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 asymmetry 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.

Scalloped wings is the Lucilia cuprina Notch homologue and a candidate for the modifier of fitness and asymmetry of diazinon resistance.

The Scalloped wings (Scl) gene of the Australian sheep blowfly, Lucilia cuprina, is shown to be the homologue of the Drosophila melanogaster Notch gene by comparison at the DNA sequence and genetic levels. A L. cuprina genomic fragment, which shows strong identity with the Notch (N) gene at the molecular level, hybridizes to the location of the Scl gene on polytene chromosomes. The two genes are functionally homologous; the dominant and recessive Notch-like phenotypes produced by mutations in the Scl gene allow these alleles to be classed as N-like or Abruptex-like. The Scl gene is under investigation as a candidate for the fitness and asymmetry Modifier (M) of diazinon resistance. We show that M affects the penetrance of wing and bristle phenotypes associated with two Scl alleles in a manner consistent with the M being an allele of Scl. In addition, we report a phenotypic interaction between the diazinon-resistance mutation, Rop-1, and the same alleles of Scl. We propose that the product of Rop-1, an esterase, may be involved in cell adhesion in developmental processes involving the Scl gene product.

Causes and consequences of esterase 6 enzyme activity variation in pre-adult Drosophila melanogaster.

We report heritable threefold differences in both larval and pupal esterase 6 activity among 17 isoallelic lines of D. melanogaster extracted from a natural population. The activity differences in the two stages are only weakly correlated with each other or with previously determined values for esterase 6 activity in adults of these lines. The pre-adult activity variation is also unrelated to polymorphisms among the lines for six esterase 6 allozymes and six restriction sites in a region encompassing the esterase 6 coding DNA and the first kbp of 5' flanking DNA. However, two insertions, of 8.0 and 6.8 kbp, located about 1.4 kbp 5' of the esterase 6 coding region are associated with low activity in larvae and, to a lesser extent, in pupae, albeit not in adults. Restriction mapping reveals similarity between the 8.0 kbp insert and the 7.4 kbp retrotransposon 17.6. The differences in larval activity among lines are positively correlated with fitness as assessed from assays of pre-adult viability and development time but no significant associations between pupal esterase 6 activity and these measures are detected. Some effects of esterase 6 allozyme differences are also found for viability and development time but these effects could be explained by linkage disequilibrium between the 8.0 kbp insert and the EST6-9 allozyme.

Associations of esterase 6 allozyme and activity variation with reproductive fitness in Drosophila melanogaster.

Previous studies have shown that the esterase 6 (EST6) enzyme of D. melanogaster is mainly produced in the sperm ejaculatory duct of the adult male and comparisons of wild-type males with laboratory null mutants have suggested that the enzyme plays a role in reproductive fitness. In this study we have compared 18 field-derived lines each isoallelic for Est6 for differences in five components of male reproductive fitness. No consistent fitness differences were found among lines differing in respect of the two major allozyme classes EST6-F and EST6-S, despite other evidence that these two classes are not selectively equivalent in the field. However, differences in reproductive fitness were found among lines differing in the minor mobility variants that segregate within EST6-F and EST6-S. A failure to distinguish among these minor forms may explain the discrepancies in previous studies on the effects of the major EST6 allozymes on reproductive fitness. The most significant associations we have found between EST6 and reproductive fitness were due to variation in EST6 activity levels. Male EST6 activity levels were found to be positively correlated with their time to first mating, negatively correlated with the numbers of eggs laid and progeny produced by their mates, and negatively correlated with the frequency with which their mates remate. We conclude that some EST6 variants differ in components of male reproductive fitness operative in laboratory cultures. However, the evidence for fitness differences is stronger for variants affecting the amount, rather than the structure of the enzyme, and the direction of the differences varies between some of the fitness components tested.

Associations between restriction site polymorphism and enzyme activity variation for esterase 6 in Drosophila melanogaster.

Thirty-five nucleotide polymorphisms were found in a 21.5-kbp region including the Est6 locus among 42 isoallelic lines extracted from a single natural population of Drosophila melanogaster. The heterozygosity per nucleotide pair was estimated to be 0.010 overall, but was lower in sequences hybridizing to transcripts than in those not hybridizing to transcripts. Eleven of 36 pairwise comparisons among the nine most common polymorphisms showed significant gametic disequilibrium. Four of these polymorphisms were also significantly associated with the major EST6-F/EST6-S allozyme polymorphism. Significant disequilibrium was generally restricted to polymorphisms less than 1-2 kbp apart. Previously reported measures of EST6 activity in virgin adult females proved not to be significantly associated with any of the six most common nucleotide polymorphisms located in the Est6 coding region or the 1.5 kbp immediately 5'. However, the 11 haplotypes for five of these polymorphisms that lie in the 1.5-kbp 5' region could explain about half of the previously reported variation among the lines for both EST6 activity and the amount of EST6 protein in virgin adult males. One particular polymorphism, for a RsaI site 530 bp 5' of the initiation codon, could explain 21% of the male activity variation among lines. This site is embedded in a large palindrome and we suggest that sequences including or close to this site may be involved in the regulation of EST6 synthesis in the ejaculatory duct of the adult male.

The number and distribution of esterase 6 alleles in populations of Drosophila melanogaster.

High resolution electrophoretic analyses of the polymorphic esterase 6 enzyme have been carried out on 133 isoallelic lines from three Australian populations of Drosophila melanogaster spanning 25 degrees of latitude. These and previous data for 157 lines from another Australian population at an intermediate latitude reveal a total of 14 polymorphic esterase 6 allozymes, falling into five major mobility classes. Two classes, EST6-F and EST6-S, contain eleven of the allozymes but one allozyme, EST6-8 within the EST6-S class, is several times more common than any other. Variation in the frequency of this single allozyme can explain most of the latitudinal clines previously reported for the major EST6-F and EST6-S classes. Thermostability analyses of 52 of the Australian lines and 13 American lines reveal at least seven more EST6 variants within five of the allozymes, bringing the total number of variants to at least 21. Of the six allozymes for which more than one line was subjected to thermostability analyses, only EST6-8 could not be partitioned into additional variants. This corroborates a previous finding that two different isolates of the Est6-8 allele have identical DNA sequences and suggests that this allele, although now the most common, has nevertheless arisen relatively recently.

Variation in the amount and activity of esterase 6 in a natural population of Drosophila melanogaster.

Forty-two homozygous lines each isoallelic for the Esterase 6 (Est-6) locus were extracted from a natural population of Drosophila melanogaster. Homogenates of 4-5 day old virgin adults of each sex from several replicate cultures of each line were assayed for EST6 activity. Depending on the line, males had from three to nine times more EST6 activity than females. Both sexes showed highly significant differences in EST6 activity among lines, with 3.2 and 2.7 fold differences between highest and lowest lines for males and females respectively. However, the variation in EST6 activity among lines was only weakly correlated across the two sexes. Female EST6 activity did not differ significantly across the six electrophoretic variants of EST6 found among the 42 lines. On the hand, a significant proportion of the variation among lines in male EST6 activity could be explained by differences among the six electromorphs. However, most of these differences were due to the relatively high activities of males from two relatively rare electromorphs and there were no significant differences in male activity among the four more common EST6 electromorphs. Radial immunodiffusion assays with polyclonal anti-EST6 antibody established that differences among lines in male EST6 activity were largely due to differences in the number of EST6 protein molecules, with negligible differences in their specific activities. It is concluded that the natural population segregates for genetic variance with large effects on the amount of EST6 protein; that there is little overlap in the variance expressed in the two sexes; and that most of the variance is different from the polymorphisms for electrophoretically detectable variants of EST6.

Molecular population genetics of structural variants of esterase 6 in Drosophila melanogaster.

Several lines of evidence indicate that natural selection operates between the major EST6-F and EST6-S allozymes of Drosophila melanogaster. In particular, consistent latitudinal clines and seasonal variation in their relative frequencies strongly suggest that they are not selectively equivalent in field populations. Several laboratory studies have found frequency-dependent fitness differences among the Est6-F and Est6-S genotypes. Moreover, the purified EST6-F and EST6-S allozymes differ in biochemical properties and the physiology of the enzyme, as a major component of the seminal fluid, suggests that these differences could affect reproductive aspects of fitness. However, molecular analyses reveal high levels of variation in the EST6 protein both within and between the EST6-F and EST6-S allozymes. Limited thermostability and more sensitive electrophoretic analyses reveal at least 17 variants of the two allozymes and sequence comparisons among 13 isolates of the Est6 gene reveal 16 nucleotide polymorphisms that would lead to amino acid differences. Two closely linked amino acid differences are strongly associated with the major difference between EST6-F and EST6-S; either or both of these are likely to cause the observed biochemical differences between EST6-F and EST6-S and may be the primary targets for the selection between these allozymes. The functional and adaptive significance of the other amino acid polymorphisms is unclear, although the data suggest that the EST6-8 haplotype within EST6-S has both arisen and proliferated relatively recently.