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Keywords:

  • Circadian clock;
  • diapause capability;
  • low-diapause variant;
  • mode of inheritance;
  • photoperiodic response;
  • post-feeding larval period;
  • pupal diapause;
  • pupariation;
  • quantitative photoperiodic time measurement system;
  • Sarcophaga similis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Abstract A variant of the flesh fly Sarcophaga similis, which does not enter pupal diapause even under diapause-inducing conditions, is established by artificial selection. Interline crosses of the wild-type and the variant revealed that the diapause capability is inherited in an incomplete dominant manner. Neither sex linkage nor a maternal factor is involved in the mode of inheritance. Genetic and genetic–environmental interactions are involved in the induction of diapause. In the wild-type, the post-feeding larval period is prolonged in response to short days. However, in the variant, the duration of the larval period under short-day conditions is identical to that under long-day conditions. Long-day responses under short-day conditions at both the larval and pupal stages in the variant indicate that the responses are established by common factors in both stages. The factors causing the long-day responses under short-day conditions at different developmental stages in the variant remain to be identified. However, it is plausible to hypothesize that common factors regulate ecdysteroid release at both the larval and pupal stages, and that malfunction of the system regulating these factors triggers such ecdysteroid release, irrespective of the photoperiod. Hence, the photoperiodic responses disappear in the variant in both stages. The adult stage of the variant has a functional circadian clock, suggesting that the nondiapause phenotype is not necessarily involved in malfunction of the circadian clock.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Most insects enter diapause in the autumn in response to short days in order to overcome adverse winter conditions. Such a photoperiodic response is widely observed in insects inhabiting the temperate zone (Danks, 1987). Although diapause is induced by seasonal stimuli, the capability to enter diapause is genetically determined. It is widely accepted that a large part of the geographic and latitudinal variation in photoperiodic responses is due to the considerable genetic polymorphism within populations (Tauber et al., 1986). There is a large literature on the genetics of capability to enter diapause for geographic strains or selected lines but most of them merely report the mode of inheritance and do not explain the role played by the detected loci in the photoperiodic induction of diapause (Tauber et al., 1986; Saunders, 2002).

Flesh flies enter pupal diapause in response to short-day conditions (Denlinger, 1971; Kurahashi & Ohtaki, 1979; Tanaka et al., 2008). They also show a clear photoperiodic response in the larval period (i.e. larvae reared under short-day conditions pupariate later when compared with those reared under long-day conditions; Henrich & Denlinger, 1982; Bradley & Saunders, 1986; Moribayashi et al., 1988, 2008). Thus, the flesh fly shows photoperiodic responses in the larval stage as well as in the pupal stage; however, the genetic relationship between these responses at different stages is unknown. In the present study, a low-diapause (ld) variant is selected from a wild-type (wt) colony of the flesh fly Sarcophaga similis (Meade). Using the variant line, the mode of inheritance of diapause capability and the genetic association between the photoperiodic responses of the larval and pupal stages are investigated. The normal functioning of the circadian clock in the ld variant is also investigated because recent studies have reported abnormal functioning of the circadian clock coincident with the nondiapause phenotype in some insect species (Hodkováet al., 2003; Pavelka et al., 2003; Goto et al., 2006).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A wt colony of S. similis (Meade) captured from the campus of Osaka City University (34°35¢N, 135°30¢E) in 2002 was maintained as described previously (Denlinger, 1972). Flies were reared under experimental photoperiodic conditions from their embryonic stage. All the experiments were performed at 20 °C unless otherwise mentioned. The diapause status was checked according to the criterion of Fraenkel & Hsiao (1968) at 20 days after larviposition.

An ld variant strain was established from the wt colony. A few adults that had not entered diapause and emerged even under diapause-inducing conditions (LD 12 : 12 h at 20 °C) were mated. The progeny were reared under the same conditions as those used for rearing the parental strains, and the selection procedure was repeated. In the fifth generation, no pupae entered diapause and no selection was possible. Therefore, further generations of the strain were maintained under diapause-averting conditions (LD 16 : 8 h at 25 °C).

Interline crosses (wt × ld and ld × wt; female indicated on the left) were performed by the mass mating of the males of one line with females of the other under various photoperiodic conditions; the progeny were continuously reared under conditions identical to those used for rearing the parental strains.

Backcrosses and hybrids for F1 crosses were obtained from the parents that were reared under LD 16 : 8 h at 25 °C and had not entered diapause in order to avoid an environmentally induced reduction in diapause due to a factor of diapause history, such as maternal effect. The progeny of these crosses were reared under LD 12 : 12 h at 20 °C to generate data for the groups: wt × (wt × ld), wt × (ld × wt), (wt × ld) × ld, (ld × wt) × ld, ld × (wt × ld), ld × (ld × wt), (ld × wt) × ld, (wt × ld) × ld, (wt × ld) × (wt × ld), and (ld × wt) × (ld × wt).

All the equations used for the present analyses were used by Henrich & Denlinger (1983). In brief, the expected diapause incidence (E) for an additive model of a backcross to the wt (BCwt), backcross to the ld (BCld), and F2 was generated with the equations:

  • image
  • image
  • image

where D indicates the observed diapause incidence of the designated population. If the observed and expected values in the F2 group are equal:

  • image
  • image

The duration of the post-feeding larval period was measured under LD 12 : 12 h and LD 16 : 8 h at 20 °C. Mature larvae just after the onset of post-feeding (wandering) stage (5 days after larviposition) were transferred to dry wood chips to induce pupariation. Puparia were counted every day.

Locomotor activities of the variant and wt flies are of interest to see whether alteration of diapause capability coincides with an alteration in the circadian clock. However, analysis of the locomotor rhythmicity of feeding-stage larvae is difficult because they burrow into the food. In addition, analysis of the post-feeding larval stage is also impossible because of its short duration, especially for the wt flies reared under LD 16 : 8 h and the ld variants reared under LD 16 : 8 and 12 : 12 h (see Results). Therefore, the locomotor activity of the adults, and not the larvae, was investigated. An activity recording system was used (Hamasaka et al., 2001). Because preliminary experiments revealed that adult flies were less active at 20 °C, a temperature of 25 °C was adopted. Adult males that emerged under LD 16 : 8 h at 25 °C were each transferred into a monitoring system. The flies had free access to 5% sucrose solution. Activity was recorded under constant darkness for 7 days. Locomotor activity was measured in terms of the number of times that a fly interrupted the infrared beam. The number was summed every 6 min. The free-running period was determined by a chi-squared periodgram analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To investigate diapause capability, the wt and ld flies as well as the F1 progenies obtained by their mating were reared under various photoperiodic conditions (Fig. 1). The wt flies showed a clear photoperiodic response for the induction of pupal diapause, with a critical day length of approximately 12.7 h. The ld variants did not enter diapause under diapause-inducing conditions of LD 12 : 12 h at 20 °C but approximately 20% of individuals did under short-day conditions (LD 10 : 14 and 8 : 16 h at 20 °C). At a temperature of 15 °C, the wt flies also showed a clear photoperiodic response; diapause incidence was 0.8% (n = 386) and 100.0% (n = 160) under LD 16 : 8 and 12 : 12 h, respectively. At this temperature, the ld variants demonstrated a weak but clear photoperiodic response (χ2 test, P < 0.01); diapause incidence was 1.5% (n = 270) and 36.1% (n = 158) under LD 16 : 8 and 12 : 12 h, respectively.

image

Figure 1. Photoperiodic response curve for the induction of pupal diapause in Sarcophaga similis for the wild-type (wt), low-diapause (ld) variant, and their F1 progeny (n = 95–360). Different letters at each photoperiod indicate that the diapause incidence was significantly different among samples (Tukey-type multiple comparison test for proportions, P < 0.01; Zar, 1999).

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The photoperiodic response curves of the F1 progenies were almost identical, and as compared with the responses of the parental strains, those of the F1 progenies were intermediate. This indicated that the genetic factors responsible for the differences in diapause induction between the strains involve neither sex linkage nor a maternal factor. Under LD 12 : 12 and 10 : 14 h, the diapause incidences in the F1 progenies tended to similar to that of the wt strain, suggesting that the diapause phenotype was an incomplete dominant trait. Under LD 13 : 11 h, however, the diapause incidences in the F1 progenies tended to similar to that of the ld strain. This implies that diapause is not merely promoted by the F1 genotype and that diapause induction is also influenced by interactions between F1 genotype and photoperiod.

The diapause incidence in the progeny from all the crosses under LD 12 : 12 h at 20 °C is shown in Table 1. Although slight differences exist among the data obtained from F1, F2, and the backcrosses, the data were divided into four groups for subsequent analyses: F1, F2, backcross to the wt strain (BCwt), and backcross to the ld strain (BCld). Figure 2 shows the actual data and the data predicted by an additive model with incomplete dominance. It is clear that genetic factors play an important role in determining the capability for diapause. However, the diapause incidence in F2, BCwt, and BCld individuals is slightly different from the predicted incidence for these populations. The chi-squared test revealed that the results obtained for the F2, BCwt, as well as BCld individuals did not fit the predictions of the additive model for these populations (P < 0.05 for F2 and P < 0.01 for BCwt and BCld; Fig. 2, Table 1). This suggested that genetic and genetic–environmental interactions are involved in diapause induction.

Table 1.  Diapause incidence of parental, F1, F2 and backcrosses using the wild-type (wt) and a variant (ld) of Sarcophaga similis under LD 12:12 h.
Cross (female × male)NDiapause (%)Expected value (additive model) (%)
  • * P < 0.05; **P < 0.01 (χ2).

  • Data from Figure 1.

  • BCwt, backcross to the wt; BCld, backcross to the ld.

wt × wt275100.0 
ld × ld2500.0 
wt × ld24279.3 
ld × wt17374.0 
F1 (Cum.)41577.1 
(wt × ld) × (wt × ld)27376.2 
(ld × wt) × (ld × wt)48665.6 
F2 (Cum.)75969.463.6*
wt × (wt × ld)25399.2 
wt × (ld × wt)45489.6 
(wt × ld) × wt41494.4 
(ld × wt) × wt42995.6 
BCwt (Cum.)155094.188.6**
ld × (wt × ld)18041.1 
ld × (ld × wt)28016.1 
(wt × ld) × ld31928.5 
(ld × wt) × ld23726.2 
BCld (Cum.)101626.838.6**
image

Figure 2. A comparison between the observed diapause incidence under LD 12 : 12 h at 20 °C among the progeny of crosses involving lines of Sarcophaga similis and the results predicted by an additive model with incomplete dominance (χ2 test, *P < 0.05, **P < 0.01). See also Table 1.

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Figure 3 shows the time of pupariation after the transfer of mature larvae to dry conditions. The wt flies reared under LD 16 : 8 h pupariated significantly faster than those reared under LD 12 : 12 h. Thus, the post-feeding larval period was significantly longer in individuals reared under LD 12 : 12 h than in those reared under LD 16 : 8 h (Steel–Dwass test, P < 0.05; Zar, 1999). However, individuals of the ld strain exhibited no such photoperiodic response (Steel–Dwass test, P > 0.05).

image

Figure 3. Time of pupariation for the wt and ld variant of Sarcophaga similis under long-day conditions (LD 16 : 8 h) and short-day conditions (LD 12 : 12 h) at 20 °C. The post-feeding larval period is represented as the mean ± SD. Different letters after the values indicate that the duration of the post-feeding larval period was significantly different among samples (Steel–Dwass test, P < 0.05; Zar, 1999). (n = 49–60).

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The wt and ld adults both showed clear circadian locomotor rhythmicity under constant darkness. The free-running period (τ), expressed as mean ± SD, was 24.8 ± 2.8 h (n = 11) and 24.1 ± 2.0 h (n = 6) for the wt and ld flies, respectively. The difference between the periods was not significant (Student's t-test, P > 0.05). Representative locomotor activities of the wt and ld flies are shown in Figure 4.

image

Figure 4. Examples of the locomotor activity rhythm of (a) wild-type (wt) and (b) low-diapause (ld) individuals of Sarcophaga similis under constant darkness for 7 days. The results are presented as a double plotted actogram and chi-squared periodgram analysis. In these examples, the free-running periods of the wt and ld flies are 23.4 and 23.5 h, respectively.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Saunders (2002) proposes a model for photoperiodic phenomena comprising ‘a photoperiodic photoreceptor' to distinguish light from dark, ‘a photoperiodic clock' to measure the length of the day or night, and ‘an output' to regulate seasonal responses such as diapause. The clock is subdivided into ‘a photoperiodic time measurement system' and ‘a counter'. A circadian clock is clearly involved in the photoperiodic clock (Saunders, 2002). Thus, it is plausible that mutations in any of the genes involved in any steps of the cascade have severe adverse effects on the induction of photoperiodic responses such as diapause. This further indicates that the loci responsible for the nondiapause phenotype are not necessarily homologous in various species or strains, as suggested by the different modes of inheritance presented in previous reports. For example, in the flesh fly Sarcophaga bullata (Henrich & Denlinger, 1983), Boettcherisca peregrina (Kurahashi & Ohtaki, 1977), the face fly Musca autumnalis (Kim et al., 1995), and the milkweed bug Oncopeltus fasciatus (Hayes et al., 1987), the diapause capability trait is inherited in an incomplete dominant manner. On the other hand, this trait follows a simple Mendelian inheritance pattern in some insect species, including the drosophilid fly Cymomyza costata (Pavelka et al., 2003) and the linden bug Pyrrhocoris apterus (Doležel et al., 2005). In these cases, a single locus has a large effect on diapause induction.

In the present study, S. similis individuals are artificially selected for diapause incidence. The ld variants do not undergo diapause under LD 12 : 12 h at 20 °C, which is the diapause-inducing condition in the wt strain. The nondiapause phenotype in the variant is established within the fifth generation, indicating that a small number of loci are involved in the phenotype. Interline crosses reveal that the capability to enter diapause is inherited in an incomplete dominant manner. The involvement of neither sex linkage nor a maternal effect is detected. The interline crosses suggest the involvement of genetic and genetic–environmental interactions in the diapause induction. Under LD 12 : 12 and 10 : 14 h, the diapause incidences of the F1 progenies tends to be similar to that of the wt but, under LD 13 : 11 h, the diapause incidences of the F1 progenies tends to be similar to that of the ld variants. The results indicate that the F1 genotype does not merely promote diapause and that diapause induction is also influenced by interactions between F1 genotype and photoperiod.

Interestingly, among the ld variants, a small proportion of individuals successfully enters diapause under LD 10 : 14 and 8 : 16 h at 20 °C and LD 12 : 12 h at 15 °C. Thus, the ld strain is able to discriminate between photoperiods and still possesses the capability to enter diapause; however, it is less sensitive to diapause-inducing stimuli. This suggests that the photoperiodic photoreceptor and photoperiodic clock still function in the ld strain.

For the sake of simplicity, the photoperiodic time-measurement system is usually discussed as a qualitative or all-or-none mechanism in the most studies (i.e. in the assumption of qualitative mechanism, the photoperiodic clock distinguishes long from shot days, or short from long nights; Denlinger et al., 2005). However, recent models for photoperiodic time measurement incorporate a quantitative concept (Saunders, 2002). Such quantitative time measurement is indeed observed in a number of insect species (Hardie, 1990; Kimura, 1990; Spieth & Sauer, 1991; Numata & Kobayashi, 1994). The photoperiodic response in the ld variant also reveals the quantitative photoperiodic time measurement in S. similis. The wt enters diapause irrespective of photoperiods when the photophase is shorter than the critical day length (CDL) but they develop without diapause when the photophase is longer than the CDL. However, the ld variant can successfully discriminate between LD 12 : 12 and LD 10 : 14 h. Thus, it is plausible that S. similis measures photoperiods quantitatively, not qualitatively, and shorter days are recognized as strong diapause-inducing conditions.

There are reports that the larvae of some flesh flies, including Sarcophaga crassipalpis (Giebultowicz & Denlinger, 1986), Sarcophaga argyrostoma (Bradley & Saunders, 1986) and B. peregrina (Moribayashi et al., 1988, 2008) exhibit a photoperiodic response in the larval period. Wt S. similis individuals also show a clear photoperiodic response in the post-feeding larval period. On the other hand, the ld strain has lost the ability to respond. Long-day responses under short-day conditions in both the larval and pupal stages of the ld variant reveal that, in both stages, the photoperiodic responses are established by common factors. In S. bullata, the selection for late pupariation (long larval period) results in a line with a higher diapause incidence than an unselected line (Henrich & Denlinger, 1982). On the basis of the results, two hypotheses are proposed: the loci that affect late pupariation are involved in diapause capability and the selected line is exposed to a larger number of short days because of its longer larval period and the increased exposure to short-day conditions directly promotes diapause according to the ‘required day number' hypothesis proposed by Saunders (1971). The short larval period produced by nondiapause selection in the present study strongly supports the former hypothesis; hence, it is possible that common genetic factors are involved in the photoperiodic responses both in the larval and pupal stages. However, there remains the possibility that the loci responsible for the short larval period are completely different from those responsible for the nondiapause phenotype but that the former is unintentionally selected by selection.

It is well known that pupariation is promoted by ecdysteroids and that diapause is induced by the lack of these compounds (Denlinger et al., 2005). In B. peregrina, nondiapause-destined mature larvae reared under long-day conditions show many small and large ecdysteroid peaks during larval–pupal and pupal–adult development. On the other hand, diapause-destined larvae reared under short-day conditions show only one small peak of ecdysteroid synthesis; thereafter, the level remains very low and the pupae enter diapause (Moribayashi et al., 1988). The striking difference between ecdysteroid titres is assumed to be caused by differences in the levels of brain hormones (e.g. prothoracicotropic hormone) that regulate ecdysone synthesis and secretion by the ring gland (Denlinger et al., 2005). In S. crassipalpis, pupariation time and developmental commitment (diapause or nondiapause) are both determined by the brain-ring gland complex (Giebultowicz & Denlinger, 1986). Delayed pupariation (long larval period) that is manifested under short-day conditions depends on delayed ecdysone secretion from the ring gland in B. peregrina (Moribayashi et al., 1988). The factors causing long-day responses under short-day conditions at different developmental stages in the ld variant remain to be identified. However, it is plausible to hypothesize that common factors regulate ecdysteroid release at both the larval and pupal stages and alteration of the system regulating the factors triggers ecdysteroid release even under short-day conditions. These hypotheses explain why the photoperiodic responses observed at different developmental stages disappear in the ld variant.

Recent studies in some insect species demonstrate that malfunction of the circadian clock produces the nondiapause phenotype (Hodkováet al., 2003; Pavelka et al., 2003; Goto et al., 2006). These results are interpreted in terms of involvement of a circadian clock into the photoperiodic clock (Saunders, 2002). In the present study, the adult stage of the ld variant has a functional circadian clock and the free-running periods of the wt and ld variant are identical. Saunders & Cymborowski (2003) also report on the linkage between circadian locomotor rhythmicity and photoperiodic response, namely the artificial selection of a line showing longer critical daylength and high larval diapause incidence even under long-day conditions in the blow fly Calliphora vicina. The selected line shows mean circadian periods for adult locomotor activity that differ very little from the original stock or from flies selected for high diapuase incidence under short-day conditions. Thus, the diapause capability is not necessarily involved in the alteration of circadian clock, and diapause capability and circadian behaviour are genetically separable (Lankinen & Forsman, 2006; see also Saunders, 2002; Bradshaw & Holzapfel, 2007). Indeed, several studies on a geographic gradient of seasonality reveal that photoperiodic responses and circadian rhythmicity have evolved independently in mice, mosquitoes and flies (Bradshaw & Holzapfel, 2007). This is reasonable because mutations in any genes downstream of a circadian clock may modify the diapause capability trait.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), a Grant-in-Aid for Young Scientists (B), 18770053, 2006-2008 to S.G.G.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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