Fishing for oil and meat drives irreversible defaunation of deepwater sharks and rays.

The deep ocean is the last natural biodiversity refuge from the reach of human activities. Deepwater sharks and rays are among the most sensitive marine vertebrates to overexploitation. One-third of threatened deepwater sharks are targeted, and half the species targeted for the international liver-oil trade are threatened with extinction. Steep population declines cannot be easily reversed owing to long generation lengths, low recovery potentials, and the near absence of management. Depth and spatial limits to fishing activity could improve conservation when implemented alongside catch regulations, bycatch mitigation, and international trade regulation. Deepwater sharks and rays require immediate trade and fishing regulations to prevent irreversible defaunation and promote recovery of this threatened megafauna group.

The effect of fishing-capture stress on the oxygen uptake rate and swimming activity of the holocephalan Callorhinchus milii.

Overfishing, capture mortality, and consequences following the release of surviving animals represent severe threats to chondrichthyans. Although holocephalans are common bycaught and discarded species, other than postrelease mortality, little is known of fishing capture stress impacts. The stress response elicited after capture, essential to increase survival chances, is energetically demanding and affects the amount of energy available for other biological activities, with potential long-term impairments. We measured the effect of 30-min simulated gillnet capture on oxygen uptake rate (MO2 ), a proxy for metabolic rate and energy use, on recovery pattern, and on swimming activity of elephant fish (Callorhinchus milii). Immediately after simulated capture, Active and Inactive MO2 , measured during swimming and resting periods, respectively, were 27.5% and 43.1% lower than precapture values. This metabolic decline is likely an adaptation for reducing the energy allocated to non-essential activities, thus preserving it to sustain the stress response and processes essential for immediate survival. Supporting this, after gillnet capture, animals decreased their swimming time by 26.6%, probably due to a reduction in the energy allocated to movement. After 7 days, swimming activity and both Inactive MO2 and Active MO2 returned to precapture values. Although metabolic decline may enhance survival chances, the associated decreased swimming activity might increase predation risk and slow the physiological recovery after a fishing event. Moreover, some of the activities involved in Inactive MO2 are fundamental for life maintenance and therefore its depression after a capture event might have long-term repercussions for life sustenance and health.

Influence of female reproductive state and of fishing-capture stress on the oxygen uptake rate of a viviparous elasmobranch.

In animals discarded after a fishing capture event, the elicited stress response necessary to ensure their survival is energetically costly. This energy is diverted from other important biological activities, including growth and reproduction, possibly impairing them. Given that elasmobranchs are among the most threatened vertebrate groups, estimating capture-induced energetic changes and comparing these variations to the energy requirements of pregnancy maintenance is necessary. In pregnant southern fiddler rays (Trygonorrhina dumerilii), we measured changes in oxygen uptake rate (MO2 ; a proxy for metabolic rate and energy usage) in response to trawling simulation and air exposure, and estimated the oxygen requirements of sustaining late-term pregnancy and embryos. MO2 was measured in pregnant females, before (prestress MO2 ) and after trawling simulation (after-capture MO2 ), and again after females gave birth (postpartum MO2 ). After-capture MO2 was 31.7% lower than MO2 measured in minimally stressed females, suggesting a reduction in energy expenditure. This reduction is likely triggered by an initially excessive energetic investment in the stress response, and is aimed at shutting down nonessential activities to redirect energy to processes fundamental for survival. Prestress MO2 was 78.5% higher than postpartum MO2 . Capture simulation decreased MO2 to values similar to those observed postpartum, suggesting a capture-induced reduction in oxygen and energy allocation to pregnancy and embryonic respiration, which could be associated with reproductive impairments. These data, by better estimating the impact of capture and discard on energetic requirements and reproductive fitness, may support the introduction of area and/or seasonal closures to fishing.

Temperature Insensitivity and Behavioural Reduction of the Physiological Stress Response to Longline Capture by the Gummy Shark, Mustelus antarcticus.

Many factors influence the physiological stress response to fisheries capture in elasmobranchs. However, the influence of sea surface temperatures (SST) and behaviour are unknown and crucial considering global fishing pressures. We investigated the effect of SST and behaviour on the physiological stress response to capture of the gummy shark, Mustelus antarcticus, and compared our results to a laboratory study using similar conditions to test whether stress responses of in situ capture are consistent with those from laboratory simulations. Capture time for 23 M. antarcticus ranged 32-241 min as measured by hook timers or time depth recorders (TDR) in SSTs ranging 12-20°C. TDR data from 13 M. antarcticus were analysed to quantify capture behaviour as the percentage of time spent moving during capture. Several physiological variables measured from blood samples obtained immediately upon the animals' landing indicated that although warmer SSTs increased metabolic rate, the stress response to capture was not exacerbated by capture duration. During capture movement occurred for an average of 10% of the time and since M. antarcticus can respire whilst stationary, restricted movement probably mitigated potential influences of increased SSTs and capture duration on the stress response. Previous laboratory findings were also shown to be indicative of in situ conditions and we thus advise that studies control for water temperature given the influence it has on variables (e.g. lactate) used to measure capture stress in elasmobranchs. We highlight the importance of seasonal water temperatures and capture behaviour when assessing the resilience to fisheries capture and the implementation of appropriate fisheries management strategies.

The adenylate energy charge as a new and useful indicator of capture stress in chondrichthyans.

Quantifying the physiological stress response of chondrichthyans to capture has assisted the development of fishing practices conducive to their survival. However, currently used indicators of stress show significant interspecific and intraspecific variation in species' physiological responses and tolerances to capture. To improve our understanding of chondrichthyan stress physiology and potentially reduce variation when quantifying the stress response, we investigated the use of the adenylate energy charge (AEC); a measure of available metabolic energy. To determine tissues sensitive to metabolic stress, we extracted samples of the brain, heart, liver, white muscle and blood from gummy sharks (Mustelus antarcticus) immediately following gillnet capture and after 3 h recovery under laboratory conditions. Capture caused significant declines in liver, white muscle and blood AEC, whereas no decline was detected in the heart and brain AEC. Following 3 h of recovery from capture, the AEC of the liver and blood returned to "unstressed" levels (control values) whereas white muscle AEC was not significantly different to that immediately after capture. Our results show that the liver is most sensitive to metabolic stress and white muscle offers a practical method to sample animals non-lethally for determination of the AEC. The AEC is a highly informative indicator of stress and unlike current indicators, it can directly measure the change in available energy and thus the metabolic stress experienced by a given tissue. Cellular metabolism is highly conserved across organisms and, therefore, we think the AEC can also provide a standardised form of measuring capture stress in many chondrichthyan species.

Frequency of multiple paternity in gummy shark, Mustelus antarcticus, and rig, Mustelus lenticulatus, and the implications of mate encounter rate, postcopulatory influences, and reproductive mode.

Elasmobranchs (sharks and rays) show an amazing diversity of reproductive modes and behaviors. Multiple paternity (MP) has been identified in all species where more than 1 litter has been investigated; yet neither direct nor indirect benefits from MP have been determined in elasmobranchs. This has led to the suggestion that MP in this group may simply be a product of convenience polyandry with variation in the frequency of MP driven by differences in mate encounter rates. Here, we use molecular markers to investigate polyandry and MP in 2 closely related and commercially important species of shark, Mustelus antarcticus and Mustelus lenticulatus. In total, 328 M. antarcticus embryos originating from 29 different mothers and 75 M. lenticulatus embryos originating from 19 different mothers were genotyped using 8 microsatellite loci. We find that MP occurs in both species. However, in both species, the majority of litters were sired by a single father. Our results do not support increased fecundity per se as a driver of MP. Further, our results do not suggest that high population densities with resulting high mate encounter rates generated by breeding aggregations necessarily lead to high frequencies of MP. Importantly, we note evidence of reproductive skew within polyandrous litters, which is a predicted outcome of postcopulatory mechanisms.

Immediate and delayed effects of gill-net capture on acid-base balance and intramuscular lactate concentration of gummy sharks, Mustelus antarcticus.

Many sharks are captured as untargeted by-catch during commercial fishing operations and are subsequently discarded. A reliable assessment of the proportion of discarded sharks that die post-release as a result of excessive physiological stress is important for fisheries management and conservation purposes, but a reliable physiological predictor of post-release mortality has not been identified. To investigate effects of gill-net capture on the acid-base balance of sharks, we exposed gummy sharks, Mustelus antarcticus, to 60 min of gill-net capture in a controlled setting, and obtained multiple blood and muscle tissue samples during a 72-h recovery period following the capture event. Overall mortality of gummy sharks was low (9%). Blood pH was significantly depressed immediately after the capture event due to a combination of respiratory and metabolic acidosis. Maximum concentrations of plasma lactate (9.9 ± 1.5 mmol L(-1)) were measured 3h after the capture event. Maximum intramuscular lactate concentrations (37.0 ± 4.6 µmol g(-1)) were measured immediately after the capture event, and intramuscular lactate concentrations were substantially higher than plasma lactate concentrations at all times. Sharks in poor condition had low blood pH and high intramuscular lactate concentration, but blood pH does not appear to be a reliable predictor of survival. Suitability of intramuscular lactate concentration as predictor of delayed mortality deserves further investigation.

Parathyroid hormone gene family in a cartilaginous fish, the elephant shark (Callorhinchus milii).

The development of bone was a major step in the evolution of vertebrates. A bony skeleton provided structural support and a calcium reservoir essential for the movement from an aquatic to a terrestrial environment. Cartilaginous fishes are the oldest living group of jawed vertebrates. In this study we have identified three members of the parathyroid hormone (Pth) gene family in a cartilaginous fish, the elephant shark (Callorhinchus milii). The three genes include two Pth genes, designated as Pth1 and Pth2, and a Pthrp gene. Phylogenetic analysis suggested that elephant shark Pth2 is an ancient gene whose orthologue is lost in bony vertebrates. The Pth1 and Pth2 genes have the same structure as the Pth gene in bony vertebrates, whereas the structure of the Pthrp gene is more complex in tetrapods compared with elephant shark. The three elephant shark genes showed distinct patterns of expression, with Pth2 being expressed only in the brain and spleen. This contrasts with localization of the corresponding proteins, which showed considerable overlap in their distribution. There were conserved sites of localization for Pthrp between elephant shark and mammals, including tissues such as kidney, skin, skeletal and cardiac muscle, pancreas, and cartilage. The elephant shark Pth1(1-34) and Pthrp(1-34) peptides were able to stimulate cAMP accumulation in mammalian UMR106.01 cells. However, Pth2(1-34) peptide did not show such PTH-like biologic activity. The presence of Pth and Pthrp genes in the elephant shark indicates that these genes played fundamental roles before their recruitment to bone development in bony jawed vertebrates.

Gestational morphogenesis of the uterine epithelium of the gummy shark (Mustelus antarcticus).

Developing embryos of the non-placental, viviparous gummy shark (Mustelus antarcticus) are supplied with yolk from external and internal yolk sacs throughout the initial stages of gestation. Yolk supplies are exhausted by the 7th month of an 11-12 month gestation. During embryonic development, there is an approximate 800% gain in dry mass. These factors suggest nutrients are transferred from the mother to the young. The results of the present study provide some insights into how this is occurring. The uteri are paired and both are functional. Using both light and transmission electron microscopy, regions of the uterus were examined throughout maturation and gestation. The layers of the uterine wall throughout the entire length are similar to the uteri of other chondrichthyans previously examined. The uterine epithelium of the body of the uterus is smooth contoured, does not form villi, and undergoes cyclical and secretory changes throughout maturity and gestation. In immature uteri, the epithelium is simple columnar with minimal periodic acid-Schiff-positive and Alcian blue-positive secretory vesicles. In mature uteri, the epithelium is highly stratified with cuboidal cells containing numerous Alcian blue-positive and periodic acid-Schiff-positive vesicles. With pregnancy, prominent changes include a reduction in the number of cell layers, a reduction in cell size, a reduction in the connective tissue intervening between epithelium and blood vessel endothelium, and an increase in blood vessel number and size, so that at term, the uterine compartment consists of a single layer of squamous cells immediately underlain by sinusoidal-like blood vessels. These features along with a small number of secretory vesicles, dilated intercellular spaces, and clear transport vesicles suggest the transepithelial transfer of water and minerals from the maternal to embryonic environment, supplemented by minimal uterine secretions. This is defined as minimal histotrophy and this article represents the first detailed description of this reproductive mode.

Microscopic organization of the sperm storage tubules in the oviducal gland of the female gummy shark (Mustelus antarcticus), with observations on sperm distribution and storage.

Oviducal gland morphology, the microscopic organization of the terminal zone, and sperm storage were described in the female gummy shark (Mustelus antarcticus). Mustelus antarcticus is a nonplacental viviparous hound shark, which displays minimal histotrophy during embryonic development. The animals examined represented all stages of maturity and gestation. The oviducal gland was found to have the same fundamental zonation as in most chondrichthyans. Using recent terminology, the oviducal gland of chondrichthyans has an anterior club zone, followed by a papillary zone, both of which produce jelly that surrounds the egg, a baffle zone that elaborates the tertiary egg envelope and a terminal zone, where sperm storage occurs. Each zone is composed of simple tubular glands that connect to transverse grooves, which extend the full width of the gland. The exception is the terminal zone, which does not have transverse grooves but consists of individual tubules. The microscopic organization and histochemical nature of the zones display similar patterns to those of other chondrichthyan genera. Tubules of the terminal zone contain four types of cell: ciliated cells, alcian blue-positive secretory cells, periodic acid-Schiff and alcian blue-negative secretory cells, and secretory columnar cells. These tubules end in recesses, the sperm storage tubules, which extend beyond the periphery of the baffle zone. Sperm were stored in the sperm storage tubules of all maturing and mature animals examined. Of note is the observation of stored sperm in an animal 1 year prior to first ovulation. Sperm were also observed throughout the uterine sphincter, body of the uterus, isthmus, and oviduct of maturing and mature animals, and in the uterine sphincter of an immature animal. These sperm represent immediately postcopulation aggregations of sperm and sperm in the process of migrating to the site of storage or to the site of fertilization.

Ultrastructure of sperm storage and male genital ducts in a male holocephalan, the elephant fish, Callorhynchus milii.

In chondrichthyes, the process of spermatogenesis produces a spermatocyst composed of Sertoli cells and their cohort of associated spermatozoa linearly arrayed and embedded in the apical end of the Sertoli cell. The extratesticular ducts consist of paired epididymis, ductus deferens, isthmus, and seminal vesicles. In transit through the ducts, spermatozoa undergo modification by secretions of the extratesticular ducts and associated glands, i.e., Leydig gland. In mature animals, the anterior portion of the mesonephros is specialized as the Leydig gland that connects to both the epididymis and ductus deferens and elaborates seminal fluid and matrix that contribute to the spermatophore or spermatozeugmata, depending on the species. Leydig gland epithelium is simple columnar with secretory and ciliated cells. Secretory cells have periodic acid-Schiff positive (PAS+) apical secretory granules. In the holocephalan elephant fish, Callorhynchus milii, sperm and Sertoli cell fragments enter the first major extratesticular duct, the epididymis. In the epididymis, spermatozoa are initially present as individual sperm but soon begin to laterally associate so that they are aligned head-to-head. The epididymis is a highly convoluted tubule with a small bore lumen and an epithelium consisting of scant ciliated and relatively more secretory cells. Secretory activity of both the Leydig gland and epididymis contribute to the nascent spermatophores, which begin as gel-like aggregations of secretory product in which sperm are embedded. Fully formed spermatophores occur in the ductus. The simple columnar epithelium has both ciliated and secretory cells. The spermatophore is regionalized into a PAS+ and Alcian-blue-positive (AB+) cortex and a distinctively PAS+, and less AB+ medulla. Laterally aligned sperm occupy the medulla and are surrounded by a clear zone separate from the spermatophore matrix. Grossly, the seminal vesicles are characterized by spiral partitions of the epithelium that project into the lumen, much like a spiral staircase. Each partition is staggered with respect to adjacent partitions while the aperture is eccentric. The generally nonsecretory epithelium of the seminal vesicle is simple columnar with both microvillar and ciliated cells.