Morphological and functional maturation of a skeletal muscle regulated by juvenile hormone
University of Ulm, Department of Neurobiology, Albert-Einstein-Allee 11, 89081 Ulm, Germany
* e-mail: uwe.rose{at}biologie.uni-ulm.de
Accepted 20 October 2003
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Summary |
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Histological and ultrastructural comparison of muscle fibres and their associated cuticular structures (apodemes) revealed dramatic growth during the first 2 weeks of reproductive development. The cross-sectional area of muscle fibres increased sevenfold, and their mass-per-length 5.3-fold. Ultrastructural examination showed growth of mitochondria, development of sarcoplasmic reticulum and increasing levels of structural organisation of myofibrils. Muscles of mature females displayed pronounced fatigue resistance, contracted more powerfully (twitch, 33.22±10.8 mN; 50 Hz, 623.66±115.77 mN) and had almost two times faster kinetics than those of immature females (twitch, 6.5±2.6 mN; 50 Hz, 14.19±2.58 mN). Together with muscular maturation, cuticular apodemes, which serve as attachment sides for ovipositor muscles, grow considerably in length and width and assume a complex surface structure. Most of the described changes were suppressed in females deprived of JH (allatectomised). The results demonstrate an adaptation of muscle properties to the requirements of reproductive behaviour that is largely regulated by juvenile hormone.
Key words: insect, locust, Locusta migratoria migratorioides, juvenile hormone, reproduction, muscle development, contraction property
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Introduction |
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Although evolutionarily remote, similar processes occur in the adult
insect, albeit regulated by a different hormone named juvenile hormone (JH).
JH, first discovered by Wigglesworth
(1934,
1936
), regulates several
aspects of insect development and reproduction. In cooperation with
20-hydroxyecdysone (20-HE), JH governs metamorphosis by inhibiting the
development of adult characters
(Riddiford, 1985
). In the
adult insect, by contrast, JH function has clearly been associated with
reproductive development (reviewed by
Wyatt, 1997
).
Early experimental data suggesting a regulatory function of JH during
reproductive development came from behavioural studies on grasshoppers
(Strong and Amerasinghe, 1977;
Hartmann, 1978) and showed that female sexual behaviour strongly depends on
corpora allata activity, the gland producing and releasing JH. Although this
effect can differ even within a single insect family
(Truman and Riddiford, 1974
;
Barth and Lester, 1973
), the
importance of JH became apparent.
By focusing on the nervous and muscular system, recent studies have
revealed a variety of developmental changes mediated by JH. Changes in the
phonotactic response of female house crickets are mediated by JH via
gene regulation (Stout et al.,
1992,
1993
). In the same species,
neurogenesis of mushroom body neurons is stimulated by JH
(Cayre et al., 1994
). Both
changes are likely to contribute to the expression of adequate sexual
behaviour (reviewed by Strambi et al.,
1997
). In the male moth Agrotis ipsilon, the sensitivity
of olfactory interneurons to sex pheromones is increased by JH
(Anton and Gadenne, 1999
),
thereby enhancing mate recognition and reproduction. Muscles are the output
elements of the information provided by the nervous system, and several
studies have demonstrated hormonal regulation of degeneration/regeneration and
structural muscle properties during metamorphosis and reproductive
development.
The remodelling of musculature during metamorphosis of holometabolous
insects involves degeneration of existing muscles
(Finlayson, 1975;
Rheuben, 1992
) and the
differentiation of new muscles (Stocker
and Nüesch, 1975
; Bate et
al., 1991
; Consoulas et al.,
1997
). These events are mainly regulated by 20-HE in the absence
of JH (Weeks and Truman, 1985
;
Schwarz and Truman, 1983
;
Luedemann and Levine, 1996; Hegstrom and
Truman, 1996
; reviewed in
Weeks and Truman, 1986
). The
importance of metamorphosis for flight muscle differentiation and development
has been shown by manipulating the levels of JH. Treatment of larval stages
with the JH analog methoprene, or implantation of corpora allata, which
shortened the length of the larval stadium, caused slowing of muscle growth
and inhibited the development of mitochondria and tracheolation in locust
(Poels and Beenakkers, 1969
;
Cotton and Anstee, 1990
) and
cricket (Novicki, 1989
).
Chemical allatectomy, however, enhanced flight muscle development and resulted
in normal flight muscles of the adultiform
(Wang et al., 1993
). By
contrast, parts of a flight steering muscle in locust (M114c) degenerate
shortly after adult emergence while the JH titer is low
(Meuser and Pflüger,
1998
). Experimentally elevated JH titers prevent muscle
degeneration. These studies indicate the importance of low JH levels during
the last larval stage for normal development of flight muscles.
In the adult insect, degeneration and regeneration of flight muscles are
clearly regulated by JH in different insect species
(Tanaka, 1994;
Borden and Slater, 1968
;
de Kort, 1990
; reviewed by
Finlayson, 1975
;
Wyatt and Davey, 1996
). The
close correlation between reproduction and flight muscle degeneration and
regeneration has been suggested to serve for the liberation of nutrients when
functional muscles are no longer needed. However, little is known about
structural and functional changes that might adapt muscle performance to the
requirements of reproductive behaviour. A recent study on the longitudinal
muscles of female locusts provided the first evidence for a functional
adaption of muscle properties controlled by JH
(Rose et al., 2001
). Changes
were shown to be segment- and gender-specific and important for oviposition
behaviour.
Oviposition behaviour in insects is not expressed before the female is
sexually mature and ensures adequate deposition of eggs. This raises the
question of whether the underlying neuromuscular system changes its properties
at the time of sexual maturation to adapt for the species-specific
requirements of oviposition. In locusts, considerable knowledge has
accumulated about various aspects of oviposition (reviewed by
Staufer and Whitman, 1997).
The female lays its eggs deep down into the soil by rhythmical digging
movements of a pair of sclerotized appendages (ovipositor valves) located at
the tip of the abdomen (Vincent,
1975
; Thompson,
1986
; Rose et al.,
2000
). The digging movements of the ovipositor are thus essential
for a successful oviposition. However, whether the muscles associated with the
ovipositor undergo structural and functional changes during reproductive
development is unknown. Among the ten pairs of ovipositor muscles
(Snodgrass, 1935
), the dorsal
and ventral ovipositor opener muscles are the largest. Their contractions open
and close the valves during oviposition digging. The patterned neural input to
these muscles, which can be elicited in embryos and larvae
(Thompson and Roosevelt,
1998
), is provided by motoneurons located in the terminal
abdominal ganglion (Thompson et al.,
1999
).
The present study addresses questions about possible structural and functional maturation of ovipositor muscles during reproductive development. I compared morphological characteristics and functional properties of the dorsal ovipositor opener muscles from immature and mature females. One of the primary goals of this work was to reveal the functional consequences of structural changes and to relate them to the behavioural requirements. A possible regulatory function of JH was assessed by manipulating JH production through allatectomy in combination with JH-analog replacement injections.
The results presented here show a pronounced growth and ultrastructural maturation of ovipositor muscle fibres during reproductive development, together with multiple changes of their contraction characteristics. Inhibition of JH production by allatectomy largely suppressed normal changes, whereas replacement injections with the JH analog methoprene almost restored the normal development. The results thus suggest a regulatory control by JH.
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Materials and methods |
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Hormonal manipulation
To determine the influence of JH on the development of muscle fibres,
female locusts were allatectomised by one of two means. Experimental animals
were either chemically allatectomised by using precocene
(7-ethoxy-6-methoxy-2,2-dimethylchromene; Sigma) or surgically allatectomised
by removing the corpora allata. Precocene (500 µg/animal, dissolved in 10
µl acetone), which chemically inactivates the corpora allata
(Bowers et al., 1976;
Pener et al., 1978
) was
topically applied onto the dorsal neck fold once at the day of adult
emergence. Control animals were treated with acetone only. The technique for
surgical allatectomy followed the method published by Strong
(1963
). In brief, animals were
anaesthetised with carbon dioxide and mounted in a dish with their dorsal side
up. The head was stretched to expose the dorsal neck membrane and the dish was
filled with ice-cold saline until the neck membrane was covered. A tear was
made in the midline of the neck membrane and, with two fine forceps, the
cephalic air sacs were displaced on one side and the corpora allata from the
other side was removed. After both corpora allata had been removed, the animal
was dismounted, blotted on filter paper and the neck membrane sealed with
histoacryl (B. Braun, Tuttlingen, Germany). The same procedure was applied to
sham-operated females, except that in this case the corpora allata were gently
pulled but not destroyed or removed. Allatectomised and sham-operated females
were then allowed to develop to an age of 1825 days. In all experiments
of this study, sham-operated and non-operated females were indistinguishable
in their anatomy and physiology and considered as mature females. However, to
be clear on this point, they were named >14 day (non-operated) and >14
day, sham-op. (sham-operated). The survival rate of animals was about 70% for
those surgically allatectomised and about 90% for precocene-treated animals.
During the experiments no differences were apparent between surgically and
chemically allatectomised females. Once the allatectomised animals reached the
appropriate age (1825 days) they usually showed clear signs of
inhibited JH production (undeveloped oocytes, cuticle lightly coloured,
considerable fat body). Those females showing no signs of inhibited JH
production were excluded from the study.
At the seventh day following adult emergence, some of the surgically allatectomised females were either injected with the JH analog methoprene (860 µg in 5 µl acetone, injected once in the abdomen) or an active corpora allata was implanted through an incision in the ventral tergo-sternal membrane of the third abdominal segment. All experimental females were individually marked and held separately from other groups.
Histology
Cuttings of fixed tissue were performed on a cryostat or ultramicrotome.
For cryostat sections, the entire valve-muscle complex
(Fig. 1A) was fixed overnight
in 2.5 mol l1 glutaraldehyde, rapidly frozen in liquid
nitrogen and subsequently cut (12 µm) on a cryomicrotom (Microm, HM500 OM;
Walldorf, Germany). Material was transferred to slides, stained with
Hematoxylin-Eosin, dehydrated and mounted in Entellan (Merck, Darmstadt,
Germany).
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Electron microscopy studies were performed on dorsal ovipositor muscle M271 taken from immature, mature or hormonally manipulated females. Muscles were fixed in situ using 2% glutaraldehyde for 30 min, post-fixed with 2% osmium tetroxide and subsequently dehydrated and embedded in Epon 812 (Fluka, Seelze, Germany). Thin sections were cut (8090 µm) and double stained with uranyl acetate and lead citrate. Sections were examined and photographed using a Zeiss EM10 electron microscope.
Semi-thin sections (1 µm) from the middle region of fixed muscles were cut, mounted on slides and stained with Methylene Blue. Sections were examined under a bright field microscope. To determine the cross-sectional area of muscle fibres, a digital picture was taken with a CCD camera (Sony ICX038AK, resolution 752x582). The mean cross-sectional area of a single fibre was determined by calculating the sum of cross-sectional areas of all measured fibres (Scion Image, 4.0.2, Scion Corporation, Frederick, USA) divided by the number of fibres.
To meaningfully compare the performance of muscle M271 in different experimental groups, the mass-per-length of the muscle was estimated. Muscles were fixed in situ for 1 h in 2.5% glutaraldehyde and carefully separated from their attachment sites with fine forceps. After measuring the length under a dissection microscope, muscles were blotted on filter paper and weighed (Sartorius, MC210P, Göttingen, Germany).
For scanning electron microscopy studies, valve apodemes were fixed in 2% glutaraldehyde, freed from adhering tissue and separated from the abdomen. After rinsing, specimens were critical-point dried, coated with goldpalladium (20 nm) and examined and photographed in a scanning electron microscope. To quantify their length and width, apodemes were measured under a dissection microscope. The length was measured between the anterior tip and their posterior attachment to the valvulae. The width was measured at the middle of the apodeme. As an overall measurement for apodemal growth the values for length and width were multiplied.
Tension recordings
Isometric tension recordings were performed on dorsal ovipositor muscle
M271 (Fig. 1A). Experimental
animals were mounted in a Sylgard-coated dish and opened dorsally. The dorsal
ovipositor muscle was fixed anteriorly with an U-shaped insect pin where it
attaches to the apodeme. The tendon at the posterior side was cut and clamped
to the lever arm of an isometric force transducer. Muscle contractions were
evoked by stimulating the motor nerve via a suction electrode.
Stimulus intensity and length of the muscle were individually adjusted to
elicit maximal muscle contraction. Between contractions, the preparation was
continuously superfused with aerated saline. The response of the transducer
was linear over the range used in the experiments and calibrated after each
experiment. Proctolin was freshly prepared from a stock solution
(103 mol l1) and bath-applied by means of
a pipette. Between different experimental trials the muscle was left unexcited
for at least 5 min to recover from previous contractions.
Statistical evaluation
Data are expressed as means ± standard deviation (S.D.).
Statistical significance was determined by parametric tests (t-test
or paired t-test where appropriate). When criteria for parametric
tests were not met the non-parametric MannWhitney rank sum test was
applied.
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Results |
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Apodeme growth
A scanning electron microscopy study was performed to quantify possible
changes in size and surface structure of isolated apodemes in greater detail.
The study revealed considerable growth of apodemes during the first two weeks
of adult development (Fig.
2A,B). Apodemes grew in length and width and assumed a complex
three-dimensional structure that is characterised by a surface contour with
pronounced edges and corrugations (Fig.
2, compare <5 day and >14 day). Apodemal area increased from
2.06±0.31 mm2 to 7.08±0.72 mm2 for
immature and mature females, respectively. Allatectomised females had apodemes
that were significantly smaller than those from mature females
(3.60±0.67 mm2; CA in
Fig. 2B). In addition, the
surface structure assumed a level of complexity that was intermediate between
that of immature and mature females. Replacement injections with methoprene
were able to significantly reverse the effect of allatectomy (5.25±0.42
mm2; CA+met in Fig.
2B). However, the values still differed considerably from those
measured in mature females.
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Muscle fibre growth
As mentioned before, ovipositor opener muscles undergo hypertrophy during
the first 2 weeks of adult life. Their mass-per-length (mass/length) increased
from 0.28 to 1.5 mg mm1
(Table 1). Allatectomy
significantly reduced this increase to 0.42 mg mm1, whereas
additional injection of the JH analog methoprene partially restored the higher
mass/length values (0.85 mg mm1) seen in mature females. To
examine muscle histology and physical dimensions in greater detail, semi-thin
sections of M271 were made and evaluated. Transverse sections obtained from
muscles of immature females revealed loosely distributed fibres with a high
degree of tracheolation and numerous nuclei
(Fig. 3A, <5 day). The mean
cross-sectional area of muscle fibres was 53.75±15.65 µm2
(Fig. 3B, <5 day). By
contrast, muscles from mature females had a much larger cross-sectional area
of 377.87±73.54 µm2
(Fig. 3B, >14 day). Their
tracheolation appeared not as pronounced as in immature females, possibly
because of the large and prominent muscle fibres
(Fig. 3A, >14 day). The
appearance of muscle fibres from allatectomised females, however, was
comparable to immature females with a cross-sectional area of
67.25±21.68 µm2 (Fig.
3B, CA). As a result of allatectomy, fatty tissue was
frequently present in cross-sections of these muscles (ft in
Fig. 3A, CA), but never
observed in sections from immature or mature females. Additional injections of
methoprene partially reversed the effects of allatectomy on the growth and
appearance of muscle fibres (cross-sectional area: 214.25±31.63
µm2, Fig. 3B,
CA+, met).
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From these experiments it became apparent that methoprene was not able to completely reverse the effect of allatectomy and therefore additional experiments were performed in which a pair of active corpora allata was implanted, instead of injecting methoprene. The intention behind this experiment was to determine whether there is a difference between methoprene (JH analog) and the natural JH (released from the corpora allata) in their action to restore the normal development. The experiments revealed muscle fibres with an appearance indistinguishable from mature females. Their mean cross-sectional area was 361.0±103.47 µm2, N=9 (data not shown) and were thus not significantly different from mature females (P>0.05, MannWhitney rank sum test).
A possible increase in the number of muscle fibres during reproductive development was accessed by relating the increase in cross-sectional area to the increased in muscle mass. Both the cross-sectional area and the mass of the muscle increased by a factor of 7.1, which suggests that the number of muscle fibres remains constant throughout maturation.
Ultrastructure
To gain further insight into possible maturational changes of muscle fibre
structure and organisation, the ultrastructure of muscles was compared. Muscle
fibres from immature females were in close contact with multiple tracheoles
and tracheae (Figs 4A,
5A). The cytoplasm associated
with the tracheoles contained numerous microtubules oriented parallel with the
cuticular tubes. Interfibrillar tracheoles were not seen. In transverse
sections, T-tubule openings and dyads were frequently present as well as
multiple, small mitochondria (Fig.
4A,C). The myofibrils had an irregular appearance and were not
well-defined. The sarcoplasm between the myofibrils covered a relatively large
area and contained multiple microtubules, elements of the sarcoplasmic
reticulum and T-tubules. Longitudinal sections showed small, elongated
mitochondria located at the level of I-band
(Fig. 4B). Although Z-lines
were clearly visible as patches of electron dense material, their alignment
was poor.
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The ultrastructure of muscle fibres from mature females was clearly different from immature females. Myofibrils showed a regular appearance and were, at the level of the A-band, clearly defined by surrounding sarcoplasmic reticulum and T-system (Fig. 4D). Well-developed dyads were regularly encountered at the A-band (Fig. 4D, arrow), whereas T-tubule invaginations were restricted to the I-band (T; Fig. 4F). Compared to immature females, mitochondria were much larger and clustered along the I-band (M; Fig. 4E,F). Tracheoles and tracheae associated with single fibres lacked the relatively large cytoplasmatic area seen in fibres from immature females, but their number remained rather constant throughout reproductive development (approx. 810 tracheoles per muscle fibre, data not shown).
The muscle fibre appearance from allatectomised females was similar to those of immature females and shared some common characteristics. Their myofibrils remained relatively small, with numerous microtubules apparent (Fig. 5B,C). The mitochondria were clustered at the level of I-band and their size was somewhat larger than those of muscle fibres from immature females (compare Figs 5B,D and 4B,C). The same was found for dyads and elements of the sarcoplasmic reticulum that appeared slightly more developed and better organised than in immature females (compare Figs 4C and 5B).
Contractions
The apparent influence of JH on the morphological development of ovipositor
muscle fibres raised questions about their functional properties. Female
locusts undergo reproductive development within the first 2 weeks of adult
life, then mate and start egg laying. When egg laying becomes necessary the
ovipositor is able to perform powerful movements for about 1525 min
while digging the oviposition hole (Rose
et al., 2000; Thompson,
1986
). To reveal possible functional changes associated with the
growth of the ovipositor opener muscle M271, various contraction parameters
were measured in immature (<5 day), mature (>14 day, sham operated) and
allatectomised (CA) females.
Generally, contraction measurements of ovipositor opener muscles displayed
a rather high degree of individual variation in all experimental groups. This
can be due to individual variation of animal size or the current status of
muscular modulation by the pentapeptide proctolin, which is known to affect
muscular contraction (Belanger and Orchard,
1993). However, most differences were robust enough to let a clear
picture emerge.
Considering the dramatic increase in cross-sectional area during reproductive development, it was not surprising that muscles from mature females generated considerably more tension during twitch and tetanic contraction than immature females (Fig. 6A,B). Twitch tension increased approx. fivefold (>14 day, sham-op., 33.22±10.8 mN; <5 day, 6.5±2.59 mN) and tetanic tension (50 Hz) approx. 44-fold (>14 day, sham-op., 623.66±115.77 mN; <5 day, 14.19 ±2.58 mN). The twitch/tetanus ratio decreased from 0.45 (<5 day) to 0.05 (>14 day, sham-op.). Removing the corpora allata shortly after adult emergence (Fig. 6B, CA) largely suppressed the ability of the muscle to exert large tension (twitch, 9.9±2.67 mN; tetanus, 61.56±17.5 mN) and revealed a twitch/tetanus ratio of 0.16. However, these values were still significantly different from immature females (twitch, P<0.05; tetanus, P<0.001, N=9, MannWhitney rank sum test). During the experiments it became apparent that contractions of muscles from immature females fused at lower stimulation frequencies (35 Hz) than in mature females (>10 Hz,Fig. 6A). A detailed evaluation of the contraction kinetics on the basis of single twitches revealed significantly shorter contraction (time-to-peak) and relaxation (50% relaxation) times for mature females (Fig. 6C). The contraction time declined from a mean value of 309.23±75.69 ms (<5 day) to 140.12±19.84 ms (>14 day, sham-op.). Similar values were measured for the 50% relaxation time (<5 day, 333.67±121.23 ms; >14 day, sham-op., 142.48±27.40 ms). Allatectomy, however, was not able to completely prevent the shift to faster contraction kinetics. The values obtained from allatectomised females were between those measured in immature and mature females (contraction, 178.32±39.15 ms; 50% relaxation, 209.48±45.72 ms). Nevertheless, these results suggest a powerful increase of contraction forces during reproductive development of female locusts that is, at least in part, influenced by JH. Associated with that increase is a shift to faster contraction kinetics.
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Proctolin modulation
It has been reported that the pentapeptide proctolin is required for a
normal function of the locust ovipositor opener muscle
(Belanger and Orchard, 1993).
During activation of motoneurons a constant realease of proctolin was shown to
maintain contractability of ovipositor muscle fibers, whereas depletion of
proctolin stores after prolonged activation led to declined muscular
contractions (Belanger and Orchard,
1993
). To determine if the effect of proctolin on the contraction
of ovipositor muscle changes with the maturational and hormonal status of the
female, stimulus evoked contractions were measured in the absence and presence
of 109 mol l1 proctolin
(Fig. 7A). Ovipositor muscles
of all three experimental groups responded to the application of
109 mol proctolin with a significant increase of tension
(Proctolin 109 mol l1;
Table 2). To test for the
existence of endogenous proctolin, 10 single test stimuli (0.3 Hz) were
applied before and after a high frequency, conditioning stimulation (50 Hz).
Conditioning stimulus lasted for 5 s and was intended to release endogenous
proctolin from motor terminals on muscle 271
(Fig. 7B). These experiments
revealed significantly increased forces (42%) in ovipositor muscles of mature
females only. By contrast, contraction forces obtained from immature females
showed a 21% decrease, whereas allatectomised females showed a 14% increase,
but both were not statistically different [Pre-stimulation (50 Hz), saline;
Fig. 7B,
Table 2]. However, in immature
females, a pronounced recovery from decreased contraction forces was observed
within the following 5 min (not shown). These experiments suggest that muscle
ability to respond to exogenous proctolin is independent of maturational
status. Pronounced modulation of muscle contractions by endogenous proctolin,
however, occurred in mature females only. To test if these results did indeed
depend on the lack of proctolin release from motor units of immature and
allatectomised females as suggested by the result, the experiment was repeated
with proctolin (109 mol l1) added to the
bath shortly after the control stimulation. If the existence of proctolin was
the limiting factor of contraction strength in immature and allatectomised
females, additional application of proctolin would be expected to increase
contractions markedly. The results however, show that proctolin was not
effective in changing the values dramatically compared to those obtained
without proctolin [Pre-stimulation (50 Hz), proctolin 109
mol l1; Table
2]. Immature females again displayed decreased tension production
which was now statistically significant. Forces measured in mature and
allatectomised females were in the range of those measured without proctolin,
but a statistically significant difference was detected for allatectomised
females only.
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Discussion |
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JH regulation of muscle development
The influence of JH on muscle development seems to be a common phenomenon
in adult insects. Flight muscles of different insect species undergo
degeneration and/or regeneration regulated by JH
(Stegwee et al., 1963;
Borden and Slater, 1968
;
Tanaka, 1994
). In the colorado
potato beetle, flight muscle regeneration, which is initiated by rising JH
titers, involves growth of muscle fibres and mitochondria and thus bears some
resemblance to the findings of the present study
(Stegwee, 1964
;
de Kort, 1990
). The
involvement of JH in regulating structural differentiation of muscle fibres
has been shown in studies on larval locust. Here, flight muscles undergo
accelerated structural differentiation after application of the JH analogue
methoprene (Cotton and Anstee,
1990
). When the last instar larvae moults to the early adult,
however, their normal flight muscle maturation is compromised (e.g. reduction
of the normal increase in the cross-sectional area of muscle fibres).
These studies clearly show that JH is involved in the regulation of
different aspects of flight muscle function and differentiation. The results
from the present study suggest in addition a pronounced structural and
functional maturation of non-flight muscles leading to fibres well adapted for
a stage-specific behaviour (e.g. oviposition). A recent study on locust
abdominal longitudinal muscles showed that this type of hormonal regulation of
muscle maturation is not an exception. Longitudinal muscles were shown to
undergo hypertrophy along with changes in their contractile properties during
reproductive development (Rose et al.,
2001). These results agree well with the data from the present
study and thus provide a further example where JH regulates morphological and
functional changes of muscles required in certain life stages (e.g.
reproduction, diapause, dispersal flight).
Many aspects of juvenile hormone action on insect tissue during development
and adult life are mysterious. In the present study allatectomy was carried
out in an attempt to remove the natural source of JH and to examine whether
this treatment inhibits the observed changes on muscle structure and function.
Most of these changes, however, were only partially inhibited by allatectomy,
except for the cross-sectional area and some aspects of the ultrastructural
maturation. This is interesting, because it indicates some kind of development
that is independent of a functional corpora allata and could be explained by
the existence of other internal sources of JH or of substances with similar
actions in insects. An alternative source of JH was located in the ovary of
Aedes, which are able to synthesise JH from farnesoic acid
(Borovsky et al., 1994).
Substances with JH activity are the thyroid hormones [especially
3',3,5-triiodo thyronine (T3)]. T3 was shown to mimic some aspects of JH
action on follicle cells of Locusta migratoria
(Davey, 2000
). The food of
locusts provides a potential source of T3 and the author suggests that thyroid
hormones are ingested by locusts. However, evidence for additional mechanisms
influencing maturation in adult insects are provided by studies investigating
the influence of metamorphosis on the subsequent maturation of insect flight
muscles in locust and crickets. Wang et al.
(1993
) reported inhibited
growth of muscles in overaged or supernumerary Schistocerca gregaria
nymphs, whereas precocious adults developed almost normal muscles. Studies on
crickets underline the importance of metamorphosis for subsequent flight
muscle maturation. Injection of the JH analog methoprene into last-instar
nymphs of the cricket Teleogryllus oceanicus slowed subsequent flight
muscle growth and blocked ultrastructural maturation
(Novicki, 1989
). It seems
reasonable to suppose therefore that parts of the structural maturation of
insect muscles are influenced by their hormonal history during metamorphosis,
although it must be pointed out that flight behaviour occurs shortly after
moulting, while oviposition is delayed until at least 2 weeks later.
Therefore, in the adult insect elevated JH titers apparently regulate
additional maturation of specific muscles to adapt them to the demands of
reproduction.
The growth described in the present study was not only restricted to the
muscles but also the apodeme, which functions as an insertion element of
muscle fibres. Smith (1964)
speculated that muscles and their integumental attachment co-develop during
ontogenesis. In the adult flesh-fly, Sarcophyga falculata, Schlein
(1972
) reported post-emergence
enlargement of an apodeme by the deposition of endocuticle and growth of
associated muscle fibres influenced by endocrine factors. The results of the
present study support the notion that the form and size of cuticular
attachment sites functionally fit their attaching muscles. In addition,
hormonal synchronisation of muscular and apodemal growth makes sense in terms
of functionality: large and powerful muscles develop considerable tension that
need to be tolerated by their cuticular attachment sites.
Although there is no doubt that JH has a pivotal role in regulating
reproductive development, the mechanisms by which JH reveals its action on the
tissue are not understood. This study was not designed to examine the
mechanisms of JH action, but it can be speculated that one, if not the
primary, role of JH is to provide muscle and apodeme with sufficient amounts
of protein needed for their growth. Studies on adult locusts showed increased
levels of persistent storage protein (PSP) during their reproductive
development (Wyatt, 1990;
Wyatt et al., 1992
). In
chemically allatectomised animals, by contrast, PSP maintained a low rate of
synthesis, whereas application of JH or an analogue elevated PSP synthesis.
The function of PSP is to provide amino acids for the construction of the
adult integument and other organs in metamorphosis
(Kanost et al., 1990
). In
adult insects, storage proteins may serve a similar function and thus limit
organ growth when their synthesis is depressed by low JH levels.
Functional maturation
Structure and performance of muscle fibres are tightly bound. Thus, the
present study aimed at revealing possible functional alterations of ovipositor
opener muscle fibres concomitant with their growth and structural maturation
during reproductive development. The most dramatic functional change was the
increase in contraction force, which was not surprising, because the maximum
force produced by a muscle should be proportional to its cross-sectional area
(Josephson, 1975). For twitch
tension, this increase can thus be largely assigned to the growth of the
muscle, since its mass-per-length values increased accordingly (mass/length,
5.6-fold; twitch tension, 5.1-fold). By contrast, tetanic tension increased
43-fold and cannot be explained solely by the growth of the ovipositor muscle.
Other factors must be responsible, of which the organisation of myofibrils is
of primary importance. The results from the present study show a poor
alignment and some kind of disorganisation of myofibrils in immature females
that will result in a lower amount of filament overlap. Since it has been
shown for invertebrate and vertebrate muscle fibres that the maximum tension
depends critically on the amount of filament overlap
(Weis-Fogh, 1956
;
Gordon at al., 1966
), this
structural feature may limit the tension produced.
The relatively high maximum tetanic tension of the ovipositor opener muscle
from mature females results in a low twitch/tetanus ratio, which is indicative
of a muscle type where graded force production is necessary
(Aidley, 1985). During the
course of oviposition, valves open and close repeatedly to dig a cavity in
which the abdomen is lowered and the eggs are eventually laid. Once the female
has performed a few cycles of digging movements, the tip of the abdomen
retracts and the valves open to press against the dug part of the oviposition
hole to stabilise the walls of the cavity
(Thompson, 1986
;
Rose et al., 2000
). Since egg
laying is performed in a substrate of different compactness, one can imagine
that the complex digging movements require muscles capable of graded
contractions, especially during the sweeping digging movements.
The brevity of muscle contraction depends to a large extent on the
development and organisation of the sarcoplasmic reticulum and the transverse
tubules (Josephson, 1975).
Thus, the increased shortening and relaxing velocities observed for the
ovipositor muscle from mature females may be the result of a well-developed
sarcoplasmic reticulum. Indeed, the results from the present study suggest a
higher degree of sarcoplasmic reticulum organisation and development for
mature, and to a lesser extent for allatectomised, females, as compared to
immature females. By contrast, a slowing of contractions during reproductive
development has been shown for abdominal longitudinal muscles of female
locusts (Rose et al., 2001
).
Here, muscles from mature females contract significantly slower than those
from immature or allatectomised females. This may be explained by the specific
functional requirements of longitudinal muscles during oviposition where they
must tolerate dramatic lengthening, which is accompanied by the fragmentation
of their Z-lines (Jorgensen and Rice,
1983
). As a consequence, longitudinal muscles display elastic
properties that give rise to slow contraction kinetics
(Rose et al., 2001
). The
ovipositor opener muscles, by contrast, representing a more conventional
muscle type, provide the driving force for rhythmic digging movements.
Proctolin
The normal function of locust ovipositor muscles has been shown to depend
critically on the pentapeptide proctolin
(Belanger and Orchard, 1993).
Endogenous proctolin is released from motoneurons and affects the tension
produced by the muscle. The experiments presented here show that exogenously
applied proctolin significantly increased force production in immature, mature
and allatectomised females. This implies the presence of a functional
modulatory receptor system, even in the immature stage. However, when the
muscular system was tested for endogenous proctolin through high-frequency
stimulation of the motor nerve prior to a test pulse, only mature females
responded with a significant increase of tension. Although this result
suggests that the structures releasing proctolin somehow improve their ability
to produce, store and/or release proctolin during reproductive development, it
seems likely that other mechanisms such as fatigue are also involved. This is
also suggested by the finding that additional proctolin, applied shortly
before the conditioning stimulus, did not reverse the effects obtained without
proctolin (compare Pre-stimulation values in
Table 2). These results would
therefore be interpreted as mainly affected by the fatigue of the muscle
fibres after the conditioning stimulus. Fatigue-resistant muscles, capable of
sustained activity, generally contain mitochondria comprising a large volume
of the fibre (Hoyle and Mc-Neill,
1968
; Strokes et al.,
1975
; reviewed in Josephson,
1975
). A look at the sizes of mitochondria of muscles from
immature, mature and allatectomised females and their fatigue-resistance
revealed a clear correlation. The ovipositor opener muscle from immature
females (small mitochondria, Fig.
4C) are non-resistant against fatigue as indicated by the fact
that, after the conditioning, stimulus twitch contraction forces declined, but
slowly recovered thereafter. Even the presumed release of endogenous proctolin
by the conditioning stimulus, or exogenously applied proctolin, had no
significant effects since fatigue seems to be the limiting factor for
contraction strength. By contrast, the ovipositor opener muscle from mature
females, which contain relatively large mitochondria
(Fig. 4F), was
fatigue-resistant and proctolin effects were clearly visible (increased
tension). The same is true for allatectomised females, although their
mitochondria were not as large as those from mature females.
The ovipositor muscles of locusts are exclusively important for reproductive behaviour in the adult female. The results of the present study suggest a pivotal role for JH in the regulation of ovipositor muscle structure and function. Although some aspects of muscle maturation appeared to be regulated independently of a functional corpora allata, the importance of JH is apparent. JH obviously synchronises muscle and apodeme development with the maturation of reproductive organs. This development changes muscle performance and adapts their properties to enable powerful, graded contractions as well as endurance. The evolutionary advantage of a retarded muscle development may lie in the savings of metabolic energy during the early developmental stages.
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