ABSTRACT
Tethered adult crickets (Gryllus bimaculatus De Geer) assume a full flight posture: they point the antennae straight ahead, hold the fore-and midlegs close to the body, stiffen the abdomen for steering and extend the hindlegs directly backwards. Tomioka and Yamaguchi (1980) have reported that about 30% of the animals displaying flight behaviour also flap their hindwings, but the remaining crickets vibrate their wings through a narrow range instead of flapping them. They distinguished the latter type of behaviour from normal flight behaviour and designated it ‘pseudo-flight behaviour’.
Degeneration of flight muscles is known to occur in young adult insects of several orders (Finlayson, 1975; Collatz and Wilps, 1986). It has been reported that in the cricket Teleogryllus oceanicus the dorsal longitudinal muscle atrophies and becomes white in appearance in late adult life (Ready and Josephson, 1982; Ready and Najm, 1985) and in the cricket Acheta domestica the dorsal longitudinal muscle increases in size during the first 2 days after the imaginal moult, but begins to degenerate on the fourth day in the presence of juvenile hormones (Chudakova and Bocharova-Messner, 1968; Srihari et al. 1975). The present experiments were designed to detect any close relationship between flight behaviour and age-related degeneration of flight muscles during adult life.
Crickets (Gryllus bimaculatus) were reared at 28°C under a 12h:12h L:D photoperiod. Maximal adult life span was about 20 days under these conditions. The flight behaviour of each individual was tested daily after the imaginal moult, as follows. The flight behaviour of the tethered cricket, held stationary in space with the pronotum glued to a supporting rod, could be elicited by exposing its head to wind. The colour and size of the following five flight muscles were examined each day after the imaginal moult (see Fig. 2A): M90 (forewing elevator muscle, muscle numbering after Furukawa et al. 1983); M99 (forewing depressor muscle); M112a (hindwing depressor muscle); M119 (hindwing elevator muscle); and M129a (hindwing depressor muscle). The wet mass of these flight muscles, fixed with 4% formaldehyde, was then measured to calculate the relative wet mass, which is the muscle mass divided by the width of the thorax. These calculations showed that there is a linear relationship between the wet mass of each muscle and the width of the thorax: the mass of each muscle increases proportionally with increase in width of the thorax, irrespective of size and sex in animals of similar age. The spike discharges of motoneurones and the electromyograms of flight muscles were recorded extracellularly using conventional methods (described by Tomioka and Yamaguchi, 1980).
Daily testing of flight behaviour after the imaginal moult revealed a close relationship between flight behaviour and age in adults of both sexes (Fig. 1A). On the first day after the imaginal moult 17 % of males and 13 % of females displayed normal flight behaviour, but by the second day 93 % of males and 96 % of females displayed it. On these days the remaining crickets displayed pseudoflight behaviour. However, the occurrence of normal flight behaviour decreased quickly after the second day, and all males displayed only pseudo-flight behaviour on and after the seventh day, while 4 % of females showed normal flight behaviour even on the tenth day.
Flight behaviour and electrical activities of flight muscles and motoneurones. (A) Relative changes in the occurrence of normal (hatched column) and pseudo-flight behaviour (open column) in both sexes after the imaginal moult. These data were obtained from 27 males and 29 females. (B) Extracellular recordings of spike discharges of the motoneurones innervating muscle M112a (top) and electromyograms of M112a (middle) and M119 (bottom). Records i and ii were obtained from a 3-day-old male displaying normal flight behaviour and a 15-day-old male displaying pseudoflight behaviour, respectively.
Flight behaviour and electrical activities of flight muscles and motoneurones. (A) Relative changes in the occurrence of normal (hatched column) and pseudo-flight behaviour (open column) in both sexes after the imaginal moult. These data were obtained from 27 males and 29 females. (B) Extracellular recordings of spike discharges of the motoneurones innervating muscle M112a (top) and electromyograms of M112a (middle) and M119 (bottom). Records i and ii were obtained from a 3-day-old male displaying normal flight behaviour and a 15-day-old male displaying pseudoflight behaviour, respectively.
As shown in Fig. 2, the mesothoracic flight muscle M90, which produces the stridulating movement of the forewing in males, showed the most distinctive sexual dimorphism among the flight muscles: the male muscle was much larger and heavier than the female muscle, although both male and female M90s were deep orange and ivory-white in appearance throughout adult life. In addition, the mass of the male M90 increased significantly during the first 5 days following the imaginal moult, while that of the female M90 did not (P<0.05, two-tailed t-test). Another mesothoracic flight muscle, M99, did not show significant sexual dimorphism in size, and both male and female M99s grew slightly during the first 5 days following the imaginal moult. The male and female M99s were deep orange and ivory-white in appearance and did not fade even by the fifteenth day.
The size of flight muscles relative to thoracic width during adult life. (A) Schematic illustration of the main inner muscles in the meso-and metathorax, viewed from the inside following removal of the left side of the thorax (d, dorsal; v, ventral). Muscle numbering follows Furukawa et al. (1983). (B) Muscle M90. (C) Muscle M99. (D) Muscle M112a. (E) Muscle M119. (F) Muscle M129a. Each open (female) or closed (male) circle was obtained from 10–11 animals. Each error bar on a graph indicates a standard deviation.
The size of flight muscles relative to thoracic width during adult life. (A) Schematic illustration of the main inner muscles in the meso-and metathorax, viewed from the inside following removal of the left side of the thorax (d, dorsal; v, ventral). Muscle numbering follows Furukawa et al. (1983). (B) Muscle M90. (C) Muscle M99. (D) Muscle M112a. (E) Muscle M119. (F) Muscle M129a. Each open (female) or closed (male) circle was obtained from 10–11 animals. Each error bar on a graph indicates a standard deviation.
Metathoracic flight muscles M112a, M119 and M129a grew significantly during the first 3 days after the imaginal moult (P<0.05); soon after muscle mass reached its maximum value, it began to decline (Fig. 2). The degree of growth and degeneration of metathoracic flight muscles in the female was greater than in the male (P<0.05). On the first day after the imaginal moult, the female muscles were the same size as the male muscles, but by the third day the female muscles were much larger. On days 3–9 two muscles, M119 and M129a, in both sexes declined in mass, though M112a continued to decline in mass beyond day 9. All of these muscles were pale orange in colour just after the imaginal moult, but the colour became deeper during muscle growth in the first 3 days after the imaginal moult. After this, the colour gradually faded to white as the muscles became thinner. The amount of cytochrome c contained in these muscles increased until the third day after the imaginal moult, then gradually decreased, and a marked decrease in the number of mitochondria, which were fairly abundant in these muscles in young adults, was apparent in the muscles of older adults (S. Shiga, S. Kogawauchi, K. Yasuyama and T. Yamaguchi, unpublished data). In view of the close relationship between the colour, the amount of cytochrome c and the number of mitochondria in crustacean muscles (Hoyle, 1983), the decrease in the amount of cytochrome c and the number of mitochondria may both contribute to the colour change observed during adult cricket life.
All flight muscles showed strong birefringence just after the imaginal moult. The birefringence observed in two muscles, M119 and M129a, however, became weaker with the decrease in mass following the fourth day after the imaginal moult, though it never completely disappeared even in fairly old adults. M112a also gradually decreased in birefringence following the fourth day, but it eventually lost birefringence in a characteristic way: without exception, the muscle fibres in the ventral part of M112a lost birefringence earlier than those in the dorsal part (Fig. 3B).
Photomicrographs of muscle M112a and its motoneurones stained with NiCl2 (whole view of dorsal upper side; anterior left side). This nerve-muscle preparation was obtained from a 19-day-old female. The photomicrographs shown in A, C and D were taken using an ordinary light microscope; the areas (150μm×200μm) centred around arrows 1 and 2 in A are enlarged in C and D, respectively. (B) A photomicrograph of the same preparation as that in A taken using a polarized-light microscope. The contour of the muscle is outlined with dots for convenience of comparison with the other photomicrographs. In A and B, a polystyrene microcapillary tube was placed near the preparation as a length scale of 1 mm to highlight birefringence. Birefringence remains only in the dorsal part of the muscle; it has disappeared from the ventral part.
Photomicrographs of muscle M112a and its motoneurones stained with NiCl2 (whole view of dorsal upper side; anterior left side). This nerve-muscle preparation was obtained from a 19-day-old female. The photomicrographs shown in A, C and D were taken using an ordinary light microscope; the areas (150μm×200μm) centred around arrows 1 and 2 in A are enlarged in C and D, respectively. (B) A photomicrograph of the same preparation as that in A taken using a polarized-light microscope. The contour of the muscle is outlined with dots for convenience of comparison with the other photomicrographs. In A and B, a polystyrene microcapillary tube was placed near the preparation as a length scale of 1 mm to highlight birefringence. Birefringence remains only in the dorsal part of the muscle; it has disappeared from the ventral part.
The nerve bundle, which includes five motoneurones innervating M112a, was forward-or back-filled with Imoll−1 NiCl2 and intensified with AgNO3. The photomicrographs in Fig. 3 show the normal innervation pattern of motoneurones not only in the dorsal part of the muscle, but also in the ventral part, which had lost birefringence. As shown in Fig. 4A, the motoneurones in young adults do not differ significantly, in either dendritic arborization or size of the somata in the meso-and metathoracic ganglia, from those in older adults. These results are supported by data from electron microscopy of muscle 112a (Fig. 4B), which indicate that a loss in birefringence is correlated closely with a loss of myofibrils; the presynaptic areas on degenerating muscle look similar to those in normal muscle.
Drawings of nickel-backfilled motoneurones innervating a Ml 12a in females (A) and electron photomicrographs of flight muscle M112a of an 11-day-old male (B). (Ai,ii) Soma profiles and dendritic arborizations of the motoneurones in the meso-and metathoracic ganglia of a 3-day-old female; (Aiii.iv) those of a 13-day-old female. Each stippled area in Ai and Aiii shows a high density of branching in which the fine processes have been omitted. Scale bar, 100 μ m. (B) The arrowhead shows the neuromuscular junction made by an axon (ax) containing synaptic vesicles onto the muscle fibre (mf), which degenerated with the disappearance of the myofilaments and the considerable decrease of sarcoplasmic volume. Note the presence of dense body structure in the axon innervating the degenerated muscle fibre. Tracheoles (tr) are also visible in the basement membrane material (bm). Scale bar, 1 μ m.
Drawings of nickel-backfilled motoneurones innervating a Ml 12a in females (A) and electron photomicrographs of flight muscle M112a of an 11-day-old male (B). (Ai,ii) Soma profiles and dendritic arborizations of the motoneurones in the meso-and metathoracic ganglia of a 3-day-old female; (Aiii.iv) those of a 13-day-old female. Each stippled area in Ai and Aiii shows a high density of branching in which the fine processes have been omitted. Scale bar, 100 μ m. (B) The arrowhead shows the neuromuscular junction made by an axon (ax) containing synaptic vesicles onto the muscle fibre (mf), which degenerated with the disappearance of the myofilaments and the considerable decrease of sarcoplasmic volume. Note the presence of dense body structure in the axon innervating the degenerated muscle fibre. Tracheoles (tr) are also visible in the basement membrane material (bm). Scale bar, 1 μ m.
Fig. IB shows simultaneous recordings of the electrical activities of the motoneurones innervating muscle Ml 12a and the electromyograms of muscles M112a and M119 during tethered flight. It is evident that the spike discharges of motoneurones appear in a 15-day-old male as well as in a 3-day-old male, whereas M112a and M119 are less active in the older adult than in the younger adult. There is no doubt that this type of age-related change in electrical activity is due to the degeneration of flight muscles, regardless of motoneurone functioning.
It is quite possible that the selective degeneration of flight muscles, which occurs shortly after growth in young adults, causes the transition from normal to pseudoflight behaviour. In the cricket Gryllus bimaculatus, males are able to copulate 4 – 6 days after the imaginal moult (Tomioka and Chiba, 1982; Nagao and Shimozawa, 1987; T. Kimura and T. Yamaguchi, unpublished data). In females, the eggs and the retractor muscles of the ovipositor mature by 6 – 8 days after the imaginal moult, though all females attract males enough to elicit courtship stridulation even on the first day after the imaginal moult (T. Kimura and T. Yamaguchi, unpublished data). These observations suggest that declining flight ability, i.e. the occurrence of pseudo-flight behaviour, with advancing age is associated with sexual readiness. Further experiments to elucidate the factors that induce selective degeneration of flight muscles, especially of M112a, which characterizes the process of degeneration, are being carried out.
ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid from the Japanese Ministry of Education, Science and Culture to TY (no. 63480022).
