Male-like behavioral patterns and physiological alterations induced by androgenic gland implantation in female crayfish
1 Department of Aquaculture, Institute of Animal Science, Agricultural
Research Organization, the Volcani Center, PO Box 6 Bet Dagan 50250,
Israel
2 Department of Life Sciences and the Institute for Applied Biosciences,
Ben-Gurion University of the Negev, PO Box 653 Beer-Sheva 84105,
Israel
* Author for correspondence (e-mail: barkia{at}volcani.agri.gov.il)
Accepted 26 February 2003
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Summary |
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Key words: androgenic gland, androgenic hormone, agonistic behavior, aggression, fighting, mating behavior, crustacea, decapoda, crayfish, Cherax quadricarinatus.
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Introduction |
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Numerous behavioral studies have been performed in crustaceans, mainly in
decapods that have complex social behavior
(Dingle, 1983;
Hyatt, 1983
), but only a few
have addressed the hormonal regulation of social behavior. Correlations were
found in the American lobster Homarus americanus between
aggressiveness and molt stage (Tamm and
Cobb, 1978
) and levels of 20-hydroxyecdysone, the active form of
the molting hormone ecdysone secreted by the Y-organ
(Bolingbroke and Kass-Simon,
2001
), and between reproductive behavior of male spider crabs
Libinia emarginata and hemolymph concentrations of methyl-farnesoate,
a juvenile-hormone-like compound secreted by the mandibular organ
(Sagi et al., 1994
). However,
no study has addressed the association between androgenic hormones and
sexually dimorphic behavior patterns. One of the reasons for this gap is that
specific sex hormones have yet to be identified in decapod crustaceans
(Sagi and Khalaila, 2001
).
Of the endocrine complex in crustaceans, the only gland that is
specifically related to sexual functions is the androgenic gland (AG). The AG
regulates male differentiation, inhibits female differentiation, and controls
male primary (e.g. spermatogenesis) and secondary (e.g. external morphology)
sexual characteristics (for reviews, see
Charniaux-Cotton and Payen,
1988; Payen, 1990
;
Sagi et al., 1997
;
Sagi and Khalaila, 2001
).
Various morphological and physiological effects of the AG in crustaceans have
been elucidated by hormonal manipulations at the whole-gland level (e.g. AG
ablation or implantation). The extent of the hormonal influence seems to
depend on the species and the developmental stage at which such manipulations
were applied. Functional sex reversal has been obtained more readily in
amphipod and isopod crustaceans, but has also been reported in gonochoristic
decapods for AG-ablated males and AG-implanted females of the freshwater prawn
Macrobrachium rosenbergii, manipulated at an early stage of
differentiation (Nagamine et al.,
1980a
,b
;
Sagi and Cohen, 1990
).
However, most of the AG implantations into immature females in decapods
resulted in inhibition of secondary vitellogenesis, oocyte degeneration and
development of various secondary male characteristics
(Charniaux-Cotton and Payen,
1988
; Sagi and Khalaila,
2001
, and references therein).
Given the broad influence of the AG on sexual characteristics, it is likely
that this gland is also involved in the regulation of male behavior, which, in
fact, is one of the sexually dimorphic traits of the animal. Effects on mating
behavior were postulated for sex-reversed M. rosenbergii individuals
that were capable of reproducing (Sagi and
Khalaila, 2001). Gleeson et al.
(1987
) suggested that
androgenic factors from the AG mediate the control of courtship behavior in
male crabs Callinectes sapidus; the suggestion was based on the
co-occurrence of spontaneous courtship displays and hypertrophy of the AGs
within a few days following eyestalk ablation. However, none of these studies
directly tested the role of the AG in the regulation of behavior.
In the present study we examined the effects of AG implantation on the behavior of C. quadricarinatus females, and focused on interactions between females. By contrasting AG-implanted and intact females across a range of morpho-anatomical, physiological and behavioral characteristics, we show directly that the aggressive and mating behaviors are among the sexually dimorphic traits that are modulated by the AG.
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Materials and methods |
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Behavior testing
The effects of AG implantation on the behavior of females were examined in
pair encounters between size-matched females. Three types of pair encounters
were conducted in two sessions. In the first session, encounters of IMP
versus IMP (N=10) and NON versus NON (N=9)
were conducted simultaneously. In the second session, which followed a period
of at least 2 weeks in isolation to minimize the effects of prior experience,
IMP and NON females that could be size-matched were used again for encounters
of IMP versus NON (N=8). Size matching was based on the mean
length of the left and right propodus of the chelipeds, since weapon size is a
most reliable criterion for fighting ability in clawed crustaceans (Barki et
al., 1992,
1997
;
Sneddon et al., 1997
),
including crayfish (Rutherford et al.,
1995
). Mean chela length ratios (large/small contestant) were
1.02±0.01 and 1.03±0.04 for contests between IMP females and
between NON females, respectively, and 1.00±0.02 for contests between
IMP and NON females (IMP/NON contestant). Molt staging was performed 3 days
prior to the encounter to ensure that all females were in the intermolt
stage.
The encounters were conducted in glass aquaria measuring 50 cmx120 cmx50 cm placed inside an enclosure of black polyethylene to minimize disturbance. The bottom was covered with 3 cm layer of aquarium gravel, and each aquarium was divided into three sections by two opaque dividers that could be remotely lifted by a string. Each of the two outer sections contained a PVC tube (75 mmx220 mm, diameter x length). The two contestants were transferred simultaneously to these sections and allowed 6 min without any physical or visual contact, for acclimation. The dividers were then lifted and the encounters were video-recorded from outside the enclosure.
Behavior, definitions and analysis
Behavior patterns observed in the encounters included typical elements of
fighting and mating. The fighting behavior of C. quadricarinatus
resembles that of other crayfish. Escalated fights begin when the two
opponents face each other in the `body up' position
(Bruski and Dunham, 1987;
Tierney et al., 2000
), with
the carapace elevated obliquely and antennae pointing upwards. In this
position the opponents perform `chela contact'
(Tierney et al., 2000
),
usually with `interlocked' chelae (Bruski
and Dunham, 1987
). During fighting the opponents engage in pushing
against each other and grasping the antennae, pereiopods and other anterior
body parts (Bruski and Dunham,
1987
; Pavey and Fielder,
1996
). The fight is terminated by the loser retreating (either
walking or tail flipping) or turning aside and lowering the body against the
substrate. The copulatory behavior of C. quadricarinatus in aquaria
was previously described in detail (Barki
and Karplus, 1999
): in general, it can be divided into three
chronological phases differing in typical behavior patterns, namely,
pre-copulation, copulation and post-copulation. In the copulation phase the
mates perform stereotyped actions that reflect cooperation, whereas the pre-
and post-copulation phases involve elements of male courtship and
dominantsubordinate relationships, respectively.
To avoid excessive aggression under our confinement conditions, we defined the termination of the encounters as the termination of fighting followed by at least one retreat by the loser, to verify its subordination. In cases of prolonged fights we stopped the encounter after 45 min of fighting. In cases where no fighting had occurred, we stopped the encounter 30 min after lifting the dividers (this criterion also included encounters with mating behavior patterns).
To compare aggression among the different types of encounters we quantified
the following parameters: (i) total duration of fighting and mean duration per
fighting bout; (ii) latencies to start of interaction (i.e. first contact) and
start of escalated fighting (response time from lifting the dividers); (iii)
frequency and duration of crawling-over (a non-aggressive behavior pattern in
which one crayfish climbs over the other). This behavior pattern was
quantified since it has previously been found to differ among males,
AG-implanted females and non-implanted females in confrontations with males
(I. Karplus, A. Sagi, I. Khalaila and A. Barki, unpublished observations).
Frequency and duration of crawling-over were standardized as means
min1 of encounter, because of the differing durations of the
encounters. Non-parametric analysis of variance (KruskalWallis test)
was applied to these data at a significance level of P<0.05. Where
an overall significant difference was found, MannWhitney
U-tests were performed for pairwise comparisons between specific
types of encounters. Because multiple tests were performed, the critical
significance level for this test was set at P<0.0167, which is the
quotient of 0.05 divided by the number of possible pairwise tests (3)
(Dunn, 1964). Mating behavior
was described quantitatively but no analysis was performed, because of the
small number of encounters that involved this behavior pattern. All analyses
were performed with the JMP 3.2 statistical software
(SAS, 1997
).
Morpho-anatomical and hemolymph vitellogenic protein
examinations
18 IMP and 19 NON females were examined about a week after the behavioral
tests. The propodus was checked externally for the development of the red
patch. Morphometric measurements with a digital caliper (±0.05 mm)
included the length and width of the propodus of the chela, the length of the
carapace, and the width of the abdomen, endopod and exopod at the second
segment. The number of simple (ovigerous) setae in 1.25 mm of the internal
endopod edge was counted. For anatomical studies, the animals were
anesthetized in ice-cold water and the ovaries were removed and weighed to the
nearest milligram. The gonadosomatic index (GSI) was calculated as the gonad
mass expressed as a percentage of body mass. Mean oocyte diameter was
calculated from a sample of 15 oocytes per ovary, measured under a light
microscope. Secondary vitellogenic cross-reactive proteins in the hemolymph
were examined by an enzyme-linked immunosorbent assay (ELISA), with an
antibody raised against the specific 106 kDa vitellogenic polypeptide
(Sagi et al., 1999).
Differences between the IMP and NON females were statistically analyzed using
the MannWhitney U-test.
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Results |
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Differences in the latencies to start of interaction and start of escalated
fighting were statistically significant (2=8.0 and 7.9,
respectively, d.f.=2, P<0.05). The first interaction occurred
soonest in IMP versus NON encounters, but the delay in these differed
significantly only from NON versus NON and not from IMP
versus IMP encounters
(Fig.1A). On the other hand,
the first fight occurred latest in IMP versus NON encounters but the
delay in this case differed significantly only from IMP versus IMP
and not from NON versus NON encounters
(Fig. 1A). Thus, the time
lapses from the start of interaction to escalated fighting were also
statistically different (
2=8.4, d.f.=2, P<0.05),
being longest in IMP versus NON, shortest in IMP versus IMP
interactions and intermediate in NON versus NON (P<0.016)
(Fig. 1A).
|
Fighting duration (both total and mean per bout) differed among encounter
types (2=10.0 and 11.7, respectively, d.f.=2,
P<0.01), being significantly lower in the IMP versus NON
than in the other two types of encounters
(Fig. 1B). Fighting duration in
the IMP versus NON encounters was lowest also after excluding the
four encounters in which there was no fighting from the analysis (8.4, 16.8
and 6.1 min, respectively to Fig.
1B), however non-significant statistically
(P>0.05).
Crawling-over was evident in seven of eight IMP versus NON
encounters, five of nine NON versus NON encounters and in none of the
IMP versus IMP encounters. The frequency
(Fig. 1C) and duration of
crawling-over were significantly different (2=13.9 and 14.7,
respectively, d.f.=2, P<0.001) and highest in the IMP
versus NON encounters. The majority of the crawling-over actions
recorded in the IMP versus NON encounters (52 of 55 climbs) were
performed by the IMP female.
Characteristic mating behavior patterns were evident in three IMP
versus NON encounters. In two pairs the IMP female performed
`thrusts' against the NON female, which is a typical male courtship pattern
(Barki and Karplus, 1999). In
the third encounter the mates exhibited the complete behavioral sequence of
copulation twice, with the IMP female performing the male behavior pattern.
The time durations of the main component of the copulation, namely `freezing'
a motionless male-beneath-female position with the ventral surfaces
brought face to face were 74.9 and 77.9 s in these two copulations,
which is within the range found previously for normal males and females under
similar conditions (112±81 s; Barki
and Karplus, 1999
), as were the time intervals from the first
physical contact (`chela contact') to `freezing' (12.9 and 14.3 s,
respectively).
Morpho-anatomical and hemolymph examinations
The specific growth rate of the IMP females in the isolated aquaria was
higher (0.62±0.11% day1) than that of the NON females
(0.46±0.07% day1) (MannWhitney
U-test, P<0.001).
AG implantation caused an increase in the expression of male secondary sexual characteristics and an inhibition of female characteristics. The red patch, a secondary male characteristic located on the outer surface of the chela propodi, developed in all the AG-implanted females whereas none of the intact females developed this characteristic. The mean relative width of the propodus of AG-implanted females was significantly greater (P<0.001) than that of the intact females (Table 1). Maternal brooding-related characteristics, namely relative abdomen and endopod widths and the number of simple setae, were significantly reduced by AG implantation (P<0.001) (Table 1).
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The gonads of AG-implanted females did not show any sign of testis-like development and a sperm duct. The ovaries were white yellowish in color compared with the green brownish ovaries of the intact females. The oocytes in the ovaries of AG-implanted females were smaller in diameter and had lower gonadosomatic index (GSI) than those of the intact females (P<0.001) (Table 1). An ELISA (enzyme-linked immunosorbent assay) test showed that the level of vitellogenic cross-reactive protein in the hemolymph of AG-implanted females was significantly lower (P<0.001) than that in the hemolymph of the intact females (Table 1).
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Discussion |
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Morpho-anatomical and physiological effects of the AG
AG implantation inhibited the development of the ovaries and of
morphological traits that facilitate maternal egg brooding, namely, wide
abdomen, large endopods of the abdominal appendages (pleopods) and simple
(ovigerous) setae specialized for egg attachment. The low GSI and small oocyte
diameter and the low level of a secondary vitellogenic-specific polypeptide in
the hemolymph indicated that secondary vitellogenesis the accumulation
of yolk protein in the ovary was arrested in AG-implanted females.
Concomitantly, AG implantation led to the development of secondary male
characteristics in the form of large claws possessing a soft red patch. In
fact, the AG-implanted females resembled intersex individuals in their
external appearance and inhibition of primary female characteristics. Such
individuals occur naturally in C. quadricarinatus populations
(Thorne and Fielder, 1991;
Medley and Rouse, 1993
).
Intersex individuals are morphologically and functionally males but possess
both male and female genital openings, a testis and sperm duct with attached
AG on the lateral half displaying the male opening, and an arrested
pre-vitellogenic ovary at the contralateral half
(Sagi et al., 1996
). However,
the AG-implanted females differed from intersex crayfish in that they lacked a
male reproductive system and genital openings. The present findings are in
keeping with those of recent studies of AG implantation into immature C.
quadricarinatus females (Sagi et al.,
1999
; Khalaila et al.,
2001
), as well as with results reported for other crayfish
(Nagamine and Knight, 1987
;
Taketomi et al., 1990
;
Taketomi and Nishikawa, 1996
;
Fowler and Leonard, 1999
). The
fact that the AG-implanted females had higher growth rates than intact females
reflects the broad influence of the AG, which extends beyond reproductive
processes. A similar growth-enhancing effect of AG implantation in females was
described for the crayfish C. destructor
(Fowler and Leonard, 1999
),
and the growth-inhibiting effect of AG removal in males was reported in the
freshwater prawn M. rosenbergii (Sagi et al.,
1990
,
1997
). This effect may be a
direct result of the involvement of the androgenic hormone in somatic growth
processes, or an indirect result of shifts in allocation of resources between
ovarian and somatic growth, because of the inhibition of the ovaries by the
AG.
Behavioral effects of the AG
The current results, coupled with previous observations in C.
quadricarinatus (Levi,
1997; I. Karplus, A. Sagi, I. Khalaila and A. Barki, unpublished
observations), indicate that both males and females fight vigorously in
same-gender contests, using the same agonistic behavioral acts. Thus, the
effect of AG implantation on the agonistic behavior was reflected mainly in
the substantially lower aggressive motivation of both AG-implanted and intact
females when they encountered one of the other rather than one of their own
types. This was reflected in lower probability for, and shorter duration of,
escalated fighting, and higher frequency of tolerance-indicative behavior
(i.e. crawling-over) in the AG-implanted versus intact females'
contests. Recently, we have examined the effects of AG implantation on
behavior in the context of malefemale interactions. The agonistic
behavior of AG-implanted females was compared with that of males and
non-implanted females in interactions with dominant males (I. Karplus, A.
Sagi, I. Khalaila and A. Barki, unpublished observations). Aggression measures
(i.e. duration of fighting periods and grasps) were found to be lower, and
tolerance measures (i.e. duration of crawling-over periods) higher, in
malenon-implanted female interactions compared with malemale
interactions, and intermediate values were found for
male-implantedfemale interactions. In this regard, encounters between
AG-implanted and intact females in the present study resembled those between
males and females. These results suggest a masculinization effect of the AG on
the agonistic behavior of implanted females.
The most striking evidence for the masculinization effect of the AG came
from the complete reversal of the mating behavior of females into typical male
behavior following AG-implantation, in spite of the absence of any other
component of a masculine reproductive system. In the present study, a pair
comprising an AG-implanted and an intact female exhibited normal sequence and
timing of copulatory behavior, with the implanted female performing the male
behavior, and in two other pairs the AG-implanted females exhibited male
courtship displays. This finding is astounding considering that we did not
detect any sign of mating behavior in malefemale encounters under
similar conditions (I. Karplus, A. Sagi, I. Khalaila and A. Barki, unpublished
observations). Mating behavior is rarely observable in C.
quadricarinatus under experimental conditions of single short encounters,
because of the difficulty in predicting receptivity of females
(Barki and Karplus, 1999). The
exceptionally large oocyte diameter in intact females in the current study
(2.33 mm, comparing with approximately 1.5 mm reported by
Khalaila et al., 2001
)
suggested that these females were more likely to be receptive. During mating,
the male and female follow a coordinated sequence of behavior patterns that
requires cooperation (Barki and Karplus,
1999
). Thus, in addition to the effect on behavior, AG
implantation into immature females led to alterations in male-specific factors
that stimulate other females to respond receptively and less aggressively.
The androgenic gland hormone
We have shown for the first time a complete picture of the effect of the
androgenic gland in crustaceans, including behavioral changes, which accompany
the physiological process. However, the basic findings of this study raise
three main questions. First, what is the nature of the androgenic hormonal
factor that controls male behavior in crustaceans? Recently, the androgenic
gland hormone (AGH) from the isopod crustacean Armadillidium vulgare
was purified and characterized on the basis of a morphological bioassay, and
the complete amino acid sequence was determined (Okuno et al.,
1997,
1999
; Martin et al.,
1998
,
1999
). The isopod AGH is a
glycosylated protein composed of two peptide chains connected each to the
other by two disulfide bridges. Attempts to identify the decapod AGH are
currently underway (for a review, see Sagi
and Khalaila, 2001
). The link between the AG and male behavior
could offer a behavioral assay suitable for studies aimed at the
identification of androgenic factors from the AG in decapods.
Secondly, what is the mode of action of this androgenic factor? Hormonal
effects on behavior are mediated by the nervous system. Although the effect of
AG implantation was evident on the adult behavior, the hormonal manipulation
was performed in young immature females. Thus, similarly to its action on
secondary male characteristics, it is possible that, at the early stage at
which the AG was implanted, the AGH induced male structural reorganization of
neural circuits underlying the generation of sexually dimorphic behaviors. The
AGH may also fill a role in modulating variations in behavior, as in the case
of the aggressive motivation during interactions. Extensive research in this
regard in crustaceans has been devoted to biogenic amines, in particular
serotonin (5-hydroxytryptamine), that function as neuromodulators in the
nervous system (for recent reviews, see
Kravitz, 2000;
Huber et al., 2001
). Increased
levels of serotonin generally correlate with increased aggression and
dominance in crustaceans. Serotonin and octopamine elicit opposite postures
that resemble those of dominant and subordinate individuals, respectively
(Livingstone et al., 1980
;
Antonsen and Paul, 1997
).
Levels of serotonin, octopamine and dopamine differed between winners and
losers among shore crabs, after fighting
(Sneddon et al., 2000
).
Infusion of serotonin into crayfish altered the timing of the decision to
withdraw, which caused fights to last longer
(Huber and Delago, 1998
).
However, as with elevations of serotonin, reduced levels of this amine
increased fighting duration in naïve juvenile lobsters
(Doernberg et al., 2001
).
Furthermore, the modulatory effect of serotonin on the tail-flip circuit
responsible for the rapid escape reaction of crayfish depends on the social
status of the individual (Yeh et al.,
1996
,
1997
). It is believed that
several other substances, such as hormones, are important in agonistic
behavior and may function in similar ways, possibly in dynamic balance with
these neuromodulators (Kravitz,
1988
,
2000
). For example, the
molting hormone 20-hydroxyecdysone (20E) appears to act through a rapid,
nongenomic mechanism that reduces synaptic efficacy in crayfish neuromuscular
junctions, and this effect can be reversed by application of serotonin
(Cooper and Ruffner, 1998
).
20E has been shown to exert differential effects on the abdominal phasic
flexor muscles (related to the escape response) and the dactyl closer muscle
of the claw (related to fighting)
(Cromarty and Kass-Simon,
1998
). This correlates to the variation in the aggressive
motivation with 20E (i.e. the tendency of the lobster to flee or fight) over
the molt cycle (Bolingbroke and Kass-Simon,
2001
).
Third, and finally, what stimulated females to respond in a similar way to
AG-implanted females as to a male? Some change must have occurred in the
AG-implanted females that provided them with male stimulus signals and/or
eliminated female stimulus signals. AG implantation might have caused changes
in urine-borne chemical cues (perhaps the AGH itself or its derivatives) used
in recognition of sex and dominance (e.g. lobsters and crayfish,
Ameyaw-Akumfi and Hazlett,
1975; Atema and Voigt,
1995
; Dunham and Oh,
1996
; Bushman and Atema,
1997
). Alternatively, the stimulating cue might have been visual,
namely the male-specific soft red patch that had developed on the claws of the
AG-implanted females, or the male-like behavior of the AG-implanted
females.
In conclusion, the results presented here strongly suggest causal relationships between an androgenic hormone derived from the AG and male behavior. Important questions remain concerning the mechanisms linking complex behavior, the nervous system and the androgenic hormone. Since androgenic hormones have not been identified yet in decapod crustaceans, further research is needed on their nature and the hormonal mechanism underlying variations in reproductive behavior.
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Acknowledgments |
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