Materials and methods Directory UMM :Journals:Journal of Insect Physiology:Vol 47.Issue1.Jan2001:

84 S. Tanaka Journal of Insect Physiology 47 2001 83–94 cannot fly but reproduces earlier and in many species is more fecund than the LW morphs Harrison, 1980; Din- gle, 1985; Roff, 1986; Zera and Denno, 1997. The gen- eral consensus is that the absence of the large wings and functional flight musculature allows more energy to be devoted to reproduction and survival Dingle, 1985. Therefore, it is reasonable to assume that there is an energetic cost involved in the development and mainte- nance of the flight capability in the migratory morph. This idea is based on an assumption that resources are limited within the organism, and thus trade-offs occur between different life-history traits Calow 1978, 1979; Stearns, 1992. However, the presence of a correlation between wing morph and reproductive characteristics does not necessarily prove their causal relationship Tanaka, 1993; Tanaka and Suzuki, 1998. To address issues concerning flight capability in wing dimorphic insects, physiological studies are necessary. Mole and Zera 1993, 1994 have compared nutritional indices and flight muscle development profiles between LW and SW morphs of the crickets Gryllus rubens and G. firmus to identify potential physiological costs of flight. Their main findings are as follows. 1 There was no direct trade-off between flight muscle and ovarian growth. 2 There was no intermorph difference in food consumption in G. rubens, but LW morphs ate more food than SW morphs in G. firmus. 3 LW morphs con- verted a lower proportion of assimilated nutrients into biomass i.e. mainly the ovary than did SW morphs. 4 Ovarian growth was greater in SW morphs than in LW morphs in G. rubens, but no difference was found in G. firmus. 5 No intermorph difference was found in the number of eggs laid in either species, in contrast to pre- vious results obtained for these Roff, 1984; Zera and Rankin, 1989 and other species Tanaka 1976, 1993. These results, together with those reported by Zera et al. 1994, provided the basis for the trade-off theories discussed in more recent articles by Zera and his col- leagues Zera and Denno, 1997; Zera et al., 1998. It seems surprising that the closely related species of G. rubens and G. firmus show such physiological incon- sistency. In fact, some of the findings related to fecundity and flight muscle development contradict earlier ones with the same and other species, as pointed out by Crno- krak and Roff 1998, Tanaka and Suzuki 1998 and Tanaka et al. 1999. In crickets, it has been known that ovarian development and food consumption are influ- enced by various factors such as temperature, mating, and the presence or absence of oviposition substrate Loher and Edson, 1973; Clifford and Woodring, 1986; Loher et al., 1987; Renucci et al., 1990. One of the consistent results in the studies by Mole and Zera 1993, 1994 is the unusually low ovipositing activity in spite of the fact that all females were mated; in the first two weeks of adult life, G. firmus females produced only 83 eggs on average and G. rubens females only 20 eggs. As pointed out by Tanaka and Suzuki 1998, this is in marked contrast to .400 for G. firmus reported by Roff 1990 and .250 reported for G. rubens by Zera and Rankin 1989. In the present study, I conducted experi- ments on egg retention to mimic this low egg-laying in a cricket. In a more recent study, Zera et al. 1994 reported intermorph differences in lipid and carbo- hydrate contents in G. firmus, but all test females were not allowed to lay eggs during the entire period of the experiment. The possible impact of suppressed ovi- positing activity on various physiological traits as well as on trade-offs was pointed out by Tanaka and Suzuki 1998 and Tanaka et al. 1999, but no study has been designed to examine these specific problems. In the present study, I investigated the effects of sup- pression of oviposition activity on food consumption, ovarian development and body weight in female adults of a wing dimorphic cricket, Modicogryllus confirmatus. Because food consumption and ovarian weight are known to be influenced by the development and histoly- sis of the flight muscle in LW morphs of this species Tanaka, 1993; Tanaka et al., 1999, a group of LW morphs in which flight muscle histolysis was induced by artificial de-alation at adult emergence Tanaka, 1994a was included to determine the role of flight muscle his- tolysis in the control of food consumption under stressful conditions. In crickets, flight muscle histolysis is com- mon in LW morphs, and in several species LW females with histolyzed flight muscles develop their ovaries as rapidly as SW females Tanaka 1976, 1986; Tanaka, 1994a,b; Zera et al., 1997. The results of these obser- vations are reported in this paper. They strongly indicate that suppression of ovipositing activity and failure to recognize the occurrence of flight muscle histolysis could 1 partly explain the above-mentioned sporadic results reported by Zera and his colleagues Mole and Zera 1993, 1994; Zera and Mole, 1994; Zera et al., 1998, and 2 call into question the validity of the bio- chemical comparison between wing morphs in crickets deprived of oviposition substrate Zera et al., 1994. Hence some of the generalizations made by Zera and Mole 1994, Zera and Denno 1997 and Zera et al. 1998 may also need re-evaluation.

2. Materials and methods

The crickets used were reared at 30 ° C and 16 h light8 h dark photoperiod, as previously described Tanaka, 1993; Suzuki and Tanaka, 1996. They were fed dry pel- lets Insect Feed, Oriental Yeast Co., Tokyo, both as nymphs and adults. In the first experiment, I examined the effect of ovi- position substrate on food consumption and body weight in SW and LW females. Newly emerged female adults of each wing morph were weighed and divided into two 85 S. Tanaka Journal of Insect Physiology 47 2001 83–94 groups. They were individually held in small plastic con- tainers diameter 10 cm; height 5 cm each having a lid with a wire-mesh-covered hole diameter 3 cm. All individuals were handled in the same way during the first 3 days. In each container, 100 mg of food and a large water vial diameter 3 cm; length 6 cm plugged with cotton-wool were provided during the first 3 days. Care was taken to prevent the food from getting wet by using the method described previously Tanaka et al., 1999. Adults of this cricket do not lay eggs during the first 3 days Tanaka, 1993. On day 3, the food left was weighed and each cricket was given a chance to mate with a sexually mature male in another container where no food or water was provided. Most individuals mated within 10 min and all mated females with a spermato- phore were returned to the original container after a 1- h mating trial and given 200 mg of food. A few females did not mate and they were discarded. In each wing-morph group, one subgroup of females was constantly provided with a large water vial plugged with cotton-wool, and the other subgroup with a small water vial diameter 1 cm; length 5 cm plugged with cotton-wool. Crickets laid eggs into the cotton plug of the large vial, but failed to do so when given the small vials. However, a few crickets of the latter subgroup managed to lay eggs and some deposited eggs on the dry floor of the container. These individuals were excluded from the analysis. All females were weighed and given more food every 2 or 3 days until day 14. Some LW females shed the hindwings during the experi- mental period. Because such de-alated LW individuals are known to histolyze the flight muscle and to start rapid reproduction Tanaka, 1993, they were separated as de-alated LW DLW from the intact LW ILW adults. In this experiment, crickets were always given plenty of food and fed ad libitum. At the end of the experiment, the total amount of food consumed was cal- culated. In a second experiment to determine ovarian develop- ment in crickets deprived of oviposition substrate 2OS and those given access to it + OS, newly emerged male and female adults were kept together in a large container 20 × 35 × 26 cm where they were fed ad libitum. As in the above experiment, some adults were allowed to lay eggs freely and the others deprived of oviposition sub- strate. In addition to the SW and ILW groups, another group, DLW, in which the hindwings were artificially removed from ILW females at adult emergence was included to test the effect of flight muscle histolysis on body weight and ovarian development. Crickets were sampled randomly from each group on days 7, 10 and 14, individually put into an air-tight tube Eppendorf, volume 1.5 ml, and stored at 220 ° C. The frozen crick- ets were thawed within a week at room temperature, weighed, and their ovaries were dissected out to deter- mine the mass by using a Mettler AT 201 balance. In a third experiment, both male and female adults were reared as a group in a large container, and 4-day- old females were individually kept in small containers and given plenty of food and a large water vial with a cotton plug. On the next day, those females which had deposited eggs were individually transferred to new con- tainers each holding 100 mg of food and a small or a large water vial. In this case, crickets of the same wing morph were divided into two subgroups of equal mean body weight to exclude the effect of body size. In addition to the SW and ILW groups, another group, SW 2o, in which one ovary was removed at adult emerg- ence from SW females, was included to examine the effect of ovarian mass on food consumption and other developmental traits. For extirpation of ovaries, newly emerged females were kept on ice for about 20 min and the right ovary was pulled out of the body with a fine forceps through a small incision made between the fourth and fifth abdominal tergites. At the end of the 2- day confinement, the food left was weighed to determine the food consumption rate, and the eggs deposited into the cotton plugs were counted for the + OS subgroups. Among the ILW group, some individuals shed their hindwings during the experiment, and such individuals were separated as DLW morphs. All crickets were stored at 220 ° C as above, and dissected later to determine weights of the whole body, crop content and ovaries. The chorionated oocytes or eggs in the ovaries were also counted. The dry weight of the dorsolongitudinal flight muscle was determined for ILW and LDW individuals according to the method of Tanaka 1993. The data were analyzed by ANOVA followed by a Games–Howell test or a t-test Statview.

3. Results