Early embryonic development of brackish water Killifish Aphanius hormuzensis (Teleostei,Aphaniidae) inhabiting coastal environment in Southern Iran

The embryonic and early developmental stages of Killifish Aphanius hormuzensis reared in a laboratory condition were investigated in detail using light microscopy. The adult male and female of A. hormuzensis were captured from the wild. They were kept in laboratory condition for 2 months and spontaneously spawned at 28 ± 1°C. The fertilized macrolecithal eggs were characterized with spherical shape, transparent and average 1.60 ± 0.20 mm in diameter. The embryonic development was started with fertilized egg activation and the first cleavage was observed in 2.5 hr ± 20 min post fertilization (hpf), followed by blastula formation and gastrulation stages in 7 and 11 hpf respectively. First somite was observed in 20 hpf and the heart beating was detected in 25 hpf. The early ontogeny of A. hormuzensis was followed by complements of organs formation and the newly hatched larva with 4.9 ± 0.10 mm in length was detected in 166 hpf. This study provides a basis for further research on reproductive biology, conservation and breeding of this species.     

Kisspeptin1 modulates odorant‐evoked fear response via two serotonin receptor subtypes (5‐HT1A and 5‐HT2) in zebrafish

Kiss1, a neuropeptide predominantly expressed in the habenula, modulates the serotonin (5‐HT) system to decrease odorant cue [alarm substance (AS)]‐evoked fear behaviour in the zebrafish. The purpose of this study was to assess the interaction of Kiss1 with the 5‐HT system as well as to determine the involvement of the 5‐HT receptor subtypes in AS‐evoked fear. We utilized 0. 28 mg/kg WAY 100635 (WAY), a selective 5‐HT_1A receptor antagonist, to observe the effects of Kiss1 administration on AS‐evoked fear. We found WAY significantly inhibited the anxiolytic effects of Kiss1 (p < 0.001) with an exception of freezing behaviour. Based on this, we utilized 92.79 mg/kg methysergide, a 5‐HT₁ and 5‐HT₂ receptor antagonist, and found that methysergide significantly blocked the anxiolytic effects of Kiss1 in the presence of the AS (p < 0.001). From this, we conclude that Kiss1 modulates AS‐evoked fear responses mediated by the 5‐HT_1A and 5‐HT₂ receptors.

     

Thermoneutral conditions correct the obese phenotype in female,but not male,Kiss1r knockout mice

Kisspeptin, a neuropeptide that activates gonadotropin-releasing hormone (GnRH) neurons, has also been implicated as a regulator of energy balance. Kisspeptin receptor (Kiss1r) knockout (KO) mice display an obese phenotype in adulthood compared to wild-type (WT) controls due to reduced energy expenditure. Additionally, experimental evidence shows that the temperature of typical rodent housing conditions (22 °C) increases the metabolism of mice above basal levels. Female Kiss1r KO mice show reduced core temperature and impaired temperature adaptation to an acute cold challenge, suggesting their temperature homeostasis processes are altered. The present study examined the phenotype of gonadectomised Kiss1r KO mice at both sub-thermoneutral and thermoneutral temperature (22 °C and 30 °C). Our results confirmed the obese phenotype in Kiss1r KO mice at 22 °C, and revealed a sexually dimorphic effect of thermal neutrality on the phenotype. In female KO mice, the obesity observed at 22 °C was attenuated at 30 °C. Plasma leptin levels were higher in KO than WT female mice at 22 °C (P < 0.001) but not at 30 °C. Importantly, the expression of Ucp1 mRNA in brown adipose tissue was lower in KO mice compared to WT mice at 22 °C (P < 0.05), but not different from WT at 30 °C. In male KO mice, a metabolic phenotype was observed at 22 °C and 30 °C. These results provide further evidence for kisspeptin-mediated regulation of adiposity via altered energy expenditure. Moreover, thermoneutral housing alleviated the obese phenotype in female Kiss1r KO mice, compared to WT, indicating the impairment in these mice may relate to an inability to adapt to the chronic cold stress that is experienced at 22 °C.     

Prolongation of somitogenesis in two anguilliform species,the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus,with refined descriptions of their early development

The embryonic development of the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus was morphologically investigated with laboratory‐reared specimens to clarify the characteristics of somitogenesis. In A. japonica, somites were first observed at 18 h post fertilization (hpf) when epiboly reached 90%. Somitogenesis progressed at a rate of 1·6 h^?1 at mean ± s.d . 22·6 ± 0·7° C and completed at 107 hpf (3 days post hatching; dph) when total number of somites (ST) reached 114, which corresponds to the species' number of vertebrae (112–119). In M. cinereus, somites were first observed at 14 hpf when epiboly completed. Somitogenesis progressed at a rate of 1·9 h^?1 at mean ± s.d . 24·4 ± 0·2° C and completed at 90 hpf (2 dph) with 149 ± 4 ST, which corresponds to the species' number of vertebrae (142–158). Both species hatched before somitogenesis was completed, at 37 hpf with 47 ST and 42 hpf with 82 ± 4 ST, respectively. The formation of other organs such as the heart, mouth and pectoral fin bud occurred during somitogenesis. Comparison with the development of zebrafish Danio rerio indicates a prolongation of somitogenesis in A. japonica and M. cinereus. Their somitogenesis rates, however, correspond well with that of D. rerio estimated at the same temperature and their developmental stages at hatching are almost equivalent to other fishes having similar yolk sizes. Therefore, the prolongation of somitogenesis in A. japonica and M. cinereus may be accounted for solely by the increased numbers of somites to be formed, not by a slow somitogenesis rate or an acceleration in organogenesis.     

Spatiotemporal expression pattern of an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus during early development,and its potential role in larval vertical migration

We have cloned and studied Hp‐ECPN, an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus. Hp‐ecpn cDNA was produced and found to contain a 1461‐bp open reading frame that encodes 486 amino acids. Accumulation of Hp‐ecpn mRNA and protein expression occurred at the 14 h postfertilization (hpf) swimming blastula stage and thereafter. The Hp‐ECPN protein was N‐glycosylated, and the amino acid sequence was similar to that of vertebrate encephalopsins. Whole‐mount immunohistochemistry revealed the presence of Hp‐ECPN in cells (ECPN cells) that appeared initially around the tip of the archenteron in 20 hpf early gastrulae. By the 54 hpf pluteus stage, ECPN cells had spread through the aboral ectoderm, and, by the eight‐arm pluteus stage, were restricted to the tips of the larval arms and the posterior end of the body. The number of ECPN cells increased under conditions of continuous light, but decreased under continuous dark. Knockdown of Hp‐ecpn mRNA using morpholino antisense oligonucleotides decreased the number of ECPN cells considerably, and inhibited the vertical swimming of the larvae. This suggested that Hp‐ECPN plays a role in photosensitive larval swimming vertical migration. In adult tissues, the ECPN cells were detected exclusively in tube feet.     

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.     

Allometric growth and functional development of the gut in developing cod Gadus morhua L. larvae

The development of the gut epithelium in cod Gadus morhua was studied during the larval period in intensive rearing systems. Cod larvae were fed enriched rotifers from mouth opening. On 17 days post‐hatch (dph) one group of larvae were fed Artemia sp. nauplii while another group were fed both rotifers and a formulated diet (co‐fed). At the end of the experiment (30 dph) larvae receiving live feed were almost three times larger than the co‐fed larvae, although no clear signs of pathological effects due to feeding regime were found in any larvae sampled for morphological studies. The midgut volume in larvae fed live feed increased by a factor of 38 during the experiment, and in particular volume increased rapidly between 24 and 30 dph. The enterocyte size increased between 12 and 24 dph from 652 ± 64 to 1479 ± 144 μm³ (mean ±s .e .). When enterocytes reached their maximum size, several morphological changes in the gut epithelium were initiated, such as increased number of mitochondria per enterocyte, increased size of the nuclei and a considerable increase in microvilli surface area. The mitochondrial membrane structures changed during the experiment, suggesting a maturation process of the mitochondria. The midgut development was strongly related to larval size rather than age. On 30 dph co‐fed larvae were equal in size to Artemia sp. fed larvae on 24 dph. This was reflected by equal values of estimated midgut volume, midgut length and total number of enterocytes and the number of mitochondria per enterocyte. The microvilli surface area, however, was significantly larger in co‐fed larvae on 24 dph compared to live‐feed larvae on 30 dph. This increase in absorptive surface was probably a response to suboptimal feeding conditions. The strong correlation between gut development and larval size and the lack of clear pathological effects, suggested that the gut tissue is flexible and can withstand periods of suboptimal nutrition at this stage.     

Dose-dependent effects of developmental mercury exposure on C-start escape responses of larval zebrafish Danio rerio

Zebrafish Danio rerio embryos were exposed to 0, 25, 50 or 75 ppb Hg²⁺ from 0 to 24 h post‐fertilization (hpf) then placed into Hg²⁺‐free water. Inductively coupled plasma‐mass spectrophotometer analysis of whole embryo Hg²⁺ content at 24 hpf showed a positive correlation with exposure regime (Pearson's one‐tailed, r²= 0·698, P < 0·01); at 5 days post‐hatch (dph), whole larval Hg²⁺ content was not detectable. Hg²⁺‐induced behavioural deficits in larvae were, therefore, due to changes during embryogenesis and not to residual Hg²⁺ in the larvae. At 5 dph, larvae were tested for responses to different frequencies but equal intensities of vibrational stimuli generated by a remotely controlled plastic hammer. Data were recorded by high‐speed videography and computer‐analysed for latency of response (ms), amplitude of the response as measured by maximum initial velocity [normalized as body (standard) lengths s^?1; V_max] and duration of behaviour from initial head movement to cessation of caudal tail movement (ms). A single mechanical stimulus resulted in behavioural outcomes that were related to embryonic Hg²⁺ uptake. Response latency increased with exposure level and displayed an increase of ×1·5–2·5 over control values (ANOVA, P < 0·01). The V_max decreased with exposure level to a low of 71% of control at the highest Hg²⁺ concentration (ANOVA, P < 0·01). Duration of behaviour displayed a biphasic response pattern in which exposure to 0, 50 or 75 ppb Hg²⁺ did not result in a significantly different response yet exposure to 25 ppb Hg²⁺ caused a significantly longer time of active response (ANOVA, P < 0·01). Repeated stimulation (1, 2 or 4 hits s^?1) resulted in a concentration‐dependent increase in response failures. Regardless of stimulation frequency, larvae exposed to 0 or 25 ppb Hg²⁺ as embryos maintained higher V_max levels for longer intervals during the testing period than those exposed as embryos to either 50 or 75 ppb Hg²⁺.     

Effects of feeding rate on the growth performance of gynogenetic albino sterlet,Acipenser ruthenus (Linnaeus, 1758) larvae

The feeding rate effects were studied on the growth performance of gynogenetic diploid larvae of sterlet Acipenser ruthenus during the first 4 weeks of exogenous feeding. The experimental rearing was conducted from 7 to 38 days post‐hatch (dph) in a circulation system. This was set up in four groups with three replicates (440 individuals/replicate), viz: AC‐control larvae fed Artemia sp., CFC‐control larvae fed compound feed, AG‐gynogenetic larvae fed Artemia sp., and CFG‐gynogenetic larvae fed compound feed. The larvae were reared in glass tanks (44 L volume, 10 individuals/L) with the temperature maintained at 18 ± 0.5°C, photoperiod of 12L:12D and water flow regime of 1‐L/min and fed 50%, 25%, 25%, and 9% of their total biomass/day during feeding. Highest TL and WBW of gynogenetic diploid larvae (AG) were observed with 50.6 ± 1.2 mm and 607.3 ± 36.1 mg (n = 30) at 38 dph. Highest TL and WBW of control larvae (AC) were recorded with 49.5 ± 3.8 mm and 600.8 ± 88.0 mg (n = 30), respectively, with 73.1% ± 11.4% survival; the lowest survival rate was at 46.4% ± 7.1% (n = 30) for the CFG group. The results indicate that the gynogenetic and normal larvae of sterlet fed with live food (Artemia nauplii) from 7 dph can achieve higher growth and survivability compared to the larvae fed with formulated test feed. Results of this study suggest that the effective rearing of sterlet larvae from 7 to 38 dph strongly depends upon the types of feed rather than the genome manipulation performed.     

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

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

Effects of elevated CO2 on life‐history traits of three successive generations of Frankliniella occidentalis and F. intonsa on kidney bean,Phaseolus vulgaris

The objective of this study was to determine how elevated CO₂ impacts on life‐history traits and life table parameters in three successive generations of invasive species Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) and its related native species, Frankliniella intonsa (Pergande), fed with kidney bean leaves grown in ambient CO₂. The oviposition period, sex ratio, net reproductive rate (R₀), intrinsic rate of increase (r_m), and finite rate of increase (λ) of F. occidentalis increased in elevated CO₂, and larval duration, survival rate, mean generation time (T), and population doubling time (DT) decreased. For F. intonsa, larval duration, survival rate, oviposition period, longevity of female adults, R₀, r_m, and λ decreased in elevated CO₂, whereas sex ratio, T, and DT increased. These results indicated that the effects of elevated CO₂ would be beneficial to F. occidentalis, whereas it would be detrimental to F. intonsa. However, the effects of elevated CO₂ on F. occidentalis and F. intonsa differed over generations. In elevated CO₂, larval duration, survival rate, oviposition period, sex ratio, r_m, and λ of F. occidentalis increased linearly through successive generations, whereas T and DT decreased linearly, which suggested that the effects of elevated CO₂ on F. occidentalis would be slowly accentuated over time. For F. intonsa, larval duration, survival rate, oviposition period, r_m, and λ decreased linearly over generations, whereas sex ratio, T, and DT increased linearly. This indicated that the effects of elevated CO₂ on F. intonsa would slowly accentuate over time. We conclude that F. occidentalis would be more adapted to elevated CO₂ than F. intonsa.     

Ontogeny of body density and the swimbladder in yellowtail kingfish Seriola lalandi larvae

The ontogeny of larval body density and the morphological and histological events during swimbladder development were investigated in two cohorts of yellowtail kingfish Seriola lalandi larvae to understand the relationship between larval morphology and body density. Larvae <3 days post hatch (dph) were positively buoyant with a mean ± s.d . body density of 1·023 ± 0·001 g cm^?3. Histological evidence demonstrated that S. lalandi larvae are initially transient physostomes with the primordial swimbladder derived from the evagination of the gut ventral to the notochord and seen at 2 dph. A pneumatic duct connected the swimbladder to the oesophagus, but degenerated after 5 dph. Initial swimbladder (SB) inflation occurred on 3 dph, and the inflation window was 3–5 dph when the pneumatic duct was still connected to the gut. The swimbladder volume increased with larval age and the epithelial lining on the swimbladder became flattened squamous cells after initial inflation. Seriola lalandi developed into a physoclist with the formation of the rete mirabile and the gas‐secreting gland comprised low‐columnar epithelial cells. Larvae with successfully inflated swimbladders remained positively buoyant, whereas larvae without SB inflation became negatively buoyant and their body density gradually reached 1·030 ± 0·001 g cm^?3 by 10 dph. Diel density changes were observed after 5 dph, owing to day time deflation and night‐time inflation of the swimbladder. These results show that SB inflation has a direct effect on body density in larval S. lalandi and environmental factors should be further investigated to enhance the rate of SB inflation to prevent the sinking death syndrome in the early life stage of the fish larvae.     

鲤鱼发育早期HPG轴和GH/IGF轴相关因子的转录起始分析

采用RT-PCR的方法,以不同发育时期的鲤鱼胚胎和幼鱼为材料,研究了与鱼类生殖相关的HPG轴以及与生长相关的GH/IGF轴中GnRH、GtH以及GH、GHR和IGF重要信号分子的转录起始特征.结果显示,sGnRH、cGnRH、GtH-Ⅰβ卢亚基和GHR于鲤鱼胚胎受精后20h开始转录,IGF-1于受精后23h开始转录,GtH-Ⅱβ亚基于受精后26h开始转录,GtH α亚基于受精后46h开始转录,GH于1dph(孵出后第1天)开始转录.其中,GHR和IGF-1均早于GH开始转录,GtH α亚基和β亚基的转录起始时间不同步.研究结果为揭示鲤鱼生殖与生长间的调控机制积累了重要的科学依据.     

Kisspeptins Modulate the Biology of Multiple Populations of Gonadotropin-Releasing Hormone Neurons during Embryogenesis and Adulthood in Zebrafish (Danio rerio)

Kisspeptin1 (product of the Kiss1 gene) is the key neuropeptide that gates puberty and maintains fertility by regulating the gonadotropin-releasing hormone (GnRH) neuronal system in mammals. Inactivating mutations in Kiss1 and the kisspeptin receptor (GPR54/Kiss1r) are associated with pubertal failure and infertility. Kiss2, a paralogous gene for kiss1, has been recently identified in several vertebrates including zebrafish. Using our transgenic zebrafish model system in which the GnRH3 promoter drives expression of emerald green fluorescent protein, we investigated the effects of kisspeptins on development of the GnRH neuronal system during embryogenesis and on electrical activity during adulthood. Quantitative PCR showed detectable levels of kiss1 and kiss2 mRNA by 1 day post fertilization, increasing throughout embryonic and larval development. Early treatment with Kiss1 or Kiss2 showed that both kisspeptins stimulated proliferation of trigeminal GnRH3 neurons located in the peripheral nervous system. However, only Kiss1, but not Kiss2, stimulated proliferation of terminal nerve and hypothalamic populations of GnRH3 neurons in the central nervous system. Immunohistochemical analysis of synaptic vesicle protein 2 suggested that Kiss1, but not Kiss2, increased synaptic contacts on the cell body and along the terminal nerve-GnRH3 neuronal processes during embryogenesis. In intact brain of adult zebrafish, whole-cell patch clamp recordings of GnRH3 neurons from the preoptic area and hypothalamus revealed opposite effects of Kiss1 and Kiss2 on spontaneous action potential firing frequency and membrane potential. Kiss1 increased spike frequency and depolarized membrane potential, whereas Kiss2 suppressed spike frequency and hyperpolarized membrane potential. We conclude that in zebrafish, Kiss1 is the primary stimulator of GnRH3 neuronal development in the embryo and an activator of stimulating hypophysiotropic neuron activities in the adult, while Kiss2 plays an additional role in stimulating embryonic development of the trigeminal neuronal population, but is an RFamide that inhibits electrical activity of hypophysiotropic GnRH3 neurons in the adult.