Differential sensitivity of Chironomus and human hemoglobin to gamma radiation.

Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study.

Chironomus ramosus larvae exhibit DNA damage control in response to gamma radiation.

PURPOSE: Chironomus ramosus is one of the recently reported radiotolerant insects. Salivary gland cells of fourth instar larvae respond to ionizing radiations with increases in the levels of antioxidant enzymes and chaperone proteins. Here we made an attempt to study the state of nuclear DNA after exposure of larvae to a lethal dose for 20% of the population (LD(20)) of gamma radiation (2200 Gy, at a dose rate 5.5 Gy/min). MATERIALS AND METHODS: Genomic DNA preparations were subjected to competitive ELISA (Enzyme linked immunosorbent assay) for detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and dynamic light scattering (DLS) to monitor any radiation-induced damage. Single salivary gland cells were subjected to alkaline single cell gel electrophoresis (ASCGE), comet assay and pulsed field gel electrophoresis (PFGE) to check for DNA double-strand breaks. RESULTS: Results from all four experimental procedures confirmed damage of nucleobases and fragmentation of nuclear DNA immediately after radiation. Some 48 h after radiation exposure, modified 8-oxodG residues returned to basal level, homodispersity of genomic DNA reappeared, the length of comet tail regressed significantly (ASCGE) and PFGE pattern matched with that of high molecular weight unirradiated DNA. CONCLUSION: Chironomus ramosus larvae showed control of DNA damage as observed over 48 h in post irradiation recovery which could be attributed to their ability to tolerate gamma radiation stress.

Effects of in vivo exposure to UV filters (4-MBC, OMC, BP-3, 4-HB, OC, OD-PABA) on endocrine signaling genes in the insect Chironomus riparius.

There is increasing evidence indicating that several UV filters might have endocrine disruptive effects. Numerous studies have evaluated hormonal effects in vertebrates, mainly reporting estrogenic and androgenic activities in mammals and fishes. There is only limited knowledge about potential endocrine activity in invertebrate hormonal systems. In this work, the effects on endocrine signaling genes of six frequently used UV filters were investigated in Chironomus riparius, a reference organism in aquatic toxicology. The UV filters studied were: octyl-p-methoxycinnamate (OMC) also called 2-ethylhexyl-4-methoxycinnamate (EHMC); 4-methylbenzylidene camphor (4-MBC); benzophenone-3 (BP-3); 4-hidroxybenzophenone (4-HB); octocrylene (OC); and octyldimethyl-p-aminobenzoate (OD-PABA). After in vivo exposure at different dosages, expression levels of the genes coding for the ecdysone receptor (EcR), the ultraspiracle (usp, ortholog of the RXR) and the estrogen-related receptor (ERR) were quantified by Real Time PCR. The EcR gene was significantly upregulated by 4-MBC, OMC/EHMC and OD-PABA, with a dose-related response following 24h exposure. In contrast, the benzophenones, BP-3 and 4-HB, as well as OC did not alter this gene at the same exposure conditions. The transcription profiles of the usp and ERR genes were not significantly affected, except for BP-3 that inhibited the usp gene at the highest concentration. To our knowledge, this is the first experimental evidence in invertebrates of a direct effect of UV filters on endocrine-related genes, and is consistent with the known effects on vertebrate hormonal receptor genes. The capability of 4-MBC, OMC/EHMC and OD-PABA to stimulate the expression of the ecdysone receptor, a key transcription factor for the ecdysone-genomic response in arthropods, suggests the possibility of a broad and long-term effect on this hormonal pathway. These findings strengthen the need for further research about the ecotoxicological implications of chronic exposure to these compounds in aquatic invertebrates.

[Assessment of radiation exposure level for hydrobionts in some special industrial ponds at the "Mayak" PA].

Evaluation of the radionuclide content in the ecosystem components (water, sediments, aquatic organisms) of industrial reservoirs-storages of liquid radioactive waste of the "Mayak" PA (reservoirs R-4, R-10, R-11, R-17, R-9) and the estimation of the absorbed dose rate in aquatic organisms of these reservoirs using the software package ERICA Assessment Tool 1.0 May 2009 have been performed. Gradient of the absorbed dose rate for the detected taxonomic groups of hydrobionts in the series of the studied reservoirs R-11 --> R-10 --> R-4 --> R-17 --> R-9 was almost equal to one order of magnitude. The estimated absorbed dose rate for phytoplankton ranged from 5.4 x 10(0) mGy/day (R-11) to 4.0 x 10(4) mGy/day (R-9), for zooplankton--from 6.4 x 10(-1) mGy/day (R-11) to 3.8 x 10(3) mGy/day (R-9), for zoobenthos (chironomids)--from 5.6 x 10(0) mGy/day (R-11) to 1.1 x 10(3) mGy/day (R-17), for fish (roach)--from 8.0 x 10(-1) mGy/day (R-11) to 1.9 x 10(1) mGy/day (R-4).

[Chromosomal and genomic mutations in Chironomus plumosus (L.) (Diptera, Chironomidae) from Novozybkov raion of Bryansk oblast].

The data on karyopool analysis of Chironomus plumosus from Novozybkov raion of Bryansk oblast, radioactively contaminated as a result of the Chernobyl accident, are presented. In the karyopool of this population, four types of heterozygous inversions (A1.2, B1.2, C1.2, D1.2) and a structurally small rearrangement in arm D, which is thought to be also associated with inversion, were identified. For inversions A1.2, C1.2, and structurally small rearrangement in arm D the cases of somatic mosaicism were described. The mean number of inversions per individual constituted 0.78. The number of genotypic combinations was 13. In 5% of the individuals chromosome B was identified, while 15.6% were polyploid (3n). In 1.8% of triploids somatic mosaicism for the level of polytene chromosomes was observed. Most of the larvae were characterized by partial asynapsis of the homologs in different regions of chromosome III. Specific features of the Chironomus plumosus karyopool from Novozybkov are thought to be associated with the habitation of a number of generations of this population on radioactively polluted territory.

Hsp70 expression in Chironomus ramosus exposed to gamma radiation.

PURPOSE: A tropical species of midge, Chironomus ramosus has been recently reported to be one of the radio-tolerant groups of organisms. The present study was undertaken to examine the protein profile and expression of Heat shock protein-70 (Hsp70) in gamma radiation stress, which has also been reported as a common biomarker for different type of stressors. MATERIALS AND METHODS: Metabolic labelling of salivary gland (SG) proteins with [(35)S]-methionine showed over-expression of a 70 kDa protein band up to 4 hours (h) of observation in the post exposure recovery period. For confirmation of the expression of Hsp70 in SG cells after gamma radiation exposure, semi-quantitative real-time-polymerase chain reaction (RT-PCR), Western blotting and immuno-fluorescence detection of Hsp70 were carried out. RESULTS: Results showed elevated levels of Hsp70 mRNA and protein in SG cells of larvae immediately after gamma radiation exposure. The levels dropped to basal values by 48 h in the recovery period. CONCLUSIONS: The present study confirmed that radio-tolerant midge, C. ramosus expressed Hsp70 upon gamma radiation exposure and Hsp70 might be one of the gamma radiation-induced stress proteins required during the early stages of radiation stress management in aquatic midge larvae. This is the first report of its kind from the juvenile stage of any aquatic insect group.

Gamma radiation tolerance of a tropical species of midge, Chironomus ramosus Chaudhuri (Diptera: Chironomidae).

PURPOSE: The Chironomid midges are known to thrive well under adverse environmental conditions and are even found inhabiting in areas contaminated by radioactive wastes. Studies were therefore undertaken to find out the radiosensitivity of different developmental stages of the Indian tropical midge, Chironomus ramosus. MATERIALS AND METHODS: In order to determine the threshold levels of lethality, eggs, larvae, pupae and adults of C. ramosus were exposed to varying dosages of gamma radiation (60Co radiation source) ranging from 0-3500 Gray (Gy) at dose-rate of 5.5 Gy/minute. The post-irradiation studies were conducted at three different time points: (a) Immediately after the end of irradiation, (b) 24 hours (h), and (c) 48 h after the end of radiation treatments. Determination of the lethal dose required to kill 50% (LD50), 90% (LD90) and 100% population was carried out using the log-probit analysis. RESULTS: Different developmental stages showed variable threshold levels of radiosensitivity. The radiation doses required to cause 100% mortality immediately after radiation exposure of egg, larva, pupa and adult stages were 1000 Gy, 3000 Gy, 3200 Gy and 3500 Gy, respectively, indicating eggs as the most sensitive stage. Detailed analysis of the LD50 values of different post-irradiation time points indicated that pupal stages were also sensitive at 48 h post-irradiation amongst all the post-embryonic stages as described in many other insects. Interestingly detailed analysis of data indicated that amongst the adult population, females were the most radioresistant, compared to the males as reported in many other insect groups in the literature. CONCLUSIONS: The Indian tropical midge C. ramosus was found to tolerate higher dose of gamma radiation as compared to other known dipteran insects. It is evident from the present findings that C. ramosus falls in the category of radiation-tolerant group of insects.

Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation.

A battery of enzymes from the eukaryotic antioxidant defense system was measured in salivary gland and in whole body extract of fourth instar larvae of Chironomus ramosus with an objective of finding any clue for the dipteran insect's capacity to tolerate heavy doses of ionizing radiation. Levels of activity of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSH-Px) were quantified in 30 days old larvae exposed to LD(20) dose of gamma radiation. Compared to controls, activity of Cu,Zn-SOD increased 3 to 4 fold and catalase 2 fold in response to ionizing radiation stress, while activities of GR and GSH-Px enzymes were decreased. Among the other SOD isoenzymes, our results showed comparable levels of Mn-SOD and Cu,Zn-SOD activity in control and irradiated groups of larvae. The increase in levels of the Cu,Zn-SOD isoenzyme was also confirmed by Western blot and zymography supported by densitometric quantification. No evidence of Fe-SOD was found in C. ramosus larvae. These findings could help to explain the persistence of natural populations of Chironomus in radioactively contaminated regions.

Physiological changes leading to anhydrobiosis improve radiation tolerance in Polypedilum vanderplanki larvae.

High tolerance against various extreme environments exhibited by some anhydrobionts might be due to being almost completely desiccated, a state where little or no chemical reactions occur. We have shown that anhydrobiotic larvae of Polypedilum vanderplanki have higher tolerance against both high- and low-linear energy transfer (LET) radiation than hydrated larvae. It is of great interest to know how the desiccating larvae gain radiation tolerance. We therefore examined effects of high-LET radiation on four kinds of larvae: (1) normal hydrated (intact) larva, (2) intermediates between the anhydrobiotic and normal hydrated state, (3) almost completely dehydrated (anhydrobiotic) larvae, and (4) immediately rehydrated larvae that are assumed to have a similar molecular profile to anhydrobiotic larvae. The intermediates and immediately rehydrated larvae survived longer after high-LET radiation than intact larvae, indicating that radiation tolerance could be enhanced even in hydrated larvae. Physiological changes toward anhydrobiosis, e.g. accumulation of protectants or increasing damage repair capacity, correlate with improved radiation tolerance in hydrated larvae. In addition, almost complete desiccation further enhanced radiation tolerance, possibly in a different way from the hydrated larvae.

Estimation of radiation tolerance to high LET heavy ions in an anhydrobiotic insect, Polypedilum vanderplanki.

PURPOSE: Anhydrobiotic larvae of Polypedilum vanderplanki are known to show an extremely high tolerance against a range of stresses. We have recently reported that this insect withstands exposure to high doses of gamma-rays (linear energy transfer [LET] 0.2 keV/microm). However, its tolerance against high LET radiation remains unknown. The aim of this study is to characterize the tolerance to high-LET radiations of P. vanderplanki. MATERIALS AND METHODS: Larval survival and subsequent metamorphoses were compared between anhydrobiotic (dry) and non-anhydrobiotic (wet) samples after exposure to 1 - 7000 Gy of three types of heavy ions delivered from the azimuthally varying field (AVF) cyclotron with LET values ranging from 16.2 - 321 keV/microm. The tolerance against 4He ions was also compared among three chironomid species. RESULTS: At all LET values measured, dry larvae consistently showed greater radiation tolerance than hydrated larvae, perhaps due to the presence of high concentrations of the disaccharide trehalose in anhydrobiotic animals, and the radiation-induced damage became evident at lower doses as development progressed. Relative biological effectiveness (RBE) values based on the median inhibitory doses reached a maximum at 116 keV/microm (12C), and the maximum RBE clearly increased as development progressed. Lower D0 (dose to reduce survival from relative value 1.00 - 0.37 on the exponential part of the survival curve), and higher Dq (quasi-threshold dose) were found in individuals exposed to 4He ions, compared to gamma-rays, and in P. vanderplanki larvae compared to non-anhydrobiotic chironomids. CONCLUSION: Anhydrobiosis potentiates radiation tolerance in terms of larval survival, pupation and adult emergence of P. vanderplanki exposed to high-LET radiations as well as to low-LET radiation. P. vanderplanki larvae might have more efficient DNA damage repair after radiation than other chironomid species.

An estimation of radiation doses to benthic invertebrates from sediments collected near a Canadian uranium mine.

A new method is described for calculating radiation doses to benthic invertebrates from radionuclide concentrations in freshwater sediment. Both internal and external radiation doses were estimated for all 14 principal radionuclides of the uranium-238 decay series. Sediments were collected from three sites downstream of a uranium mining operation in northern Saskatchewan, Canada. Sediments from two sites, located approximately 1.6 and 4.4 km downstream from mining operations, yielded absorbed doses to both larval midges, Chironomus tentans, and adult amphipods, Hyalella azteca, of 59-60 and 19 mGy/year, respectively, compared to 3.2 mGy/year for a nearby control site. External beta radiation from protactinium-234 (234Pa) and alpha radiation from uranium (U) contributed most of the dose at the impacted sites, whereas polonium-210 (210Po) was most important at the control site. If a weighting factor of 20 was employed for the greater biological effect of alpha vs. beta and gamma radiation, then total equivalent doses rose to 540-560 mGy/year at the site closest to uranium operations. Such equivalent doses are above the 360-mGy/year no-observed-effect level for reproductive effects in vertebrates from gamma radiation exposure. Data are not available to determine the effect of such doses on benthic organisms, but they are high enough to warrant concern. Detrimental effects have been observed in H. azteca at similar uranium concentration in laboratory toxicity tests, but it remains unclear whether the radiotoxicity or the chemotoxicity of uranium is responsible for these effects.

Influence of UV radiation on four freshwater invertebrates.

Laboratory tests confirmed a negative and variable response of the following four species to artificial UV radiation: Cypridopsis vidua, an ostracode; Chironomus riparius, a midge larvae; Hyalella azteca, an amphipod; and Daphnia magna, a daphnid. Severe damage occurred at UV-B irradiance ranging from 50 to 80% of incident summer values. Under constant exposure to UV and photosynthetically active radiation (PAR) the acute lethal response was recorded at 0.3, 0.8, 0.8 and 4.9 W m-2 UV-B for D. magna, H. azteca, C. riparius and C. vidua, respectively. Sublethal UV-B damage to invertebrates included impaired movement, partial paralysis, changes in pigmentation and altered water balance (bloating). A series of UV-B, UV-A and PAR treatments, applied separately and in combination, revealed a positive role for both UV-A and PAR in slowing down UV-B damage. Mean lethal concentration values of the species typically more tolerant to UV and PAR (Cypridopsis, Chironomus) decreased conspicuously when both UV-A and PAR were eliminated. For UV-B-sensitive species (Hyalella, Daphnia) these differences were notably smaller. We suggest that this gradation of sensitivity among the tested species demonstrates potential differences in repairing mechanisms which seem to work more efficiently for ostracodes and chironomids than for amphipods and daphnids. Manipulations with a cellulose acetate filter showed that lower range UV-B (280-290 nm), produced by FS-40 lamps, may cause excessive UV damage to invertebrates.