Ultrastructural changes in sarcosomes after exposure of adult Calliphora erythrocephala to lethal and sub-lethal high temperatures

The effects of lethal and sub-lethal high temperatures on the morphology of intact flight muscle sarcosomes of adult Calliphora erythrocephala are described. Treatment of adult flies to lethal temperatures results in ultrastructural changes in the organisation of the cristae and the appearance of electron opaque inclusions. These changes have not been observed after sub-lethal heat treatment, when 50% of the animals recover. It is suggested that changes in the ultrastructure of the intact sarcosomes may be correlated with changes in their ability to couple oxidative phosphorylation with α-glycerophosphate. Age-dependent changes in the sensitivity of sarcosomes are related to changes in the heat death point of the animal and suggest that the impairment of sarcosomal function may be one of the primary lesions in heat death of adult C. erythrocephala.     

Studies on the biochemical basis of susceptibility and resistance of the housefly to fluoroacetate

1. Administration of fluoroacetate to sensitive houseflies in amounts close to the L.D.₅₀ range (0.25–0.3 μg/fly) brought about a prompt elevation of their citrate content. With about 10-fold higher doses of fluoroacetate a concurrent increase of both citrate and pyruvate levels took place in the fly tissues.

2. Incubation of sarcosomes of the sensitive housefly strain in the presence of oxidizable substrates and fluoroacetate resulted in accumulation of citrate, inhibition of respiration and uncoupling of oxidative phosphorylation. The magnitude of the effects varied considerably with the different substrates used, being particularly pronounced with pyruvate and malate and inappreciable with succinate and -glycerophosphate.

3. The respiratory inhibition induced by a brief exposure in the cold of housefly sarcosomes to fluoroacetate, persisted after the sarcosomes had been washed free from fluoroacetate. The toxic effect of fluoroacetate on the respiratory chain could be prevented by an excess of simultaneously added acetate.

4. The susceptibility of the respiratory function of the sarcosomes to fluoroacetate inhibition was abolished by sonication. The unresponsiveness of the sonicated sarcosomes to fluoroacetate was attended by a loss of their respiratory chain phosphorylation activity.

5. Sarcosomes derived from a partially resistant housefly strain, when incubated in the presence of fluoroacetate, failed to accumulate citrate, but displayed the characteristic respiratory-inhibition response. Sarcosomes from a highly resistant strain showed no impairment of their functional capacity by fluoroacetate. However, all the different housefly strains tested proved to be equally sensitive to the deleterious effect of fluorocitrate on sarcosomal respiration.

6. The possible biochemical mechanisms underlying the toxicity of fluoroacetate in the housefly are considered with particular reference to the altered response of the target systems exhibited by the fluoroacetate-resistant strains.     

Genetic variation for resistance and acclimation to high temperature stress in Drosophila buzzatii

Genetic variation for resistance to a high temperature stress under saturated humidity was examined within and among three Drosophila buzzatii populations from Australia. Further, the acclimation of this species to high temperatures was tested by prelreating flies for a shorter, sublethal, time period under conditions that lead to expression of heat shock proteins. Genetic variation for temperature resistance was present among lines for flies either pretreated to high temperature or not. Pro-treating increased survival, with the benefit significantly higher if pretreating was performed 24 h rather than 96 h before exposure to the potentially lethal stress. For (lies pretreated at both times, resistance to heat stress was even greater. The lack of a significant treatment by line interaction term suggested that all lines were similarly plastic for acclimation following previous exposure(s) to a high temperature. Significantly more males survived the heat stress than females, and, within each sex, larger flies were generally more heat resistant than smaller ones. Additionally, the lines from the population that naturally encounters the highest temperatures were generally more resistant to high temperature stress.     

Strong responses of Drosophila melanogaster microbiota to developmental temperature

Physiological responses to changes in environmental conditions such as temperature may partly arise from the resident microbial community that integrates a wide range of bio-physiological aspects of the host. In the present study, we assessed the effect of developmental temperature on the thermal tolerance and microbial community of Drosophila melanogaster. We also developed a bacterial transplantation protocol in order to examine the possibility of reshaping the host bacterial composition and assessed its influence on the thermotolerance phenotype. We found that the temperature during development affected thermal tolerance and the microbial composition of male D. melanogaster. Flies that developed at low temperature (13°C) were the most cold resistant and showed the highest abundance of Wolbachia, while flies that developed at high temperature (31°C) were the most heat tolerant and had the highest abundance of Acetobacter. In addition, feeding newly eclosed flies with bacterial suspensions from intestines of flies developed at low temperatures changed the heat tolerance of recipient flies. However, we were not able to link this directly to a change in the host bacterial composition.     

Heat tolerance in Drosophila subobscura along a latitudinal gradient: Contrasting patterns between plastic and genetic responses

Susceptibility to global warming relies on how thermal tolerances respond to increasing temperatures through plasticity or evolution. Climatic adaptation can be assessed by examining the geographic variation in thermal‐related traits. We studied latitudinal patterns in heat tolerance in Drosophila subobscura reared at two temperatures. We used four static stressful temperatures to estimate the thermal death time (TDT) curves, and two ramping assays with fast and slow heating rates. Thermal death time curves allow estimation of the critical thermal maximum (CT_max), by extrapolating to the temperature that would knock down the flies almost “instantaneously,” and the thermal sensitivity to increasing stressful temperatures. We found a positive latitudinal cline for CT_max, but no clinal pattern for knockdown temperatures estimated from the ramping assays. Although high‐latitude populations were more tolerant to an acute heat stress, they were also more sensitive to prolonged exposure to less stressful temperatures, supporting a trade‐off between acute and chronic heat tolerances. Conversely, developmental plasticity did not affect CT_max but increased the tolerance to chronic heat exposition. The patterns observed from the TDT curves help to understand why the relationship between heat tolerance and latitude depends on the methodology used and, therefore, these curves provide a more complete and reliable measurement of heat tolerance.     

Mitochondria in the flight muscles of insects. II. Effects of the medium on the size form,and organization of isolated sarcosomes

1. The sarcosomes of Drosophila and the blowfly, Phormia, are dense, spherical, homogeneous bodies when isolated from flight muscle and promptly examined under the phase contrast, oil immersion objective. 2. Their average diameter in newly emerged flies is about 1 micro. This value increases rapidly during the 1st week of adult life and then becomes constant at approximately 2.5 micro. At each age the variation in sarcosome diameter conforms approximately to a normal distribution. 3. The degree to which isolated sarcosomes retain their initial size and organization is remarkably conditioned by the composition and the hydrogen ion concentration of the medium in which they are teased and suspended. In suboptimal media three major categories of change were encountered: (1) swelling, with or without compaction of the contents (as in distilled water and salt solutions); (2) shrinkage to rod-like, pleomorphic forms (as in blood serum); and (3) fuzzy degeneration (as in sugar solutions). 4. The membrane that surrounds each sarcosome becomes plainly visible in swollen sarcosomes. A continuation of swelling is accompanied by the escape of the sarcosomal contents, the vacated membrane persisting as a spherical, optically empty ghost. 5. Sarcosomes appear to behave like osmometers when suspended in various aqueous solutions. Solutes which penetrate the membrane show only transient effects in preventing the osmotic entry of water. 6. Under this analysis we find the membrane to be more or less freely permeable to the ions of sodium, potassium, calcium, magnesium, chloride, and phosphate, to non-electrolytes smaller than hexoses, to phosphorylated hexoses, and to several intermediates of the citric acid cycle. 7. The sarcosomal membrane appears to be less permeable to non-electrolytes larger than pentoses, provided that such molecules are not phosphorylated. 8. The membrane shows a higher permeability to ATP than to ADP. The significance of this observation is considered with respect to the ADP-ATP shuttle between sarcosomes and muscle fibrils. 9. Simple solutions of electrolytes or non-electrolytes cause more or less conspicuous changes in the microscopic appearance of sarcosomes. Prolonged preservation was achieved only in more complicated media containing protein. It is concluded that the Donnan equilibrium is the source of the principal osmotic forces regulating the movement of water through the sarcosomal membrane. 10. The optimal medium for the preservation of isolated sarcosomes was an intracellular Ringer solution containing 2.5 per cent crystalline bovine albumin in 0.16 M potassium phosphate buffer at pH 7.0.     

Field Studies of Entomophthora (Zygomycetes: Entomophthorales)—Induced Behavioral Fever in Musca domestica (Diptera: Muscidae) in Denmark

House flies were collected over 3 days (three to five times per day) from specific sites on a dairy farm with a range of high to low temperatures. Flies were held individually to determine whether the distribution of fungus-infected (Entomophthora muscae and E. schizophorae) house flies differed according to the stage of infection and temperature. All but 2 of 396 infected flies (99.5%) had E. muscae. More E. muscae-infected flies collected from cool areas were in later stages of infection (i.e., dying 0–2 days after capture), whereas flies collected on sun-exposed surfaces tended to be in earlier stages of infection (i.e., dying 6–8 days after capture). Most flies died 3–5 days after capture and were consequently in the middle stages of infection. A mark and release experiment was conducted to determine whether E. schizophorae-inoculated flies frequented surfaces with higher temperatures than did uninfected control flies. About 3000 yellow-marked house flies inoculated with E. schizophorae and 3000 blue-marked control flies were released in an enclosed swine farrowing barn. Significantly more inoculated flies were recorded on the heat lamps than flies in the control group. The results suggest that behavioral fever occurs in the field for flies infected with both E. muscae and E. schizophorae and that flies can cure themselves of infection through the use of artificial heat sources.     

Mitochondria in the flight muscles of insects. III. Mitochondrial cytochrome c in relation to the aging and wing beat frequency of flies

1. In the present study a correlation has been sought between aging, flight muscle mitochrondria (sarcosomes), cytochrome c, and flight ability in the blowfly, Phormia regina. 2. During the 1st week of adult life, individual sarcosomes increase in mass from 2.7 x 10^–7 µg. dry weight at the time of emergence, to 8.5 x 10^–7 µg. by the 7th day. During this period of growth, the number of sarcosomes per fly (6.7 x 10⁸) remains constant. When mature, the sarcosomes account for 32.6 per cent of the total muscle dry weight, or close to 40 per cent on a wet weight basis. 3. It appears probable that the high content of flight muscle cytochromes is entirely localized in the sarcosomes. The cytochromes continue to be synthesized and increase in titer within the sarcosomes for 7 days after adult emergence. 4. As determined spectroscopically, the various cytochrome components at all times maintain a constant ratio both to one another and to the sarcosomal dry weight. This suggests the possibility that the cytochrome system may be synthesized as a single entity. 5. The wing-beat frequency of Drosophila funebris and Phormia varies with the age of these flies, being lowest at the time of emergence and maximum after the 6th day. 6. The relations between wing-beat frequency, respiration during flight, and sarcosomal cytochrome c content are discussed. On the basis of some likely assumptions it is calculated that the cytrochrome c turnover number is over 5,000, and that the cytochrome c turns over once for every two wing-beat cycles.     

Local adaptation of reproductive performance during thermal stress

Considerable evidence exists for local adaptation of critical thermal limits in ectotherms following adult temperature stress, but fewer studies have tested for local adaptation of sublethal heat stress effects across life‐history stages. In organisms with complex life cycles, such as holometabolous insects, heat stress during juvenile stages may severely impact gametogenesis, having downstream consequences on reproductive performance that may be mediated by local adaptation, although this is rarely studied. Here, we tested how exposure to either benign or heat stress temperature during juvenile and adult stages, either independently or combined, influences egg‐to‐adult viability, adult sperm motility and fertility in high‐ and low‐latitude populations of Drosophila subobscura. We found both population‐ and temperature‐specific effects on survival and sperm motility; juvenile heat stress decreased survival and subsequent sperm motility and each trait was lower in the northern population. We found an interaction between population and temperature on fertility following application of juvenile heat stress; although fertility was negatively impacted in both populations, the southern population was less affected. When the adult stage was also subject to heat stress, the southern population exhibited positive carry‐over effects whereas the northern population's fertility remained low. Thus, the northern population is more susceptible to sublethal reproductive consequences following exposure to juvenile heat stress. This may be common in other organisms with complex life cycles and current models predicting population responses to climate change, which do not take into account the impact of juvenile heat stress on reproductive performance, may be too conservative.     

A comparison of the responses of tropical and temperate flies (Diptera: Sarcophagidae) to cold and heat stress

Summary Two flesh fly species from the tropical lowlands (Peckia abnormis and Sarcodexia sternodontis) were more susceptible to both cold-shock and heatshock injury than temperate flies (Sarcophaga crassipalpis and S. bullata) and a fly from a tropical high altitude (Blaesoxipha plinthopyga). A brief (2-h) exposure to 0°C elicits a protective response against subsequent cold injury at–10°C in the temperate flies and in B. plinthopyga but no such response was found in the flies from the tropical lowlands. However, both tropical and temperate flies could be protected against heat injury (45°C) by first exposing them to a mild heat shock (2 h at 40°C). The supercooling point is not a good indicator of cold tolerance: supercooling points of pupae were similar in all species, ranging from–18.9 to–23.0°C, and no differences were found between the tropical and temperate species. Among the temperate species, glycerol, the major cryoprotectant, can be elevated by short-term exposure to 0°C, but glycerol could not be detected in the tropical flies. Low-temperature (0°C) exposure also increased hemolymph osmolality of the temperate species, but no such increase was observed in the tropical lowland species. Adaptations to temperature stress thus differ in tropical and temperate flesh flies: while flies from both geographic areas share a mechanism for rapidly increasing heat tolerance, only the temperate flies appear capable of responding rapidly to cold stress. The presence of a heat shock response in species that lack the ability to rapidly respond to cold stress indicates that the biochemical and physiological bases for these two responses are likely to differ.     

Genetic and environmental factors in the resistance of Drosophila subobscura adults to high temperature shock

Summary Survival time following a high temperature shock of Drosophila subobscura adults in dry air has shown great variability. This experimental condition involved desiccation as the first cause of death. Here survival is studied under saturation humidity, so that the mortality may be imputed only to thermic stress. We analyze the influence of culture temperature and crowding on resistance for different sex and age of the adults. The results show strong influences of these environmental factors on heat shock resistance and show interactions with the age and sex of the adults. We suggest that these facts could be due to acclimatization and/or to adaptation. The acclimatization would occur during development and would affect physiological processes related to aging of the flies. The adaptation would take place for selection, acting through differential mortality before the heat shock. Of course, other processes could be significant. Whatever the causal explanation, it will be necessary in any future research related with heat shock resistance to take these factors into account.     

Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus

Although Leishmania metacyclic promastigotes are generally considered resistant to human complement, studies of in vitro-cultured axenic stationary promastigotes using serum concentrations that approximate physiological plasma conditions indicate complement sensitivity. Natural Leishmania infection is caused by sand fly-inoculated promastigotes, whose complement resistance has not been analyzed systematically. We compared Leishmania susceptibility to human complement in L. infantum promastigotes derived from in vitro cultures and from sand flies. Phlebotomus perniciosus sand flies were fed with axenic promastigotes, L. infantum-infected U-937 cells, or spleen cells from L. infantum-infected hamsters. On selected days post-feeding, flies were dissected and promastigotes isolated; in addition, axenic promastigotes were obtained from culture at equivalent days of growth. In near-physiological serum concentration and temperature conditions, measurement of real-time kinetics of propidium iodide uptake showed that 90% of axenic- and sand fly-derived promastigotes were rapidly killed by complement. We found no substantial differences between promastigotes from axenic culture, those isolated from flies on different post-feeding days, or those generated in flies fed with distinct inocula. The results indicate that Leishmania susceptibility to human complement is independent of promastigote developmental stage in the sand fly mid-gut and in axenic culture.     

Predictability rather than amplitude of temperature fluctuations determines stress resistance in a natural population of Drosophila simulans

The adaptability of organisms to novel environmental conditions depends on the amount of genetic variance present in the population as well as on the ability of individuals to adjust their phenotype through phenotypic plasticity. Here, we investigated the phenotypic plasticity induced by a single generation's exposure to three different temperature regimes with respect to several life‐history and stress‐resistance traits in a natural population of Drosophila simulans. We studied a constant as well as a predictably and an unpredictably fluctuating temperature regime. We found high levels of phenotypic plasticity among all temperature regimes, suggesting a strong influence of both temperature fluctuations and their predictability. Increased heat tolerance was observed for flies developed in both types of fluctuating thermal environments compared with flies developed in a constant environment. We suggest that this was due to beneficial hardening when developing in either fluctuating temperature environment. To our surprise, flies that developed in constant and predictably changing environments were similar to each other in most traits when compared to flies from the unpredictably fluctuating environment. The unpredictably changing thermal environment imposed the most stressful condition, resulting in the lowest performance for stress‐related traits, even though the absolute temperature changes never exceeded that of the predictably fluctuating environment. The overall decreased stress resistance of flies in the unpredictably fluctuating environment may be the consequence of maladaptive phenotypic plasticity in this setting, indicating that the adaptive value of plasticity depends on the predictability of the environment.     

Seasonal changes between one- and two-phasic response of plant leaves to heat stress

Summary Plant leaves normally respond to increasing injurious temperatures with a more or less continuous increase of tissue damage. A discontinuous response comprising a first lethal or sublethal temperature range which is followed by a non-injurious temperature range and a second, the lethal temperature range appeared to be an exception. The data with Populus deltoides x simonii show that a two-phasic response is most pronounced after heat shocks of 15 s and was no longer detected after exposure to heat longer than 9 min. In the course of the growing season the two-phasic stress response appears from the beginning of August until leaf fall in late October. Similar results with Convolvulus arvensis and Ligustrum vulgare show that this stress response is not particular of this hybrid of Populus. The wintergreen leaves of Ligustrum do not continue the two-phasic response until leaf fall in spring. Thus, the two-phasic response cannot be related to leaf senescence, however, it seems to indicate a transitional state of the heat tolerance in the period between late summer and late fall. In this state, the socalled primary thermostability of the organs decreases with decreasing natural heat hardening, however, sublethal heat shocks then have hardening or adaptive effects.     

Characteristics of p53 protein stabilization in cells of patients with ataxia-telangiectasia after gamma-irradiation

Stabilization of P53 protein in cells isolated from patients with a grave hereditary disease ataxia-telangiectasia (AT), characterized by strongly enhanced sensitivity to ionizing radiation and impairment of cell cycle control after DNA damage, was studied. The level of expression of these reactions by patients may vary, and it tends to be linked with the severity of the disease. In all AT strains studied, both acquired by the authors and obtained from foreign colleagues, we observed the alteration of timing and character of stabilization of P53 protein, after the action of ionizing radiation in sublethal dosage, as compared to that in cells from healthy donor.     

The effect of environmental temperature on olfactory perception in Drosophila melanogaster

Olfaction provides chemical information to an animal about its environment. When environmental conditions change, individuals should be able to adequately maintain function. Temperature may influence olfaction in a double manner, as it modifies the concentrations of gaseous compounds and affects biological processes. Here, we address acclimatization to environmental temperature in the olfactory system of Drosophila melanogaster using heat and cold treatments. Because the consequences of temperature shifts persist for some time after the treatment's end, comparison of olfactory behaviors at the same temperature in treated and untreated flies allows us to infer the biological effects of temperature in olfaction.At intermediate odorant concentrations heat always generates a reduction of olfactory sensitivity, as they would be expected to compensate for the increase of volatiles in the air. Cold produces the opposite effect. These changes are observed in both sexes and in natural populations as well as in standard laboratory stocks.Short applications suffice to cause detectable olfactory perception changes, but even prolonged temperature treatments have only a transitory effect. Together, these results suggest that olfaction in Drosophila underlies acclimatization to environmental temperature. However, sensitivity changes are not immediate and may cause imperfect adjustment of olfactory function for short time periods.     

A comprehensive assessment of geographic variation in heat tolerance and hardening capacity in populations of Drosophila melanogaster from eastern Australia

We examined latitudinal variation in adult and larval heat tolerance in Drosophila melanogaster from eastern Australia. Adults were assessed using static and ramping assays. Basal and hardened static heat knockdown time showed significant linear clines; heat tolerance increased towards the tropics, particularly for hardened flies, suggesting that tropical populations have a greater hardening response. A similar pattern was evident for ramping heat knockdown time at 0.06 °C min^?1 increase. There was no cline for ramping heat knockdown temperature (CT_max) at 0.1 °C min^?1 increase. Acute (static) heat knockdown temperature increased towards temperate latitudes, probably reflecting a greater capacity of temperate flies to withstand sudden temperature increases during summer in temperate Australia. Larval viability showed a quadratic association with latitude under heat stress. Thus, patterns of heat resistance depend on assay methods. Genetic correlations in thermotolerance across life stages and evolutionary potential for critical thermal limits should be the focus of future studies.     

Mitochondria in the flight muscles of insects. I. Chemical composition and enzymatic content

1. The "indirect" thoracic muscles of adult dipterous and hymenopterous insects consist of a unique type of muscle characterized by the presence of numerous spherical, intracytoplasmic bodies termed "sarcosomes." 2. When the muscle is teased or ground, the sarcosomes are liberated as a turbid suspension of bodies ranging from 1 to 4 µ in diameter. A method is described for the isolation of sarcosomes by a simple differential centrifugation. 3. The cytochemical, chemical, and enzymatic properties of sarcosomes were examined for the purpose of appraising their relation to the cytoplasmic bodies of other tissues. 4. Fresh sarcosomes are slowly but selectively stained by the mitochondrial reagents, Janus green B and pinacyanol. Fixed sarcosomes give a positive reaction with Regaud's mitochondrial stain. 5. Chemical analyses show that approximately 29 per cent of the dry weight of sarcosomes consists of lipids and 60 per cent of protein. Microbiological assay indicates the presence of about 1 gamma of riboflavin per milligram of nitrogen. These values resemble those reported for isolated mitochondria of vertebrate liver and kidney. 6. When examined spectroscopically the sarcosomes, like the vertebrate mitochondria, show a high titer of cytochromes a, b, and c. 7. The titer of cytochrome oxidase varies systematically with the adult age of the insect. A similar relation is observed for the enzyme catalase. 8. Isolated sarcosomes show significant titers of succinoxidase, α-glycerophosphate dehydrogenase, malic dehydrogenase, and pyruvic dehydrogenase. The following dehydrogenases could not be demonstrated: xanthine, phenylalanine, glycine, lactic, choline, glutamic, and alcohol. These results are compared with those previously reported for vertebrate mitochondria. 9. In view of their manifold points of biochemical similarity, it is concluded that the sarcosomes are the mitochondria of this highly specialized muscular tissue.     

Strategic differences in thermal adaptation between two Drosophila species,D. virilis and D. immigrans

Summary Preadult viability and developmental time at four different temperatures, heat and cold resistances of adult flies, effects of acclimatization on heat resistance, and preferred temperature of adult flies were compared between two species of Drosophila, D. virilis and D. immigrans. Four Japanese local populations were surveyed for each species. As compared with immigrans, virilis was higher in its ability to tolerate both heat and cold stresses and was viable over a broader temperature range. On the other hand, immigrans revealed a superior ability to acclimatize and a rigid preference for gradually changing thermal environment. Differences between geographical populations are remarkable for heat tolerance in virilis and cold tolerance in immigrans. In conclusion, thermal adaptation of virilis seems to be based on the high tolerance to extreme temperatures and that of immigrans mainly on the behavioural preference for viable temperatures.