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The pattern of reactivation of diapausing larvae in the blowfly, Calliphora vicina
J. Insect Physiol., 1974, Vol. 20, pp. 2487 to 2496. PergamonPress. Printed in Great Btitain
THE PATTERN OF REACTIVATION OF DIAPAUSING LARVAE IN THE BLOWFLY, CALLIPHORA VICINA E. B. VINOGRADOVA Department of Entomology, Zoological Institute Academy of Sciences, Leningrad, U.S.S.R. (Received 20 April 1974) Abstract-In Calliphora vicina from Belomorsk (North Karelia) the pattern of reactivation of diapausing larvae at different temperatures (4, 12.5, 15.5, 20, and 25C) was studied experimentally. Most favourable in this respect are the first and last temperatures. Photoperiodic reactivation was established. The rate of pupation under a long-day regime is two to four times greater when compared with that observed under a short-day regime. In C. vi&a maternally operating photoperiods determined not only the ratio of diapausing and non-diapausing larvae in the progeny but also the peculiarities of the diapause. A more intensive diapause is typical for the progeny of short-day-treated flies. The diapause of the progeny of long-day-treated flies is less intense and can be easily terminated as a result of the action of the above-mentioned factors.
INTRODUCTION BLOWFLY Calliphora vi&a is widely distributed and is extensively used for different purposes as a laboratory subject. Investigations of the ecological regulation of its seasonal development have been started only recently. The onset of larval diapause in C. vicina from Gorky was shown experimentally to depend not only on the developmental temperature but also on the maternally operating photoperiod. The larvae can terminate their diapause at low and high temperatures (ZINOVJEVA and VINOGRADOVA, 1972; VINOGRADOVA and ZINOVJEVA, 1972). This article deals with detailed experimental investigations on the influence of different temperatures and photoperiods upon the termination of larval diapause in C. vicina. The dependence of the pattern of reactivation on the diapauseinducing conditions has also been studied. THE
MATERIALS
AND METHODS
A strain from Belomorsk (North Karelia), which is situated near the northern limit of the C. vicina area of distribution, was used for these experiments. The flies were kept in gauze cages (15 x 30 x 30 in.) and given meat, sugar, and water. The larvae were fed on meat. The experiments were conducted in photothermostats with automatically regulated photoperiods and temperatures. Temperature fluctuation did not exceed + O*YC. Luminescent lamps were used for lighting. 2487
E. B. VINOGRADOVA
2488
RESULTS
To obtain diapausing larvae, flies after emergence were reared at 20°C and in different photoperiodic conditions (20, 18, 17, 16, 1.5, and 12 hr of light per day). The eggs and hatched larvae were reared at 12.5 or 15.5”C combined with a short (12 hr) or long (20 hr) daylength. At these temperatures the first flies emerge in 30 to 35 days. As this period proves to be sufficient for the completion of the development without a diapause it might be presumed that the development of the larvae which did not pupate during this period is to some degree arrested.
IO-
IllIIIIIiII11IIIIlllllll 20
30
40
50
60
70
80
90
100
110
120
130
Days
FIG. 1. The dynamics of pupation of the larvae of C. flies were kept at 20°C and exposed to 18 hr of light 12 hr of light per day; - - - -, larvae developed under under a rCgime of 12 hr of per day; --,
vicina at 15.5”C. 0, The per day; 0, at 20°C and a rCgime of 20 hr of light light per day.
Larval development at 15.5”C is shown in Fig. 1. The dynamics of the pupation which reflects the physiological state of the larvae depends on the photoperiod experienced by both parents and the larvae themselves. Thus in the progeny of the long-day-exposed females (18 hr of light per day) most of the larvae pupate on the thirtieth day: 98.2 per cent with a long daylength and 83.7 per cent with a But by this time in the progeny of the short-day-exposed short daylength. females only 58 and 21 per cent, respectively, of the larvae have pupated. The dynamics of the increase in the number of pupating larvae during the period of 2 to 4 months shows that their development is arrested to a different degree as compared with that normally observed. While the pupation of some larvae is only slightly retarded, the development of others is severely inhibited, as is The development is renewed spontaneously. characteristic of the diapause. Apparently a temperature of 15.5”C is favourable both for diapause induction in some individuals, mainly in the progeny of the short-day-exposed females, and for diapause development. Reactivation occurs slowly, and 87 to 93 per cent of the larvae pupate by the 105th to 121st day. There is no great difference between pupation under a short-day and under a long-day regime.
REACTIVATION
IN DIAPAUSING
BLOWFLY
2489
LARVAE
In the next series of experiments, the development of the progeny of the females which were exposed to different daylengths were reared at 12.5”C and with a short day (Table 1). The combination of this temperature and photoperiod is more
TABLE I-THE
INFLUENCE
OF MATERNAL PHOTOPERIODS OFTHEPROGENYINC.'UiCi?ZU
ON
Daylength Diapausing (hr of light per day) No. of larvae (%) 12 15 16 17 18
1628 1518 1628 1641 2549
95.1 85.0 84.6 20.8 33.1
20
3099
2.7
LARVAL
DIAPAUSE
larvae
favourable for the induction of larval diapause in C. vicina. Diapause incidence varies with the photoperiod operating during oiigenesis. In the progeny of females exposed to a long daylength (20, 18, and 17 hr of light per day) most of the larvae pupate on the thirtieth day, but 12, 15, and 16 hr of light are taken by the flies as a short-day regime and induce 84 to 95 per cent of individuals to enter diapause. Incidentally, on the forty-fifth day as compared with the thirtieth day, the number of puparia in all photoperiodic regimes hardly increases which indicates that the differentiation of the progeny into developing and diapausing parts is complete. Thus in C. vi&a from Belomorsk the critical daylength lies between 16 and 17 hr of light per day. All the larvae produced by the females exposed to different photoperiods, with the exception of those exposed to 20 hr of light per day, were used in further reactivation experiments. First, the reactivation of diapausing larvae at 12.5, 15.5, and 20°C in combination with long (20 hr of light per day) and short (12 hr) photoperiod was investigated. One-month-old larvae were used in the experiments. In most experiments there was 100 to 170 larvae, but in those on the progeny of the females exposed to 17 hr of light there were 40 larvae. The dynamics of the pupation served to indicate that reactivation was in progress. The total number of pupated larvae and the length of time during which they pupated permit us to estimate the intensity of the diapause and the most suitable reactivation conditions. A temperature of 12.5”C is not so favourable for termination of the diapause (Fig. 2). The number of pupae increases slowly during 4 months. Differentiation between the curves in the two groups, characterizing the reaction of the larvae, is observed. The first group includes the larvae produced by long-day-exposed females and the-second one corresponds to the progeny of the short-day-treated females. By the 79th day about 60 per cent of the larvae of the first group and
E. B. VINOGRADOVA
2490
IO
20
30
40
50
60
70
80
SO
100
110
120
Days
FIG. 2. The dynamics of pupation of diapausing larvae at 12~5°C and with 12 hr of light per day. The flies were exposed to 18 hr of light per day, e ; 17 hr, A; 16 hr, A; 15 hr, 0; and 12 hr, 0.
only 8 per cent of the larvae of the second one pupate. Only from this time is a visible increase of the puparia in the progeny of the short-day-exposed flies observed, which is connected with the beginning of mass reactivation. At 15.5”C the larvae terminate their diapause faster and the dependence of pupation on the maternally operated photoperiod is shown more clearly (Fig. 3a, b). For instance, with a short daylength the speed of pupation of the larvae produced by the flies exposed to 15 and 16 hr of light begins to increase considerably on about the 65th day, i.e. 2 weeks earlier as compared with the 12*5”C regime. But in the progeny of the long-day-treated flies about 80 to 95 per cent of the larvae pupate by this time. At 15.5”C the stimulating influence of a long-day regime on the termination of larval diapause is observed. This can be clearly seen if the dynamics of the al g
b
loo
A
so
.E 80 t g 70 ic 60 b 50 i% CI 40 -G p: 30 Lg 20 IO z e0
FIG. 3a.
REACTIVATION
IN
DIAPAUSING
BLOWFLY
LARVAE
2491
Days FIG.
3b.
3. The dynamics of pupation of diapausing larvae at 15.5°C. The larvae were exposed to (a) 12 hr of light per day and (b) 20 hr of light per day. Other designations are the same as in Fig. 2. FIG.
of the progeny of long-day-treated flies in long-day conditions (Fig. 3b) is compared with that in short-day conditions (Fig. 3a). In the first case the number of reactivated larvae increases rapidly and reaches 8.5 per cent on the 35th day. In the second case only about 40 per cent of the larvae pupate during this period. At 20°C all the above-mentioned factors are observed even more clearly (Fig. 4a, b). The increase of temperature accelerates the processes of reactivation and The pupation curves become more the long day strengthens this tendency. abrupt. Thus under a long-day regime in the progeny of the long-day-treated flies about 40 to 60 per cent of the larvae pupate in 4 days and about 85 per cent in 7 days’ time. On the other hand, the pupation of the larvae produced by short-day-treated flies occurs only slowly and reaches the same level only after 35 to 40 days. The differences in diapause termination between larvae produced by long-daytreated flies and those produced by short-day-treated flies are evident at all the temperatures studied and under both short- and long-day regimes. These results show that there is a definite difference in the physiological state of diapausing larvae of different origins in spite of the fact that the manifestation of their diapause occurs in the same conditions. These differences are probably connected with the photoperiods experienced by the parental generation. Next we examined reactivation at 25°C. This high temperature terminates the diapause very quickly and no essential difference between the action of longand short-day regimes is observed. Thus in the progeny of the short-day-treated flies correspondingly 57 and 58 per cent of the larvae pupate on the 4th day, 81 and 71 per cent on the 11th day, and 91 and 84 per cent on the 18th day. pupation
81
E. B. VINOGRADOVA
2492
IO
20
so
40
50
60
(b)
Doys
FIG. 4. The dynamics of pupation of diapausing larvae at 20°C. The larvae were exposed to (a) 12 hr of light per day and (h) 20 hr of light per day. Other designations are the same as in Fig. 2.
TABLE ~-THE
DEPENDENCE OF THE REACTIVATION OF DIAPAUSING LARVAEOF C. r~i&zu ON THEDURATIONOF COOLINGAT 4°C Duration of cooling (months) 1
Daylength during oSgenesis of the females (hr of light per day) 18 17 15 12
No. Reactivated larvae of larvae (%) 80 40 100 175
68.7 75.0 17.0 4.5
2
3
No. Reactivated of larvae larvae (%)
No. Reactivated larvae of larvae (%)
85 40 100 150
87.0 100.0 850 83.3
79 39 96 116
100.0 100.0 84.3 99.1
REACTIVATION
IN
DIAPAUSING
BLOWFLY
LARVAE
2493
Special experiments were carried out to deal with the reactivation of C. vicin~ diapausing at low temperatures just above zero. For this purpose l-month-old larvae produced by females which were kept under different photoperiodic regimes were cooled to 4°C and then transferred into conditions favourable for morphogenesis (15.5% and short-day length). The pupae were counted on the tenth day. The dependence of reactivation on the length of the cooling period is shown in Table 2. One month’s cooling proved to be sufficient for reactivation of 69 to 75 per cent of the individuals in the progeny of the long-day-exposed flies. But in the progeny of the short-day-treated flies, a more intensive diapause is manifest and it takes longer period of cooling (2 or 3 months) to terminate their diapause. DISCUSSION
The results of these and earlier experimental investigations (VINOGRADOVA and ZINOVJEVA, 1972; ZINOVJEVAand VINOGRADOVA,1972) throw some light on the ecological regulation of the seasonal development of C. vicina. Larval diapause is controlled by the temperature and photoperiodic regimes during larval development and by the maternally operating photoperiod. The above-mentioned factors interact closely and the importance of each of them in diapause induction can vary depending on the actual ecological situation and on population peculiarities. In C. vi&a larval diapause is possible within certain temperature limits. Single diapausing larvae can appear if their development occurs at 20°C but a temperature of 15°C and lower is necessary for the induction of diapause in most of the individuals (more than 50 per cent). Special experiments have established the approximate time of the onset and manifestation of diapause. In about half of the larvae this occurs when they are approximately 15 days old and in the other half diapause is later, up to 1 month old (ZINOVJEVAand VINOGRADOVA,1972). The physiological state of the larvae is under maternal control. A maternally operating short photoperiod considerably strengthens predisposition to diapause in their progeny, but a maternally operating long photoperiod promotes continuous development of the larvae even if the ambient temperature is lowered. Such dependence of the larval physiological state on the maternally operating photoperiods is observed only with definite temperature limits, beginning approximately with 16°C and going up to 9°C. Further lowering of the temperature induces all the larvae to enter diapause, independent of the photoperiodic regime during the life of the females. The critical daylength determining the time of the appearance of diapausing insects in nature is adaptive and as a rule changes within the distribution area of a species (DANILEVSKY, 1961). A similar situation has been revealed in C. oicina. In the northern population from Belomorsk the photoperiodic threshold lies between 16 and 17 hr of light per day, but in the more southern population from Gorky it lies between 15 and 16 hr (VINOGRRDOVAand ZINOVJEVA, 1972). A more gradual decrease in the number of diapausing larvae with increase in the duration of illumination and connected with this a considerable percentage of diapausing
2494
E. B. VINOGRADOVA
individuals (20-33%), even in the progeny of long-day-treated females, is typical of C. vicina from Belomorsk. But in the strain from Gorky the increase of daylength by 1 hr within the photoperiodic threshold results in an abrupt decrease in the number of diapausing larvae from 99 to 3 per cent. Comparison of the photoperiodic induction in two geographical strains of C. vicina shows that the degree of maternal influence on the diapause in the progeny varies within this species. It has been shown previously that, within definite temperature limits, in the strain from Gorky the physiological state of the larvae is determined exclusively by the maternally operating photoperiod. But the strain from Belomorsk differs in this respect ; the physiological state of its larvae is under a double control and depends on both the imaginal and larval photoperiodic regimes, but the first plays the leading r61e. An analysis of the literature dealing with maternal induction of diapause in the progeny shows that during certain stages in the development of the daughter generation insects which have been studied retain a definite degree of independence in their reaction to environmental factors (VINOGRADOVA,1973). Therefore the physiological state of the progeny is a result of all those factors acting during the development of the maternal and daughter generations. The reactivation of C. viciw from Belomorsk has been shown to depend on the Investigations of the ability of different temperatemperature and photoperiods. tures (from 4 to 25°C) to break the larval diapause show that it is the extreme temperatures that are the most favourable for diapause development. This is confirmed both by the considerable percentage of reactivated larvae and by the short period needed for pupation to occur. As a rule at low temperatures just above zero it takes a considerable time to terminate the larval diapause. Thus in the progeny of the short-day-treated females most of the larvae pupate after being cooled at 4°C for 2 or 3 months if the temperature is raised to that favourable for But 1 month’s cooling at 4°C is enough to terminate a less inmorphogenesis. tensive diapause in the progeny of long-day-treated females. Similar data concerning the duration of the period of reactivation were obtained for. another blowfly, Lucilia cmsar (RING, 1968). A n increase of temperature from 12.5 to 25°C accelerates the processes of reactivation and correspondingly the rate of pupation. For instance, with a short daylength in the progeny of short-dayexposed females at 12.5”C mass pupation begins only on the 79th day, at 15*5”C on the 60th, at 20°C on the 7th to 15th, and at 25°C on the 2nd or 3rd day. Experiments on C. vicina have revealed that photoperiodic reactivation is possible. Long photoperiods stimulate resumption of development. Under a long-day regime the rate of pupation is two to four times greater when compared with that observed under a short-day regime. At 25°C reactivation occurs so rapidly that the difference between the short and long daylengths is not manifest. Up to the present time photoperiodic reactivation has been established in numerous insects (about 25 species) which hibernate mainly as a larva or an adult (MUELLER, 1965; DANILEVSKY and SHELDESHOVA,1968). No such data are available so far for flies, but photoperiodic reactivation is known in other Diptera, namely in some
REACTIVATION OFDIAPAUSING BLOWFLY LARVAE
2495
mosquitoes with larval diapause-AZdes triseriatus (BAKER, 1935 ; LOVE and WHELCHEL, 1955 ; VINOGRADOVA,1969), Anopheles p1umbeu.q A. claw&r (VINOGRADOVA,1969), Weyomyia smithii (SMITH and BRUST,1971), A. barberi, Orthopodomyia signifera (BAKER, 1935), Chaoborus americanus (BRADSHAWand SHAPPIRIO, 1966; BRADSHAW,1969), Toxorhynchites rutilus (JENNERand MCCRARY, 1964), and also Chironomus tentans (ENGELMANNand SHAPPIRIO,1965). Considerable differences are observed in the process of reactivation in the progeny which originates from females exposed to different photoperiods. Long photoperiods induce only a small percentage of the larvae to enter diapause (2-33 per cent), but short photoperiods induce most of the individuals (84-95 per cent) to enter diapause. In the first case the diapause is less intensive and can be easily terminated as a result of the influence of a long-day regime or an increase in temperature. A more intensive diapause is typical for the progeny of short-daytreated females and it takes a more prolonged influence of environmental factors to break this diapause. Thus the photoperiods to which the females have been exposed determine not only the ratio of non-diapausing and diapausing larvae in the progeny but also the intensity of the diapause too. The only case of an analogous inhibitory influence of a maternal origin is observed in the moth Orgyia antiqua (MASLENNIKOVA,1972) where the intensity of the egg diapause depends on the photoperiod of the female.
REFERENCES
BAKERF. C. (1935) The effect of photoperiodism
on resting treehole mosquito larvae.
Can. Ent. 67, 149-153.
BRADSHAW W. E. (1969) Major environmental factors inducing the termination of larval diapause in Chaoborzls americanus (Diptera, Culicidae). Biol. Bull., Woods Hole 136, 2-8. BRADSHAW W. E. and SHAPPIRIO D. G. (1966) The rble of food, photoperiod and temperature in the termination of the larval diapause in Chaobovus (Diptera, Cubcidae). Am. Zool. 6, 508.
DANILEVSKY A. S. (1961) Photoperiodism and Seasonal Development of Insects. Leningrad State University.
In Russian.
DANILEVSKY A. S. and SHELDESHOVA G. G. (1968) Adaptive significance of photoperiodic and cold reactivation. In Photoperiodic Adaptations of Insects and Mites, pp. 80-90. Leningrad State University.
In Russian.
ENGELMANN W. and SHAPPIRIO D. G. (1965) Photoperiodic control of the maintenance and termination of larval diapause in Chironomus tentans. Nature, Lond. 207, 548-549. JENNER CH.E. and MCCRARYA. B. (1964) Photoperiodic control of larval diapause in the giant mosquito Toxorhynchites rut&s. Am. Zool. 4, 434. LOVE J. and WHELCHEL J. G. (1955) Photoperiodism and the development of Aedes triseriatus. Ecology 36, 340-342.
MASLENNIKOVA V. A. (1972) Influence of hormonal balance of diapausing insects on their reactivation. In Problems of Photoperiodism and Diapause in Insects, pp. 229-241. Leningrad State University.
In Russian.
MULLERH. J. (1965) Probleme der Insektendiapause. Verb. deutsch. Zool. Ges. 192-222. RING R. A. (1968) Termination of diapause in larva of Lucilia Caesar L. (Diptera, Calliphoridae).
Can. J_ Zool. 46, 335-344.
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E. B. V~NOGRADOVA
SMITH S. M. and BRUST R. A. (1971) Photoperiodic control of the maintenance and termination of larval diapause in Weyomyia smithii Coq. (Diptera, Culicidae) with notes on osgenesis in the adult female. Can.J. 2001. 49, 1065-1073. VINOGRADOVA E. B. (1969) Diapause of Bloodsucking Mosquitoes and its Regulation. Nauka, Leningrad. In Russian. VINOGRADOVA E. B. (1973) Maternal influence on the diapause of the progeny in insects. In 25th Annual Readings in Memory of IV. A. Cholodkowsky, pp. 39-66. Leningrad. In Russian. VINOGRADOVA E. B. and ZINOVJEVAK. B. (1972) Maternal induction of larval diapause in the blowfly, Calliphora vicina. J. Insect Physiol. 18,2401-2409. ZINOVJ~VAK. B. and VINOGRADOVA E. B. (1972) The control of seasonal development in parasites of blowflies-II. Ecological control of winter adaptations in CaZZiphora vi&a R.-D. (Diptera, Calliphoridae). In Host-Parasite Relationships in Insects, pp. 90-99. Nauka, Leningrad. In Russian.
THE PATTERN OF REACTIVATION OF DIAPAUSING LARVAE IN THE BLOWFLY, CALLIPHORA VICINA E. B. VINOGRADOVA Department of Entomology, Zoological Institute Academy of Sciences, Leningrad, U.S.S.R. (Received 20 April 1974) Abstract-In Calliphora vicina from Belomorsk (North Karelia) the pattern of reactivation of diapausing larvae at different temperatures (4, 12.5, 15.5, 20, and 25C) was studied experimentally. Most favourable in this respect are the first and last temperatures. Photoperiodic reactivation was established. The rate of pupation under a long-day regime is two to four times greater when compared with that observed under a short-day regime. In C. vi&a maternally operating photoperiods determined not only the ratio of diapausing and non-diapausing larvae in the progeny but also the peculiarities of the diapause. A more intensive diapause is typical for the progeny of short-day-treated flies. The diapause of the progeny of long-day-treated flies is less intense and can be easily terminated as a result of the action of the above-mentioned factors.
INTRODUCTION BLOWFLY Calliphora vi&a is widely distributed and is extensively used for different purposes as a laboratory subject. Investigations of the ecological regulation of its seasonal development have been started only recently. The onset of larval diapause in C. vicina from Gorky was shown experimentally to depend not only on the developmental temperature but also on the maternally operating photoperiod. The larvae can terminate their diapause at low and high temperatures (ZINOVJEVA and VINOGRADOVA, 1972; VINOGRADOVA and ZINOVJEVA, 1972). This article deals with detailed experimental investigations on the influence of different temperatures and photoperiods upon the termination of larval diapause in C. vicina. The dependence of the pattern of reactivation on the diapauseinducing conditions has also been studied. THE
MATERIALS
AND METHODS
A strain from Belomorsk (North Karelia), which is situated near the northern limit of the C. vicina area of distribution, was used for these experiments. The flies were kept in gauze cages (15 x 30 x 30 in.) and given meat, sugar, and water. The larvae were fed on meat. The experiments were conducted in photothermostats with automatically regulated photoperiods and temperatures. Temperature fluctuation did not exceed + O*YC. Luminescent lamps were used for lighting. 2487
E. B. VINOGRADOVA
2488
RESULTS
To obtain diapausing larvae, flies after emergence were reared at 20°C and in different photoperiodic conditions (20, 18, 17, 16, 1.5, and 12 hr of light per day). The eggs and hatched larvae were reared at 12.5 or 15.5”C combined with a short (12 hr) or long (20 hr) daylength. At these temperatures the first flies emerge in 30 to 35 days. As this period proves to be sufficient for the completion of the development without a diapause it might be presumed that the development of the larvae which did not pupate during this period is to some degree arrested.
IO-
IllIIIIIiII11IIIIlllllll 20
30
40
50
60
70
80
90
100
110
120
130
Days
FIG. 1. The dynamics of pupation of the larvae of C. flies were kept at 20°C and exposed to 18 hr of light 12 hr of light per day; - - - -, larvae developed under under a rCgime of 12 hr of per day; --,
vicina at 15.5”C. 0, The per day; 0, at 20°C and a rCgime of 20 hr of light light per day.
Larval development at 15.5”C is shown in Fig. 1. The dynamics of the pupation which reflects the physiological state of the larvae depends on the photoperiod experienced by both parents and the larvae themselves. Thus in the progeny of the long-day-exposed females (18 hr of light per day) most of the larvae pupate on the thirtieth day: 98.2 per cent with a long daylength and 83.7 per cent with a But by this time in the progeny of the short-day-exposed short daylength. females only 58 and 21 per cent, respectively, of the larvae have pupated. The dynamics of the increase in the number of pupating larvae during the period of 2 to 4 months shows that their development is arrested to a different degree as compared with that normally observed. While the pupation of some larvae is only slightly retarded, the development of others is severely inhibited, as is The development is renewed spontaneously. characteristic of the diapause. Apparently a temperature of 15.5”C is favourable both for diapause induction in some individuals, mainly in the progeny of the short-day-exposed females, and for diapause development. Reactivation occurs slowly, and 87 to 93 per cent of the larvae pupate by the 105th to 121st day. There is no great difference between pupation under a short-day and under a long-day regime.
REACTIVATION
IN DIAPAUSING
BLOWFLY
2489
LARVAE
In the next series of experiments, the development of the progeny of the females which were exposed to different daylengths were reared at 12.5”C and with a short day (Table 1). The combination of this temperature and photoperiod is more
TABLE I-THE
INFLUENCE
OF MATERNAL PHOTOPERIODS OFTHEPROGENYINC.'UiCi?ZU
ON
Daylength Diapausing (hr of light per day) No. of larvae (%) 12 15 16 17 18
1628 1518 1628 1641 2549
95.1 85.0 84.6 20.8 33.1
20
3099
2.7
LARVAL
DIAPAUSE
larvae
favourable for the induction of larval diapause in C. vicina. Diapause incidence varies with the photoperiod operating during oiigenesis. In the progeny of females exposed to a long daylength (20, 18, and 17 hr of light per day) most of the larvae pupate on the thirtieth day, but 12, 15, and 16 hr of light are taken by the flies as a short-day regime and induce 84 to 95 per cent of individuals to enter diapause. Incidentally, on the forty-fifth day as compared with the thirtieth day, the number of puparia in all photoperiodic regimes hardly increases which indicates that the differentiation of the progeny into developing and diapausing parts is complete. Thus in C. vi&a from Belomorsk the critical daylength lies between 16 and 17 hr of light per day. All the larvae produced by the females exposed to different photoperiods, with the exception of those exposed to 20 hr of light per day, were used in further reactivation experiments. First, the reactivation of diapausing larvae at 12.5, 15.5, and 20°C in combination with long (20 hr of light per day) and short (12 hr) photoperiod was investigated. One-month-old larvae were used in the experiments. In most experiments there was 100 to 170 larvae, but in those on the progeny of the females exposed to 17 hr of light there were 40 larvae. The dynamics of the pupation served to indicate that reactivation was in progress. The total number of pupated larvae and the length of time during which they pupated permit us to estimate the intensity of the diapause and the most suitable reactivation conditions. A temperature of 12.5”C is not so favourable for termination of the diapause (Fig. 2). The number of pupae increases slowly during 4 months. Differentiation between the curves in the two groups, characterizing the reaction of the larvae, is observed. The first group includes the larvae produced by long-day-exposed females and the-second one corresponds to the progeny of the short-day-treated females. By the 79th day about 60 per cent of the larvae of the first group and
E. B. VINOGRADOVA
2490
IO
20
30
40
50
60
70
80
SO
100
110
120
Days
FIG. 2. The dynamics of pupation of diapausing larvae at 12~5°C and with 12 hr of light per day. The flies were exposed to 18 hr of light per day, e ; 17 hr, A; 16 hr, A; 15 hr, 0; and 12 hr, 0.
only 8 per cent of the larvae of the second one pupate. Only from this time is a visible increase of the puparia in the progeny of the short-day-exposed flies observed, which is connected with the beginning of mass reactivation. At 15.5”C the larvae terminate their diapause faster and the dependence of pupation on the maternally operated photoperiod is shown more clearly (Fig. 3a, b). For instance, with a short daylength the speed of pupation of the larvae produced by the flies exposed to 15 and 16 hr of light begins to increase considerably on about the 65th day, i.e. 2 weeks earlier as compared with the 12*5”C regime. But in the progeny of the long-day-treated flies about 80 to 95 per cent of the larvae pupate by this time. At 15.5”C the stimulating influence of a long-day regime on the termination of larval diapause is observed. This can be clearly seen if the dynamics of the al g
b
loo
A
so
.E 80 t g 70 ic 60 b 50 i% CI 40 -G p: 30 Lg 20 IO z e0
FIG. 3a.
REACTIVATION
IN
DIAPAUSING
BLOWFLY
LARVAE
2491
Days FIG.
3b.
3. The dynamics of pupation of diapausing larvae at 15.5°C. The larvae were exposed to (a) 12 hr of light per day and (b) 20 hr of light per day. Other designations are the same as in Fig. 2. FIG.
of the progeny of long-day-treated flies in long-day conditions (Fig. 3b) is compared with that in short-day conditions (Fig. 3a). In the first case the number of reactivated larvae increases rapidly and reaches 8.5 per cent on the 35th day. In the second case only about 40 per cent of the larvae pupate during this period. At 20°C all the above-mentioned factors are observed even more clearly (Fig. 4a, b). The increase of temperature accelerates the processes of reactivation and The pupation curves become more the long day strengthens this tendency. abrupt. Thus under a long-day regime in the progeny of the long-day-treated flies about 40 to 60 per cent of the larvae pupate in 4 days and about 85 per cent in 7 days’ time. On the other hand, the pupation of the larvae produced by short-day-treated flies occurs only slowly and reaches the same level only after 35 to 40 days. The differences in diapause termination between larvae produced by long-daytreated flies and those produced by short-day-treated flies are evident at all the temperatures studied and under both short- and long-day regimes. These results show that there is a definite difference in the physiological state of diapausing larvae of different origins in spite of the fact that the manifestation of their diapause occurs in the same conditions. These differences are probably connected with the photoperiods experienced by the parental generation. Next we examined reactivation at 25°C. This high temperature terminates the diapause very quickly and no essential difference between the action of longand short-day regimes is observed. Thus in the progeny of the short-day-treated flies correspondingly 57 and 58 per cent of the larvae pupate on the 4th day, 81 and 71 per cent on the 11th day, and 91 and 84 per cent on the 18th day. pupation
81
E. B. VINOGRADOVA
2492
IO
20
so
40
50
60
(b)
Doys
FIG. 4. The dynamics of pupation of diapausing larvae at 20°C. The larvae were exposed to (a) 12 hr of light per day and (h) 20 hr of light per day. Other designations are the same as in Fig. 2.
TABLE ~-THE
DEPENDENCE OF THE REACTIVATION OF DIAPAUSING LARVAEOF C. r~i&zu ON THEDURATIONOF COOLINGAT 4°C Duration of cooling (months) 1
Daylength during oSgenesis of the females (hr of light per day) 18 17 15 12
No. Reactivated larvae of larvae (%) 80 40 100 175
68.7 75.0 17.0 4.5
2
3
No. Reactivated of larvae larvae (%)
No. Reactivated larvae of larvae (%)
85 40 100 150
87.0 100.0 850 83.3
79 39 96 116
100.0 100.0 84.3 99.1
REACTIVATION
IN
DIAPAUSING
BLOWFLY
LARVAE
2493
Special experiments were carried out to deal with the reactivation of C. vicin~ diapausing at low temperatures just above zero. For this purpose l-month-old larvae produced by females which were kept under different photoperiodic regimes were cooled to 4°C and then transferred into conditions favourable for morphogenesis (15.5% and short-day length). The pupae were counted on the tenth day. The dependence of reactivation on the length of the cooling period is shown in Table 2. One month’s cooling proved to be sufficient for reactivation of 69 to 75 per cent of the individuals in the progeny of the long-day-exposed flies. But in the progeny of the short-day-treated flies, a more intensive diapause is manifest and it takes longer period of cooling (2 or 3 months) to terminate their diapause. DISCUSSION
The results of these and earlier experimental investigations (VINOGRADOVA and ZINOVJEVA, 1972; ZINOVJEVAand VINOGRADOVA,1972) throw some light on the ecological regulation of the seasonal development of C. vicina. Larval diapause is controlled by the temperature and photoperiodic regimes during larval development and by the maternally operating photoperiod. The above-mentioned factors interact closely and the importance of each of them in diapause induction can vary depending on the actual ecological situation and on population peculiarities. In C. vi&a larval diapause is possible within certain temperature limits. Single diapausing larvae can appear if their development occurs at 20°C but a temperature of 15°C and lower is necessary for the induction of diapause in most of the individuals (more than 50 per cent). Special experiments have established the approximate time of the onset and manifestation of diapause. In about half of the larvae this occurs when they are approximately 15 days old and in the other half diapause is later, up to 1 month old (ZINOVJEVAand VINOGRADOVA,1972). The physiological state of the larvae is under maternal control. A maternally operating short photoperiod considerably strengthens predisposition to diapause in their progeny, but a maternally operating long photoperiod promotes continuous development of the larvae even if the ambient temperature is lowered. Such dependence of the larval physiological state on the maternally operating photoperiods is observed only with definite temperature limits, beginning approximately with 16°C and going up to 9°C. Further lowering of the temperature induces all the larvae to enter diapause, independent of the photoperiodic regime during the life of the females. The critical daylength determining the time of the appearance of diapausing insects in nature is adaptive and as a rule changes within the distribution area of a species (DANILEVSKY, 1961). A similar situation has been revealed in C. oicina. In the northern population from Belomorsk the photoperiodic threshold lies between 16 and 17 hr of light per day, but in the more southern population from Gorky it lies between 15 and 16 hr (VINOGRRDOVAand ZINOVJEVA, 1972). A more gradual decrease in the number of diapausing larvae with increase in the duration of illumination and connected with this a considerable percentage of diapausing
2494
E. B. VINOGRADOVA
individuals (20-33%), even in the progeny of long-day-treated females, is typical of C. vicina from Belomorsk. But in the strain from Gorky the increase of daylength by 1 hr within the photoperiodic threshold results in an abrupt decrease in the number of diapausing larvae from 99 to 3 per cent. Comparison of the photoperiodic induction in two geographical strains of C. vicina shows that the degree of maternal influence on the diapause in the progeny varies within this species. It has been shown previously that, within definite temperature limits, in the strain from Gorky the physiological state of the larvae is determined exclusively by the maternally operating photoperiod. But the strain from Belomorsk differs in this respect ; the physiological state of its larvae is under a double control and depends on both the imaginal and larval photoperiodic regimes, but the first plays the leading r61e. An analysis of the literature dealing with maternal induction of diapause in the progeny shows that during certain stages in the development of the daughter generation insects which have been studied retain a definite degree of independence in their reaction to environmental factors (VINOGRADOVA,1973). Therefore the physiological state of the progeny is a result of all those factors acting during the development of the maternal and daughter generations. The reactivation of C. viciw from Belomorsk has been shown to depend on the Investigations of the ability of different temperatemperature and photoperiods. tures (from 4 to 25°C) to break the larval diapause show that it is the extreme temperatures that are the most favourable for diapause development. This is confirmed both by the considerable percentage of reactivated larvae and by the short period needed for pupation to occur. As a rule at low temperatures just above zero it takes a considerable time to terminate the larval diapause. Thus in the progeny of the short-day-treated females most of the larvae pupate after being cooled at 4°C for 2 or 3 months if the temperature is raised to that favourable for But 1 month’s cooling at 4°C is enough to terminate a less inmorphogenesis. tensive diapause in the progeny of long-day-treated females. Similar data concerning the duration of the period of reactivation were obtained for. another blowfly, Lucilia cmsar (RING, 1968). A n increase of temperature from 12.5 to 25°C accelerates the processes of reactivation and correspondingly the rate of pupation. For instance, with a short daylength in the progeny of short-dayexposed females at 12.5”C mass pupation begins only on the 79th day, at 15*5”C on the 60th, at 20°C on the 7th to 15th, and at 25°C on the 2nd or 3rd day. Experiments on C. vicina have revealed that photoperiodic reactivation is possible. Long photoperiods stimulate resumption of development. Under a long-day regime the rate of pupation is two to four times greater when compared with that observed under a short-day regime. At 25°C reactivation occurs so rapidly that the difference between the short and long daylengths is not manifest. Up to the present time photoperiodic reactivation has been established in numerous insects (about 25 species) which hibernate mainly as a larva or an adult (MUELLER, 1965; DANILEVSKY and SHELDESHOVA,1968). No such data are available so far for flies, but photoperiodic reactivation is known in other Diptera, namely in some
REACTIVATION OFDIAPAUSING BLOWFLY LARVAE
2495
mosquitoes with larval diapause-AZdes triseriatus (BAKER, 1935 ; LOVE and WHELCHEL, 1955 ; VINOGRADOVA,1969), Anopheles p1umbeu.q A. claw&r (VINOGRADOVA,1969), Weyomyia smithii (SMITH and BRUST,1971), A. barberi, Orthopodomyia signifera (BAKER, 1935), Chaoborus americanus (BRADSHAWand SHAPPIRIO, 1966; BRADSHAW,1969), Toxorhynchites rutilus (JENNERand MCCRARY, 1964), and also Chironomus tentans (ENGELMANNand SHAPPIRIO,1965). Considerable differences are observed in the process of reactivation in the progeny which originates from females exposed to different photoperiods. Long photoperiods induce only a small percentage of the larvae to enter diapause (2-33 per cent), but short photoperiods induce most of the individuals (84-95 per cent) to enter diapause. In the first case the diapause is less intensive and can be easily terminated as a result of the influence of a long-day regime or an increase in temperature. A more intensive diapause is typical for the progeny of short-daytreated females and it takes a more prolonged influence of environmental factors to break this diapause. Thus the photoperiods to which the females have been exposed determine not only the ratio of non-diapausing and diapausing larvae in the progeny but also the intensity of the diapause too. The only case of an analogous inhibitory influence of a maternal origin is observed in the moth Orgyia antiqua (MASLENNIKOVA,1972) where the intensity of the egg diapause depends on the photoperiod of the female.
REFERENCES
BAKERF. C. (1935) The effect of photoperiodism
on resting treehole mosquito larvae.
Can. Ent. 67, 149-153.
BRADSHAW W. E. (1969) Major environmental factors inducing the termination of larval diapause in Chaoborzls americanus (Diptera, Culicidae). Biol. Bull., Woods Hole 136, 2-8. BRADSHAW W. E. and SHAPPIRIO D. G. (1966) The rble of food, photoperiod and temperature in the termination of the larval diapause in Chaobovus (Diptera, Cubcidae). Am. Zool. 6, 508.
DANILEVSKY A. S. (1961) Photoperiodism and Seasonal Development of Insects. Leningrad State University.
In Russian.
DANILEVSKY A. S. and SHELDESHOVA G. G. (1968) Adaptive significance of photoperiodic and cold reactivation. In Photoperiodic Adaptations of Insects and Mites, pp. 80-90. Leningrad State University.
In Russian.
ENGELMANN W. and SHAPPIRIO D. G. (1965) Photoperiodic control of the maintenance and termination of larval diapause in Chironomus tentans. Nature, Lond. 207, 548-549. JENNER CH.E. and MCCRARYA. B. (1964) Photoperiodic control of larval diapause in the giant mosquito Toxorhynchites rut&s. Am. Zool. 4, 434. LOVE J. and WHELCHEL J. G. (1955) Photoperiodism and the development of Aedes triseriatus. Ecology 36, 340-342.
MASLENNIKOVA V. A. (1972) Influence of hormonal balance of diapausing insects on their reactivation. In Problems of Photoperiodism and Diapause in Insects, pp. 229-241. Leningrad State University.
In Russian.
MULLERH. J. (1965) Probleme der Insektendiapause. Verb. deutsch. Zool. Ges. 192-222. RING R. A. (1968) Termination of diapause in larva of Lucilia Caesar L. (Diptera, Calliphoridae).
Can. J_ Zool. 46, 335-344.
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SMITH S. M. and BRUST R. A. (1971) Photoperiodic control of the maintenance and termination of larval diapause in Weyomyia smithii Coq. (Diptera, Culicidae) with notes on osgenesis in the adult female. Can.J. 2001. 49, 1065-1073. VINOGRADOVA E. B. (1969) Diapause of Bloodsucking Mosquitoes and its Regulation. Nauka, Leningrad. In Russian. VINOGRADOVA E. B. (1973) Maternal influence on the diapause of the progeny in insects. In 25th Annual Readings in Memory of IV. A. Cholodkowsky, pp. 39-66. Leningrad. In Russian. VINOGRADOVA E. B. and ZINOVJEVAK. B. (1972) Maternal induction of larval diapause in the blowfly, Calliphora vicina. J. Insect Physiol. 18,2401-2409. ZINOVJ~VAK. B. and VINOGRADOVA E. B. (1972) The control of seasonal development in parasites of blowflies-II. Ecological control of winter adaptations in CaZZiphora vi&a R.-D. (Diptera, Calliphoridae). In Host-Parasite Relationships in Insects, pp. 90-99. Nauka, Leningrad. In Russian.