The aquaculture of freshwater prawn, Macrobrachium malcolmsonii (Milne Edwards), at farmers'level in Pakistan

Abstract. Artisanal culture of Macrobrachium malcolmsonii (Milne Edwards) was studied on two sites in Pakistan. The juvenile prawns of M. malcolmsonii were collected randomly from the lower belt of the river Indus, transported and stocked in ponds at Mirpur Sakro and Chilya, Thatta, Sindh, Pakistan after acclimation. They were fed on supplementary feed containing 15% protein once daily. The stocked prawns grew from a mean weight of 0·5–2·0g to 47·5–83·5g in ponds under the conditions described herein, so a total production of 216·5–1037·5kg/ha was achieved.     

Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r‐MCP43) of giant freshwater prawn,M. rosenbergii (de Man) for immunological diagnostic methods

White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6‐histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD‐infected post‐larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti‐rMCP43 was found to be capable of detecting MrNV in WTD‐infected post‐larvae as early as at 24 h post‐infection. The antiserum raised against r‐MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti‐rMCP43 and pure r‐MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT‐PCR to test the efficiency of antiserum raised against r‐MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV‐positive coded samples as detected by RT‐PCR.     

Assessment of cocoa input needs using soil types and soil spectral analysis

In this study, diffuse reflectance spectroscopy (DRS) approach was examined for making input recommendations in the smallholder cocoa farms of Papua New Guinea (PNG). Soil samples were collected from four provinces of PNG. Soil samples from four different depths (0–10, 10–30, 30–60 and 60–90 cm) of 32 profiles in each of these site were used to create a database of soil chemical and physical properties. Spectral reflectance values at 1 nm interval covering visible to shortwave‐infrared (350–2,500 nm) were collected for each of these soil samples to develop partial least squares regression models. Soil textural fractions, soil organic carbon contents and available N were well predicted by the DRS approach with R² values larger than 0.75. Moderate to poor estimation efficiencies were observed for remaining parameters. Nevertheless, the estimated soil attributes and their corresponding measured soil parameters were used as inputs to an input recommendation model of soil diagnosis to create input recommendation for a targeted cocoa yield of 1,000 kg dry cocoa beans ha^‐1 Resulting input recommendations were similar for both of these input sources (measured and DRS‐estimated) suggesting that the DRS approach may provide an easy way to create input recommendations.     

Development and characterization of five novel cell lines from snubnose pompano,Trachinotus blochii (Lacepede, 1801), and their application in gene expression and virological studies

Five novel permanent cell lines have been established from gill, heart, kidney, eye and fin of snubnose pompano, Trachinotus blochii. They were designated as snubnose pompano gill (SPG), snubnose pompano heart (SPH), snubnose pompano kidney (SPK), snubnose pompano eye (SPE) and snubnose pompano fin (SPF), respectively. All these cell lines were characterized and cryopreserved successfully at different passage levels. Cell lines were passaged every alternate day; SPG, SPH, SPK, SPE and SPF cell lines attained passage levels of 68, 74, 82, 79 and 106, respectively, since the initiation of their development in 2019. The cell lines grew well in Leibovitz's 15 medium containing 15% foetal bovine serum at 28°C. Immunophenotyping of the cell lines revealed the presence of fibronectin and pancytokeratin. No mycoplasma contamination was found. The transfection study revealed the gene expression efficiency of these cell lines by expressing the green fluorescent protein (GFP). The authentication on origin of cell lines from T. blochii was confirmed by amplification of species-specific mitochondrial cytochrome oxidase I gene. The results showed the susceptibility of these cell lines to fish nodavirus (FNV) and tilapia lake virus (TiLV) and resistance to cyprinid herpesvirus 2 (CyHV-2). The FNV infection in the cell lines was confirmed by RT-PCR, Western blot, ELISA and immunocytochemistry, while TiLV infection was confirmed by RT-PCR assay. These results revealed that these cell lines are suitable for virological and foreign gene expression studies.     

Characterization and identification of leaf morphology of <Emphasis Type="Italic">Populus deltoides</Emphasis> Bartr. clones

Leaves are of fundamental importance to plants, representing their facility to generate power and are the sensing units of plants towards the environment. An attempt was made to characterize and compare the variations of leaf morphology of various Populus deltoides Bartr. clones by studying the winter buds and other leaf parameters of fully developed leaves. To achieve these objectives, forty-three exotic and indigenous clones of P. deltoides Bartr. were evaluated for different parameters. On the basis of various morphological characteristics the results reveal that each clone has a distinct color pattern of leaves. Different colors observed in these clones varied from light green through green to dark green. Two distinct lengths of the leaf apex were found, i.e., short and long; as well both acuminate and acute apex types were found. Erratic distribution of serration of leaves was also found. In this study, the morphological traits of leaves provided discriminatory grounds for separating various populations of P. deltoides Bartr. clones. Winter bud studies indicate that different clones vary considerably with regard to shape, color, shape of leaf scars and exudation.     

Aflatoxins and ochratoxin A contamination in rice,corn and corn products from Punjab,Pakistan

In present study, a total of 275 retail cereal samples including rice, corn and corn products was analyzed for the presence of aflatoxins and ochratoxin A (OTA) using HPLC equipped with a fluorescence detector. The data has shown that 38 out of 68 samples of rice, 37/105 of corn and 43/102 samples of corn products have been found contaminated with AFs, average level of AFB₁ and total AFs, 8.23 and 19.54, 7.90 and 12.08, and 5.47 and 7.85 μg/kg, respectively. Furthermore, 34, 28 and 26 numbers of rice, corn and corn product samples were found to be contaminated with OTA with an average level of 12.94, 5.29 and 3.69 μg/kg, respectively. The samples of rice, corn and corn products found above the permissible European Union (EU) limit for AFB₁ and total AFs were 18, 13 and 14 and 28, 14 and 20%, respectively; however, the samples of rice, corn and corn products above the EU limit were 40, 14 and 15%, respectively.     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.     

Development,distribution and expression of a DNA vaccine against nodavirus in Asian Seabass,Lates calcarifier (Bloch, 1790)

Fish nodavirus (betanodavirus), a viral pathogen responsible for viral nervous necrosis (VNN) was isolated from infected Asian sea bass (Lates calcarifer). The distribution, clearance and expression of nodavirus vaccine, on the basis of DNA vaccine (pFNCPE42 DNA‐pcDNA3.1) construction, were analysed in tissues of the Asian seabass by PCR, RT‐PCR, ELISA and Immunohistochemistry. Fish immunized with a single intramuscular injection of 20 μg of the pFNCPE42‐DNA vaccine showed a significant increase in the serum antibody level in the 3rd week after vaccination, compared to control eukaryotic expression vector pcDNA3.1 vaccinated fish. Results from PCR studies indicated that the vaccine‐containing plasmids were distributed in heart, intestine, gill, muscle and liver 10 days after vaccination. Clearance of pFNCPE42‐DNA vaccine was studied at 10, 25, 50, 75 and 100 days of post vaccination (d p.v). At 100 days p.v. pFNCPE42‐DNA was cleared from muscle of vaccinated sea bass. In vitro and in vivo expression of fish nodavirus capsid protein gene (FNCP) was determined by fluorescent microscopy. Asian seabass was immunized with pFNCPE42‐DNA vaccine at a dose of 20 μg per fish and were challenged with betanodavirus by intramuscular injection. The vaccinated seabass was protected from nodaviral infection and 77.33% of relative percent survival (RPS) was recorded.     

Enhanced <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">tuberosi</Emphasis> Resistance in Transgenic Potato Expressing a Rice GLP Superoxide Dismutase Gene

Germin like proteins (GLPs) are a large group of related and ubiquitous plant proteins which are considered to be involved in different processes important for plant development and defense. Multiple functional copies of this gene family have been reported in a number of species (wheat, barley, rice, soybean mosses and liverwort), and their role is being evaluated by gene regulation studies and transgenic approaches. To analyze the role of a rice (Oryza sativa) root expressed germin like protein1 OsRGLP1, for its antifungal activity, transgenic potato plants were developed. These transgenic potato plants were molecularly characterized and biologically assessed after inoculation with Fusarium oxysporum f. sp. tuberosi. Functional analysis showed high accumulation of H₂O₂, increased Superoxide Dismutase (SOD) activity and no oxalate oxidase activity (OxO) in transgenics in comparison to nontransformed control. This increased SOD activity, resistance to heat and sensitivity to H₂O₂ suggest it is a Fe-like SOD. OsRGLP1 expression in potato plants exhibited enhanced resistance in comparison to nontransformed wild type plants suggesting its role in providing protection against Fusarium oxysporum f. sp. tuberosi through elevated SOD level. Overall, results suggest that OsRGLP1 is a candidate for the engineering of potato plants with increased fungal tolerance however, the greater height and tuber number was observed. This phenotype associated with the resistance needs to be evaluated to determine if this is a positive or negative feature.     

Maturity indices of Indian horse-chestnut (<Emphasis Type="Italic">Aesculus indica</Emphasis> Colebr.) seeds under temperate Kashmir conditions

Our study on maturity indices of Aesculus indica Colebr.seeds was conducted under temperate Kashmir conditions in 2006.Seed collection was started from 15 July and continued until the maturation of seeds in December.The seeds were harvested fortnightly from identified trees and on each collection date maturity indices,viz.seed color,seed weight,moisture content,seed dimension,specific gravity and germination percentage were recorded.The study revealed that at the time of maturity (Nov.–Dec.) the seed color ...