Sex-linked Recessive Lethal Mutations Research Articles

Contribution of transposable elements to transgenerational effects of chronic radioactive exposure of natural populations of Drosophila melanogaster living for a long time in the zone of the Chernobyl nuclear disaster

The accident at the Chernobyl Nuclear Power Plant (ChNPP) led to the negative impact of chronic radioactive contamination on populations of organisms associated with the transgenerational transmission of genome instability. When the destabilization of genome, different genetic damages occur, the accumulation of which leads to the formation of mutations, morphological anomalies, and mortality in the offspring. The mechanisms underlying the manifestation of transgenerational events in the offspring of irradiated parents are not well understood. In this study, for the first time, the features of the influence of transposable elements (TEs) on the long-term biological consequences of the ChNPP are considered. In this work, specimens of D. melanogaster obtained from natural populations in 2007 in the areas of the ChNPP with heterogeneous radioactive contamination were studied. The descendants from these populations were maintained in laboratory (inbred) conditions for 160 generations. A stable transgenerational transmission of dominant lethal mutations (DLMs) to the offspring of all studied populations was shown. The DLM frequencies strongly were correlated with the level of survival of offspring. The mean frequencies of recessive sex-linked lethal mutations varied at the level of spontaneous point mutations. The simultaneous presence of P, hobo and I elements indicates that the studied populations do not have a definite cytotype, their phenotypic status is unstable. The behavior of TEs in the genomes of offspring depends not only on parental exposure, but also on origin of population, distance to the ChNPP, and inbred conditions. The obtained results confirm the hypothesis that TEs are involved in transgenerational transmission and accumulation of mutations by the offspring of irradiated parents. The TEs pattern present in the Chernobyl genomes of D. melanogaster is a peculiar of epigenetic mechanism for the regulation of plasticity and adaptation of populations living for many generations under conditions of a technogenically caused radiation background.

Transgenerational effects in offspring of chronically irradiated populations of Drosophila melanogaster after the Chernobyl accident.

The zone of the Chernobyl nuclear disaster represents the largest area of chronic low-intensity radioactive impact on the natural ecosystems. The effects of chronic low-dose irradiation for natural populations of organisms and their offspring are unknown. The natural populations of Drosophila melanogaster sampled in 2007 in Chernobyl sites with different levels of radiation contamination were investigated. The offspring of specimens from these populations were studied under laboratory conditions to assess the effects of parental irradiation on the mutation process and survival of the offspring. Transgenerational effects of radioactive contamination were observed at the level of gross chromosomal rearrangements (dominant lethal mutations). The frequency of point/gene mutations (recessive sex-linked lethal mutations) of the offspring of the irradiated parents corresponded to the actual level of spontaneous mutations. The survival rate of offspring decreased over 160 generations and significantly correlated with the dominant lethal mutation levels. Our results provide a compelling evidence that other factors (distance from the Chernobyl Nuclear Power Plant, time after the initial exposure, selection site and origin of population) can affect the changes in the levels of the studied parameters along with the parental radiation exposure. They can also make a significant contribution to the health of the offspring of animals exposed to radioactive contamination. These data should be useful for future radioecological studies which will clarify the true mechanisms of transgenerational inheritance and generation of mutations to the offspring of chronically irradiated animals and their reactions to the interaction of various environmental factors.

Protective effects of tea polyphenols and \u03b2-carotene against \u03b3-radiation induced mutation and oxidative stress in Drosophila melanogaster

BackgroundThe commonly consumed antioxidants β-carotene and tea polyphenols were used to assess their protective effects against γ-radiation induced sex-linked recessive lethal (SLRL) mutation and oxidative stress in Drosophila melanogaster. Third instar larvae and adult males of wild-type Oregon-K (ORK) were fed on test agents for 24 and 72 h respectively before exposure to 10Gy γ-irradiation. The treated/control flies were used to assess the induction of SLRLs. We also evaluated antioxidant properties of these phytochemicals in the third instar larvae.ResultsDifferent stages of spermatogenesis in adult males showed a decrease in γ-radiation induced SLRL frequencies upon co-treatment with test agents. A similar trend was observed in larvae. Furthermore, a significant increase in antioxidant enzymatic activities with a decrease in malondialdehyde content was observed.Conclusionβ-carotene and tea polyphenols have exerted antigenotoxic and antioxidant effects in Drosophila. This study demonstrated the suitability of Drosophila as an alternative to mammalian testing for evaluating the antigenotoxic and antioxidant activity of natural products.

Ameliorative effects of gallic acid, quercetin and limonene on urethane-induced genotoxicity and oxidative stress in Drosophila melanogaster

The main objective of our present work was to ascertain the efficacy of Drosophila melanogaster model for assessing antigenotoxic and antioxidant effects of dietary phytochemicals gallic acid (GA), quercetin (QC) and limonene (Lim) against urethane (URE), a genotoxic environmental carcinogen. Oregon-K (ORK) adult male flies were fed GA, QC and Lim in combination with URE (20 mM) in 10% sucrose for 72 h. Third instar larvae were fed instant medium containing the above phytochemicals and URE for 24 h. Sex-linked recessive lethal (SLRL) test and assays for estimating glutathione content (GSH), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (MDA content) were performed. Adult feeding experiments demonstrated that co-treatment of flies with URE and the test phytochemicals has significantly decreased the frequencies of SLRL mutations in all the germ cell stages when compared to that with URE alone. Larval feeding experiments also showed a similar pattern. The above results correlate well with antioxidative potentials of the test agents where we observed the elevated enzymatic levels with a significant reduction in MDA level in Drosophila larvae. The results further suggest that the dietary phytochemicals have an antioxidant and antimutagenic property which can be assessed using D. melanogaster.

Study of the mutagenic effects of genetically modified soybean in Drosophila and mice

Using genetically modified (GM) soybean as a food substrate during the Drosophila breeding, as well as the addition of GM soybean in the standard mouse feed, revealed no mutagenic effect according to the criteria of recessive sex-linked lethal mutations (RSLLMs) and morphoses in Drosophila and anomalous sperm in mice.

The study of genetically modified soybeans mutagenic effects in drosophila and mouse

The use of genetically modified soybeans (GM) as a food substrate during Drosophila reproduction, as well as the addition of GM-soybeans in the standard mice meal showed no mutagenic effect on criteria recessive sex-linked lethal mutations (RSPLM) and dominant mutations (DM) in Drosophila and abnormal sperm in mice.

Sodium Copper Chlorophyllin (SCC) Induces Genetic Damage in Postmeiotic and Somatic Wing Cells of Drosophila melanogaster

There is no apparent evidence to indicate that sodium copper chlorophyllin (SCC) is mutagenic. The aim of the present study was thus to determine the mutagenic effect of SCC, in postmeiotic germ cells of the adult male Drosophila. This investigation was based on the ability to examine whether SCC induced sex-linked recessive lethal mutations (SLRL), as well as the somatic mutation and recombination test (SMART). Four different SCC concentrations were used: 0, 45, 69, 80, and 100 mM. For SLRL, two broods were generated to test sperm and primarily spermatids. Results showed a significant frequency of recessive lethal mutations compared with control sperm cells with SCC at 69, 80, and 100 mM. In contrast, the frequency of somatic mutations rose by 0.21 only with 100 mM of SCC. These findings provide evidence that SCC is a weak mutagen in both cell lines. The differential response may be attributed to repair mechanisms that are active in somatic cells but almost absent in germ cells.

Induction of complete and mosaic sex-linked recessive lethal mutations by cigarette smoke filtrate in Drosophila melanogaster

The genotoxic effects of cigarette smoke filtrate (SF) on the germ-line stages were examined in Drosophila melanogaster using the sex-linked recessive lethal test, which detects a broad spectrum of genetic alterations and proved to show correlations between mutagenicity and carcinogenicity of the tested chemicals. SF was extracted from fiberglass filter cartridges; each used in smoking 15 cigarettes. The proper SF concentrations (0.2 µL) in 0.45% NaCl saline were injected intraperitoneally in 2- to 3-day-old wild-type males, alongside with controls injected with 0.2 µL of saline. The genotoxicity effects of SF were examined in all spermatogenesis stages of treated males. Results showed that SF was toxic with an median lethal dose value of approximately 0.2% and induced significant sterility effects. The mutagenicity of SF (0.2%) was significantly stage specific and induced complete sex-linked recessive lethal mutations in the broods representing the spermatocytes and late and early spermatogonia, and induced mosaic mutations in the untreated progeny in the brood representing late spermatogonia. These results indicated, for the first time, that SF induces mosaic mutations, which could result from DNA instabilities and labile permutations that can be replicated and passed to future generations before being fixed into mutations in the untreated progeny of treated males, or originating from mutations that result in increasing hyperplasia of the gonad that subsequently produce the actual mutations in later cell cycles. Such delayed mutagenic effects of SF indicated that SF and, consequently, cigarette smoking have much greater genotoxicity than what was previously predicted.

A U-Shaped Dose–Response Relationship between X Radiation and Sex-Linked Recessive Lethal Mutation in Male Germ Cells ofDrosophila

We reported previously that low-dose X irradiation of DNA repair-proficient immature sperm of wild-type Drosophila melanogaster at a low dose rate (50 mGy/min) resulted in a mutation frequency that was lower than that in the sham-irradiated group. Therefore, a U-shaped dose-response relationship was suggested. Here we show that the dose-response curve is actually U-shaped by carrying out a large-scale sex-linked recessive lethal assay using Drosophila. No reduction of the mutation frequency was observed in a strain mutant for the nucleotide excision repair gene mei-9a (Drosophila homologue of human XPF). Introduction of a chromosome fragment containing mei-9+ into the mei-9a mutant strain restored the reduction of the mutation frequency in the low-dose-irradiated group. These results showed that DNA repair was responsible for the U-shaped dose-response relationship in Drosophila.

Genotoxicity testing of the methanol extract of the plant Cotinus coggygria and gallic acid on Drosophila melanogaster

The genotoxic activity of methanol extract obtained from the stem of Cotinus coggygria Scop. and synthetic gallic acid were investigated using the Drosophila sex-linked recessive lethal test (or SLRL test). In the tested methanol extract of C. coggygria (1 g), 62.50 mg of pyrocatechol equivalent of phenols was detected. Also, 46.76 mg of flavonoids and 15.75 mg of nonflavonoids were observed in 1 g of dry weight of extract. Methanol extract of C. coggygria in a concentration of 5% and 5% synthetic gallic acid were shown to be clearly genotoxic, inducing sex-linked recessive lethal mutations on the X-chromosome of Drosophila melanogaster males in all three broods.

Reduction in Mutation Frequency by Very Low-Dose Gamma Irradiation of Drosophila melanogaster Germ Cells

To determine whether the linear no-threshold (LNT) model for stochastic effects of ionizing radiation is applicable to very low-dose radiation at a low dose rate, we irradiated immature male germ cells of the fruit fly, Drosophila melanogaster, with several doses of (60)Co gamma rays at a dose rate of 22.4 mGy/h. Thereafter, we performed the sex-linked recessive lethal mutation assay by mating the irradiated males with nonirradiated females. The mutation frequency in the group irradiated with 500 microGy was found to be significantly lower than that in the control group (P < 0.01), whereas in the group subjected to 10 Gy irradiation, the mutation frequency was significantly higher than that in the control group (P < 0.03). A J-shaped dose-response relationship was evident. Molecular experiments using DNA microarray and quantitative reverse transcription PCR indicated that several genes known to be expressed in response to heat or chemical stress and grim, a positive regulator of apoptosis, were up-regulated immediately after irradiation with 500 microGy. The involvement of an apoptosis function in the non-linear dose-response relationship was suggested.

Reduction of Background Mutations by Low-Dose X Irradiation of Drosophila Spermatocytes at a Low Dose Rate

A sex-linked recessive lethal mutation assay was performed in Drosophila melanogaster using immature spermatocytes and spermatogonia irradiated with X rays at a high or low dose rate. The mutation frequency in the sperm irradiated with a low dose at a low dose rate was significantly lower than that in the sham-irradiated group, whereas irradiation with a high dose resulted in a significant increase in the mutation frequency. It was obvious that the dose-response relationship was not linear, but rather was U-shaped. When mutant germ cells defective in DNA excision repair were used instead of wild-type cells, low-dose irradiation at a low dose rate did not reduce the mutation frequency. These observations suggest that error-free DNA repair functions were activated by low dose of low-dose-rate radiation and that this repaired spontaneous DNA damage rather than the X-ray-induced damage, thus producing a practical threshold.

Influence of the yellow Locus on Sensitivity of Drosophila Germ Cells to Chemical Mutagens

The effect of the yellow (y) locus on germ cell sensitivity to the alkylating agent ethyl methanesulfonate (EMS) has been studied in Drosophila. Since DNA repair is one of the most important factors that control cell sensitivity to mutagens, the approaches used in our experiments aimed at evaluating the relationship between germ-cell mutability and activity of DNA repair. Germ-cell mutability and repair activity were assessed using several parameters, the most important of which was the frequency of the recessive sex-linked lethal mutations (RSLLM). In one series of experiments, the adult males of various genotypes (Berlin wild; y; y ct v; y mei-9a) were treated by mutagenic agents and then crossed to Basc females. Comparative analysis of germ-cell mutability as dependent on genotype and the stage of spermatogenesis showed that the yellow mutation significantly enhanced the premeiotic cell sensitivity to EMS, presumably, due to the effect on DNA repair. In the second series of experiments, the effect of the maternal DNA repair was studied and, accordingly, mutagen-treated Basc males were crossed to females of various genotypes including y and y mei-9a ones. The crosses involving y females yielded F1 progeny with high spontaneous lethality, whereas in F2, the frequency of spontaneous mutations was twice higher. The germ cell response to EMS depended also on female genotype: the effect of yellow resulted in increased embryonic and postembryonic lethality, whereas the RSLLM frequency decreased insignificantly. The latter result may be explained by elimination of some mutations due to 50% mortality of the progeny. The results obtained using the above two approaches suggest that the yellow locus has a pleiotropic effect on the DNA repair systems in both males and females of Drosophila.

An assessment of the genotoxicity of 2-hydroxy-1,4-naphthoquinone, the natural dye ingredient of Henna

2-Hydroxy-1,4-naphthoquinone (HNQ; Lawsone; CAS 83-72-7) is the principal natural dye ingredient contained in the leaves of Henna ( Lawsonia inermis). Published genotoxicity studies on HNQ suggested it was a weak bacterial mutagen for Salmonella typhimurium strain TA98 or was more clearly mutagenic for strain TA 2637, both in the presence of metabolic activation. HNQ was unable to induce sex-linked recessive lethal mutations in Drosophila melanogaster. However, a small increase in micronucleus frequency was reported in the bone marrow of mice at a single mid-range dose level, 24 h after intraperitoneal injection. In view of the wide use of Henna hair dyes it was deemed necessary to conduct a thorough investigation, under Good Laboratory Practice conditions, of the genotoxicity of HNQ. HNQ was non-mutagenic in bacterial (Ames test) or mammalian (V79 hprt) assays. It was borderline positive in a mouse lymphoma tk mutation assay and a chromosome aberration test (CHO cells), results that may reflect a similar clastogenic mechanism. Negative in vivo genotoxicity results were noted in the rat hepatocyte in vivo/in vitro UDS test, in peripheral lymphocytes (chromosome aberrations) of rats receiving repeated oral doses of HNQ at the MTD for 28 days, and in mouse and hamster bone marrow chromosome aberration tests. However small, but statistically significant increases in the incidence of bone marrow micronuclei were observed in two out of five tests at 72 h after dosing, but not at 24 or 48 h. There was evidence of haematotoxicity at 72 h, which may have been enhanced by the vehicle (DMSO) used in the positive tests. As erythropoiesis and administration of haematotoxic agents are known to induce small increases in the frequency of bone marrow micronuclei, typically at delayed sampling times, the data suggest that the positive 72 h response produced by HNQ is consistent with stimulation of haematopoiesis subsequent to haematological toxicity of HNQ, and not due to a DNA-reactive mechanism. Overall, the weight of evidence suggests that Henna and HNQ pose no genotoxic risk to the consumer.

Genetic and reproductive toxicity of butadiene and isoprene

Butadiene (BD) and its 2-methyl analogue, isoprene, have been extensively studied in animals and BD in population studies. Both chemicals are metabolised by liver cytochrome P450 dependent monogenases to monoepoxide and diepoxide intermediates. The diepoxide intermediates of both compounds were mutagenic in Salmonella typhimurium. However, unlike the monoepoxide of BD, the monoepoxides of isoprene were not mutagenic. It appears that they have no alkylating capacity. BD did not induce somatic cell mutation and recombination or sex-linked recessive lethal mutation in Drosophila melanogaster and isoprene produced no increase in chromosomal aberrations in CHO cells in vitro. Comparative concentrations of haemoglobin adducts in the blood of mice and rats after exposure to BD indicated that reaction with blood may decrease the levels of reactive intermediates available to tissues in rats, but not in mice contributing to greater potency of BD in the mouse. For isoprene, the adducts reach approximately the same concentrations in both species. DNA adducts have also been detected in testicular and lung cells of mice after BD exposure. The level of epoxybutene haemoglobin adducts was significantly elevated in BD-exposed workers, but lower than in rats and mice. In conjunction with the toxicology and carcinogenesis studies for BD and isoprene, additional mice were included for the evaluation of cytogenetic effects. Both chemicals produced increases in sister chromatid exchanges in bone marrow cells and in the frequency of micronuclei in normochromatic and polychromatic erythrocytes, but only BD produced an increase in the percent of bone marrow cells with chromosomal aberrations. At similar doses, the effects with BD were 2–3 times larger than with isoprene. There were also increased hprt mutation frequencies in rats and mice after BD exposure. Biomonitoring studies with hprt mutations in lymphocytes showed conflicting results, with both positive and negative findings. BD has been shown to be positive in one human cytogenetic biomonitoring study and not in three others, but chromosomal aberrations were increased in BD-exposed workers after challenge with gamma rays. Re-analysis of GSTTI null individuals showed positive results. There was an increase in spermatid micronuclei in mice by BD and its metabolites and in rats only by its metabolites. The cytotoxic response of germ cells in mice is greater than in rats. Dominant lethal mutations have been induced by BD and diepoxybutane, but not by epoxybutene. There was some evidence of congenital malformations in mice after BD exposure and there was a linear concentration-related induction of heritable translocations in mice. There was no induction of dominant lethal mutations or congenital malformations in rats. Using the heritable translocation data in mice, it has been determined that if a worker is continually exposed over 5 or 6 weeks to 20–25 ppm of BD, the risk of producing a child with a balanced reciprocal translocation is twice as high as the background risk. Since genetic damage cannot be measured directly in human germ cells, risk to such cells can also be estimated from germ cells and somatic cells of the mouse and human somatic cells using the parallelogram approach. Using doubling doses, the fourth corner of the parallelogram was calculated as a doubling dose for human germ cells of 4390 ppm/h. However, it is still questioned if man is more like rat than mouse in terms of sensitivity to exposure. Similar germ cell data do not exist for isoprene. In conventional developmental studies, where rats and mice were exposed to BD, maternal toxicity was shown in rats but there was no evidence of developmental toxicity or teratogenic effects and there was a small effect on sperm morphology. After exposure to isoprene, there was no adverse effect on rat dams or other reproductive indices. In mice, there was reduced foetal body weight and decreased maternal weight gain and isoprene also affected ovarian follicles. There was a reduction in testicular function parameters such as testicular weight and sperm motility.

Effect of Gamma Radiation and Sex-Linked Recessive Lethal Mutations on Sperm Transfer in Ephestia kuehniella (Lepidoptera: Pyralidae)

Sperm quality and sperm competition play important roles in determining the efficiency of genetic methods for suppression of lepidopteran populations. Herein we have examined competitiveness of mutant and irradiated males of Ephestia kuehniella by counting eupyrene (fertile) and apyrene (non-fertile) sperm transferred to the female during copulation. Mutant BL-2 males, trans-heterozygous for two sex-linked recessive lethal mutations sl-2 and sl-15, produced 50% fewer of both types of sperm as compared to with WT-C (wild-type) males. However, the ratio of apyrene to eupyrene sperm remained the same in both male types (9.5:1). Irradiation of mature male pupae, heterozygous for either sl-2 or sl-15 mutations, with doses between 150 and 350 Gy showed dose-dependent effects on the amount of sperm transferred and on the total length of mating times. As the treatment dose increased the volume of sperm transferred by the male decreased and the mating times got longer. In the F1 descendants of the treated males, males were found to transfer either a relatively normal or a very small volume of sperm, which could reflect changes in gamete segregation and in chromosomal aberrations that are inherited. The dose of 175 Gy is suggested as optimal for irradiation of Ephestia kuehniella.

Reproductive compensation and human genetic disease.

The effects of reproductive compensation on the population genetics of sex-linked recessive lethal mutations are investigated. Simple equations are presented which describe these effects, and so complement existing population genetic theory. More importantly, this type of mutation is responsible for several severe human genetic diseases such as Duchenne muscular dystrophy. It is argued that the applications of three modern reproductive technologies--effective family planning, in utero diagnosis with termination, and embryo sexing--will lead to reproductive compensation. The adoption of any of these technologies may rapidly elevate the frequencies of those mutations which are lethal in childhood. This increase is large, in the order of 33% upwards, and occurs rapidly over two to five generations. It also depends on the source of mutations, the effect being larger if most mutations are paternal. In utero diagnosis and/or embryo sexing increase the frequency of the mutation, but simultaneously decrease disease incidence by preventing the birth of affected offspring. In contrast, effective family planning may rapidly increase both mutation frequency and disease incidence.

Radiation-Induced Inherited Sterility Combined with a Genetic Sexing System in Ephestia kuehniella (Lepidoptera: Pyralidae)

The Mediterranean flour moth, Ephestia kuehniella Zeller, is the only lepidopteran pest in which a genetic sexing technique is available. We investigated the potential of using genetic sexing to enhance the effectiveness of F 1 sterility in population suppression. The irradiation dose-response relationship in the parental (P) generation of the moth was determined and related to the level of inherited sterility expressed in the F 1 generation. One generation before irradiation, males of the BL-2 strain, which are trans-heterozygous for 2 sex-linked recessive lethal mutations (sl-2 and sl-15), were mated to females of the wild-type strain WT-C. Progeny of these matings consisted exclusively of males (sl/ +). All female progeny died because they were hemizygous for either sl-2 or sl-15. The sl/ + males were treated with 8 doses of gamma rays ranging from 100 to 350 Gy and mated to WT-C females. The resulting data on the P and F 1 generations revealed that E kuehniella is extremely radioresistant. Irradiation at all doses had little influence on the mating success of sl/+ males and on fecundity of parental pairs. At lower doses, most embryonic mortality originated from the expression of lethal alleles sl-2 and sl-15. The egg hatch significantly decreased starting at 150 Gy. but effects of irradiation became evident only at doses higher than 200 Gy. In reciprocal F 1 matings. the level of induced inherited sterility correlated positively with the irradiation dose. F 1 males exhibited a higher level of induced sterility than F 1 females; it indicates that induced mutations have a sex-dependent impact. Optimal doses for the induction of F 1 sterility in E. kuehniella with the use of genetic sexing are in the range of 175-200 Gy.

Induction of delayed mutations by benzene and ethylene dibromide in drosophila

Two carcinogens, ethylene dibromide and benzene, were used to induce delayed (germinal mosaic) sex-linked recessive lethal mutations in spermatozoa and spermatids of adult Drosophila males. Significant numbers of delayed mutations (in F3) were scored in absence of conventional (in F2) mutations. A large proportion of nonlethal F2 cultures carried delayed mutations, so much so that, in some cultures, all F2 females were carriers of mutations. The mechanism through which single strand damage to treated X chromosomes can result in such delayed lethals is discussed. These observations indicate that the delayed mutation test should be used for testing the mutagenicity of environmental compounds, especially carcinogens, which tested negative in the conventional sex-linked recessive lethal mutation test. The data will support the relationship between mutagenesis and carcinogenesis and, also will further enhance the sensitivity of the Drosophila mutation assay.

Mutagenicity testing of nine herbicides and pesticides currently used in agriculture

Nine herbicides and pesticides were tested for their mutagenicity using the Drosophila sex-linked recessive lethal mutation assay. These are Ambush, Treflan, Blazer, Roundup, 2,4-D Amine, Crossbow, Galecron, Pramitol, and Pondmaster. All of these are in wide use at present. Unlike adult feeding and injection assays, the larvae were allowed to grow in medium with the test chemical, thereby providing long and chronic exposure to the sensitive and dividing diploid cells, i.e., mitotically active spermatogonia and sensitive spermatocytes. All chemicals induced significant numbers of mutations in at least one of the cell types tested. Some of these compounds were found to be negative in earlier studies. An explanation for the difference in results is provided. It is probable that different germ cell stages and treatment regimens are suitable for different types of chemicals. larval treatment may still be valuable and can complement adult treatment in environmental mutagen testing.