Identification,characterization, and expressions profile analysis of growth hormone receptors (GHR1 and GHR2) in Hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus polyphekadion ♂)

Growth hormone is an essential hormone that plays essential roles in growth, metabolism, cellular differentiation, immunity and reproduction in fish, by means of the growth hormone receptors. The encoding cDNA growth hormone receptors (GHR1 and GHR2) were cloned and characterized from Hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus polyphekadion♂). Sequence analysis of the cloned GHR1 was observed as containing 2176, which comprised an ORF of 1842 bp, 5 UTR of 6 bp and 3 UTR of 328 bp, with 612 amino acids encoding proteins, while GHR2 was observed as containing 1824 bp that encompassed an ORF of 708 bp, 5 UTR of 48 bp and 3 UTR of 1068 bp with 235 amino acids encoding proteins. Relative mRNA expression of GHR1 and GHR2 in the liver and muscle was found to be highest respectively. Our findings provide vital statistics of GHRs likely to play a significant role in the growth of the fish.     

Evaluation of the effects of different stocking densities on growth and stress responses of juvenile hybrid grouper ♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus in recirculating aquaculture systems

This study was aimed at evaluating the physiological and metabolic responses of juvenile hybrid grouper ♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus to stocking density. Hybrid grouper juveniles (mean ± SE = 25.43 ± 2.36 g live mass) were stocked for 22 weeks in a recirculating aquaculture system (RAS) under four different densities: low stocking density (LD; 1.03 kg m⁻³), medium stocking density (MD; 2.06 kg m⁻³), high stocking density (HD; 3.09 kg m⁻³) and extra-high stocking density (EHD; 4.11 kg m⁻³). Biometric variables were recorded and plasma, liver, intestine and stomach samples were taken for biochemical analysis at the end of the experimental period. Final stocking densities were 6.27, 16.04, 23.77 and 28.32 kg m⁻³, respectively, with significant differences in growth performance. Our results showed that the best growth rates and feed utilisation occurred in the MD group. Higher plasma cortisol and glucose levels and lower triglyceride levels reflected the stress responses in the EHD group. Moreover, the activity of aspartate and alanine transaminases was elevated in the HD and EHD groups due to enhanced gluconeogenesis. The activity of the digestive enzyme pepsin significantly increased in the MD group. We found that 2.06–3.09 kg m⁻³ is the most suitable starting density for culturing juvenile hybrid grouper in recirculating aquaculture systems.     

Antiviral effects of β-defensin derived from orange-spotted grouper (Epinephelus coioides)

Defensins are a group of small antimicrobial peptides playing an important role in innate host defense. In this study, a β-defensin cloned from liver of orange-spotted grouper, Epinephelus coioides, EcDefensin, showed a key role in inhibiting the infection and replication of two kinds of newly emerging marine fish viruses, an enveloped DNA virus of Singapore grouper iridovirus (SGIV), and a non-enveloped RNA virus of viral nervous necrosis virus (VNNV). The expression profiles of EcDefensin were significantly (P < 0.001) up-regulated after challenging with Lipopolysaccharide (LPS), SGIV and Polyriboinosinic Polyribocytidylic Acid (polyI:C) in vivo. Immunofluorescence staining observed its intracellular innate immune response to viral infection of SGIV and VNNV. EcDefensin was found to possess dual antiviral activity, inhibiting the infection and replication of SGIV and VNNV and inducting a type I interferon-related response in vitro. Synthetic peptide of EcDefensin (Ec-defensin) incubated with virus or cells before infection reduced the viral infectivity. Ec-defensin drastically decreased SGIV and VNNV titers, viral gene expression and structural protein accumulation. Grouper spleen cells over-expressing EcDefensin (GS/pcDNA-EcDefensin) support the inhibition of viral infection and the upregulation of the expression of host immune-related genes, such as antiviral protein Mx and pro-inflammatory cytokine IL-1β. EcDefensin activated type I IFN and Interferon-sensitive response element (ISRE) in vitro. Reporter genes of IFN-Luc and ISRE-Luc were significantly up-regulated in cells transfected with pcDNA-EcDefenisn after infection with SGIV and VNNV. These results suggest that EcDefensin is importantly involved in host immune responses to invasion of viral pathogens, and open the new avenues for design of antiviral agents in fisheries industry.     

Morphogenesis of sense organs and behavioural changes in larvae of the brown-marbled grouper Epinephelus fuscoguttatus (Forsskål)

The morphogenesis of sense organs and related behavioural changes in the hatchery-reared brown-marbled grouper Epinephelus fuscoguttatus larvae were examined to gain better understanding of its early life history because ecological field observations for grouper species is difficult. The newly hatched larvae (2.1 mm total length) had developing eyes and otic vesicles, a pair of free neuromast on the head and ciliated olfactory epithelium. At 3 days post hatching (dph), the eyes became fully pigmented with pure-cone retinae, the semicircular canals formed in the inner ear, and the larvae (2.8 mm) were able to swim horizontally, preying on rotifers. Retinal rods and the intra-oral taste buds at pharyngeal appeared next. The olfactory lamellae and the head lateral line system then formed, and the inner ears developed completely in the larvae during the metamorphosis period (15–40 dph; 5.1–18.1 mm). At settlement (50 dph; 32.8 mm), the fish possessed taste buds in the mouth entrance region, and the lateral line system developed completely. The sensory development correlates well with the known aspects of its life history at sea whereby the larvae can feed early and avoid predators during the passive drift, are able to swim shoreward to search nursery ground along the metamorphosis stage and survive in seagrass beds at settlement.     

New isolate of Loma psittaca (Microsporidia: Glugeidae) infecting the stomach wall of cultured hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) in South China

Loma psittaca, previously described as inhabiting the intestinal mucosa of an anadromous fish, Colomesus pisttacus, from the Amazon Basin, is reported as being found for the first time in a marine fish, the hybrid grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀), from Lingshui city, Hainan Province, China, expanding the geographical distribution and host range of this parasite. Numerous whitish xenomas (0.5–0.7 mm in diameter) of this new isolate of L. psittaca were found distinctly in the muscle layer of the host stomach wall. Electron microscopic observations showed a monokaryotic nucleus in all developmental stages. Round or elongated multinucleate merogonial plasmodia surrounded by numerous mitochondria were observed initially, subsequently transforming into uninucleate sporonts through multiple fissions. Sporonts, each with a large centrally positioned nucleus, further developed into sporoblasts. Each sporoblast mother cell gave rise to two uninucleate sporoblasts by binary fission. Mature spores were ellipsoidal, measuring 4.0 ± 0.3 (3.7–4.3) μm in length and 2.2 ± 0.2 (2.1–2.5) μm in width. Spores possessed a mushroom-like anchoring disk, a bipartite polarplast, isofilar polar filaments arranged in 12–14 turns in one row, and a trilaminar spore wall. The obtained partial SSU rRNA gene sequence of the new isolate was 1330 bp in length and showed 99.4% sequence similarity with an estuary isolate of L. psittaca previously reported in South America. SSU rRNA gene-based phylogenetic analyses demonstrated that the two L. psittaca isolates first clustered together and then formed a dichotomy that included the digestive-tract-infecting Loma species, L. acerinae, with high support values within group I.     

Parasite diversity as an indicator of environmental change? An example from tropical grouper (Epinephelus fuscoguttatus) mariculture in Indonesia

Fish parasites are used to monitor long-term change in finfish grouper mariculture in Indonesia. A total of 210 Epinephelus fuscoguttatus were sampled in six consecutive years between 2003/04 and 2008/09 and examined for parasites. The fish were obtained from floating net cages of a commercially run mariculture facility that opened in 2001. The fauna was species rich, consisting of ten ecto- and 18 endoparasite species. The ectoparasite diversity and composition was relatively stable, with the monogeneans Pseudorhabdosynochus spp. (83-100% prevalence, Berger-Parker Index of 0·82-0·97) being the predominant taxon. Tetraphyllidean larvae Scolex pleuronectis and the nematodes Terranova sp. and Raphidascaris sp. 1 were highly abundant in 2003/04-2005/06 (max. prevalence S. pleuronectis 40%, Terranova sp. 57%, Raphidascaris sp. 1 100%), and drastically reduced until 2008/09. These parasites together with the prevalence of Trichodina spp., ecto-/endoparasite ratio and endoparasite diversity illustrate a significant change in holding conditions over the years. This can be either referred to a definite change in management methods such as feed use and fish treatment, or a possible transition of a relatively undisturbed marine environment into a more affected habitat. By visualizing all parameters within a single diagram, we demonstrate that fish parasites are useful bioindicators to monitor long-term change in Indonesian grouper mariculture. This also indicates that groupers can be used to monitor environmental change in the wild. Further taxonomic and systematic efforts in less sampled regions significantly contributes to this new application, supporting fish culture and environmental impact monitoring also in other tropical marine habitats.     

Comparative analysis of the aquaculture potential of hybrid Tilapia zillii (male) × T. guineensis (female) (Teleostei: Cichlidae) in floating cages,cement tanks and earth ponds

The effect of three rearing systems, floating cages, cement tanks and earth ponds, on the growth rate, feeding efficiency and mortality rates of hybrid tilapia — Tilapia zillii (male) × T. guineensis (female) — was evaluated for 233 days. Fish of average weight 12.59g were stocked at a density of 20 fish m^?3 and were fed a 30% protein pelleted commercial feed. The pH, degree of aeration and transparency were monitored. The mortality rate, final mean weight, daily weight gain, and feed conversion ratios were significantly different among rearing systems. The highest potential was in earth ponds (2.86%, 171.70g, 0.74g d^?1, 5.42, respectively), the cages were of intermediate potential (7.50%, 137.22g, 0.57g d^?1, 7.70) and the lowest potential was in cement tanks (31.86%, 45.23g, 0.15g d^?1, 10.28). These differences could be linked to the relevant physical and chemical characteristics of the three different rearing systems.     

Immunohistochemical localization of transforming growth factor α in the major salivary glands of male and female rats

Summary The three major salivary glands of normal male and female Fischer 344 rats of different ages were examined for the localization of epidermal growth factor (EGF) and transforming growth factor (TGF) by immunohistochemical staining. EGF was demonstrated only in the granulated convoluted tubule (GCT) cells of the submandibular gland, the results confirming the previous reports, and most abundantly in adult males and pregnant females. TGF stain was localized in all three glands and was found throughout the entire duct system, excluding acinar cells. The myoepithelial cells of the sublingual gland were also reactive with the TGF antibody. The specificity of the staining was confirmed by negative staining reaction with the absorbed antibody and by radio-immunoassay and Western blot methods. This is the first report describing the presence of TGF in the rat salivary glands.     

Genetic regulation of the class conversion of dsDNA-specific antibodies in (NZB × NZW)F1 hybrid

To investigate the possible effects of NZW genes on the class conversion of dsDNA-specific antibodies in NZB X NZW (B/W)F1 hybrids, we measured IgM, IgG1, and IgG2 dsDNA-specific antibodies, using the Crithidia luciliae kinetoplast immunofluorescence test, in NZB, NZW, B/W F1 hybrid, B/W F1 X NZB backcross, and B/W F1 X NZW backcross mice at 4, 7, and 10 months of age. The highest serum levels of IgM dsDNA-specific antibodies were observed in NZB mice at the ages tested; however, the amounts of IgG1 and IgG2 antibodies were scanty. In contrast, a large amount of both IgG1 and IgG2 dsDNA-specific antibodies was produced in B/W F1 hybrids, in which the serum IgM antibodies were lower than those observed in NZB mice. NZW mice were virtually negative for these antibodies. Progeny testing suggested that a combined effect of two unlinked dominant genes of the NZB strain determines the production of dsDNA-specific antibodies and that these genes only act to produce IgM antibodies. These traits are to a great degree modified by the NZW loci in B/W F1 hybrids, and a combined effect of two unlinked dominant genes leads to conversion of the class of the antibodies from IgM to IgG, which, in turn, increases the serum levels of dsDNA-specific antibodies. The F1 hybrid of C57BL/6 and NZW strains produced no dsDNA-specific antibodies, indicating that the relevant NZB predisposing genes are required for the NZW gene action. Linkage studies showed that one of such NZW genes is to some extent linked to the H-2 complex on chromosome 17, but not to Mup-1 (chromosome 4) or a coat color locus (chromosome 2). The appearance of IgG dsDNA-specific antibodies correlated well with the incidence of renal disease in B/W F1 X NZB backcross mice.     

The reproductive history of a hybrid female (Macaca mulatta ×Macaca fuscata)

In 1968 a female hybrid (Macaca mulatta ×Macaca fuscata) was born into the freeranging Arashiyama B troop of Japanese macaques (Macaca fuscata). At the age of 4 years this hybrid had her first offspring and by 1978 had accumulated eight descendants (five daughters, one son and two grandchildren). In this report the reproductive history of the hybrid is discussed and compared to the other non-hybrid members of the troop.     

Seasonal changes in daily torpor patterns of free-ranging female and male Daubenton’s bats (Myotis daubentonii)

Daily torpor can provide significant energy and water savings in bats during cold ambient temperatures and food scarcity. However, it may reduce rates of foetal and juvenile development. Therefore, reproductive females should optimize development by minimizing times in torpor. To test this hypothesis, the use of torpor by female and male free-ranging Daubenton’s bats (Myotis daubentonii) during reproduction (gestation, lactation, and post-lactation period) was investigated in 1998 and 1999. Temperature-sensitive radio transmitters were attached to the bats to measure skin temperature. Simultaneously, ambient temperature was recorded. While both sexes became torpid during daytime, male bats used daily torpor (>6°C below individual active temperature) significantly more often during reproductive period (mean: 78.4 % of day time in May and 43 % in June) than females. Female bats went into daily torpor, particularly in late summer when juveniles were weaned (mean: 66.6 % of daytime). Lowest skin temperatures occurred in a female bat with 21.0°C during post-lactation. Skin temperatures of male bats fluctuated from 16.8°C in torpor to 37.2°C during times of activity. There was a significant effect of reproductive period on skin temperature in females whereas mean ambient temperature had no significant effect. However, mean ambient temperature affected mean skin temperatures in males. Our findings indicate that female Daubenton’s bats adopt their thermoregulatory behaviour in particular to optimize the juvenile development.     

Electrophoretic confirmation of the intergeneric hybrid ×Ruttyruspolia (Acanthaceae)

A plant collected in South Africa in the early 1960's has been considered an intergeneric hybrid with the parental taxa beingRuspolia hypocrateriformis (Vahl)Milne-Redhead var.australis Milne-Redhead andRuttya ovata Harv. The intermediate morphology of the plant provided the strongest evidence of its hybrid origin. The natural hybrid, named formally as ×Ruttyruspolia A. Meeuse & de Wet, is highly sterile. Crosses between the two presumed parental taxa produced two plants that are very similar to the putative natural hybrid. We had examined the presumed parental species and the natural and artificial hybrids using enzyme electrophoresis. The two parental species are highly differentiated at genes specifying soluble enzymes; they have a genetic identity of 0.51. They have no common alleles at two genes, and contain alternative alleles in very different frequencies at two loci.Ruttya andRuspolia exhibit both unique and common alleles at two additional genes. The natural and artificially produced plants of ×Ruttyruspolia are identical electrophoretically and contain alleles unique to each of the parental species at two genes. In addition, individuals of ×Ruttyruspolia combine alternative high frequency alleles from each parent at two loci. Allozymes provide strong confirming evidence for the hybrid origin of naturally occurring ×Ruttyruspolia because the products of specific alleles either unique to or highly characteristic of the two putative parental taxa are found combined in ×Ruttyruspolia.     

Isozyme analysis of progeny derived from (Allium fistulosum ×Allium cepa) ×Allium cepa

Summary Relatively large quantities of seed were obtained from the interspecific backcross (A. fistulosum xA. cepa) ×A. cepa allowing, for the first time, an extensive study of the heritable traits exhibited by backcross progeny. Two backcross populations, BC1034 and BC1040, distinguished by differentA. fistulosum parents, were characterized for the isozyme markersIdh-1, Adh-1, andPgi-1. Statistical methods are described to calculate cell probabilities for a mixed population of F₂ and BC₁ progeny, using an estimate of the fraction of F₂ progeny in the population derived from the isozyme data. Cell probability distributions were calculated for a mixed population with independent pairs of loci and a mixed population with nonindependent pairs of loci. The isozyme lociIdh-1 andPgi-1 appear to be linked, with a map distance estimated at 33 centimorgans (cM) in BC1034 and 42 cM in BC1040. The probability distribution model for linked loci did not account for all of the distorted segregation ratios inIdh-1 ×Adh-1 orPgi-1 ×Adh-1. The cytological literature does not support linkage betweenIdh-1 ×Adh-1 orPgi-1 ×Adh-1. The distorted segregation ratios for these pairs of loci are likely the result of genetic incompatibilities between the two species.Journal Article No. 1578, Agricultural Experiment Station, New Mexico State University, Las Cruces, NM, USA     

Apoptotic cell death induces temperature-sensitive lethality in hybrid seedlings and calli derived from the cross of Nicotiana suaveolens×N. tabacum

Hybrid lethality expressed in the interspecific hybrid of Nicotiana suaveolens Lehm. ×N. tabacum L. cv. Hicks-2 is one of the mechanisms for reproductive isolation and it is temperature-sensitive. Apoptotic changes were detected in the cells of hybrid seedlings and calli expressing lethality at 28 °C but not under high-temperature conditions (36 °C), when the lethality is suppressed. Condensation of chromatin, fragmentation of nuclei and cytoplasmic reduction are the cytological changes associated with apoptosis leading to hybrid lethality. Fragmentation of nuclei was correlated with the lethal symptoms in both hybrid seedlings and calli, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid seedlings and calli showing lethal symptoms revealed a specific ladder pattern suggesting nucleosomal fragmentation which is one of the biochemical changes of apoptosis. In-situ detection using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in distinct stages on each organ of hybrid seedlings and centripetally in hybrid calli. From these results, we confirmed that cell death inducing hybrid lethality was indeed apoptosis. Received: 23 December 1999 / Accepted: 5 April 2000     

Annual growth rings and long-term growth patterns of mangrove trees from the Bragança peninsula,North Brazil

Ring analysis was carried out on 39 Rhizophora mangle trees from two salineand one brackish forest sites on apeninsula in north Brazil. All trees showedconstant growth over their entire lifespan, however the distinctiveness of growthrings was greater in trees from the salineareas than in trees from the brackish site.The mean radial increments form a patternof three distinct groups (`fast', `medium',and `slow' growth). Although all threegrowth groups contained trees from eachstudy site, trees from the brackish and thefrequently inundated saline area presenteda significantly higher growth rate (3.3 mmy^-1) than the trees growing in asaline, seldom inundated area. Radiocarbonanalysis showed that R. mangle formsannual rings in the region. The mean age ofeach forest, derived from the mean stemradius, the growth rates, and the treeaffiliation to each growth group, explainsforest's density and basal area. For thetrees from a saline area belonging to themedium growth group (mean increment 2.5 mmy^-1), the cambial growth correlatessignificantly with the precipitation in thetransition months between the dry and therainy season. The slowest growing trees(1.2 mm y^-1) showed a closerelationship between the ring width and thenumber of months with rainfall < 50 mm.Based on these results, we propose that thelocal abiotic factors influence theindividual growth rates but their effect onthe forest structure is modified by bioticfactors, such as neighborhoodcompetition.     

Morphological and Molecular Genetic Verification of Interspecific Hybrid Salix × zhataica (Salicaceae) from Central Yakutia

Russian Journal of Genetics - The results of morphological and molecular genetic analysis confirming the hybrid nature of Salix × zhataica Efimova, Shurduk et Ahti are presented. In...     

Construction and analysis of cDNA libraries from the antennae of male and female cotton bollworms Helicoverpa armigera (Hübner) and expression analysis of putative odorant-binding protein genes

Two high-quality cDNA libraries were constructed from female and male antennae of the cotton bollworm Helicoverpa armigera (Hübner). The titers were approximately 2.0 × 10? pfu/ml for females and 2.3 × 10? pfu/ml for males, and this complies with the test requirement. From the libraries, 1750 male ESTs and 1640 female ESTs were sequenced and further analyzed. We identified 15 olfactory genes (12 are new), and 14 of them have the characteristic six conserved cysteine residues. With the exception of OBP9, all the genes were classified as classical OBP genes. By alignment and cluster analysis, the 14 classical OBPs were divided into pheromone binding protein (PBP) genes, odorant binding protein (OBP) genes, general odorant binding protein 1 (GOBP1) genes, general odorant binding protein 2 (GOBP2) genes and antennae binding protein (ABP) genes. Among these genes, we obtained three PBP genes (PBP1-PBP3) including two new PBP genes, one new ABP gene, nine new OBP genes (OBP1-OBP9), one known GOBP1 gene and one known GOBP2 gene. Furthermore, the expression patterns of these 14 classical OBP genes were investigated in various tissues by real-time quantitative polymerase chain reaction (qPCR). The results indicated that some OBP genes are expressed differently in different sexes and tissues, but most of them are highly expressed in antennae.