Effect of culture density and antioxidants on naupliar production and gene expression of the cyclopoid copepod, Paracyclopina nana

Although attempts have been made to use mass cultures of marine copepods as live foods in marine aquaculture, some limitations such as low density culture still exist. The brackish water cyclopoid copepod, Paracyclopina nana has the potential for mass culturing as live food. In this study, we not only investigated the effect of culture density on the naupliar production and specific gene expressions of P. nana, but also the effect of several antioxidants under the conditions of a high density culture. The naupliar production of the copepod decreased with increasing culture density. The expression of glutathione reductase (GR), selenium-dependent glutathione peroxidase (SeGPx), glutathione S-transferase kappa (GST kappa), heat shock protein 40 (Hsp40), and Hsp70 genes of P. nana increased in the high density treatment but vitellogenin genes (Vg1 and Vg2) showed downregulation. In the condition with 20 inds./mL, vitamin C had a significant decrease but sodium selenite induced the naupliar production of P. nana greatly. The expressions of GR, SeGPx, Hsp70, and Vg genes increased with the vitamin C treatment. Sodium selenite caused a decrease of SeGPx and Hsp40 but GST kappa increased in the treatment with 20 inds./mL. These results suggest that sodium selenite is a positive antioxidant which can increase the culture efficiency of the copepod.     

Molecular characterization and expression pattern of Spodoptera litura (Lepidoptera: Noctuidae) vitellogenin, and its response to lead stress

Vitellogenin (Vg) cDNA from Spodoptera litura Fabricius was cloned and sequenced. The open reading frame (ORF) of Vg cDNA was 5247 nucleotides in length (GenBank Accession no. EU095334), which encoded for a protein of 1748 amino acids. S. litura Vg comprised three conserved regions (Vitellogenin-N domain, DUF1943 and von Willebrand factor type D domain (VWD)), a 17 amino-acid signal peptide and a RXXR cleavage signal (RTIR). The highly conserved GL/ICG motif, the DGXR motif and cysteine residues were found in the C-terminus of the Vg. Vg mRNA was found specifically in the female fat body. Vg expression was first transcribed in 6th day female pupae and levels increased with insect development. The maximum level of Vg mRNA appeared in 24-h-old adults. When S. litura larvae were exposed to lead (Pb) (25-200 mg Pb/kg), there was a significant inhibition in Vg of female adults. The start of Vg expression was advanced ahead by Pb, from 6th day pupae to 3rd day or 4th day pupae. Low levels of Vg in male adults were also induced by low concentrations of Pb (12.5 and 25 mg Pb/kg). These data show that Pb stress elicits an important Vg response in S. litura.     

Near anoxia and sulfide as possible factors influencing the spatial distribution of Acartia tonsa and Acartia clausi: Comparative evaluation of egg tolerance

In many coastal and estuarine areas the planktonic copepods Acartia tonsa and Acartia clausi show a spatial separation with A. tonsa restricted to brackish waters and confined environments and A. clausi inhabiting areas more influenced by sea water. The hatching and viability of A. tonsa and A. clausi eggs exposed to anoxia and anoxia/sulfide (conditions that are frequent in bottom waters of the most confined areas) was evaluated to determine if these stress factors play a role in the distribution of these species. Subitaneous eggs, spawned by laboratory reared organisms, were incubated in near anoxia (< 7.59 × 10^− 3 mmol O₂ L^− 1) or anoxia/sulfide (∼ 1 mmol L^− 1) for different periods (1, 4, 8 and 15 days), then transferred to normoxic conditions. The exposure of the eggs to near anoxia and sulfide appears to induce the same response (quiescence) in both species. Exposure times ≤ 8 days to near anoxia or anoxia/sulfide did not affect egg viability, while 15 day exposure caused significant declines in hatching success of both species. A significant difference between the effects of near anoxia and anoxia/sulfide was observed when incubation lasted 15 days; hatching of eggs exposed to sulfide being higher than that of eggs exposed to near anoxia for both species. No significant differences were observed between the two species in hatching success of eggs exposed to both near anoxia and anoxia/sulfide (with the exception of eggs incubated in near anoxia for 4 days). The results indicate that the impact of anoxia and sulfide on the eggs of the two Acartia species cannot be a factor explaining the spatial distribution in coastal and brackish environments of these copepods.Feeding experiments on A. clausi were also performed. Suitability of different algal species to rear this copepod was evaluated and the results were compared with data previously obtained for A. tonsa. Differences in feeding needs between A. clausi and A. tonsa are discussed.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Nuclear and chloroplast DNA sequences data support the origin of Potamogeton intortusifolius J.B. He in China as a hybrid between P. perfoliatus Linn. and P. wrightii Morong

Internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequence data were used to test the hypothesis that natural populations of Potamogeton intortusifolius J.B. He in China originated from hybridization between P. perfoliatus Linn. and P. wrightii Morong. Based on ITS sequences data, P. intortusifolius possessed heterozygous rDNA genotypes which confirmed the hybrid origin of P. intortusifolius. Chloroplast rbcL gene sequences of P. intortusifolius from Yichang population revealed the same chloroplast haplotype as P. perfoliatus and the samples of P. intortusifolius from Weinan population had the same chloroplast haplotype as P. wrightii, which indicated that both putative parental species had been the maternal parent and that the two populations of P. intortusifolius had independent origins. This study confirms P. intortusifolius as a reciprocal hybrid. Because P. × intortusifolius in China has the same hybrid origin as P. × anguillanus Koidz. in Japan, it is suggested that P. × intortusifolius should be a synonym of P. × anguillanus.     

Factors affecting horizontal transmission of the microsporidium Amblyospora albifasciati to its intermediate copepod host Mesocyclops annulatus

Factors that directly impact horizontal transmission of the microsporidium Amblyospora albifasciati to its intermediate copepod host, Mesocyclops annulatus were examined in laboratory bioassays. Results were evaluated in relation to life history strategies that facilitate persistence of the parasite in natural populations of its definitive mosquito host, Ochlerotatusalbifasciatus. A moderately high quantity of meiospores from mosquito larvae was required to infect adult female copepods; the IC50 was estimated at 3.6 × 10⁴ meiospores/ml. Meiospore infectivity following storage at 25 °C was detected up to 30 days, while meiospores stored at 4 °C remained infectious to copepods for 17 months with virtually no decline in infectivity. Uninfected female M. annulatus are long-lived; no appreciable mortality was observed in field-collected individuals for 26 days, with a few individuals surviving up to 70 days. The pathological impact of A. albifasciati infection on M. annulatus resulted in a 30% reduction in survivorship after 7 days followed by gradual progressive mortality with no infected individuals surviving more than 40 days. This moderate level of pathogenicity allows for a steady continual release of spores into the environment where they may be ingested by mosquito larvae. Infected female copepods survived in sediment under conditions of desiccation up to 30 days, thus demonstrating their capacity to function as a link for maintaining A. albifasciati between mosquito generations following periods of desiccation. The susceptibility of late stage copepodid M. annulatus to meiospores of A. albifasciati and subsequent transstadial transmission of infection to adult females was established.     

Optimisation of exopolysaccharide production by Gomphidius rutilus and its antioxidant activities in vitro

The production conditions of the Gomphidius rutilus exopolysaccharides (GREP) in submerged culture were optimised, and the antioxidant activities of GREP in vitro were evaluated. The optimal culture medium constituents were determined as follows: 30 g/L sucrose, 3.0 g/L soybean meal, 0.25 g/L MgSO₄, 1.5 g/L K₂HPO₄, 0.5 g/L KH₂PO₄, 0.03 g/L ZnSO₄, and 0.01 g/L FeSO₄. The optimum parameters for the liquid fermentation were as follows: temperature, 25 °C; cultivation time, 6 d; initial pH, 8.0; volume of medium, 150 mL; and rotary speed, 180 rpm. GREP content and dry cell weight in optimised conditions were 540.1 ± 15.9 mg/L and 8.2 ± 0.3 g/L, respectively. GREP content under the optimised conditions was 2.5 times than that under the basic culture medium and initial conditions. GREP demonstrated positive antioxidant potential on superoxide anion radical, 1,1-diphenyl-2-picrylhydrazyl, and hydroxyl radical scavenging, and reducing power.     

Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin (Budorcas taxicolor Bedfordi)

A bacterial strain with high cellulase activity was isolated of feces sample of Golden Takin (Budorcas taxicolor Bedfordi). The bacterium was classified and designated Bacillus subtilis LN by morphological and 16SrDNA gene sequence analysis. Two putative cellulase genes, CelL15 and CelL73, were simultaneously cloned from the isolated strain by PCR. The putative gene CelL15 consisted of an open reading frame (ORF) of 1470 nucleotides and encoded a protein of 490 amino acids with a molecular weight of 54 kDa. The CelL73 gene consisted of an open reading frame (ORF) of 741 nucleotides and encoded a protein of 247 amino acids with a molecular weight of 27 kDa. Both genes were purified and cloned into pET-28a for expression in Escherichia coli BL21 (DE3). The ability of E. coli to degrade cellulose was enhanced when the two recombinants were cultured together.     

Influence of oxygen and temperature on growth and metabolic performance of Paralichthys lethostigma (Pleuronectiformes: Paralichthyidae)

In this study, we apply Fry's classification of environmental factors to demonstrate the limiting effects of oxygen and its interaction with temperature on the growth of juvenile P. lethostigma. We also evaluated the properties of two metabolic indices, marginal metabolic scope (MMS) and limiting oxygen concentration (LOC), as indicators of metabolic scope. We found that oxygen limitation has its greatest impact near the optimum temperature for growth of the species. At 29 °C a reduction from 6.00 mg/L to 4.00 mg/L caused a 50% reduction in growth rate while at 27 °C the reduction had no significant effect on growth rate. The results are particularly relevant because these temperatures and oxygen concentrations are commonly observed in nursery areas during summer months. At all temperatures fish from the lowest oxygen treatment (1.75 mg/L) had negative growth rates. Comparisons between daily oscillating oxygen treatments and constant treatments failed to demonstrate significant effects. At temperatures past the optimum, growth rates between the 6.00 mg/L and 4.00 mg/L treatments were not statistically different. LOC was significantly affected by temperature, oxygen, and their interaction. Estimates were positively correlated with oxygen treatment (R² > 0.71) and negatively correlated with temperature at moderate and low oxygen concentrations (R² > − 0.84). MMS was significantly affected by temperature and oxygen and was significantly correlated with oxygen treatment (R² > − 0.91), but correlations with temperature were not as clear. In conclusion, oxygen and temperature interactions have significant effects on metabolic scope and growth rates of fish, well above the accepted hypoxia threshold of 2.00 mg/L and MMS has proved a useful estimator of the metabolic scope of the organism within an environment.     

A cytosolic glutathione s-transferase,GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties

Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione s-transferase-theta (GST-θ) from freshwater prawn Macrobrachium rosenbergii. A full length cDNA of GSTT (1417 base pairs) was isolated and characterized bioinformatically. Exposure to virus (white spot syndrome baculovirus or M. rosenbergii nodovirus), bacteria (Aeromonas hydrophila or Vibrio harveyi) or heavy metals (cadmium or lead) significantly increased the expression of GSTT (P < 0.05) in hepatopancreas. Recombinant GST-θ with monochlorobimane substrate had an optimum activity at pH 7.5 and 35 °C. Furthermore recombinant GST-θ activity was abolished by the denaturants triton X-100, Gua-HCl, Gua-thiocyanate, SDS and urea in a dose-dependent manner. Overall, the results suggest a potential role for M. rosenbergii GST-θ in detoxification and possibly conferring immune protection.     

Biological conversion of olive pomace into compost by using Trichoderma harzianum and Phanerochaete chrysosporium

Olive pomace was composted by using a reactor for a period of 50 days in four bioreactors. Urea was added to adjust C/N ration between 25–30. At the end of 50 days of composting using Trichoderma harzianum and Phanerochaete chrysosporium, cellulose and lignin were highly degraded. It was found that after 30 days, P. chrysosporium and T. harzianum degraded approximately 71.9% of the lignin and 59.25% of the cellulose, respectively. The percent of ash content in the raw waste mixture was 13%. This percentage increased from 13% to 18.55% in treatment bioreactors and from 13% to 13.55% in control reactors during 50 day of composting process. The amount of CO₂ produced by the treated sample was 3 mg of CO₂/g organic carbon which is indicated that the treated sample was considered as stable compost. The results proved that the use of accelerating agents was found to be efficient in producing mature stable with nearly non-phytotoxicity compared to control sample in less than 50 days.     

In silico and in vitro comparative activity of novel experimental derivatives against Leishmania major and Leishmania infantum promastigotes

This in silico and in vitro comparative study was designed to evaluate the effectiveness of some biurets (K1 to K8) and glucantime against Leishmania major and Leishmania infantum promastigotes. Overall, eight experimental ligands and glucantime were docked using AutoDock 4.3 program into the active sites of Leishmania major and Leishmania infantum pteridine reductase 1, which were modeled using homology modeling programs. The colorimetric MTT assay was used to find L. major and L. infantum promastigotes viability at different concentrations of biuret derivatives in a concentration and time-dependent manner and the obtained results were expressed as 50% and 90% of inhibitory concentration (IC₅₀ and IC₉₀). In silico method showed that out of eight experimental ligands, four compounds were more active on pteridine reductase 1. K3 was the most active against L. major promastigotes with an IC₅₀ of 6.8 μM and an IC₉₀ of 40.2 μM, whereas for L. infantum promastigotes was K8 with IC₅₀ of 7.8 μM. The phenylethyl derivative (K7) showed less toxicity (IC₅₀s > 60 μM) in both Leishmania strains. Glucantime displayed less growth inhibition in concentration of about 20 μM. In silico and especially docking results in a recent study were in accordance with the in vitro activity of these compounds in presented study and compound K3, K2 and K8 showed reasonable levels of selectivity for the Leishmania pteridine reductase 1.     

Disinfection and toxicological assessments of pulsed UV and pulsed-plasma gas-discharge treated-water containing the waterborne protozoan enteroparasite Cryptosporidium parvum

We report for the first time on the comparative use of pulsed-plasma gas-discharge (PPGD) and pulsed UV light (PUV) for the novel destruction of the waterborne enteroparasite Cryptosporidium parvum. It also describes the first cyto-, geno- and ecotoxicological assays undertaken to assess the safety of water decontaminated using PPGD and PUV. During PPGD treatments, the application of high voltage pulses (16 kV, 10 pps) to gas-injected water (N₂ or O₂, flow rate 2.5 L/min) resulted in the formation of a plasma that generated free radicals, ultraviolet light, acoustic shock waves and electric fields that killed ca. 4 log C. parvum oocysts in 32 min exposure. Findings showed that PPGD-treated water produced significant cytotoxic properties (as determined by MTT and neutral red assays), genotoxic properties (as determined by comet and Ames assays), and ecotoxic properties (as determined by Microtox™, Thamnotox™ and Daphnotox™ assays) that are representative of different trophic levels in aquatic environment (p < 0.05). Depending in part on the type of injected gas used, PPGD-treated water became either alkaline (pH ≤ 8.58, using O₂) or acidic (pH ≥ 3.21, using N₂) and contained varying levels of reactive free radicals such as ozone (0.8 mg/L) and/or dissociated nitric and nitrous acid that contributed to the observed disinfection and toxicity. Chemical analysis of PPGD-treated water revealed increasing levels of electrode metals that were present at ≤ 30 times the tolerated respective values for EU drinking water. PUV-treated water did not exhibit any toxicity and was shown to be far superior to that of PPGD for killing C. parvum oocysts taking only 90 s of pulsing [UV dose of 6.29 μJ/cm²] to produce a 4-log reduction compared to a similar reduction level achieved after 32 min PPGD treatment as determined by combined in vitro CaCo-2 cell culture-qPCR.     

Isolation and functional characterization of DNA damage repair protein (DRT) from Lepidium latifolium L.

We have isolated and in silico characterized a cold regulated plastocyanin encoding gene from Lepidium latifolium L designated as LlaDRT. Its cDNA sequence (JN214346) consists of a 504 bp ORF, 48 and 205 bp of 5′ and 3′ UTR regions, respectively encoding a protein of 17.07 KDa and pI 4.95. In silico and phylogenetic analysis of LlaDRT suggested that the protein has features of a typical plastocyanin family member and of a nearest relative of the predominant isoform of Arabidopsis (PETE2) plastocyanin. Validation of stress response of LlaDRT by qPCR under different abiotic stress regulators viz salicylic acid, jasmonic acid, calcium chloride, ethylene and abscisic acid revealed its possible regulation and crosstalk amongst different pathways.