ab oratory
limited
l
a
n imals
Guidelines
Guidelines for the Care and Welfare of
Cephalopods in Research –A consensus
based on an initiative by CephRes, FELASA
and the Boyd Group
Graziano Fiorito
1,2
, Andrea Affuso
1,3
, Jennifer Basil
4
,
Alison Cole
2
, Paolo de Girolamo
5,6
, Livia D’Angelo
5,6
,
Ludovic Dickel
7
, Camino Gestal
8
, Frank Grasso
9
, Michael Kuba
10
,
Felix Mark
11
, Daniela Melillo
1
, Daniel Osorio
12
, Kerry Perkins
12
,
Giovanna Ponte
2
, Nadav Shashar
13
, David Smith
14
, Jane Smith
15
and Paul LR Andrews
16,2
Abstract
This paper is the result of an international initiative and is a first attempt to develop guidelines for the care
and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of
700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investiga-
tors, animal care committees, facility managers and animal care staff which will assist in improving both the
care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered
include: implications of the Directive for cephalopod research; project application requirements and the
authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and
severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and
transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting require-
ments, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling,
feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers,
physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and
treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and
confirmation of death. Sections covering risk assessment for operators and education and training require-
ments for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare
requirements for the main laboratory species currently used are summarised in Appendices. Knowledge
gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.
1
Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy
2
Association for Cephalopod Research ‘CephRes’, Italy
3
Animal Model Facility - BIOGEM S.C.A.R.L., Ariano Irpino (AV),
Italy
4
Biology Department, Brooklyn College - CUNY Graduate Center,
Brooklyn, NY, USA
5
Department of Veterinary Medicine and Animal Productions -
University of Naples Federico II, Napoli, Italy
6
AISAL - Associazione Italiana per le Scienze degli Animali da
Laboratorio, Milano, Italy
7
Groupe me
´
moire et Plasticite
´
comportementale, University of
Caen Basse-Normandy, Caen, France
8
Instituto de Investigaciones Marinas (IIM-CSIC), Vigo, Spain
9
BioMimetic and Cognitive Robotics, Department of Psychology,
Brooklyn College - CUNY, Brooklyn, NY, USA
10
Max Planck Institute for Brain Research, Frankfurt, Germany
11
Integrative Ecophysiology, Alfred Wegener Institute for Polar and
Marine Research, Bremerhaven, Germany
12
School of Life Sciences, University of Sussex, Sussex, UK
13
Department of Life Sciences, Eilat Campus, Ben-Gurion
University of the Negev, Beer, Sheva, Israel
14
FELASA, Federation for Laboratory Animal Science Associations
15
The Boyd Group, Hereford, UK
16
Division of Biomedical Sciences, St George’s University of
London, London, UK
Corresponding author:
Graziano Fiorito, Research – CephRes, via dei Fiorentini 21, 80133
Napoli Italy.
Email: cephres@cephalopodresearch.org
Laboratory Animals
2015, Vol. 49(S2) 1–90
! The Author(s) 2015
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DOI: 10.1177/0023677215580006
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Keywords
Cephalopods, Directive 2010/63/EU, animal welfare, 3Rs, invertebrates
Contributors
People listed here provided data, information and com-
ments, and contributed to different extents during the
preparation of this work.
The following list is arranged by country in alpha-
betical order; different contributors are merged by
Institution.
France
Christelle Alves, Cecile Bellanger, Anne-Sophie
Darmaillacq, Ce
´
line Gaudin
Groupe me
´
moire et Plasticite
´
comportementale,
EA4259, University of Caen Basse-Normandy,
Caen, France
Joe
¨
l Henry
Physiologie de la reproduction des Mollusques,
University of Caen Basse-Normandy, Caen, France
Germany
Tamar Gutnick
Max Planck Institute for Brain Research, Frankfurt,
Germany
Italy
Anna Di Cosmo
Department of Biology - University of Naples
Federico II, Napoli, Italy
Carlo Di Cristo
Department of Biological and Environmental
Sciences - University of Sannio, Benevento, Italy
Viola Galligioni
CIBio - Centre for Integrative Biology, Trento, Italy
& Association for Cephalopod Research ‘CephRes’,
Italy
Anna Palumbo
Stazione Zoologica Anton Dohrn, Napoli, Italy
Perla Tedesco
Department of Biological and Environmental
Science and Technologies - University of Salento,
Lecce, Italy
Letizia Zullo
Istituto Italiano di Tecnologia, Department of
Neuroscience and Brain Technologies, Genoa, Italy
Portugal
Anto
´
nio Sykes
C. Mar – Centre of Marine Sciences, Universidade
do Algarve, Faro, Portugal
Spain
Roger Villanueva Lo
´
pez
Renewable Marine Resources Department - Institut
de Cie
`
ncies del Mar, Barcelona, Spain
United Kingdom
Ngaire Dennison
Home Office, Animals in Science Regulation Unit,
Dundee, Scotland, UK
Penny Hawkins
RSPCA Research Animals Department,
Southwater, West Sussex, UK
United States of America
Gregory J. Barord, Heike Neumeister, Janice Simmons,
Roxanna Smallowitz
Biology Department, Brooklyn College - CUNY
Graduate Center, Brooklyn, NY, USA
Jean Geary Boal
Biology Department, Millersville University,
Millersville, PA, USA
Roger Hanlon, William Mebane
Marine Resources Center, Marine Biological
Laboratory, Woods Hole, MA, USA
Judit R Pungor
Hopkins Marine Station of Stanford University,
Pacific Grove, CA, USA
James B. Wood
Waikiki Aquarium, University of Hawaii-Manoa,
Honolulu, HI, USA
1. Introduction
Cephalopods (i.e. nautilus, cuttlefish, squid and
octopus) have been used for diverse scientific pur-
poses across Europe for over 100 years.
1,2
However,
until recently, scientific procedures involving cephalo-
pods have not been covered by EU regulations, with
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the exception of procedures using Octopus
vulgaris in the United Kingdom (see discussion in
Smith et al.
3
).
The inclusion of ‘live cephalopods’ (Article 1, 3b) in
EU Directive 2010/63/EU on the ‘protection of animals
for scientific purposes’ represents a landmark. It is the
first time that an entire class of invertebrates, covering
approximately 700 known species,
4,5
has been included
in laboratory animal legislation throughout the EU.
The decision was largely based upon a review of the evi-
dence for sentience and capacity to experience pain,
suffering, distress and lasting harm (PSDLH) in ceph-
alopods
6
(see also Table 1) which is now supported by
more recent circumstantial (for reviews see
7,8
) and
objective evidence
9–11
for the existence of nociceptors
in cephalopods. Annexes III and IV to the EU
Directive provide general guidance on care and accom-
modation requirements and methods of humane killing
for all species covered by the Directive, but specific
guidance is restricted to vertebrates, and there are no
specific details for cephalopods.
Prompted by the need for guidelines on these and
other matters covered by the Directive, members of
the international cephalopod research community
have met on several occasions over the past 3 years
and have produced publications aimed at cephalopod
researchers, on: i. requirements of the EU Directive,
implementation, ethics and project review;
3
ii. PSDLH, anaesthesia and humane killing;
8
and
iii. implications for neuroscience research and the
Three Rs, i.e. Replacement, Reduction, Refinement.
2
This work has led to the development of a set of
consensus Guidelines for the Care and Welfare of
Cephalopods in Research which aim to assist research-
ers in complying with the Directive, and are the subject
of this paper. These guidelines have been developed as a
joint initiative between CephRes (www.cephalopodre-
search.org), FELASA (www.felasa.eu) and the Boyd
Group UK (http://www.boyd-group.demon.co.uk/).
The Guidelines for the Care and Welfare of
Cephalopods in Research, which should be regarded as
a starting point for future developments, begin with a set
of general principles of good practice, representing the
present state of knowledge that may reasonably be
applied to all cephalopods. These are followed by a tabu-
lated set of specific guidelines (see Appendices) for typical
cephalopod species, currently used in EU laboratories,
which also reflect well-established principles.
1.1 What is a cephalopod?
For the purpose of these guidelines, cephalopods are
defined as all living species that are members of the
molluscan class Cephalopoda.
4,5,12
The term ‘live ceph-
alopod’ is not defined in the Directive, but guidance
indicates that these animals are covered by the
Directive from ‘when they hatch’.
13,14
Cephalopods are characterised by bilateral body
symmetry, a prominent head and a set of arms, includ-
ing tentacles in Decapods, which are considered as mus-
cular hydrostats and derived from the primitive
molluscan foot.
15–21
The class contains two, only dis-
tantly related, living subclasses: Nautiloidea (repre-
sented by Nautilus and Allonautilus) and Coleoidea,
which includes cuttlefish, squid and octopuses.
20,22
In the Nautiloidea, the external shell, common to the
molluscan Bauplan, still exists, whereas in the
Coleoidea it has been internalised or is absent. The var-
iety of species that compose the taxon is reflected in the
diversified habitats they have adapted to: oceans, ben-
thic and pelagic zones, intertidal areas and deep sea,
polar regions and the tropics.
23–26
Understanding the requirements of a particular species
in relation to its natural habitat is fundamental in main-
taining healthy laboratory populations of cephalopods.
Assumptions for housing, care and use of these animals
based on fish, whilst appropriate in some circumstances,
should be made with great caution as the evolutionary
convergence between fish and cephalopods
24,27
does not
reflect the actual requirements of different species.
Generally, cephalopods have a high metabolic rate,
grow rapidly and are short-lived.
28,29
These animals are
exothermic, highly adapted to the marine aquatic envir-
onment and are therefore unlikely to tolerate rapid or
significant changes in the quality or temperature of the
water they are housed in. They react rapidly to environ-
mental changes/external stimuli with immediate physio-
logical consequences that can be relatively long lasting.
Such changes, as well as having potential welfare impli-
cations, will also impact upon experimental results.
Cephalopods are considered among the most
‘advanced’ invertebrates, having evolved many charac-
teristic features such as relatively large, highly differen-
tiated multi-lobular brains, a sophisticated set of
sensory organs, fast jet-propelled locomotion, and com-
plex and rich behavioural repertoires.
25,30–37
1.2 What the Directive 2010/63/EU means
for cephalopod research
The entry into force of the Directive 2010/63/EU (here-
after referred to as ‘the Directive’)
38,39
means that, from
1st January 2013, scientific research and testing invol-
ving ‘live cephalopods’ is regulated by a legal frame-
work at both EU and Member State levels, and as a
consequence all scientific projects that cross the thresh-
old set for regulation (i.e. involve procedures that may
cause PSDLH equivalent to, or higher than that caused
by the insertion of a hypodermic needle in line with
good veterinary practice) will require authorisation by
Fiorito et al. 3
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Table 1. Summary of evidence for the capacity of cephalopods to experience pain based upon the criteria used by the EFSA 2005 panel (as the basis for recom-
mending the inclusion of cephalopods in revision of Directive 86/609/EEC), and here updated with more recent studies. See also Andrews et al. for review
8
and
additional references.
Criterion used by EFSA 2005 panel Judgement Comment and references
Presence of receptors sensitive to noxious stimuli,
located in functionally useful positions on (or) in the
body, and
YES Circumstantial evidence, e.g. cutaneous free nerve endings
491
available at time of EFSA
report. Recent neurophysiological afferent recording studies
10,11
have provided direct
evidence for presence of mechano-nociceptors in both squid and octopus.
Connected by nervous pathways to the ‘lower’ parts of
the nervous system
LIKELY
(but not proven)
Evidence that peripheral afferent axons project to brain from the arms and
mantle,
492,493
but modality not identified although likely to include nociceptors if
present.
Possession of higher brain centres [in the sense of
integration of brain processing], especially a structure
analogous to the human cerebral cortex
YES Most complex brain structure amongst invertebrates and clear hierarchical
organisation.
34,35
Vertical lobe approximates to the hippocampus in mammals and is unique in inverte-
brates in having gyri.
26,142
Studies in progress investigating self-awareness and consciousness, as discussed in
Edelman and coworkers.
494,495
Possession of nervous pathways connecting the noci-
ceptive system to the higher brain centres
LIKELY
(but not proven)
Evidence for ascending afferent projections from ‘lower’ to ‘higher’ brain regions
including the vertical lobe, but no neurophysiological studies showing projection of
signals from nociceptors.
492,493
Indirect evidence from behavioural studies for projection of signals from nociceptors to
higher brain regions (see below).
Receptors for opioid substances found in the central
nervous system especially the brain
LIKELY
(but not proven)
Not studied directly. Opioid system is highly conserved in evolution.
496
Limited evi-
dence for presence of enkephalins
497,498
and opioid receptors.
497–502
Analgesics modify the animal’s response to stimuli
that would be painful for a human
Not studied Investigation of candidate substances is required using a combination of neurophysio-
logical recording from nociceptive afferents and behavioural studies.
An animal’s response to stimuli that would be painful
for a human is functionally similar to the human
response (that is, the animal responds so as to avoid
or minimise damage to its body)
YES Good evidence of learned avoidance of punishment (e.g. electric shock) but assumes
that this stimulus activates nociceptors and not some other afferent modality that
evokes an aversive but non-painful sensation (e.g.
26,503
). Limited supportive evidence
from behavioural studies of predatory behaviour.
26,36,72,504–507
Equivocal evidence for wound-directed behaviours.
9,10,189
An animal’s behavioural response persists and it
shows an unwillingness to resubmit to a painful pro-
cedure; the animal can learn to associate apparently
non-painful with apparently painful events.
YES Evidence for: peripheral mechano-nociceptor sensitisation (at least 48 h) following
injury to either an arm or fin; contralateral afferent sensitisation (but see Alupay
et al.
10
); hyper-responsiveness to visual stimuli following arm injury.
9,11
4 Laboratory Animals 49(S2)
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Figure 1. Schematic overview summarising the major components of a project application and stages of project approval under Directive 2010/63/EU. Note that the
details of the project approval process may differ across member states. For details see text and review in Smith et al
3
.
Fiorito et al. 5
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