Cycles of protein synthesis during pupal diapause in the flesh fly,Sarcophaga crassipalpis

Protein synthesis is cyclic during pupal diapause in Sarcophaga crassipalpis. These cycles are in phase with infradian MO₂ cycles, which have a periodicity of about 4 days at 25°C. Mean incorporation of [³⁵S]methionine by diapausing pupae was 5.4% during the 2 days of highest MO₂ but dropped to 1.7% during the 2 days of low MO₂. Diapausing pupae treated with a juvenile hormone analog prior to pupariation had a constant high MO₂ similar to peak values observed in untreated pupae, and such pupae consistently incorporated [³⁵S]methionine at a high rate (7.7%). [³⁵S]Methionine incorporation by nondiapausing pupae and pharate adults was eightfold higher than the peak rates observed during diapause. Autoradiography of in vivo labeled proteins indicated quantitative and qualitative changes in the synthesis of proteins by diapausing pupae during different phases of the MO₂ cycle. Brains from diapausing pupae labeled in vitro showed higher incorporation at the peak of the MO₂ cycle than at the nadir of the cycle, but no such differences were detected for integument, fat body, or fat body supernatant. Theses differences in tissue response indicate that control of protein synthesis during diapause is not cell autonomous, but is a function of the metabolism of the intact organism.     

Deuterium oxide alters pupal diapause response in the flesh fly, Sarcophaga crassipalpis

ABSTRACT. Deuterium oxide averts pupal diapause in the flesh fly Sarcophaga crassipalpis Macquart when fed to larvae or when applied topically to photosensitive embryos exposed to short daylength. Deuterium oxide was not effective in promoting diapause when presented to embryos or larvae reared at long daylength. The effect of deuterium oxide appears to be cumulative in the larval state: increasing exposure time progressively reduces diapause response. If flies reared on deuterium oxide are exposed to continuous darkness, the diapause response remains high, thus implying that the physiological capacity for diapause is not disrupted. We suggest that deuterium oxide exerts its effect on the circadian rhythm controlling diapause induction.     

Photoperiodic induction of pupal diapause in the flesh fly,Sarcophaga crassipalpis: embryonic sensitivity

Summary The last two days of embryonic development are crucial in programming pupal diapause in the flesh fly,Sarcophaga crassipalpis. Short daylength (greater than 10 1/2h of darkness) during this interval permits expression of diapause while long daylength during this brief sensitive stage eliminates the potential for diapause. Length of scotophase rather than photophase programs the diapause although three hours of light is needed to separate tandem dark periods. Early in the scotophase, photosensitivity is restricted to blue light (less than 540 nm). The scotophase can be divided into 4 phases according to the effect of light breaks on diapause expression. During Phase I (0–6 h after scotophase onset) embryos are highly sensitive to light interruption and diapause is effectively eliminated. A period of insensitivity to light, Phase II, extends from 6–hh after onset of scotophase. Light breaks at 10–11h coincide with the critical scotophase length and result in a partial reduction of diapause. In Phase IV, the scotophase reaction is complete and diapause competence is preserved even in the presence of light. Although light breaks result in elimination of diapause throughout Phase I, recovery time from a 1 h light break (length of darkness needed to counter the effect of a light break) differs dramatically depending upon when the light break is presented. Early in Phase I (0–3h) recovery from light interruption is rapid, while late in Phase I (4–6h), the effects of light are not readily reversible. The scotophase reaction thus appears to follow a step-wise progression rather than represent a simple linear response. We present a molecular model that could account for the dynamics of the scotophase reaction.     

Role of the brain and ring gland in regulation of pupal diapause in the flesh fly Sarcophaga crassipalpis

Larvae of Sarcophaga crassipalpis photoperiodically programmed for pupal diapause pupariate later than larvae programmed for continuous development. Pupariation time is determined by the brain-ring gland complex as evidenced by transplantation experiments in which the timing of pupariation was transferred from one larva to another by transplantation of the brain-ring gland complex. The developmental commitment (diapause or nondiapause) of the larva also can be transferred with the brain-ring gland complex if the recipient's own neuroendocrine system is suppressed by ring gland extirpation. Thus, photoperiodic programming of the brain-ring gland complex is not only responsible for developmental commitment but also for determining the duration of the prepupal period. Surgical experiments with pupae indicate that an intact brain-ring gland complex is required for diapause termination and initiation of adult development. Pupae fail to break diapause if either the brain or the ring gland is removed or if their nervous connections are severed.     

High temperature and hexane break pupal diapause in the flesh fly, Sarcophaga crassipalpis, by activating ERK/MAPK

Pupal diapause in the flesh fly, Sarcophaga crassipalpis, can be terminated by exposure to high temperatures or, artificially, with a topical application of organic solvents. To analyze the molecular mechanisms involved in diapause termination we explored the possibility that the mitogen-activated protein kinases (MAPK) are involved in this response. Levels of phospho-ERK increased within 10 min after hexane application. Extracellular signal-regulated kinase (ERK) was also activated when pupae were transferred from 20 to 25 degrees C, thus suggesting that ERK activation is a likely component of the signal transduction pathway used to initiate development in response to diapause-terminating signals. 20-Hydroxyecdysone and cyclic GMP terminate diapause in this fly, and the juvenile hormone analog methoprene shortens the diapause, but none of these agents activated ERK. ERK was readily activated in isolated abdomens treated with hexane, thus we conclude that ERK is directly activated by the hexane treatment. ERK activation was evident in the brain, epidermis, midgut and fat body, but not in the ventral nerve mass or ring gland, thus suggesting that ERK does not act directly on the ring gland to promote ecdysteroid synthesis but exerts its effect through stimulation of the brain.     

Mendelian inheritance of pupal diapause in the flesh fly, Sarcophaga bullata

Pupal diapause (dormancy) in the flesh fly, Sarcophaga bullata, is induced by short-day photoperiods and low temperature. In this study, the inheritance mode of diapause was investigated by crossing a nondiapausing (nd) strain of S. bullata with 2 diapausing strains having different diapause capacities. The results consistently indicated that diapause incidence is inherited in a simple Mendelian pattern, thus a single gene or a small gene cluster linked to the photoperiodic clock controls the seasonal response of diapause. The fact that the nd strain lacked daily rhythmicity in adult eclosion and showed altered expression of 2 circadian clock genes suggests that the photoperiodic and circadian clocks are related through a shared molecular component in S. bullata.     

Induction and decay of thermosensitivity in the flesh fly,Sarcophaga crassipalpis

When pharate adults of the flesh fly Sarcophaga crassipalpis are exposed to 40°C for 4 h they become more tolerant of high temperatures that are normally lethal (thermotolerance). In contrast, a 1-h exposure to 45°C decreases tolerance to a subsequent high temperature challenge (thermosensitivity). While control flies experience little mortality when held at 35°C for 24–48 h the thermosensitized flies die when exposed to 35°C. Sensitivity to a second thermal challenge slowly decays over a 72-h period. The acquisition of thermotolerance prevents the development of thermosensitivity. Brains from thermosensitized flies cultured at 43°C express the 72-kDa heat-shock protein and normal protein synthesis is inhibited. This implies that development of thermosensitivity is not associated with a loss in the capacity to express the 72-kDa heat-shock protein.Abbreviations ICN ICN Biomedicals, Inc. PO Box 19536, Irvine, CA 92713-9921 - LD light dark cycle - LT₅₀ time required to kill 50% of the test animals - SDS sodium dodecyl sulfate - TRIS Tris(hydroxymethyl)aminomethane     

Sexual difference in the photoperiodic induction of pupal diapause in the flesh fly Sarcophaga similis

The flesh fly Sarcophaga similis enters pupal diapause in response to short days, but averts diapause under long days. This species shows a sexual difference in the photoperiodic induction of diapause, with females having shorter critical daylength than males. Here, we proposed two hypotheses to explain this sexual difference. First, we proposed a sexual difference in the qualitative evaluation of photoperiods. This hypothesis assumes under the external coincidence model that although the photoinducible phase of both sexes locates at late scotophase, in males, it locates at a slightly earlier phase. However, the results of night interruption experiments clearly ruled out this hypothesis. Because we verified that S. similis evaluated photoperiods quantitatively, we next proposed a sexual difference in the quantitative evaluation of photoperiods. This hypothesis incorporates concepts of a hypothetical substance accumulation that shows a diapause‐inducing effect and an internal threshold that serves as a reference to determine the diapause/nondiapause developmental program. In long‐day exposure experiments and night interruption experiments, females consistently showed a lower incidence of diapause than males. Thus, the present study data satisfactorily meet the second hypothesis, that is a sexual difference in the quantitative evaluation of photoperiods exists in S. similis.     

Influence of maternal age on incidence of pupal diapause in the flesh fly, Sarcophaga bullata

ABSTRACT. Females of the flesh fly, Sarcophaga bullata Parker, produce an increasingly higher number of diapausing progeny in successive broods. Though a maternal effect completely eliminates the capacity for diapause in the first brood of females with an embryonic and larval history of short day, diapause is restored at low levels in later broods. Exposure to long daylength at the onset of adult life does not alter the diapause response of later broods, thus suggesting that the age effect cannot be modified by daylength manipulation. The age response implies that changes in maternal physiology exert an important regulatory control on the diapause fate of the pupa.     

Anoxia blocks thermotolerance and the induction of rapid cold hardening in the flesh fly, Sarcophaga crassipalpis

Abstract. Anoxia induced by nitrogen or carbon dioxide, or hypoxic/hypobaric conditions generated by a partial vacuum sensitizes red-eye pharate adults of Sarcophaga crassipalpis Macquart to a high temperature exposure that is normally nonlethal (40C for 2–3 h). Thermotolerance induced by a2h exposure to 40C (under aerobic conditions) doubles the pharate adults' tolerance to 45C but provides no protection against a combined exposure to 45C and anoxia, and only modest protection against a combined exposure to 40C and anoxia. Under aerobic conditions, exposing pharate adults to 0C for 2 h increases their tolerance to -10C (rapid cold hardening). Rapid cold hardening at 0C is not induced under anoxia. These results imply that tolerance to high temperatures and rapid cold hardening are dependent on aerobic processes and suggest that certain forms of temperature stress can be further exacerbated with anoxia.     

Cold tolerance in diapausing and non-diapausing stages of the flesh fly, Sarcophaga crassipalpis

ABSTRACT. Supercooling points (SCP) and low temperature tolerance were determined for larval, pupal and adult stages of Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae). No stage tolerates tissue-freezing. Ontogenetic changes in SCP profiles are similar for comparable developmental stages of diapause and non-diapause groups. Feeding larvae have SCPs near -7°C which decrease to -11°C in the postfeeding wandering phase of the final larval instar. The lowest SCPs are recorded for pupae at -23°C. The capacity to survive at -17°C varies with age of the diapausing pupae: 10-day-old pupae are less cold tolerant than pupae that have been in diapause for 45–80 days. Although the SCP of non-diapausing pupae is as low as in diapausing pupae, non-diapausing pupae are extremely sensitive to low temperature exposure and do not survive to adult eclosion when exposed to -17°C for as little as 20 min. The use of hexane to break pupal diapause has no effect on SCPs or low temperature tolerance.     

Thermoperiodic regulation of the circadian eclosion rhythm in the flesh fly, Sarcophaga crassipalpis

We recorded the eclosion time of the flesh fly, Sarcophaga crassipalpis, at different depths in the outdoor soil and under temperature cycles with various amplitudes in the laboratory, to examine the timing adjustment of eclosion in response to temperature cycles and their amplitudes in the pupal stage. In the soil, most eclosions occurred in the late morning, which was consistent with the eclosion time under pseudo-sinusoidal temperature cycles in the laboratory. The circadian clock controlling eclosion was reset by temperature cycles and free-ran with a period close to 24 h. This clock likely helps pupae eclose at an optimal time even when the soil temperature does not show clear daily fluctuations. The eclosion phase of the circadian clock progressively advanced as the amplitude of the pseudo-sinusoidal temperature cycle decreased. This response allows pupae located at any depth in the soil to eclose at the appropriate time despite the depth-dependent phase delay of the temperature change. In contrast, the abrupt temperature increase in square-wave temperature cycles reset the phase of the circadian clock to the increasing time, regardless of the temperature amplitude. The rapid temperature increase may act as the late-morning signal for the eclosion clock.     

Effects of environmental factors on circadian activity in the flesh fly, Sarcophaga crassipalpis

Abstract.The diel locomotor activity patterns of wandering larvae in the flesh fly, Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae), were examined using a novel apparatus and shown to be primarily diurnal, but with a minority (37%) showing nocturnal activity. In response to the environmental stress of heat shock, a significantly larger proportion (72%) of the larvae became nocturnal. In comparison, adult circadian activity also was predominantly diurnal, but not correlated with the larval activity patterns. In addition, adult patterns showed age-related changes in entrainment and free running period. Finally, the phase of circadian-gated adult eclosion was shown to be entrained by a 3-day exposure to light–dark cycles delivered prior to pupariation, with the phase maintained throughout pupal–adult metamorphosis under constant dark conditions. These results demonstrate that environmental changes may have profound effects on the expression of 24-h activity patterns and circadian rhythms during different life stages throughout development.     

GABA and picrotoxin alter expression of a maternal effect that influences pupal diapause in the flesh fly, Sarcophaga bullata

A maternal effect that operates in the flesh fly, Sarcophaga bullata Parker (Diptera: Sarcophogidae), prevents the expression of pupal diapause in the progeny of females that have been reared under short day conditions. This set of experiments tests the possibility that the maternal effect can be prevented by the use of various environmental stresses or chemical treatments administered to the mother. High and low temperature shocks, food deprivation and reducing the size of the mother were all ineffective in altering transmission of the diapause-suppressing maternal effect. Several chemical agents, however, were effective. Gamma-aminobutyric acid (GABA) and one of its antagonists, picrotoxin, elicited opposite responses when injected into female flies. Whereas GABA suppressed the incidence of diapause in the female's progeny, picrotoxin increased the diapause incidence, thus suggesting the possibility of a central role for GABA in regulation of the maternal effect. Octopamine and pilocarpine injected into females also were effective in countering the maternal effect and thus permitting expression of diapause in the female's progeny.