Purified glutathione S-transferases from parasites as candidate protective antigens.

Glutathione S-transferase (GST) activity was detected in larvae of the Australian sheep blowfly Lucilia cuprina, and in the nematode Haemonchus contortus. A specific inhibitor of the enzyme was shown to affect survival of both species of parasite in vitro. GST from both parasites has been purified and partially characterized. Antisera raised to the purified enzymes were shown to inhibit the enzyme activity in vitro. However, the antisera had no effect on the survival of either parasite.     

Heterorhabditis spp. and Steinernema (= Neoaplectana) spp.: temperature, and aspects of behavior and infectivity

The infectivity of several species/strains of nematodes of the genera Heterorhabditis and Steinernema for postfeeding, 3rd instar larvae of the sheep blowfly, Lucilia cuprina, was tested in sand at various temperatures. Of the two genera, the steinernematids were more active at lower temperatures and parasitized L. cuprina over a greater temperature range. The temperature range of infectivity for L. cuprina differed between nematodes of the same genus and between strains of the same species. Parasitization of L. cuprina and Galleria mellonella occurred in a temperature range that was greater than that permitting nematode development and reproduction. Different strains of the same species were found to have different temperature limits for development and reproduction. Developmental rate was different for each nematode species tested with the heterorhabditids taking longer to complete their life cycle than did the steinernematids.     

Polytene chromosomes of the Old World screwworm fly (Chrysomya bezziana) and its evolutionary relationships with Lucilia cuprina and Cochliomyia hominivorax (Diptera: Calliphoridae).

Standard polytene chromosome maps for the Old World screwsworm fly, Chrysomya bezziana, are presented. Good quality polytene chromosomes obtainable from pupal trichogen cells allow detailed analysis of autosomal euchromatin. The sex chromosomes are represented by irregular heterochromatic structures resembling those described previously in trichogen polytene chromosomes of the Australian sheep blowfly, Lucilia cuprina. A high degree of homology with the banding pattern of L. cuprina polytene chromosomes allowed direct recognition of approximately 60% of the L. cuprina complement in the C. bezziana maps. A further 13% may be homologous. The extensive homology observed is discussed in relation to the rate of chromosome rearrangement and conservation of karyotype elements in the evolution of Calliphorid flies. The observed conservation in polytene banding patterns should facilitate construction of phylogenies over a number of generic groups.     

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.     

Infrared spectra of carbon monoxide bound to mitochondria from diverse species and tissues reveal structurally similar cytochrome c oxidase dioxygen reaction sites

Infrared bands for CO bound to mitochondria from bovine and porcine hearts, bovine brain, rat kidney, and blowfly flight muscle and to intact blowfly flight muscle have been measured in the carbon-oxygen stretch region. Each spectrum contains a narrow band near 1963 cm-1 similar to the major band found earlier for the carbonyl cytochrome c oxidase purified from bovine heart. A second band near 1959 cm-1 ascribed to a less stable conformer of the purified oxidase carbonyl is also detected in mitochondria. These spectra support very similar CO (and O2) binding sites among all the oxidases examined whether the enzyme is purified or is still within mitochondria or intact tissue and therefore suggest that the reduced heme A ligand binding site has been highly conserved during evolution.     

Progress towards the development of a transgenic strain of the Australian sheep blowfly (Lucilia cuprina) suitable for a male-only sterile release program

The Australian sheep blowfly Lucilia cuprina is the most important pest species involved in cutaneous myiasis (flystrike) of sheep in Australia and New Zealand. In New Zealand L. cuprina is primarily controlled through the application of insecticides. However, there is an increased interest in biological methods of control of this species. We have proposed to develop a genetically modified strain of L. cuprina that would be ideal for a male-only sterile release program. To that end we have developed a method for making transgenic L. cuprina using a piggyBac vector and an EGFP marker gene. We have also developed in Drosophila melanogaster a 2-component genetic system for controlling female viability. Females carrying both components of the system die unless fed a diet that contains tetracycline. We anticipate that the female-killing system will need to be optimised for L. cuprina in order to make a strain with the properties required for a male-only release program.     

Fatal association between dieldrin-resistant and susceptible Australian sheep blowflies, Lucilia cuprina.

A novel phenomenon of interactions between genotypes of the dieldrin-resistance (Rdl) locus of the Australian sheep blowfly (Lucilia cuprina) is described. Susceptible adult flies exposed to dieldrin-resistant (Rdl/Rdl or Rdl/S) adults, raised from larvae grown on media containing sublethal concentrations of dieldrin, display mortality related to the concentration on which the resistant flies developed. The resistant flies excrete quantities of dieldrin that are toxic to susceptible flies. These observations provide an additional mechanism to those previously identified for the rapid evolution of resistance to dieldrin by L. cuprina.     

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.     

The immune response of the sheep popliteal lymph node to a purified phenoloxidase from larval cuticle of the sheep ectoparasite, Lucilia cuprina

The popliteal lymph node of the sheep can mount a strong immune response to a phenoloxidase, enzyme A, purified from larval cuticle of a major sheep ectoparasite, the sheep blowfly, Lucilia cuprina. Continuous sampling from a cannulated efferent lymphatic allowed monitoring of changes in both cellular output (total cells and large blast cells) and specific antibody production (by ELISA) following primary and secondary challenge with antigen. Anti-enzyme A antibodies in lymph selectively precipitated enzyme A but not a second cuticular phenoloxidase, enzyme B, a finding that will prove useful in immunolocalization of the enzymes and in elucidating their origins. The implications for immunization of sheep against L. cuprina are discussed.     

Suppression of populations of Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), with a novel blowfly trap

The Australian sheep blowfly, Lucilia cuprina , initiates more than 85% of fly strikes on sheep in Australia with an estimated average annual cost of A$280 million to the Australian sheep industry. LuciTrap^® is a commercially available, selective trap for L. cuprina consisting of a plastic bucket with multiple fly entry cones and a synthetic attractant. The impact of LuciTrap on populations of L. cuprina on sheep properties in five Australian states was evaluated by comparing L. cuprina populations on paired properties with and without LuciTraps over seasons when significant fly populations could be expected. Twenty-four comparisons (trials) were conducted over 4 years. During times of 'higher fly density' (when the 48 h geometric mean of trap catches on the control property was greater than five L. cuprina ), the overall geometric mean trap catches for control and trapped properties differed significantly ( P  < 0.001) with mean trap catches of 19.4 and 7.74 L. cuprina , respectively. The selectivity of the LuciTrap was confirmed with 59% of all trapped flies being L. cuprina . Chrysomya spp. and Calliphora spp. constituted 9.3% and 1.1% of the catches with a variety of other flies (mainly Sarcophagidae and Muscidae ) providing the remainder (31%). Lucilia sericata was only trapped in Tasmania and made up 7.7% of the Lucilia spp. catch in that state. Seventy-two per cent of the trapped L. cuprina were female. The deployment of LuciTrap on sheep properties at one trap per 100 sheep from the beginning of the anticipated fly season suppressed the populations of L. cuprina by 60% compared with matched control properties. The LuciTrap is a selective and easy to use fly trap and constitutes an effective, non-insecticidal tool for use in integrated management programs for L. cuprina .     

Peritrophins of adult dipteran ectoparasites and their evaluation as vaccine antigens

Several peritrophins of larvae of Lucilia cuprina (sheep blowfly) have demonstrated potential as vaccine antigens, and some have been characterised and cloned. These proteins are tightly associated with the peritrophic matrix, a chitinous tube or sac lining the lumen of the gut of most insects. The peritrophins require strong denaturants for their removal from peritrophic matrix. We now report the preliminary characterisation of peritrophins of the adult stage of L. cuprina and Haematobia irritans exigua (buffalo fly). Similar SDS-PAGE profiles were obtained for proteins extracted in SDS or urea from isolated adult peritrophic matrices of both species. Radioiodination of urea-extracted peritrophins improved sensitivity, indicating numerous proteins of 15-75 kDa. Direct radioiodination of L. cuprina peritrophic matrix preferentially labelled high molecular weight complexes and proteins of 80-90 kDa. Two-dimensional gel analyses of a urea extract of adult L. cuprina peritrophic matrix revealed that most proteins were moderately acidic. Antibodies produced against SDS-extracted peritrophins, or against sonicated peritrophic matrices of these two flies were crossreactive. The sera also appeared to recognise SDS-extracted components of Triton X-100 treated and washed adult peritrophic matrix of the mosquito, Aedes vigilax (Skause). This profile altered as the peritrophic matrix matured. In concordance with extracts from the adult L. cuprina and H.i. exigua peritrophic matrices, proteins in the 50-75 kDa region were immunodominant. The vaccine potential of the peritrophins of these Diptera were examined following vaccination of cattle and rabbits with adult H.i. exigua or L. cuprina peritrophins. When the adult life stages of H.i. exigua or two mosquitoes, A. vigilax and A. aegypti (Linnaeus), were fed on the sera or blood of vaccinated hosts, there were no detrimental effects to any life cycle stages of these Diptera.     

Prasinons A and B: Potent Insecticides from Streptomyces prasinus

Two metabolites which have high activity against sheep blowfly larvae (Lucilia sericata and L. cuprina) were found to be produced by Streptomyces prasinus NCIB 10719. These substances were isolated from culture filtrate by solvent extraction and chromatography and named prasinons A and B. Fermentation factors affecting the formation of these substances are described together with their physical, chemical, and biological properties.     

Asymmetry – where evolutionary and developmental genetics meet

The mechanisms responsible for the fine tuning of development, where the wildtype phenotype is reproduced with high fidelity, are not well understood. The difficulty in approaching this problem is the identification of mutant phenotypes indicative of a defect in these fine-tuning control mechanisms. Evolutionary biologists have used asymmetry as a measure of developmental homeostasis. The rationale for this was that, since the same genome controls the development of the left and right sides of a bilaterally symmetrical organism, departures from symmetry can be used to measure genetic or environmental perturbations. This paper examines the relationship between asymmtry and resistance to organophosphorous insecticides in the Australian sheep blowfly, Lucilia cuprina. A resistance gene, Rop-1, which encodes a carboxylesterase enzyme, also confers a significant increase in asymmetry. Continued exposure of resistant populations to insecticide has selected a dominant suppressor of the asymmetry phenotype. Genetic evidence indicates that the modifier is the L. cuprina Notch homologue.     

Ultrastructural localization of phenoloxidases in cuticle and haemopoietic tissue of the blowfly Lucilia cuprina

The ultrastructural localization of two types of biochemically characterized phenol oxidase activity is described in the larva of the sheep blowfly, Lucilia cuprina. Cuticular tyrosinase activity (enzyme A) is seen in epicuticular filaments and procuticle. Procuticle activity can be detected only after a presumed process of activation takes place in damaged cuticle. By using either the dopamine reaction or inducing melanization by hot-water treatment, tyrosinase is readily shown in haemopoietic tissue which, in L. cuprina, occurs subdermally as well as being associated with the dorsal vessel. The adaptation of the diaminobenzidine technique, used to stain laccase in electrophoretic gels, to ultrastructural cytochemistry has made it possible to demonstrate enzymic activity probably due to laccase (enzyme B). The laccase activity is present in the inner epicuticle of late wandering third instar larvae (about to pupariate) but is not present in the epicuticle of younger larvae.     

Ecological analysis of field trials conducted to assess the potential of sex-linked translocation strains for genetic control of the Australian sheep blowfly, Lucilia cuprina (Wiedemann)

Field trials were conducted with translocation/eye colour (TE) strains of L. cuprina to measure the mating ability of the males under field conditions and assess their potential for suppressing sheep blowfly populations. Rates of increase in L. cuprina were highest in spring (3.9-9.1 per generation), consistently low during summer (0.1-0.6 per generation) and somewhat higher during autumn (1.1-3.4 per generation). The TE strains released had the potential to prevent population increases of this magnitude. Their failure to do so during these trials resulted from their low mating competitiveness (0.33) relative to that of field-reared males (1.0), inadequacy of the larval release method and the limited capacity of the experimental mass-rearing facility.     

Predicting insecticide resistance: mutagenesis, selection and response

Strategies to manage resistance to a particular insecticide have usually been devised after resistance has evolved. If it were possible to predict likely resistance mechanisms to novel insecticides before they evolved in the field, it might be feasible to have programmes that manage susceptibility. With this approach in mind, single-gene variants of the Australian sheep blowfly, Lucilia cuprina, resistant to dieldrin, diazinon and malathion, were selected in the laboratory after mutagenesis of susceptible strains. The genetic and molecular bases of resistance in these variants were identical to those that had previously evolved in natural populations. Given this predictive capacity for known resistances, the approach was extended to anticipate possible mechanisms of resistance to cyromazine, an insecticide to which L. cuprina populations remain susceptible after almost 20 years of exposure. Analysis of the laboratory-generated resistant variants provides an explanation for this observation. The variants show low levels of resistance and a selective advantage over susceptibles for only a limited concentration range. These results are discussed in the context of the choice of insecticides for control purposes and of delivery strategies to minimize the evolution of resistance.     

Association of exogenous DNA with cattle and insect spermatozoa in vitro

Spermatozoa isolated from domestic cattle (Bos taurus), the Australian sheep blowfly (Lucilia cuprina), and the honeybee (Apis mellifera) are capable of binding exogenous radiolabeled linear DNA. Both motile and nonmotile bovine sperm exhibit four distinct patterns of DNA association. Following treatment with DNase I, the relative proportion of one of these patterns increases specifically in living sperm, suggesting that a small proportion of DNA that associates with bovine sperm may be sequestered within the sperm head.     

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.     

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).     

The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly.

Organophosphate (OP) insecticide resistance in certain strains of Musca domestica is associated with reduction in the carboxylesterase activity of a particular esterase isozyme. This has been attributed to a 'mutant ali-esterase hypothesis', which invokes a structural mutation to an ali-esterase resulting in the loss of its carboxylesterase activity but acquisition of OP hydrolase activity. It has been shown that the mutation in Lucilia cuprina is a Gly137-->Asp substitution in the active site of an esterase encoded by the Lc alpha E7 gene (Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997. A single amino acid substitution converts a carboxylesterase to an organophosphate hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94, 7464-7468). We now report the cloning and characterisation of the orthologous M. domestica Md alpha E7 gene, including the sequencing of cDNAs from the OP resistant Rutgers and OP susceptible sbo and WHO strains. The Md alpha E7 gene has the same intron structure as Lc alpha E7 and encodes a protein with 76% amino acid identity to Lc alpha E7. Comparisons between susceptible and resistance alleles show resistance in M. domestica is associated with the same Gly137-->Asp mutation as in L. cuprina. Bacterial expression of the Rutgers allele shows its product has OP hydrolase activity. The data indicate identical catalytic mechanisms have evolved in orthologous Md alpha E7 and Lc alpha E7 molecules to endow diazinon-type resistance on the two species of higher Diptera.