Ontogeny of lymphoid organs and development of IgM-bearing cells in Atlantic halibut (Hippoglossus hippoglossus L.)

In teleost fish, the head kidney, thymus, and spleen are generally regarded as important immune organs. In this study, the ontogeny of these organs was studied in Atlantic halibut (Hippoglossus hippoglossus), larvae at various stages of development. We observed that the kidney was present at hatching, the thymus at 33 days post hatch (dph), while the spleen was the last organ to be detected at 49 dph. All three lymphoid organs were morphologically well developed during late metamorphic stages. Real time RT-PCR analysis showed that IgM mRNA expression could be observed at 66 dph and later, which correlates well with in situ hybridisation data showing that a few IgM positive cells could be detected in the anterior kidney and spleen from 66 dph. Our data also showed that the highest levels of IgM mRNA could be detected in halibut spleen. Immunostaining using a monoclonal antibody against halibut IgM detected IgM positive cells at 94 dph in both the head kidney and the spleen, which is much later than the IgM mRNA. Numerous cells expressing both IgM mRNA and protein could be detected in the spleen and anterior kidney and also to some extent in thymus specimens from adult halibut.     

Molecular characterization of trypsinogens and development of trypsinogen gene expression and tryptic activities in grass carp (Ctenopharyngodon idellus) and topmouth culter (Culter alburnus)

This study examined the gene structures and expression of trypsinogens, as well as the trypsin activities of the grass carp Ctenopharyngodon idellus (herbivorous) and the topmouth culter Culter alburnus (carnivorous), which are commercially important freshwater species of the family Cyprinidae in China. Isolated full-length trypsinogen cDNA clones were 869 bp and 857 bp. The deduced amino acid sequences were 242 aa and 247 aa long, both containing the highly conserved residues essential for serine protease catalytic and conformational maintenance. The results from isoelectric and phylogenetic analyses suggest that grass carp trypsinogen is grouped with teleost trypsinogen group I, while topmouth culter trypsinogen is grouped with group II. The expression pattern of trypsinogen mRNA was similar between these two species, appearing 2 days post-hatching (dph) and reaching peaks at 11 and 23 dph. The trypsin-specific activities in both species were detected 2 dph and reached the major peaks at 8 dph, however the minor peaks were observed at 20 dph in the grass carp and 17 dph in the topmouth culter. The trypsin-specific activity was significantly higher in the grass carp than in the topmouth culter, which may be attributed to the nature of their different nutritional habits.     

A finite interval of initial swimbladder inflation in Latris lineata revealed by sequential removal of water‐surface films

A finite interval of initial swimbladder inflation in striped trumpeter Latris lineata larvae occurred over 4 days at 16° C. Water‐surface films were removed on different days to form treatments: 4, 8, 9, 10, 11 and 12 days post hatching, dph (day 4, 8, 9, 10, 11 and 12 treatments, respectively). No swimbladder inflation was recorded prior to water‐surface film removal. When the water‐surface films were removed in day 4 and 8 treatments, initial swimbladder inflation was first recorded in larvae 9 dph at mean ± s .e . 35·0 ± 5·4%(n = 4) and 45·0 ± 7·9%, respectively. Water‐surface film removal at days 9, 10 and 11, resulted in initial swimbladder inflation the following day at 62·5 ± 2·5, 62·5 ± 7·2 and 11·3 ± 5·5% in larvae 10, 11 and 12 dph, respectively. No swimbladder inflation was recorded following water‐surface film removal on day 12. There was no significant difference in initial inflation among larvae in day 4, 8, 9 and 10 treatments, ranging from 65·0 ± 4·1 to 73·8 ± 6·9%(P > 0·05). Initial inflation was significantly lower in the day 11 treatment (11·3 ± 5·5%)(P < 0·05). During the inflation interval (9–12 dph) swimbladders displayed one of three morphologies; liquid dilation, gas inflated and collapsed. Collapse of the swimbladder lumen was first apparent in larvae without swimbladder inflation from 11 dph and progressively developed thereafter in all larvae with non‐inflated swimbladders. Larvae >6·1 mm standard length lost the ability to undergo initial swimbladder inflation. This study demonstrates that the interval for initial swimbladder inflation in striped trumpeter is short, finite and related to larval size. The end of the inflation interval was marked by onset of abnormal swimbladder morphologies, but not to closure of the pneumatic duct.     

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic cells are generally the most oxidized. Glutathione is considered the most important cellular redox buffer and the average reduction potential (E_h) of a cell or organism can be calculated from the concentrations of glutathione (GSH) and glutathione disulfide (GSSG). In this study, triplicate groups of cod larvae at various stages of development (3 to 63 days post-hatch; dph) were sampled for analyses of GSSG/2GSH concentrations, together with activities of antioxidant enzymes and expression of genes encoding proteins involved in redox metabolism. The concentration of total GSH (GSH+GSSG) increased from 610±100 to 1260±150 μmol/kg between 7 and 14 dph and was then constant until 49 dph, after which it decreased to 810±100 μmol/kg by 63 dph. The 14- to 49-dph period, when total GSH concentrations were stable, coincides with the proposed period of metamorphosis in cod larvae. The concentration of GSSG comprised approximately 1% of the total GSH concentration and was stable throughout the sampling series. This resulted in a decreasing E_h from −239±1 to −262±7 mV between 7 and 14 dph, after which it remained constant until 63 dph. The changes in GSH and E_h were accompanied by changes in the expression of several genes involved in redox balance and signaling, as well as changes in activities of antioxidant enzymes, with the most dynamic responses occurring in the early phase of cod larval development. It is hypothesized that metamorphosis in cod larvae starts with the onset of mosaic hyperplasia in the skeletal muscle at approximately 20 dph (6.8 mm standard length (SL)) and ends with differentiation of the stomach and disappearance of the larval finfold at 40 to 50 dph (10–15 mm SL). Thus, metamorphosis in cod larvae seems to coincide with high and stable total concentrations of GSH.     

Differentiation of ambisexual gonads and immunohistochemical localization of P450 cholesterol side-chain cleavage enzyme during gonadal sex differentiation in the protandrous anemonefish, Amphiprion clarkii

To clarify the relationship between steroid hormones and sex differentiation of the protandrous anemonefish Amphiprion clarkii, we histologically examined its gonadal differentiation. From hatching to 30 days post hatching (dph), all of the gonads surveyed were sexually undifferentiated. The gonads of all fish first differentiated into ovaries at 60 dph, and the oocytes gradually developed and increased in number as the ovaries grew up until 183 dph. Some cysts of differentiated spermatogenic germ cells appeared in the ovaries at 214 dph, and ambisexual gonads with both ovarian and testicular tissues formed by 273 dph. Using immunohistochemistry, we then investigated the expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), during gonadal sex differentiation. P450scc-immunopositive reactions first appeared in sexually undifferentiated gonads at 30 dph. Beginning at 60 dph, the number of strongly positive cells increased throughout the differentiation of the ovaries and continued to increase during the testicular differentiation until 210 dph. Immunopositive cells were observed more frequently in ovarian tissue than in testicular tissue in the ambisexual gonads at 270 dph. These results suggest that endogenous steroid hormones are important for the sex differentiation, including the primary sex differentiation and subsequent testicular differentiation, of the anemonefish.     

Estimating gonadal development using a non-destructive measurement of ovarian length in live Tasmanian striped trumpeter, Latris lineata (Forster, 1801)

Sexually mature female striped trumpeter Latris lineata (Forster, 1801) were sampled monthly for two spawning seasons until the start of gonadal recrudescence, and then fortnightly until ovulations ceased. Oocyte size and ovarian length, measured by inserting a semi-rigid biopsy catheter to the full extent of insertion, were recorded at each sample time. Ovarian length was expressed as proportion of fork length to provide a gonad index (GI). In non-ovulating females, there was little change in GI throughout the year. However, in ovulating females, GI increased from 18.3 five months before the first spawning season to 27.6 at the peak of the season in October, decreasing to 19.1 the following May and then increasing again to a maximum of 31.1 the following October, in concert with annual changes in reproductive condition. There was a positive linear correlation between GI and oocyte size during the period of oocyte growth ( r  = 0.75, n = 302). Based on the range of GI values for each stage in oocyte development (primary, cortical alveoli, vitellogenic, maturing and hydrated), GI was 90% accurate at assessing fish as pre-vitellogenic and 83% accurate at assessing fish as undergoing final oocyte maturation. This study demonstrated that measurement of GI by catheterization provides a rapid and non-destructive method for assessing maturational status of striped trumpeter broodstock.     

Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes

The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes.The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P < 0.04, P < 0.001, respectively) and provoked increased mRNA FST expression in oocytes matured in vitro compared to immature oocytes (P < 0.01) and those vitrified without calcium (P < 0.004). CTSB mRNA expression was increased in immature oocytes and oocytes vitrified with calcium compare to mature oocytes (P < 0.02). The developmental potential of vitrified oocytes was impaired compared to non-vitrified oocytes, being more evident in oocytes vitrified with calcium.In summary, vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these oocytes.     

Expression and activity of trypsin and pepsin during larval development of the spotted rose snapper Lutjanus guttatus

The present study aimed to describe and understand the development of the digestive system in spotted rose snapper (Lutjanus guttatus) larvae from hatching to 40 days post-hatch (dph). The mouth opened between 2 and 3 dph, at that moment the digestive tract was barely differentiated into the anterior and posterior intestine, although the liver and pancreas were already present. Gastric glands were observed until 20 dph, followed by the differentiation of the stomach between 20 and 25 dph. Trypsinogen expression and trypsin activity were detected at hatching, increasing concomitantly to larval development and the change in the type of food. Maximum levels of trypsinogen expression were observed at 25 dph, when animals were fed with Artemia nauplii, and maximum trypsin activity was detected at 35 dph, when larvae were fed with an artificial diet. On the other hand, pepsinogen gene expression was detected at 18 dph, two days before pepsin enzymatic activity and appearance of gastric glands. Maximum pepsin activity was also observed at 35 dph. These results suggest that in this species weaning could be initiated at an earlier age than is currently practiced (between 28 and 30 dph), since larvae of spotted rose snapper develop a functional stomach between days 20 and 25 post-hatch.     

Histological development of the digestive system of Mayan cichlid Cichlasoma urophthalmus (Günther 1862)

The ontogeny of the digestive tract in Cichlasoma urophthalmus was studied by means of optical microscopy from hatching to 30 days post‐hatching (dph; 855 degree days, dd). The development of the digestive system in this precocial species was a very intense and asynchronous process, which proceeded from both distal ends interiorly. At hatching, the digestive tract consisted of a straight tube with a smooth lumen dorsally attached to the yolk‐sac. The digestive accessory glands were already differentiated and eosinophilic zymogen granules were visible in the exocrine pancreas. At the onset of exogenous feeding between 5 and 6 dph (142.5–171.0 cumulative thermal units, CTU), the buccopharynx, oesophagus, intestine, liver and pancreas were almost completely differentiated, with the exception of the gastric stomach that completed its differentiation between 11 and 14 dph (313.5–399.0 CTU). The development of gastric glands at 14 dph and the differentiation of the stomach in the fundic, cardiac and pyloric regions at 19 dph (541.5 CTU) were the last major events in digestive tract development and designated the onset of the juvenile period. Remnants of yolk were still detected until 16 dph (456.0 CTU), indicating a long period of mixed nutrition that lasted between 10 and 11 days (285.0–313.5 CTU). The results of the organogenesis of larvae complement previous data on the functionality of the digestive system and represent a useful tool for establishing the functional systemic capabilities and physiological requirements of larvae to ensure optimal welfare and growth under aquaculture conditions, which might be useful for improving current larval rearing practices for this cichlid species.     

RAG-2 promotes heptamer occupancy by RAG-1 in the assembly of a V(D)J initiation complex

V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.     

Molecular characterization and expression of Kiss2/Kiss2r during embryonic and larval development in (Megalobrama amblycephala Yih, 1955)

In order to test whether Kiss/Kissr systems have potential roles in regulating the embryonic and larval development in teleosts, in this study the Kiss2/Kiss2r full‐length cDNA was cloned from blunt snout bream (Megalobrama amblycephala Yih, 1955) by rapid amplification of cDNA ends and their expression patterns were detected in different tissues of adult and developmental stages for the embryonic and larval periods via quantitative real‐time PCR. Both Kiss and Kissr genes full‐length cDNA sequences of M. amblycephala were obtained and phylogenetic analysis results indicated that these genes belong to the Kiss2/Kiss2r clade. Bioinformatics analyses revealed that there was a conserved decapeptide in M. amblycephala Kiss2 gene putative amino acids, but only two transmembrane domains were predicated in Kiss2r. Tissue distribution analyses showed that both genes were widely expressed in the tissues tested, with high levels in the muscle, gonad and pituitary. At different developmental stages, the mRNA expression of Kiss2/Kiss2r was highest in the blastocyst/15 hpf stage and lowest in the 30 hpf /blastocyst stages for the embryonic period, highest in 7 dph/15 dph and lowest in 30 dph/30 dph for larval period, respectively. These results suggest that the Kiss2/Kiss2r system has varied potential for influencing embryonic and larval development in fish species.     

Early development and allometric growth in the armoured catfish <Emphasis Type="Italic">Corydoras aeneus</Emphasis> (Gill, 1858)

An ontogenetic series of in-captivity bred Corydoras aeneus was used, in order to study the developmental changes in the external morphology. Allometric growth of several body parts was studied, attempting to reveal important steps in the species’ early life history. Based on the external morphology, the different stages during early development of C. aeneus were identified, according to Balon (Journal of the Fisheries Research Board of Canada 32:1663–1670, 1975). After hatching, at a SL of 3.5 mm, the developmental state corresponded to an eleutherembryonic phase, followed by the protopterygiolarval phase (4.4–5.7 mm SL), the pterygiolarval phase (5.7–14.0 mm SL) and the juvenile period. In addition, an overall growth curve and inflexion points were determined. As such, ontogenetic changes in growth coefficients k (in SL = b age ^k ) were determined. Log transformed data were used for a piecewise linear regression method, as per regression spline smoothing procedures. This way, the growth curve could be divided into six different intervals of growth rate. Initially, the slope was 0.05 until 0.7 dph, then increasing to 0.18 until 4 dph, and 0.36 until 10 dph. After this, growth rate reached a maximum of 0.76 until 24 dph, slowed down to 0.47 until 37 dph and then finally again slowed down to 0.36. A similar growth analysis was also done on the different body parts and these results were compared to both morphological and data from literature. This led to the conclusion that the inflexion points found during the early development of C. aeneus matched the different key-events known in teleost early life history and development. The transition from endo- to exogenous feeding, at the moment a functional branchial respiratory system becomes increasingly important, was the first point at which allometries changed together with functional demands. A second, similar congruence occurred at the transition to the pterygiolarval phase, when priorities shift towards locomotory needs. Finally, our results also indicated a transition to a carangiform swimming mode at approximately 8 mm SL. Handling editor: K. Martens     

Developmental competence of mature yak vitrified–warmed oocytes is enhanced by IGF-I via modulation of CIRP during in vitro maturation

The objective of this study was to investigate whether developmental competence of mature vitrified–warmed yak (Bos grunniens) oocytes can be enhanced by supplemented insulin-like growth factor I (IGF-1) during in vitro maturation (IVM), and its relationship with the expression of cold-inducible RNA-binding protein (CIRP). In experiment 1, immature yak oocytes were divided into four groups, and IVM supplemented with 0, 50, 100 and 200 ng/mL IGF-1 was evaluated; the mRNA and protein expression levels of CIRP in mature oocytes in the four groups were evaluated using quantitative real-time PCR and western blotting analyses. In experiment 2, the mature yak oocytes in the four groups were cryopreserved using the Cryotop (CT) method, followed by chemical activation and in vitro culture for two days and eight days to determine cleavage, blastocyst rates, and total cell number in the blastocysts. Mature yak oocytes without vitrification served as a control group. The outcomes were as following: (1) the expression of CIRP in the matured oocytes was up-regulated in the IGF-1 groups and was highest expression was observed in the 100 ng/mL IGF-1 treatment group. (2) In the vitrified–warmed groups, the rates of cleavage and blastocyst were also highest in the 100 ng/mL IGF-1 treatment group (81.04 ± 1.06%% and 32.16 ± 1.01%), which were close to the rates observed in groups without vitrification (83.25 ± 0.85% and 32.54 ± 0.34%). The rates of cleavage and blastocyst in the other vitrified–warmed groups were 70.92 ± 1.32% and 27.33 ± 1.31% (0 ng/mL); 72.73 ± 0.74% and 29.41 ± 0.84% (50 ng/mL); 72.43 ± 0.61% and 27.61 ± 0.59% (200 ng/mL), respectively. There was no significant difference in the total cell number per blastocysts between the vitrified–warmed groups and group without vitrification. Thus, we conclude that the enhancement in developmental competence of mature yak vitrified–warmed oocytes after the addition of IGF-1 during IVM might result from the regulation of CIRP expression in mature yak oocytes prior to vitrification.