Isolation and characterization of immunoglobulin of the Indian major carp, rohu [Labeo rohita (Ham.)

Information on the structure and character of immunoglobulin of fishes is essential in health management. A study was carried out to characterize the serum immunoglobulin (IgM) of the Indian major carp, rohu Labeo rohita (Ham.). Rohu (500g) were immunised with bovine serum albumin (BSA) and the anti-BSA antibody was purified employing BSA-CL agarose affinity column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified Ig in a 3% gel under non-reduced conditions revealed a single protein having a molecular weight of 850kDa. Analysis of the purified serum in 10% SDS-PAGE under reduced conditions revealed that the immunoglobulin contained heavy and light chains with molecular weights of 85 and 23kDa, respectively. A polyclonal mouse anti-rohu IgM was prepared and used in an immunodot test which showed a specific reaction of the crude rohu anti-BSA antiserum and the purified anti-BSA IgM with BSA. Results indicate that the immunoglobulin of L. rohita is tetrameric IgM, similar to that of other fishes.     

Molecular characterization of toll-like receptor 2 (TLR2), analysis of its inductive expression and associated down-stream signaling molecules following ligands exposure and bacterial infection in the Indian major carp, rohu (Labeo rohita)

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various TLR types, TLR2 is involved in recognizing specific microbial structures such as peptidoglycan (PGN), lipoteichoic acid (LTA), zymosan etc., and after binding them it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce various cytokines. In this report, TLR2 gene was cloned and characterized in rohu (Labeo rohita), which is highly commercially important fish species in the farming-industry of Indian subcontinent. Full-length rohu TLR2 (rTLR2) cDNA comprised of 2691 bp with a single open reading frame (ORF) of 2379 bp encoding a polypeptide of 792 amino acids (aa) with an estimated molecular mass of 90.74 kDa. Structurally, it comprised of one leucine-rich repeat region (LRR) each at N-terminal (LRR-NT; 44-55 aa) and C-terminal (LRR-CT; 574-590 aa), 21 LRRs in between C and N-terminal, one trans-membrane (TM) domain (595-612 aa), and one TIR domain (645-790 aa). Phylogenetically, rohu TLR2 was closely related to common carp and exhibited significant similarity (93.1%) and identity (88.1%) in their amino acids. During embryogenesis, rTLR2 expression was detected as early as ∼7 h post fertilization indicating its importance in embryonic innate immune defense system in fish. Basal expression analysis of rTLR2 showed its constitutive expression in all the tissues examined, highest was in the spleen and the lowest was in the eye. Inductive expression of TLR2 was observed following zymosan, PGN and LTA exposure and Streptococcus uberis and Edwardsiella tarda infections. Expression of immunoregulatory cytokine interleukin (IL)-8, in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with inductive expression of TLR2. In vitro studies showed that PGN treatment induced TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) expression, NF-κB (nuclear factor kappa B) activation and IL-8 expression. Blocking NF-κB resulted in down-regulation of PGN mediated IL-8 expression indicating the involvement of NF-κB in IL-8 induction. Together, these findings highlighted the important role of TLR2 in immune surveillance of various organs, and in augmenting innate immunity in fish in response to pathogenic invasion. This study will be helpful in developing preventive measures against infectious diseases in fish.     

Expression analysis of heat shock protein genes during Aeromonas hydrophila infection in rohu,Labeo rohita,with special reference to molecular characterization of Grp78

Heat shock protein (Hsp) genes are stress-related genes that activate the host immune system during infection. Hsp genes expression in fish, studied during bacterial infections, is mostly confined to Hsp70 and Hsp90. The present study is an expression analysis of seven Hsp genes: Apg2, Hsp90, Hsp70, glucose-regulated protein 78 (Grp78), heat shock cognate 70 (Hsc70), Grp75, and Hsp30 during Aeromonas hydrophila infection in rohu, Labeo rohita. Forty-eight rohu juveniles were challenged with 3 × 10⁷ cfu bacteria per 20 g of body weight intraperitoneally. The expression of these genes was studied in infected liver, anterior kidney, and spleen tissues of rohu at different time periods: 0, 1, 3, 6, 12, 24, 48, 72 h, 7, and 15 days post-infection by qPCR. The Hsp gene modulation was greater in liver as compared to spleen and kidney tissues. Induced expression of Apg2, Hsp90, Grp78, Grp75, and Hsc70 was noticed during peak periods (3 to 24 h post-challenge) of bacterial infectivity. Hsp70 was found to be down-regulated during the process of infection. In contrast to the other six genes, Hsp30 showed a variable expression pattern in all three tissues. Grp78 was found to be the most highly (1,587-fold) expressed gene in liver at 12 h post-challenge. Further, molecular characterization of Grp78 revealed it to be a highly conserved Hsp gene, essential not only during infection but also during early developmental stages of rohu, and its expression was noticed in all organs studied except in gill tissues, which indicated its potential immune regulatory role during infection process.     

Establishment of a leukocyte cell line derived from peritoneal macrophages of fish,Labeo rohita (Hamilton, 1822)

A continuous leukocyte cell line with phagocytic activity was established from peritoneal macrophages of rohu, Labeo rohita (LRPM). LRPM was initiated from adherent mononuclear leukocytes isolated from peritoneal cavity of rohu, without use of any growth factors or feeder cells. These cells exhibited maximum growth at 30 °C in L-15 medium containing 20 % foetal bovine serum, and has been subcultured for more than 60 passages till date. The cells showed 85 % viability after 6 months of storage in liquid nitrogen. The species of origin of the LRPM was confirmed by the amplification and sequencing of 655 bp fragment of cytochrome oxidase subunit I of mitochondrial DNA. Functionally, LRPM showed phagocytic activity of yeast cells and fluorescent latex beads as evaluated by phase contrast and scanning electron microscopy, respectively. Immuno-modulators such as bacterial lipopolysaccharide and phorbol myristate acetate resulted in functional activation of LRPM; and enhanced their microbicidal activity through release of reactive oxygen species and nitric oxide. Culture supernatant from activated cells also revealed lysozyme activity. Cells of LRPM were positive for alpha-naphthyl acetate esterase enzyme indicating macrophage lineage. Our results indicate that this cell line can be a useful in vitro tool to study the role of macrophages in teleost immune system and to evaluate the effects of new aquaculture drugs. The LRPM cell line represents the first reported leukocyte cell line of peritoneal origin from any freshwater species of fish.     

Isolation of enriched carp spermatogonial stem cells from Labeo rohita testis for in vitro propagation

The in vitro culture system for spermatogonial stem cells (SSCs) is a powerful tool for exploring molecular mechanisms of male gametogenesis and gene manipulation. Very little information is available for fish SSC biology. Our aim was to isolate highly pure SSCs from the testis of commercially important farmed carp, Labeo rohita. The minced testis of L. rohita was dissociated with collagenase. Dissociated cells purified by two-step Ficoll gradient centrifugation followed by magnetic activated cell sorting (MACS) using Thy1.2 (CD90.2) antibody dramatically heightened recovery rate for spermatogonial cells. The purified cells were cultured in vitro conditions for more than two months in L-15 media containing 10% fetal bovine serum (FBS), 1% carp serum, and other nutrients. The proliferative cells were dividing as validated by 5-bromo-2′-deoxyuridine (BrdU) incorporation assay and formed colonies/clumps with the typical characteristics of SSCs A majority of enriched cell population represented a Vasa⁺, Pou5f1/pou5f1⁺, Ssea-1⁺, Tra-1-81⁺, plzf⁺, Gfrα1/gfrα1⁻, and c-Kit/c-kit⁻ as detected by immunocytochemical and/or quantitative real-time polymerase chain reaction (RT-PCR) analyses. Thus, Thy1⁺ SSCs were enriched with greater efficiency from the mixed population of testicular cells of L. rohita. A population of enriched spermatogonial cells could be cultured in an undifferentiated state. The isolated SSCs could provide avenue for undertaking research on basic and applied reproductive biology.     

The hatching of common carp (Cyprinus carpio L.) embryos in response to exposure to different concentrations of cryoprotectant at low temperatures

The hatching performance of embryos of the common carp (Cyprinus carpio L.) was examined after 1, 7, 14, 21, or 28 days of storage at -8, -6, -4, -2, 0, 2, or 4 degrees C with different concentrations of methanol (0.5-7.0 M in 0.5 M steps) or varying concentrations of methanol in 0.1 M sucrose or trehalose. Preserved embryos failed to hatch after storage at -8 and -6 degrees C, regardless of the duration of storage or the concentrations tested. Likewise, there was no hatching out above 5.0 M concentration of methanol, even with the addition of sucrose or trehalose. After storage at 2 or 4 degrees C, the hatching rate was higher with mixtures of methanol (1.5 M) and trehalose (0.1 M) than with methanol plus sucrose or methanol alone. At 4 degrees C, the solution containing 1.5 M methanol supplemented with trehalose gave the highest hatching response of embryos stored for 14 days. Comparison of hatching after 24h of storage at the effective temperatures (-4, -2, 0, 2, and 4 degrees C) revealed that low concentrations of methanol were effective at high temperatures and high concentrations at sub-zero temperatures. The combination of 0.1 M trehalose with 1.5 M methanol gave the highest percentage hatching out both at 4 and 2 degrees C. At 0 degrees C, the highest percentage hatching occurred with 0.1 M trehalose plus 2.5 M methanol and at -2 and 4 degrees C, the best results were with 0.1 M trehalose plus 3.0 M methanol.     

Effect of different cryoprotectants and vitrificant solutions on the hatching rate of turbot embryos (Scophthalmus maximus)

Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, in order to achieve a vitrifiable medium, a high concentration of permeable cryoprotectants must be employed, and the incorporation of high molecular weight compounds should also be considered. The toxicity of these permeable and non-permeable agents has to be assessed, particularly when high concentrations are required. In the present study, permeable and non-permeable cryoprotectant toxicity was determined in turbot embryos at two development stages (F stage-tail bud and G stage-tail bud free). Embryos treated with pronase (2mg/ml, 10 min at 22 degrees C) were incubated in dimethyl sulfoxide (Me2SO), methanol (Meth.) or ethylene glycol (EG) in concentrations ranging from 0.5 to 6M for periods of 10 or 30 min, and in 5, 10, and 15% polyvinylpyrrolidone (PVP), 10, 15, and 20% sucrose or 0.1, 1, and 2% X-1000 for 2 min. The embryos were then washed well and incubated in seawater until hatching. The toxicity of permeable cryoprotectants increased with concentration and exposure time. There were no significant differences between permeable cryoprotectants. However, embryos tolerated higher concentrations of Me2SO than other cryoprotectants. Exposure to permeable cryoprotectants did not affect the hatching rate except at G stage with X-1000 treatment and 20% sucrose. Taking into account the cryoprotectant toxicity and the vitrification ability of cryoprotectant mixtures, three vitrification solutions (V1, V2, and V3), and one protocol for stepwise incorporation were designed. The tested solutions contained 5M Me2SO+2M Meth+1M EG plus 5% PVP, 10% sucrose or 2% X-1000. The hatching rate of embryos that had been exposed to the the vitrification solutions was analyzed and no significant differences were noticed compared with the controls. Our results demonstrate that turbot embryos can be subject to this cryoprotectant protocol without deleterious effect on the hatching rate.     

Family association between immune parameters and resistance to Aeromonas hydrophila infection in the Indian major carp, Labeo rohita

Seven innate immune parameters were investigated in 64 full-sib families (the offspring of 64 sires and 45 dams) from two year-classes of farmed rohu carp (Labeo rohita). Survival rates were also available from Aeromonas hydrophila infection (aeromoniasis) recorded in controlled challenge tests on a different sample of individuals from the same families. Due to strong confounding between the animal additive genetic effect and the family effects (common environmental + non-additive genetic), reliable additive (co)variance components and hence heritabilities and genetic correlations could not be obtained for the investigated parameters. Therefore, estimates of the association of challenge test survival with the studied immune parameters were obtained as product moment correlations between family least square means. These correlations revealed statistically significant (p < 0.05) negative correlations of survival with bacterial agglutination titre (−0.48), serum haemolysin titre (−0.29) and haemagglutination titre (−0.34); and significant positive correlation with ceruloplasmin level (0.51). The correlations of survival to aeromoniasis with myeloperoxidase activity, superoxide production and lysozyme activity were found to be not significantly different from zero (p > 0.05). Assuming that the negatively correlated candidate traits are not favourable as indirect selection criteria, the results suggest that ceruloplasmin level could potentially be a marker for resistance to aeromoniasis in rohu. The use of this immune parameter as an indirect selection criterion for increased resistance to aeromoniasis in rohu will, however, require that the parameter shows significant additive genetic variation and a significant genetic correlation with survival. Further studies are therefore needed to obtain a reliable heritability estimate for ceruloplasmin and its genetic correlation with survival from aeromoniasis.     

Lectin like properties and differential sugar binding characteristics of C-reactive proteins purified from sera of normal and pollutant induced Labeo rohita

Different forms of C-reactive proteins (CRPs) have been purified to electrophoretic homogeneity from the sera of Labeo rohita confined in freshwater (CRP_N) and water polluted with nonlethal doses of cadmium (CRP_Cd) or mercury (CRP_Hg). CRP_N[emsp4 ], CRP_Cd[emsp4 ], and CRP_Hg show remarkable differences in their electrophoretic mobility but exhibit strong immunological cross reactivity. All these CRPs exhibit variable agglutination properties with erythrocytes from diverse sources in presence of Ca⁺², which could be inhibited by a variety of sugars showing specificity for galactose. Inhibition results show that the potency of galactose as an inhibitor increases about 4 fold in the process of transformation of CRP_N to CRP_Cd and CRP_Hg[emsp4 ]. In case of CRP_N[emsp4 ], Gal (11) Gal and oNO₂ phenyl -Gal show highest inhibitory potency while oNO₂-phenyl -Gal is the most potent inhibitor for CRP_Cd and CRP_Hg but the potency of Gal (11) Gal reduced drastically. 6-phosphate D-Gal and stachyose are 20 times weaker inhibitors than D-Gal for induced CRP mediated agglutination, in contrast, these sugars are only 6 times weaker for CRP_N[emsp4 ]. Dissociation constants of the binding of CRP_N with phosphoryl choline (PC) and galactose are about 9[emsp4 ]mM and PC binding causes a change in the and conformations of these CRPs.     

Dietary pyridoxine enhances thermal tolerance of Labeo rohita (Hamilton) fingerlings reared under endosulfan stress

A preliminary experiment was carried out to study the effect of dietary pyridoxine (PN) on thermal tolerance of Labeo rohita fingerlings exposed to endosulfan (1/10th 96 h LC₅₀=0.2 ppb) stress, reared at 26.0±0.5 °C to assess its culture potential in different agro-climatic zones. Two hundred seventy fingerlings were randomly distributed into six treatment groups in triplicate. Five iso-caloric and iso-nitrogenous purified diets were prepared with graded levels of pyridoxine. Six treatment groups were T₀ (10 mg PN+without endosulfan), T₁ (0 mg PN+endosulfan), T₂ (10 mg PN+endosulfan), T₃ (50 mg PN+endosulfan), T₄ (100 mg PN+endosulfan) and T₅ (200 mg PN+endosulfan). After feeding for 60 days, critical temperature maxima (CTmax), lethal temperature maxima (LTmax), critical temperature minima (CTmin) and lethal temperature minima (LTmin) were determined in each group. There was significant (P<0.05) effect of dietary pyridoxine on temperature tolerance (CTmax, LTmax, CTmin and LTmin) of the groups fed diets supplemented with 100 and 200 mg PN/kg diet compared to other experimental groups. Positive correlations were observed between CTmax and LTmax (R²=0.85) as well as between CTmin and LTmin (R²=0.97). The effect was more prominent on lower thermal tolerance limit (CTmin and LTmin). The overall results obtained in this preliminary study indicated that pyridoxine supplementation at 100 mg PN/kg diet enhances the thermal tolerance of endosulfan exposed L. rohita fingerlings.     

The effect of Euglena viridis on immune response of rohu, Labeo rohita (Ham.)

The study evaluated the effect of dietary doses of Euglena viridis on the immune response and disease resistance of Labeo rohita fingerlings against infection with the bacterial pathogen Aeromonas hydrophila. L. rohita fingerlings were fed with diet containing 0 (Control), 0.1 g, 0.5 g, 1.0 g Euglena powder kg⁻¹ dry diet for 90 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined after 30, 60 and 90 days of feeding. Fish were challenged with A. hydrophila 90 days post-feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with Euglena showed increased levels of superoxide anion production, lysozyme, serum bactericidal activity, serum protein and albumin (P < 0.05) compared with the control group. Following challenge with A. hydrophila less survivability was observed in the control group (56.65%) than the group fed the experimental diets. The group fed 0.5 g Euglena kg⁻¹ dry diet showed the highest percentage survival (75%). These results indicate that Euglena stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.     

Efficiency of osmotic and chemical treatments to improve the permeation of the cryoprotectant dimethyl sulfoxide to Japanese whiting (Sillago japonica) embryos

Insufficient cryoprotectant permeation is one of the major obstacles for successful fish embryo cryopreservation. The purpose of this study was to test the effectiveness of osmotic and chemical treatments to enhance cryoprotectant uptake by fish embryos. Japanese whiting Sillago japonica embryos at the somites and tail elongation stages were treated with hyperosmotic sugar solutions (1 M trehalose and sucrose) for 2-6 min, or a permeating agent (2-6 mg/mL pronase) for 30-120 min, and then impregnated with 10-15% DMSO in artificial sea water or aqueous solutions containing inorganic salts (0.125-0.25 M MgCl₂ and CaCl₂). The viability of the embryos after the treatments was estimated from hatching rates and the internal DMSO concentration was measured by HPLC. Treatment with trehalose for 3 min prior to impregnation with DMSO enhanced the uptake of the cryoprotectant by 45% without significantly affecting embryo viability, whereas pronase had no noticeable effect on cryoprotectant permeation. Incorporation of DMSO into the embryos was enhanced by 143-170% in the presence of 0.25 M MgCl₂ and 0.125 M CaCl₂ compared to sea water. A combination of treatments with trehalose and MgCl₂ was even more effective in promoting DMSO permeation (191% compared to untreated embryos). Tail elongation embryos were less tolerant of the treatments, but had higher DMSO impregnation. In conclusion, the use of trehalose (as dehydrating agent) and MgCl₂/CaCl₂ (as a vehicle during impregnation) greatly promoted cryoprotectant uptake and may be a promising aid for the successful cryopreservation of fish embryos.     

Effect of dietary β-1,3 glucan on immune responses and disease resistance of healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita Hamilton)

The immunostimulant β-1,3 glucan was fed at 0·1% in feed for 7 days to healthy and aflatoxin B₁(AFB₁)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial. The effects of AFB₁, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied. A single intraperitoneal injection of AFB₁at 1·25 mg kg⁻¹body weight) caused a significant (P< 0·05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan. Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control. Feeding of glucan to AFB₁-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A. hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB₁exposed fish. Although feeding of glucan was able to increase specific immunity, al measured through haemagglutination titre against sheep red blood cells, and bacterial (E. tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen.     

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 °C, 17 °C and 25 °C respectively. Thereafter, a recovery period of 24 h at 17 °C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immunomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 °C and 24 h of air exposure at 17 °C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 °C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 °C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 °C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops.     

Biochemical and biophysical aspects of the tolerance of anhydrobiotic crustacean embryos to very high temperatures

1.

Heat tolerance of dry cysts of the fairy shrimp Branchipus schaefferi and the brine shrimp, Artemia franciscana was studied using rapid (∼100 °C/min) and slow (∼4 °C/min) heating.     

Response of Lymantria dispar L. (Lepidoptera: Lymantriidae) to Bacillus thuringiensis subsp. kurstaki at different ingested doses and temperatures

We examined mortality and feeding inhibition response of Lymantria dispar L. (Lepidoptera: Lymantriidae) larvae to ingested doses of Bacillus thuringiensis subsp. kurstaki as a function of dose, instar and temperature. We developed generalized (logistic) linear mixed models and a mixture survival model, commonly used in medical statistics, to analyze the complex data set. We conducted bioassays of Foray 48B with larvae from the NJSS laboratory stock, using droplet imbibing or force-feeding to ensure dose ingestion. The dose causing mortality in 50% of the test population (LD₅₀) under standard test conditions (22 °C) ranged from 0.019 International Units (IU)/larva for first instar larvae (L₁) to 1.6 IU/larva for L₄. Temperature affected larval mortality in two ways. Mortality occurred sooner and progressed more rapidly with increasing temperature (13-25 °C) at each dose level and instar, while the maximum level of mortality attained by each instar decreased with increasing rearing temperature. The mechanisms underlying this effect are being investigated. Larvae that survived exposure to B. thuringiensis resumed feeding after a period that was dependent on instar, dose, and temperature. The equations describing observed mortality and feeding recovery responses were used to construct a simulation model, which was able to predict both processes, and which forms the basis for a process-oriented model that can be used as a decision support tool in aerial sprays.     

Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s

Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by pluteus. On the contrary, the same treatment with cadmium induces continuous HSP70/72 synthesis and produces irregular gastrula embryos which then degenerate. Moreover, a long treatment induces over control embryos a slight increase in the amount of constitutive HSP75 during development while lead treatment depresses constitutive HSP75 at early stages and doubles its quantity at late stages.     

Shape and function of the Bicoid morphogen gradient in dipteran species with different sized embryos

The Bicoid morphogen evolved approximately 150 MYA from a Hox3 duplication and is only found in higher dipterans. A major difference between dipteran species, however, is the size of the embryo, which varies up to 5-fold. Although the expression of developmental factors scale with egg length, it remains unknown how this scaling is achieved. To test whether scaling is accounted for by the properties of Bicoid, we expressed eGFP fused to the coding region of bicoid from three dipteran species in transgenic Drosophila embryos using the Drosophila bicoid cis-regulatory and mRNA localization sequences. In such embryos, we find that Lucilia sericata and Calliphora vicina Bicoid produce gradients very similar to the endogenous Drosophila gradient and much shorter than what they would have produced in their own respective species. The common shape of the Drosophila, Lucilia and Calliphora Bicoid gradients appears to be a conserved feature of the Bicoid protein. Surprisingly, despite their similar distributions, we find that Bicoid from Lucilia and Calliphora do not rescue Drosophila bicoid mutants, suggesting that that Bicoid proteins have evolved species-specific functional amino acid differences. We also found that maternal expression and anteriorly localization of proteins other than Bcd does not necessarily give rise to a gradient; eGFP produced a uniform protein distribution. However, a shallow gradient was observed using eGFP-NLS, suggesting nuclear localization may be necessary but not sufficient for gradient formation.     

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.