Phylogenetic of Marble Goby (Oxyeleotris marmorata Blkr.) in South Sumatra Based on Cytochrome C Oxidase Subunit I (COI) Gene

Marble goby (Oxyeleotris marmorata) in Indonesia, spread across Sumatra, Kalimantan and Papua. The purpose of this study was to utilize a sequence of mitochondrial DNA Cytochrome Oxidase Subunit I gene, to analyze the phylogenetic tree and genetic distance between cultured and captured populations. This research was conducted on March-August 2020. The methods used in barcoding species were DNA isolation, amplification using PCR (Polymerase Chain Reaction) and sequencing of the COI mtDNA gene. The domesticated samples (n=3) were collected from Gandus Fish Seed Center (GFSC), while the wild samples (n=3) were captured from the Musi River in Beruge Village, Babat Toman District, both in Musi Banyuasin Regency. The sequenced COI mtDNA gene fragments were obtained from the PCR method. Three samples performed good PCR results, while the other three didn’t amplify properly. After the editing process, the COI gene sequencing produced a nucleotide length of 610 bp. Based on the BLAST (Basic Local Alignment Search Tool), domesticated marble goby (OMD2, OMD3) was in the same cluster with marble goby from the Musi River (OMS3). The genetic distance indicated that two specimens of domesticated marble goby were 100% identical, while the wild (OMS3) indicated a genetic distance of 0.0066 to domesticated species.

Assessment of Responses of Common Carp, Cyprinus carpio, Exposed to Sub-Lethal Concentrations of Lead, by Molecular and Mucosal Biomarkers

Heavy metals are one of the most persistent pollutants in waters, and molecular biomarkers as bioindicators could uncover early warning signals of stress suffered by organisms exposed to these pollutants. This research was conducted to explore the impact of lead contaminant on expression levels of hsp70 gene as well as protein pattern and innate immune indicators of skin mucus in Common carp as a biological model. The fish were exposed to different concentrations of Pb(NO3)2 as a source of lead (0, 7.875, 15.75, and 31.5 mg/L of lead) for 14 days. After the completion of the experiment, the liver and gill tissue plus mucus samples were collected from the fish. Next, the effects of exposure to sublethal doses of lead were investigated on expression levels of hsp70 gene, protein pattern, and innate immune indicators of skin mucus. The results indicated that the expression level of hsp70 gene significantly increased (at 7.5% & 30% LC50) in the liver tissue compared to the control group (P<0.05). The protein pattern of treatment samples was different from that of the control group. Also, in the mucus innate immune parameters, the levels of alkaline phosphatase increased significantly at high concentrations of lead contamination in comparison with the control group (P<0.05). The protein levels first revealed an ascending trend (P<0.05), and then a descending trend (at 30% LC50), though it was not a significant downward trend compared with the control group (P>0.05). The results suggested that these physiological indicators in Cyprinus carpio could be used as molecular and mucosal biomarkers for ecotoxicological studies.

First Record of a Novel Mitochondrial DNA Haplotype of a Marble Trout Lineage of Salmo Trutta in Turkey

A new record of a marble trout mtDNA haplotype known to be restricted to Adriatic basin (called marmoratus lineage within Salmo trutta complex) has been reported from Eşen Stream in the Aegean Sea basin of southeastern Turkey, based on sequence data of the mitochondrial DNA control region. The results of this study showed a single unique haplotype from this population, called MATR1. Phylogenetic analyses of this haplotype along with other haplotypes belonging to different mitochondrial DNA lineages of the S. trutta complex confirmed the existence of the marmoratus lineage in Turkey, suggesting a possible river capture between the Adriatic and Aegean Sea basins until the last (Würmian) marine regression.

Bacterial Examination of Wild and Cultured Fish Present in the Same Aquatic Ecosystem, and the Antibiotic Resistance of the Isolated Bacteria

This study investigated the bacteria present in natural and cultured fish species from the same aquatic system, and difference of their antibiotic resistance. A total of 129 fish, Georgian shemaya (Alburnus derjugini), black sea salmon (Salmo labrax) and rainbow trout (Oncorhynchus mykiss), were sampled on a monthly basis between the months of October 2016 and September 2017 in Kürtün Dam Lake. A total of 41 bacterial isolates were isolated from the fishes. The bacterial species were identified by molecular methods (PCR) using universal primers for bacteria. Acinetobacter lwoffii, Acinetobacter sp., Aeromonas sobria, and Pseudomonas sp. were isolated from both wild and cultured fish. Yersinia ruckeri was isolated from cultured fish, which showed severe mortality rate and typical symptoms. Various antibiotics including ampicillin (AMP10µg), gentamicin (CN10 µg), oxytetracycline (T30 µg), amoxicillin/clavulanic acid (AMC10µg), enrofloxacin (ENR5µg), trimethoprim/sulfamethoxazol (TMP-SMZ25µg), florfenicol (FFC30µg), sulfamethoxazol (SMZ25µg) and erythromycin (E15µg) were used for determination of the bacterial resistances. The highest resistances were determined against ampicillin (56%), sulfamethoxazol (46.3%) and oxytetracycline (34.1) in all bacterial isolates. While the bacteria isolated from wild fish did not show resistance to enrofloxacin and amoxicillin/clavulanic acid, those from cultured fish did not show resistance to gentamicin and erythromycin.

A Cost-effective DNA Isolation Strategy from Crustaceans Enables the First Molecular Phylogenetic Identification of Moina macrocopa from India

We report the first barcode identification of water flea species Moina macrocopa (strain JSK1) from the Indian subcontinent. The strain isolated from a freshwater pond near Mumbai by collective efforts of CUBE (Collaborative Undergraduate Biology Education), was morphologically characterized as Moina species. Keeping up with the growing demand of affordable science in colleges and to avoid hazardous technologies, we established a home grown porcelain-abrasion DNA extraction strategy for crustaceans, and compared it against routinely used protocols employing liquid nitrogen or insect-DNA isolation kits. Our method successfully yielded high quality genomic DNA from Moina, comparable to published protocols as further analysed by restriction digestion, ligation and PCR efficiency. We also sequenced two barcoding loci namely, COI and ITS sequences, duly submitted and curated by Genbank (accession numbers MH734122.1 and MH745035.1). Molecular phylogenetic analyses identified the Indian species as Moina macrocopa and related it closely to the Russian clade. Protein prediction analyses of the variations in COI revealed that the missense mutations lie in the aliphatic membrane helices and should not affect the catalytic capacity of cytochrome oxidase. The strain JSK1 is currently disseminated and maintained collectively by CUBE students across India, towards developing it as a model system.

Clinical Investigation and Molecular Diagnosis of Microsporidian Enterocytozoon hepatopenaei (EHP) in Shrimps from Selangor, Malaysia

In recent years, shrimp aquaculture production in Southeast Asia countries was highly infected by, microsporidian parasite, EHP. Recently, shrimps from farms located in Selangor were encountering growth retardation and it highly concerned us to carry out a clinical investigation in both farms (Location A and B). EHP infected P. monodon samples were collected from both locations. A total of 43 shrimp samples were collected and diagnosed via PCR using the 18S rRNA gene. Environmental parameters were found relatively higher than the advised values. It indicates that the farms are highly infected and toxic which had led to growth retardation and mortality. However, no significant differences were observed (except water temperatures) between the locations. The phylogenetic analysis expressed EHP detected from Malaysia formed in one clade and all the reference sequences clustered based on geographical continents (Asian-Latin America). Minimum-spanning network analysis shows that Malaysia samples Location A are genetically related with other Asian EHP samples even though the total positive rate of EHP infection was lower. However, samples from Location B were not clustered within the EHP clade and shows the highest genetic variance within Malaysia and among Asian samples. This preliminary investigation results confirmed the incidence of EHP outbreak in Malaysia.

In Silico Prediction of Molecular Interaction Within PmCBP-VP24 Complex to Understand Initial Instigation of WSSV into Shrimps

White Spot Disease is one of the most devastating diseases of shrimps. Molecular interaction between shrimp receptor protein PmCBP (Chitin binding protein of Peneaus monodon) and viral envelop protein VP24 is obligatory for binding of the White Spot Syndrome Virus to the shrimp digestive tract, and failure of this anchoring leads to an ineffectual infection. This is a first study that throws light on the molecular interaction of PmCBP-VP24 complex and provides important clues for initial steps of ingression of the virus into shrimps.

Genetic Differentiation of the Blue Swimming Crab Portunus pelagicus Along the Coastal Thai Waters Revealed by SSCP Analysis of Cytochrome c Oxidase Subunit I

The basic information on genetic diversity and population structure is essential for the construction of appropriate management schemes leading to sustainable fisheries of the blue swimming crab (Portunus pelagicus). Here, genetic heterogeneity of P. pelagicus (N=174) was examined by single-strand conformational polymorphism (SSCP) analysis of mitochondrial cytochrome c oxidase subunit I (PpCOI270). Seven SSCP genotypes were found across all investigated samples. The average genetic distance between pairs of geographic samples was 0.0014-0.7247. Significant geographic heterogeneity (P<0.05) and restricted levels of female gene flow between paired samples (0.03-1.60 individuals per generation) were observed except between Chanthaburi - Prachuap Kriri Khan and Ranong - Krabi (P>0.05; 6.54 and 16.17 individuals per generation) located in the same coastal regions. Therefore, the gene pool of P. pelagicus in Thai waters was genetically differentiated to different stocks even though it is biologically regarded as a potential dispersal species. Five geographic samples of P. pelagicus in Thai waters could be differentiated to three genetic stocks; Chanthaburi and Suratthani (stock A), Prachuap Khiri Khan (stock B) and Ranong and Krabi (stock C).

Associated Vibrio Species in Shrimp Vibriosis from Traditional Brackish Water Pond in the North Coastal of Central Java, Indonesia

Indonesian shrimp cultures are threatened by vibriosis. Some traditional brackish water ponds remained along the north coast of Central Java after the disease outbreaks destroyed the shrimp culture. This study aimed to discover the Vibrio diversity associated with shrimp vibriosis in traditional brackish water ponds. An exploratory method with purposive sampling was used in this study. Twenty-four shrimps presumably infected with vibriosis were collected from two district regions on the north coast of Central Java in July–September 2018. The bacteria associated in shrimp vibriosis were isolated from the telson and inner part of the hepatopancreas with TCBS medium. Forty-one bacteria associated with shrimp vibriosis were obtained, and then repetitive-polymerase chain reaction (rep-PCR) was performed to obtain Vibrio strains. On the basis of rep-PCR results, five respresentative strains were selected for further study. The results of 16S rDNA sequence analysis showed that the JKP03, JKP05, JKP19, JKM01, and JKM06 isolates were closely related to Vibrio rotiferianus, Vibrio diabolicus, Vibrio parahaemolyticus, Vibrio alginolyticus, and Shewanella algae, respectively. Vibrio biodiversity in shrimp vibriosis was high. These results confirmed that traditional shrimp farming was susceptible to vibriosis. Therefore, control methods such as vaccines, probiotics, and immunostimulant formulas must be developed to prevent and control the outbreak of shrimp vibriosis in traditional brackish water pond.

Karyology of Rhodeus amarus (Block, 1782) (Teleostei, Acheilognathidae) from Turkey

The aim of this study was to determine chromosomal characteristics of Rhodeus amarus (Block, 1782) from Turkey by conventional procedures (Giemsa, C-banding and Ag-NOR staining). Metaphase chromosomes were obtained from the head kidney cells. The diploid number was found as 48 and the fundamental number as 76. Chromosomes were morphologically characterized as metacentric (four pairs), submetacentric (10 pairs) and subtelo-acrocentric (10 pairs). C-bands were found to occur on the pericentromeric regions of most of the chromosomes and a single AgNOR was observed on Silver stained metaphases. The results may expand the knowledge on chromosomal features of bitterlings.