|Year : 2022 | Volume
| Issue : 2 | Page : 145-153
Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.)
Vinaya Shetty1, NJ Shetty2, SK Jha3, RC Chaubey4
1 Centre for Applied Genetics, J. B. Campus, Bangalore University, Bengaluru, India; Department of Entomology, Texas A&M University, College Station, Texas, USA
2 Centre for Applied Genetics, J. B. Campus, Bangalore University, Bengaluru, India
3 Environmental Assessment Division, Bhabha Atomic Research Centre, Mumbai, India
4 Radiation Biology and Health Science Division (BRNS-DAE), Bhabha Atomic Research Centre, Mumbai, India
|Date of Submission||11-Oct-2021|
|Date of Acceptance||26-Nov-2021|
|Date of Web Publication||08-Sep-2022|
N J Shetty
Centre for Applied Genetics, J. B. Campus, Bangalore University, Bengaluru 560056
Source of Support: None, Conflict of Interest: None
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.
Keywords: gamma radiation; heat shock proteins; Hsc70; Hsp83; Aedes aegypti
|How to cite this article:|
Shetty V, Shetty N J, Jha S K, Chaubey R C. Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.). J Vector Borne Dis 2022;59:145-53
|How to cite this URL:|
Shetty V, Shetty N J, Jha S K, Chaubey R C. Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.). J Vector Borne Dis [serial online] 2022 [cited 2022 Nov 28];59:145-53. Available from: https://www.jvbd.org/text.asp?2022/59/2/145/335770
| Introduction|| |
Aedes aegypti is a well-known insect vector responsible for transmitting the viruses that cause dengue fever and the more severe dengue hemorrhagic fever. Numerous techniques are being employed to restrict its growth and spread. One among them is the Sterile Insect Technique (SIT), a new-age approach that involves the generation of sterile males to check insect populations, usually using gamma radiation, and has been successfully applied on several insect species, including mosquito vectors,,.
Intentional irradiation aside, there is also increasing evidence to suggest that gamma radiation is an environmental stress factor that impacts all living organisms ubiquitously. Insects perceive stress signals and respond with diverse strategies to defend against the stress, one of which is the regulation of protein expression. Studies have confirmed that gamma radiation exposure can increase the levels of oxidative stress in insects, disturb the protein’s functional activity, and intensify the activity of protein oxidation processes,,. There are a considerable number of studies investigating the effects of gamma radiation on Aedes, including its ability to induce sterility. Radiation-induced male sterility has been demonstrated in many mosquito species, including Ae. aegypti,,. There are, however, very few reports of the molecular changes induced by gamma radiation that contribute to sterility.
Heat shock proteins (HSPs) are commonly studied in the context of insect stress management,, and gamma irradiation has been known to induce the activity of HSPs in different organisms,. In vertebrates for instance, various radiation-responsive HSP genes have been identified from cells, tissues or individuals, and their roles have been characterized at the cellular and molecular levels,,. The main role of HSPs is well known as molecular chaperones that promote proper assembly of denatured proteins, and hsp genes respond uniquely to different kinds of stress,. It has been reported that HSPs play an important role in temperature stress-induced male sterility, male gametogenesis, and embryonic development in several drosophila species,,. The HSPs in Ae. aegypti AeaHsp70 and AeaHsp83 have been proved to be important markers of temperature-induced stress. They are postulated to function as critical proteins to protect and enhance the survival of Ae. aegypti larvae and pupae subjected to temperature stress,. Heat shock cognate 70 (Hsc70), an essential member of the HSP70 family, is an important stress-resistance protein found in insects and is known to protect them from environmental stresses. The Heat Shock Proteins (HSPs), especially Hsp83/90 and Hsc70, are abundantly expressed in insects and are known modulators of insect survival,,. Although, the practical application of measuring hsp gene products may be limited because their levels are altered by various external stimuli,,,, it has been suggested that they can serve as biomarkers of environmental contaminants, such as pesticides and metallic pollutants,. There is however no mention in literature of HSPs in the context of gamma radiation induced stress in Aedes.
To this end, the current study was designed to determine the gamma radiation induced expression of heat shock proteins (Hsc70 and Hsp83) in Ae. aegypti. A dose equivalent to 50Gy that produced above 90% of sterility in the total irradiated males were selected for this study. An MS-MS based proteomic approach was chosen, because it would allow the detection of unique peptides leading to a more specific identification of the HSPs induced. Further, this approach would allow the identification of few other proteins that are also possibly regulated in response to gamma radiation induced stress.
| Material & Methods|| |
Aedes aegypti larvae collected from the J.P Nagar area of Bengaluru, India were reared at 25 ± 10C and 75 ± 5% relative humidity under 14-hour photoperiod in the insectary of the Centre for Applied Genetics, Bangalore University, India following standard protocol.
Gamma irradiation experiments
Experiments were performed in three replicates along with a control. Overall, a total of 120 adult males, 2–3 days of age, were irradiated with 50Gy of gamma radiation from a60Co (Theratron 780-C machine) source with a dose rate of 253.56cGy/min, at the Kidwai Memorial Institute of Oncology, Bengaluru, India. The mosquitoes were placed in plastic boxes (5 X 4 X 2.5 cm) covered with fine net cap during irradiation. A dosimetry was used to quantify the dose received by the irradiated insects and confirm that all the doses delivered lay within a 5% error range. After irradiation, the mosquitoes were reared as described until further analysis. Another set of non-irradiated 60 adult males were maintained as controls (20 males in triplicates).
Preparation of crude homogenates
Whole-body homogenates were prepared by pooling twenty irradiated males each at two different time intervals, i.e., 6- and 12-hour post irradiation. A control set was prepared in a similar manner. The homogenized pellet was mixed with 500μl ice cold extraction buffer (50mM KPO4 buffer at pH 7.4), followed by centrifugation at 16,300g at 40C for 15 min. The clear supernatant was transferred to a new eppendorf tube and kept at -800C for approximately one week until analysis. Total protein concentration was determined by Bradford protein assay, using bovine serum albumin as the standard.
Sodium Dodecyl Sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE)
Fifteen micrograms of protein from each sample (both experimental and control), were loaded onto a 10% SDS gel along with a standard protein maker ranging 10–250kD (Precision Plus Protein Standards (Dual Colour), Bio-Rad), and was run as described by Laemmli. A total of three such gels were prepared to serve as technical replicates.
The gels were stained using colloidal Coomassie blue stain 250 (Bio-Rad), de-stained to remove excess stain, and then scanned, using a densitometric scanner to determine the concentration and molecular weight of each characteristic band relative to the protein molecular weight marker.
In-gel digestion and LC-MS/MS analysis
In-gel digestion, tryptic peptide extractions, and Nano-LC-MS/MS were conducted by the mass spectrometry facility, Centre for Cellular and Molecular Platforms (CCAMP), NCBS, Bengaluru, India following the methods of Shevchenko et al..
The three SDS gel slabs were rinsed with water for a few hours, and the bands of interest labelled as A, B, C, D, E and F [Figure 1], were excised with a clean scalpel. Each band was cut into cubes (~1cubic millimetre) and transferred into labelled micro centrifuge tubes. Gel fragments from each tube were subjected to in gel digestion using Trypsin (Promega, Madison, WI) for approximately 12 h at 37°C. The digested peptides were reconstituted in 15μL of the 0.1% formic acid and 1μL of the same was injected on column and subjected to a 70 min RPLC gradient, followed by analysis on LTQ-Orbitrap-MS. The identity of the generated data was searched using MASCOT as the search engine against Swiss-prot, A. aegypti and TrEMBL databases.
|Figure 1: A representative image of the Sodium Dodecyl Sulphate (SDS) Poly Acrylamide Gel Electrophoresis (PAGE) for proteins of post-irradiated adult males (at 6h and 12h time intervals) and control group of Aedes aegypti. The bands excised at each time point corresponding to molecular weight ranges ~60-75 and ~80-95 (kDa) are marked with letters A, B, C, D, E and F.|
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Progenesis LC-MS software was used for ion intensity-based label-free quantification and data analysis. Raw-files were imported and all replicate runs were aligned and normalized by the software to generate fold changes. Differentially expressed proteins exhibiting a fold change greater than 2-fold between the irradiated samples at two different time intervals (at 6- and 12-hr post-irradiation) and the controls were considered for analysis. Only those proteins identified at a false discovery rate of 1% or less were included. The unknown proteins were searched against nr database. The analyses were exported into an Excel spreadsheet containing the identified protein list including their corresponding scores, normalized fold change values (relative protein expression), ANOVA p-value (reliability of the measured differences are calculated between each group, are calculated using summed peptide ion abundances) and number of peptides matched to each protein.
Ethical statement: Not applicable
| Results & Discussion|| |
This study is an account of a mass spectrometry-based relative quantification of proteins from SDS-PAGE bands of proteins extracted from adult male Ae. aegypti whole body lysate at 6 and 12 hours post gamma irradiation, corresponding to the ~60–75kDa and ~80–95kDa, respectively [Figure 1]. The analysis revealed a significant difference (p<0.05) in the relative abundance of a total of 21 proteins identified by MASCOT when the 6- and 12-hour post-irradiation samples were compared to the control. The results with the more than two (>2) fold change values derived from the Progenesis-based analysis are captured in [Table 1] & [Table 2]. Hsc70 and Hsp83 were both found to be upregulated in Ae. aegypti upon gamma irradiation. Interestingly, the study also revealed an up-regulation of enzymes such as Arginine kinase known to be upregulated in dipterans during stress, Esterase B1 implicated in insecticide resistance and downregulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 that are involved in ubiquitin-mediated protein degradation.
|Table 1: Differential expression profiles of selected proteins (MW ranges ~ 60–75kDa and ~80–95kDa) at 6h post irradiated Ae. aegypti|
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|Table 2: Differential expression profiles of selected proteins (MW ranges ~ 60–75kDa and ~80–95kDa) at 12h post irradiated Ae. aegypti|
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The response of cells or organisms to heat shock and other stressors is connected to the induction of heat shock proteins (HSPs). In addition to their roles in protecting cells from stress, almost all HSPs are constitutively expressed in organisms at normal conditions, where they function as chaperones to ensure the correct folding of proteins and assist the translocation of proteins across intracellular membranes,,. While there have been studies correlating the expression of heat shock protein to temperature shock, there are no reports of the HSP response to gamma irradiation in Aedes.
Beyond its role as molecular chaperones, some members of the HSP family have been reported to be involved in the development of sterility. Hsp70, for instance, plays a crucial role in spermatogenesis and thus has been linked to male sterility in Drosophila,. Hsp70 family of the proteins located within the cytoplasm, one of the members of this family, Hsc70 constitutively expressed in spermatogonial cells. It was also noted that Hsc70 is required as members of a chaperone complex for activating the ecdysone receptor which controls the successive stages of the insect’s life cycle and also contributes to other processes such as reproduction,. Likewise, Huang et al. found an inverse correlation between thermal protection and fecundity with respect to the overexpression of Hsp70 in Liriomyza huidobrensis. Temperature-induced male sterility has been reported in Drosophila buzzatii as well. Also, the involvement of Hsp83 in spermatogenesis has been recorded.
Although gamma radiation based sterility has been extensively studied in Ae. aegypti particularly because of work on SIT,,, the plausible role of HSPs in the pathogenesis of sterility has not been looked at into. An earlier study from our lab recorded above 90% male sterility to 50Gy of gamma radiation exposure in Ae. aegypti. Thus further investigation is necessary to validate the possible secondary role of HSPs in the induction of radiation-induced male sterility in the said species.
Hsp83 identified in the bands A and C corresponding to the ~80–95kDa bands of samples collected at 6- and 12-hours post-irradiation, respectively, was significantly up-regulated (~3 fold) at both time points, when compared to the control (band E) [Figure 1]. RNA expression studies have revealed a 40-to-50-fold increase in Hsp83 levels in Ae. aegypti larvae and pupae post heat shock treatment to 42oC. Of course, the stress response with respect to gamma radiation and temperature cannot be compared in this case because there is a known lack of absolute correlation between mRNA and protein expression levels. AeaHsp83 are important markers of stress and have been postulated to function as critical proteins involved in the protection and enhanced survival of Ae. aegypti. A ~3- fold increase in the expression levels of this protein at 50 Gy exposures could explain the survivability of adults exposed to high doses of gamma radiation. Also, this increase in AeaHsp83 expression may be associated with the sterility that is induced at this dose.
Hsp genes in the same organism are likely to respond differently to different stimuli. A heat shock study in Ae. aegypti, for instance, has shown that exposure to higher temperature has little effect on the RNA expression of AeaHsc70. In our study, however, the analysis of gel bands B and D identified Hsc70 (Heat shock cognate 70) to be significantly (P<0.05) more abundant post-exposure to the gamma radiation. An approximate 2 and 2.5 fold up-regulation were observed following 6 and 12 hours of irradiation, respectively, when compared to control (band F). Although no studies on gamma-radiation induced HSP genes in mosquitoes are documented, there are a few studies on other insects. A study of the Indian meal moth, Plodia interpunctella, for instance, has also reported an increased level of Hsc70 in response gamma radiation exposure. Hsc70 expression appears to be triggered by gamma radiation and not temperature stress in Ae. aegypti. However, the gamma radiation induced expression of Hsc70 seems to be conserved across various species of insects. Another study has shown that Chironomus ramosus larvae expressed Hsp70 upon gamma-radiation exposure and has postulated Hsp70 might be one of the gamma radiation-induced stress proteins required during the early stages of radiation stress management. Hsc70 thus does play a role in the tolerance of high doses of radiation induced stress in Ae. aegypti and hence could be considered as a potential biomarker for the detection of high doses of radiation induced stress.
The results of this study become more interesting in the light of the fact that several families of heat shock proteins known to be expressed in mosquitoes may have a cumulative role in determining the susceptibility to the virus. In Anopheles gambiae, it has been shown that the expression of Hsc70B is induced not only by heat shock but also during an arbovirus infection, and Hsc70B protein is expressed to cope with cellular stress imposed during infection. On the other hand, it was also shown that the Hsc70B protein product has important roles in homeostasis and suppression of o’yong-nyong virus replication in the vector, An. gambiae. In this context, a 2-3 fold up regulation of Hsc70 and Hsp83 would thus indicate that at this dose, it may contribute to the susceptibility of Ae. aegypti to viruses.
Apart from the HSPs, the experiment also picked up few other proteins whose expressions were dysregulated upon irradiation. 19 other differentially expressed proteins, which have important biological functions, were identified, providing a basis for understanding their possible roles in response to gamma radiation stress. Of these, 13 proteins were more abundant when compared to that from control, while the remaining 6 proteins were less abundant [Table 1] & [Table 2]. The differentially expressed proteins identified from this study were ATP citrate synthase isoform X1, activity of metabolic enzyme pyruvate carboxylase, metabolic enzyme pyruvate kinase, glutamate semialdehyde dehydrogenase, tropomyosin invertebrate, protease m1 zinc metalloprotease, alanyl tRNA synthetase (exhibiting the highest fold change among all identified), arginine kinase, dihydropyrimidine dehydrogenase, Pyrroline 5 carboxylate dehydrogenase, Enzyme esterase B1, Nucleoside diphosphate kinase, and Putative calreticulin. The activities of proteins were low until 6-hour period and showed a gradual increase after 12-hour post irradiation over the control group. Although these findings are beyond the scope of our study, we feel it is important to discuss a few of them which are particularly relevant in the context of insect stress management.
Arginine kinase, which plays a role in the regulation of energy metabolism in cells, by catalyzing the formation of ATP from ADP (or vice versa) showed a 2.80-fold increase at 6 hours post radiation treatment. This enzyme is known to be prevalent in systems with fluctuating energy demands, acting as an energy buffering system and an energy shuttle, delivering ATP generated by mitochondria to high energy requiring processes, such as membrane turnover. Its presence is crucial when emerging from a dormant state and oxidative stress, acting as a modulator of energetic reserves under such conditions,.
Increased expression of arginine kinase, has been reported in other Dipterans exposed to infection,,. It seemed to modulate the adaption of insects to adverse environments,. Hence an upregulation of this enzyme post gamma radiation may play an important role in the survival of Ae. aegypti.
A 2.38-fold upregulation was observed in another enzyme Esterase B1 in Ae. aegypti at 12-hours post-irradiation. Interestingly, elevated levels of Esterase B1 have been observed in several insecticide resistant strains of Ae. aegypti, possibly to overcome insecticide induced stress,. An overproduction of esterase B1 has also been recorded in insecticide resistance strains of few species of Culex against organophosphates. An increased activity of esterase was noted in a pyrethroid resistant population of Anopheles albimanus Wiedemann. If esterase levels are an indication of insecticide resistance, then this implies that gamma radiation could influence the susceptibility of Ae. aegypti to insecticides. Although, of course, this would warrant more temporal analyses to conclude the expression levels of the enzyme beyond the 12 hours’ time point before such a correlation is drawn.
Two proteins required for embryonic, larval and germline development, 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin activating enzyme E1, were found to be downregulated in our dataset. The ubiquitin-proteasome system, through influencing protein stability plays an essential role in cell cycle regulation, stress, DNA repair, and carcinogenesis,,. The polyubiquitinated proteins generated by ubiquitin-conjugating system serve as substrates for the proteasome,. In short, proteins modified through one of the ubiquitin-activating enzyme E1are further targeted by the 26S proteasome for degradation. The results of this study are therefore in agreement with a previous study that reported that oxidative stress induces the loss of activity of 26S proteosome.
| Conclusion|| |
Response patterns of Ae. aegypti adults to gamma radiation stress conditions are complex, as the differentially abundant proteins are involved in multiple functional categories. The present study has identified a number of differentially expressed proteins, which can be chosen for further validation, and understanding the specific role of these proteins will provide new insights into the adaptive mechanisms of Ae. aegypti in response to gamma radiation stress. Our experiments revealed that gamma irradiation influenced the expression profile of HSP genes in Ae. aegypti. Higher expressions of these proteins suggest that they could be involved in the enhancement of the survival of Ae. aegypti and thus might be a potentially useful tool as a molecular marker for detection of radiation-induced stress.
Conflict of interest: None
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[Table 1], [Table 2]