axon & eye photoreceptor cell | conditional - heat sensitive, with Scer\GAL4GMR.PF
eye | heat sensitive, with Scer\GAL4GMR.PF
glial cell & brain & pupa | conditional ts, with Scer\GAL4repo
wing, with Scer\GAL4Hml.Δ
Adulthood-only expression of shi1.UAS under the control of Scer\GAL4esg.PU leads to a significant increase in the number of mitotic cells (phospho-H3 staining) in the adult midgut, as compared to controls. (The temporal regulation of expression is dependent on Gal80[ts] and induced by temperature shift to 29[o]C for 5-6 days during adulthood).
Scer\GAL4He.PZ-driven expression of shi1.Scer\UAS does not induce increase in the number of circulating hemocytes in third instar larvae relative to controls.
Expression of shi1.Scer\UAS under the control of Scer\GAL4ppk.PG at the permissive temperature of 25[o]C results in mild pruning defects class IV dendritic arborizing neurons at the pupal stage.
Expression of temperature-sensitive shi1.Scer\UAS under the control of Scer\GAL4109(2)80 in somatic clones results in compromised dendrite growth (slightly reduced total dendrite length, significantly increased number of dendrite crossings) in the class IV dendritic arborizing (da) neurons of third instar larvae (shifted to non-permissive temperature at 48hrs after egg laying and examined at 96hrs) when compared to controls.
Long-term memory is impaired in 10-day-old flies when neurotransmission from α/β mushroom body neurons is blocked at the time of retrieval, through expression of shi1.Scer\UAS under the control of Scer\GAL4c739. Blocking synaptic release across the time of conditioning, and during the 0-3 hour time window immediately after conditioning does not affect long term memory.
30-day-old flies expressing shi1.Scer\UAS in the α/β mushroom body neurons, under the control of Scer\GAL4c739, behave exactly as 10-day-old flies, exhibiting impairment in long-term memory with temperature shifts at retrieval.
Long term memory induced by 5x spaced conditioning is impaired when the synaptic output of DPM neurons is blocked through expression of shi1.Scer\UAS in the DPM neurons under the control of Scer\GAL4c316 in 10-day-old flies. No effect is observed when a synaptic blockade in DPM neurons is imposed after training during the early consolidation time window and at memory retrieval. Blocking the output of DPM neurons during 5xspaced conditioning has no effect on memory expression at 24h after conditioning in flies at 30 days of age.
The synaptic blockade during 5xspaced conditioning in Scer\GAL4VT64246>shi1.Scer\UAS flies significantly impairs memory expression at 24 h after conditioning at 10 but not at 30 days of age.
The synaptic blockade of the DPM neurons through expression of shi1.Scer\UAS under the control of Scer\GAL4VT64246 during 5x massed conditioning has no effect on memory tested at 24 h for both 10-day-old and 30-day-old flies.
Inhibition of shi in epidermal cells through overexpression of the temperature sensitive allele shi1.Scer\UAS under the control of Scer\GAL45-56 prevents phagocytic clearance of ddaC neuron dendrite debris following pupal stage dendritic pruning. The remaining dendrite fragments maintain the dendrite branching patterns at the basal side of epidermal cells. Similarly, when larval dendrites are injured (severed with an infrared laser) expression of shi1.Scer\UAS potently blocks the spreading of dendritic debris into larval epidermal cells and its subsequent degradation.
Mushroom body gamma neurons expressing shi1.Scer\UAS under the control of Scer\GAL4Tab2-201Y do not show axon pruning defects at the restrictive temperature.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4FoxP.1.5 show severely disturbed movement at elevated temperatures. Within minutes of being transferred to the elevated temperature they drop to the floor of the vial and show extremely uncoordinated and abnormal walking behaviour, are unable to climb and show spastic movements along the bottom of the vial. They eventually walk less and often stop moving, but they do not paralyse. The righting reflex is also impaired (they take longer to right themselves after being flipped on their backs).
Expression of shi1.Scer\UAS in octopaminergic neurons, under the control of Scer\GAL4Tdc2.PC results in water learning that is indistinguishable from that of control flies.
Expression of shi1.Scer\UAS under the control of either Scer\GAL40273 or Scer\GAL4GMR58E02, respectively, to block during training either the entire population of ~130 or a subpopulation of ~90 dopaminergic neurons in the protocerebral anterior medial cluster, results in significant impairment in water memory formation at 32[o]C but not at the permissive 23[o]C.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL40273 drink significantly less water during the 2-min training cycle. However, the magnitude of the decreased drinking is unlikely to account for the abolition of memory performance as these flies still consume a quantity of water that is comparable to that of wild-type flies at 23[o]C and is sufficient to form robust 3-min water memory.
Flies in which the protocerebral anterior medial cluster neurons were blocked for 30 minutes, through expression of shi1.Scer\UAS under the control of Scer\GAL40273 or Scer\GAL4GMR48B04, during memory testing display memory performance that is indistinguishable from controls.
Expression of shi1.Scer\UAS under the control of Scer\GAL4GMR48B04 results in a significant defect in water learning. This defect is not observed at the permissive 23[o]C temperature and water consumption and olfactory acuity.
Expression of shi1.Scer\UAS under the control of Scer\GAL40104 does not impair water learning.
Expression of shi1.Scer\UAS in around 55 dopaminergic neurons in the protocerebral anterior medial cluster, under the control of Scer\GAL4GMR48B04, results in a significant defect in water learning. The addition of Scer\GAL80GMR58E02, removing expression in this subset of the dopaminergic neurons, restores wild-type learning performance.
Expression of shi1.Scer\UAS under the control of Scer\GAL4GMR48B04 converts the behavior of naive thirsty flies from water approach to water avoidance. This behavioral reversal is not evident at the permissive temperature.
Expression of shi1.Scer\UAS under the control of Scer\GAL4GMR48B04 has no effect on water avoidance in sated flies, suggesting that these flies perceive water normally and that output from R48B04 neurons is only required for water approach in thirsty flies.
Addition of Scer\GAL80GMR58E02 to flies expressing shi1.Scer\UAS under the control of Scer\GAL4GMR48B04 suppresses the behavioral defect of naive thirsty flies (back to water approach from water avoidance).
Addition of Scer\GAL800104 to flies expressing shi1.Scer\UAS under the control of Scer\GAL4GMR48B04 abolishes naive water-seeking behavior in thirsty flies.
Suppression of the bgr;'2 neurons, through the addition of Scer\GAL800104 to flies expressing shi1.Scer\UAS under the control of Scer\GAL4GMR48B04 restores water-seeking behavior.
Expression of shi1.Scer\UAS in the L4 lamina cells under the control of Scer\GAL4VT40547 results in a selective loss of tangential cell response to moving ON and OFF edges.
Expression of shi1.Scer\UAS in the Tm2 lamina cells under the control of Scer\GAL4VT40547 results in a selective loss of tangential cell response to moving ON and OFF edges.
Late third instar larvae expressing shi1.Scer\UAS at 30[o]C show decreased cytoneme numbers compared with controls.
Blockade of astrocyte endocytic function during early pupal stages by expression of shi1.Scer\UAS, under the control of Scer\GAL4alrm.PD results in suppression of synaptic clearance from the neuropil and suppression of mushroom body γ neuron axon clearance.
Expression of shi1.Scer\UAS under the control of Scer\GAL4qvr.PW (using a temperature pulse of 287[o]C from ZT0-ZT6 to inactivate shi) results in an increase in sleep relative to controls.
Expression of shi1.Scer\UAS at 32[o]C in all Kenyon cells (KCs) (using the split-GAL4 MB010B driver lines Scer\GAL4DBD.52H09.T:Zzzz\ZipRREEL and Hsap\RELAAD.13F02.T:Zzzz\ZipEERRL) strongly reduces CO[[2]] avoidance in starved flies. The effect is dependent of the duration of starvation, with the effect becoming significant after flies have been starved for 24 hours, and becoming almost completely abolished at 42 hours. No effect is seen in well-fed flies. When these flies are exposed to a combination of CO[[2]] and vinegar as strong attraction to the mixture is seen.
Inactivating α'/β' and a small fraction of α/β core neuron input by expressing shi1.Scer\UAS at 32[o]C using the split-GAL4 MB186B driver lines (Scer\GAL4DBD.34A03.T:Zzzz\ZipRREEL and Hsap\RELAAD.52H09.T:Zzzz\ZipEERRL) reduces CO[[2]] avoidance behaviour in starved, but not well-fed, flies.
Inactivation of α/β KC by expressing shi1.Scer\UAS using Scer\GAL4GMR67B04 has no effect on CO[[2]] avoidance, either in starved or well-fed flies.
Expression of shi1.Scer\UAS in all Kenyon Cells under the control of Scer\GAL4ey-OK107 results in a reduction in CO[[2]] avoidance in starved but not well-fed flies. Expression under the control of either Scer\GAL4Mef2.247 or Scer\GAL4D52H has no effect on the response to CO[[2]].
Expression of shi1.Scer\UAS in the bilateral ventral projection neuron under the control of Scer\GAL4GMR53A05 results in complete diminishment of CO[[2]] avoidance in both 24 and 42 hours starved flies compared to controls. Almost complete abolishment of the CO[[2]] response is seen by 24 hours. A normal response to CO[[2]] is seen in fed flies.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tdc2.PC at 30[o]C have significantly increased ethanol resistance compared to controls.
Expression of shi1.Scer\UAS under the control of Scer\GAL4tank-4-12 at 30[o]C increases ethanol sensitivity in males and in females (time required for 50% of flies to sedate upon exposure to 60% ethanol is reduced compared to controls). No significant effect on ethanol sensitivity is seen in females expressing shi1.Scer\UAS under the control of Scer\GAL4tank-4-12 at 30[o]C.
D. melanogaster males expressing shi1.Scer\UAS under the control of Scer\GAL4Gr32a.3.776 at the restrictive temperature court D. virilis females as avidly as conspecific D. melanogaster females, a phenotype that is not seen at the permissive temperature or in control wild-type D. melanogaster males.
Flies expressing shi1.Scer\UAS in PN[[v]]-1 neurons (under the control of any of Scer\GAL4VT33008, Scer\GAL4VT1606, Scer\GAL4VT31497 or Scer\GAL4VT48643) show an impaired response to 0.5% CO[[2]] at 30[o]C. No impairment is seen at 21[o]C. 0.5% CO[[2]] avoidance is normal when shi1.Scer\UAS is expressed in PN[[v]]-2 (Scer\GAL4NP7243), PN[[v]]-3 (Scer\GAL4VT12760) or PN[[v]]-4 (Scer\GAL4Mmp2-NP0509) neurons at 30[o]C.
Flies expressing shi1.Scer\UAS in PN[[v]]-2 neurons (under the control of Scer\GAL4NP7243) show an impaired response to 2% CO[[2]] at 30[o]C. No impairment is seen at 21[o]C. 2% CO[[2]] avoidance is normal when shi1.Scer\UAS is expressed in PN[[v]]-1 (any of Scer\GAL4VT33008, Scer\GAL4VT1606, Scer\GAL4VT31497 or Scer\GAL4VT48643)), PN[[v]]-3 (Scer\GAL4VT12760 or Scer\GAL4GH146) or PN[[v]]-4 (Scer\GAL4Mmp2-NP0509) neurons at 30[o]C. The avoidance response also remains intact when shi1.Scer\UAS is expressed in PN[[v]]-2 and PN[[v]]-3 neurons simultaneously.
Expression of shi1.Scer\UAS under the control of Scer\GAL4E564 increases spontaneous proboscis extensions more than 5-fold at the restrictive temperature of 32[o]C compared to the permissive temperature.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4NP6510 (flies raised at 31[o]C for 12 hours and 25[o]C for 12 hours each day) do not show a significant decline in locomotor performance in a startle-induced negative geotaxis (SING) assay (assayed 1, 2 and 3 weeks after eclosion).
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4c305a and incubated at 31[o]C for 10 minutes prior to testing show a significant reduction in locomotor performance in a SING assay compared to unheated controls.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Mef2.247 and incubated at 31[o]C for 10 minutes prior to testing show a slight, but significant, increase in locomotor performance in a SING assay compared to unheated controls.
The inactivation of Ptth-expressing neurons by expression of shi1.Scer\UAS under the control of Scer\GAL80ts.αTub84B and Scer\GAL4NP0423 affects light avoidance with 8 to 10 hours delay in third instar larvae.
Thermo-suppression of neuronal activity by expressing shi1.Scer\UAS under the control of Scer\GAL4NP5272 partially suppresses the aversive memory seen in wild type flies in response to electric shock. Memory at two hours after shock is significantly impaired but two minute and nine hour memory are unaffected.
Thermo-suppression of neuronal activity by expressing one copy of shi1.Scer\UAS under the control of Scer\GAL45-HT1B.PA causes a gradual suppression over time of the aversive memory seen in wild type flies in response to electric shock. Memory at nine hours after shock is significantly impaired but two minute and two hour memory are both unaffected.
Thermo-suppression of neuronal activity by expressing shi1.Scer\UAS under the control of Scer\GAL4c061 suppresses the aversive memory seen in wild type flies in response to electric shock. Memory is significantly impaired at all time points tested (two minutes, two hours and nine hours after shock). Reflexive avoidance is normal. The phenotype is not seen if the temperature shift occurs after the electric shock.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tdc2.PC and trained and tested at 31[o]C (restrictive temperature) show no defects in 3 hour memory after olfactory appetitive conditioning with sucrose.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tdc2.PC and trained and tested at 31[o]C show significantly impaired 3 minute memory after olfactory appetitive conditioning with arabinose.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tdc2.PC and trained and tested at 31[o]C show no defects in 3 minute memory after olfactory appetitive conditioning with sorbitol.
3 minute memory after olfactory appetitive conditioning with arabinose is blocked and 3 minute memory after olfactory appetitive conditioning with sucrose is significantly reduced compared to wild type in flies expressing shi1.Scer\UAS under the control of Scer\GAL40104 and trained and tested at 31[o]C.
1 day memory after spaced training in an olfactory associative conditioning assay is blocked in flies expressing shi1.Scer\UAS under the control of either Scer\GAL4E0946, Scer\GAL4G0338 or Scer\GAL4G0431 which are transferred to 30[o]C for 1 hour during the test one day after the training, but is normal if the flies are transferred to 30[o]C from 30 minutes before training to the first 8 hours after training, 8 to 16 hours after training or 16 to 24 hours after training.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Trh.PB show reduced flight ability in a cylinder drop test when they are raised at the non-permissive temperature of 29[o]C either from the beginning of the pupal stage or from 2 days after eclosion onwards. The defect is more severe in those flies raised at 29[o]C from the beginning of the pupal stage.
Air-puff stimulated flight responses of the dorsal longitudinal indirect flight muscle (DLM) of tethered flies expressing shi1.Scer\UAS under the control of Scer\GAL4Trh.PB and raised at the non-permissive temperature of 29[o]C from the beginning of the pupal stage are different to wild type. In 7/15 cases, no electrophysiological response was seen, while the remaining flies showed sustained electrophysiological responses similar to controls. In flies raised at 29[o]C from 2 days after eclosion onwards, 5/15 flies initiated an electrophysiological response but could not maintain it, while the remaining flies were normal.
Expression of shi1.Scer\UAS under the control of Scer\GAL4ppk25.PS at the restrictive temperature of 30[o]C for 1 hour results in males with a 10-fold lower courtship index than controls. The total behavioural index of the males (fraction of time spent courting, walking or preening) and the gustatory response to sucrose is normal in these males.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4GH146 show significant odorant-selective short-term habituation and a significant difference between ethyl butyrate exposure at room temperature (which is normal short-term habituation and 32[o]C (when dynamin function and presynaptic transmission from Scer\GAL4GH146 projection neurons is inhibited, leading to no habituation).
Exposure of flies expressing shi1.Scer\UAS in the mushroom bodies under the control of Scer\GAL4Mef2.247 (which blocks neurotransmission from these neurons) to ethyl butyrate at 32[o]C, has no significant effect on habituation compared to genetic and temperature controls.
Expression of shi1.Scer\UAS under the control of either Scer\GAL4Rh3.PP, Scer\GAL4hth-GAL4 or Scer\GAL4Rh6+DRA abolishes the polarotactic response of flies to linearly polarised UV light presented dorsally.
Expression of shi1.Scer\UAS under the control of Scer\GAL4Rh2.PB reduces the polarotactic response of flies to linearly polarised UV light presented dorsally.
Expression of shi1.Scer\UAS under the control of Scer\GAL4Rh3.PP reduces the polarotactic response of flies to linearly polarised UV light presented ventrally.
Expression of shi1.Scer\UAS under the simultaneous control of Scer\GAL4ninaE.PT, Scer\GAL4Rh5.PT and Scer\GAL4Rh6.PD strongly reduces the polarotactic response of flies to linearly polarised green light presented ventrally.
Expression of shi1.Scer\UAS under the control of either Scer\GAL4Oamb.KI or Scer\GAL4Tdc2.PC at the restrictive temperature of 29[o]C results in impaired courtship conditioning in males compared to controls.
Expression of shi1.Scer\UAS under the control of Scer\GAL45-40 at the restrictive temperature results in 100% larval paralysis.
Expression of shi1.Scer\UAS under the control of either Scer\GAL4iav.PK or Scer\GAL4ppk.1.9 at the restrictive temperature significantly alters navigational behaviour in response to gentle touch, with many larvae failing to change their moving direction in response to 1mN tactile stimuli. The withdrawal response after a 1mN tactile stimulus is also reduced compared to wild type. Navigational behaviour and withdrawal response to a gentle touch of 7mN are normal in these larvae.
Expression of shi1.Scer\UAS under the control of either Scer\GAL4GMR91F06 or Scer\GAL4tutl-GAL4 at the restrictive temperature significantly alters navigational behaviour of larvae in response to gentle touch, while expression driven by Scer\GAL4GMR60G12 has no effect on navigational behaviour.
Escape behaviour when presented with a looming stimulus is strongly suppressed compared to wild type in flies expressing shi1.Scer\UAS under the control of either Scer\GAL4Foma1 or Scer\GAL421D at the non-permissive temperature.
The 24 hour arabinose induced olfactory-memory performance of flies expressing shi1.Scer\UAS at the restrictive temperature under the control of Scer\GAL4Gr66a.PD is statistically indistinguishable from that of wild-type controls.
Severing of the proximal dendrites at the glial wrapping boundary or within the wrap is not seen in ddaC neurons at 10 hours after puparium formation (APF) in animals carrying shi1.Scer\UAS and Scer\GAL4repo and incubated at 29[o]C from 3-10 hours APF, in contrast to controls which show complete severing of the proximal dendrites at the glial wrapping boundary in 80% of ddaC neurons at this stage. Instead, proximal dendrites are often severed at unwrapped segments in the mutant animals. Despite this delay of early steps in dendrite pruning, pruning is completed by 14 hours APR in these mutant animals.
Perturbing neurotransmission in dopaminergic neurons through expression of shi1.Scer\UAS under the control of either Scer\GAL4ple.PF or Scer\GAL4Ddc.PL at the restrictive temperature of 30[o]C does not affect conditioned aversion to ethanol, but blocks the formation of a conditioned preference. No effects are observed at the permissive temperature (24[o]C).
Temporal expression of shi1.Scer\UAS in dopaminergic neurons (under the control of Scer\GAL4ple.PF) in such a way that neural activity is silenced only during training, when ethanol is presented simultaneously with an odor cue, has no effect on the development of conditioned preference.
Impairing neurotransmission through expression of shi1.Scer\UAS in the entire body (Scer\GAL4ey-OK107), in a combination of γ and αβ neurons (Scer\GAL4Mef2.247 and Scer\GAL4Tab2-201Y), or in just the αβ neurons (Scer\GAL45-66a) during training and testing for ethanol conditioning, disrupts both conditioned aversion and preference. There are no differences in conditioned responses in these groups at the permissive temperature with the exception of the highly expressed Scer\GAL4ey-OK107, an effect that is likely a result of low levels of expression at the permissive temperature. In addition, olfactory control tests reveal that manipulating mushroom body neurotransmission does not significantly impair odor attraction.
Silencing a combination of γ and αβ neurons (through expression of shi1.Scer\UAS under the control of Scer\GAL4ey-OK107, Scer\GAL4Mef2.247 and Scer\GAL4Tab2-201Y) during acquisition blocks conditioned preference. Acquisition is not affected by silencing of αβ neurons alone (Scer\GAL45-66a), suggesting that the γ neurons, rather than αβ neurons, are important for acquisition. In contrast, silencing the α'β' neurons (through expression of shi1.Scer\UAS under the control of Scer\GAL4ey-OK107 and Scer\GAL44-59) during consolidation or the αβ neurons (Scer\GAL45-66a) during testing blocked conditioned preference.
Expression of shi1.Scer\UAS in olfactory receptor neurons under the control of Scer\GAL4Mz709 at a restrictive temperature blocks endocytosis in ensheathing glia and arrests the clearance of severed ORN axons. Plasticity is blocked in these flies at the restrictive temperature.
Expression of shi1.Scer\UAS specifically in astrocytes under the control of Scer\GAL4alrm.PD at the restrictive temperature blocks endocytosis in these cells but does not block the induction of plasticity in the nervous system.
Larvae in which shi1.Scer\UAS is expressed under the control of Scer\GAL4GMR.PFa avoids light normally at 20[o]C but are blind at 30[o]C. This blindness can be genetically rescued using Scer\GAL80Cha.PK to inhibit Scer\GAL4 function selectively in cholinergic cells. Larvae in which Scer\GAL4GMR.PFa is inhibited with Scer\GAL80Cha.PK display wild-type light avoidance at the non-permissive temperature.
Expression of shi1.Scer\UAS under the control of Scer\GAL4Tdc2.PC does not completely eliminate octopaminergic function. However, expression of shi1.Scer\UAS and rearing of the animals at the restrictive temperature of 29[o]C is sufficient to elicit marked abnormalities in the innervation of muscles by type II endings. These include markedly reduced type II arbors, lack of innervation of muscles by type II arbors, thinning of type II neurites, and the lack of Tbh in some type II boutons.
Blocking neurotransmission through expression of shi1.Scer\UAS at the restrictive temperature under the control of Scer\GAL4Hn.493 or Scer\GAL4c316 affects memory consolidation but not learning or retrieval of 3 hour memory. In contrast, blocking neurotransmission from Scer\GAL4Hn.819 or Scer\GAL4Ddc.PL neurons does not affect learning, consolidation, or retrieval of 3 hour memory.
Three-hour memory performance is decreased after blocking neurotransmission from DPM neurons with shi1.Scer\UAS expressed under the control of Scer\GAL4Hn.493, Scer\GAL4c316 or Scer\GAL45015 immediately after training for 1 hour. Performance is reduced further by blocking anesthesia-sensitive memory via application of cold-induced anesthesia 2 hours after training.
Blocking synaptic transmission in neurons through expression of shi1.Scer\UAS in subsets of neurons controlling olfaction (Scer\GAL4Orco.2.642.T:Hsim\VP22), hearing and hygrosensation (Scer\GAL4nan.PK), and vision (Scer\GAL4GMR.PF) does not affect nociceptive behaviours.
Blocking synaptic transmission in neurons through expression of shi1.Scer\UAS in pain-expressing neurons (Scer\GAL4pain-GAL4) blocks thermal nociception. These flies do not exhibit defects in general coordination (as assessed by negative geotactic response).
Expression of shi1.Scer\UAS in the mushroom body, under the control of Scer\GAL4ey-OK107, Scer\GAL4Mef2.247 or Scer\GAL4c316, has no effect on the avoidance response to noxious heat.
Expression of shi1.Scer\UAS under the control of Scer\GAL4repo.PU during the adult stage under constant darkness conditions results in the flies becoming arrhythmic within 2 days of expression (the temperature sensitive Scer\GAL80ts.αTub84B allele is used to control the timing of expression by shifting the adults to 30[o]C at the beginning of the subjective daytime on day 1 of constant darkness). This arrhymicity is reversible upon shifting to 23[o]C. Prolonged exposure (4-5 days) to 30[o]C results in lethality in these animals.
Expression of shi1.Scer\UAS specifically during the adult stage under the control of either Scer\GAL4alrm.PD or Scer\GAL4Eaat1.PR under constant darkness conditions reduces the percent rhythmicity of flies in locomotor activity assays compared to controls (the temperature sensitive Scer\GAL80ts.αTub84B allele is used to control the timing of expression).
Flies expressing shi1.Scer\UAS under the control of either Scer\GAL4GH146, Scer\GAL4NP2631, Scer\GAL4NP5288 or Scer\GAL4c316 show a significant impairment in 3 hour memory in an appetitive olfactory memory assay when they are trained at the permissive temperature and then immediately shifted to the restrictive temperature of 31[o]C for 2 hours. Expression of shi1.Scer\UAS under the control of Scer\GAL4NP0225 does not result in a significant defect in 3 hour memory when flies are tested under these conditions.
The defect in 3 hour memory which is seen in flies expressing shi1.Scer\UAS under the control of either Scer\GAL4GH146, Scer\GAL4NP2631 or Scer\GAL4NP5288 in an appetitive olfactory memory assay when they are trained at the permissive temperature and then immediately shifted to the restrictive temperature of 31[o]C for 2 hours is not seen if the flies are also carrying Scer\GAL80Cha.PK.
Flies expressing shi1.Scer\UAS under the control of either Scer\GAL4GH146 or Scer\GAL4NP5288 do not show a defect in 24 hour memory in an appetitive olfactory memory assay (flies are trained at the permissive temperature and then immediately shifted to the restrictive temperature of 31[o]C for 2 hours). Flies expressing shi1.Scer\UAS under the control of Scer\GAL4c316 show a significant defect in 24 hour memory when tested under these conditions.
The odor avoidance (to 3-octanol and to 4-methylcyclohexanol) of flies expressing shi1.Scer\UAS under the control of either Scer\GAL4Mz160 or Scer\GAL4NP2492 at the restrictive temperature is normal.
Expression of shi1.Scer\UAS under the control of either Scer\GAL4Mz160 or Scer\GAL4NP2492 at the restrictive temperature during training and consolidation does not impair 2 hour memory in an aversive olfactory conditioning assay. However, expression of shi1.Scer\UAS under the control of either Scer\GAL4Mz160 or Scer\GAL4NP2492 at the restrictive temperature during memory retrieval results in a strong memory impairment 2 hours after a single cycle of conditioning.
Expression of shi1.Scer\UAS under the control of Scer\GAL4Mz160 at the restrictive temperature during memory retrieval does not result in an defect in memory 2 hours after training in an appetitive conditioning assay.
The defect in 2 hour memory which is seen when flies carrying shi1.Scer\UAS and Scer\GAL4Mz160 are kept at the restrictive temperature during memory retrieval is suppressed if the flies are also carrying either Scer\GAL80Cha.PK or Scer\GAL80NP2492 (which blocks Scer\GAL4 expression).
Expression of shi1.Scer\UAS under the control of either Scer\GAL4Mz160 or Scer\GAL4NP2492 at the restrictive temperature during memory testing results in a strong impairment of memory 24 hours after both massed and spaced conditioning. Expression of shi1.Scer\UAS under the control of either Scer\GAL4Mz160 or Scer\GAL4NP2492 at the restrictive temperature during spaced training and the following 2 hours has no effect on 24 hour memory.
Expression of shi1.Scer\UAS under the control of Scer\GAL4GMR71D08 at the restrictive temperature during memory testing results in a strong impairment of 2 hour memory.
Dendrite growth is completely arrested in the dorsal dendritic arborisation (da) neurons in larvae expressing shi1.Scer\UAS under the control of Scer\GAL4109(2)80 which are raised at the non-permissive temperature of 30[o]C. At 18[o]C, da neurons show a normal dendritic pattern in these larvae, while at intermediate temperatures, a reduction in dendrite branch number is seen, which is correlated with rising temperature. The terminal branches are progressively shortened and often form clusters at higher temperatures.
Wild-type larvae display differences in their LN(v) dendrite length when exposed to constant darkness versus constant light. Reducing the activity of the Bolwig organ by means of expressing shi1.Scer\UAS at the restrictive temperature under the control of Scer\GAL4GMR.PF causes dendrite expansion regardless of constant light exposure.
The behavioural defects of flies carrying shi1.Scer\UAS and Scer\GAL4GC16 are suppressed by the presence of Scer\GAL80Ir64a.PA.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Ddc.PL at a restrictive temperature (30[o]C), where synaptic vesicle recycling is blocked, experience more than double the number of encounters than control flies in 'fight chambers' (with a headless female, food and light). The average duration of each encounter is greatly shortened throughout the duration of a fight when compared to fights between flies of the same genotype at the permissive temperature (19[o]C). Low-intensity front fencing events more than triple in number in the experimental flies at 30[o]C. Despite an increase in the number of interactions between the flies, fights do not escalate to mid-intensity levels (no lunges or hold is observed) at restrictive temperatures. Escaltion is seen at permissive temperatures. No significant differences are found in geotaxis, phototaxis or courtship index compared to controls.
Expression of shi1.Scer\UAS in serotonergic neurons under the control of Scer\GAL4l.Trh.PK does not affect the number or average duration of fight encounters between males at either permissive or restrictive temperatures compared to controls. These flies do however show an increase in the numbers of low-intensity fencing events during the first 20mins of a fight at restrictve temperatures compared to the same genotype at permissive temperatures. At 19[o]C, shi1.Scer\UAS-Scer\GAL4l.Trh.PK flies exhibit large and significant reductions in lunge rate and numbers. The number of 'holds' also drops at the restrictive temperature, indicating a drop in mid-intensity aggression. Thus, at the restrictive temperature of 30[o]C, fights between pairs of experimental flies could escalte to mid-intensity levels, but the freuqency of usage of these behavioral patterns is dramatically reduced. As a consequence, hierarchical relationships, which require lunging behavior, are establed in a smaller percentage of fights (67% at the permissive temperature vs. 10% at the restrictive temperature).
shi1.Scer\UAS-Scer\GAL4l.Trh.PK flies, as controls, show anticipated temperature-induced increases in locomotion. At restrictive temperatures, no changes in courtship behavior are found.
shi1.Scer\UAS-Scer\GAL4l.Trh.PK flies show an unusual pattern of movement that isn't seen in control flies. When moving, these flies suddenly collapse, roll to the side, then spring back up and continue to do whatever they were doing. Despite this, these flies initiate lunging behavior in 60% of fights with wild-type males, indicating that their 'willingness to fight' is not compromised by the motor dysfunction phenotype.
Expression of shi1.Scer\UAS in DA neurons under the control of Scer\GAL4ple.PF does not affect fighting behavior at the permissive temperature. At restrictive temperature, however, the flies do not land on the food cup, Instead, they are in almost constant motion while in the fighting chamber. Courtship index is significantly reduced in these flies at the restrictive temperature.
Expression of shi1.Scer\UAS in the posterior region of the third-instar larval eye disc under the control of Scer\GAL4GMR.PF does not affect R-cell localization at the permissive temperature (i.e. 18[o]C). However, when larvae are reared at the restrictive temperature (i.e. 32[o]C), they display a severe R-cell nuclear mislocalization phenotype.
Male and female flies expressing shi1.Scer\UAS under the control of Scer\GAL4c739 at the restrictive temperature (30[o]C) cross the centre of the assay arena less often than controls in an assay of walking behaviour.
Male and female flies expressing shi1.Scer\UAS under the control of Scer\GAL4NP2320 at the restrictive temperature (30[o]C) display an increase in the number of start/stop actions compared to controls during a walking assay.
Males expressing shi1.Scer\UAS under the control of Scer\GAL4c584 at the restrictive temperature (30[o]C) travel shorter distances and have a lower mean walking speed than controls in a walking assay. Females expressing shi1.Scer\UAS under the control of Scer\GAL4c584 at the restrictive temperature (30[o]C) show no significant difference in distance travelled and mean walking speed compared to controls.
Pre-incubation at the restrictive temperature of 30[o]C induces paralysis of flies carrying shi1.Scer\UAS under the control of Scer\GAL4elav-C155.
The integrity and performance of the mechanical feedback amplification provided by Johnson's Organ neurons is unaffected by expression of shi1.Scer\UAS under the control of Scer\GAL4elav-C155.
Non-linear, intensity-dependent amplification and nerve responses to pure-tone noise are unaffected by expression of shi1.Scer\UAS under the control of Scer\GAL4elav-C155.
Expression of shi1.Scer\UAS under the control of either Scer\GAL4NP1131 or Scer\GAL4Tab2-201Y has no effect on general sensory acuity compared to controls.
Larvae in which synaptic output has been blocked by expressing shi1.Scer\UAS under the control of Scer\GAL4c305a, Scer\GAL4Tab2-201Y or Scer\GAL4NP1131 at the restrictive temperature exhibit a significant reduction in performance in larval odor-sugar learning tests compared to shi1.Scer\UAS/+ controls. Expression under the control of Scer\GAL4c305a is not significantly different from Scer\GAL4c305a/+ controls and no reduction in performance is seen at the permissive temperature except for in shi1.Scer\UAS Scer\GAL4NP1131 larvae, in where a significant increase is seen compared to Scer\GAL4NP1131/+ controls. Expression of shi1.Scer\UAS under the control of either Scer\GAL4Tab2-201Y or Scer\GAL4NP1131 in combination with Scer\GAL80Mef2.PT (to prevent Scer\GAL4 activity in the mushroom body) has no effect on larval odor-sugar learning behavior. Expression of shi1.Scer\UAS using any of Scer\GAL4Mef2.247, Scer\GAL4D52H, Scer\GAL4c305a and Scer\GAL4c503 has no effect on learning behavior.
Embryos expressing shi1.Scer\UAS under the control of Scer\GAL4elav.PU at 34[o]C show defects in glial cell migration. The axonal scaffold remains largely intact.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4ple.PF show a partial impairment in punishment learning when trained and tested at 34-36[o]C. Relief learning scores of these flies are indistinguishable from that of controls when trained and tested at 34-36[o]C.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Ddc.PL show normal punishment learning scores when trained and tested at 34-36[o]C. Relief learning scores of these flies are indistinguishable from that of controls when trained and tested at 34-36[o]C.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tdc2.PC show normal reward learning ability when trained and tested at 34-36[o]C. Relief learning scores of these flies are indistinguishable from that of controls when trained and tested at 34-36[o]C.
per oscillations are approximately 4-8 hours delayed in flies where shi1.Scer\UAS is expressed under the control of Scer\GAL4P2.4.Pdf. The DN1 clock also appears to be phase-delayed, with its peak and trough shifted by 4-8 hours, whereas the LN[[d]] clock does not exhibit a clear delay.
Flies expressing shi1.Scer\UAS under the control of either Scer\GAL4c673a or Scer\GAL4fru-GAL4 at 30[o]C have an increase in fat levels compared to control flies. The abdominal fat cells have large fat droplets surrounded by thin rings of cytoplasmic material. These flies show a reduction in CO[[2]] emission compared to control flies.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4c673a at 30[o]C show increased food intake compared to control flies.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4fru-GAL4 at 30[o]C show decreased food intake compared to control flies.
Flies expressing shi1.Scer\UAS under the control of one of Scer\GAL4Ilp2.215-3, Scer\GAL4ple.PF, Scer\GAL4eg-Mz360 or Scer\GAL4Ddc.PL at 30[o]C show not change in fat content compared to control flies.
Flies expressing shi1.Scer\UAS under the control of both Scer\GAL4c673a and Scer\GAL4fru-GAL4 simultaneously at 30[o]C are lethargic and die within 5 days after expression.
Flies expressing shi1.Scer\UAS under the control of either Scer\GAL4c673a or Scer\GAL4fru-GAL4 at 30[o]C show a reduced rate of fat store depletion compared to control flies under starvation conditions.
The increase in stored fat seen in flies expressing shi1.Scer\UAS under the control of either Scer\GAL4c673a or Scer\GAL4fru-GAL4 at 30[o]C can be reversed if the flies are returned to room temperature.
Scer\GAL4Tab2-201Y/shi1.Scer\UAS and Scer\GAL4OK301/shi1.Scer\UAS larvae show normal aversive memory when both training and testing are performed at the permissive temperature (25[o]C). Memory is also normal when the larvae are trained at the restrictive temperature (30[o]C) and tested at the permissive temperature (25[o]C).
Olfactory memory is completely impaired in Scer\GAL4Tab2-201Y/shi1.Scer\UAS and Scer\GAL4OK301/shi1.Scer\UAS larvae when aversively trained at 25[o]C and tested at 30[o]C.
Memory performance is abolished when Scer\GAL4ple.PF/shi1.Scer\UAS larvae are aversively trained at the restrictive temperature (31[o]C) and tested at the permissive temperature (25[o]C).
Memory performance is normal when Scer\GAL4Tdc2.PC/shi1.Scer\UAS larvae are aversively trained at the restrictive temperature (31[o]C) and tested at the permissive temperature (25[o]C).
Memory performance is abolished when Scer\GAL4Tdc2.PC/shi1.Scer\UAS larvae are appetitively trained with sucrose at restrictive temperature and tested at permissive temperature (25[o]C).
Memory performance is not abolished when Scer\GAL4ple.PF/shi1.Scer\UAS larvae are appetitively trained with sucrose at restrictive temperature and tested at permissive temperature (25[o]C).
Expression of shi1.Scer\UAS in the mushroom body under the control of Scer\GAL45-120 and Scer\GAL45-98 results in a strong reduction in oviposition preference at 30[o]C. Positional aversion to 5% acetic acid is unaffected in experimental and control flies.
Expression of shi1.Scer\UAS in the lateral neurons under the control of Scer\GAL4P2.4.Pdf does not affect egg laying or positional responses to 5% acetic acid at the restrictive temperature of 30[o]C.
Expression of shi1.Scer\UAS in the ellipsoid body ring neurons under the control of Scer\GAL44-67 or Scer\GAL42-72 results in positional aversion to 5% acetic acid at 30[o[C and 23[o]C. Egg-laying preference in these flies is essentially unchanged.
Expression of shi1.Scer\UAS in the ellipsoid body ring neurons and the mushroom body under the control of both Scer\GAL44-67 and Scer\GAL45-120 results in disruption of oviposition and positional preference that are essentially the sum of those seen with the individual GAL4 lines.
Blocking of DA neuron function by shifting Scer\GAL4c061; Scer\GAL80Mef2.PT; shi1.Scer\UAS flies to 31[o]C for 1 hour does not enhance memory performance in fed flies. Memory performance of Scer\GAL4c061; Scer\GAL80Mef2.PT/Scer\GAL80ple.PK; shi1.Scer\UAS flies is statistically indistinguishable from controls.
Expression of shi1.Scer\UAS in the eye under the heat-induced control of Scer\GAL4LL54 at 31[o]C for 6 hours at during pupal development results in wild-spread death of interommatidial pigment cells.
Time to copulation in shi1.Scer\UAS/Scer\GAL80Gad1 females is significantly shorter at 30[o]C than at 23[o]C.
Virgin females expressing shi1.Scer\UAS under the control of Scer\GAL4ppk.PG at the restrictive temperature of 32[o]C show a reduction in sexual receptivity and an increase in egg-laying rate compared to control virgin females. This effect is significantly suppressed if the females also carry Scer\GAL80ppk.PY.
At the restrictive temperature, expression of shi1.Scer\UAS using Scer\GAL4rafl-1, Scer\GAL4osk-norka, Scer\GAL4c305a or Scer\GAL4c739 results in attenuation of brain responses at 20-30 Hz in response to a moving visual stimulus.
Exposure of shi1.Scer\UAS, Scer\GAL4rafl-1 or shi1.Scer\UAS, Scer\GAL4osk-norka flies to the restrictive temperature only during training in the visual novelty paradigm results in selective loss of the at 20-30 Hz brain response to visual novelty.
Expression of shi1.Scer\UAS using Scer\GAL4rafl-1 at the restrictive temperature immobilizes the flies.
At the restrictive temperature, flies expressing shi1.Scer\UAS using Scer\GAL4c309, Scer\GAL4c305a or Scer\GAL4c739 display significantly increased optomotor responsiveness.
Overexpression of shi1.Scer\UAS with Scer\GAL4ort.C1-4 significantly reduces the UV light attractiveness over green light at nonpermissive, but not permissive, temperatures. This reduction was smaller than that caused by expressing shi1.Scer\UAS simultaneously with Scer\GAL4Rh4.PT and Scer\GAL4Rh3.PP.
Flies overexpressing shi1.Scer\UAS by Scer\GAL4ort.C2 exhibit attenuated UV light preference over green light at nonpermissive, but not permissive, temperatures.
Expression of shi1.Scer\UAS under the control of Scer\GAL4ninaE.PT at the restrictive temperature (34[o]C) results in severe defects in visual motion processing behaviours; all measurable aspects of direction-selective response to sparse and dense stimuli in translation and rotation indices are eliminated in these flies.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL421D at the restrictive temperature (34[o]C) show defects in visual motion processing behaviours; the flies move robustly "With" the direction of all stimuli tested, including sparse stimuli that normally cause an "Against" response. Translational response is compromised consistently on all conditions, but different aspects of rotational responses are preserved, depending on the specific stimulus used. Under sparse and dense stimuli, suppression of both forward movement and turning is eliminated but a directional bias in turns is not eliminated.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Foma1 at the restrictive temperature (34[o]C) show defects in visual motion processing behaviours; the flies move normally in the absence of motion stimuli and respond robustly to a sparse motion stimulus, they are significantly impaired in their response to the dense stimulus. In particular, they fail to move "With" the dense stimulus. 80% of the response in translation on the sparse stimulus is preserved in the flies, and translation response on the dense stimulus is not affected. Turns are suppressed more strongly on the sparse stimulus, and only weakly, if at all, on the dense stimulus. The flies lose almost all normally elicited directionality in turning, reversing the remaining small turn bias relative to that of their controls on both sparse and dense stimuli.
Expression of shi1.Scer\UAS under the control of either Scer\GAL4ple.PF or Scer\GAL4Ddc.PL at the restrictive temperature (32[o]C) results in significantly reduced intermale courtship after repeated ethanol exposure compared to the level of intermale courtship seen in control males under these conditions.
Expression of shi1.Scer\UAS under the control of Scer\GAL4Or83b.7.087 in all olfactory sensory neurons results in the disappearance of the increase in proboscis extension in response to sucrose.
Aggression is reduced within 15 minutes of shifting to the restrictive temperature in flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tdc2.PC, when a pair of flies are placed in a small closed chamber containing a food pad in the centre. The fighting latency is longer at 31[o]C than at 23[o]C, and the frequencies of lunging and high-intensity fighting (holding, boxing and tussling) are lower at 31[o]C in the mutant flies.
shi1.Scer\UAS; Scer\GAL4yuri-c263 flies show a small but significant increase in gravitaxis. shi1.Scer\UAS; Scer\GAL4esg-c287a flies show a small but significant decrease in gravitaxis. shi1.Scer\UAS; Scer\GAL4c105 and shi1.Scer\UAS; Scer\GAL4Aph-4-c232 flies also show a significant decrease in gravitaxis, with the strongest effect being seen in the former. In all cases, there is no significant change in locomotor activity of the mutant flies compared to controls.
Gravitaxis is normal in shi1.Scer\UAS; Scer\GAL417d or shi1.Scer\UAS; Scer\GAL4H24 flies.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Cha.7.4 show rapid paralysis when transferred to the restrictive temperature (30[o]C). The flies become progressively more sensitive to paralysis and need longer to recover with increasing age.
Expression of shi1.Scer\UAS under the control of Scer\GAL45-40, Scer\GAL4NP2225 or Scer\GAL4NP5092 causes a striking defect in the crawling pattern. The peristaltic wave associated with movement is delayed, with excessively tight muscle contractions. This slow propagation results in a tenfold decrease in wave frequency, from 2 waves per second to under 0.2 waves per second. In addition, the PNS-inhibited larvae have a postural defect, causing them to roll onto their side during crawling. The Scer\GAL45-40 x shi1.Scer\UAS larvae also have an increased proportion of backwards waves, and excessive curling of the head.
Expression of shi1.Scer\UAS under the control of Scer\GAL49-201av, or Scer\GAL48-73 in the chordotonal organs does not disrupt the larval crawling pattern and reveals only minor defects in crawling.
Expression of shi1.Scer\UAS under the control of Scer\GAL4clh201 or Scer\GAL4109(2)80 neurons produces crawling defects similar to inhibiting the entire PNS. The defects include delayed, slow peristaltic wave, with excessively tight muscle contractions, rolling, and with Scer\GAL4109(2)80 x shi1.Scer\UAS, a predominance of backward waves.
Expression of shi1.Scer\UAS under the control of Scer\GAL41003.3, Scer\GAL4clh24 or Scer\GAL47-33 results in no strong crawling defects.
Inhibiting the bd neurons through expression of shi1.Scer\UAS under the control of Scer\GAL4clh8 or Scer\GAL48-113 results in mild propagation defects. Inhibition of the class I mds along with Scer\GAL42-21-driven expression of shi1.Scer\UAS produces mild propagation defects. When the bd and class I drivers are combined (Scer\GAL4clh8 and Scer\GAL42-21) and both classes of neurons are inhibited by shi1.Scer\UAS, much more severe propagation defects, with slow, tight waves, are observed.
Expression of shi1.Scer\UAS under the control of both Scer\GAL48-113 and Scer\GAL42-21 causes severe disruptions in the larval crawling pattern.
At the restrictive temperature of 31[o]C, the aversive olfactory conditioning scores of shi1.Scer\UAS driven by Scer\GAL4c305a, Scer\GAL4c320 or Scer\GAL4c739 are significantly reduced compared to controls or testing at the non-restrictive temperature of 25[o]C. Transferring flies expressing shi1.Scer\UAS by Scer\GAL4c320 or Scer\GAL4c305a to the restrictive temperature during or for 1hr immediately after the training period significantly reduced both aversive and appetitive odour conditioning scores when compared to controls. Transferring flies expressing shi1.Scer\UAS with Scer\GAL4c739 to the restrictive temperature during the memory retrieval period significantly reduced aversive olfactory conditioning scores when compared to controls.
Flies with shi1.Scer\UAS being driven by Scer\GAL4c320 have a statistically significant olfactory acuity defect at 31[o]C compared to controls when trained against methylcyclohexanol.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4c305a exhibit normal odour and shock acuity at both the permissive (25[o]C) and the restrictive (31[o]C), indistinguishable from controls.
Expression of shi1.Scer\UAS by Scer\GAL4c316, and transferring the animals to 31[o]C for one hour immediately following training, significantly reduces 3hr aversive odour memory compared to controls.
Under non-permissive temperatures, no significant head yaw nor roll responses to a moving striped drum are observed in flies expressing shi1.Scer\UAS by Scer\GAL4NP6298, Scer\GAL4NP5214 or Scer\GAL4NP0723.
Transferring flies expressing shi1.Scer\UAS with Scer\GAL4NP6298 to restrictive temperatures for 15 minutes abolishes the walking optomotor response.
Flies expressing shi1.Scer\UAS with Scer\GAL4NP5214 or Scer\GAL4NP0723 are reluctant to start flight, and Scer\GAL4NP6298 stop shortly after initiation of flight, in a striped drum at non-permissive temperatures.
Flies expressing shi1.Scer\UAS with Scer\GAL4NP6298 do not elicit a collision-avoidance response at non-permissive temperatures.
Expression of shi1.Scer\UAS with Scer\GAL421D abolishes optomotor yaw torque response at the non-permissive temperature when pattern contrast is 0.1% or 1%, but is not significantly different from controls with pattern contrast of 3% or above.
Expression of shi1.Scer\UAS coincidentally by Scer\GAL421D and Scer\GAL4CG14200-NP1086 significantly reduces the optomotor yaw torque response at non-permissive temperatures with pattern contrasts of 0.1%, 1% and 5%, but not at 10%.
The optomotor yaw torque response of tethered flying flies expressing shi1.Scer\UAS with Scer\GAL421D at low light intensities (I = 5 x 10[-4] cd/m[2], and a pattern wavelength of 18[o]) is abolished at non-permissive temperatures, unlike controls. Extensive dark adaptation before the experiment does not improve the score, and responses do not improve throughout the 3 minute experimental procedure. These flies respond to an increased background luminescence of I = 10[-3] cd/m[2], though this response is reduced to about half that of controls.
Flies expressing shi1.Scer\UAS with Scer\GAL421D in low light intensities and doubled spatial wavelength (I = 5 x 10[-4] cd/m[2], and lambda = 36[o]) show significant optomotor yaw torque response, whereas simultaneous expression with Scer\GAL421D and Scer\GAL4CG14200-NP1086 does not.
Flies expressing shi1.Scer\UAS with Scer\GAL4ninaE.PT at non-permissive temperatures are motion blind in landmark orientation tests and do not fixate on narrow landmarks.
Flies expressing shi1.Scer\UAS with Scer\GAL4NP6298, Scer\GAL4NP5214 or Scer\GAL4NP0723 at non-permissive temperatures show significantly reduced fixation on a 10[o] stripe, compared to controls, in a landmark orientation assay. Walking activity is lower, and walking trajectories less straight. Flies expressing with Scer\GAL4NP6298 show no preferred orientation when the stripe width is reduced to 5[o], or are presented with a stripe width of 10[o] but 75% pattern contrast.
Flies expressing shi1.Scer\UAS coincidentally by Scer\GAL4NP6298 and Scer\GAL4CG14200-NP1086, at non-permissive temperatures show reduced fixation on a 10[o] stripe, compared to controls, in a landmark orientation assay.
Targeted expression of shi1.Scer\UAS driven by Scer\GAL4αTub84B.PP results in dramatic alterations in the architecture of the CSD interneuron (CSDn). The extent of branching of presynaptic terminals in the contralateral lobe is strikingly reduced compared with wild-type.
Third instar larvae expressing shi1.Scer\UAS under the control of Scer\GAL45-40 show a progressive decrease in linear locomotion speed the longer the duration at the restrictive temperature, becoming completely immobilised after 20 minutes at 37[o]C. There is a progressive increase in the duration of the locomotive peristalsis wave and a corresponding decrease in peristalsis wave frequency during the first 20 minutes at 37[o]C, while there is no significant reduction in stride length. The immobilised larvae still show mouth hook movements. The locomotion defect is reversible upon shifting to the permissive temperature.
Third instar larvae expressing shi1.Scer\UAS under the control of Scer\GAL421-7 at the restrictive temperature show a significant decrease in locomotion speed compared to wild type, with the majority of larvae being paralysed after 20 minutes at the restrictive temperature. There is a progressive increase in the duration of the locomotive peristalsis wave and a corresponding decrease in peristalsis wave frequency during the first 20 minutes at 37[o]C, while there is no significant reduction in stride length. The segmental nerves show progressively more arrhythmic and irregular motor neuron firing with increasing exposure to 37[o]C: the burst duration becomes prolonged and irregular. The transition of motor bursting from one segment to the next (that normally accompanies the posterior-to-anterior rhythmic peristaltic contraction) is lost after 15 minutes at 37[o]C: after this time the burst duration of the most posterior segment (L7) is prolonged and the segmental nerve of the immediately anterior segment (L6) remains silent.
Third instar larvae expressing shi1.Scer\UAS under the control of Scer\GAL4Cirl.1018 show normal locomotion speed.
1 day memory after spaced training in an olfactory associative conditioning assay is abolished in flies expressing shi1.Scer\UAS under the control of Scer\GAL4ey-OK107, but is normal in flies expressing shi1.Scer\UAS under the control of Scer\GAL4Feb170 when the flies are shifted to 29[o]C for the spaced training and maintained at this restrictive temperature for 6 hours after the spaced training.
1 day memory after spaced training in an olfactory associative conditioning assay is abolished in flies expressing shi1.Scer\UAS under the control of either Scer\GAL4ey-OK107 or Scer\GAL4Feb170 when the flies are maintained at 18[o]C for 23 hours after training, and then shifted to 29[o]C one hour before testing.
Silencing of synaptic transmission from mushroom bodies by expression of shi1.Scer\UAS under the control of either Scer\GAL4c309, Scer\GAL4247 (both drive expression in all lobes of the mushroom bodies) or Scer\GAL4Tab2-201Y (in the γ-lobes) leaves spontaneous odor intensity discrimination and avoidance of individual odors unchanged. However, at 30[o]C spontaneous odor identity, conditioned odor intensity and identity discrimination are severely compromised.
Flies expressing shi1.Scer\UAS under the control of one of Scer\GAL4247, Scer\GAL4Ddc.PL or Scer\GAL4ple.PF at the restrictive temperature show defective choice performance after training with position/colour cues when subjected to a visual choice test with conflicting cues.
Flies expressing shi1.Scer\UAS under the control of one of Scer\GAL4Aph-4-c507 at the restrictive temperature show normal choice performance after training with position/colour cues when subjected to a visual choice test with conflicting cues.
shi1.Scer\UAS; Scer\GAL4NP1017 adults starved of water for 2 hours, show a significantly reduced proboscis extension response at the restrictive temperature (30oC) when their prothoracic legs are exposed to water compared to identically treated controls. These animals rapidly recover some, but not all of their response to water on returning to the permissive temperature (22oC). Responses of these animals to sugar and salt appear normal.
Flies that express shi1.Scer\UAS in dorsal paired medial neurons, driven by Scer\GAL4c316, show impaired reward odor memory when raised at 31oC. Scer\GAL4c316>shi1.Scer\UAS expression during training does not affect memory, but expression for 30 minutes after training significantly reduces reward-odor memory. Similarly, expression of shi1.Scer\UAS in dorsal paired medial neurons, driven by Scer\GAL4Mz717, for 60 minutes at 31oC after training significantly reduces reward-odor memory.
Fewer than 10% of shi1.Scer\UAS Scer\GAL4fru virgin females copulate within a 20 minute period, whereas approximately 50% of control females copulate in the same period. This is not due to a passive failure to accept the male, but rather an active rejection behaviour in which the female extends her ovipositor to prevent mating. In addition, these virgin females are induced to lay their unfertilized eggs. These rejection and egg-laying behaviours are characteristic of mated females.
Expression of shi1.Scer\UAS, under the control of Scer\GAL4Ccap.PP acutely suppresses synaptic activity in NCCAP for 1 hour after eclosion. shi1.Scer\UAS expression can suppress synaptic transmission from approximately 5hrs prior to eclosion. Approximately 93% of new eclosed adults expressing shi1.Scer\UAS under the control of Scer\GAL4Ccap.PP fail to completely expand their wings. Expression of temperature-sensitive shi1.Scer\UAS under the control of Scer\GAL4dimm-929 in flies which are raised at 18oC and transferred to 34oC immediately after eclosion for 1hr before being returned to 18oC, results in complete wing expansion failure in less than 1% of animals.
Eggs laid by shi1.Scer\UAS; Scer\GAL4EQ1 mothers transiently shifted to 34'C are mildly dorsalized.
Expression of shi1.Scer\UAS under the control of one of Scer\GAL4sca-C253, Scer\GAL4c309, Scer\GAL4c747, Scer\GAL4c758 or Scer\GAL430Y results in a decrease in the total sleep at 29oC (the non-permissive temperature for shi1.Scer\UAS) compared to controls.
Sleep is reduced during light and dark periods in flies expressing shi1.Scer\UAS under the control of Scer\GAL4sca-C253 at 29oC.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Mef2.247 at 29oC show reduced sleep only during the early morning.
Flies expressing shi1.Scer\UAS under the control of one of Scer\GAL4sca-C253, Scer\GAL4c309 or Scer\GAL430Y show a reduction in the average sleep bout length and a lower sleep consolidation index compared to controls at 29oC.
Compared to control males, males expressing shi1.Scer\UAS under the control of either Scer\GAL4ey-OK107, Scer\GAL430Y or Scer\GAL4c747 at the restrictive temperature (30[o]C) show an increase in the latency to initiate courtship and reduced courtship activity towards immature virgin females.
The frequency of short courtship bouts (less than 60 seconds) towards immature virgin females is strikingly increased and the mean duration of courtship bouts is greatly reduced compared to controls in males expressing shi1.Scer\UAS under the control of Scer\GAL4c747 at 30[o]C. Mean duration of courtship bouts toward newly emerged males is unaffected.
Compared to control males, males expressing shi1.Scer\UAS under the control of either Scer\GAL4J183, Scer\GAL4OK348 or Scer\GAL4104Y at 30[o]C show a slight but statistically significant reduction in the courtship index towards immature virgin females, while the latency to initiate courtship is unaffected.
The mean duration of courtship bouts towards both immature virgin females and towards newly emerged males is reduced compared to controls in males expressing shi1.Scer\UAS under the control of Scer\GAL4J183 at 30[o]C.
Expression of shi1.Scer\UAS under the control of either Scer\GAL4Aph-4-c232 or Scer\GAL4C41 at the 30[o]C has no effect on the courtship of males towards immature virgin females.
Courtship behaviour towards immature males is not affected in males expressing shi1.Scer\UAS under the control of either Scer\GAL4c747 or Scer\GAL4ey-OK107 at 30[o]C.
Compared to control males, males expressing shi1.Scer\UAS under the control of either Scer\GAL4J183 or Scer\GAL4104Y at 30[o]C show a reduction in the courtship index towards immature males, while the latency to initiate courtship is unaffected.
Flies expressing shi1.Scer\UAS under the control of either Scer\GAL4ey-OK107 or Scer\GAL4c747 at 30[o]C show increased locomotor activity compared to controls.
Expression of shi1.Scer\UAS at 29[o]C in the cholinergic neurons under the control of Scer\GAL4Cha.7.4 leads to severe paralysis in larva, although mature postsynaptic densities still form.
Flies that express shi1.Scer\UAS in both dopaminergic and serotonergic neurons, driven by Scer\GAL4Ddc.PL, show a decrease in the 20-30 Hz response to a visual stimulus when heated to 38oC. These flies produce a normal response when returned to room temperature. The same phenotype is seen when flies express shi1.Scer\UAS exclusively in dopaminergic neurons, driven by Scer\GAL4ple.PF. Flies expressing shi1.Scer\UAS, driven by either Scer\GAL4Ddc.PL or Scer\GAL4ple.PF, also show a significant decrement in visual fixation behaviour at 38oC, while they display normal fixation behaviour at 22oC. Flies expressing shi1.Scer\UAS, driven by either Scer\GAL4Ddc.PL or Scer\GAL4ple.PF, show an altered response to methamphetamine compared to wild-type flies. Methamphetamine administration rescues the 20-30 Hz response to a visual stimulus in the transgenic flies at 380C, while the drug attenuates the response in wild-type flies.
Strong expression of shi1.Scer\UAS in the mushroom bodies, under the control of Scer\GAL4Tab2-201Y or Scer\GAL4OK301 at permissive temperatures does not affect 30 minute memory. Memory performance is also unchanged when larvae are trained and kept at the permissive temperature 10 minutes before the test. However, when larvae are trained and kept at the permissive temperature and shifted to the restrictive temperature 10 minutes before the test, memory performance is significantly impaired.
The naive olfactory response of shi1.Scer\UAS/Scer\GAL4OK301 larvae, but not of shi1.Scer\UAS/Scer\GAL4Tab2-201Y is higher than wild-type, while no changes in temperature significantly alter the olfactory and gustatory acuities of shi1.Scer\UAS/Scer\GAL4OK301 or shi1.Scer\UAS/Scer\GAL4Tab2-201Y larvae.
Flies expressing shi1.Scer\UAS, under the control of Scer\GAL4NP1535, show normal proboscis extension reflex responses to 100 mM sucrose at the permissive temperature (22oC). When these flies are transferred to 30oC, the PER response is significantly reduced. This reduction is recovered when flies are returned to 22oC.
When shi1.Scer\UAS is driven by Scer\GAL4fru.P1, when animals are placed at 31oC transiently, courtship behaviour is abolished. Grooming, walking and flight behaviours are normal.
Expression of shi1.Scer\UAS, under the control of Scer\GAL4Cha.7.4 at a restricted temperature (30oC) for 10 minutes, can result in cholinergically disabled flies that have a substantially slowed return to baseline electroretinogram levels after a 3-second light pulse. This effect is not observed with the flies are maintained at the permissive temperature (19oC).
The courtship index score for shi1.Scer\UAS; Scer\GAL4fru-GAL4/+ males raised at the permissive temperature (19oC) and then shifted to the restrictive temperature (29oC) 1-2 hr before testing with a single wild-type virgin female is less than 50% of the score for identically treated wild-type or Scer\GAL4fru-GAL4/+ males. These males do not court other males and perform normally in a battery of other behavioural tests (locomoter, flight, phototaxis, odor sensitivity, taste sensitivity and taste discrimination).
Approximately half of males expressing shi1.Scer\UAS under the regulation of Scer\GAL4c309 at 30oC, generate almost no tone pulses; at 25oC, all males of this type sing vigorously with acoustical parameters in the normal range. Those mutant males that do generate pulses at 30oC do this at lower amplitudes compared to sounds produced at 25oC, whereas the pulse amplitude for control animals does not change with temperature. Males carrying either shi1.Scer\UAS or shi1.Scer\UAS/Scer\GAL4c309 demonstrate lower intrapulse frequencies compared to wild-type. Males expressing shi1.Scer\UAS under the regulation of Scer\GAL4c309, exhibit a dramatic reduction in the rate of pulse production at 30oC compared to 25oC. After heat-shock at 30oC for 30minutes, males expressing shi1.Scer\UAS under the regulation of Scer\GAL4c309 require an equivalent time to initiate mating compared to the behavior of shi1.Scer\UAS (without Scer\GAL4c309), despite a nominal 2-fold latency difference. After heat-shock at 30oC for 30minutes, approximately 83% of males expressing shi1.Scer\UAS under the regulation of Scer\GAL4c309 initiate courtship, with only 17% copulating, compared to approximately 90% copulation at the permissive temperature (25oC). Of the 83% of males that initiate courtship, 30% exhibit normal abdominal bending directed at the female genitalia.
Males expressing shi1.Scer\UAS under the regulation of Scer\GAL4c309 at 30oC, exhibit a significant decrease in courtship towards females compared to low-temperature controls. Males expressing shi1.Scer\UAS under the regulation of Scer\GAL4c309 at 30oC, exhibit a similar level of general locomotion as controls and indeed court wild-type males at anomalously high levels. Males expressing shi1.Scer\UAS under the regulation of Scer\GAL4c309 at 30oC, exhibit an approximate 9-fold reduction in wing extension, and fly subnormally. Approximately 3% of control males adhere to the top 5cm of the cylindrical test apparatus and 86% adhere to the bottom 30cm, compared to control values of 44% and 25%, respectively. Males expressing shi1.Scer\UAS under the regulation of Scer\GAL4c309 at 30oC, generate almost no tone pulses; at 25oC, all males of this type sing vigorously with acoustical parameters in the normal range. shi1.Scer\UAS-affected males at 30oC generate an average of only one pulse every 2 minutes, compared to 18 pulses per minute at 25oC. Males expressing shi1.Scer\UAS under the regulation of Scer\GAL4c309 at 30oC, exhibit a courtship chaining index of 35%. This phenotype is substantially reduced to approximately wild-type levels when Scer\GAL80Cha.PK is co-expressed. Reducing the extent to which Scer\GAL4c309 drives expression of shi1.Scer\UAS in heat-treated males by expression of Scer\GAL80Cha.PK results in a minimal diminution of courtship directed at females, compared to wild-type or shi1.Scer\UAS/Scer\GAL4c309 30oC values. Reducing the extent to which Scer\GAL4c309 drives expression of shi1.Scer\UAS in heat-treated males by expression of Scer\GAL80Cha.PK does not affect the level of male courting, which remains approximately five times higher than in controls.
shi1.Scer\UAS when driven by Scer\GAL4NPFR1.6.6 or Scer\GAL4npf.1 at the permissive temperature mutant animals exhibit a response to alcohol vapour no different from controls. When preincubated at 30oC 60 minutes before assay, the mutant flies are consistently more resistant to alchohol than controls.
At the permissive temperature of 23oC, 120 minute fasted third instar larvae expressing shi1.Scer\UAS (under the regulation of Scer\GAL4npf.1 or Scer\GAL4NPFR1.6.6) display normal feeding responses to both liquid and solid foods. However, if third instar larvae are incubated at 30oC for 15 minutes, they exhibit attenuated feeding response to the solid but not liquid food. Foraging activity is completely restored upon return to the permissive temperature.
At the permissive temperature of 23oC, high frequency stimulation induces normal HFMR (high-frequency-stimulation-induced miniature release) in the neuromuscular junctions of animals expressing shi1.Scer\UAS under the control of Scer\GAL4Mhc.PW. However, at the restrictive temperature of 31oC, HFMR is suppressed in animals expressing shi1.Scer\UAS under the control of Scer\GAL4Mhc.PW (in contrast to controls). This suppression of HFMR at 31oC is not irreversible, because a second high frequency stimulation after recovery to the permissive temperature induces a normal HFMR in the mutant animals. Basic synaptic properties are normal at the neuromuscular junction of animals expressing shi1.Scer\UAS under the control of Scer\GAL4Mhc.PW at both 23oC and 31oC.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4c316, exhibit a normal 3 hour memory following odorant training when shifted to the non-permissive temperature for up to 15 minutes after training. However, when these flies are subjected to the non-permissive temperature between 30 to 60 minutes following training, their 3 hour memory is abolished. However, when a 30 minute temperature shift is imposed between 150 and 180 minutes after training, there is no significant effect on 3 hour memory.
The propensity of males expressing shi1.Scer\UAS under the control of Scer\GAL455B to father offspring and to repress female remating is substantially decreased when pairs of copulating flies are shifted from the permissive to the restrictive temperature early, but not later, in copulation. The intensity of the defect in both phenotypes seems to be coupled. The copulation duration of males expressing shi1.Scer\UAS under the control of Scer\GAL455B increases with the duration that the copulating pair of flies spent at the permissive temperature.
Animals expressing shi1.Scer\UAS under the control of Scer\GAL4repo that are raised at the restrictive temperature (29oC) during early pupal development show abnormal morphology of glial cells surrounding the mushroom body; in wild-type pupae, glial processes infiltrate into the mushroom body lobes by 6 hours after puparium formation (APF), but the dorsal lobes of animals expressing shi1.Scer\UAS under the control of Scer\GAL4repo at the restrictive temperature are devoid of any glial processes, even at 18 hours APF. In addition, the mutant animals do not show axon pruning in the mushroom body that is normally seen during early pupal development (disappearance of clustered varicosities and loss of axon branches is not seen). These mutant animals do not survive to adulthood. A small number of animals expressing shi1.Scer\UAS under the control of Scer\GAL4repo that are raised at the restrictive temperature (29oC) during early pupal development and then shifted to 25oC at 24 hours APF eclose. These animals have abnormal axon branches beside the α and β lobes of the mushroom body shortly after hatching.
When expression is driven by Scer\GAL4MJ286, Scer\GAL4MJ146, Scer\GAL4c747, Scer\GAL4c309, Scer\GAL440B, Scer\GAL47B, Scer\GAL453B or Scer\GAL429B male courtship behavior is reduced though for Scer\GAL440B, Scer\GAL47B and Scer\GAL453B the general effects on locomoter/peripheral behaviour obscure the relevance. When expression is driven by Scer\GAL4c747 wing extension and vibration duration during courtship are increased. When expression is driven by Scer\GAL4c309 or Scer\GAL4MJ286 wing extension and vibration duration during courtship are decreased.
Expression driven in different subsets of mushroom body lobes blocks synaptic transmission in those places when at the restrictive temperature. 2 hour memory relies on all mushroom alpha, beta and gamma lobes. ARM relies more heavily on alpha/beta lobes. LTM retrieval requires output from alpha/beta lobes. gamma lobes do not play a role in LTM retrieval.
At the permissive temperature of 25oC, both immediate (3 min) memory and 3 hr memory of Scer\GAL4c316;shi1.Scer\UAS flies are statistically indistinguishable from wild-type.
At the restrictive temperature of 31oC, immediate (3 min) memory of Scer\GAL4c316;shi1.Scer\UAS flies is statistically indistinguishable from wild-type. However, 3 hr memory is statistically lower than wild-type.
To block DPM neuron output during training, Scer\GAL4c316;shi1.Scer\UAS and control flies are incubated at 31oC for 15 min prior to and during training. Flies are returned to 25oC immediately following training, and 3 hr memory tested at 25oC. Blocking DPM output during training does not affect memory. The memory of Scer\GAL4c316;shi1.Scer\UAS flies is indistinguishable from control flies that are trained at the restrictive temperature.
The 3 hour memory of Scer\GAL4c316;shi1.Scer\UAS flies trained at 25oC, and then shifted to 31oC 15 minutes before testing, is indistinguishable from control flies, suggesting that DPM output is not required for memory recall.
Scer\GAL4c316;shi1.Scer\UAS flies placed at 31oC for 15 minutes prior to training (and returned to 25oC immediately after), that are then treated to 31oC for 15 minutes before testing exhibit memory that is no worse than that of flies receiving either manipulation alone and is indistinguishable from that of control flies.
Incubation of Scer\GAL4c316;shi1.Scer\UAS flies at 31oC fpr 2 hours, then returning them to 25oC for 15 minutes before training does not have an effect on 3 hour memory.
Temperature manipulations do not significantly impair odor or shock acuity of Scer\GAL4c316;shi1.Scer\UAS flies flies.
Blocking DPM output with Scer\GAL4c316;shi1.Scer\UAS specifically reduces benzaldehyde immediate memory and leaves OCT immediate memory intact.
The OCT and MCH performance of Scer\GAL4c316;shi1.Scer\UAS flies is unaffected by temperature and is indistinguishable from the memory of wild-type flies.
Blocking DPM output during acquisition does not affect 1 hr OCT memory - Scer\GAL4c316;shi1.Scer\UAS and Scer\GAL4Mz717;shi1.Scer\UAS fly memory is indistinguishable from wild-type. However, DPM blockade in Scer\GAL4c316;shi1.Scer\UAS and Scer\GAL4Mz717;shi1.Scer\UAS flies severely impairs 1 hr benzaldehyde memory. In contrast, DPM blockade during acquisition does not significantly affect memory with OCT-MCH. Furthermore, olfactory acuity and the response to electric shock of Scer\GAL4c316;shi1.Scer\UAS and Scer\GAL4Mz717;shi1.Scer\UAS flies is unaffected by temperature.
Like wild-type, shi1.Scer\UAS; Scer\GAL4per.PK males raised at 25oC show a reduction in mating behaviour 5 days after courtship conditioning for 7 hours. This reduction is eliminated in males of this genotype (but not in wild-type) raised at 30oC. As having these flies at 30oC during testing is sufficient to eliminate this reduction, but earlier time spent at 30oC is not, it seems likely that this phenotype is due to a failure of memory retrieval, rather than formation or storage.
When shi1.Scer\UAS is driven by Scer\GAL4103Y, Scer\GAL4c747 or Scer\GAL4OK107 the avoidance response seen by flies to Drosophila stress odorant (dSO) is unaffected. When shi1.Scer\UAS is driven by Scer\GAL4Gr21a.9.323 at non-permissive temperatures the avoidance response seen by flies to ~1% CO2 is not seen. A response to dSO still occurs. When shi1.Scer\UAS is driven by Scer\GAL4GH146, Scer\GAL4Or47b.7.467, Scer\GAL4GH146 or Scer\GAL4Or83b.2.642.T:Hsim\VP22 a normal avoidance response to dSO is seen. When shi1.Scer\UAS is driven by Scer\GAL4c761 at non-permissive temperatures the avoidance response seen by flies to dSO and ~1% CO2 is reduced.
Expression of shi1.Scer\UAS under the control of Scer\GAL4GMR.PF at 23oC results in extensive degeneration of photoreceptors R1 to R6, characterised by loss of rhabdomeres and accumulation of multivesicular bodies and vacuoles in the photoreceptor cells.
When shi1.Scer\UAS is expressed under the control of Scer\GAL4A307 and the temperature is shifted to the restrictive temperature for 24
hours during the first quarter of pupal development, an unusual overgrowth
of the giant fiber (GF) axons is observed in the thoracic neuromeres
in the resulting adults. In 61% of cases, the GF axons branch throughout
the second and third neuromeres. In a minority of cases, the GFs grow
straight through the target region and terminate in the subesophageal
neuromere in the brain. Correlated with the anatomy, 60% of the GFs
reach the target area and are connected to the dorsal longitudinal
muscle (DLM) (as assayed physiologically), although the GFs that are
able to drive the dorsal longitudinal motoneuron (DLMn) do so much
less reliably than in controls. If the GF axons are examined immediately
after the temperature shift, rather than in the adult, they mostly
terminate in a "retraction bulb", which can be in the connective (36%
of the GFs) or the first thoracic neuromere (36% of the GFs) or in
the brain (in a minority of cases). Loss or shrinkage of the tergotrochanteral
muscle (TTM) fibres is seen in these animals.
When shi1.Scer\UAS is expressed under the control of Scer\GAL4A307
and the temperature is shifted to the restrictive temperature for 24
hours during 25-50% of pupal development, the resulting adult giant
fiber axon usually terminates in the target area but the ending is
abnormal. Most adult GF axons lack the large laterally directed presynaptic
terminal ("bendless" phenotype). Almost all GFs are connected to the
target area (either to the DMLn or the tergotrochanteral motoneuron
(TTMn) as assayed physiologically), but the GF-TTMn connection is severely
disrupted (being either absent or weakened). Temperature shifts to
the restrictive temperature between 17-50% of pupal development occasionally
result in adult specimens with GF terminals in the brain. If the GF
axons are examined immediately after a temperature shift at 33% of
pupal development, rather than in the adult, then 64% of the axons
terminate in retraction bulbs, which can be located either in the
first or second thoracic neuromere. In some cases, the retraction
bulbs show single long extensions that resemble "retraction tails"
that reach the target area. If the GF axons are examined immediately
after a temperature shift at 50% of pupal development, rather than
in the adult, then the presynaptic terminal is retracted only slightly
from the target area.
When shi1.Scer\UAS is expressed under the control of Scer\GAL4A307
and the temperature is shifted to the restrictive temperature for 24
hours at 62.5 or 75% of pupal development, there is only a minor affect
on the resulting adult GF anatomical phenotype; typically the bend
is present and is normal in size, although it may contain irregularities
in shape. However, when assayed physiologically in the adult, the
TTM exhibits long latencies and low following frequencies. If the
GF axons are examined immediately after a temperature shift at 67%
of pupal development, rather than in the adult, there are minor defects
in the synaptic terminal but no retraction.
When shi1.Scer\UAS is expressed under the control of Scer\GAL4A307
and the temperature is shifted to the restrictive temperature for 24
hours very late in pupal development (for example at 87.5% of pupal
development) there is no impact on the GFs or their ability to drive
the TTMn or DLMn in the adult.
When shi1.Scer\UAS is expressed under the control of Scer\GAL4c17
and the temperature is shifted to the restrictive temperature (for
24 hours) at 50% pupal development, the GF axons in the resulting adults
occasionally (17%) have a bendless phenotype and the GF-TTMn connection
is physiologically slightly disrupted. Temperature shift at 62.5%
of pupal development results in adults that have GF axons that are
anatomically wild-type and physiologically slightly abnormal.
80-100% of flies expressing shi1.Scer\UAS under the control of Scer\GAL4Ccap.PP at 29oC have defects in wing expansion.
The ability of flies expressing shi1.Scer\UAS under the control of Scer\GAL4Mef2.247 to learn to avoid odors that they have previously experienced with an electric shock and to learn to be attracted to odors that have been combined with a sugar reward is the same as wild type at 26oC. However, at 34oC electric shock memory is impaired while sugar memory is abolished completely. If shi1.Scer\UAS;Scer\GAL4Mef2.247 flies are trained at 26oC and tested at 34oC, sugar memory is decreased but if these flies are trained at 34oC and tested at 26oC, sugar memory is not different to wild type. These flies have the same ability as wild type to respond spontaneously to electric shocks, sugar and the odorants EA and IA. Expression of shi1.Scer\UAS, under control of the dopaminergic neuron-specific Scer\GAL4ple.PF driver, causes impairment of electric shock-associated odorant memory at 34oC. This phenotype also occurs if flies are trained at 34oC and tested at 26oC. Sugar-associated odorant memory is not affected.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tab2-201Y or Scer\GAL4238Y are repulsed by high concentrations of odorants (ethyl acetate, mango, methyl cyclohexanol, ethyl propionate or benzaldehyde) at both the permissive (18oC) and restrictive temperature (30oC), as occurs in wild-type flies. Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tab2-201Y or Scer\GAL4238Y at 18oC are attracted to low concentrations of the same odorants (ethyl acetate, mango, methyl cyclohexanol, ethyl propionate or benzaldehyde), but the attraction is altered to various degrees in these flies if the temperature is raised to 30oC (this is in contrast to wild-type flies which show no alteration in odour attraction at 30oC compared to 18oC). In flies expressing shi1.Scer\UAS under the control of Scer\GAL4238Y at 30oC, attraction to all odours is abolished or significantly reduced. In flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tab2-201Y at 30oC, attraction to ethyl acetate, mango or methyl cyclohexanol is diminished, but attraction to ethyl propionate or benzaldehyde is not significantly affected. Flies expressing shi1.Scer\UAS under the control of Scer\GAL4OK66 at 30oC show disruption in both attraction (at low concentrations) and repulsion (at high concentrations) to ethyl acetate. At high concentrations, when repulsiveness of benzaldehyde (BA) or octanol (OCT) has reached saturation, when wild-type flies are forced to choose between 10% BA and 10% BA + 40% OCT, they strongly prefer 10% BA + 40% OCT. At 18oC, flies expressing shi1.Scer\UAS under the control of Scer\GAL4238Y also strongly prefer 10% BA + 40% OCT, but at 30oC the preference for 10% BA + 40% OCT is largely abolished in these flies.
Expression of shi1.Scer\UAS under the control of Scer\GAL4Tab2-201Y does not result in mushroom body defects. Expression of shi1.Scer\UAS under the control of Scer\GAL4OK107 results in gross defects in the whole brain.
shi1.Scer\UAS; Scer\GAL4c309 males raised at 19oC, show male-male courtship behaviour but decreased levels of courtship with females when transferred to 30oC. Their male-male courtship behaviour includes: orientation toward and following a male partner, tapping the partner's abdomen with the forelegs, unilateral wing vibration, licking of the partner's genitalia and attempted copulation. Much higher levels of head-to-head interactions occur between mutant males at 30oC than at 19oC. These behavioural effects are seen within minutes of transfer to 30oC, and subside within a few minutes of transfer back to 19oC.
Mutant males at 30oC exhibit these behaviours towards wild-type males, but their advances are not reciprocated. None of these phenotypes are seen when expression is instead driven with Scer\GAL4OK107, Scer\GAL4c747, Scer\GAL4c492b or Scer\GAL417d.
The male-male courtship behaviours seen in shi1.Scer\UAS; Scer\GAL4c309 flies at 30oC are suppressed by the presence of Scer\GAL80Cha.PK.
Spike-like potentials are seen when local field potential (LFP) recordings are made from the differential activity between one electrode place in between the mushroom bodies at approximately the level of the base of the calyces versus one placed in the lamina or medulla of the optic lobes. These spike like potentials are significantly repressed when synaptic transmission in mushroom body neurons is disrupted by expression of shi1.Scer\UAS with Scer\GAL4c309. This effect is not seen when shi1.Scer\UAS is driven in motoneurons with Scer\GAL4D42. LFP activity recorded between the left and right optic lobes is normal in shi1.Scer\UAS; Scer\GAL4c309 flies. Heat induced paralysis has no effect on any of these phenotypes.
Expression of shi1.Scer\UAS, under the control of either Scer\GAL4247 or Scer\GAL4c747 (with two copies of shi1.Scer\UAS) blocks memory performance at 34[o]C. There is a significant reduction in 3 min memory performance when the flies are tested at the restrictive temperature. Thirty minute memory scores are largely unaltered at the permissive temperature. However, if the flies are trained at the permissive temperature and tested at the restrictive temperature, there is a severe decrement in performance.
Expression of two copies of shi1.Scer\UAS under the control of Scer\GAL4c747 at the permissive temperature and tested after 3 hours having been shifted to the restrictive temperature only 15 mins earlier, results in no memory performance, in contrast to controls.
Flies expressing shi1.Scer\UAS in the mushroom bodies through Scer\GAL4247 and Scer\GAL4c747 trained and exposed to extinguishing stimuli at the restrictive temperature and then tested at the permissive temperature show obvious extinction of their olfactory memories. In contrast, expression of shi1.Scer\UAS using Scer\GAL4GH146 during the extinction procedure does not lead to memory extinction.
When expression is driven by Scer\GAL4MJ85b flies rapidly become paralysed after being shifted to the restrictive temperature and recover locomotion within one minute after a shift back to the permissive temperature. When expression is driven in the mushroom body by Scer\GAL4c747 or Scer\GAL4c309 locomotion and sensorimotor responses to odor and footshock were normal at permissive and restrictive temperatures. Associative learning (produced by pairing odour and footshock in pavlovian conditioning) were normal at permissive temperature. However when shifted to restrictive temperature 30 mins before training, olfactory learning was abolished. When shifted to restrictive temperature for 30 mins and then back to permissive temperature for 30 mins before training performance was normal. When expression is driven by Scer\GAL4c747 or Scer\GAL4c309, and flies are shifted to restrictive temperature for 30 mins, trained, immediately returned to permissive temperature and tested for retention of learning after 30 mins performance is normal. This indicates that synaptic transmission from mushroom body neurons is not required for acquisition of an odour-footshock association. When expression is driven by Scer\GAL4c747 or Scer\GAL4c309, and flies are trained at the permissive temperature, immediately shifted to restrictive temperature for 30 mins, returned to permissive temperature for 5 mins before testing retention at 30 mins, performance is normal. This indicates that synaptic transmission from mushroom body neurons is not required for storage of early memory. When expression is driven by Scer\GAL4c747 or Scer\GAL4c309, and flies are trained at the permissive temperature, immediately shifted to restrictive temperature, then testing 5 minute retention at restrictive temperature, performance is defective. Flies show disrupted synaptic transmission in mushroom body neurons. Defective retrieval is also evident in a reversal-retention protocol.
At 22oC, flies expressing shi1.Scer\UAS under the control of either Scer\GAL4GMR.PF or Scer\GAL4Cha.7.4 do not show any obvious abnormalities in morphology or behaviour.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Cha.7.4 become motionless within 2 minutes after transfer to 30oC. After transfer to 30oC, the flies gradually become motionless without increased motor activities. The temperature-sensitive paralysis is reversible upon transfer to 22oC; the flies regain their activity and start to walk within 1 minute.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4GMR.PF do not show paralysis at 30oC.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4GMR.PF show a normal response to gravity (measured using a countercurrent apparatus) at both 19o and 30oC.
At 19oC, flies expressing shi1.Scer\UAS under the control of Scer\GAL4GMR.PF show a normal response to light when startled; they move towards the light (into the distal tubes) when it is placed at the opposite end of the starting position in a countercurrent apparatus, and they stay in the starting tubes when the light is at the starting point. However, at 30oC, these flies show no significant preference for the distal tubes when the light is placed at the opposite end of the starting position and they show a similar distribution pattern in the countercurrent apparatus when the light is at the starting point, indicating that they cannot distinguish light at 30oC.
The electroretinograms (ERGs) of flies expressing shi1.Scer\UAS under the control of Scer\GAL4GMR.PF are normal at 18oC. However, after a 10 minute light stimulation at 31oC, the on- and off-transients of the ERG are completely absent and signal amplitudes of the sustained component are reduced.
At the permissive temperature, larvae expressing shi1.Scer\UAS under the control of either Scer\GAL4GMR.PF or Scer\GAL4Cha.7.4 show a strong tendency to avoid light (as do wild-type larvae). However, at the restrictive temperature of 30oC, the larvae show distinct abnormalities. Larvae expressing shi1.Scer\UAS under the control of Scer\GAL4GMR.PF retain normal locomotor activity at 30oC, but are distributed evenly on the test plates, indicating that they cannot distinguish light from dark. Larvae expressing shi1.Scer\UAS under the control of Scer\GAL4Cha.7.4 show greatly reduced locomotor activity after 10 minutes at 30oC.
Approximately 15% of animals expressing shi1.Scer\UAS under the control of Scer\GAL4GMR.PF develop into adults when maintained at 30oC during development. All of these adults show severe defects in eye morphology, with the eye being reduced to a small misshapen rudiment.
When animals expressing shi1.Scer\UAS under the control of Scer\GAL4GMR.PF are incubated at 30oC during the last 48 hours of larval development, developing photoreceptors are still present, but they do not form a normal topographic array in the lamina and medulla.
Flies expressing shi1.Scer\UAS under the control of Scer\GAL4c309, Scer\GAL4OK107, or Scer\GAL4c747 show diminished odour avoidance at the restrictive temperature. Flies expressing shi1.Scer\UAS under the control of Scer\GAL4Tab2-201Y show normal 3-minute memory at both the permissive and restrictive temperature. Flies expressing shi1.Scer\UAS under the control of Scer\GAL4c739 or Scer\GAL4Mef2.247 show normal odour and shock avoidance but show a disruption in 3-minute memory at the restrictive temperature. Memory at 3 hours is abolished in flies expressing shi1.Scer\UAS under the control of Scer\GAL4c739 when they have been trained at the permissive temperature and then tested at the restrictive temperature. Flies expressing shi1.Scer\UAS under the control of Scer\GAL4c739 trained at the restrictive temperature and then tested at the permissive temperature show a significant difference in memory at 3 hours compared to control flies carrying only Scer\GAL4c739, but no significant difference compared to control flies carrying only shi1.Scer\UAS, indicating a general effect of heat on lines carrying shi1.Scer\UAS.
When expression is driven by Scer\GAL4c316 at 25oC flies learn and remember indistinguishably from wild type flies. However at 34o flies have undetectable memory though initial learning is as for wild type.
Scer\GAL4ppk.PG, shi1.UAS has abnormal neuroanatomy | pupal stage phenotype, enhanceable by par-1HMS00405/Scer\GAL4ppk.PG
Scer\GAL4c17, shi1.UAS has abnormal neurophysiology | heat sensitive phenotype, enhanceable by Scer\GAL4c17/Sema1aUAS.cYa
Scer\GAL4c17, shi1.UAS has abnormal neuroanatomy | heat sensitive phenotype, enhanceable by Scer\GAL4c17/Sema1aUAS.cYa
Scer\GAL4esg.PU, shi1.UAS has increased occurrence of cell division | adult stage phenotype, non-enhanceable by SH3PX1HMJ21552, Scer\GAL4esg.PU
Scer\GAL4c316, shi1.UAS has abnormal memory | adult stage phenotype, non-enhanceable by Dscam1exon17.2.UAS.GFP
Scer\GAL4elav.Switch.PO/shi1.UAS is an enhancer of short lived | RU486 conditional phenotype of Scer\GAL4elav.Switch.PO, Zzzz\poly-GA200.UAS
shi1.UAS/Scer\GAL4ppk.PG is an enhancer of abnormal neuroanatomy | pupal stage phenotype of Scer\GAL4ppk.PG, par-1HMS00405
shi1.UAS/Scer\GAL4VT030604 is an enhancer | heat sensitive of abnormal memory | adult stage phenotype of rsh1
shi1.UAS/Scer\GAL4c739 is an enhancer of abnormal heat stress response phenotype of Pgam51
Scer\GAL4c17/shi1.UAS is an enhancer of abnormal neurophysiology | heat sensitive phenotype of Scer\GAL4c17, Sema1aUAS.cYa
Scer\GAL4c17/shi1.UAS is an enhancer of abnormal neuroanatomy | heat sensitive phenotype of Scer\GAL4c17, Sema1aUAS.cYa
shi1.UAS, Scer\GAL4elav.Switch.PO is a non-enhancer of short lived | RU486 conditional phenotype of Hsap\APP2xAPP.SP.UAS, Scer\GAL4elav.Switch.PO
shi1.UAS/Scer\GAL4VT049246 is a non-enhancer of abnormal memory | adult stage phenotype of rsh1
shi1.UAS, Scer\GAL4esg.PU is a suppressor | partially of increased occurrence of cell division | adult stage | progressive phenotype of SH3PX1HMJ21552, Scer\GAL4esg.PU
Scer\GAL4GMR57C10/shi1.UAS is a suppressor of bang sensitive phenotype of parabss1
Scer\GAL4109(2)80/shi1.UAS is a suppressor | partially | somatic clone of abnormal neuroanatomy | somatic clone | third instar larval stage phenotype of RopG27
shi1.UAS, Scer\GAL4elav.Switch.PO is a non-suppressor of short lived | RU486 conditional phenotype of Hsap\APP2xAPP.SP.UAS, Scer\GAL4elav.Switch.PO
shi1.UAS/Scer\GAL4VT049246 is a non-suppressor of abnormal memory | adult stage phenotype of rsh1
Scer\GAL4ppk.PG, shi1.UAS has larval multidendritic class IV neuron | pupal stage phenotype, enhanceable by par-1HMS00405/Scer\GAL4ppk.PG
Scer\GAL4c17, shi1.UAS has giant fiber neuron | heat sensitive phenotype, enhanceable by Scer\GAL4c17/Sema1aUAS.cYa
Scer\GAL4esg.PU, shi1.UAS has adult midgut epithelium phenotype, non-enhanceable by SH3PX1HMJ21552, Scer\GAL4esg.PU
Scer\GAL4GMR.PF, shi1.UAS has rhabdomere R1 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has rhabdomere R2 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has rhabdomere R3 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has rhabdomere R4 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has rhabdomere R5 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has rhabdomere R6 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has photoreceptor cell R1 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has photoreceptor cell R2 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has photoreceptor cell R3 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has photoreceptor cell R4 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has photoreceptor cell R5 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has photoreceptor cell R6 phenotype, suppressible by CDaseUAS.cAa
Scer\GAL4GMR.PF, shi1.UAS has photoreceptor neuron phenotype, suppressible by lace[+]/lacek05305
shi1.UAS/Scer\GAL4ppk.PG is an enhancer of larval multidendritic class IV neuron | pupal stage phenotype of Scer\GAL4ppk.PG, par-1HMS00405
shi1.UAS/Scer\GAL4ppk.PG is an enhancer of dendrite | pupal stage phenotype of Scer\GAL4ppk.PG, par-1HMS00405
Scer\GAL4c17/shi1.UAS is an enhancer of giant fiber neuron | heat sensitive phenotype of Scer\GAL4c17, Sema1aUAS.cYa
shi1.UAS, Scer\GAL4esg.PU is a suppressor | partially of adult midgut epithelium | progressive phenotype of SH3PX1HMJ21552, Scer\GAL4esg.PU
Scer\GAL4109(2)80/shi1.UAS is a suppressor | partially | somatic clone of larval multidendritic class IV neuron | somatic clone | third instar larval stage phenotype of RopG27
shi1.UAS is a suppressor | partially of egg phenotype of CblL.UAS, Scer\GAL4EQ1
shi1.UAS is a suppressor | partially of egg chorion phenotype of CblL.UAS, Scer\GAL4EQ1
shi1.UAS is a suppressor | partially of dorsal appendage phenotype of CblL.UAS, Scer\GAL4EQ1
Scer\GAL4Tub.PU/shi1.UAS is a non-suppressor of eye disc | somatic clone | third instar larval stage phenotype of Vps43B1
Scer\GAL4Tub.PU/shi1.UAS is a non-suppressor of photoreceptor | somatic clone | third instar larval stage phenotype of Vps43B1
The penetrance of the pruning defects observed in class IV dendritic arborizing neurons of pupae expressing par-1HMS00405 under the control of Scer\GAL4ppk.PG is significantly enhanced by co-expression of shi1.Scer\UAS, which on its own also induce mild pruning defects.
Expression of shi1.Scer\UAS under the control of Scer\GAL4GMR57C10 partially suppresses the bang sensitivity of hemizygous parabss1 flies when incubated for 3 min at 28[o]C, but not at 23[o]C.
shi1.Scer\UAS under the control of Scer\GAL4GMR57C10 partially suppresses the bang sensitivity of parabss1/+ flies at 23[o]C.
Expression of shi1.Scer\UAS under the control of Scer\GAL4tub.PU does not suppress the photoreceptor loss seen in Vps43B1 mutant eye disc clones.
Expression of temperature-sensitive shi1.Scer\UAS under the control of Scer\GAL4109(2)80 in RopG27/RopG27 somatic clones ameliorates the loss of dendritic terminal branches displayed by RopG27/RopG27 class IV dendritic arborizing (da) neurons in third instar larvae (shifted to non-permissive temperature at 48hrs after egg laying and examined at 96hrs). Compared to controls kept under the same temperature regimen the shi1.Scer\UAS expressing RopG27/RopG27 class IV da neurons show increased number of dendrite crossings.
Expression of shi1.Scer\UAS under the control of Scer\GAL4c739 enhances the sensitivity to heat stress seen in Pgam51 mutant flies.
w1118; shi1.Scer\UAS flies establish more dominance relationships compared to w1118(CS); shi1.Scer\UAS pairs at both 19[o]C and 30[o]C.
Expression of shi1.Scer\UAS with Dscamexon17.2.Scer\UAS.T:Avic\GFP by Scer\GAL4c316, and transferring the animals to 31[o]C for one hour immediately after training significantly reduces 3hr aversive odour memory compared to controls.
The fusion and reduction of dorsal appendages seen in eggs laid by CblL.Scer\UAS; Scer\GAL4EQ1 mothers is partially suppressed by shi1.Scer\UAS.
Co-expression of shi1.Scer\UAS in feminised males expressing traScer\UAS.cFa under the control of Scer\GAL455B by shifting copulating pairs of flies to the restrictive temperature either late or early during copulation has little influence of the fertility of the males but highly decreases their ability to repress female remating.
The degeneration of photoreceptor cells seen in flies expressing shi1.Scer\UAS under the control of Scer\GAL4GMR.PF at 23oC is suppressed by coexpression of CDaseScer\UAS.cAa; rhabdomeres are largely intact, there are fewer vacuolated cells and the trapezoidal arrangement of the rhabdomeres is retained. The degeneration of photoreceptor cells seen in flies expressing shi1.Scer\UAS under the control of Scer\GAL4GMR.PF is substantially suppressed by lacek05305/+.
shi1.Scer\UAS and Sema-1aScer\UAS.cYa show an interaction when expressed under the control of Scer\GAL4c17; even in the absence of a temperature shift to the restrictive temperature only half the adults have anatomically normal giant fiber (GF) axons, with the remainder having either a bendless phenotype or failing to reach the target area. Only 45% of the GF axons are physiologically normal. When shi1.Scer\UAS and Sema-1aScer\UAS.cYa are co-expressed under the control of Scer\GAL4c17 and animals are shifted to the restrictive temperature (for 24 hours) at 37.5% of pupal development, 44% of the GF axons in the resulting adults terminate in the thorax and are anatomically defective and most of the reamining GF axons terminate in the brain. None of the GF axons are physiologically normal in these adults. When shi1.Scer\UAS and Sema-1aScer\UAS.cYa are co-expressed under the control of Scer\GAL4c17 and animals are shifted to the restrictive temperature (for 24 hours) at 50% of pupal development, most of the GF axons in the resulting adults exit the brain but the presynaptic terminals in the thorax are defective. The disrupted axon terminals are unusually large and are often filled with membrane-bound vesicles. None of the GF axons are physiologically normal in these adults. When shi1.Scer\UAS and Sema-1aScer\UAS.cYa are co-expressed under the control of Scer\GAL4c17 and animals are shifted to the restrictive temperature (for 24 hours) at 62.5% of pupal development, none of the GF axons show a bend when examined immediately after the temperature shift, suggesting that all immature synapses have retracted.
The male-male courtship behaviours seen in shi1.Scer\UAS; Scer\GAL4c309 flies at 30oC are suppressed by the presence of Scer\GAL80Cha.PK.
Kitamoto
T.Kitamoto
