Heat shock response and metabolic stress in the tropical estuarine copepod Pseudodiaptomus annandalei converge at its upper thermal optimum

Highlights

• Cellular and metabolic responses to thermal stress in a tropical estuarine copepod.

• Maximal mRNA gene (hsp70 and hsp90) expressions occur at 32–33 °C.

• Physiological and molecular responses to thermal stress converge at 32.3 °C.

• This convergence is 3–4 °C above the mean annual temperature of its habitat (29 °C).

• An early preparative HSR strategy is proposed for Pseudodiaptomus annandalei.

Abstract

Heat shock response (HSR), in terms of transcription regulation of two heat shock proteins genes hsp70 and hsp90), was analysed in a widespread tropical copepod Pseudodiaptomus annandalei. The mRNA transcripts of both genes were quantified after copepods at a salinity of 20 underwent an acclimation process involving an initial acclimation temperature of 29 °C, followed by gradual thermal ramping to the target exposure temperature range of 24–36 °C. The respective cellular HSR and organismal metabolism, measured by respiratory activity at exposure temperatures, were compared. The fold change in mRNA expression for both hsp70 and hsp90 (8–9 fold) peaks at 32 °C, which is very close to 32.4 °C, the upper thermal optimum for respiration in the species. Unexpectedly, the modelled HSR curves peak at only 3 °C (hsp90) and 3.5 °C (hsp70) above the mean water temperature (29.32 °C) of the copepod in the field. We propose that copepods in tropical waters adopt a preparative HSR strategy, early at the upper limit of its thermal optimum, due to the narrow thermal range of its habitat thus precluding substantial energy demand at higher temperatures. However, the model suggests that the species could survive to at least 36 °C with short acclimation time. Nevertheless, the significant overlap between its thermal range of hsp synthesis and the narrow temperature range of its habitat also suggests that any unprecedented rise in sea temperature would have a detrimental effect on the species.

Introduction

The calanoid copepod Pseudodiaptomus annandalei Sewell, 1919 is one of the most common or abundant calanoid copepods found in tropical and subtropical estuarine waters (Chen et al., 2006, Beyrend-Dur et al., 2011), brackish waters (Golez et al., 2004) and mangroves (Hwang et al., 2010, Chew et al., 2015). This species is not only important as a natural food source for estuarine fish (Chew et al., 2012), but is also used as a larval feed in fish farms (Chen et al., 2006, Rayner et al., 2015). Despite its ecological and commercial importance, it is only recently that simulative experiments have been used to study the molecular or organismal response to environmental stressors in tropical copepods (Beyrend-Dur et al., 2011, Jiang et al., 2013, Lehette et al., 2016). Manipulative experiments of this nature are useful to not only increase copepod production (in aquaculture), but also to better understand how these marine organisms which constitute vital trophic links in marine food webs (Kiørboe, 1997, Chew et al., 2012), respond to thermal stress (e.g. Jiang et al., 2009; Beyrend-Dur et al., 2011). For instance, laboratory experiments have shown that adult P. annandalei experience thermal stress above 32 °C as the respiration rate at 36 °C almost doubled that at 32 °C (Lehette et al., 2016). Subsequent experiments on the developmental acclimation of P. annandalei could confirm that its upper thermal optimum most likely occurred at 32.4 °C, beyond which thermal stress in respiration sets in (Lehette and Chong, 2016). This temperature is also likely to be close to the pejus temperature. Both experimental results on adults, and developmental acclimation, suggest that such experimental data could help to improve climate change predictive models (Lehette and Chong, 2016).

The oxygen consumption rate provides an indication of organismal metabolism (Clarke and Fraser, 2004, Seibel and Drazen, 2007), including the thermal tolerance mechanism which, in part, involves oxygen transport (Pӧrtner, 2002). Although respiration rate has been used as a physiological indicator of thermal stress in animals (i.e. at the organismal level), recent studies suggest the usefulness of more direct bio-indicators at the cellular level, such as the families of biochemical (Anestis et al., 2010, Jung and Lee, 2012) and molecular markers that mainly involve heat shock proteins (HSPs) such as HSP70, HSP90, and HSP100 (Feder and Hofmann, 1999, Shabtay and Arad, 2005).

The HSPs have been widely used as stress markers in aquatic organisms, e.g. molluscs (e.g. Farcy et al., 2009; Liu and Chen, 2013), sponges (Krasko et al., 1997), corals (Fang et al., 1997), barnacles (Berger and Emlet, 2007) and copepods (Rhee et al., 2009, Xuereb et al., 2012). HSPs are a large, highly conserved group of molecular chaperones that are needed for folding, unfolding, assembly, disassembly and degradation of proteins, especially during conditions involving thermal or osmotic stress and chemical exposure, in which proteins are potentially damaged (Ellis, 2013, Saibil, 2013). However, considerable variability in the heat shock response (HSR) is observed in marine organisms depending on the species, tissue, HSP family, developmental stage, type of stressor and geographical location (Feder and Hofmann, 1999, Pӧrtner et al., 2007, Tomanek, 2008).

The first gene sequence for hsp70 in a copepod was reported by Voznesensky et al. (2004) for Calanus finmarchicus (Gunnerus, 1770). Since then, other temperate copepod species are increasingly being used as test models for the assessment of thermal and other environmental stresses (e.g. Tartarotti and Torres, 2009; Xuereb et al., 2012). The HSR, as expressed by hsp70, was the most pronounced among ten different hsp gene transcripts investigated in the intertidal benthic copepod Tigriopus japonicus Mori, 1938 (Rhee et al., 2009). The only hsp study on P. annandalei does not relate to HSR but to the expression of functional genes in laboratory-reared copepods exposed to nickel toxicity (Jiang et al., 2013), and this study involved a subtropical population. To our present knowledge, no hsp studies have been conducted on copepods found in tropical regions. Furthermore, with a few exceptions (e.g. Li et al., 2015; Rahlff et al., 2017), integrative studies that explore how copepods respond to heat shock to restore homeostasis, at both the cellular and organismal levels, are rare. Such studies are needed to provide greater confidence in our understanding of the HSR, and whether it could be combined with physiological methods to accurately predict climate change effects.

In this study, we assessed whether the mRNA expression of the hsp70 and hsp90 genes could be used as useful stress markers in laboratory-acclimated P. annandalei exposed to a gradual temperature shift from the natural environmental temperature of 29 °C to temperatures that ranged from 24° to 36°C. Here, we hypothesize that the copepod's hsp protective response should peak at, or close to, 32 °C (the upper thermal optimum) given the narrow thermal range (26.5–32.1 °C) of its tropical estuarine habitat (Ramarn et al., 2012, Yong et al., 2016), and a metabolic energy requirement that increases very substantially above 32 °C (Lehette et al., 2016).