The amino acid sequence of cytochrome c from the blowfly Lucilia cuprina.

The amino acid sequence of cytochrome c isolated from the sheep blowfly Lucilia cuprina has been determined by comparison of the compositions of the tryptic peptides to those predicted from the published sequences of cytochromes c from other insects. Cytochrome c from L. cuprina differs at a single residue when compared to cytochrome c from the screw worm fly Haematobia irritans, a species belonging to the same order as the blowfly. This substitution, proline for alanine, has been located at position 44 in the protein chain.     

The amino acid sequence of cytochrome c from the blowfly Lucilia cuprina

The amino acid sequence of cytochrome c isolated from the sheep blowfly Lucilia cuprina has been determined by comparison of the compositions of the tryptic peptides to those predicted from the published sequences of cytochromes c from other insects. Cytochrome c from L. cuprina differs at a single residue when compared to cytochrome c from the screw worm fly Haematobiairritans, a species belonging to the same order as the blowfly. This substitution, proline for alanine, has been located at position 44 in the protein chain.     

Hypersensitivity responses and repeated infections with Lucilia cuprina, the sheep blowfly.

, , and 1992. Hypersensitivity responses and repeated infections with Lucilia cuprina, the sheep blowfly. International Journal for Parasitology 22: 1175–1177. Sheep repeatedly infected with L. cuprina at 2- but not 4-week intervals developed partial resistance to infection after five infections, as measured by larval recovery. However, resistance did not persist for more than three infections. Skin weal responses were measured after injection of larval products simultaneously with each infection. The only correlation between weal size and larval recoveries occurred at infection 1 and indicated a relationship between skin sensitivity and innate rather than acquired resistance. The results suggest that resistance to L. cuprina can develop after repeated infections but that it is short lived and requires frequent larval exposure. A role for hypersensitivity responses was not confirmed by the weal responses but was suggested by the size of wound developed per larva recovered.     

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.     

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.     

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.     

Monitoring and selection of resistance to pyrethroids in the Australian sheep blowfly, Lucilia cuprina

Field and laboratory populations of the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Calliphoridae), were surveyed by bioassay for possible resistance to the synthetic pyrethroids, a group of insecticides under development for blowfly control. A normal distribution of LC50 values was found using deltamethrin, the test pyrethroid, with no indication of specific resistance despite widespread use of deltamethrin on sheep to control the sheep body louse, Damalinia ovis (Schrank) (Trichodectidae). There was no cross-resistance to deltamethrin from existing organophosphate (OP) resistance nor from previous use of DDT. Selection with deltamethrin on a combined field strain, CSF85, increased the LC50 gradually over the first twenty generations until it stabilized at approximately 25x that of the unselected CSF85. This laboratory-induced resistance extended to other pyrethroids, cypermethrin (16x), cyhalothrin (25x) and cycloprothrin (10x), and increased the existing resistance of CFS85 to the OP diazinon (11x) and the carbamate, butacarb (83x).     

Relationship between protein ingestion and sexual receptivity in females of the Australian sheep blowfly Lucilia cuprina

The receptivity of females of Luciliu cuprina to mating attempts is increased by protein feeding. Females became maximally receptive after consuming a quantity of protein which was insufficient to allow full ovarian development in any individual, and which was 25 % of that needed to produce full ovarian development in most flies. Receptivity increased progressively over 3 days following a protein meal taken on the day after emergence, but was almost maximal within 24 h when females were given a protein meal on the fifth day after emergence. Topical application of the insect growth regulator Altosid caused a marked increase in the sexual receptivity of non-protein fed females.     

Characterisation of proteases involved in egg hatching of the sheep blowfly, Lucilia cuprina

A number of proteases were identified in the egg shell washings (ESW) collected during the egg hatching of Lucilia cuprina (sheep blowfly). Characterization of these proteases indicated a pH optima in a similar pH range that was optimal for L. cuprina egg hatching. Mechanistic characterization of these proteases indicated that they were predominantly of the serine class. Several protease inhibitors were tested for their ability to inhibit L. cuprina egg hatching in vitro. Egg hatching was significantly (P<0.05) inhibited by PMSF (61%), 1,10-Phenanthroline (42%) and Pepstatin (29%). The inhibition of egg hatching by PMSF showed a strong concentration dependence, with its effects ranging from inhibition at high concentrations to enhancement of egg hatching at low concentrations. Addition of ESW to unhatched eggs, significantly (P<0.05) enhanced their rate of hatching above untreated control eggs. This enhancement of egg hatching was significantly (P<0.05) reversed by the protease inhibitors Elastatinal (40%), 1,10-Phenanthroline (40%) and PMSF (38%). These studies indicate a role for serine and/or metallo-proteases in facilitating L. cuprina egg hatch.     

Organisation and expression of a cluster of yolk protein genes in the Australian sheep blowfly, Lucilia cuprina

The Australian sheep blowfly Lucilia cuprina is a major pest for the Australian and New Zealand sheep industries. With the long-term aim of making a strain of L. cuprina suitable for a genetic control program, we previously developed a tetracycline-repressible female lethal genetic system in Drosophila. A key part of this system is a female-specific promoter from a yolk protein (yp) gene controlling expression of the tetracycline-dependent transactivator (tTA). Here we report the sequence of a 14.2 kb genomic clone from L. cuprina that contains a cluster of three complete yp genes and one partial yp gene. The Lcyp genes are specifically expressed in females that have received a protein meal. A bioinformatic analysis of the promoter of one of the yp genes (LcypA) identified several putative binding sites for DSX, a known regulator of yp gene expression in other Diptera. A transgenic strain of L. cuprina was made that contained the LcypA promoter driving the expression of the Escherichia coli lacZ reporter gene. Transgenic females express high levels of β-galactosidase after a protein meal. Thus the LcypA promoter could be used to obtain female-specific expression of tTA in transgenic L. cuprina.     

Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina

Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina. International Journal for Parasitology16: 69–75. Resistance to blowfly larvae infections developed in sheep exposed to at least four consecutive infections. Half of the sheep treated showed significant levels of resistance, the others remained susceptible. This resistance took the form of a decreased yield of third instar larvae in comparison to controls and sheep which remained susceptible. In addition an increased sensitivity to larvae developed, as shown by the area of wound obtained per maggot recovered, by the early appearance in resistant sheep of exudate from the infection site and by skin reactions to larval products. Radioimmunoassays demonstrated high levels of serum antibody against larval excretory/secretory antigens, though the response did not peak until after four infections. Resistant animals showed somewhat lower antibody titres than susceptible sheep. Consecutive infections of only 50 larvae failed to induce resistance to larger challenge infections. It is suggested that consecutive infections of larger numbers of maggots induce a hypersensitivity response which may effect larval survival especially of first and second instar maggots.     

The influence of oocyte resorption on ovarian development rates in the Australian sheep blowfly, Lucilia cuprina

Rates of ovarian development in L. cuprina are determined by ambient temperatures and females require a minimum of 57 day degrees above 8°C to mature their first complement of eggs. The number of oocytes that a female can mature depends on her size and the amount of protein-rich material ingested. Under field conditions, females usually obtain sufficient protein to reach maturity but rarely mature their full egg complements (Vogt et al., 1985), i.e., most females resorb some of their oocytes. Oocyte resorption prolongs the maturation period by approximately 0.3 day degrees/oocyte resorbed.A model of ovarian development rates is presented which incorporates resorption delays and uses ambient temperature regimes to estimate the physiological ages and maturation rates of field females.

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Résumé Les taux de développement ovarien de L. cuprina sont déterminés par la température ambiante, et la femelle exige un minimum de 57 degrés-jours audessus de 8°C pour développer son premier lot d'oeufs. Le nombre d'ovocytes qu'une femelle peut former dépend de sa taille et de la quantité d'aliments riches en protéines absorbées. Dans les conditions de la nature, les femelles obtiennent normalement suffisamment de protéines pour atteindre la maturité mais rarement l'ensemble de leur contingent d'oeufs se développe totalement, c'est à dire que la majorité des femelles résorbé une partie de ses ovocytes. La résorption des ovocytes prolonge la période de maturation d'environ 0,3 dégre-jour par ovocyte résorbé. Un modèle de taux de développement ovarien est proposé qui incorpore les retards dus à la résorption et utilise les régimes de température ambiante pour évaluer les âges physiologiques et les taux de maturation des femelles dans la nature.

     

Synthesis and deposition of chitin in larvae of the Australian sheep blowfly,Lucilia cuprina

Chitin synthesis in third-instar Lucilia cuprina larvae cultured at 23 °C was investigated using in vivo and in vitro systems, the latter with whole and with homogenized integuments. Synthesis was at a maximum between 24 and 48h after ecdysis from the second instar. Chitin was deposited in layers, and labeled GlcNAc was rapidly cleared from the hemolymph. In in vitro homogenate systems, the rapid conversion of UDP-([¹⁴C]GlcN)Ac to ([¹⁴C]GlcN)Ac and its 1-phosphate derivative contributed to the low incorporation of this precursor into chitin. The extent of the conversion was reduced by the addition of KCN or phenylthiourea. In in vivo and in vitro tissue systems the level of incorporation of ([¹⁴C]ClcN)Ac was higher than that of UDP-([¹⁴C]GlcN)Ac. However, in in vitro homogenate systems there was no difference unless UTP was added when the level of incorporation of only ([¹⁴C]GlcN)Ac was increased (by a factor of 9). Incorporation of UDP-([¹⁴C]GlcN)Ac, but not that of ([¹⁴C]GlcN)Ac, was decreased when larvae were deprived of food. Soluble oligosaccharides were detected in in vitro homogenate systems. They were formed during chitin synthesis and may represent newly initiated chitin chains. A reappraisal of current ideas on chitin synthesis in insects is needed.     

Quantitative aspects of the effect of mating on readiness to lay in the Australian sheep blowfly,Lucilia cuprina

Virgin females of Lucilia cuprinararely lay eggs, whereas mated females do so readily. This effect of mating is due entirely to increased readiness to lay, and not to any effect on ovarian development. An investigation was made of how readiness to lay was affected by matings which differed in terms of the male's chemical and mechanical contribution. Individual males were mated, during 1 day, to a succession of females whose readiness to lay was determined 1 or 8 days after mating. On both days, the proportion of females laying was inversely related to the number of females with which the male had previously mated. A high proportion of females that had mated with previously unmated or oncemated males laid at both 1 and 8 days after mating. However, this proportion tended to decline between day 1 and day 8 in females that had mated with males with two or more previous matings, and this effect was most evident in females mated with males that had previously mated with four or more females. When matings were manually terminated as soon as coupling had occurred, the proportion laying remained as low as in virgins. This proportion progressively increased as mating duration increased from 2 to 6 min. The proportion that laid after mating terminated at 6 or 8 min was as high as that for females from full-term matings (mean duration, 12.5 min). The results are generally similar to those obtained in parallel experiments on the effect of mating on sexual receptivity in this species and, therefore, indicate that the physiological bases for the two effects of mating might be the same.     

Immunization of sheep against infection with larvae of the blowfly Lucilia cuprina

Sheep were repeatedly exposed to a second stage excretory-secretory antigen preparation of Lucilia cuprina by intradermal injection (S group) or intranasal aerosol (I group) in an attempt to induce immunity to the larvae. Hypersensitivity responses to the injections were monitored and correlated with larval numbers at subsequent challenge. Intradermal injections showed that the immediate or IgE-mediated and the intermediate or Arthus response were the major skin hypersensitivity reactions to the larval antigens. At challenge there was no significant reduction in larval numbers between the S and the control group, however the Arthus reaction did show some correlation with larval recoveries in the S group. There was a significant reduction in larval numbers (P < 0.005 Mann-Whitney U Test) between the I and the control group. In addition, the animals which showed respiratory hypersensitivity to the aerosol during the immunization period had the lowest larval recoveries. The results of a second challenge in the I group did not show continued protection. It is suggested that the protective response was suppressed by exposure to antigens at the first challenge infection. Exudate samples recovered during the infection were analysed for protein amount and the results were correlated with larval survival. They suggested that two separate mechanisms of resistance are operating in this experiment. The first occurs early in the infection, is probably associated with immediate hypersensitivity, and may control the initiation of protein leakage. The second occurs later in the infection and may result in the leakage of proteins able to control larval survival.     

Gold labelling of RNA in virus-induced mitochondrial vesicles in the sheep blowfly Lucilia cuprina.

Membranous vesicles are thought to be the replication site for viral RNA of many plant and animal viruses. A relatively rare site of virus-associated vesiculation is that of the mitochondrial outer membrane. In this study, virus-induced mitochondrial vesicles of the blowfly, Lucilia cuprina, were labelled with ribonuclease/gold and an antibody against double stranded RNA (anti-polyinosinic:polycytidylic acid). Both methods showed the presence of RNA in the vesicles thus indicating they may be a site for viral RNA replication in Lucilia.     

Aminopeptidases as potential targets for the control of the Australian sheep blowfly, Lucilia cuprina.

A series of experiments were carried out to investigate the role of proteinase enzymes in the growth of larvae of the sheep blowfly, Lucilia cuprina. First, instar larvae were incubated on an artificial growth media in the presence of various concentrations of inhibitors of all the major proteinase classes. Inhibitors of serine proteinases and aminopeptidases were found to cause significant growth inhibition and in some cases death of the larvae within 24 h, suggesting that these enzymes were the major classes involved in protein digestion in the gut of the insect. A second group of experiments analysed the effects of two inhibitors from the same or different proteinase classes in the growth media. Synergistic inhibition of larval growth was observed with the incorporation of inhibitors of serine proteinases and aminopeptidases. The results suggest that these classes of proteinases are both central to protein digestion in this insect, probably in the gut, and that the inhibition of both types of activity leads to an almost complete blockade of digestion. Testing in vivo gave similar results with infections on sheep skin inhibited by either serine proteinase or aminopeptidase enzyme inhibitors and the combination of both stopped the infection process. The role of aminopeptidases in larval metabolism and as potential targets for blowfly control agents is examined.     

Juvenile hormone synthesis by ring glands of the blowfly Lucilia cuprina

The major radiolabelled product released from ring gland and brain-ring gland complexes of third instar larval and pre-pupal stages of the sheep blowfly Lucilia cuprina upon incubation with L-[methyl-³H]methionine corresponded to one diastereomer of juvenile hormone III bisepoxide (JHB₃). Endocrine glands incubated with the juvenile hormone precursor 2E,6E-farnesoic acid released increased quantities of JHB₃, together with significant amounts of juvenile hormone III but not the isomeric methyl 2E-6,7-epoxyfarnesoate. Synthesis of JHB₃ was developmentally and neurally regulated. Ring glands and brain-ring gland complexes from third instar larvae released more JHB₃ than comparable preparations from pre-pupae, while isolated corpus allatum segments of the gland were more active than intact brain-gland complexes. These results reinforce the emerging status of JHB₃ as the characteristic juvenile hormone of dipteran insects. Arch. Insect Biochem. Physiol. 34:239–253, 1997. © 1997 Wiley-Liss, Inc.