CHANGES IN THE GUT MICROBIOME OF A TROPICAL COPEPOD (Acartia sp) IN RESPONSE TO WARMING

Vu Dang Ha Quyen¹, Pham Phuong Linh²,  Bui Văn Canh², Tran Quang Sáng¹, Minh-Hoang Le², Dang Thuy Binh¹, Khuong V. Dinh^2,3,*

¹Institute for Biotechnology and Environment, Nha Trang University, 02 Nguyen Dinh Chieu Street, Nha Trang, Vietnam

²Cam Ranh Centre for Tropical Marine Research and Aquaculture, Institute of Aquaculture, Nha Trang University, No 2 Nguyen Dinh Chieu Street, Nha Trang City, Viet Nam

³School of Biological Sciences, Washington State University, Pullman, WA, USA

^*Corresponding author: quyenvdh@ntu.edu.vn

Copepods are a major food source for the early life stages of marine animals, and therefore play a key role in the transfer of energy and resources in the marine food web. Copepods are also particularly vulnerable to the warmer climate. Recent advancements in molecular ecology have shed light on the critical role of gut microbiomes in shaping the susceptibility of host animals to warming, yet it is unknown how the gut microbiome of marine copepods may be altered in response to a warming environment. For the first time, we addressed this fundamental gap of knowledge by assessing changes in the diversity of the gut microbiome in the copepod Acartia sp. Copepods were reared in three temperatures 26, 30, and 34°C, covering the typical thermal range and thermal extreme on the coast of Vietnam. Gut microbiomes of nauplii, copepodites and adults were analysed using metabarcoding analyses (high throughput DNA sequencing of V1–V9 of 16S rRNA hypervariable regions). We found 1,262,987 reads of the amplicon sequence corresponding to 392 total operational taxonomic units at 97% similarity were recorded. The gut content of all copepods contained Cyanobacteria, Proteobacteria, Bacteroidetes, Planctomycetes, Actinobacteria, and Acidobacteria. Importantly, thermophilic Proteobacteria was found dominantly at high temperatures (30°C and 34°C). At 34°C, Vibrio was the only dominant group, accounting for 70% of species found in copepod guts. These results may suggest 1) the role of Proteobacteria and Vibrio in the response of copepods to thermal stress, or 2) it may just simply reflect the higher thermal optimum of Proteobacteria and Vibrio with no link to the copepod fitness of copepods. The next logical step is to explore the functional role of these thermophilic groups in relation to changes in copepod fitness, which will fundamentally advance our mechanistic understanding of the adaptability of tropical copepods, and more general marine invertebrates to the warming climate.

Keywords: copepods, Acartia sp., temperature, metagenomic, climate change, marine heatwaves

EFFECT OF DENSITY AND FOOD ON GROWTH RATE AND SURVIVAL RATE OF DOG CONCH Strombus canarium (Linnaeus, 1758)         

Vu Trong Dai^1*, Pham Thi Khanh¹,  Nguyen Manh Dung¹, Truong Viet Hoang¹

¹Institute of Aquaculture, Nha Trang University

^*Corresponding author: daivt@ntu.edu.vn  

Dog conch Strombus canarium belongs to the class Gastropoda, and contains a high nutritional value and potential economic aquaculture species in Vietnam. This research was conducted to determine the effect of density and food on growth and survival rate of dog conch in Khanh Hoa province. The results showed that at the density of 100 individual/m², the growth rate and survival rate of dog conch was highest (0.28 mm/day and 82.3%, respectively) and significantly higher than those at the other treatments. The growth and survival rate of dog conch presented a significant difference when fed different food. The growth and survival rate was highest (0.35 mm/day and 87.4%, respectively) in the treatment fed TA1 (p<0.05).

Keywords: growth rate, food, density, dog conch

EFFECTS OF HCG AND LHRH-A ON REPRODUCTIVE PERFORMANCE OF MALABAR SNAPPER (Lutjanus malabaricus)

Minh V. Nguyen¹*, Phan Van Ut¹, Nguyen Dac Kien²

¹ Institute of Aquaculture, Nha Trang University, Nha Trang, Vietnam

² Institute of Biotechnology and Environment, Nha Trang University, Nha Trang, Vietnam

*Corresponding author: minhnguyen@ntu.edu.vn

The current study aims to evaluate the impact of hormone injection HCG and LHRH-a on  reproductive performance of Malabar snapper. Malabar snapper Broodstock (2⁺, BW=2,0-3,0 kg) were reared in seacages at Hon Thi, Nha Phu Lagoon, Khanh Hoa, Vietnam. The broodstock were monthly checked for evaluating maturation status, and when the ovary in the female obtained stage IV(c) the fish were selected for hormone injection. The concentrations of HCG and/or LHRHA for the females were as followed, (1) HCG 1.300 IU, 2) HCG 1.000 IU + 50 µg LHRHa/kg, and 3) LHRHa 100 µg/kg. While the males were injected with a half of those dose (3 fish each dose).

The results showed that all the doses successfully stimulated spawning in female Malabar snapper. The relative fecundity was 138,312 ± 150 eggs/kg, fertilization rate 77,7%, hatching rate 76,8%. The eggs were incubated at 27.8-29.7^oC, 33‰ and hatched after 15-16 h. The survival rate of the 3 day post hatched larvae was 85%, while only about 4% of the larvae went through metamorphosis. Juvenile Malabar (70 days) was 5.8±0.2 cm in length and 6.2± 0.4g in body weight, with 78% of survival rate.

Keywords: Malabar snapper, reproduction, hormone injection.

ELECTROPORATION AS AN EFFECTIVE DELIVERY SYSTEM TO INTEGRATE CATHELICIDIN GENE IN CHANNEL CATFISH GENOME BY CRISPR/CAS9 KNOCK-IN SYSTEM

Rhoda Mae Simora^1,2*, Max Bangs², De Xing², Wenwen Wang², Xiaoli Ma², Baofeng Su² and Rex Dunham²

¹Institute of Fish Processing Technology, College of Fisheries and Ocean Science, University of the Philippines Visayas, Miagao, Iloilo 5023,

²School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL 36849

*Corresponding author: rcsimora@up.edu.ph

CRISPR/Cas9-based gene knock out in mammalian cells particularly in teleost has proven to be very efficient with mutation rates, but precise insertion of exogenous DNA or gene knock-in via the homology directed repair pathway remains elusive.  While microinjection is more routinely used to introduce CRISPR components into fish embryos, it is labor-intensive and time-consuming, as embryos need to be injected individually.  A common alternative to microinjection is electroporation, which uses a rapid and high-voltage electric pulse to deliver DNA into embryos.  Here, we utilized single electroporation, where CRISPR components were co-delivered with the donor DNA into fertilized eggs, and double electroporation, where both sperm and fertilized eggs received the CRISPR components and donor DNA.  In this study, we designed double-stranded DNA constructs driven by zebrafish ubiquitin (dsDNA Ubi 40ng/µl) and carp β-actin (dsDNA β-actin 40ng/µl) promoters, as well as plasmid DNA construct driven by zebrafish ubiquitin promoter (plasmid Ubi 50ng/µl) carrying cathelicidin gene, a disease-resistant gene derived from American alligator.  We succeeded in integrating with high efficiency an exogenous cathelicidin gene into chromosome 1 of channel catfish genome.  Highest integration rates were found using microinjection, both in dead fry with plasmid Ubi as donor DNA (61.5%) as well as in alive fingerlings with sdDNA Ubi donor (31.8%).  Moreover, electroporation also proved to be an efficient method to deliver transgene as revealed by 44.4 % integration rate in dead fry and 12.5 % in alive fish using single electroporation technique.  Overall, we have successfully generated a CRISPR knock-in transgenic channel catfish carrying cathelicidin gene using microinjection and electroporation strategies. 

Keywords: CRISPR/Cas9, electroporation, microinjection, channel catfish, cathelicidin

PHYSICAL RELATIONSHIP ON FECUNDITY AND HATCHING RATE OF MUD SPINY LOBSTER UNDER HATCHERY CONDITIONS

Wasana Arkronrat^1*, Vutthichai Oniam¹, Rungtiwa konsantad¹, Piyawat Promraksa², Prapaporn Deemark³, Supakant Chaichotranunt⁴

¹Klongwan Fisheries Research Station, Faculty of Fisheries, Kasetsart University, Prachuap Khiri Khan 77000, Thailand

²Andaman Coastal Research Station for Development, Faculty of Fisheries, Kasetsart University, Ranong 85120, Thailand

³Coastal Aquaculture Technology Research Group, Coastal Aquaculture Research and Development Division, Department of Fisheries, Bangkok 10900, Thailand

³Prachuap Khiri Khan Coastal Aquaculture Research and Development Center, Department of Fisheries, Prachuap Khiri Khan 77000, Thailand

^*Corresponding author: ffiswna@ku.ac.th

Mud spiny lobster Panulirus polyphagus (Herbst, 1793) is an important commercial marine crustacean species. It is distributed throughout the coastal waters of the tropical regions of the eastern Indian Ocean to the Pacific Ocean. There is high demand for this species for local consumption  and  export  in  many  countries  including  Thailand.  Nevertheless,  studies on P. polyphagus production under hatchery conditions are very limited. The study and utilization of ovigerous female P. polyphagus is important first steps for the development of female brooder management and seed production. Therefore, the objectives of this study were to investigate the physical relationship on fecundity and hatchability of P. polyphagus under hatchery conditions. Ovigerous female P. polyphagus were caught by local fishers using lobster traps in the coastal area of Bang Hin sub-district, Kapoe district, Ranong province, Thailand during March to August 2022. P. polyphagus were transferred to the hatchery of the Klongwan Fisheries Research Station, Prachuap Khiri Khan province, and individually reared in 500-L fiberglass tanks (incubation tanks). The relationships between carapace length (CL) or body weigh (BW) and fecundity (F) or hatching rate (HR) were examined using a simple linear correlation analysis and Pearson’s correlation coefficient. A probability plot was used to test for normality before performing ANOVA. The results showed that average CL, BW and egg batches weight (EBW) of ovigerous female P. polyphagus were 91.0±8.6 mm, 666.2±183.7 g and 48.6±22.7 g, respectively. The F estimates range from 140,826 to 1,531,842 eggs with brood size highly correlated to CL and BW (n = 48). For hatchability study under hatchery conditions (salinity ranged from 30-33 ppt, DO ranged from 3.47-4.94 mg/L, water temperature ranged from 27.0-28.3 °C, pH ranged from 7.99-8.65, total ammonia ranged from 0.03-0.64 mg-N/L, nitrite-nitrogen ranged from 0.02-1.12 mg-N/L and alkalinity ranged from 131-162 mg/L as CaCO3), the development of eggs to hatching lasted 14 to 27 days. The average HR of this lobster was 33.9±17.7 % and the average number of newly hatched larvae (primary phyllosomal larva) was 231,708±163,552 larvae. But hatchability was not related with CL and BW. The knowledge gained from this study will be useful for seed production of P. polyphagus.

Keywords: Mud spiny lobster, Panulirus polyphagus, Fecundity, Hatchability

REPRODUCTIVE DEVELOPMENT OF FEMALE WALLAGO CATFISH (Wallago attu) IN CAPTIVITY

Duc Nghia Vo¹*, Van Huy Nguyen¹, Duc Thanh Nguyen¹, Thi Thu An Le¹,

Matthew J. Wylie², P. Mark Lokman³, Anh Tuan Nguyen¹

¹University of Agriculture and Forestry, Hue University, 102 Phung Hung Street, Hue, Vietnam

²The New Zealand Institute for Plant and Food Research Limited, 293–297 Port Nelson, Nelson 7010, New Zealand

³Department of Zoology, University of Otago, PO Box 56, Dunedin 9054, New Zealand

^*Corresponding author: voducnghia@huaf.edu.vn

The wallago catfish (Wallago attu) is a new potential fish for aquaculture in Viet Nam. However, data related to the reproductive cycle of W. attu in captivity are not available. To provide reliable indicators for oocyte maturation (OM) and the spawning season of the captive W. attu, this study investigated the temporal variation in hepatosomatic and gonadosomatic indices, oocyte diameter and color (greenish vs yellowish), germinal vesicle migration, and plasma concentrations of estradiol-17β and vitellogenin in female broodstock in association with changes in light density, temperature and amount of rainfall during the reproductive cycle. The results of this study displayed a clear seasonality in all the investigated parameters. The highest amount of E2 (2.6 ± 3.5 ng/mL) was found in April, followed by a peak of Vtg (543 ± 43 ng/mL) in June. Meanwhile, the largest mean oocyte diameter (1.70 ± 0.02 mm) was observed in June. The shortest distance between the germinal vesicle and the edge of the oocyte (0.20 ± 0.01 mm) was recorded in July. Correspondingly, the amount of rainfall increased remarkably in July from 43.9 mm to over 200 mm in August. Taken together, we conclude that OM and the onset of the spawning season of captive W. attu occur in July and August, respectively. The percentage of greenish oocytes increased significantly over sampling time points. Therefore, the changes in the color of oocytes combined with oocyte diameter could be considered as promising indicators to predict the OM and spawning season of captive W. attu.

Keywords: Wallago attu, oocyte diameter, plasma estradiol-17β, vitellogenin, spawning, captivity, broodstock

THE TECHNICAL PROCEDURE OF SEED PRODUCTION OF SHORT-NECKED CLAM Paphia undulata           

Vu Trong Dai^1*, Ngo Anh Tuan¹, Ngo Thị Thu Thao²

¹Institute of Aquaculture, Nha Trang University; ²College of Aquaculture and Fisheries

^*Corresponding author: daivt@ntu.edu.vn

Short-necked clam Paphia undulata is a high nutritional value and potential economic aquaculture species in Vietnam. The technical procedure of seed production of this clam was successful built in Khanh Hoa province. The broodstock of short-necked clam responded and gave higher affected by temperature shock as optimal stimulated spawning methods. In the veliger stage: larval rearing 1- 3 individual/mL at a salinity of 31‰ and algae used as live feed were the most suitable rearing conditions for the growth and survival rate of larvae. In the spat stage and seed: salinity of 31‰ combined with algae and formulated food were the most suitable condition for growth and survival rate. The stocking density of 2 individual/cm² combined with no bottom substance was the most suitable for growth and survival rate. The most appropriate transportation method of seed was closed method with a density of 10,000 seed/bag, a transportation time of 6 hours, which presented the highest survival rate and lowest oxygen consumption.

Keywords: growth rate, food, density, short-necked clam, stimulated method

REPRODUCTIVE BIOLOGY OF Pangasius macronema BLEEKER, 1850

Tran Dong Phuong An*^1,2, Nguyen Van Trieu², Bui Minh Tam², Tran Ngoc Hoai Nhan², Pham Thanh Liem²  

¹Department of Science and Technology, Can Tho City

²College of Aquaculture and Fisheries, Can Tho University,
Campus 2, 3/2 street, Ninh Kieu District, Can Tho City, Vietnam

* Corresponding author: tdpan1812@gmail.com

Pangasius macronema (or ca sac soc in Vietnamese) is a high-value food fish and is widely distributed in the lower Mekong Basin including Laos, Thailand, Cambodia, and the Mekong Delta (Vietnam). This species becomes a new candidate for aquaculture due to its high value and well adaption to rearing conditions. However, domestication of this species is still limited because of lacking reproductively biological information. In this study, some reproductive characteristics of P. macronema were determined. Fish samples were collected along Hau River from February 2020 to January 2021 by using various fishing tools. Gonad development was observed by both external morphology and histological sections. Length, fish body weight, gonads weight, and egg diameter were used to determine spawning season and length of maturity. Results obtained from 1,080 fish showed that the highest GSI (6.93%) occurred in March and then reduced from May to July; Fulton and Clack’s indices were relatively low from February to November; meanwhile, ratios of stage IV gonads reached more than 75% in May and June, those data revealed that spawning season occurred from May to July. However, stage V and stage VI were not found within the collected samples. The mean of absolute fecundity was of 6,007±4,484 eggs/fish and relative fecundity was 187.179±133.164 eggs/kg. The egg of P. macronema was a demersal egg with an egg diameter of 0.797±0.089 mm. Results also showed that the length of maturity (L_m) of P. macronema was 10.7 cm in males and 11,1 cm in females.

Keywords: Pangasius macronema, reproductive biology, spawning season