Resource allocation in yolk-feeding fish

An insight is made into the main processes that occur in fish during endogenous feeding period. The ways in which yolk absorption rate can be measured are evaluated. Essential amino acids and polyunsaturated fatty acids are preferentially retained for incorporation into body tissue. Profound physiological and anatomical changes in yolk and a sequence of slow, fast, and a second period of slow absorption occur during the endogenous feeding period. Attempts to quantify the ontogenetic sequence are reviewed. Various methods of body size assessment are compared, and sources of bias in individual and population growth estimates are discussed. Several calorimetric methods are compared of which direct calorimetry using an oxygen bomb is the reference method. An advanced elemental analysis (CHNS) is a reliable technique that is adequate for early stages. Indices of growth potential are reviewed including a comparison of different measures, models and approaches used to estimate growth. Changes in body hydration, caloric value, content of lipids, protein, free amino acids (FAA) and minerals, and in content of RNA and DNA occur in early ontogeny. Ways to quantify metabolic rate are identified. Mean relative respiration rate of initial egg before activation is very low, about 20 mm³ g⁻¹ h⁻¹. Ontogenetic sequence in absolute metabolic rate of fish embryos and yolk-feeding larvae involves an increase through hatching to a peak at the time of first feeding ability, and a decrease under starvation. Models predicting the relationship between oxygen consumption and age in yolk-feeding fish are reviewed. Sequence of metabolic fuels begins with use of small molecules as carbohydrates, soon switched to FAA. Later lipids are progressively used, they provide energy for swimming activity. After yolk depletion body protein-bound amino acids are mobilised. In this review I focused on the major environmental variables as temperature, oxygen, salinity, pH, toxic xenobiotics, light, UV radiation, magnetic field and substrate, along with intrinsic factors as egg or body size, sex and genetic factors. A question was posed on how the extrinsic and intrinsic factors determine yolk absorption, growth and metabolic rates in yolk-feeding fish. Special attention is devoted to fish body size attained exclusively on yolk. A considerable variety of body size responses to temperature was found, for which several explanations are forwarded. Methodological progress made recently is characterised and the most conspicious advances in understanding of fish early life history are highlighted. Information derived from these studies can be used in management of fish populations in the field and to optimise activities in aquaculture.     

Microbial contamination of embryos and semen during long term banking in liquid nitrogen

We report on microbial contamination of embryos and semen cryopreserved in sealed plastic straws and stored for 6-35 years in liquid nitrogen. There were 32 bacterial and 1 fungal species identified from randomly drawn liquid nitrogen, frozen semen, and embryos samples stored in 8 commercial and 8 research facility liquid nitrogen (LN) tanks. The identified bacteria represented commensal or environmental microorganisms and some, such as Escherichia coli, were potential or opportunistic pathogens for humans and animals. Stenotrophomonas maltophilia was the most common contaminant identified from the samples and was further shown to significantly suppress fertilization and embryonic development in vitro. Analysis of the strains by pulsed field gel electrophoresis revealed restriction patterns with no relatedness indicating that there was no apparent cross-contamination of S. maltophilia strains between the germplasm and liquid nitrogen samples. In addition, no transmission of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) from infected semen and embryos straws to clean germplasm stored in the same LN tanks or LN was detected.     

Vitamin E in early stages of sea bass (Dicentrarchus labrax) development

This study reports titration of vitamin E levels in the sea bass (Dicentrarchus labrax) using high-pressure liquid chromatography. The first part of the work is devoted to vitamin E detection in: (1) plasma of maturing females and males characterized by different body sizes; (2) seminal fluid and eggs; and (3) developing embryos of sea bass fed with vitamin E. In the second part of the study, variations of vitamin E levels during larval development are analyzed. The results show a direct correlation between plasma vitamin E content and body size for both adult male and female sea bass. High vitamin E levels were found in seminal fluid, in eggs before and after fertilization, and in embryos during development and at hatching, whereas vitamin E level was low in dead embryos and in embryos with limited survival. During larval development, the vitamin E content decreased slowly but steadily during the first four days of larval growth; subsequently, it progressively increased from day 9 to day 40. In teratogenic larvae, vitamin E content was significantly higher than in normal larvae. This study provides evidence on how vitamin E exerts an antioxidant defense in sea bass reproduction.     

Skeletal heterochrony is associated with the anatomical specializations of snakes among squamate reptiles

Snakes possess a derived anatomy, characterized by limb reduction and reorganization of the skull and internal organs. To understand the origin of snakes from an ontogenetic point of view, we conducted comprehensive investigations on the timing of skeletal elements, based on published and new data, and reconstructed the evolution of the ossification sequence among squamates. We included for the first time Varanus, a critical taxon in phylogenetic context. There is comprehensive delay in the onset of ossification of most skeletal elements in snakes when compared to reference developmental events through evolution. We hypothesize that progressing deceleration accompanied limb reduction and reorganization of the snake skull. Molecular and morphological studies have suggested close relationship of snakes to either amphisbaenians, scincids, geckos, iguanids, or varanids. Likewise, alternative hypotheses on habitat for stem snakes have been postulated. Our comprehensive heterochrony analyses detected developmental shifts in ossification for each hypothesis of snake origin. Moreover, we show that reconstruction of ancestral developmental sequences is a valuable tool to understand ontogenetic mechanisms associated with major evolutionary changes and test homology hypotheses. The “supratemporal” of snakes could be homolog to squamosal of other squamates, which starts ossification early to become relatively large in snakes.     

Direct somatic embryogenesis from cotyledon and cotyledonary node explants in bishop’s weed <Emphasis Type="Italic">Trachyspermum ammi</Emphasis> (L.) sprague

A three-stage procedure for embryogenesis in Trachyspermum ammi was developed from cotyledon and cotyledonary node explants cultured in Murashige and Skoog (MS) liquid medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Globular somatic embryos without intervening callus phase developed in 4 wk. The development of embryos to heart and torpedo stages required second-stage subculture of the explants (along with developing embryos) in liquid medium with lower concentrations of 2,4-D. Further development of embryos required a third-stage subculture in hormone-free liquid medium supplemented with 100 mg l⁻¹ casein hydrolysate. Regeneration of complete plantlets occurred after the fully developed somatic embryos were transferred to solidified half-strength MS medium supplemented with 1 mg l⁻¹ gibberellic acid.     

DYRK2 and GSK-3 phosphorylate and promote the timely degradation of OMA-1, a key regulator of the oocyte-to-embryo transition in C. elegans

Oocyte maturation and fertilization initiates a dynamic and tightly regulated process in which a non-dividing oocyte is transformed into a rapidly dividing embryo. We have shown previously that two C. elegans CCCH zinc finger proteins, OMA-1 and OMA-2, have an essential and redundant function in oocyte maturation. Both OMA-1 and OMA-2 are expressed only in oocytes and 1-cell embryos, and need to be degraded rapidly after the first mitotic division for embryogenesis to proceed normally. We report here a distinct redundant function for OMA-1 and OMA-2 in the 1-cell embryo. Depletion of both oma-1 and oma-2 in embryos leads to embryonic lethality. We also show that OMA-1 protein is directly phosphorylated at T239 by the DYRK kinase MBK-2, and that phosphorylation at T239 is required both for OMA-1 function in the 1-cell embryo and its degradation after the first mitosis. OMA-1 phosphorylated at T239 is only detected within a short developmental window of 1-cell embryos, beginning soon after the proposed activation of MBK-2. Phosphorylation at T239 facilitates subsequent phosphorylation of OMA-1 by another kinase, GSK-3, at T339 in vitro. Phosphorylation at both T239 and T339 are essential for correctly-timed OMA-1 degradation in vivo. We propose that a series of precisely-timed phosphorylation events regulates both the activity and the timing of degradation for OMA proteins, thereby allowing restricted and distinct functions of OMA-1 and OMA-2 in the maturing oocyte and 1-cell embryo, ensuring a normal oocyte-to-embryo transition in C. elegans.     

RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis

The RNA interference technique is a powerful tool to understand gene function. Intriguingly, RNA interference cannot only be used for cells in vitro, but also in living organisms. Here, we have adapted the method for use in the chick embryo. However, this technique is limited by the uncertainty in predicting the RNAi transfection efficiency and site in the embryo. Hence, we elaborated a modified vector system, pEGFP-shRNA, which can coexpress enhanced green fluorescent protein (EGFP) and short hairpin RNA (shRNA) simultaneously to facilitate analysis of gene silencing in chicken embryos. We tested the silencing of two highly conserved genes (cAxin2, cParaxis), which play crucial roles in chicken embryonic developmental processes. For each target gene, four to five small DNA inserts, each of them encoding one shRNA, were selected and cloned individually to the vector downstream of the Pol III promoter (either human H1 or U6 promoter), which shared with highly conserved motifs in human and chicken. The pEGFP-shRNA constructs were electroporated into the neural tube or somites. After subsequent re-incubation of 24 h, the EGFP expression, with green fluorescent signal, indicated the transfected regions in the neural tube or somites. The EGFP expressing embryos were further submitted into the process of in situ hybridization for examination of the silencing effects. The results show that the EGFP signal in transfected areas correlated with the silencing of the target genes (cAxin2, cParaxis). The cAxin2 expression was inhibited by shRNAs of either targeting the RGS domain or the DAX domain coding region. The cParaxis mRNA level in transgenic somites and the related migratory myogenic population was also reduced. The results suggest that our novel dual expression EGFP-shRNA system opens a new possibility to study gene function in a convenient and efficient way.     

Developing embryos of Sesbania sesban have unique potential to photosynthesize under high osmotic environment

This study has been carried out to investigate the photosynthetic activities in developing embryos of Sesbania sesban under a highly osmotic environment. In S. sesban, the embryo turns green/chlorophyllous at the early heart shape stage. Interestingly, despite being deeply embedded within the supporting tissues (several layers of pod wall, seed coat and endosperm) and developing in a highly osmotic environment, the growing embryo of the developing seed showed the presence of various components of photosynthetic machinery besides being chlorophyllous. The shade-adaptive nature of the photosynthetic machinery of the embryo is evident from (a) low chlorophyll a/b ratio, (b) photosystem (PS) II attaining maximal activity at low photon flux density and (c) lesser plastoquinone pool. The photosynthetic potential of the growing embryo seems to contribute towards seed filling as it has the potential not only to harvest light energy but also to fix CO2 as efficiently as other photosynthetic parts of S. sesban. In fact, ribulose-1,5 bisphosphate carboxylase purified from embryos manifested subunit composition similar to that of leaves. The PS II activity in leaves, cotyledonary leaves and pod wall declined sharply with increase in the level of NaCl and sucrose above 150 and 300 mmol, respectively. Amazingly, PS II activity in developing embryos was maximal in the presence of 250 mmol NaCl or 500 mmol sucrose and remained high even when NaCl and sucrose levels were increased to 500 and 1000 mmol, respectively. We hypothesize that the developing embryos have some factor(s) which protect(s) the photosynthetic machinery in an environment of high osmotic strength.