Karl H. Joplin

Cold shock elicits expression of heat shock proteins in the flesh fly, Sarcophaga crassipalpis

Abstract When exposed to brief cold shocks of −10 or −18°C, pharate adults (red eye stage) of the flesh fly, Sarcophaga crassipalpis are unable to eclose as adults. The injury does not cause immediate death: even an 8 h exposure to −18°C allows pharate adult development to proceed to the point of eclosion. Though development continues following cold shock, we observed a dose-dependent decrease in the rate of oxygen consumption, indicating that damage has occurred. The rate of protein synthesis is slow during the first hour after cold shock but quickly recovers. The previously described heat shock proteins, molecular weights 92 and 72 kDa, are expressed in both the brain and integument during recovery from cold shock and are expressed for longer times with increased severity of the cold shock. The cold shock induced 72 kDa protein is immunologically related to the heat shock 70 protein family of Drosophila melanogaster. In addition to the heat shock proteins, three proteins with molecular weights of 78, 45 and 23 kDa, are induced in the integument, but not in the brain, during recovery from cold shock. We previously demonstrated that at high temperatures different developmental stages synthesize two different members of the heat shock 70 protein family, a 65 and a 72 kDa protein. We detect no such developmental switch in response to cold shock. These results demonstrate that heat shock proteins can be induced by extremes of both high and low temperatures but the nature of the stress (heat or cold shock) determines which proteins are induced.

Alteration of the eclosion rhythm and eclosion behavior in the flesh fly, Sarcophaga crassipalpis, by low and high temperature stress

Abstract Heat shock (45°C), cold shock (−10°C), and indirect chilling injury (a prolonged exposure to 2°C) did not interfere with the continuation of pharate adult development in the flesh fly, Sarcophaga crassipalpis, but such flies failed to eclose properly when the exposure was of sufficient duration. In all three forms of injury, development following the temperature treatments was also retarded. Among flies that were less severely affected and still capable of emerging as adults, the circadian time of adult eclosion shifted from near dawn to near the middle of the photophase, thus suggesting that the neurally-based clock is among the systems most vulnerable to heat-shock and cold-shock injury. Tensiometric records of ptilinum expansion revealed important differences in the nature of the injury caused by the different temperature stresses. Heat-shocked flies and those subjected to indirect chilling injury displayed the two behavioral programs normally associated with adult eclosion, the program for obstacle removal (POR) and the program for forward movement (PFM), but such flies failed to eclose because the muscle contractions generated by these motor patterns were insufficient for successful eclosion. In contrast, cold-shocked flies retained the capacity for strong muscle contraction, but the centrally-generated POR and PFM programs were altered. As the duration of cold shock increased, both patterns became more erratic; the PFM program was then lost completely, and in the most severe cases of cold-shock injury, flies lost the capacity to generate both programs. This suggests that neuronal damage is the likely cause of injury inflicted by cold shock.

Cold Shock and Heat Shock

Cold shock is the stress inflicted by a brief and rapid exposure to low, but nonfreezing, temperatures. When the shock is sufficiently severe, the organism sustains injury that may ultimately result in death. This form of stress has received little attention in insects, but it has been well recognized in bacteria, blue-green algae, yeasts, protozoans, higher plants, mammalian spermatozoa and embryos, and in cultures of plant and animal cells (review by Morris et al., 1983; Watson and Morris, 1987). Cold shock, also referred to as “direct chilling injury,” is dependent on the rate of cooling: greater injury is caused by more rapid cooling. The temperature threshold causing injury will vary between species and strains, but consistently this form of injury is observed in the absence of ice formation and at temperatures well above the supercooling point. The actual cause of injury elicited by cold shock remains elusive, but some form of membrane damage is likely. The normal integrity of the cell membrane may be altered by phase transitions of lipids within the membrane (Quinn, 1985) or by thermoelastic stress (McGrath, 1987).

Developmental and tissue specific control of the heat shock induced 70 kDa related proteins in the flesh fly, Sarcophaga crassipalpis

Heat shock (40–43°C) induces two proteins in Sarcophaga crassipalpis that are immunologically related to the heat shock 70 protein family in Drosophila. These Sarcophaga heat shock proteins, Mr of 65 and 72 kDa, show developmental and tissue-specific expression. Heat shock protein 65 is expressed in the brain and integument of third-instar larvae at 43°C. Expression of heat shock protein 65 in both tissues ceases at pupariation and heat shock protein 72 is the protein induced at 43°C throughout the rest of development. Tissue specificity for the expression of these two heat shock proteins can be observed in 3-day-old adult males: brain and integument express heat shock protein 72 while the terminalia and flight muscle express heat shock protein 65. Both a developmental and temperature switch can be seen in the male terminalia: day-1 terminalia express heat shock protein 72 at either 40 or 43°C, on day-2 heat shock protein 72 is expressed at 40°C but heat shock protein 65 is expressed at 43°C, thereafter the terminalia express primarily heat shock protein 65 at either heat shock temperature. Control of heat shock protein expression in S. crassipalpis is thus considerably more complex than the Drosophila literature suggests.

Redundant complexity : A critical analysis of intelligent design in biochemistry

Biological systems exhibit complexity at all levels of organization. It has recently been argued by Michael Behe that at the biochemical level a type of complexity exists--irreducible complexity--that cannot possibly have arisen as the result of natural, evolutionary processes and must instead be the product of (supernatural) intelligent design. Recent work on self-organizing chemical reactions calls into question Behes analysis of the origins of biochemical complexity. His central interpretative metaphor for biochemical complexity, that of the well-designed mousetrap that ceases to function if critical parts are absent, is undermined by the observation that typical biochemical systems exhibit considerable redundancy and overlap of function. Real biochemical systems, we argue, manifest redundant complexity--a characteristic result of evolutionary processes.

Diapause specific proteins expressed by the brain during the pupal diapause of the flesh fly, Sarcophaga crassipalpis

Abstract Proteins synthesized by pupal brains of Sarcophaga crassipalpis were examined during diapause and nondiapause conditions using pulse labelling and 2-dimensional electrophoresis. In vitro cultured brains from diapausing pupae synthesized fewer proteins than brains from nondiapausing pupae or pharate adults. Brains from older diapausing pupae (60 days) synthesized slightly more proteins than brains from pupae in early diapause (14 days). A cluster of about 15 brain proteins appears to be specific to diapausing pupae. In contrast, no differences could be detected in the brain proteins of the photosensitive first-instar larvae exposed to long (nondiapause) or short (diapause) daylengths.

Cellular differences in ring glands of flesh fly pupae as a consequence of diapause programming

The ultrastructure of the ring gland (corpus cardiacum (CC), prothoracic gland (PG) and corpus allatum (CA)) was examined in diapausing and nondiapausing flesh fly pupae. The diapause developmental state, which is environmentally regulated and coordinated by the brain-ring gland complex, is associated with differences in the ultrastructure of PG and CA cells but not in the CC. During diapause the PG and CA cells have extensive arrays of rough endoplasmic reticulum and spherical mitochondria. The PG cells also contain lipid droplets surrounded by an electron dense amorphous coat not seen in PG cells from nondiapausing pupae. In nondiapausing pupae, the PG and CA cells contain large amounts of ribosomes throughout the cytoplasm but very little rough endoplasmic reticulum and elongated mitochondria. The fact that ring glands from diapausing pupae readily incorporate (35)S-methioninc indicates that the gland is actively synthesizing proteins, thus the contrasts in ring gland ultrastructure are not due to cellular quiescence during diapause but reflect fundamental cellular and physiological differences between the diapause and nondiapause developmental program.