The extent of genetic diversity among Vanilla species: Comparative results for RAPD and ISSR

Vanilla is a large genus of about 110 species in the orchid family (Orchidaceae), including the species Vanilla planifolia from which commercial vanilla flavoring is derived. Since most species of vanilla are considered rare and endangered there is an urgent need to conserve them through genetic analysis and propagation/conservation studies on this crop.The present study investigated the genetic diversity among nine leafy- and leaf-less Vanilla species employing 30 decamer RAPD primers and 10 ISSR primers. The species under study were diverse and displayed a range of variability (0–66% and 0–81% for RAPD and ISSR, respectively). A total of 154 RAPD polymorphic markers (83.24%, h = 0.378) and 93 ISSR polymorphic markers (86.11%, h = 0.363) were used to generate a genetic similarity matrix followed by the cluster analysis. Specific groupings were revealed by each cluster analysis with slight variation between two different markers. Among the nine species studied, V. planifolia, Vanilla aphylla and Vanilla tahitensis revealed very low level of variation within their collections, thus indicating a narrow genetic base. The large genetic distance of Vanilla andamanica from other species suggests its different origin. A close genetic affinity was observed between the pairs V. planifolia, V. tahitensis and Vanilla albida, V. aphylla. These are the first comparative results for RAPD and ISSR reporting inter-relationship among nine cultivated, wild and hybrid Vanilla species.     

Effects of organic selenium supplemented diet on growth performance and antioxidant response in Cirrhinus mrigala

The present study was conducted to evaluate the …effect of dietary selenium on growth performance and antioxidant enzymes activity in Cirrhinus mrigala for a period of 45 days. Four numbers of experimental diets were prepared, T_c for Control (0 mg organic selenium/kg of diet), T₁ (2.5 mg organic selenium/kg of diet), T₂ (5.0 mg organic selenium/kg of diet) and T₃ (10 mg organic selenium/kg of diet). The water quality parameters (temperature, DO, pH, conductivity, hardness, ammonia, nitrate and nitrite) were found to be insignificant (p > .05) among all the treatments. Superoxidase dismutase (SOD) was found to be significantly highest in T₂ (6.10 ± 0.07) when compared with the other treatments. Similarly, catalase (CAT) activity in T₁ (13.21 ± 0.13) was significantly (p < .05) different with that of Control, T₂ and T₃. Present study also revealed that 2.5mg organic selenium/kg supplemented diet (T₁) showed highest results in net weight gain (15.55 ± 1.36 g), net weight gain percentage (185.88 ± 8.98%) and SGR (2.35 ± 0.06 g) compared with other experimental diet.     

Changes in ovary and testis and breeding fitness of the climbing perch,Anabas testudineus (Bloch, 1792), exposed to sub‐lethal concentrations of monocrotophos

Monocrotophos (MCP) is one of the organophosphate pesticides extensively used in agriculture and animal husbandry. The present study reports the effects of sub‐lethal concentrations of MCP on gonadosomatic index (GSI), gonadal histology and breeding fitness of the fish Anabas testudineus. The fish were exposed to three sub‐lethal concentrations of monocrotophos (T1: 3.5 mg/L, T2: 5.3 mg/L and T3: 10.6 mg/L) for 45 days with 12 fish (males and females in equal numbers) in each tank, and one group was kept as control (C) without any treatment. GSI decreased in all treatment groups in comparison with control. Significant decrease in fertilization and hatching rate was observed at all concentrations in comparison with control on 15, 30 and 45 days of monocrotophos exposure. The decreased vitellogenesis, disruption of follicular wall and oocyte atresia were observed in all the treated females at 30 and 45 days of exposure. Rupture of seminiferous tubules was noticed in all the treated males irrespective of exposure periods. The results thus indicated that monocrotophos could deleteriously impact the gonadal structure and function in A. testudineus, which could seriously impact the reproductive success of the animal.     

Molecular phylogeny in Indian Citrus L. (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA

The taxonomy and phylogeny of Indian Citrus is revisited using PCR-RFLP of the trnD-trnT and rbcL-ORF 106 regions as well as sequence data analysis of the trnL-trnF intergenic spacer region of cpDNA. The study was based on 50 accessions of Citrus genotypes, collected from wild, semi-wild and domesticated stocks. Of the 13 restriction enzymes (RE) used for restriction digestion of the polymerase chain reaction (PCR) amplicons, four (Hinf I, Msp I, Alu I, Hae III) generated 47 restriction fragments, of which 24 (51%) were polymorphic. PCR-RFLP data showed a genetic distance ranging from 0 to 0.79 among 50 accessions of Citrus, and a cluster analysis, based on Neighbor-Joining (NJ) method, placed all the accessions in eight major clusters. Analysis of trnL-trnF sequences from 23 representative accessions of Citrus showed a pair-wise sequence divergence rate in the range of 0–0.064. NJ, minimum evolution (ME) and maximum parsimony (MP) analyses of trnL-trnF sequences produced phylogenetic trees, which placed all the 23 accessions in five clusters. PCR-RFLP analysis resulted in a well resolved phylogenetic tree with branches supported by moderate to high bootstrap values, while the trnL-trnF sequence-based trees showed only moderate to low bootstrap support for the internal tree branches, indicating uncertain origin of some Citrus genotypes. This study shows that the trnL-trnF spacer sequence data can detect genetic variation in Indian Citrus genotypes, but the utility of the data in inferring phylogeny at intra and inter-specific levels is limited probably by factors such as hybridization, bud mutations, apomixis and polyploidy. However, PCR-RFLP and trnL-trnF data supported the recognition of C. maxima, C. medica, and C. reticulata as the basal species of edible Citrus.     

Cryopreservation of silver barb Puntius gonionotus (Bleeker) spermatozoa: effect of extender composition,cryoprotective agents and freezing rate on their postthawing fertilization ability

The effects of extender composition, cryoprotectant concentration and freezing and thawing on the fertilization efficiency of cryopreserved spermatozoa of Puntius gonionotus were evaluated. Computer‐aided motility analysis of semen was conducted to check the suitability of spermatozoa for cryopreservation after mixing with different extenders and cryoprotective agents (CPAs). Extender‐4 with an osmolality 260 mOsmol kg⁻¹and pH 7.6 was used for the cryopreservation study. Among the CPAs, dimethyl sulphoxide (DMSO) was least toxic and more than 60% fertilization was achieved when used at 1.4 M at 0 °C for 10 and 30 min, whereas the toxicity of all CPAs to spermatozoa was evident when tested at 30 °C. Semen frozen at −16 °C min⁻¹ with 1.4 M DMSO showed 70% fertilization, which was significantly higher (P<0.05) than other freezing rates. Samples thawed at 35 °C water showed a fertilization rate comparable with that of fresh semen. Computer‐assisted semen analysis of fresh and frozen semen after thawing showed variations in different types of motility in spermatozoa and in their class. There was no significant difference in motility before or after cryopreservation; however, significant differences could be observed in the average path velocity (VAP), straight line velocity (VSL) and curve linear velocity (VCL). Semen of silver barb could be cryopreserved with extender‐4 by addition of 1.4 M DMSO to a final cryopreservation medium (MED 2) cooled at a rate of −16 °C min⁻¹, stored in liquid nitrogen (−196 °C) and utilized after thawing at 35±2 °C.     

Genetic variations in survival of rohu carp (Labeo rohita, Hamilton) after Aeromonas hydrophila infection in challenge tests

A challenge test against Aeromonas hydrophila was conducted using 2062 rohu carp (Labeo rohita) fingerlings obtained from 52 dams and 87 sires (87 full-sib families) of two year-classes (2003 and 2004). Attempts to establish a cohabitant challenge model were not successful. Therefore, fish were challenged by intraperitoneal injection with A. hydrophila in two replicate tanks per year-class, and dead fish were collected hourly. The mortality reached its peak at 16–22 h after challenge and had almost completely stopped after 58 h. The test was terminated after 382 h at which the average survival was 43.9 and 48.9% in the two 2003 year-class tanks, and 77.1 and 35.8% in the two 2004 year-class tanks. Heritability estimates for survival were obtained from sire and dam threshold models (THR) and sire and dam linear repeatability models (LINR). For both types of models the heritability estimates based on data from single tanks were not consistent. For both year-classes, data from one of the two challenged tanks demonstrated significant additive genetic variation in survival during the A. hydrophila infection, whereas the heritability estimates were not significantly different from zero for the other tank. Further, genetic correlation between survivals in the two replicate tanks in each year-class was not significantly different from zero. The differential results from the replicate tanks demonstrate that additional challenge test experiments are needed before firm conclusions can be drawn about the magnitude of additive genetic variation for survival to aeromonasis in rohu carp. A cohabitant challenge model that allows the testing of important defence mechanisms in the skin and mucous membranes of the fish might have been more appropriate. To establish a valid cohabitant challenge model for rohu carp should be given high priority.     

Rearing performance of Clarias batrachus larvae: Effect of age at stocking on growth and survival during fingerling production

An experiment was conducted to evaluate the rearing performance of different larval stages for optimum growth and survival during fingerling production of Clarias batrachus with an aim to reduce the rearing time of the catfish larvae in a hatchery system. The larvae were reared for 5, 10 and 15 days before stocking them in the nursery tanks. No significant (P < 0.05) difference could be found in length increment at the end of first week of rearing. But the total length was reduced during rest three weeks in five days old larvae compared to that of other two higher age groups. In 5 days old larvae the reduced weight (P < 0.05) was observed during the entire rearing period. However, the specific growth rate (SGR) did not vary among the groups. The survival rate as well as total biomass was decreased while stocking five days old larvae for rearing, compared to that of other two age groups.     

Modeling the water and nitrogen transports in a soil–paddy–atmosphere system using HYDRUS-1D and lysimeter experiment

Efficient water and fertilizer use is of paramount importance both in rain-fed and irrigated rice cultivation systems to tread off between the crop water demand during the dry spell and the fertilizer leaching. This lysimeter study on paddy in a lateritic sandy loam soil of the eastern India, to simulate the water and solute transports using the HYDRUS-1D model, reveals that this model could very well simulate the soil depth-specific variations of water pressure heads and nitrogen (N) concentrations with the efficiency of >86 and 89%, respectively. The change in the level of water ponding depth did not have a significant effect on the time to peak and the temporal variability of N concentration in the bottom soil layer. The lysimeter-scale water balance analysis indicated that the average deep percolation loss and crop water use were 35.01 ± 2.03 and 39.74 ± 1.49% of the total water applied during the crop growth period, respectively. Similarly, the amount of N stored in the plant and lost through soil storage, deep percolation, and other losses (mineralization, denitrification, and gaseous N loss to the atmosphere through plant leaves) were 1.60 ± 0.16, 0.17 ± 0.04, 12.00 ± 0.48, and 86.23 ± 0.41% of the total applied nitrogen, respectively. The simulation results reveal that a constant ponding depth of 3 cm could be maintained in paddy fields to reduce the N leaching loss to 7.5 kgN/ha.