The fecundity, embryonic development, growth and viability of captive Chiloscyllium punctatum were investigated over two breeding and laying seasons. Chiloscyllium punctatum is an oviparous species, which lays rectangular eggs from late July to February. The six adult female C. punctatum in the open ocean display tank at UnderWater World, Mooloolaba, Australia laid a total of 692 eggs between July 2004 and February 2006, with 228 of those being viable. This equates to each female laying 115·3 eggs over the two seasons, with 38 viable. The embryos are first visible to the naked eye at c. 18 days post deposition (dpd), while pre‐hatching is first seen c. 35 dpd, and the embryos hatch at an average of 153 dpd (temperature 21–25° C). Embryonic growth is most rapid from 99 dpd until hatching. An average of 21·4% of the eggs hatched over the two seasons.
The development of the eye of the oviparous brown banded bamboo shark, Chiloscyllium punctatum, was monitored from egg deposition through adulthood. The order and timing of retinal cell differentiation were assessed by light and transmission electron microscopy. As in other vertebrates, the ganglion cells are the first to differentiate, in this case by 81 days post-egg deposition (dpd). The order then deviates from what is typically quoted for vertebrates, with the Müller and amacrine cells differentiating morphologically around the same time, followed by the bipolar cells (101 dpd) and finally the horizontal cells and photoreceptors (124 dpd). The neural retina is fully differentiated and synaptic connections are formed approximately 1 month prior to hatching, which occurs at about 158 dpd. The mature retina is duplex, with a peak rod to cone ratio of approximately 12:1. The eye and lens of C. punctatum continue to grow throughout life and become less aspherical with growth; the equatorial (nasotemporal) lens diameter is 12% larger than the axial (anterior-posterior) lens diameter in embryos and 8% larger in adults. Access to developmental stages and the protracted gestational period of C. punctatum make it a highly valuable model for developmental studies of the visual system. This study also provides an evolutionary perspective on retinal neurogenesis in an elasmobranch.
The development of the visual system inanamniotic vertebrates is a continual process, allowing for ontogenetic changes in retinal topography and spatial resolving power. We examined the number and distribution of retinal ganglion cells in wholemounted retinae throughout the protracted embryonic development (∼5 months) of a chondrichthyan, i.e. the brown-banded bamboo shark Chiloscyllium punctatum, from the beginning of retinal cell differentiation (approximately halfway through embryogenesis) to adulthood. We also identified and quantified the number of apoptosed cells within the ganglion cell layer to evaluate the contribution of apoptosis to changes in retinal topography. C. punctatum undergoes rapid changes in ganglion cell distribution during embryogenesis, where high levels of apoptosis, especially around the retinal periphery, result in relative increases in ganglion cell density in the central retina which progressively extend nasally and temporally to form a meridional band at hatching. After hatching, C. punctatum forms and maintains a horizontal streak, showing only minor changes in topography during growth, with basal levels of apoptosis. The total number of retinal ganglion cells reaches 547,881 in adult sharks, but the mean (3,228 cells·mm-2) and peak (4,983 cells·mm-2) retinal ganglion cell densities are highest around the time of hatching. Calculated estimates of spatial resolving power, based on ganglion cell spacing (assuming a hexagonal mosaic) and assessment of the focal length from cryosections of the eye, increase from 1.47 cycles·degree-1 during embryogenesis to 4.29 cycles·degree-1 in adults. The increase in spatial resolving power across the retinal meridian would allow this species to hunt and track faster, more mobile prey as it reaches maturity.
The epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) are commonly found in periodically hypoxic environments. The ecophysiological time available for these animals to safely exploit these niches during different seasonal temperatures was examined. The time to loss of righting reflex (T (LRR)) was examined in response to an open ended anoxic challenge at three seasonal temperatures (23, 25 and 27°C). Ventilation rates were measured in an open ended anoxic challenge at 23°C and during 1.5 h of anoxia followed by 2 h of re-oxygenation at 23 and 25°C. The mean T (LRR) of epaulette and grey carpet sharks was inversely proportional to temperature. The T (LRR) was similar between species at 23°C; however, grey carpet sharks had significantly reduced T (LRR) at higher temperatures. During the standardised anoxic challenge, epaulette sharks entered into ventilatory depression significantly earlier at 25°C. During re-oxygenation, epaulette sharks exposed to anoxia at 23°C had no significant increase in ventilation rates. However, after anoxic challenge and re-oxygenation at 25°C, epaulette sharks showed a significant increase in ventilation rates during re-oxygenation. Grey carpet sharks displayed no evidence of ventilatory depression during anoxia. However, during re-oxygenation, grey carpet sharks had significantly elevated ventilation rates above pre-experimental levels and control animals. These data demonstrate that the anoxia tolerance times of both species were temperature dependent, with a significant reduction in the T (LRR) occurring at higher temperatures. Epaulette sharks had a significantly greater T (LRR) at higher temperatures than grey carpet sharks, which did not enter into a ventilatory depression.
Recent advances in microwave chemical fixation (MCF) and/or high pressure freezing (HPF) combined with transmission electron microscopy have resulted in superior ultrastructural detail in a variety of tissue types. To date, selachian tissue has been fixed and processed using only standard chemical fixation (CF) methods, and the resulting ultrastructure has been less than ideal. In this study, we compared the ultrastructure of the fragile retinal tissue from the brown banded bamboo shark, Chiloscyllium punctatum, obtained using CF, MCF, and HPF methods. For all fixation protocols, ultrastructural preservation was improved by keeping the tissue in oxygenated Ringer solution until the time of fixation. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF. Although HPF occasionally resulted in very high quality ultrastructure, microwave fixation was almost comparable, quicker and far more consistent.
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