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.     

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.     

Reproductive and developmental consequences of a diapause maternal effect in the flesh fly, Sarcophaga bullata

Abstract Progeny of Sarcophaga bullata produced from mothers with a history of short day will not enter pupal diapause even if they are reared in a strong diapause-inducing environment (LD 12:12 h at 20^oC). Short-day exposure and diapause commitment are normally inseparable, but this maternal effect provides a tool for examining separately the effect of photoperiod and diapause commitment. Duration of the wandering period of the third instar is longer in diapause-destined larvae than in non-diapause-destined larvae, and fecundity of flies that have experienced pupal diapause is lower than in long-day flies that have not been through diapause. The puparia of diapausing pupae contain more hydrocarbons than puparia of nondiapausing pupae, and this contributes to higher rates of net transpiration for the nondiapausing flies. Flies showing the maternal effect (short-day experience but no diapause) show an intermediate response: length of wandering, fecundity rate and quantities of puparial hydrocarbon are between the extremes observed in the other two groups of flies. Thus, the maternal effect switches the developmental programme to nondiapause, but the progeny retain some characteristics of diapause. Evidence from reciprocal crosses indicates that the photoperiodic history of the female, rather than the male, is responsible for the influence on fecundity.     

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.     

Changes in microRNA abundance may regulate diapause in the flesh fly,Sarcophaga bullata

Diapause, an alternative developmental pathway characterized by changes in developmental timing and metabolism, is coordinated by molecular mechanisms that are not completely understood. MicroRNA (miRNA) mediated gene silencing is emerging as a key component of animal development and may have a significant role in initiating, maintaining, and terminating insect diapause. In the present study, we test this possibility by using high-throughput sequencing and qRT-PCR to discover diapause-related shifts in miRNA abundance in the flesh fly, Sarcophaga bullata. We identified ten evolutionarily conserved miRNAs that were differentially expressed in diapausing pupae compared to their nondiapausing counterparts. miR-289-5p and miR-1-3p were overexpressed in diapausing pupae and may be responsible for silencing expression of candidate genes during diapause. miR-9c-5p, miR-13b-3p, miR-31a-5p, miR-92b-3p, miR-275-3p, miR-276a-3p, miR-277-3p, and miR-305-5p were underexpressed in diapausing pupae and may contribute to increased expression of heat shock proteins and other factors necessary for the enhanced environmental stress-response that is a feature of diapause. In S. bullata, a maternal effect blocks the programming of diapause in progeny of females that have experienced pupal diapause, and in this study we report that several miRNAs, including miR-263a-5p, miR-100-5p, miR-125-5p, and let-7-5p were significantly overexpressed in such nondiapausing flies and may prevent entry into diapause. Together these miRNAs appear to be integral to the molecular processes that mediate entry into diapause.     

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.     

A maternal effect that eliminates pupal diapause in progeny of the flesh fly, Sarcophaga bullata

Flesh flies that have experienced pupal diapause produce progeny that will not enter diapause even when reared in a strongly diapause-inducing environment. The effect is determined, not by diapause itself, but by the short days previously received by the larvae during the programming of pupal diapause. Reciprocal cross matings indicate that the effect is transmitted solely by the female parent. Though the embryos develop within the uterus of the female, the maternal effect is transmitted prior to the onset of embryogenesis, probably during oögenesis. Only by rearing a generation in long-day (nondiapausing) conditions can the capacity for pupal diapause be restored in the progeny. The effect is likely to provide an adaptive mechanism for preventing an untimely diapause response among the progeny of overwintering females that emerge early in the spring.     

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.     

Selection for late pupariation affects diapause incidence and duration in the flesh fly, Sarcophaga bullata

ABSTRACT. This artificial selection study with the flesh fly, Sarcophaga bullata Parker, tested the hypothesis that phenotypic variability in the length of the larval stage (under non-diapause conditions) is largely a consequence of genetic variability. Selection for late pupariation resulted in a line that pupariated significantly later and also developed more slowly during other stages of the life cycle. In a diapause-inducing environment, the selected line pupariated later, showed a higher incidence of pupal diapause, and remained in diapause longer than the unselected line. This is the first experimental evidence in S.bullata to show that diapause incidence and duration are related. The relationship between developmental rate and diapause traits may stem from the pleiotropic effects of genes associated with late pupariation, or from one or more genes associated with late pupariation being closely linked to genes that affect diapause.     

Genomic organization in the flesh fly Sarcophaga bullata

The genome of the flesh fly Sarcophaga bullata has been characterized both cytologically and biochemically. S. bullata has a haploid DNA level of 0.61 picograms which is five times larger than the haploid genome size of Drosophila melanogaster. Reassociation kinetics of Sarcophaga DNA shows that its sequence organization is very similar to that of D. melanogaster in having a very large proportion of single copy DNA (81%) and only small amounts of highly and moderately repetitive DNA (9% and 6%, respectively). cRNAs from all three sequence classes were prepared and their cytological distributions on diploid and polytene cells determined by in situ hybridization. The cytological distribution of the highly repetitive probe was found to be restricted to the centromeric heterochromatin of two of the five autosomes and this sequence class was also found to be markedly underreplicated in polytene foot-pad cells. No highly repetitive DNA was localized on either of the sex chromosomes, but only on the two large centromeric regions of chromosomes C and E. Moderately repetitive DNA was found uniformly distributed on all of the autosomes in both testis and polytene foot-pad squashes. As in the case of the highly repetitive sequence probe, no moderately repetitive DNA was detected on either the X or Y chromosomes. Moderately repetitive DNA in Sarcophaga was also shown to have the Drosophila type pattern of sequence interspersion with a moderately repetitive element of 5,000 nucleotides adjacent to a unique element of greater than 10,000 nucleotides. The Sarcophaga genome is the largest for which this type of interspersion has so far been demonstrated.     

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.     

Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata

The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluroescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromosome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.     

Seasonal variation in generation time, diapause and cold hardiness in a central Ohio population of the flesh fly, Sarcophaga bullata

Abstract.

• 1 Generation time, diapause phenology and cold tolerance of the flesh fly, Sarcophaga bullata, were examined under confined natural conditions in central Ohio. In this locality, the fly can complete a maximum of four generations annually.
• 2 Very few pupae entered diapause in the first and second generations (May to July in 1988). In the third generation (August) 37% of the pupae entered an overwintering diapause, as did all pupae from the fourth generation (September).
• 3 The adult eclosion date in the spring and annual generation time can be predicted accurately from degree day data.
• 4 Cold tolerance of the field-overwintering portion of the population was high. After 30 days under field conditions, diapausing pupae readily survived a 7-day exposure to — 17°C. Glycerol appears to be the major cryoprotectant in S.bullata, and glycerol concentrations in the field population (95–142 mm ) remained high throughout the winter.
• 5 In contrast, diapausing flies reared under laboratory conditions (20°C, 12:12 LD) were less cold tolerant, and glycerol concentrations were lower (6.9–21.2 mm ). Field conditions thus promote the acquisition of high levels of cold tolerance, presumably as a consequence of the accumulation of higher concentrations of glycerol.
• 6 In spite of differences in the cold tolerance of laboratory and field flies, the supercooling points of the two groups of flies were nearly the same, thus implying that the supercooling point is not a good indicator of cold tolerance.

     

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.     

Oleic acid is elevated in cell membranes during rapid cold-hardening and pupal diapause in the flesh fly, Sarcophaga crassipalpis

The integrity of cellular membranes is critical to the survival of insects at low temperatures, thus an advantage is conferred to insects that can adjust their composition of membrane fatty acids (FAs). Such changes contribute to homeoviscous adaption, a process that allows cellular membranes to maintain a liquid-crystalline state at temperatures that are potentially low enough to cause the membrane to enter the gel state and thereby lose its ability to maintain homeostasis. Flesh flies (Sarcophaga crassipalpis) were subjected to two experimental conditions that elicit low temperature tolerance: rapid cold-hardening and diapause. FAs were isolated and analyzed using gas chromatography-mass spectrometry. FAs changed in response to both rapid cold-hardening and diapause. In response to rapid cold-hardening (8 h at 4 degrees C), the proportion of oleic acid (18:1n-9) in pharate adults increased from 30% to 47% of the total FA pool. The proportion of almost every other FA was reduced. By entering diapause, pupae experienced an even greater increase in oleic acid proportion, to 58% of the total FA pool. Oleic acid not only promotes membrane fluidity at low temperature but also allows the cell membrane to maintain a liquid crystalline state if temperatures increase.     

Genetic analysis of diapause capability and association between larval and pupal photoperiodic responses in the flesh fly Sarcophaga similis

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

Manifestations of a pupal diapause in two species of flies,Sarcophaga argyrostoma and S. bullata

Sarcophaga argyrostoma shows under certain conditions a state of arrested development, recognized as a diapause, in the pupa shortly after the head has become evaginated and before the antennae of the adult fly become visible, i.e. about 40 hr after formation of the puparium. Diapause occurs in: (1) Cultures raised and maintained at 13·5°C and (2) cultures raised at 18°C under conditions of short days—8 hr light/16 hr darkness (LD 8 : 16). Diapause also arises when larvae are raised at higher temperatures and the prepupae or young pupae transferred to 13·5°C. Diapause does not ensue from transference from 23 to 18°C, LD 8 : 16. In both cases of diapause development starts spontaneously after 6 to 12 months. Upon transference to higher temperatures development in the 13·5°C diapause starts almost immediately, whereas in the 18°C, LD 8 : 16 diapause it starts only after a delay the length of which depends on the temperatures employed. The 18°C, LD 8 : 16 diapause is also shortened by chilling at 6°C for 1 to 2 months. The diapause in S. bullata is manifested in exactly the same morphological state as in S. argyrostoma, but differs from it in that it has so far not been induced by short days, and in the 13·5°C diapause is terminated spontaneously after shorter periods. In certain instances arrested development occurred in the state when the antennae are just visible.