A significant proportion of cry^b/cry^b, Rh7¹/Rh7¹ adults or cry^b/cry⁰¹, Rh7¹/Rh7¹ adults previously trained under 12h:12h light:dark cycles fail to maintain rhythmicity under constant darkness conditions, while the rhythmic individuals present a significantly longer period, as compared to controls. The defects of cry^b, Rh7¹ double homozygotes to maintain circadian rhythmicity under constant darkness is partially suppressed by the following conditions: the presence of Rh7^{+tCH322-180G19}; the expression of Rh7^Scer\UAS.cNa under the control of either Scer\GAL4^P2.4.Pdf or Scer\GAL4^tim.PE; the expression of Rh7^{Scer\UAS.T:Zzzz\FLAG} under the control of Scer\GAL4^P2.4.Pdf; or the expression of Rh3^Scer.UAS.cVa, Rh4^Scer.UAS.cVa or Rh5^Scer.UAS.cVa under the control of Scer\GAL4^P2.4.Pdf.

Following a 6 hour delay in their light-dark cycle, cry^b Rh5² flies and cry^b Rh6¹ flies show a similar delay in resynchronizing their evening activity peak as cry^b flies. Triple mutant cry^b Rh5² Rh6¹ flies resynchronize over a longer period than either the cry^b single or double mutants.

Following a 6 hour delay in their light-dark cycle, cry^b norpA³⁹ flies show a longer delay in resynchronizing their evening activity peak (5-7 days) compared to cry^b flies. The addition of Rh5² or Rh6¹ to this background extends the time required for resynchronization to beyond 7 days. The addition of both Rh5² and Rh6¹ to the cry^b norpA³⁹ background results in minimal (if any) tendency to resynchronize circadian behaviour.

Following a 6 hour advancement in their light-dark cycle, cry^b norpA³⁹ flies show a longer delay in resynchronizing their circadian behaviour (several days) compared to either single mutant. Introducing either Rh5² or Rh6¹ to this background interferes more strongly with resynchronization, while the quadruple cry^b norpA³⁹ Rh5² Rh6¹ mutant shows the strongest phenotype.

ninaE¹⁷ cry^b double mutants need more days to adapt to a new phase of green light (472-603nm) and Y-FL (475-724nm) compared to cry^b single mutants.

A Rh6¹ mutant background does not affect the ability of cry^b mutants to adapt to a new phase of green light (472-603nm) and Y-FL (475-724nm).

A Rh5³ mutant background does not affect the ability of cry^b mutants to adapt to a new phase of green light (472-603nm) and Y-FL (475-724nm).

The retrainment of ninaE¹⁷ Rh6¹ cry^b to 6 hour delayed green light (472-603nm) and Y-FL (475-724nm) is significantly slower than that of ninaE¹⁷ cry^b mutants. Indeed some flies do not adapt to the new photoperiod within 7 days.

ninaE¹⁷ Rh5³ Rh6¹ cry^b and ninaE¹⁷ Rh5⁴ Rh6¹ cry^b quadruple mutants are not retrained by green light (472-603nm) and Y-FL (475-724nm).

In contrast to wild-type animals, per⁰¹; ; cry^b double mutants expressing Scer\GAL80^Pdf.PS and Scer\GAL4^Mai179 driven per^Scer\UAS.cBa display robust locomotor activity rhythms, with a long 25.7h period under constant light conditions.

Under constant darkness, cry^b Pdf⁰¹ double mutants display locomotor rhythms that are similar to cry^b mutants, but with a short 22.8h period. Conversely, cry^b Pdf⁰¹ double mutants display locomotor rhythms that are similar to Pdf⁰¹ single mutants under constant darkness, with a fraction of the flies displaying a weak short-period rhythmicity as reported for Pdf⁰¹.

Flies expressing sgg^EP1576 under the control of Scer\GAL4^tim.PE in a cry^b background have a locomotor activity period of 22.6 hours under constant darkness conditions, which is longer (and closer to that of wild type) than the locomotor activity period of 20.3 hours seen under these conditions in flies expressing sgg^EP1576 under the control of Scer\GAL4^tim.PE in wild-type background.

tim^ls/+, jet^c/+ (Veela/+); cry^b/+ flies exhibit robust rhythmicity in constant light, this phenotype is stronger than in tim^ls/+, jet^c/+ (Veela/+) flies and is not seen in cry^b/+ flies.

The main difference between cry^b and eya¹;cry^b double mutants is the absence of the compound eyes in the eya¹;cry^b flies.

Under LD conditions eya¹;cry^b double mutant flies exhibit the typical bimodal activity pattern consisting of M and E activity bouts, as in wild-type. After transfer into LL conditions, eya¹;cry^b flies remain rhythmic and free-run with a period close to 24 hours (whereas wild-type flies become immediately arrhythmic).

eya¹;cry^b double mutants do not exhibit internal desynchronisation under LL conditions, and free-run with a wild-type-like period.

per⁰¹; cry^b double mutants exhibit a functional clock with a well-defined evening activity peak anticipating `lights off' that is absent in per<sup>01</sup> mutants. The evening peak in activity also arrives earlier during the light phase than the wild-type, possibly suggesting an underlying short-period oscillation. tim<sup>01</sup>;cry<sup>b</sup> double mutants and per<sup>01</sup>;tim<sup>01</sup>;cry<sup>b</sup> triple mutants do not show any locomotor anticipation of lights off at 18<sup>o</sup>C, although some residual behaviour is recovered at 29<sup>o</sup>C. tim<sup>01</sup>;cry<sup>b</sup> double mutants and per<sup>01</sup>;tim<sup>01</sup>;cry<sup>b</sup> triple mutants exhibit no change in the phase relationship of the locomotor peak to the lights off signal, as the peak activity moves progressively later as it tracks increasing length light-dark cycles. Hence cry<sup>b</sup> suppresses the effect of per<sup>01</sup> on circadian activity. This is confirmed by the similar behaviour of per<sup>01</sup>;tim<sup>01</sup>;cry<sup>b</sup> to per<sup>01</sup>;cry<sup>b</sup> mutants. Under constant darkness or constant light, the rhythmicity of per<sup>01</sup>;cry<sup>b</sup> breaks down during the first day. In per<sup>01</sup>;cry<sup>b</sup> mutants, brief light pulses administered at 18<sup>o</sup>C during the night phase at ZT15 and ZT21 in light-dark 12:12 cycles give advances in the phase of locomotor activity. Thus the `clock' of these flies `remembers' a previously administered light pulse, even in light-dark cycles.

Overexpression of 5-HT1B^Scer\UAS.cYa under the control of Scer\GAL4^tim.PE in a cry^b homozygous background does not have a discernible effect on the flies light sensitivity due to the strong phenotype produced by the cry^b mutation. However, ~87% of the cry^b heterozygotes overexpressing 5-HT1B^Scer\UAS.cYa under the control of Scer\GAL4^tim.PE are rhythmic in constant bright light, while only 13% of the control flies are rhythmic under these conditions. Fast Fourier Transform analysis indicates that cry^b heterozygous flies overexpressing 5-HT1B^Scer\UAS.cYa under the control of Scer\GAL4^tim.PE have very strong behavioural rhythms in constant bright light, although the average period is lengthened to about 28 hours.

Reducing 5-HT1B expression, through induction of 5-HT1B^IR.Scer\UAS, under the control of Scer\GAL4^tim.PE, or increasing cry levels through cry^Scer\UAS.cDa expression, eliminates the strong behavioural rhythms in constant bright light observed in cry^b heterozygous flies overexpressing 5-HT1B^Scer\UAS.cYa under the control of Scer\GAL4^tim.PE.

A large number (~43%) of cry^b flies overexpressing 5-HT1B^Scer\UAS.cYa under the control of Scer\GAL4^tim.PE are arrhythmic in dark-dark conditions, as compared to 7% in control cry^b flies.

When the locomotor activity of wild-type flies are monitored at 18 and 29^oC, an earlier evening peak is seen at 18^oC. This is not seen in norpA⁴¹, cry^b mutants.

W^GMR.PG; cry^b flies show anomalous rhythmicity in when kept in constant light, like cry^b flies. The longer period of rhythmicity seen when cry^b are kept in constant light of high intensity is suppressed when the flies are also mutant for either W^GMR.PG, gl^60j or norpA³⁶.

gl^60j, cry^b and W^GMR.PG; cry^b flies fail to entrain to light-dark cycles of even the highest amplitude.

so¹; cry^b double mutants have the same partially penetrant eye phenotype as so¹ single mutants. When these mutants have eye remnants, they can re-entrain to to 6 hour phase advances of the LD cycle in green or red light, but not blue light. When these mutants are completely eyeless, they are unable to re-entrain at any wavelength and their evening activity peak occurs after lights off, while flies with eye remnants show a peak of activity two hours before lights off, as do wild-type flies. Unlike so¹ single mutants, so¹; cry^b double mutants rarely show antidromic phase shifting.

gl^60j cry^b double mutant flies do not entrain at all to 12 hour light-12 hour dark (LD) cycles. At 100 lux, approximately 70% of the flies show free-running behaviour. The average activity shows a small peak after lights off at 100 lux and at 1000 lux virtually all flies react to lights off with a pronounced activity peak. At 1000 lux, the activity level is generally lower during the light phase than during the dark phase. A free-running circadian activity component is present in 81% of the activity records, and after transfer into constant darkness conditions rhythmic locomotor behaviour usually continues from this free-running component and not from the lights-off peak. Approximately 30% of gl^60j cry^b double mutant flies show poor rhythmicity, even under LD conditions. At 100 lux, about 20% of the flies are arrhythmic, and about 10% show a complex rhythmicity characterised by several small but significant peaks in the periodograms. gl^60j cry^b double mutants show neither period changes nor any increase in activity level or intervals between morning and evening locomotor activity peaks after transfer from constant darkness to constant light conditions.

In light-dark (LD) conditions per^s,cry^b double mutant flies show a temperature dependant defect in locomotor activity cycles. At 18^oC all flies show a periodic component of about 24 hours (as seen in wild-type) but about 60% of them also display a minor 19 hour component. At 28^oC 79% of the rhythmic flies display the endogenous 19 hour period as the main rhythmic component. When per^s,cry^b double mutant flies are entrained under an light-dark (LD) 10:10 regime - the entrainment breaks down very rapidly during the first two cycles of the new light/dark regime, with their daytime activity advancing by about 90 minutes in each successive day.

norpA³⁹; cry^b double mutants take very long to entrain to a new LD regime, some flies do not even reach a new phase by the eleventh day after the shift.

Double mutants of cry^b with norpA³⁹, neither of which cause entrainment problems alone, exhibit entrainment problems in the initial LD regime. Only 50% of those that entrain to the initial LD cycle were able to synchronize to a new light regime that applies 16-lux blue light. At lower light intensities nearly all of the double mutants fail to entrain to the new LD cycles. Double mutant per^s, cry^b flies have complex behavior under LD conditions. The double mutant shows both entrained and free running behavioral components. The double mutants are active at different times essentially every day, so their average activity plot shows a monotonic decrease in behavior throughout the daytime, whereas singly mutant per^s flies exhibit a pronounced midday peak of activity.

A Dple\cry1^{Scer\UAS.T:Hsap\MYC} transgene driven by Scer\GAL4^tim.PE successfully restores both naive and trained responses to magnetic field in a wavelength-dependent manner in the cry^b background.

Expression of Dple\cry2^{Scer\UAS.T:Hsap\MYC} under the control of Scer\GAL4^tim.PE successfully restores both naive and trained responses to magnetic field in cry^b mutants, and it does so in a wavelength-dependent manner.

cry^b mutant flies expressing Dple\cry1^{W328F.Scer\UAS} under the control of Scer\GAL4^tim.PE show appropriate, significant naive and trained responses to magnetic field.

Expression of Dple\cry1^Scer\UAS.cZa under the control of Scer\GAL4^tim.PE partially suppresses the cry^b circadian phase defects.

Expression of Dple\cry2^Scer\UAS.cZa under the control of Scer\GAL4^tim.PE fails to suppress the circadian phase defects in cry^b mutants.