Annie Christianus

Use of intestinal Pseudomonas aeruginosa in fish to detect the environmental pollutant benzo[a]pyrene

This study examined the potential of Pseudomonas aeruginosa abundance in the intestines of fish as an indicator of exposure to benzo[a]pyrene (BaP). P. aeruginosa populations were enumerated in juvenile African catfish (Clarias gariepinus) injected intramuscularly three days previous with 0, 10, 30, 40, 50 or 70mg/kg of BaP. Hepatic EROD and GST activities and biliary fluorescent aromatic compounds (FACs) 1-OH BaP, 3-OH BaP, 7,8-D BaP and BaP were quantified to investigate agreements between the new indicator and established fish biomarkers. The shape of bacterial population (logarithm of colony-forming unit) dose-response curve generally matched those of biliary FACs concentrations. Conversely, the EROD and GST dose-response curves were generally the mirror images of the bacterial population curve. Changes in intestinal P. aeruginosa population appear to be an indirect effect of BaP exposure because exposure to 0-100μg/ml BaP had no effect on P. aeruginosa populations grown on agar plates containing BaP. Using intestinal P. aeruginosa population of fish as a universal indicator of BaP pollution in aquatic environments is discussed.Conversely, the EROD and GST dose-response curves were generally the mirror images of the bacterial population curve.

The effects of intramuscular and intraperitoneal injections of benzo[a]pyrene on selected biomarkers in Clarias gariepinus

This study investigated the dose-dependent and time-course effects of intramuscular (i.m.) and intraperitoneal (i.p.) injection of benzo[a]pyrene (BaP) on the biomarkers EROD activity, GST activity, concentrations of BaP metabolites in bile, and visceral fat deposits (Lipid Somatic Index, LSI) in African catfish (Clarias gariepinus). Intraperitoneal injection resulted in 4.5 times higher accumulation of total selected biliary FACs than i.m. injection. Hepatic GST activities were inhibited by BaP via both injection methods. Dose-response relationships between BaP injection and both biliary FAC concentrations and hepatic GST activities were linear in the i.p. injected group but nonlinear in the i.m. injected fish. Hepatic EROD activity and LSI were not significantly affected by BaP exposure by either injection route. We conclude that i.p. is a more effective route of exposure than i.m. for future ecotoxicological studies of PAH exposure in C. gariepinus.

Bacteria flora associated with different body parts of hatchery reared juvenilePenaeus monodon, tanks water and sediment

Bacteria flora of intestine and hepatopancreas, body surface and muscles of juvenilePenaeus monodon along with its rearing water and sediment was analyzed. Juvenile shrimp were reared in four tanks in the Hatchery complex, Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia. Water quality parameters were measured every day. Samples were collected aseptically and homogenized before being inoculated in Tryptone Soy agar, Thiosulphate Citrate Bile Salt agar, MacConkey agar andPseudomonas-isolating agar. There was no significant difference between water quality parameters and shrimp body weight of replicate tanks. Total plate count for water and totalVibrio count for rearing water and digestive system were within previous reported ranges. Eight different genera were isolated in which 7 genera were identified. Gram negative bacteria were dominant (72%)Vibrio was the most dominant genera followed byShewanella andBurkholderia. Clavibacter followed byStaphylococcus were the most dominant gram positive bacteria. No coliform bacteria was detected in the shrimp body parts and rearing environment. Incidence ofShewanella in the digestive system was significantly higher than sediment, rearing water and muscles. This may be implied its ability to colonize in the digestive tract of juvenileP. monodon.

Ovarian development of a river catfish Hemibagrus nemurus (Valenciennes, 1840) in captivity.

Hemibagrus nemurus is a riverine catfish with high economic and nutritive values. Investigations on ovarian development of this fish were carried out to determine the mode of ovarian development and describe the oocyte developmental stages. Histological studies were done on ovaries using light microscopy and scanning electron microscopy. Fish were sampled monthly for a period of six months (August 2009 to January 2010). The mean oocyte diameter (OD) ranged from 871 ± 161.41 µm to 1,167 ± 26.77 µm and the highest OD was in November. Oocyte size-frequency distribution showed a polymodal distribution. The mean gonadosomatic index (GSI) ranged from 1.14 ± 0.87% to 7.06 ± 1.40% and highest GSI was in November. The ovaries exhibited three phases of oocyte growth, which were primary growth, secondary growth and maturation phases. Based on histological criteria, the oocyte developmental stages were divided into seven stages as chromatin nucleolar, early perinucleolar, late perinucleolar, cortical alveolar, vitellogenesis, mature oocyte and germinal vesicle migration stages. All the seven stages of oocyte development were observed in the ovaries. Oogonia were always present throughout the developmental stages. The ovaries had more than two stages of oocyte development. This is the first report on the mode of ovarian development of H. nemurus. These findings indicated that H. nemurus has asynchronous mode of ovarian development and is capable of spawning several times in a year under favourable conditions.

Artificial neural network modeling of biomarkers to infer characteristics of contaminant exposure in Clarias gariepinus

This study examined the potential of artificial neural network (ANN) modeling to infer timing, route and dose of contaminant exposure from biomarkers in a freshwater fish. Hepatic glutathione S-transferase (GST) activity and biliary concentrations of BaP, 1-OH BaP, 3-OH BaP and 7,8D BaP were quantified in juvenile Clarias gariepinus injected intramuscularly or intraperitoneally with 10-50 mg/kg benzo[a]pyrene (BaP) 1-3 d earlier. A feedforward multilayer perceptron (MLP) ANN resulted in more accurate prediction of timing, route and exposure dose than a linear neural network or a radial basis function (RBF) ANN. MLP sensitivity analyses revealed contribution of all five biomarkers to predicting route of exposure but no contribution of hepatic GST activity or one of the two hydroxylated BaP metabolites to predicting time of exposure and dose of exposure. We conclude that information content of biomarkers collected from fish can be extended by judicious use of ANNs.

Evaluation of in vitro Vibrio static activity of Shewanella algae isolated from healthy Penaeus monodon

To conquer disease problem in shrimp industries, probiotic biocontrol is a well known remedy now. The antagonistic ability of separated isolates from different parts of juvenile Penaeus monodon were screened against shrimp Vibrio pathogens; Vibrio parahaemolyticus and Vibrio alginolyticus. The most antagonistic effect was observed for an isolate that was primarily identified as Shewanella algae using conventional methods followed by Biolog microlog software. Since production of antagonistic agents rely on cultural conditions, antagonistic ability of candidate probioic against the mentioned Vibrios was assessed using Response Surface Methodology, with central composite design in which four independents variables were assumed: temperature (10 - 50°C), pH (6 -10), NaCl concentration (0 - 50%) and time (12 – 60 h). The coefficients of multiple determinations (R2), for the responses of antagonistic effect of S. algae against V. parahaemolyticus and V. alginolyticus values were 0.807 and 0.805, respectively. Concentration of the NaCl exhibited least influence on the antibacterial effect of candidate probiotic while the other independent variables exhibited different degree of affect. The candidate probiotic revealed a reasonable antibacterial response in quite a wide range of temperature and pH in which the maximum levels were in the same range of optimum shrimp culture.

Isolation by the 5′ anchored PCR technique and characterization of eighteen microsatellite loci in horseshoe crab (Tachypleus gigas)

The horseshoe crab, a well-known ‘living fossil’, is represented by two species (Tachypleus gigas and Carcinoscorpius rotundicauda) in peninsular Malaysia. One of these, T. gigas inhabits shallow marine waters and migrates to intertidal sandy beaches for breeding during high tides at every full and new moon throughout the year (Hajeb et al. 2009). This species is being used as bait by local fishermen to catch crabs or shrimps by netting or cage fishing methods. Due to its low commercial value in comparison to other marine economic species such as fishes, crabs and shrimps, most of the animals caught in fishing nets or cages are released immediately back to sea by fishermen or left to die in the sun. In certain states of Malaysia, such as Kelantan, Penang and Johore, adult female horseshoe crabs are sold to local seafood restaurants and in fish markets due to consumption of their eggs by the local people. Horseshoe crab populations are rapidly declining around the world due to various reasons such as water pollution, loss of their living and spawning habitats and human exploitation of adult horseshoe crabs (Li et al. 2009; Shin et al. 2009). Assessing the genetic variability of horseshoe crab is important as it has direct benefit for the conservation and management of wild populations of the species. However, there is no information available on the population genetic structure and diversity of T. gigas in Malaysia. To address this research need, we have isolated and developed the first set of polymorphic microsatellite markers for T. gigas by using the 5′ anchored PCR technique. A total of 130 individuals of horseshoe crabs were collected from five locations along the coastal areas of peninsular Malaysia, namely Port Dickson (Negeri Sembilan), Pantai Bersih (Penang), Sungai Muar (Johor), Pantai Balok

Bacteria and Fungi Identified on Horseshoe Crabs, Tachypleus gigas and Carcinoscorpius rotundicauda in the Laboratory

In Malaysia, reduction of horseshoe crab sightings in their natural habitat has prompted studies on the ecology of this invertebrate and potential threats to its survival. In this study, eggs and newly hatched Tachypleus gigas, collected from a natural spawning ground in Banting, Selangor (Malaysia), were conditioned and cultured in the laboratory to test the potential of fungal or bacterial infections to occur and impair development and growth. During the culture period, evidence of bacterial and fungal infestation was observed. Identified species included bacteria (Shewanella putrefaciens, Bacillus cereus, Corynebacterium sp. and Enterococcus faecalis) and fungi (Aspergillus sp., Aspergillus niger, Peniclillium sp., Gliocladium sp.). Eggs infected with bacteria or fungi usually turned reddish, grey or black and ceased to develop. Larvae became passive and coated with black spore and in some cases died soon after molting into the larval stage. The species with greatest potential to affect the growth and survival of horseshoe crab eggs and larvae sampled in this study were S. putrefaciens (a Gram-negative bacterium) and A. niger (a fungi). The suite of species identified during this study were different from those previously identified on horsehose crabs in culture, suggesting possible species, location, and/or condition-specific variation in communities of infectious microbes brought in from the natural environment and propagated in culture. Infected horseshoe crabs, therefore, may require different management or treatment strategies and additional study.