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.

Characterization of highly toxic indigenous strains of mosquitocidal organism Bacillus sphaericus.

Three indigenous isolates of Bacillus sphaericus (ISPC-5, ISPC-6 and ISPC-8), along with standard 2362 and 1593 strains, were evaluated for spore viability and mosquitocidal activity. Among these, ISPC-8 was the most viable and virulent isolate, exhibiting a significantly higher total viability count (TVC) and lower LC(50) values. The TVC of the standard strains ranged from 4.0 to 9.2 x 10(8) spores mL(-1), whereas it was 1.3 x 10(9) spores mL(-1) for ISPC-8. The LC(50) values of ISPC-8, 2362 and 1593 against Culex quinquefasciatus were 0.68 x 10(3), 1.22 x 10(3) and 1.85 x 10(3) spores mL(-1), respectively. The ISPC-8 was further assessed for host spectrum and found to be more active against C. quinquefasciatus, followed by Culex tritaeniorhynchus, Aedes albopictus and Aedes aegypti. The ISPC-8 strain was thus found to be a promising isolate for developing biopesticides. Among the indigenous strains, only ISPC-8 was found to have binary toxin genes (binA and binB). Comparative sequence analysis revealed that the BinA (41.9 kDa) protein of ISPC-8 differs by one amino acid (R197M), whereas BinB (51.4 kDa) differs by two amino acids (H99P, P174S) as compared with 1593 and 2362 strains. The purified binary proteins of ISPC-8 showed an LC(50) value of 6.32 ng mL(-1) against C. quinquefasciatus larvae after 48 h.

Expression, purification and characterization of the Cry2Aa14 toxin from Bacillus thuringiensis subsp. kenyae.

An indigenous strain HD-550 of Bacillus thuringiensis subsp. kenyae was found to be toxic to lepidopteran as well as dipteran insects. The cry2Aa gene (classified as cry2Aa14) from this isolate was cloned and expressed in Escherichia coli. Only a little amount of the expressed Cry2Aa14 protein was observed in soluble fraction under normal induction condition. The inclusions were non-toxic to test insects, whereas solubilized Cry2Aa14 was highly toxic to lepidopteran and dipteran insects. Cry2Aa14 protein was expressed as thioredoxin (trx) fusion protein for improving the yield of active protein. An enhancement of nearly 15% was observed in the yield of active Cry2Aa14. The TrxA-Cry2Aa14 protein purified from the solubilized fraction also showed toxicity profile similar to the wild-type protein. The LC(50) values of Cry2Aa14 and TrxA-Cry2Aa14 protein against Spodoptera litura was 694 and 696 ng/cm(2), respectively, while for Culex quinquefasciatus the LC(50) values were 894 and 902 ng/ml, respectively. The broad spectrum toxicity of the Cry2Aa14 thus indicates that this protein could be an important component in integrated pest management. Further, the trx tag clearly led to higher yield, which facilitates protein purification for biophysical and biochemical characterization.

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.

Purification and characterization of mosquitocidal Bacillus sphaericus BinA protein.

Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal binary toxin during sporulation. The binary toxin is composed of toxic BinA (41.9kDa) and receptor binding BinB (51.4kDa) polypeptides and is active against vectors of filariasis, encephalitis and malaria. The toxin has been tested with limited use for the control of vector mosquitoes for more than two decades. The binA gene from a local ISPC-8 strain of B. sphaericus that is highly toxic to Culex and Anopheles mosquito species was cloned into pET16b and expressed in Escherichia coli. The purified BinA protein differs by one amino acid (R197M) from BinA of the highest toxicity strains 1593/2362/C3-41. Majority of the expressed protein was observed in inclusion bodies. BinA inclusions alone from E. coli did not show toxic activity, like reported previously. However, the active form of BinA could be purified to homogeneity from the soluble fraction of E. coli cell lysate, grown at reduced temperature after isopropyl beta-d-thiogalactopyranoside induction. The purified BinA protein with and without poly-histidine tag showed LC(50) dose of 82.3 and 66.9ngml(-1), respectively, at 48h against Culex quinquefasciatus larvae. The secondary structure of BinA is expected to be mainly beta strands as estimated using far-UV circular dichroism. The estimates matched well with the secondary structure predictions using amino acid sequence. This is the first report of large-scale purification and accurate toxicity estimation of soluble B. sphaericus BinA. This can help in design and synthesis of improved bacterial insecticide.

Characterization of the cry1Ac17 gene from an indigenous strain of Bacillus thuringiensis subsp. kenyae.

An indigenously isolated strain of Bacillus thuringiensis subsp. kenyae exhibited toxicity against lepidopteran as well as dipteran insects. The lepidopteran active cry1Ac protoxin gene coding sequence of 3.5 kb from this strain was cloned into vector pET28a(+). However, it could not be expressed in commonly used Escherichia coli expression hosts, BL21(DE3) and BL21(DE3)pLysS. This gene is classified as cry1Ac17 in the B. thuringiensis toxic nomenclature database. The coding sequence of this gene revealed that it contains about 3% codons, which are not efficiently translated by these expression hosts. Hence, this gene was expressed in a modified expression host, Epicurian coli BL21-Codonplus (DE3)-RIL. The expression of gene yielded a 130-kDa Cry1Ac17 protein. The protein was purified and its toxicity was tested against economically important insect pests, viz., Helicoverpa armigera and Spodoptera litura. LC(50) values obtained against these insects were 0.1 ng/cm(3) and 1231 ng/cm(2), respectively. The higher toxicity of Cry1Ac17 protein, compared to other Cry1Ac proteins, toward these pests demonstrates the potential of this isolate as an important candidate in the integrated resistance management program in India.