Chromosomal responses of blowfly Lucilia cuprina to heat and heavy metal stress

Chromosomal responses to heat and heavy metal shocks were studied in the pupal trichogen polytene chromosomes of Lucilia cuprina. Heat shock induced seven distinct puffs on different chromosomes at the following loci: 1C2, 21A1, 21C, 23B, 24B, 42A and 95B3. Arsenate and mercury, two of the most common toxic environmental chemical pollutants also, induced almost the same set of puffs except 20B2, which appeared to be induced by metals only and 95B3, which was not induced by arsenate. The findings suggest that a common set of gene loci encoding the heat shock proteins is responsive to these diverse environmental stresses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the heat shock response inEnterococcus faecalis

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacteriumEnterococcus faecalis. The heat resistance (60°C or 62.5°C, 30 min) of log phase cells ofE. faecalis grown at 37°C was enhanced by exposing cells to a prior heat shock at 45°C or 50°C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72 kDa) and GroEL (63 kDa) heat shock proteins ofEscherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La)E. coli proteins and to theBacillus subtilis ⁴³ factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.     

Measuring fitness and intergenic interactions: The evolution of resistance to diazinon inLucilia cuprina

Resistance at the diazinon-resistance locus (Rop-1) is recessive with respect to fitness. Selection initially occurs at concentrations lower than those required to controlLucilia cuprina. The presence of theRop-1 allele initially disrupted development so that in the absence of diazinon, carriers were at a relative selective disadvantage. Continued use of the chemical, subsequent to resistance evolving, selected a modifier to ameliorate this effect. Modified resistant phenotypes show similar developmental stability and relative fitness to susceptible individuals. Frequency-dependent interactions are observed between resistant and susceptible phenotypes of theRop-1 locus. The interactions are determined by the concentration of diazinon and range from competitive to facilitative. The results are discussed in the context of the contribution insecticide resistance systems can make to the study of general evolutionary phenomena.     

Causes and correlates of loss and recovery of sexual receptivity inLucilia cuprina females after their first mating

After an initial mating, females of the Australian sheep blowfly, Lucilia cuprina(Diptera: Calliphoridae), rejected mating attempts by males for several days. Almost all (>98%) females were unreceptive 24 h after mating. When tested 9 days after mating receptivity had returned in 24% of females denied the opportunity to oviposit and 45% of females given repeated opportunities to oviposit on liver. Those mated females that regained receptivity mated as readily as virgins. Gravid first-, second-, or third-cycle females that did not oviposit when presented with liver had a higher receptivity than those that laid. These non-layers laid after remating. Injection of 1 male equivalent of an extract of male accessory reproductive glands into virgin females switched off their receptivity within 5 h for at least 8 days. An extract of testis reduced receptivity 5 h after injection but had no detectable effect 3 days after injection. Both extracts increased the tendency of virgin females to oviposit. There was no difference in the sperm stores of females that refused to lay a second egg mass and comprised a high percentage of receptive females and those given no opportunity to lay this egg mass.     

Genetics of the hemolymph esterases ofLucilia cuprina (Diptera: Calliphoridae)

When hemolymph from adults ofLucilia cuprina was partitioned on native polyacrylamide gels, nonspecific esterase staining demonstrated 10 bands with up to six bands in an individual. The bands derive from alleles at two loci,E _HA (five alleles) andE _HB (four alleles).E _HA is located on chromosome 4, 16.3 map units fromsv (singed vibrissae) and 22.1 map units fromra (radial vein gaps).E _HB is located on chromosome 5, 34.0 map units fromto ² (topaz² eyes) and 7.2 map units frommv (M1-veinless).     

Basic features of the Staphylococcal heat shock response

The major heat shock proteins of Staphylococcus aureus had apparent M_rs of 84,000, 76,000, and 60,000, and other prominent proteins of M_rs 66,000, 51,000, 43,000 and 24,000 were also induced. Staphylococcus epidermidis showed a similar response. These proteins were also induced by CdCl₂, ethanol and apparently osmotic stress (1.71 M NaCl or 2.25 M sucrose). Most of the proteins sedimented with the membrane fraction, but the M_r 60,000 protein remained in the cytoplasm.     

Small heat shock proteins and adaptation of various Drosophila species to hyperthermia

The dynamics and the level of accumulation of small heat shock proteins (sHSP group 21–27) after a heat exposure were studied in three Drosophila species differing in thermotolerance. The southern species Drosophila virilis, having the highest thermotolerance, surpassed thermosensitive D. lummei and D. melanogaster in the level of sHSPs throughout the temperature range tested. The results suggest an important role of sHSPs in the molecular mechanisms of adaptation to adverse environmental conditions, particularly to hyperthermia.     

Insecticide resistance and malathion carboxylesterase in the sheep blowfly,Lucilia cuprina

Resistance to the organophosphorus insecticide malathion in genetically related strains of the Australian sheep blowflyLucilia curprina was examined. Separate lines of blowflies were established by homozygosis of the fourth chromosome of the parental RM strain. Both the RM and the derived resistant (der-R) strains are approximately 100 times more resistant to malathion than the related susceptible der-S strain, resistance being correlated with a 45- to 50-fold increase in a malathion carboxylesterase (MCE) activity. MCE has a pH optimum ranging between 6.6 and 8.0 and is strongly inhibited by the carboxylesterase inhibitors triphenyl phosphate, paraoxon, and diiospropylfluorophosphate. Subcellular fractionation revealed that MCE was localized predominantly to the cytosol and mitochondria in both resistant and susceptible blowflies. A single MCE was purified to homogeneity from RM blowflies. It has a pI of 5.5, is a monomer of 60.5 kDa, and hydrolyzes malathion with aV _max of 755 nmol/min/mg protein and aK _m of 11.0 µM. L. cuprina have thus evolved a remarkable MCE which is faster and more efficient at hydrolyzing a specific insecticide than any other insect esterase yet described.     

Preliminary study of heat shock proteins in nemerteans

Nemerteans experience varying environmental temperatures during low tide exposures. Inducible heat shock proteins (hsps) have been reported for most organisms following both artificial heat stress and natural environmental temperature variations. This preliminary study reports the presence of hsps in the phylum Nemertea. A lethal temperature of 36 °C was determined for Paranemertes peregrina Coe, 1901. The nemerteans were exposed to a temperature of 34 °C for 2 h. After a 2 h recovery time, the worms were then analyzed for hsps by SDS–PAGE and western immunoblot protocols. Control worms were allowed to acclimate to ambient temperatures (13–15 °C) before hsp analysis. Hsp70 and hsp90 were detected in both the control and heat-shocked worms in highly variable concentrations, and the latter group had significantly elevated hsp70 levels. In addition, the analysis detected different isoforms of hsp70. The detection of hsps indicates a possible role in nemertean physiology during response to thermal stress, and potentially to other environmental challenges.     

Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.     

Genetics of lucilin,a storage protein from the sheep blowfly,Lucilia cuprina (Calliphoridae)

Lucilin, the main storage protein of larval fat body and hemolymph in the sheep blowfly, Lucilia cuprina, has been isolated as a series of trimers composed of subunits of 83,000±5% daltons. Extensive electrophoretically detectable polymorphism of lucilin subunit patterns occurs in wild and laboratory populations of Lucilia; from four to nine bands are seen in any one individual. Evidence from genetic, electrophoretic, immunological, and structural studies suggests the existence of a series of 12 or more closely related structural loci (designated Luc-1 to Luc-12) which may have arisen through gene duplication. Codominant allelic variation has been found at several of these loci. Luc-1 and Luc-3, and probably the other structural loci of the series, are located on chromosome 2.Financial support for this work was largely obtained through the Australian Research Grants Committee (Grant D65/15167). J. A. T. held an Honorary Fellowship at the Australian National University during 1972–1973.     

Field trial of a compound chromosome strain for genetic control of the sheep blowfly Lucilia cuprina

Summary In 1979–80 a field trial of a compound chromosome (CC) strain of the Australian sheep blowfly Lucilia cuprina was conducted in the isolated Brindabella Valley, N.S.W. New genetic material was introduced into the strain before release by inducing 104 new CC elements by irradiation of recently captured field strains, and combining the resulting strains. Weekly releases, averaging 1.1 million larvae per week, were begun in November 1979 and continued to May 1980. Field-inseminated females were trapped weekly and their genotypes and those of their mates were determined through genetic testing. The proportion of wild X wild matings declined from 16% in December 1979 to 1% in April 1980. During this period the proportion of CC X CC matings rose from 50% to 90%. Larvae sampled from infested sheep had compound chromosomes, indicating that compound chromosomebearing females can successfully oviposit in the field. Trapping of flies resumed at the start of the 1980–81 season, without further releases. Progeny tests revealed the presence of both CC and wild flies. The proportions of CC X CC matings among field-inseminated females were 90% in October, 44% in November, nil in December, and 12% in January. No CC X CC matings were detected in 33 field-inseminated females trapped and tested during April, and 70 tested males reared from myiasis samples in April 1981 proved to be wild type. These results indicate that the CC strain overwintered in the field and strongly suggest that it bred in the field for at least one generation following the spring emergence before being eliminated from the population.     

Small heat shock proteins in the development of thermotolerance in Pisolithus sp.

Small heat shock proteins (HSPs) have been shown to confer thermotolerance in many organisms. Here, we demonstrate that small HSPs (sHSPs) can also be involved in development of thermotolerance in Pisolithus sp. In heat shock response, Pisolithus isolate RV82 synthesized proteins of molecular mass 28, 26 and 15–18 kDa. These group of proteins are synthesized when mycelial mass are exposed to heat shock temperature (42 °C) for short period (30 min) and incubated back at 28 °C, the optimal temperature for growth. Our results show sHSPs are an important biochemical alteration in ectomycorrhizal fungi under thermal stress.     

The relationship between fecundity and oocyte resorption in field populations of Lucilia cuprina

In the Australian sheep blowfly, Lucilia cuprina, the number of ovarioles, and hence the maximum number of eggs developed in each ovarian cycle, is a linear function of adult size (headwidth). Field females have a mean headwidth of 3.0 mm (range=2.30–3.50 mm) and a mean potential fecundity/cycle of 232 eggs (range=111–318 eggs). Realised fecundity is influenced by the quality and amount of protein in the adult diet. All field females appear to have the same minimum protein requirement for egg maturation, i.e., whatever their size (potential fecundity) females must obtain sufficient protein to mature at least 110 eggs in order to become gravid. Females whose protein intake exceeds this minimum but falls short of the amount needed for maturation of their full egg complements resorb some of their oocytes and mature the remainder. Since potential fecundity increases with fly size, large females resorb more oocytes than small females under suboptimal protein regimes, i.e., the proportion of oocytes resorbed increases with fly size. Under field conditions, females rarely matured full complements of eggs, which indicates a general shortage of protein-rich material. The mean reduction in fecundity of field females during these studies was 53.4 eggs/cycle (23.2%).

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Résumé Chez Lucilia cuprina Wied., le nombre d'ovarioles, et ainsi le nombre maximum d'oeufs développés au cours de chaque cycle ovarien, est une fonction linéaire de la taille de l'adulte (largeur de la tête). La largeur moyenne de la tête des femelles de la nature est 3 mm (2,3 à 3,5) et la fécondité potentielle moyenne par cycle de 232 oeufs (111 à 318).La fécondité réelle est influencée par la qualité et la quantité de protéines dans le régime alimentaire. Toutes les femelles de la nature semblent avoir les mêmes exigences minimales en protéines pour la maturation des ovocytes, c'est à dire que quelle que soit leur taille (fécondité potentielle) les femelles doivent obtenir les protéines suffisantes à la formation d'au moins 110 ovocytes pour devenir gravides. Les femelles dont l'absorption de protéines dépasse ce minimum, mais est insuffisant pour permettre la maturation de la totalité de leurs ovocytes, en résorbent quelques uns et conduisent à maturité le reste. Puisque la fécondité potentielle augmente avec la taille des mouches, les grosses femelles résorbent plus d'ovocytes que les petites avec un régime suboptimal en protéines, c'est à dire que la proportion d'ovocytes résorbés augmente avec la taille des mouches. Dans les conditions de la nature, la totalité des ovocytes parvient rarement à maturité, ce qui indique une limitation générale en aliments riches en protéines. La réduction moyenne de la fécondité chez les femelles de la nature a été au cours de cette étude de 53.4 oeufs par cycle (23,2%).

     

Modification of developmental instability and fitness: Malathion-resistance in the Australian sheep blowfly,Lucilia cuprina

The evolution of resistance to malathion byLucilia cuprina initially results in an increase in fluctuating asymmetry. Resistant flies are at a selective disadvantage, relative to susceptibles, in the absence of the insecticide. A fitness/asymmetry modifier of diazinon-resistant phenotypes ameliorates these effects resulting in malathion-resistant phenotypes of relative fitness and asymmetry similar to susceptibles. For the nine genotypic combinations of the modifier and malathion-resistance alleles, developmental time increases linearly with increasing asymmetry. Percentage egg hatch decreases linearly with increasing asymmetry. The initially disruptive effect of the malathion-resistant allele was partially dominant, the effect of the modifier dominant. The results are discussed in terms of developmental perturbation, asymmetry estimation and relative fitness to consider whether it is adequate to use changes in fluctuating asymmetry alone as measures of developmental instability. It is suggested that in some circumstances antisymmetry may indicate developmental instability and that the diazinon/malathion-resistance systems inL. cuprina may allow the relative importance of genetical and/or environmental developmental perturbations to be ascertained.