Why is grouper larval rearing difficult?: an approach from the development of the feeding apparatus in early stage larvae of the grouper,Epinephelus coioides

The osteological development of elements forming the oral cavity was examined in early stage larvae of the grouper,Epinephelus coioides, from hatching to 242.5 hours after hatching. By the time of initial mouth opening, at 54 hours after hatching, the fundamental elements, composed of the trabecula, some components of the lower branchial and hyoid arches, the quadrate and symplectic-hyomandibular cartilages, maxilla and Meckel's cartilage, had appeared. No further elements were observed until 165 hours after initial mouth opening, except some components in the lower branchial arch and head region. The appearance of new elements and initial ossification of existing cartilage occurred thereafter, but all elements related to feeding either had not appeared or had not started ossifying until 188.5 hours after initial mouth opening. Based on the morphology and developmental modes of these elements, the feeding mode of grouper larvae was considered to be “sucking/grasping.” However, the appearance and ossification of elements occurred slowly, with no transitional phase from sucking to grasping modes of feeding being observed during the study; such delayed development of the feeding-related bony elements was considered to be a cause of the difficulty in rearing early stage grouper larvae.     

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.     

Effects of a Potential Host Gut-Derived Probiotic,Bacillus subtilis 6-3-1, on the Growth,Non-specific Immune Response and Disease Resistance of Hybrid Grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂)

Probiotics and Antimicrobial Proteins - A potential host-derived probiotic, Bacillus subtilis 6-3-1, was successfully screened from 768 isolates from the intestines of healthy hybrid grouper...     

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.     

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.     

芳香化酶抑制剂letrozole对赤点石斑鱼(Epinephelus akaara)性逆转的作用

在繁殖季节,采用腹部埋植方式,用非类固醇型芳香化酶抑制剂（aromatase inhibitor,AI）letrozole以5mg／kg体重的剂量处理2龄雌性赤点石斑鱼（每4周埋植1次,共埋植2次）,检查埋植后性腺组织结构、血清性类固醇激素以及脑和性腺芳香化酶活性的变化。结果显示：一次埋植AI即可有效诱导雌性赤点石斑鱼发生不同程度的性逆转;性腺成熟指数明显下降;性腺中卵细胞退化,精原细胞增殖,出现大量精母细胞和精子细胞;性逆转雄鱼的精巢在组织结构上与正常雄鱼精巢没有明显差异,部分鱼成为功能性雄鱼。第一次埋植AI后4周轻微挤压腹部有14．3％的鱼可排精,精子活力与正常雄鱼相同。第二次埋植后明显提高性逆转效果,排精率在第6、8周分别达到35．3％和48．4％。此外,埋植AI后性腺芳香化酶活性显著降低,化脑郝芳香化酶活性的变化不明显;血清11-酮基睾酮（11-ketotestosterone,11-KT）浓度显著增加,雌二醇（estradiol-17β,E2）水平显著降低,而睾酮（testosterone,T）含量无明显变化。这些结果表明,AI主要通过抑制内源性E2的产生并提高11-KT水平,从而诱导赤点石斑鱼由雌性转变为雄性。     

β-葡聚糖刺激下的斜带石斑鱼(Epinephelus coioides)转录组分析

斜带石斑鱼(Epinephelus coioides)是一种重要的海水经济养殖鱼类,然而病害是制约其养殖产量的一个重要因素。近年来-葡聚糖作为一种新型的天然免疫增强剂在水产养殖业中得到广泛应用。本研究采用腹腔注射0.2 mL-葡聚糖(浓度0.05 g/mL)刺激斜带石斑鱼,在刺激6 h、72 h后分别取头肾、血液淋巴细胞、胸腺和脾脏组织进行转录测序。结果一共组装了175 568条unigene,平均长度是1 507 bp。通过Blast比对数据库进行功能注释,一共注释了121 629条unigene。六大数据库注释得到的unigene数量分别为:91 955条(Nt)、66 089条(Nr)、14 126条(GO)、24 018条(COG)、9 673条(KEGG)和54 148条(Swiss Prot)。根据基因差异表达分析筛选出6类免疫相关的显著性差异基因71个,涉及抗原加工呈递、细胞粘附分子、补体系统、细胞因子、模式识别受体和吞噬作用。对差异基因进行GO富集分析,筛选出免疫相关条目7条,包括免疫应答、B细胞受体信号通路、趋化因子受体活性、白细胞趋化性、趋化因子调节信号通路、白细胞介素-1受体绑定和溶酶体组份。随机挑选7个显著性差异的免疫基因进行qRT-PCR验证,验证结果与测序结果变化趋势一致。本实验结果为研究-葡聚糖调节鱼类免疫系统的作用机制提供数据参考。     

Orange-spotted grouper (Epinephelus coioides) TLR2, MyD88 and IL-1β involved in anti-Cryptocaryon irritans response

Cryptocaryon irritans is one of the most important ectoparasites of marine fish, and can have a devastating effect on aquacultured fish populations. The role of TLR signaling pathways in anti-parasitic immune responses is poorly understood in fish. In this paper, we first cloned Epinephelus coioides MyD88 full-length cDNA (EcMyD88) and its respective gene. The open reading frame (ORF) of cDNA is 873 bp encoding 291 amino acid residues. Similar to other species, the EcMyD88 gene contains of five conserved exons and four diverse introns. The constitutive expression of EcMyD88 was detected in the gill, trunk kidney, head kidney, spleen, and heart in high concentrations, while the skin, brain, liver, and muscles contained much lower titers, indicating that EcMyD88 may play a crucial role in host innate immunologic surveillance. To identify the potential role of TLR pathways in fish anti-C. irritans immune responses, we chose three important molecules involved in anti-parasite responses, TLR2, MyD88 and IL-1β to indicate TLR pathway’s signal-in, signal transduction, and signal-out functions, respectively. The expression profile of these three genes was detected in grouper infected by C. irritans. Results showed these molecules each experience significant changes within the skin, gill (two infected mucosal sites), head kidney and spleen (two systematic immune organs) after C. irritans infection. These findings indicate the TLR signaling pathway may play an important role in host defense against C. irritans.     

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.     

Is the Octomacridae the sister family of the Diplozodae?

Diplozoidae and Octomacridae are usually considered as sister families. Essentially this is because they are the only polyopisthocotyleans parasitising primary freshwater teleosts. Because of the lack of phylogenetically informative morphological characters to explore the pattern of colonisation of the primary continental freshwater teleosts and in order to understand the appearance of the "natural parabiosis" of Diplozoidae, a molecular phylogeny was inferred by comparing newly obtained partial 28S and 18S rDNA gene sequences of Eudiplozoon nipponicum and Diplozoon homoion with other already available sequences. The phylogenetic analysis seems to show that Diplozoidae and Octomacridae are not sister groups. Thus, the colonisation of primary freshwater teleosts by these two families could be independent.     

Did the evolution of the phytoplankton fuel the diversification of the marine biosphere?

We discuss the possible links between the fossil record of marine biodiversity, nutrient availability and primary productivity. The parallelism of the fossil records of marine phytoplankton and faunal biodiversity implicates the quantity (primary productivity) and quality (stoichiometry) of phytoplankton as being critical to the diversification of the marine biosphere through the Phanerozoic. The relatively subdued marine biodiversity of the Palaeozoic corresponds to a time of relatively low macronutrient availability and poor food quality of the phytoplankton as opposed to the diversification of the Modern Fauna through the Mesozoic–Cenozoic. Increasing nutrient runoff to the oceans through the Phanerozoic resulted from orogeny, the emplacement of Large Igneous Provinces (LIPs), the evolution of deep-rooting forests and the appearance of more easily decomposable terrestrial organic matter that enhanced weathering. Positive feedback by bioturbation of an expanding benthos played a critical role in evolving biogeochemical cycles by linking the oxidation of dead organic matter and the recycling of nutrients back to the water column where they could be re-utilized. We assess our conclusions against a recently published biogeochemical model for geological time-scales. Major peaks of marine diversity often occur near rising or peak fluxes of silica, phosphorus and dissolved reactive oceanic phosphorus; either major or minor ⁸⁷Sr/⁸⁶Sr peaks; and frequently in the vicinity of major (Circum-Atlantic Magmatic Province) and minor volcanic events, some of which are associated with Oceanic Anoxic Events. These processes appear to be scale-dependent in that they lie on a continuum between biodiversification on macroevolutionary scales of geological time and mass extinction.     

Imperfect Batesian mimicry—the effects of the frequency and the distastefulness of the model

Batesian mimicry is the resemblance between unpalatable models and palatable mimics. The widely accepted idea is that the frequency and the unprofitability of the model are crucial for the introduction of a Batesian mimic into the prey population. However, experimental evidence is limited and furthermore, previous studies have considered mainly perfect mimicry (automimicry). We investigated imperfect Batesian mimicry by varying the frequency of an aposematic model at two levels of distastefulness. The predator encountered prey in a random order, one prey item at a time. The prey were thus presented realistically in a sequential way. Great tits (Parus major) were used as predators. This experiment, with a novel signal, supports the idea that Batesian mimics gain most when the models outnumber them. The mortalities of the mimics as well as the models were significantly dependent on the frequency of the model. Both prey types survived better the fewer mimics there were confusing the predator. There were also indications that the degree of distastefulness of the model had an effect on the survival of the Batesian mimic: the models survived significantly better the more distasteful they were. The experiment supports the most classical predictions in the theories of the origin and maintenance of Batesian mimicry.     

Phytochemicals: the good, the bad and the ugly?

Phytochemicals are constitutive metabolites that enable plants to overcome temporary or continuous threats integral to their environment, while also controlling essential functions of growth and reproduction. All of these roles are generally advantageous to the producing organisms but the inherent biological activity of such constituents often causes dramatic adverse consequences in other organisms that may be exposed to them. Nevertheless, such effects may be the essential indicator of desirable properties, such as therapeutic potential, especially when the mechanism of bioactivity can be delineated. Careful observation of cause and effect, followed by a coordinated approach to identify the responsible entities, has proved extremely fruitful in discovering roles for phytochemical constituents. The process is illustrated by selected examples of plants poisonous to animals and include the steroidal alkaloid toxin of Veratrum californicum (Western false hellebore), piperidine alkaloids of Lupinus species (lupines), and polyhydroxy indolizidine, pyrrolizidine and nortropane alkaloids of Astragalus and Oxytropis species (locoweeds), Castanospermum australe (Moreton Bay chestnut) and Ipomoea species (morning glories).