“Ice and Fire”: Thermal Stress Dynamics and Adaptations in Drosophila melanogaster

Abstract

Throughout my thesis, I have subjected Drosophila melanogaster to cold stress (across mul ple life stages, where appropriate) and inves gated its effects in terms of behaviour and various life history traits of the fly such as developmental me, survival, and fecundity. In this context, I have also studied popula ons of D. melanogaster that were selected for cold stress tolerance and their controls, so that I can understand both the immediate and evolu onary responses to cold stress. I have tried to unveil molecular and physiological mechanisms underlying the associated adapta ons. Outline of the thesis (a) Impact of thermal stress on locomo on in D. melanogaster Behavioural traits such as locomo on and flight are central to life as they influence ma ng, foraging and avoidance of stressful environments. Behavioural traits are also essen al for thermoregula on for ectotherms who cannot maintain body temperature internally. However, behavioural traits are highly temperature sensi ve and are progressively impaired outside the opmum temperature ranges (Berrigan & Partridge, 1997). Consequently, thermal stress impacts behavioural traits such as locomo on and flight way before any effects can be observed in terms of survivorship (Kjærsgaard et al. 2015). Thus, any environmental change that reduces locomotory performance has consequences for mortality rates and lifeme reproduc ve success, more so in the light of climate change. Previous research has shown that thermal stress (both heat and cold) results in deficits in the locomotory performance of ectotherms (Garcia & Teets, 2019; Kjærsgaard et al., 2010). Although the underlying mechanisms of heat stress and cold stress are yet to be fully understood, it is clear that cold stress dysregulates the ion balance, resul ng in neuromuscular dysfunc ons (Andersen et al., 2017), affec ng the complex behaviours dependent on it. In D. melanogaster, cold stress can induce temporary paralysis (Andersen et al., 2015), disrupt ma ng behaviour (Singh et al., 2015), and reduce reproduc ve output (Rinehart et al., 2000). These deficits can be reduced/mi gated with selec on or acclima on (Anderson et al., 2005; Armstrong et al., 2012; Gerken et al., 2016; Singh et al., 2015). However, the evolved cold resistance or hardening has not yet been fully explored at the level of behavioural traits. Therefore, in the first part of my thesis, I inves gated the effects of cold stress and selec on for cold stress resistance using locomotory performance as the focal trait of the study. Through this study, I have explored the following ques ons: 1) How do cold shock and heat shock affect the climbing ability of D. melanogaster? 2) How does selec on for increased cold resistance impact the climbing ability? 3) How does selec on for increased cold resistance affect climbing ability a er heat shock? 4) How does rapid cold/heat hardening affect locomotory performance? (b) Evolu on of female reproduc ve traits in response to selec on for increased cold resistance Exposure to low temperatures is known to influence the reproduc ve fitness of many arthropods, including ma ng behaviour, male fer lity, sperm compe ve ability, female fer lity, and fecundity (Chakir et al., 2002; Mensch et al., 2017; Mocke & Matsumoto, 2014; Singh et al., 2015, 2016; Singh & Prasad 2016). Exposure to low temperatures directly affects gamete viability, as it makes sperm and eggs inviable (Novitski & Rush, 1949). Addi onally, exposure to cold shock also kills the sperm stored in female reproduc ve organs (Novitski & Rush, 1949), resul ng in reduced female fer lity and egg viability post-cold shock (Singh et al., 2015). To regain fer lity, females first need to eliminate inviable sperm and gather new sperm by rema ng (Singh et al., 2015). This highlights the importance of sperm processing post-cold shock (Lefevre & Jonsson, 1962). In addi on to the direct impact of gamete viability, cold stress can also affect reproduc ve output by decreasing reproduc ve frequency, as reported in D. melanogaster (Best et al., 2012), the adzuki bean beetle, Callosobruchus chinensis (Katsuki & Miyatake, 2009) and cricket, Acheta domes cus (Kindle et al., 2006). Various studies have reported the evolu on of life history traits in response to selec on, where the popula on selected for resistance to cold stress evolved increased survivorship post-cold shock (Chen & Walker, 1993; Tucić, 1979). However, very few studies have explored the evolu on of reproduc ve traits in response to selec on. Singh et al. (2015) reported increased egg viability, ma ng frequency, and male ma ng ability in popula ons selected for cold shock. Therefore, it is likely that adapta on to cold shock also involves changes in the reproduc ve physiology of the females. For that, I inves gated the process of sperm dumping in females selected for resistance to cold shock, a trait essen al in rema ng post-cold stress. I hypothesized that selected females are be er at sperm handling post-cold shock compared to control females. (c) Evolu on of life stage-specific thermal tolerance Holometabolous insects like Drosophila melanogaster have dis nct developmental stages with varying thermal requirements for their juvenile and adult forms. These stages are directly dependent on temperature, and understanding the cold tolerance of early development stages is crucial for esma ng species' ability to cope with thermal changes. In Drosophila, cold resistance varies across developmental stages, with larval stages being more sensi ve than both pupae and adults (Jensen et al., 2007; Tucić, 1979). However, the poten al of pre adult stages for cold adapta ons is unexplored and unclear. Addi onally, each developmental stage might have a different capacity for phenotypic plas city due to morphological and physiological differences. The adult stage in Drosophila melanogaster has been recognized to rapidly cold harden in response to pre-exposure to mild cold shock, but li le is known about evolu onary responses to subzero temperatures across different life stages. This study inves gates the inherent cold tolerance of each stage and the poten al gene c link between selec on for cold tolerance at the adult stage and the evolu onary response at the other stages. In addi on to that, I also explored the capacity of pre-adult stages for rapid cold hardening. (d). Gene expression in response to cold adapta on In response to changes in environmental temperature, most organisms undergo metabolic rate adjustments along with various other physiological changes. Ectotherms, which cannot regulate their body temperature and alter their metabolic rates internally, have evolved intricate mechanisms to cope with exposure to low temperatures (Lee, 1991). Some of these mechanisms include the synthesis of an freeze proteins (Duman, 2001) and the accumula on of cryoprotectants such as glycerol and trehalose, and other low molecular weight polyols and sugars, which are known to protect against cold-induced damage and improve supercooling. Addi onally, ectotherms also upregulate an array of genes to enhance their cold tolerance. For instance, recent discoveries have highlighted the cri cal role of Heat Shock Proteins (HSps) in cold adapta ons. While HSPs are tradi onally associated with heat stress, mul ple studies have reported their induc on in response to various physiological stresses such as cold (Goto & Kimura, 1998), desicca on (Tammariello et al., 1999), inbreeding (Kristensen et al., 2006), and high larval density (Sørensen & Loeschcke, 2001). In a pioneering study, Burton et al. (1988) reported the synthesis of 70 kDa heat shock proteins (Hsps) during recovery from chronic exposure to 0°C in the absence of a tradi onal heat shock. A er this study, the cold-induced up-regula on of the Hsps complex has been consistently verified in drosophilids at both mRNA and protein levels. Addi onally, Colinet et al. (2010) observed that reducing the expression of small Hsp22 and Hsp23 genes through RNA interference (RNAi) led to an increased chill coma recovery me. These findings, combined with studies on various other insect species, collec vely support the no on that Hsps play a crucial role in enhancing cold tolerance in insects (Ště na et al., 2015). Apart from these, several other Hsps (Hsp22, Hsp23, Hsp26, Hsp27, Hsp40, Hsp68, Hsp70Aa, and Hsp83) are also reported to be involved in cold tolerance in D. melanogaster (Colinet et al., 2010b). Apart from HSPs, a few other genes, such as Frost (Fst) and Stv, are also known to be involved in recovery from chill injury (Colinet et al., 2010a). Through this study, I have tried to explore the gene c basis of evolved cold resistance. Towards this, I have studied the temporal expression of mul ple Hsp genes and frost gene in popula ons adapted to cold shock and their controls.