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).     

Molecular mechanism of synthetic pyrethroid and organophosphate resistance in field isolates of <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> tick collected from a northern state of India

The frequently used chemical control method to manage Rhipicephalus microplus is limited by the emergence of resistance populations. Understanding of resistance mechanisms is essential to develop strategy for sustainable management. The present study was focused on working out the molecular mechanisms of resistance against synthetic pyrethroids (SPs) and organophosphates (OPs) in field isolates of R. microplus collected from six districts of Uttar Pradesh, India. Adult immersion test with discriminating concentrations (AIT-DC) was used to determine resistance status of isolates to SPs (deltamethrin, cypermethrin) and OPs (diazinon, coumaphos). All the six isolates were found resistant to SPs with resistance factor (RF) of 2.9–58.6 and to one of the OP compounds, diazinon having RF of 3.5–13.7 but susceptible to coumaphos (RF?<?1.4). Three R. microplus genes, viz. para-sodium channel domain II S4-5 linker, carboxylesterase (372 bp) and acetylcholinesterase 2 (1692 bp) were sequenced and compared with respective sequences of reference susceptible IVRI-I, reference OP resistant population (IVRI-III), IVRI-IV and multi-acaricide resistant population (IVRI-V) of R. microplus. A C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to L64I amino acid substitution was detected in all six isolates. The G1120A mutation in the carboxylesterase gene could not be detected in any isolate. Five nucleotide substitutions viz., G138A, G889A, T1090A, C1234T and G1403A were identified in the acetylcholinesterase 2 gene leading to four amino acid substitutions. The findings of the study corroborate the role of mutation in sodium channel and acetylcholinesterase 2 genes in SP and OP resistance in this part of India.     

Biochemistry of Esterases Associated with Organophosphate Resistance in Lucilia cuprina with Comparisons to Putative Orthologues in Other Diptera

Esterase activities associated with organophosphate insecticide resistance in the Australian sheep blowfly, Lucilia cuprina, are compared with similar activities in other Diptera. The enzymes making the major contribution to methyl butyrate hydrolysis (ali-esterase) in L. cuprina, M. domestica, and D. melanogaster comigrate during electrophoresis. The enzymes in L. cuprina and D. melanogaster correspond to the naphthyl acetate hydrolyzing E3 and EST23 isozymes of those species. These and previously published data suggest that the ali-esterases of all three species are orthologous. Strains of L. cuprina fall into four groups on the basis of quantitative determinations of their ali-estesterase, OP hydrolase, and malathion carboxylesterase activities and these groups correspond to their status with respect to two types of OP resistance. Strains susceptible to OPs have high ali-esterase, low OP hydrolase, and intermediate MCE activities; those resistant to malathion but not diazinon have low ali-esterase, intermediate OP hydrolase, and high MCE activities; those resistant to diazinon but not malathion have low ali-esterase, high OP hydrolase, and low MCE activities; those resistant to both OPs have low ali-esterase, high OP hydrolase, and high MCE activities. The correlated changes among the three biochemical and two resistance phenotypes suggest that they are all properties of one gene/enzyme system; three major allelic variants of that system explain OP susceptibility and the two types of OP resistance. Models are proposed to explain the joint contribution of OP hydrolase and MCE activities to malathion resistance and the invariant association of low ali-esterase and elevated OP hydrolase activities in either type of resistance.     

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 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.     

Multiple Mutations and Gene Duplications Conferring Organophosphorus Insecticide Resistance Have Been Selected at the Rop-1 Locus of the Sheep Blowfly, Lucilia cuprina

Sequences of the esterase gene E7 were compared across 41 isogenic (IV) strains of the sheep blowfly, Lucilia cuprina, and one strain of the sibling species, L. sericata. The 1.2-kb region sequenced includes sites of two insecticide resistance mutations. Gly137Asp confers resistance to organophosphorus insecticides (OPs), particularly preferring diethyl OPs such as diazinon, while Trp251Leu prefers dimethyl OPs, and particularly malathion, with the additional presence of carboxylester moieties. We found that there are just eight haplotypes among the 41 chromosomes studied: two Gly137Asp containing haplotypes, two Trp251Leu containing haplotypes, and four susceptible haplotypes, including the L. sericata sequence. While phylogenetic analysis of these haplotypes suggests that the Asp137 and Leu251 mutations each arose at least twice, evidence for recombination was detected across the region, therefore single origins for these resistance mutations cannot be ruled out. Levels of linkage disequilibrium in the data are high and significant hitchhiking is indicated by Fay and Wu s H test but not the Tajima test. A test of haplotype diversity indicates a paucity of diversity compared with neutral expectations. Both these results are consistent with a very recent selective sweep at the LcE7 locus. Interestingly, gene duplications of three different combinations of OP resistant haplotypes were identified in seven of the isogenic (IV) strains. All three types of duplication involve an Asp137 and a Trp251 haplotype. To examine whether more haplotypes existed before the hypothesised selective sweep, fragments of E7 surrounding the resistance mutations were amplified from pinned material dating back to before OPs were used. Four new sequence haplotypes, not sampled in the survey of extant haplotypes, were obtained that are all associated with susceptibility. This is suggestive of a higher historical level of susceptible allelic diversity at this locus.Reviewing Editor: Dr. Rasmus Nielsen     

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b₅ enhanced the k_cat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min⁻¹) with little effect on K_M (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species.     

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.     

Correlated insecticide cross-resistance in azinphosmethyl resistant codling moth (Lepidoptera: Tortricidae)

Resistance to several classes of insecticides was correlated with azinphosmethyl resistance in codling moth, Cydia pomonella (L.), in California. In tests of laboratory and field populations, cross-resistance was positively correlated with azinphosmethyl and two organophosphates (diazinon, phosmet), a carbamate (carbaryl), a chlorinated hydrocarbon (DDT), and two pyrethroids (esfenvalerate and fenpropathrin). Additionally, negatively correlated cross-resistance was identified between azinphosmethyl and two other organophosphates, chlorpyrifos and methyl parathion. Patterns of resistance observed in laboratory colonies were confirmed with field bioassays. In bioassays of field populations, azinphosmethyl resistance was observed to increase from 1991 to 1993, although levels of resistance remained < 13-fold. Because orchards with azinphosmethyl resistance have had difficulties with suppression of codling moth, and cross-resistance was found for all tested classes of insecticides, strategies for managing resistance will need to be developed so as to protect current and future control tactics. The two insecticides with negatively correlated cross-resistance are discussed as potential tools for resistance management.     

Development and evaluation of male-only strains of the Australian sheep blowfly, <Emphasis Type="Italic">Lucilia cuprina</Emphasis>

The Australian sheep blowfly Lucilia cuprina (Wiedemann) is a major pest of sheep in Australia and New Zealand. From the 1960s to the 1980s there was a major effort to develop "field female killing" or FFK strains of L. cuprina that could be used for a cost-effective genetic control program. The FFK strains carried eye color mutations that were lethal to females in the field but not under conditions in the mass rearing facility. Males did not die in the field as normal copies of the eye color genes had been translocated to the Y chromosome and an autosome. Although the FFK strains showed some promise in field tests, a genetic control program in mainland Australia was never implemented for several reasons including instability of the FFK strains during mass rearing. A stable transgenic strain of L. cuprina that carried one or more dominant repressible female lethal genes offered the potential for efficient genetic control of blowfly populations. Here I review our research on tetracycline-repressible female lethal genetic systems, Lucilia germ-line transformation and sex determination genes that ultimately led to the successful development of transgenic "male-only" strains of L. cuprina. The technology developed for L. cuprina should be directly transferable to other blowfly livestock pests including L. sericata and the New World and Old World screwworm. 29     

Control of the sheep blowfly in Australia and New Zealand – are we there yet?

The last 50 years of research into infections in Australia and New Zealand caused by larvae of the sheep blowfly, Lucilia cuprina, have significantly advanced our understanding of this blowfly and its primary host, the sheep. However, apart from some highly effective drugs it could be argued that no new control methodologies have resulted. This review addresses the major areas of sheep blowfly research over this period describing the significant outcomes and analyses, and what is still required to produce new commercial control technologies. The use of drugs against this fly species has been very successful but resistance has developed to almost all current compounds. Integrated pest management is becoming basic to control, especially in the absence of mulesing, and has clearly benefited from computer-aided technologies. Biological control has more challenges but natural and perhaps transformed biopesticides offer possibilities for the future. Experimental vaccines have been developed but require further analysis of antigens and formulations to boost protection. Genetic technologies may provide potential for long-term control through more rapid indirect selection of sheep less prone to flystrike. Finally in the future, genetic analysis of the fly may allow suppression and perhaps eradication of blowfly populations or identification of new and more viable targets for drug and vaccine intervention. Clearly all these areas of research offer potential new controls but commercial development is perhaps inhibited by the success of current chemical insecticides and certainly requires a significant additional injection of resources.     

A genomics-informed,SNP association study reveals FBLN1 and FABP4 as contributing to resistance to fleece rot in Australian Merino sheep

Background  

Fleece rot (FR) and body-strike of Merino sheep by the sheep blowfly Lucilia cuprina are major problems for the Australian wool industry, causing significant losses as a result of increased management costs coupled with reduced wool productivity and quality. In addition to direct effects on fleece quality, fleece rot is a major predisposing factor to blowfly strike on the body of sheep. In order to investigate the genetic drivers of resistance to fleece rot, we constructed a combined ovine-bovine cDNA microarray of almost 12,000 probes including 6,125 skin expressed sequence tags and 5,760 anonymous clones obtained from skin subtracted libraries derived from fleece rot resistant and susceptible animals. This microarray platform was used to profile the gene expression changes between skin samples of six resistant and six susceptible animals taken immediately before, during and after FR induction. Mixed-model equations were employed to normalize the data and 155 genes were found to be differentially expressed (DE). Ten DE genes were selected for validation using real-time PCR on independent skin samples. The genomic regions of a further 5 DE genes were surveyed to identify single nucleotide polymorphisms (SNP) that were genotyped across three populations for their associations with fleece rot resistance.     

A single nucleotide polymorphism in the acetylcholinesterase gene of the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae) is associated with chlorpyrifos resistance

The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based on predatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae), resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant strains of K. aberrans. No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).     

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.     

Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae)

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve “maggot therapy”. We have previously reported the germ-line transformation of L. cuprina and the design of a “female killing system” that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.     

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.     

INTERACTION BETWEEN GENOTYPES AT THE DIAZINON-RESISTANCE LOCUS OF THE AUSTRALIAN SHEEP BLOWFLY,LUCILIA CUPRINA. FACILITATION OF DEVELOPMENT OF SUSCEPTIBLE GENOTYPES

The survival of larvae with susceptible (+ +) genotypes at the diazinon-resistance locus of the Australian sheep blowfly, Lucilia cuprina is facilitated by prior conditioning of diazinon-containing media by heterozygote (R+) or resistant (RR) larvae. The effect is not observed on media without diazinon. Conditioning by + + genotypes does not affect subsequent development of susceptible larvae and the viability of R + and RR larvae is unaffected by conditioning of the media. Similar results are observed when the medium is conditioned with crushed larvae of the three genotypes. The development of + + larvae is enhanced only on media containing diazinon to which R + or RR crushed larvae are added. The results for other comparisons are equivalent for conditioned or unconditioned media. The number of crushed ++, R +, or RR larvae added to the medium containing diazinon does not affect the proportion of + + eggs that develop through larval and pupal stages to emerge as adults. The rate of development of the + + genotype is, however, positively correlated with the number of crushed R + or RR larvae added.     

Carbaryl resistance in Mexican strains of the southern cattle tick (Acari: Ixodidae)

Susceptibility to carbaryl in six Mexican strains of the southern cattle tick, Boophilus microplus (Canestrini), was evaluated with the Food and Agricultural Organization larval packet test. Tick strains from the cattle fever tick quarantine zone in Texas were more susceptible to carbaryl than to coumaphos or diazinon. Compared with the susceptible reference (Gonzalez) strain, Mexican tick strains demonstrated 10.9-59.5-fold resistance to carbaryl. Significant cross-resistance was found between carbaryl and the organophosphate acaricides coumaphos and diazinon. Bioassay results with synergists suggested that metabolic detoxification mechanisms did not play a major role in carbaryl resistance. Resistance to carbaryl was likely conferred by insensitive acetylcholinesterase. The implications of carbaryl resistance in tick eradication and control also are discussed.     

Susceptibility of larvae of the sheep blowfly Lucilia cuprina to various Heterorhabditis spp., Neoaplectana spp., and in undescribed steinernematid (Nematoda)

The susceptibility of third state larvae of the sheep blowfly Lucilia cuprina in sand to 11 species and strains of Heterorhabditis and Neoaplectana and to one species of an undescribed steiner-nematid was tested at various dosages. Larvae were susceptible to all, although far less so to one strain of N. bibionis and to the undescribed steinernematid. Estimates of LD₅₀ and LD₉₀ are presented for each effective strain. Heterorhabditis spp. were able to reproduce in L. cuprina larvae subjected to low dosages of effective-stage juveniles whereas Neoaplectana spp. were not able to reproduce at any dosage. The possibility exists for using Heterorhabditis species as a control agent against L. cuprina larvae after these have left the sheep to pupate in the soil.     

Habitat preferences and carcase colonization by sheep blowflies in the northern North Island of New Zealand

Abstract. In urban Auckland, from September 1991 to May 1992, only six specimens of the Australian sheep blowfly, Lucilia cuprina, were collected in farmed parkland and in garden habitats. The common green blowfly, Lucilia sericata, was the most common calliphorid trapped in these habitats. Neither of the two Lucilia species were found in native bush remnants in urban or rural areas where Calliphora hilli was dominant. Similarly very few L.cuprina (0.3% of the total) were trapped in rural rubbish tips in January and February where the majority of blowflies were again L.sericata. These results are compared with data collected from pastoral habitats, where L.cuprina is the major cause of flystrike. Lucilia cuprina was reared from five out of ninety-nine carcases found in rural areas. Calliphora stygia and L.sericata were the most common blowflies colonizing carrion and were reared from 59% and 51% of the carcases respectively.