Survival rates and physiological recovery responses in the lesser-spotted catshark (Scyliorhinus canicula) after bottom-trawling

Comparative Biochemistry and Physiology, Part A 233 (2019) 1–9

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Survival rates and physiological recovery responses in the lesser-spotted catshark (Scyliorhinus canicula) after bottom-trawling C. Barragán-Méndeza,1, I. Ruiz-Jaraboa,

T

⁎,1

, J. Fuentesb, J.M. Manceraa, I. Sobrinoc

a Department of Biology, Faculty of Marine and Environmental Sciences, University of Cádiz, International Campus of Excellence of the Sea (CEI-MAR), Av. República Saharaui s/n, E-11510, Puerto Real, Cádiz, Spain b Centre for Marine Sciences (CCMar), University of Algarve, Gambelas Campus, 8005-139 Faro, Portugal c Spanish Institute of Oceanography (IEO), Oceanographic Centre of Cádiz, Puerto Pesquero, Muelle de Levante, s/n, PO Box 2609, E-11006 Cádiz, Spain

A R T I C LE I N FO

A B S T R A C T

Keywords: Bottom-trawling Capture stress Discard survival Elasmobranchs Fisheries physiological recovery Scyliorhinus canicula

In 2019, Europe will adopt a no-discards policy in fisheries. This entails the landing of captured species unless strong evidence is provided supporting their survival and recovery after fishing. Thus, research on this topic is gaining momentum. Bottom-trawling, as a non-selective fishing method, is characterized by a high proportion of discards including vulnerable key species, such as demersal sharks. Their survival may also depend on capture depth. By paralleling onboard and laboratory experiments with the small-spotted catshark, Scyliorhinus canicula, we offer a robust experimental design to assess the survival of discarded sharks. Catsharks were captured by bottom-trawling at two depths (shallow ~89 m and deep ~479 m). Blood samples were collected following trawl capture and analyzed for stress biomarkers (lactate, osmolality, phosphate, urea). During recovery in onboard tanks, behavior was video-recorded and fish were re-sampled after 24 h. A second experiment was conducted in laboratory facilities to simulate air-exposure after trawling and to analyze the physiological recovery. Our results showed that 95.7% of the animals survived 24 h after trawling. We confirmed that trawling elicited acute stress responses in catshark but that they managed to recover. This was demonstrated by lactate concentrations that were 2.6 mM upon capture, but recovered to assumed baselines after 24 h (0.2 mM). Non-invasive video monitoring revealed behavioral differences with depth, whereby those captured at 89 m depth required longer to recover than those captured at 479 m depth. Implementation of standardized survival studies by fishery managers can benefit from holistic physiological approaches, such as the one proposed here.

1. Introduction European Union policy regarding fisheries aims at a progressive elimination of discards (EU Delegated Regulation N° 2015/2439). Framed under the new Common Fisheries Policy and according to the Article 15 of the Regulation (EU) N° 1380/2013, discards should be introduced as landings by the year 2019. This compulsory landing obligation will impact all species regulated by quotas or minimum sizes. However, as reported in the regulation, species with solid scientific evidence indicating high survival rates and physiological recovery, could be released into the sea and not landed as discards. The terms “solid” and “high” are under discussion by the International Council for the Exploration of the Sea (ICES), managers and international stakeholders, but refer to those studies including the best scientific approaches available (Lewin et al., 2018; Radford et al., 2018; Uhlmann

et al., 2016; van der Reijden et al., 2017; WKMEDS, 2014). Knowledge of post-release survival of discards holds major implications for sustainability of fisheries and is essential for resource management. It should be mentioned here that in the southern Spain, an exemption to the landing obligation was approved for the hookline voracera in the Strait of Gibraltar (EU Commission Delegated Regulation 6794/2018) based on a study evaluating the physiological recovery of captured blackspot seabream. Discards in fisheries are those parts of a catch that are not retained onboard, but returned to the sea regardless of whether they are dead or alive (Catchpole et al., 2014). According to the Food and Agriculture Organization of the United Nations (FAO), Northeastern Atlantic European waters (FAO area 27), generate the impressive discard amount of 1.4 million tons per year (Kelleher, 2005). Most of these are generated by demersal bottom-trawling fisheries, which are non-selective fishing

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Corresponding author at: Department of Biology, Faculty of Marine and Environmental Sciences, University of Cádiz, International Campus of Excellence of the Sea (CEI-MAR), Av. República Saharaui s/n, E-11510, Puerto Real, Cádiz, Spain. E-mail address: [email protected] (I. Ruiz-Jarabo). 1 These authors have contributed equally to this work https://doi.org/10.1016/j.cbpa.2019.03.016 Received 10 October 2018; Received in revised form 16 March 2019; Accepted 18 March 2019 Available online 21 March 2019 1095-6433/ © 2019 Elsevier Inc. All rights reserved.

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methods. Specifically, demersal fisheries in the Southwestern Atlantic coast of Europe record discards ranging from 43% to 90% of total captures (Kelleher, 2005). In this geographical area, discards are particularly strongly represented by demersal shark species (Coelho and Erzini, 2008). This group is more sensitive to fishing activities than other fish due to their life-history strategies (sexual maturation requiring years, and few offspring), and due to over-exploitation it would require a few decades to recover present populations (Ragonese et al., 2013; Stevens et al., 2000). However, the number of studies focused on post-capture survival for this taxonomic group are limited (Ellis et al., 2016; Wilson et al., 2014). Survival of discards is influenced by many factors such as the species, size of the animal, fishing gear, fight/capture and air-exposure duration (Cook et al., 2018; Methling et al., 2017; Wilson et al., 2014). Survival of captured fish has been related to animal vitality, injuries or impairment of their reflexes (Morfin et al., 2017; Olsen et al., 2012; Uhlmann et al., 2016), and some approaches were conducted including post-capture video recording of animals maintained in enclosures lowered to the seafloor (Talwar et al., 2017). However, immediate survival rate only provides a crude evaluation of the injury experienced by captured fish, as they have been subjected to acute physiological stress that may affect their behavior, reproduction, or eventually lead to death (Wedemeyer et al., 1990) even days after being released (Gallagher et al., 2014). Moreover, stress and injuries due to capture processes can temporarily alter behavior in released fish, increasing risk of predation (Raby et al., 2013). Considering all the above, it becomes clear that fisheries constitute a source of acute stress in fish (Frick et al., 2010a; Olsen et al., 2012; Skomal and Mandelman, 2012). The established primary stress responses in teleost fish include the release of catecholamines and corticosteroid hormones (Reid et al., 1998; Wendelaar Bonga, 1997). 1αhydroxycorticosterone is the described corticosteroid hormone in elasmobranch fish (Anderson, 2012; Hazon and Balment, 1998), although the relationship of this hormone in the stress response is not clear in sharks. In turn, in teleost fish these hormones elicit secondary responses ranging from changes in the cardiorespiratory system to the release of energy metabolites into the blood (Mommsen et al., 1999; Schreck and Tort, 2016), including other relevant secondary responses, such as hydromineral imbalance related to changes in plasma osmolality and electrolytes (Deck et al., 2016; Frick et al., 2010a) that can lead to death if sustained (Dapp et al., 2016) without recovery of homeostatic levels. Other responses described in sharks are related to blood pH depression, driven in part by lactic-acidosis and changes in plasma phosphate levels (Lambert et al., 2018; Skomal and Mandelman, 2012). Physiological stress responses in sharks depend on the species, capture gear and capture duration (Frick et al., 2010b), and include hyperglycemia, acidemia and profound disturbances to ionic, osmotic and fluid volume homeostasis (Skomal and Mandelman, 2012). Most studies to date include post-capture analysis of the onset of anaerobic glycolysis coupled to the production of lactate (Gallagher et al., 2014; Skomal and Mandelman, 2012), or other metabolic stress indicators (Guida et al., 2016). Capture-induced exhaustion may lead to weakened condition with reduced chance of survival if released. Therefore, validation of recovery of these animals becomes critical. Tracking released sharks is typically employed in long-term post release survival estimations (Gallagher et al., 2014; Moyes et al., 2006). However, this procedure is expensive and of limited value for small species, and the need for alternative approaches for this type of research is relevant. Our main objective was to evaluate the survival, as well as the physiological and behavioral recovery capacity of a commonly discarded shark species in Europe, the small-spotted catshark (Scyliorhinus canicula), after an episode of bottom trawling. This is a small demersal species inhabiting Atlantic waters from Norway to Senegal and the Mediterranean Sea found in depths ranging from 10 m to 780 m. Since air exposure has been described as one of the most important factors in

the stress response in small-spotted catshark (Murray et al., 2015), we conducted a first experiment aiming to evaluate the survival rates after different air exposure times (in a range covering 0 h to 3 h air-exposure, mimicking commercial procedures) aboard a fishing vessel. Capture depth of fishing operations can lead to different physiological responses in sharks (Treberg and Speers-Roesch, 2016); therefore, a second objective of this study was to evaluate possible differences depending on this factor. Thus, a second experiment was conducted aboard a vessel, where animals captured at two depths (~89 m and ~479 m) were allowed to recover for 24 h in flow-through water tanks. In this experiment, stress biomarkers in blood (lactate, osmolality, urea and phosphate) were analyzed altogether with the characterization of the behavioral recovery responses after capture by means of non-invasive underwater video recording. A third experiment was conducted in laboratory facilities. The objective of this experiment was to characterize the physiological responses in discarded catshark exposed to the same air stressors as those captured by bottom-trawling, in order to better understand the recovery responses in this species. The information obtained here can be of value for fisheries management in Europe, providing a better understanding of stress physiology. 2. Material and methods 2.1. Ethics statement This study was performed aboard a research bottom-trawling vessel (Experiments 1 and 2, air-exposure and depth survival and recovery experiments, respectively), and in a research laboratory in Spain (Experiment 3, where an acute-stress situation, such as that experienced by catsharks aboard a commercial fishing vessel, was mimicked) in accordance with the Guidelines of the European Union (2010/63/UE) and the Spanish legislation (RD 1201/2005 and law 32/2007) for the use of laboratory animals. According to RD1201/2005, the experimental procedures were reviewed and approved by the University of Cádiz's (UCA) Ethics Committee. 2.2. Geographical location, vessel and tow characteristics Animals were collected from hauls carried out during a bottomtrawl survey off the Southwestern Atlantic Spanish waters (Fig. 1). Hauls were conducted between 59 m and 550 m depth during the ARSA1116 cruise, onboard the O/V “Miguel Oliver” (length: 70 m; engine power: 2 × 1000 kw) in November 2016 following described international procedures (Anon, 2015). The sampling gear, which included a 44/60 BAKA trawl, was towed at 3 knots for 1 h along a specific isobath (at the same depth) for each haul. The arrival and departure of the net at the bottom, as well as its horizontal and vertical openings (on average, 20.8 m and 1.9 m, respectively) were measured using a MARPOL system, ensuring that all trawls were conducted under similar gear conditions. The start and finish positions of each trawl were recorded using the Global Positioning System (GPS). A conductivity-temperature-pressure instrument (CTD) was placed in the net, while another probe registered surface water continuously, so that temperature and salinity at the bottom (13.01–17.03 °C and 35.92–36.80 psu salinity) and at the surface (18.81–21.19 °C and 36.44–36.64 psu salinity) were recorded during these processes. 2.3. Onboard air-exposure experiment A first experiment was performed to evaluate survival rates of smallspotted catshark captured by bottom-trawling and exposed to air. This experiment was conducted aboard a fishing vessel and catsharks were discarded on the fishing deck and allowed to asphyxiate. These animals could be retained on the fishing deck under commercial fishing procedures for up to 1.25 h in northern Spain (Rodriguez-Cabello et al., 2001), while this time is even longer (up to 3–4 h) in the Gulf of Cádiz 2

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Fig. 1. Sampled area off the Southwestern Atlantic Spanish waters. Triangles indicate shallow water hauls, while squares indicate deep water hauls.

the same position (still belly-up) after 5 h, or that had ceased to breathe (no opercula or mouth movements), were considered dead (moreover, their bodies became rigid due to rigor mortis). All the animals (dead and alive) were released into the wild (as close to the area of capture as possible) after this experiment based on common procedures aboard oceanographic vessels in Spain.

(southern Spain/Portugal) before being discarded into the ocean (personal observation). Thus, a range of air exposure times (1, 1.5, 2, 2.5 and 3 h) were tested. Four tows were conducted in four days between 9.00 UTC and 12.00 UTC covering a range from 59 to 550 m depth. Once the catches arrived at the fishing deck, they were introduced onto a pit connected to a lower deck, where the catch was sorted by the crew, simulating commercial fishing activities. Following this treatment, a total of 130 adult catsharks of both sexes (71 males and 59 females) were randomly kept, to provide 30 to 40 animals from each tow (317 ± 8 g body weight and 45.7 ± 0.4 cm total length, mean ± SEM). Catsharks were measured in length and weight, and individually labelled with a rubber band placed on the caudal fin. Groups of 10 catsharks were retained outside the water in the same baskets used to sort the fish during commercial activities for established periods ranging from 1 to 3 h. Following the air exposure event the animals were placed belly-up in the bottom of a 350-L tank (0.72 m2) with constant flow of seawater (collected from the surface of the sea during navigation) with additional air supply. In a previous test conducted two days before this study, in the same vessel and geographic area (unpublished data), we had seen (in fish held for 36 h in water tanks aboard) that mortality occurs in this species captured by bottomtrawling before the first 5 h after capture. In this previous experiment, breathing rates (mouth and opercula movements) were monitored every 10 min during the first hours, and every hour after that in 30 catsharks captured at different depths and exposed to air for 15 min to 2 h (after triage activities). If animals were not breathing, we touched their caudal fin looking for body movements. Animals were considered dead if they ceased to breathe and move their bodies. Thus, mortality was monitored constantly during the first 5 h. Animals that remained in

2.4. Onboard depth and recovery experiment A second experiment was conducted to evaluate the physiological effects of bottom-trawling and the recovery response of catsharks onboard. Seven tows were conducted (three at 89 ± 27 m and four at 479 ± 26 m, mean ± SEM), all performed at 07.00 UTC in order to avoid putative effects of circadian rhythm variations in the animals. Time of net rolling from the bottom to the surface was 13 ± 1 min and 18 ± 0 min for the shallow and deep trawls, respectively. Once the catches arrived at the fishing deck (between 08.30 and 09.00 UTC), they were introduced onto a pit connected to a lower deck, as described above. Samples of 10 adult catsharks of both sexes were randomly kept from each tow, considering a total of 70 animals (43 males and 27 females) from seven tows (431 ± 12 g body weight and 49.8 ± 0.4 cm total length). Catsharks were measured in length and weight with a wet tissue covering their eyes (this appears to calm the animals), individually labelled with a rubber band placed on the caudal fin and blood was collected by caudal puncture with 1-mL heparinized syringes with 21G needles, as described before (Ruiz-Jarabo et al., 2018). Plasma was obtained after centrifugation of whole blood (10,000 g, 4 min; Centrifuge 5810R, Eppendorf) and immediately frozen at −20 °C (by employing a refrigerated mixture that immediately freezes the 3

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samples). These samples were maintained at −20 °C for < 9 days, transferred to the Department of Biology (University of Cádiz, Spain) and then maintained at −80 °C. All 10 animals from each tow were then placed into a 350-L tank (0.72 m2) with their backs facing the bottom, with constant flow of seawater (collected from the surface of the sea during navigation) with additional air supply. Recovery behavior was video-recorded (see below). The total time of air exposure registered for each animal averaged 18 min (17:35 ± 8:41 min), values in good agreement with those reported for this species in a commercial fishery (Revill et al., 2005). There were no significant differences in air exposure time between trawls. After 24 h in the tank, animals were captured by hand, their eyes covered with a wet tissue and blood samples were collected for each catshark in < 1 min in order to avoid physiological deviations due to post-capture stress (Lawrence et al., 2018) and then released into the wild. Animals were not fed during captivity and were not anesthetized. Anesthesia has been shown to affect stress-related blood variables in sharks (Frick et al., 2009), and the use of anesthesia could introduce an additional bias to the data and be fatal for the animals immediately after the trawling procedure. During the experiment, salinity and temperature in the tanks varied between 36.44 and 36.64 psu and 18.81–21.19 °C, respectively. Thus, while salinity was maintained the same as that of the natural environment where these animals were captured, temperature aboard was 4 °C above that of the natural environment at the bottom.

(November; latitude 36° 31′ 34″N) and temperature (ambient temperature of approximately 19 °C) and randomly allocated into 6 tanks of 400-L with a surface area of 0.72 m2, covered by a fine-mesh tissue to shade the aquarium and acclimated for 17 days. All maintenance procedures were previously described in a paralleled study (BarragánMéndez et al., 2018). To evaluate air exposure effects, three tanks were selected as undisturbed controls, and catsharks from the other three tanks were exposed to air. For this procedure, animals were captured by hand, as this species remains resting in the bottom of the tank during daylight, and can be easily captured without the employment of nets. While no major disturbance is caused to catsharks in the same tank, the captured animals are placed in dry tanks for 18 min. This is the minimum time this species is exposed to air when captured by bottom-trawling in the Gulf of Cádiz (personal observation) and other areas (Rodriguez-Cabello et al., 2001), and allowed to recover in water tanks thereafter. Blood samples were collected at 0 h and 24 h after air exposure with 2–3 animals from each tank (in triplicate, n = 7–8 per group) to parallel “onboard” experiments. Moreover, an additional sampling was conducted after 5 h to evaluate recovery responses according to previous studies in S. canicula and S. stellaris (Murray et al., 2015; Piiper et al., 1972). The first sampling points for both experimental groups were established at 08.30–09.30 UTC, as performed in the vessel. Sampling in the laboratory was performed following the procedure used aboard the vessel using the same method as described previously in Section 2.4. Animals were then anesthetized in 0.1% v/v 2-phenoxyethanol (P1126, Sigma-Aldrich). Euthanasia was performed by severing the head with a sharp knife. All procedures lasted less than four minutes per tank. Weight and length of the animals were then recorded.

2.5. Video analysis onboard Recovery responses after trawling and air exposure during the onboard experiment were video-tracked for 1.5 h after placing the fish into the tanks. Two cameras (Full HD 1080p) were positioned for each tank: one underwater, held at 8 cm from the bottom, and the other above the water surface. Due to the lack of references describing shark recovery responses, after viewing all videos we decided to categorize the recordings (see Supplementary Video S1) as follows: I) Equilibrium recovery: the first movement of the fish after being placed into the tank, allowing the animal to lie down with its belly on the bottom (as they were initially placed with their backs touching the bottom of the tank); II) “Shelter” search: although there were no shelter areas in the tanks, we describe the second recovery response as the moment when catsharks conduct a few body movements placing their snout close to the wall or other animals (we considered that they were looking for shelter, and stopped moving when their snouts were in touch with something), and then they remain still; III) Tank bottom recognition: fish swim along the bottom of the tank for > 3 s, but always with some part of their bodies in touch with it; and IV) Water column swimming behavior: fish completely abandon the bottom to swim upwards in the water column, exploring the tank. The time it took for each fish to reach each response was recorded. To prevent potential observation bias, videos were independently analyzed three times each by two trained observers, and averages for individual catshark responses calculated. All time measurements were recorded with an accuracy of 1 s.

2.7. Plasma variables Osmolality was measured in 20 μL samples with a Vapro 5520 Osmometer (Wescor, USA). Plasma lactate, phosphate and urea levels were measured using commercial kits from Spinreact (Lactate ref. 1,001,330; Phosphate ref. 1,001,155; Urea ref. 1,001,323, Spinreact SA, Sant Esteve de Bas, Spain) adapted for 96-well microplates. Previous studies analyzed these and other plasma variables in elasmobranch species following similar procedures (Lambert et al., 2018; Martins et al., 2018; Talwar et al., 2017). All assays were performed using a BioTek PowerWave 340 Microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA) using KCjunior Data Analysis Software for Microsoft Windows XP, unless otherwise stated. 2.8. Statistics Normality and homogeneity of variances were analyzed using Shapiro-Wilk's test and Levene's test, respectively. Correlations between time of air exposure and measured parameters were analyzed using linear regression in the recovery experiment onboard, but as no relationship was described for them (p > 0.05), time of air exposure was not included as a factor of variance in further analyses. Differences between groups for each behavioral response were tested using a nested two-way ANOVA with depth (shallow and deep) and trawl (as the nested variable) as the factors of variance. Differences between groups for plasma variables in the onboard experience were tested using twoway repeated-measures ANOVA with depth (shallow and deep) and time (repeated measures at 0 and 24 h) as the factors of variance. A two-way ANOVA was performed for the air-exposure laboratory experiment, with group (control and air exposure) and time (0, 5 and 24 h) as the factors of variance. When necessary, data were logarithmically transformed to fulfill the requirements of ANOVA. When ANOVA yielded significant differences, Tukey's post-hoc test was used to identify significantly different groups. Student's t-test was employed to evaluate differences in the video responses between depths. Statistical significance was accepted at p < 0.05. All the results are

2.6. Laboratory air-exposure experiment An additional experiment was conducted in laboratory-controlled conditions. We aimed at testing the physiological effects of air exposure mimicking capture conditions after bottom trawling. Under laboratory conditions it was possible to better control environmental variables and to avoid interference due to onboard handling. Forty-five (45) smallspotted catshark adults of both sexes (380.6 ± 12.0 g body weight and 50.7 ± 0.5 cm total length; 26 males and 19 females) were captured by bottom trawling in shallow waters (between 50 and 100 m depth) as described above and maintained in the fish husbandry facility of the Faculty of Marine and Environmental Sciences (Puerto Real, Cádiz, Spain) until the beginning of the experiment. Fish were transferred to a flow-through seawater (38 psu) system under natural photoperiod 4

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Fig. 3. Behavioral variables of small-spotted catshark, S. canicula, after bottom trawling. Fish captured at two depths were studied: one trawled in shallow waters (~89 m, black bars) and the other in deep waters (~479 m, white bars). Responses described are: i) body balance recovery (balance), ii) search for shelter (shelter), iii) exploration of the tank floor (explore), and iv) free swimming in the water column (swim). Data are expressed as mean ± SEM (n = 30). Asterisks (*) indicate significant differences between capture depth for the same response (p < .05, two-way nested ANOVA).

Fig. 2. Survival rates (%) of small-spotted catshark, S. canicula, captured by bottom trawling and exposed to different air exposure times. The lethal time 50 (LT50) is derived from the time-response curve (sigmoidal dose-response equation) and represents the time at which 50% of the catsharks died. Data are expressed as mean ± SEM. Every sampling point is constituted by at least three trawls (each trawl constitutes a single sample), with 10 fish per trawl, except those of 2.5 and 3 h of air exposure, which have only one trawl per time unit.

Table 1 P-values from the two-way repeated-measures ANOVA analysis of plasma lactate, osmolality, phosphate and urea concentrations in small-spotted catshark, S. canicula, after bottom trawling. Fish captured at two depths were studied: one trawled in shallow waters (~89 m, n = 30) and the other in deep waters (~479 m, n = 40). Samplings were performed at time 0 h (prior to the introduction into recovery tanks) and 24 h later.

given as mean ± SEM, unless stated otherwise. All tests were performed using Statistica 7 software for Windows.

3. Results In the first and second experiments (aboard a fishing vessel), smallspotted catsharks captured by bottom trawling in the Gulf of Cádiz (SW Spain) showed the same mortality rates when exposed to air for 18 min or for 1 h (95.7 ± 2.0 and 95.2 ± 3.0, respectively). The survival rate diminishes thereafter reaching 0.0% survival after 3 h of air exposure. When exposed to air under the conditions described in this study, 50% of the S. canicula individuals died within 1 h and 42 min, constituting the lethal time 50 (LT50) (Fig. 2). In the second experiment (effect of depth of capture and further recovery), mortality was observed in two out of 30 animals captured in shallow waters (the reasons were unknown), while one fish out of 40 died from those captured in deeper waters (it was severely injured by trawling, with great wounds covering its head and through the gills).

Variable

Depth

Time

Depth x Time

Lactate (mmol L−1) Osmolality (mOsm Kg−1) PO42− (mmol L−1) Urea (mmol L−1)

0.94 < 0.05 0.47 < 0.05

< < < <

0.81 0.73 < 0.02 0.84

0.00001 0.00001 0.00001 0.01

3.2. Metabolic responses P-values of the plasma variables analyzed in the onboard experiment are shown in Table 1, while those of the experiment conducted in ground facilities are shown in Table 3. Lactate, as one of the most representative secondary stress biomarkers in sharks after capture, was 10-fold higher in plasma of all fish (captured at all depths, without significant differences between them, p = 0.94) at 0 h when compared to fish sampled at 24 h (2.6 ± 0.2 mM at 0 h, and 0.2 ± 0.0 mM at 24 h; p < 0.005; Fig. 4A) in the onboard study. Similar concentrations were also described in those fish exposed to air in the experiment conducted in the laboratory (2.8 ± 0.2 mM at 0 h, and 0.2 ± 0.0 mM after 24 h recovery; p < 0.005; Fig. 4B). Furthermore, plasma lactate decreased gradually in the air-exposed group compared to the untouched-control group in the experiment conducted in the laboratory (with values of 1.16 ± 0.25 mM 5 h after air exposure; Fig. 4B), thus reaching similar levels in both groups 24 h after air exposure (p = 0.64). All fish sampled in both experiments (onboard a fishing vessel and in ground facilities), presented similar lactate levels 24 h after the challenge either by trawling or air-exposure (p = 0.39, Student's t-test).

3.1. Video analysis Behavioral recovery responses recorded for 1.5 h after introducing the animals in the tanks (Fig. 3) indicated that catshark captured in shallower waters (~89 m depth) recover faster than those captured in deep waters (~479 m depth). The results from the two-way nested ANOVA revealed that trawl, as the nested variable, had no significant effects on the depth of capture (p > 0.05). Shallow-inhabiting fish placed their bellies on the bottom of the tanks (equilibrium recovery) in 9 ± 3 s, while those living in deeper waters did this in 30 ± 5 s. Time required to look for a shelter area was three times higher for deep-living catsharks (01:06 ± 00:15 min against 03:24 ± 00:41 min for shallowand deep-living catsharks, respectively). However, exploring the bottom of the tank showed no statistical differences in time for both groups, thus requiring between 08:58 ± 01:14 min for those shallowinhabitants to 13:00 ± 02:14 min for the deeper group. Free swimming in the water column took 19:30 ± 03:43 min on average for all fish in the first group; while only 20 fish out of 30 (67%) presented this behavior in deeper trawl catsharks (time response 43:17 ± 06:07 min), this response being statistically different between the experimental groups (p < 0.05).

3.3. Osmoregulatory consequences Osmoregulation was disturbed in catsharks in response to the stress imposed by trawling and/or air exposure. Plasma osmolality was statistically higher just after trawling, independently of the depth of capture (843 ± 10 and 864 ± 7 mOsm kg−1 in catsharks captured in 5

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Fig. 4. Plasma lactate levels in small-spotted catshark, S. canicula, after bottom trawling (A, onboard) and an air exposure experiment performed under laboratory conditions (B). Data are expressed as mean ± SEM. The onboard figure shows black bars for the animals at time 0 h, just after bottom trawling, and white bars indicate 24 h of recovery in tanks. Asterisks (*) in the onboard figures indicate significant differences between 0 and 24 h within each depth group (p < .05, two-way repeated-measures ANOVA followed by a Tukey's post hoc test, n = 30–40). The figure showing the laboratory condition experiments includes black bars for the group exposed to air for 18 min, and white bars for the control-unexposed group. Different uppercase letters in the laboratory condition experiment figure indicate significant differences with time for the air-exposed group, while asterisks (*) indicate significant differences between both groups at each time (p < .05, two-way ANOVA followed by a Tukey's post hoc test, n = 7–8).

Fig. 5. Plasma osmolality values in small-spotted catshark, S. canicula, after bottom trawling (A, onboard) and an air exposure experiment performed under laboratory conditions (B). Data are expressed as mean ± SEM. Further details as in legend of Fig. 4.

catsharks captured in shallow and deep waters, respectively, with no statistical differences between them, p = 0.30), reaching similar levels in both groups when recovered 24 h later (3.5 ± 0.2 and 4.0 ± 0.3 mM 1 in catsharks captured in shallow and deep waters, respectively, with no statistical differences between them, p = 0.88). Levels of plasma phosphate in the laboratory experiment (Table 4) showed an increase in the air-exposed group 5 h after the stress (2.4 ± 0.2 and 3.4 ± 0.4 mM in the control and air-exposed groups, respectively; p < 0.05). Urea, as an important osmolite in sharks, showed no differences in concentration as a result of time in the onboard experience (although a higher 5% was observed between 0 and 24 h for both groups), while differences were observed with depth of capture (399 ± 8 and

shallow and deep waters, respectively, with no statistical differences between them, p = 0.42), and decreased after 24 h recovery (p < 0.05) in onboard tanks (790 ± 12 and 818 ± 10 mOsm kg−1 in catsharks captured in shallow and deep waters, respectively, with no statistical differences between them, p = 0.19; Fig. 5A). The experiment performed in the laboratory confirmed this osmotic imbalance immediately after air exposure (showing plasma osmolality values of 923 ± 5 and 968 ± 8 mOsm kg−1 in the control and air-exposed groups, respectively, at time 0 h; p < 0.05; Fig. 5B), recovering control values within the first 5 h (p = 0.98). Plasma levels of phosphate and urea in the onboard experience are shown in Table 2. Higher phosphate levels (p < 0.05) were described in all the animals just after trawling (6.3 ± 0.5 and 5.3 ± 0.3 mM 1 in

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4.1. Behavioral recovery responses are depth-dependent

Table 2 Plasma phosphate and urea concentrations (mmol L−1) in small-spotted catshark, S. canicula, after bottom trawling. Fish captured at two depths were studied: one trawled in shallow waters (~89 m, n = 30) and the other in deep waters (~479 m, n = 40). Samplings were performed at time 0 h (prior to the introduction into recovery tanks) and 24 h later. Data are shown as mean ± SEM. Different upper- and lowercase letters indicate significant differences between shallow and deep groups at time 0 h and 24 h, respectively. Asterisks (*) indicate significant differences within each group between 0 and 24 h (p < .05, two-way repeated-measures ANOVA followed by a Tukey's post hoc test). Variable

2−

Shallow

−1

(mmol L ) PO4 Urea (mmol L−1)

By using a non-invasive technique, such as video analysis, we are able to infer physiological and behavioral consequences of capture stress through examination of swimming responses. Video-recording is an effective technique to study fish behavior in lab-maintained sharks (Pistevos et al., 2015) and teleosts (de Vrieze et al., 2014); however, to the best of our knowledge, this is the first time this approach has been used to analyze recovery responses onboard in a shark species after being captured. In our study, catsharks from deeper waters require longer times than fish caught in shallower waters to achieve behavioral recovery responses associated to a trawling event (Fig. 3). This difference indicates that both groups of sharks have undergone dissimilar capture processes, such as time of net rolling (13 min and 18 min for the shallow and deep trawls, respectively), decompression side-effects/ barotrauma (~89 and ~479 m up to the water surface), buffeting and compression received in the net due to the catch biomass. This may cause other unknown factors; or different physiological responses exist between both groups that may be related to sub-populations inhabiting different depths. The latter hypothesis should be cautiously addressed and future studies are required to confirm or reject it. Notwithstanding the time taken for this physiological recovery depends on the depth of capture, 95.7% of all the animals included in the second experiment managed to survive after 24 h, showing consistency with results from previous studies (Revill et al., 2005).

Deep

0h

24 h

0h

24 h

6.3 ± 0.4 415 ± 5 A

3.6 ± 0.2 * 399 ± 8 a

5.2 ± 0.3 384 ± 5 B

4.0 ± 0.3 * 366 ± 6 b

Table 3 P-values from the two-way repeated-measures ANOVA analysis of plasma lactate, osmolality, phosphate and urea concentrations in small-spotted catshark, S. canicula, after 18 min of air exposure in ground facilities. Variable −1

Lactate (mmol L ) Osmolality (mOsm Kg−1) PO42− (mmol L−1) Urea (mmol L−1)

Depth

Time

Depth x Time

< 0.00001 0.051 < 0.05 < 0.001

< < < <

< 0.00001 < 0.001 0.16 0.58

0.001 0.01 0.00001 0.05

4.2. Energy requirements Our measurements of plasma lactate as a well-documented consequence of stress in elasmobranchs, including S. canicula, due to enhanced anaerobic glycolysis in muscle and other tissues (BarragánMéndez et al., 2018; Skomal, 2007), are in line with the range described in previous studies (3–15 mM) performed in other shark species after capture (Cliff and Thurman, 1984; Gallagher et al., 2014; Mandelman and Farrington, 2007; Moyes et al., 2006), returning to basal/low levels (< 1 mM) in captivity or after a period of recovery (Cliff and Thurman, 1984; Gallagher et al., 2014), as confirmed in this study. A major issue in this field is to evaluate basal conditions of plasma lactate in unstressed wild animals. However, conducting experiments in controlled ground facilities may be a useful approach to establishing basal energy metabolism conditions in sharks and other chondrichthyans (Frick et al., 2010b, 2012; Martins et al., 2018), potentially providing the best option for understanding baseline stress in wild animals. Moreover, the duration of the capture process (duration of the stressor) significantly affected blood lactate levels in different shark species (Gallagher et al., 2014) due to anaerobic catabolism of carbohydrates. Thus, anaerobic metabolism results in the formation of lactic acid in the blood, and lactate is a valuable metric in determining the aerobic status of an organism (reviewed in Lawrence et al., 2018). Given these facts, as plasma lactate concentrations in the experiments conducted in the present study are well matched after the stressful challenges imposed, we could state that trawling capture and air-exposure induced stress responses in S. canicula, as was described before for the Atlantic stingray, Hypsanus sabinus (Lambert et al., 2018). The strength of our results, as we have combined a field experiment supported by a laboratory-controlled experiment, reinforces the idea that measured lactate levels after 24 h in the onboard experiment are those of unstressed or mildly-stressed sharks. Thus, we offer an alternative to those studies that faced the problem of obtaining blood samples from “unstressed” sharks, and offer solid “baseline values” of lactate and other variables that were subjected to the specific conditions of each study (Frick et al., 2010a; Gallagher et al., 2014).

Table 4 Plasma phosphate and urea concentrations (mmol L−1) in small spotted catshark, S. canicula, after 18 min of air exposure in ground facilities. Data are expressed as mean ± SEM (n = 7–8). No differences were observed for any group during time. Asterisks (*) indicate significant differences between both groups at a single time (p < .05, two-way ANOVA followed by a Tukey's post hoc test). Variable PO4

2−

(mmol L−1) Urea (mmol L−1)

Group

0h

5h

24 h

Control Air exposure Control Air exposure

2.4 ± 0.1 2.9 ± 0.2 515 ± 17 476 ± 15

2.4 ± 0.2 3.4 ± 1.1 * 477 ± 14 412 ± 24 *

1.7 ± 0.1 1.7 ± 0.2 519 ± 13 484 ± 10

366 ± 6 mM 1 in catsharks captured in shallow and deep waters, respectively, p < 0.05). Thus, the shallow water group presented 8 to 9% higher plasma urea concentrations than fish captured in deeper waters. In contrast, for the laboratory-conditions experiment (Table 4), urea showed a significant decrease in air-exposed fish compared to the control-group 5 h after the stress situation (477 ± 14 and 412 ± 24 mM in the control and air-exposed groups, respectively; p < 0.05). 4. Discussion Here we provide strong evidence showing that the small-spotted catshark manages to survive and successfully return to its plasma homeostatic levels after a trawling event. These results could not be extended to all small sharks, but have paved the way to future studies in other species. Recovery behavior and energy management of the stress responses induced by fishing vary depending on the depth at which catsharks were captured. Nonetheless, plasma levels of stress-related markers appear to return to low, presumably close to the basal levels, in fish captured at ~89 and ~479 m depth in < 24 h. It should be noted that the observed results are restricted to a vessel deploying its gear for 60 min, which may be considered as mild conditions compared to commercial bottom-trawl vessels in the area (between 120 and 150 min, authors´ personal observation).

4.3. Acid-base and ionic imbalances Typical secondary responses to acute stress in sharks are transient 7

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Competitividad), and project SUREDEPAR (Programa Pleamar, Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente) to JMM and by the Ministry of Science and Higher Education and European Social Funds through the Portuguese National Science Foundation (FCT) by Project PTDC/MAR-BIO/3034/2014 to JF. CCMar is supported by FCT through project UID/Multi/04326/2013. The authors are indebted to Candelaria Burgos for her invaluable assistance with the map, to Dr. González and to Martha Bonnet Dunbar for the technical assistance with the English grammar, and to Dr. Novelli, Dr. Navarro and people aboard the ARSA surveys for their help during this experience.

disruptions to acid-base balance, including depression in blood pH, which is driven in part by lactic-acidosis (Lambert et al., 2018; Skomal and Mandelman, 2012). In order to counteract this acidosis, blood phosphate levels increased in the present study, in agreement with previous reports (Manire and Hueter, 2001). Inorganic phosphate accounts for most of the intracellular buffering in the white muscle of fish (Okuma and Abe, 1992), and leakage from damaged muscle cells into the blood may contribute to buffering plasma acid-base imbalance, shown by measurements of plasma levels of phosphate. Effects of capture stress on other blood constituents are imbalances in other electrolytes, such as an increase in plasma osmolality (Frick et al., 2010a; Skomal, 2007), which is confirmed in the present study, matching with previous results in Squalus acanthias (Mandelman and Farrington, 2007) or Prionace glauca (Moyes et al., 2006). The osmotic strategy of marine sharks includes the maintenance of high levels of plasma osmolality through elevated levels of organic salts, such as urea (Evans and Kormanik, 1985; Smith, 1929). Some studies have reported transient declines in plasma urea concentrations attributed to stressinduced compromises in the post-capture period (Frick et al., 2010a; Mandelman and Farrington, 2007; Skomal and Mandelman, 2012), as observed in our results. Furthermore, it should be mentioned that levels of urea and depth of capture may be somehow related as seen by the results derived from the present study. This could be due to the differentiated energy metabolism strategies described in sharks depending on their inhabiting depth (Speers-Roesch and Treberg, 2010), although further studies are necessary to confirm this hypothesis.

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5. Conclusions In conclusion, our study offers a novel approach to characterizing survival and recovery processes of captured fish after bottom-trawling or other fishing methods. We report that S. canicula captured by 60 min bottom-trawling in the Southwestern Atlantic waters of Europe manage to successfully recover within the first 24 h. The survival rates are closely related to time of air exposure. Moreover, depth of capture influenced the recovery of the animals, with catsharks captured at lowers depths requiring longer periods to recover than those captured in shallower waters (the reasons were unknown and future studies may be directed towards the analysis of barotrauma in captured sharks). Finally, by using a comprehensive set of physiological stress biomarkers, like those employed in this study, future approaches may be able to derive models that effectively link physiology to survival in captured sharks. Including both laboratory and field studies is necessary to fully characterize survival rates in these highly-vulnerable species captured and discarded worldwide. Moreover, future studies may be conducted under commercial conditions (trawling for > 2 h). The information provided in this study may serve fisheries stakeholders and managers, although more information is required related to the exact timing of physiological recovery of this (and other) shark species, or the effects of direct sun exposure, air temperature, stress induced by barotrauma. More studies are thus required to properly evaluate the effects of capture, the recovery processes and to calculate survival rates of sharks. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpa.2019.03.016. Acknowledgements The survey data have been collected by the Spanish Oceanographic Institute integrated in the sampling program co-funded by the European Union through the European Maritime and Fisheries Fund (EMFF) within the National Program of collection, management and use of data in the fisheries sector and support for scientific advice regarding the Common Fisheries Policy. This work was partially supported by funds from the PADI Foundation (App # 28467) to IR-J, projects AGL201348835-C2-R and AGL2016-76069-C2-1-R (Ministerio de Economía y 8

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