Journal of Experimental Marine Biology and Ecology 447 (2013) 1422

Contents lists available at SciVerse ScienceDirect

Journal of Experimental Marine Biology and Ecology

journal homepage: www.elsevier.com/locate/jembe

Special issue: Cephalopod Biology

Pathogens and immune response of cephalopods

Sheila Castellanos-Martnez, Camino Gestal , 1
Instituto de Investigaciones Marinas (IIM-CSIC), Vigo, Spain

a r t i c l e

i n f o

Article history:
Received 4 December 2011
Received in revised form 2 May 2012
Accepted 25 January 2013
Keywords:
Cephalopods
Diseases
Genomic approach
Innate immune response
Pathogens
Symbionts

a b s t r a c t
Cephalopod mollusks are an important marine resource for sheries, and have received marked attention for
studies on organismal biology; they are also good candidates for aquaculture. Wild and reared cephalopods
are affected by a wide variety of pathogens, mainly bacteria, protozoa and metazoan parasites. Cephalopods
do not have acquired immunity and immunological memory; therefore vaccination cannot be used to protect
them against infectious diseases. Their defense mechanisms rely only on their innate immunity. In this review, we will summarize and update knowledge on the most common pathogens, the diseases they cause,
and on symbionts. In addition, we provide a general overview of the cephalopod immune system, response
to pathogens with a short discussion on the gene expression involved in the immune response by these
animals.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Scientic interest for cephalopods is increased over the last century
for, at least, a couple of reasons: i. their value as experimental animals
for biomedical and behavioral research (for review see for example:
Grant et al., 2006; Hanlon and Messenger, 1996; Hochner, 2008;
Hochner et al., 2006; Mather, 1995); ii. their position in the world
marked as a major shery resource (e.g. Boyle and Rodhouse, 2005).
Since the decline of traditional sheries (Balguerias et al., 2000;
Caddy and Rodhouse, 1998), cephalopods have gained attention in
aquaculture practice. Among other cephalopods, Octopus vulgaris has
been considered the candidate species in European aquaculture because of its easy acclimatization to farming conditions, its rapid growth
and its good value for the market (Vaz-Pires et al., 2004). The octopus
on-growing is currently developed in Galicia (NW Spain) on an industrial scale (Garcia and Garcia, 2011; Garcia et al., 2004).
Despite the benets, one of the disadvantages of aquaculture is the
increase in the incidence of pathologies produced by bacteria and/or
transmissible parasites that could be a serious risk for the production.
On the other hand, the EU Directive on the use of animals for experimental purposes European Parliament and Council of the European
Union, 2010, requires capture of animals from the wild to be minimized. Thus, it becomes urgent that practices of culturing animals

This article is part of a special issue on Cephalopod Biology published under the
auspices of CephRes-ONLUS (www.cephalopodresearch.org).
Corresponding author. Tel.: + 34 986 231930x285.
E-mail address: cgestal@iim.csic.es (C. Gestal).
1
Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientcas,
Eduardo Cabello 6, 36208 Vigo, Spain. Tel.: +34 986 231930x285.
0022-0981/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jembe.2013.02.007

be further expanded. Finally, an important outcome of the Directive

2010/63/EU is that appropriate care and maintenance of cephalopods
for research purposes will require proper knowledge and practice in
terms of assessment of health and disease prevention of the animals.
These considerations prompted this review.
Cephalopods, like the rest of mollusks, lack a specic immune response and do not posses immunological memory. Thus, they rely on
the innate immune system in facing with diseases (for review see: Ford,
1992; Gestal et al., 2008; Malham and Runham, 1998). Despite the increasing economic and scientic importance of cephalopods, the research
effort on pathogens and pathologies suffered by cephalopods is less than
54% of the effort directed to other commercially important marine resources (Pascual and Guerra, 2001). Similarly, available knowledge on
the immune response in these animals is still limited (Ford, 1992).
2. Common pathogenic agents
Knowledge on the most common pathogenic agents identied in
cephalopods has been reviewed in the classic Diseases of Marine
Animals with contributions by Hanlon and Forsythe (1990a, b) and
Hochberg (1990).
2.1. Bacteria
Bacterial infections have been reported, for example, on the squid's
ns after injuries produced during capture (Hanlon et al., 1983), or on
lesions suffered by octopus in captivity (Hanlon et al., 1984). This may
cause the spread of the infection to other organisms in the tank or in
the system. Hanlon et al. (1984) observed skin ulcers in reared Octopus
joubini and Octopus briareus; these appeared rst on the dorsal mantle
and then spread to head and arms. Four stages of ulceration have been

S. Castellanos-Martnez, C. Gestal / Journal of Experimental Marine Biology and Ecology 447 (2013) 1422

identied, the rst being a noticeable lighting of chromatophore

patterns due to infection. Skin appears to be wrinkled, showing hyperplasia of the epidermis and increased mucus production. Gram negative
bacteria Vibrio alginolyticus, Vibrio damsela, Pseudomonas stutzeri, and
Aeromonas caviae were isolated from O. joubini ulcers; while Vibrio
parahaemolyticus, V. damsela, and P. stutzeri where isolated from the ulcers of O. briareus (Hanlon et al., 1984).
Ford et al. (1986) found bacteria as natural populations on the mantle
of wild-caught Lolliguncula brevis in the absence of evident damages to
the host. In addition, it appears that wild squids have less bacterial population than those reared in tanks. The bacteria recorded on wild squids
were mostly the Gram negative: Aeromonas sp., Flavobacterium sp., Pseudomonas sp., Proteus sp., V. parahaemolyticus, V. alginolyticus, Vibrio sp. and
Flexibacter. All of these were also found on the normal skin of squid reared
in tanks. However, V. alginolyticus, Vibrio metschnikovii, Aeromonas sp.,
and Pseudomonas sp. were the only ones noticed on ulcerated skin of
squids. Bacillus sp. and Streptococcus sp. were found in wild squids and
also in those kept in laboratory both in conditions where the animals
appeared with and without ulcerations (Ford et al., 1986).
Mantle and arms are not the only parts of the body of cephalopods
susceptible to bacterial infection. The gills are in contact with the environment all the time and therefore exposed to numerous pathogenic
agents. For example, the gills of O. vulgaris have been found infected
by Rickettsiales-like organisms. These are observed like basophilic
intracytoplasmatic microcolonies (about 102 m long) appearing within epithelial cells of the gills. Gill cells became hypertrophic and necrosis
was occasionally observed. No signicant harm has been observed in
the host, but bacteria are able to have a detrimental effect on the respiratory gaseous exchange of the octopus (Gestal et al., 2008).
One of the most recent records of bacterial infections in wild cephalopods, was reported in O. vulgaris kept in oating cages at the Ria of
Vigo (Farto et al., 2003). The specimens show lesions in the mantle
and some of them died once in laboratory. Lesions on the mantle
have been attributed to the bacteria Cytophaga-like and Pseudomonas,
which were isolated from the damaged tissue. However, the Gram
negative bacteria Vibrio lentus, originally isolated from reared
Mediterranean oysters (Macan et al., 2001), was isolated for
the rst time from the branchial heart of octopuses. Experimental infections performed by a challenge bath of V. lentus (72 h, 2108 cfu/ml) induced mortality in 50% of octopuses after the rst 6 h. The lesion showed
a typical round pattern in the arms or the head. No variations in mortality rate were recorded after 9 h post-infection, which is assumed to be a
result of inter-individual differences in the immune system (Farto et al.,
2003). Low salinity (29%) reported in the area was suggested be a stressor that may impair immune response of animals against the opportunistic pathogen V. lentus (Farto et al., 2003; Ford et al., 1986).

2.2. Protozoa: the eimeriorin coccidia Aggregata spp.

Coccidians of the genus Aggregata are a source of a severe disease
in cephalopods. The protozoan infects the digestive tract of the host
(Hochberg, 1990), mainly the caecum, thus impairing the absorption
of nutrients (Boucher-Rodoni et al., 1987).
The genus Aggregata is distributed all around the world. A total of
10 species have been described to date (Gestal et al., 2010) infecting
cuttleshes, squids and octopuses and also octopuses inhabiting
deep-sea hydrothermal vents (Table 1).
Traditional identication and characterization of Aggregata species
have relied primarily on differences in morphological features such as
size and shape of sporogonial stages and host specicity. Nowadays,
molecular techniques provide useful methods for taxonomic studies,
and are important tools in solving problems of species delimitation.
So far, molecular characterization of A. octopiana and A. eberthi has
been carried out by sequencing the 18S rRNA gene (Kopecn et al.,
2006; Gestal, pers. comm.).

15

Table 1
A tabularized overview of Aggregata species reported to infect cephalopods.
Cephalopod host

Species of Aggregata

References

Sepia ofcinalis
Todarodes sagittatus
Martialia hyadesi
Octopus vulgaris
Octopus bimaculoides
Octopus doeini
Octopus tehuelchus
Enteroctopus megalocyathus
Vulcanoctopus hydrothermalis

A. eberthi
A. sagittata
A. andresi
A. octopiana
A. millerorum
A. dobelli
A. valdessensis
A. patagonica
A. bathytherma

Dobell (1925)
Gestal et al. (2000)
Gestal et al. (2005)
Gestal et al. (1999b)
Poynton et al. (1992)
Poynton et al. (1992)
Sardella et al. (2000)
Sardella et al. (2000)
Gestal et al. (2010)

The intracellular protozoan Aggregata spp. has a heteroxenous life

cycle which requires a crustacean intermediate host to develop its
merogonic stage, while cephalopods are the denitive hosts in which
the parasite develops gamogony and sporogony stages (Hochberg,
1990). The infection by Aggregata spp. has been recorded being almost
exclusive of O. vulgaris and Sepia ofcinalis in Spain (Pascual et al.,
1996a) reaching about 82 106 sporocyst per gram of infected tissue.
Taking in consideration the elevated infection prevalence and intensity,
and the important pathological effects, parasites of the genus Aggregata
are known to be the main epizootiological agents in wild and cultured
octopus stocks (Gestal et al., 2007b).
Infections by Aggregata initiate in the mucosal folds where the tissue ruptures at the basal membrane and detachment of the epithelial
cells is produced. As a consequence, the mucosal folds of the intestine
and caecum suffer atrophy; at the intracellular level, displacement of
the nucleus host cell to one side is visible (Gestal et al., 2002a;
Poynton et al., 1992). All the infected tissues show hemocytic inltration and a pericyst reaction in both (gamogony and sporogony) infective stages (Gestal et al., 2002a; Licciardo et al., 2005; Mladineo and
Jozic, 2005). The capsule formed is originally composed of attened
hemocytes and then connective tissue elements appear (Tripp,
1974). In senescent octopuses, the infection is predominated by
sporogonial (few merogonial) stages that extend widely in the tissue,
showing scarce hemocyte inltration or brotic reactions, signs of a
weak immune system (Pascual et al., 2010). During severe infective
episodes the pathology is even extended to the mantle and gill's connective and epithelial tissues with similar signs of damage (Mladineo
and Bocina, 2007).
The injury caused by the protozoan also has an effect at a biochemical level. The infection produces decrease of the pH of the digestive tissue infected, and as a consequence an inaccurate functioning of the
digestive enzymes, such as maltase and leucin-aminopeptidase, thus
producing a malabsorption syndrome (Gestal et al., 2002b). In addition,
heavily infected specimens show poor conditions reected in Fulton's
condition index, low DNA/RNA ratio, RNA/protein conversion and
even decrease of the number of circulating hemocytes (Gestal et al.,
2007b).
Although pathologies induced by this protozoan are not fatal, it severely weakens the cephalopod host making it more vulnerable to
other biotic and abiotic stressors (Pascual et al., 2007).
The case of chronic infection by the coccidia Aggregata spp. in the
cephalopod host offers the opportunity to study the mechanisms of immune response of the cephalopods at different developmental stages of
the host life cycle, and different intensities of infection. Then, this should
be established like a model study of the hostparasite relationship.

2.3. Metazoa
Since the review written by Hochberg (1990), the research added
in the last years has contributed to increase the knowledge on the
biology of cephalopods, their trophic relationships (Gonzlez et al.,
2003) and the importance of parasites as marine tags (Pascual and

16

S. Castellanos-Martnez, C. Gestal / Journal of Experimental Marine Biology and Ecology 447 (2013) 1422

Hochberg, 1996) to resolve cephalopod migrations (Semmens et al.,

2007).
Cephalopods are secondary, third or paratenic hosts for trematodes digenea, cestodes and nematodes (Hochberg, 1990). According
to the ecological and behavioral features of the cephalopod's life
cycle, they occupy an ecological niche that makes them favorable to
be infected by specic groups of parasites. Those parasites are transmitted to the denitive host: shes, marine mammals or birds
(Clarke, 1996; Gonzalez et al., 2003).
2.4. Cestoda
Among the cestodes carried by cephalopods, plerocercoids of
Phyllobothrium sp. and Pelichnibothrium speciosum, Tentacularia
coryphaenae, and Hepatoxylon trichiuri infect the digestive tract of
Dosidicus gigas from the central East Pacic and Chile (PardoGandarillas et al., 2009; Shukhgalter and Nigmatullin, 2001). In addition, new data has also shown the presence of those plerocercoids in
Illex argentinus from the Atlantic (Nigmatullin and Shukhgalter, 1990;
Sardella et al., 1990), and in Illex coindetii from the Adriatic Sea (Petric
et al., 2011). In Spain, the plerocercoid cestodes Dinobothrium sp.,
Nybelinia yamagutii, and Nybelinia lingualis have been identied in
the ommastrephid squids I. coindetii and Todaropsis eblanae (Pascual
et al., 1996b), and also in Todarodes sagittatus, O. vulgaris, Loligo
vulgaris, Sepia orbignyana and Eledone cirrhosa (Pascual et al., 1996a).
2.5. Trematoda
Little research has been done in digenetic trematodes since
Overstreet and Hochberg (1975) and Hochberg (1990). Most of the
new records belong to unidentied Didimozoidae metacercaria infecting
the digestive tract of D. gigas from the NW Pacic (Nigmatullin et al.,
2009; Shukhgalter and Nigmatullin, 2001). The intensity of infection is
variable, from one to even 1500 larvae (Nigmatullin et al., 2009), and
prevalence has been reported to be up to 86% in squids with mantle
length (ML) ranging 30431 mm (Nigmatullin et al., 2009; Shukhgalter
and Nigmatullin, 2001).
The species Derogenes varicus and Hirudinella ventricosa were identied from the digestive tract of I. argentinus by Nigmatullin and
Shukhgalter (1990), and metacercaria of didymozoid Monilicaecum sp.
have been recorded in I. argentinus paralarvae from Brazil (Vidal and
Haimovici, 1999). However, the infection through an intermediate
host in paralarvae smaller than 3.7 mm ML was discarded, because
crustacean prey has not been recorded in paralarvae's digestive tract
(Vidal and Haimovici, 1998). In contrast, large paralarvae could be
infected by an intermediate host such as copepods (Vidal and
Haimovici, 1999).
The identication of larval stages of helminthes is difcult because
they do not show adult features. As a consequence, they are usually
recorded only as genera, except some Trypanorhyncha cestodes that
can be recognized because the tentacular armature (Randhawa,
2011). Therefore, molecular tools are used in order to identify larval
stages and to complement morphological descriptions with molecular characterization based on the large subunit ribosomal DNA
(Agust et al., 2005; Randhawa, 2011). Digenetic parasite species are
usually distinguished based on the sequences of internal transcribed
ribosomal spacer regions (ITS), which are well conserved and useful
for determining species boundaries (Nolan and Cribb, 2005).
2.6. Nematoda
Nematode parasites use marine mammals as denitive hosts as
well as birds that feed on infected sh, whereas crustaceans are the
rst intermediate hosts, and cephalopods, mainly squids, play a role
of intermediate or paratenic hosts (Mattiucci and Nascetti, 2008).

Humans are accidental hosts, and when infected they can manifest
allergies and digestive disorders (Audicana et al., 2002).
As before, only larval stages occur in cephalopods; therefore, an
accurate taxonomic identication is unavailable since adult features
are needed (Hochberg, 1990). Furthermore, the existence of sibling species makes essential genetic identication by sequencing genomic DNA,
ITS regions or mitochondrial DNA (Mattiucci and Nascetti, 2008).
Nematodes are certainly widespread commonly inside a connective
tissue capsule mainly infecting the digestive tract, but they can also be
found in the gonads (Pascual et al., 2007). Records from Ommastrephes
bartramii and D. gigas from the southeastern Pacic include nematode
species Anisakis physeteris, Prorrocaecum sp., Contracaecum, and Anisakis
types I and II, distinguished by the presenceabsence of mucron and tail
shape (Nigmatullin et al., 2009; Pardo-Gandarillas et al., 2009). From
Galician waters, Anisakis simplex s. str. has been recorded in the cuttlesh
S. ofcinalis, Sepia elegans and squids Allotheuthis subulata, T. eblanae,
I. coindetii and T. sagittatus (Abollo et al., 2001; Pascual et al., 1999).
The highest prevalence of Anisakis larvae was harbored by T. sagittatus
(34%) and T. eblanae, which were also found in the sh Merluccius
merluccius, Prionace glauca, Lophius piscatorius, Belone belone and
Scorpaena scrofa (Abollo et al., 2001). I. coindetii is infected by Anisakis
peggrefi in the Adriatic Sea (Petric et al., 2011) and by A. simplex and
Hysterothylacium sp. in the Tyrrenhian Sea (Gestal et al., 1999a).
The numerous nematode records in cephalopods usually register
the taxonomic identity and site of infection. However, the tissue
host reaction is scarcely mentioned, which could be due to the absence of gross pathology associated to the parasites that are encysted
in a capsule of connective tissue mainly in the stomach, gonads and
nidamental glands (Abollo et al., 1998). The histological inspection
of infected organs exposes displacement of host tissue in direct contact with the nematode larvae. A variable inammatory reaction is
visible by the hemocytes that move towards the place of infection,
and in addition, secretion of mucus and cell debris is clearly observed
(Abollo et al., 1998; Pascual et al., 1995).
2.7. Crustacea
Crustaceans are macroparasites that mainly inhabit the mantle cavity and the gills of cephalopods, although they can also be found over
external surfaces of the body such as arms or head (Hochberg, 1990).
The mantle cavity and gills are suitable sites for harboring the copepod Pennella such as in I. coindetii, L. vulgaris, A. subulata, Alloteuthis
media or the octopod E. cirrhosa (Gestal et al., 1999a). Post-embryonic
stages of Pennella have a seasonal infestation pattern with a peak
in winter and spring. In addition, parasites are visibly aggregated in
I. coindetii perhaps due to reproductive reasons (Pascual et al., 2001).
Post-embryonic stages of Pennella sp. do not have a negative effect on
the cephalopod growth, but affect specimens heavily infected (Pascual
et al., 1997). Infections of Pennella sp. have been reported to affect the
condition of O. vulgaris, E. cirrhosa and S. ofcinalis (Pascual et al.,
1996a). The common octopus, O. vulgaris, is also infected by the copepod Octopicola sp., the squid I. coindetii harbor Stellicola hochbergi, and
Doridicola cf. agilis was found in the squid T. sagittatus (Pascual et al.,
1996a).
Few isopods are also hosted in the mantle cavity of cephalopods
(Hochberg, 1990), but they are assumed as accidental or temporary
infections (Pascual et al., 2002). The cymothoid isopods mainly inhabit the gill chambers, skin and sh ns (Hochberg, 1990). In addition, Bello and Mariniello (1998) provided the rst record of the
cymothoid isopod Livoneca sinuata found in the mantle cavity of
Sepiola ligulata from the Adriatic Sea.
Cymothoids establish unusual associations with jellysh, crustaceans and other organisms, although this association seems to be frequent with cephalopods. Therefore, the nding of several specimens
of L. sinuata in the mantle cavity of L. vulgaris from Turkey cannot be
considered rare (Trilles and Oktener, 2004). The gills of this squid host

S. Castellanos-Martnez, C. Gestal / Journal of Experimental Marine Biology and Ecology 447 (2013) 1422

were found infected by the isopod Anilocra physodes, although the parasite probably was acquired during trawl nets (Gestal et al., 1999a).
Cryptoniscus larvae of Epicaridea isopods were found embedded as
minute opaque external marks (surrounded by a membrane) at the
beginning of the esophagus in Loligo gahi. Due to the location of the
parasite, it probably does not obstruct the feeding activity, but it
could destroy the musculature and as a consequence, affect mastication (Pascual et al., 2002).
3. Most common endosymbionts
3.1. Bacteria, Vibrio scheri
A special case is the association between the Hawaiian Bobtail
Squid Euprymna scolopes and the bacteria V. scheri, a symbiosis
that initiate as soon as the squid hatches (McFall-Ngai, 1994). The
bacteria enter into the light organ of the animal and packed tightly
in the crypts. Once the symbiosis is established, the bacteria start to
emit light, and as a consequence the squid looks luminescent on its
ventral side, thus matching downwelling moonlight and/or starlight,
as a strategy to avoid predators (Nyholm and McFall-Ngai, 2004).
The immune system of E. scolopes is educated to let the colonization
only by V. scheri. Inside the squid's light organ, the peptidoglycan of
V. scheri and environmental bacteria stimulates mucus secretion
from the supercial ciliated epithelia, but only alive Gram-negative bacteria aggregates tightly within the mucus matrix; the outer membrane
proteins of V. scheri prevent adhesion by host hemocytes and evade
phagocytic response (Nyholm et al., 2009). In addition, V. scheri promotes irreversible apoptosis of ciliated epithelial cells in the crypts. Furthermore, ducts are constricted to discourage further colonization by
environmental symbionts, and the cell cytotoxic response is attenuated
as part of the symbiotic role played by V. scheri (Altura et al., 2011;
McFall-Ngai et al., 2010).

17

Despite the fact that dicyemids are commonly found in cephalopods, it is not clear if dicyemids damage the host. Apparently, no
damage is produced by dicyemids; it has been supposed that they
contribute to eliminate ammonium from the host urine (Hochberg,
1990). However, dicyemids could be a problem when the population
rises so much that it blocks the renal sac ducts. On the basis of these
considerations dicyemids are considered endosimbyonts (Furuya
and Tsuneki, 2003; Hochberg, 1990).
Dicyemid have been reported from a variety of hosts and sites, such
as Australia (Finn et al., 2005), the Atlantic and the Mediterranean Sea
(Gestal et al., 1997), Mexico (Castellanos-Martinez et al., 2011), and
principally from Japan (Furuya, 2008, 2009, 2010).
Whether dicyemids are primitive animals or not is still debated.
They present tubular cristae, a double-stranded ciliary necklace and
lack of collagenous connective tissue; on the basis of these characteristics, dicyemids were classied as Protozoa by Cavalier-Smith (1993)
arguing that multicellularity is not a conclusive feature to classify
them in Animalia. Therefore, dicyemids were classied as multicellular
protozoa. On the other hand, Furuya et al. (1997) demonstrated the
presence of two types of cellular junctions (adherent and gap junctions)
in vermiforms and infusoriform embryos, which is a feature absent in
colonial organisms. Hence, although dicyemids have no tissues, they
present basic types of cell junctions similar to multicellular animals,
and therefore, dicyemids should not be considered as primitive organisms (Furuya et al., 1997).
The basic morphology of dicyemids has been assumed to be derived from the endosymbiotic life style (Stunkard, 1954). Data deduced from several studies including 18S and DoxC (Kobayashi
et al., 1999, 2009), serotonin (putatively involved in the functioning
of cilia and developmental processes: Czaker, 2006), Pax6 (Aruga
et al., 2007) and innexin (Suzuki et al., 2010), conrm the view that
dicyemids are simplied Bilateria derived from a triploblastic ancestor,
belonging to Lophotrochozoans but not related to platyhelminthes
(Suzuki et al., 2010).

3.2. Dicyemida (Mesozoa)

Dicyemids are endosymbionts that inhabit the renal sacs of benthic cephalopods (octopuses and cuttleshes, Hochberg, 1990), but
also the squid Todarodes pacicus (Furuya and Tsuneki, 2003) and
branchial heart appendages of Rossia pacica (Furuya et al., 2004b).
Typically vermiforms, they have a low number of cells (840) and
lack of organs and cavities (Furuya and Tsuneki, 2003). The name
Dicyemida refers to the two stages in their life cycle: nematogen,
characterized by asexuate reproduction, and rhombogen (sexuate,
Hochberg, 1990). The zygote develops into an infusoriform embryo
which is released into the host urine at the end of its development;
therefore, it is expelled towards the sea (Furuya and Tsuneki, 2003).
Presumably, an elevated density of dicyemid population stimulates the shift between asexual and sexual reproduction; alternatively
this shift may be linked to the reproductive status of the host. A biochemical message is suggested to signal to the dicyemids the onset of
the reproductive phase in the cephalopod host (Hochberg, 1990).
This let vermiforms to reproduce before the cephalopod host dies.
Vermiform stages are composed by a central long cell, the axial cell,
enveloped by a single layer of ciliated cells (McConnaughey, 1983). The
anterior part, the calotte, is composed by 410 cells which are important
for taxonomic identication (Furuya and Tsuneki, 2003; Hochberg,
1990). The shape of the calotte is different among species, presumably
as an adaptation to attach to specic zones inside the renal sacs
(Furuya and Tsuneki, 2003). In contrast, infusoriform embryos are morphologically complex, and they are typically composed by 3739 cells,
which are species specic (Furuya et al., 2004a). Renal sacs provide
space and food for the maintenance of dicyemids. These attach their calotte into the crypts of the renal sacs, while the rest of the body oats in
the host urine to take nutrients through the ciliated cells along the body
via endocytosis (Hochberg, 1990).

4. Response to diseases: overview on the cephalopod

immune system
Cephalopods are advanced mollusks with a well developed circulatory system; a systemic and two accessory hearts (branchial hearts)
distribute the hemolymph through arteries and capillaries to the
whole body (Schipp, 1987; Wells and Smith, 1987). Branchial hearts
contribute to the production of hemocyanin, and elimination of particles (Beuerlein et al., 1998, 2002). Similar to other mollusks, cephalopods have a non adaptive (or innate) immune system. They do not
have immunoglobulins and therefore they do not have extended protection against pathogens for future infections. Thus, the cephalopod
immune system works on the basis of cellular factors. The hemocytes respond by phagocytosis, encapsulation, inltration or cytotoxic
activities to infections and destroy or isolate pathogens. In addition,
molecules dissolved in the serum (opsonins, agglutinins, lysozyme)
also contribute to the immune response (Ford, 1992).
4.1. Cellular elements and their role
The hemocytes play a major role in the internal defense, by the
recognition and the elimination of foreign materials, as well as shell
and wound repair (Cheng, 1975). In cephalopods the hemocytes
(also named leukocytes), are produced in the white bodies located
behind the eyes in the orbital pits of the cranial cartilages (Cowden,
1972). A white body is constituted by two primary lobes, of unequal
size, organized into several secondary lobes and a large number of
small lobules providing a glandular appearance to the organ (Claes,
1996). Its embryonic development has not been studied yet
(Cowden and Curtis, 1973). Inside the organ, strings of leukopoietic
cells are found in different developmental stages (Cowden, 1972).

18

S. Castellanos-Martnez, C. Gestal / Journal of Experimental Marine Biology and Ecology 447 (2013) 1422

Their leukopoietic function is deduced from the ultrastructural similarities between the putative nal stage of their cells and the circulating blood cells, which correspond to a single cell line; hence, only one
type of hemocyte is found in the peripheral hemolymph (Claes, 1996;
Cowden and Curtis, 1973, 1981).
At least two kinds of hemocytes have been identied in bivalves and
gastropods (hyalinocytes and granulocytes) according to the presence or
absence of granules, which are characterized by staining afnity (Chu,
2000; Lpez et al., 1997; Salimi et al., 2009). Furthermore, there is an
agreement about the function of hemocytes to repair tissue damage,
nutrient transport and digestion, and internal defense against non-self
material (Cheng, 1975; Chu, 2000). However, in the case of cephalopods,
those tasks are performed by the single type of cell present in the circulating hemolymph (Claes, 1996; Cowden and Curtis, 1981).
Wound repair involves the movement and aggregation of hemocytes at the injured site to prevent bleeding, until epithelial cells
grow over the wound to complete the healing (Chu, 2000). In cephalopods hemocytes are capable to form a plug which is accompanied
by vasoconstriction and collagen synthesis to help to repair a lesion
(Ferl, 1988). Pascual et al. (2006) reported the nding of specimens
of Octopus hubbsorum with a large knob on the dorsal mantle and
nodules with smooth surface on the sucker's base. The histological
analysis showed a mass proliferation of dense brous tissue between
the dermis and muscle layers suggesting that the tissue has been
repaired by the hemocytes (Chu, 2000). Because the knob is located
in the area where octopuses are usually hooked, the authors showed
the capability of the hemocytes to restore the tissue injured in octopuses that escaped from shermen catches.
As in their molluskan relatives (Lpez et al., 1997) the cellular defense by cephalopod hemocytes involves phagocytosis as well as production of oxygen and nitrogen radicals (Ford, 1992; Malham and
Runham, 1998; Malham et al., 1997; Rodrguez-Domnguez et al.,
2006). Phagocytosis of microbial agents and non-self materials is an important defense reaction (Cheng, 1975; Chu, 2000). In addition, a low
number of circulating cells can be a sign of stress (Malham et al.,
1998). Changes in the number, morphology or viability of hemocytes
can be used as an indicator of the organism's health (Ellis et al., 2011)
since variations have been found in relation to parasitic infections (da
Silva et al., 2008) or contamination (Mayrand et al., 2005).
Malham et al. (1997) recorded 80% of phagocytic hemocytes in
E. cirrhosa when challenged with not opsonized Vibrio anguillarum.
Longest exposure to the bacteria led to a higher percentage of phagocytosis, mainly if the bacteria were pre-incubated in cephalopod hemolymph
free of cells, which suggests that phagocytosis is assisted by opsonizing
elements (Malham et al., 1997). However, Rodrguez-Domnguez et al.
(2006) recorded 50% of phagocytosis in hemocytes of O. vulgaris challenged with zymosan (not opsonized). In addition, phagocytosis did
not increase with the incubation time (Rodrguez-Domnguez et al.,
2006). A similar result was recorded by ow cytometry after challenging O. vulgaris hemocytes with uorospheres (maximum level
of phagocytosis = 55%, Castellanos-Martinez and Gestal, 2011). In
both cases, the incubation was performed at 15 C.
Microorganisms also trigger the phagocytic reaction. However,
this is not effective when biotic and/or abiotic particles are larger
than hemocytes. Under such circumstances hemocytes surround the
particle forming various layers of cells, isolating it and limiting potential damage (Chu, 2000), even the defensive response is not capable
to eliminate the intruder (Tripp, 1963). This is commonly found in
cephalopods infected by helminthes and nematodes, due to their
large size (even when in larval forms).
4.2. Citotoxicity
4.2.1. Reactive oxygen intermediates (ROIs)
Destruction of pathogens through phagocytosis or under hemocyte
stimulation is complemented with the production of oxidative

chemicals. Most frequently, these are represented by reactive oxygen

intermediates (ROIs), collectively known as respiratory burst. In these
conditions, an increased uptake and consumption of oxygen and stimulation of NADPH oxidase occurs (Chu, 2000). The initial metabolite is superoxide anion (O2), which is dismutated to hydrogen peroxide
(H2O2), and converted to other toxic ROIs such as hydroxyl radical
(OH) and single oxygen 1O2 (Bugg et al., 2007). ROIs are usually measured in the cellular fraction; this allows investigating any change in
hemocyte functionality mediated by bactericidal or pathogen activity
(Ellis et al., 2011). The most common assays utilized to measure ROIs
are based on nitroblue tetrazolium (NBT) reduction and luminoldependent chemiluminiscence (LDCL). In the rst case, oxygen radicals
(O2) can reduce yellow, water soluble, NBT to an insoluble dark
blue formazan visible under microscope or spectrophotometer after
extracting formazan from the cells (Anderson, 1994; Pipe, 1992). LDCL
is utilized to measure the activity of myeloperoxidase/hydrogen peroxide (MPO/H2O2) system; luminol generates excited aminophthalate
anions that relax to the ground state with the production of light
(Anderson, 1994).
Radicals are released into the extracellular medium to kill pathogenic agents. ROI production is a common defense mechanism noticed in bivalves as Mytilus edulis (Pipe, 1992), Crassosstrea virginica
(Anderson, 1994), Mytilus galloprovincialis (Arumugam et al., 2000)
and Mercenaria mercenaria (Bugg et al., 2007). Few records are available for cephalopods. Malham et al. (2002) showed that hemocytes of
E. cirrhosa produce intracellular superoxide in response to stress. The
superoxide production increases after octopus exposure to the air for
5 min, indicating that this radical is produced by the animal also in
response to this kind of stress (Malham et al., 2002). By applying
the reduction of ferricytochrome C, Novoa et al. (2002) measured
the production of superoxide after stimulation of the circulating hemocytes and white body cells with Escherichia coli lipopolysaccharide
(LPS), zymosan and PMA. The response was obtained in the white
bodies and by circulating hemocytes using PMA and LPS, but the
highest reaction was recorded when stimulated with zymosan
(Novoa et al., 2002).
Currently, ow cytometry is a widely used tool to measure molluskan
hemocyte immune response through the detection of uorescence produced by each cell (Bugg et al., 2007). Flow cytometry is advantageous
since it allows almost real-time measurement of the response and to analyze the response of each cell within a big sample (Davey, 2002). The
oxidative activity measured in O. vulgaris hemocytes after challenging
the cells with zymosan showed that the uorescence produced by the
hemocytes varied from 111.6% to 129.5% (Castellanos-Martinez and
Gestal, 2011).
Since oxidative activity is activated in response to pathogens, it is
reasonable to expect that parasites affecting cephalopods use strategies to deactivate the hemocyte cytotoxic response. A recent study revealed that ROIs decrease in O. vulgaris hemocytes when the infection
by the protozoan A. octopiana increases (Castellanos-Martinez and
Gestal, 2011).

4.2.2. Nitric oxide

Nitric oxide (NO) is considered part of the innate immune response
and is synthesized after parasite infection (Rivero, 2006). NO results
from the oxidation of L-arginine to citrulline by the enzyme nitric
oxide synthase (NOS) which is present in mammals as neuronal, inducible and endothelial isoforms. NO is a signaling molecule with a physiological function in vasodilatation, secretor control, intestinal relaxation,
macrophage cytotoxicity, regulation of developmental processes, neurotransmission and neuromodulation (Jacklet, 1997; Palumbo, 2005).
Furthermore, NO has been detected in the central nervous system of
Polyplacophora, gastropods and cephalopods (Palumbo, 2005). In cephalopods, nitric oxide synthase has been suggested to play a role in tactile
learning (Robertson et al., 1994); the presence of NOS in the brain of S.

S. Castellanos-Martnez, C. Gestal / Journal of Experimental Marine Biology and Ecology 447 (2013) 1422

ofcinalis let to hypothesize the role of NO as a messenger molecule

(Di Cosmo et al., 2000; Di Cristo et al., 2007).
NO is a highly-reactive free radical gas that is not stored and readily
diffuses through membranes (Jacklet, 1997), so it is an effective agent
against pathogens. A reduction of NO was observed in erythrocytes
infected with Leishmania donovani in contrast to control cells
(Chowdhury et al., 2010). Similarly, NO produced by O. vulgaris hemocytes seems to decrease with the increase of infection by A. octopiana.
A special case is the symbiotic relationship between the squid
E. scolopes and the bacteria V. scheri (see above). This relationship is
so intimate that biochemical communication avoids possible attacks
by cytotoxicity of cells (McFall-Ngai et al., 2010). NOS has been detected
in the cells lining the ducts and antechambers of the crypts of the light
organ including in the mucus; a weak level has been observed in the
deep crypts and in ciliated epithelial tissue (Davidson et al., 2004).
Apparently NO works by limiting the aggregating population of
V. scheri cells, inducing the bacteria to turn on genes related to resistance to NO (McFall-Ngai et al., 2010). In fact, NOS and NO, are attenuated in the symbiotic areas of the light organ as early as 6 h after
colonization, and this level appears irreversible when the symbiosis is
completely established (Davidson et al., 2004). Moreover, NO attenuation seems to be required to induce apoptosis in ciliated epithelial
cells, a strategy that prevents colonization by environmental bacteria
(Altura et al., 2011; Nyholm and McFall-Ngai, 2004). Attenuation of
NO perhaps occurs through the activation of V. scheri hnoX gene that
encodes for a heme NO/oxygen-binding (H-NOX) protein. H-NOX belongs to a family of putative sensor proteins present in bacteria, thus
V. scheri could use it for facilitating colonization (Wang et al., 2010).
4.3. Humoral factors
Marine mollusks lack specic immune response and immunoglobulins; instead factors with agglutinating, opsonic, lytic, antimicrobial and
protease-inhibition activities are present in the serum. Those factors are
part of the mollusk humoral defense (Chu, 2000). Humoral factors complement the cellular activity. After the internalization of a particle or
pathogen, this is enclosed in a vacuole (phagosome) where killing and
destruction takes place by toxic radicals (oxygen or nitrogen) or enzymes like acid phosphatase, peroxidase, -glucuronidase, NADH oxidase and lysozyme (Cheng, 1975; Chu, 2000).
The cephalopod cell-free hemolymph is the carrier of oxygen,
which is delivered to the whole organism (Wells and Smith, 1987),
but it is also the carrier of the humoral components that include
lectins, such as the 260-kDa lectin described in O. vulgaris (Rgener
et al., 1985). Recently, Alpuche et al. (2010) described a lectin of
66 kDa (OmA) found in Octopus maya which was not been identied
before. It was found to be a homolog to the type A hemocyanin from
Octopus doeini. Due to the specicity of the lectin to galactosamine,
mannose and fucose, it was suggested that it could play a role in the
immune response by recognizing and agglutinate oligosaccharides
from cells and perhaps also from pathogens (Alpuche et al., 2010). In
the presence of rat erythrocytes the hemagglutinating activity of the
new OmA lectin from O. maya was elevated (Alpuche et al., 2010).
Antibactericidal activity of sera from O. maya was conrmed using
beef erythrocytes, but sera from S. ofcinalis and Sepioteuthis
lessoniana resulted to have higher agglutinating success over a wide
range of bacteria (Fisher and Dinuzzo, 1991).
Enzymes like lysozyme are part of the defense mechanism. Its effectiveness against a broad variety of bacteria is due to the catalyzed
hydrolysis of N-acetylmuramic acid (14) N-acetylglucosamine links
of the polymeric chains in the bacterial cell wall. Lysozyme is highly
concentrated in leukocytes, neutrophilic granulocytes and macrophages (Grossowicz et al., 1979), but the enzyme has also been
found in the serum of C. virginica (McDade and Tripp, 1967) and in hemocytes and tissue from the octopus E. cirrhosa (Malham et al., 1998).
The lysozyme activity was higher in hemocytes of octopuses infected

19

by V. anguillarum when measured immediately after injection; activity

that was reduced after 4 and 24 h (Malham et al., 1998).
5. Genomic approach: molecular tools and gene expression
technologies applied to pathogens to cephalopod hosts
For many pathogens of cephalopod mollusks, current diagnostic
techniques are rather limited, and screening has been restricted to histological and ultrastructural examination. Protozoans and anisakids are
the most studied cephalopod pathogens. Most recently, molecular techniques for detecting and identifying pathogens in cephalopods have
been developed as valid and suitable tools. They are expected to be increasingly utilized in pathogen monitoring programs.
However, the routine use of DNA-based diagnostic tools is hampered
by a number of major concerns. Not all regions of the pathogen DNA are
equally useful as targets for molecular detection. It is necessary to identify
regions of the genome that may prove useful for species differentiation. In
addition, molecular techniques could be useful to determine if strains
demonstrate genetic and/or virulence differences (Gestal et al., 2008).
To date, most of the studies involved in the research of the host
immune system are devoted to understanding how the immune system responds against pathogens beyond the functional response. Because mollusks lack of adaptive immune response they can only face
pathogens with their innate immunity, being the hemocytes the main
effectors (Cheng, 1975).
Expressed sequence tags (ESTs) have been sequenced from redundant, normalized libraries and are currently used as a valuable molecular tool. They have been successfully applied to nd genes which are
involved in different physiological processes (e.g. respiratory chain,
cell communication, cell defense). ESTs have also been successful to determinate genes differentially expressed, mainly in bivalves of aquaculture interest naturally infected by parasites such as Ruditapes decussatus
infected by Perkinsus olseni (Prado-Alvarez et al., 2009) or in those
experimentally challenged with bacteria such as R. decussatus (Gestal
et al., 2007a) or M. galloprovincialis (Li et al., 2010; Pallavicini et al.,
2008). Although cephalopods are important subjects in aquaculture
practice (Vaz-Pires et al., 2004), they are currently reared in the Atlantic
Galician coast in oating cages (Garca Garca et al., 2004), there is still a
lack of knowledge about the molecular bases that regulate the cephalopod immune response.
Molecular tools have been successfully applied to study the symbiotic relationship between E. scolopes and the bacteria V. schery. In
recent years 11 cDNA libraries were generated from light organs of
a pool of juvenile squids E. scolopes with and without the colonizing
bacteria V. scheri. From a total of 13,962 non-redundant ESTs 9,728
had a signicant hit to a protein in the NCBI nonredundant protein
database. From these, 6061 correspond to annotated ESTs; 2793 to
hypotetical and 874 to unknown proteins (Chun et al., 2006).
The data available from the cDNA library has lead to identify genes
related to the immune system such as the complement factor C3 in
tissues (light organ without core, central core, mantle, arm muscle,
gills and white body) of juvenile and adult squids. The lowest level
of C3 transcript was detected in arm tissue and hemocytes even
when those cell are the primary site of synthesis (Castillo et al., 2009).
Transcripts encoding proteins in the Toll/NF-B pathway have been
also identied. The analysis showed only one Toll-Like Receptor (TLR)
that probably works as a global microbe receptor. The MyD88-like transcript has not been identied from E. scolopes EST database; it was identied in C. gigas encoding a protein that links the TLR with the IRAK
protein. This result suggests a similar Toll/NF-kB pathway present in
mollusks, but further investigation is required (Goodson et al., 2005).
6. Closing remarks
As summarized above, few data have been added over the last
20 years to the classical knowledge available for cephalopod parasitology.

20

S. Castellanos-Martnez, C. Gestal / Journal of Experimental Marine Biology and Ecology 447 (2013) 1422

Most of the studies utilized parasites to characterize the ecology of

squid stocks leaving aside the effect of parasites on the stocks' health.
However, the increasing economic importance of cephalopods in
worldwide sheries and aquaculture raises the interest not only to develop research on zoonotic pathogens, but also to decipher the host
parasite relationship as seen from the side of the cephalopod immune
response.
Recent advancements in molecular biology allowed to explore, for
example, gene expression in relation with immune response to diseases or parasite infections in cephalopods. In all the above studies,
the identity of the transcripts was determined by matching the experimental sequence to the genomic data. However, despite the fact that
several thousands of redundant nucleotide records are counted for
mollusks in GeneBank, and the economic value of many cephalopod
species and the relatively reduced costs of building full-genome sequences of organism programs, no cephalopod has been sequenced
until now; only oysters have been selected for genome sequencing
among bivalves (Hedgecock et al., 2005). A research effort is needed!
The availability of the full-genome sequence data would be of great
help in the identication of many important genes related to the immune system and the response to diseases in cephalopods.
Acknowledgments
This work has been partly funded by a research grant from the
Galician Council Xunta de Galicia (10PXIB402116PR). Sheila CastellanosMartnez has been supported by a scholarship from CONACyT. [SS]
References
Abollo, E., Gestal, C., Lopez, A., Gonzlez, A.F., Guerra, A., Pascual, S., 1998. Squid as
trophic bridges for parasite ow within marine ecosystems: the case of Anisakis
simplex (Nematoda: Anisakidae), or when the wrong way can be right. S. Afr. J.
Mar. Sci. 20, 223232.
Abollo, E., Gestal, C., Pascual, S., 2001. Anisakis infestation in marine sh and cephalopods from Galician waters: an updated perspective. Parasitol. Res. 87, 492499.
Agust, C., Aznar, F.J., Olson, P.D., Littlewood, D.T.J., Kostadinova, A., Raga, J.A., 2005.
Morphological and molecular characterization of tetraphyllidean merocercoids
(Platyhelminthes: Cestoda) of striped dolphins (Stenella coeruleoalba) from the
Western Mediterranean. Parasitology 130, 461474.
Alpuche, J., Pereyra, A., Mendoza-Hernandez, G., Agundis, C., Rosas, C., Zenteno, E.,
2010. Purication and partial characterization of an agglutinin from Octopus
maya serum. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 156, 15.
Altura, M., Stabb, E., Goldman, W., Apicella, M., McFall-Ngai, M.J., 2011. Attenuation of
host NO production by MAMPs potentiates development of the host in the squidvibrio symbiosis. Cell. Microbiol. 13, 527537.
Anderson, R.S., 1994. Hemocyte-derived reactive oxygen intermediate production in
four bivalve mollusks. Dev. Comp. Immunol. 18, 8996.
Aruga, J., Odaka, Y.S., Kamiya, A., Furuya, H., 2007. Dicyema Pax6 and Zic: tool-kit genes
in a highly simplied bilaterian. BMC Evol. Biol. 7.
Arumugam, M., Romestand, B., Torreilles, J., Roch, P., 2000. In vitro production of superoxide and nitric oxide (as nitrite and nitrate) by Mytilus galloprovincialis haemocytes
upon incubation with PMA or laminarin or during yeast phagocytosis. Eur. J. Cell Biol.
79, 513519.
Audicana, M.T., Ansotegui, I.J., de Corres, L.F., Kennedy, M.W., 2002. Anisakis simplex:
dangerous dead and alive? Trends Parasitol. 18, 2025.
Balguerias, E., Quintero, M.E., Hernandez-Gonzalez, C.L., 2000. The origin of the Saharan
Bank cephalopod shery. ICES J. Mar. Sci. 57, 1523.
Bello, G., Mariniello, L., 1998. Occurrence of Livoneca sinuata (Isopoda, Cymothoidae) in
the mantle cavity of Sepiola ligulata (Cephalopoda, Sepiolidae). Arch. Fish Mar. Res.
46, 3742.
Beuerlein, K., Schimmelpfennig, R., Westermann, B., Ruth, P., Schipp, R., 1998.
Cytobiological studies on hemocyanin metabolism in the branchial heart complex
of the common cuttlesh Sepia ofcinalis (Cephalopoda, Dibranchiata). Cell Tissue
Res. 292, 587595.
Beuerlein, K., Lohr, S., Westermann, B., Ruth, P., Schipp, R., 2002. Components of the
cellular defense and detoxication system of the common cuttlesh Sepia
ofcinalis (Mollusca, Cephalopoda). Tissue Cell 34, 390396.
Boucher-Rodoni, R., Boucaud-Camou, E., Mangold, K., 1987. Feeding and digestion.
Cephalopod Life Cycles. Academic Press London, London, pp. 85108.
Boyle, P., Rodhouse, P., 2005. Cephalopods: Ecology and Fisheries. Blackwell Science,
Ames, Iowa, pp. 1452.
Bugg, D.A., Hegaret, H., Wikfors, G.H., Allam, B., 2007. Oxidative burst in hard clam
(Mercenaria mercenaria) haemocytes. Fish Shellsh Immunol. 23, 188196.
Caddy, J.F., Rodhouse, P.G., 1998. Cephalopod and groundsh landings: evidence for
ecological change in global sheries? Rev. Fish Biol. Fish. 8, 431444.

Castellanos-Martinez, S., Gestal, C., 2011. Immune response of Octopus vulgaris against
the infection by the gastrointestinal parasite Aggregata octopiana. J. Shellsh. Res.
30, 997998.
Castellanos-Martinez, S., Carmen Gomez, M., Hochberg, F., Gestal, C., Furuya, H., 2011. A
new dicyemid from Octopus hubbsorum (Mollusca: Cephalopoda: Octopoda).
J. Parasitol. 97, 265269.
Castillo, M.G., Goodson, M.S., McFall-Ngai, M.J., 2009. Identication and molecular
characterization of a complement C3 molecule in a lophotrochozoan, the Hawaiian
bobtail squid Euprymna scolopes. Dev. Comp. Immunol. 33, 6976.
Cavalier-Smith, T., 1993. Kingdom protozoa and its 18 phyla. Microbiol. Rev. 57, 953994.
Cheng, T.C., 1975. Functional morphology and biochemistry of molluscan phagocytes.
Ann. N. Y. Acad. Sci. 266, 343379.
Chowdhury, K.D., Sen, G., Biswas, T., 2010. Regulatory role of nitric oxide in the reduced
survival of erythrocytes in visceral leishmaniasis. Biochim. Biophys. Acta Gen. Subj.
1800, 964976.
Chu, F.L., 2000. Defense mechanisms of marine bivalves. In: Fingerman, M.,
Nagabhushanam, R. (Eds.), Recent advances in marine biotechnology. Immunology
and Pathology, 5. Science Publishers Inc., Eneld, UK, pp. 142.
Chun, C.K., Scheetz, T.E., Fatima Bonaldo, M., Brown, B., Clemens, A., Crookes-Goodson,
W.J., Crouch, K., DeMartini, T., Eyestone, M., Goodson, M.S., Janssens, B., Kimbell,
J.L., Koropatnick, T.A., Kucaba, T., Smith, C., Stewart, J.J., Tong, D., Troll, J.V.,
Webster, S., Winhall-Rice, J., Yap, C., Casavant, T.L., McFall-Ngai, M.J., Soares, M.,
2006. An annotated cDNA library of juvenile Euprymna scolopes with and without
colonization by the symbiont Vibrio scheri. BMC Genomics 7, 154.
Claes, M.F., 1996. Functional morphology of the white bodies of the cephalopod
mollusc Sepia ofcinalis. Acta Zool. 77, 173190.
Clarke, M.R., 1996. The role of cephalopods in the world's oceans: general conclusions
and the future. Philos. Trans. R. Soc. Lond. B 351, 11051112.
Cowden, R.R., 1972. Some cytological and cytochemical observations on leukopoietic
organs, the white bodies of Octopus vulgaris. J. Invertebr. Pathol. 19, 113119.
Cowden, R.R., Curtis, S.K., 1973. Observations on living cells dissociated from leukopoietic
organ of Octopus briareus. Exp. Mol. Pathol. 19, 178185.
Cowden, R.R., Curtis, S.K., 1981. Cephalopods. In: Ratcliffe, N.A., Rowley, A.F. (Eds.),
Invertebrate blood cells. General Aspects, Animals Without True Circulatory
Systems to Cephalopods, 1. Academic Press, London, UK, pp. 301323.
Czaker, R., 2006. Serotonin immunoreactivity in a highly enigmatic metazoan phylum,
the pre-nervous Dicyemida. Cell Tissue Res. 326, 843850.
da Silva, P.M., Comesana, P., Fuentes, J., Villalba, A., 2008. Variability of haemocyte and
haemolymph parameters in European at oyster Ostrea edulis families obtained
from brood stocks of different geographical origins and relation with infection by
the protozoan Bonamia ostreae. Fish Shellsh Immunol. 24, 551563.
Davey, H.M., 2002. Flow cytometric techniques for the detection of microorganisms.
Methods Cell Sci. 24, 9197.
Davidson, S.K., Koropatnick, T.A., Kossmehl, R., Sycuro, L., McFall-Ngai, M.J., 2004. NO
means yes in the squid-vibrio symbiosis: nitric oxide (NO) during the initial
stages of a benecial association. Cell. Microbiol. 6, 11391151.
Di Cosmo, A., Di Cristo, C., Palumbo, A., d'Ischia, M., Messenger, J.B., 2000. Nitric oxide
synthase (NOS) in the brain of the cephalopod Sepia ofcinalis. J. Comp. Neurol.
428, 411427.
Di Cristo, C., Fiore, G., Scheinker, V., Enikolopov, G., d'Ischia, M., Palumbo, A., Di Cosmo,
A., 2007. Nitric oxide synthase expression in the central nervous system of Sepia
ofcinalis: an in situ hybridization study. Eur. J. Neurosci. 26, 15991610.
Dobell, C.C., 1925. The life history and chromosome cycle of Aggregata eberthi (Protozoa:
Sporozoa: Coccidia). Parasitology 17, 1136.
Ellis, R., Parry, H., Spicer, J.I., Hutchinson, T., Pipe, R., Widdicombe, S., 2011. Immunological
function in marine invertebrates: responses to environmental perturbation. Fish
Shellsh Immunol. 30, 12091222.
European Parliament, Council of the European Union, 2010. Directive 2010/63/EU of
the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientic Purposes. Council of Europe, Strasbourg.
Farto, R., Armada, S.P., Montes, M., Guisande, J.A., Perez, M.J., Nieto, T.P., 2003. Vibrio
lentus associated with diseased wild octopus (Octopus vulgaris). J. Invertebr. Pathol.
83, 149156.
Ferl, J.P., 1988. Wound healing after arm amputation in Sepia ofcinalis (Cephalopoda:
Sepioidea). J. Invertebr. Pathol. 52, 380388.
Finn, J.K., Hochberg, F.G., Norman, M.D., 2005. Phylum Dicyemida in Australian waters:
rst record and distribution across diverse cephalopods hosts. Phuket Mar. Biol.
Cent. Res. Bull. 66, 8396.
Fisher, W.S., Dinuzzo, A.R., 1991. Agglutination of bacteria and erythrocytes by serum
from six species of marine mollusks. J. Invertebr. Pathol. 57, 380394.
Ford, L.A., 1992. Host defense mechanisms of cephalopods. Annu. Rev. Fish Dis. 2, 2541.
Ford, L.A., Alexander, S.K., Cooper, K.M., Hanlon, R.T., 1986. Bacterial populations of
normal and ulcerated mantle tissue of the squid, Lolliguncula brevis. J. Invertebr.
Pathol. 48, 1326.
Furuya, H., 2008. A new dicyemid from Sepiella japonica (Mollusca: Cephalopoda:
Decapoda). J. Parasitol. 94, 223229.
Furuya, H., 2009. Two new dicyemids from Sepia longipes (Mollusca: Cephalopoda:
Decapoda). J. Parasitol. 95, 681689.
Furuya, H., 2010. A new dicyemid from Benthoctopus sibiricus (Mollusca: Cephalopoda:
Octopoda). J. Parasitol. 96, 11231127.
Furuya, H., Tsuneki, K., 2003. Biology of dicyemid mesozoans. Zool. Sci. 20, 519532.
Furuya, H., Tsuneki, K., Koshida, Y., 1997. Fine structure of dicyemid mesozoans, with
special reference to cell junctions. J. Morphol. 231, 297305.
Furuya, H., Hochberg, F.G., Tsuneki, K., 2004a. Cell number and cellular composition in
infusoriform larvae of dicyemid mesozoans (Phylum Dicyemida). Zool. Sci. 21,
877889.

S. Castellanos-Martnez, C. Gestal / Journal of Experimental Marine Biology and Ecology 447 (2013) 1422
Furuya, H., Ota, M., Kimura, R., Tsuneki, K., 2004b. Renal organs of cephalopods: a habitat
for dicyemids and chromidinids. J. Morphol. 262, 629643.
Garca Garca, J., Rodrguez Gonzlez, L., Garca Garca, B., 2004. Cost analysis of octopus
ongrowing installation in Galicia. Span. J. Agric. Res. 2, 531537.
Garcia, J.G., Garcia, B.G., 2011. Econometric model of viability/protability of octopus
(Octopus vulgaris) ongrowing in sea cages. Aquacult. Int. 19, 11771191.
Garcia, J., Gonzalez, L., Garcia, B., 2004. Cost analysis of octopus ongrowing installation
in Galicia. Span. J. Agric. Res. 2, 531537.
Gestal, C., Abollo, E., Arias, G., Pascual, S., 1997. Dicyema typus Van Beneden, 1876
(Dicyema: Dicyemidae), un mesozoo parsito del rin de Octopus vulgaris
Curier, 1797 (Mollusca: Cephalopoda) del noroeste de Espaa. Rev. Iber. Parasitol.
57, 8587.
Gestal, C., Belcari, P., Abollo, E., Pascual, S., 1999a. Parasites of cephalopods in the northern
Tyrrhenian Sea (western Mediterranean): new host records and host specicity. Sci.
Mar. 63, 3943.
Gestal, C., Pascual, S., Corral, L., Azevedo, C., 1999b. Ultrastructural aspects of the sporogony of Aggregata octopiana (Apicomplexa, Aggregatidae), a coccidian parasite
of Octopus vulgaris (Mollusca, Cephalopoda) from NE Atlantic coast. Eur. J. Protistol.
35, 417425.
Gestal, C., Guerra, A., Abollo, E., Pascual, S., 2000. Aggregata sagittata n. sp. (Apicomplexa:
Aggregatidae), a coccidian parasite from the European ying squid Todarodes
sagittatus (Mollusca: Cephalopoda). Syst. Parasitol. 47, 203206.
Gestal, C., Abollo, E., Pascual, S., 2002a. Observations on associated histopathology with
Aggregata octopiana infection (Protista: Apicomplexa) in Octopus vulgaris. Dis.
Aquat. Org. 50, 4549.
Gestal, C., de la Cadena, M.P., Pascual, S., 2002b. Malabsorption syndrome observed in
the common octopus Octopus vulgaris infected with Aggregata octopiana (Protista:
Apicomplexa). Dis. Aquat. Org. 51, 6165.
Gestal, C., Nigmatullin, C.M., Hochberg, F.G., Guerra, A., Pascual, S., 2005. Aggregata
andresi n. sp. (Apicomplexa: Aggregatidae) from the ommastrephid squid Martialia
hyadesi in the SW Atlantic Ocean and some general remarks on Aggregata spp. in
cephalopod hosts. Syst. Parasitol. 60, 6573.
Gestal, C., Costa, M.M., Figueras, A., Novoa, B., 2007a. Analysis of differentially
expressed genes in response to bacterial stimulation in hemocytes of the carpetshell clam Ruditapes decussatus: identication of new antimicrobial peptides.
Gene 406, 134143.
Gestal, C., Guerra, A., Pascual, S., 2007b. Aggregata octopiana (Protista: Apicomplexa): a
dangerous pathogen during commercial Octopus vulgaris ongrowing. ICES J. Mar.
Sci. 64, 17431748.
Gestal, C., Roch, P., Renault, T., Pallavicini, A., Paillard, C., Novoa, B., Oubella, R., Venier,
P., Figueras, A., 2008. Study of diseases and the immune system of bivalves using
molecular biology and genomics. Rev. Fish. Sci. 16, 133156.
Gestal, C., Pascual, S., Hochberg, F., 2010. Aggregata bathytherma sp. nov. (Apicomplexa:
Aggregatidae), a new coccidian parasite associated with a deep-sea hydrothermal
vent octopus. Dis. Aquat. Org. 91, 237242.
Gonzalez, A.F., Pascual, S., Gestal, C., Abollo, E., Guerra, A., 2003. What makes a cephalopod a suitable host for parasite? The case of Galician waters. Fish. Res. 60,
177183.
Gonzlez, A.F., Pascual, S., Gestal, C., Abollo, E., Guerra, A., 2003. What makes a cephalopod a suitable host for parasite? The case of Galician waters. Fish. Res. 60, 177183.
Goodson, M.S., Kojadinovic, M., Troll, J.V., Scheetz, T.E., Casavant, T.L., Soares, M.B.,
McFall-Ngai, M.J., 2005. Identifying components of the NF-kappa B pathway in
the benecial Euprymna scolopes Vibrio scheri light organ symbiosis. Appl. Environ. Microbiol. 71, 69346946.
Grant, P., Zheng, Y., Pant, H.C., 2006. Squid (Loligo pealei) giant ber system: a model
for studying neurodegeneration and dementia? Biol. Bull. 210, 318333.
Grossowicz, N., Ariel, M., Weber, T., 1979. Improved lysozyme assay in biological-uids.
Clin. Chem. 25, 484485.
Hanlon, R.T., Forsythe, J.W., 1990a. Diseases of Mollusca: Cephalopoda. Diseases caused
by microorganisms. In: Kinne, O. (Ed.), Diseases of marine animals. Introduction,
Cephalopoda, Annelida, Crustacea, Chaetognatha, Echinodermata, Urochordata,
III. Biologische Anstalt, Helgoland, Hamburg, Germany, pp. 2346.
Hanlon, R.T., Forsythe, J.W., 1990b. Diseases of Mollusca: Cephalopoda. Structural abnormalities and neoplasia. In: Kinne, O. (Ed.), Diseases of marine animals. Introduction,
Cephalopoda, Annelida, Crustacea, Chaetognatha, Echinodermata, Urochordata, III.
Biologische Anstalt, Helgoland, Hamburg, Germany, pp. 203228.
Hanlon, R.T., Messenger, J.B., 1996. Cephalopod Behaviour. Cambridge University Press,
Cambridge, pp. 1232.
Hanlon, R.T., Hixon, R.F., Hulet, W.H., 1983. Survival, growth, and behavior of the loliginid
squids Loligo plei, Loligo pealei, and Lolliguncula brevis (Mollusca, Cephalopoda) in
closed seawater systems. Biol. Bull. 165, 637685.
Hanlon, R.T., Forsythe, J.W., Cooper, K.M., Dinuzzo, A.R., Folse, D.S., Kelly, M.T., 1984.
Fatal penetrating skin ulcers in laboratory-reared octopuses. J. Invertebr. Pathol.
44, 6783.
Hedgecock, D., Gaffney, P.M., Goulletquer, P., Guo, X.M., Reece, K., Warr, G.W., 2005.
The case for sequencing the Pacic oyster genome. J. Shellsh. Res. 24, 429441.
Hochberg, F.G., 1990. Diseases of Mollusca: Cephalopoda. Diseases caused by Protistans
and Metazoans. In: Kinne, O. (Ed.), Diseases of marine animals. Introduction,
Cephalopoda, Annelida, Crustacea, Chaetognatha, Echinodermata, Urochordata,
III. Biologische Anstalt, Helgoland, Hamburg, Germany, pp. 47202.
Hochner, B., 2008. Octopuses. Curr. Biol. 18, R897R898.
Hochner, B., Shomrat, T., Fiorito, G., 2006. The octopus: a model for a comparative analysis of the evolution of learning and memory mechanisms. Biol. Bull. 210, 308317.
Jacklet, J.W., 1997. Nitric oxide signaling in invertebrates. Inv. Neurosci. 3, 114.
Kobayashi, M., Furuya, H., Holland, P.W.H., 1999. Evolution: Dicyemids are higher
animals. Nature 401, 762. http://dx.doi.org/10.1038/44513.

21

Kobayashi, M., Furuya, H., Wada, H., 2009. Molecular markers comparing the extremely
simple body plan of dicyemids to that of lophotrochozoans: insight from the
expression patterns of Hox, Otx, and brachyury. Evol. Dev. 11, 582589.
Kopecn, J., Jirk, M., Obornk, M., Tokarev, Y.S., Luke, J., Modry, D., 2006. Phylogenetic
analysis of coccidian parasites from invertebrates: search for missing links. Protist
157, 173183.
Li, H., Venier, P., Prado-Alvarez, M., Gestal, C., Toubiana, M., Quartesan, R., Borghesan, F.,
Novoa, B., Figueras, A., Roch, P., 2010. Expression of Mytilus immune genes in response to experimental challenges varied according to the site of collection. Fish
Shellsh Immunol. 28, 640648.
Licciardo, G., Garziano, A., Nocera, G., Gaglio, G., Marino, F., De Vico, G., 2005. Contributo alla
conoscenza dell'azione patogena di Aggregata octopiana (Apicomplexa: Aggregatidae)
in Octopus vulgaris nel sud del Mar Tirreno. Ittiopatologia 2, 193198.
Lpez, C., Carballal, M.J., Azevedo, C., Villalba, A., 1997. Morphological characterization of
the hemocytes of the clam, Ruditapes decussatus (Mollusca: Bivalvia). J. Invertebr.
Pathol. 69, 5157.
Macan, M.C., Ludwig, W., Aznar, R., Grimont, P.A.D., Schleifer, K.H., Garay, E., Pujalte,
M.J., 2001. Vibrio lentus sp. nov., isolated from Mediterranean oysters. Int. J. Syst.
Evol. Microbiol. 51, 14491456.
Malham, S.K., Runham, N.W., 1998. A brief review of the immunology of Eledone
cirrhosa. S. Afr. J. Mar. Sci. 20, 385391.
Malham, S.K., Runham, N.W., Secombes, C.J., 1997. Phagocytosis by haemocytes from
the lesser octopus Eledone cirrhosa. Iberus 15, 111.
Malham, S.K., Coulson, C.L., Runham, N.W., 1998. Effects of repeated sampling on the
haemocytes and haemolymph of Eledone cirrhosa (Lam.). Comp. Biochem. Physiol.
A: Mol. Integr. Physiol. 121, 431440.
Malham, S.K., Lacoste, A., Gelebart, F., Cueff, A., Poulet, S.A., 2002. A rst insight into
stress-induced neuroendocrine and immune changes in the octopus Eledone
cirrhosa. Aquat. Living Resour. 15, 187192.
Mather, J.A., 1995. Cognition in cephalopods. Adv. Study Behav. 24, 317353.
Mattiucci, S., Nascetti, G., 2008. Advances and trends in the molecular systematics of
anisakid nematodes, with implications for their evolutionary ecology and hostparasite co-evolutionary processes. Adv. Parasitol. 66, 47148.
Mayrand, E., St Jean, S.D., Courtenay, S.C., 2005. Haemocyte responses of blue mussels
(Mytilus edulis L.) transferred from a contaminated site to a reference site: can
the immune system recuperate? Aquac. Res. 36, 962971.
McConnaughey, B.H., 1983. Mesozoa. In: Adiyodi, K.G., Adiyodi, R.G. (Eds.), Reproductive biology of invertebrates. Oogenesis, Oviposition and Oosorption, I. John Wiley
and Sons, New York, NY, pp. 135145.
McDade, J.E., Tripp, M.R., 1967. Mechanism of agglutination of red blood cells by oyster
hemolymph. J. Invertebr. Pathol. 9, 523.
McFall-Ngai, M.J., 1994. Animalbacterial interactions in the early life history of marine
invertebrates: the Euprymna scolopes/Vibrio scheri symbiosis. Am. Zool. 34,
554561.
McFall-Ngai, M.J., Nyholm, S.V., Castillo, M.G., 2010. The role of the immune system in
the initiation and persistence of the Euprymna scolopesVibrio scheri symbiosis.
Semin. Immunol. 22, 4853.
Mladineo, I., Bocina, I., 2007. Extraintestinal gamogony of Aggregata octopiana in the
reared common octopus (Octopus vulgaris) (Cephalopoda: Octopodidae). J. Invertebr.
Pathol. 96, 261264.
Mladineo, I., Jozic, M., 2005. Aggregata infection in the common octopus, Octopus
vulgaris (Linnaeus 1758), Cephalopoda: Octopodidae, reared in a ow-through
system. Acta Adriat. 46, 193199.
Nigmatullin, C.M., Shukhgalter, O.A., 1990. Helmintofauna y aspectos ecolgicos de las
relaciones parasitarias del calamar (Illex argentinus) en el Atlntico Sudoccidental.
Frente Maritimo 7A, 5768.
Nigmatullin, C.M., Shchetinnikov, A.S., Shukhgalter, O.A., 2009. On feeding and helminth
fauna of neon ying squid Ommastrephes bartramii (Lesueur, 1821) (Cephalopoda:
Ommastrephidae) in the southeastern Pacic. Rev. Biol. Mar. Oceanogr. 44, 227235.
Nolan, M.J., Cribb, T.H., 2005. The use and implications of ribosomal DNA sequencing
for the discrimination of digenean species. Adv. Parasitol. 60, 101163.
Novoa, B., Tafalla, C., Guerra, A., Figueras, A., 2002. Cellular immunological parameters
of the octopus, Octopus vulgaris. J. Shellsh. Res. 21, 243248.
Nyholm, S.V., McFall-Ngai, M.J., 2004. The winnowing: establishing the squid-vibrio
symbiosis. Nat. Rev. Microbiol. 2, 632642.
Nyholm, S.V., Stewart, J.J., Ruby, E.G., McFall-Ngai, M.J., 2009. Recognition between
symbiotic Vibrio scheri and the haemocytes of Euprymna scolopes. Environ.
Microbiol. 11, 483493.
Overstreet, R.M., Hochberg, F.G., 1975. Digenetic trematodes in cephalopods. J. Mar.
Biol. Assoc. UK 55, 893910.
Pallavicini, A., Costa, M.M., Gestal, C., Dreos, R., Figueras, A., Venier, P., Novoa, B., 2008.
High sequence variability of myticin transcripts in hemocytes of immunestimulated mussels suggests ancient host-pathogen interactions. Dev. Comp.
Immunol. 32, 213226.
Palumbo, A., 2005. Nitric oxide in marine invertebrates: a comparative perspective.
Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 142, 241248.
Pardo-Gandarillas, M.C., Lohrmann, K.B., Valdivia, A.L., Ibez, C.M., 2009. First record of
parasites of Dosidicus gigas (d'Orbigny, 1835) (Cephalopoda: Ommastrephidae)
from the Humboldt Current system off Chile. Rev. Biol. Mar. Oceanogr. 44, 397408.
Pascual, S., Guerra, A., 2001. Vexing question on sheries research: the study of cephalopods and their parasites. Iberus 19, 8795.
Pascual, S., Hochberg, F.G., 1996. Marine parasites as biological tags of cephalopod
hosts. Parasitol. Today 12, 324327.
Pascual, S., Gonzlez, A., Arias, C., Guerra, A., 1995. Histopathology of larval Anisakis
simplex B (Nematoda, Anisakidae), parasites of short-nned squid in the SE
North Atlantic. Bull. Eur. Assoc. Fish Pathol. 15, 160161.

22

S. Castellanos-Martnez, C. Gestal / Journal of Experimental Marine Biology and Ecology 447 (2013) 1422

Pascual, S., Gestal, C., Estevez, J.M., Rodriguez, H., Soto, M., Abollo, E., Arias, C., 1996a.
Parasites in commercially-exploited cephalopods (Mollusca, Cephalopoda) in
Spain: an updated perspective. Aquaculture 142, 110.
Pascual, S., Gonzalez, A., Arias, C., Guerra, A., 1996b. Biotic relationships of Illex coindetii
and Todaropsis eblanae (Cephalopoda, Ommastrephidae) in the Northeast Atlantic:
evidence from parasites. Sarsia 81, 265274.
Pascual, S., Gestal, C., Abollo, E., 1997. Effect of Pennella sp. (Copepoda, Pennellidae) on the
condition of Illex coindetii and Todaropsis eblanae (Cephalopoda, Ommastrephidae).
Bull. Eur. Assoc. Fish Pathol. 7, 9195.
Pascual, S., Gonzlez, A.F., Arias, C., Guerra, A., 1999. Larval Anisakis simplex B
(Nematoda: Ascaridoidea) of short-nned squid (Cephalopoda: Ommastrephidae)
in north-west Spain. J. Mar. Biol. Assoc. UK 79, 6572.
Pascual, S., Gonzlez, A.F., Gestal, C., Abollo, E., Guerra, A., 2001. Epidemiology of
Pennella sp. (Crustacea: Copepoda), in exploited Illex coindetti stock in the NE
Atlantic. Sci. Mar. 65, 307312.
Pascual, S., Vega, M.A., Rocha, F.J., Guerra, A., 2002. First report of an endoparasitic
epicaridean isopod infecting cephalopods. J. Wildl. Dis. 38, 473477.
Pascual, S., Rocha, F., Guerra, A., 2006. Gross lesions in the Hubb octopus Octopus
hubbsorum. Mar. Biol. Res. 2, 420423.
Pascual, S., Gonzalez, A., Guerra, A., 2007. Parasites and cephalopod sheries uncertainty:
towards a waterfall understanding. Rev. Fish Biol. Fish. 17, 139144.
Pascual, S., Gonzlez, A.F., Guerra, A., 2010. Coccidiosis during octopus senescence: preparing for parasite outbreak. Fish. Res. 106, 160162.
Petric, M., Mladineo, I., Sifner, S.K., 2011. Insight into the short-nned squid Illex
coindetii (Cephalopoda: Ommastrephidae) feeding ecology: is there a link between
helminth parasites and food composition? J. Parasitol. 97, 5562.
Pipe, R.K., 1992. Generation of reactive oxygen metabolites by the hemocytes of the
mussel Mytilus edulis. Dev. Comp. Immunol. 16, 111122.
Poynton, S.L., Reimschuessel, R., Stoskopf, M.K., 1992. Aggregata dobelli n. sp. and
Aggregata millerorum n. sp. (Apicomplexa, Aggregatidae) from two species of octopus (Mollusca, Octopodidae) from the Eastern North Pacic-Ocean. J. Protozool. 39,
248256.
Prado-Alvarez, M., Gestal, C., Novoa, B., Figueras, A., 2009. Differentially expressed
genes of the carpet shell clam Ruditapes decussatus against Perkinsus olseni. Fish
Shellsh Immunol. 26, 7283.
Randhawa, H.S., 2011. Insights using a molecular approach into the life cycle of a tapeworm infecting great white sharks. J. Parasitol. 97, 275280.
Rivero, A., 2006. Nitric oxide: an antiparasitic molecule of invertebrates. Trends
Parasitol. 22, 219225.
Robertson, J.D., Bonaventura, J., Kohm, A.P., 1994. Nitric oxide is required for tactile
learning in Octopus vulgaris. Proc. R. Soc. Lond. B 256, 269273.
Rodrguez-Domnguez, H., Soto-Ba, M., Iglesias-Blanco, R., Crespo-Gonzlez, C., AriasFernndez, C., Garca-Estvez, J., 2006. Preliminary study on the phagocytic ability
of Octopus vulgaris Cuvier, 1797 (Mollusca: Cephalopoda) haemocytes in vitro.
Aquaculture 254, 563570.

Rgener, W., Renwrantz, L., Uhlenbruck, G., 1985. Isolation and characterization of a
Lectin from the hemolymph of the cephalopod Octopus vulgaris (Lam) inhibited
by Alpha-D-lactose and N-acetyl-lactosamine. Dev. Comp. Immunol. 9, 605616.
Salimi, L., Jamili, S., Motalebi, A., Eghtesadi-Araghi, P., Rabbani, M., Rostami-Beshman,
M., 2009. Morphological characterization and size of hemocytes in Anodonta
cygnea. J. Invertebr. Pathol. 101, 8185.
Sardella, N.H., Roldn, M.I., Tanzola, D., 1990. Helmintos parsitos del calamar (Illex
argentinus) en la subpoblacin Bonaerense-Norpatagnica. Frente Maritimo 7A,
5358.
Sardella, N.H., Re, M.E., Timi, J.T., 2000. Two new Aggregata species (Apicomplexa:
Aggregatidae) infecting Octopus tehuelchus and Enteroctopus megalocyathus
(Mollusca: Octopodidae) in Patagonia, Argentina. J. Parasitol. 86, 11071113.
Schipp, R., 1987. General morphological and functional characteristics of the cephalopod circulatory system. An introduction. Experientia 43, 474477.
Semmens, J.M., Pecl, G.T., Gillanders, B.M., Waluda, C.M., Shea, E.K., Jouffre, D., Ichii, T.,
Zumholz, K., Katugin, O.N., Leporati, S.C., Shaw, P.W., 2007. Approaches to resolving
cephalopod movement and migration patterns. Rev. Fish Biol. Fish. 17, 401423.
Shukhgalter, O.A., Nigmatullin, C.M., 2001. Parasitic helminths of jumbo squid
Dosidicus gigas (Cephalopoda: Ommastrephidae) in open waters of the central
east Pacic. Fish. Res. 54, 95110.
Stunkard, H.W., 1954. The life-history and systematic relations of the Mesozoa. Q. Rev.
Biol. 29, 230244.
Suzuki, T.G., Ogino, K., Tsuneki, K., Furuya, H., 2010. Phylogenetic analysis of dicyemid
mesozoans (phylum Dicyemida) from innexin amino acid sequences: dicyemids
are not related to Platyhelminthes. J. Parasitol. 96, 614625.
Trilles, J.P., Oktener, A., 2004. Livoneca sinuata (Crustacea; Isopoda; Cymothoidae) on
Loligo vulgaris from Turkey, and unusual cymothoid associations. Dis. Aquat. Org.
61, 235240.
Tripp, M.R., 1963. Cellular responses of mollusks. Ann. N. Y. Acad. Sci. 113, 467.
Tripp, M.R., 1974. Molluscan immunity. Ann. N. Y. Acad. Sci. 234, 2327.
Vaz-Pires, P., Seixas, P., Barbosa, A., 2004. Aquaculture potential of the common octopus (Octopus vulgaris Cuvier, 1797): a review. Aquaculture 238, 221238.
Vidal, E.A.G., Haimovici, M., 1998. Feeding and the possible role of the proboscis and
mucus cover in the ingestion of microorganisms by rhynchoteuthion paralarvae
(Cephalopoda: Ommastrephidae). Bull. Mar. Sci. 63, 305316.
Vidal, E.A.G., Haimovici, M., 1999. Digestive tract parasites in rhynchoteuthion squid
paralarvae, particularly in Illex argentinus (Cephalopoda: Ommastrephidae). Fish.
Bull. 97, 402405.
Wang, Y.L., Dufour, Y.S., Carlson, H.K., Donohue, T.J., Marletta, M.A., Ruby, E.G., 2010. HNOX-mediated nitric oxide sensing modulates symbiotic colonization by Vibrio
scheri. Proc. Natl. Acad. Sci. U.S.A. 107, 83758380.
Wells, M.J., Smith, P.J.S., 1987. The performance of the Octopus circulatory system: a triumph of engineering over design. Experientia 43, 487499.