Volume 52, Number 2
Paul L.R. Andrews
All animals are equal, but some animals are more equal than others.
– George Orwell, Animal Farm
The above well-known quotation from Animal Farm in some ways illustrates humans’ ambivalent view of the relative status of invertebrates and vertebrates in a number of settings: in the research laboratory (differing legal protection), in wildlife conservation (e.g., the appeal of beetles vs. giant pandas), in the home (e.g., swatting flies vs. the humane disposal of mammalian vermin), and in cuisine (e.g., methods for cooking crustaceans vs. chicken). Even the terms “spineless” and “lacking a backbone” are used pejoratively.
Invertebrates are clearly considered by many to be at the “lower end” of a scale of creatures that puts humans at the extreme “upper end,” although the units on the y-axis of this graph are a matter for debate. And within the Vertebrate and Invertebrate subphyla, terminology implying some sort of ranking is often used even in academic publications—fish, which account for about half of vertebrate species, are referred to as “lower” vertebrates (e.g., Sneddon 2004) and Octopus vulgaris (a cephalopod) as an “advanced” invertebrate (e.g., Wells 1978). But Packard (1972) makes a compelling case that the “lower” vertebrate and “advanced” invertebrate are more than a match for each other in evolutionary competition.
The demarcation in the way invertebrates are viewed, and as a consequence treated, extends to the laboratory. While this issue of the ILAR Journal is very welcome and may mark a turning point, it is notable that this single issue covers about 95% of animal species (the more than 1 million invertebrate species) whereas previous issues have been devoted to amphibians, fish, and birds (ILAR 2007, 2009, 2010). This reflects the relative paucity of information in the area of invertebrate welfare and the relative youth of this topic as an area for research, with little study in major areas critical for research, such as criteria for general anesthesia.
Legislation also reflects the invertebrate/vertebrate divide. The United Kingdom enacted one of the first pieces of national legislation covering animal experimentation, the Cruelty to Animals Act of 1876 (Tansey 1998). This Act of Parliament permitted “the advancement of new discovery of physiological knowledge by experiments calculated to give pain” but it applied only to nonhuman vertebrates. It is not unreasonable to suggest that the exclusion of invertebrates indicates that, in contrast to vertebrates, they were considered to have a lesser (if any) perception of pain. Smyth (1978) comments that “invertebrates look far less like us,” so humans may find little to recognize in common with “us” in their appearance or behavior and therefore find it harder to empathize or connect at any level.
The 1876 UK legislation also reflects the species that were commonly used in physiological studies at the time (dogs, cats, rabbits, and frogs). In 1986 the legislation was revised and became the Animals (Scientific Procedures) Act; that revision did not include invertebrates but an amendment in 1993 gave O. vulgaris the same legal protection as vertebrates with respect to experimental procedures (however, to date there have been no studies on O. vulgaris under the authority of the Act).
In the European Union the October 2010 revised Directive (2010/63/EU) “on the protection of animals used for scientific purposes” covers “live cephalopods” under Article 1, 3b (however, decapod crustacea—e.g., crabs, lobsters—were included in drafts of the new EU legislation but not in the adopted directive). Member states are required to transpose it into national legislation by November 2012 and apply it by January 2013. Some of the challenges that will need to be addressed in the European Union to comply with this directive are considered in the articles in this issue.
Debating and solving the ethical and welfare issues that are often taken for granted when dealing with laboratory vertebrates in general and mammals in particular for “advanced invertebrates” will provide a useful learning process that will inform best practice when considering invertebrates that lack legal protection. The difficulties likely to be encountered should not be underestimated. Even Russell and Burch (1959, 6) in their classic Principles of Humane Experimental Technique avoided the issue: “The higher invertebrates perhaps deserve a review to themselves, but they raise many problems which would gravely complicate an account which can otherwise be quite general and confident.”
The papers in this special issue cover four major aspects of invertebrates in the laboratory: (1) the use of invertebrates in biomedical and related research, (2) the culture and maintenance of invertebrates, (3) evidence for pain and suffering and their alleviation, and (4) attitudes and their influence on regulation and oversight. Each of these will be reviewed briefly before concluding this introductory overview with some comments about future directions.
Use of Invertebrates in Biomedical and Related Research
…[T]he solution of a physiological or pathological problem often depends solely on the appropriate choice of the animal for the experiment so as to make the result clear and searching.
– Claude Bernard (1949/1865)
There is little doubt that invertebrate species have made major contributions to biomedical research even if judged only by their contribution to Nobel Prizes in the last 50 years, as illustrated in the following examples:
- Caenorhabditis elegans: genetic regulation of organ development and programmed cell death (Brenner, Horvitz, and Sulston; 2002);
- sea urchin and clam eggs: key regulators of the cell cycle (Hartwell, Hunt, and Nurse; 2001);
- Aplysia: signal transduction in the nervous system (Carlsson, Greengard, and Kandel; 2000);
- honeybee: organization and elicitation of individual and social behavior patterns (von Frisch, Lorenz, and Tinbergen; 1973);
- squid: ionic mechanism involved in excitation and inhibition in the peripheral and central portions of the nerve cell membrane (Eccles, Hodgkin, and Huxley; 1963).
Wilson-Sanders (2011) provides a comprehensive overview of the diverse species of invertebrates used in biomedical and related research, with tables summarizing their utility in studies of developmental biology, genetics, and diseases.
While the emphasis is on Drosophila melanogaster and C. elegans primarily because of their tractability for genetic and molecular studies, it would be unwise to neglect the potential of the diversity of over a million species of invertebrates to contribute to biomedical research as model organisms to reveal fundamental biological processes including those involved in disease. Among the reasons to consider invertebrates for use in research are their
(1) simpler system than that of vertebrates. This is the argument often applied to studying nervous systems (Usherwood and Newth 1975) such as that of Aplysia californica and using species such as the honeybee as behavioral models.
(2) unique or larger structure than that of vertebrates. For example, the squid giant axon (originally mistaken for a blood vessel and rediscovered by Young in 1933) and giant synapse revealed fundamental insights into the operation of neurones (see Bullock and Horridge 1965 for references). The brain-controlled skin chromatophore system of cephalopods provides another example of a unique biological system (Hanlon and Messenger 2008).
(3) properties not readily exhibited by vertebrates. Pupation in insects provided important insights into the Hox (homeotic) family of genes fundamental to the organization of body plans. The striking capacity for tissue regeneration is seen in many invertebrates but particularly in echinoderms, as exemplified by arm regeneration in brittlestars (Amphiura filiformis; Bannister et al. 2005) and regeneration of the gut in sea cucumbers (Holothuria glaberrima; Mashanov et al. 2010). It is likely that interest in these properties will increase because of the growth of research in tissue engineering for medical applications.
Several drivers are likely to increase the use of invertebrates in research, including the possibility of “replacing” vertebrate models with invertebrates (“relative substitution”; Russell and Burch 1959), although this requires an assessment of sentience so that an animal with “higher” sentience is replaced by one with “lower” (e.g., how would an ethical committee approach the replacement of a trout by an octopus to answer the same biomedical problem?); the relatively low cost of some invertebrates; and different regulatory and ethical considerations (but see below). However, the overriding justification should be a scientific one based on a “cost-benefit” analysis—that is, an assessment of the “cost” to the animal and the benefit, in the broadest sense, of doing the research.
Culture and Maintenance of Invertebrates
The use of invertebrates in research immediately raises the question of their sourcing and maintenance, as discussed by Smith and colleagues (2011). This is a crucially important aspect of the laboratory use of invertebrates as most research requires a supply of “standardized” animals throughout the year and journals require increasingly detailed information about the animals used in a reported study (see below). Sourcing and maintenance (including the correct environmental conditions to ensure optimal health, welfare, and, if required, reproduction) may be relatively simple for D. melanogaster and C. elegans, but for marine species obtained from the wild they may be more of a challenge. Advances have been made in the culture of many aquatic invertebrates including cuttlefish (Sepia officinalis and S. pharaonis) and squid (Sepioteuthis lessoniana), but laboratory studies on O. vulgaris and Eledone cirrhosa usually involve wild-caught animals (e.g., Malham et al. 2002).
The inclusion of cephalopods in the revised EU legislation (as mentioned above) will necessitate the development of guidelines covering all aspects of their provision, maintenance, and welfare. Furthermore, bearing in mind the differences between the squid, cuttlefish, octopus, and nautilus, it is likely that each species will need its own set of guidelines. It will also be necessary to develop humane methods for handling (e.g., atraumatic weighing of cephalopods to monitor welfare may not be that simple and even in familiar mammalian species such as mice different handling techniques have a major impact on stress and anxiety; Hurst and West 2010) and for anesthesia and euthanasia, together with criteria for anesthesia and identification of pain and distress.
Evidence of Pain and Suffering and Methods for Their Alleviation
Elwood (2011), Crook and Walters (2011), and Cooper (2011) review different aspects of these difficult and somewhat controversial topics, an understanding of which is essential for both ensuring animal welfare during experimentation and minimizing suffering. Elwood (2011) and Crook and Walters (2011) draw attention to the difference between nociception and pain perception and the survival advantage of the ability to detect, avoid, and learn from noxious stimuli that have the potential to damage tissue. The general hierarchical organization of the central nervous system is well established in vertebrates, but it is appropriate to exercise caution about applying preconceived notions to invertebrate nervous systems.
Defining Nociception and Pain
Nociceptors can be identified by a growing range of molecular markers (e.g., those in the transient receptor potential cation channel subfamily V member 1 [TRPV1], or capsaicin, receptor family) and more classically by recording from the afferent nerve with careful characterization of stimulus intensity-response relationships. It is worth recalling that the behavioral consequences triggered by nociceptor activation are encoded in the primary afferent signal transmitted from the tissue to the central nervous system. In vertebrates responses to activation of nociceptors range from localized responses (e.g., edema, vasodilatation) in the affected tissue via axon collaterals if present, to reflex responses mediated via the spinal cord (e.g., limb withdrawal reflex, scratch reflex) and brainstem resulting in a coordinated response often involving several body systems. Complex endocrine responses to “stress” mediated via the hypothalamic-pituitary-adrenal axis require the more rostral projection of the information encoded in the nociceptive afferent, and conscious perception (feeling pain and the associated emotional aspects) requires projection to the thalamus and cerebral cortex.
Humans tend to think of the effects of noxious mechanical, thermal (hot and cold), and chemical stimuli (including pH) on the surface epithelium (“skin”), but both eating and breathing (air or water) can expose the “interior” of the animal to noxious stimuli that may evoke reflex response such as vomiting and coughing (both complex reflex motor responses mediated via the brain stem and widely present in vertebrates) to remove the irritant, and, in the case of ingested toxins, the unpleasant sensation of nausea (requiring cerebral cortical processing in humans) that is important in the genesis of learned aversions (Stern et al. 2011). Researchers have reported ejection of ingested toxic food in the sea anemone (Lindquist and Hay 1995) and gastropod Pleurobranchaea (McClellan 1983) and of gastric contents in the squid Sepioteuthis sepioidea (Garcia-Franco 1992). Painful sensations can also arise from noxious stimulation of the viscera (e.g., gut pain) and could be as much a cause of reduced food intake in an invertebrate as in a vertebrate.
Assessing and Treating Pain/Nociception in Invertebrates
How can scientists determine whether invertebrates “feel” pain? In considering the issue of assessing pain in animals, Bateson (1991) wrote, “We may feel confident about a mammal or even a bird. But what about a locust or an octopus?” For vertebrates there is generally good knowledge of comparative brain neuroanatomy, allowing a degree of reverse engineering based on knowledge of human brain pain pathways to determine whether similar pathways exist in other vertebrates. Even among vertebrates, however, conclusions about “higher” brain functions based on comparative neuroanatomy have been challenged when considering whether fish feel pain (Rose 2002). Furthermore, as imaging techniques have improved it is becoming clear that even among mammals there are differences between primates and nonprimates in the brain pathways involved in processing information from nociceptors (e.g., Craig 2002). Thus investigators should not underestimate the difficulty in identifying functionally analogous pathways in invertebrates with fundamentally differently organized central nervous systems. It is worth recalling that until the late 1980s there was a common view that human neonates did not feel pain (Fitzgerald and McIntosh 1989).
Molecular, neurophysiological, and behavioral studies have provided evidence for responses to noxious mechanical stimuli in the hermit anemone (Calliactis parasitica) and the California sea slug and to noxious mechanical, thermal (heat), and chemical stimuli in the medicinal leech (Hirudo medicinalis), D. melanogaster, and C. elegans (St. John Smith and Lewin 2009 for review).
Behavioral studies provide important insights into the question of pain perception, but knowledge of the way information is processed is important and, together with molecular and physiological (biomarkers of stress such as heart rate, endocrine, and metabolic changes) studies, will be key to identifying endogenous pathways capable of modulating nociception, the transmitters of which provide targets for analgesics (e.g., opioids, cannabinoids, steroids).
Anesthetic and analgesic techniques for invertebrates are relatively poorly developed in contrast to those for vertebrates (and especially mammals), although as Cooper (2011) points out there may be more information in the world literature and a systematic approach is needed for collecting, assessing, and applying that information. There is certainly a need for some consensus on criteria for general (surgical) anesthesia in all invertebrates used in the laboratory and for decapod crustacea and molluscs in particular. Anesthesia has been used only for short-duration manipulation or surgical interventions. Techniques for sustained general anesthesia and maintenance of physiological systems would permit in vivo neurophysiological or functional brain imaging studies, and the lack of such methods limits knowledge of the central processing of nociceptive inputs, especially in cephalopods.
Finally, although pain (usually assumed to be cutaneous) is perhaps the most common focus from a welfare perspective, animals are susceptible to numerous other unpleasant experiences such as anxiety, asphyxia, dyspnoea, fear, headache, itching, and photophobia. Researchers, animal care staff, and IACUC members should be mindful of all such stressors in considering the welfare of invertebrates in the laboratory.
Attitudes and Their Influence on Regulation and Oversight
“Slugs Displace Bunnies in the Lab” (Davis 2002). This headline accompanied a news article reporting the possibility of replacing the Draize test using rabbits with an irritancy test measuring defensive secretions from slug skin. Although many would view this as a positive development, a spokesman for the British Union for the Abolition of Vivisection is quoted as calling the use of any animal “morally unacceptable” (Davis 2002). This example illustrates the difficulty not only in making judgments about the use of animal in research but also particularly in determining the “relative positions” of vertebrates and invertebrates. This is a complex area and Mather (2011) tackles it by considering the philosophical basis for attitudes toward invertebrates. She describes the contractarian, utilitarian, and rights-based approaches, using diverse examples ranging from the treatment of invertebrates in the kitchen, commercial fisheries, public aquariums, and the laboratory. She accords decapod crustacea and cephalopods special consideration (as is the case in several other articles in this special issue). Mather (2011) concludes that “as invertebrates are better understood, people—whatever their value system—will come to appreciate and take better care of them.”
It is to be hoped that greater understanding will translate to the laboratory and this is likely to be facilitated by ethical review and regulatory frameworks, which are reviewed in the final article, by Harvey-Clark (2011). He presents two IACUC case studies that clearly reveal the particular challenges of research using invertebrates and then provides a useful resource to engage researchers and regulators in confronting some of the key issues.
Scientific journals have a key role to play in encouraging adoption of best practice in the welfare of animals used in research by ensuring that experiments on invertebrates are properly reported. The recently published “ARRIVE” guidelines for reporting animal research (Animal Research: Reporting In Vivo Experiments; Kilkenny et al. 2010, www.nc3rs.org.uk/ARRIVE) provide a clear list of the essential elements that need to be reported for in vivo experiments and, although primarily aimed at studies in vertebrates, can be readily adapted for use in invertebrates (especially decapod crustacea and cephalopods). Comprehensive reporting of methodological aspects is important to enable assessment of welfare and facilitate the systematic gathering of information on ethics, experimental design, housing, husbandry, and adverse events induced by experimental procedures. Many of these aspects are considered in the following articles and especially that by Harvey-Clark (2011).
There seems to be some agreement that in laboratory procedures involving invertebrates in general, and decapod crustacea and cephalopods in particular, the “precautionary principle” should operate at least until there is definitive evidence of their ability to suffer, recognizing that pain may be only one component of suffering. The agreed special position of cephalopods (particularly octopuses and cuttlefish) is already reflected in some national legislation (e.g., Canada). In the European Union it is likely that over the next 2 years such legislation will spur research to address a range of issues—from optimal anesthetic and handling protocols to recognition of signs of pain and distress analogous to those developed for mammals (e.g., Morton and Griffiths 1985)—as cephalopods, at least, will have the same legal protection as is afforded to vertebrates.
Understanding the functioning of phylogenetically ancient brains in highly evolved animals with a fundamentally different organization (at least anatomically) from vertebrates represents a major intellectual challenge and may also prompt reconsideration of some prevailing ideas of consciousness (see Edelman and Seth 2009 for discussion).Available online (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:EN:PDF); this and other websites cited in this Introduction were accessed between February 22 and April 11, 2011. Information about these Nobel Prizes is available online at http://nobelprize.org/nobel_prizes/medicine/laureates. In the European Union it is likely that the forthcoming legislation will drive the development of a consensus view on many of these aspects for cephalopods.
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Paul L.R. Andrews, BSc, PhD, is Professor of Comparative Physiology in the Division of Biomedical Sciences at St. George’s University of London in the United Kingdom.
Address correspondence and reprint requests to Prof. Paul L.R. Andrews, Division of Biomedical Sciences, St. George’s University of London, Cranmer Terrace, London, SW17 0RE, UK or email firstname.lastname@example.org.