Morphological and physiological specialization for digging in amphisbaenians, an ancient lineage of fossorial vertebrates
1 Departamento de Fisiologia, Instituto de Biociências, Universidade
de São Paulo, Rua do Matão Travessa 14 No. 321, CEP 05508-900,
São Paulo, SP, Brazil
2 Laboratório de Biologia Celular, Instituto Butantan, Avenida Vital
Brazil 1500, 05503-900, São Paulo, SP, Brazil
3 School of Science and the Environment, Coventry University, James Starley
Building, Priory Street, Coventry CV1 5FB, UK
4 Departamento de Morfologia, Instituto de Biociências, Universidade
Estadual Paulista, CEP: 18618-000, Botucatu, Brazil
5 Department of Zoology and Entomology, The University of Queensland, St
Lucia, QLD 4072, Australia
* Author for correspondence (e-mail: navas{at}usp.br)
Accepted 20 April 2004
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Summary |
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Key words: Amphisbaenia, reptile, muscle, digging, Leposternon microcephalum
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Introduction |
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Amphisbaenians exploit tropical fossorial habitats in America and Africa
and are predators of small animals (Gans,
1968). They burrow by compressing the substrate against the walls
of the tunnel by means of powerful horizontal, vertical or torsional movements
of the head (Gans, 1978
).
Their ability to sustain burrowing at the low O2 and high
CO2 concentrations that appear to characterize fossorial
microhabitats (McNab, 1966
)
seems associated with a low rate of O2 uptake
(
O2) for
reptiles (Abe and Johansen,
1987
; Kamel and Gatten,
1983
), significant cutaneous oxygen uptake
(Abe and Johansen, 1987
),
elevated blood oxygen affinity (Johansen
et al., 1980
), small red blood cells and high haematocrit
(Ramirez et al., 1977
) and
significant amounts of myoglobin, particularly in the muscles associated with
digging (Weber et al., 1981
).
These traits suggest that aerobic metabolism might be relevant to support the
efforts associated with production and maintenance of an extensive tunnel
matrix and, possibly, with chasing eventual intruders
(Gans, 1968
). Therefore,
amphisbaenian digging muscles must produce forceful, powerful and enduring
contractions. Because trade-offs between power output and fatigue resistance
have been detected at the whole-muscle
(Wilson et al., 2002
) and
whole-organism (Van Damme et al.,
2002
) levels, amphisbaenian digging muscles pose intriguing
physiological questions. Additionally, as muscle performance is affected by
the specific protein isoforms expressed within the fibres
(Moss et al., 1995
), and the
mitochondria and sarcoplasmic reticulum supporting myofibrillar activity
(Rome and Lindstedt, 1998
),
similar trade-offs are also likely to exist at the individual-fibre level.
The purpose of our study is to integrate a diversity of approaches to
increase our understanding of the physiology underlying amphisbaenian digging
behaviour, to explore the influence of morphology on force production and
required effort, and to discuss our findings in terms of animal evolution,
ecology, morphology and kinematics. We focus on the Brazilian species
Leposternon microcephalum [Amphisbaenidae;
Fig. 1; see Gans
(1971) for a detailed
description], a shovel-snouted amphisbaenian that uses only upward strokes of
the head to excavate and exhibits a large mass of dorsal musculature
associated with upward soil compression strokes. Specifically, we aim to
describe the kinematics of head-first digging, measure the maximum digging
forces that live animals can produce, describe the metabolic and morphological
characteristics of the main muscle associated with digging, analyse the
influence of animal diameter on the power output of the digging muscles and
evaluate the work requirements of digging for different animal diameters.
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Materials and methods |
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Recording of digging activity
Three individuals were filmed in a set-up that consisted of a rectangular
glass container partially filled with sifted soil
(Fig. 2A), barely wider than
the thickest individual. Animals were induced to burrow by tapping the base of
their tail, and the digging behaviour was recorded with a Panasonic television
camera. The video sequences were later observed and analysed at 60 frames
s-1 using a time-lapse videocassette recorder system.
|
Measurement of digging forces
Maximum digging forces for each individual were measured at room
temperature (25±1°C) using a custom-built apparatus consisting of a
strain gauge (model 1030; UFI, Morro Bay, CA, USA) and a sheet of plastic
(Fig. 2B). Animals were induced
to push the strain gauge upwards (so as to imitate a soil compression stroke)
by gentle squeezing of the posterior end of the animal. The forces produced
were calculated from chart recordings of strain gauge voltage output. The
strain gauge was calibrated by hanging objects of known weight from it at the
same point on the strain gauge that each individual was induced to push
against. The maximum diameter of the head of each individual was used to
calculate the cross-sectional area of the head region, assuming the head to be
a perfect circle. Each individual performed between five and eight digging
movement trials against the strain gauge. The maximum digging force was the
peak force imposed on the strain gauge during any one digging movement.
Study muscle
This study focuses on the longissimus dorsi, an extensive dorsal muscle in
L. microcephalum that starts at the base of the head (proximal part)
and runs backwards along the dorsal body of the animal (distal part). This
muscle is believed to be homologous to the muscle carrying the same name in
snakes, turtles and lizards, although it seems to be less exaggerated in
amphisbaenians that use lateral head movements while digging, such as
Amphisbaena alba (Gans,
1978). The longissimus dorsi is bilateral, with well-defined right
and left sides that are separated by a central tendon.
Collection of muscle samples
To collect samples for both muscle fibre and biochemical analyses, animals
were placed on a balance to measure body mass to the nearest 0.01 g and then
anaesthetized and killed with pentobarbital sodium (50 mg kg-1,
intraperitoneally) before dissection. Animals were quickly dissected and the
dorsal trunk muscle was photographed. Two samples from each of the proximal,
medial and distal parts of the muscle (equal thirds) were quickly removed and
frozen immediately in liquid nitrogen and then stored at 85°C until
analysis. The time taken between anaesthetic injection and completion of the
dissection was always less than 10 min. Samples of the right dorsal muscle
were subsequently used for biochemistry, and samples from the left dorsal
muscle were used for histochemistry.
Muscle histochemistry
Serial transverse sections were obtained by producing 79 µm-thick
muscle samples using a cryostat at 20°C. Serial sections for each
muscle sample were either reacted for haematoxylin-eosin (HE), NADH-TR
(nicotinamide adenine dinucleotide tetrazolium reductase) or myofibrillar
ATPase. For ATPase reactions, adjacent sections were subjected to either acid
(pH 4.5) or alkaline (pH 10.4) pre-incubations, following the procedures
detailed in Dubowitz and Brooke
(1973). The HE treatment was
used to dye muscle tissue to verify section quality and overall fibre
morphology. NADH-TR is an oxidative enzyme, present in both the sarcoplasmic
reticulum and the mitochondria, that transfers hydrogen to a dark tetrazolium
salt. This reaction, therefore, gives a positive result (dark) for oxidative
fibres and a negative result (light) for glycolytic fibres but also identifies
fibres of mixed metabolic characteristics (intermediate colouration), usually
referred to as oxidative-glycolytic. The alkaline myofibrillar ATPase
identifies myosin ATPase by the breakdown of ATP and subsequent formation of
calcium phosphate and, after treatment with cobalt chlorate and ammonium
sulphite, cobalt sulphite (which is dark) is produced. This reaction is
considered positive (dark) for fast fibres and negative (light) for slow
fibres (opposite results are expected from acid pre-incubation). Thus, it is
possible to classify the fibres as oxidative or glycolytic and, regarding
general velocity of contraction, as fast or slow. The NADH-TR and myofibrillar
ATPase reactions are complementary because slow fibres tend to be highly
oxidative and fast fibres tend to be either glycolytic or
oxidative-glycolytic.
The results of histochemical reactions were analysed from digital images obtained from laminae using a compound microscope attached to a computerized image analysis system (Stereo Investigator 2000). For each section obtained, all fibres of six non-overlapping and randomly selected fields (at 10x magnification) were typed and quantified, including proportions and area. Fibres were classified as either fast or slow according to the results of the myofibrillar ATPase reaction and as either highly oxidative or moderately oxidative from the NADH-TR reaction (no glycolytic fibres were found). One intact section per reaction was analysed for each muscle region in each of the five individuals.
Muscle biochemistry
Muscle samples were homogenized using a Teflonglass homogenizer
(Marconi Ltd, Piracicaba, SP, Brazil) in ice-cold 20 mmol l-1
imidazole (pH 7.4) buffer with 2 mmol l-1
ethylenediaminetetraacetic acid (EDTA), 20 mmol l-1 NaF, 1 mmol
l-1 phenylmethylsulfonyl fluoride (PMSF) and 0.1% Triton X-100. The
homogenates were then submitted to sonication using a U-200S control unit
(IKA-Labor Technik, Staufen, Germany) for three 10 s intervals and directly
used in the assays. Measurements were made of the maximal activities of
pyruvate kinase (PK) and lactate dehydrogenase (LDH), key enzymes in the
glycolytic pathway, and citrate synthase (CS), which plays a major role in the
TCA cycle. Measurements were obtained at 25°C with a Beckman DU-70
spectrophotometer (Fullerton, CA, USA), following the changes in the
absorbance of nicotinamide adenine dinucleotide reduced form (NADH) at 340 nm
or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at 412 nm, under
substrate saturation and in the absence of inhibitory conditions. All
reactions were initiated by addition of substrate. Results were expressed in
µmol min-1 g-1 wet muscle mass. Established enzyme
protocols were used (Bergmeyer,
1983), with minor modifications as follows:
PK (E.C. 2.7.1.40) 100 mmol l-1 imidazole (pH 7.0), 10 mmol l-1 MgCl2, 100 mmol l-1 KCl, 2.5 mmol l-1 ADP, 0.02 mmol l-1 fructose-1,6-biphosphate (F1,6P2), 0.15 mmol l-1 NADH, 12 U ml-1 LDH, muscle sample homogenate and 3.6 mmol l-1 phospho(enol)pyruvate (omitted for control);
LDH (E.C. 1.1.1.27) 100 mmol l-1 imidazole (pH 7.0), 5 mmol l-1 dithiothreitol (DTT), 15 mmol l-1 NADH, muscle sample homogenate and 1 mmol l-1 pyruvate (omitted for control);
CS (E.C. 4.1.3.7) 50 mmol l-1 Tris (pH 8.0), 0.1 mmol l-1 DTNB, 0.2 mmol l-1 acetyl-CoA, muscle sample homogenate and 0.9 mmol l-1 oxalacetate (omitted for control).
Statistics
Conventional analyses were used after evaluating the suitability of data
for parametric approaches. To avoid statistical problems related to the
parametric evaluation of ratios, the count of fibres of a given type was
analysed in the context of an analysis of covariance (ANCOVA), using total
fibre count as a covariate. To test hypotheses regarding differentiation of
muscle type along the longitudinal axis of the muscle, fibre count of a given
type per muscle region was nested within reaction type (mATPase and
NADH+) in an ANCOVA.
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Results |
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Forces developed
L. microcephalum were able to produce forces approaching 24 N
during simulated digging trials. Although the sample size was small, the
amount of force produced by individuals was clearly and positively correlated
with head width (Fig.
4;P<0.01).
|
Overall characteristics of the digging muscle
The longissimus dorsi of L. microcephalum connects dorsally to a
thick dorsal tendon and ventrally to the rib cage by an array of connective
tissue. Its diameter decreases at the distal part and it is composed of
symmetrical pennated fibres that originate bilaterally at the dorsal tendon,
with a pennation angle that ranges from 20 to 35°, and run diagonally and
backwards to the ribs. Fibres are well organized in fascicles and joined
together by a thin layer of loose connective tissue that becomes more dense
when proximal to the tendon. The fibres are either polygonal or round and
exhibit an acidophilus sarcoplasm and a central or peripheral basophilic
nucleus. The muscle of all individuals changed in colour longitudinally, being
dark red proximally and becoming lighter distally. Detailed illustrations of
amphisbaenian muscle and skeletal morphology can be found elsewhere
(Gans, 1973;
Gasc, 1981
).
Fibre morphological and biochemical profile
Two main fibre types were observed (Fig.
5). The NADH reaction allowed classification of fibres as strongly
oxidative or moderately oxidative, whereas the ATPase reaction indicated the
presence of both fast and slow fibres. Fibres identified as either fast or
moderately oxidative were far more common than alternative types at any muscle
section. Fibres identified as either fast or moderately oxidative appeared to
be the same and were far more common than alternative types at any muscle
section. The counts for both reaction types were similar (ANCOVA,
F1,22=0.034, P=0.854). The proportion of
alternative fibre types (slow or highly oxidative) was somewhat higher in the
proximal part than in other parts of the muscle, but this difference was
clearly non-significant (nested ANCOVA, F4,22=0.647,
P=0.635). Despite limited histochemical differentiation, fibres
exhibited remarkable morphological and biochemical differences along the
longitudinal axes of the dorsal muscle. At the proximal part, fibres exhibited
small areas (918±302 µm2, mean ± S.D.)
in comparison with fibres at the distal part (1606±491
µm2; F1,997=713, P<0.001; see
Fig. 5). Despite the inability
of the histochemical approach to identify differences in the metabolic profile
of fibres along the longitudinal axis of the digging muscle, proximal fibres
had a distinct biochemical profile. Proximal fibres were characterized by a
significantly higher activity of CS and a significantly lower activity of PK,
in comparison with either medial or distal parts
(Fig. 6). Theactivity of LDH
did not differ among muscle sections despite much greater absolute values in
comparison with other enzymes.
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Implications of a model of work during head-first digging
Amphisbaenians must compress portions of substrate to an exiguous thickness
to open and increase the length of a tunnel of similar diameter to that of the
body. We developed a mechanical model (see details in the Appendix) that
addresses changes in the amount of work (w1) required to
compress substrate in head-first diggers as their diameter changes. Our
analysis leads to the following proportionality, in which
k0 is a constant of compressibility of granular soils,
h is the diameter of the animal, and J is a linear measure
related to the minimum thickness of a given type of substrate when it is
compressed:
![]() | (1) |
The ratio h/J indicates how thick the layer J would be when expressed as a fraction of the diameter of an animal. The relationship between w1 and h/J, then, addresses the amount of work required by an amphisbaenian of a given size to open a tunnel of body diameter by compressing detached substrate to an insubstantial thickness. The parameter J will increase with animal diameter because the amount of substrate to be compressed will also increase, and higher forces will be required for the head upstroke while digging. The key point is that the work required for compression increases exponentially after a given animal diameter (h), thus imposing great physical challenges to animals of large diameters (Fig. 7).
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Discussion |
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Various aspects of muscle and fibre morphology enhance force and work
production in the longissimus dorsi of L. microcephalum. First,
pennation increases the effective cross-sectional area of the muscle, hence
increasing force production achieved at a given whole-muscle shortening
velocity as muscle fibre velocity decreases as a proportion of whole-muscle
velocity (for a review, see Lieber and
Fridén, 2000). Second, the curvilinear shape of the muscle
probably allows for the elongation of fibres diagonally and distally,
following the tubular shape of the body. Fibre elongation would also result in
increased forces (see fig. 9b in Lieber
and Fridén, 2000
). Third, an annular fibre distribution
will result, during contraction, in increased radial coelomic pressures in the
body and in proximal body stiffening; two events that grant appropriate
dispersion of reaction forces through the body of the animal during a
soil-compression stroke.
According to the histochemical tests conducted, the longissimus dorsi of
L. microcephalum mainly exhibits fibres that are both fast and
moderately oxidative, and fibre type proportion remains fairly constant along
the longitudinal axis. This finding contrasts with the distinct change in
biochemical profile derived from the enzymatic analysis and suggests that
metabolic differentiation occurs within the dominant type of fibres. It is
also possible, however, that improved histochemical tools for amphisbaenian
muscle might, in the future, allow for higher resolution of fibre typing. The
dominance of moderately oxidative fibres is ecologically relevant, because
strictly glycolytic fibres, despite high power production, are probably
inappropriate for sustained digging as they are prone to fatigue. Strictly
aerobic fibres, on the other hand, would enhance sustained activity but would
be comparatively less powerful and would be more sensitive to oxygen stress,
an ecological trait that might affect some amphisbaenians (oxygen availability
has never been measured in amphisbaenian underground environments). The fibres
identified as fast and moderately oxidative are usually characterized by a
relatively high capacity for both oxidative and glycolytic carbohydrate usage
(Hochachka and Somero, 2002);
their presence apparently reflects a compromise in the functional properties
of the muscle and allows for the production of sustained and significant
forces in various behavioural and ecological contexts related to a fossorial
life.
The biochemical analysis allows for interesting insights regarding
metabolic specialization in the longissimus dorsi of L.
microcephalum. First, this muscle exhibits low activity of LDH in
comparison with truly specialized glycolytic muscles in ectotherms (for
example: Baldwin et al., 1995;
Bennett and Dawson, 1972
;
Mendiola et al., 1991
;
Miller et al., 1993
;
Somero and Childress, 1980
).
Indeed, the glycolytic capacity of the longissimus dorsi of L.
microcephalum is somewhat lower than that of the white portion of leg
muscles associated with locomotion in some lizards
(Bennett and Dawson, 1972
;
Garland et al., 1987
;
Gleeson, 1983
), snakes
(Wilkinson and Nemeth, 1989
)
and crocodiles (Baldwin et al.,
1995
). However, in the present study, PK/LDH ratios ranged from
0.27 (proximal) to 0.55 (distal), values that suggest relatively high
glycolytic fluxes and a high capacity to incorporate carbohydrates into
oxidative pathways (Hochachka et al.,
1983
). Although this muscle does not classify as highly aerobic,
CS activity in the proximal part is high in comparison with that of the
skeletal muscles of other reptiles (Bennett
and Dawson, 1972
; Garland et
al., 1987
; Gleeson,
1983
). Additionally, the CS/LDH ratio, an estimator of the
reliance of muscle on aerobic pathways
(Hochachka et al., 1983
),
changes from 0.023 (distal) to 0.036 (proximal); the latter value is
comparable with that of ectotherm aerobic muscles, such as the red muscle of
tuna fish (
0.04, Guppy et al.,
1979
) or the vastus lateralis of high elevation humans (
0.04;
Hochachka et al., 1983
;
Kayser et al., 1996
). The
oxidative capacity of the muscle seems quite enhanced at the proximal region,
which is also characterized by an apparent increase in myoglobin
concentration, increase in fibre density and decrease in fibre diameter. A
smaller fibre area probably allows for more intricate capillary network,
facilitates oxygen diffusion and enhances fine-tuned movements of the
head.
The unambiguous trend towards improved aerobic capacity at the proximal part of the longissimus dorsi of Leposternon microcephalum, coupled with apparently enhanced oxygen transportation and storage, strongly suggests functional differentiation of the muscle along the longitudinal axes. The functional significance of the muscle's metabolic heterogeneity is probably related to differential participation of parts of the muscle in the head soil-compression stroke. Our model suggests that, given the elastic properties of the substrate, increased contraction time, higher endurance and less power are required along most of the soil-compression stroke. By contrast, short-term and high-power contractions are required for final soil compression. So, one interpretation is that proximal fibres, more aerobic and able to withstand fatigue, are particularly relevant for initial soil compression, whereas distal fibres, more glycolytic in nature, are recruited to compensate for the higher power requirement of final soil compression. Additionally, the relative importance of different parts of the muscle, and the probability of recruitment, might vary as the animal moves through different substrates. The fibres further back in the muscle might gain importance when substrate viscosity increases. Additionally, proximal fibres are probably used more often and in a more diverse array of behavioural contexts, as amphisbaenians use their heads for prey capture and territorial defence.
In conclusion, to build underground galleries, the digging muscles of Leposternon microcephalum must be designed to produce forceful, powerful and repeated contractions of the head. These functional requirements pose problems that might be further complicated by the unsuitability of highly aerobic physiological solutions due to power requirement and perhaps oxygen stress. Similar problems are probably experienced by head-first diggers in other taxa. Digging muscles are restricted to a tubular body shape, and force production cannot be indefinitely enhanced by increasing muscle (= body) diameter because the work required for digging also increases with animal diameter, and at a non-linear rate. For the species studied, the solutions to these problems have been: (1) maintenance of small body diameters and increased muscle mass by elongation of the body; (2) enhancement of force production through intense muscle fibre pennation and fibre elongation around the body axis; additionally, a pennate muscle design is probably important to allow for partial contraction of the muscle, otherwise the inactive part would be stretched by the active part, decreasing the magnitude of the external forces produced; (3) reliance on fast and moderately oxidative fibres, able to use both aerobic and oxygen-independent pathways, and that apparently enable animals to modulate carbohydrate metabolism according to oxygen availability and energetic demands; and (4) muscular metabolic heterogeneity along the longitudinal axes. Muscular metabolic heterogeneity is quite unusual in ectotherm vertebrates and is possibly a finely tuned mechanism to maintain energy homeostasis during digging, itself a heterogeneous process in terms of force requirements. Regarding biochemistry, the digging muscles of this and perhaps other amphisbaenian species do not depart from what has been observed in other squamate skeletal muscles. However, our integrative study indicates that the exploitation of a highly specialized habitat in amphisbaenids is based on a complex set of morphological and physiological muscle traits that is probably very ancient in the lineage of vertebrate evolution.
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Appendix. A model of work during head-first digging |
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![]() | (A1) |
![]() | (A2) |
The minimum estimate of the work component related to the soil compression
w1 is defined by the first law of thermodynamics,
considering no heat exchange. Then:
![]() | (A3) |
The minus sign in the above equation indicates that work is applied to the
volume. From equation A2, and
using the technique of change of variables to integrate
equation A3 in h
(instead of in V), we obtain:
![]() | (A4) |
Here, h0 and h are, respectively, linear
dimensions related to the volume of the substrate before and after
compression, for which we have no information. However, if we assume a linear
magnitude J to be related to the maximum possible compression
applicable to an amount of a given substrate, then the maximum volume
displaced would be proportional to J:
![]() | (A5) |
![]() | (A6) |
Thus, the ratio of actual:maximum volume is given by:
![]() | (A7) |
Finally, we return to the minimum estimate of the work component
w1 and relate it to the linear dimension of the animal (i.e. the
mean body diameter h), and the following relation of proportionality
expressed in Equation 1 (main
text) applies:
![]() | (A8) |
Notice that if h tends to zero, the work of compression tends to zero as well. As h rises, the work required grows and tends to minus infinity in a non-linear way. The compression is limited to J, and, as a consequence, at some value of body diameter h, the increase in required work for digging would surpass the augmented force due to a higher cross-sectional area of the muscles, related to h2 (see Fig. 7).
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Acknowledgments |
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Footnotes |
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