Morphological and morphometric aspects of early life stages of piabanha Brycon gouldingi (Characidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Embryonic development and larval stages of Steindachneridion parahybae (Siluriformes: Pimelodidae) - implications for the conservation and rearing of this endangered Neotropical species

Steindachneridion parahybae is a freshwater catfish endemic to the Paraiba do Sul River and is classified as an endangered Neotropical species. An increasing number of conservation biologists are incorporating morphological and physiological research data to help conservation managers in rescue these endangered species. This study investigated the embryonic and larval development of S. parahybae in captivity, with emphasis in major events during the ontogeny of S. parahybae. Broodstocks were artificially induced to reproduce, and the extrusion occurred 200-255 degree-hours after hormonal induction at 24 degrees C. Larval ontogeny was evaluated every 10 minutes under microscopic/stereomicroscopic using fresh eggs samples. The main embryogenic development stages were identified: zygote, cleavage, including the morula, blastula, gastrula phase, organogenesis, and hatching. The extruded oocytes showed an average diameter of 1.10 +/- 0.10 mm, and after fertilization and hydration of eggs, the average diameter of eggs increased to about 1.90 +/- 0.60 mm, characterized by a large perivitelline space that persisted up to embryo development, the double chorion, and the poles (animal and vegetative). Cell division started about 2 minutes after fertilization (AF), resulting in 2, 4, 8 (4 x 2 arrangement of cells), 16 (4 x 4), 32 (4 x 8) and 64 (2 x 4 x 8) cells. Furthermore, the blastula and gastrula stages followed after these cells divisions. The closed blastopore occurred at 11 h 20 min AF; following the development, the organogenetic stages were identified and subdivided respectively in: early segmentation phase and late segmentation phase. In the early segmentation phase, there was the establishment of the embryonic axis, and it was possible to distinguish between the cephalic and caudal regions; somites, and the optic vesicles developed about 20 h AF. Total hatching occurred at 54 h AF, and the larvae average length was 4.30 +/- 0.70 mm. Gradual yolk sac reduction was observed during the first two days of larval development. The first feeding occurred at the end of the second day. During the larval phase, cannibalism, heterogeneous larval growth and photophobia were also observed. This information will be important in improving the artificial reproduction protocols of S. parahybae in controlled breeding programs.

Embryonic development and larval stages of Steindachneridion parahybae (Siluriformes: Pimelodidae): implications for the conservation and rearing of this endangered Neotropical species

Steindachneridion parahybae is a freshwater catfish endemic to the Paraíba do Sul River and is classified as an endangered Neotropical species. An increasing number of conservation biologists are incorporating morphological and physiological research data to help conservation managers in rescue these endangered species. This study investigated the embryonic and larval development of S. parahybae in captivity, with emphasis in major events during the ontogeny of S. parahybae. Broodstocks were artificially induced to reproduce, and the extrusion occurred 200-255 degree-hours after hormonal induction at 24°C. Larval ontogeny was evaluated every 10 minutes under microscopic/stereomicroscopic using fresh eggs samples. The main embryogenic development stages were identified: zygote, cleavage, including the morula, blastula, gastrula phase, organogenesis, and hatching. The extruded oocytes showed an average diameter of 1.10 ± 0.10 mm, and after fertilization and hydration of eggs, the average diameter of eggs increased to about 1.90 ± 0.60 mm, characterized by a large perivitelline space that persisted up to embryo development, the double chorion, and the poles (animal and vegetative). Cell division started about 2 minutes after fertilization (AF), resulting in 2, 4, 8 (4 x 2 arrangement of cells), 16 (4 x 4), 32 (4 x 8) and 64 (2 x 4 x 8) cells. Furthermore, the blastula and gastrula stages followed after these cells divisions. The closed blastopore occurred at 11 h 20 min AF; following the development, the organogenetic stages were identified and subdivided respectively in: early segmentation phase and late segmentation phase. In the early segmentation phase, there was the establishment of the embryonic axis, and it was possible to distinguish between the cephalic and caudal regions; somites, and the optic vesicles developed about 20 h AF. Total hatching occurred at 54 h AF, and the larvae average length was 4.30 ± 0.70 mm. Gradual yolk sac reduction was observed during the first two days of larval development. The first feeding occurred at the end of the second day. During the larval phase, cannibalism, heterogeneous larval growth and photophobia were also observed. This information will be important in improving the artificial reproduction protocols of S. parahybae in controlled breeding programs.

Ethinylestradiol differentially interferes with IGF-I in liver and extrahepatic sites during development of male and female bony fish

Growth and sexual development are closely interlinked in fish; however, no reports exist on potential effects of estrogen on the GH/IGF-I-axis in developing fish. We investigate whether estrogen exposure during early development affects growth and the IGF-I system, both at the systemic and tissue level. Tilapia were fed from 10 to 40 days post fertilization (DPF) with 17alpha-ethinylestradiol (EE(2)). At 50, 75, 90, and 165 DPF, length, weight, sex ratio, serum IGF-I (RIA), pituitary GH mRNA and IGF-I, and estrogen receptor alpha (ERalpha) mRNA in liver, gonads, brain, and gills (real-time PCR) were determined and the results correlated to those of in situ hybridization for IGF-I. Developmental exposure to EE(2) had persistent effects on sex ratio and growth. Serum IGF-I, hepatic IGF-I mRNA, and the number of IGF-I mRNA-containing hepatocytes were significantly decreased at 75 DPF, while liver ERalpha mRNA was significantly induced. At 75 DPF, a transient decline of IGF-I mRNA and a largely reduced number of IGF-I mRNA-containing neurons were observed in the female brain. In both sexes, pituitary GH mRNA was significantly suppressed. A transient downregulation of IGF-I mRNA occurred in ovaries (75 DPF) and testes (90 DPF). In agreement, in situ hybridization revealed less IGF-I mRNA signals in granulosa and germ cells. Our results show for the first time that developmental estrogen treatment impairs GH/IGF-I expression in fish, and that the effects persist. These long-lasting effects both seem to be exerted indirectly via inhibition of pituitary GH and directly by suppression of local IGF-I in organ-specific cells.

Impact of copper mine tailings (stamp sand) on survival and development of aquatic organisms near Gay, Michigan

Heavy metal-rich copper mine tailings, called stamp sands, were dumped by mining companies directly into streams and along the Lake Superior shoreline, degrading Keweenaw Peninsula waterways. One of the largest disposal sites is near Gay, Michigan, where tailings have been moved along the shoreline by currents since mining ceased. As a result, the smallest sand particles have been washed into deeper water and are filling the interstitial spaces of Buffalo Reef, a critical lake trout spawning site. This research is the first to investigate if stamp sand is detrimental to survival and early development of eggs and larvae of lake sturgeon, lake trout, and Northern leopard frogs, and also examines if the presence of stamp sands influences substrate selection of earthworms. This study found that stamp sand had significantly larger mean particle sizes and irregular shapes compared to natural sand, and earthworms show a strong preference for natural substrate over any combination that included stamp sand. Additionally, copper analysis (Cu2+) of surface water over stamp sand and natural sand showed concentrations were significantly higher in stamp sand surface water (100 μg/L) compared to natural sand surface water (10 μg/L). Frog embryos had similar hatch success over both types of sand, but tadpoles reared over natural sand grew faster and had higher survival rates. Eggs of lake sturgeon showed similar hatch success and development over natural vs. stamp sand over 17 days, while lake trout eggs hatched earlier and developed faster when incubated over stamp sand, yet showed similar development over a 163 day period. Copper from stamp sand appears to impact amphibians more than fish species in this study. These results will help determine what impact stamp sand has on organisms found throughout the Keweenaw Peninsula which encounter the material at some point in their life history.

DEVELOPMENT OF A PREDICTIVE COMBUSTION MODEL OF A SPARK IGNITED ENGINE WITH GASOLINE DIRECT INJECTION, VARIABLE VALVE TIMING, DURATION AND LIFT TECHNOLOGIES

There is a need by engine manufactures for computationally efficient and accurate predictive combustion modeling tools for integration in engine simulation software for the assessment of combustion system hardware designs and early development of engine calibrations. This thesis discusses the process for the development and validation of a combustion modeling tool for Gasoline Direct Injected Spark Ignited Engine with variable valve timing, lift and duration valvetrain hardware from experimental data. Data was correlated and regressed from accepted methods for calculating the turbulent flow and flame propagation characteristics for an internal combustion engine. A non-linear regression modeling method was utilized to develop a combustion model to determine the fuel mass burn rate at multiple points during the combustion process. The computational fluid dynamic software Converge ©, was used to simulate and correlate the 3-D combustion system, port and piston geometry to the turbulent flow development within the cylinder to properly predict the experimental data turbulent flow parameters through the intake, compression and expansion processes. The engine simulation software GT-Power © is then used to determine the 1-D flow characteristics of the engine hardware being tested to correlate the regressed combustion modeling tool to experimental data to determine accuracy. The results of the combustion modeling tool show accurate trends capturing the combustion sensitivities to turbulent flow, thermodynamic and internal residual effects with changes in intake and exhaust valve timing, lift and duration.

Estrogen receptor subtype beta2 is involved in neuromast development in zebrafish (Danio rerio) larvae

Estrogens are known to play a role in both reproductive and non-reproductive functions in mammals. Estrogens and their receptors are involved in the development of the central nervous system (brain development, neuronal survival and differentiation) as well as in the development of the peripheral nervous system (sensory-motor behaviors). In order to decipher possible functions of estrogens in early development of the zebrafish sensory system, we investigated the role of estrogen receptor beta(2) (ERbeta(2)) by using a morpholino (MO) approach blocking erbeta(2) RNA translation. We further investigated the development of lateral line organs by cell-specific labeling, which revealed a disrupted development of neuromasts in morphants. The supporting cells developed and migrated normally. Sensory hair cells, however, were absent in morphants' neuromasts. Microarray analysis and subsequent in situ hybridizations indicated an aberrant activation of the Notch signaling pathway in ERbeta(2) morphants. We conclude that signaling via ERbeta(2) is essential for hair cell development and may involve an interaction with the Notch signaling pathway during cell fate decision in the neuromast maturation process.

H2B histone gene expression during {\it Xenopus laevis\/} development

Histone gene expression is replication-independent during oogenesis and early embryogenesis in amphibians; however, it becomes replication-dependent during later embryogenesis and remains replication-dependent through adulthood. In order to understand the mechanism for this switch in transcriptional regulation of histone gene expression during amphibian development, linker-scanning mutations were made in a Xenopus laevis H2B histone gene promoter by oligonucleotide site-directed mutagenesis and assayed by microinjection into oocytes and embryos. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CCAAT, and ATF motifs, required for maximal transcription in both oocytes and embryos. Factors binding to the CCAAT and ATF motifs are present in oocytes and embryos and increase slightly in abundance during early development. A sequence (CTTTACAT) in the frog H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is additionally required for maximal H2B transcription in frog embryos. Oocytes and embryos contain multiple octamer-binding proteins that are expressed in a sequential manner during early development. Sequences encoding three novel octamer-binding proteins were isolated from Xenopus cDNA libraries by virtue of their similarity with the DNA binding (POU) domain of the ubiquitously expressed transcription factor Oct-1. The protein encoded by one of these genes, termed Oct-60, was localized mainly in the cytoplasm of oocytes and was also present in early embryos until the gastrula stage of development. Proteins encoded by the other two genes, Oct-25 and Oct-91, were present in embryos after the mid-blastula stage of development and decreased by early neurula stage. The activity of the Xenopus H2B octamer motif in embryos is not specifically associated with increased binding by Oct-1 or the appearance of novel octamer-binding proteins but requires the presence of an intact CCAAT motif. We found that synergistic interactions among promoter elements are important for full H2B promoter activity. The results suggest that transcription of the Xenopus H2B gene is replication-dependent when it is activated at the mid-blastula stage of development and that replication-dependent H2B transcription is mediated by Oct-1. ^

Horn Growth and Reproduction in a Long-Lived Male Mammal: No Compensation for Poor Early-Life Horn Growth

Secondary sexual traits in males of polygynous species are important determinants of reproductive success. It is, however, unknown if and how the development of continuously growing traits at different life-stages is related to reproduction in long-lived male mammals. In this study, we evaluated the relationship of early and late horn growth on social status and reproduction in long-lived male Alpine ibex (Capra ibex). For this, we analysed individual horn growth and assessed its effect on dominance and reproduction. No evidence was detected for compensatory horn growth, as late-life horn growth positively depended on early-life horn growth in males. Still, individuals with longer horn segments grown during early adulthood experienced a stronger age-dependent length decline in annual horn growth during the late development. Accordingly, a divergence between individual growth potential and realized horn growth late in life has to be assumed. Residual age-specific horn length and length of early grown horn segments both positively affected dominance and reproductive success, whereas, contrary to our expectation, no significant effect of the length of horn segments grown during the late development was detected. Suspected higher somatic costs incurred by high-quality males during their late development might at least partly be responsible for this finding. Overall, our study suggests that the total length of horns and their early development in long-lived male Alpine ibex is a reliable indicator of reproductive success and that individuals may be unable to compensate for poor early-life growth performance at a later point in life.

Impacts of ocean warming and acidification on the larval development of the barnacle Amphibalanus improvisus

The world's oceans are warming and becoming more acidic. Both stressors, singly or in combination, impact marine species, and ensuing effects might be particularly serious for early life stages. To date most studies have focused on ocean acidification (OA) effects in fully marine environments, while little attention has been devoted to more variable coastal ecosystems, such as the Western Baltic Sea. Since natural spatial and temporal variability of environmental conditions such as salinity, temperature or pCO2 impose more complex stresses upon organisms inhabiting these habitats, species can be expected to be more tolerant to OA (or warming) than fully marine taxa. We present data on the variability of salinity, temperature and pH within the Kiel Fjord and on the responses of the barnacle Amphibalanus improvisus from this habitat to simulated warming and OA during its early development. Nauplii and cyprids were exposed to different temperature (12, 20 and 27°C) and pCO2 (nominally 400, 1250 and 3250 µatm) treatments for 8 and 4 weeks, respectively. Survival, larval duration and settlement success were monitored. Warming affected larval responses more strongly than OA. Increased temperatures favored survival and development of nauplii but decreased survival of cyprids. OA had no effect upon survival of nauplii but enhanced their development at low (12°C) and high (27°C) temperatures. In contrast, at the intermediate temperature (20°C), nauplii were not affected even by 3250 µatm pCO2. None of the treatments significantly affected settlement success of cyprids. These experiments show a remarkable tolerance of A. improvisus larvae to 1250 µatm pCO2, the level of OA predicted for the end of the century.

Effects of elevated pCO2 on the post-settlement development of Pocillopora damicornis

Ocean acidification represents a key threat to the recruitment of scleractinian corals. Here, we investigated the effect of increased partial pressure of carbon dioxide (pCO2) on the early development of Pocillopora damicornis by rearing the recruits for 12 days at 3 pCO2 levels (446, 896 and 1681 µatm). Results showed that increased pCO2 exerted minor effects on symbiont density and maximum quantum yield (Fv/Fm), while significantly enhanced the relative electron transport through photosystem II (PSII) of Symbiodinium. Notably, calcification and biomass of recruits decreased sharply by 34% and 24% respectively in 896 µatm, and tended to remain constant as pCO2 was raised from 896 to 1681 µatm. Furthermore, recruits in 1681 ?atm, with comparable surface area as those in 896 µatm, produced fewer buds. These findings indicated that juvenile P. damicornis under high pCO2 would enhance electron transport rate and suppress asexual budding to favor skeletal and tissue growths, which are more critical for their persistence and survival in a high pCO2 environment. This work suggested the physiological plasticity of juvenile corals under short-term exposure to elevated pCO2.

Impact of CO2-driven acidification on the development of the sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea)

We evaluated the impact of ocean acidification on the early development of sea cucumber Apostichopus japonicus. The effect of pH-levels (pH 8.04, 7.85, 7.70 and 7.42) were tested on post-fertilization success, developmental (stage duration) and growth rates. Post-fertilization success decreased linearly with pH leading to a 6% decrease at pH 7.42 as compared to pH 8.1. The impact of pH on developmental time was stage-dependent: (1) stage duration increased linearly with decreasing pH in early-auricularia stage; (2) decreased linearly with decreasing pH in the mid-auricularia stage; but (3) pH decline had no effect on the late-auricularia stage. At the end of the experiment, the size of doliolaria larvae linearly increased with decreasing pH. In conclusion, a 0.62 unit decrease in pH had relatively small effects on A. japonicus early life-history compared to other echinoderms, leading to a maximum of 6% decrease in post-fertilization success and subtle effects on growth and development.

Elevated pCO2 causes developmental delay in early larval Pacific oysters, Crassostrea gigas

Increasing atmospheric CO2 equilibrates with surface seawater, elevating the concentration of aqueous hydrogen ions. This process, ocean acidification, is a future and contemporary concern for aquatic organisms, causing failures in Pacific oyster (Crassostrea gigas) aquaculture. This experiment determines the effect of elevated pCO2 on the early development of C. gigas larvae from a wild Pacific Northwest population. Adults were collected from Friday Harbor, Washington, USA (48°31.7' N, 12°1.1' W) and spawned in July 2011. Larvae were exposed to Ambient (400 µatm CO2), MidCO2 (700 µatm), or HighCO2 (1,000 µatm). After 24 h, a greater proportion of larvae in the HighCO2 treatment were calcified as compared to Ambient. This unexpected observation is attributed to increased metabolic rate coupled with sufficient energy resources. Oyster larvae raised at HighCO2 showed evidence of a developmental delay by 3 days post-fertilization, which resulted in smaller larvae that were less calcified.

Early expression of antiinsulin autoantibodies of humans and the NOD mouse: Evidence for early determination of subsequent diabetes

With the development of an insulin autoantibody (IAA) assay performed in 96-well filtration plates, we have evaluated prospectively the development of IAA in NOD mice (from 4 weeks of age) and children (from 7 to 10 months of age) at genetic risk for the development of type 1 diabetes. NOD mice had heterogeneous expression of IAA despite being inbred. IAA reached a peak between 8 and 16 weeks and then declined. IAA expression by NOD mice at 8 weeks of age was strongly associated with early development of diabetes, which occurred at 16–18 weeks of age (NOD mice IAA+ at 8 weeks: 83% (5/6) diabetic by 18 weeks versus 11% (1/9) of IAA negative at 8 weeks; P < .01). In man, IAA was frequently present as early as 9 months of age, the first sampling time. Of five children found to have persistent IAA before 1 year of age, four have progressed to diabetes (all before 3.5 years of age) and the fifth is currently less than age 2. Of the 929 children not expressing persistent IAA before age 1, only one has progressed to diabetes to date (age onset 3), and this child expressed IAA at his second visit (age 1.1). In new onset patients, the highest levels of IAA correlated with an earlier age of diabetes onset. Our data suggest that the program for developing diabetes of NOD mice and humans is relatively “fixed” early in life and, for NOD mice, a high risk of early development of diabetes is often determined by 8 weeks of age. With such early determination of high risk of progression to diabetes, immunologic therapies in humans may need to be tested in children before the development of IAA for maximal efficacy.

The Optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.