Protein Synthesis and Degradation in the Flight Muscles of Adult Crickets (Gryllus Bimaculatus)

The ability to fly is one of the most remarkable characteristics of insects. Some species are ready to fly soon after adult emergence, while others require several days to complete the maturation of their flight muscles. Although flight may confer obvious selective advantages, a number of species lose their ability to fly either by shedding their wings or by histolysing the flight muscle after adult emergence (Johnson, 1969). In general, the loss of flight ability coincides with the initiation of reproductive activity, and examples supporting this relationship have been reported and discussed from the ecological point of view (Dingle, 1985). However, the physiological mechanisms controlling flight muscle histolysis and ovarian development in relation to migratory activity are poorly understood. The present study was conducted to provide the baseline data needed to approach this problem.

Adults of the cricket Gryllus bimaculatus increase the mass of their flight muscles during the first 3 days after final ecdysis and decrease it thereafter through selective degeneration of the metathoracic muscles (Shiga et al. 1991). Changes in the mass of flight muscles are likely to be caused by changes in the rates of synthesis and degradation of the proteins comprising the tissue. However, no information is available about how these rates change during the course of the development and histolysis of the flight muscles. In an aphid, Acyrthosiphon pisum, flight muscle histolysis is induced after a period of flight and feeding (Johnson, 1959), and it has recently been suggested that some factors produced in the flight muscles are involved in the programmed cell death of the muscle (Kobayashi and Ishikawa, 1994a); however, how these factors are implicated in the degradation of muscle proteins is unknown. One of the main purposes of the present study was to investigate the possible existence of up-regulated proteins involved in the process of flight muscle histolysis in G. bimaculatus.

Soon after we commenced our study, we noticed relatively large individual variations in the pattern of changes in flight muscle mass, which made it difficult to determine whether the flight muscle from a given cricket was in the process of developing or degenerating. Wing-shedding or de-alation is a behaviour commonly observed in various groups of insects. It is often closely correlated both with the initiation of flight muscle histolysis and with reproduction (Johnson, 1969). In crickets, artificial de-alation or removal of the hind wings at adult emergence induces flight muscle histolysis (Tanaka, 1976, 1986, 1991, 1993, 1994; Roff, 1989). For example, crickets with intact wings increase the dry mass of their flight muscle by 75 % during the first few days of adult life, while those artificially de-alated at adult emergence all reduce it to a low level over the same period (Tanaka, 1991). In the present study, we observed the developmental profiles of thoracic muscles in intact adults, examined the effect of artificial de-alation on flight muscle histolysis and determined the relative importance of protein synthesis and degradation in G. bimaculatus. Because it has often been suggested that flight and ovarian development are mutually exclusive in migratory insects (Johnson, 1969), the relationship between flight muscle development and ovarian development was also investigated.

The laboratory culture of Gryllus bimaculatus De Geer used in the present study was established from a stock culture at the Toyosato Museum of Entomology, Tsukuba, in 1990; it is maintained on insect pellets (Oriental Yeast Co., Tokyo) and exposed to a 16 h:8 h light:dark cycle at a temperature of 30 °C and at 25 % relative humidity. Crickets were reared in groups of 80–120 individuals in plastic containers (30 cmX48 cmX 30 cm), each holding water vials (90 ml) plugged with cotton as a water supply and as ovipositing sites. For observations, newly emerged crickets (<24 h after adult emergence) were transferred from these containers into small cylindrical plastic cups (8 cm diameter X 4.5 cm height), each containing food and a small water vial (5 ml) with a cotton plug on the bottom. Crickets failed to use the small vial as an oviposition site, so ovipositing activity was strongly inhibited in the present observations. Crickets were either kept intact or artificially dealated. Artificial de-alation was applied to 0-or 1-day-old individuals by gently pulling their hind wings with forceps. These wings came off easily and no appreciable amount of blood was shed.

A pair of the dorso-longitudinal muscles (DLMs), both sides of the dorso-ventral muscles (DVMs) in the mesothorax and one side of the DVM in the metathorax were dissected out from each cricket in ice-cold saline, 0.9 % NaCl, and rinsed with fresh saline several times to wash off the haemolymph. Samples were homogenized in 0.2 mol l⁻¹ sodium phosphate buffer (pH 7.2) with a powered homogenizer (S-203, Ikeda Rika) and the homogenates were centrifuged at 9000 g for 30 min at 4 °C. The protein content of the supernatant was measured using a protein assay kit (Bio-Rad Laboratories). To assess ovarian development, excised ovaries were weighed after being dried in an oven at 110 °C for 24 h.

SDS–PAGE (using gradient gels ranging from 5 % to 20 % polyacrylamide) was performed as described by Laemmli (1970). Gels were stained with 0.1 % Coomassie Brilliant Blue R250 in 40 % methanol, 10 % acetic acid and destained in 10 % methanol, 7.5 % acetic acid. Cytochrome c from horse heart (Wako Pure Chemical Industries) was run as a molecular mass marker. Fluorograms were processed according to Chamberlain (1979). Dried gels were exposed to Fuji X-ray film at −80 °C followed by development with a Fuji X-ray film developer.

DLMs were dissected out and cultured in sterile Ringer’s solution containing 5 μCi of [³H]methionine (3.08 TBq mmol⁻¹, Amersham) in a tube (1.5 ml) for 2 h at 30 °C. After incubation, the tubes containing the DLMs were immediately cooled on ice. The DLMs were then washed twice with ice-cold fresh saline and homogenized in lysis buffer [0.05 mol l⁻¹ Tris–HCl, pH 8.5, 0.25 mol l⁻¹ NaCl, 1 mmol l⁻¹ phenylmethylsulphonyl fluoride (PMSF), 0.1 % Triton X-100, 0.1 % sodium deoxycholate (DOC)]. The homogenate was centrifuged at 9000 g for 15 min at 4 °C. The supernatant was stored at -80 °C as DLM extract.

To measure the radioactivity incorporated into the DLMs, 10 μl of the DLM extract was put on a 24 mm disc of Whatman 3MM filter paper. After drying completely using a hairdryer, the disc was put in cold 10 % trichloroacetic acid (TCA) and washed three times with cold 5 % TCA, once with ethanol/ether (3:1, v/v) and once with ether. Dried discs were put into scintillant (Pharmacia CNS II) and the radioactivity was counted using an Aloka scintillation counter.

To compare the rates of protein synthesis in developing and degenerating muscles, DLMs taken from intact and de-alated crickets at days 0, 1 and 2 were exposed to pulse–chase labelling. DLMs were incubated in sterile Ringer’s solution containing 5 μCi of [³⁵S]methionine (47.1 TBq mmol⁻¹, Amersham) for 1 h at 30 °C. The muscles were then washed three times with fresh Ringer’s solution. One half of a pair of the DLMs was homogenized immediately after incubation, centrifuged at 9000 g for 30 min at 4 °C and kept at −80 °C until analysed by fluorography. The other half was also processed in the same way after a 3 h chase incubation in Ringer’s solution containing cold methionine at 30 °C.

Developmental profiles of the protein content of DLMs (Fig. 1), metathoracic DVMs (Fig. 2) and mesothoracic DVMs (Fig. 3) in crickets with intact wings were obtained. The protein content of DLMs showed relatively large temporal and individual variation in both sexes (Fig. 1). In general, it increased over the first 4 or 5 days, and then either remained at a fairly constant level or decreased. Metathoracic DVMs showed a similar profile (Fig. 2), but the initial increase in protein content appeared to occur over the first 3–4 days. No further increase was observed, and most individuals tended to show a decrease in protein content thereafter. Mesothoracic DVMs, however, showed no reduction in protein content comparable to that shown by DLMs even 14 days after emergence. The developmental profiles of metathoracic DVMs and DLMs were highly correlated (r=0.683 in males, P<0.01; r=0.682 in females, P<0.01 N=60), but the relationship was not perfect. A similar temporal pattern in protein content was also observed for mesothoracic DVMs in males, but not in females in which the protein content was kept relatively constant over the 14 days following adult emergence (Fig. 3). The maximal protein content of mesothoracic DVMs in females was about 25 % of that in males.

Ovarian development, as determined by changes in dry mass, became apparent 4 days after adult emergence and showed large individual variation thereafter (Fig. 4). The females with reduced DLMs (<0.5 mg) on day 7 onwards in Fig. 1 had large ovaries (>115 mg), indicating a negative relationship between the two traits.

De-alation at day 1 influenced flight muscle development. As shown in Fig. 5, the protein content of both DLMs and metathoracic DVMs declined significantly after de-alation (Kruskal–Wallis test; P<0.01), whereas it did not in intact crickets (P>0.05). In contrast, mesothoracic DVMs were not affected by de-alation (P>0.05), indicating that no histolysis had taken place.

Table 1 illustrates the effects of de-alation on the protein content of DLMs and on ovarian dry mass 5 days after emergence (4 days after de-alation). The mean protein content of DLMs was significantly reduced in de-alated crickets compared with intact ones. The opposite relationship was observed for mean ovarian dry mass, which was significantly greater in the de-alated group. A significant negative relationship was observed between protein content and ovarian dry mass (r=−0.406; P<0.01), although the variation was relatively large and some crickets with reduced DLMs possessed undeveloped ovaries and vice versa (data not shown).

In intact crickets, SDS–PAGE separation of DLM proteins showed rather similar protein profiles over the first 5 days following adult emergence (Fig. 6). When crickets were dealated immediately after adult emergence, the amounts of several proteins, including cytochrome c, began to decline by day 3 and they disappeared by day 5. However, there was no evidence for the appearance of any new protein prior to, or during, muscle histolysis.

The rate of protein synthesis by DLMs of intact females was measured in vitro after adult emergence (Fig. 7). A high rate of [³H]methionine incorporation was observed on the day of adult emergence, but the rate declined rapidly over the next 3 days. This result was consistent with the pattern of growth of this muscle as determined by total protein content (Figs 1 and 5).

Fig. 8 compares the rates of protein synthesis of developing and degenerating DLMs. A half-pair of developing DLMs taken from a day 0, intact cricket showed a high rate of incorporation of radioactivity after a 1 h incubation with [³⁵S]methionine. A considerably reduced, yet detectable, level of incorporated radioactivity was observed in the DLMs from day 1 or day 2 intact crickets. A similar pattern was also seen in the other half-pairs of DLMs, which were incubated with Ringer’s solution containing cold methionine for 3 h after the initial 1 h pulse incubation. These muscles showed a greater incorporation of radioactivity than those that experienced the pulse incubation alone, indicating that protein synthesis exceeded protein degradation during the 3 h chase incubation. In degenerating DLMs, low levels of incorporation of [³⁵S]methionine were detected on the day after de-alation (day 1), but incorporation was negligible on day 2. Because the radioactivity in the DLM did not increase after the chase incubation in the cold medium, it appeared that de-alation at adult emergence not only stopped protein synthesis but also triggered muscle breakdown.

The present observations indicate that the thoracic muscles of G. bimaculatus continue to develop after adult emergence. The protein content of the thoracic muscles increased during the first 2 days of adult life in both sexes, except in the case of the mesothoracic DVMs in females. This pattern of development is consistent with the previous observations of Shiga et al. (1991) in which the development of the muscles of this species was determined from the fresh mass. According to Shiga et al. (1991), the flight muscles begin to degenerate soon after reaching a maximal size in all individuals, and the degree of degeneration is greater in the DLMs than in the mesothoracic DVMs. This difference may be related to the fact that the DLMs are exclusively used for moving the wings but the DVMs are also used in walking. In the present study, however, while the DLMs in some individuals degenerated shortly after adult emergence (as in the study of Shiga et al. 1991), other individuals retained the muscles without undergoing degeneration for up to 2 weeks. This difference may be due to the differences in cricket strains or rearing conditions between the two studies.

Artificial de-alation effectively induced flight muscle degeneration in all individuals, as has been observed in other crickets (Tanaka, 1986, 1991, 1993, 1994; Roff, 1989). When female adults of G. bimaculatus were artificially de-alated at day 1, the protein content of the DLMs and metathoracic DVMs decreased to a low level within 4 days of de-alation; in contrast, intact individuals maintained high levels of protein in those muscles. Mesothoracic DVMs were not affected by dealation or ageing, supporting the finding of Shiga et al. (1991) that selective degeneration occurs in the flight muscles of this cricket. These results indicate that artificial de-alation can be used to study the process of flight muscle histolysis in this cricket, even though individuals of this species do not shed their wings naturally.

The mass of flight muscles is determined by a balance between protein synthesis and degradation. Using an in vitro culturing technique, we demonstrated that protein synthesis in DLMs was quite active at adult emergence, but its rate rapidly declined by the third day, when a minimal level was reached. This would explain the observation that the total protein content of DLMs increases during the first 4–5 days of adult life and levels off thereafter. The low rate of protein synthesis observed after the third day was probably the minimal level required to maintain the mass of this muscle.

We used a pulse-chase technique to investigate the relative importance of protein synthesis and degradation in histolysing muscles. Compared with developing DLMs removed from intact crickets, DLMs taken from de-alated crickets showed reduced rates of protein synthesis in vitro. Furthermore, the proteins synthesized in the medium containing [³⁵S]methionine during the first 1 h were not detectable after additional culturing in non-radioactive methionine containing medium for 3 h. This could suggest that, in the DLM, not only is the rate of protein synthesis reduced but also that protein degradation is initiated 1 day after de-alation, leading to a rapid decrease in the total protein content of the muscle.

Davis et al. (1989) found that haemolymph proteins produced after insemination trigger flight muscle histolysis in queens of the fire ant Solenopsis spp. Kobayashi and Ishikawa (1994a,b) found that some specific proteins, such as ubiquitin-like proteins, are produced in the flight muscle at the onset of muscle breakdown in an aphid (Acyrthosiphon pisum). In G. bimaculatus, our analysis of flight muscle proteins using SDS–electrophoresis provided no evidence to indicate any up-regulation of proteins that might be involved in flight muscle histolysis. This suggests that the physiological mechanism regulating flight muscle histolysis in this cricket does not require synthesis of any new protein in the muscle. Further investigation using other techniques, such as two-dimensional PAGE and antibody staining for ubiquitin, is necessary to confirm this possibility.

The role of juvenile hormone in ovarian development and flight muscle histolysis has been studied in several insects (Pener, 1985). In crickets, this hormone stimulates both ovarian development and flight muscle histolysis. Recently, this was confirmed experimentally in the cricket Modicogryllus confirmatus, in which isolated thorax-abdomens were treated with various doses of juvenile hormone III and its analogue (Tanaka, 1994). Although it has been demonstrated that juvenile hormone III stimulates ovarian development in G. bimaculatus (Koch and Hoffmann, 1985), its role in flight muscle development and histolysis has yet to be investigated.

Many insects reproduce after migratory flight, and it is therefore reasonable to find the initiation of flight muscle histolysis coinciding with that of ovarian development in these insects. In several species of crickets, artificial de-alation induces flight muscle histolysis and rapid ovarian development (Tanaka, 1976, 1986, 1991). Under conditions where food is limited, crickets with histolysing flight muscles produce more eggs than those with well-developed flight muscles, indicating that some nutrient derived from the muscle is used for egg development (Tanaka, 1993, 1994). In G. bimaculatus, dealated females had ovaries with a greater dry mass than those of intact females (Table 1). However, the relationship between ovarian mass and protein content of DLM was not as clear as in the other crickets mentioned above. This might be because the crickets in this study were given plentiful food, which may have obscured the influence of flight muscle histolysis on ovarian development. G. bimaculatus seems to provide an excellent system in which to study the biochemical mechanisms controlling flight muscle development and histolysis as well as ovarian development.

We would like to thank Mr Y. Uemura (Toyosato Museum of Entomology) for providing Gryllus bimaculatus. The suggestions of both anonymous referees are appreciated.