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.     

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.     

Computer simulation of genetic control. Comparison of sterile males and field-female killing systems

Summary A computer program, GENCON, designed to simulate genetic control using field-female killing systems, is described. These systems incorporate sex-linked translocations and conditional lethal mutations. Genetic death in field populations is caused by semisterility of the translocation and by homozygosis of the mutations in females and non-translocation males of field origin. Simulations using the program compare the effectiveness, in populations regulated by density, of genetic control using this type of system with control using sterile-male release. At high release rates, sterile males cause more rapid suppression and earlier eradication than sex-linked translocation strains. However, if releases are interrupted before eradication, the rate of recovery of density-dependent populations is more rapid following sterile-male release than following suppression with translocation strains. In such populations, the cumulative population suppression (number of individuals killed) is greater with translocation-strain release than with sterile-male release. At low release rates, sex-linked translocation strains can be much more effective at suppressing and eradicating density-dependent populations than sterile males. In continental Australia, eradication of the sheep blowfly Lucilia cuprina is probably not practicable. A suppression campaign using sex-linked translocation strains could yield a higher benefit to cost ratio than one using sterile males.     

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%).

     

Homozygous-viable pericentric inversions for genetic control of Lucilia Cuprina

Summary The isolation of homozygous-viable pericentric inversions for inclusion in field-female killing (FK) systems in Lucilia cuprina is described. From 7,236 irradiated chromosomes screened, 16 pericentric inversions were isolated. Four of these were viable as homozygotes. One of these, In (3LR) 14, possesses the properties required for inclusion in FK systems (tight linkage of one inversion break-point to the white-eye gene and substantial genetic exchange within the inversion in heterozygous females).     

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.     

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.     

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.     

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

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

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.     

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.     

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.     

Readiness to lay in the blowfly Lucilia cuprina is unaffected by oviposition site-deprivation or egg-load

Oviposition by Lucilia cuprina Wiedemann (Diptera, Calliphoridae) was examined in relation to period of oviposition site-deprivation and egg-load. Effects of oviposition site-deprivation were examined by comparing oviposition performance of individual females that had matured their batch of oocytes within the previous 24 h with that of females which had reached ovarian maturity 8 days previously. Egg-load was manipulated by causing females of this anautogenous species to consume different amounts of protein-rich material. In no-choice experiments, individual females of the different categories were given access for 4 h to oviposition substrate, soaked with (i) liver exudate, (ii) the exudate diluted 16-fold or (iii) the undilated exudate containing the oviposition deterrent sodium chloride at a concentration of 2 M. These solutions elicited oviposition from different proportions of females, but neither these proportions, nor the interval between introduction of the oviposition site and the initiation of oviposition, was significantly affected by the period of oviposition site-deprivation or the number of eggs matured by the females.

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Résumé L'effet de la privation de lieu de ponte a été étudié en comparant les pontes de femelles isolées ayant formé leurs ufs mûrs dans les 24 heures précédentes, à celles de femelles ayant atteint leur maturité sexuelle 8 jours avant. La rétention ovocytaire est provoquée en faisant consommer aux femelles de cette espèce anautogène différentes quantités d'aliments riches en protéines. La ponte de femelles dont le contingent total de leurs ovocytes s'est développé, — c'est-à-dire 260 —, après consommation ad libitum de foie de mouton pendant 48 heures, a été comparée à celle de femelles ayant formé 190 ovocytes mûrs après ingestion d'une quantité limitée de jus de foie.Dans des expériences sans choix, les femelles isolées de différences catégories ont eu accès pendant 4 heures au substrat de ponte trempé: 1) dans du jus de foie, 2) dans du jus dilué 16 fois, 3) dans du jus de foie non dilué mais contenant NaCl (inhibiteur de la ponte) à la concentration de 2 M. Le jus non dilué a provoqué une forte stimulation, induisant la ponte de 80% des femelles. Le jus dilué et celui contenant NaCl n'ont induit la ponte que de 40% des femelles avec des niveaux de stimulation bien plus faibles. La date d'introduction du lieu de ponte et le taux de rétention des ovocytes mûrs n'ont eu auçun effet sur la proportion de femelles réagissant à ces 3 types de stimulation.

     

Thehermit transposable element of the Australian sheep blowfly,Lucilia cuprina, belongs to thehAT family of transposable elements

We report the cloning ofhermit, a member of thehAT family of transposable elements from the genome of the Australian sheep blowfly,Lucilia cuprina. Hermit is 2716 bp long and is 49% homologous to the autonomoushobo element,HFL1, at the nucleic acid level.Hermit has 15 bp terminal inverted repeats that share 10 bp with the terminal inverted repeats ofHFL1. Conceptual translation reveals a 583 residue open reading frame (ORF) that is 64% similar and 42% identical to theHFL1 ORF. However, the sequence of thehermit element contains two frameshifts within the putative ORF, indication thathermit is an inactive element. Analysis ofL. cuprina strains from within and outside Australia suggested thathermit is present as a single copy in all the genomes analysed.     

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.     

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.

     

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     

Chromosomal inversions and genetic control revisited: the use of inversions in sexing systems for higher Diptera

Summary Genetic sexing systems based on sex-linked translocations and deleterious mutations are subject to breakdown from genetic recombination in males. Including inversions in these strains may provide a solution to this problem, by ensuring selective elimination of recombinant products. Inversions could be used either in coupling to or in repulsion to the translocation. The latter system, requiring homozygous-viable inversions, would be more difficult to construct, but would offer several advantages not available with coupled translocation/inversion systems. A system proposed for the blowfly Lucilia cuprina is outlined, which combines homozygous-viable pericentric inversions in repulsion to existing sex-linked translocations. This system should both stabilize the genetic sexing system and increase the suppressive potential of such strains.     

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

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