Measuring recovery of bycatch turtles from hypoxia using RAMP

Midland painted turtle, Harding 2009

Reflex impairment was tested in recovering freshwater painted turtles (Chrysemys picta) that were exposed to hypoxia as bycatch in fyke net fisheries (LeDain et al. 2013).

"Adapting the use of RAMP from fish to gauge the severity of anoxia on painted turtles was effective. All the reflexes used in this study indicated some level of impairment following submergence, with some reflexes being more sensitive than others. The tactile responses to the limbs and head were the most insensitive to submergence: they were often the only remaining reflexes after submergence (Table 1). Thus, the absence of tactile responses could be indicative of turtles requiring assisted recovery the most. Other species of turtles may require different types of reflex responses that suit their morphology better (e.g., loggerhead turtles, Caretta caretta, are too large to use a hand holding ‘‘escape response’’). However, a tactile response is a universal reflex that can be used in all species. Employing a presence/absence scoring reduced subjectivity and will ease the training of commercial fishers, researchers, and government and nongovernment agencies on the use of the RII. In sum, we feel the RII is a very useful tool to assess turtle condition."

The authors suggest that tests for reflex impairment (RII, reflex impairment index) modeled after RAMP methodology can be useful for assessing assisted recovery and potential mortality in turtle bycatch.

"Although reduction of turtle mortality can be achieved through educating fishers on proper handling and recovery methods for turtles, the first course of action should be checking nets frequently and implementing seasonal or area restrictions. Bycatch reduction devices (turtle excluders and escape modifications) are also effective in reducing the number of turtles caught in nets while maintaining fish catch (Guillory and Prejean 1998; Lowry et al. 2005; Fratto et al. 2008; Hart and Crowder 2011; Larocque et al. 2012c). However, since turtle bycatch can still occur despite these conservation measures, albeit at reduced levels, examining reflex impairment is an effective and inexpensive way to discern whether turtles require assisted recovery after incidental capture in submerged nets."

Using RAMP to estimate fisheries discard mortality in the southern New England flatfish complex

Yellowtail flounder NOAA

Winter flounder NOAA

Windowpane flounder NOAA

RAMP curves were estimated for yellowtail flounder, winter flounder, and windowpane flounder in the southern New England flatfish complex (Barkley et al. 2012).  Fish were treated with experimental trawl and air exposures and sublethal and lethal effects on reflex impairment noted.  Seven reflex actions were tested and responses were combined into RAMP scores.

RAMP curves were calculated (see also for anatomy of a RAMP curve):

RAMP was determined to be a useful method for predicting discard mortality in the flatfish complex and can be used for rapid, real time, onboard sampling of discard mortality rates:

"Implications

The utility of RAMP is the ability to test the reflexes of fish caught and use the RAMP score to predict mortality using the reflex impairment-mortality relationship. Creating reflex impairment-mortality relationships opens up the possibilities to expand RAMP sampling and to gain a more accurate representation of the total commercial discard mortality rates. These reflex methods can be applied to a subsample of fish during commercial fishing trips to allow for a more representative discard mortality estimate. The RAMP methods are also not limited to a particular gear type, so fish caught in the large-mesh otter trawl fishery as well as fish caught in the scallop fishery can be assessed using the same reflexes and can be compared to the same reflex impairment-mortality relationship. This allows for the estimation of discard mortality over a wide spectrum of gears, tow-times, and time on deck.

Summary of conclusions

- The suite of seven reflexes is a reliable indicator of survivability in yellowtail and winter flounder.

• Tow-time was not as important as a stressor on flounder as air exposure. 
• The results indicate that the discard mortality of yellowtail and winter flounder may be reduced onboard fishing vessels by limiting the amount of time the fish are on a dry deck.
• Windowpane flounder are less hearty then yellowtail and winter flounder, and may not be able to survival discarding.
• The reflex impairment-mortality relationships developed from this project for yellowtail flounder and winter flounder can be used to estimate the discard mortality rate of fish, based on at-sea RAMP sampling over multiple gear types."

Realtime knowledge of discard mortality rates can be used in adjusting fishing practices to decrease and avoid discard mortality, as well as for testing new fishing gears designed to reduce bycatch capture and discarding.

C&R fishing, physiology, RAMP, and fitness outcomes

A study by Cooke et al. 2013 reviewed the physiological consequences of C&R (catch and release) fishing. They concluded that:

"An underlying tenet of catch-and-release studies that incorporate physiological tools is that a link exists between physiological status and fitness. In reality, finding such relationships has been elusive, with further extensions of individual-level impacts to fish populations even more dubious."

They presented a conceptual scheme for the development of physiological stress in C&R fishes:

Cooke et al. 2013, Figure 1. "Schematic of the general stress response to fisheries capture. The thick black solid line labelled ‘general response’ provides an example of a typical response of a physiological indicator of stress, such as plasma cortisol, to a fisheries capture event. Following the initial response, a negative feedback occurs and recovery is initiated. The stressors connected by a bracket to the general response line exemplify the multiple, interactive and potentially cumulative stressors involved in a fisheries capture event, all of which contribute to the general stress response and are dependent on environmental conditions and the initial condition of the individual fish. The thick black broken line represents a disrupted recovery pattern, where recovery to routine physiological condition does not occur and there are life history consequences. The grey broken line represents an example recovery profile for individuals held in facilitated recovery gear, where the general physiological response is muted and recovery to routine condition is accelerated."

Observations of physiological stress in fishes have measured short-term changes associated with C&R angling.  However Cooke et al. 2013 conclude that:

"Beyond the problems noted previously, there is mounting evidence that, taken alone, conventional blood chemistry measures may not be definitive enough to forecast long-term survival following fisheries-related injuries and stressors (see Skomal & Bernal 2010; Pankhurst 2011; Renshaw et al. 2012), although another possible explanation is that researchers are failing to use the appropriate physiological indices (Renshaw et al. 2012; see section below on use of a limited set of metrics). Although now technically feasible to attempt to link physiological condition to fate, it has thus far failed to enhance C&R science with respect to long-term outcomes. However, with shorter-term outcomes (e.g. behavioural endpoints) and when used for conducting mechanistic laboratory studies to complement field studies, physiology has yielded valuable insight."

Prediction of delayed mortality has been successful with observations of reflex impairment (RAMP) in fishes from C&R fisheries. Cooke et al. 2013 note:

“Unlike traditional physiological tools, there has been considerable success in using a simple reflex impairment index [reflex assessment mortality predictors (RAMP) score] to predict delayed mortality for fish released from commercial fishing gears and subsequently monitored in large tanks (summarised in Davis 2010) or released into the wild with telemetry tags (Raby et al. 2012). The success of RAMP for predicting mortality is likely attributed to its holistic nature: underlying physiological impairments are integrated into whole-animal responses that can easily be assessed in a quantitative way. In the context of C&R, Campbell et al. (2010) developed a condition index for red snapper, Lutjanus campechanus Poey, that combined reflex impairment with indicators of barotrauma and was associated with immediate mortality and proxy indicators of post-release predation risk (post-release mortality was not assessed directly).

Additional research is needed to develop predictors of fate in C&R science and the logical focus should be on fisheries for which significant mortality is observed that seems to be independent of physical injury (e.g. deep hooking). Reflex assessment mortality predictors will not replace traditional physiological metrics, but it is a valid and inexpensive complement and could be incorporated into any study of C&R mortality even if the project team has little or no experience in physiological research.”

In communicating the results of C&R fishing to management and fishing communities, Cooke et al. 2013 found:

“Although physiological tools can play an important role in understanding and mitigating the sublethal consequences of C&R on fishes (Cooke et al. 2002; Wikelski & Cooke 2006; Arlinghaus et al. 2007a), it is important that the findings of physiological studies be interpreted correctly and used appropriately. It is difficult to translate the physiological results of C&R research into best practices given the limitations listed previously. Where investigators have identified physiological consequences of C&R, findings must therefore be interpreted cautiously with results not extrapolated beyond the boundaries of their study design. For instance, Wedemeyer and Wydoski (2008) examined the physiological response of some economically important salmonids to C&R fishing, and they interpreted many significant trends between angling duration and blood parameters as ‘transient’ effects, ‘generally mild’ and of ‘little physiological consequence’, without fully exploring a broader suite of metrics (e.g. cortisol) shown to be associated with angling stress in other recreational fishes. Moreover, their study was restricted to moderate water temperatures, like many C&R studies (reviewed in Gale et al. in press). The results of their study were then noticed by the angling community, which further extrapolated the findings on angling web sites, message boards and blogs, inferring that C&R in general has negligible consequences on trout and without considering how factors not explored in their study such as water temperature could alter the outcome for the fish. Consequently and likely quite unintentionally on the part of researchers, peer-to-peer communication pathways common within the recreational angling community could foster a shift of the social norm about the potential conservation value of C&R. When management implications arising from C&R physiological studies are presented in the peer reviewed literature, authors should thus provide appropriate caveats, context and draw conclusions carefully. Although the interpretation of physiological data can be subjective, it is suggested that such findings always be viewed in the context of the broader stress response and recovery probable for a given species/population (Fig. 1).”

Catch and release largemouth bass, University of Illinois

Cooke et al. have summarized important steps to take for minimizing mortality in C&R fisheries in an effort to standardize these protocols according to scientific principles (see Arlinghaus et al. 2007, Pelletier et al. 2007):

"General guidelines for catch-and-release recreational angling to conserve fishery resources:

Minimize angling duration

The duration of the angling event increases the physiological disturbance from which the fish has to recover. Angling results in a combination of aerobic and anaerobic exercise that causes a number of physiological changes such as the depletion of energy reserves, accumulation of lactate, and alterations in acid/base status. Studies have shown that these physiological disturbances are generally more severe with increasing angling duration. In addition, the length of time required for physiological variables to return to resting levels tends to increase with angling duration.Therefore, anglers should try to land fish as quickly as possible to minimize the duration of the exercise and the related physiological disturbance.There are techniques for achieving shorter angling durations, such as choosing equipment that matches the size of fish that are expected to be encountered.

Minimize air exposure

Air exposure is harmful to fish. Air exposure occurs upon capture when a fish is removed from the hook, weighed, measured and/or held for photo opportunities. When a fish is exposed to air, the gill lamellae collapse causing the gill filaments to stick together. This has several negative physiological implications. It can cause severe anoxia. Fish that are exposed to air typically experience greater acid/base disturbance (fluctuation of pH in the blood) than those which are not. When fish are exposed to air for a significant length of time, they require a much longer time to return to their normal state. Furthermore, extended air exposure (beyond a species-specific timing threshold) can eventually result in permanent tissue damage or death. Although different fish species vary in their tolerance to air exposure, it is recommended to minimize the duration of the air exposure whenever possible.

Avoid angling in extreme water temperatures

Most fish are ectothermic (they cannot regulate their own body temperature) so the environment regulates their temperature. Any changes in the ambient water temperature can have a significant impact on their cellular function, protein structure, enzyme activity, diffusion rates and metabolism. In addition, the amount of dissolved oxygen in water is lower at higher water temperatures. Angling stressors tend to be magnified at higher water temperatures as reflected in strong relationships between water temperature and mortality for several species. On the other hand, extremely cold temperatures likely also have detrimental effects, although this has been poorly studied to date. Although individual species exhibit different thermal tolerances, catch-and-release angling has the potential to be harmful at extreme water temperatures. In some jurisdictions, there are restrictions on angling when water temperatures exceed some threshold. Since water temperature exerts control over almost all physiological processes in fish, extreme water temperatures are undoubtedly conditions in which fishes are most vulnerable and where angling should be avoided.

Use barbless hooks and artificial lures/flies

Hooks are used to capture fishes. Therefore, hook design is an important element to consider when attempting to reduce hooking related injuries and mortality. Hooks with barbs can lead to greater injury than barbless hooks and even contribute to mortality, although the literature accounts are disparate. However, barbless hooks can minimize the amount of harm caused by reducing tissue damage at the point of hook entry and by reducing the amount of time required to remove a hook. Since there is no barb, the hook can easily be removed. Some studies show that circle hook can be an effective tool in catch-and-release fisheries, when used properly under certain conditions [see section on circle hooks]. The type of bait used is another important factor in fish injuries. Live/organic baits (e.g., worms) used on hooks can be ingested and the hook becomes lodged into the viscera. This makes it hard to remove the hook and it will likely cause damage to the vital organs/tissue during the process. Artificial lures or flies do not get ingested as much so there is minimal damage to the vital organs/tissues from the hook(s). Barbless hooks and artificial lures/flies can greatly reduce handling time, hooking injuries and the likelihood of mortality.

Refrain from angling fish during reproductive period
The reproductive period is the time during which fish attept to produce off-spring and is thus critical for sustaining fish populations. Angling fish during their reproductive period canl reduce the number of off-spring that could contribute the population. Some species, like the largemouth bass, provide parental care and protection for their off-spring. If this dad is removed from the nest, even for a brief moment, its off-spring become extremely vulnerable to predators. Thus, angling immediately prior to or during the reproductive period could affect fitness and should be avoided."

Approaches for modeling and predicting bycatch mortality

Modeling and prediction of bycatch mortality can be approached in several ways. Efforts can be focused on prediction from knowledge of controlling environmental factors and fishing processes encountered by animals. Alternatively, efforts can be focused on prediction from knowledge of animal condition that integrates effects of fishing factors. A third hybrid approach combines information about animal condition and controlling fishing factors.

Fishing is conducted in freshwater and seawater, with catch retained, discarded, released, or escaped from commercial, recreational, catch and release, and subsistence fisheries. In all cases, knowledge of target and non-target fishing mortality is essential for management and conservation of fisheries stocks and ecosystems. Fishing occurs under a variety of environmental and operational conditions. Examples of fishing gears include trawls, seines, traps, dredges, hook and line, gill nets, lift nets, and falling gear. While immediate mortality is evident for non-target bycatch discards, delayed mortality is generally hidden from view for discards and escapees from fishing gears and operations.

Fishing factors include a range of types for discards and escapees. Master controlling variables include temperature, air exposure, gear injury, fatigue and exhaustion, fish size, barotrauma, and predators. Synergistic effects of combinations of factors can be significant controllers of mortality.

Davis 2002 capture and discard

Suuronen 2005 capture and escape

Bycatch mortality can be modeled by experimental determination of relationships among environmental and operational factors and mortality rates of various species, either under laboratory or field conditions. Since there are an almost infinite number of factor combinations in a fishery, it is important to prioritize the stress and mortality effects of factors and factor combinations. Primary effects are then modeled for mortality rates. 

Effects of fish size, fishing gear type, and temperature on sablefish mortality, AFSC

A second approach to modeling bycatch mortality is to shift focus from environmental and operational fishing conditions to a more limited set of predictors for mortality based on animal condition. These include wounding, physiological impairment, and reflex impairment. Reflex impairment and RAMP have been found to be the most efficient and inclusive predictors of immediate and delayed mortality.

AFSC

RAMP curves for the relationships between reflex impairment and species mortality are constructed under simulated or actual fishing conditions that are expected.

AFSC

Using constructed RAMP curves, bycatch mortality rates can be measured and predicted in fishing operations through time and space by sampling fish from fisheries.

Georges Bank scallop fishery bycatch

Hybrid combinations of the two modeling approaches can be used if data are available. Animals that are captured or escape from fishing gears can be sampled for RAMP while environmental and operational conditions are noted. Then relationships among these factors can be modeled.

Effect of air exposure on Atlantic cod reflex impairment and mortality, Humborstad et al. 2009

Survival of bycatch species escaping purse seines

Many species of schooling fish are caught using purse seines.

Purse seine

Fish in schools caught by purse seines are generally in aggregations of one species.  However there can also be fish that are too small, too many, or other species such as turtles, porpoise, and tuna that are not designated for capture in the fishery. For management purposes, these non-target animals are considered bycatch and are discarded, either from the fishing gear or from the catching vessel.  

Porpoise and tuna bycatch in purse seine

What are the mortality rates for bycatch species that are discarded or escape from purse seines? Experiments have been designed to answer this question for mackerel that are "slipped" from a purse seine because too many are caught in a net set.

Huse and Vold 2010

Mortality rates of mackerel were related to a stress index of crowding density and time.

Huse and Vold 2010  Stress indices (fish density (kg m⁻¹) times crowding time) from Lockwood et al. (1983) (diamonds) and from our own experiments (triangles). The exponential line is fitted to the data from Lockwood et al.  

Reflex impairment and RAMP can be used in field survival and mortality experiments. Observations of reflex impairment for fishes, turtles, and porpoise can be made in holding nets and related to vitality and mortality using RAMP calculations. Note that the mortality curve for mackerel is similar to RAMP curves for other species.  

Use of RAMP in field experiments and fishing operations can result in large amounts of real time, high quality data on discarded or escaped bycatch vitality and mortality.  This data is key for the effective management of fisheries stocks and conservation of ocean ecosystems.

Survival of bycatch species escaping trawls

New fishing trawl gears are designed to facilitate the escape of animal species that would otherwise be landed as bycatch and discarded. Knowledge of animal behavior such as startle, rheotaxis, phototaxis, avoidance, and sheltering is used to facilitate escape of potential bycatch species from trawls.

He, editor

Survival of animals that escape from trawls is an important aspect of conservation. Any mortality of escapees must be accounted for as bycatch mortality in fisheries management.

Rahikainen et al. 2004

Measurement of survival and mortality rates for escapees is a difficult research problem. One approach is to design trawls and nets that can sample escapees for later observation of behavior, recovery from capture, or mortality.

 Suuronen 2005

Reflex actions and volitional behavior can be observed in cages after animals have escaped from trawls and through their recovery period. RAMP can validated during these experiments and used to predict mortality rates for animals observed to escape from fishing gears.

Project Survival

Banning fishery discards and using RAMP

European Union fishery ministers have agreed to phase out the practice of discarding unwanted or regulated animals (bycatch) from landed catches.  The practice of discarding bycatch can be tremendously wasteful of fishery resources including fish, elasmobranchs, invertebrates, birds, amphibians, reptiles, and mammals.

Discarding, Richard

Banning discarding from fisheries requires total retention of animals caught, which must be landed and processed.  As many of these discarded species are of low economic value, efforts are made to design fishing gears that avoid catching bycatch species in the first place.

Suuronen 2005

A key assumption in the ethical design of fishing gears that do not catch bycatch and discarded species is that animals survive gear encounters. Escaping animals must have significant survival rates after gear encounters if they are to continue contributing to recruitment and ecosystem function. If animals escape from fishing gears and do not survive, they are the same problem as discards in fisheries, except that they are hidden.

Suuronen 2005

Measurement of mortality rates for discards and for animals that escape from fishing gears is vital to the management of fisheries, as they represent a significant form of fishing mortality. Discard and escapee mortality rates have been difficult to measure and new, effective methods are needed.

Viability estimates for Pacific halibut bycatch, based on vitality codes (1-4) for injury and activity have been incorporated into fisheries management for several years.  Recent research results by Benoît et al. 2012 on discard mortality have suggested methods based on fishery-scale sampling with semi-quantitative vitality codes (excellent-1, good-2, poor-3, and moribund-4) and conditional reasoning.

Benoît et al. 2012. Post-capture survival probability over time (h) for five southern Gulf of St. Lawrence marine fish taxa (panels), as a function of their pre-holding vitality class score (colours). The shaded areas represent the 95% confidence band for the Kaplan–Meier empirical survival curve for each vitality class, plotted up to the time at which the last observation was made for a given taxon and vitality level. The lines represent the fits of the selected model for each species and vitality class. For cod and plaice, the fits for models M3 and M4 are presented using solid lines and dashed lines respectively (note that these lines largely overlap). The location of the circles along the line and the size of the circles indicate respectively the times at which observations were censored and the proportion of censored observations for the taxon and vitality level at that time.

Reflex impairment measured by RAMP is a quantitative measure of vitality that gives increased resolution and accuracy to the determination of health and survival of discards and animals encountering and escaping fishing gears. Future research on this subject can benefit from the incorporation of fishery-scale sampling of RAMP for discards and for animals escaping from fishing gears.

The banning of discarding will make the evaluation of mortality rates for animals escaping from fishing gears especially important. "Out of sight and out of mind" will not be a viable strategy with regards to evaluating fishing mortality for gears engineered to enhance escape of bycatch species.

Using RAMP to help automate aquaculture operations

Net pen for fish culture (NOAA).

Aquaculture of animals in tanks and net pens requires monitoring and maintenance of vitality, health, and normal behavior for efficient and economic operations. Presently, health is monitored by sampling for disease outbreaks, while vitality and behavior are observed by aquaculture technicians during the course of their daily activities of feeding, cleaning, and operation of facilities.

Inside net pen for fish culture (NOAA).

In tanks and net pens, animals can swim and feed normally. They can also respond to stimuli administered inside their rearing environment, such as light flashes, sound bursts, food scent, and touch. Responses to these stimuli can be in the form of reflex actions such as startle, orientation, depth distribution, aggregation, and dispersal. These reflex responses can be observed remotely and automatically using video, infra-red, and sonar technology in light and dark conditions. Reflex responses can be recorded and summed as RAMP scores for measures of impairment and correlation with mortality.

RAMP can be a quantitive measure of animal state and be used to help automate aquaculture monitoring and maintenance. When impaired responses to stimuli are observed, alarms can be triggered and technical staff can be alerted to changes in animal vitality, health, and behavior. Then on site alteration of operations can bring rearing conditions back to nominal states and return animals and their reflex actions to vitality and health.

Future research in aquaculture can consider the use of RAMP and automated reflex testing for development of efficient operation protocols and quality assurance. RAMP can also be used as a research tool for testing and validating new designs for aquaculture operations that optimize animal vitality and health.

Field validation of dungeness crab RAMP underway

Crabs tell us about their vitality, using the language of reflex impairment and RAMP.

Yochum

Field validation of dungeness crab RAMP measures for discard mortality are underway off Newport, Oregon.  These field trials with combined RAMP measurements, fishery conditions, and mark and recapture experiments are a large scale field effort to develop RAMP tools for quantification of bycatch mortality (NOAA 2013).

Undersize Dungeness crab marked with a green T-bar spaghetti tag and released as part of the RAMP field validation experiment. The tag is inserted through the suture at the back of the carapace so that it can be retained through a molt. Picture and caption from Stoner and Yochum, 2013.

Pink and Chum Salmon reflex impairment at spawning grounds

A study by Raby et al. 2013 has shown remarkable resilience by Pink and Chum Salmon to simulated fisheries capture stress incurred upon arrival at spawning grounds.  These salmon species were observed to have low mortality and successful spawning after being stressed by exposure to exhaustive exercise, air, and injury.  In the authors words:

"Our study results provide evidence that, after reaching spawning areas, both Pink and Chum Salmon may be resilient to certain forms of capture-related exhaustion stress. Short of producing immediate mortality through extended anoxia, Pink and Chum Salmon are apparently able to recover from substantial physiological disturbance related to capture and ultimately spawn. Natural prespawn mortality rates for Pink Salmon (6.5%) and Chum Salmon (3.2%) in the channel during the study year (R. Stitt, Fisheries and Oceans Canada, personal communication) were nearly identical to the prespawn mortality rates for fish subjected to our capture and tagging procedures."

One of the objectives of the study was to test for possible relationships between reflex impairment (RAMP) and mortality.  However because of the general lack of mortality in these salmon species just prior to spawning, increasing reflex impairment as RAMP was related to increasing intensity of capture stressors and not to mortality or spawning success.   

Patterns of impairment are shown by the authors:

"The pattern of impairment of individual reflexes with successively increasing levels of overall reflex impairment (RAMP scores) was largely consistent. Tail grab and body flex were by far the two most easily impaired reflexes (Tables 2, 3). However, in Chum Salmon, tail grab impairment predominated body flex impairment at low RAMP scores (0.2, 0.4) compared with Pink Salmon. Orientation was typically the third reflex to become impaired for both species when overall RAMP score increased beyond 0.4 (Tables 2, 3). We did not commonly observe VOR impairment, which was almost always the last reflex impaired."

The authors suggest that radical shifts in metabolism during preparation for spawning may be responsible for the observed resilience to simulated capture:

"We hypothesize that Pink and Chum Salmon are resilient to capture-related exhaustion upon reaching spawning areas because of a combination of low water temperature (about 12^◦C in this study) and a physiological shift towards increased use of anaerobic pathways during their final weeks of life. The capture and release of fish arriving at the spawning ground does not appear to influence survival, in contradiction to the results of other studies, which focused on earlier components of Pacific salmon spawning migrations."

The possible shifting of metabolic pathways in prespawning Pink and Chum Salmon, associated with lack of mortality at high levels of reflex impairment is a cautionary tale for use of RAMP to predict vitality, mortality, and spawning fitness. The relationships between reflex impairment, metabolic pathways, and life history traits are important subjects for future research and validation.  

RAMP and bonefish recovery from capture stress

Bonefish are typically captured in a high value capture and release sport fishery.  Released bonefish can be subjected to high rates of predation if predators are present and stress levels are high.  Recovery from capture stress is important for survival and maintenance of bonefish fishery stocks.  Recovery technology including holding bonefish in bags or live wells prior to release has been evaluated by Brownscombe et al. 2013.  They used measurements of reflex impairment (RAMP), locomotory activity, and predation to test recovery from capture stress and survival after release.

Bonefish Key West Flats Fishing

Practical application of RAMP was suggested by Brownscombe et al. 2013. "Bonefish anglers may be able to use RAMP to assess bonefish condition, and make educated decisions on whether to release the fish, or retain it for a short period to facilitate recovery. Likewise, if water temperatures and bonefish impairment scores are very high, responsible anglers can recess until conditions are more favorable."

Here are some excerpts from their paper regarding the validation of RAMP to measure capture stress and predict delayed mortality after release:

"The primary objective of this study was to evaluate the effectiveness of retaining bonefish in recovery bags for reducing short-term locomotory impairment when subjected to angling-related stressors, and whether potential improvements in swimming ability translated to increased survival.

Reflex indicators have recently been deemed effective predictors of mortality (Davis, 2010; Raby et al., 2012), and could be used by anglers to evaluate in which instances fish would benefit from recovery. We predicted that fish retained in recovery bags would exhibit lower reflex impairment, as well as higher locomotory ability and survival than those immediately released.

We validated the use of reflex action mortality predictors (RAMP) (Davis, 2005, 2010) to assess bonefish vitality after 0, 2, 4 and 6 min of air exposure. The 0-minute assessments (n = 30) occurred prior to air exposure on fish from all treatments, while bonefish in the 2-minute treatment (n = 20) were those used in recovery bag experiments (see below), and 4, 6 min treatments (n = 5) were conducted on alternate fish. Five predictors were measured; tail grab, equilibrium (orientation), body flex, head complex, and vestibular-ocular response (VOR). These predictors were chosen because Raby et al. (2012) found that they were strong predictors of coho salmon (Oncorhynchus kisutch) mortality after being caught in commercial nets, and all these predictors can be easily and quickly measured by bonefish anglers. RAMP was assessed in the same manner by Raby et al. (2012). The presence of a tail grab response was assessed by grabbing the fish's tail while it is submerged in water; it was considered impaired if the fish did not attempt to swim away from the handler. Equilibrium was assessed by rolling the fish upside down in water; impairment was indicated when the fish was unable to right itself within 3 s. Body flex was tested by holding the fish by the middle of the body in air; it was considered impaired if the fish made no attempt to struggle free. Head complex was considered impaired if while holding fish in air, a regular pattern of ventilation of the fish's operculum was not observed for at least 5 s. VOR was assessed by rolling the fish back and forth in air; it was considered impaired if its eyes did not roll to maintain the same pitch and track the angler. Higher RAMP scores indicated greater impairment.

Based on the responsiveness of bonefish to the RAMP indices, we used them to evaluate the utility of the recovery bags. After air exposure, bonefish released with accelerometers had similar RAMP scores between immediate release (2.8 ± 0.14) and recovery (2.7 ± 0.14) treatments. However, after retention in a recovery bag for 15 min, all bonefish had RAMP scores of zero (i.e., full recovery).

Our results demonstrate that retaining bonefish in recovery bags for 15 min reduced locomotory impairment upon release during the critical time period where most predation occurs, and this practice has the potential to increase survival after catch-and-release angling. Presumably, retaining bonefish in a live well with ambient oxygen levels (Shultz et al., 2011) would have a similar benefit if an angler had access to a boat.

The five impairment indicators we tested on bonefish provided a gradient in impairment scores that related to the degree of stressor (i.e., 0–6 min of air exposure). RAMP scores have been correlated with stressor duration and mortality for a number of fish species (Davis, 2005, 2007; Davis and Ottmar, 2006; Humborstad et al., 2009; Raby et al., 2012). Indeed, the duration of a stressful event increases the level of physiological disturbance in bonefish (Suski et al., 2007; Donaldson et al., 2008), while longer handling times and air exposure durations result in higher post-release predation rates (Danylchuk et al., 2007a). In this study, bonefish that were equipped with accelerometers exhibited moderate impairment scores after 2 min of air exposure, while no impairment was detected after 15 min of retention in a recovery bag, and fish from the recovery treatment exhibited significantly higher levels of activity upon release. Therefore RAMP scores appear to be a good indication of bonefish vitality. 

The impairment indicators tail grab, equilibrium, and body flex were the first to become impaired in bonefish, and impairment levels within these predictors did not vary with increased stress duration. This was likely because bonefish were all highly impaired at the lowest level of stress we inflicted. Indeed, a previous study found roughly that 50% of bonefish lose equilibrium after angling events (Danylchuk et al., 2007a), while 95% of bonefish lost equilibrium after simulated angling stress (2 min of air exposure) in this study. These three predictors may provide an indication of impairment levels with lesser degrees of stress. Head complex was the next to become impaired at 4 min of air exposure, followed by VOR at 6 min. Therefore head complex and VOR predictors are indicative of very high levels of physiological disturbance in bonefish. This predictor-specific pattern of impairment in bonefish is nearly identical to that of coho salmon (see Raby et al., 2012)."

Mortality sources and the limits of RAMP

Exposure of animals to stressors can result in changes to physiology, behavior, and injury that can result in stress, impaired reflex actions, morbidity, and delayed mortality.  Stressors in fishing, aquaculture, net penning, aquarium trade, research settings, and other ecosystems are present in a number of ways as departures from nominal temperature, light, oxygen, food, xenobiotics, injury, crowding, disease, social interactions, and predators.

While reflex impairments and RAMP can accurately assess vitality and stress levels and predict delayed mortality, these measures are solely dependent on the internal state of an animal at the time of observation.  When other external stressors and sources of mortality are present after an animal is assessed with RAMP, predictions of delayed mortality may not be accurate.

In open, wild ecosystems, important sources of mortality in animals can be predation, lack of food or feeding ability, and impairment of social behavior that is protective (schooling, shoaling, and shelter seeking).  The presence of any or all of these stressors can alter mortality rates predicted by RAMP.  Use of RAMP for predicting delayed mortality in open systems is probably limited to short term delayed mortality.

In closed, human managed ecosystems, external sources for delayed mortality can be controlled and eliminated after RAMP measurements and RAMP predictions of delayed mortality can be accurate over longer time periods.

Reflex impairment in dogs, birds, and turtles

Reflex impairment in animals treated by veterinarians (dogs, cats, birds, rabbits, and livestock) is widely tested as part of a neurological examination to determine the potential presence and location of neurological impairment. The neurological exam consists of tests on mentation, posture and gait, cranial nerves, proprioception, spinal reflexes, and sensory pain perception.  Detailed summaries of these test procedures can be found here and here. In the veterinarian context, results of these neurological exams are generally confined to determination of whether the nervous system is affected in a disease process and to provide an accurate anatomic diagnosis when the nervous system is affected.  Consideration of contributions to disease by neurologic, medical, and orthopedic sources are differentiated into separate testing protocols for the purpose of formulating diagnosis and treatment plans. 

 Clippinger et al. 2007

Vernau et al. 2007

The veterinarian sequence of neurological testing in dogs and cats has been applied to sea turtles.  Results of the study showed that many of the neurological methods for dogs and cats can be adapted for use in sea turtles. The authors concluded that a standardized neurologic examination resulted in an accurate assessment of neurologic function in impaired sea turtles and could help in evaluating effects of rehabilitation efforts and suitability for return to their natural environment. Another study made a detailed assessment of chelonian health that included measuring reflex impairment as part of emergency and critical care. Measured reflex actions included head lift, cloacal or tail touch, eye touch, and nose touch.

Freshwater turtles have been tested for reflex impairment in an effort to evaluate the effects of submergence and increased temperature in bycatch mortality of three species.

Stoot et al. 2013

The RAMP results from reflex impairment testing in fish and invertebrates suggest that the neurological and reflex state of an animal includes the effects of injury and infection when related to fitness outcomes such as recovery, vitality, morbidity, and potential mortality. This inclusion of fitness effects probably results from the fact that the RAMP method is a scoring system that expresses the proportion of whole animal impairment, calculated based on the presence or absence of a suite of reflex actions.  Shifting focus and perspective from individual mechanistic explanations for disease to comprehensive whole animal measures for vitality can help link reflex impairment with fitness outcomes.

Further study and reflection on human and veterinarian medicine approaches to neurological testing can probably inform selection of reflexes to be used in the RAMP approach for reflex testing.  The interaction of medical and RAMP perspectives for quantifying disease states may result in advances towards understanding how nervous system and reflex function can be a comprehensive indicator of disease and vitality states, combining the effects of injury, infection, and nerve impairment.

Choices for testing reflex impairment in RAMP

Reflexes can be tested in unrestrained or restrained animals. Tests can be adapted to experimental or operational conditions needed in specific situations. A reflex action is scored not impaired (0) when strong or easily observed and scored impaired (1) when not present, weak, or there is doubt about presence. Reflex impairment scores for an individual animal are then summed and divided by the total observable impairments possible to calculate proportion impairment. Impairment score is then correlated with mortality to produce RAMP. RAMP can be expanded when appropriate to include testing for reflex impairment, barotrauma, and injury.

Below are some examples of previous reflex testing in fish, crustaceans, and turtles. Davis 2010 has shown several types of fish reflexes with impairment after stress induction. In free swimming fish, studied reflexes included orientation where the fish should normally be upright, righting reflex where the fish returns to an upright position and the startle response in which the fish shows rapid forward motion in response to stimuli (Lutnesky and Szyper 1990; Artigas et al. 2005; Davis and Ottmar 2006). In restrained fish, studied reflexes included body flex upon restraint where the fish attempts to escape when restrained, dorsal fin erection in which the fins become erect when the fish is restrained, operculum and mouth closure where the operculum or mouth clamps shut when lifted or opened, the gag response where the fish opens its mouth and flexes the body when the throat is stimulated and the vestibular–ocular response (VOR) shown by eye rolls when the body is rotated around the long axis (Trumble et al. 2000; Davis 2007). Other studies of reflexes in free swimming fish have included atonic immobility, dorsal light reaction, and optomotor and optikinetic responses (Douglas and Hawryshyn 1990; McCormack and McDonnell 1994; Wells et al. 2005; Hasegawa 2006).

Raby et al. 2012 used a reflex impairment index for coho salmon modified from the previously developed RAMP method (Davis 2005, 2007). Immediately prior to release, all tagged and biopsied fish were tested for the presence of five reflexes that were consistently present in control, excellent condition fish. Each reflex was assessed categorically (0 = unimpaired, 1 = impaired) in a conservative matter – that is, if the handler had doubt as to whether the reflex was present, it was recorded as being impaired. Reflexes tested were the following: tail grab, body flex, head complex, vestibular-ocular response (VOR) and orientation. Presence of the tail grab response was assessed by the handler attempting to grab the tail of the fish with the fish submerged in water (in a fish bag or holding trough); a positive response was characterized by the fish attempting to burst-swim immediately upon contact. The body flex response was tested by holding the fish out of water using two hands wrapped around the middle of the body. The fish actively attempting to struggle free was characterized as a positive response. Head complex was noted as positive if, when held out of water, the fish exhibited a regular pattern of ventilation (for ∼5 s) observable by watching the opening and closing of the lower jaw. VOR was observed by turning the fish on its side (i.e. on a lengthwise axis) out of water. Positive VOR was characterized by the fish’s eye rolling to maintain level pitch, tracking the handler. Finally, upon release, each fish was placed upside-down in the river just below the surface: a positive orientation reflex was noted if the fish righted itself within 3 s. The entire reflex assessment took ≤ 20 s to complete and was always conducted on fish upon release. If a fish was too vigorous to allow researcher handling and assessment of reflexes, it was assigned an unimpaired status for all reflexes. The reflex actions included in our protocol are thought to be sufficiently varied that they involve different neurological pathways and/or muscle groups such that there are no redundancies. For example, some of the reflexes are part of the autonomous nervous system (head complex, i.e. respiration), while others clearly are not (tail grab, body flex). Moreover, using this RAMP protocol with Pacific salmon, no two reflexes in the suite of five are consistently present/absent together (see Results; G. Raby, unpublished data). From the reflex results for each fish, we calculated a RAMP score: a simple proportion of the five measured reflexes that were impaired in an individual fish (0 = no reflexes impaired, 1 = all reflexes impaired; Davis 2007).

Humborstad et al. 2009 in Atlantic cod.

Barkley and Cadrin 2012 in yellowtail flounder.

Campbell et al. 2009 in red snapper.  Following exposure to rapid decompression, and removal from the hyperbaric chamber, external symptoms of barotrauma were noted and the reflex responses tested.  The barotrauma – reflex(BtR) score developed was modified from the RAMP procedure developed by Davis and Ottmar (2006). All barotrauma incidences observed and reflex responses tested were categorical in nature (1 = unimpaired state, 0 = impaired state). Fish were examined for a suite of barotraumas, including expanded abdominal cavity (tightened air bladder), stomach everted and protruded from the oral cavity, intestine protrusion out of the anus, exophthalmia (eyes bulging), subcutaneous hemorrhaging, and activity level. Reflex response testing was performed out of the water over 1 min, with the fish restrained so that each test could be done in isolation. Reflex responses tested included gag, opercular, dorsal spine, vestibular-ocular (VO), and tail-flex responses. The gag response was tested by inserting a narrow probe into the oesophagus; a positive response was noted as involuntary-muscle contractions intended to dislodge the probe from the oesophagus. The opercular response was measured by observing whether the fish actively attempted to ventilate the gills (gilling). Active gilling was considered a positive response. The dorsal-spine response was tested by moving a probe over the dorsal spine, causing it to fold back. Positive dorsal spine response was noted when spines returned to an erect defensive position. VO response was observed by turning the fish along its lateral axis. Positive VO was noted when the eye rotated within the orbit and refocused on a fixed position (the observer). Tail-muscle flex was tested by inserting a syringe needle into the hypaxial musculature of the subject. Positive tail-flex response was noted if the muscle contracted. If the subject demonstrated hypaxial-muscle contraction (tail flapping) before insertion of the needle, it was considered positive and no needle insertion took place. To calculate the BtR score, the total number of barotraumas and reflex responses present was summed, divided by the total number possible, and subtracted from 1 [BtR = 1 - (summed individual responses/total responses possible)]. A BtR score close to 0 indicated a fish with low impairment, and a score close to 1 indicated reflex impairment or elevated level of trauma. Following BtR observations, the subject was placed in a test arena for predation simulation and measurement of performance responses. For the thermocline-exposed fish (T), the test arena had previously been heated 7oC above ambient, and for the non-thermocline-exposed fish (NT), it was kept at ambient temperature. Predator attack was simulated from behind a screen by rapidly thrusting a dipnet directly at the lateral surface of the fish. Predator simulations were administered at 0, 5, 10, and 15 min post-decompression, which allowed investigation of the time-course of impairment and estimation of recovery times. Response variables measured were closest simulated predator approach distance (AD, cm), maximum burst swimming speed attained (BSS, cm/s), and the amount of time the fish spent reacting to the stimuli, or the duration of the response (Dur, s). A video camera (Sony DCR-TRV117, 32 frames/s) was mounted above the testing arena to film the sequence of predator attack simulation and subject response. A grid was placed on the bottom of the test arena to assist in calculations of AD and swimming speed. 

Stoner 2012 in crustaceans.

Stoner 2012 in spot prawns.

Stoot et al. 2013 in turtles.