Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.).

We aimed to assess the effect of gamma radiation on the expression of heat shock proteins Hsc70 and Hsp83 in Aedes aegypti. Adult males were irradiated with 50Gy of gamma radiation, and changes in the expression of proteins in SDS-PAGE gel bands corresponding to molecular weights ~60-75kDa and ~80-95kDa were analyzed at two different time points 6 and 12-hour post-irradiation, using a temporal mass spectrometry based semi-quantitative analysis. A 2-3-fold increase was observed in both proteins Hsc70 and Hsp83, at both time points. In addition, the experiment also revealed the overexpression of several other molecules such as Arginine Kinase - known to be upregulated in certain insects during stress, Esterase B1- implicated in insecticide resistance, and also down-regulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 - both known to be involved in ubiquitin-mediated protein degradation. The results taken together with existing data on Hsp83 and Hsc70, indicate that these proteins may enhance the survival of Ae. aegypti following gamma radiation and could serve as molecular markers for the detection of radiation-induced stress.

Evaluation of gamma radiation-induced DNA damage in Aedes aegypti using the comet assay.

The study was undertaken to evaluate gamma radiation-induced DNA damage in Aedes aegypti. The comet assay was employed to demonstrate the extent of DNA damage produced in adult male A. aegypti exposed to seven different doses of gamma radiation, ranging from 1 Gy to 50 Gy. DNA damage was measured as the percentage of comet tail DNA. A significant linear increase in DNA damage was observed in all samples; the extent of damage being proportional to the dose of gamma radiation the organism received, except in those treated with 1 Gy. The highest amount of DNA damage was noticed at 1 h postirradiation, which decreased gradually with time, that is, at 3, 6 and 12 h postirradiation. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the postirradiation time increased. The comet assay serves as a sensitive and rapid technique to detect gamma radiation-induced DNA damage in A. aegypti. This could be used as a potential biomarker for environmental risk assessment.

Effect of gamma radiation on life history traits of Aedes aegypti (L.).

Aedes aegypti is an important vector for Dengue and Dengue hemorrhagic fever. Considering its medical importance and its relevance as a model system, this study was undertaken to evaluate the impact of different doses of gamma radiation for three generations of A. aegypti. Two to three days old virgin males of A. aegypti were irradiated with 15 doses of gamma radiation, ranging from 1 to 50 Gy and were immediately mass mated with the same aged virgin females. Observations were made for changes on their life history traits, particularly fecundity, hatchability, adult emergence, sex ratio and longevity, for three generations. Adult males exposed 30, 35, 40, 45 and 50 Gy doses showed a significant decrease in fecundity in F0 generations. While hatchability was observed to have decreased with increasing radiation doses from 3 Gy onwards in the F1 generation, samples irradiated with 30, 35, 40, 45 and 50 Gy maintained significant decline in hatchability in their succeeding generations, F2 and F3 also. Similarly, a decline was observed in adult emergence from 3 Gy onwards in all three generations. A male favoring sex ratio distortion was observed at the doses of 35, 40, 45 and 50 Gy in all three generations. Following exposure to 4 Gy, parental males and the resultant progeny showed increased longevity by 10.56 and 8.66 days respectively. Similarly, the F1 generations of samples irradiated with 30, 35 and 40 Gy exhibited an increase in longevity by 7.16, 7.44 and 6.64 days respectively. Dose response curve for fertility among the three generations was drawn and presented. The effect of radiological exposure on the life history traits of A. aegypti varies with dose for the three generations studied. These results have potential implications in mutational studies and risk assessment and also contribute to a better understanding towards employment of the sterile insect technique in A. aegypti, plausibly paving the way to an effective mosquito genetic control program.

Evaluation of genotoxicity of the acute gamma radiation on earthworm Eisenia fetida using single cell gel electrophoresis technique (Comet assay).

Earthworms (Eisenia fetida) most suitable biological indicators of radioactive pollution. Radiation-induced lesions in DNA can be considered to be molecular markers for early effects of ionizing radiation. Gamma radiation produces a wide spectrum of DNA. Some of these lesions, i.e., DNA strand breaks and alkali labile sites can be detected by the single-cell gel electrophoresis (SCGE) or comet assay by measuring the migration of DNA from immobilized nuclear DNA. E. fetida were exposed to different doses of gamma radiation, i.e., 1, 5, 10, 20, 30, 40 and 50Gy, and comet assay was performed for all the doses along with control at 1, 3 and 5h post irradiation to evaluate the genotoxicity of gamma radiation in this organism. The DNA damage was measured as percentage of comet tail DNA. A significant increase in DNA damage was observed in samples exposed to 5Gy and above, and the increase in DNA damage was dose dependent i.e., DNA damage was increased with increased doses of radiation. The highest DNA damage was noticed at 1h post irradiation and gradually decreased with time, i.e., at 3 and 5h post irradiation. The present study reveals that gamma radiation induces DNA damage in E. fetida and the comet assay is a sensitive and rapid method for its detection to detect genotoxicity of gamma radiation.

Inheritance Pattern of Temephos Resistance, an Organophosphate Insecticide, in Aedes aegypti (L.).

The present paper reports the mode of inheritance of resistance in laboratory induced temephos resistant and susceptible strains of Ae. aegypti. Homozygous resistant and susceptible strains of Ae. aegypti were generated by selective inbreeding at a diagnostic dose of 0.02 mg/L of temephos. Genetic crosses were carried out between these strains to determine the inheritance pattern of temephos resistance. The log-dosage probit mortality relationships and degree of dominance (D) were calculated. The dosage-mortality (d-m) line of the F 1 generation was nearer to the resistant parent than the susceptible one. The "D" value was calculated as 0.15 indicating that the temephos resistant gene is incompletely dominant. The d-m lines of the F 2 generation and progeny from the backcross exhibited clear plateaus of mortality across a range of doses indicating that temephos resistance is controlled by a single gene. Comparison of the mortality data with the theoretical expectations using the χ (2) test revealed no significant difference, confirming a monogenic pattern of inheritance. In conclusion, the study provides evidence that the temephos resistance in Ae. aegypti follows an incompletely dominant and monogenic mode of inheritance.

Effects of acute gamma radiation on the reproductive ability of the earthworm Eisenia fetida.

Earthworms are the most suitable biological indicators of radioactive pollution because they are the parts of nutritional webs, and are present in relatively high numbers. Four months old Eisenia fetida were exposed to different doses of gamma radiation, namely 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 & 60 Gy to study the effects of radiation on different reproductive parameters. The number of cocoons laid and the hatchlings emerged were recorded for all the selected doses. There was no reduction in cocoon production, however; decreasing size and weight of the cocoons was observed from the samples exposed to 20 Gy and above doses. Significant reductions in the hatchlings were recorded in earthworms exposed to 10 Gy and above doses. The dose response curves for a percentage reduction in hatchlings were constructed. Exposure to radiation dose of 1 and 2 Gy did not show any reduction, however, there was ≈10%, ≈50% and ≈90% decrease in the hatchlings in samples exposed to 3, 15 and 45, 50, 55 and 60 Gy doses respectively. Delayed hatchability was also reported at al exposure level. Histology of irradiated earthworms revealed that the structural damage in the seminal vesicles was prominent at the exposed dose of 3 Gy onwards with complete degeneration on exposure to 60 Gy of gamma radiation.

Differential expression of glutathione s-transferase enzyme in different life stages of various insecticide-resistant strains of Anopheles stephensi: a malaria vector.

BACKGROUND & OBJECTIVES: Interest in insect glutathione s-transferases (GSTs) has primarily focused on their role in insecticide resistance. These play an important role in biotransformation and detoxification of many different xenobiotic and endogenous substances including insecticides. The GST activity among 10 laboratory selected insecticide resistant and susceptible/control strains of Anopheles stephensi was compared using the substrates 1-chloro-2,4-dinitrobenzene (CDNB). The difference in the GST activities of different life stages of diverse insecticide resistant strains was compared and presented. METHODS: About 100 larvae, pupae, adult males, adult females and eggs (100 µg in total weight) were collected and used for the experiment. The extracts were prepared from each of the insecticide-resistant strains and control. Protein contents of the enzyme homogenate and GST activities were determined. RESULTS: Deltamethrin and cyfluthrin-resistant strains of An. stephensi showed significantly higher GST activity. Larvae and pupae of DDT-resistant strain showed peak GST activity followed by the propoxur-resistant strain. On contrary, the GST activity was found in reduced quantity in alphamethrin, bifenthrin, carbofuran and chloropyrifos resistant strains. Adults of either sexes showed higher GST activity in mosquito strain resistant to organophosphate group of insecticides namely, temephos and chloropyrifos. INTERPRETATION & CONCLUSION: The GST activity was closely associated with almost all of the insecticides used in the study, strengthening the fact that one of the mechanisms associated with resistance includes an increase of GST activity. This comparative data on GST activity in An. stephensi can be useful database to identify possible underlying mechanisms governing insecticide-resistance by GSTs.

Chromosomal inversions among insecticide-resistant strains of Anopheles stephensi Liston, a malaria mosquito.

Polytene chromosomes were prepared from the ovarian nurse cells of semi-gravid females of ten insecticide-resistant strains of Anopheles stephensi. Altogether, 16 heterozygous paracentric inversions, namely b/+ (11D-16C) in alphamethrin; i/+ (14B-18A) and h/+ (27B-28A) in DDT; j/+ (14A-16B) in chlorpyrifos; k/+ (11D-16B) in cyfluthrin; l/+ (11A-16C) in deltamethrin; m/+ (14B-15C) and e/+ (32A-33B) in bifenthrin; n/+ (12D-14B), f/+ (33A-36A) and g/+ (33C-34A) in propoxur; o/+ (11A-12D), h/+ (37A-37C) and i/+ (31C-32C) in temephos; d/+ (33D-35C) in carbofuran and a/+ (41C-43B) in neem strains, were reported. No inversions were observed in X chromosome so far. The frequency of inversions in different insecticides was found to be highest in the 2R arm, followed by the 3R arm. Such inversions were not reported in the corresponding susceptible strains or in the parental stocks.

Autosomal inheritance of alphamethrin, a synthetic pyrethroid, resistance in Anopheles stephensi-Liston, a malaria mosquito.

Anopheles stephensi­Liston (Culicidae: Diptera) is an important urban malarial vector in the Indian sub-continent, accounting for about 15% of the total annual malaria incidence. Chemical control represents a key strategy in the management of this insect vector. However, owing to erratic and continuous application of insecticides, resistance has become a common phenomenon among them and their control has become an uphill task. The genetics of alphamethrin, a synthetic pyrethroid resistance was studied to determine its mode of inheritance. The late third instar larvae were selectively inbred for 27 and ten generations to synthesize homozygous resistant (R) and susceptible (S) stocks, respectively, to the diagnostic dose of 0.12 mg l−1. The log-dosage probit mortality relationships and degree of dominance (D) were calculated. Resistance was observed in both sexes, the dosage-mortality (d-m) line of F 1 was towards the resistant parent and the 'D' value was found to be 0.8 indicating alphamethrin resistant (amr) gene to be autosomal and incompletely dominant. The d-m lines of F 2/backcross exhibited a clear plateau of mortality across a range of doses indicating monogenic resistance. The null hypothesis for monogenic resistance was tested from mortality data of backcross progeny compared with theoretical expectations using the χ2 test and was found to be non-significant. Understanding genetics of insecticide resistance is significant in prediction and management of resistant insects. The amr genes can be used as genetic marker in A. stephensi, which can be used in several applications in conducting basic and applied genetic research.

Genetic Study of Propoxur Resistance-A Carbamate Insecticide in the Malaria Mosquito, Anopheles stephensi Liston.

Anopheles stephensi Liston (Diptera: Culicidae) is the urban vector of malaria in the Indian subcontinent and several countries of the Middle East. The genetics of propoxur resistance (pr) in An. stephensi larvae was studied to determine its mode of inheritance. A diagnostic dose of 0.01 mg/L as recommended by WHO was used to establish homozygous resistant and susceptible strains. Reciprocal crosses between the resistant and susceptible strains showed an F(1) generation of incomplete dominance. The progenies of backcrosses to susceptible parents were in 1 : 1 ratio of the same phenotypes as the parents and hybrids involved. The dosage mortality (d-m) lines were constructed for each one of the crosses, and the degree of dominance was calculated. It is concluded that propoxur resistance in An. stephensi larvae is due to monofactorial inheritance with incomplete dominance and is autosomal in nature.

The genetics of green thorax, a new larval colour mutant, non-linked with ruby - eye locus in the malaria mosquito, Anopheles stephensi.

BACKGROUND & OBJECTIVES: Anopheles stephensi, an important vector of malaria continues to be distributed widely in the Indian subcontinent. The natural vigour of the species combined with its new tolerance, indeed resistance to insecticides has made it obligatory that we look for control methods involving genetic manipulation. Hence, there is an immediate need for greater understanding of the genetics of this vector species. One of the requirements for such genetic studies is the establishment of naturally occurring mutants, establishment of the genetic basis for the same and use of such mutants in the genetic transformation studies and other genetic control programme(s). This paper describes the isolation and genetic studies of a larval colour mutant, green thorax (gt), and linkage studies involving another autosomal recessive mutant ruby- eye (ru) in An. stephensi. METHODS: After the initial discovery, the mutant green thorax was crossed inter se and pure homozygous stock of the mutant was established. The stock of the mutant ruby- eye, which has been maintained as a pure stock in the laboratory. Crosses were made between the wild type and mutant, green thorax to determine the mode of inheritance of green thorax. For linkage studies, crosses were made between the mutant green thorax and another autosomal recessive mutant ruby-eye. The percentage cross-over was calculated for the genes linkage relationship for gt and gt ru. RESULTS: Results of crosses between mutant and wild type showed that the inheritance of green thorax (gt) in An. stephensi is monofactorial in nature. The gt allele is recessive to wild type and is autosomal. The linkage studies showed no linkage between ru and gt. INTERPRETATION & CONCLUSION: The mutant gt represents an excellent marker for An. stephensi as it is expressed in late III instar stage of larvae and is prominent in IV instar and pupal stages with complete penetrance and high viability. The said mutant could be easily identified without the aid of a microscope. This mutant can be used extensively to conduct basic and applied research. The mutant has been maintained in two large cages in our laboratory.

Dark larva a new autosomal larval colour mutant in the malaria mosquito, Anopheles stephensi Liston.

Anopheles stephensi is an important urban malaria vector, which is widely distributed throughout the Indian subcontinent. The said vector species has developed resistance to various insecticides and therefore, it is desirable to develop alternative strategies including genetic methods for its control. One of the requirements for such strategy is to establish morphological mutants and to establish the genetic basis of the same. Such mutant markers could be used in the construction of genetically modified strain/s in the genetic control programme of An. stephensi. The dark colour expresses in all larval stages and pupae with full penetarence and uniform expression in both the sexes. The viability of the mutant is as good as the wild type. The genetic studies of the dark larva revealed that the gene da is mono-factorial, autosomal and recessive to the wild type. The gene da is an excellent marker for An. stephensi.

An evaluation of clinical and histopathological status in paucibacillary leprosy patients after completion of fixed duration therapy.

The present study was carried out involving 25 patients with paucibacillary leprosy who attended the outpatient department of dermatology of Father Muller's Medical College Hospital during the period January 2001 to March 2002. All the patients were examined clinically and histopathologically at the beginning and at the end of six months of MDT and relevant data recorded. Clinicopathological correlation with histopathological classification before MDT was 72% and 68% at the end of MDT in our study. At the end of treatment 4 (16%) cases were clinically active and 8 (32%) were histopathologlcally active. The study showed that active cases were significantly reduced as a result of MDT, both clinically and histopathologically. The histopathological activity that outlasts MDT may be due to the bacillary fragments that persist; but clinical activity coupled with histopathological activity seen in 2 patients at the end of 6 months of MDT was possibly an indicator of relapse and these patients and similar others need to be followed up for a longer duration. In this study, resolution of granuloma and clinical activity after completion of MDT were assessed.

Giant lesions in histoid leprosy--an unusual presentation.

A forty-year-old man presented with multiple, asymptomatic, raised lesions of one-year duration. Well-defined, non-tender papules and nodules were seen on the normal looking skin. Few giant lesions were seen over the lower legs and feet. Diffuse infiltration of the face and ears was present. Bilateral ulnar, radial cutaneous, sural and right superficial peroneal nerves were thickened. Slit-skin smear from a nodule had a BI of 6+. Biopsy showed features of histoid leprosy. The lesions had developed de novo, without previous dapsone monotherapy.

An evaluation of clinical and histopathological status in paucibacillary leprosy patients after completion of sixed duration therapy

The presente study was carried out involving 25 patients with paucibacillary leprosy who attended the outpatient department of dermatology of Father Muller's Medical College Hospital during the period January 2001 to March 2002. All the patients were examined clinically and histopathologically at the beginning and at the end of six months of MDT and relevant data recorded. Clinicopathological correlation with histopathological classification before MDT was 72% and 68% at the end of MDT in our study. At the end of treatment 4 (16%) cases were clinically active and 8 (32%) were histopathologically active. The study showed that active cases were siginificantly reduced as a result of MDT, both clinically and histopatologically. The histopathological activity that outlasts MDT may be due to the bacillary fragments that persist; but clincial activity coupled with histopathological activity seen in 2 patients at the end of 6 months of MDT was possibly an indicator of relapse and these patients and similar others need to be follewed up for a longer duration. Is this study, resolution of granuloma and clinical activity after completion of MDT were assessed

Ruby-eye, a new autosomal mutant in the malaria mosquito, Anopheles stephensi Liston.

BACKGROUND & OBJECTIVES: Anopheles stephensi, an important vector of malaria continues to be distributed widely in the Indian subcontinent. This vector species has developed resistance for various insecticides. Therefore, it is desirable to develop alternate strategy, which does not involve resistance. In order to develop such strategy, it is mandatory that genetic studies of concerned vector species should be established. This paper describes the isolation and genetic studies of an eye colour mutant, ruby-eye (ru), and linkage studies involving another autosomal recessive mutant greyish brown larva (grb ru) in A. stephensi. METHODS: The stocks of mutants ruby-eye (ru), greyish brown (grb ru) and wild type mosquitoes were maintained in the laboratory. Crosses were made between the wild type and mutant to determine the mode of inheritance of ruby-eye. For linkage studies crosses were made between the mutant ruby-eye and another autosomal recessive mutant greyish brown larva. The percentage cross over was calculated for the genes linkage relationship for ru and grb ru. RESULTS: Results of crosses between mutant and wild type show that the inheritance of ruby-eye in A. stephensi is monofactorial in nature. The ru allele is recessive to wild type and is autosomal. The linkage studies showed no linkage between grb and ru. INTERPRETATION & CONCLUSION: The mutant ru represents an excellent marker for A. stephensi as it expresses in all the life stages with complete penetrance and high viability. This mutant can be used extensively to conduct basic and applied research.

Brown larva: an allele of the green larva mutation in the malaria mosquito, Anopheles stephensi.

We isolated a new larval color mutant, brown larva (b), from the Bangalore, India strain of Anopheles stephensi Liston. The gene b is an autosomal recessive with uniform expression and complete penetrance. We conducted extensive crosses to establish allelism between brown larva (b) and green larva (g) reported previously in An. stephensi from our laboratory. The wild-type is dominant to green larva, which, in turn, is dominant to brown larva. These larval color mutants belong to an allelic series.

Chromosomal translocations and inherited semisterility in the malaria vector Anopheles stephensi Liston.

Anopheles stephensi males were irradiated with 3500 rads of gamma rays at the rate of 140 rads/min to induce chromosomal aberrations. Seven reciprocal translocations were isolated, including four sex-linked and three autosomal. The presence of translocations were confirmed by cytological analysis.