolla sinica sp. nov. (Nematoda: Enoplida), from Yangma Island of the Yellow Sea, China. Zootaxa 2022, 5200, 344–354. [Google Scholar] [CrossRef]
- Rho, H.S.; Lee, H.; Lee, H.J.; Min, W. A new free-living marine nematode species of the genus Belbolla (Enoplida, Enchelidiidae) from a subtidal zone of the East Sea, Korea, with some ecological and biogeographical information. Environ. Biol. Res. 2020, 38, 578–585. [Google Scholar] [CrossRef]
- Guo, Y.Q.; Warwick, R.M. Three new species of free-living nematodes from the Bohai Sea, China. J. Nat. Hist. 2001, 35, 1575–1586. [Google Scholar] [CrossRef]
- Wang, W.Q.; Wang, M. The Mangroves of China; Science Press: Beijing, China, 2007; pp. 68–86. [Google Scholar]
- Jonge, V.N.; Bouwman, L.A. A simple density separation technique for quantitative isolation of meiobenthos using the colloidal silica Ludox-TM. Mar. Biol. 1977, 42, 143–148. [Google Scholar] [CrossRef]
- Somerfield, P.J.; Warwick, R.M.; Moens, T. Chapter 6: Meiofauna Techniques. Methods for the Study of Marine Benthos.; Blackwell Scientific Publications: Oxford, UK, 2005; pp. 269–272. [Google Scholar]
- McIntyre, A.D.; Warwick, R.M. Meiofauna Techniques. Methods for the Study of Marine Benthos; Blackwell Scientific Publications Oxford Britain: Oxford, UK, 1984; pp. 217–244. [Google Scholar]
- Shih, Y.J.; Chen, Y.Z.; Guo, Y.Q. A new species of free-living marine nematode (Ptycholaimellus: Chromadoridae: Chromadorida: Nematoda) from mangrove wetlands in China. Zool. Stud. 2022, 61, 20. [Google Scholar]
- Bhadury, P.; Austen, M.C.; Bilton, D.T.; Lambshead, P.J.D.; Rogers, A.D.; Smerdon, G.R. Evaluation of combined morphological and molecular techniques for marine nematode (Terschellingia spp.) identification. Mar. Biol. 2008, 154, 509–518. [Google Scholar] [CrossRef]
- Bhadury, P.; Austen, M.C.; Bilton, D.T.; Lambshead, P.J.D.; Rogers, A.D.; Smerdon, G.R. Development and evaluation of a DNA-barcoding approach for the rapid identification of nematodes. Mar. Ecol. Prog. Ser. 2006, 320, 1–9. [Google Scholar] [CrossRef]
- Juario, J.V. New Free-Living Nematodes from the Sublittoral Zone of the German Bight; University of Hamburg: Hamburg, Germany, 1974. [Google Scholar]
- Allgén, C.A. Pacific free-living marine nematodes (Papers from Dr. Th. Mortensen’s Pacific expedition 1914-16. LXXVI). Vidensk. Meddr. Fra. Dan. Nat. 1951, 113, 263–411. [Google Scholar]
- Inglis, W.G. Two new species of free-living marine nematodes from the west coast of Scotland. Hydrobiologia 1961, 18, 284–292. [Google Scholar] [CrossRef]
- Gagarin, V.G.; Thanh, N.V. Two new nematode species (Nematoda) from the mangroves of the Yen River Delta, Vietnam. Inland Water Biol. 2016, 9, 39–47. [Google Scholar] [CrossRef]
- Timm, R.W. The marine nematodes of the bay of Bengal. Proc. Pak. Acad. Sci. 1961, 1, 25–88. [Google Scholar]
- Rho, H.S.; Lee, H.J.; Lee, H.; Choi, C.G. Two new free-living marine nematodes of the genus Belbolla (Nematoda, Enoplida, Enchelidiidae) from a shallow subtidal benthic habitat of the outermost islands of Korea. Korean J. Environ. Biol. 2021, 39, 423–434. [Google Scholar] [CrossRef]
- Huang, Y.; Zhang, Z. Three new species of the genus Belbolla (Nematoda: Enoplida: Enchelidiidae) from the Yellow Sea, China. J. Nat. Hist. 2005, 39, 1689–1703. [Google Scholar] [CrossRef]
- Belogurov, O.I.; Fadeeva, L.S.; Belogurova, L.S. Studies of nematodes of the subfamily Eurystomininae (Enoplida, Enchelidiidae) from the Far East Seas of the USSR. Zool. Zhurnal 1983, 62, 15–24. [Google Scholar]
- Belogurov, O.I.; Belogurova, L.S. Morphology of Belbolla intarma sp. n., diagnosis and a table for the determination of the species of the genus Belbolla. Biol. Morya 1980, 4, 74–77. [Google Scholar]
- Nasira, K.; Shahina, F.; Shamim, S. Descriptions of Bathyeurystomina minima sp. n. and Belbolla longispiculata sp. n. with observations on Pareurystomina vaughtae and Eurystomina indica (Enoplida: Enchelidiidae) from Pakistan. Int. J. Nematol. 2014, 24, 87–96. [Google Scholar]
- Micoletzky, H. Freilebende marine nematoden vond den Sunda-Inseln: I. Enoplidae (Papers from Dr. Th. Mortensens Pacific expedition 1914-16. LIII. Vidensk Meddr Dan. Nat. 1930, 87, 232–339. [Google Scholar]
- Luc, M.; De Coninck, L.A.P. Nématodes libres marins de la région de Roscoff. Arch. Zool. Expérimentale Générale 1959, 98, 103–165. [Google Scholar]
- Cobb, N.A. One Hundred New Nemas (Type Species of 100 New Genera); Waverly Press: Baltimore, MD, USA, 1920; pp. 208–311. [Google Scholar]
- Gagarin, V.G.; Tu, N.D. Adoncholaimus minor sp.n. and Belbolla vietnamica sp.n. (Nematoda, Enoplida) from mangrove forest of the Yen River Estuary in Vietnam. Int. J. Nematol. 2016, 26, 1–8. [Google Scholar]
- Kumar, A.; Sen, D.; Bhadury, P. Unraveling free-living marine nematode community structure from a biodiversity-rich tropical coastal setting based on molecular approaches. Mar. Biodivers. 2015, 45, 537–547. [Google Scholar] [CrossRef]
- Pereira, T.J.; Fonseca, G.; Mundo-Ocampo, M.; Guilherme, B.C.; Rocha-Olivares, A. Diversity of free-living marine nematodes (Enoplida) from Baja California assessed by integrative taxonomy. Mar. Biol. 2010, 157, 1665–1678. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Macheriotou, L.G.K.B.; Nguyen, D.T.; Phuong Nguyen, T.X.; Noppe, F.; Armenteros, M.; Boufahja, F.; Rigaux, A.; Vanreusel, A.; Derycke, S. Metabarcoding free-living marine nematodes using curated 18S and CO1 reference sequence databases for species-level taxonomic assignments. Ecol. Evol. 2019, 9, 1211–1226. [Google Scholar] [CrossRef] [Green Version]
- Zhang, S.; Lin, S.Q.; Meng, F.X.; Wu, M.; Wang, C.S. Nematode (Enoplida) diversity in sediment samples collected from the Clarion Clipperton Fracture Zone. Acta Ecol. Sin. 2017, 37, 1630–1638. [Google Scholar]
- Tytgat, B.; Nguyen, D.T.; Nguyen, T.X.P.; Pham, T.M.; Long, P.K.; Vanreusel, A.; Derycke, S. Monitoring of marine nematode communities through 18S rRNA metabarcoding as a sensitive alternative to morphology. Ecol. Indic. 2019, 107, 105554. [Google Scholar] [CrossRef]
- Holterman, M.; Schratzberger, M.; Helder, J. Nematodes as evolutionary commuters between marine, freshwater and terrestrial habitats. Biol. J. Linn. Soc. 2019, 128, 756–767. [Google Scholar] [CrossRef] [Green Version]
Figure 1.
Belbolla mangrove sp. nov. (A) Lateral view of male head; (B) lateral view of female anterior part; (C) lateral view of female head; (D) lateral view of male body; (E) lateral view of ovaries; (F) lateral view of male posterior body part, showing the spicule and precloacal supplement; (G) lateral view of female tail end. Scale bars: 10 (A,C,F,G); 20 (B,E); 50 μm (D).
Figure 1.
Belbolla mangrove sp. nov. (A) Lateral view of male head; (B) lateral view of female anterior part; (C) lateral view of female head; (D) lateral view of male body; (E) lateral view of ovaries; (F) lateral view of male posterior body part, showing the spicule and precloacal supplement; (G) lateral view of female tail end. Scale bars: 10 (A,C,F,G); 20 (B,E); 50 μm (D).
Figure 2.
Belbolla mangrove sp. nov. (A) Lateral view of male head end; (B) lateral view of labial papillae; (C) lateral view of amphideal fovea; (D) lateral view of male head end; (E) lateral view of male pharynx; (F) lateral view of ovaries; (G) lateral view of male posterior body part, showing the spicule and precloacal supplement; (H) lateral view of female tail end; (I) lateral view of male tail end. Scale bars: 10 (A–D); 20 (E,G–I); 50 μm (F).
Figure 2.
Belbolla mangrove sp. nov. (A) Lateral view of male head end; (B) lateral view of labial papillae; (C) lateral view of amphideal fovea; (D) lateral view of male head end; (E) lateral view of male pharynx; (F) lateral view of ovaries; (G) lateral view of male posterior body part, showing the spicule and precloacal supplement; (H) lateral view of female tail end; (I) lateral view of male tail end. Scale bars: 10 (A–D); 20 (E,G–I); 50 μm (F).
Figure 3.
Neighbor-joining tree based on a 643 bp region of the 18S rDNA gene.
Table 1.
GenBank accession numbers of the three male individuals of the new species, Belbolla mangrove sp. nov., discovered in this study.
Table 1.
GenBank accession numbers of the three male individuals of the new species, Belbolla mangrove sp. nov., discovered in this study.
| No | Site | Habitat | Longitude | Latitude | Gender | GenBank Accession Number of 18S rDNA | Amplified Fragment Length (bp) |
|---|---|---|---|---|---|---|---|
| 1 | Jiulong River Estuary, Zhangzhou City, Fujian Province | mangrove | 117.95° E | 24.45° N | male | MZ229775 | 930 bp |
| 2 | Jiulong River Estuary, Zhangzhou City, Fujian Province | mangrove | 117.95° E | 24.45° N | male | MZ229776 | 935 bp |
| 3 | Ximen Island, Wenzhou City City, Zhejiang Province | mangrove | 121.17° E | 28.34° N | male | MZ229777 | 853 bp |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
Zhu, H.; Guo, Y.; Zhou, H.; Shih, Y.-J.; Li, F.; Lee, F.W.F.; Xu, S.J.; Tam, N.F.Y. A New Species of Free-Living Nematode (Enoplida: Enchelidiidae) from the Mangrove Wetlands of China. J. Mar. Sci. Eng. 2023, 11, 1412. https://doi.org/10.3390/jmse11071412
AMA Style
Zhu H, Guo Y, Zhou H, Shih Y-J, Li F, Lee FWF, Xu SJ, Tam NFY. A New Species of Free-Living Nematode (Enoplida: Enchelidiidae) from the Mangrove Wetlands of China. Journal of Marine Science and Engineering. 2023; 11(7):1412. https://doi.org/10.3390/jmse11071412
Chicago/Turabian StyleZhu, Huilan, Yuqing Guo, Haichao Zhou, Yi-Jia Shih, Fenglan Li, Fred Wang Fat Lee, Steven Jingliang Xu, and Nora Fung Yee Tam. 2023. "A New Species of Free-Living Nematode (Enoplida: Enchelidiidae) from the Mangrove Wetlands of China" Journal of Marine Science and Engineering 11, no. 7: 1412. https://doi.org/10.3390/jmse11071412
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.
