FIRST NEAR COMPLETE MITOGENOME OF THE RHIZOCEPHALAN PARASITE, Sacculina beauforti

Gusti-Afiz G.R.N.*, Fazhan H., Ikhwanuddin M., and Waiho K.

¹Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia

*Corresponding author: gusti@umt.edu.my

The genetic information of rhizocephalan's parasite, especially its mitogenome, is important nowadays due to some reports of infection of the rhizocephalan’s parasite on the aquaculture crab species and other commercial crab species. Some of these parasite group is known to infect crustaceans and caused sterilization and damage to the gonad of the crabs. The parasite infection is species-specific which will cause a sudden outbreak if the infected organisms are accidentally introduced into aquaculture facilities. There is no reliable cure for this parasite up to now, the only solution is to mass killing all the infected crabs. Due to the limited information on these parasites, we sequenced for the first time the nearly complete mitochondrial genome sequence of the rhizocephalan parasite under the family Sacculinidae. The sequencing was made using Next-Generation Sequencing (NGS) rather than using the conventional method which needs the primers as the starter template of the sequencing. The nearly complete mitogenome is 15,380 bp for Sacculina beauforti, which consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and one major incomplete of the control region annotated using the online server. Comparative analysis of the order of the genes with other rhizocephalan showed a similar arrangement with other parasites from the same rhizocephalan group. Phylogenetic analysis of the 13 protein-coding genes showed a closed relationship based on the monophyletic clade observed between the parasites from the same family after adding the mitogenome of the copepods as the outgroups. 

Keywords: rhizocephalan,  mitogenome,  sacculinidae,  parasite

HSP70 KNOCKDOWN AND ITS EFFECT ON GENE TRANSCRIPT EXPRESSION OF THE WHITE-LEG SHRIMP Penaeus vannamei

Nurul Aifaa Azman^1, Mhd Ikhwanuddin² Yeong Yik Sung¹

¹Institute of Marine Biotechnology (IMB), Universiti Malaysia Terengganu 21030 Kuala Nerus Terengganu, Malaysia

²Institute of Tropical Aquaculture and Fisheries, University Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia

^*Corresponding author: Yeong@umt.edu.my

The white-leg shrimp Penaeus vannamei is an important aquaculture species, thus there is a growing interest in mass production in intensive culture conditions. Despite the tremendous economic benefits of intensified shrimp farming, the industry has faced a number of challenges, including diseases that brings about mass mortalities. Vibriosis, caused by a gram-negative bacterium occurring in marine environments, is likely to be the most severe of the shrimp diseases. Antibiotics and other chemical-based therapies used to combat vibriosis have resulted in a number of health and environmental hazards. As a result, there is a rising interest in alternative ways that are sustainable, safe, and ecologically beneficial. Interestingly, Hsps, namely Hsp70 (either produced by the host or provided exogenously), has been found as a possible immunostimulant in shrimp that aids in the enhancement of shrimp resistance to bacterial infection, including vibriosis. In this work, RNAi was employed to suppress Hsp70 expression in shrimp larvae in order to investigate the function of these proteins. Specifically, dsRNA capable of knocking down Hsp70 was produced, and appropriate amounts delivered into tissues by micro-injection were determined. The effect of Hsp70 knockdown on the transcriptome was investigated, with a focus on the potential pathway affected by this condition.

Keywords: Penaeus vannamei, Hsps, RNAi, Vibrio

PHENOTYPIC AND TRANSCRIPTOMIC RESPONSES OF THE BLACK TIGER SHRIMP Penaeus monodon POSTLARVAE TO VARYING DIETARY Κ-CARRAGEENAN INCLUSIONS

Yashier U. Jumah^1* and Augusto E. Serrano Jr.²

¹Aquaculture Department, College of Fisheries, Mindanao State University - Tawi-Tawi College of Technology and Oceanography, Sanga-Sanga, Bongao, Tawi-Tawi 7500, BARMM, Philippines

²Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao 5023, Iloilo, Philippines

*Corresponding Author: ajofflink@gmail.com

Carrageenan is an immunostimulant that needs further investigation for its nutritional value in the shrimps. Thus, the study was conducted to determine the phenotypic and transcriptomic profile of the carrageenan-supplemented black tiger shrimp Penaeus monodon postlarvae. There were five dietary κ-carrageenan inclusions [i.e., 0.0, 0.15, 0.30, 0.45, and 0.60 g kg^-1]  and each was divided in  triplicate groups. Plots were laid out in a completely randomized design stocked with post larvae 20 (PL 20; ~0.01g) in a recirculating system for 30 days. Phenotypes were evaluated after the feeding trial experiment. A group of carrageenan-treated shrimp with the highest growth performance and the control were selected for transcriptomic analysis. Whole body samples were stored in RNAlater™ at -20℃ and RNA extraction was done using TRIzol™ plus reagent kit (Invitrogen, USA) following the manufacturer’s instructions. Results showed that the growth indices of shrimps in the 0.15 and 0.3 g kg^-1 were significantly higher than that of the control group (p<0.05); while the values of those in the 0.45 and 0.60 g kg^-1 groups were not significantly different from those of the control group. Bioinformatics showed that the top 20 up-regulated metabolic pathways are belong to carbohydrate and lipid metabolisms which supported the observation that the carrageenan-treated groups exhibited better growth and efficiency performance than the control group. In conclusion, the study demonstrated that diets containing dietary k-carrageenan have a growth-promoting effect through carbohydrate and lipid assimilation in the black tiger shrimp P. monodon postlarvae.

Keywords: annotated genes, total RNA, gene expression, signal transduction pathways, immunostimulant

ELUCIDATING THE FUNCTIONS OF HEAT SHOCK PROTEIN 70 IN THE BRINE SHRIMP Artemia franciscana USING RNAI

Mimi Iryani Mat Taib^1,2*, Thomas H. MacRae³, Yeong Yik Sung²,

¹Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

²Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

³Department of Biology, Dalhousie University, Halifax, NS, B3H 4R2, Canada

^*Corresponding author: m.iryani@umt.edu.my

Artemia franciscana cysts contain an abundant amount of the small heat shock proteins such as ArHsp21, ArHsp22, artemin, and particularly p26 that contributes to stress tolerance and embryo development. However, little is known of the role of ATP-dependent molecular chaperone, Hsp70 in this organism. In this study, RT-PCR and immunoprobing of western blots with an antibody specific to Hsp70 respectively demonstrated the knockdown of Hsp70 mRNA and protein in cysts and nauplii released from females injected with Hsp70 dsRNA. The loss of Hsp70 does not affect cysts development and nauplii morphology, with the latter developed normally to adult stage, observations similar to the control. Although Hsp70 interference had no apparent effect on the development of diapause-destined embryos, the released cysts displayed reduced tolerance to desiccation and freezing, suggesting a role of Hsp70 in cysts stress tolerance. Over 28 days of culture, nauplii lacking Hsp70 showed higher mortality at the end of the experiment, however the effect is insignificant. Hsp70 also contributes to nauplii tolerance against Vibrio campbellii infection, with the survival of Hsp70 knockdown nauplii reduced approximately 31% when compared to the control (p<0.05). This study provides new insights into the roles of molecular chaperone Hsp70 in development, growth, survival and stress tolerance of the brine shrimp A. franciscana.

Keywords: Hsp70, Artemia franciscana, RNA Interference, Vibrio campbellii

MOLECULAR DETECTION OF PHYCODNAVIRUSES USING DNA POLYMERASE B (pol B) GENE FROM SPONGE SPECIES AND SEAWATER SURROUNDING TERENGGANU ISLANDS

Logajothiswaran Ambalavanan^*1, Shumpei Iehata¹ and Sandra Catherine Zainathan^1,2

¹Faculty of Fisheries and Food Science, University of Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

²Institute of Marine Biotechnology, University of Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

* Corresponding author: loga_5427@yahoo.com

Sponges are important in nutrient cycles in coral reef systems. Sponges provide a potential for biotechnology because sponges contain microbial symbionts such as bacteria and virus which helps in nutrient cycling. Viruses are ubiquitous biotic components of pelagic and benthic ecosystems and are important in the ecosystem by carrying out nutrient recycling. Viruses such as Phycodnaviruses are responsible for infecting marine brown algae which have a symbiotic relationship with the marine sponges. The virus affecting the marine brown algae causes the reduction of marine brown algae, thus reducing the oxygen and nutrient transfer from algae to the sponge. Thus, the aim of this study was to detect the presence or absence of the Phycodnaviruses in marine sponges which include Xestospongia sp., Aaptos sp., Petrosia sp. and Theonella sp. in Terengganu islands, particularly from Bidong and Karah Islands using the DNA polymerase B (pol B) method. A total of 110 sponge samples from four species, water samples (n=24) from each island, sediment (n=8), phytoplankton (n=24) (diluted DNA 10x) were collected from both locations. These samples were preceded for PCR analysis according to Short and Suttle (2003). The primers were based on DNA polymerase B (pol B) of Phycodnaviridae. Out of 166 samples of each sponge tissue, sediment, water sample and plankton (filtered with 0.22um), the PCR protocol by Short and Suttle (2003) demonstrated 13 positive pooled samples for the presence of Phycodnavirus in sponge tissue (n = 8) and water samples (n = 5) from Karah Island. Sequence analysis of DNA pol B gene showed 93% and 92% similarities with unknown Phycodnavirus clone KBVp-12 DNA polymerase for water samples. These results indicated the first detection of Phycodnavirus in marine sponge species, Xestospongia sp. and Aaptos sp., and water sample in Malaysia.

Keywords: Marine sponge, Phycodnavirus, DNA pol B gene marker, PCR analysis, Bidong and Karah Island, Terengganu.

NEW REPORT: REVEALING BIOSYNTHETIC GENE CLUSTER OF MARINE BACTERIUM, Pseudoalteromonas viridis BBR56

Desy Putri Handayani, Alim Isnansetyo^*, Indah Istiqomah

Department of Fisheries, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia

*Corresponding Author: isnansetyo@ugm.ac.id

The BBR56 strain was isolated from seawater of Dutungan Island, Barru, South Sulawesi. This bacterium was grown in Zobell 2216E agar medium for 24 h at 30 °C. DNA was isolated using Promega Genomic DNA Mini Kit TM050. The quantity of genomic DNA was determined using both NanoDrop spectrophotometers and Qubit 4.0 fluorometer. A kit from Oxford Nanopore Technology was used for library preparation. MinKNOW software version 20.06.9 was operated for GridION sequencing. Base-calling was determined using Guppy version 4.0.11 with high accurate mode. All FASTQ files were filtered using Filtlong software and visualized using NanoPlot. De-novo assembly was performed using Flye software version 2.8.1. Medaka software was used for polishing the assembled sequence. DFAST software was used for annotation. The assembled genomes and their annotation were performed using both Busco and CheckM software. This bacterium had 98.37% similarities of 16S rRNA gene sequence with P. viridis G-1387. It has a red color, circular, opaque, entire, and effusive elevation colony. The genome size of this bacterium was 5,5 Mb (GC content 49.3%) and consisted of 2 chromosomes (4,248240 bp of Chromosome 1 and 1,301,391 bp of Chromosome 2), 4,505 CDSs, 4,444 coding genes, 31 rRNAs, 113 tRNAs, 4ncRNAs, 4 CRISPRs, and 61 pseudogenes. The complete genome sequence has been deposited at GenBank/EMBL/DDBJ under the Accession Number Chromosome 1 and Chromosome 2 (CP072425-CP072426), BioProject PRJNA716373, and Biosample SAMN18435505. The raw sequencing reads have been deposited in the Sequence Read Archive (SRA) under accession number SRR14179986. Biosynthetic gene clusters were identified using Anti-SMASH and BAGEL4. The BBR56 genome harbors several important BGCs, namely non-ribosomal peptides, polyketide-like betalactone, Lant class I, Sactipeptide, and RiPP-like, and prodigiosin. These results can be used as a reference for utilizing BBR56 bacteria as a producer of secondary metabolites. 

Keywords: antiSMASH, Bagel4, marine bacterium, P. viridis BBR56, secondary metabolites