Variation in host preferences of malaria mosquitoes is mediated by skin bacterial volatiles.

The host preferences of the anthropophilic mosquito species in the Anopheles gambiae complex (Diptera: Culicidae) are mediated by skin bacterial volatiles. However, it is not known whether these mosquitoes respond differentially to skin bacterial volatiles from non-human host species. In this study, the responses of two malaria mosquito species in the An. gambiae complex, Anopheles gambiae s.s. (hereafter, An. gambiae) and Anopheles arabiensis, with different host preferences, to volatiles released from skin bacteria were tested. Skin bacteria collected from human, cow and chicken skin significantly increased trap catches; traps containing bacteria collected from human skin caught the highest proportions of An. gambiae and An. arabiensis. Traps with bacteria of human origin caught a significantly higher proportion of An. gambiae than of An. arabiensis, whereas bacterial volatiles from the chicken attracted significantly higher numbers of An. arabiensis than of An. gambiae. Additionally, An. gambiae showed a specialized response to volatiles from four specific bacteria, whereas An. arabiensis responded equally to all species of bacteria tested. Skin bacterial volatiles may therefore play important roles in guiding mosquitoes with different host preferences. The identification of these bacterial volatiles can contribute to the development of an odour blend that attracts mosquitoes with different host preferences.

Ancestral state reconstruction analysis of hymenopteran sex determination mechanisms.

We provide the first phylogenetic evidence supporting complementary sex determination (CSD) as the ancestral mechanism for haplodiploidy in the Hymenoptera. It is currently not possible, however, to distinguish the evolutionary polarity of single locus (sl) CSD and multiple-locus (ml) CSD given the available data. In this light, we discuss the seemingly maladaptive hypothesis of ml-CSD ancestry, suggesting that collapse from ml-CSD to sl-CSD should remain a viable evolutionary hypothesis based on (i) likely weakening of frequency-dependent selection on sex alleles under ml-CSD and (ii) recent findings with respect to the evolutionary novelty of the complementary sex determiner gene in honeybees. Our findings help provide a phylogenetically informed blueprint for future sampling of sex determination mechanisms in the Hymenoptera, as they yield hypotheses for many unsampled or ambiguous taxa and highlight taxa whose further sampling will influence reconstruction of the evolutionary polarity of sex determination mechanisms in major clades.

Diploid males sire triploid daughters and sons in the parasitoid wasp Cotesia vestalis.

In the Hymenoptera, males develop as haploids from unfertilized eggs and females develop as diploids from fertilized eggs. In species with complementary sex determination (CSD), however, diploid males develop from zygotes that are homozygous at a highly polymorphic sex locus or loci. We investigated mating behavior and reproduction of diploid males of the parasitoid wasp Cotesia vestalis (C. plutellae), for which we recently demonstrated CSD. We show that the behavior of diploid males of C. vestalis is similar to that of haploid males, when measured as the proportion of males that display wing fanning, and the proportion of males that mount a female. Approximately 29% of diploid males sired daughters, showing their ability to produce viable sperm that can fertilize eggs. Females mated to diploid males produced all-male offspring more frequently (71%) than females mated to haploid males (27%). Daughter-producing females that had mated to diploid males produced more male-biased sex ratios than females mated to haploid males. All daughters of diploid males were triploid and sterile. Three triploid sons were also found among the offspring of diploid males. It has been suggested that this scenario, that is, diploid males mating with females and constraining them to the production of haploid sons, has a large negative impact on population growth rate and secondary sex ratio. Selection for adaptations to reduce diploid male production in natural populations is therefore likely to be strong. We discuss different scenarios that may reduce the sex determination load in C. vestalis.

Complementary sex determination in the parasitoid wasp Cotesia vestalis (C. plutellae).

In the Hymenoptera, single locus complementary sex determination (sl-CSD) describes a system where males develop either from unfertilized haploid eggs or from fertilized diploid eggs that are homozygous at a single polymorphic sex locus. Diploid males are often inviable or sterile, and are produced more frequently under inbreeding. Within families where sl-CSD has been demonstrated, we predict that sl-CSD should be more likely in species with solitary development than in species where siblings develop gregariously (and likely inbreed). We examine this prediction in the parasitoid wasp genus Cotesia, which contains both solitary and gregarious species. Previous studies have shown that sl-CSD is absent in two gregarious species of Cotesia, but present in one gregarious species. Here, we demonstrate CSD in the solitary Cotesia vestalis, using microsatellite markers. Diploid sons are produced by inbred, but not outbred, females. However, frequencies of diploid males were lower than expected under sl-CSD, suggesting that CSD in C. vestalis involves more than one locus.

The dioxin TCDD protects against aflatoxin-induced mutation in female rats, but not in male rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant and a potent carcinogen in laboratory rodents. When combined with other environmental toxins, it has been shown to increase the (geno)toxicity of some compounds. In this study, the effect of TCDD on the mutagenicity of aflatoxin-B1 (AFB1) was examined in the rat liver using a lacI transgenic rodent mutation assay. AFB1 induces GC-->TA transversions. Since TCDD is known to have a differential effect in male and female rodents, both sexes were studied. The data showed that a 6-week pre-exposure to TCDD had no significant effect on the frequency of aflatoxin-induced mutation in the liver of male rats. However, the TCDD treatment completely prevented the aflatoxin-induced transversion mutations in female animals.

Protection by dietary compounds against mutation in a transgenic rodent.

One of the most relevant biomarkers of genotoxicity and, potentially, carcinogenesis is the occurrence of mutations. Data indicate that carcinogens are highly specific with regard to their target tissue in inducing both tumors and mutations. This specificity may reflect the dependence on tissue-specific metabolic activation, the organ-specific environment or both. Ideally, therefore, mutation should be determined in a real animal rather than in a cell culture system. The lacI transgenic rodent model provides such a system. We have used this model to investigate tissue, species and sex specificity of mutation induced by selected dietary carcinogens and to examine how some compounds may alter the induction of mutation. We have studied mutation using several chemicals, including the dietary heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the environmentally important aromatic hydrocarbon benzo[a]pyrene and the food contaminant aflatoxin B1. We have shown that the mutagenic potency of these chemicals can be modulated by other dietary compounds, including green tea and conjugated linoleic acid, and the dioxin 2,3,7,8-tetrachlorodibenzo[b,e][1,4]dioxin (TCDD). These results demonstrate that the lacI transgenic rodent is a useful model for the study of chemoprevention in vivo.

Msh2 DNA mismatch repair gene deficiency and the food-borne mutagen 2-amino-1-methy1-6-phenolimidazo [4,5-b] pyridine (PhIP) synergistically affect mutagenesis in mouse colon.

Msh2 deficiency and food-borne carcinogen PhIP have been implicated as genetic and environmental factors, respectively, in human colon carcinogenesis. It is not clear whether loss of one or both alleles of Msh2 gene increases the mutational sensitivity in colon when exposed to environmental carcinogens. In the current study, Msh2(+/-)/lacI and Msh2(-/-)/lacI double transgenic mice were treated with PhIP and mutations in the lacI gene were studied in the colon. The spontaneous mutation frequency (MF) is approximately eightfold higher in Msh2(-/-) mice than in Msh2(+/+) mice, while Msh2(+/-) mice display similar levels of spontaneous mutation as the Msh2 wild type mice. PhIP induced a significant increase in MF in all genotypes of mice. However, induced MF is much higher in Msh2(-/-) mice compared to Msh2(+/+) and Msh2(+/-) mice. Msh2(+/-) mice displayed an increased level of G:C>T:A transversions and -1 frameshifts upon PhIP treatment. In contrast, loss of both Msh2 alleles mainly results in increased frequency of G:C>A:T transitions when exposed to PhIP. These results suggest that a defect in mismatch repair may result in an enhanced sensitivity from exposure to a dietary carcinogen. It also provides insight into interaction between genetic and environmental factors in human carcinogenesis.

Protective effect of green tea against benzo[a]pyrene-induced mutations in the liver of Big Blue transgenic mice.

We assessed the ability of green tea to protect against benzo[a]pyrene (B[a]P)-induced mutations in the liver of lacI transgenic male C57BL/6 Big Blue mice. The mice were given a 2% Japanese green tea hot water extract as their sole source of drinking water for 10 weeks. After 7 weeks, they received a total dose of 150 mg/kg B[a]P. Treatment with B[a]P resulted in a two-fold higher lacI mutant frequency than the untreated controls (8.6+/-0.8 x 10(-5) versus 4.0+/-0.7 x 10(-5), P=0.01). B[a]P increased the frequency of its characteristic mutation (GC-->TA transversions) nearly five-fold, from 0.75 x 10(-5) to 3.7 x 10(-5). In mice treated with green tea, the induced B[a]P mutant frequency decreased by 63%, while GC-->TA transversions were reduced by 54%. Thus, we report evidence that green tea extract significantly suppressed B[a]P-induced mutation by lowering its specific transversion mutation in the lacI transgene in vivo. Further studies will address the correlation between the modulation of metabolic enzymes and the protection against induced mutation by green tea.

Mutations induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in cecum and proximal and distal colon of lacI transgenic rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a food-borne mutagen and carcinogen that induces tumors of the colon and the prostate gland in male rats and of the mammary gland in female rats. In this study we describe the frequency and specificity of PhIP-induced mutations in the cecum, proximal colon and distal colon of male and female lacI transgenic rats. This is the first report of mutational data from discrete regions of the colon. After 61 days of treatment with 200 p.p.m. PhIP mixed into the diet, PhIP-induced mutant frequencies were elevated 7-fold in the cecum and 14- to 21-fold in the colon of male and female rats compared with untreated controls. PhIP-induced mutant frequencies increased significantly (overall trend, P < 10(-4)) along the length of the colon of both males and females, with cecum < proximal colon < distal colon. A total of 754 PhIP mutants (363 male, 391 female) were sequenced to provide the mutational spectra for each of the three tissue sections from males and females. These mutational spectra consisted predominantly of G:C-->T:A and G:C-->C:G transversions and deletions of G:C base pairs. There were no significant differences between the mutational spectra with respect to sex or position in the colon. Therefore, we surmise that following induction of mutations by PhIP in male and female colons, non-mutagenic factors, possibly hormonal, preferentially influence the formation of tumors in the colon of male rats.

Mutagenicity of TCDD in Big Blue transgenic rats.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a significant environmental contaminant resulting from such industrial processes as pulp and paper production. TCDD is a suspected human carcinogen and its ability to induce cancer in laboratory rodents is well documented. Its mechanism of tumor initiation, however, is not well understood and in vitro mutagenicity studies have yielded inconsistent results. In this study, Big Blue lacI transgenic rats were used to assess the mutagenicity of TCDD in both male and female animals. After 6 weeks of exposure to 2 microg/kg TCDD neither an increase in mutation frequency nor any change in mutation spectrum was observed in either male or female animals.

Spontaneous mutation of the lacI transgene in rodents: absence of species, strain, and insertion-site influence.

Comparison of spontaneous mutation spectra derived from different transgenic constructs can provide valuable insights for interpreting the mechanisms of spontaneous mutation. In this study, spontaneous mutation frequencies and spectra of the lacI transgene are compared in the liver of C57BL/6, B6C3F1, and BC-1 mice and F344 rats. Before correction for clonal expansion, the mutant frequency varied from 2.6 +/- 0.45 to 5.0 +/- 2.4 x 10(-5). Correction for potential clonal expansion reduced the range in mutation frequency to between 2.3 +/- 0.45 and 3.5 +/- 2.0 x 10(-5). There is thus no statistical difference in spontaneous mutation frequency between the different strains and species. G:C --> A:T transitions and to a lesser extent, G:C --> T:A transversions dominate the mutational spectra in all of these animals. In three strains of mice, G:C --> A:T transitions account for 50.7-53.3% of mutation, 81.7-83.8% of which involve CpG sites, whereas G:C --> T:A transversions account for 17.8-32.9% of mutations with 43.2-50.0% found at CpG sites. In rats, G:C --> A:T transitions account for 38.0% of the spectra, 70.0% of which involve CpG sites, whereas G:C --> T:A transversions account for 23.0% of the spectra, 70.0% of which involve CpG sites. The distribution of other classes of mutations is also very similar. We conclude that, despite reports about species and strain differences in induced mutation, spontaneous mutations in the lacI transgene appear to be similar, regardless of genomic location, rodent strain, or species. In addition to insights into spontaneous mutation, this study also provides essential data for comparison with and interpretation of induced mutations.

Modulation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mutation in the cecum and colon of big blue rats by conjugated linoleic acid and 1,2-dithiole-3-thione.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent mutagen and suspected human carcinogen present in cooked protein-rich food. It preferentially induced colon tumors in male rats and mammary tumors in female rats. In the present study, the in vivo antimutagenic efficacy of two dietary compounds, conjugated linoleic acid (CLA) and 1,2-dithiole-3-thione (DTT), against PhIP was explored using 1acI transgenic Big Blue rats. Five- or six-week-old male Big Blue rats were fed a diet containing CLA (0.5%, wt/wt) or DTT (0.005%, wt/wt) starting one week before exposure to 200 ppm PhIP for 61 days. PhIP treatment induced a approximately 8- to 16-fold increase in the mutation frequency (MF) in the colon. The induced MF was significantly lower in the cecum than in the proximal and distal colon (approximately 52 x 10(-5) vs. 100 x 10(-5), p < 0.008). CLA and DTT significantly reduced the PhIP-induced MF in the distal colon (p < 0.05) by 14% and 24%, respectively. The frequency of -1 frameshift mutations was lower in the distal colon of CLA- or DTT-treated rats. This protective effect was not observed in the cecum or in the proximal colon. In contrast, the PhIP-induced MF in the cecum (specifically, the frequency of -1 frameshifts and GC-->TA transversions) was elevated by 43% after treatment with CLA. In conclusion, CLA and DTT modulate PhIP-induced mutagenesis in a tissue-specific manner, and different modulation pathways are employed by CLA and DTT.

Mutagenicity in lung of big Blue((R)) mice and induction of tandem-base substitutions in Salmonella by the air pollutant peroxyacetyl nitrate (PAN): predicted formation of intrastrand cross-links.

Peroxyacetyl nitrate (PAN) is a ubiquitous air pollutant formed from NO(2) reacting with acetoxy radicals generated from ambient aldehydes in the presence of sunlight and ozone. It contributes to eye irritation associated with photochemical smog and is present in most urban air. PAN was generated in a chamber containing open petri dishes of Salmonella TA100 (gas-phase exposure). After subtraction of the background mutation spectrum, the spectrum of PAN-induced mutants selected at 3.1-fold above the background mutant yield was 59% GC-->TA, 29% GC-->AT, 2% GC-->CG, and 10% multiple mutations - primarily GG-->TT tandem-base substitutions. Using computational molecular modeling methods, a mechanism was developed for producing this unusual tandem-base substitution. The mechanism depends on the protonation of PAN near the polyanionic DNA to release NO(2)(+) resulting in intrastrand dimer formation. Insertion of AA opposite the dimerized GG would account for the tandem GG-->TT transversions. Nose-only exposure of Big Blue((R)) mice to PAN at 78ppm (near the MTD) was mutagenic at the lacI gene in the lung (mutant frequency +/-S.E. of 6.16+/-0.58/10(5) for controls versus 8.24+/-0.30/10(5) for PAN, P=0.016). No tandem-base mutations were detected among the 40 lacI mutants sequenced. Dosimetry with 3H-PAN showed that 24h after exposure, 3.9% of the radiolabel was in the nasal tissue, and only 0.3% was in the lung. However, based on the molecular modeling considerations, the labeled portion of the molecule would not have been expected to have been bound covalently to DNA. Our results indicate that PAN is weakly mutagenic in the lungs of mice and in Salmonella and that PAN produces a unique signature mutation (a tandem GG-->TT transversion) in Salmonella that is likely due to a GG intrastrand cross-link. Thus, PAN may pose a mutagenic and possible carcinogenic risk to humans, especially at the high concentrations at which it is present in some urban environments.

Mutant frequencies and mutation spectra of dimethylnitrosamine (DMN) at the lacI and cII loci in the livers of Big Blue transgenic mice.

The lacI gene in Big Blue transgenic rodents has traditionally been used as a surrogate gene for in vivo mutations. Recently, a more efficient and less expensive assay involving direct selection in the smaller lambda cII gene has been developed. Little is known, however, about the comparative sensitivity of the two loci or their influence on the recovered mutation spectrum following mutagen treatment. We have compared the mutation frequency (MF) and mutational spectrum (MS) of lacI and cII from the same DNA samples isolated from the liver of control and dimethylnitrosamine (DMN)-treated mice. A three-fold (p<0.01) increase in the MF was observed at both loci in the DMN-treated group compared to the corresponding control groups. While the DMN-induced mutation spectrum at lacI was significantly different from its corresponding spontaneous mutation spectrum (p<0.001), the mutation spectrum at cII (p>0.28) was not. The mutation spectra at the two loci from the DMN-treated mice resembled each other but the 4, 2.5 and 12-fold increase in the mutation frequency of A:T>T:A transversions, single base deletions and deletions of more than four base pairs, respectively, at lacI, altered the spectra significantly (p<0.007). The number of mutations of these classes at cII was also increased, but the fractions were lower than at lacI. The spontaneous mutation spectra at the cII and lacI loci resembled each other except for the seven-fold increase in G:C

Tris(2,3-dibromopropyl)phosphate causes a gradient of mutations in the cortex and outer and inner medullas of the kidney of lacI transgenic rats.

Tris(2,3-dibromopropyl)phosphate (TDBP) is a kidney carcinogen in rats in which exposure results in tumors specifically in the outer medulla. We have previously shown that TDBP induces mutation in the rat kidney. Here we demonstrate that TDBP induces mutation in the kidney of the F344 Big Blue lacI transgenic rat in a gradient with the highest induction (6.4-fold) in the cortex and lowest induction (2.2-fold) in the inner medulla, when given at 2000 ppm in the feed for 45 days. Similar results were obtained at 100 ppm, although the gradient effect was less pronounced. Because exposure to TDBP results in increased cell proliferation in the outer medulla, our results suggest that tissue-specific targeting of TDBP-induced kidney tumors reflects the combination of cell proliferation and mutation induction. This is also the first known case when transgenic animals have been used to study mutation at the suborgan level.

Interpretation of mutational spectra from different genes: analyses of PhIP-induced mutational specificity in the lacI and cII transgenes from colon of Big Blue rats.

The cII assay provides an alternative choice to the lacI transgene for mutational studies involving Big Blue(R) transgenic mice and rats, or permits the evaluation of mutational responses in both genes. Here, we compare the mutational response of the cII gene from colon of Big Blue(R) F344 rats treated with a dietary mutagen and animal carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to those previously determined in the lacI transgene from colon of the same group of animals. A cursory inspection of PhIP-induced mutational spectra (MS) in cII and lacI suggests that the two transgenes respond differently to PhIP-induced mutation. However, a more thorough analysis of the MS in the two transgenes, including consideration of the number of mutational target sequences in each gene and nearest neighbor analyses of mutated nucleotides, indicates that PhIP-induced mutational specificity is similar in both genes. The evaluation of PhIP-induced mutational responses in these two transgenes serves as a model for intergenic mutational analyses.

Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice.

Mutation frequency and specificity were determined as a function of age in nuclear DNA from liver, bladder, and brain of Big Blue lacI transgenic mice aged 1.5-25 months. Mutations accumulated with age in liver and accumulated more rapidly in bladder. In the brain a small initial increase in mutation frequency was observed in young animals; however, no further increase was observed in adult mice. To investigate the origin of mutations, the mutational spectra for each tissue and age were determined. DNA sequence analysis of mutant lacI transgenes revealed no significant changes in mutational specificity in any tissue at any age. The spectra of mutations found in aging animals were identical to those in younger animals, suggesting that they originated from a common set of DNA lesions manifested during DNA replication. The data also indicated that there were no significant age-related mutational changes due to oxidative damage, or errors resulting from either changes in the fidelity of DNA polymerase or the efficiency of DNA repair. Hence, no evidence was found to support hypotheses that predict that oxidative damage or accumulation of errors in nuclear DNA contributes significantly to the aging process, at least in these three somatic tissues.

Prostate mutations in rats induced by the suspected human carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Male lacl transgenic rats were fed a diet containing 200 ppm of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine present in cooked meats. PhIP was found to be a powerful prostate mutagen. After 61 days of treatment, the lacl mutant frequency was 71 x 10(-5), >20-fold higher than the spontaneous mutant frequency of 3.2 x 10(-5). The predominant PhIP-induced mutations were G:C->T:A transversions and deletions of G:C bp. The results directly link PhIP-induced mutations with the earlier observation of PhIP-induced prostate cancer in rats and suggest that exposure to dietary PhIP could be a risk factor in the incidence of human prostate cancer.

Mutational analysis of the liver, colon and kidney of Big Blue rats treated with 2-amino-3-methylimidazo[4,5-f]quinoline.

The heterocyclic aromatic amine (HAA) 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ) induces intestinal tumours and hepatocellular carcinomas in rats, but no tumourigenic effects have been identified in the kidney. The tissue-specific mutagenicity of IQ was studied at the lacI locus in the liver, colon and kidney of Big Blue transgenic rats. At the highest dosing regime of IQ (20 mg/kg for 5 consecutive days) the mean mutant frequencies were significantly increased above background (P < 0.05) and were highest in the liver (12.9 +/- 6.2 x 10(-5)), followed by colon (7.4 +/- 1.4 x 10(-5)) and kidney (5.9 +/- 0.8 x 10(-5)). The mutational spectra from the livers of IQ-treated rats was statistically significantly different to that from the livers of control rats (P < 0.01). The lacI mutation spectra of the liver, colon and kidney from IQ-treated rats were similar. These were characterized by an increase in GC transversions in the liver and colon and an increase in the proportion of 1 bp G:C deletions in the liver and kidney. A single G deletion in the sequence 5'-CGGGA-3', characteristic of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine exposure, was detected in the liver and colon. A 2 bp GC deletion was identified at an identical position in the liver, colon and kidney. The colon was the only organ to contain two larger deletions of 13 and 33 bp. A preference was observed for base substitution mutations at guanine in the sequence 5'-CGC/T-3' and for 1 bp deletions at the guanine doublet in the sequence 5'-CGGA-3', especially in the liver and colon. Using the lacI gene as marker in the Big Blue rat model, the mutations identified in the IQ spectra have similarities to those identified for other HAAs studied in the same experimental system, but not to mutations identified in IQ-induced tumours.

Mutational spectrum of dimethylnitrosamine in the liver of 3- and 6-week-old lacI transgenic mice.

We determined the spectrum of mutations in the lacI gene in the liver of Big Blue(R) transgenic mice after exposure to five daily doses of 2 mg/kg dimethylnitrosamine (DMN) at 3 and 6 weeks of age. This dose has been reported to increase the mutant frequency 9-fold when the animals are 3 weeks old. The lacI mutations recovered when treated at 3 weeks consist of mainly G:C --> A:T transitions, predominantly at non-CpG sites, and thus are consistent with mutagenesis by DMN. No increase in mutant frequency was reported when the mice were treated at 6 weeks of age. As we have previously shown that changes in mutational spectrum can be detected even when no statistically significant increase in mutant frequency is seen, we also examined the spectrum after treatment at 6 weeks. No changes from the spontaneous spectrum were detected. The comparison of the outcome of DMN treatment at 3 and 6 weeks confirms a change in metabolic activation, adduct removal, or mutation fixation between 3 and 6 weeks of age.