Biodiversity of microbial communities on some species in the class Gastropoda common in coastal waters in Central Vietnam
Gastropoda is a class in coastal waters in Central Vietnam. The interaction between microorganisms and Gastropoda mainly consists of symbiotic and parasitic relationships. Biodiversity of microbial communities on some species of Gastropoda is evaluated.
| Рубрика | Биология и естествознание |
| Вид | статья |
| Язык | английский |
| Дата добавления | 23.07.2021 |
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Biodiversity of microbial communities on some species in the class Gastropoda common in coastal waters in Central Vietnam
Thanh Binh Chu1, Thi Thu Hong Do1, Xuan Thinh Le2, Cao Cuong Ngo1, Thi Thu Huyen Pham1, Thi Thuy Duong Hoang1, Tai Tu Nguyen1, Temir A. Britaev1
1Vietnam - Russia Tropical Center
2National Institute of Hematology and Blood Transfusion
Abstract
Gastropoda is a large class in coastal waters in Central Vietnam. The interaction between microorganisms and Gastropoda mainly consists of symbiotic and parasitic relationships. In this study, biodiversity of microbial communities on some species of Gastropoda is evaluated, thereby predicting their interaction. From 12 samples of Gastropoda including 3 species: Trochus maculatus, Cypraea eglantica, Chicoreus bruneus, 101 microorganism strains including 79 bacterial strains (78.2%), 18 yeast strains (17.8%) and 4 actinomycetes strains (4.0%) were isolated. There were 15 strains (including 8 yeast strains, 5 bacterial strains and 2 actinomycetes strains) selected to identify based on sequence analysis of the D1/D2 region (yeast) and 16S rRNA gene (bacteria and actinomycetes). Based on the identification results, it is possible to predict the nutritional relationship between microorganisms and species of Gastropoda.
Keywords: Gastropoda; biodiversity; microorganisms; Central Vietnam
Биоразнообразие микробных сообществ некоторых видов класса Гастропода, распространенного в прибрежных водах в центральной части Вьетнама
Т.Б. Тьу1, Т.Т.Х. До1, С.Т. Ле2, К.К. Нго1, Т.Т.Х. Фам1, Т.Т.З. Хоанг1, Т.Т. Нгуен1, Т.А. Бритаев1
1Российско-вьетнамский тропический научно-исследовательский и технологический центр
2Национальный институт гематологии и переливания крови Социалистическая Республика Вьетнам, Ханой, округ Кау Зай, ул. Фам Ван Бач
Аннотация. Гастропода - это большой класс моллюсков в прибрежных водах в центральной части Вьетнама. Взаимодействие между микроорганизмами и гастроподой в основном состоит из симбиотических и паразитических отношений. В настоящем исследовании оценивается биоразнообразие микробных сообществ некоторых видов гастроподы, что позволяет прогнозировать их взаимодействие. Из 12 образцов гастроподы, включающих 3 вида: Trochus maculatus, Cypraea eglantica, Chicoreus bruneus, изолирован 101 штамм микроорганизмов, из них 79 штаммов бактерий (78,2%), 18 штаммов дрожжей (17,8 %) и 4 штамма актиномицетов (4,0 %). Было отобрано 15 штаммов (включая 8 штаммов дрожжей, 5 штаммов бактерий и 2 штамма актиномицетов) для идентификации на основе анализа последовательностей области D1/D2 (дрожжи) и гена 16S рРНК (бактерии и актиномицеты). На основании результатов идентификации можно предсказать питательную взаимосвязь между микроорганизмами и видами гастроподы.
Ключевые слова: гастропода; биоразнообразие; микроорганизмы; центральная часть Вьетнама
Introduction
Scientists have studied the relationship between bacteria and Gastropoda since the 1980s [10]. The interaction between microorganisms and Gastropoda mainly consists of symbiotic and parasitic relationships. Symbiotic relationships between microorganisms and Gastropoda include nutrient exchange, supply of organic compounds, production of vitamins and amino acids, resistance to pathogenic microorganisms, habitats, etc. Symbiotic bacteria usually belong to autotrophic bacteria such as Thioprofundum lithotrophicum, Thiohalophilus thiocy- anatoxydans, Methylosphaera hansonii, Methylosbacter luteus, etc. [5]. Microorganisms can also produce toxins and cause serious diseases.
Ha Tinh and Ninh Thuan are two provinces in Central Vietnam. The coastal area of these two provinces is the habitat of many species in the class Gastropoda [7]. Research on the relationship between microorganisms and Gastropoda in coastal marine ecosystems contributes to building a database for the conservation, development and sustainable exploitation of these animals.
Materials and methods
Materials. Samples. 12 samples of Gastropoda were collected in coastal waters of Ha Tinh and Ninh Thuan.
Table 1
List of samples
|
No. |
Species in the class Gastropoda |
Code sample |
Place |
|
|
1 |
Trochus maculatus |
H1, H3 |
Ha Tinh |
|
|
N1, N3 |
Ninh Thuan |
|||
|
2 |
Cypraea eglantica |
H2, H5 |
Ha Tinh |
|
|
N2, N5 |
Ninh Thuan |
|||
|
3 |
Chicoreus bruneus |
H4, H6 |
Ha Tinh |
|
|
N4, N6 |
Ninh Thuan |
|||
|
Total |
12 |
Media. Hansen medium for yeast [4]. MPA medium for bacteria [6]. SCA medium for actinomycetes [6].
Methods. Isolation method. Animal samples were peeled, collected the gastrointestinal tract, then diluted with salt water 9%o. The diluent was cultured on a petri containing medium suitable for each microorganism group. Petri plates were put in incubators at the suitable temperature [8].
Grouping method. The strains were cultivated on suitable media and conditions. Then they were differentiated by their macro- and micro-morphologies according to C.P. Kurtzman et al. [8]
DNA extraction. The experiment was carried out using Zymo Research Kit (USA). biodiversity microbial communities gastropoda
PCR. PCR is established for each microorganism group using the suitable primers and heating programs. Specifically, NL1/NL4 primers for yeast, 1492R/27F for bacteria and actinomycetes [3].
Sequence analysis. The sequencing results were compared to related data in Genbank by the BLAST search on NCBI.
Results and discussions
Isolation results. 101 microorganism strains were isolated from 12 samples collected from Ha Tinh and Ninh Thuan (Table 2).
Table 2 showed that bacteria had the greatest amount. Actinomycetes was the smallest of microorganism group. Fungi had not been found in isolated samples. Yeast was found mainly in samples collected from Ha Tinh, while actinomycetes were found mainly in samples in Ninh Thuan. Sample N1 (Trochus macula- tus) had the existence of 3 microorganism groups. Sample N5 (Cypraea eglantica) only isolated bacteria. Other samples had the existence of two microorganism groups. The number of microbial strains isolated on each species in the class Gastropoda was also different (Figure 1).
Table 2
List of microorganism strains
|
No. |
Sample |
Strains code |
|||
|
Yeast |
Bacteria |
Actinomycetes |
|||
|
1 |
H1 |
H1.1Y, H1.4Y, H1.6Y |
H1.2B, H1.3B, H1.7B, H1.8B |
- |
|
|
2 |
H2 |
H2.1Y, H2.2Y |
H2.3B, H2.4B, H2.5B, H2.6B, H2.7B, H2.8B, H2.9B, H2.10B, H2.11B, H2.12B, H2.13B |
- |
|
|
3 |
H3 |
H3.8Y |
H3.1B, H3.3B, H3.4B, H3.5B, H3.6B, H3.7B, H3.9B, H3.10B |
- |
|
|
4 |
H4 |
-- |
H4.1B, H4.3B, H4.4B, H4.5B, H4.6B |
H4.2A |
|
|
5 |
H5 |
H5.1Y, H5.2Y, H5.11Y |
H5.3B, H5.6B, H5.7B, H5.8B, H5.9B, H5.10B, H5.12B, H5.13B, H5.14B |
- |
|
|
6 |
H6 |
H6.1Y, H6.2Y, H6.12Y |
H6.3B, H6.4B, H6.5B, H6.6B, H6.9B, H6.11B, H6.15B, H6.16B |
- |
|
|
7 |
N1 |
N1.1Y, N1.2Y |
N1.3B, N1.5B, N1.7B |
N1.4A, N1.6A |
|
|
8 |
N2 |
N2.3Y, N2.5Y |
N2.1B, N2.2B, N2.4B, N2.6B |
- |
|
|
9 |
N3 |
N1.1Y |
N3.2B, N3.3B, N3.4B, N3.5B, N3.6B, N3.7B, N3.8B, N3.9B, N3.10B, N3.11B |
- |
|
|
10 |
N4 |
- |
N4.1B, N4.2B, N4.3B, N4.5B, N4.6B, N4.7B, N4.8B, N4.9B |
N4.4A |
|
|
11 |
N5 |
- |
N5.4B, N5.5B, N5.15B, N5.16B |
- |
|
|
12 |
N6 |
N6.12Y |
N6.7B, N6.8B, N6.13B, N6.14B |
- |
|
|
Total |
18 (17.8%) |
79 (78.2%) |
4 (4.0%) |
Figure 1. Number of microorganism strains in each species
Trochus maculatus and Chicoreus bruneus were 2 species with the existence of 3 microorganism groups (yeast, bacteria and actinomycetes). Actinomycetes had not been found in Cypraea eglantica.
Grouping based on characteristics of colonies and cells. Characteristics of colonies and cells play an important role in the classification of microorganisms. In this study, microorganism strains were grouped based on colony and cell characteristics in order to provide more accurate assessments of the diversity of microbial communities.
Bacteria. Based on colony and cell characteristics, 79 bacteria strains were divided into 5 groups (Table 3).
The isolated bacterial strains had a low biodiversity. The majority of bacteria group is the group with milky white colonies, slippery surface, viscous, spherical cells, separated (group 1 - 40.5 %). The results of bacteriological isolation are similar to the results of previous studies on the existence of bacteria in the species of Gastropoda [1].
Table 3
Grouping results of bacteria
|
Group |
Quantity (percent) |
Characteristic |
||
|
Colony |
Cell |
|||
|
1 |
32 (40.5%) |
Milky white, smooth surface, viscous |
Spherical cells, separated |
|
|
2 |
8 (10.1%) |
Red, smooth surface |
Rod cells, separated |
|
|
3 |
11 (13.9%) |
White, smooth surface |
Spherical cells |
|
|
4 |
7 (8.9%) |
Pink, smooth surface |
Rod cells |
|
|
5 |
21 (26.6%) |
Yellow, smooth surface |
Rod cells |
Yeast. Yeast was the microorganism group with the highest biodiversity. 18 yeast strains were divided into 8 groups (Table 4).
Table 4
Grouping results of yeast
|
Group |
Strains code |
Characteristic |
||
|
Colony |
Cell |
|||
|
1 |
H6.1Y, H5.11Y |
Milky white, spongy |
Spherical cells, separated, reproduce by budding |
|
|
2 |
N1.2Y, H1.6Y |
White, smooth |
Spherical cells, separated, reproduce by budding |
|
|
3 |
N3.1Y, N6.12Y |
Creamy white, spongy |
Spherical cells, reproduce by budding |
|
|
4 |
H1.1Y, H3.8Y, N1.1Y, H2.2Y |
Black, wet, viscous |
Spherical cells, arranged in chains |
|
|
5 |
H6.12Y, N2.5Y |
Dark brown, wet, viscous |
Rod and spherical cells, arranged in chains |
|
|
6 |
H2.1Y, H5.1Y, H5.11Y |
White, wrinkled surface |
Spherical cells, reproduce by budding |
|
|
7 |
H1.4Y |
Black, wet, viscous |
Rod cells, arranged in chains |
|
|
8 |
N2.3Y, H5.2Y, H6.2Y |
Creamy white, spongy |
Spherical cells, separated, reproduce by budding |
Yeast is a polymorphic microorganism group, characteristics of colonies (color, shape) and cells that can change in the life cycle [3]. Therefore, it is necessary to use other classification methods (biochemical characteristics, molecular biology) to be able to more accurately assess the biodiversity of yeast.
Actinomycetes. 4 actinomycetes strains were divided into 2 groups according to the colony and cell characteristics described in Table 5.
Table 5
Grouping results of actinomycetes
|
Group |
Strains code |
Characteristic |
||
|
Colony |
Cell |
|||
|
1 |
N1.6A, N4.4A |
Brownish, dry, hard, thick |
Rod cells, branching |
|
|
2 |
H4.2A, N1.4A |
Brown, small, dry, thin |
Rod cells, branching |
According to the references, there are currently no reports of the existence of actinomycetes in the species of Gastropoda [9]. However, in this study, the number of actinomycetes isolated was very low, and the survey was only conducted on 12 animal samples. A larger number of animal samples should be surveyed to confirm the existence of actinomycetes in the species of Gastropoda.
Based on the grouping results, 15 microorganism strains representing each group were sequenced (including 8 yeast strains, 5 bacterial strains and 2 actino- mycetes strains).
Figure 2. Colony characteristics of microorganism strains
Identification results. Microbial identification is considered a highly reliable classification method. Based on the identification results, it is possible to give the accurate assessment of biodiversity of microbial community. In this study, microorganism strains representing each group were sequenced with the corresponding genes. In yeast strains, DNA sequencing of the D1/D2 region of the large subunit of the 28S ribosomal RNA gene was evaluated with NL1/NL4 primers. 16S rRNA gene sequences were used to study bacteria and actinomycetes with 1492R/27F primers.
Bacteria. The results of bacterial identification showed that 5 bacteria strains identified could belong to 3 genera: Burkholderia, Cellulosimicrobium, Rhodococcus. Specially, genus Cellulosimicrobium includes cellulose-degrading bacteria. M.A. Dar et al. (2015) also published the existence of cellulose-degrading bacteria in Achati- na fulica. He suggested that the interaction between Cellulosimicrobium and species of Gastropoda could be symbiotic relationships [1].
Figure 3. Phylogenetic tree of bacteria, based on 16S rRNA gene sequences
Yeast. In the grouping by characteristics of colonies and cells, yeast is a microorganism group with the highest biodiversity (they were divided into 8 groups). However, the yeast identification results showed that they consisted of 4 genera: Meyerozyma, Aureobasidium, Pichia, Candida. According to the references, these 4 yeast genera can be found in some marine animals including sea snails (a spe-
cies of Gastropoda) [3]. However, there has been no report on the interaction between these yeast genera and species of Gastropoda.
Figure 4. Phylogenetic tree of yeast, based on the D1/D2 region sequences
Actinomycetes. Combining the results of grouping based on characteristics of colonies and cells with sequencing results, it could be confirmed that 4 isolated actinomycetes strains belonging to genus.
Figure 5. Phylogenetic tree of actinomycetes, based on 16S rRNA gene sequences
Streptomyces. Genus Streptomyces is a common genus in marine sediments [9]. As mentioned, there are currently no reports of the existence of actinomycetes in species belonging to Gastropoda. 4 isolated actinomycetes strains in this study might have entered the gastrointestinal tract of animal species along with their food.
Conclusions
Survey of 12 animal samples in the class Gastropoda (including species: Trochus maculatus, Cypraea eglantica, Chicoreus bruneus), 101 strains of microorganisms were isolated. Bacteria is the group with the highest number of strains, yeast is the group with the highest biodiversity.
Actinomycetes belong to genus Streptomyces; yeasts belong to the genera: Meyerozyma, Aureobasidium, Pichia, Candida; bacteria belong to the genera: Burkholderia, Cellulosimicrobium, Rhodococcus. Genus Cellulosimicrobium and species of Gastropoda may have symbiotic interaction.
References
[1] Dar MA, Pawar KD, Jadhav JP, Pandit RS. Isolation of cellulolytic bacteria from the gastrointestinal tract of Achatina fulica (Gastropoda: Pulmonata) and their evaluation for cellulose biodegradation. International Biodeterioration & Biodegradation. 2015; 98: 73-80.
[2] Dar MA, Pawar KD, Pandit RS. Gut Microbiome Analysis of Snails: A Biotechnological Approach. Organismal and Molecular Malacology, Sajal Ray, IntechOpen. 2017. doi: 10.5772/68133
[3] Duarte AWF, Pagnocca F, Chaud L, Dayo-Owoyemi I, Pessoa A, Felipe MGA, Sette LD. Taxonomic assessment and enzymes production by yeasts isolated from marine and terrestrial Antarctic samples. Extremophiles: Microbial Life Under Extreme Conditions. 2013.
[4] Eaton AD, Clesceri LS, Greenberg AW. (eds.) Standard Methods for the Examination of Water and Wastewater. 21st Ed. Washington: APHA; 2005.
[5] Guus R, Irene LG. On the evolutionary ecology of symbioses between chemosynthetic bacteria and bivalves. Appl. Microbiol. Biotechnol. 2012;94: 1-10.
[6] Greenberg AE, Trussell RR, Clesceri LS. (Eds). Standard Methods for the Examination of Water and Wastewater. 16th/19th Ed. Washington: APHA; 1985.
[7] Lutaenko KA, Kwang-Sik Choi, Thai NC. Coastal Marine Biodiversity of Vietnam: Regional and Local Challenges and Coastal Zone Management for Sustainable Development. Final Report submitted to APN. 2011.
[8] Kurtzman CP, Fell JW, Boekhout T, Robert V. Methods for isolation, phenotypic characterization and maintenance of yeasts. In Kurtzman CP, Fell JW, Boekhout T. (eds.) The yeasts: a taxonomic study. 5th ed. San Diego: Elsevier; 2011. p. 88-110.
[9] Kurtboke I. Microbial Resources: From Functional Existence in Nature to Applications. London: Academic Press; 2017.
[10] Taylor JD, Glover EA. Chemosymbiotic bivalves. In: Landman NH, Jones DS. (eds.) The vent and seep biota. The Netherlands: Springer; 2010. p. 107-135.
Список литературы
[1] Dar M.A., Pawar K.D, Jadhav J.P., Pandit R.S. Isolation of cellulolytic bacteria from the gastrointestinal tract of Achatina fulica (Gastropoda: Pulmonata) and their evaluation for cellulose biodegradation // International Biodeterioration & Biodegradation. 2015. Vol. 98. Pp. 73-80.
[2] Dar M.A., Pawar K.D., Pandit R.S. Gut Microbiome Analysis of Snails: A Biotechnological Approach // Organismal and Molecular Malacology, Sajal Ray, IntechOpen. 2017. doi: 10.5772/68133
[3] Duarte A. W.F., Pagnocca F., Chaud L., Dayo-Owoyemi I., Pessoa A., Felipe M.G.A., Sette L.D. Taxonomic assessment and enzymes production by yeasts isolated from marine and terrestrial Antarctic samples // Extremophiles: Microbial Life Under Extreme Conditions. 2013.
[4] Standard Methods for the Examination of Water and Wastewater. 21st ed. / ed. by A.D. Eaton, L.S. Clesceri, A.W. Greenberg. Washington: APHA, 2005.
[5] Guus R., Irene L.G. On the evolutionary ecology of symbioses between chemosynthetic bacteria and bivalves // Appl. Microbiol. Biotechnol. 2012. Vol. 94. Pp. 1-10.
[6] Standard Methods for the Examination of Water and Wastewater. 16th/19th ed. / ed. by A.E. Greenberg, R.R. Trussell, L.S. Clesceri. Washington: APHA, 1985.
[7] Lutaenko K.A., Kwang-Sik Choi, Thai N.C. Coastal Marine Biodiversity of Vietnam: Regional and Local Challenges and Coastal Zone Management for Sustainable Development. Final Report submitted to APN. 2011.
[8] Kurtzman C.P., Fell J. W., Boekhout T., Robert V. Methods for isolation, phenotypic characterization and maintenance of yeasts // The yeasts: a taxonomic study. 5th ed. / ed. by C.P. Kurtzman, J.W. Fell, T. Boekhout. San Diego: Elsevier, 2011. Pp. 88-110.
[9] Kurtboke I. Microbial Resources: From Functional Existence in Nature to Applications. London: Academic Press, 2017.
[10] Taylor J.D., Glover E.A. Chemosymbiotic bivalves // The vent and seep biota / ed. by N.H. Landman, D.S. Jones. The Netherlands: Springer, 2010. Pp. 107-135.
Сведения об авторах
Тьу Тхань Бинь - научный сотрудник микробиологической лаборатории, Институт биотехнологии, Российско-вьетнамский тропический научно-исследовательский и технологический центр.
До Тхи Тху Хонг - научный сотрудник микробиологической лаборатории, Институт биотехнологии, Российско-вьетнамский тропический научно-исследовательский и технологический центр.
Ле Суан Тхинь - главный техник банка стволовых клеток, Национальный институт гематологии и переливания крови.
Нго Као Кыонг - научный сотрудник микробиологической лаборатории, Институт биотехнологии, Российско-вьетнамский тропический научно-исследовательский и технологический центр.
Тхи Тху Хуен - научный сотрудник микробиологической лаборатории, Институт биотехнологии, Российско-вьетнамский тропический научно-исследовательский и технологический центр.
Хоанг Тхи Тхую Зыонг - заведующий лабораторией экологии водной среды, Институт тропической экологии, Российско-вьетнамский тропический научно-исследовательский и технологический центр.
Нгуен Тай Ту - научный сотрудник лаборатории экологии водной среды, Институт тропической экологии, Российско-вьетнамский тропический научно-исследовательский и технологический центр.
Бритаев Темир Аланович - исследователь, Институт тропической экологии, Российско- вьетнамский тропический научно-исследовательский и технологический центр; доктор биологических наук, профессор, приглашенный лектор биологического факультета Московского государственного университета имени М.В. Ломоносова и биологического факультета Университета Модены и Реджо-Эмилии (Италия).
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