Archives of Insect Biochemistry and Physiology 32:641-649 (1 996)

Genetic Evidence That Mutants of the Methoprene-tolerant Gene of Drosophila melanogaster Are Null Mutants Thomas G . Wilson Department of Biology, Colorado State University, Fort Collins

The Methoprene-tolerant (Met) mutation of Drosophila melanogaster results in resistance to juvenile hormone (JH) or JH analogs and appears to alter JH re- ception during late larval development. Several alleles of Met have been re- covered from methoprene selection screens after mutagenesis wi th ethyl methanesulfonate, X-rays, or transposable genetic elements. The phenotype of flies carrying any of these alleles is similar-resistance to the toxic and mor- phogenetic effects of methoprene-but otherwise is essentially wild-type. Un- derstanding the function of the Met gene requires that we know whether these alleles are hypomorphic, producing some functional gene product, or amorphic, producing no functional gene product. This determination was made by com- paring the methoprene-resistance phenotype produced by representative Met alleles with that produced by a chromosome carrying a deficiency that deletes the Met gene. The level of resistance to either the toxic or the morphogenetic effect of methoprene was similar among flies heterozygous for either the defi- ciency chromosome or for any of the alleles. The results provide genetic evi- dence that the Met alleles recovered to date are amorphic and suggest that the Met gene may not be mutable to a more severe Met allele that affects the viability, development, or reproduction of the flies. o 1996 Wiiey-Liss, Inc.

Key words: juvenile hormone, methoprene, hormone receptor gene, insecticide resistance

INTRODUCTION

Understanding the role and mechanism of action of juvenile hormone (JH") in insects has been a frustratingly slow task for insect physiologists. One relatively unexploited approach is a genetic one, that of identifying mutants which have blocked JH synthesis, enhanced JH degradation, or insensitive

Acknowledgments: I thank Greg Thomas for technical assistance and Sandoz, Inc., for supplying methoprene. This work was funded by NSF grant IBN-9419774.

Received September 15, 1995; accepted February 15, 1996.

Address reprint requests to Thomas G . Wilson, Department of Biology, Colorado State Univer- sity, Fort Collins, CO 80523.

*Abbreviations used: J H = juvenile hormone; Met = Methoprene-tolerant.

0 1996 Wiley-Liss, Inc.

642 Wilson

JH reception during development. This approach is most feasible for geneti- cally well-understood insects such as Drosophila melanogaster, Tribolium castaneum, or Musca dornestica. Of these insects Drosophila has proven the most useful. Each of two mutants, apterous and cricklet, possesses a phenotype in the adult stage that is suggestive of JH deficiency (Butterworth and King, 1965; Postlethwait and Weiser, 1973; Shirras and Bownes, 1989; but see Altaratz et al., 1991). The apterous mutation has been better studied and shown to be JH deficient, at least in the adult stage (Bownes, 1989; Altaratz et al., 1991; Dai and Gilbert, 1993).

A different type of JH mutant, Methoprene-tolerant (Met), was recovered in my laboratory in a genetic screen for resistance to the JH analog methoprene (Wilson and Fabian, 1987) and was genetically characterized (Wilson and Fabian, 1986). Met flies show high resistance to both JH I11 and to several JH analogs (Wilson and Fabian, 1986; Wilson, unpublished results); this resis- tance occurs to both the toxic effects of methoprene as well as to the morpho- genetic disruptions caused by exposure to sublethal doses of methoprene during early pupal development, which is subsequently manifested as ab- normal morphology of the adult abdomen (Ashburner, 1970).

The biochemistry of Met resistance has been explored. Possible mechanisms of resistance due to enhanced secretion or metabolism, tissue sequestration, and reduced cuticular penetration of JH were ruled out by direct experimen- tation (Shemshedini and Wilson, 1990). However, when binding of JH to a target tissue was examined, Met flies were found to possess a cytosolic high- affinity JH binding protein that has a 10-fold lower binding affinity for JH 111 than that from Met' flies (Shemshedini and Wilson, 1990). Indirect evidence suggests that this binding protein may be a JH receptor (Shemshedini et al., 1990). Thus, Met may offer an approach to better understand JH signaling in this insect. The Met gene is being cloned by transposon tagging with a P- element transposable genetic element (Turner and Wilson, 1995), but these results at present are incomplete.

Alleles of Met have been recovered following mutagenesis by the alkylat- ing agent ethyl methanesulfonate (Wilson and Fabian, 1987), by X-rays (Wil- son, unpublished data), and by P-element transposable genetic elements (Wilson, 1993). Although resistance to methoprene is high in flies bearing any of these alleles, the remaining phenotype is surprisingly similar to wild- type (Wilson and Fabian, 1986). This result is unexpected for an altered gene product involved in JH reception: one might expect a more severe pheno- type, such as female sterility, considering the role for JH in vitellogenic oo- cyte development in Drosophila (Postlethwait and Weiser, 1973; Wilson, 1982). Resolving this dilemma requires a better understanding of the nature of the Met alleles. It is possible that the mild nonconditional phenotype of Met flies results from a minor change in the Met gene and subsequent partial activity of the Met gene product (a hypomorphic allele) that functions in JH recep- tion at a level near that of wild-type. On the other hand, any or all of the Met alleles could be drastically altered, resulting in nonfunctional gene product (an amorphic allele), but sufficient redundancy in the JH reception pathway allows another gene product to compensate for the loss of Met gene product, resulting in a mild nonconditional phenotype. Such functional redundancy

Methoprene-tolerant Mutants of Drosophila 643

is not uncommon in Duosophifa; for example, it was recently shown for genes involved in cell death in these flies (Grether et al., 1995).

How might one distinguish between these two possibilities? One approach is an examination of the phenotype of flies that are heterozygous for a chro- mosome carrying a deficiency that includes the Met region. Such a chromo- some would obviously not produce Met' gene product. Several chromosomes have been identified that carry large deficiencies for the 10C-D cytogenetic region, where Met is located (Wilson and Fabian, 1986). An examination of flies carrying any of these deficiency chromosomes as homozygotes or hemizygotes is uninformative, because these flies usually die early in em- bryonic development due to loss of one or more vital genes also included in the deficient region. However, flies that are heterozygous for these deficien- cies survive and can be assessed for Met resistance. In previous work (Wil- son and Fabian, 1986) Met/Df flies were found to have high resistance to methoprene, but Met'/Df flies were not examined. If the Met alleles are hy- pomorphic, then one would expect Met'/Met flies to have more functional gene product than Met+/Df flies and thus be less resistant to methoprene; if amorphic, then the two genotypes should have similar resistance phenotypes. In the following work I compared the resistance of flies heterozygous for either Met or for a Met-deficiency chromosome and conclude that the extant Met alleles are amorphic, having a null phenotype.

MATERIALS AND METHODS Drosophila Strains and Culture Conditions

Oregon-RC was obtained from the Mid-America Drosophila Stock Center, Bowling Green State University, Ohio, and was used as the wild-type strain in this work as in several previous studies with Met (Wilson and Fabian, 1986). An X-chromosome (bearing Met') derived from this strain is designated + in this study. The Met allele was derived following ethyl methanesulfonate mu- tagenesis of Oregon-RC (Wilson and Fabian, 1986). The MetK1' allele is a P-ele- ment insertional allele (Wilson and Turner, 1992; Wilson, 1993) derived from a yellow vermilion stock. MetD2' is an allele recovered in a screen (Wilson and Fabian, 1987) following X-ray treatment of vermilion males with 3000 R. The deficiency chromosome Df(Z)rn259-4 is deficient for the region 1OC2-3 to 10El- 2 (Mortin and Lefevre, 1979); this deficiency chromosome was obtained from the Drosophila Stock Center at Indiana University, Bloomington, Indiana, and is fully described in Lindsley and Zimm (1992). Flies were raised in un- crowded conditions at 25 f 1°C on a standard agar-yeast-cornmeal diet with propionic acid added to retard fungal growth. All cultures were maintained under a LD 12h:12h photoperiod.

Methoprene Treatment Methoprene was obtained from Sandoz, Inc., as the biologically active iso-

mer ZR-2008. Thirty newly hatched larvae were transferred to food contained in 25 by 75 mm plastic vials (Sarstedt Inc., Newton, NC). Methoprene was dissolved in ethanol and applied in a volume of 25 p1 of solution to the sur- face of the food within one day following the transfer of larvae. Since the

644 Wilson

toxicity of methoprene is a function of larval density (Wilson and Chaykin, 1985), the physical transfer of larvae minimizes variability due to larval den- sity. Since the applied methoprene remains in the top portion of the food, this method of methoprene treatment does not allow precise definition of methoprene concentration in the food. However, we have found that the repro- ducibility of resistance measurement by this method is similar to that of other methods of application in which methoprene is either incorporated into Droso- phila Instant Food (Carolina Biological Supply Co., Burlington, NC) at a de- fined concentration or is topically applied in acetone solution to white puparia.

Although the sensitive period for JH analog effectiveness is late larval-early pupal (Postlethwait, 1973), the lethal period is pharate adult development to early adulthood. When cultures are treated with a lethal dose of a JH analog, many pharate adults do not eclose, and those that manage to eclose are weak and quickly become immobilized in the food. Therefore, toxicity was evalu- ated by determining the number of adults that eclosed and survived for at least one day and is expressed as pupal mortality, similar to previous work (Wilson and Fabian, 1986). Morphogenetic effects of methoprene were evalu- ated by examining the morphology of sternite bristle patterns (Bouchard and Wilson, 1987).

RESULTS AND DISCUSSION Toxicity of Methoprene

In previous work (Wilson and Fabian, 1986) we found that Met homozy- gotes or hemizygotes were as much as 100-fold more resistant to methoprene or JH I11 than were Met' flies. Riddiford and Ashburner (1991) topically ap- plied the JH analog pyriproxyfen to Met homozygotes and found resistance relative to either of two wild-type strains (Canton-S and Oregon-R), although it was less than 100-fold. Since different wild-type strains have variable re- sistance (Wilson and Thurston, 1988), the resistance difference observed be- tween these studies is likely due to different wild-type strains used as the standard to represent susceptibility as well as the use of different JH analogs. The susceptible flies used in the original study were FM7 balancer chromo- some homozygotes (Wilson and Fabian, 1986), which turned out to be more susceptible to methoprene than the Oregon-RC strain used in the present study. Oregon-RC is more representative of a "typical" susceptible strain (Wil- son and Thurston, 1988, and unpublished data).

Three different alleles of Met, produced by three different mutagenizing agents, were used to represent the Met mutation. Heterozygotes with any of these alleles or with the deficiency chromosome were produced by crossing with the Oregon-RC strain; this cross also served to produce a more uniform genetic background for comparing the various strains.

The deficiency chromosome DffI)rn259-4 was used as a chromosome car- rying a bona fide Met null. This chromosome is deficient for the region 10C2- 3 to 10E1-2 and as such clearly deletes the Met gene, whose location has been determined by both recombination (Wilson and Fabian, 1986) and in situ hy- bridization (Turner and Wilson, 1995) to the 10C5 region. By comparing the resistance of +/Of flies with those of +/Met , the relative amount of func-

Methoprene-foleranf Mutants of Drosophila 645

tional gene product contributed by the Met chromosome could be assessed. If Met is a hypomorph, producing some gene product, then the resistance phenotype of +/Met should be less severe (resulting in less resistance) than in +/Df flies. If Met is an amorph, producing little or no gene product, then the phenotype of +/Met should be similar to +/Df flies (Fig. 1).

The results show that Met homozygotes and Met/Df heterozygotes were highly resistant to methoprene, and Oregon-RC (+/+I flies were susceptible, as expected, when resistance was measured using four different diagnostic amounts of methoprene applied to the food (Table 1). +/Of flies were found to be partially resistant, surviving 0.005 pl/vial of methoprene well and 0.01 pl/vial more poorly, and these concentrations were most effective at discrimi- nating among the genotypes. +/Met flies bearing any of the three alleles of Met showed resistance similar to that of +/Of flies. This similarity suggests that each of the three Met alleles produces little or no functional Met' gene product.

If Met flies are null, then one would expect the Met homozygotes to show similar resistance to Met/Df flies. A comparison of these two phenotypes shows this result to be generally true, although Met homozygotes are slightly less resistant than MetlDfflies (Table 1). This difference may be due to varia- tion in the genetic backgrounds between the two strains. To test this possibil- ity, Met/MetDZ9 heterozygotes, which have a genetic background different from that of Met /Df heterozygotes, were constructed. When tested on methoprene, the resistance of these flies was found to be very similar to that of Met/Df flies (Table l), as would be expected if neither allele produces functional Met' gene product. These results further support the contention that the Met al- leles are null.

Exuected Resistance from Met Hvuomoruhic or Amorphic Alleles

Genotype Gene Contribution Functional Gene Product

1.0

0.5

+/Met - (morph) +-

0.5

Resistans

None

Moderate

Low

Moderate

Fig. 1. Expected resistance from a Met mutation that is either a hypomorph or an amorph. Boxed portion of the chromosome represents a Met' gene, which contributes 0.5 of the total gene product found in diploid wild-type flies. The hypomorphic Met allele is depicted as con- tributing a fraction of functional Met' gene between none (0) and full (0.5). The amorphic allele, depicted as a line, contributes no functional gene product.

646 Wilson

TABLE 1. Survival of Flies Carrying Various Met Alleles or a Met Deficiency Chromosome on Methoprene Food*

Methoprene concentration (nl/ food vial) Genotvue n 0.0 0.0025 0.005 0.01 0.05

+/+ 222 82 (3.8) 10 (5.3) 0 0 0 MetlMet 184 78 (4.6) 78 (7.9) 67 (2.6) 47 (5.4) 25 (7.5) Met/Df 193 81 (2.4) 79 (4.3) 75 (3.8) 57(2.1) 36 (6.6) +/Met 228 84 (7.6) 73 (6.2) 52 (2.4) 38 (6.1) 5 (2.7) +/ MetK17 189 87 (6.7) 63 (9.6) 38 (5.3) 26 (9.3) 0 +/MetDZ9 260 90 (2.6) 80 (4.8) 47 (11.4) 25 (5.9) 0 +/Of 189 80 (5.9) 77 (7.0) 58 (2.9) 22 (49) 0 Met/MetD’g 203 82 (7.5) 83 (3.3) 66 (4.5) 61 (2.9) --39 (7.51

“Each survival value represents the percentage survival of pupae raised as larvae o n a par- ticular concentration of methoprene. Each survival value represents the mean of three sepa- rate determinations w i t h the standard error value given in parentheses. Of represents the Df(Iim259-4 chromosome. The + X-chromosome was derived f rom the Oregon-RC strain. The number of pupae examined of the given genotype for al l concentrations of methoprene i s represented by n. For heterozygotes the maternally derived chromosome is listed first.

Resistance to Morphogenetic Defects Methoprene and other JH analogs result in sternal bristle defects, even at

sublethal doses (Ashburner, 1970). This morphogenetic effect consists of a disruption of the sternite bristle patterns and is characterized by misshapen, misoriented, and smaller/absent bristles (Bouchard and Wilson, 1987). At higher methoprene doses the bristles on all sternites are affected, but at lower doses only sternite 7, the most posterior sternite that possesses bristles, is markedly affected (Bouchard and Wilson, 1987, and unpublished data). Al- though the molecular basis for the bristle pattern disruption by methoprene is unclear, Met flies are resistant to this effect (Wilson and Fabian, 1986).

Do +/Of flies show resistance to this morphogenetic effect of methoprene

A B C D

Fig. 2. Sternite bristle patterns of various Met mutant and +/+ adults surviving treatment with methoprene as described in Materials and Methods. Only sternites 6 (large arrow) and 7 (small arrow) are shown. The bristles shown in the upper half of each panel consists entirely of those of sternite 6, which typically carries 16-20 bristles, while the bristles shown in the bottom half belong to sternite 7 (usually carrying 6-8 bristles), shown in the center portion and flanked by 1-4 tergite bristles (marked by an asterisk in A). (A) MeVMet treated with 0.05 pl/vial of methoprene, (B) +/Df treated with 0.01 pl/vial, (C) +/Met treated with 0.01 p h i a l , and (D) +/+ treated with 0.0025 yl/vial. Note the severe disruption of the bristle pattern on both sternites in D compared to those in A and note the less severely disrupted bristle patterns and especially the similar appearance of these patterns in B and C.

Methoprene-folerant Mutants of Drosophila 647

at a level similar to that of +/Met flies? The surviving females from the me- thoprene tests shown in Table 1 were examined for evidence of this resis- tance. As expected (Wilson and Fabian, 1986), Oregon-RC survivors on 0.0025 pl/vial showed grossly disrupted bristle patterns of sternites 6 and 7 (Fig. 2D), and Met homozygous and Met/Df heterozygous survivors showed with- out exception normal bristle patterns on these sternites when cultured on any of the methoprene concentrations (Fig. 2A). +/Of survivors on 0.01 PI/ vial showed an intermediate effect consisting of abnormal bristles on the most methoprene-sensitive sternite, sternite 7, but reasonably normal bristle pat- terns on the remaining anterior sternites (Fig. 2B); survivors on 0.0025 p1/ vial and sometimes on 0.005 pl/vial showed normal patterns on all sternites. When +/Met heterozygotes for each allele were examined, their sternite bristle patterns (Fig. 2C) were indistinguishable from those of +/Of survivors. Fi- nally, Met/MefDZ9 heterozygous survivors showed normal bristle patterns at all concentrations (not shown). Thus, each of the Met alleles shows a resistance phenotype suggestive of a null allele for this effect of methoprene as well.

The results of this study suggest that the Met alleles recovered to date are either null or produce very little functional gene product. These results are consistent with the results of earlier studies of the Met phenotype (Wilson and Fabian, 1986) and with JH binding results showing biphasic binding in +/Met heterozygotes (Shemshedini and Wilson, 1990). How such a drastic change at the gene level can produce such a minor change at the phenotypic level for fitness of Met flies has been discussed previously (Shemshedini and Wilson, 1990; Minkoff and Wilson, 1992). Of several possible explanations, we favor the ”functional redundancy” explanation: flies protect critical path- ways and mechanisms with alternative gene products that allow survival and limited reproduction. Although a genetic analysis points to Met alleles having a null phenotype, the final determination must await molecular clon- ing of the Mef’ gene and its use to detect Met’ transcripts in flies carrying each of the alleles.

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Methoprene-tolerant Mutants of Drosophila 649

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