Assessment of reflex impairment and mortality in discarded deep-sea giant isopods

Giant isopod, Wikipedia

Giant isopods were subjected to simulated capture and discarding by Talwar et al. (2016). Reflex impairment and mortality were induced by capture, exposure to air, and time at surface before discarding. Reflex actions tested are included in Table 1.

Talwar et al. (2016)

Six reflex actions were tested in control animals. Impairment of antennae extension and pleopod movement were not associated with mortality and were removed from the mortality analysis. Figure 1 shows the relationship between increasing reflex impairment and increasing mortality. 

Talwar et al. (2016)

Note that an impairment score of 0 was associated with 50% mortality. Clearly this score does not mean that animals were not impaired. Stressed animals were initially impaired without associated mortality, as indicated by the loss of antennae extension and pleopod movement.  Removal of these two reflex actions from scoring and the mortality analysis may have produced a tighter analysis, but fails to show the sublethal effects of the experimental stressors. 

Why observe several reflex actions together to measure animal vitality?

Philipchircop 2012

Why observe several reflex actions together to measure animal vitality? The short answer is that animals are whole beings; a summary collection of component parts and their interactions in response to stimuli.

Animals are constructed of biochemical and behavioral components that interact to form a whole; capable of responding to stressors. The interactions of stressors and behavior are also important for prediction of vitality impairment and survival. Reflex actions are fixed behavior patterns that include biochemical, muscle, organ, and nerve functions.

Efforts to identify factors that can control vitality and predict post-release survival and mortality of captured animals generally strive to identify single important variables. For example, temperature changes, injury, exhaustion, and hypoxia can control vitality and survival. For simplicity, single factors are statistically modeled as predictors for survival. Factor interactions are rarely considered because of their complexity.

Patterns of vitality impairment vary with species and contexts. Observing impairment of several reflex actions and possible injury in a defined context integrates the effects of multiple stressors, contexts, and their interactions on animal impairment and survival. Measurement of single reflex action impairment can miss the range of vitality that spans from excellent to moribund. 

Stoner 2012 (crabs)

Below are several examples of the cascading nature of impairment observed as individual reflex actions cease to function in a spectrum of stressor intensities. Reflex actions with higher proportion of impairment are impaired before those with lower percentage. Note that patterns of impairment vary with taxa and context.

Davis 2010 (walleye pollock, coho salmon, northern rock sole, Pacific halibut)

Stoot et al. 2013 (turtles)

Uhlmann et al. 2016 (plaice, sole)

Forrestal 2016 (triggerfish)

Forrestal 2016 (yellowtail snapper)

Danylchuk et al. 2014 (lemon shark)

McArley and Herbert 2014 (snapper)

Sampson et al. 2014 (mottled mojarra)

Stoner 2009 (Tanner crab, snow crab)

Stoner 2012 (spot prawn)

Triage for captured and released fisheries species: research and survival

Will they survive? (The Guardian, 2013)

Vitality impairment can be linked to post-capture mortality in fisheries bycatch. Vitality impairment can be estimated by direct observation of animal activity, responsiveness, and injury. For each critical fisheries species in crabs, fishes, sharks, and turtles, reflex actions that are consistently present in healthy, uninjured individuals are listed as control levels. Impairment is signified by loss of reflex action types and addition of injury types after capture.  

Reflex actions are fixed, consistent animal behavior patterns that can be triggered by perception of external stimuli (light, sound, smell, gravity, touch). Stimulation of reflex actions is not controlled by body size, motivation, strength of stimulus, or fear. Reflex action traits summed as a whole animal can be an expression of vitality (Davis 2010). In contrast, volitional behavior can be altered by body size, motivation, strength of stimulus, fear, cognition, and as such is not a controlled measure of vitality.

With the species reflexes and potential injury lists, observations of captured animals can be made in commercial and sport fisheries. Patterns of significant impairment can be determined and related to fishing context and species (Raby et al. 2015). These patterns help identify the relative effects of fishing gears, handling, and physical factors (air, temperature, light, pressure) on impairment and potential survival and mortality.

Figure shows overlap between information on animal physiology and fisheries biology, adapted from Horodysky et al. 2015 and modified to show vitality information. Measures of vitality include reflex impairment and injury, which are whole animal measures that are ecologically relevant, linking physiological and population level research and hypothesis testing. Volitional behavior is coordinated whole animal movements beginning with perception and motivation, followed by attraction and aversion to various stimuli (injury, threat, food, shelter, species mates, migration).

Patterns of vitality impairment can guide research questions and priorities to triage fisheries for treatment of bycatch mortality and enhancement of survival. Vitality impairment can measure the efficacy of engineering fishing gears to increase bycatch survival. 

Observing vitality impairment

Animal vitality can be measured by observing species traits associated with activity, responsiveness, and injury. For each species, a group of reflex actions can be observed that are consistently present in healthy animals. As vitality becomes impaired, reflex action traits disappear and injury traits may begin to appear. 

Activity, responsiveness, and injury for measurement of vitality impairment (Benoît et al. 2010). 

Fisheries show gradients of stressors associated with capture, handling, and release or escape. Discard mortality, survival, and vitality impairment are controlled by stressor gradients.

Gradients of mortality and simulated stressors in sablefish fisheries; water temperature and gear type including trawl (time), longline, pot. Smaller fish are more sensitive to stressors (AFSC).

Vitality impairment gradients are associated with stressors and can be used to predict survival and delayed mortality for populations of impaired animals. Vitality impairment gradients can be measured by identifying classes of health condition; excellent, good, poor, and moribund based on rapid observation and impression of animal injury and activity (Benoît et al. 2015). 

The resolution for observations of vitality impairment gradients can be increased by including more information. RAMP is an example of this approach (Davis and Ottmar 2006, Davis 2010). A list can be made of reflex actions present in control animals and possible injuries. Then presence or absence of listed traits is observed after exposure to stressors. Increasing impairment is associated with stress effects and morbidity.

Reflex actions observed in snapper by McArley and Herbert 2014.

Relationships between vitality impairment and survival or delayed mortality can be experimentally determined. Then predictions of stress effects in other settings with similar stressors can be made by measuring vitality impairment associated with stressors, without the need to hold or tag animals. Vitality impairment can be rapidly observed in sampled populations as an additional factor to evaluate stressor effects and is a useful indicator of animal health and stress status, that can be validated experimentally.

Reflex impairment and mortality for individuals (A) and groups (B) of Atlantic cod with 95% confidence intervals (Humborstad et al. 2009). 

Elements of vitality testing and delayed mortality in fisheries

Conceptual diagram outlining elements for vitality testing and delayed mortality in fisheries. Fish are captured and environment sampled. Fish become stressed which is measured as impairment from control health by observing reflex actions and injury types. Stressed fish are held for captive observation to determine delayed mortality. Bias and error can be introduced by initial impressions of vitality before testing reflex actions and injury, by differing observer scoring opinions, and by holding conditions that are stressful for the fish. 

Scoring vitality impairment is most difficult when observer decision is used. Training observers is a key part of RAMP development. Reflex actions (RA) are clearly present in control animals, and observers do not need decisions to score present. As impairment increases, scoring RA requires increasing observer decisions about whether sampled RA are present. The decision can be based on how control RA appear to trained observers. Each observer will have different opinions that can be influenced by their initial impressions of the animal and of the stressor treatments the animal has been exposed to.

Initially after stress induction, RA impairment increases and mirrors stress levels, while mortality is not evident. When animals reach a critical impairment level, replicates begin to show mortality, which increases rapidly over small changes in RA score. At highest levels of impairment decisions are less frequent as the animal ceases general movement and responsiveness.

Observer bias and RAMP

Cognitive bias (The Daily Omnivore, 2012)

Subjective scores for animal behavior can be biased by observer opinions about experimental treatment differences and resulting outcomes (Tuyttens et al. 2014). The research paper title expresses a fundamental bias of human perception and belief: “Observer bias in animal behaviour research: can we believe what we score, if we score what we believe?” The problem is to separate belief from observation. This may be accomplished by clearly defining and adhering to consistent protocols for behavior observation and analysis.

RAMP relies on subjective scoring for presence or absence of reflex actions or injury types. Control fish have a suite of reflex actions that are consistently and clearly present when tested for. When an observer begins to notice the weakening or complete loss of a reflex action, that action is scored as absent (impaired). There will be variation among observers in the decisions about when reflex actions are impaired and bias will vary with experimental protocol. 

Because RAMP is an aggregate vitality impairment index summed from control reflex actions and potential injury types, a RAMP score includes the observer bias for each included reflex action and injury. Close correspondence of RAMP scores and mortality is noted at low and high scores because observers clearly know when fish are active and when fish are severely injured and impaired. Relationship of mortality and RAMP is more variable at intermediate levels of impairment and mortality in part because observer opinion about impairment is more variable. To reduce observer bias, RAMP for a species must be designed to include reflex actions and injury types that can be clearly separated into present or absent scores. Also experimental treatments can be administered without informing observers.   

Vitality of a stressed fish is readily observed. We are primarily seeing the activity, responsiveness, and injury presented by the animal. The most widely used vitality index in commercial fisheries is for the halibut fisheries of the northeast Pacific Ocean (AFSC Observer Manual 2015), based on Appendices S-X for trawl, pot, and longline fisheries.  For trawl and pot fisheries, three levels of vitality (excellent, poor, and dead) are scored by observing injury types and spontaneous activity, startle response to touch, and operculum clamping. For longline fisheries, vitality is scored by observing injury types. Mortality rates are assigned to vitality impairment scores using tagging experiments (Williams 2014).

Vitality impairment codes (Benoît et al. 2010).

Benoît et al. (2010) constructed a fishery vitality index with four levels of impairment (excellent, good, poor, moribund) that are scored by observing injury types, spontaneous body movement, startle to touch, and operculum clamping. Their vitality index and the halibut vitality index use the progressive increase of injury and impairment of activity to score vitality impairment. Benoît et al. (2010) corrected for observer bias by using a random effects term in their statistical model. 

Reflex actions scored for presence or absence in RAMP for snapper (McArley & Herbert 2014).

The RAMP vitality index alters impairment scoring to only include presence or absence of a larger number of injury and reflex actions. This shift attempts to introduce more information about activity and injury types that may be associated with mortality and to reduce decisions about degree of impairment for individual activity and injury traits. Impairment is observed as a progressive increase in the number of reflex actions that become absent and the number of injury types that become present when compared to control animals. Because observer bias can be introduced in scoring, observer protocols must be well defined with clear rules for presence or absence of traits. Observer judgements about correspondence between experimental treatments and outcomes could also be eliminated by careful experimental design.

Belly up: Righting reflex action time to recovery correlated with delayed mortality?

Upside down fish in market tank (Hong Kong)

RAMP incorporates presence/absence of several reflex actions and injuries to measure vitality impairment and potential delayed mortality. A simpler method may be possible by measuring time for recovery of orientation when fish are placed upside down in water. This method can be tested.

Place a fish upside down in water and observe the time until the fish returns to normal orientation. This duration is a measure of vitality impairment. Longer recovery times indicate greater vitality impairment and data can be included in statistical models for relationships among fishery stressors, injury, righting time, and delayed mortality. We can test the relationship between righting impairment and delayed mortality. 

Righting reflex action is a central behavior that is the nexus of neural, muscle, and organ actions and is intimately linked with loss of physiological regulation associated with stressor exposure.  Olfactory impairment is another example of a central function that is correlated with delayed mortality (in humans, Pinto 2014).

Body orientation is a sensitive measure of fish consciousness. Presence or absence of righting can be included in the RAMP score. Loss and recovery of orientation is a well known symptom for induction of and recovery from fish anesthesia and is used as an indicator of morbidity and delayed mortality in stress experiments (Davis and Ottmar 2006, Szekeres et al. 2014, Raby et al. 2015).  

Measuring replicate animals for the time to righting recovery and delayed mortality after a stressor experiment can test the correlation between righting impairment and delayed mortality. If the correlation between righting and delayed mortality is valid and strong, then we have a rapid method for predicting discard mortality on board fishing vessels without need for holding or tagging fish to confirm their survival. Research groups on fishing vessels can observe fish during catching, landing, sorting, and discarding under differing stressors; seasons, water temperatures, tow durations, catch quantities, species mixes, and sorting times.

The importance of vitality in fishing experiments

Key fishing stressor factors, Davis, 2002

Knowledge of key factors controlling fisheries is necessary for sustainable management of fishery stocks. Scientific hypothesis testing in the form of fishing experiments is a necessary component of fisheries knowledge development and validation. Fishing experiments are performed in the field by simulating actual fishing conditions, by actual fishing, and during survey cruises. Fishing experiments can be used to identify key stressor factors that control and contribute to the survival and mortality of captured, discarded, or escaped animals as well as identifying the key factors controlling fishing gear capture efficiency and selectivity.

Trawl captured animals, Robert A. Pawlowski, NOAA Corps

While field fishing experiments represent realistic conditions, they are a matrix of confounded factors which cannot be easily separated into mechanistic hypothesis tests and explanations of factor importance. Effects of factors are often synergistic and prior animal stressor history can alter relative effects of subsequent exposure to factors, e.g., depth changes, injury, elevated temperature, air exposure, and size and species differences.

Flow chart of experimental fishing stressor factors, Davis and Olla 2001

Simulated fishing experiments with factors in controlled laboratory conditions is one way to test hypotheses about mechanistic effects of individual factors and their interactions. However these laboratory experiments are generally viewed as not realistic to field conditions and they are used to identify factors that may be important in the field. Furthermore, modern requirements of animal care laws and committees restrict the use of laboratory fishing experiments by not allowing human application of experimental stressor factors on animals and the use of mortality outcomes. These same laws and committees do not have jurisdiction over field fishing experiments. 

Laboratory trawl tow tank, NOAA RACE

Given that factors are confounded in field fishing experiments, how can we test for effects of factors in the traditional mechanistic hypothesis test? We can test for changes in animal vitality. Since vitality has been shown to be correlated with survival and mortality, it is a useful indicator of animal outcomes before and after exposure to experimental stressor factors. For example, we generally do not know the exposure of animals to stressors prior to experimental manipulation of factors. Not knowing the complete stressor profile is not an obstacle since the animal knows the complete stressor profile and presents vitality levels that have integrated the effects of that profile. Then we can expose animals to additional stressor factors and measure further changes in vitality from their initial levels. 

Important to shift mechanistic thinking from needing to know the effects of individual factors to knowing the effects of fishing variability. Manipulations of time in air and elevated temperature represent differences in fisher sorting and handling behavior on deck and are appropriate for defining the effects of fishing variability. Effects of variation in tow time and catch quantity can be manipulated and are included in the mix of animals landed. The questions of associations among individual fishing stressor factors is left for another day and are more of interest to mechanistic scientists than to managers and fishers. Fishing variability will give a picture of the fishery and its potential effects on animal vitality. By measuring animal vitality, which integrates the effects of stressor factors, you have measured a key master variable that indicates the important effects of fishing.

Vitality is the key variable that can be used to indicate and predict delayed survival and mortality outcomes for discards and escapees from fishing. The relationships between vitality and survival and mortality are defined by captive observation or tagging and biotelemetry experiments. During exposure of animals it is important to insure that all stressor types normally in the fishery in question are present for the population of tested animals (e.g., temperature, air exposure, fatigue, injury) and that a full range (0-100%) of vitality impairment and mortality are observed. Then relationships can be calculated for each species of interest that do not extrapolate beyond available data ranges and that apply to the fishery of interest. These relationships can then be used to predict survival and mortality for animals under any condition of interest in the fishery without the need for further captive observations or tagging.

Consider how scientific peer-reviewers may see this shift from mechanistic thinking and develop thoughts that elaborate the importance of vitality from the animal’s point of view. Some resistance is expected from mechanists who believe that they can attribute cause and effect to individual factors. There is always a matrix of interactions, even under the most restrictive and controlled experimental conditions. There are always interactions and synergisms to account for. As a result, there are associations among factors, rather than cause and effect. In other words, there are causes and conditions associated with effects. 

From the fishing experiment perspective, we set up fishing conditions that are real or that simulate fishing and then measure animal vitality, which is an integrated measure of the effects of interacting factors. It is useful to identify the important stressors by experimentally changing them in fishing experiments; changes in time in air, trawl time, trap retrieval time, depth, season, temperature, catch amount, and injuries. Always remember that there is a hidden context of conditions, i.e., the animals are prestressed by other factors not being controlled. But this hidden context can be accounted for by observing and comparing vitality impairment among animals observed in all treatments, including simply captured animals without additional stressor exposures (using positive controls). This experimental approach is useful both for fishers who wish to modify fishing gear and practices, as well as managers who wish to observe animal vitality and correlate that with mortality and survival.

Measuring reflex action impairment in sole and plaice; preliminary steps to making RAMP

Collection of fish with beam trawl, Jochen Depestele

The beginning steps for measuring reflex action impairment and making a RAMP are detailed in “Calibration tests for identifying reflex action mortality predictor reflexes for sole (Solea solea) and plaice (Pleuronectes platessa): preliminary results” authored by Depestele, J. et al. 2014. Experiments were designed to collect sole and plaice using short hauls of a beam trawl, to test their reflex actions, and to identify consistent reflexes for making RAMP. These experiments followed steps for making RAMP.

A short video demonstrates testing plaice for reflex actions including righting, eye roll (vestibular-ocular response), evade, operculum, mouth, and tail grab. Fish are shown in a series of increasing impairment.

Conclusions from the study included:

Preliminary investigations have been undertaken on-board the RV Belgica to assess the potential presence of a range of reflexes in sole and plaice. A wide range of potential reflexes was investigated prior and during the sea trial, leading to a final selection of seven reflexes with a good potential of being consistently present in fish in a favorably vital condition. Fish in a “perfect” condition could not be retrieved, but 22 individuals of plaice and sole were selected from short hauls and their survival potential was evaluated during 70 hours in on-board holding facilities. Only one sole died, and indicated hence that the control fish for the calibration test serve purpose.

Holding tanks for fish on board RV Belgica, Jochen Depestele

The final selected reflex actions were very similar for sole and plaice, except for one. Forced opening of sole’s operculum did not reveal much resistance of the fish, while holding plaice by its head did not induce curling of the fish. The most consistent reflex actions for sole were called “stabilize, mouth, and tail grab”, followed by the “vestibular-ocular response”. Vital individuals seemingly dig into the sand or stabilize themselves onto the floor of the water-filled box. They also keep their mouth closed when trying to open it with a probe. When fish have stabilized, they respond clearly to grabbing their tail or even tickling it. The “head” reflex was easy to assess, though not always present. However, it is clear that vital soles curled around one’s hand when they had been in holding tanks. This was not that obvious for fish that were just released from the codend. Natural righting was observed regularly, although some individuals remained at their backs for >5 sec and did not return to their natural position at all or only after stimulating them. The consistency could thus be questioned, but good candidate reflexes were proposed for sole, and should be further evaluated. The most consistent reflexes of plaice were the turning of the eyes when the fish was turned around longitudinally. The resistance of plaice to forced opening of the operculum was a clear reaction as well. Not fully consistent, but nevertheless a good indication of the reflexes was the “evade” response and the “tail grab”. When the tail is touched or grabbed in a “good” way (which might require some practice), then the fish swim away, or at least the fins stimulate propulsion. The mouth of plaice was easily opened, but mostly the individuals tried to close it or seemingly opposed to the forced movement.

Our investigations confirmed that on-board holding facilities result in high survival of plaice and sole from very short hauls (<20min). Investigated individuals were non-randomly selected and thus it was not surprising that their physical injuries were limited. These individuals were suitable for developing the reflexes, although they were limited in number (22 for plaice and 22 for sole) and they also did not range over a wide variety of fish conditions (e.g. limited length variability). The seven reflexes from these preliminary investigations are therefore proposed as candidates for the development of a RAMP score for sole and plaice.

The tests of the reflexes were run directly after releasing fish from the codend. When examining the survival from fish that were accommodated for some time (e.g. 48 hours), we noted that they reacted more strongly and had much clearer responses to the reflex tests. In particular the tail grab worked very nicely for sole when their status (alive or dead) was tested. Therefore we suggest that the proposed reflexes are tested once more on surviving individuals of short hauls after an accommodation period of >24hours. Consistency of the outcome of the reflex tests is expected to be improved when the impairment from the catching process is accounting for. Other recommendations for follow-up tests relate to the registration of potential environmental and biological confounding factors.

Survival of the fittest

Bob van Marlen, IMARES

Work is in progress at ICES to define methods for estimating discard survival in reference to the new European Commission Common Fisheries Policy. The ICES newsletter states, “Under the recently reformed European Commission Common Fisheries Policy (CFP), the practice of discarding fish will be phased out, replaced instead with landing obligations. Under the landing obligation, all catches of regulated species must be landed and counted against quotas unless it has been scientifically proven that the species can survive the discarding process. Species that display a high level of discard survivability will be awarded an exemption, meaning that fishers can return these fish to the sea. Unregulated and protected species will continue to be released.”

The ICES WKMEDS focuses on developing guidelines and identifying best practice for undertaking experiments to investigate the survival of organisms discarded from the catches of commercial fisheries. 

Importantly ICES states, “Techniques for estimating survival under review include captive observation, vitality assessment, and tagging and biotelemetry, each with its own advantages and disadvantages. By using a combination of techniques, as WKMEDS suggests, clear synergies can be achieved and challenges overcome.”

"It's an exciting time", state the workshop Chairs Michael Breen and Tom Catchpole, "this group will be central to an international community that are working together to address the important issue of discard survival."

The WKMEDS group is producing a synthesis of previous discard survival and mortality research with the goal to develop an integrated approach for estimating discard survival. The integrated approach is designed to guide scientists and managers in their evaluation of potential candidate fisheries and species for discarding exceptions to the landing obligation of the CFP. The integrated approach incorporates information about the role of fishing conditions as stressors, species sensitivity to stressors, vitality impairment of captured species, estimated discard survival rates, and cost-benefit analysis of methods for survival assessment in fisheries as well as impacts of discarding on fisheries stocks.

Quest for a discard survival predictive scoring system to use on board fishing vessels

Releasing tagged Atlantic cod, John Clarke Russ

The European Union Common Fisheries Policy (CFP) ban on discarding allows for animals to be discarded if “scientific evidence demonstrates high survival rates”. Estimating discard survival for fisheries has become a priority for implementation of the CFP. Limited data on discard survival and mortality is available and methods for estimation have not been standardized. Ideally, a standardized numerical scoring system can be developed and validated, based on readily observable responses and symptoms present in animals that are candidates for discarding and survival. 

RAMP is an example of a predictive scoring system for vitality, survival, and mortality, based on animal reflex actions, barotrauma symptoms, and injury that can be observed in fishing operations where real time decisions must be made about potential discarding. See post for RAMP development and validation; also Davis (2010) and Stoner (2012) for reviews of RAMP method. Other uses for RAMP are in live fisheries, aquaculture, and pollution research and monitoring.

For inspiration and alternative perspectives, examples of validated mortality predictive scoring systems can be found in human and veterinarian intensive care unit (ICU) settings, where patients present with symptoms and disease likely to result in morbidity and mortality (Rockar et al. 1994, Bouch and Thompson 2008, Timmers et al. 2011). Measurements of blood plasma and urine variables commonly made in ICU settings are not contemplated for RAMP since they are not readily made on board fishing vessels.

Below is an example of human ICU mortality prediction using the SAPS II scoring system. Note the similarity to RAMP curves for mortality prediction.

SAPS II mortality predictive scoring system, ClinCalc

Celinski and Jonas (2004) discussed scoring systems developed for the human ICU environment:

“How are scoring systems developed? All available data types and variables can potentially be used to create a scoring system. However, to make it useful, variables have to be selected to be appropriate for the predictive properties of the scoring system. The information must be unambiguous, mutually exclusive, reliable and easy to determine and collect. Ideally, the variables should be frequently recorded or measured.

The variables can be selected using clinical judgement and recognized physiological associations or by using computerized searching of data (collected from patient databases) and relating it to outcome. The variables are then assigned a weighting in relation to their importance in the predictive power of the scoring system (again by using clinical relevance or computerized databases).

Logistic regression analysis, a multivariate statistical procedure, is then used to convert a score to a predicted probability of the outcome measured (usually morbidity or mortality) against a large database of comparable patients. Lastly, the scoring system must be validated on a population of patients independent from those used to develop the scoring system.”

For discard survival prediction, groups of animals, rather than individuals, are the appropriate unit for consideration since proportion mortality is the determined outcome during index development and validation. These groups can represent various scales of resolution in fisheries of interest, i.e., single tows or traps, sets of longline, trap, or gill-net, daily catch.

Jean-Roger Le Gall (2005) discussed the appropriate use of ICU severity scoring systems:

“A good severity system provides an accurate estimate of the number of patients predicted to die among a group of similar patients; however, it does not provide a prediction of which particular patients will in fact die. Using a well-calibrated severity model, we can reasonably expect that approx. 75% of patients with a probability of mortality of 0.75 will die, but we cannot know in advance which of those patients will be among the 25% who will live. Furthermore, these 25% will not have falsified the odds but will have confirmed the validity of the probabilities. 

       The possibility that clinical decisions can be augmented by having an objective (although not always more accurate) assessment of a patient’s severity of illness is appealing. Physicians are interested in severity systems for individual patients as an adjunct to their informed but subjective opinion. Using these tools as part of the decision-making process is reasonable and prudent. Using these tools to dictate individual patient decisions is not appropriate. Decisions will and should remain the responsibility of the individual physician and should be based on a number of criteria, one of which is severity as estimated by a well calibrated scoring system.”

Stacy et al. (2013) discussed development and appropriate use of a predictive scoring system for survival in Kemp’s ridley sea turtles:

“Three mortality prediction indices (MPI) scoring systems were developed using different combinations of blood analytes, with anticipation that at least one of the three would be more accurate in predicting mortality in sea turtles within 7 days after admission. Turtles with higher scores were categorized as physiologically deranged to a degree that could result in death, and turtles that received lower scores were categorized as physiologically stable and likely to survive. Categorization of each turtle was then compared to the known outcome for that individual.

Receiver operating characteristic (ROC) analysis was used to assess the diagnostic performance of each MPI scoring system (Greiner et al., 2000; Giguere et al., 2003). The ROC analysis produces a plot that is used to estimate the area under a ROC curve, which is a summary statistic of diagnostic accuracy. A perfect test [i.e., sensitivity (SE) = 100% and specificity (SP) = 100%] will produce an area under the curve (AUC) = 1. The AUC can be used to distinguish a non-informative test (AUC = 0.5), a less accurate (0.5 < AUC ≤ 0.7), moderately accurate (0.7 < AUC ≤ 0.9), highly accurate (0.9 < AUC < 1), and perfect test (AUC = 1)."

ROC analysis of Kemp's ridley sea turtle mortality predictive scoring system, Stacy et al. 2013

"It is clear that results of mortality prediction indices (MPI) scoring systems cannot be used indiscriminately to make euthanasia decisions, because this would result in euthanasia of some turtles with a falsely positive MPI score that would otherwise survive. As with other health scoring systems in human and veterinary medicine, the MPI scores should not prevent clinicians from providing care to an individual, and euthanasia decisions should only be made in light of numerous other clinical factors, including neurological status, vision, ability to forage, ability to swim, pain and suffering, and duration of illness. Finally, MPI scores may be useful when applied retrospectively in a stranding event for comparison of various treatment outcomes within a facility or among different facilities. Thus, the MPI could provide an objective assessment tool of treatment success and contribute to the advancement of medical care in sea turtles.”

Assumptions for use of RAMP

Loggerhead sea turtle escaping trawl, NOAA

Here is a list of key assumptions for the use of RAMP. The list is probably not exhaustive and can be added to as new perspectives and research warrant. These assumptions have been experimentally tested and validated to various degrees by peer-reviewed published research. Further validation is useful and helps to better define possible error terms in RAMP curves. Healthy, control animals are assumed to have a full complement of reflex actions present. See choices for reflex action testing. 

Vitality is inversely related to reflex impairment. Animal vitality is an abstract concept for which we have strong intuitive notions related to observing absence of injury and presence of behavior, including activity and responsiveness. Reflex actions are fixed response patterns to stimuli that clearly reflect internal state without confounding factors. By using reflex actions to quantitatively measure vitality, the confounding effects of volitional behavior and motivation that are often more related to external conditions can be eliminated. Also animals may not be injured, yet show reflex impairment and reduced vitality associated with other factors (e.g., temperature, exhaustion, hypoxia, and xenobiotics).

Reflex impairment is directly related to stressor types and intensities.  Stressors have been shown to induce reflex impairment, interpreted as symptoms of stress. Therefore reflex impairment is a useful measure of stress. Reflex impairment integrates the effects of stress in whole animal responses that are ecologically meaningful for vitality and fitness outcomes. An impaired animal can have morbidity or decreased predator avoidance, feeding, sheltering, migrating, and reproducing.

Reflex impairment occurs immediately after exposure to stressors. Time course studies for several species have shown immediate impairment after exposure to stressors.  Animals with lower levels of stress can then recover full reflex actions hours to days after exposure to stressors. Reflex actions are sensitive measures of sublethal acute and chronic stress as well as predictors of delayed mortality.    

RAMP curve is different for each species and related to stressor sensitivity. Each species has reflex responses that are evolved for habitat types in which they occur. Differences in reflex types and responsiveness among species are apparent in body types, predator avoidance, habitat choices, and feeding strategies. Some species are easily injured and reflex impaired, while others resist injury or are relatively insensitive to environmental insults (e.g., temperature, hypoxia, and hydrostatic pressure).

RAMP curve used for a species is experimentally derived by inclusion of appropriate types of stressors and animal sizes, ages, and sex. RAMP curves must be derived from reflex impairment observed in animals experimentally exposed to combinations of stressors present in systems of interest. Also animals representing size, age, and sex of interest should be included in impairment experiments. The experiments should result in animals with reflex impairment that ranges from 0 to 100%, with accompanying mortality. The curve must include the complete range of impairment and mortality to avoid extrapolation beyond available data. 

RAMP curve is stable for a species and comprehensive experimentally tested conditions. The stable RAMP curve, with defined conditions of reflex types and testing, can be used among widely different situations for measuring animal vitality, survival, and delayed mortality. Exceptions have been noted for larvae or juveniles with ontogenetically delayed development of reflex actions and spawning anadromous adults which show altered sensitivity to stressors.

Reflex actions in RAMP are given equal weighting rather than weighted differently. Reflex impairment used in a RAMP curve is the result of summing several reflex actions. This approach views the whole animal as the important entity of vitality and fitness. Different reflex actions may be affected by different stressor types. In stressor systems of interest, there are relatively unlimited sets of stressor combinations. Therefore, no a priori expectations of importance for specific reflex actions are made and all measured reflex actions can be equally important. However, the order of reflex action impairment relative to stressor intensity can give valuable information about species sensitivity and associated life history characteristics. 

Observers are assumed to objectively score presence or absence of reflex action in a replicable manner. This assumption is satisfied by using the “rule of doubt”. If any doubt exists about the presence of a reflex action, the action is scored as absent. If the reflex action is present without a doubt, it is scored as present. Further controlled comparisons of reflex scoring among observers is warranted to better define possible observer error terms.

RAMP mortality and survival predictions are dependent on the accuracy of captive holding, tagging, and biotelemetry experiments. To calculate RAMP curves, animals are observed for delayed mortality after initial exposure to experimental stressors. Mortality observed with captive holding is simply related to initial stress, assuming that holding conditions are not stressful.  Mortality observed with tagging or biotelemetry includes sources other than initial stress (e.g., additional stressors, predation, disease, and food limitation)(Thorsteinsson 2002).

Sedna, mother of all sea creatures, K. Sagiatok