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Seasonal changes in the photoperiodic response regulating diapause in a tropical beetle, Stenotarsus rotundus
0022-1910/88$3.00+ 0.00 Copyright 0 1988Pergamon Press plc
J. Insecr Physiol.Vol. 34, No. 12, pp. 1135-I142, 1988 Printed in Great Britain. All rights reserved
SEASONAL CHANGES IN THE PHOTOPERIODIC RESPONSE REGULATING DIAPAUSE IN A TROPICAL BEETLE, STENOTARSUS ROTUNDUS SEIJI ‘I”ANAKAt, DAVID L. DENLINGER and HENK WOLDA* Department of Entomology, Ohio State University, Columbus, OH 43210, U.S.A. and ‘+Smithsonian Tropical Research Institute, Balboa, Republic of Panama (Received 20 June 1988) Abstract-Adulls of Stenotarsus rotundus aggregate at the base of a palm tree and remain in a photoperiodical:y regulated diapause for up to 10 months each year in a tropical lowland forest, at 9”N. To examine seasonal changes in the photoperiodic response, beetles collected from the field at different times of the year were exposed to different photoperiods, and their gonad and flight muscle development were examined. The physiological responses of the adults suggest that diapause may be divided into three phases. During the initial phase (June-September), some beetles developed gonads in response to long photoperiods (13 h light-l 1 h dark), but the number of such individuals decreased towards the end of this phase. At short photoperiods (12 h light-12 h dark), no development occurred. During the intermediate phase (October-December). beetles showed no rapid development at either photoperiod. However, diapause development before the winter solstice proceeded more rapidly at short photoperiods than at long photoperiods. During the final phase (January-April), gonad and flight muscle development occurred earlier and more rapidly at long photoperiods. Thus, the beetle’s response to photoperiodic changes throughout the season serves to coordinate the seasonal cycle of development in this tropical species. Kev Word Index: Stenofarus rotundus.Endomvchidae, imaginal diapause, photoperiod, flight muscles, gonad development INTRODUCTION Imaginal diapause of the fungus beetle, Stenotarsus rotundus (Endomychidae, Coleoptera), lasts up to IO months in the tropical lowland forest on Barro Colorado Island, Panama (Wolda and Denlinger, 1984; ‘I‘anaka et al., 1987b). This interval covers 6
months of the wet season (June-December) and the entire 4-month dry season (January-April) during which average ambient temperature is approx 25°C. Diapausing beetles cluster in a large aggregation (40,000-70,000 individuals) at the base of a palm tree and the metabolic rate of the aggregating beetles is extremely low (Tanaka et al., 1988). During their final 3 months in the aggregation site, beetles show a series of physiological changes leading to diapause termination (Tanaka et al., 1987a). Males initiate gonad development in late January. Fat consumption of both sexes increases in February, and flight-muscle regeneration takes place during March and April. Female gonad development begins in March, and all femabzs contain small oijcytes by the time the dry season ends in April. After the first heavy rains of the wet season, the beetles become active, mate, and disperse from the tree by flight. Although their reproduction site is unknown, new adults come back to the same tree 2 months later and form another aggregation (Wolda and Denlinger, 1984; Tanaka et al., 1987~). Photoperiod is the primary factor controlling tPresent address: The National Institute of Sericultural and Entomological Research, l-2 Ohwashi, Tsukuba-shi, Ibaraki 305, Japan
diapause termination in S. rotundus (Tanaka et al., 1987a). Although daylength on Barro Colorado Island (9”N) increases only by a half hour between January and April, beetles can perceive the small changes in daylength during the dry season and respond by terminating diapause. The rate of diapause termination is a graded response which is proportional to the absolute length of the photoperiod. As daylength increases during the dry season, the diapause termination process is accelerated, as observed in the lacewing, Chrysopu downesi (Tauber and Tauber, 1976a,b). This, together with a response to high humidity, enables the fungus beetle to prepare for an active reproductive period at the onset of the wet season. The above scenario for the control of diapause termination poses an important question about the induction and maintenance of diapause in S. rotundus: How can diapause be induced or maintained before September when the daylength is equal to or longer than the daylength in April? In the tropical locust, Nomuducris septemfusciutu, imaginal diapause occurs either at a long photoperiod or a short photoperiod (Norris, 1959, 1964, 1965). A longer diapause is induced at a short photoperiod if the immature stages have been exposed to a long photoperiod. The reverse conditions, i.e. a long photoperiod preceded by a short photoperiod, is known to prevent diapause, but the locusts do not usually encounter such conditions in nature during the critical stages for diapause determination. Thus, all individuals enter diapause in the field. Before reproduction, diapausing locusts experience increasing daylengths,but whether or not diapause ends in
1135
1136
SEIJI TANAKA et
response to such changes in natural daylength is not known. Several temperate species of carabid beetles require short days before they become ready to complete gonad development in long days (Thiele, 1966, 1975, 1977; Krehan, 1970; Ferenz, 1975, 1977). Because a sequence of short days and long days occurs only after the winter, reproduction can occur only in the spring. Likewise, the photoperiodic control of development in the temperate cricket, Pteronemobius nitidus (Tanaka, 1978, 1979, 1983) and the antlion, Hagenomia micans (Furunishi and Masaki, 1982, 1983) prevents larval maturation in the fall and delays adult emergence until the spring. To elucidate the environmental control of diapause in S. rotundus, we examined the photoperiodic response of this species at different times of the year and found that the beetles remained sensitive to photoperiod while at the aggregation site. However, their response to different photoperiods varies with the season and appears to be different from any of the examples cited above. The present paper describes the results of our observations and discusses the photoperiodic control of diapause in S. rotundus. MATERIALS
AND METHODS
al.
individual. In each individual of both sexes, the dorso-longitudinal flight muscles were removed and the thickness of the largest three fibres were measured using an ocular micrometer. Diapausing beetles have whitish, thin flight muscle fibres < 50 pm in thickness, while beetles capable of flight have pinkish, well developed flight muscle fibres 287.5 pm. In the present study, the proportion of individuals with well-developed flight muscles was determined for each experimental treatment. To examine the response of male gonads to different photoperiods, male beetles collected on November 7, 1985, were exposed to various durations and combinations of long (13 h light), intermediate (12: h light) and short (12 h light) photoperiods at 25°C. Beetles were then killed in Kahle’s solution, and testis-lobe length was determined as described above. For this test, the proportion of males that started testis development was also determined. In the field, testis lobes remained small (47 mmu) until early January, thus, males with a mean testis lobe length 28 mmu were regarded as having initiated testis development. RESULTS
Insects and stu& site
Responses to short days
Adults of S. rotundus were collected from a common palm tree, Oenocarpus panamanus, on Barro Colorado Island (9”9’19”N), Panama. This particular individual tree has been used by this insect as an aggregation site every year since 1980. While the amount of rainfall greatly varies both seasonally and annually, the average daily air temperature on the island is relatively constant at about 25°C in the forest floor near the study site (Wolda and Denlinger, 1984). Thus, we used 25°C for all experiments in the laboratory. The longest and shortest daylengths, including civil twilight in Panama (9”N), are about 13 and 11.8h. respectively (Beck, 1982). Therefore, we used 13 h light-l 1 h dark and 12 h light-12 h dark as long-day and short-day conditions, respectively.
When beetles started arriving at the palm tree in June, their gonads were undeveloped (Fig. lA, B and C). Transfer of beetles from the field to short days (12 h light) at any time between June and February did not induce oijcyte development. When transferred to short days on February 4, 46.7% of the females started developing oiicytes by May 5 (Fig. IA). In the field, all females had developing oiicytes by May 5 (n = 15). The difference in proportion between these groups was statistically significant (x2 = 10.9; P < 0.05) indicating that the formation of oiicytes was delayed in short days. Males initiated gonad maturation earlier than females, both in the field and laboratory (Fig. IC). Beetles kept in short days for 1 or 2 months before February 4 had smaller testis lobes than those collected on February 4. As in females, short days retarded gonad development. Flight muscles were well developed in all individuals on June 20 (Fig. ID). When beetles were transferred to short days. flight-muscle histolysis occurred in all individuals within 30 days. Some beetles were still observed arriving at the aggregation site in September, but very few beetles possessed well-developed flight muscles from October to February. When beetles were collected on February 4 and kept in short days (for 3 months) until May 5, 22% had well developed flight muscles while those collected from the field and examined on May 5 (n = 30) had all well developed flight muscles (x2 = 30.7; P < 0.05). This confirmed our previous observation (Tanaka et al., 1987a) that flight-muscle development is also delayed by short days.
Experimental procedures
To examine the seasonal changes in the response to photoperiod, beetles were collected from the tree on different dates of 1985 and 1986. At the time of each collection, 35 beetles were killed and stored in Kahle’s solution (Tanaka, 1986). The rest were divided into 2 groups and exposed to short days and long days for various lengths of time at 25 f I’C (r.h. about 100%). Beetles were kept in plastic containers (470 ml vol.) with folded filter paper and a water-vial plugged with absorbent cotton. Thirty beetles (15 males and I5 females) were removed from each photoperiod every month and killed in Kahle’s solution to examine the state of their gonads and flight muscles. Details of the scoring system for gonad and flight muscle development have been described (Tanaka et al., 1987a). In brief, three randomly selected ovarioles were removed from each female and the lengths of the primary oiicytes measured using an ocular micrometer [1 micrometer unit (mmu) = 12.5 pm]. In males, testis lobe length was determined for 3 randomly selected lobes from each
Responses to long days
In long-day conditions, some females developed oocytes even before October (Fig. IE). The proportion of such females increased when exposure to long
Photoperiodism in a tropical beetle
1137
experiments and in the field, gonad development in long days occurred earlier in males than in females. Flight muscles were well developed in all beetles collected in June. When such individuals were transferred to long days on June 20, flight muscles degenerated in 44% of the beetles within 30 days, but some beetles retained flight muscles as long as 90 tiays (Fig. 1H). A similar result was obtained from beetles collected in August. In this case, however, flight muscles had already degenerated before the beetles were collected. Thus, the well-developed flight muscles observed after 30 or 60 days of long-day exposure probably resulted from regeneration rather than retention of the muscles. The response decreased in September beetles and none of the beetles responded during October and November. In subsequent months, transfer to long days accelerated the onset of muscle development, and increased length of long-day exposure increased the incidence of flightmuscle development. Some degeneration was noted late in the season (beetles collected in February and dissected in May), presumably due to depletion of the energy reserves.
days was extended from 30 days to 60 days, but a 90-day exposure yeilded no further increase in this proportion (Fig. 1E) and, indeed, oiicytes again became smaller after (1 90-day exposure (Fig. 1F). Females collected in October and November did not initiate oacyte development within 90 days, even in long days, but some development eventually occurred if the females were subjected to even greater periods of long-day exposure [(a 120 days) Tanaka et al., 1987a]. From December onward, oijcyte formation and growth was observed in long days. The incidence of females with developing oiicytes increased with increased time of long-day exposure. Oacyte length reached a maximum of about 12mmu and then decreased, probably due to depletion of the fat body reserves. The response of male gonads was slightly different from that of female gonads. Testis lobes progressively increased in size with increased length of exposure to long days until reaching a maximal length of 22 mmu (Fig. 1G.). A 30-day exposure to long days was not effective in stimulating testis-lobe development before January. From September to November testis-lobe size was not influenced by a 60-day exposure to long days (P > O.OS), but the same treatment significantly increased testis lobe size in beetles collected in August (t = 3.52, P < 0.05) and December (t = 6.43; P < 0.05). As observed in the short-day
Comparison with natural daylengths
To determine whether the effects of different photoperiods are constant throughout diapause,
S D Females
Mean
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oocyte
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Fig. I(a)
SEIJITANAKA et
1138
al.
G=)
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0
50
(HI
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Fig. l(b) Fig. 1. Gonad and flight muscle development in cycles of 12h light-12 h dark (SD) or 13h light-l 1 h dark (LD) after transfer from the aggregation site on different dates in 1985 and 1986. A and E, proportion of females with primary oBcytes; B and F, mean primary oijcyte length; C and G, mean testis lobe length; D and H, proportion of beetles with well developed flight muscles. N = 15 males and 15 females. Beetles were dissected
on the day of collection
(O),
or 30 (Q), 60 (a),
beetles at different phases of diapause were exposed to long days or short days and the responses to these photoperiods were compared with those to natural daylengths. OBcyte development. Among beetles collected between October and April, oiicyte development occurred most rapidly in long days (Fig. 2). Exposure to long days from October to February induced oocyte development in 33% of the females (group 12). All females had developing odcytes when kept in long days through March (group 7), but oijcytes of some females started degenerating during the next 30 days at this photoperiod (group 1). Among beetles exposed to short days or natural daylengths from October to March, little or no oocyte development was observed (groups 7 and 11). However, an earlier transfer to long days did not always elicit a higher response: when transferred to long days on December 6, the proportion of females that developed oiicytes by March 6 was 42.9% (group 8) while it was 66.7 and 73.3% in the beetles transferred to long days on January 6 and February 4,
or 90 (0)
days after collection.
respectively (groups 9 and 10). Differences among these 3 groups were not statistically significant (P > 0.05). The results indicate that exposure to long days between December and February was no more effective in shortening diapause than the naturally prevailing short daylength. The same conclusion was also reached when mean oijcyte length was compared among these groups (data not shown). In contrast, long days experienced before or after this period (December &February 4) were quite effective in shortening the diapause period. All females exposed to long days beginning October 6 developed oiicytes by March 6 (group 7), while less than half the females did so when their transfer to long days was delayed until December 6 (group 8) (x2 = 11.61; P < 0.05). Transfer to long days on February (group 10) or March 6 (group 5) induced rapid development of oiicytes as compared with the result obtained for beetles continuously exposed to natural daylengths (groups 11 and 6), suggesting that the final stage of diapause development was accelerated by long days.
Photoperiodism GROUP
16 17 1%
Date ONDJFMA ?,?54$1?0
Testis lobe length
?P with oocytes 50
in a tropical beetle
1139
Beetles with well develmed flight muscles
100
m Ca H I ‘II 6 654650 ONDJ FMA
50 (“1.)
1oc
-
10
20
’ o-
‘v
100 0 w
(1=12.5~m)
100 W.1
Fig. 2. Gonad and flight-muscle development in cycles of 12 h light-12 h dark (e), 13 h light-l 1 h dark (O), or natural daylength (a). Photoperiodic regimes are given on the left: 0, natural daylength; q, 12 h light-12 h dark or 13 h light-l 1 h dark. N = 15 males and 15 females. Mean and standard deviation are given for testis lobe lengths. Testis development
As with female gonads, rapid development of male gonads occurred in long days (Fig. 2). Some beetles transferred to long days in October started testis development by January (group 16) while testes of those transferred to long days in December (group 17) or continuously ‘exposed to natural daylengths (group 18) remained undeveloped. Among beetles examined in February (groups 12-1 S), the earlier the time of transfer to long days the larger the mean testis-lobe size. By March 6, most males were near maturity (groups 7-‘11). During the next 30 days, testis-lobe size increased slightly in the males transferred in March (group 5), while in beetles transferred earlier, lobe size remained unchanged (groups 1, 3 and 4) or decreased ((group 2). In short days, beetles did not increase testis-lobe size by February (groups 12, 13 and 14 in Fig. 2) while some of those collected from the field in February had apparently initiated testis development (group 15). Among beetles collected in December or later and kept in short days until March or April, a positive correlatio:n was obtained between mean testis-lobe length and the time of transfer to short days (r = 0.98 and 0.93 for beetles examined in March and April, respectively; P < 0.05). However, beetles transferred to short days in October developed .testis lobes more rapidly than those transferred 2 months later (t = 4.40 and 3.05 for group 1 vs 2 and group 7 vs 8, respectively; P < 0.05). Natural daylengths in October and November are nearly equivalent to those in January and February, thus the results may indicate that the photoperiods promoting rapid testis development
after December are not as effective as a shorter photoperiod before December. To test this, groups of 15 males collected from the field on November 7, 1985, were exposed to various combinations of long, intermediate, and short photoperiods at 25°C. Testis lobes remained small (< 8 mmu) at any constant photoperiod by December 22 (groups 1, 2 and 3 in Fig. 3), but they started developing by January 31 (groups 4, 5 and 6). In this case, the proportion of males that initiated testis development increased with photoperiod, and the differences between the three groups were statistically significant (X*-test; P < 0.05). However, when exposed to different photoperiods during the first 45 days and then transferred to an intermediate photoperiod more beetles started testis development when the initial photoperiod was shorter (x2 = 4.46 and 13.39 for groups 7 vs 8 and groups 7 vs 9, respectively; P < 0.05). A similar tendency was also observed among the beetles maintained until February 10 (groups 10, 11 and 12), although no statistically significant differences were observed either in testis lobe length or in the proportion of males with developing testes. These results further confirm the above results, and suggest that a long photoperiod, which is favourable for testis development after December 22, is less effective than shorter photoperiods in accelerating diapause development before December 22. Flight muscles
The proportion of beetles with well-developed flight muscles was generally lower in males than in females. However, the pattern of response to different
SEIJI TANAKA el al.
1140
MALES WITH
DATE
GROUP NCN 7 krn-%s?
DEC
DEVELOPING
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TESTIS LOBE LENGTH
JAN FEB
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Fig. 3. Effects of various lengths and combinations of short (S) (12 h light-12 h dark), intermediate (I) (124 h light-l lf h dark), and long (L) 13 h light-l 1 h dark photoperiods on testis development of males collected on November 7, 1985. I mmu = 12.5 pm. Beetles that had testis lobes 2 8 mmu in length were regarded as those with developing testes. Where an analysis of variance showed a significant difference
among groups (S, significant; NS, non-significant at 5%), a Duncan multiple range test was performed; numbers followed by different letter significant at 5%. N = 15, except for groups l-3 (n = 10). photoregimes was quite similar in both sexes (Fig. 2). The pattern was also similar to the pattern of response observed in oijcyte development. Flight muscles tended to degenerate relatively quickly once they attained full development. Some groups of beetles maintained in long days until April 5 (groups IL4 in Fig. 2) thus included a mixture of beetles with well-developed flight muscles and those with degenerating flight muscles. DISCUSSION
Based on changes in the physiological condition of S. rotundus and the dynamics of its response to daylength, diapause in this species may be divided into 3 phases: the initial, intermediate and final phases as shown in Fig. 4. In the initial phase, beetles arrive at the aggregation site, flight muscles degenerate, and the metabolic rate decreases from 30@400pl/g/h to less than 5Opl/g/h (at 25°C) (Tanaka et al., 1988). During the intermediate phase, beetles remain inactive in the aggregation and their metabolic rate remains very low (Wolda and Denlinger, 1984). During the final phase they occasionally move up and down the tree, apparently in response to moisture during the dry season, but otherwise they remain inactive in aggregation. At the end of this phase, beetles mate and disperse from the tree. Beetles in the initial and final phases respond much more rapidly to a long photoperiod than during the intermediate phase. However, the response to the same long photoperiod appears to be different in the initial and final phases. Beetles during the final phase terminate diapause more quickly at long photoperiod
when a transfer from the field is delayed. But, during the initial phase, the response to a long photoperiod is greatest at the beginning of the phase and progressively declines. In females, longer exposure to a long photoperiod during the initial phase does not always elicit a higher response, as it does during the final phase (Fig. 1E and F). Finally, some beetles that have just arrived at the tree undergo flight-muscle histolysis at the same long photoperiod that always stimulates flight-muscle development during the final phase. During the initial phase beetles may use a threshold response to photoperiod rather than the graded response observed during the final phase. If this is the case, the threshold for induction or maintenance of diapause could be very close to 13 h light-l 1 h dark in June and may increase as the season advances. This could explain the gradually decreasing proportions of individuals that avert or terminate diapause when a transfer from the field to 13 h light-l 1 h dark is delayed. Beetles come to the aggregation site over a few months, but our preliminary observations suggest no significant differences in the response to photoperiod between early and late arrivers (Tanaka et al., unpubl. observ.). The fact that none of the beetles initiated gonad development in June and July may suggest that the daylength actually perceived by them during this season is slightly shorter than 13 h light11 h dark. Our unpublished observations show that there is no significant difference in effect between 13 h light and 13.5 h light in beetles collected in August, thus supporting the idea that beetles show a threshold response during the initial phase. An alternative explanation is that the daylengths actually read by the beetles in June are nearly equal
Photoperiodism
1141
in a tropical beetle TEMPERATURE
2 5°C 13 H DAYLENGTH 12
PHASE
!TT
INITIAL
INTERMEDIATE
FINAL
MOhTH SEASON
Fig. 4. Seasonal changes in the responsiveness to long days of Stenotarsus rotundus. During the initial phase, some beetles develop gonads and flight muscles at 13 h light-l 1 h dark, but the numbers decrease towards the end of this phase. Beetles during the intermediate phase show no rapid development of gonads or flight muscles. During the final phase, beetles gradually recover their responsiveness to long days and show a graded response to photoperiod. Temperature and daylength data on Barro Colorado Island are based on Tanaka et al. (1987a).
to 13 h light-l 1 h da:rk and it is a decrease in daylength that induces diapause in this species. Several examples in which diapause occurs only under a decreasing photoperiod include the lygaeid bug, Neocoryphus bicrucis (Solbrek, 1979) the wood louse, Armadillidium vulgare (Mocquard et al., 1980) and the phytoseiid mite, Amblyseius longispinosus (Hanamura, 1982). A systematically designed experiment to test this hypothesis in S. rotundus is yet to be done. During the intemlediate phase, beetles do not terminate diapause rapidly either at a short or long photoperiod. Among those collected in October, the first sign of the initi#ation of gonad development is seen only after 3 months for males (Fig. 1G) and 4 months for females at 13 h light-l 1 h dark (Tanaka et al., 1987a). At l;! h light-12 h dark, the beetles require another 2-3 months to start gonad development (Tanaka et al., 1987a). From these results, we conclude that diapause during the intermediate phase is controlled by a graded response, as it is during the final phase. However, because of its high intensity, diapause in this species would probably persist throughout this phase under any photoperiodic conditions. Imaginal diapause of the temperate lacewing, Chrysopa carnea, is thought to be maintained by a graded response to photoperiod (Tauber and Tauber, 1973, 1976~; Tauber et al., 1986). In this case, however, all Chrysopa adults collected from the field were tested at a relatively high temperature (24°C). Thus, how much the graded response to photoperiod actually contributes to the maintenance of diapause relative to the role of low temperature prevailing in the fall and wimer (through February) remains unknown. By examining the responses of various organs to combinations of different photoperiods, we have found that control of diapause in S. rotundus involves a rather complicated response to seasonally changing daylengths: the pattern of response changes as diapause progresses. For example, testis development during the final phase proceeds more rapidly at a long photoperiod than at a short photoperiod. However, when beetles collected in November were exposed to
different photoperiods for 45 days before they were transferred to 124 h light-l :f h dark, more gonad development occurred when the initial photoperiod was shorter (Fig. 3.). Thus, the rather deep diapause of S. rotundus decreases in intensity more rapidly as the daylength shortens until late December and the beetles become ready for diapause termination in response to increasing daylengths during the final phase. Shifts in optimal daylength for diapause development, as observed in S. rotundus, may be compared with temperature responses of some temperate insects. For example, the optima1 temperature for diapause development in eggs of the chrysomelid beetle, Atrachya menestriesi, is 7.5”C during the early stage but decreases to 0°C after they experience 7.5”C for 10 days (Ando, 1978). In eggs of the cricket, Allonemobius fasciatus, the optimal temperature is relatively high during the early stage, decreases to 6°C and increases again towards the end of the diapause period (Tanaka, 1987). With this type of response, diapause can persist until a cold period comes. Once these insects experience cold, the intensity of diapause quickly diminishes, preparing them for postdiapause development in the spring. During the final phase, S. rotundus shows a graded response to photoperiod for diapause termination (Tanaka et al., 1987a). In this sense, this beetle is similar
to
the
temperature
lacewing,
C. downesi
(Tauber and Tauber, 1976~). However, unlike the lacewing in which adults require short days before they become responsive to increasing daylengths, neither a period of short days nor an increase in photoperiod is an absolute requirement for S. rotundus.
Gonad development in S. rotundus apparently begins earlier in males than in females. However, the flight muscles regenerate at the same time which, in females, coincides with the time of oiicyte development (Tanaka et al., 1987a). Our previous studies suggest that both flight muscle and oijcyte development are stimulated by juvenile hormone (Wolda and Denlinger, 1984) while testis development is regulated
SEIJI TANAKA et al.
1142
by other factor(s) (Tanaka et al., 1987b). The corpora allata, which increase in size during flight muscle development, remain small when testis development is in progress. A juvenile hormone analogue (methoprene) and 20-hydroxyecdysone have no influence on testis development, although the former stimulates development of flight muscles in both sexes and oocytes in females. We have not yet identified what factor(s) stimulates testis development. However, it appears that the developmental stimulus for testis development always precedes the release of juvenile hormone during the diapause termination process. We have never encountered a case in which males, during the final phase, had well developed flight muscles without developing their testes, but this combination of developmental traits was readily observed during the initial phase. The changes in responsiveness to daylength that we observed suggest the following phenological sequence: the beetles are reproductively active during the relatively long days between mid-April and July. Migration from their reproductive habitat to the aggregation site begins shortly before the summer solstice (Wolda and Denlinger, 1984; Tanaka et al., 1987~) and under the maximal daylength conditions young beetles initiate a variety of physiological changes leading to the onset of diapause. If the beetles are exposed experimentally to long photoperiod (13 h light-l 1 h dark) at that time, they can respond by retaining or even regenerating their flight muscles, and by maturation of the gonads. The beetles become progressively more refractory to stimulation by long daylengths, and they regain their responsiveness to long daylengths after experiencing the short days around the winter solstice. As daylength increases in January and February the process of diapause termination accelerates. The beetles are thus primed to reproduce, and the onset of the rains, usually in mid April, triggers mating and dispersal from the aggregation site. Acknowledgemenl-This from the Smithsonian
study Institute
was supported by a grant Scholarly Studies program.
REFERENCES Ando Y. (1978) Studies on egg diapause in the false melon beetle, Atrachya menetriesi Faldermann (Coleoptera: Chrysomelidae). Bull. Fur. Agric. Hirosaki Univ. 30, 131-21.5. In Japanese. Beck S. D. (1982) insect Photoperiodism. 2nd edn. Academic Press, New York. Ferenz H. J. (1975) Photoperiodic and hormonal control of reproduction in male beetles, Pterostichus nigrita. J. Insect Physiol. 21, 33 l-341. Ferenz H. J. (1977) Two-step photoperiodic and hormonal control of reproduction in the female beetle, Pterostichus nigrita. J. Insect Physiol. 23, 671676. Furunishi S. and Masaki S. (1982) Seasonal life cvcle in two species of ant-lion (Neuroptera: Myrmeleontidae). Japan. J. Ecol. 32, 7- 13. Furunishi S. and Masaki S. (1983) Photoperiodic control of development in the ant-lion, Hagenomyia micans (Neuroptera: Myrmeleontidae). Ent. Gen. 9, 51-62. Hanamura T. (1982) The diapause of the preceous mite, Ambi.vseius longispinosus Acarina, Phytoseiidae). Bull. Fruif Tree Res. Stn, Ser. E. 1982, 77-90. In Japanese. Krehan J. (1970) Die Steuerung von Jahresrhythmik und Diapause bei Larval-und Imagoiibenvinterern der Gattung Pterostochus (Col., Carab.). Oecologia 6,58-105.
Mocqard J. P., Pavese A. and Juchault P. (1980) Determinisme de la reproduction saisonnitre des femelles d’ Armadiliidium vulgare Latr. (Crustace. Isopode, Onisco’ide). 1. Action de la temperature et de la photoperiode. Ann. Sci. natn. Zool. 14, 91-97. Norris M. J. (1959) Influence of daylength on imaginal diapause in the red locus, Nomadacris septemfasciata (Serv.). Entomologia exp. appl. 2, 154168. Norris M. J. (1964) Environmental control of sexual maturation in insects. Symp. R. ent. Sot.. Land. 2, 56-65. Norris M. J. (1965) The influence of constant and changing photoperiods on imaginal diapause on imaginal diapause in the red locusts (Nomadacris septemfasciata Serv.). J. Insect Physiol. 11, 1105-1119. Solbreck C. (1979) Induction of diapause in a migratory seed bug, Neacoryphus bicrucis (Say) (Heteroptera, Laygaeidae). Oecologia 43, 4149. Tanaka S. (1978) Effects of changing photoperiod on nymphal development in Pteronemobius nitidus (Orthoptkra: Gryllidak). Konryu 46, 135-15 1. Tanaka S. (1979) Multinle . .Dhotoneriodic control of the seasonal life cycle in Pteronemob’ius nitidus (Orthoptera: Gryllidae). Kontyu 47, 465475. Tanaka S. (1983) Seasonal control of nymphal diapause in the spring ground cricket, Pteronemobius nitidus. In Diapause and Life Cycle Strategies in Insects (Edited by Brown V. K. and Hodek I.), pp. 35- 53. Dr W. Junk, The Hague. Tanaka S. (1986) Sexual dimorphism in Stenofarsus rotundus. Coleop. Bull. 40, 4547. Tanaka S. (1987) Changing responses to temperature and moisture of diapausing and developing eggs of Allonemobius fasciatus (Orthoptera: Gryllidae). J. Insect Physiol. 33, 6355641. Tanaka S., Denlinger D. L. and Wolda H. (1987a) Daylength and humidity as environmental cues for diapause termination in a tropical beetle. Physiol. Ent. 12,2133224. Tanaka S., Wolda H. and Denlinger D. L. (1987b) Seasonality and its physiological regulation in three neotropical insect taxa from Barro Colorado Island, Panama. Insect Sci. Appl. 8, 507-514. Tanaka S., Wolda H. and Denlinger D. L. (1987~) Abstinence from mating by sexually mature males of the fungus beetle, Stenotarsus rotundus, during a tropical dry season. Biotropica 19, 252-254. Tanaka S., Wolda H. and Denlinger D. L. (1988) Group size affects the metabolic rate of a tropical beetle. Physiol. Ent. 13, 239-241. Tauber M. J. and Tauber C. A. (1973) Quantitative response to daylength during diapause in insects. Nature 244, 296297. Tauber M. J. and Tauber C. A. (1976a) Developmental requirements of the univoltine species, Chrysopa downesi: Photoperiodic stimuli and sensitive stages. J. Insect Physiol. 22, 331-335. Tauber M. J. and Tauber C. A. (1976b) Environmental control of univoltinism and its evolution in an insect species. Can. J. Zool. 54, 26&266. Tauber M. J. and Tauber C. A. (1976~) Insect seasonality: Diapause maintenance, termination, and postdiapause development. A. Rev. Ent. 21, 81-107. Tauber M. J., Tauber C. A. and Masaki S. (1986) Seasonal Adaptations of Insects. Oxford University Press. Thiele H. U. (1966) Einfliisse der Photoperiode auf die Diapause von Carabiden. Z. Ang. Ent. 58, 143-149. Thiele H. U. (1973) Remarks about Mansingh’s and Muller’s classifications of dormancies in insects. Can. Ent. 105, 925- 928. Thiele H. U. (1977) Measurement of daylength as a basis for photoperiodism and annual periodicity in the carabid beetle, Pterostichus nigrita F. Oecologia 30, 33 l-348. Wolda H. and Denlinger D. L. (1984) Diapause in a large aggregation of a tropical beetle. Ecol. Em. 9, 217-230.
J. Insecr Physiol.Vol. 34, No. 12, pp. 1135-I142, 1988 Printed in Great Britain. All rights reserved
SEASONAL CHANGES IN THE PHOTOPERIODIC RESPONSE REGULATING DIAPAUSE IN A TROPICAL BEETLE, STENOTARSUS ROTUNDUS SEIJI ‘I”ANAKAt, DAVID L. DENLINGER and HENK WOLDA* Department of Entomology, Ohio State University, Columbus, OH 43210, U.S.A. and ‘+Smithsonian Tropical Research Institute, Balboa, Republic of Panama (Received 20 June 1988) Abstract-Adulls of Stenotarsus rotundus aggregate at the base of a palm tree and remain in a photoperiodical:y regulated diapause for up to 10 months each year in a tropical lowland forest, at 9”N. To examine seasonal changes in the photoperiodic response, beetles collected from the field at different times of the year were exposed to different photoperiods, and their gonad and flight muscle development were examined. The physiological responses of the adults suggest that diapause may be divided into three phases. During the initial phase (June-September), some beetles developed gonads in response to long photoperiods (13 h light-l 1 h dark), but the number of such individuals decreased towards the end of this phase. At short photoperiods (12 h light-12 h dark), no development occurred. During the intermediate phase (October-December). beetles showed no rapid development at either photoperiod. However, diapause development before the winter solstice proceeded more rapidly at short photoperiods than at long photoperiods. During the final phase (January-April), gonad and flight muscle development occurred earlier and more rapidly at long photoperiods. Thus, the beetle’s response to photoperiodic changes throughout the season serves to coordinate the seasonal cycle of development in this tropical species. Kev Word Index: Stenofarus rotundus.Endomvchidae, imaginal diapause, photoperiod, flight muscles, gonad development INTRODUCTION Imaginal diapause of the fungus beetle, Stenotarsus rotundus (Endomychidae, Coleoptera), lasts up to IO months in the tropical lowland forest on Barro Colorado Island, Panama (Wolda and Denlinger, 1984; ‘I‘anaka et al., 1987b). This interval covers 6
months of the wet season (June-December) and the entire 4-month dry season (January-April) during which average ambient temperature is approx 25°C. Diapausing beetles cluster in a large aggregation (40,000-70,000 individuals) at the base of a palm tree and the metabolic rate of the aggregating beetles is extremely low (Tanaka et al., 1988). During their final 3 months in the aggregation site, beetles show a series of physiological changes leading to diapause termination (Tanaka et al., 1987a). Males initiate gonad development in late January. Fat consumption of both sexes increases in February, and flight-muscle regeneration takes place during March and April. Female gonad development begins in March, and all femabzs contain small oijcytes by the time the dry season ends in April. After the first heavy rains of the wet season, the beetles become active, mate, and disperse from the tree by flight. Although their reproduction site is unknown, new adults come back to the same tree 2 months later and form another aggregation (Wolda and Denlinger, 1984; Tanaka et al., 1987~). Photoperiod is the primary factor controlling tPresent address: The National Institute of Sericultural and Entomological Research, l-2 Ohwashi, Tsukuba-shi, Ibaraki 305, Japan
diapause termination in S. rotundus (Tanaka et al., 1987a). Although daylength on Barro Colorado Island (9”N) increases only by a half hour between January and April, beetles can perceive the small changes in daylength during the dry season and respond by terminating diapause. The rate of diapause termination is a graded response which is proportional to the absolute length of the photoperiod. As daylength increases during the dry season, the diapause termination process is accelerated, as observed in the lacewing, Chrysopu downesi (Tauber and Tauber, 1976a,b). This, together with a response to high humidity, enables the fungus beetle to prepare for an active reproductive period at the onset of the wet season. The above scenario for the control of diapause termination poses an important question about the induction and maintenance of diapause in S. rotundus: How can diapause be induced or maintained before September when the daylength is equal to or longer than the daylength in April? In the tropical locust, Nomuducris septemfusciutu, imaginal diapause occurs either at a long photoperiod or a short photoperiod (Norris, 1959, 1964, 1965). A longer diapause is induced at a short photoperiod if the immature stages have been exposed to a long photoperiod. The reverse conditions, i.e. a long photoperiod preceded by a short photoperiod, is known to prevent diapause, but the locusts do not usually encounter such conditions in nature during the critical stages for diapause determination. Thus, all individuals enter diapause in the field. Before reproduction, diapausing locusts experience increasing daylengths,but whether or not diapause ends in
1135
1136
SEIJI TANAKA et
response to such changes in natural daylength is not known. Several temperate species of carabid beetles require short days before they become ready to complete gonad development in long days (Thiele, 1966, 1975, 1977; Krehan, 1970; Ferenz, 1975, 1977). Because a sequence of short days and long days occurs only after the winter, reproduction can occur only in the spring. Likewise, the photoperiodic control of development in the temperate cricket, Pteronemobius nitidus (Tanaka, 1978, 1979, 1983) and the antlion, Hagenomia micans (Furunishi and Masaki, 1982, 1983) prevents larval maturation in the fall and delays adult emergence until the spring. To elucidate the environmental control of diapause in S. rotundus, we examined the photoperiodic response of this species at different times of the year and found that the beetles remained sensitive to photoperiod while at the aggregation site. However, their response to different photoperiods varies with the season and appears to be different from any of the examples cited above. The present paper describes the results of our observations and discusses the photoperiodic control of diapause in S. rotundus. MATERIALS
AND METHODS
al.
individual. In each individual of both sexes, the dorso-longitudinal flight muscles were removed and the thickness of the largest three fibres were measured using an ocular micrometer. Diapausing beetles have whitish, thin flight muscle fibres < 50 pm in thickness, while beetles capable of flight have pinkish, well developed flight muscle fibres 287.5 pm. In the present study, the proportion of individuals with well-developed flight muscles was determined for each experimental treatment. To examine the response of male gonads to different photoperiods, male beetles collected on November 7, 1985, were exposed to various durations and combinations of long (13 h light), intermediate (12: h light) and short (12 h light) photoperiods at 25°C. Beetles were then killed in Kahle’s solution, and testis-lobe length was determined as described above. For this test, the proportion of males that started testis development was also determined. In the field, testis lobes remained small (47 mmu) until early January, thus, males with a mean testis lobe length 28 mmu were regarded as having initiated testis development. RESULTS
Insects and stu& site
Responses to short days
Adults of S. rotundus were collected from a common palm tree, Oenocarpus panamanus, on Barro Colorado Island (9”9’19”N), Panama. This particular individual tree has been used by this insect as an aggregation site every year since 1980. While the amount of rainfall greatly varies both seasonally and annually, the average daily air temperature on the island is relatively constant at about 25°C in the forest floor near the study site (Wolda and Denlinger, 1984). Thus, we used 25°C for all experiments in the laboratory. The longest and shortest daylengths, including civil twilight in Panama (9”N), are about 13 and 11.8h. respectively (Beck, 1982). Therefore, we used 13 h light-l 1 h dark and 12 h light-12 h dark as long-day and short-day conditions, respectively.
When beetles started arriving at the palm tree in June, their gonads were undeveloped (Fig. lA, B and C). Transfer of beetles from the field to short days (12 h light) at any time between June and February did not induce oijcyte development. When transferred to short days on February 4, 46.7% of the females started developing oiicytes by May 5 (Fig. IA). In the field, all females had developing oiicytes by May 5 (n = 15). The difference in proportion between these groups was statistically significant (x2 = 10.9; P < 0.05) indicating that the formation of oiicytes was delayed in short days. Males initiated gonad maturation earlier than females, both in the field and laboratory (Fig. IC). Beetles kept in short days for 1 or 2 months before February 4 had smaller testis lobes than those collected on February 4. As in females, short days retarded gonad development. Flight muscles were well developed in all individuals on June 20 (Fig. ID). When beetles were transferred to short days. flight-muscle histolysis occurred in all individuals within 30 days. Some beetles were still observed arriving at the aggregation site in September, but very few beetles possessed well-developed flight muscles from October to February. When beetles were collected on February 4 and kept in short days (for 3 months) until May 5, 22% had well developed flight muscles while those collected from the field and examined on May 5 (n = 30) had all well developed flight muscles (x2 = 30.7; P < 0.05). This confirmed our previous observation (Tanaka et al., 1987a) that flight-muscle development is also delayed by short days.
Experimental procedures
To examine the seasonal changes in the response to photoperiod, beetles were collected from the tree on different dates of 1985 and 1986. At the time of each collection, 35 beetles were killed and stored in Kahle’s solution (Tanaka, 1986). The rest were divided into 2 groups and exposed to short days and long days for various lengths of time at 25 f I’C (r.h. about 100%). Beetles were kept in plastic containers (470 ml vol.) with folded filter paper and a water-vial plugged with absorbent cotton. Thirty beetles (15 males and I5 females) were removed from each photoperiod every month and killed in Kahle’s solution to examine the state of their gonads and flight muscles. Details of the scoring system for gonad and flight muscle development have been described (Tanaka et al., 1987a). In brief, three randomly selected ovarioles were removed from each female and the lengths of the primary oiicytes measured using an ocular micrometer [1 micrometer unit (mmu) = 12.5 pm]. In males, testis lobe length was determined for 3 randomly selected lobes from each
Responses to long days
In long-day conditions, some females developed oocytes even before October (Fig. IE). The proportion of such females increased when exposure to long
Photoperiodism in a tropical beetle
1137
experiments and in the field, gonad development in long days occurred earlier in males than in females. Flight muscles were well developed in all beetles collected in June. When such individuals were transferred to long days on June 20, flight muscles degenerated in 44% of the beetles within 30 days, but some beetles retained flight muscles as long as 90 tiays (Fig. 1H). A similar result was obtained from beetles collected in August. In this case, however, flight muscles had already degenerated before the beetles were collected. Thus, the well-developed flight muscles observed after 30 or 60 days of long-day exposure probably resulted from regeneration rather than retention of the muscles. The response decreased in September beetles and none of the beetles responded during October and November. In subsequent months, transfer to long days accelerated the onset of muscle development, and increased length of long-day exposure increased the incidence of flightmuscle development. Some degeneration was noted late in the season (beetles collected in February and dissected in May), presumably due to depletion of the energy reserves.
days was extended from 30 days to 60 days, but a 90-day exposure yeilded no further increase in this proportion (Fig. 1E) and, indeed, oiicytes again became smaller after (1 90-day exposure (Fig. 1F). Females collected in October and November did not initiate oacyte development within 90 days, even in long days, but some development eventually occurred if the females were subjected to even greater periods of long-day exposure [(a 120 days) Tanaka et al., 1987a]. From December onward, oijcyte formation and growth was observed in long days. The incidence of females with developing oiicytes increased with increased time of long-day exposure. Oacyte length reached a maximum of about 12mmu and then decreased, probably due to depletion of the fat body reserves. The response of male gonads was slightly different from that of female gonads. Testis lobes progressively increased in size with increased length of exposure to long days until reaching a maximal length of 22 mmu (Fig. 1G.). A 30-day exposure to long days was not effective in stimulating testis-lobe development before January. From September to November testis-lobe size was not influenced by a 60-day exposure to long days (P > O.OS), but the same treatment significantly increased testis lobe size in beetles collected in August (t = 3.52, P < 0.05) and December (t = 6.43; P < 0.05). As observed in the short-day
Comparison with natural daylengths
To determine whether the effects of different photoperiods are constant throughout diapause,
S D Females
Mean
with
primary
oocytes
oocyte
length
10 _. E ZL _ Isr.
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Fig. I(a)
SEIJITANAKA et
1138
al.
G=)
10
0
50
(HI
0
Fig. l(b) Fig. 1. Gonad and flight muscle development in cycles of 12h light-12 h dark (SD) or 13h light-l 1 h dark (LD) after transfer from the aggregation site on different dates in 1985 and 1986. A and E, proportion of females with primary oBcytes; B and F, mean primary oijcyte length; C and G, mean testis lobe length; D and H, proportion of beetles with well developed flight muscles. N = 15 males and 15 females. Beetles were dissected
on the day of collection
(O),
or 30 (Q), 60 (a),
beetles at different phases of diapause were exposed to long days or short days and the responses to these photoperiods were compared with those to natural daylengths. OBcyte development. Among beetles collected between October and April, oiicyte development occurred most rapidly in long days (Fig. 2). Exposure to long days from October to February induced oocyte development in 33% of the females (group 12). All females had developing odcytes when kept in long days through March (group 7), but oijcytes of some females started degenerating during the next 30 days at this photoperiod (group 1). Among beetles exposed to short days or natural daylengths from October to March, little or no oocyte development was observed (groups 7 and 11). However, an earlier transfer to long days did not always elicit a higher response: when transferred to long days on December 6, the proportion of females that developed oiicytes by March 6 was 42.9% (group 8) while it was 66.7 and 73.3% in the beetles transferred to long days on January 6 and February 4,
or 90 (0)
days after collection.
respectively (groups 9 and 10). Differences among these 3 groups were not statistically significant (P > 0.05). The results indicate that exposure to long days between December and February was no more effective in shortening diapause than the naturally prevailing short daylength. The same conclusion was also reached when mean oijcyte length was compared among these groups (data not shown). In contrast, long days experienced before or after this period (December &February 4) were quite effective in shortening the diapause period. All females exposed to long days beginning October 6 developed oiicytes by March 6 (group 7), while less than half the females did so when their transfer to long days was delayed until December 6 (group 8) (x2 = 11.61; P < 0.05). Transfer to long days on February (group 10) or March 6 (group 5) induced rapid development of oiicytes as compared with the result obtained for beetles continuously exposed to natural daylengths (groups 11 and 6), suggesting that the final stage of diapause development was accelerated by long days.
Photoperiodism GROUP
16 17 1%
Date ONDJFMA ?,?54$1?0
Testis lobe length
?P with oocytes 50
in a tropical beetle
1139
Beetles with well develmed flight muscles
100
m Ca H I ‘II 6 654650 ONDJ FMA
50 (“1.)
1oc
-
10
20
’ o-
‘v
100 0 w
(1=12.5~m)
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Fig. 2. Gonad and flight-muscle development in cycles of 12 h light-12 h dark (e), 13 h light-l 1 h dark (O), or natural daylength (a). Photoperiodic regimes are given on the left: 0, natural daylength; q, 12 h light-12 h dark or 13 h light-l 1 h dark. N = 15 males and 15 females. Mean and standard deviation are given for testis lobe lengths. Testis development
As with female gonads, rapid development of male gonads occurred in long days (Fig. 2). Some beetles transferred to long days in October started testis development by January (group 16) while testes of those transferred to long days in December (group 17) or continuously ‘exposed to natural daylengths (group 18) remained undeveloped. Among beetles examined in February (groups 12-1 S), the earlier the time of transfer to long days the larger the mean testis-lobe size. By March 6, most males were near maturity (groups 7-‘11). During the next 30 days, testis-lobe size increased slightly in the males transferred in March (group 5), while in beetles transferred earlier, lobe size remained unchanged (groups 1, 3 and 4) or decreased ((group 2). In short days, beetles did not increase testis-lobe size by February (groups 12, 13 and 14 in Fig. 2) while some of those collected from the field in February had apparently initiated testis development (group 15). Among beetles collected in December or later and kept in short days until March or April, a positive correlatio:n was obtained between mean testis-lobe length and the time of transfer to short days (r = 0.98 and 0.93 for beetles examined in March and April, respectively; P < 0.05). However, beetles transferred to short days in October developed .testis lobes more rapidly than those transferred 2 months later (t = 4.40 and 3.05 for group 1 vs 2 and group 7 vs 8, respectively; P < 0.05). Natural daylengths in October and November are nearly equivalent to those in January and February, thus the results may indicate that the photoperiods promoting rapid testis development
after December are not as effective as a shorter photoperiod before December. To test this, groups of 15 males collected from the field on November 7, 1985, were exposed to various combinations of long, intermediate, and short photoperiods at 25°C. Testis lobes remained small (< 8 mmu) at any constant photoperiod by December 22 (groups 1, 2 and 3 in Fig. 3), but they started developing by January 31 (groups 4, 5 and 6). In this case, the proportion of males that initiated testis development increased with photoperiod, and the differences between the three groups were statistically significant (X*-test; P < 0.05). However, when exposed to different photoperiods during the first 45 days and then transferred to an intermediate photoperiod more beetles started testis development when the initial photoperiod was shorter (x2 = 4.46 and 13.39 for groups 7 vs 8 and groups 7 vs 9, respectively; P < 0.05). A similar tendency was also observed among the beetles maintained until February 10 (groups 10, 11 and 12), although no statistically significant differences were observed either in testis lobe length or in the proportion of males with developing testes. These results further confirm the above results, and suggest that a long photoperiod, which is favourable for testis development after December 22, is less effective than shorter photoperiods in accelerating diapause development before December 22. Flight muscles
The proportion of beetles with well-developed flight muscles was generally lower in males than in females. However, the pattern of response to different
SEIJI TANAKA el al.
1140
MALES WITH
DATE
GROUP NCN 7 krn-%s?
DEC
DEVELOPING
TESTES
TESTIS LOBE LENGTH
JAN FEB
5.9 + 0.6
1
151
2
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5
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Fig. 3. Effects of various lengths and combinations of short (S) (12 h light-12 h dark), intermediate (I) (124 h light-l lf h dark), and long (L) 13 h light-l 1 h dark photoperiods on testis development of males collected on November 7, 1985. I mmu = 12.5 pm. Beetles that had testis lobes 2 8 mmu in length were regarded as those with developing testes. Where an analysis of variance showed a significant difference
among groups (S, significant; NS, non-significant at 5%), a Duncan multiple range test was performed; numbers followed by different letter significant at 5%. N = 15, except for groups l-3 (n = 10). photoregimes was quite similar in both sexes (Fig. 2). The pattern was also similar to the pattern of response observed in oijcyte development. Flight muscles tended to degenerate relatively quickly once they attained full development. Some groups of beetles maintained in long days until April 5 (groups IL4 in Fig. 2) thus included a mixture of beetles with well-developed flight muscles and those with degenerating flight muscles. DISCUSSION
Based on changes in the physiological condition of S. rotundus and the dynamics of its response to daylength, diapause in this species may be divided into 3 phases: the initial, intermediate and final phases as shown in Fig. 4. In the initial phase, beetles arrive at the aggregation site, flight muscles degenerate, and the metabolic rate decreases from 30@400pl/g/h to less than 5Opl/g/h (at 25°C) (Tanaka et al., 1988). During the intermediate phase, beetles remain inactive in the aggregation and their metabolic rate remains very low (Wolda and Denlinger, 1984). During the final phase they occasionally move up and down the tree, apparently in response to moisture during the dry season, but otherwise they remain inactive in aggregation. At the end of this phase, beetles mate and disperse from the tree. Beetles in the initial and final phases respond much more rapidly to a long photoperiod than during the intermediate phase. However, the response to the same long photoperiod appears to be different in the initial and final phases. Beetles during the final phase terminate diapause more quickly at long photoperiod
when a transfer from the field is delayed. But, during the initial phase, the response to a long photoperiod is greatest at the beginning of the phase and progressively declines. In females, longer exposure to a long photoperiod during the initial phase does not always elicit a higher response, as it does during the final phase (Fig. 1E and F). Finally, some beetles that have just arrived at the tree undergo flight-muscle histolysis at the same long photoperiod that always stimulates flight-muscle development during the final phase. During the initial phase beetles may use a threshold response to photoperiod rather than the graded response observed during the final phase. If this is the case, the threshold for induction or maintenance of diapause could be very close to 13 h light-l 1 h dark in June and may increase as the season advances. This could explain the gradually decreasing proportions of individuals that avert or terminate diapause when a transfer from the field to 13 h light-l 1 h dark is delayed. Beetles come to the aggregation site over a few months, but our preliminary observations suggest no significant differences in the response to photoperiod between early and late arrivers (Tanaka et al., unpubl. observ.). The fact that none of the beetles initiated gonad development in June and July may suggest that the daylength actually perceived by them during this season is slightly shorter than 13 h light11 h dark. Our unpublished observations show that there is no significant difference in effect between 13 h light and 13.5 h light in beetles collected in August, thus supporting the idea that beetles show a threshold response during the initial phase. An alternative explanation is that the daylengths actually read by the beetles in June are nearly equal
Photoperiodism
1141
in a tropical beetle TEMPERATURE
2 5°C 13 H DAYLENGTH 12
PHASE
!TT
INITIAL
INTERMEDIATE
FINAL
MOhTH SEASON
Fig. 4. Seasonal changes in the responsiveness to long days of Stenotarsus rotundus. During the initial phase, some beetles develop gonads and flight muscles at 13 h light-l 1 h dark, but the numbers decrease towards the end of this phase. Beetles during the intermediate phase show no rapid development of gonads or flight muscles. During the final phase, beetles gradually recover their responsiveness to long days and show a graded response to photoperiod. Temperature and daylength data on Barro Colorado Island are based on Tanaka et al. (1987a).
to 13 h light-l 1 h da:rk and it is a decrease in daylength that induces diapause in this species. Several examples in which diapause occurs only under a decreasing photoperiod include the lygaeid bug, Neocoryphus bicrucis (Solbrek, 1979) the wood louse, Armadillidium vulgare (Mocquard et al., 1980) and the phytoseiid mite, Amblyseius longispinosus (Hanamura, 1982). A systematically designed experiment to test this hypothesis in S. rotundus is yet to be done. During the intemlediate phase, beetles do not terminate diapause rapidly either at a short or long photoperiod. Among those collected in October, the first sign of the initi#ation of gonad development is seen only after 3 months for males (Fig. 1G) and 4 months for females at 13 h light-l 1 h dark (Tanaka et al., 1987a). At l;! h light-12 h dark, the beetles require another 2-3 months to start gonad development (Tanaka et al., 1987a). From these results, we conclude that diapause during the intermediate phase is controlled by a graded response, as it is during the final phase. However, because of its high intensity, diapause in this species would probably persist throughout this phase under any photoperiodic conditions. Imaginal diapause of the temperate lacewing, Chrysopa carnea, is thought to be maintained by a graded response to photoperiod (Tauber and Tauber, 1973, 1976~; Tauber et al., 1986). In this case, however, all Chrysopa adults collected from the field were tested at a relatively high temperature (24°C). Thus, how much the graded response to photoperiod actually contributes to the maintenance of diapause relative to the role of low temperature prevailing in the fall and wimer (through February) remains unknown. By examining the responses of various organs to combinations of different photoperiods, we have found that control of diapause in S. rotundus involves a rather complicated response to seasonally changing daylengths: the pattern of response changes as diapause progresses. For example, testis development during the final phase proceeds more rapidly at a long photoperiod than at a short photoperiod. However, when beetles collected in November were exposed to
different photoperiods for 45 days before they were transferred to 124 h light-l :f h dark, more gonad development occurred when the initial photoperiod was shorter (Fig. 3.). Thus, the rather deep diapause of S. rotundus decreases in intensity more rapidly as the daylength shortens until late December and the beetles become ready for diapause termination in response to increasing daylengths during the final phase. Shifts in optimal daylength for diapause development, as observed in S. rotundus, may be compared with temperature responses of some temperate insects. For example, the optima1 temperature for diapause development in eggs of the chrysomelid beetle, Atrachya menestriesi, is 7.5”C during the early stage but decreases to 0°C after they experience 7.5”C for 10 days (Ando, 1978). In eggs of the cricket, Allonemobius fasciatus, the optimal temperature is relatively high during the early stage, decreases to 6°C and increases again towards the end of the diapause period (Tanaka, 1987). With this type of response, diapause can persist until a cold period comes. Once these insects experience cold, the intensity of diapause quickly diminishes, preparing them for postdiapause development in the spring. During the final phase, S. rotundus shows a graded response to photoperiod for diapause termination (Tanaka et al., 1987a). In this sense, this beetle is similar
to
the
temperature
lacewing,
C. downesi
(Tauber and Tauber, 1976~). However, unlike the lacewing in which adults require short days before they become responsive to increasing daylengths, neither a period of short days nor an increase in photoperiod is an absolute requirement for S. rotundus.
Gonad development in S. rotundus apparently begins earlier in males than in females. However, the flight muscles regenerate at the same time which, in females, coincides with the time of oiicyte development (Tanaka et al., 1987a). Our previous studies suggest that both flight muscle and oijcyte development are stimulated by juvenile hormone (Wolda and Denlinger, 1984) while testis development is regulated
SEIJI TANAKA et al.
1142
by other factor(s) (Tanaka et al., 1987b). The corpora allata, which increase in size during flight muscle development, remain small when testis development is in progress. A juvenile hormone analogue (methoprene) and 20-hydroxyecdysone have no influence on testis development, although the former stimulates development of flight muscles in both sexes and oocytes in females. We have not yet identified what factor(s) stimulates testis development. However, it appears that the developmental stimulus for testis development always precedes the release of juvenile hormone during the diapause termination process. We have never encountered a case in which males, during the final phase, had well developed flight muscles without developing their testes, but this combination of developmental traits was readily observed during the initial phase. The changes in responsiveness to daylength that we observed suggest the following phenological sequence: the beetles are reproductively active during the relatively long days between mid-April and July. Migration from their reproductive habitat to the aggregation site begins shortly before the summer solstice (Wolda and Denlinger, 1984; Tanaka et al., 1987~) and under the maximal daylength conditions young beetles initiate a variety of physiological changes leading to the onset of diapause. If the beetles are exposed experimentally to long photoperiod (13 h light-l 1 h dark) at that time, they can respond by retaining or even regenerating their flight muscles, and by maturation of the gonads. The beetles become progressively more refractory to stimulation by long daylengths, and they regain their responsiveness to long daylengths after experiencing the short days around the winter solstice. As daylength increases in January and February the process of diapause termination accelerates. The beetles are thus primed to reproduce, and the onset of the rains, usually in mid April, triggers mating and dispersal from the aggregation site. Acknowledgemenl-This from the Smithsonian
study Institute
was supported by a grant Scholarly Studies program.
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