Enzyme activities of pharyngeal jaw musculature in the cichlid Tramitichromis intermedius: implications for sound production in cichlid fishes
Boston University Marine Program, Marine Biological Laboratory, 7 MBL
Street, Woods Hole, MA 02543, USA
Present address: Department of Organismal Biology and Anatomy, University of
Chicago, 1027 E. 57th Street, Chicago, IL 60637, USA
* Author for correspondence (e-mail: plobel{at}mbl.edu)
Accepted 12 August 2002
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Summary |
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Key words: Cichlidae, pharyngeal jaw, citrate synthase, 3-hydroxyacyl-CoA dehydrogenase, lactate dehydrogenase, sexual dimorphism, muscle, sound production, stridulation
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Introduction |
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The pharyngeal jaw complex of cichlid fishes has been studied from several
different perspectives, such as evolutionary biology (e.g.
Casciotta and Arratia, 1993;
Kaufman and Liem, 1982
),
feeding biology (e.g. Aerts et al.,
1986
; Claes and De Vree,
1991
; Liem, 1973
,
1978
,
1979
) and biomechanics (e.g.
Galis, 1992
;
Galis and Drucker, 1996
;
Lauder, 1983
). Beginning with
Liem (1973
), most of these
studies have examined and compared the anatomy and function of these different
muscles and their behavior during feeding. To the best of our knowledge, no
study has yet compared pharyngeal jaw muscles from a cellular perspective. The
importance of such an approach is that it allows an insight into new patterns
unseen by potential limitations of previously applied techniques
(Galis, 1992
).
Within any organism, there is a diversity of muscle types and functions
(see Rome and Lindstedt,
1997), with capabilities resulting from a balance of the cellular
components (Lindstedt et al.,
1998
). One way of determining differences in function between
muscles is by comparing levels of enzyme activity
(Bass et al., 1969
). This
enables discrimination of muscle type based on extrinsic factors
(Josephson, 1975
). By assaying
key metabolic enzymes within muscle cells, one can determine the main
energetic pathway used (aerobic versus anaerobic) within these
muscles and compare performance capabilities quantitatively (e.g.
Bass et al., 1969
;
Bevier, 1995
;
Taigen et al., 1985
). By
exploring the enzyme activities of a suite of muscles, it is then possible to
survey and compare differences in performance capabilities and deduce
differences in their function.
Elucidation of the functional properties of these muscles allows us to address three main questions: (1) are there differences in function between the different pharyngeal jaw muscles; (2) are there differences in pharyngeal jaw muscle physiology between males and females; and (3) are the pharyngeal jaws involved in sound production?
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Materials and methods |
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Captive-raised male and female T. intermedius were kept in a 5501 aquarium and fed a mixed diet consisting of flake food, small pellets and brine shrimp several times each day.
Muscle preparation
Sexually mature T. intermedius individuals were euthanized with an
overdose of ethanol-buffered MS-222 (MBL Animal Care Protocol No. 01-13).
Under a stereomicroscope, the muscles responsible for the principal movements
of the pharyngeal jaws from both left and right sides were dissected
(sensu Liem, 1973):
levator externus II (LE2), levator externus III (LE3), levator externus IV
(LE4), levator posterior (LP), protractor pectoralis (PP), pharyngeohyodius
(PH), pharyngeohyodius cleithralis externus (PHCE), pharyngeohyodius
cleithralis internus (PHCI) and retractor dorsalis (RD)
(Fig. 1). A small sample of
axial muscle (Ax) just anterior to the caudal peduncle was also dissected to
serve as an example of fast-twitch glycolytic muscle
(Greer-Walker and Pull, 1975
;
Mosse and Hudson, 1977
;
Rome et al., 1988
). Muscles
were weighed and then homogenized on ice using a glassglass homogenizer
in 10 volumes of cold buffer (7.5 mmoll-1 Tris; 1
mmoll-1 EDTA; pH 7.6). Samples were then put through two
freezethaw cycles to ensure rupturing of mitochondria and stored at
0°C until analysis.
|
Analysis of enzyme activity
Muscle samples were kept on ice immediately before analysis to prevent
denaturation of the enzymes. Supernatant was drawn from the crude homogenate
and added to the reaction mixture. Enzyme activities were determined using the
protocols outlined below and assayed using a Perkin-Elmer Lambda 3B UV/V is
spectrophotometer.
Citrate synthase (CS): 0.25 mmoll-1 5,5'-dithiobis(2-nitrobenzoic acid); 0.3 mmoll-1 acetyl-CoA; pH 8.0. The reaction was catalyzed with 0.5 mmoll-1 oxalacetic acid and assayed at 412 nm.
3-Hydroxyacyl-CoA dehydrogenase (HOAD): 100 mmoll-1 triethanolamine; 5 mmoll-1 EDTA; 0.15 mmoll-1 NADH; pH 7.0. The reaction was catalyzed with 0.1 mmoll-1 acetoacetyl-CoA and assayed at 340 nm.
L-lactate dehydrogenase (LDH): 50 mmoll-1 triethanolamine; 5 mmoll-1 EDTA; 0.15 mmoll-1 NADH; pH 7.6. The reaction was catalyzed with 2.4 mmoll-1 pyruvate and assayed at 340 nm.
Enzyme activities were calculated as µmol product min-1
g-1 tissue (Bergmeyer,
1974). In addition to the determination of individual enzyme
activities within tissues, relative levels of ß-oxidation (CS activity
divided by HOAD activity; Olson, 2001) and the relative anaerobic capacity
(LDH activity divided by CS activity; Bass
et al., 1969
) were determined to distinguish muscle type.
Statistics
Intermuscular and intersexual activities were compared using a two-way
analysis of variance (ANOVA). Statistical tests were performed using StatView
Software (SAS Systems, Cary, NC, USA), with significance set at
P<0.05.
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Results |
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HOAD activities (Fig. 2B) were significantly different among muscles (P<0.0001, F=8.056, d.f.=9) and between males and females (P=0.0184, F=5.078, d.f.=1). Differences in HOAD activity in female muscles were found between LE2 and Ax, LE2 and LE4, LE2 and LP, LE2 and PH, LE2 and PHCE, LE2 and PHCI, LE2 and PP, LE2 and RD, LE3 and LE4, LE3 and LP, LE3 and PHCI, LE3 and PP, and LE3 and RD (StudentNewmanKeuls test, P<0.05). No significant differences in HOAD activity were found among the muscles of males. LE2 had significantly different levels of activity between males and females (StudentNewmanKeuls test, P<0.05).
LDH activities (Fig. 2C) were significantly different among muscles (P<0.0001, F=4.651, d.f.=9). For males, differences in LDH activity were found between LE2 and PH, LE4 and PH, and PP and PH (StudentNewmanKeuls test, P<0.05). There were no differences in LDH activity among females, nor were there any differences in anaerobic capacity between males and females.
Males have a significantly higher overall relative capacity for ß-oxidation than females (P<0.0001, F=18.822, d.f.=1; Fig. 3A). For males, significant differences in HOAD/CS activities were found between Ax and LP, Ax and PP, LE2 and LP, LE2 and PP, LE3 and PP, LE3 and LP, LE4 and LP, LE4 and PP, LP and PH, LP and PHCE, LP and PHCI, LP and RD, PH and PP, PP and PHCE, PP and PHCI, and PP and RD (StudentNewmanKeuls test, P<0.05). There were no differences in activity among the muscles of females. HOAD/CS activities for both LP and PP were different between males and females (StudentNewmanKeuls test, P<0.05).
|
Male pharyngeal jaw muscles have a significantly higher relative anaerobic capacity than females (P<0.0001, F=53.530, d.f.=1; Fig. 3B). Significant differences for males existed between Ax and LP, LE2 and LP, LE2 and PP, LE3 and LP, LE3 and PP, LE4 and LP, LP and PH, LP and PHCE, LP and RD, PH and PP, PP and PHCE, and PP and PHCI (StudentNewmanKeuls test, P<0.05). There were no differences in relative anaerobic capacity among the muscles of females, but significant differences existed for both LP and PP between males and females (StudentNewmanKeuls test, P<0.05).
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Discussion |
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Based upon the fact that only male T. intermedius produce sounds,
and both males and females were fed the same diet, the observed physiological
dimorphism in the capacity for aerobic/anaerobic activity could be explained
by a role for the muscle in sound production in males. Other sonic fishes,
such as the toadfish Opsanus beta and Opsanus tau (family
Batrachoididae), have been shown to display sexually dimorphic performance
capacities of their homologous sound-producing muscles (Walsh et al.,
1987,
1989
). By demonstrating such
sexual dimorphism in the physiology of the pharyngeal musculature in T.
intermedius, this study corroborates acoustic and behavioral data,
supporting the hypothesis that this apparatus is used in sound production in
male cichlid fish.
The pharyngeal jaws play an important role in feeding in many teleost
species (Lauder, 1983), and
functional duality of the pharyngeal jaws for feeding and sound production has
been shown in the grunt Haemulon plumieri (family Haemulidae;
Burkenroad, 1930
) and the
Japanese parrot fish Oplegnathus fasciatus (family Oplegnathidae;
Nakazato and Takemura, 1987
)
and suggested in the crevalle jack Caranx hippos (family Carangidae;
Taylor and Mansueti, 1960
).
The highly maneuverable design of the pharyngeal jaw apparatus in the
cichlids, resulting from the lower pharyngeal sling (Liem,
1973
,
1986
), where the jaws are
principally occluded by the LE4 and LP
(Liem, 1991
), may contribute
to the amplitude modulation in the acoustic signal
(Lobel, 2001
). By contrast,
the pharyngeal jaws of the damselfish (Pomacentridae) are occluded by
contraction of the PP, which raises the cleithrum
(Galis and Snelderwaard,
1997
). This comparatively limited movement of the pomacentrid
pharyngeal jaws may result in the limitation for only pulse patterning of the
acoustic signal (Lobel,
2001
).
Previous studies have suggested that the RD is the sonic muscle in fish
that use the pharyngeal jaws for sound production. In O. fasicatus,
Nakazoto and Takemura (1987) identified the RD as the primary muscle
responsible for stridulation of the pharyngeal jaws during sound production.
However, they only implanted an electromyographic electrode in the dilator
operculi and inferred the role of the sonic muscle through anatomical
dissections. Lanzing (1974)
also suggested that this muscle was the `sonic muscle' in Mozambique tilapia
Oreochromis mossambicus (=Tilapia mossambica) but this was
inferred only from histological sections of a young juvenile individual. Both
of these `sonic muscles' originate on the vertebral column and insert on the
posterior section of the upper pharyngeal jaw; both muscles match the position
and description of the RD (Winterbottom,
1974
). The data from the present study indicate that the T.
intermedius RD is designed for high levels of anaerobic performance, but
this muscle is not sexually dimorphic. Although the RD does contribute to the
movement of the upper pharyngeal jaw in T. intermedius, its role as a
principal muscle in sound production is questionable.
It is possible that the enzyme activities in cichlid pharyngeal musculature
might differ with diet. The pharyngeal toothplates of cichlids have been show
to vary with diet (e.g. Greenwood,
1973), and variation in the performance of the pharyngeal muscles
might follow a similar pattern. These performance capabilities may be
determined by diet (i.e. in molluscivores, insectivores and piscivores, where
different properties are necessary for the demands of different food
processing) or this could be a pattern seen in all cichlid pharyngeal muscles.
Comparison of the pharyngeal muscle performance capabilities in other cichlid
species with diverse ecologies or evolutionary histories may elucidate the
universality of the aerobicanaerobic ratios across the Cichlidae.
The relationship between the pharyngeal jaws for feeding and sound
production may have profound evolutionary implications. The importance in
feeding (Liem, 1973,
1991
) and the adaptability of
the pharyngeal jaws for processing different food types (e.g.
Greenwood, 1965
;
Huysseune, 1995
;
Smits et al., 1996
;
Witte et al., 1990
) allow
cichlids to exploit different trophic niches
(Sage and Selander, 1975
).
Furthermore, acoustic communication may be an important mechanism for sexual
selection in cichlid fish (Lobel,
1998
,
2001
). With the pharyngeal jaw
serving as a possible mechanism for sound production; trophic biology and
reproductive biology could be directly linked by this structure. Consequently,
the dual use of the pharyngeal jaw may serve as a mechanism mediating the
sympatric speciation of cichlid fishes (sensu
Kornfield and Smith,
2000
).
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Acknowledgments |
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