Isolation and Characterisation of Culturable Thermophilic Cyanobacteria from Perak Hot Springs and their Plant Growth Promoting Properties Effects on Rice Seedlings (Oryza sativa L.).

Malaysia is home to a number of hot springs that are rich in microbial diversity including the photosynthetic cyanobacteria. Although this microbial community has been characterised based on metagenomics approach, the culturable thermophilic isolates have not been isolated and characterised extensively. Compared to the mesophiles, information on plant growth promoting (PGP) properties of these thermophiles remain largely untapped. As the amount of arable land for microbial bioprospecting is decreasing due to extensive human activities, the search for alternative source for microbial strains with PGP properties is important for the development of potential biofertilisers. This study sought to isolate and characterise culturable cyanobacteria strains from two local hot springs - Sungai Klah (SK) and Lubuk Timah (LT) located in Perak using morphological and molecular methods. The IAA production from the axenic cultures were measured. The PGP properties were also measured by priming the rice seeds with cyanobacterial water extracts. A total of six strains were isolated from both hot springs. Strains LTM and LTW from LT were identified as Leptolyngbya sp. whereas strains SEM, SEH, STH and STM were identified as Thermosynechococcus elongatus. All six strains produced IAA ranged from 670.10 pg/µL to 2010 pg/µL. The water extracts were found to increase the seed amylase activity of the rice seeds from 5th day of germination (DAG) to 10th DAG. In general, the IAA production and increased seed amylase activity might have contributed in enhancing the longest root length, shoot length and root-to-shoot (RS) ratio. To conclude, the thermophilic cyanobacteria from hot springs can be further exploited as a novel source of PGP microbes for the development of biofertilsers.

Development of the muscular and nervous systems during the larval ontogeny of the stalked barnacle, Octolasmis angulata Aurivillius 1894 (Cirripedia: Thoracicalcerea: Poecilasmatidae).

The advancements in microscopic techniques have stimulated great interest in the muscular and neural architectures of invertebrates, specifically using muscle and neural structures to infer phylogenetic relationships. Here, we provide the data on the development of the muscular and nervous systems during the larval development of stalked barnacle, Octolasmis angulata using the phalloidin F-actin and immunohistochemical labelling (e.g. acetylated α-tubulin and serotonin) and confocal laser scanning microscopy analysis. All naupliar stages shared the same muscle and neural architectures with only the discrepancy in size. The nauplii have a complex muscle arrangement in their feeding apparatus and naupliar appendages. Most naupliar muscles undergo histolyse during the cyprid metamorphosis. The cyprid muscles form beneath the head shield at the end of nauplius VI. The naupliar and cyprid central nervous systems exhibit the typical tripartite brain comprising the protocerebrum, deutocerebrum and tritocerebrum. The serotonin-like immunoreactivity is mainly found in the naupliar brain, mandibular ganglia, cyprid brain and posterior ganglia. Our study revealed that numerous muscle and neural architectures in the naupliar and cyprids have phylogenetic significance, but future studies on the myoanatomy and neuroanatomy of other barnacle species are necessary to determine the homology of these structures.

Targeting PirAvp and PirBvp Toxins of Vibrio parahaemolyticus with Oilseed Peptides: An In Silico Approach.

Acute hepatopancreatic necrosis disease (AHPND), caused by PirAvp- and PirBvp-releasing Vibrio parahaemolyticus strains, has resulted in massive mortality in shrimp aquaculture. Excessive use of antibiotics for AHPND management has led to antibiotic resistance, highlighting the urgency to search for alternatives. Using an in silico approach, we aimed to discover PirAvp/PirBvp-binding peptides from oilseed meals as alternatives to antibiotics. To search for peptides that remain intact in the shrimp digestive tract, and therefore would be available for toxin binding, we focused on peptides released from tryptic hydrolysis of 37 major proteins from seeds of hemp, pumpkin, rape, sesame, and sunflower. This yielded 809 peptides. Further screening led to 24 peptides predicted as being non-toxic to shrimp, fish, and humans, with thermal stability and low water solubility. Molecular docking on the 24 peptides revealed six dual-target peptides capable of binding to key regions responsible for complex formation on both PirAvp and PirBvp. The peptides (ISYVVQGMGISGR, LTFVVHGHALMGK, QSLGVPPQLGNACNLDNLDVLQPTETIK, ISTINSQTLPILSQLR, PQFLVGASSILR, and VQVVNHMGQK) are 1139-2977 Da in mass and 10-28 residues in length. Such peptides are potential candidates for the future development of peptide-based anti-AHPND agents which potentially mitigate V. parahaemolyticus pathogenesis by intercepting PirAvp/PirBvp complex formation.

Synergistic Antimicrobial Effect of a Seaweed-Probiotic Blend Against Acute Hepatopancreatic Necrosis Disease (AHPND)-Causing Vibrio parahaemolyticus.

The outbreak of acute hepatopancreatic necrosis disease (AHPND) has caused great economic losses to the shrimp culture sector. However, the use of antibiotics to fight this disease has resulted in negative impacts on human health and the environment. Thus, the use of natural alternatives to antibiotics may be a better solution. In this study, four Bacillus species obtained from the guts of shrimps (Fenneropenaeus penicillatus and Penaeus monodon) showed antimicrobial activity against the AHPND-causing Vibrio parahaemolyticus strain 3HP using the cross-streaking and agar spot methods. Two of the Bacillus isolates, B2 and BT, also showed good probiotic properties, exhibiting tolerance to bile, good adhesion to shrimp mucus, non-hemolytic, susceptibility to antibiotics and being safe towards hosts. Moreover, a seaweed-probiotic blend (a combination of Bacillus B2 and 20 mg/ml of the red seaweed Gracilaria sp.) exhibited synergistic in vitro inhibition against V. parahaemolyticus strain 3HP, with an observed inhibition zone of 5.0 mm. The broth co-culture experiment results further indicated that the seaweed-probiotic blend inhibited V. parahaemolyticus through competitive exclusion. The in vivo challenge trials also confirmed that this seaweed-probiotic blend significantly reduced the mortality of shrimps post-challenge with the AHPND-causing V. parahaemolyticus strain 3HP (p < 0.05) compared to the negative control (mortality rate = 13.88% vs 72.19%). Thus, this seaweed-probiotic blend may serve as an alternative to antibiotics in controlling the outbreak of AHPND.

First development of the Malaysian River Integrity Index (MyRII) based on biological, chemical and physical multi-metrics.

Anthropogenic pressures are causing substantial degradation to the freshwater ecosystems globally and Malaysia has not escaped such a bleak scenario. Prompted by the predicament, this study's objective was to pioneer a river assessment system that can be readily adopted to monitor, manage and drive improvement in a wholesome manner. Three sets of a priori metrics were selected to form the Ichthyofaunal Quality Index (IQI: biological), Water Quality Index (WQI: chemical) and River Physical Quality Index (RPQI: physical). These indices were further integrated on equal weighting to construct a novel Malaysian River Integrity Index (MyRII). To test its robustness, the MyRII protocol was field tested in four eco-hydrological zones located in the Kampar River water basin for 18 months to reveal its strengths, weaknesses, and establish the "excellent", "good", "average", "poor" and "impaired" thresholds based on the "best performer" reference site in an empirical manner. The resultant MyRII showed a clear trend that corresponded with different levels of river impairment. Test site zone A which was a reference site with minimal disturbance achieved the highest MyRII (88.95 ± 4.29), followed by partially disturbed zone B (61.95 ± 5.90) and heavily disturbed zone C (50.00 ± 4.29). However, the MyRII in zone D (59.9 ± 6.39), which was a heavily disturbed wetland that was disjointed from the river, did not conform to such trend. Also unveiled and recognized, however, are some unexpected nuances, limitations and challenges that emerged from this study. These are critically discussed as precautions when interpreting and implementing the MyRII protocol. This study adds to the mounting body of evidence that water resource stakeholders and policymakers must look at the big picture and adopt the "balanced ecosystem" mind-set when assessing, restoring and managing the rivers as a freshwater resource.

Water quality variation during a strong El Niño event in 2016: a case study in Kampar River, Malaysia.

El Niño and Southern Oscillation (ENSO) is a natural forcing that affects global climate patterns, thereon influencing freshwater quality and security. In the advent of a strong El Niño warming event in 2016 which induced an extreme dry weather in Malaysia, water quality variation was investigated in Kampar River which supplies potable water to a population of 92,850. Sampling points were stratified into four ecohydrological units and 144 water samples were examined from October 2015 to March 2017. The Malaysian Water Quality Index (WQI) and some supplementary parameters were analysed in the context of reduced precipitation. Data shows that prolonged dry weather, episodic and sporadic pollution incidents have caused some anomalies in dissolved oxygen (DO), total suspended solids (TSS), turbidity and ammoniacal nitrogen (AN) values recorded and the possible factors are discussed. The month of March and August 2016 recorded the lowest precipitation, but the overall resultant WQI remained acceptable. Since the occurrence of a strong El Niño event is infrequent and far between in decadal time scale, this paper gives some rare insights that may be central to monitoring and managing freshwater resource that has a crucial impact to the mass population in the region of Southeast Asia.

First evidence for temporary and permanent adhesive systems in the stalked barnacle cyprid, Octolasmis angulata.

Although there have been extensive studies on the larval adhesion of acorn barnacles over the past few decades, little is known about stalked barnacles. For the first time, we describe the larval adhesive systems in the stalked barnacle, Octolasmis angulata and the findings differ from previous reports of the temporary (antennulary) and cement glands in thoracican barnacles. We have found that the temporary adhesives of cyprid are produced by the clustered temporary adhesive glands located within the mantle, instead of the specialised hypodermal glands in the second antennular segment as reported in the acorn barnacles. The temporary adhesive secretory vesicles (TASV) are released from the gland cells into the antennule via the neck extensions of the glands, and surrounded with microtubules in the attachment disc. Cement glands undergo a morphological transition as the cyprid grows. Synthesis of the permanent adhesives only occurs during the early cyprid stage, and is terminated once the cement glands reach maximum size. Evidence of the epithelial invaginations on the cement glands supports the involvement of exocytosis in the secretion of the permanent adhesives. This study provides new insight into the larval adhesives system of thoracican barnacles.

Gill monogeneans of Nile tilapia (Oreochromis niloticus) and red hybrid tilapia (Oreochromis spp.) from the wild and fish farms in Perak, Malaysia: infection dynamics and spatial distribution.

Tilapia is one of the commercially important fish in Malaysia as well as in other parts of the world. An understanding of monogenean infection dynamics in tilapia fish may assist us in searching for some intervention measures in reducing the loss of fish caused by parasitic diseases. The present study aimed (1) to compare infection level of monogeneans between the wild and cultured Oreochromis niloticus, and between the cultured O. niloticus and cultured red hybrid tilapia, and (2) to examine the spatial distribution of monogenean species over the gills of the different host species. From a total of 75 fish specimens, six species of monogeneans from two genera: Cichlidogyrus (C. halli, C. mbirizei, C. sclerosus, C. thurstonae, C. tilapiae) and Scutogyrus (S. longicornis) were identified. Data showed that the infection level of cultured O. niloticus was higher than that of the wild O. niloticus, however, the former was lower than that of the cultured red hybrid tilapia. Higher species richness of monogeneans was observed in the cultured red hybrid tilapia as compared to the others. Results for spatial distribution showed that the monogeneans have no preference on the left or right sides of the gills. However, C. halli, C. mbirizei, and C. tilapiae showed preferences on specific gill arches in the cultured O. niloticus and red hybrid tilapia. In general, the gill arch IV harboured the least number of monogeneans. The susceptibility of monogenean infection between the different types of tilapia is discussed.

Ultrastructure of head organs (anterior adhesive apparatus) and posterior secretory systems of Caballeria liewi Lim, 1995 (Monogenea, Ancyrocephalidae).

Caballeria liewi Lim, 1995, uses adhesive secretions from the head organs and posterior secretory systems to assist in locomotion and attachment. Ultrastructural investigations show that the head organs of C. liewi consist of three pairs of antero-lateral pit-like openings bearing microvilli and ducts leading from two types of uninucleated gland cells (located lateral to the pharynx), one type producing rod-like (S1) bodies with an electron-dense matrix containing less electron-dense vesicles and the second type producing oval (S2) bodies with a homogeneous electron-dense matrix. Interlinking band-like structures are observed between S1 bodies and between S2 bodies. S1 body is synthesised in the granular endoplasmic reticulum, transported to a Golgi complex to be packaged into vesicles and routed into ducts for exudation. The synthesis of the S2 body is unresolved. Haptoral secretions manifested externally as net-like structures are derived from dual electron-dense (DED) secretory body produced in the peduncular gland cells. The DED body consists of a less electron-dense oval core in a homogeneous electron-dense matrix. On exocytosis into the pyriform haptoral reservoir, DED bodies are transformed into a secretion with two types of inclusions (less electron-dense oval and electron-dense spherical inclusions) in an electron-dense matrix. The secretions are further transformed (as small, oval, electron-dense bodies) when transported to the superficial anchor grooves, and on exudation into the gill tissues, the secretions become an electron-dense matrix. Secretory bodies associated with uniciliated structures, anchor sleeves and marginal hooks are also observed.

Isolation and identification of gastrointestinal microbiota from the short-nosed fruit bat Cynopterus brachyotis brachyotis.

Studies on the microbial ecology of gut microbiota in bats are limited and such information is necessary in determining the ecological significance of these hosts. Short-nosed fruit bats (Cynopterus brachyotis brachyotis) are good candidates for microbiota studies given their close association with humans in urban areas. Thus, this study explores the gut microbiota of this species from Peninsular Malaysia by means of biochemical tests and 16S rRNA gene sequences analysis. The estimation of viable bacteria present in the stomach and intestine of C. b. brachyotis ranged from 3.06×10(10) to 1.36×10(15)CFU/ml for stomach fluid and 1.92×10(10) to 6.10×10(15)CFU/ml for intestinal fluid. A total of 34 isolates from the stomach and intestine of seven C. b. brachyotis were retrieved. A total of 16 species of bacteria from eight genera (Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Pantoea, Pseudomonas and Serratia) were identified, Enterobacteriaceae being the most prevalent, contributing 12 out of 16 species isolated. Most isolates from the Family Enterobacteriaceae have been reported as pathogens to humans and wildlife. With the possibility of human wildlife transmission, the findings of this study focus on the importance of bats as reservoirs of potential bacterial pathogens.

Attachment ability of a clamp-bearing fish parasite, Diplozoon paradoxum (Monogenea), on gills of the common bream, Abramis brama.

Monogeneans, which are mainly fish ectoparasites, use various types of haptoral (posterior) attachment apparatus to secure their attachment onto their hosts. However, it remains unclear how strongly a monogenean can attach onto its host. In the present study, we aimed for the first time to (1) measure pull-off forces required to detach a pair of clamp-bearing monogeneans, Diplozoon paradoxum, from gills of Abramis brama and (2) determine the contribution of muscles to the clamp movements. A mean force of 6.1±2.7 mN (~246 times the animals' weight) was required to dislodge a paired D. paradoxum vertically from the gills. There were significant differences (P<0.05, Tukey test) between the widths of clamp openings in D. paradoxum treated in three different solutions: the widest clamp openings were observed in the monogeneans treated in 100 mmol l(-1) potassium chloride solution (58.26±13.44 µm), followed by those treated in 20 mmol l(-1) magnesium chloride solution (37.91±7.58 µm), and finally those treated in filtered lake water (20.16±8.63 µm). This suggests that the closing of the clamps is probably not due to the continuous contraction of extrinsic muscles but is caused by the elasticity of the clamp material and that muscle activity is required for clamp opening.

Resilin-like protein in the clamp sclerites of the gill monogenean Diplozoon paradoxum Nordmann, 1832.

Resilin is a soft and elastic protein, which is found in many exoskeletal structures of arthropods. Proteins with similar chemical properties have been described for other invertebrates including monogenean fish parasites. However, for the latter taxon no clear microscopic evidence for a resilin-like protein has been shown so far. Here, we present the results of detailed microscopic analyses of the clamp sclerites (attachment devices) of the monogenean Diplozoon paradoxum. Toluidine blue, which is known to stain resilin, selectively stained the material in the clamp sclerites. In addition, when exposed to UV light, this material exhibited an intense blue autofluorescence. The emission spectrum of this autofluorescence has its maximum at 424 nm and is nearly identical to emission spectra of blue autofluorescences observed in 2 well-studied structures containing high proportions of resilin in the exoskeleton of the locust Schistocerca gregaria. The results strongly indicate that the sclerite material of D. paradoxum contains a resilin-like protein. The presence of such a protein likely enhances the attachment efficiency of the clamp sclerites and increases their lifetime.