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.     

Diapause hormone in the corn earworm, Helicoverpa zea: optimum temperature for activity, structure-activity relationships, and efficacy in accelerating flesh fly pupariation

Diapause hormone (DH) effectively terminated pupal diapause in Helicoverpa zea. This effect was temperature-dependent, with an optimum of 21 degrees C. The dose-response curve indicated an ED50 of DH for diapause termination of approximately 100 pmol. The core sequence and essential amino acids were determined by bioassays using modified and truncated DH analogs. A C-terminal hepta-peptide, LWFGPRLa, was the core sequence required for diapause termination. Activity was lost when Alanine was substituted for any of the amino acids in the hepta-peptide, with the exception of Glycine. A fragment series of analogs suggested that the amide and Arginine were the most important components needed for terminating diapause. Leucine, Tryptophan, and Phenylalanine at the N-terminus of the hepta-peptide were also critical for activity. The C-terminal Leucine was less important: deletion resulted in decreased activity, although it could not be substituted by Alanine. The fact that a portion of the DH sequence is similar to the pyrokinin that accelerates fly pupariation prompted us to also evaluate the capability of DH to accelerate development in the flesh fly, Sarcophaga bullata. The threshold dose of DH essential to accelerate fly pupariation was 5 pmol for immobilization/retraction and longitudinal contraction and 10 pmol for tanning, approximately one or two orders of magnitude lower than the effective dose required for diapause termination in H. zea. Tensiometric measurements revealed that DH affected neuromuscular patterns of pupariation behavior and associated cuticular changes in a manner similar to that of the fly pyrokinins and their analogs.     

Ontogenetic patterns of cold-hardiness and glycerol production in Sarcophaga crassipalpis

Developmental patterns of low-temperature tolerance and glycerol production were determined for larval, pupal and adult stages of the flesh fly Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae). Both diapause and non-diapause-destined flies were reared at relatively high temperatures, 20° or 25°C, prior to testing. Cold tolerance was greatest for diapause pupae aged 12–35 days after pupariation. Among non-diapause-destined flies, pupae exhibited a greater level of low temperature tolerance than larvae or adults. Although diapause pupae were more tolerant than non-diapause pupae maximal cold tolerance was not attained in either group until 10 days after pupariation. Non-diapause-destined feeding and wandering larvae had higher glycerol levels than larvae destined for diapause. During the first 6 weeks after pupariation glycerol titres increased steadily in diapause pupae. Rapid loss of glycerol is associated with the termination of pupal diapause.     

A comparison of the pupariation acceleration activity of pyrokinin-like peptides native to the flesh fly: Which peptide represents the primary pupariation factor?

Five native pyrokinin-like peptides (Neb-PK-1, Neb-PK-2, Neb-PVK-1, [L9]Neb-PVK-2, [I9]Neb-PVK-2) identified in the neuropeptidome of the flesh fly Neobellieria bullata were compared for their quantitative and/or qualitative effects on puparium formation (pupariation). In a standard pupariation bioassay, both Neb-PVK-1 and [I9]Neb-PVK-2 proved inactive, whereas [L9]Neb-PVK-2 demonstrated only weak activity. In contrast, both Neb-PK-1 and Neb-PK-2 demonstrated potent threshold doses, with Neb-PK-2 about 10-fold more active than Neb-PK-1. Analysis of neuromuscular activity during pupariation using a tensiometric technique demonstrates that the two native Neb-PKs accelerate the onset of immobilization and cuticular shrinkage more than motor programs associated with retraction of the anterior segments and longitudinal body contraction. It was further determined that the sensitivity of various components of the pupariation process to these peptides decreases in the following order: immobilization>cuticular shrinkage>motor program for anterior retraction>motor program for longitudinal contraction congruent to tanning of cuticle of the newly formed puparium. A paradoxical situation was observed whereby the motor programs of pupariation are temporally dissociated from actual morphogenesis of the puparium. The tensiometric data suggest that the most likely candidate for a primary pupariation factor is Neb-PK-2, rather than Neb-PK-1.     

Fraenkel's pupariation factor identified at last

Thirty-five years ago, Zdarek and Fraenkel demonstrated that nervous tissue extracts influenced development by accelerating pupariation in the grey flesh fly, Neobellieria bullata. We have now identified this pupariation factor as SVQFKPRLamide, designated Neb-pyrokinin-2 (Neb-PK-2). To achieve this, the central nervous system of N. bullata wandering stage larvae, that is, preceding pupariation, were dissected and extracted before HPLC separation. Chromatographic fractions were screened with a bioassay for pupariation accelerating activity. Only one fraction showed huge pupariation activity. Mass spectrometry revealed the presence of a pyrokinin, whose primary sequence could not be unequivocally determined by tandem mass spectrometry. However, this Neb-pyrokinin appeared to be very prominent in the ring gland from which it was subsequently purified and identified. Synthetic Neb-PK-2 accelerates pupariation with a threshold dose of only 0.2 pmol and therefore, Neb-pyrokinin is considered to be the genuine pupariation factor. The immunohistochemical distribution pattern of Neb-PK-2 is very similar to that of Drosophila pyrokinin-2, from which it differs by only one amino acid residue. Hence, the recently identified G-protein coupled receptors (CG8784, CG8795) for Drosophila pyrokinin-2 might play an important role in puparium formation.     

Egg development and diapause: ecophysiological and genetic basis of phenological polymorphism and adaptation to varied hosts in the green oak tortrix,Tortrix viridana L. (Lepidoptera: Tortricidae)

Experiments concerned 8 tortrix populations associated with varied oak species. They showed that the egg development included a phase of diapause. Completion of embryogenesis at 20 degrees C was used as a criterion for whether diapause was completed. Under semi-natural conditions diapause terminated in late autumn or early winter, then eggs developed continuously, without postdiapause winter quiescence, even in severe cold. The eggs from the populations associated with holm or cork oak completed diapause then hatched later than those from the populations associated with sessile or pubescent oak. Reciprocal crossbreedings confirmed that this phenological polymorphism was genetically determined. Under constant temperatures the physiological state of diapausing eggs, assessed by measuring their cold requirements to complete diapause by exposure to 8 degrees C, varied gradually with increasing age. This diapause development was strongly temperature-dependent. Cold requirements of diapausing eggs were much higher in a late-hatching than in an early-hatching population. This explains the phenological polymorphism of the tortrix: the more eggs need cold, the later they complete diapause in autumn, and the later they hatch in spring. Egg cold requirements varied widely within populations too, which resulted in large variations in the date of diapause end among individuals. The date of egg hatch was influenced by the temperatures occurring during diapause and postdiapause, but apparently not by photoperiod.     

The physiological basis of anterior inhibition of puparium formation in ligated fly larvae

Fly larvae ligated after the critical period of ecdysone release frequently fail to pupariate in the anterior part. Evidence is presented against anterior inhibition being caused by a hormonal imbalance, such as a decreased titre of ecdysone, an increased titre of juvenile hormone, and/or a lack of the neurohormonal factors that accelerate the process of pupariation. It is unlikely that the anterior inhibition results from a sustained stimulus from the sense organs, or from the presence of an inhibitory substance. The oxygen consumption in the inhibited anterior parts is 25 per cent of that in the anterior parts that pupariate. This suggests that the inhibition of pupariation is a result of oxygen deficiency. When anteriorly inhibited larvae are subjected to pure oxygen the inhibited anterior ends that would never tan in air commence to tan. The dissection of anteriorly inhibited larvae showed that the tracheal trunks were severed at the point of ligation and that the tracheae were filled with haemolymph. In the pupariated posterior parts the tracheae showed melanotic plugs at the severed ends of the tracheae preventing the flooding. Reducing the injury, by careful ligation, or by immobilizing the anterior ends by cold or tetrodotoxin reduces the incidence of anterior inhibition. Thus, anterior inhibition results from a lack of oxygen caused by the injury to the tracheal system during ligation.     

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.     

A nondiapausing variant of the flesh fly, Sarcophaga bullata, that shows arrhythmic adult eclosion and elevated expression of two circadian clock genes, period and timeless

We describe a variant of the flesh fly, Sarcophaga bullata, which fails to enter pupal diapause in response to short daylength and low temperatures. This fly also has an arrhythmic adult eclosion pattern: rather than eclosing in early photophase, the variant ecloses arrhythmically throughout the photophase and scotophase. The loss of both diapause (photoperiodic response) and the gating of adult eclosion (presumably a circadian response) suggests that the same clock system is involved in these two responses. An examination of the expression patterns of the clock genes period and timeless demonstrates that both genes are present in the nondiapausing variant, but surprisingly, both genes are expressed at higher levels. This abnormality we observe, possibly the consequence of an upstream clock gene malfunction or a malfunction of the autoregulatory loop, results in disruption of a component of the clock system that is apparently needed for both photoperiodism and circadian rhythmicity.     

Haemolymph ecdysteroid titres in diapause- and non-diapause-destined larvae and pupae of Sarcophaga argyrostoma

No differences were observed between the rates of development of larvae and pupae from diapause- and non-diapause-destined lines of Sarcophaga argyrostoma except that those destined for diapause have a longer post-feeding, wandering, larval phase associated with a lower haemolymph ecdysteroid titre, as measured by radioimmunoassay. Following pupariation, both cultures show a high haemolymph titre associated with larval/pupal apolysis. The developing culture displays an ecdysteroid peak at 72 h after pupariation which may be involved with pupal/adult apolysis and the initiation of pharate-adult development. This peak is reduced in the diapause-destined culture. Following the initiation of pharate adult development, there is a very large peak at 85–90 h. Those pupae entering diapause display very low titres as a result of the failure of the brain/prothoracic gland axis to release ecdysone. There are no quantitative or qualitative differences between the titres of specific ecdysteroids in the prepupae of the two lines as determined by reverse-phase high-performance liquid chromatography. A preliminary examination of the levels of free and conjugated ecdysteroids has provided the basis for proposing a mechanism of ecdysone metabolism in this insect.     

Disruption of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae), by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

The action of venom from the ectoparasitic wasp, Nasonia vitripennis, was monitored by examining alterations in patterned muscular movements characteristic of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata. Venom injected into larvae prior to pupariation caused a dose-dependent delay in pupariation. Eventually, such larvae did pupariate, but puparia were abnormally formed. Barographic records revealed that all elements of pupariation behavior were present in venom-injected larvae, but pupariation behavior was not well synchronized with tanning, thus implying that the venom caused disruption in the temporal organization of central motor programs. When larvae were ligated and injected with venom posterior to the ligature, no response was evident in the posterior region, suggesting that the venom does not directly stimulate muscles or neuromuscular junctions. Injection of exogenous ecdysteroid into venom-injected larvae restored some elements of pupariation behavior, consistent with ecdysone's role in stimulating the release of anterior retraction factor and puparium tanning factor, two factors that are released from the CNS to regulate pupariation. When the venom was injected into newly emerged imagoes, the duration of extrication behavior was shortened, whereas all phases of post-eclosion behavior were lengthened. These observations imply that the venom affects CNS centers that regulate the muscular systems engaged in extrication and post-eclosion behavior.     

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.     

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.     

Effects of temperature and photoperiod on the termination of larval diapause in Lucilia sericata (Diptera: Calliphoridae)

Larvae of the blow fly, Lucilia sericata (Meigen), enter diapause in the third instar after cessation of feeding. The effects of temperature and photoperiod on the termination of diapause were examined. The diapause terminated spontaneously under the diapause-inducing condition of 20 degrees C and LD 12:12, although pupariation was not synchronous. Diapause development proceeded under a low temperature of 7.5 degrees C. Transfer to long-day conditions of LD 16:8 or to a high temperature of 25 degrees C induced prompt and synchronous pupariation. Low temperatures in winter probably play a predominant role in the termination of diapause under natural conditions.     

Induction and development of winter larval diapause in a drosophilid fly,Chymomyza costata

Photoperiodic response during induction of larval hibernal diapause of Chymomyza costata was characterized and the course of diapause development was analyzed in the laboratory. C. costata becomes sensitive to photoperiodic stimuli during an unspecified stage of its early development (embryo, 1st larval instar); the sensitivity gradually increases during the 2nd and early 3rd larval instars and reaches its maximum just before the moment when it abruptly ceases at the age of 15-19 days after oviposition. Diapause intensifies during a period of 2-3 weeks after induction and, later, is maintained without apparent development until death (between 150 and 250 days) under 18 degrees C and a short-day photoperiod (L10:D14, SD). Diapause may be terminated in a horotelic process by exposure to a low temperature (2 degrees C) during which larvae subsequently (1) synchronize their post-diapause development (requires up to 14 days of chilling), (2) lose photoperiodic sensitivity (2 months), and finally (3) terminate diapause (5 months). Alternatively, diapause may be terminated in a tachytelic process by exposure to a high temperature (18 degrees C) and long-day photoperiod (L16:D8, LD) during which no synchronization occurs and pupariation takes place after a mean of 25.2 days (with a broad range from 8 to more than 50 days). Larvae that are transferred from LD to SD during their sensitive period switch their developmental programming from pupariation to diapause. Proliferation of adult primordial structures (imaginal discs, neuroblasts) slows down within 1 day after transfer. In contrast, whole body growth continues for at least 3 days before its rate slows down and matches the rate characteristic for SD conditions.     

Developmental changes in dopamine levels in larvae of the fly Chymomyza costata: comparison between wild-type and mutant-nondiapause strains

Dopamine and two related catecholamines, L-3,4-dihydroxyphenylalanine (DOPA) and N-acetyl dopamine (NADA), were analyzed in whole body and tissue samples taken throughout late larval development of the drosophilid fly Chymomyza costata, which enters facultative diapause as a mature 3rd instar larva in response to short photophase. Wild-type (W) and mutant-nondiapause (M) strains reared under diapause inducing (d) and preventing (nd) photophases were compared. Developmental changes in the whole body of dopamine levels showed some general features irrespective of fly strain and rearing conditions: sharp major peaks during moult from second to third instar larva and during pupariation; lower minor peaks around the middle of both 2nd and 3rd instars. Significant differences between the strains and conditions were also found: dopamine levels were lower throughout the 2nd instar and during the 2nd to 3rd instar moult of mutant strain larvae (M/d) as compared to wild-type larvae (W/nd and W/d); while the late 2nd and late 3rd instar larvae destined to diapause (W/d) maintained relatively high dopamine concentrations, their counterparts destined to continuous development (W/nd and M/d) significantly decreased dopamine levels prior to the 2nd to 3rd instar moult or pupariation. Possible relationship between the dopamine levels and diapause induction/onset in C. costata larvae is discussed. Integument contained more than 90% of the dopamine found in the whole body. The gut and central nervous tissues showed relatively low pools of dopamine, only trace amounts were detected in haemolymph and no dopamine was found in fat body. DOPA levels were low and stable throughout larval development of both W and M strains and under both conditions. NADA levels peaked during second halves of 2nd and 3rd instars of both strains, then dropped to trace levels and were elevated again during 2nd to 3rd instar moult as well as in tanned prepupae. No elevation of NADA levels was recorded in 3rd instar W/d larvae which entered diapause.     

Parental effects: physiological age, mating pattern, and diapause duration on diapause incidence of progeny in the cabbage beetle, Colaphellus bowringi Baly (Coleoptera: Chrysomelidae)

The cabbage beetle, Colaphellus bowringi Baly, is a short-day species that has both a facultative summer and winter adult diapause. In this study, we systematically investigated the effects of parental physiological age, mating pattern (including mating frequency and delayed mating), and diapause duration on the diapause incidence of progeny in the beetle. As parents aged, progeny diapause incidences showed considerable inter-parental variability and an abnormally varying pattern, but their predicting line was approximate to horizontal when reared under constant conditions. There was significant difference in progeny diapause incidence between multiple-mating and once-mating treatments. Higher diapause incidences in the progeny were observed in delayed-mating treatments. Progeny diapause incidences increased as diapause duration in the parental generation increased, and hibernating individuals produced more diapausing progeny than aestivating ones. Reciprocal cross tests between the post-diapause adults and non-diapause adults with no diapause history showed that the effect of parental diapause duration on the progeny diapause incidence was determined by both mothers and fathers but the mothers appeared to have a stronger effect than the fathers. Our results suggest that the parental age effect in Colaphellus bowringi might not be simply age-dependent, but may be controlled by the interaction of multiple factors, including physiological, environmental, behavioral and genetic factors. We discuss the ecological and physiological significance of these findings.