Summary of Biology doctoral thesis: Research on ragworm (Nereididae: Tylorrhynchus) in soil ecosystem in the northern coastal area of Vietnam

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The objective of the thesis is to research Ragworm (Nereididae: Tylorrhynchus) in soil macrofauna community structure in the northern coastal area of Vietnam and identify some living environment conditions of the Ragworm in the study area. Ruoi habitats in the study area to provide scientific and practical basic for their conservation and development.

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Nội dung Text: Summary of Biology doctoral thesis: Research on ragworm (Nereididae: Tylorrhynchus) in soil ecosystem in the northern coastal area of Vietnam

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MINISTRY OF VIETNAM ACADEMY EDUCATION AND OF SCIENCE AND TRAINING TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ---------------------------- NGUYEN THI HA RESEARCH ON RAGWORM (NEREIDIDAE: TYLORRHYNCHUS) IN SOIL ECOSYSTEM IN THE NORTHERN COASTAL AREA OF VIETNAM Major: Theory of Zoology Major code: 9 42 01 03 SUMMARY OF BIOLOGY DOCTORAL THESIS Ha Noi - 2020 2
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The work was completed at: Academy of Science and Technology - Vietnam Academy of Science and Technology. Science of supervisor 1: Prof.Dr. of science Vu Quang Manh Science of supervisor 2: PhD. Le Hung Anh Science reviewer 1: Science reviewer 2: Science reviewer 3: The thesis will be defended in front of the Institute's doctoral thesis evaluation council, meeting at the Academy of Science and Technology - Vietnam Academy of Science and Technology at … hour, date… month…year 2020. The thesis can be found at: - Library of Academy of Science and Technology - Vietnam National Library 4
INTRODUCTION 1. Reasons to choose a topic With a coastline of more than 3,200 km, along with dense estuary systems and monsoon tropical climate created for Vietnam in general and northern Vietnam in particular has estuary coastal ecosystem speciality and have the highest biological productivity. Groups include Mollusca, Arthropoda, Annelida and are component soil macrofauna community structure in the northern coastal area of Vietnam. In particular, in phylum Annelida, it is impossible not to mention genus Tylorrhynchus of the familia Nereidida, ordo Phyllodocida, class Polychaeta which has important role in the estuarine and marine ecosystems. In the world, polychaeta are found in Japan, South China, Singapore, Indonesia ... with the number of discovered species of more than 5000 species divided into 86 families. This group have important role in the food chain and net, additional ragworm are also considered as an indicator group of organisms to assess the quality of the water and bottom environment. In our country, the appearance of ragworm has been mentioned by Nguyen Cong Tieu and Nguyen Cong Tru from the seventeenth and eighteenth centuries, many authors have published a list of hundreds of polychaeta species which appeared in Vietnam, especially there are species that have the ability to go deep into the mainland. However, questions about the Ragworm that are favored by the Vietnamese people as a nutritious food source have not been studied: i) Are they in the list of published polychaeta in Vietnam? ii) In addition, with the change of time, space, climate changes, and 5
living environment, the Ragworm in different provinces of Northern Vietnam have differences, morphological changes or is not?. additional, during the their life cycle, the ragworm has a time to live in the soil but there has not been any research to consider the Ragworm as a component in soil macrofauna community structure in the northern coastal area of Vietnam. With the above scientific and practical basis, a basic survey of the research on the Ragworm as a component in soil macrofauna community structure in ecosystem is necessary. On that basis, we choose to research topic " Research on ragworm (Nereididae: Tylorrhynchus) in soil ecosystem in the northern coastal area of Vietnam”. 2. The purpose of the thesis The objective of the thesis is to research Ragworm (Nereididae: Tylorrhynchus) in soil macrofauna community structure in the northern coastal area of Vietnam and identify some living environment conditions of the Ragworm in the study area. Ruoi habitats in the study area to provide scientific and practical basic for their conservation and development. 3. Research content 1/ Taxonomy of ragworm (Nereididae: Tylorrhynchus) by the traditional morphological combined DNA molecular biotechnology, in soil ecosystems in the coastal areas of Northern Vietnam. 2/ Research Ragworm in soil macrofauna community structure according to five types of habitats, the five vertical deep layers in the soil and the four seasons in the study area. 6
3/ Surveying some characteristics of the Ragworm habitat, contributing to their development and conservation in the study area. 4. The meaning of the thesis Scientific significance: For the first time, Ragworm (Nereididae: Tylorrhynchus) has been studied as a component in soil macrofauna community structure in the northern coastal area of Vietnam. The results of the thesis provide the taxonomic position of the ragworm, new data on the relationship of the Ragworm with other Macrofauna groups s in soil in terms of composition, density, biomass and some elements of the habitat of the Ragworm in the study area Practical significance: The practical significance of the thesis is that the data obtained on taxonomy, biological characteristics, ecology of the ragworm in soil macrofauna community structure in the northern coastal area of Vietnam are the basis for further studies to propose a number of conservations and special to properly use, develop and exploit the resources of the Ragworm in the northern Vietnam in general and Hai Duong in particular. In addition, these data contribute to build books, related curricula and teaching topics at undergraduate and postgraduate level. 7
Chapter 1: Overview 1.1. Situation of research on polychaeta worms in the world Study on classification of polychaete by morphological characteristics and DNA molecular genetic methods Taxonomy is the first stage in biodiversity research. Studies on the taxonomy of polychaeta worms have been carried out quite early in the world and include some typical works such as the study of Uschakov (1955) in the Far East that recorded 400 species, study in the Japanese of Imajima & Hartman (1964) with 467 species, Indian waters with Fauvel (1953), Chinese waters with Wubaoling (1986) or South African waters studied Day's rescue (1967). In the world, classification of organisms by DNA molecular genetic methods was conducted from the late nineteenth century. However, if the group of polychaeta alone had 225794 sequences solved. In particular, the Nereididae family has 15418 sequences and is implemented in many countries around the world such as the US, Germany, Sweden and China. vv. Research on the biology and ecology of polychaete (Polychaeta) Research on the biology and ecology of polychaeta in the world can include the work of some authors such as: Zenkevich (1965), Gidholm (1969), Pearson & Rosenberg (1987), Alogi (1989, 1990). ). These authors have raised a number of characteristics on the growth, development and reproduction of polychaete, the relationship between polychaete with other organisms, their habitats and vice versa. 8
Other studies on polychaetes According to Giangrande et al (2005), Castrol & Micheal (1997): Most polychaeta are a source of protein food, an indicator for assessing environmental quality, besides they have important role in the chain and food wed. Therefore, according to Uschakov (1955), some polychaeta have been raised and exploited as protein-rich food for shrimps and crabs, as bait for export for sea tourists. 1.2. Situation of research on the ragworm (Nereididae: Tylorrhynchus) in the world Research on the taxonomy of ragworm (Nereididae: Tylorrhynchus) by morphological characteristics and DNA molecular genetic methods In the world, the study on taxonomy of ragworm (Nereididae: Tylorrhynchus) by morphological characteristics has been done by some authors such as Fauvel (1953), Day (1967), Uschakov (1955) or Imajima (1972), the authors gave some morphological characteristics of the head, limb and silk of ragworm. In addition, only China in the world that has conducted ragworm ( Nereididae: Tylorrhynchus) classification by molecular biology technique, with the sequencing of the mitochondrial genomic DNA sequence of the Tylorrhynchus heterochaetus has been published on Genbank. Research on the biology and ecology of the ragworm (Nereididae: Tylorrhynchus) In the world, research on the biology and ecology of ragworm has been conducted and can be referred to as research by Kent (2016) and Imajima et al (1964, 1972). These authors have 9
given the nutritional characteristics, reproductive properties, reproductive forms and sexism of ragworm. 1.3. Situation of research on Polychaeta in Vietnam Study on classification of polychaete by morphological characteristics and molecular biology techniques (DNA) The study has summarized the studies on polychaeta taxonomy by morphological characteristics in ecosystems: coral reefs, seagrass beds, soft-bottom tidal areas, mangrove forests, aquaculture ponds . In Vietnam, for ragworm, the classification using DNA molecular biology techniques has not been implemented. Research on the biology and ecology of polychaete In Vietnam, research on the biology and ecology of polychaete has been carried out by some authors such as works of Pham Dinh Trong (1999, 2003, 2018), Do Van Nhuong et al (2007), Phan Thi Kim Hong (2009, 2012). The majority of studies refer biomass and density fluctuation of ragworm in different seasons and ecosystems. Other studies on polychaetes Research on the role and importance of polychaete has been done by a number of authors such as Theo Nguyen Van Chung (1994), Pham Dinh Trong and Do Van Nhuong (2001, 2003, 2004). Studies show that polychaete make an important contribution to biodiversity, which is an indicator group for assessing aquatic environment quality and the bottom layer. Besides, this group is an important role in the chain and food wed. According to Pham Dinh Trong (1997, 2018), the current marine resources in general as well as their food sources are benthos, 10
polychaete, and ragworm (Tylorrhynchus) in particular, are in serious decline. Therefore, to conserve biodiversity, in recent years our country has established 16 marine conservation areas and 10 biosphere reserves in the world. In addition, some ragworm are now being researched and raised as live feeds for shrimp, crab and fish hatcheries or bait for tourists (Pham Dinh Trong and Tran Huu Huy, 2005). 1.4. Situation of research on ragworm (Nereididae: Tylorrhynchus) in Vietnam Research on the taxonomy of ragworm (Nereididae: Tylorrhynchus) by the morphological characteristics and molecular biology techniques of DNA Based on the morphological characteristics Dang Ngoc Thanh et al (1980) confirmed in Vietnam that the presence of a species of Ragworm, Tylorrhynchus heterochaetus. However, there are no documents to confirm that the most common species of ragworm are used for food in different provinces of Vietnam is a species. Are they on the list of published polychaete species? And according to the changes of time, space, weather and climate factors, whether or not there is difference of ragworm in the different provinces, these issues have not been studied. In addition, in Vietnam, there has not been any research done to classify ragworm by DNA molecular biology technique. Research on the biology and ecology of ragworm (Nereididae: Tylorrhynchus) Research on biology and ecology of Ragworm has been done by some authors such as Dang Ngoc Thanh et al (1980) Pham Dinh Trong (1999, 2000, 2001, 2018), Nguyen Quang Chuong (200, 2009). The study of the authors has mentioned some aspects such as: 11
season which has ragworm, size, path of reproductive migration of ragworm. However, during the life cycle, ragworm have time to live in the soil but there have not been any studies considering ragworm as a component in soil macrofauna community structure. The study refers to the environmental indicators in the habitat of which has ragworm (Nereididae: Tylorrhynchus) Research by Nguyen Quang Chuong (2009) shows that the species Ragworm (Tylorrhynchus heterochaetus) lives in an environment with a bottom of mud or sandy mud, pH from 7.2 to 7.8, salinity of 0 - 5 ‰. Or Pham Dinh Trong (2018) shows that in the habitat which has ragworm, the pH of water ranges from 7-9, the total amount of dissolved solids and salinity ranges from 0.2 to 0.3 ‰. Other studies on ragworm (Nereididae: Tylorrhynchus) According to Tran Thi Thanh Binh et al. (2016) the species (Tylorrhynchus heterochaetus) has high economic value and a nutritious food source for humans. Pham Dinh Trong (2018) shows that Ragworm has an important role in improving organic humus, organisms that indicate environmental pollution and was a valuable medicine. According to Nguyen Van Khang (1991) and Pham Dinh Trong (1999, 2018) ragworm which has natural were declining, which is caused by the gradual loss of habitats and their habitat being compromised and polluted. Therefore, Pham Dinh Trong (2018) in his research has devised a complete process of 5 steps to renovate and design the Ragworm field. CHAPTER 2: SUBJECTS, LOCATION, TIME AND RESEARCH METHODS 2.1. Object and scope of the study 12
The object of the thesis research is Ragworm (Annelida: Polychaeta: Phyllodocida: Nereididae: Tylorrhynchus). Scope of the study: Research on taxonomy of ragworm (Nereididae: Tylorrhynchus) by by traditional morthrphological and DNA molecular genetic methods. Ragworm (Nereididae: Tylorrhynchus) have been surveyed and studied as an important component in soil macrofauna community structure in the northern coastal area of Vietnam. 2.2. Research location Research locations of the project include five provinces and cities in the the northern coastal area of Vietnam: Quang Ninh, Hai Duong, Hai Phong, Thai Binh and Nam Dinh. 2.3. Research time The study was conducted from 2015 to 2019. The specimens were collected in two years (2016-2017 and 2017-2018) in 8 surveys with the support of the Ministry of Education and Technology's Science and Technology project, code B2016-SPH-24. Number of sample: morphological analysis specimens of ragworm included 305 individuals (70 males, 235 females), DNA molecular genetic analysis specimens included 5 individuals; large invertebrates sample of about 4200 specimens (3209 quantitative samples, 991 qualitative specimens). 2.4. Research methods 2.4.2. Research methods in the field Qualitative samples were collected expanded at points in the study area to supplement the groups of macrofauna. Quantitative samples were collected in Hai Duong province. according to the four seasons in a year (spring, summer, autumn, 13
winter), according to space: five habitats (Thai Binh river bank, Ragworm rearing rice field, Dike, Grassland, Cultivated field); five soil vertical layers in ecosystem, Each habitat takes five pits, a pit has an area of 50 cm × 50 cm, 50 cm deep, each depth is 10 cm: deep layer (-1): 0-10 cm, depth (-2): 10-20 cm, depth (-3): 20-30 cm, depth (-4): 30-40 cm and depth (-5): 40-50 cm. 2.4.3. Research methods in the laboratory Classification of morphology characteristics of ragworm according to Fauvel (1953); Day (1967); Gallardo (1967); Imajima (1964, 1972); Fauchald K (1977); Mortimer (2003). Classification of other macrofauna according to Dang Ngoc Thanh et al. (1980). Density and biomass of macrofauna are calculated per 1m2. Classification by DNA molecular biology technique: Selection of 5 representative samples of ragworm which were collected in Ragworm rearing rice field habitat in five provinces and cities in the Northern coastal area of Vietnam were analyzed by DNA sequences of mitochondrial gene COI ( cytochrome oxidase subunit I) and used 3 reference sequences including 2 sequences belonging to the Chinese Tylorrhynchus heterochaetus (KM111507.1, NC 025561.1) and 1 sequence from the Portuguese Arenicola (KM042101.1) genealogy tree. Molecular biology techniques (DNA) are carried out at the Department of Molecular Biology and Conservation, Institute of Ecology and Biological Resources. The technique of collecting, preserving and analyzing physical and chemical samples in environment of water and soil according to guidance of Vietnam Regulation 6663-1: 2011, Vietnam Standard 5297: 1995. Soil samples were analyzed in a 14
laboratory of the Institute of Chemistry under Vietnam Academy of Science and Technology. Processing data using Word and Excel 2010 and PRIMER 5 software in analyzing biodiversity indicators; BioEdit 7.0.0 (Hall, 1997) and MEGA 6.0.6 (Tamura et al., 2007) were performed in DNA sequence analysis and spss 16 was made to compare differences of morphological indicators among populations of the sampling areas. CHAPTER 3: RESEARCH RESULTS 3.1. Taxonomy of ragworm (Nereididae: Tylorrhynchus) in soil ecosystem in the northern coastal area of Vietnam 3.1.1 Morphological characteristics classification of Ragworm (Nereididae: Tylorrhynchus) Morphological classification of 305 individuals of ragworm (70 males, 235 females) were collected in the study area. The results showed that the morphological characteristics of ragworm (Tylorrhynchus) were similar to the description of the species Tylorrhynchus heterochaetus of Dang Ngoc Thanh et al (1980) with the following characteristics: Adult body is about 35-135 mm long with 23-74 segments, body width ranges from 2 mm to 10 mm, body weight is about 0.06 to 4.51 gr. The body is divided into 2 parts: the head (Prostomium), the body (Metastomium) and the tail end (Pygidium). The head (Prostomium) consists of 2 pairs of eyes, 1 pair of tentacles, 1 pair of palps, 4 pairs of cirri in which the topmost pair is the longest. The pharynx has soft tender papillae, the upper surface is less than the underside, and there are strong jaws, each side has 7- 10 conical teeth, the first one is far from the tip. 15
Body (Metastomium): Consists a lot of segments with a relatively homogeneous structure, each segment has 2 limbs. The limb (Parapodia) is a special appendage that is transformed from the wall of the body used for swimming and cows. The limb included notopodium neuropodium). Dorsal branches of lateral limbs are flattened lobes. Branches of the ventral surface forming a sickle shape. Each side has 2 silk tufts (silk threads include many different small hard hairs): back silk, belly silk, in each silk has strong buttress (aciculum) clear; 2 cirri: back cirri and belly cirri in which the cirri belly is slender, thin, short, the back cirri has a round lobe and a crown at the tip. The posterior part of the body of the lateral limbs is larger than the front, the dorsal silk and the belly are denser. Limb is not transformed into gill. In this study, by observing the limb of the body from the anterior to the posterior part of the body, it shows the appearance of 7 types of silk, including: 1/ aciculum ; 2/ sickle-shape; 3/paddle- shaped silk (oar-shape); 4/dorsal homogomph falciger dorsal; 5 homogomph spiniger bristle; 6/ heterogomph falciger and 7/ lyriform. Thus, compared with previous studies on the morphology of Tylorrhynchus heterochaetus in Northern Vietnam by previous authors such as Fauvel (1953), Dang Ngoc Thanh et al (1980), in this study, at the lateral part of Ragworm have been described, adding a picture of a lyre in the middle of the body. The reason of the study of the previous authors only discovered 6 types of silk on the limb because the authors only observed the anterior and posterior parts of the body, and the middle part of the body with the presence of the lyriform are not mentioned by the authors. The morphological characteristics of adult (Nereididae: Tylorrhynchus) male and female of ragworm 16
To distinguish the sex of normal Ragworm based on color. Visually, the male Ragworm are milky white and the females are blue or light blue. However, the analytical results in the thesis distinguishes the sexes by observing the genital organs on a microscope. Table 3.1: Results of some measurement criteria of ragworm (Nereididae: Tylorrhynchus) in coastal provinces in Northern Vietnam Province Province Quang Hai Hai Phong Thai Binh Nam Ninh Duong Đinh ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ Targets n=2 n=3 n=1 n=4 n=2 n=3 n=4 n=5 n=4 n=5 2 9 5 9 5 5 6 6 Max 2,3 3,36 1,6 4,11 2,62 4,51 0,7 2,9 0,5 1,9 3 9 6 6 9 0,2 0,4 0,1 0,0 0,3 0,1 Min 0,25 0,48 0,49 0,46 5 1 6 6 9 5 1,0 1,0 0,5 0,4 0,7 Mas 6 3 0,8 (g) Avera ± 1,17 ± 1,57 1,27 1,43 8 ± 9 5 ge ± ± ± ± ± 0,5 ± ± (g/indi 0,4 0,57 0,3 0,93 0,53 0,72 0,2 4 0,0 0,3 vidual 6 6 5 6 4 ) 1,13±0,54b 1,44±0,87c 1,36±0,66c 0,79±0,53 0,73±0,34 a a Max 95 96 105 135 115 129 80 125 65 120 Min 38 35 65 58 50 48 60 40 48 40 Len 68, 68,7 85, 92,9 79,3 80,0 69, 70, 64 2 87 4 2 6 68 86 58, 45 gth Avera 75 (m ± ± ± ± ± ± ± ± ± ± ge(g/i 14, 15,7 11, 22,1 15,4 17,6 8,5 16, 14, m) ndivid 6,5 4 2 59 9 5 3 9 4 ual) 68,69±15,3 91,28±20,6 79,75±16,9b 69,73±16, 69,67±14, 9a c 66a 4a Wid Max 10 10 7 10 6 7 5 10 5 8 th( Min 3 2 3 4 2 2 3 3 4 3 mm Avera 5,8 5,85 4,8 5,6 4,4 4,14 4,2 5,1 4,7 5,2 17
) ge(m 6 ± ± 9 ± ± 5 8 5 1 m/ ± 2,06 1,11 ± 1,06 1,02 ± ± ± ± indivi 2,1 1,3 0,8 1,4 0,4 1,1 dual) 6 3 6 5,85±2,09c 5,48±1,37b 4,25±1,05a 5,12±1,44 5,18±1,14 c b b Max 71 72 65 74 72 64 51 57 53 62 Seg Min 44 40 52 48 48 44 43 29 36 23 men 57, t 59, 47, 43, 43, Avera 77 55,7 58,2 84 56,8 55,4 25 2 75 43, num ge(Se ± 7 4 6 88 ber ± ± ± ± ± ± ± ± ± gment 3,53 5,3 2,8 7,4 6,0 (Seg / 6,9 7,24 5,66 5,7 9,1 men 6 3 6 2 6 Indivi t) dual) 56,49±7,26 59,45±5,06 56,03±5,77b 43,47±7,3 43,47±9a b c 4a Male/female 1/1,8 1/3,3 1/1,4 1/14 1/14 ratio (Note: In the same row of different letters in the same row, there is a statistically significant difference (p
Results of comparing 5 research sequences with 2 sequences of Chinese Tylorrhynchus heterochaetus and Portuguese Arenicola defodiens sequence showed that: 5 Vietnamese sequences were 99% similar to 2 Chinese sequences and similar 80% of the Portuguese sequence. Vietnam's five sequences differ from China's two sequences at two nucleotide positions, number 12 (G-A) and 328 (T-C) (Table 3.3). Genetic distance between 5 Vietnamese sequences with 2 Chinese sequences from 0.3-0.7% and with the Arenicola sequence defodiens KM042101.1 from 27.1-27.5%. In addition, 5 Vietnamese sequences and 2 Chinese sequences are on the same derivative branch, the Portuguese Arenicola defodiens KM042101.1 sequence is outside the branch. Table 3.3: Position of Nucleotides was different in the COI genome between the 5 Reregens (Nereididae: Tylorrhynchus) studied. The positions of the Positions nucleotides are different Sequence 12 12 32 35 37 1 8 Sequence of Ragworm (Nereididae: Tylorrhynchus) Hai G Phong A C C T Sequence of Ragworm (Nereididae: Tylorrhynchus) . Quảng Ninh . T . . Sequence of Ragworm (Nereididae: Tylorrhynchus) . Nam Định . . . . Sequence of Ragworm (Nereididae: Tylorrhynchus) . . T T . 19
Thái Bình Sequence of Ragworm (Nereididae: Tylorrhynchus) . Hải Dương T . . . Tylorrhynchus heterochaetus KM111507.1 China A . . . C Tylorrhynchus heterochaetus NC 025561.1 China A . . . C Thus, from the results of the analysis of morphological and DNA, it can be affirmed that the ragworm in the coastal ecosystem of Northern Vietnam and favored by people as a food source is a Tylorrhynchus heterochaetus and has classified positions are: Regnum: Animals Phylum: Annelida Subclass: Errantia Class: Polychaeta Ordo: Phyllodocida Family: Nereididae Genus: Tylorrhynchus Grube (1866), Species: Tylorrhynchus heterochaetus (Quatrefages, 1866) Original name (Orig. Name): Nereis heterocheta Quatrefages (1866). Ceratocephale osawai Izuka (1903) Chinonereis edestus Chamberlin (1924) Nereis heterocheta Quatrefages (1866) Tylorhynchus heterochaetus [auctt.] Tylorrhynchus chinensis Grube (1866) Tylorrhynchus hetereochaetosus [auct. misspelling] Tylorrhynchus heterochaetus [auct. misspelling] Tylorrhynchus sinensis Dawydoff (1952) 3.2. Ragworm (Tylorrhynchus heterochaetus) in soil macrofauna community structure in the northern coastal area of Vietnam 20