Elsevier

Fish & Shellfish Immunology

Volume 105, October 2020, Pages 203-208
Fish & Shellfish Immunology

Full length article
Heat shock increases hydrogen peroxide release from circulating hemocytes of the snail Biomphalaria glabrata

https://doi.org/10.1016/j.fsi.2020.07.029Get rights and content

Highlights

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    Heat shock increases NOX2 mRNA expression in snail hemocytes.

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    Heat shock increases hydrogen peroxide production by snail hemocytes.

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    Heat shock induced hydrogen peroxide production is HSP-90 dependent.

Abstract

Planorbid freshwater snails are important intermediate hosts for parasitic diseases caused by parasitic worms, most notably schistosomiasis. There are numerous reports of snails, specifically Biomphalaria glabrata, having compromised defences against schistosomes after being exposed to thermal stress. Environmental modifications to the defenses of schistosome transmitting snails could have negative ramifications for human disease risk in the context of climate change. Here the effects of heat shock on the production of hydrogen peroxide, a primary anti-microbial effector in many molluscs, were examined. The present findings show that heat shock increases NADPH oxidase 2 mRNA levels and hydrogen peroxide produced by snail hemocytes, and that both of these phenotypes could be reversed by an HSP-90 inhibitor. These findings indicate that snail defense systems are altered by heat shock at a molecular level in B. glabrata, and that snail immunity to many pathogens may be altered by the rapid variations in temperature that are associated with global climate change.

Introduction

Freshwater planorbid snails are obligate intermediate hosts for various parasitic worms which can cause both human and veterinary diseases. Schistosomiasis is the most detrimental disease caused by parasitic worms, because it can affect numerous mammalian species and is the cause of extensive human illness and hundreds of thousands of deaths per year [1]. The snail Biomphalaria glabrata (Bg) is a notable intermediate host of the schistosome parasites that cause schistosomiasis, and has a primary role the transmission of Schistosoma mansoni (Sm) in the Americas. Understanding the physiology and defense systems of schistosome transmitting snails, like B. glabrata, may be important for the development of methods to control snail populations and schistosome infection or transmission [2,3].

The production of reactive oxygen species (ROS) by hemocytes in B. glabrata has been shown to be vital for snail defense against schistosome infection [[4], [5], [6], [7], [8], [9], [10], [11]]. In hemocytes, NADPH oxidase 2 (NOX2) dependent ROS are a substrate for Cu/Zn superoxide dismutase's (SOD1) production of hydrogen peroxide, which is a potent anti-microbial effector and one of the primary mechanisms by which hemocytes kill schistosome larvae [6,8,12,13]. Genetic or environmental factors that alter the efficiency of hydrogen peroxide production by hemocytes can modify the resistance of Bg to Sm infection, and thus may help to determine the magnitude of transmitted parasites to mammalian hosts [4,6,9]. It is important to note that the activity of intraphagosomal NOX2, and the subsequent enzymatic activity by antimicrobial enzymes including SOD1, can alter the efficiencies of many immune pathways and are completely essential for anti-microbial defenses in most organisms [12,14]. Most importantly, superoxide, hydrogen peroxide, and hypochlorous acid are primary anti-microbial effectors, and any alteration to the activities of the enzymes that produce these compounds can have drastic effects on immune defense [14].

Recently, it has been shown that alterations in water temperature can modify the physiology of Bg and/or its ability to resist schistosome infection [[15], [16], [17], [18], [19], [20], [21]]. Specifically, heat shock of resistant Bg can increase their susceptibility to schistosome infection, though this phenotype is partially dependent on the Bg strain/genotype and duration of heat shock [[15], [16], [17],21,22]. Heat Shock Proteins (HSP) are produced when cells experience a diverse array of environmental stressors. One such stress, transient elevated temperatures or ā€˜heat shockā€™, has been shown to induce the expression of numerous heat shock proteins, which trigger responses in multiple systems and activate numerous pathways. Generally, these responses promote stabilization of proteins, and help to regulate immune responses [23]. In the context of snails, heat shock of resistant snails increases the expression of HSPs, specifically HSP-70 and 90 [15,17]. Chemical inhibition of HSP-90 was shown to prevent the reversal of the resistant phenotype in BS-90 snails [15]. These findings have implicated HSPs in schistosome defense, and also suggest that the increasing likelihood of heat shock, in the context of global climate change, may increase the susceptibility of Bg to schistosomes. No physiological mechanisms currently explain why Bg can become more susceptible to infection when they are exposed to elevated temperatures.

Given that Guadeloupean B. glabrata (BgGUA) snails do not exhibit modified infection phenotypes to schistosomes after heat shock [22], one would not expect that their primary immune defense (hemocyte produced ROS) would be altered by heat shock events. Contrary to this prediction, this study links one essential mechanism of hemocyte defense with heat shock exposure. The presented findings show that heat shock of BgGUA increases hydrogen peroxide production by hemocytes in an HSP-90 dependent manner. These results also demonstrate that this elevated hydrogen peroxide production is likely the result of increased NOX2 mRNA levels, rather than increased SOD1 mRNA levels. Thus, there may be more substrate for SOD1 to convert to hydrogen peroxide rather than more SOD1 mRNA expression. Though elevated hydrogen peroxide is associated with schistosome resistance in Bg, and may help to explain the resilient infection phenotype in heat shocked BgGUA, extensive ROS production is a double-edged sword. Excessive ROS can contribute to dysregulation immune responses as well as bystander damage to self tissue. Therefore, these findings may also implicate heat shock with bystander damage during immune responses.

Section snippets

Animals and ethics

BgGUA were collected in 2005 from Guadeloupe, and were housed and maintained identically as previously described [[24], [25], [26], [27], [28]]. In brief, snails were housed in 12ā€“25-gallon glass tanks temperature in 26Ā Ā°C dechlorinated water. Experiments adhered to the Public Health Service Domestic Assurance for humane care and use of laboratory animals (PHS Animal Welfare Assurance Number A3229-01), as Animal Care and Use Proposal 4360; which was approved by the Oregon State University

BgGUA hemocytes exhibit elevated hydrogen peroxide production after heat shock

PMA induces the canonical hydrogen peroxide pathway beginning by activating protein kinase C and mitogen-activated protein kinases [10,37]. Subsequently, NOX2 is activated and produces superoxide which is then converted to hydrogen peroxide by SOD1. These findings verify that PMA-induced hydrogen peroxide production can be inhibited by DPI exposure in BgGUA hemocytes, and that PMA stimulation is sufficient for hemocyte hydrogen peroxide production (Fig. 1a) [4,9,10,13,38]. PMA stimulated

Discussion

The interplay between global climate change and infectious disease risk is complex and will likely be very disease specific. Mathematical models of schistosome infection currently postulate that warming trends may increase global schistosomiasis risk primarily because they suggest that there will be an increase in the range of intermediate snail species (like Bg) outside of their current control areas [[42], [43], [44]]. Additionally, direct experimental approaches, addressing the hypothesis

CRediT authorship contribution statement

Euan R.O. Allan: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Michael S. Blouin: Funding acquisition, Project administration, Resources, Supervision, Writing - original draft, Writing - review & editing.

Acknowledgements

This work was supported by the National Institutes of Health [AI109134] awarded to M.B. and the SGU Small Research Grant Initiative [SRGI-18022] awarded to E. A.

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