Thompson FISH495

Development of non-invasive stress biomarkers in octopuses

Rachel ThompsonJune 1, 2009

Abstract

Octopuses are an important part of the Pacific Northwest ecosystem. Recently,local populations have been experiencing declines due to pollution and large-scaleclimate processes. Non-invasive sampling techniques were developed in an attempt tocharacterize the physiological condition of adult octopuses in a controlled environment.Potential biomarkers of stress (proteins in epidermal mucus and behavior patterns) wereidentified, and could be useful in identifying a stressed state in an octopus in the wild or in captivity. Larval developmental patterns were established by analysis of the expressionof two important developmental genes, orthodenticle-like protein and hedgehog. Thisstudy serves to provide researchers and aquarists with baseline physiological data thatcould be used to identify a stressed state in an adult octopus, or an altered developmental pattern in larvae, using techniques that are less invasive than hemolymph or tissuesampling.

Introduction

Octopuses are advanced invertebrates with a strong tie with the Pacific Northwest.Two local species are the Red Octopus (

Octopus rubescens

) and the Giant PacificOctopus (

Enteroctopus dofleini

). Both are benthic species, with ranges along the EasternPacific Ocean from Mexico to Alaska. Recently, populations have been declining due toseveral factors, including pollution and large-scale climate processes affecting the PugetSound region (Rigby et al. 2005, Villanueva and Norman 2008). Octopus species areimportant prey items for higher predators in Puget Sound. Their conservation is essentialto the maintenance of the region’s trophic structure (Onthank and Cowles 2008).

Octopuses are oviparous organisms that brood and care for their eggs duringdevelopment (Kaneko et al. 2006). Red Octopus larvae hatch approximately 6 to 8 weeksafter they are laid, and enter the water column as planktonic predators (von Boletzky2003). The timing and success of development is dramatically affected by environmentalvariables, such as changes in temperature and exposure the chemicals (Clarke et al.2009). In a laboratory setting, increased temperatures (to 23 degrees Celsius from 18degrees Celsius) accelerated the development of octopus hatchlings, and shortened their life spans by up to 20% (Forsythe et al. 1988). The effect on adult octopuses has yet to bedetermined. However, as cephalopods are poikilothermic organisms, their physiologyduring all life stages is dramatically influenced by water temperature. The challenge liesin identifying and quantifying these influences.Responses to stress in octopuses can be obvious, such as color changing whenfaced by a predator, but indicators of chemical or temperature stress are not often visual.Stress can impair immune function in many invertebrates, leading to decreased diseaseresistance (Malham et al. 2003). The cephalopod immune system consists of humoral andcellular mechanisms (Ford 1992). The common stress response of animals is intended tomaintain homeostasis when threatened by an environmental change. In cephalopods, thisresponse releases catecholamines into the hemolymph, which shut down body functionsrelated to growth, reproduction and immunity with the intent of allocating energyresources towards more immediate concerns (Malham et al. 2002). Specific effects of stressors, such as exposure to air, include decreased hemocyte counts, and a decrease in phagocytotic activity in those remaining cells. Since immune defenses are down-regulated in this manner while the animal is in a stressed condition, it is left moresusceptible to disease.

The introduction of temperature and chemical stressors can have significanteffects on the physiology of aquatic organisms, including but not limited to, disruption of development, reproduction and growth (Zala and Penn 2004). It has also been found thatexposure to chemical pollution, specifically endocrine-disrupting chemicals (EDCs), suchas those found in urbanized areas of the Puget Sound, can result in abnormal behavior invertebrates (Zala and Penn 2004). EDCs can have adverse effects on a range of behaviorsthat are controlled by hormones, including reproductive and sexual behavior, activitylevel, aggression, communication and motivation (Zala and Penn 2004). It is thereforelikely that analyzing changes in behavior patterns could be used as an indicator, or biomarker, for detecting harmful environmental contaminants (Zala and Penn 2004). Ithas been suggested that behavior might be a more useful indicator or “biomarker” thanstandard assays. Behavioral assays are non-invasive, inexpensive and potentially more powerful than other methods, since behavior is the outcome of many complexdevelopmental and physiological processes (Zala and Penn 2004). On the other hand, behavior can be difficult to measure and highly variable. The combination of behavioraland physiological data could provide a comprehensive method for characterizing theoctopus stress response.Gene expression patterns are reflective of the timing of particular events indevelopment. Many developmental genes are highly conserved in the animal kingdom(Ingham and McMahon 2001). Two important genes are those that code for orthodenticle-like protein and hedgehog, both of which play critical roles in body patterning andmorphogenesis. Analysis of gene expression changes can provide insight into the physiological state of an organism. For example, gene expression “rhythms” in rockyintertidal mollusks allow them to adapt to low-tide heat stress events (Gracey et al. 2008).