Early Larval Development Research Articles

Embryotoxicity of TiO2 nanoparticles to Mytilus galloprovincialis (Lmk)

Few data exist on the ecotoxicological effects of nanosized titanium dioxide (nTiO2) towards marine species with specific reference to bivalve molluscs and their relative life stages. Mytilus galloprovincialis Lamarck was selected to assess the potential adverse effects of nTiO2 (0–64 mg/L) on its early larval development stages (pre-D shell stage, malformed D-shell stage and normal D-shell stage larvae) considering two exposure scenarios characterised by total darkness (ASTM protocol) and natural photoperiod (light/dark). This approach was considered to check the presence of potential effects associated to the photocatalytic properties of nTiO2. Parallel experiments were carried on with the bulk reference TiCl4. The toxicity of nTiO2 showed to be mainly related to its “nano” condition and to be influenced by the exposure to light that supported the increase in the number of pre-D shell stage (retarded) larvae compared to the malformed ones especially at the maximum effect concentrations (4 and 8 mg nTiO2/L). The non-linear regression toxicity data analysis showed the presence of two EC50 values per exposure scenario: a) EC(50)1 = 1.23 mg/L (0.00–4.15 mg/L) and EC(50)2 = 38.56 mg/L (35.64–41.47 mg/L) for the dark exposure conditions; b) EC(50)1 = 1.65 mg/L (0.00–4.74 mg/L) and EC(50)2 = 16.39 mg/L (13.31–19.48 mg/L) for the light/dark exposure conditions. The potential implication of agglomeration and sedimentation phenomena on ecotoxicological data was discussed.

Characterization of embryogenesis and early larval development in the Pacific triton, Charonia tritonis (Gastropoda: Caenogastropoda)

The Pacific triton, Charonia tritonis, is one of the largest carnivorous gastropods in coral reef ecosystems. This species has been in danger of extinction for many years due to unsustainable fishing. The scarcity of information about its life history greatly limits its artificial propagation and in this study, we present information on embryogenesis and early larval development. Females produced 550–600 egg capsules during each spawn, each of which contained approximately 3000 yolky eggs that measured 428.0 ± 16.10 μm in diameter. Formation and retraction of a small polar lobe were identified during egg maturation and the first two divisions, and absorption of the polar lobe into the 3D cell was observed during the eight-cell stage. The early embryo developed into a stereoblastula by spiral cleavages, and gastrulation by epiboly ended by days 5–7. The trochophore, protoconch, and eyespots appeared at 9–12, 15–18, and 20–22 days, respectively. An average of 2000 free-swimming veligers with a shell length of 664.2 ± 49.81 μm were released from each capsule after 59 ± 4.0 days incubation at 24 ± 0.5 °C. The vela were reduced by the shedding of cilia after eight weeks, although the left tentacle was still absent after 20 weeks.

The time course of L-gulono-gamma-lactone oxidase expression during the larval ontogeny of fish (Acipenser persicus)

In our previous study, for the first time in any fish species, the mRNA expression of L-Gulono-γ-lactone oxidase (GULO) was investigated during early development of Persian sturgeon, Acipenser persicus, and it was demonstrated that GULO mRNA was expressed throughout the development of Persian sturgeon. Accordingly, it was suggested that the initial production of GULO and probably ascorbate might occur before hatching during the embryonic stage. To evaluate the correspondence between transcript and protein level, GULO activity of Persian sturgeon during ontogenetic development of embryos and larvae was investigated. The GULO activity was detected in all time-points of the development of Persian sturgeon from embryos to the late larval stage indicating the start of ascorbate synthesis before hatching. However, there was a marked difference in the mRNA/protein activity ratio at different times. As the GULO mRNA and protein production peaks were observed during a time course from embryonic to early larval development before the start of the exogenous feeding, it can be concluded that the vitamin C requirement must initially be fulfilled by endogenous formation.

Smurf-mediated differential proteolysis generates dynamic BMP signaling in germline stem cells during Drosophila testis development

Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.

In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import.

BackgroundCarbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. Importantly, GDP-L-fucose is the only nucleotide-sugar donor used by fucosyltransferases and its availability represents a bottleneck in fucosyl-glycotope expression.MethodsA homology-based genome-wide bioinformatics approach was used to identify and molecularly characterize the enzymes that contribute to GDP-L-fucose synthesis and Golgi import in S. mansoni. Putative functions were further investigated through molecular phylogenetic and immunocytochemical analyses.ResultsWe identified homologs of GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER), which constitute a de novo pathway for GDP-L-fucose synthesis, in addition to a GDP-L-fucose transporter (GFT) that putatively imports cytosolic GDP-L-fucose into the Golgi. In silico primary sequence analyses identified characteristic Rossman loop and short-chain dehydrogenase/reductase motifs in GMD and GMER as well as 10 transmembrane domains in GFT. All genes are alternatively spliced, generating variants of unknown function. Observed quantitative differences in steady-state transcript levels between miracidia and primary sporocysts may contribute to differential glycotope expression in early larval development. Additionally, analyses of protein expression suggest the occurrence of cytosolic GMD and GMER in the ciliated epidermal plates and tegument of miracidia and primary sporocysts, respectively, which is consistent with previous localization of highly fucosylated glycotopes.ConclusionsThis study is the first to identify and characterize three key genes that are putatively involved in the synthesis and Golgi import of GDP-L-fucose in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, molecular phylogenetics, and developmental expression in intramolluscan larval stages.

Dynamics of yolk sac and oil droplet utilization and behavioural aspects of swim bladder inflation in burbot, Lota lota L., larvae during the first days of life, under laboratory conditions

The study concerns investigation of early larval development of burbot, Lota lota. As part of a two-year study, the first 15 days (until the end of yolk sac resorption) of burbot larvae development under controlled conditions were examined. The aim of the study was to observe the moment of swim bladder inflation and the behavioural aspects of this process, as well as the analysis of yolk and oil droplet resorption and the beginning of exogenous nutrition. It was observed that larvae began to inflate their swim bladder on the 3rd day post-hatch. On 5 DPH, none of the larvae without an inflated bladder was able to swim up the distance separating it (10 cm) from the water surface. Since 9 DPH, 50 % of larvae started exogenous feeding, and the absence of yolk was observed on 13 DPH and oil droplet on 14 DPH, while on 15 DPH, 100 % of feeding larvae were observed. The presented results indicate that the analyzed period is critical for burbot, and it is characterized by a high mortality rate (over 60 %). Additionally, the results suggest that, under controlled conditions, the latest moment when burbot larvae should be given exogenous food is 9–10 DPH and until the moment of the swim bladder inflation, larvae should be kept in tanks with a low water depth (up to 10 cm). The data presented in this study could have a significant influence on the efficiency of larvae rearing, both for aquaculture and for restocking purposes.

Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous nonprotein antioxidant defense molecule is the tripeptide glutathione (γ-glutamylcysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0–5 days postfertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione using HPLC and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0–120h of zebrafish development (including mature oocytes, fertilization, midblastula transition, gastrulation, somitogenesis, pharyngula, prehatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12h postfertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12h, and then oscillated around −190mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (−220mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis.

In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the fucosyltransferase multigene family.

Fucosylated glycans of the parasitic flatworm Schistosoma mansoni play key roles in its development and immunobiology. In the present study we used a genome-wide homology-based bioinformatics approach to search for genes that contribute to fucosylated glycan expression in S. mansoni, specifically the α2-, α3-, and α6-fucosyltransferases (FucTs), which transfer L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. We identified and in silico characterized several novel schistosome FucT homologs, including six α3-FucTs and six α6-FucTs, as well as two protein O-FucTs that catalyze the unrelated transfer of L-fucose to serine and threonine residues of epidermal growth factor- and thrombospondin-type repeats. No α2-FucTs were observed. Primary sequence analyses identified key conserved FucT motifs as well as characteristic transmembrane domains, consistent with their putative roles as fucosyltransferases. Most genes exhibit alternative splicing, with multiple transcript variants generated. A phylogenetic analysis demonstrated that schistosome α3- and α6-FucTs form monophyletic clades within their respective gene families, suggesting multiple gene duplications following the separation of the schistosome lineage from the main evolutionary tree. Quantitative decreases in steady-state transcript levels of some FucTs during early larval development suggest a possible mechanism for differential expression of fucosylated glycans in schistosomes. This study systematically identifies the complete repertoire of FucT homologs in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, developmental expression, and evolutionary history.

Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

BackgroundNutritional symbioses play a central role in insects’ adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology.ResultsWe used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch over to the late embryonic stages in pea aphid development.ConclusionsOur data show that, in the development of A. pisum, a specific host gene set regulates the biosynthetic pathways of amino acids, demonstrating how the regulation of gene expression enables an insect to control the production of metabolites crucial for its own development and symbiotic metabolism.

Precise Lamination of Retinal Axons Generates Multiple Parallel Input Pathways in the Tectum

The axons of retinal ganglion cells (RGCs) form topographic connections in the optic tectum, recreating a two-dimensional map of the visual field in the midbrain. RGC axons are also targeted to specific positions along the laminar axis of the tectum. Understanding the sensory transformations performed by the tectum requires identification of the rules that control the formation of synaptic laminae by RGC axons. However, there is little information regarding the spatial relationships between multiple axons as they establish laminar and retinotopic arborization fields within the same region of neuropil. Moreover, the contribution of RGC axon lamination to the processing of visual information is unknown. We used Brainbow genetic labeling to visualize groups of individually identifiable axons during the assembly of a precise laminar map in the larval zebrafish tectum. Live imaging of multiple RGCs revealed that axons target specific sublaminar positions during initial innervation and maintain their relative laminar positions throughout early larval development, ruling out a model for lamina selection based on iterative refinements. During this period of laminar stability, RGC arbors undergo structural rearrangements that shift their relative retinotopic positions. Analysis of cell-type-specific lamination patterns revealed that distinct combinations of RGCs converge to form each sublamina, and this input heterogeneity correlates with different functional responses to visual stimuli. These findings suggest that lamina-specific sorting of retinal inputs provides an anatomical blueprint for the integration of visual features in the tectum.

Reproduction of the Diadematidae sea urchin Centrostephanus rodgersii in a recently colonized area of northern New Zealand

Reproduction of the sea urchin Centrostephanus rodgersii at the Mokohinau Islands (northeast New Zealand) was studied in terms of the gametogenetic cycle and sex ratio, and larval range of thermal tolerance was determined. A comparison with the reproductive patterns in populations located in New South Wales (NSW), the historical range of the species, and Tasmania, where the species has only recently arrived (≈ 50–60 years ago), was made. Results indicate that C. rodgersii in New Zealand is capable of growing gonads, completing a gametogenetic cycle and producing viable gametes. The timing of this process is similar to the population in Sydney (central NSW), where spawning is probably triggered by the winter solstice and lasts from July to September. The sex ratio was skewed towards females (1.6 females for each male), which may be indicative of the recent arrival of the species in New Zealand. Larvae obtained from this population were able to develop at a measurable rate after 72 h only between 16°C and 25°C. Ambient temperatures during spawning at the Mokohinau Islands (15.7–16.8°C) appear to be at the lower end of the range of temperatures suitable for early larval development.

Characterization of the Calcium Binding Protein Family in Zebrafish

Calcium Binding Proteins (CaBPs), part of the vast family of EF-Hand-domain containing proteins, modulate intracellular calcium levels. They thereby contribute to a broad spectrum of biological processes – amongst others cell migration, gene expression and neural activity. In this study we identified twelve members of this protein family in zebrafish including one gene (cabp4b) currently not present in the zebrafish genome assembly. To gain insight into their biological functions, we carried out a detailed analysis of the expression patterns of these genes during zebrafish late embryonic and early larval development. We detected specific transcription for most of them in different neuronal cell populations including the neuroretina, the inner ear and the notochord. Our data supports potential roles for CaBPs during neuronal development and function and provides a starting point for genetic studies to examine CaBPs' function in these tissues and organs.

Teratogenic Effects of 4-Nonylphenol on Early Embryonic and Larval Development of the Catfish Heteropneustes fossilis

Alkylphenol polyethoxylates (APEs), which are widely used in detergents, paints, herbicides, insecticides, and in many other formulations, have been widely detected in aquatic environments. 4-Nonylphenol (NP) is an important APE detected at microgram levels per litre (0.1-336 μg/L) in water. The objective of the present study was to evaluate NP's toxic effects at low and high sublethal concentrations (0.1 and 1 μg/L) on embryonic development of the catfish Heteropneustes fossilis at different time intervals. The data show that fertilization rate was decreased and cleavage and blastula were severely affected leading to complete mortality of embryos. NP exposure resulted in various body malformations in larvae, such as vertebral deformations, e.g., fin blistering/necrosis, axial deformities (lordosis, kyphosis, and scoliosis) of the spine in the abdominal and caudal region, tail curved completely backward, shortened body, severe spinal and yolk sac malformations, C-shaped severe spinal curvature, cranial malformation with undeveloped head, and failure of eye development. The level of body malformations increased with the concentration and exposure time. After 72 h of exposure, all larvae were dead at both concentrations. Scanning electron microscope study showed that epidermal cells (keratinocytes) were severely damaged in both low- and high-dose treatments throughout development, leading to development of numerous depressions representing sinking holes on the skin. Mucous glands increased significantly in treatment groups compared with control groups. The present study highlights the severe teratogenic effects of NP. The prevalence of the contaminant, if not checked, can lead to decreased population and ultimate disappearance of the species.

Developmental transcription of genes putatively associated with growth in two sturgeon species of different growth rate

In the present study, we surveyed developmental changes in the transcription of growth hormone (gh), insulin-like growth factor-I (igf-I), ghrelin (ghrl) and vascular endothelial growth factor (vegf) genes in the largest freshwater fish, European sturgeon (Beluga, Huso huso) and compared the same parameters to that of its phylogenically close moderate-sized species, Persian sturgeon (Acipenser persicus). The transcripts of gh, igf-I, ghrl and vegf were detected at all developmental time-points of Persian sturgeon and Beluga from embryos to juvenile fish. Changes in normalized gh, igf-I, ghrl and vegf transcription by using the geometric average of genes encoding ribosomal protein L6 (RPL6) and elongation factor (EF1A) over the time of development of Persian sturgeon and Beluga were statistically significant (P<0.05). Our results showed that the mRNA expression levels of both igf-I and ghrl were low during early larval development and then increased significantly to the late larval time-points when larvae started exogenous feeding. In both Beluga and Persian sturgeon, after a low mRNA expression during the embryonic stage, the transcript levels of vegf displayed an increasing trend during yolk-sac fry, consistent with organogenesis. The vegf level remained constantly high in the time of exogenous feeding. The highest detection of gh transcripts coincided with the end of the embryonic stage (hatching time) in Persian sturgeon and 3days-post-hatching (dph) in Beluga. In Persian sturgeon, the gh transcript started to decrease to the rest of the developmental time-points, whereas in Beluga gh transcript had a marked second increase from the time of exogenous feeding (20-dph). This Beluga specific increase in gh transcription may be associated with the marked growth rate and extraordinary size of this fish species.

Duplication and maintenance of the Myb genes of vertebrate animals

SummaryGene duplication is an important means of generating new genes. The major mechanisms by which duplicated genes are preserved in the face of purifying selection are thought to be neofunctionalization, subfunctionalization, and increased gene dosage. However, very few duplicated gene families in vertebrate species have been analyzed by functional tests in vivo. We have therefore examined the three vertebrate Myb genes (c-Myb, A-Myb, and B-Myb) by cytogenetic map analysis, by sequence analysis, and by ectopic expression in Drosophila. We provide evidence that the vertebrate Myb genes arose by two rounds of regional genomic duplication. We found that ubiquitous expression of c-Myb and A-Myb, but not of B-Myb or Drosophila Myb, was lethal in Drosophila. Expression of any of these genes during early larval eye development was well tolerated. However, expression of c-Myb and A-Myb, but not of B-Myb or Drosophila Myb, during late larval eye development caused drastic alterations in adult eye morphology. Mosaic analysis implied that this eye phenotype was cell-autonomous. Interestingly, some of the eye phenotypes caused by the retroviral v-Myb oncogene and the normal c-Myb proto-oncogene from which v-Myb arose were quite distinct. Finally, we found that post-translational modifications of c-Myb by the GSK-3 protein kinase and by the Ubc9 SUMO-conjugating enzyme that normally occur in vertebrate cells can modify the eye phenotype caused by c-Myb in Drosophila. These results support a model in which the three Myb genes of vertebrates arose by two sequential duplications. The first duplication was followed by a subfunctionalization of gene expression, then neofunctionalization of protein function to yield a c/A-Myb progenitor. The duplication of this progenitor was followed by subfunctionalization of gene expression to give rise to tissue-specific c-Myb and A-Myb genes.

Lactobacillus rhamnosus Accelerates Zebrafish Backbone Calcification and Gonadal Differentiation through Effects on the GnRH and IGF Systems

Endogenous microbiota play essential roles in the host’s immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host’s development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment) with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf) exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP), higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group). We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

Embryonic and Early Larval Development in Hatchery‐Reared Common Snook

AbstractTo gain an improved understanding of the early life history of common snook Centropomus undecimalis and refine hatchery production techniques for this species, a combination of digital photography and histological techniques were used to document the embryonic and early larval development of hatchery‐reared individuals. Embryo development from fertilization to hatching took 15 h at 28°C. Larvae at 2 d posthatch showed fully pigmented eyes, and histological sections of the digestive tract revealed the presence of cellular structures indicative of a functional gut. This suggests that common snook larvae have the mechanical ability to detect, capture, and digest prey at 2 d posthatch.

Dynamic expression profiles of virus-responsive and putative antimicrobial peptide-encoding transcripts during Atlantic cod (Gadus morhua) embryonic and early larval development

Early life stage mortality is one of the problems faced by Atlantic cod aquaculture. However, our understanding of immunity in early life stage fish is still incomplete, and the information available is restricted to a few species. In the present work we investigated the expression of immune-relevant transcripts in Atlantic cod during early development. The transcripts subjected to QPCR analysis in the present study were previously identified as putative anti-viral or anti-bacterial genes in Atlantic cod using suppression subtractive hybridization (SSH) libraries, QPCR, and/or microarrays. Of the 11 genes involved in this study, only atf3, cxc chemokine and gaduscidin-1 were not detected at the transcript level in all developmental stages investigated from unfertilized egg to early larval stage. Adam22, hamp, il8, irf1, irf7, lgp2, sacsin, and stat1 transcripts were detected in unfertilized egg and 7h post-fertilization (~2-cell stage) embryos, showing maternal contribution of these immune-relevant transcripts to the early embryonic transcriptome. The Atlantic cod genes included in this study presented diverse transcript expression profiles throughout embryonic and early larval development. For example, adam22 and sacsin transcripts rose abruptly during blastula/gastrula stage and were then expressed at relatively high levels through subsequent embryonic and early larval developmental stages. A peak in irf1 and irf7 transcript expression during early segmentation suggests that these interferon pathway genes play developmental stage-specific roles during cod embryogenesis. Stat1 had increasing transcript expression throughout blastula/gastrula, segmentation, and early larval developmental stages. Atf3, cxc chemokine, gaduscidin-1, and il8 transcripts rose approximately 2–3 fold during hatching, supporting the hypothesis that there is preparation at the immune-relevant transcript expression level to deal with environmental pathogens that may be encountered during early larval development. The specific roles that interferon pathway and other immune-relevant genes play in early life stage cod, and the potential impact of their dynamic transcript expression on immune competence of Atlantic cod embryos and larvae, remain unclear and warrant further study.

Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides, and their expression in tissues during early development

In this study, we cloned two trypsinogens of the orange-spotted grouper, Epinephelus coioides, and analyzed their structure, expression, and activity. Full-length trypsinogen complementary (c)DNAs, named T1 and T2, were 900 and 875 nucleotides, and translated 242 and 244 deduced amino acid peptides, respectively. Both trypsinogens contained highly conserved residues essential for serine protease catalytic and conformational maintenance. Results from isoelectric and phylogenetic analyses suggested that both trypsinogens were grouped into trypsinogen group I. Both trypsinogens had similar expression patterns of negative relationship with body weight; expression was first detected at 1 day post-hatching (DPH) and exhibited steady-state expression during early development at 1-25 DPH. Both expression and activity levels significantly increased after 30 DPH due to metamorphosis. Grouper larval development is very slow with insignificant changes in total length and body weight before 8 DPH. The contribution of live food to an increase in the trypsin activity profile may explain their importance in food digestion and survival of larvae during early larval development.

C-Type Protoconchs and Planktotrophy in Small Eastern North AmericanFargoa(Pyramidellidae)

C-type protoconchs have been thought always to reflect a “direct” or short pelagic development, an idea deriving from Thorson's (1946) study of the early development (besides planktonic veligers) of only one pyramidellid species. The small-shelled genus Fargoa Bartsch (1955), some ectoparasitic on serpulid polychaetes such as Hydroides, apparently uniformly has C-type protoconchs. The eggs and early larval development of Fargoa dianthophila (Wells and Wells, 1961) (shells < 1.8 mm) and F. bartschi (Winkley, 1909) (< 4.0 mm) were studied in the laboratory at Woods Hole, Massachusetts. The eggs were only 53 to 57 µm and 55 to 60 µm in diameter, respectively, and as would be expected from eggs this size both species are definitely planktotrophic. Few other pyramidellids or other “opisthobranchs” lay eggs this small, and the single Brachystomia (Monterosato, 1884) species studied by Thorson (1946) was also planktotrophic. The protoconch diameters of F. dianthophila and F. bartschi are, respectively, about 260 to 315 µm and about 210 to 230 µm, i.e., surprisingly large. This variation parallels neither the nearly uniform egg size nor the disparate adult shell sizes. No attempt was made to rear the larvae in the laboratory because there were only a few newly hatched veligers and these protoconchs indicated much growth in the plankton. These Fargoa veligers may remain planktonic for longer times than planktotrophic Boonea Robertson (1978). One protoconch, an egg capsule containing a late blastula, and a newly hatched swimming veliger are illustrated. Small pyramidellids appear to lay the smallest eggs, but this seems not to be true of other “opisthobranchs.”