Philosophy of using RAMP to measure vitality, survival, and mortality of animals

Blue sharks and food, WASC

Mortality can occur over varying time frames after an animal is exposed to potentially lethal stressors. The problem of mortality prediction is made more difficult by animal mobility, as animals can become hidden from observation, especially over longer time frames. Then indicator measures must be used to predict cryptic delayed mortality. What is an effective indicator for predicting mortality? Do we observe the animal immediately after stress induction and before leaving our presence, or do we observe the conditions in which the animal was stressed? 

A common approach to predicting delayed animal mortality is to observe the conditions in which stress is induced and use this information as an indicator for mortality.  Animals are experimentally exposed to important stressors and their combinations in a matrix of interactions. Then animals are sampled for mortality after holding them captive for short periods or tagging, releasing, and recapturing or using biotelemetry over longer time frames. Mortality, and its inverse, survival are then modeled from sampled combinations of risk factors. Since there are relatively unlimited sets of risk factors and their interactions, indicator models for mortality based on stressors often will not give realistic estimates or not include important conditions for stress induction.

Alternatively, animal impairment can be observed as an indicator for delayed mortality after exposure to risk factors. Reflex impairment occurs immediately in an animal when it’s neural, muscular, or organ systems are stressed. Summing presence or absence of several reflex actions calculates an index called RAMP (reflex action mortality predictor) which is a direct measure of reflex impairment and vitality. Correlation of RAMP with immediate and delayed mortality make it an indicator for mortality and survival. With RAMP, the approach of predicting mortality is based on direct observations of animal vitality. The animal continually integrates all the effects of experienced risk factors as reflex impairment and communicates it’s health state, vitality, and fitness through the language of RAMP. Other types of animal impairment that have been tested as potential indicators for mortality include physiological variables (cortisol, glucose, lactate, and electrolytes) and injury.  However these measures are not consistently correlated with delayed mortality.

In an effort to ameliorate mortality risk factors, a hybrid approach can be used for predicting cryptic delayed mortality that conserves and integrates information. Instead of asking the question “Does the animal die?” we can ask “When, where, and under what conditions does the animal die?” Animals are observed in experimentally controlled conditions of mortality risk (Davis 2002, Suuronen 2005). Then initial stressor conditions are sampled, as well as time courses for animal impairment and delayed mortality. Relationships between stressor factors, animal impairment, and delayed mortality can be identified and modeled. The resulting knowledge base can be used to test hypotheses about importance of mortality risk factors and efficacy of predicting cryptic delayed mortality using animal impairment as an indicator. Previous research has shown that reflex impairment measured as RAMP is a powerful predictor for cryptic delayed mortality (Davis 2010). After validation, RAMP can be used to test the effects of experimental or natural changes in mortality risk factors such as design of fishing gears, aquaculture rearing conditions, aquarium trade, pollution exposure, climate change, and other potentially risky situations.

Trawl bycatch reduction device, FRDC

The problem of using indicators to predict cryptic delayed mortality is simplified by shifting from modeling mortality in potentially unlimited sets of risk factors to direct, real time measurement of animal impairment and prediction of delayed mortality. This shift in focus to reflex impairment allows for real time testing of animal fitness in systems of interest and is a cheaper, more efficient use of limited research resources than using risk factor indicators for mortality prediction.

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.

Science and reflex impairment testing using RAMP

RAMP is a method for testing whole animal reflex impairment and can be used in scientific inquiry.  The method of science includes three key elements for the explanation and prediction of observations through hypotheses.  Scientific hypotheses are 1) confirmable, meaning that they can make unexpected predictions about outcomes of experiments; 2) falsifiable, meaning that they can be tested by specific experiments; and 3) unique, meaning that they are the simplest and most plausible explanations for experimental observations.

Scientific hypotheses and explanations that fail these three tests are abandoned as not consistent with rational thought and discourse.  There are, of course, unlimited non-rational explanations for observations and perceptions and these are the purview of imagination, art, religion, philosophy, and politics as subjects of opinion and not necessarily open to testing, validation, and changing minds through discourse.  Lots of fun can obtain from imagination and humans consistently enjoy imaginative play outside of the realm of scientific inquiry.

The RAMP method certainly contains the three tests for scientific inquiry.  The RAMP reflex tests are specific observations of animal responses that are stable and repeatable under control conditions and that change as animal vitality is impaired.  These tests are designed as a quantitative language to communicate whole animal vitality states to observers.  RAMP can be used to confirm new predictions about animal vitality, morbidity and mortality.  Animals can be experimentally exposed to stressors (light, sound, temperature, hypoxia, injury, handling, and xenobiotics) in a wide variety of ecologically and economically relevant settings.  Specific stress outcomes (vitality, recovery, morbidity, and mortality) can be predicted using RAMP and then validated by observations through time and space.  RAMP can be used to falsify specific hypotheses concerning relationships among stressors, animal vitality, and stress outcomes.  RAMP can be used to identify unique explanations for experimental observations of animal outcomes in stressful conditions because of its singular expression of animal vitality, without being confounded with other factors, i.e., motivation, avoidance, attraction, size, sex, and species.

Additionally, RAMP is an interesting formulation of specific animal neurological, muscle, and organ reflex actions.  While RAMP accurately measures whole animal reflex impairment, the mechanistic details of reflex actions remain to be studied, as little is known about how stressors affect neurological function and associated reflex actions in different phyla.  The fact that reflex impairment is almost immediate after exposure to acute stressors suggests that neurological changes are important primary stress responses.  However, quantification of neurological dynamics is difficult and expensive because of their ephemeral nature.  Although rapid neurological changes in a stressed animal may be short-lived, RAMP data show that these changes exert profound control of reflex actions and later fitness outcomes.

Reflex impairment related to pollutant concentrations

Reflex impairment can be used to test for the behavioral and fitness outcomes of exposure to possible pollutants.  A recent study tested effects of triclosan on reflex impairment in an estuarine fish, Atlantic croaker:

"The effects of triclosan on reflex responses and anti-predator behavior in an estuarine fish

Tiffany L. Hedrick-Hopper and Sandra L. Diamond, Department of Biological Sciences, Texas Tech University, Lubbock, TX Background/Question/Methods Triclosan is a common antibacterial compound found in an increasing number of personal care products including toothpastes, deodorants, and soaps. Despite partial removal by wastewater treatment plants, an increasing amount of triclosan is entering watersheds where it can have significant effects on aquatic organisms. Even at low levels, triclosan negatively impacts thyroid homeostasis in anurans and fish, and it can decrease startle responses and activity levels in anurans. The purpose of this research was to investigate the effects of triclosan on reflex responses and anti-predator behavior in juvenile Atlantic croaker (Micropogonias undulatus), an estuarine fish. Sixty Atlantic croaker were held in individual tanks and randomly assigned to be fed a diet of either normal food pellets or pellets impregnated with 50 ppm triclosan for 14 days. Both prior to and immediately following the 14 day exposure, fish were tested for a suite of reflex action mortality predictors (RAMP) and were subjected to a video-recorded 30 second simulated predator attack. Videos were then analyzed for the specific strategies (run, hide, cut across tank, turn gambit) employed by the fish before and after exposure. Results/Conclusions We found that fish exposed to triclosan were significantly more likely than control fish to exhibit reflex impairment. Specifically fish lost the dorsal spine erection response, meaning that they did not raise their dorsal fin when the fin was flattened. Reflex impairment is correlated with increases in overall fish stress and mortality outcomes. Treated fish also experienced significant shifts in their anti-predator strategies. Triclosan-exposed fish spent significantly more time in their post-exposure test hiding from the simulated predator than fish in the control group. In some cases, fish continued to stay stationary even as the simulated predator touched them. The results of this study indicate that triclosan does impair fish reflexes and creates shifts in the strategies used by croaker to escape their predators. Since these schooling fish have been shown to exhibit dominance hierarchies, triclosan may affect social patterning in Atlantic croaker. These behavioral effects may have important implications not only for croaker and similar fish species but also for croaker predators such as bottlenose dolphins as contaminated fish may be easier prey, leading to increased predator body burdens." This study used free-swimming fish and video analysis of reflex responses.  The RAMP approach can easily be adapted to pollutant research and aquaculture settings for efficient, real-time monitoring of supply waters and sediments, as well as health conditions for animal rearing.  In the basic research context, neurobiological studies of zebrafish have made use of reflex testing in pharmacology and toxicology laboratory settings.