Genomic knockout of hsp23 both decreases and increases fitness under opposing thermal extremes in Drosophila melanogaster
Graphical abstract
Introduction
A key concept in understanding the mechanistic basis of environmental adaptation is the idea that genes upregulated or downregulated under certain conditions help organisms to cope with those conditions. In animals, the products of upregulated genes may help to detoxify plant secondary metabolites (Schweizer et al., 2017), trigger colour changes to protect against predators (Yoda et al., 2021), and counter the effects of stressful climatic conditions (Gonzalez-Tokman et al., 2020). There are numerous examples in the literature of differentially regulated genes affecting the ecology of species, such as the expansion of host range in green peach aphids (Myzus persicae) (Mathers et al., 2017), the development of high temperature tolerance in corals such as Acropora hyacinthus (Barshis et al., 2013) and Porites astreoides (Kenkel et al., 2013), dealing with thermal and humidity stresses in Tribolium castaneum (Koch and Guillaume 2020), and dealing with predators in guppies (Poecilia reticulata) (Ghalambor et al., 2015).
With respect to changes in gene expression in response to climatic stresses, one group of genes that has been intensively studied is the heat shock protein or hsp gene group. The hsp genes are upregulated particularly under heat stress, but they are also upregulated under many other conditions including cold stress, conditions that induce diapause, desiccation stress, starvation, and anoxia (King and MacRae 2015; Rinehart et al., 2000). Small heat shock proteins (sHSPs) are key members of the HSP family, which are ubiquitously expressed in organisms from all three domains of life, especially after being exposed to environmental stress (Li et al., 2009). Most shsps are highly conserved in both sequence and expression pattern and might have diverged in function across insects to diverse environments (Li et al., 2009). They mainly function as molecular chaperones to protect proteins from being denatured under heat and cold stress (Li et al., 2009; Sun and MacRae 2005; Van Montfort et al., 2002; Wang et al., 2017). In addition, sHSPs can also protect against other stresses such as drought, oxidation, hypertonic stress, UV, and heavy metals (Dasgupta et al., 1992; Waters et al., 2008).
Although shsps and other hsps are typically upregulated under stress, it has been known for some time that this upregulation can come with costs (Sorensen et al., 2003). The relationship between expression levels and fitness variation can be complex (Chen et al., 2018; Evans 2015; Feder and Walser 2005; Koch and Guillaume 2020), particularly because transcript level abundance may not always be indicative of protein abundance and enzyme activity. Stressful conditions may trigger plastic increases in gene expression that are not necessarily adaptive (Ghalambor et al., 2015; Koch and Guillaume 2020) and that depend on genetic background, so it is always important to follow up gene expression studies with functional characterization of the impact of specific genes (Sarup et al., 2011).
Among all the shsp genes, hsp23 is thought to play a key role in temperature tolerance and thermal adaptation in insects. Overexpression of muscle-specific hsp23 may promote proteostasis and protect muscle from heat stress in Drosophila melanogaster (Kawasaki et al., 2016). Moreover, overexpression of hsp23 gene in female ovaries produces offspring embryos with improved thermal tolerance (Lockwood et al., 2017). In other species such as Leishmania donovani, the scuttle fly Megaselia scalaris and the whitefly Bemisia tabaci, hsp23 expression is necessary for surviving extreme temperature treatments (Diaz et al., 2015; Hombach et al., 2014; Malewski et al., 2015). In two invasive Bactrocera species, the expression of hsp23 is closely related to heat hardening (Gu et al., 2019). Apart from heat stress, studies involving RNAi and other approaches have also emphasized the role of hsp23 in cold tolerance (Colinet et al. 2010a, 2013; Rinehart et al., 2007).
A challenge in assessing the phenotypic effects associated with genes like hsp23 is that techniques used to modify expression of a gene may have other effects. While RNAi can knock down the expression of a specific gene, off-target effects can lead to erroneous conclusions about its function (Heigwer et al., 2018; Kulkarni et al., 2006; Ma et al., 2006). The major source of off-target effects is the loaded RNA-induced silencer exogenous complex consisting of dsRNA, Dicer-2 and R2D2 which can interact with unintended homologous target sequences, such as with incomplete-base pairing through shRNAs or siRNA and partial homology to other transcripts by long dsRNAs (Heigwer et al., 2018; Iwasaki et al., 2010; Ma et al., 2006). In addition, RNAi may provide insufficient downregulation to generate a significant phenotypic response. For example, suppression of the period gene in transgenic flies only caused a 50% reduction in protein level, insufficient to reliably produce mutant phenotypes (Kalidas and Smith 2002; Martinek and Young 2000). In recent analyses of the knockdown efficiency of RNAi lines from a publicly available resource, 90% of RNAi lines exhibited residual gene expression of 25% or more, which can give rise to hypomorphic phenotypes (Heigwer et al., 2018; Perkins et al., 2015). Some RNAi systems can also be inefficient; for example some Drosophila GAL4 strains only express low levels of GAL4 or have insufficient time to allow for turnover of mRNA and protein (Heigwer et al., 2018).
Despite this, gene expression studies involving RNAi and other approaches have emerged as important methods for analysing gene function particularly in D. melanogaster. For shsps, gene knockdown experiments have suggested that hsp22 and hsp23 genes contribute to adaptive responses to fluctuating thermal conditions and particularly in recovery of adults from chill coma (Colinet et al., 2010a). Genetic elimination of the hsp70 up-regulation response decreases survival when the larvae are exposed to severe cold shocks and there is no compensatory response (Štětina et al., 2015). Expression of hsp22, hsp23, hsp68 and hsp70Aa has been linked to age-related plasticity in the induction of cold resistance (Colinet et al., 2013). In flesh flies, suppression of the expression of hsp23 and hsp70 has a profound effect on the survival of pupae at low temperatures (Rinehart et al., 2007). In Pyrrhocoris apterus, hsp70 influences repair of injuries caused by cold stress (Koštál and Tollarová-Borovanská 2009). Expression studies suggest that the shsp genes also play important roles in normal development and other physiological responses (Jagla et al., 2018; Sun and MacRae 2005), with changing patterns of hsp23 expression across time and tissues (Jagla et al., 2018).
CRISPR provides an alternative approach to introduce mutations at sites targeted by an RNA-guided endonuclease in conjunction with a target-specific small guide RNA (sgRNA). It has revolutionized functional genomics studies, providing the capacity to variously create allelic variation in, or completely eliminate the expression of, a specific gene of interest (Doudna and Charpentier 2014; Taning et al., 2017). Due to its high specificity and applicability, CRISPR/Cas9-mediated gene editing has been employed in many organisms including insects and cells, both for fundamental research of gene function and applied research in the modification of organisms of economic importance (Taning et al., 2017). As with RNAi, CRISPR/Cas9 can generate mutations in off-targets (Fu et al., 2013; Hruscha et al., 2013; Zhang et al., 2015). The likelihood of this can be minimized in the design of sgRNAs used and in sequencing those regions with homology to the sgRNAs in mutants isolated to identify any off-target mutations that may arise (Manghwar et al., 2020; Taning et al., 2017). A variety of methods and fly strains have been developed to simplify CRISPR in D. melanogaster where it is routinely used to create somatic and germline variation (Allen et al., 2021; Perry et al., 2021; Port et al., 2014).
To date studies on hsp23 in Drosophila have not analysed the phenotypic impact of a loss of gene function. Here, we used CRISPR/Cas9 to generate a hsp23-knockout mutant in D. melanogaster. This strain was used to test whether hsp23 has essential functions in thermal responses and development. Since the upregulation of hsp genes is thought to be adaptive in stressful environments, we examined whether the hsp23 knockout mutant had reduced fitness under environmental conditions that trigger the upregulation of hsp23. Our work sheds light on the potential effects of hsp23 expression on thermally linked phenotypic plasticity, and provides insights into its roles in stress tolerance and hardening responses. It also highlights the challenges of undertaking gene-based assessments when assessing adaptive plasticity hypotheses.
Section snippets
D. melanogaster strains and genomic deletion of Hsp23
The fly strains used in the experiments are listed in Table 1. Most strains had been kept on cornmeal media at 25 °C, 12:12 L:D cycle and 65% RH for a generation following Richardson et al., (2016).
Genomic sequence of hsp23 was obtained from FlyBase (https://flybase.org). The genomic region harbouring potential sgRNAs at 5′ and 3′ ends were PCR checked and sequenced in the AC9 background (Table 2). sgRNAs targeting the 5′ end and the 3′ end (Table 2) were evaluated using the CRISPR Optimal
Characterization of hsp23 gene knockouts
The pCFD5 vector was chosen for its ability to express multiple sgRNAs ubiquitously (Port and Bullock 2016). The pCFD5(hsp23sg1sg2) plasmid was created, containing two sgRNAs flanking the CDS of hsp23. Each of the sgRNAs is followed by a gRNA core sequence for Cas9 integration and is separated by tRNA sequences. pCFD5 (hsp23sg1sg2) plasmids were injected into 25709 embryos and integrated into the attP site on chromosome 2 through ϕC31-mediated genome integration. The resulting pCFD5(hsp23)
Discussion
We were successful in using CRISPR/Cas9 to knock out the hsp23 gene. In previous studies, RNAi has been used to understand the function of hsp23 but this only results in partial knockdown and transient silencing of gene expression (Heigwer et al., 2018). When investigating the effects of hsp23 on chill coma recovery in D. melanogaster, the use of the act-GAL4/+ line only resulted in 52% knockdown of hsp23 expression (Colinet et al., 2010a). A similar issue was noted in B. dorsalis where feeding
Acknowledgements
We thank Ying Ting Yang and Perran A. Ross for technical assistance. Fly strains were sourced from the Bloomington Drosophila Stock Centre. Funding for this research was provided through Australian Research Council awarded to AAH (DP120100916, www.arc.gov.au/grants).
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