Sea bass Dicentrarchus labrax nervous necrosis virus isolates with distinct pathogenicity to sea bass larvae.

Reproduction of nodavirus disease was performed by experimental infection of sea bass eggs during fertilization or at larval stage 4 with 2 genetically distinguishable nodavirus strains (Sb1 and Sb2) isolated from sea bass collected along the Atlantic and Mediterranean French coast. The pathogenicity of the virus strains was assigned after detection of the virus by ELISA and immunohistochemistry (IHC). The Atlantic (Sb1) strain was more pathogenic than the Mediterranean (Sb2) strain during the fertilization step whilst both strains were pathogenic following experimental exposure of 4 d old larvae. Virus lesions developed in the brain 4 to 6 d following experimental exposure. Experimental ELISA proved very sensitive for detecting the nodavirus in Sb1 or Sb2 experimentally infected larvae, as well as in naturally infected sea bass larvae collected in French hatcheries or in barramundi larvae reared in the Pacific area. The development of an ELISA specific for the 2 nodavirus strains isolated from the sea bass should be useful for the detection of the virus, in addition to other techniques recommended by the Office International des Epizooties (OIE).     

Induction of Tetraploid Gynogenesis in the European Sea Bass (Dicentrarchus labrax L.)

A preliminary study on tetraploid gynogenetic induction in the European sea bass was performed by pressure-blocking the second polar body release and the first cleavage in eggs fertilized with ultraviolet-irradiated sperm. Fertilization of eggs with genetically inactivated sperm produced only haploid development that terminated around hatching. Pressure treatments (8.500 psi for 2 min) applied at 6 and 65 min after fertilization (a.f.) produced variable levels (7–95%) of tetraploid larvae at hatching. A small proportion of mosaics (3.8n/4.2n) was also recorded.     

Trypsin activity and physiological aspects in larval rearing of European sea bass (Dicentrarchus labrax) using live prey and compound diets

The aim of this study was to evaluate the potential of a compound diet as a live prey substitute for feeding European sea bass larvae ( Dicentrarchus labrax L.). The effect of a commercial diet (Nippai ML feed) and live prey ( Artemia nauplii) on tryptic enzyme activity, protein content, growth (standard length) and survival rates of sea bass larvae were tested during a 27-day rearing experiment. Sea bass larvae were divided into two groups. The live food group (control group) was fed exclusively on newly hatched Artemia nauplii (Inve AF grade), the test group was fed exclusively with the compound diet from day 15 onwards. As trypsin has been demonstrated to be a useful indicator for evaluating digestibility of food and the nutritional condition of fish larvae, individual tryptic enzyme activity was determined in both feeding groups. Larvae older than 14 days after hatching and fed on live food showed a significantly higher tryptic enzyme activity than larvae fed the compound diet. A similar relationship between tryptic activity and standard length in both test groups was detected only in small larvae (standard length < 7 mm). The usefulness of proteolytic enzyme activity measurements in larval fish research, as well as its use in aquaculture nutrition research, was confirmed. Protein content, increase in length and survival rates of the sea bass larvae were additionally determined in order to evaluate an influence on the diet. The protein content of larvae fed the Artemia nauplii was higher and the growth of larvae fed the compound diet was reduced. Larval mortality was not affected by the diet given.     

Distribution of Japanese temperate bass, <Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>, eggs and pelagic larvae in Ariake Bay

We collected eggs and larvae of the Japanese temperate bass, Lateolabrax japonicus, and present horizontal and temporal changes of distribution relative to development and growth during the species pelagic life history in Ariake Bay. Sampling was conducted from the inner to central region (11 sampling stations) of Ariake Bay using a plankton net (80 cm diameter, 0.5-mm mesh) from November 2000 to February 2001. Both eggs and larvae were collected most abundantly in mid-December. The CPUE of eggs in the surface layer was higher than the middle layer, which is in contrast to that at the larval stage. Most eggs were collected around the central and western regions of the bay. The distribution of eggs shifted vertically to the middle layer with development. Yolk-sac larvae were collected in the central region of the bay, and preflexion and flexion larvae were more abundantly collected in the inner region of the bay. The body length of larvae around the inner bay was larger than in the central region. The pelagic life history can be summarized as follows: eggs are distributed around the central region of the bay and eggs and larvae expand their distribution to the inner and shallower waters with growth. We conclude that the shift of vertical distribution in pelagic stages and the hydrographic features of the middle layer form one of the mechanisms enabling the inshore migration of L. japonicus.     

Nitrogen distribution and excretion during embryonic post-yolk sac development inZoarces viviparus

In the viviparous teleostZoarces viviparus (L.) embryonic post-yolk sac development in the ovary is characterized by significant increases in dry weight and nitrogen per embryo, thus indicating an extensive matrotrophic relationship. In the ovarian fluid surrounding the embryos during their intraovarian development, sources of nitrogen were shown to derive from amino acids, urea, ammonia and various cellular components. The level of urea in the ovarian fluid increased significantly from 3.68±0.25 mol urea-N·ml^-1 during early post-yolk sac embryonic development to 6.14±0.44 in late development. The corresponding level of ammonia-N in the ovarian fluid increased from 0.25 to 0.45 mol·ml^-1. An estimation of embryonic nitrogen loss was made by measuring urea and ammonia-N excretion in vitro by post-yolk sac embryos or larvae (i.e. seawater-acclimated embryos). Urea-N constituted an average of 65% of the total nitrogen excreted by the embryos into the ambient medium during a 5-h time-course compared to only 35% in the larvae. Urea-N was excreted at maximum rates during the first hour of the experiment, 0.54±0.09 mol N·g^-1·h^-1 by embryos and 0.35±0.02 by larvae, and then declined to lower levels in both embryos and larvae. A decline after 1 h was also observed for excretion rates of ammonia. In conclusion, the capacity for urea excretion by post-yolk sac embryos ofZ. viviparus may be of adaptive significance for their prolonged stay in ovario. The capacity for excretion of urea seems to decrease after acclimation to sea water.Abbreviations aa amino acid(s) - bm body mass - dw dry weight - NPS ninhydrin-positive substances - sw sea water - ww wet weight     

Ontogenic change in tissue osmolality and developmental sequence of mitochondria-rich cells in Mozambique tilapia developing in freshwater

We investigated a change in tissue fluid osmolality and developmental sequences of mitochondria-rich (MR) cells during embryonic and larval stages of Mozambique tilapia, Oreochromis mossambicus, developing in freshwater. Tissue osmolality, representing body fluid osmolality, ranged from 300 to 370 mOsm/kg during embryonic and larval stages. This suggests that tilapia embryos and larvae are also able to regulate body fluid osmolality to some extent, although the levels are somewhat higher and fluctuate more greatly in embryos and larvae than in adults. Na⁺/K⁺-ATPase-immunoreactive MR cells were first detected in the yolk-sac membrane 3 days before hatching (day − 3), followed by their appearance in the body skin on day − 2. Subsequently, MR cells in both the yolk-sac membrane and body skin increased in number, and most densely observed on days − 1 and 0. Whereas yolk-sac and skin MR cells decreased after hatching, MR cells in turn started developing in the gills after hatching. Thus, the principal site for MR cell distribution shifted from the yolk-sac membrane and body skin during embryonic stages to the gills during larval stages, and tilapia could maintain continuously their ion balance through those MR cells during early life stages.     

Convergent maternal provisioning and life-history evolution in echinoderms

In marine invertebrates, the frequent evolution of lecithotrophic nonfeeding development from a planktotrophic feeding ancestral developmental mode has involved the repeated, independent acquisition of a large, lipid-rich, usually buoyant egg. To investigate the mechanistic basis of egg-size evolution and the role of maternally provisioned lipids in lecithotrophic development, we identified and quantified the egg lipids in six sea urchin species and five sea star species encompassing four independent evolutionary transformations to lecithotrophy. The small eggs of species with planktotrophic development were dominated by triglycerides with low levels of wax esters, whereas the larger eggs of lecithotrophs contain measurable triglycerides but were dominated by wax ester lipids, a relatively minor egg component of planktotrophs. Comparative analysis by independent contrasts confirmed that after removing the influence of phylogeny, the evolution of a large egg by lecithotrophs was correlated with the conspicuous deposition of wax esters. Increases in wax ester abundance exceeded expectations based solely on changes in egg volume. Wax esters may have roles in providing buoyancy to the egg and for postmetamorphic provisioning. Experimentally reducing the amount of wax esters in blastula stage embryos of the lecithotroph Heliocidaris erythrogramma resulted in a viable but nonbuoyant larvae. During normal development for H. erythrogramma, wax ester biomass remained constant during development to metamorphosis (five days postfertilization), but decreased during juvenile development before complete mouth formation (12 days postfertilization) and was further reduced at 18 days postfertilization. The function of wax esters may be specific to the lecithotrophic developmental mode because there were negligible wax esters present in competent pluteus larvae of Strongylocentrotus drobachiensis, a planktotrophic species. These data suggest that this seminal evolutionary modification, the production of a large egg, has been accomplished in part by the elaboration of a preexisting oogenic component, wax esters. The modification of preexisting oogenic processes may facilitate the observed high frequency of transformations in larval mode in marine invertebrates.     

Carryover effects of predation risk on postembryonic life-history stages in a freshwater shrimp

For organisms with complex life histories it is well known that risk experienced early in life, as embryos or larvae, may have effects throughout the life cycle. Although carryover effects have been well documented in invertebrates with different levels of parental care, there are few examples of predator-induced responses in externally brooded embryos. Here, we studied the effects of nonlethal predation risk throughout the embryonic development of newly spawned eggs carried by female shrimp on the timing of egg hatching, hatchling morphology, larval development and juvenile morphology. We also determined maternal body mass at the end of the embryonic period. Exposure to predation risk cues during embryonic development led to larger larvae which also had longer rostra but reached the juvenile stage sooner, at a smaller size and with shorter rostra. There was no difference in hatching timing, but changes in larval morphology and developmental timing showed that the embryos had perceived waterborne substances indicative of predation risk. In addition to carryover effects on larval and juvenile stages, predation threat provoked a decrease of body mass in mothers exposed to predator cues while brooding. Our results suggest that risk-exposed embryos were able to recognize the same infochemicals as their mothers, manifesting a response in the free-living larval stage. Thus, future studies assessing anti-predator phenotypes should include embryonic development, which seems to determine the morphology and developmental time of subsequent life-history stages according to perceived environmental conditions.     

Hormonally-regulated differential expression of the two duplicated insecticyanin genes,ins-a and ins-b,during development of the tobacco hornworm,Manduca sexta

The effects of juvenile hormone (JH) and 20-hydroxyecdysone (20E) on the developmental expression of the two insecticyanin genes, ins-a and ins-b, were investigated with two gene-specific probes. Removal of the corpora allata (-CA, source of JH) clearly delayed and down-regulated the epidermal expression of these genes but enhanced their expression in the fat body during the early development of the fifth instar. Application of JH I to the -CA larvae at the time of head capsule slippage completely restored the normal epidermal expression pattern of the two genes in the early fifth instar, then INS-a mRNA declined prematurely whereas INS-b mRNA remained similar to that in the intact larvae. By contrast, in the fat body of -CA larvae, the exogenous JH had little effect on the levels of INS-a mRNA, but enhanced expression of INS-b mRNA relative to intact larvae. Culture of epidermis from day 1 fifth instar larvae with 40 ng/ml 20E for up to 24 h accelerated the loss of INS-a mRNA without affecting the levels of INS-b mRNA. Both mRNAs declined in isolated larval abdomens over a 24 h period, and this decline was slowed by 1 g methoprene (a JH analog). Together these results indicate that JH controls the levels of the two mRNAs in both the epidermis and fat body, with additional factors involved in regulating these genes in the fat body during the molt and in the epidermis during the growth phase.     

The relationship among egg size,density and food level on larval development in the wood frog (Rana sylvatica)

Summary Although inter- and intraspecific variation in egg size among amphibians has been well documented, the relationship between egg size and fitness remains unclear. Recent attempts to correlate egg size intraspecifically with larval developmental patterns have been equivocal. In this study the development of larvae derived from large eggs and small eggs, from a single population in Maryland were compared under a range of food levels and larval population densities. Both food level and density had significant effects on the length of the larval period and size at metamorphosis. However, the response among larvae derived from different egg sizes was not additive. At low densities and high food levels, larvae from small eggs had longer larval periods and a larger size at metamorphosis than larvae derived from large eggs. In contrast, at high densities larvae from small eggs had longer developmental periods but were smaller at metamorphosis than larvae from large eggs. In addition, larvae from small eggs were more sensitive to density irrespective of food level. These results suggest that optimal egg size is correlated with environmental factors, which may explain the maintenance of both geographic and within population variation in egg size commonly observed in amphibians.     

家蚕五龄幼虫血淋巴内维生素的研究

本实验对家蚕Bombyx mori五龄健康的雌雄幼虫、氟中毒的雌幼虫、细胞质多角体病毒(CPV)感染的雄幼虫血淋巴内的烟酸、烟酰胺、吡哆辛(VB₆)、硫胺素(VB₁)及核黄素(VB₂)含量,采用离子对反相色谱法进行了定量测定.在五龄幼虫发育阶段,血淋巴内每种维生素含量均是雌蚕高于雄蚕,健康者高于病态者,且病蚕含维生素的量持续下降;烟酸和烟酰胺浓度一直减少,可能是部分烟酸、烟酰胺在酶作用下与某些蛋白质结合,部分烟酸、烟酰胺形成NADP酶的缘故;VB₆、VB₁和VB₂浓度增加,是幼虫大量摄食、贮存能量和营养,供日后生命循环需要的结果.     

DEVELOPMENTAL CONSEQUENCES OF AN EVOLUTIONARY CHANGE IN EGG SIZE: AN EXPERIMENTAL TEST

Larvae of two species of sea urchins (Strongylocentrotus droebachiensis and S. purpuratus) differ in initial form and in the rate of development. To determine whether these differences are attributable to the large interspecific difference in egg size, we experimentally reduced egg size by isolating blastomeres from embryos. The rate of development of feeding larvae derived from isolated blastomeres was quantified using a novel morphometric method. If the differences early in the life histories of these two species are due strictly to differences in egg size, then experimental reduction of the size of S. droebachiensis eggs should yield an initial larval form and rate of development similar to that of S. purpuratus. Our experimental manipulations of egg size produced three clear results: 1) smaller eggs yielded larvae that were smaller and had simpler body forms, 2) smaller eggs resulted in slower development through the early feeding larval stages, and 3) effects of egg size were restricted to early larval stages. Larvae from experimentally reduced eggs of the larger species had rates of development similar to those of the smaller species. Thus, cytoplasmic volumes of the eggs, not genetic differences expressed during development, account for differences in larval form and the rate of form change. This is the first definitive demonstration of the causal relationship between egg size (parental investment per offspring) and life-history characteristics in marine benthic invertebrates. Because larval form influences feeding capability, the epigenetic effects of egg size on larval form are likely to have important functional consequences. Adaptive evolution of egg size may be constrained by the developmental relationships between egg size and larval form: evolutionary changes in egg size alone can result in concerted changes in larval form and function; likewise evolutionary changes in larval form and function can be achieved through changes in egg size. These findings may have broader implications for other taxa in which larval morphology and, consequently, performance may be influenced by changes in egg size.     

Lipid and fatty acid composition during embryo and larval development of puye Galaxias maculatus Jenyns, 1842, obtained from estuarine, freshwater and cultured populations

Galaxias maculatus eggs and larvae obtained from broodfish captured either in an estuarine or a freshwater environment, as well as from cultured broodstock were analysed to compare their lipid and fatty acid profiles. Results showed a lower lipid content in embryos and larvae from estuarine populations than those from fresh water, denoting the influence of environmental conditions. The n-3:n-6 ratio was higher in eggs from estuarine and cultured populations, being in the range of marine fishes, whereas for eggs from freshwater fish was lower and typical of freshwater fishes. The polyunsaturated fatty acids (PUFA), particularly docosahexaenoic acid (22:6n-3) and eicosapentaenoic acid (20:5n-3), were higher in eggs and larvae of broodstock coming from culture or estuarine environments than in those from fresh water. Moreover, these fatty acids markedly increased after hatching in larvae coming from estuarine populations, suggesting the effect of the environment on fatty acid profiles to physiologically prepare the larvae to adapt to higher salinity conditions. Linoleic acid (18:2n-6) content was higher in fresh water fish and its reduction during embryo and larval development was accompanied by a significant increase of arachidonic acid (20:4n-6), which was not observed in embryos or larvae from broodstock fish from estuary or aquaculture origin. Both environment and diet of broodstock fish affected lipid and fatty acid composition of G. maculatus embryo and larvae as well as their changes during development.     

Osmotic changes during the development of eggs and larvae of the lumpsucker, Cydopterus lumpus L.

The demersal eggs of Cyclopterus appear to osmoregulate like the pelagic eggs of cod and plaice. Unfertilized eggs in ovarian fluid exhibited ovoplasm osmolarities similar to those of adult blood and ovarian fluid (356–359 mosmol kg⁻¹). Yolk osmolarities remained virtually constant from fertilization and during development (356–366 mosmol), with a slight decrease near hatching (to 332 mosmol). Yolk and body fluids of larvae (338 mosmol) had osmoconcentrations similar to egg yolk values near hatching. Yolk osmoconcentration of unfertilized eggs remained unchanged during the first 12 h in sea water, with a slow increase thereafter. Fertilized eggs of bad quality cultures exhibited higher yolk osmoconcentrations than eggs of good quality. Cyclopterus eggs were found to develop normally and survive in 20–34%o salinity, larvae seemed to have the same salinity range.     

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.