ICES Journal of
Marine Science
ICES Journal of Marine Science (2015), 72(Supplement 1), i164 –i169. doi:10.1093/icesjms/fsu249
Contribution to the Supplement: ‘Lobsters in a Changing Climate’
Original Article
Caribbean spiny lobsters equally avoid dead and clinically
PaV1-infected conspecifics
Rebeca I. Candia-Zulbarán1,2*, Patricia Briones-Fourzán 2, Enrique Lozano-Álvarez2,
Cecilia Barradas-Ortiz 2, and Fernando Negrete-Soto 2
1
Universidad Nacional Autónoma de México, Posgrado en Ciencias del Mar y Limnologı́a, Ciudad Universitaria, México DF, Mexico
Universidad Nacional Autónoma de México, Instituto de Ciencias del Mar y Limnologı́a, Unidad Académica de Sistemas Arrecifales, Puerto Morelos,
Quintana Roo, Mexico
2
*Corresponding author: tel: +52 998 8710009; e-mail: [email protected]
Candia-Zulbarán, R. I., Briones-Fourzán, P., Lozano-Álvarez, E., Barradas-Ortiz, C., and Negrete-Soto, F. Caribbean spiny lobsters
equally avoid dead and clinically PaV1-infected conspecifics. – ICES Journal of Marine Science, 72: i164 – i169.
Received 30 August 2014; revised 15 December 2014; accepted 20 December 2014; advance access publication 11 January 2015.
Social behaviour in Caribbean spiny lobsters (Panulirus argus) is mediated by conspecific chemical cues. These lobsters can be attracted to shelters
emanating chemical cues from conspecifics but tend to avoid shelters emanating chemical cues from injured conspecifics, dead conspecifics, and
conspecifics with visible signs of a potentially lethal disease caused by the pathogenic Panulirus argus virus 1 (PaV1). However, previous studies have
not controlled for the presence of PaV1 (i.e. subclinical infection) in grossly “healthy” lobsters, although visible signs of disease do not appear until
several weeks after infection. We conducted a controlled experiment using a set of 2 m-long Y-mazes to examine and contrast the response of
P. argus lobsters to shelters emanating chemical cues from conspecifics in four different conditions: uninfected, subclinically PaV1-infected (i.e.
infected but not diseased), clinically PaV1-infected (i.e. infected and diseased), and dead. Using polymerase chain reaction, we tested for PaV1
in all grossly healthy lobsters and used exclusively uninfected lobsters in intermolt as focal lobsters. Focal lobsters similarly avoided shelters emanating chemical cues from clinically infected (80% avoidance) and from dead conspecifics (85% avoidance), but their response to chemical
cues from uninfected and from subclinically infected conspecifics did not differ significantly from random. These results indicate that PaV1diseased lobsters produce chemical cues that are as repellent to conspecifics as are chemicals emanating from dead conspecifics, and that subclinically
infected lobsters either do not emit the repellent chemicals or they do so at sub-threshold levels. However, the nature of the repellent chemicals and
whether they originate from the pathogen or the host remains to be determined.
Keywords: avoidance behaviour, chemical cues, disease, Panulirus argus, Panulirus argus virus 1.
Introduction
The Caribbean spiny lobster, Panulirus argus (Latreille, 1804) is one
of the most valuable fishing resources in the Western Central
Atlantic and constitutes by itself over 50% of the world catch of
spiny lobsters (Phillips et al., 2013). Panulirus argus has a complex
life cycle and undergoes several habitats shifts during its benthic
life. The post-larvae settle in vegetated habitats where the small juveniles remain for a few months and exhibit asocial behaviour.
However, larger juveniles eventually shift from the vegetation to occupying structured crevice-type shelters, a habitat shift that coincides with a change from asocial to social behaviour (Childress
and Herrnkind, 2001a, b). The most ubiquitous example of sociality
in P. argus and other spiny lobsters is gregarious sheltering, which is
mediated by intraspecific chemical cues released in the urine
(Ratchford and Eggleston, 1998; Horner et al., 2006, 2008). For an
individual seeking shelter, following conspecific scents into a den
both reduces its time of exposure and allows it to assess the
quality of the den (Nevitt et al., 2000; Childress and Herrnkind,
2001a), while congregating in dens can increase per capita survival
through either a “dilution effect” or “group defense behavior”
(Eggleston et al., 1990; Childress and Herrnkind, 2001a, b;
Briones-Fourzán et al., 2007). However, gregariousness is not generated by chemical attraction per se unless the benefits of aggregation
outweigh the costs (Loehle, 1995; Childress, 2007). Benefits of
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Panulirus argus avoid dead and clinically PaV1-infected conspecifics
i165
aggregation for P. argus would include efficient use of available shelters, decreased predation risk, and increased survival, persistence,
foraging ranges, and reproductive opportunities (Childress and
Herrnkind, 2001a; Dolan and Butler, 2006; Briones-Fourzán et al.,
2007; Childress, 2007). Costs would include an increase in competition and intraspecific aggression as well as in transmission of parasites and pathogenic disease (Loehle, 1995; Lafferty et al., 2004;
Childress, 2007).
Catches of P. argus have been declining steadily since 2000,
mostly due to a combination of overexploitation, changes in environmental and ecological conditions impacting the lobster
habitats (Ehrhardt et al., 2011). However, another potential
cause for this decline is the disease caused by a pathogenic virus,
P. argus virus 1 (PaV1; Shields and Behringer, 2004), which has
become an important source of mortality for the juveniles of
P. argus since its detection in 1999 – 2000 (Moss et al., 2013). In
the laboratory, transmission of PaV1 was found to occur by
contact between healthy and diseased lobsters and through
water, at least over short distances (Butler et al., 2008). However,
experiments conducted by Behringer et al. (2006) showed that
healthy lobsters avoided sharing shelters with visibly diseased conspecifics and that the latter were avoided before becoming infectious, which might help reduce contact transmission rates of PaV1
and keep prevalence levels relatively low. More recently, Anderson
and Behringer (2013) showed that avoidance was chemical in
nature. This would appear akin to avoidance of conspecific alarm
odours (haemolymph-borne chemicals emanating from injured conspecifics), which is strongly exhibited by P. argus (Briones-Fourzán
et al., 2008; Shabani et al., 2008).
Aggregation behaviour is partly communicated by chemical
cues released in the urine of conspecifics, which also communicates social status. Thus, urine-borne signals released by P. argus
lobsters at short distances from conspecifics elicit avoidance
behaviour (similar to haemolymph, Shabani et al., 2008) but
attract conspecifics when released at greater distances (Horner
et al., 2006; Briones-Fourzán et al., 2008); therefore, the choice
of shelter will greatly depend on the context on which chemical
cues are transmitted from conspecifics (Shabani et al., 2009).
Also, the progression of PaV1 infection can affect mobility of lobsters, with heavily infected lobsters becoming lethargic (Shields
and Behringer, 2004; Behringer et al., 2008), but to our knowledge,
previous studies with PaV1 have not tested for the presence of the
virus in presumably healthy individuals. This is important because
visible signs of disease do not appear until many weeks after infection (Behringer et al., 2006). Moreover, subclinically infected
lobsters (i.e. positive for PaV1 by polymerase chain reaction
(PCR) but with no visible signs of disease) can be as abundant in
natural habitats as visibly diseased lobsters (Huchin-Mian et al.,
2013) or even more so (Behringer et al., 2011), and hence uninfected lobsters might not be able to avoid being near potentially infectious conspecifics.
Therefore, the aim of the present study was to contrast the behavioural response of healthy, uninfected individuals of P. argus lobsters
to shelters emanating chemical scents from conspecifics in four
conditions: uninfected with PaV1, subclinically infected with
PaV1, clinically infected with PaV1, and dead (freshly killed and
crushed), controlling for the presence of PaV1 in focal lobsters.
Unlike previous studies wherein experimentally infected lobsters
were used, we used exclusively lobsters that were infected due to
viral exposure under natural conditions. We expected focal lobsters
to exhibit attraction to uninfected lobsters, avoidance of clinically
infected and dead lobsters, and a neutral response to subclinically
infected lobsters, as we have found uninfected lobsters very close
to subclinically infected lobsters in field observations (HuchinMian et al., 2013; RIC-Z, unpublished data).
Material and methods
Lobster collection
The experiments were conducted in the Unidad Académica de
Sistemas Arrecifales, Universidad Nacional Autónoma de México,
at Puerto Morelos, Mexico (20854′ N, 86854′ W). Using scuba
diving, we collected grossly healthy and visibly diseased juvenile lobsters by hand over the Puerto Morelos reef lagoon and transferred
them to the laboratory within 1 h of capture. Visibly diseased lobsters (i.e. clinically infected with PaV1) exhibit a milky-white
haemolymph that is clearly visible through the transparent membrane between the cephalothorax and abdomen. Diseased and
“healthy” lobsters were held in separate tanks provided with multiple hollow concrete blocks for shelter. During the holding period
(≤1 week), lobsters were fed every other day with mussels. The
holding tanks and all experimental units (see below) received seawater from an open flow system and were in the open but under
shade. The seawater was pumped from the Puerto Morelos reef
lagoon and was treated with ozone before passing to an elevated reservoir for distribution to the tanks and also after being used in the
tanks.
Experimental set-up
We used a set of four fibreglass Y-mazes 2 m in length, which has
been estimated as the distance over which spiny lobster behaviours
driven by chemoreception of urine probably function (Horner et al.,
2006, 2008; Shabani et al., 2009). Each Y-maze (Figure 1) had two
independent head tanks (Briones-Fourzán et al., 2008). All tanks
Figure 1. Schematic representation of experimental Y-mazes used to
test for response by uninfected P. argus lobsters to shelters emanating
chemical cues from conspecifics in four different conditions.
i166
were filled to a standpipe height of 0.3 m. Thus, each head tank
(0.6 m long × 0.5 m wide × 0.5 m tall) held 90 l of water,
whereas the Y-maze (2.0 m long × 0.8 m wide × 0.6 m tall) contained 500 l. A panel (1.0 m long × 0.6 m tall) divided half the
length of the Y-maze into two equal arms. Seawater flowed into
the head tanks and then from each head tank to an arm of the
Y-maze at a rate of 2 l min – 1. The water then mixed in an open
area before flowing out through the standpipe located behind the
start area of the Y-maze. Using dye visualizations, we estimated
the downstream velocity as 0.52 + 0.03 cm s – 1 (mean + SD, n ¼ 3).
Two identical shelters were placed one in each arm of the Y-maze
at a distance of 1.8 m from the start area. One of the shelters received
water that had flowed through the head tank containing the stimulus
(see below), whereas the other received plain seawater that had
flowed through the head tank that held no stimulus (control). A
semicircular wire-mesh screen was positioned at the start area
(Figure 1) to prevent lobsters from using the corners in the start
area of the Y-maze as refuge. The head tanks and Y-mazes were completely opaque to preclude visual contact between lobsters and were
mounted on different surfaces to eliminate the transference of vibrations. Also, to prevent transference of acoustic cues potentially produced by the stimuli, the water from the head tanks fell from a height
of 5 cm above the water surface of the Y-maze. Before the experiment, 16 lobsters (50.0 + 15.4 mm CL, mean + SD) were individually tested in the Y-mazes with no chemical stimulus (i.e. the two
shelters receiving plain seawater from the head tanks) to test for potential errors in the Y-maze design or orientation that may bias the
choice trials. Each shelter was selected by 50% of the lobsters, indicating no potentially confounding effects of device orientation or
design.
Testing for DNA of PaV1 by PCR
We tested the haemolymph of all grossly healthy lobsters for the
presence of DNA of PaV1 via PCR assays. After swabbing the exoskeleton with 70% ethanol, 300 ml of haemolymph was withdrawn from the base of one of the fifth pereopods using a sterile
1 ml disposable syringe fitted with a 30 G needle and immediately
fixed in 96% ethanol and stored at –208C. DNA was extracted
from haemolymph samples following salt precipitation protocols
similar as described by Aljanabi and Martı́nez (1997). DNA precipitation was achieved by adding 200 ml sodium acetate 3 M, pH 5.2,
instead of NaCl. DNA integrity was assessed by electrophoresis in
1% agarose gels.
The DNA of PaV1 was amplified by PCR in a 25 ml reaction containing 1 ml extracted DNA, 0.33 mM of each primer 45aF and
543aR (Montgomery-Fullerton et al., 2007), 2.5 mM MgCl2
(Promega), 0.6× reaction buffer (Promega), 0.4 mM dNTP
mixture (Promega), and 0.75 U Taq DNA polymerase (Promega).
The thermal cycling conditions were 1 cycle for 948C for 10 min followed by 30 cycles of 948C for 30 s, 638C for 30 s, 728C for 1 min;
followed by 728C for 10 min. The presence of the expected 499 bp
PaV1 amplicon was determined by resolving 5 ml of the PCR
product and 3 ml of loading buffer in a 2% agarose gel containing
0.1% ethidium bromide and DNA visualization using UV illumination (MiniBis Prow). As negative and positive controls, we used
ultrapure water and haemocyte DNA extracted from lobsters
heavily infected with PaV1, respectively (Huchin-Mian et al., 2013).
Experimental treatments
Based on the PCR results, grossly healthy lobsters were categorized
into uninfected (i.e. negative to PaV1 by PCR) and subclinically
R. I. Candia-Zulbarán et al.
infected lobsters (i.e. positive to PaV1 by PCR but with no visible
signs of disease). The experiment consisted of four treatments that
differed in the condition of the lobsters used as sources of chemical
stimuli. These lobsters were denoted “stimulus”. The stimuli in three
treatments consisted of uninfected lobsters, subclinically infected
lobsters, and clinically infected lobsters, whereas the stimuli in the
fourth treatment consisted of one half of a dead lobster, freshly
killed lobster, and bisected lengthwise. We used only one-half of a
dead lobster per trial to minimize the sacrifice of animals. Only lobsters that tested negative for PaV1 by PCR were used as focal lobsters
and all focal lobsters were used only once.
Trials were conducted overnight. In each trial, the stimulus was
randomly assigned to the left or right head tank and placed into
the head tank 30 min before dark, and the focal lobster was then
placed in the start area of the Y-maze, where it was allowed to acclimatize for 2 h within a mesh cylinder (0.45 m in diameter, 0.40 m in
height). The cylinder was then removed, leaving the focal lobster free
to roam the Y-maze. Between 09:00 and 10:00 h the following
morning, we recorded the position of the focal lobster in the
Y-maze and checked the flow rate into the head tanks. The focal
lobster was then removed, measured with calipers (carapace
length, CL, from between the rostral horns to the posterior
margin of the carapace, +0.1 mm), and moult-staged by microscopic examination of the tip of one pleopod (see Lyle and
MacDonald, 1983). To ensure that no scents remained after each
trial, the head tanks and Y-mazes were drained and thoroughly
brushed, and then water was allowed to flow at a high rate until
the beginning of the next trial.
We ran at least 20 replicate trials in each treatment but discarded
those in which the water flow at the end of the trial differed between
the head tanks by more than +0.50 l min21 or the focal lobster was
not in intermolt (Briones-Fourzán et al., 2008). We also discarded
six trials in which the stimuli (clinically infected lobsters) died
during the night. To avoid potentially confounding effects of
season, we interspersed trials from the four treatments across the
experimental period.
Irrespective of the stimulus, the result of a trial was denoted
“attraction” if the focal lobster chose the shelter subjected to chemical cues from the stimulus or “avoidance” if the focal lobster chose
the shelter receiving plain seawater; thus, results are expressed as
percentages of lobsters exhibiting attraction vs. avoidance. We
used the score method with continuity correction (Newcombe,
1998) to compute 95% confidence intervals for percentages.
Results from each treatment were subjected to a two-tailed binomial
test wherein the probability of choosing either shelter was 50% (p ¼
0.5, a ¼ 0.05), and results from the four treatments were contrasted
in a 2 × 4 contingency table.
Results
The overall mean size (+SD) of focal lobsters from all valid trials
was 48.8 + 13.3 mm CL (n ¼ 77) and did not vary significantly
with treatment (one-way analysis of variance, F ¼ 1.418, d.f. ¼ 3,
74; p ¼ 0.244). The focal, uninfected lobsters were not significantly
attracted to shelters with chemical cues emanating from uninfected
conspecifics (63.2% attraction, n ¼ 19, p ¼ 0.359) and did not significantly avoid shelters with chemical cues emanating from subclinically infected conspecifics (44.7% avoidance, n ¼ 18, p ¼ 0.814;
Figure 2). In contrast, focal lobsters significantly avoided shelters
emanating chemical cues from clinically PaV1-infected conspecifics
(80% avoidance, n ¼ 20, p ¼ 0.012) as well as shelters emanating
Panulirus argus avoid dead and clinically PaV1-infected conspecifics
Figure 2. Results of Y-maze experiment investigating the effects of
chemical cues from P. argus lobsters in four different conditions on
shelter choice by uninfected conspecifics. Horizontal dotted line
denotes random shelter choice (50%). Error bars denote 95%
confidence intervals. p-values were based on two-tailed binomial
tests (a ¼ 0.05).
scents from dead conspecific (85% avoidance, n ¼ 20, p ¼ 0.003;
Figure 2).
The full contingency table analysis contrasting the four treatments yielded a significant result (x2 ¼ 14.748, d.f. ¼ 3, p ¼
0.002). A subdivided x2 analysis (Zar, 1999) showed no significant
difference in the response of focal lobsters to uninfected vs. subclinically infected conspecifics (x2 ¼ 0.222, d.f. ¼ 1, p ¼ 0.638), or
in the response of focal lobsters to clinically infected vs. dead conspecifics (x 2 ¼ 0.173, d.f. ¼ 1, p ¼ 0.667).
Discussion
We contrasted the response of focal lobsters uninfected with PaV1 to
chemical scents emanating from uninfected, subclinically infected
(i.e. infected but not diseased), clinically infected (i.e. infected and
diseased, and infectious), and dead conspecifics in a single experiment. Focal lobsters significantly avoided shelters with chemical
cues from clinically infected conspecifics and from dead conspecifics, consistent with the results of Anderson and Behringer (2013)
and Briones-Fourzán et al. (2008), respectively, but their response
to shelters with scents from subclinically infected lobsters and
from uninfected conspecifics did not differ from random. The
latter result was unexpected, as in previous experiments with
P. argus in Y-mazes, the focal lobsters had been significantly
attracted to shelters emanating chemical cues from healthy conspecifics (Ratchford and Eggleston, 1998; Briones-Fourzán et al., 2008).
However, more recently, Anderson and Behringer (2013) also
obtained a non-significant result testing attraction to chemical
cues from non-diseased lobsters. Moreover, according to
Childress et al. (this issue), conspecific attraction in P. argus
appears declining Caribbean-wise, the causes for which remain to
be determined.
Spiny lobsters have been extensively studied regarding the
mechanisms of aggregation and avoidance, and these studies have
shown that in general, aggregation is mediated by urine-borne chemicals and avoidance is mediated by haemolymph-borne chemicals
(Horner et al., 2006, 2008; Shabani et al., 2008, 2009; Aggio and
Derby, 2011). Yet, results from Anderson and Behringer (2013)
and from our study clearly show that avoidance of diseased lobsters
by healthy conspecifics is driven by chemoreception of chemicals in
i167
the urine. The avoidance of diseased lobsters was similar in magnitude to avoidance of “alarm” chemicals in the haemolymph of
injured conspecifics, which is an effective anti-predator strategy
for gregarious species (Dicke and Grostal, 2001; Briones-Fourzán
et al., 2008). However, haemolymph-borne alarm chemicals elicit
aversion for a limited period after injury because they degrade
within a few hours (Ferner et al., 2005); thus, according to Aggio
and Derby (2011), in overnight experiments using dead specimens
as stimuli, the actual chemical stimulus may be changing over time.
Yao et al. (2009) suggest that some studies attributing avoidance to
alarm odours emanating from dead or injured conspecifics may in
fact involve “necromones”, i.e. biochemicals given off by a decomposing organism and hence reliably associated with death, potential
sources of infections, and perhaps other pathologies. Unlike alarm
odours, which degrade rapidly and are species-specific, necromones
(e.g. certain unsaturated fatty acids such as oleic and linoleic acid)
can persist for days (Diez et al., 2013) and are highly conserved, as
they have been shown to repel animals in many diverse taxa, including crustaceans (Yao et al., 2009). However, given the conserved
nature of necromones, death recognition is generally coupled to
additional cues conveying relatedness (Yao et al., 2009). Therefore,
whether the avoidance of dead conspecifics shown by focal lobsters
in our experiment reflects avoidance to alarm odours or aversion to
necromones remains unclear.
Similarly, it remains to be determined whether the aversive chemicals emanating from PaV1-diseased lobsters originate from the
pathogen or from the host (Aggio and Derby, 2011). Some hosts
can chemically detect certain pathogens, but examples from marine
host-pathogen systems are scant (Toth et al., 2004; Wisenden et al.,
2009). Alternatively, PaV1 may alter the chemical cues emanating
from overtly diseased lobsters by altering their body chemistry in a
specific or in a non-specific manner. In the first case, the chemicals
produced by diseased lobsters would convey a specific signal for the
PaV1 disease. In the latter case, the chemicals would convey a nonspecific signal of “sickness” and the uninfected lobsters may simply
discriminate against “sick” conspecifics, and not against a specific
disease. For example, some fish discriminate against conspecifics
infested with parasitic trematodes, although these parasites cannot
be transmitted from fish to fish (see Krause et al., 1999; Wisenden
et al., 2009). However, when clinical signs of PaV1 appear, the infection has already become systemic, extending to the hepatopancreas,
gill, heart, hindgut, glial cells around the ventral nerves, cuticular
epidermis, and foregut (Li et al., 2008). In heavily infected lobsters,
the hepatopancreas exhibits focal necrosis, ischaemia, and atrophy,
which leads to metabolic wasting (Shields and Behringer, 2004).
Changes in haemolymph constituents in lobsters experimentally
infected (Li et al., 2008) and naturally infected with PaV1
(Pascual-Jiménez et al., 2012) reflect increasing tissue degradation
and catabolism of the hepatopancreas. Therefore, it is also possible
that visibly diseased lobsters exude chemicals associated with decomposition that are repelling to conspecifics.
Behringer et al. (2006) found that experimentally infected lobsters were avoided even before they became infectious, which
would appear at odds with our finding that subclinically infected
lobsters were not significantly avoided. However, although the
PCR assay that we used serves to detect the DNA of PaV1 in the
haemolymph of lobsters, it does not allow determining the viral
load or the progression of the disease in infected lobsters.
Therefore, whether subclinically infected lobsters do not emit repelling chemicals or emit these chemicals at subthreshold levels may
depend on the progression of the infection process.
i168
In summary, healthy lobsters do avoid visibly diseased conspecifics overnight in laboratory conditions and their response is similar
to that exhibited towards dead conspecifics. However, this does not
imply that all healthy lobsters will invariably avoid sharing shelters
with diseased conspecifics in the wild. For example, healthy lobsters
often cohabit with diseased conspecifics in “casitas”, large artificial
shelters deployed over otherwise shelter-poor habitats to increase
biomass of P. argus lobsters in some Caribbean countries
(Lozano-Álvarez et al., 2008; Briones-Fourzán et al., 2012), an occurrence for which Lozano-Álvarez et al. (2008) offered two potential explanations. One explanation is that the large shelter area
provided by casitas (1 –2 m2) may allow cohabitation of healthy
and diseased lobsters without physical contact. However, this explanation is not supported by results of our experiment wherein
16 of 20 focal lobsters avoided chemical cues from diseased lobsters
from a distance of up to 2 m (the length of the Y-mazes). Similarly, in
a field experiment testing distribution of juvenile lobsters in a radial
array of shelters located at 0.5, 1, and 2 m from a centrally located
diseased conspecific, lobsters moved on average 1 m away from
the diseased lobsters over time (Anderson and Behringer, 2013).
The other explanation is that in shelter-poor areas, where the risk
of predation for juvenile lobsters is higher compared with shelterrich areas, healthy lobsters make a trade-off between avoiding
sharing shelters with diseased conspecifics and avoiding predation
risk (Lozano-Álvarez et al., 2008). Indeed, avoidance behaviours
may be costly, both energetically and in the form of trade-offs
with conflicting demands (Loehle, 1995; Wisenden et al., 2009)
and, as noted by Shabani et al. (2009), the choice of shelter is
complex and will depend heavily on the context on which chemical
cues are transmitted from conspecifics. More experiments are
needed to examine the trade-off between disease avoidance and predation avoidance under different ecological contexts.
Acknowledgements
We greatly acknowledge the help provided by J. P. Huchin-Mian,
I. Segura-Garcı́a, and T. Islas-Flores in laboratory activities and
PCR assays. A. Espinosa-Magaña, R. Martı́nez-Calderón,
R. Muñoz de Cote-Hernández, and M. Ortiz-Matamoros helped
to collect and maintain the experimental lobsters and/or with the
experimental trials. M. Villanueva-Méndez allowed us the use of
his laboratory facilities. Comments by three anonymous reviewers
greatly enhanced this manuscript. Funds for this study were provided by Universidad Nacional Autónoma de México and
Consejo Nacional de Ciencia y Tecnologı́a, México (CONACYT,
grants no. 82724 and 101200, and a Doctoral studentship for
RIC-Z). Annual permits to collect lobsters were issued by
Comisión Nacional de Acuacultura y Pesca.
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