Cold-hardiness: a component of the diapause syndrome in pupae of the flesh flies, Sarcophaga crassipalpis and S. bullata

ABSTRACT. Diapausing pupae of Sarcophaga crassipalpis Macquart and S. bullata Parker reared at 20 or 25C readily survive exposure to - 10C for at least 25 days. In contrast, non-diapausing pupae produced by a variety of means are consistently intolerant of the low temperature. Non-diapausing pupae are not immediately killed by exposure to -10C: pupae exposed to the low temperature for up to 3 days proceed with pharate adult development but ultimately die before adult eclosion. Unlike many temperate zone insects, diapausing flesh fly pupae do not require a period of chilling for induction of cold-hardiness, and the attribute of cold-hardiness cannot be separated from other features of the diapause syndrome. Some cold-hardiness is already acquired during the third larval instar: diapause-destined larvae exposed to -10C are more successful in pupariating than non-diapause-destined larvae of the same age.     

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.     

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.     

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.     

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.     

Pupariation in flies: A tool for monitoring effects of drugs,venoms, and other neurotoxic compounds

A complex Sarcophaga bullata pupariation assay was used to evaluate the neurotropic effects of several drugs, venoms, and insecticides. The assay consists of tests for (1) immediate effects on the intact larva, (2) effects on ligated (ie, isolated from the central nervous system) larval abdomens, (3) morphogenetic effects on the puparium, and (4) effects on stereotyped pupariation behavior. The latter are monitored barographically by recording changes in hemocoelic pressure. Of 62 compounds screened, 18 showed morphogenetic activity at a threshold dose of 5 μg or less, 11 at a dose of 50 μg, four at a dose of 100 μg, and 29 showed no morphogenetic activity. From a comparison of the putative pharmacological actions of the tested compounds with their morphogenetic effects, certain generalizations can be made: Agents that paralyze neuromuscular systems at the peripheral level (eg, tetrodotoxin), or suppress or modify basic motor patterns centrally (eg, veratrine sulphate), cause retention of larval morphological characters in the puparium. Compounds that stimulate convulsive contractions of segmental musculature (mostly cholinergic drugs like eserine sulphate, nicotine, organophosphate insecticides) cause retention of larval segmentation on longitudinally contracted puparia. Five compounds (venom of the scorpion, Leirus quinquestriatus, pyrethrins, protoveratrine A, and kainic and quisqualic acids) stimulate musculature of the denervated abdomen. Barographic monitoring of changes in pupariation behavior appears to be a most sensitive and informative test. It reveals great differences in the ways in which compounds producing seemingly identical morphogenetic effects affect and modify behavior, thus making pharmacological classification more accurate.     

Cold shock and heat shock: a comparison of the protection generated by brief pretreatment at less severe temperatures

Abstract Brief exposure to low (0^oC) or high (40^oC) temperature elicits a protective response that prevents injury when the flesh fly, Sarcophaga crassipalpis Macquart, is subjected to more severe cold (-10^oC) or heat (45^oC). Both the low and high temperature responses were found in all developmental stages of the fly, but were most pronounced in the pupal and pharate adult stages. The protective responses generated by brief exposure to 0 or 40^oC appear similar in that both result in a rapid acquisition of cold or heat tolerance and a loss of protection after the flies are returned to 25^oC. The protection generated by chilling is obvious within 10 min of exposure to 0^oC while a 30 min exposure to 40^oC is required to induce the high temperature protection. High temperature protects against cold shock injury within a narrow range (around 36^oC) but we have no evidence that low temperature can protect against heat injury. We previously demonstrated that the rapid increase in cold tolerance correlates with concomitant increases in glycerol concentration, but in this study we found no significant elevation in glycerol in heat-shocked flies. Thus the physiological and biochemical bases for the rapid responses to cold and heat appear to be different.     

How does an eclosing fly deal with obstacles?

ABSTRACT. Stereotyped eclosion behaviour of Sarcophaga bullata Parker (Diptera, Sarcophagidae) consists of two different repetitive sequences of movements. One, which is instrumental for making forward progress in extricating the body from the puparium or for digging through the soil, is called the programme for forward movement (PFM). The other, which is performed as a response to an obstruction, is called the programme for obstruction removal (POR). The initial and final phases of each repetitive sequence are identical in both programmes, the sequences differing only in the middle portion. In the POR, the locomotor elements of the PFM sequence (peristalsis) are substituted by a train of repetitive expansions of the ptilinum, which are caused by simultaneous contractions of the abdominal and thoracic muscles. These ptilinal movements function as an air hammer against the obstruction. Switching from the PFM to POR can be experimentally induced by a rough mechanical stimulation of the head (squeezing, pushing against a barrier) but not by gentle tactile stimulation of the ptilinum or antennae. The critical period for induction of the POR is in the phase of the PFM sequence at which the two programmes diverge. A constant mechanical stimulus is necessary for a sustained performance of the whole POR. A hypothetical scheme of the neural basis of this behaviour is suggested.     

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.     

EFFECT OF ECDYSTERONE ON VITELLOGENIN CONCENTRATION IN HAEMOLYMPH OF MALE AND FEMALE SARCOPHAGA BULLATA

Using Polyacrylamide gel electrophoresis, cycloheximide, incorporation of ³H-labelled amino acids and immunological methods, we have demonstrated that injection of ecdy- sterone induces de novo synthesis and release of vitellogenin in both sexes of Sarcophaga bullata. Vitellogenin concentrations were measured by the Mancini-radial immunodiffusion technique. In males a dose as low as 1 ng always makes vitellogenin appear in the haemolymph but very reproducible results are only obtained when doses varying from 10 to 250 ng were injected. In this range, the dose-response curve was linear on a semi- logarithmic scale.

In females, vitellogenin concentration remained low until a few hours after liver feeding and thereafter it rose sharply and reached its maximum about 24 h after the protein meal. 100 μg 6-hydroxydopamine HCl, injected before liver feeding in 4-day-old females, inhibited vitellogenin synthesis and yolk deposition, probably by interfering with the release of a brain hormone. This inhibitory effect on vitellogenin synthesis, but not that on yolk deposition, could be overruled by injection of ecdysterone. Juvenile hormone was ineffective on both. Females, ovariectomized on day 2 or 3, accumulated vitellogenin in their haemolymph, indicating that the continuous presence of the ovaries was not required for vitellogenin synthesis. The possible relation between the gonadotrophs hormone from the brain, vitellogenin synthesis and moulting hormone metabolism is discussed.     

Photoperiodic sensitivity and diapause induction during ovarian,embryonic and larval development of the flesh fly,Sarcophaga argyrostoma

Sensitivity to the daily photoperiod, particularly with respect to pupal diapause induction, was studied during ovarian, embryonic, and larval development of the flesh flySarcophaga argyrostoma. Large flies were shown to have a greater number of primary follicles in their ovaries and to be capable of limited ovarian maturation in the absence of exogenous protein (autogeny). Such ovarian development occurred independently of photoperiod. However, long days experienced during embryogenesis caused more rapid development, and earlier larviposition, than short days. Short days during embryonic and subsequent larval development also induced pupal diapause, whereas long days led to continuous or non-diapause development of the pupae. Pupal diapause could not be induced by photoperiods during the vitellogenic phase of ovarian development. InSarcophaga argyrostoma, a maternal effect preventing pupal diapause among the progeny of files with a diapause history was not observed.     

Cold-hardiness and development of eggs of Rhopalosiphum insertum

Abstract. 1. The eggs of Rhopalosiphum insertum (Walker) showed a seasonal increase in cold-hardiness under field conditions. Their supercooling point fell from -35°C in November to below -40°C in January, then rose to-35°C or above by March.
2. Laboratory experiments demonstrated that both temperature and date affected cold-hardiness of the eggs. The supercooling points of eggs kept at 16 h photoperiod or in darkness did not, however, differ significantly.
3. Eggs brought from the field into warm, long-day conditions would not hatch until after mid-January. After this date, per cent hatch was significantly greater in 16 h photoperiod than in darkness; it did not differ between eggs kept at 5 or 0°C, but was reduced at -5°C.
4. It is concluded that eggs of Rinserturn are in diapause until mid-January, and that hatching rate and cold-hardiness are determined by separate environmental factors.     

Identification and Purification of Sulfotransferases for 20-Hydroxysteroid from the Larval Fat Body of a Fleshfly,Sarcophaga peregrina

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC₅₀=0.61 μM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3′-phosphoadenosine 5′-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [³⁵S]-3′-phosphoadenosine 5′-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina.     

The selection for early and late pupariation in the flesh-fly, Sarcophaga argyrostoma, and its effect on the incidence of pupal diapause

ABSTRACT. Artificial selection from the first and last larvae of Sarcophaga argyrostoma (Robineau-Desvoidy) to form puparia gives rise to two strains, 'fast' (F) and 'slow' (S), 'fast' pupariating about 3 days earlier than 'slow' in continuous light, 25°C. The two strains differ only in the time from larval wandering to pupariation; other aspects of development are identical. In light-dark cycles (1°C) the distributions of pupariation times in 'fast' are unimodal and nearly normal, whereas those for 'slow' are multimodal and with a marked skew, especially in short daylengths. Pupariation times in 'fast'×'slow' hybrids, an F₂ generation, and a backcross [(F×S)×S] are intermediate between 'fast' and 'slow' but incline towards 'fast'. It is concluded that control of pupariation time is polygenic, and that 'slow' contain considerable residual variation. When compared with the unselected stock, 'fast' and 'slow' both produce a reduced incidence of pupal diapause in short daylengths, and 'fast' show a shorter critical daylength. These effects are interpreted in terms of a modified version of Gibbs' (1975) photoperiodic 'counter' hypothesis.     

Afrotropical Sarcophagidae in a carrion fly community

Abstract. Collections of Afrotropical Sarcophagidae were made at a single locality. Seventeen species in two genera were found: Heteronychia with two species, and Sarcophaga with fifteen species. All male flies were identified, together with a few females, wherever possible. The number of male flies collected exceeded that of females. A small proportion of females captured were gravid.     

Visualizing the postembryonic development of Sarcophaga peregrina (flesh fly) by NMR microscopy

The postembryonic development of the flesh fly was studied using high resolution nuclear magnetic resonance imaging. Because this development occurs in a puparium, this process cannot be observed directly using standard histological techniques. The remodelling of histolysing larval tissues to developing imaginal tissues including the yellow body, a transient alimentary structure, and the integration of the developed adult structure were revealed in the images. Most surprisingly it was found that a large gas space that forms in the central region of the prepupa moves to the dorso-anterial region in less than 5 min due to the larval–pupal apolysis together with separation of the developing pupal epidermis from the puparium.