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Fish mycotic disease outbreaks occur due to infections with oomycete pathogens such as Saprolegnia spp. and Aphanomyces invadans, and cause large-scale fish production losses. Despite its negative impact on aquaculture, little is known about the diversity of oomycete pathogens. The aim of this study was to identify the diversity of pathogenic oomycetes causing infections in major aquaculture zones of Mymensingh and Jessore regions in Bangladesh. A total of 449 water and infected fish samples were collected from 28 fish farms in both regions of which 29 samples were able to grow out with mycelia on the Potato Dextrose Agar (PDA)/Glucose Peptone Yeast Agar (GPYA) plates. Sequence database searches using the rRNA Internal Transcribed Spacer (ITS) region revealed that 15 belonged to Pythium spp., 12 were Aphanomyces invadans and two corresponded to Saprolegnia parasitica. Five isolates of Pythium spp. were identified to the species level: one was closely related to Pythium catenulatum, four to Pythium rhizo-oryzae, rest were identified up to genus. The Pythium spp. were only isolated from water samples whereas A. invadans and S. parasitica were found in fish lesions. Phylogenetic analysis revealed that a single A. invadans clone exists in the sampled area. The results obtained confirm the existence of pathogenic oomycetes in Bangladesh fish farms and this will pave future research on diversity, prevention and control measures. Keywords: Oomycete, Saprolegnia, Aphanomyces invadans, Internal transcribed spacer (ITS), Phylogeny

Effects of supplementary feeds on growth, survival, and production of red fin mahseer, Tor tor (Hamilton) was assessed in captive condition under a monoculture system. Experiments were conducted for a period of 12 months where two feeds varying in protein contents were used in treatments. One laboratory formulated feed with 36.67% protein and a commercial feed with 28% protein were characterized as treatment-1 (T1) and treatment-2 (T2), respectively. At the same time water quality parameters were measured. The net increment in weight after 12 months was 671.60 g and 509.80 g in T1 and T2 respectively. The feed conversion ratio (FCR) was 4.72 and 5.26, and the specific growth rate (SGR) was 1.39 and 0.10 % day-1 in T1 and T2 respectively. In both treatments 100% fish were survived and provided the net production (kg/ha) 2807.06 and 2557.78 in T1 and T2, respectively. The water quality parameters were within the suitable range for fish culture. Between the feeds evaluated, feed with 36.67% protein (T1) proved best result considering the growth and production performance of the fish. Overall growth performance of T. tor was satisfactory; however, further research is needed. J. Bangladesh Agril. Univ. 16(3): 533–538, December 2018

Sumithion, an organophosphate pesticide, has been used extensively in agriculture as well as in broodfish and nursery ponds in Bangladesh. Although it is being applied for beneficial purposes but eventually it exhibits some short and long term toxic effects to the aquatic ecosystem. Therefore, the present study investigated the toxic effects of sumithion on the breeding performance and subsequent viability of embryos and larvae of Heteropneustes fossilis. A total of 400 adult H. fossilis were equally stocked in two ponds. Sumithion at the dose of 0.24 ppm was applied for four months every 7 days interval in one pond while the rest one was considered as control. No significant (p>0.05) difference was found in growth between the treated and control H. fossilis; however, fertilization and hatching rates of the eggs obtained from the treated group were significantly (p>0.05) lower than that of control. Subsequently, the embryos and larvae produced from the broods of the control pond were exposed to 5 different concentrations of sumithion (0.5, 1.0, 2.0, 4.0, and 8.0 ppm) against a control (0 ppm). Each concentration was assessed in five replicates having 200 eggs. Data obtained from the sumithion acute toxicity tests were evaluated using the Probit Analysis. The mortality of embryos increased with the increasing sumithion concentrations from 0 to 8.0 ppm and the significant (p>0.05) differences were found only among the treated concentrations above 2.0 ppm. The 24 h LC50 value for embryos was estimated to be 7.803 ppm. Dose-response decreased in hatching success and recorded as 90.8, 85.0, 76.2, 73.0, 65.0, and 51.2 % for control and 5 different concentrations of sumithion, respectively (p

The Patharghata Fishery Ghat in Chittagong is one of the largest fish landing centers in Bangladesh. The study aimed to explore different types of gears and crafts used from the landing center, fish species caught by the gears, and certain degree of socio-economic condition of the fishermen. Data were collected from local fishermen through on the spot inspections, Personal Interview (PI), and Focus Group Discussion (FGD). The gears used from the landing center were classified as Large Mesh Drift Gill Nets (DGNs), Marine Set Bag Nets (MSBNs), and Trammel Nets. Mainly 7 types of DGNs having the mesh sizes ranging from 2.5 to 16 inches had been used by the fishermen of which Lakkha Jal, Lal jal and Char Shuta Jal were the most commonly used DGNs to catch Hilsha, Lakhua, Pomfret etc. Hilsha Shad (Tenualosa ilisha) was the most dominant species and about 60% of different nets were used to catch this species. Only single type of artisanal fishing craft (trawlers) was found in the landing center and is propelled by inboard engine. The capacity of the trawlers varied from 8 to 22 metric tons. In case of socio-economic condition, most of the fishermen belonged to the age groups of 21-30 and 31-40 years; each represented 31% of the total fishermen. About 91% of fishermen were found to be Muslim and rest 9% were Hindu. The average members of the nuclear and joint families were 3.78 and 7.83 per household, respectively; however, these groups of people were being deprived in their society. The study also revealed that about 67% of fishermen had no formal education and 27% had primary and rest 6% had secondary level of education. The annual income of the fishermen ranged from BDT 50,000 to 100,000 and the main expenditure of their earnings were for food (80%), followed by physical treatment (10%) of their family members. They had hardly any savings. Lack of knowledge and government support, and illiteracy were the major problems of the fishermen in the landing center.Asian J. Med. Biol. Res. December 2016, 2(4): 712-726

Seaweed polysaccharides have been widely used as viscosifier, gelling agents, and stabilizer in the various application fields, e.g., food, pharmaceutical, nutraceutical, and chemical industries. Applications of seaweed polysaccharides are further expanding to versatile directions, e.g., biofuels, bioactive compounds, and functional materials for medical and basic researches. Production of functional oligo- and monosaccharides by the use of specific enzymes is also expected to improve the value of seaweed polysaccharides. The enzymes that depolymerize seaweed polysaccharides are distributed largely among seaweed-associating organisms like marine invertebrates and bacteria. Among them, herbivorous marine gastropods such as abalone and sea hare are the most prominent producers of polysaccharide-degrading enzymes. To date, various kinds of polysaccharide-degrading enzymes have been isolated from the digestive fluid and hepatopancreas of these animals. Among them, alginate lyase, β-1,3-glucanase, mannanase, and cellulase are the major constituents of their digestive fluid. In this chapter, the authors describe the general methods for the preparation and activity assay of the gastropod polysaccharide-degrading enzymes and provide basic knowledge for their primary structures.

Previously we isolated the major alginate lyase isozyme LbAly35 from a marine snail Littorina brevicula and showed that this enzyme was significantly heat stable in a broad pH range compared with other molluscan alginate lyases (Hata et al., Fish Sci 75:755–763, 2009). LbAly35 showed practically no similarity to other molluscan alginate lyases in the N-terminal amino-acid sequence of 20 residues and no cross-reactivity with anti-abalone alginate lyase antiserum. These led us to consider that the primary structure of LbAly35 is considerably deviated from other molluscan enzymes. Thus, in the present study, we first compared the thermal stability of LbAly35 with an abalone alginate lyase, HdAly, and found that the first order inactivation rate constants for LbAly35 at 40 and 45 °C were 1/20 and 1/45 of those for HdAly, respectively. Then, we cloned cDNAs encoding LbAly35 and characterized its deduced amino-acid sequence comparing with those of other molluscan alginate lyases. The cDNAs were amplified by polymerase chain reaction (PCR) and 5′- and 3′-RACE PCRs from the L. brevicula hepatopancreas cDNA using degenerated primers synthesized on the basis of partial amino-acid sequences of LbAly35. The cDNA covering the entire translational region of LbAly35 comprised 1,093 bp and encoded an amino-acid sequence of 296 residues. The amino-acid sequence consisted of an initiation methionine, a putative signal peptide for secretion (22 residues), a propeptide-like region (10 residues), and a mature LbAly35 domain of 263 residues. Although the N-terminal region of LbAly35 was significantly deviated from those of other molluscan alginate lyases, the catalytic domain of LbAly35 showed ~45 % identity to other molluscan enzymes which had been classified under polysaccharide-lyase-family-14 (PL-14). In addition, the amino-acid residues crucially important for the catalytic actions of PL-14 enzymes were also conserved in LbAly35. Accordingly, LbAly35 was regarded as a member of PL-14 as other molluscan alginate lyases despite the significant deviation of its N-terminal region.

A β-D-mannosidase (EC 3.2.1.25) with a molecular mass of approximately 100 kDa was purified from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by column chromatographies on TOYOPEARL Butyl-650 M, TOYOPEARL DEAE-650 M, and Superdex 200 10/300 GL. This enzyme, named AkMnsd in the present study, showed optimal activities at pH 4.5 and 40 °C and was stable at the acidic pH range from 2.0 to 6.7 and the temperature below 38 °C. The Km and Vmax values for AkMnsd determined at pH 6.0 and 30 °C with p-nitrophenyl β- d -mannopyranoside were 0.10 mM and 3.75 μmol/min/mg, respectively. AkMnsd degraded various polymer mannans as well as mannooligosaccharides liberating mannose as a major degradation product. Linear mannan from green alga Codium fragile was completely depolymerized by AkMnsd in the presence of AkMan, an endolytic β-mannanase, which we previously isolated from the same animal (Zahura et al., Comp. Biochem. Physiol. B 157, 137–148 (2010)). A cDNA encoding AkMnsd was amplified from the Aplysia hepatopancreas cDNA by the PCR using degenerated primers designed on the basis of N-terminal and internal amino-acid sequences of AkMnsd. The cloned AkMnsd cDNA consisted of 2985 bp and encoded an amino-acid sequence of 931 residues with the calculated molecular mass of 101,970 Da. The deduced sequence of AkMnsd showed 20–43% amino-acid identity to those of glycoside-hydrolase-family 2 (GHF2) β-mannosidases. The catalytically important amino-acid residues determined in GHF2 enzymes were completely conserved in AkMnsd. Thus, AkMnsd is regarded as a new member of GHF2 mannosidase from marine gastropod.

Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5′- and 3′-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed ∼40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg; however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1.

Alginate lyase (EC 4.2.2.3) is an enzyme that splits glycosyl linkages of alginate chain via β-elimination producing unsaturated oligoalginates. This enzyme is widely distributed in herbivorous marine mollusks, brown algae, and marine and soil bacteria. In the present study, we determined the general properties and partial amino-acid sequences of alginate lyases from three Archeogastropoda, i.e., Haliotis discus hannai, H. iris, and Omphalius rusticus, and one Mesogastropoda, i.e., Littorina brevicula, in order to enrich the information about functional and structural diversity in gastropod alginate lyases. The alginate lyases were extracted from hepatopancreas of these animals and purified by ammonium sulfate fractionation followed by conventional column chromatography. Single alginate lyases with molecular masses of approximately 28 kDa, 34 kDa, and 34 kDa were isolated from H. discus, H. iris, and O. rusticus, respectively. While three alginate lyases with molecular masses of 35 kDa, 32 kDa, and 28 kDa were isolated from L. brevicula. These enzymes were identified as poly(M) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate. Western blot analysis using an antiserum raised against H. discus enzyme suggested that H. iris, and O. rusticus enzymes shared similar primary/higher order structure with H. discus enzyme, but the L. brevicula enzymes did not. H. discus, H. iris, and O. rusticus enzymes were classified to polysaccharide-lyase family-14 by the analysis of partial amino-acid sequences, while the L. brevicula enzymes were not.

Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28 kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5′- and 3′-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129 bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43–53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)-block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate.