Adaptive response to alkylating agents in the Drosophila sex-linked recessive lethal assay

The adaptive response to alkylating agents was studied in Drosophila assays under various treatment procedures. Pre-treatment of males as well as treatment of females with low doses of EMS (0.05-0.1 mM) did not affect sex-linked recessive lethal (SLRL) rates induced by high doses of this mutagen (10 mM, various feeding duration) in mature sperm cells. Pre-treatment of males with a low dose of MMS (0.1 mM) enhanced mutagenesis induced by the high dose of EMS (10 mM) at different stages of spermatogenesis, the observed effects exceeding the additive action of both mutagens. On the contrary, larval pre-treatment with the adaptive dose of EMS (0.05 mM) resulted in resistance of their germ cells to higher doses of EMS (1 mM). Specifically, offspring production increased while dominant lethality in F(1) as well SLRL frequency in F(2) was significantly reduced as compared with the effects of larval exposure to the challenge dose. Under the conditions tested, the adaptive response of germ cells to alkylating agents was demonstrated in larvae, but not in adult flies.     

Modification of the repair processes in chemical mutagenesis in Drosophila melanogaster]

The effect of 1,4-dihydroisonicotinic acid derivative (1,4-DHINA), glutapyrone, on the frequency of sex-linked recessive lethals (SLRL), which were induced by ethyl methanesulfonate (EMS) in spermatozoa, was studied in Drosophila males and females under different exposure conditions. This test was used to analyze repair processes in EMS mutagenesis. Glutapyrone manifested the protective effect after precultivation of males at the stage of larvae and females with glutapyrone. This preparation was shown to have no effect on the frequency of EMS-induced mutations under other conditions (precultivation and subsequent cultivation of mature males with glutapyrone). These data confirmed a key role of indirect mechanisms responsible for the effect of the examined antimutagen. Glutapyrone may be assumed to improve the synthesis or functioning of enzymes involved in the repair of O6-ethylguanine. After adaptive treatment of Drosophila with low doses of the mutagen, glutapyrone increased the fertility of parents but did not suppress chemical mutagenesis.     

Effect of the yellow locus on sensitivity of drosophila sex 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.     

Efficiency of different methods of EMS application for the induction of dominant lethals in germ cells of medfly, Ceratitis capitata (Wied.), males

EMS (ethyl methanesulfonate) fed to adult Mediterranean fruit flies in 10% sugar water was found to be the most effective treatment for the induction of dominant lethals in male germ cells. This application procedure showed a direct regression between the log concentration of EMS and the probit F1 egg lethality, provided a reasonably uniform uptake of EMS by the exposed males, and was non-toxic at the relevant concentrations. The same application procedure, but employing 1% sugar water, was also non-toxic to the treated males but resulted in large variations in the rate of uptake of the mutagen, thus producing no clear correlation between the concentration of EMS and dominant lethality. Injection of adult males with EMS caused high parental mortality and caused a severe reduction in mating propensity at concentrations below that causing dominant lethality.

Dominant lethality was observed in all treatment procedures as a reduction in egg hatchability, whereas adult emergence from surviving pupae was never affected. A small, but significant, reduction in pupal production from hatched eggs was observed in the treatment involving “egg/larval feeding” and in all adult treatments, but in no case could this be correlated to the concentration of EMS.

The high levels of radioactivity, observed in the testes of males treated with ¹⁴C-labelled EMS through feeding of adults (10% sugar), in spermathecase of females mated to these males and in resultant F1 eggs, suggest that a major portion of the label reaching the testes was associated with the sperm itself rather than with other parts of the testes or the seminal fluid.     

Frequency and spectrum of chlorophyll-deficient mutations in rice after treatment with radiation and alkylating agents

Three varieties of rice were treated with gamma rays and two alkylating agents EMS and DES, alone and in combinations, with a view to finding out the frequency and spectrum of chlorophyll mutations in relation to the genotype and the nature of the mutagen.Chlorophyll mutation frequency was enhanced with increasing dose but dropped at very high doses (doses that induced over 90% seedling lethality in M₁). The fall was attributed to either the increased mutated sector and diplontic selection after exposure to very high doses or relatively high resistance of some of the seeds.Among chlorophyll mutants in M₂ induced by radiations as well as alkylating agents, the albina type formed the majority class. EMS induced a significantly higher proportion of albinas than did gamma rays.     

Buthionine sulfoximine mediated enhancement of γ-radiation induced mutation frequency in Drosophila melanogaster

Experiments were carried out to investigate whether or not depletion of the glutathione (GSH) level in Drosophila melanogaster larvae with buthionine sulfoximine (BSO) treatment can result in the modulation of the frequency of sex-linked recessive lethal (SLRL) mutations induced by γ-radiation. Third instar larvae were fed on BSO for 24 h before exposure to 10 Gy γ-radiation. Immediately after this the larvae were divided into two batches, which were used to determining the GSH level and the induction of SLRL mutations respectively. The results obtained suggest that the depletion of the GSH level with BSO can lead to an enhancement in the frequency of SLRL mutations (significant at the 5% level). In a subsequent experiment in which adult Drosophila melanogaster male flies were fed on BSO for 72 h before irradiation, a significant increase was observed in the incidence of SLRL mutations.     

DNA-content in isolated nuclei of postembryonic stages of progeny from normal and irradiated males of Tetranychus urticae Koch (Acari,Tetranychidae)

The 1C DNA-content of isolated nuclei of postembryonic stages of Tetranychus urticae stained with the classic and a modified Schiffs reagent was cytophotometrically estimated as 0.1 pg, a low value in animals. For many tissues of this arrhenotokous species the ploidy ratio between males and females is 12, indicating the absence of sex-related differences in ploidy. — In addition, DNA measurements were performed to evaluate irradiation-experiments, starting with X-irradiation of mature sperm in males with doses known from previous work to induce chromosomal fragments that are subject to loss and missegregation in the embryonic mitotic stages of the female progeny despite the presumed holokinetic nature of the chromosomes. The DNA-content of the nuclei of the surviving postembryonic preadult stages did not indicate the occurrence of nuclei with in-between male/female values, ruling out loss and missegregation of fragments as important factors in postembryonic lethality. Abnormally low DNA-values in some adult females could be attributed to development of embryos before oviposition caused by radiation-induced effects.     

Radiation biology and inherited sterility in false codling moth (Lepidoptera: Tortricidae)

False codling moth, Cryptophlebia leucotreta (Meyrick), male and female mature pupae and newly emerged adults were treated with increasing doses of gamma radiation and either inbred or out-crossed with fertile counterparts. For newly emerged adults, there was no significant relationship between dose of radiation and insect fecundity when untreated females were mated to treated males (N female by T male). However, fecundity of treated females mated to either untreated (T female by N male) or treated males (T female by T male) declined as the dose of radiation increased. A similar trend was observed when mature pupae were treated. The dose at which 100% sterility was achieved in treated females mated to untreated males (T female by N male) for both adults and pupae was 200 Gy. In contrast, newly emerged adult males treated with 350 Gy still had a residual fertility of 5.2% when mated to untreated females, and newly emerged adult males that were treated as pupae had a residual fertility of 3.3%. Inherited effects resulting from irradiation of parental (P1) males with selected doses of radiation were recorded for the F1 generation. Decreased F1 fecundity and fertility, increased F1 mortality during development, and a significant shift in the F1 sex ratio in favor of males was observed when increasing doses of radiation were applied to the P1 males.     

Somatic cell mutagenicity in Drosophila melanogaster in comparison with genetic damage in early germ-cell stages

With the intention of assessing the general performance, sensitivity and the underlying mechanisms of somatic cell mutagenicity assays in Drosophila, a study was undertaken to compare the effectiveness of 5 procarcinogens and 4 direct-acting agents in the white/white-coral eye mosaic assay (SMART) with their activity in early (premeiotic) male and female germ-cell stages, after exposure of Drosophila larvae. The outcome indicated a lack of agreement in the results from recessive lethal assays (SLRL) in comparison with the somatic mutation and recombination test (SMART). The procarcinogens 2-naphthylamine (NA), 3-methylcholanthrene (MC), 9,10-dimethylanthracene (DA) and 7,12-dimethylbenz[a]anthracene (DMBA), and the direct-acting mutagens bleomycin (BM), methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), were quite efficient in producing somatic recombination and mutations in white/white-coral larvae, as opposed to only weak effects in early germ-cell stages. 2-Acetylaminofluorene (2AAF) showed marginal effects in both germ cells and somatic tissue after exposure of female larvae, but was inactive in testis. The discrepancy in mutational response between somatic cells and premeiotic germ cells is most impressive for MMS and BM. There is sufficient evidence for attributing a good sized proportion of the encountered variation to efficient error-free DNA repair of premutational damage and to segregational elimination during meiosis of deleterious mutations: (1) The efficient point mutagen ENU was the but one agent producing high levels of viable genetic alterations in early germ cells and in somatic cells. A similar behaviour was previously described for diethylnitrosamine, which ethylates DNA in the same fashion as ENU. (2) In early germ-cell stages of mei-9L1 male larvae, MMS induced multiple mutations (putative clusters) at a low dose differing by a factor 20-40 from those needed to produce an equivalent response in repair-competent strains. This is consistent with the concept of an active excision repair in premeiotic cells. (3) In the case of EMS, next to DNA repair, germinal selection seems to restrict the realization of EMS-induced genetic damage in premeiotic cells. (4) Bleomycin-induced chromosome aberrations caused high mortality rates in males (hemizygous for an X-chromosome) but not in females. MMS and BM, agents known to show preference for chromosome aberration induction, produced 3-6-fold higher rates of somatic mutational events (SME) in female genotypes as compared with the other sex.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

The effect of theyellow (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 sex-linked recessive lethals (RSLLM). In one series of experiments, the adult males of various genotypes (Berlin K; y; y ct v; and y mei-9 ^a) were treated by mutagenic agents and then crossed to Bascfemales. Comparative analysis of germ-cell mutability as dependent on genotype and the stage of spermatogenesis showed that theyellow 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 Bascmales were crossed to females of various genotypes including y and y mei-9 ^a 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 F₁. The results obtained using the above two approaches suggest that theyellow locus has a pleiotropic effect on the DNA repair systems in both males and females of Drosophila.     

Hybrid Lethal Systems in the Drosophila Melanogaster Species Complex. II. the Zygotic Hybrid Rescue (Zhr) Gene of D. Melanogaster

Hybrid females from Drosophila simulans females X Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as larvae. We have recovered a mutation in melanogaster that rescues the former hybrid females. It was located on the X chromosome at a position close to the centromere, and it was a zygotically acting gene, in contrast with mhr (maternal hybrid rescue) in simulans that rescues the same hybrids maternally. We named it Zhr (Zygotic hybrid rescue). The gene also rescues hybrid females from embryonic lethals in crosses of Drosophila mauritiana females X D. melanogaster males and of Drosophila sechellia females X D. melanogaster males. Independence of the hybrid embryonic lethality and the hybrid larval lethality suggested in a companion study was confirmed by employing two rescue genes, Zhr and Hmr (Hybrid male rescue), in doubly lethal hybrids. A model is proposed to explain the genetic mechanisms of hybrid lethalities as well as the evolutionary pathways.     

A novel system of fertility rescue in Drosophila hybrids reveals a link between hybrid lethality and female sterility

Hybrid daughters of crosses between Drosophila melanogaster females and males from the D. simulans species clade are fully viable at low temperature but have agametic ovaries and are thus sterile. We report here that mutations in the D. melanogaster gene Hybrid male rescue (Hmr), along with unidentified polymorphic factors, rescue this agametic phenotype in both D. melanogaster/D. simulans and D. melanogaster/D. mauritiana F(1) female hybrids. These hybrids produced small numbers of progeny in backcrosses, their low fecundity being caused by incomplete rescue of oogenesis as well as by zygotic lethality. F(1) hybrid males from these crosses remained fully sterile. Hmr(+) is the first Drosophila gene shown to cause hybrid female sterility. These results also suggest that, while there is some common genetic basis to hybrid lethality and female sterility in D. melanogaster, hybrid females are more sensitive to fertility defects than to lethality.     

Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes

Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.     

MMS sensitive strains in Drosophila melanogaster

Larval populations of Drosophila melanogaster were treated with the monofunctional alkylating agent methyl methyl methanesulfonate (MMS). Four different stocks were tested. The method methyl used permits the direct registration of MMS-induced lethality of every genotype present in the treated population. Up to 5-fold differences in sensitivity between different genotypes were obeserved. Larvae of the wild-type strain. Antibes, which was reported to have increased UV sensitivity in embryonic stages, were MMS sensitive. In another stock, marked with spa^pol, female larvae were about twice as sensitive as male larvae.     

Radiation biology and inherited sterility of light brown apple moth (Lepidoptera: Tortricidae): developing a sterile insect release program

The radiation biology of two geographically isolated populations of the light brown apple moth [Epiphyas postvittana (Walker)] was studied in Australia and New Zealand as an initiation of a SIT/F1 sterility program. Pharate and < or = 2 d pre-emergence pupae were exposed to increasing radiation doses up to a maximum dose of 300 Gy. Fertility and other life history parameters were measured in emerging adults (parental) and their progeny (F1-F3 adults). Parental fecundity was significantly affected by increasing irradiation dose in pharate pupae only. For both populations, parental egg fertility declined with increasing radiation. This was most pronounced for the irradiated parental females whose fertility declined at a higher rate than of irradiated males. At 250 Gy, females < or = 2 d preemergence pupae produced few larvae and no adults at F1. No larvae hatched from 250 Gy-irradiated female pharate pupae. At 300 Gy, males still had residual fertility of 2-5.5%, with pharate pupae being the more radio-sensitive. Radiation-induced deleterious inherited effects in offspring from irradiated males were expressed as increased developmental time in F1 larvae, a reduction in percent F1 female survival, decreased adult emergence and increased cumulative mortality over subsequent generations. Males irradiated at > or = 150 Gy produced few but highly sterile offspring at F1 and mortality was > 99% by F2 egg.     

Genotoxic damage induced by isopropanol in germinal and somatic cells of Drosophila melanogaster

Isopropanol (isopropyl alcohol, 2-propanol, IPA) is a volatile solvent widely used in domestic or industrial environments and reported as innocuous in various test systems. The aim of this work was to search for in vivo genotoxic effects of IPA in Drosophila melanogaster, studying its ability to induce nondisjunction (ND) in females, sex linked recessive lethals (SLRL) in males, and somatic mutation and/or recombination (SMART) in larvae. Treatments were acute (60min) and were administered via inhalation. IPA had low toxicity in adult flies (75% IPA mortality index, MI=12.7% (females) and 2.6% (males)) and larvae (MI=14.3%, 75% IPA). Female fertility was severely affected during the first 24h (brood I, BI) after treatment, but, afterwards, control values were recovered. IPA induced a 50-fold increase of ND (%) in 24h old females, and a six-fold rise in 4-5 d old BI offspring. Nondisjunction frequencies (%) in the offspring of broods II to V (24h in each case) were similar to control values. IPA doses of 25% and 50% (v/v), tested in 24h old females, showed a significant dose-dependent increase of ND(%)in BI only, with control values in subsequent broods. Flies gave normal offspring when kept in regular media for 24h before mating. The eye spot test (SMART) showed a significant increase at 50% IPA (p<0.05, m=2), but the response was not dose-dependent. IPA failed to induce SLRL in any of the spermatogenesis stages tested. These findings suggest that the main effect of IPA is to induce chromosomal malsegregation; IPA must be present at the resumption of M-phase I after fertilization, to exert these effects. The alcohol does not affect DNA directly, but perturbations of the nuclear membrane may be responsible for induction of ND.     

Effects of substerilizing doses of gamma radiation on adult longevity and level of inherited sterility in Teia anartoides (Lepidoptera: Lymantriidae)

The effects of substerilizing doses of gamma radiation on the longevity and level of inherited sterility in the Australian moth Teia anartoides Walker were determined. Six day-old male pupae were treated with 0, 100, and 160 Gy of gamma radiation by using a 1.25 MeV Cobalt60 irradiation source. Laboratory studies of male longevity showed that radiation had little impact in adult moths of the P1, F1, and F2 generations. Inherited deleterious effects resulting from irradiation were observed in the progeny of F1 and F2 generations. Outcrosses between substerile parental males or their highly sterile male progeny to wild-type females did not affect female fecundity. However, adverse effects were observed for these crosses in the rates of successful egg hatch and postembryonic development. Fertility was always greater in out-crosses involving a P1 male than in any of the F1 out-crosses. F1 males were always more sterile than F1 females, and the level of sterility for the F1 and F2 generations was higher than that of the controls. The incidence of larval and pupal mortality was higher in the F2 than the F1 generation. A dose of 100 Gy had the highest success in inducing deleterious effects that were inherited through to the F2 generation. Our results indicated that the use of partially sterilizing doses of radiation has good potential as a selective strategy for management or eradication of T. anartoides.     

Study of genotoxicity and antigenotoxicity of the <Emphasis Type="Italic">Cotinus coggygria</Emphasis> Scop. methanol extract by <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> sex-linked recessive lethal test

The genotoxic and antigenotoxic effects of Cotinus coggygria Scop. methanol extract was investigated using the Drosophila sex-linked recessive lethal (or SLRL) test. The results presented here show that the methanol extract of Cotinus coggygria in a concentration of 5% and artificial chemical agent ethyl methanesulfonate EMS (0.75 ppm) induce recessive lethal mutations on X-chromosome on Drosophila melanogaster in all broods (I, II and III). Post-treatment with lower concentration of the methanol extract of Cotinus coggygria (2%) was effective in reducing genotoxicity of mutagen.     

Irradiation of Anastrepha obliqua (Diptera: Tephritidae) revisited: optimizing sterility induction

The effects of irradiation doses increasing from 0 to 100 Gy (1 Gy is energy absorbed in J kg(-1) of irradiated material) on fertility, flight ability, survival, and sterile male mating performance were evaluated for mass-reared Anastrepha obliqua (Macquart). High sterility values (> 98.2%) for irradiated males were obtained for doses as low as 25 Gy. Egg hatch was inhibited for irradiated males crossed with irradiated females at a low dose of 20 Gy. However, we estimated that to achieve 99.9% sterility (standard goal of many sterile insect technique programs), irradiation doses had to be increased to a dose between 50 and 75 Gy. At doses of 25 Gy and greater, we observed a decreasing trend in adult flight ability and an increasing trend in adult mortality. Such differences were greater for pupae irradiated at a young age compared those irradiated 24 h before emergence. Our single most relevant finding was that sterility induction (i.e., oviposition of nonfertilized eggs) was two times greater for males irradiated at low doses (40 Gy) than for males irradiated at high doses (80 Gy) when used at a 3:1:1 sterilized male to fertile male to fertile female ratio. Males irradiated at high doses may have been outcompeted by unirradiated males when courting unirradiated females. Implications of our findings for sterile insect technique programs are discussed.     

Induction of male recombination in Drosophila melanogaster by chemical treatment

Acridine orange (AO), ethidium bromide (EB), ethyl methanesulfonate (EMS) and 8- ethoxycaffeine ( EOC ) were fed to larvae of Drosophila melanogaster in order to test their capacity for the induction of meiotic recombination in males. Our results show that AO and EB increase significantly the male recombination frequencies. No relationship between chromosome breakage ability and male recombination induction was found since EMS and EOC , two effective chromosome-breaking agents, were unable to increase the male recombination.