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Application of molecular markers in Malaysian fisheries genetic resources

Tan, Soon Guan, Bhassu, Subha, Rosly Hassan

Buletin Persatuan Genetik Malaysia, 2005;11(2):0-0.
MyJurnal

The Malaysian fish production is about 1.5 million metric tonnes and 86.9% of this comes from the marine sector and 13.1% from the inland sector. This included fish production by capture and culture. (DOF, 2002). Fisheries genetic resources need to have a value in terms of economic, ecological and social uses and they need to characterized. This is the mandate to FAO and it is also necessary for fisheries management and aquaculture development. The vast aquatic diversity that exists in Malaysia consist of numerous taxa of marine and freshwater fishes, crustaceans, mollusks, plants and animals. These figures could be an underestimate to the actual figure. The levels of genetic diversity includes ecosystems, communities, population, genotypes and individual genes. A knowledge of the genetic background of a species and its population structure is essential for its management, breeding and conservation programmes in fisheries. Problems like how to choose the right candidates for breeding, identifiying and monitoring lines, families and individuals, monitoring and control of inbreeding, inheritance of simple traits and genetic improvement through selection for favourable gene and gene combinations can potentially be answered through the use of molecular markers in the management of fisheries genetic resources.
Fulltext Host, pathogen and environment: a bacterial gbpA gene expression study in response to magnesium environment and presence of prawn carapace and commercial chitin

Tiruvayipati S, Bhassu S

Gut Pathog, 2016;8:23.
PMID: 27231485 DOI: 10.1186/s13099-016-0105-5

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium which is found largely in estuarine and coastal waters. The bacteria has been a main focus in gastro-intestinal infections caused primarily due to the consumption of contaminated seafood. It was shown to survive in magnesium concentrations as high as 300 mM which are toxic to various other micro-organisms. Several genes of V. parahaemolyticus were studied, among which gbpA (N-acetyl glucosamine binding protein) was reported in Vibrio cholerae.
Fulltext Host, pathogen and the environment: the case of Macrobrachium rosenbergii, Vibrio parahaemolyticus and magnesium

Tiruvayipati S, Bhassu S

Gut Pathog, 2016;8:15.
PMID: 27114742 DOI: 10.1186/s13099-016-0097-1

Macrobrachium rosenbergii is well-known as the giant freshwater prawn, and is a commercially significant source of seafood. Its production can be affected by various bacterial contaminations. Among which, the genus Vibrio shows a higher prevalence in aquatic organisms, especially M. rosenbergii, causing food-borne illnesses. Vibrio parahaemolyticus, a species of Vibrio is reported as the main causative of the early mortality syndrome. Vibrio parahaemolyticus infection in M. rosenbergii was studied previously in relation to the prawn's differentially expressed immune genes. In the current review, we will discuss the growth conditions for both V. parahaemolyticus and M. rosenbergii and highlight the role of magnesium in common, which need to be fully understood. Till date, there has not been much research on this aspect of magnesium. We postulate a model that screens a magnesium-dependent pathway which probably might take effect in connection with N-acetylglucosamine binding protein and chitin from V. parahaemolyticus and M. rosenbergii, respectively. Further studies on magnesium as an environment for V. parahaemolyticus and M. rosenbergii interaction studies will provide seafood industry with completely new strategies to employ and to avoid seafood related contaminations.
RETRACTED: Molecular characterization and expression analysis of lily type lectin-1 (OfLTL-1) in rock bream (Oplegnathus fasciatus)

Arockiaraj J, Bhassu S

Fish Shellfish Immunol, 2010 Dec 28.
PMID: 21193051

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
Fulltext A new insight to biomarkers related to resistance in survived-white spot syndrome virus challenged giant tiger shrimp, Penaeus monodon

Mohd Ghani F, Bhassu S

PeerJ, 2019;7:e8107.
PMID: 31875142 DOI: 10.7717/peerj.8107

The emergence of diseases such as white spot disease has become a threat to Penaeus monodon cultivation. Although there have been a few studies utilizing RNA-Seq, the cellular processes of host-virus interaction in this species remain mostly anonymous. In the present study, P. monodon was challenged with WSSV by intramuscular injection and survived for 12 days. The effect of the host gene expression by WSSV infection in the haemocytes, hepatopancreas and muscle of P. monodon was studied using Illumina HiSeq 2000. The RNA-Seq of cDNA libraries was developed from surviving WSSV-challenged shrimp as well as from normal healthy shrimp as control. A comparison of the transcriptome data of the two groups showed 2,644 host genes to be significantly up-regulated and 2,194 genes significantly down-regulated as a result of the infection with WSSV. Among the differentially expressed genes, our study discovered HMGB, TNFSF and c-Jun in P. monodon as new potential candidate genes for further investigation for the development of potential disease resistance markers. Our study also provided significant data on the differential expression of genes in the survived WSSV infected P. monodon that will help to improve understanding of host-virus interactions in this species.
Fulltext Signature selection forces and evolutionary divergence of immune-survival genes compared between two important shrimp species

Soo TCC, Bhassu S

PLoS One, 2023;18(1):e0280250.
PMID: 36634148 DOI: 10.1371/journal.pone.0280250

In recent years, shrimp aquaculture industry had grown significantly to become the major source of global shrimp production. Despite that, shrimp aquaculture production was impeded by various shrimp diseases over the past decades. Interestingly, different shrimp species demonstrated variable levels of immune strength and survival (immune-survival) ability towards different diseases, especially the much stronger immune-survival ability shown by the ancient shrimp species, Macrobrachium rosenbergii compared to other shrimp species. In this study, two important shrimp species, M. rosenbergii and Penaeus monodon (disease tolerant strain) (uninfected control and VpAHPND-infected) were compared to uncover the potential underlying genetic factors. The shrimp species were sampled, followed by RNA extraction and cDNA conversion. Five important immune-survival genes (C-type Lectin, HMGB, STAT, ALF3, and ATPase 8/6) were selected for PCR, sequencing, and subsequent genetics analysis. The overall genetic analyses conducted, including Analysis of Molecular Variance (AMOVA) and population differentiation, showed significant genetic differentiation (p<0.05) between different genes of M. rosenbergii and P. monodon. There was greater genetic divergence identified between HMGB subgroups of P. monodon (uninfected control and VpAHPND-infected) compared to other genes. Besides that, based on neutrality tests conducted, purifying selection was determined to be the main evolutionary driving force of M. rosenbergii and P. monodon with stronger purifying selection exhibited in M. rosenbergii genes. Potential balancing selection was identified for VpAHPND-infected HMGB subgroup whereas directional selection was detected for HMGB (both species) and ATPase 8/6 (only P. monodon) genes. The divergence times between M. rosenbergii and P. monodon genes were estimated through Bayesian molecular clock analysis, which were 438.6 mya (C-type Lectin), 1885.4 mya (HMGB), 432.6 mya (STAT), 448.1 mya (ALF3), and 426.4 mya (ATPase 8/6) respectively. In conclusion, important selection forces and evolutionary divergence information of immune-survival genes between M. rosenbergii and P. monodon were successfully identified.
Fulltext Differential STAT gene expressions of Penaeus monodon and Macrobrachium rosenbergii in response to white spot syndrome virus (WSSV) and bacterial infections: Additional insight into genetic variations and transcriptomic highlights

Soo TCC, Bhassu S

PLoS One, 2021;16(10):e0258655.
PMID: 34653229 DOI: 10.1371/journal.pone.0258655

Diseases have remained the major issue for shrimp aquaculture industry for decades by which different shrimp species demonstrated alternative disease resistance or tolerance. However, there had been insufficient studies on the underlying host mechanisms of such phenomenon. Hence, in this study, the main objective involves gaining a deeper understanding into the functional importance of shrimp STAT gene from the aspects of expression, sequence, structure, and associated genes. STAT gene was selected primarily because of its vital signalling roles in stress, endocrine, and immune response. The differential gene expressions of Macrobrachium rosenbergii STAT (MrST) and Penaeus monodon STAT (PmST) under White Spot Syndrome Virus (WSSV) and Vibrio parahaemolyticus/VpAHPND infections were identified through qPCR analysis. Notably, during both pathogenic infections, MrST demonstrated significant gene expression down-regulations (during either early or later post-infection time points) whereas PmST showed only significant gene expression up-regulations. Important sequence conservation or divergence was highlighted through STAT sequence comparison especially amino acid alterations at 614 aa [K (Lysine) to E (Glutamic Acid)] and 629 aa [F (Phenylalanine) to V (Valine)] from PmST (AY327491.1) to PmST (disease tolerant strain). There were significant differences observed between in silico characterized structures of MrST and PmST proteins. Important functional differentially expressed genes (DEGs) in the aspects of stress, endocrine, immune, signalling, and structural were uncovered through comparative transcriptomic analysis. The DEGs associated with STAT functioning were identified including inositol 1,4,5-trisphosphate receptor, hsp90, caspase, ATP binding cassette transmembrane transporter, C-type Lectin, HMGB, ALF1, ALF3, superoxide dismutase, glutathione peroxidase, catalase, and TBK1. The main findings of this study are STAT differential gene expression patterns, sequence divergence, structural differences, and associated functional DEGs. These findings can be further utilized for shrimp health or host response diagnostic studies. STAT gene can also be proposed as a suitable candidate for future studies of shrimp innate immune enhancement.
Fulltext Biochemical indexes and gut microbiota testing as diagnostic methods for Penaeus monodon health and physiological changes during AHPND infection with food safety concerns

Soo TCC, Bhassu S

Food Sci Nutr, 2022 Aug;10(8):2694-2709.
PMID: 35959249 DOI: 10.1002/fsn3.2873

Severe shrimp disease outbreaks have a destructive impact on shrimp aquaculture and its associated downstream food processing industries. Thus, it is essential to develop proper methods for shrimp disease control, which emphasizes the importance of food safety. In this study, we performed biochemical tests and gut microbiome analysis using uninfected control and Vp AHPND-infected Penaeus monodon samples. Biochemical tests were performed to assess the phenoloxidase (PO) activity, respiratory Burst (RB) activity, nitrite concentration, superoxide dismutase (SOD) activity, total hemocyte count (THC), and total protein concentrations. Overall, upregulations were detected in these biochemical tests, which showed the activation of the immune response in P. monodon during acute hepatopancreatic necrosis disease (AHPND) infection, especially at 6 hpi and 12 hpi. Besides that, shrimp gut samples were collected and pooled (n = 3), followed by DNA extraction, PCR amplification targeting the V3/V4 16S ribosomal RNA (rRNA) region, next-generation sequencing (NGS), and bioinformatics analysis. Proteobacteria was the most abundant phylum in both samples. The Rhodobacteraceae family and Maritimibacter genus were proposed to be vital forshrimp health maintenance. Vp AHPND bacterial colonization and secondary Vibrio infections were postulated to have occurred based on the higher abundances of Vibrionaceae family and Vibrio genus in the Vp AHPND-infected sample. Firmicutes phylum together with Photobacterium and Aliiroseovarius genera were inferred to be pathogenic or related factors of AHPND infections. In conclusion, physiology (immune response activation) and gut microbiome changes of disease tolerant P. monodon during AHPND infection were identified. Both biochemical tests and 16S rRNA analysis are proposed as a combined strategy for shrimp health diagnosis for ensuring shrimp health maintenance, disease control, and food safety.
Potential muscle activity disturbance in Penaeus monodon during Acute Hepatopancreatic Necrosis Disease (AHPND) infection: Inference through gene expression, calcium concentration, and MicroRNA

Soo TCC, See SA, Bhassu S

J Invertebr Pathol, 2020 11;177:107497.
PMID: 33130047 DOI: 10.1016/j.jip.2020.107497

Global shrimp aquaculture farmers have suffered major economic losses due to disease outbreaks. A notable shrimp disease is Acute Hepatopancreatic Necrosis Disease (AHPND), which is caused by a new strain of Vibrio parahaemolyticus bacteria (VpAHPND) that mainly inhabits the shrimp gut and damages the hepatopancreas. Fewer studies have investigated whether this disease will affect shrimp muscle functioning or cause any muscle damage. We challenged Penaeus monodon shrimp with VpAHPND bacteria using an immersion method. Expression of Dystrophin gene, an important regulatory gene for maintenance of muscle integrity, was quantified from muscle samples using qRT-PCR. Additional verification was conducted by determining calcium concentration and bta-miR-4286 and dre-miR-107b miRNAs expression. P. monodon dystrophin gene demonstrated the highest expression level during AHPND infection when muscle calcium concentration was detected at its lowest level at 6 h post-infection (hpi). The highest muscle calcium concentration, determined at 36 hpi, was supported by higher bta-miR-4286 miRNA expression and lower dre-miR-107b miRNA expression in VpAHPND-infected samples compared to uninfected samples at the same time point. We deduced an interactive relationship between dystrophin gene expression, calcium concentration, and miRNA expression in P. monodon muscle tissues triggered by the invading VpAHPND bacterium.
Rapid in-situ detection kit (RisK): Development of loop-mediated isothermal amplification (LAMP) assay for the rapid identification of selected invasive alien fish in Malaysian freshwaters

Vythalingam LM, Hossain MAM, Bhassu S

Mol Cell Probes, 2021 02;55:101683.
PMID: 33259896 DOI: 10.1016/j.mcp.2020.101683

Invasive alien fish species have become a silent treat towards the ecosystem especially the native fish population in Malaysia. There has been a need to develop rapid identification methods that can aid management teams in identifying fish species that are not native to our ecosystem. Current visual identification methods are highly tedious and require time, delaying action towards curbing the invasion. The LAMP assay successfully identified six popular invasive fish species in Malaysia. None of the LAMP assays showed false positives and the Limit of Detection of the LAMP primers were highly sensitive and could detect DNA samples up to 1 × 10-15 ng/μl. The LAMP primers designed were highly specific to the target species and did not amplify non target species. DNA sequencing was done to ensure the accuracy of LAMP assay results. This study demonstrates that LAMP is a suitable tool in species identification efforts of invasive fish species in Malaysia.
In vitro and in vivo thermal stress induces proliferation of Blastocystis sp

Thergarajan G, Govind SK, Bhassu S

Parasitol Res, 2018 Jan;117(1):177-187.
PMID: 29188368 DOI: 10.1007/s00436-017-5688-3

Blastocystis sp. is known to be the most commonly found intestinal protozoan parasite in human fecal surveys and has been incriminated to cause diarrhea and abdominal bloating. Binary fission has been widely accepted as the plausible mode of reproduction for this parasite. The present study demonstrates that subjecting the parasites in vitro to higher temperature shows the proliferation of parasite numbers in cultures. Transmission electron microscopy was used to compare the morphology of Blastocystis sp. subtype 3 isolated from a dengue patient having high fever (in vivo thermal stress) and Blastocystis sp. 3 maintained at 41 °C (in vitro thermal stress) and 37 °C (control). Fluorescence stains like acridine orange (AO) and 4',6'-diamino-2-phenylindole (DAPI) were used to demonstrate the viability and nuclear content of the parasite for both the in vitro and in vivo thermal stress groups of parasites. Blastocystis sp. at 37 °C was found to be mostly vacuolar whereas the in vitro thermal stressed isolates at 41 °C were granular with electron dense material seen to protect the granules within the central body. Parasites of the in vivo thermal stressed group showed similar ultrastructure as the in vitro ones. AO and DAPI staining provided evidence that these granules are viable which develop into progenies of Blastocystis sp. These granular forms were then observed to rupture and release progenies from the mother cells whilst the peripheral cytoplasmic walls were seen to degrade. Upon exposure to high temperature both in vitro and in vivo, Blastocystis sp. in cultures show higher number of granular forms seen to be protected by the electron dense material within the central body possibly acting as a protective mechanism. This is possibly to ensure the ability to survive for the granules to be developed as viable progenies for release into the host system.
Fulltext Characterisation of Asian snakehead murrel Channa striata (Channidae) in Malaysia: an insight into molecular data and morphological approach

Song LM, Munian K, Abd Rashid Z, Bhassu S

ScientificWorldJournal, 2013;2013:917506.
PMID: 24396312 DOI: 10.1155/2013/917506

Conservation is imperative for the Asian snakeheads Channa striata, as the species has been overfished due to its high market demand. Using maternal markers (mitochondrial cytochrome c oxidase subunit 1 gene (COI)), we discovered that evolutionary forces that drove population divergence did not show any match between the genetic and morphological divergence pattern. However, there is evidence of incomplete divergence patterns between the Borneo population and the populations from Peninsular Malaysia. This supports the claim of historical coalescence of C. striata during Pleistocene glaciations. Ecological heterogeneity caused high phenotypic variance and was not correlated with genetic variance among the populations. Spatial conservation assessments are required to manage different stock units. Results on DNA barcoding show no evidence of cryptic species in C. striata in Malaysia. The newly obtained sequences add to the database of freshwater fish DNA barcodes and in future will provide information relevant to identification of species.
Molecular classification and phylogenetic relationships of selected edible Basidiomycetes species

Avin FA, Bhassu S, Shin TY, Sabaratnam V

Mol Biol Rep, 2012 Jul;39(7):7355-64.
PMID: 22327649 DOI: 10.1007/s11033-012-1567-2

Morphological identification of edible mushrooms can sometimes prove troublesome, because phenotypic variation in fungi can be affected by substrate and environmental factors. One of the most important problems for mushroom breeders is the lack of a systematic consensus tool to distinguish different species, which are sometimes morphologically identical. Basidiomycetes as one of the largest groups of edible mushrooms have become more important in recent times for their medicinal and nutritional properties. Partial rDNA sequences, including the Internal Transcribed Spacer I-5.8SrDNA-Internal Transcribed Spacer II, were used in this study for molecular identification and assessment of phylogenetic relationships between selected edible species of the Basidiomycetes. Phylogenetic trees showed five distinct clades; each clade belonging to a separate family group. The first clade included all the species belonging to the Pleurotaceae (Pleurotus spp.) family; similarly, the second, third, fourth, and fifth clades consist of species from the Agaricaceae (Agaricus sp.), Lyophllaceae (Hypsigygus sp.), Marasmiaceae (Lentinula edodes sp.) and Physalacriaceae (Flammulina velutipes sp.) families, respectively. Moreover, different species of each family were clearly placed in a distinct sub-cluster and a total of 13 species were taken for analysis. Species differentiation was re-confirmed by AMOVA analysis (among the populations: 99.67%; within: 0.33%), nucleotide divergence, haplotyping and P value. Polymorphism occurred throughout the ITS regions due to insertion-deletion and point mutations, and can be clearly differentiated within the families as well as genera. Moreover, this study proves that the sequence of the ITS region is a superior molecular DNA barcode for taxonomic identification of Basidiomycetes.
Differential transcriptome analysis of the disease tolerant Madagascar-Malaysia crossbred black tiger shrimp, Penaeus monodon hepatopancreas in response to acute hepatopancreatic necrosis disease (AHPND) infection: inference on immune gene response and interaction

Soo TCC, Devadas S, Mohamed Din MS, Bhassu S

Gut Pathog, 2019;11:39.
PMID: 31372182 DOI: 10.1186/s13099-019-0319-4

Background: Penaeus monodon is the second most widely cultured marine shrimp species in the global shrimp aquaculture industry. However, the growth of P. monodon production has been constantly impaired by disease outbreaks. Recently, there is a lethal bacterial infection, known as acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus AHPND strain (VpAHPND), which led to mass mortalities in P. monodon. Unfortunately, there is still insufficient knowledge about the underlying immune response of P. monodon upon AHPND infection. The present study aims to provide an insight into the antibacterial immune response elicited by P. monodon hepatopancreas towards AHPND infection.
Methods: We have employed high-throughput RNA-Seq technology to uncover the transcriptome changes of P. monodon hepatopancreas when challenged with VpAHPND. The shrimps were challenged with VpAHPND through immersion method with dissected hepatopancreas samples for the control group (APm-CTL) and treatment group at 3 (APm-T3), 6 (APm-T6), and 24 (APm-T24) hours post-AHPND infection sent for RNA-Seq. The transcriptome de novo assembly and Unigene expression determination were conducted using Trinity, Tgicl, Bowtie2, and RSEM software. The differentially expressed transcripts were functionally annotated mainly through COG, GO, and KEGG databases.
Results: The sequencing reads generated were filtered to obtain 312.77 Mb clean reads and assembled into 48662 Unigenes. Based on the DEGs pattern identified, it is inferred that the PAMPs carried by VpAHPND or associated toxins are capable of activating PRRs, which leads to subsequent pathway activation, transcriptional modification, and antibacterial responses (Phagocytosis, AMPs, proPO system). DAMPs are released in response to cell stress or damage to further activate the sequential immune responses. The comprehensive interactions between VpAHPND, chitin, GbpA, mucin, chitinase, and chitin deacetylase were postulated to be involved in bacterial colonization or antibacterial response.
Conclusions: The outcomes of this research correlate the different stages of P. monodon immune response to different time points of AHPND infection. This finding supports the development of biomarkers for the detection of early stages of VpAHPND colonization in P. monodon through host immune expression changes. The potential genes to be utilized as biomarkers include but not limited to C-type lectin, HMGB1, IMD, ALF, serine proteinase, and DSCAM.
Fulltext Pollination implications of the diverse diet of tropical nectar-feeding bats roosting in an urban cave

Lim VC, Ramli R, Bhassu S, Wilson JJ

PeerJ, 2018;6:e4572.
PMID: 29607265 DOI: 10.7717/peerj.4572

Background: Intense landscaping often alters the plant composition in urban areas. Knowing which plant species that pollinators are visiting in urban areas is necessary for understanding how landscaping impacts biodiversity and associated ecosystem services. The cave nectar bat,Eonycteris spelaea, is an important pollinator for many plants and is often recorded in human-dominated habitats. Previous studies of the diet ofE. spelaearelied on morphological identification of pollen grains found in faeces and on the body of bats and by necessity disregarded other forms of digested plant material present in the faeces (i.e., plant juice and remnants). The main objective of this study was to examine the diet of the nectarivorous bat,E. spelaea,roosting in an urban cave at Batu Caves, Peninsular Malaysia by identifying the plant material present in the faeces of bats using DNA metabarcoding.
Methods: Faeces were collected under the roost ofE. spelaeaonce a week from December 2015 to March 2016. Plant DNA was extracted from the faeces, Polymerase chain reaction (PCR) amplified atITS2andrbcLregions and mass sequenced. The resultant plant operational taxonomic units were searched against NCBI GenBank for identification.
Results: A total of 55 species of plants were detected from faeces ofE. spelaeaincludingArtocarpus heterophyllus, Duabanga grandifloraandMusaspp. which are likely to be important food resources for the cave nectar bat.
Discussion: Many native plant species that had not been reported in previous dietary studies ofE. spelaeawere detected in this study includingBauhinia strychnoideaandUrophyllum leucophlaeum, suggesting thatE. spelaearemains a crucial pollinator for these plants even in highly disturbed habitats. The detection of many introduced plant species in the bat faeces indicates thatE. spelaeaare exploiting them, particularlyXanthostemon chrysanthus,as food resources in urban area. Commercial food crops were detected from all of the faecal samples, suggesting thatE. spelaeafeed predominantly on the crops particularly jackfruit and banana and play a significant role in pollination of economically important plants. Ferns and figs were also detected in the faeces ofE. spelaeasuggesting future research avenues to determine whether the 'specialised nectarivorous'E. spelaeafeed opportunistically on other parts of plants.
Fulltext Effect of Blastocystis sp. in dengue patients-Increase in the treatment cost and exacerbation of symptoms

Thergarajan G, Kumar S, Bhassu S, Omar SFBS, Rampal S

PLoS One, 2019;14(3):e0211034.
PMID: 30893309 DOI: 10.1371/journal.pone.0211034

Increasing incidences of dengue have become a global health threat with major clinical manifestation including high fever and gastrointestinal symptoms. These symptoms were also expressed among Blastocystis sp. infected individuals, a parasite commonly seen in human stools. This parasite has been previously reported to replicate faster upon exposure to high temperature. The present study is a hospitalized-based cross-sectional study involved the collection of faecal sample from dengue patients. Stool examination was done by in vitro cultivation to isolate Blastocystis sp. Growth pattern of all the positive isolates were analyzed to identify the multiplication rate of Blastocystis sp. isolated from dengue patients. Distribution of Blastocystis sp. among dengue patients was 23.6%. Dengue patients who were positive for Blastocystis sp. infection denoted a significantly higher fever rate reaching 38.73°C (p<0.05) compared to the non-Blastocystis sp. infected patients (38.44°C). It was also found that Blastocystis sp. infected patients complained of frequenting the toilet more than five times a day (p<0.05) compared to those who were non-Blastocystis sp. infected. At the same time, the duration of hospitalization was significantly longer (p<0.05) for Blastocystis sp. infected dengue patients compared to the non-Blastocystis sp. infected patients. Besides, Blastocystis sp. isolated from dengue patients (in vivo thermal stress) showed a higher growth rate compared to the non-dengue isolated which was exposed to high temperature (in vitro thermal stress). Our findings suggest that presence of Blastocystis sp. during dengue infection could trigger the increase of temperature which could be due to highly elevated pro inflammatory cytokines by both parasitic and virus infection. This could justify why the temperature in Blastocystis sp. infected dengue patients is higher compared to the non-Blastocystis sp. infected patients. Higher temperature could have triggered a greater parasite multiplication rate that contributed to the aggravation of the gastrointestinal symptoms.
Fulltext MicroRNA Regulation in Infectious Diseases and Its Potential as a Biosensor in Future Aquaculture Industry: A Review

SiouNing AS, Seong TS, Kondo H, Bhassu S

Molecules, 2023 May 26;28(11).
PMID: 37298833 DOI: 10.3390/molecules28114357

An infectious disease is the most apprehensive problem in aquaculture as it can lead to high mortality in aquatic organisms and massive economic loss. Even though significant progress has been accomplished in therapeutic, prevention, and diagnostic using several potential technologies, more robust inventions and breakthroughs should be achieved to control the spread of infectious diseases. MicroRNA (miRNA) is an endogenous small non-coding RNA that post-transcriptionally regulates the protein-coding genes. It involves various biological regulatory mechanisms in organisms such as cell differentiation, proliferation, immune responses, development, apoptosis, and others. Furthermore, an miRNA also acts as a mediator to either regulate host responses or enhance the replication of diseases during infection. Therefore, the emergence of miRNAs could be potential candidates for the establishment of diagnostic tools for numerous infectious diseases. Interestingly, studies have revealed that miRNAs can be used as biomarkers and biosensors to detect diseases, and can also be used to design vaccines to attenuate pathogens. This review provides an overview of miRNA biogenesis and specifically focuses on its regulation during infection in aquatic organisms, especially on the host immune responses and how miRNAs enhance the replication of pathogens in the organism. In addition to that, we explored the potential applications, including diagnostic methods and treatments, that can be employed in the aquaculture industry.
Fulltext Molecular divergence and species delimitation of the cultivated oyster mushrooms: integration of IGS1 and ITS

Avin FA, Bhassu S, Tan YS, Shahbazi P, Vikineswary S

ScientificWorldJournal, 2014;2014:793414.
PMID: 24587752 DOI: 10.1155/2014/793414

Identification of edible mushrooms particularly Pleurotus genus has been restricted due to various obstacles. The present study attempted to use the combination of two variable regions of IGS1 and ITS for classifying the economically cultivated Pleurotus species. Integration of the two regions proved a high ability that not only could clearly distinguish the species but also served sufficient intraspecies variation. Phylogenetic tree (IGS1+ITS) showed seven distinct clades, each clade belonging to a separate species group. Moreover, the species differentiation was tested by AMOVA and the results were reconfirmed by presenting appropriate amounts of divergence (91.82% among and 8.18% within the species). In spite of achieving a proper classification of species by combination of IGS1 and ITS sequences, the phylogenetic tree showed the misclassification of the species of P. nebrodensis and P. eryngii var. ferulae with other strains of P. eryngii. However, the constructed median joining (MJ) network could not only differentiate between these species but also offer a profound perception of the species' evolutionary process. Eventually, due to the sufficient variation among and within species, distinct sequences, simple amplification, and location between ideal conserved ribosomal genes, the integration of IGS1 and ITS sequences is recommended as a desirable DNA barcode.
Fulltext A checklist of the bats of Peninsular Malaysia and progress towards a DNA barcode reference library

Lim VC, Ramli R, Bhassu S, Wilson JJ

PLoS One, 2017;12(7):e0179555.
PMID: 28742835 DOI: 10.1371/journal.pone.0179555

Several published checklists of bat species have covered Peninsular Malaysia as part of a broader region and/or in combination with other mammal groups. Other researchers have produced comprehensive checklists for specific localities within the peninsula. To our knowledge, a comprehensive checklist of bats specifically for the entire geopolitical region of Peninsular Malaysia has never been published, yet knowing which species are present in Peninsular Malaysia and their distributions across the region are crucial in developing suitable conservation plans. Our literature search revealed that 110 bat species have been documented in Peninsular Malaysia; 105 species have precise locality records while five species lack recent and/or precise locality records. We retrieved 18 species from records dated before the year 2000 and seven species have only ever been recorded once. Our search of Barcode of Life Datasystems (BOLD) found that 86 (of the 110) species have public records of which 48 species have public DNA barcodes available from bats sampled in Peninsular Malaysia. Based on Neighbour-Joining tree analyses and the allocation of DNA barcodes to Barcode Index Number system (BINs) by BOLD, several DNA barcodes recorded under the same species name are likely to represent distinct taxa. We discuss these cases in detail and highlight the importance of further surveys to determine the occurences and resolve the taxonomy of particular bat species in Peninsular Malaysia, with implications for conservation priorities.
Virus-like particles of Macrobrachium rosenbergii nodavirus produced in bacteria

Goh ZH, Tan SG, Bhassu S, Tan WS

J Virol Methods, 2011 Jul;175(1):74-9.
PMID: 21536072 DOI: 10.1016/j.jviromet.2011.04.021

Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.

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