Flesh Fly Sarcophaga Crassipalpis Research Articles

Effects of gender, age, and nutrition on circadian locomotor activity rhythms in the flesh fly Sarcophaga crassipalpis

In many animal species, circadian rhythms of behavior are not constant throughout the lifetime of the individual but rather exhibit at least some degree of plasticity. In the present study, we have examined the potential influences of gender, age, and nutrition (presence or absence of liver) on the expression of circadian locomotor activity rhythms in the flesh fly Sarcophaga crassipalpis. We found no significant differences in endogenous circadian period under constant dark conditions with respect to gender, nutrition, or age for the duration of our experiments. On the other hand, both male and female flesh flies, as expected, were predominantly diurnal under light-dark cycles, but the pattern of entrainment differed between the sexes. Females also displayed higher activity levels than males. Also, in contrast with males, female activity levels increased with age. Moreover, females exhibited an extraordinary, but transient (one to three days), departure from diurnality which we characterize as “extended dark activity” (EDA). This phenomenon appeared as a continuous bout of locomotor activity that extended at least three hours into the early half of the dark phase at levels at least twice the median of the overall locomotor activity for the individual fly. EDA occurred as an age-dependent response to liver consumption, never appearing prior to day 4 post-eclosion but, thereafter, transpiring within one or two days after a 48-h exposure to liver. These results suggest a linkage between physiological events associated with egg provisioning and locomotor activity in the anautogenous flesh fly. Furthermore, our findings identify the existence of multiple influences on the expression of circadian clock-regulated behavior.

Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species

The imidazole derivative KK-42 is a synthetic insect growth regulator known previously to be capable of averting embryonic diapause in several Lepidoptera, but whether it also affects diapauses occurring in other developmental stages remains unknown. In the present study, we examined the effect of KK-42 on pupal diapause in two species of Lepidoptera, the Chinese oak silkworm Antheraea pernyi and the corn earworm Helicoverpa zea, and in one species of Diptera, the flesh fly Sarcophaga crassipalpis. In A. pernyi, KK-42 delayed pupal diapause termination under the long day conditions that normally break diapause in this species. Likewise, in H. zea, KK-42 delayed termination of pupal diapause, a diapause that, in this species, is normally broken by high temperature. KK-42-treated pupae of these two species eventually terminated diapause and successfully emerged as adults, but the timing of diapause termination was significantly delayed. KK-42 also significantly increased the incidence of pupal diapause in H. zea and S. crassipalpis when administered to larvae that were environmentally programmed for diapause, but it was not capable of inducing pupal diapause in H. zea if larvae were reared under environmental conditions that do not normally evoke the diapause response. Experiments with H. zea showed that the effect of KK-42 on pupal diapause was dose- and stage-dependent, but not temperature-dependent. Results presented here are consistent with a link between KK-42 and the ecdysteroid signaling pathway that regulates pupal diapause.

Extensive Reorganization of Behavior Accompanies Ontogeny of Aggression in Male Flesh Flies

Aggression, costly in both time and energy, is often expressed by male animals in defense of valuable resources such as food or potential mates. Here we present a new insect model system for the study of aggression, the male flesh fly Sarcophaga crassipalpis, and ask whether there is an ontogeny of aggression that coincides with reproductive maturity. After establishing that reproductive maturity occurs by day 3 of age (post-eclosion), we examined the behavior of socially isolated males from different age cohorts (days 1, 2, 3, 4, and 6) upon introduction, in a test arena, with another male of the same age. The results show a pronounced development of aggression with age. The change from relative indifference to heightened aggression involves a profound increase in the frequency of high-intensity aggressive behaviors between days 1 and 3. Also noteworthy is an abrupt increase in the number of statistically significant transitions involving these full-contact agonistic behaviors on day 2. This elevated activity is trimmed back somewhat by day 3 and appears to maintain a stable plateau thereafter. No convincing evidence was found for escalation of aggression nor the establishment of a dominance relationship over the duration of the encounters. Despite the fact that aggressive interactions are brief, lasting only a few seconds, a major reorganization in the relative proportions of four major non-aggressive behaviors (accounting for at least 96% of the total observation time for each age cohort) accompanies the switch from low to high aggression. A series of control experiments, with single flies in the test arenas, indicates that these changes occur in the absence of the performance of aggressive behaviors. This parallel ontogeny of aggressive and non-aggressive behaviors has implications for understanding how the entire behavioral repertoire may be organized and reorganized to accommodate the needs of the organism.

The Flesh Fly Sarcophaga (Liopygia) crassipalpis Macquart 1839 as an Invader of a Corpse in Calabria (Southern Italy)

The first aim of forensic entomology is to help investigators estimate the time of death. Its application has become widespread in many countries because the entomological evidence on corpses (especially Diptera and Coleoptera) can be used to estimate the minimum Post Mortem Interval (mPMI) at both indoor and outdoor crime scenes.

Early changes in the pupal transcriptome of the flesh fly Sarcophagha crassipalpis to parasitization by the ectoparasitic wasp, Nasonia vitripennis

We investigated changes in the pupal transcriptome of the flesh fly Sarcophaga crassipalpis, 3 and 25 h after parasitization by the ectoparasitoid wasp, Nasonia vitripennis. These time points are prior to hatching of the wasp eggs, thus the results document host responses to venom injection, rather than feeding by the wasp larvae. Only a single gene appeared to be differentially expressed 3 h after parasitization. However, by 25 h, 128 genes were differentially expressed and expression patterns of a subsample of these genes were verified using RT-qPCR. Among the responsive genes were clusters of genes that altered the fly's metabolism, development, induced immune responses, elicited detoxification responses, and promoted programmed cell death. Envenomation thus clearly alters the metabolic landscape and developmental fate of the fly host prior to subsequent penetration of the pupal cuticle by the wasp larva. Overall, this study provides new insights into the specific action of ectoparasitoid venoms.

Transitions in the heartbeat pattern during pupal diapause and adult development in the flesh fly, Sarcophaga crassipalpis

The heartbeat of diapausing pupae of the flesh fly Sarcophaga crassipalpis was investigated using electrocardiographic methods including gravimetry, thermography, and optocardiography. During deep diapause, characterized by minimum metabolic activity, the heart exhibited discontinuous bouts of exclusively unidirectional, anterograde pulsations (40–60contractions/min) that lasted only a few seconds. These bouts of cardiac pulsations were separated by periods of rest lasting 5–30min. During infradian peaks of metabolic activity (4-day cycles) that occur throughout diapause, periods of rest were shortened and frequency of the anterograde heartbeat increased more than two-fold. Throughout diapause, the heart consistently exhibited a simple, bi-phasic pattern of pulsations generated by bouts of anterograde heartbeats interspersed with periods of cardiac rest. When the fly broke diapause and initiated pharate adult development, a new tri-phasic pattern was observed: the new pattern incorporated heartbeat reversal, as noted by the appearance of retrograde pulsations that directed hemolymph in a posterior direction. These retrograde heartbeats occurred exclusively in the abdominal portion of the dorsal vessel and were not observed in the head or thorax. The transition to pharate adult development was also accompanied by the appearance of extremely strong extracardiac pulsations that served a respiratory function. Although these pulsations made it more challenging to record heartbeat patterns in pharate adults, we observed progressive shortening of the resting periods and a continual increase in the rate of both anterograde and retrograde pulsations, a trend that was further magnified in the adult fly (anterograde contractions up to 300/min and retrograde contractions approximately 125/min). These results imply that the circulatory function of the heart is homeostatically regulated and is responsive to developmental changes and the diverse metabolic rate demands of larvae, diapausing pupae, pharate adults and adult flies.

Insulin signaling and the regulation of insect diapause

A rich chapter in the history of insect endocrinology has focused on hormonal control of diapause, especially the major roles played by juvenile hormones (JHs), ecdysteroids, and the neuropeptides that govern JH and ecdysteroid synthesis. More recently, experiments with adult diapause in Drosophila melanogaster and the mosquito Culex pipiens, and pupal diapause in the flesh fly Sarcophaga crassipalpis provide strong evidence that insulin signaling is also an important component of the regulatory pathway leading to the diapause phenotype. Insects produce many different insulin-like peptides (ILPs), and not all are involved in the diapause response; ILP-1 appears to be the one most closely linked to diapause in C. pipiens. Many steps in the pathway leading from perception of daylength (the primary environmental cue used to program diapause) to generation of the diapause phenotype remain unknown, but the role for insulin signaling in mosquito diapause appears to be upstream of JH, as evidenced by the fact that application of exogenous JH can rescue the effects of knocking down expression of ILP-1 or the Insulin Receptor. Fat accumulation, enhancement of stress tolerance, and other features of the diapause phenotype are likely linked to the insulin pathway through the action of a key transcription factor, FOXO. This review highlights many parallels for the role of insulin signaling as a regulator in insect diapause and dauer formation in the nematode Caenorhabditis elegans.

Flesh Fly Sarcophaga crassipalpis Macquart (Insecta: Diptera: Sarcophagidae)

Sarcophaga crassipalpis is commonly used in laboratories to study gene expression, diapause processes, and physiological processes. It also has a significant impact in other areas of science including parasitology and forensic entomology. This 6-page fact sheet was written by Lazaro A. Diaz and Phillip E. Kaufman, and published by the UF Department of Entomology and Nematology, October 2011. Reviewed September 2014. EENY503/IN905: A Flesh Fly Sarcophaga crassipalpis Macquart (Insecta: Diptera: Sarcophagidae) (ufl.edu)

Rapid cold-hardening blocks cold-induced apoptosis by inhibiting the activation of pro-caspases in the flesh fly Sarcophaga crassipalpis

Apoptosis plays important roles in the selective elimination of sub-lethally damaged cells due to various environmental stresses. The rapid cold-hardening (RCH) response protects insects from the otherwise lethal consequences of injury due to cold-shock. We recently demonstrated that cold shock induces apoptotic cell death in insects and that RCH functions to specifically block cold-shock-induced apoptosis. In the present study we used isolated fat body, midgut, muscle, and Malpighian tubules from adult flesh flies Sarcophaga crassipalpis to test the following hypotheses: (1) cold-induced apoptosis varies among different tissues and (2) RCH blocks the apoptotic pathway by preventing the activation of pro-caspases. Cold-shock induced substantial amounts of apoptotic cell death that matched with tissue damage as determined using vital dyes. RCH treatment significantly reduced apoptotic cell death in all tested tissues. Caspase-3 (executioner) activity was 2-3 times higher in the cold- and heat-shocked groups than in control and RCH groups. Likewise, the activity of caspase-9 (initiator) showed a similar trend as for caspase-3 in all tissues but midgut. In addition, cold-shock and heat-shock treatments also increased caspase-2 activity 2-3 folds in both soluble and membrane fractions of fat body and muscle extracts compared to controls.

Heat shock response to hypoxia and its attenuation during recovery in the flesh fly, Sarcophaga crassipalpis

In this study, pharate adults of the flesh fly Sarcophaga crassipalpis were exposed to two, four, seven, or ten days of severe hypoxia (3% oxygen) to evaluate its impact on emergence and the expression of genes encoding heat shock proteins (Hsps) and heat shock regulatory elements. A four-day exposure to hypoxia significantly reduced survival, but more than seven days was required to reach the LD 50. Eight genes encoding Hsps, at least one from each major family of Hsps (Hsp90, Hsp70, Hsp60, Hsp40, and sHsps) and two genes encoding proteins involved in Hsp regulation (heat shock factor, hsf, and sirtuin) were cloned, and expression levels were assessed during and after hypoxia using qRT-PCR. Most, but not all hsps studied, were significantly up-regulated during hypoxia, and expression levels for most of the hsps reverted to control levels a few hours after return to normoxia. Hsp70 was the most responsive to hypoxia, increasing expression several hundred fold. By contrast, hsp90 and hsp27 showed little response to hypoxia but did respond to recovery. Neither hsf nor sirtuin were elevated by hypoxia, an observation consistent with their assumed post-transcriptional regulatory roles. These data demonstrate a strong Hsp response to hypoxia, suggesting an important role for Hsps in responding to low oxygen environments.

Does it pay to delay? Flesh flies show adaptive plasticity in reproductive timing

Life-history plasticity is widespread among organisms. However, an important question is whether it is adaptive. Most models for plasticity in life-history timing predict that animals, once they have reached the minimal nutritional threshold under poor conditions, will accelerate development or time to reproduction. Adaptive delays in reproduction are not common, especially in short-lived species. Examples of adaptive reproductive delays exist in mammalian populations experiencing strong interspecific (e.g., predation) and intraspecific (e.g., infanticide) competition. But are there other environmental factors that may trigger an adaptive delay in reproductive timing? We show that the short-lived flesh fly Sarcophaga crassipalpis will delay reproduction under nutrient-poor conditions, even though it has already met the minimal nutritional threshold for reproduction. We test whether this delay strategy is an adaptive response allowing the scavenger time to locate more resources by experimentally providing supplemental protein pulses (early, mid and late) throughout the reproductive delay period. Flies receiving additional protein produced more and larger eggs, demonstrating a benefit of the delay. In addition, by tracking the allocation of carbon from the pulses using stable isotopes, we show that flies receiving earlier pulses incorporated more carbon into eggs and somatic tissue than those given a later pulse. These results indicate that the reproductive delay in S. crassipalpis is consistent with adaptive post-threshold plasticity, a nutritionally linked reproductive strategy that has not been reported previously in an invertebrate species.

Mechanisms of suspended animation are revealed by transcript profiling of diapause in the flesh fly

Diapause is a widespread adaptation to seasonality across invertebrate taxa. It is critical for persistence in seasonal environments, synchronizing life histories with favorable, resource-rich conditions and mitigating exposure to harsh environments. Despite some promising recent progress, however, we still know very little about the molecular modifications underlying diapause. We used transcriptional profiling to identify key groups of genes and pathways differentially regulated during pupal diapause, dynamically regulated across diapause development, and differentially regulated after diapause was pharmacologically terminated in the flesh fly Sarcophaga crassipalpis. We describe major shifts in stress axes, endocrine signaling, and metabolism that accompany diapause, several of which appear to be common features of dormancy in other taxa. To assess whether invertebrates with different diapause strategies have converged toward similar transcriptional profiles, we use archived expression data to compare the pupal diapause of S. crassipalpis with the adult reproductive diapause of Drosophila melanogaster and the larval dauer of Caenorhabditis elegans. Although dormant invertebrates converge on a few similar physiological phenotypes including metabolic depression and stress resistance, we find little transcriptional similarity among dormancies across species, suggesting that there may be many transcriptional strategies for producing physiologically similar dormancy responses.

Isolation of diapause-regulated genes from the flesh fly, Sarcophaga crassipalpis by suppressive subtractive hybridization

Subtractive suppressive hybridization (SSH) was used to characterize the diapause transcriptome of the flesh fly Sarcophaga crassipalpis. Through these efforts, we isolated 97 unique clones which were used as probes in northern hybridization to assess their expression during diapause. Of these, 17 were confirmed to be diapause upregulated and 1 was diapause downregulated, while 12 were shown to be unaffected by diapause in this species. The diapause upregulated genes fall into several broad categories including heat shock proteins, heavy metal responsive genes, neuropeptides, structural genes, regulatory elements, and several genes of unknown function. In combination with other large-scale analyses of gene expression during diapause, this study assists in the characterization of the S. crassipalpis diapause transcriptome, and begins to identify common elements involved in diapause across diverse taxa.

Gene discovery using massively parallel pyrosequencing to develop ESTs for the flesh fly Sarcophaga crassipalpis

BackgroundFlesh flies in the genus Sarcophaga are important models for investigating endocrinology, diapause, cold hardiness, reproduction, and immunity. Despite the prominence of Sarcophaga flesh flies as models for insect physiology and biochemistry, and in forensic studies, little genomic or transcriptomic data are available for members of this genus. We used massively parallel pyrosequencing on the Roche 454-FLX platform to produce a substantial EST dataset for the flesh fly Sarcophaga crassipalpis. To maximize sequence diversity, we pooled RNA extracted from whole bodies of all life stages and normalized the cDNA pool after reverse transcription.ResultsWe obtained 207,110 ESTs with an average read length of 241 bp. These reads assembled into 20,995 contigs and 31,056 singletons. Using BLAST searches of the NR and NT databases we were able to identify 11,757 unique gene elements (E<0.0001) representing approximately 9,000 independent transcripts. Comparison of the distribution of S. crassipalpis unigenes among GO Biological Process functional groups with that of the Drosophila melanogaster transcriptome suggests that our ESTs are broadly representative of the flesh fly transcriptome. Insertion and deletion errors in 454 sequencing present a serious hurdle to comparative transcriptome analysis. Aided by a new approach to correcting for these errors, we performed a comparative analysis of genetic divergence across GO categories among S. crassipalpis, D. melanogaster, and Anopheles gambiae. The results suggest that non-synonymous substitutions occur at similar rates across categories, although genes related to response to stimuli may evolve slightly faster. In addition, we identified over 500 potential microsatellite loci and more than 12,000 SNPs among our ESTs.ConclusionOur data provides the first large-scale EST-project for flesh flies, a much-needed resource for exploring this model species. In addition, we identified a large number of potential microsatellite and SNP markers that could be used in population and systematic studies of S. crassipalpis and other flesh flies.

Rapid elevation of Inos and decreases in abundance of other proteins at pupal diapause termination in the flesh fly Sarcophaga crassipalpis

We analyzed changes in brain proteins 24 h after pupal diapause termination in Sarcophaga crassipalpis by a combination of 2-dimensional gel electrophoresis and mass spectrometry. The proteome analysis revealed significant changes in 20 proteins, 11 of which represented ≥ 2.5-fold changes. Three proteins were present only in the brains of diapausing pupae. Among the most abundant proteins that showed a change, 1 was more abundant, 7 were less abundant, and 2 were absent following diapause termination. The protein that increased in abundance following diapause termination showed highest identity to myo-inositol-1-phosphate synthase (Inos). Proteins that decreased at diapause termination included those showing highest identities to fatty acid binding protein, CG2331-PA, twinstar, catalase, and a histone. Proteins absent at diapause termination included ribosomal protein L17A and one unnamed protein. An increase of Inos protein level was confirmed using Western blot analysis. Attempts to terminate diapause by injection of several Inos-related metabolites failed, thus suggesting that the elevation of Inos at diapause termination is downstream of the physiological regulation that initiates development.

Sex‐specific differences in spatial behaviour in the flesh fly Sarcophaga crassipalpis

Abstract Territoriality in the flesh fly Sarcophaga crassipalpis (Diptera: Sarcophagidae) is studied in the laboratory. In rectangular enclosures, male flies exhibit a lower tolerance (occupation of the same physical space) of same‐sex conspecifics than do females. In circular arenas, male flies show significantly higher levels of spatial separation among themselves (as determined from nearest neighbour analyses) than do females: males show a slight tendency towards a uniform distribution, whereas females exhibit a slight tendency towards clustering. The male spatial behaviour occurs during the photophase but not the scotophase, suggesting that visual cues are required for maintenance of inter‐individual spacing. No significant differences in male spacing behaviour occur between subjective day and subjective night in either constant dark or constant light conditions, suggesting that spatial patterning is not driven by a circadian rhythm.

Life‐history plasticity after attaining a dietary threshold for reproduction is associated with protein storage in flesh flies

Body condition affects the timing and magnitude of life history transitions. Therefore, identifying proximate mechanisms involved in assessing condition is critical to understanding how these mechanisms affect the expression of life history plasticity. Nutrient storage is an important body condition parameter, likely playing roles in both attaining minimum body-condition thresholds for life history transitions and expression of life history traits.We manipulated protein availability for females of the flesh fly Sarcophaga crassipalpis to determine whether reproductive timing and output would remain plastic or become fixed. Liver was provided for 0, 2, 4, or 6 days of adult pre-reproductive development. Significantly, liver was removed after the feeding threshold had been attained and females had committed to producing a clutch.We also identified the major storage proteins and monitored their abundances, because protein stores may serve as an index of body condition and therefore may play an important role in life history transitions and plasticity.Flesh flies showed clear post-threshold plasticity in reproductive timing. Females fed protein for 2 days took ~30% longer to provision their clutch than those fed for 4 or 6 days. Observations of oogenesis showed the 2-day group expressed a different developmental program including slower egg provisioning.Protein availability also affected reproductive output. Females fed protein for 2 days produced ~20% fewer eggs than females fed 4 or 6 days. Six-day treated females provisioned larger eggs than 4-day treated females, followed by 2-day treated females with the smallest eggs.Two storage proteins were identified, LSP-1 and LSP-2. LSP-2 accumulation differed across feeding treatments. The 2- and 4-day treatment groups accumulated LSP-2 stores but depleted them during provisioning of the first clutch, whereas the 6-day group accumulated the greatest quantity of LSP-2 and had substantial LSP-2 stores remaining at the end of the clutch. This pattern of accumulation and depletion suggests that LSP-2 could play roles in both provisioning the current clutch and future clutches, making it a good candidate molecule for affecting reproductive timing and allotment. LSP-1 was not associated with post-threshold plasticity; it was carried over from larval feeding into adulthood and depleted uniformly across all feeding groups.

Rapid cold hardening elicits changes in brain protein profiles of the flesh fly, Sarcophaga crassipalpis

Rapid cold hardening (RCH) refers to the enhanced cold tolerance acquired by a brief exposure to a moderately low temperature. Although ecological aspects of this response have been well documented in insects, less is known about the physiological and biochemical mechanisms elicited by RCH. In this study we used two-dimensional electrophoresis to detect differences in brain protein abundance in pharate adults of the flesh fly Sarcophaga crassipalpis, in response to a 2 h RCH exposure at 0 degrees C. Fourteen high abundance proteins that responded to RCH were selected for mass spectrometric identification. Three proteins that increased in abundance during RCH included ATP synthase subunit alpha, a small heat shock protein (smHsp), and tropomyosin-1 isoforms 33/34. Eleven proteins that decreased in abundance or were missing following RCH included several proteins involved in energy metabolism, protein degradation, transcription, actin binding, and cytoskeleton organization. That several proteins increased in abundance during RCH underscores the dynamics of the RCH mechanism and suggests that more than one physiological response likely contribute to RCH. The increase in ATP synthase suggests an elevation of ATP during RCH, and the smHsp increase suggests that at least one of the Hsps is actually mobilized during RCH, rather than after RCH as previously assumed.

Mating affects reproductive investment into eggs, but not the timing of oogenesis in the flesh fly Sarcophaga crassipalpis

We examined the effects of mating on reproductive investment and the timing of oogenesis in the flesh fly Sarcophaga crassipalpis by exposing females to males or not. All females exposed to males were mated within a few days and we found that mating affected reproductive investment. Virgin females not exposed to males produced a large clutch of eggs ( approximately 91), but females exposed to males and mated produced 10% more. There was no effect of mating on egg length or mass. There was also no effect of mating on the timing of oogenesis. Females in both treatments provisioned their eggs at the same rate with yolk first becoming visible in the oocytes on day three of adulthood and complete provisioning of eggs occurring by the seventh day of adulthood. We examined the biochemical basis of egg provisioning by identifying the yolk proteins and quantifying their blood titer during the oogenic period in both, females exposed to males and mated and those not exposed to males. There was no difference in the timing of the first appearance, peak titer, or disappearance of yolk proteins in the blood between the two treatments. However, consistent with our observation of greater egg production in mated females, these females contained a greater peak yolk protein titer.

P38 MAPK is a likely component of the signal transduction pathway triggering rapid cold hardening in the flesh fly Sarcophaga crassipalpis

Rapid cold hardening (RCH) is an adaptation enabling insects to quickly respond to low temperature, but little is known about the molecular events that trigger this response. In this study of the flesh fly Sarcophaga crassipalpis, we explore a possible role for mitogen-activated protein kinases (MAPKs) in the low temperature signaling that elicits RCH. We report that p38 MAPK from S. crassipalpis, which shows high cDNA sequence homology to p38 MAPKs from other insects and mammals, is rapidly activated at temperatures around 0 degrees C, temperatures that are most effective for inducing RCH. By contrast, low temperature does not activate either extracellular signal-regulated kinase (ERK) or Jun N-terminal kinase (JNK). An increase in phospho-p38 MAPK was observed within 10 min following exposure to 0 degrees C and reached its maximum level in 2 h. When flies were transferred from 0 to 25 degrees C, the level of phospho-p38 MAPK decreased immediately and reached trace levels by 3 h. Nondiapausing flies were much more responsive to p38 MAPK activation than cold-hardy diapausing pupae. Thus, p38 MAPK activation and RCH both show the same narrow ranges of temperature sensitivity, temporal profiles of activation and decay, and developmental specificity. These correlations suggest that p38 MAPK plays a potential role in regulating the induction of RCH. The p38 MAPK response was not dependent upon the brain, as evidenced by high activation in isolated abdomens exposed to low temperature.