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.     

Genetic instability in mass-rearing colonies of a sex-linked translocation strain of Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) during a field trial of genetic control

Summary Genetic breakdown occurred in a strain of Lucilia cuprina constructed for the purpose of genetic control of this pest. The strain incorporated autosomal recessive eye colour mutations linked in repulsion with a translocation involving the Y chromosome (male-determining) and two autosomes. In the original strain females had white eyes and males were wild type. The spontaneous breakdown involved a failure of the sex-limited inheritance of the eye colour mutations. Characteristically the frequency of white-eyed males increased rapidly in the strain, whereas the frequencies of the three other phenotypically recognizable breakdown products did not. This suggested that the white-eyed males had a selective advantage over both the wild type males and the other breakdown products. Genetic analysis revealed that recombination, which is normally rare in L. cuprina males, is considerably more frequent in the presence of a Y-autosome translocation, but that recombination alone was insufficient to account for the rate of increase of the white-eyed males in the colony. Genetic and cytological analysis of the breakdown products revealed that reversion of the multi-break translocation also occurred, and that many of the white-eyed males had either only a Y-single-autosome translocation or no translocation at all; thus these males were more fertile than the wild type multi-translocation males. In addition, under colony cage conditions the white-eyed males may have had a behavioural advantage in competition with the wild type males.     

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.     

The effects of azadirachtin A on the morphology of the ring complex of Lucilia cuprina (Wied) larvae (Diptera: Insecta)

This study investigated the effect of azadirachtin A (AZA) on the ultrastructure of the endocrine glands (the prothoracic gland, the corpus allatum, and the corpus cardiacum) responsible for controlling moulting and ecdysis in Lucilia cuprina larvae. All constituent endocrine glands within the ring complex exhibited ultrastructural change after exposure of larvae to AZ A in the diet. The effects on the nucleus were the most noticeable and included: (1) crenulation of nuclear shape, (2) clumping of heterochromatin, and (3) pyknosis. The degenerative changes to the nuclei appeared to precede changes within the cytoplasm. It was concluded that the degeneration of all three endocrine glands within the ring complex would contribute to a generalised disruption of neuroendocrine function.     

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.     

Developmental study of thermotolerance and the heat shock response inLucilia cuprina (Weidemann)

The ability to withstand thermal stress in a laboratory population of the blowflyLucilia cuprina (measured as per cent adult survival following varying periods of exposure to elevated temperature up to a maximum of 48°C) was in the order pupa > larva > adult. Pre-exposure to a mild heat shock (37°C) induced tolerance to temperatures which were otherwise lethal. An analysis of heat shock-induced protein synthesis during development at similar elevated temperatures presented patterns corresponding to the above observations on thermotolerance. The induced level of synthesis of major heat shock proteins (viz., 79, 69, 28, 20 and 19 kDa) were greater in larval tissues than in most of the adult tissues except gonads. The response varied between young (2 days) and old (30 days) adults in a tissue-specific manner. In general, heat shock protein 69 kDa was most abundant in all the tissues studied. Control as well as heat shocked Malpighian tubules of adults uniquely showed two major [³⁵S]methionine labelled bands corresponding to approximately 62 and 64 kDa. Immunoblots showed the 62 kDa protein to cross react with an antibody againstHelioihis HSP60. Although the synthesis of the 62 kDa polypeptide was prominent only in Malpighian tubules of adult blowflies, nearly equal levels of this HSP60 family polypeptide were present in all tissues (control as well heat shocked) except the larval salivary glands.     

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.     

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%).

     

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.     

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.     

Genetic control of Lucilia cuprina: analysis of field trial data using simulation techniques

Summary An analytical version of the genetic control simulation program GENCON has been used to further analyze the data obtained during field trials of genetic control of the sheep blowfly, Lucilia cuprina, in 1976–79. In the simulations, population trends from a nonrelease area were used as an estimate of the rates of increase that would have occurred in the target population if there had been no releases. Genetic data from the target area (frequencies of matings by released males) were used to predict the frequencies of descendants of released males, the resulting genetic death, and the effects of this on population trends. In simulations that assumed no migration and full survival and competitiveness of all field-reared descendants of released males (translocation-bearing males and males and females heterozygous for deleterious mutations), neither the predicted genetic changes nor the predicted population trends agreed well with the observed data. Further simulations suggested that reduced survival or competitiveness of field-reared descendants did not account for this disagreement, but that immigration of wild flies into the test areas was probably a major contributor to the failure to achieve suppression. However, immigration alone was not sufficient to explain all the differences between observed and expected results. Other plausible contributors to this failure were: (1) lower survival of translocation males due to the effects of a dieldrin resistance allele carried on the translocation, and (2) increased survival of immature stages of L. cuprina at low population densities.     

The genetic bases of high-level resistance to diflubenzuron and low-level resistance to cyromazine in a field strain of the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae)

Abstract  Several genes on chromosomes IV and VI have a significant influence on high-level resistance to diflubenzuron in a strain of the Australian sheep blowfly from Tara, Queensland. Low-level resistance to cyromazine in the same strain is due to genes on these chromosomes with a gene (gene complex) in the sv marker region of chromosome IV being particularly important. For both insecticides, genetic background influences resistance status. If the results of the Tara strain prove typical for those of other populations, resistance to diflubenzuron in the Australian sheep blowfly has potentially significant consequences for woolgrowers.     

The use of bridging systems to increase genetic variability in compound chromosome strains for genetic control of Lucilia cuprina (Wiedemann)

Summary Genetic variability in the non-compound portion of the genomes of compound-chromosome (CC) strains intended for genetic control can be increased by the use of bridging strains which can be crossed to both CC and normal strains. Two bridging systems are described for chromosome-5 CC strains of Lucilia cuprina. The first system relies on the established viability and fertility of males trisomic for chromosome 5R. Males carrying the (5L.YL)23 half-translocation, a C(5R), and a normal chromosome 5 were crossed successfully to a CC strain and a normal strain. The second system uses a pair of reciprocal whole-arm 4;5 translocations to generate gametes disomic for 5R and nullosomic for 5L, which in combination with C(5L)-bearing gametes form viable near-euploid offspring with only small duplications and deficiencies. These offspring (C(5L); (4L.5R)357; (4R.5R)194; (4L.4R)) were crossed successfully with both CC and T(4;5)357/ + individuals. The latter were in turn crossed successfully with normal strains. The T(Y;5)23 system allows replacement of the non-CC genome with wild material more rapidly than the T(4;5)357/T(4;5)194 system, but unlike the latter does not allow replacement of the Y chromosome in the CC strain. The double translocation system is currently being used in L. cuprina.     

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.     

Functions of the white and topaz loci of Lucilia cuprina in the production of the eye pigment xanthommatin

The white and topaz eye color mutants of L. cuprina are defective in the production of the brown screening pigment xanthommatin. Both white and topaz mutants were found to be unable to accumulate xanthommatin precursors in the larval malpighian tubules, correlating with their reduced early pupal level of this metabolite. In addition, white mutants showed reduced rates of accumulation of kynurenine and 3-hydroxykynurenine in the adult eyes. Another mutant strain, grape, was also defective in its ability to accumulate these xanthommatin precursors in the eyes, although accumulation was normal in the larval tubules. In contrast, the topaz mutants were found to be normal in eye accumulation, although tubule accumulation was markedly abnormal. These properties of the white and topaz mutants of L. cuprina are compared with those of the white and scarlet mutants of D. melanogaster, and it seems likely that in the two species these genes are involved with the uptake or storage of xanthommatin precursors in specific tissues.This work was supported by Grant D2 75/15248 from the Australian Research Grants Committee.     

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     

Cloning and characterization of thewhite andtopaz eye color genes from the sheep blowflylucilia cuprina

Summary Clones carrying thewhite andtopaz eye color genes have been isolated from genomic DNA libraries of the blowflyLucilia cuprina using cloned DNA from the homologouswhite andscarlet genes. respectively, ofDrosophila melanogaster as probes. On the basis of hybridization studies using adjacent restriction fragments, homologous fragments were found to be colinear between the genes from the two species. The nucleotide sequence of a short region of thewhite gene ofL. cuprina has been determined, and the homology to the corresponding region ofD. melanogaster is 72%; at the derived amino acid level the homology is greater (84%) due to a marked difference in codon usage between the species. A major difference in genome organization between the two species is that whereas the DNA encompassing theD. melanogaster genes is free of repeated sequences. that encompassing theirL. cuprina counterparts contains substantial amounts of repeated sequences. This suggests that the genome ofL. cuprina is organized on the short period interspersion pattern. Repeated sequence DNA elements, which appear generally to be short (less than 1 kb) and which vary in repetitive frequency in the genome from greater than 10⁴ copies to less than 10² copies, are found in at least two different locations in the clones carrying these genes. One type of repeat structure, found by sequencing, consists of tandemly repeating short sequences. Restriction site and restriction fragment length polymorphisms involving both thewhite andtopaz gene regions are found within and between populations ofL. cuprina.