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Aims: High demand for frog meat in Malaysia especially the American bullfrog (Rana catesbeiana) has promoted intensive farming of the animal. However, the farming of American bullfrog is restricted by the occurrence of diseases. This study reports the first isolation of Elizabethkingia meningoseptica from specimens of American bullfrog that suffer from cataract and ‘red-leg’ syndrome.Methodology and Result: The pathogen was isolated from eyes and internal organs (liver, kidney and spleen) of thediseased bullfrog specimens. All the bacterial isolates were subjected to phenotypic characterization and antibiotic susceptibility assay, and further identified by using the 16S rDNA sequencing analysis. We designed two pair of specific PCR primers (22-25 mers) which are complimentary to the β-lactamase gene in the reference strain ofE. meningoseptica ATCC49470. The result showed all the bacterial isolates shared similar phenotypic characters and antibiotic susceptibility. BLAST analysis of the 16S rDNA sequences indicated that the bacterial isolates had very high sequence homology (100%) with E. meningospetica ATCC49470 and E. meningoseptica isolates from mosquito. The two PCR primers were very specific to E. meningoseptica isolates of this study. Conclusion, significance and impact of study: This is the first isolation and characterization of bacterial pathogen, E. meningoseptica in cultured American bullfrog (Rana catesbeina) that suffered from eye cataract and ‘red-leg’syndrome in Sabah, Malaysia. It is suspected that one of the possible transmission routes of the bacterial pathogen could be via mosquito bites. The findings suggest that there is urgent requirement for standard guideline of good farming practice to be adopted in frog farms throughout the country. Such a guideline can help in minimizing economic losses, preventing transmission of the zoonotic bacterial pathogen to farm workers, and sustaining the industry in Malaysia andupgrading frog meat quality for international market.

Aims: Vibrio harveyi causes vibriosis to Asian seabass (Lates calcarifer). The disease spreads rapidly among fish stocked in the same cage. It causes high mortality especially in weak and small sized fish stocked at high density in poorly managed net cage. Study to determine the virulence levels of the bacterial pathogen in various aquaculture animals is a key to prevent vibriosis in marine aquaculture. Methodology and Result: Isolation of bacteria from diseased Asian seabass was done using tryptic soy agar (TSA)and thiosulphate citrate bile sucrose agar (TCBS) plates. Virulence of two strains of Vibrio harveyi (VHJR4 and VHJR7)was tested against clinically healthy aquaculture animals. The analysis revealed that the two bacterial strains differ in pathogenicity. The V. harveyi strain VHJR7 was virulent to Asian seabass at 1.40 x 104 c.f.u. g-1, humpback grouper(Cromileptis altivelis) at LD50 8.33 x 103 c.f.u. g-1 and black tiger shrimp (Penaeus monodon) at LD50 3.26 x 104 c.f.u. g-1,respectively. The V. harveyi strain VHJR4 was not virulent to Asian seabass and humpback grouper but it caused mortality to black tiger shrimp at LD50 1.32 x 106 c.f.u. g-1. Phenotypically, the two strains shared most of the biochemical features except that the V. harveyi strain VHJR7 was a urease positive and grew at 8.5 % NaCl, and at 10 °C. The percentage similarity of nucleotide sequences of 16S rDNA in V. harveyi VHJR4 and V. harveyi VHJR7 was higher (99%) but reduced at 95 % in hemolysin gene. Conclusion, significance and impact of study: Pathogenic strain of V. harveyi causes mortality and affects aquaculture production of Asian seabass. Hence, vaccine development against the bacterial pathogen is urgently needed for sustainability of Asian seabass aquaculture in Malaysia.

Magallana bilineata is a commercially important oyster species in Malaysia. Despite its economic significance, the biology and ecology of this species, particularly its reproductive cycle, are poorly understood in the region. Therefore, this study aimed to investigate the reproductive cycle of M. bilineata in Mengkabong Bay, Tuaran, Sabah and its correlation with environmental factors. A total of 105 oyster specimens were collected and examined to determine the sex ratio, gonad developmental stages, maturity index (MI), and condition index (CI). The results revealed a sex ratio of 1:2.36 (♂:♀) in the oyster population of the bay, with a significantly higher proportion of females (P < 0.05). Hermaphroditism was detected in 1.90% of the samples. M. bilineata was found to spawn throughout most months in the bay, with the highest MI and CI values recorded in November 2019 when most oysters were in mature or maturing stages. Salinity was found to be the primary factor influencing male gonad maturation, while temperature was the primary factor influencing female gonad maturation. Understanding the reproductive biology of this species is crucial for predicting its future survival and facilitating assisted spawning and hatchery spat production, thereby ensuring the conservation and sustainability of the oyster fishery in Malaysia.

Asian green mussel is commercially farmed in tidal waters in several enclosed bays in Sabah, Malaysia. In this study, two areas on the west coast of Sabah – Ambong Bay and Marudu Bay – were selected for the monitoring of the growth and mortality rates of green mussels farmed in suspension raft. Both growth and survival rates were then correlated with physicochemical parameters (dissolved oxygen, pH, salinity, temperature, water transparency, chlorophyll-a), nutrients (phosphate (PO43-), ammonia (NH3-N), nitrate (NO3-N) and nitrite (NO2-N) and condition index of mussel from each study site, as well as between the study sites. The twelve-month growth study (September 2017 to August 2018) was started with a total of 180 mussel specimens (90 at each site). The initial size (mean) of the mussel seed used was 47.7 ± 3.5 mm and 51.1 ± 3.9 mm for Marudu Bay and Ambong Bay, respectively. Mussels in Marudu Bay attained mean size of 73.47 ± 11.05 mm (SGR 0.17% ± 0.22) compared to 64.05 ± 7.44 mm (SGR 0.11% ± 0.22) for Ambong Bay at the end of the experiment. The cumulative mortality rates were 9.2% ± 4.9 and 55.5% ± 30.0 for Marudu Bay and Ambong Bay, respectively. The Pearson correlation indicated a significant positive relationship between mortality and water transparency (r = 0.684, p

The hard clam (Meretrix meretrix) is a popular edible shellfish in South and Southeast Asia, being heavily exploited by hand dredging in Marudu Bay, Malaysia. The current study was performed to evaluate the shell damages caused by this artisanal dredging gear. Samplings were conducted in triplicates at two sites using both hand dredging and hand collecting (control) sampling methods. The shell length and total weight, damage areas and breakage patterns of each clam were recorded. A questionnaire survey was conducted with local bivalve harvesters to gather information on the fishing pressure in Marudu Bay. Results revealed that the efficiency (fishing yield) of hand dredging gear was three times higher than hand collecting. However, hand dredging gear causes lethal shell damages to small hard clams (

Omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) have many health benefits to human. Increasing evidence have shown that climate change reduces the availability of plankton n-3 LC-PUFA to primary consumers which potentially reduces the availability of n-3 LC-PUFA to human. Since marine bivalves are an important source of n-3 LC-PUFA for human beings, and bivalve aquaculture completely depends on phytoplankton in ambient water as food, it is important to understand the impact of climate change on the lipid nutritional quality of bivalves. In this study, fatty acid profile of different bivalves (mussels, oysters, clams, scallops and cockles) from different regions (tropical, subtropical and temperate) and time (before 1990, 1991-1995, 1996-2000, 2001-2005, 2006-2010, 2011-2015, 2016-2020) were extracted from published literature to calculate various lipid nutritional quality indicators. The results of this study revealed that the effects of global warming and declines in aragonite saturation state on the lipid content and lipid indices of bivalves are highly dependent on the geographical region and bivalves. In general, global warming has the largest negative impact on the lipid content and indices of temperate bivalves, including decreasing the PUFA/SFA, EPA + DHA and n-3/n-6. However, global warming has a much smaller negative impact on lipid content and lipid indices in other regions. The declines of aragonite saturation state in seawater promotes the accumulation of lipid content in tropical and subtropical bivalves, but it compromised the PUFA/SFA, EPA + DHA and n-3/n-6 of bivalves in all regions. The findings of this study not only fill the knowledge gap of the impact of climate change on the lipid nutritional quality of bivalves, but also provide guidance for the establishment of bivalve aquaculture and fisheries management plans to mitigate the impact of climate change.

Bivalves have high diversity, widely distributed in various aquatic environments, including saltwater, brackish water and freshwater. Bivalves are known to rich in polysaccharides and have wide applications in functional foods, pharmaceuticals, and industrial research. Despite many relevant reports are available, the information is poorly organized. Therefore, in this study, we conducted a comprehensive scientific review on the potential bioactivity of polysaccharides derived from bivalves. In general, the polysaccharides derived from bivalves possess various bioactive properties, including anticancer, antioxidant, anticoagulant and immunomodulatory activities. The bioactivity of these biomolecules highly depends on the bivalve species, extraction methods, purification methods, dosages, etc. The information in this study can provide an overview of the bioactivities of bivalve polysaccharides. This is very useful to be used as a guide for identifying the health benefits of polysaccharides derived from different bivalve species., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fin whales are a cosmopolitan species found in the largest water masses of the world. In Malaysia, as well as other tropical countries in the Southeast Asian region, literature on fin whales is limited, and as a result, there is confusion regarding their distribution range in the region. This study utilizes the fresh tissue of the skin and blubber of a dead fin whale that was stranded in Sabah (Borneo, Malaysia) on the coast of the South China Sea to confirm the species identity, possible properties of the species' diet, and any trace element contamination. The DNA profile results confirmed that the whale belonged to Balaenoptera physalus . Further investigation of its cytochrome b gene sequence indicated that it was closely related to the southern fin whale ( Balaenoptera physalus quoyi ). This finding indicates that fin whales indeed migrate to warm tropical waters and that their continuous global distribution spans the equatorial region. The dominant fatty acids, such as C18:0, C16:1, C18:1N9T and C16:0 profiles, were consistent with the pelagic plankton diet that the whale would have had during its migration in the tropical waters of the South China Sea. The whales are likely pelagic feeders and thus need to be offshore, which would explain why they are rarely seen in shallow coastal areas during migration in these waters. The concentrations of K, Ca, Sc, Mg and Al ranged from 0.45 μg g -1 to 7.80 μg g -1 , while Cr, Cd, As and Pb were either very low or could not be detected. This is consistent with concentrations of trace elements previously reported for other baleen whale genera from the Southern Ocean. Our study demonstrates the importance of the South China Sea as a migration route for the southern fin whale, since it is a rich food source with relatively low contaminant levels. The South China Sea is therefore well-suited to ensure these whales' survival during migration.

The common methods that are presently used to identify Vibrio harveyi include microscopic examination and biochemical, immunological and PCR-based assays. These methods require technical expertise, and can be time-consuming. A rapid method is required for the high-throughput screening of large number of samples. As such, we have developed a rapid, simple yet sensitive and specific detection method based on the use of the loop-mediated isothermal amplification (LAMP) of DNA. A set of six primers, i.e., two outer, two inner and two loop primers, was designed based on the in silico analysis of a large pool of 39 strains of the toxR gene sequence of V. harveyi . The addition of the loop primers decreased the reaction time of the LAMP by more than half. Furthermore, with the application of SYBR Green, the result can be obtained as quickly as in 10 to 15 min without the need of gel electrophoresis. The specificity of the method primers was then determined by performing LAMP with Vibrio and non- Vibrio samples. LAMP has a greater sensitivity than PCR reaction. The sensitivity of PCR was at 0.6 pg concentration of V. harveyi recombinant plasmid DNA standard, while LAMP was able to detect lower amounts even at 0.6 fg. The development of the LAMP assay will provide a valuable tool for the high-throughput rapid detection of V. harveyi contamination both in laboratories and in the field.

Marine leech Zeylanicobdella arugamensis (Piscicolidae), an economically important parasite is infesting predominantly cultured groupers, hybrid groupers and other fish in Southeast Asian countries. In this study, we tested the anti-parasitic potential of a medicinal plant Nephrolepis biserrata found in Sabah, East Malaysia against Z. arugamensis. Various concentrations of methanol extracts of the plant were tested experimentally against Z. arugamensis and disinfestation of the leech from its primary host hybrid groupers. The composition of methanol extract of N. biserrata was determined through LC-QTOF analysis. The significant anti-parasitic activity of 100% mortality of leeches was observed with the exposure of N. biserrata extracts. The average time to kill the leeches at concentrations of 25, 50 and 100 mg/ml was 25.11 ± 3.26, 11.91 ± 0.99, and 4.88 ± 0.50 min., respectively. Further, at various low concentrations of N. biserrata 2.5, 5 and 10 mg/ml, hybrid groupers were disinfested in an average time of 108.33 ± 12.65, 65.83 ± 9.70 and 29.16 ± 5.85 min., respectively. The tandem mass spectrometry data from LC-QTOF indicated some hits on useful bioactive compounds such as terpenoids (ivalin, isovelleral, brassinolide, and eschscholtzxanthin), flavonoids (alnustin, kaempferol 7,4'-dimethyl ether, and pachypodol), phenolics (piscidic acid, chlorogenic acid, and ankorine), and aromatic (3-hydroxycoumarin). Thus N. biserrata can act as a potential biocontrol agent.

The region of northern Borneo is home to the current state of Sabah, Malaysia. It is located closest to the southern Philippine islands and may have served as a viaduct for ancient human migration onto or off of Borneo Island. In this study, five indigenous ethnic groups from Sabah were subjected to genome-wide SNP genotyping. These individuals represent the "North Borneo"-speaking group of the great Austronesian family. They have traditionally resided in the inland region of Sabah. The dataset was merged with public datasets, and the genetic relatedness of these groups to neighboring populations from the islands of Southeast Asia, mainland Southeast Asia and southern China was inferred. Genetic structure analysis revealed that these groups formed a genetic cluster that was independent of the clusters of neighboring populations. Additionally, these groups exhibited near-absolute proportions of a genetic component that is also common among Austronesians from Taiwan and the Philippines. They showed no genetic admixture with Austro-Melanesian populations. Furthermore, phylogenetic analysis showed that they are closely related to non-Austro-Melansian Filipinos as well as to Taiwan natives but are distantly related to populations from mainland Southeast Asia. Relatively lower heterozygosity and higher pairwise genetic differentiation index (F ST ) values than those of nearby populations indicate that these groups might have experienced genetic drift in the past, resulting in their differentiation from other Austronesians. Subsequent formal testing suggested that these populations have received no gene flow from neighboring populations. Taken together, these results imply that the indigenous ethnic groups of northern Borneo shared a common ancestor with Taiwan natives and non-Austro-Melanesian Filipinos and then isolated themselves on the inland of Sabah. This isolation presumably led to no admixture with other populations, and these individuals therefore underwent strong genetic differentiation. This report contributes to addressing the paucity of genetic data on representatives from this strategic region of ancient human migration event(s)., (© 2018 John Wiley & Sons Ltd/University College London.)

The causative agent responsible for vibriosis in tropical fish aquaculture, Vibrio harveyi, has become a major bacterial pathogen. Studies suggest that this bacterium has developed resistance to antibiotics commonly used in aquaculture. In view of this situation and the requirement for the proposed postantibiotic era, bacteriophage therapy seems to be a promising control strategy for fish vibriosis. In this study, a lytic Vibrio phage VhKM4 belonging to a member of large, marine Myoviridae was successfully isolated. It exhibited bacteriolysis to both V. harveyi VHJR7 and V. parahaemolyticus ATCC 17802. The latent period of the VhKM4 phage was recorded at 60 min. It also recorded average burst size of approximately 52 plaque-forming units per infected cell. A strong bacteriolytic activity at low multiplicity of infection of 0.01 indicates the effectiveness of this large marine myovirid against fish pathogenic strain of V. harveyi VHJR7. Received June 16, 2016; accepted October 7, 2016.

Vibrio parahaemolyticus has long been known pathogenic to shrimp but only recently it is also reported pathogenic to tropical cultured marine finfish. Traditionally, bacterial diseases in aquaculture are often treated using synthetic antibiotics but concern due to side effects of these chemicals is elevating hence, new control strategies which are both environmental and consumer friendly, are urgently needed. One promising control strategy is the bacteriophage therapy. In this study, we report the isolation and characterization of a novel vibriophage (VpKK5), belonging to the family Siphoviridae that was specific and capable of complete lysing the fish pathogenic strain of V. parahaemolyticus. The VpKK5 exhibited short eclipse and latent periods of 24 and 36 min, respectively, but with a large burst size of 180 pfu/cell. The genome analysis revealed that the VpKK5 is a novel bacteriophage with the estimated genome size of 56,637 bp and has 53.1% G + C content. The vibriophage has about 80 predicted open reading frames consisted of 37 complete coding sequences which did not match to any protein databases. The analysis also found no lysogeny and virulence genes in the genome of VpKK5. With such genome features, we suspected the vibriophage is novel and could be explored for phage therapy against fish pathogenic strains of V. parahaemolyticus in the near future., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This paper describes the complete sequence of a giant lytic marine myophage, Vibrio phage ValKK3 that is specific to Vibrio alginolyticus ATCC(®) 17749™. Vibrio phage ValKK3 was subjected to whole genome sequencing on MiSeq sequencing platform and annotated using Blast2Go. The complete sequence of ValKK3 genome was deposited in DBBJ/EMBL/GenBank under accession number KP671755.

This paper describes the complete sequence of a novel lytic marine siphophage, VpKK5, that is specific to Vibrio parahemolyticus., (Copyright © 2015 Lal and Ransangan.)

This study determined the effect of light intensity and photoperiod on the dry cell weight and total amount of carotenoids in four isolates of purple non-sulfur bacteria obtained from shaded and exposed microhabitats of a mangrove ecosystem in Kota Kinabalu, Sabah, Malaysia. The initial isolation of the bacteria was carried out using synthetic 112 medium under anaerobic conditions (2.5 klx) at 30 ± 2°C. On the basis of colony appearance, cell morphology, gram staining, motility test, and 16S rRNA gene sequencing analyses, all four bacteria were identified as Afifella marina. One of the bacterial isolates, designated as Af. marina strain ME, which was extracted from an exposed mud habitat within the mangrove ecosystem, showed the highest yield in dry cell weight (4.32± 0.03 g/l) as well as total carotenoids (0.783 ± 0.002 mg/g dry cell weight). These values were significantly higher than those for dry cell weight (3.77 ± 0.02g/l ) and total carotenoid content (0.706 ± 0.008 mg/g) produced by the isolates from shaded habitats. Further analysis of the effect of 10 levels of light intensity on the growth characteristics of Af. marina strain ME showed that the optimum production of dry cell weight and total carotenoids was achieved at different light intensities and incubation periods. The bacterium produced the highest dry cell weight of 4.98 g/l at 3 klx in 72 h incubation, but the carotenoid production of 0.783 mg/g was achieved at 2.5 klx in 48 h incubation. Subsequent analysis of the effect of photoperiod on the production of dry cell weight and total carotenoids at optimum light intensities (3 and 2.5 klx, respectively) revealed that 18 and 24 h were the optimum photoperiods for the production of dry cell weight and total carotenoids, respectively. The unique growth characteristics of the Af. marina strain ME can be exploited for biotechnology applications.

Betanodavirus is the causative agent of the viral nervous necrosis (VNN) or viral encephalopathy and retinopathy disease in marine fish. This disease is responsible for most of the mass mortalities that occurred in marine fish hatcheries in Malaysia. The genome of this virus consists of two positive-sense RNA molecules which are the RNA1 and RNA2. The RNA1 molecule contains the RdRp gene which encodes for the RNA-dependent RNA polymerase and the RNA2 molecule contains the Cp gene which encodes for the viral coat protein. In this study, total RNAs were extracted from 32 fish specimens representing the four most cultured marine fish species in Malaysia. The fish specimens were collected from different hatcheries and aquaculture farms in Malaysia. The RNA1 was successfully amplified using three pairs of overlapping PCR primers whereas the RNA2 was amplified using a pair of primers. The nucleotide analysis of RdRp gene revealed that the Betanodavirus in Malaysia were 94.5-99.7% similar to the RGNNV genotype, 79.8-82.1% similar to SJNNV genotype, 81.5-82.4% similar to BFNNV genotype and 79.8-80.7% similar to TPNNV genotype. However, they showed lower similarities to FHV (9.4-14.2%) and BBV (7.2-15.7%), respectively. Similarly, the Cp gene revealed that the viruses showed high nucleotide similarity to RGNNV (95.9-99.8%), SJNNV (72.2-77.4%), BFNNV (80.9-83.5%), TPNNV (77.2-78.1%) and TNV (75.1-76.5%). However, as in the RdRp gene, the coat protein gene was highly dissimilar to FHV (3.0%) and BBV (2.6-4.1%), respectively. Based on the genome analysis, the Betanodavirus infecting cultured marine fish species in Malaysia belong to the RGNNV genotype. However, the phylogenetic analysis of the genes revealed that the viruses can be further divided into nine sub-groups. This has been expected since various marine fish species of different origins are cultured in Malaysia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Culture of Asian seabass, Lates calcarifer (Bloch) is a popular aquaculture activity in Malaysia. This fish is in high demand and fetches a good price in the local market. The seed for this fish is commercially produced by induced spawning in hatcheries. However, the seed supply is affected by frequent mass mortality of larvae aged between 15 and 60 dph. The clinical signs shown by the affected larvae include lethargy, loss of appetite, uncoordinated swimming, unusual spiral movement pattern and dark coloration. Histological examination of brain and eye of the affected specimens revealed extensive cell vacuolation in larvae aged 15-25 dph. Partial nucleotide sequence of the nervous necrosis virus coat protein gene of the affected larvae showed 94.0-96.1% homology to the nucleotide sequences of coat protein gene from nervous necrosis virus isolated from other countries in the Southeast Asia and Australia. This study provides scientific evidence based on molecular technique that many episodes of mass mortality in seabass larvae in Sabah is associated with the viral nervous necrosis. Because no effective treatment has been reported for this infection, stringent biosecurity measures must be adopted for exclusion of the pathogen from the culture system., ((c) 2010 Elsevier B.V. All rights reserved.)

The grow out of Asian seabass Lates calcarifer in marine net-cages is a popular aquaculture activity in Malaysia. Production of this species is greatly affected by the occurrence of vibriosis, which causes heavy mortality. Generally, young fish are more susceptible; they exhibit anorexia and skin darkening, followed by heavy mortality. The acutely affected older fish may also exhibit bloody lesions around the anus and the base of the fins. Twenty-one bacterial isolates obtained from internal organs (kidney, heart, spleen and liver) of the affected specimens were subjected to phenotypic characterization, testing for antibiotic susceptibility, and 16S ribosomal DNA sequencing. The sequencing result showed that all of the bacterial isolates belonged to Vibrio harveyi. The phenotypic characterization, however, identified 4 of the bacterial isolates as V. harveyi, 16 as V. parahaemolyticus, and 1 as V. alginolyticus. These findings suggest that biochemical features alone cannot be reliably used to identify bacterial pathogens, including V. harveyi, in aquaculture. Antibiotic susceptibility assays showed that some antibiotics, including oxytetracycline, nitrofurantoin, furazolidone, streptomycin, sulfamethoxazole, chloramphenicol, nalidixic acid, and oxolinic acid were effective against V. harveyi. Considering the side effects of these antibiotics, however, their use is not recommended in the aquaculture of Asian seabass.