Climate change has driven the Earth’s surface temperatures to increase over the past decades, and simultaneously led to the widespread prevalence of heatwaves (IPCC, 2019). Specifically, regional extremes of atmospheric temperatures above any historical baseline could be termed heatwaves (Hobday et al., 2016). Heatwaves are projected to increase the frequency, intensity, and duration (Frölicher et al., 2018; Perkins-Kirkpatrick and Lewis, 2020) in current era of unprecedented climate change, and cause devastating ecological consequences and economic losses (Smith et al., 2021). Intertidal ecosystems are among the most ecologically and economically productive in the world, but are frequently attacked by intensifying heatwaves (Leung et al., 2019). Atmospheric temperature changes at low tide in the intertidal zone are far more dramatic and extreme than sea surface temperatures (Helmuth et al., 2016; He et al., 2022). Organisms inhabiting intertidal habitats are consequently susceptible to intensifying heatwaves (Balogh and Byrne, 2020; Scanes et al., 2023). Nevertheless, compared with native organisms in the intertidal ecosystems, the fate of highly invasive organisms frequently attacked by heatwaves has received much less attention (Crespo et al., 2021), constraining our capability to assess whether extreme weather events like heatwaves might facilitate their invasions.

Biological invasion represents one of the most urgent ecological threats to global biodiversity (Bellard et al., 2017). Especially, the swiftly spreading invasive mytilid mussels have caused serious fouling issues in intertidal ecosystems worldwide, affecting the local biodiversity, fishery productivity and ecosystem functioning (Amini et al., 2017; Zhao et al., 2020). Charru mussels, Mytella strigata, native to the Atlantic coast of South America from Venezuela to Argentina, represent one of the most typically invasive fouling mytilid species (Sanpanich and Wells, 2019). Over the last decade, M. strigata has rapidly invaded many Asian countries from the tropical Western Atlantic, such as China, the Philippine, and Singapore (Mediodia et al., 2017; Lim et al., 2018; Ma et al., 2022). In the intertidal zone, M. strigata can form dense mussel beds by adhering to various substrates and conspecifics (as shown in Figure 1), preventing almost all other epifauna and infauna from surviving and reducing the area of suitable habitats for motile organisms (Wangkulangkul et al., 2022; Xu et al., 2023b). The strong attachment of byssus to the substratum is the key in sustaining the rapid invasion and fouling of mytilid mussels (Li et al., 2018). Under the context of climate change, consequently, gaining a better understanding of responses of mussel byssus production to extreme weather events such as heatwaves is critical for mussel invasion assessment.

Mussel byssus is an exogenous attachment structure, which is composed of byssal thread and adhesive plaque and secreted by byssal gland, as schematically illustrated in Figure 1. Several proteins synthesized in byssal glands have been identified for byssus formation. For example, Mfp-1 (mussel foot protein-1) is mostly located over the first layer of the byssal thread, Mfp2-4 involved in adhesive plaque attachment, and the precursor collagen known for the building of the byssal framework (Lin et al., 2007; Lee et al., 2011). Element such as Zn²⁺, Fe³⁺, Ca²⁺, Al³⁺ can cross-link with foot proteins through polyphenol oxidase, and play major roles in maintaining the structure and mechanical properties (ductility and rigidity) of byssus (Zhao and Waite, 2006; Li et al., 2018). At the molecular aspect of biological organization, differential expressions of genes in byssal glands can affect the quality and efficiency of byssus production (Li et al., 2017). Ample evidence suggests that the strength of byssus attachment could be dependent on environmental conditions, including food availability, seawater temperature, salinity and acidity (e.g., O’Donnell et al., 2013; Schmitt et al., 2015a; Li et al., 2017; Shang et al., 2021; Huang et al., 2022). Nevertheless, to our knowledge, very little research has been focused on how extreme weather events, especially heatwaves, affect the byssus production of highly invasive mytilid mussels.

The current study aimed at obtaining a deeper understanding of how intensifying heatwaves affect the byssus production of the highly invasive mussel M. strigata that has rapidly invaded the intertidal ecosystems of the South China Sea. Considering the remarkable physiological capacity of M. strigata to cope with heatwaves (Xu et al., 2023a), it is reasonable to hypothesize that at the individual level of biological organization the ability of M. strigata to secrete byssus and attach to the substratum may not be affected by intensifying heatwaves that occur frequently in the South China Sea. It would appear that this species might continue to rapidly invade intertidal ecosystems in an unprecedented climate change era if that were the case.

As demonstrated in Figure 3, exposure of M. strigata to simulated heatwaves significantly affected its byssus production. The duration and intensity of heatwaves, along with their interaction, dramatically affected the number of byssal threads (p < 0.05; Figure 3A). On days 3 and 5, mussels stressed by heatwaves secreted significantly smaller amount of byssal threads than those thrive at ambient temperatures (p < 0.05). The length of byssal threads was significantly affected by the duration and intensity of simulated heatwaves (p < 0.05; Figure 3B), however, no significance interaction was observed between the two factors (p > 0.05). In line with results in the number of byssal threads, the duration and intensity of simulated heatwaves and their interaction significantly affected the diameter (p < 0.05; Figure 3C) and the volume (p < 0.05; Figure 3D) of byssal threads, in particular with significant impacts mostly pronounced on days 3 and 5 (p < 0.05). More specifically, on days 3 and 5, the effect was significant (p < 0.05), but there was no effect on day 1 (p > 0.05). The interaction between heatwaves intensity and duration indicated that the combination of heatwaves intensity and duration had a significant effect on the number, diameter, and volume of byssal threads.

Under the electron microscope, as shown in Figure 4, the diameter of byssal threads secreted from heatwaves-stressed mussels (Figure 4A) was evidently smaller than those formed under ambient temperature (Figure 4B). Exposure of mussels to simulated heatwaves made adhesive plaque much smaller than those produced by unstressed mussels (Figures 4C, D).

Compared with mussels grown under ambient conditions, concentrations of Zn²⁺ and Fe³⁺ in byssal threads secreted by heatwaves-stressed mussels were significantly increased (p < 0.05; Figure 5), and total amounts of Ca²⁺ and Al³⁺ incorporated into byssal threads were significantly decreased (p < 0.05; Figure 5).

As demonstrated in Figure 6, when heatwaves attacked, PPO activity in byssal glands was not significantly influenced by the duration and intensity of simulated heatwaves and their interactions (p > 0.05).

Relative expression levels of the genes involved byssus production in response to simulated heatwaves were shown in Figure 7. Specifically, following exposure to heatwaves, the expression of the foot protein gene Mfp1-3 was up-regulated, but the expressions of genes Mfp-4 and the precursor collagen (PreCol) were down-regulated. Likewise, the expression of cell apoptosis genes increased when heatwaves prevailed. The expressions of extracellular matrix-receptor interaction genes and neuroactive ligand-receptor interaction genes exhibited similar patterns in response to heatwaves, i.e., their levels were consistently down-regulated in heatwaves-stressed mussels.

Considering the survival and physiological tolerance of M. strigata to heatwaves which have frequently occurred in the South China Sea, as demonstrated in Xu et al. (2023a), it is reasonable to hypothesize that M. strigata might have already evolved to cope with impacts of heatwaves on the byssus production. Yet, at various biological levels of organization, it is evident that the capacity of M. strigata to produce byssus was significantly impaired when subjected to heatwaves, thereby offering no support to our previous hypothesis. Heatwaves are among the most important determinant factors capable of constraining the fate of highly invasive fouling species against the background of climate change (Zhao et al., 2019; Xu et al., 2023b), due to their disastrous impacts on organisms and ecosystems (Smale et al., 2019). In particular, heatwaves can facilitate the introduction and establishment of invasive species by enhancing the habitat disturbance of native species (Griffin, 2016). To explain these species- and even habitat-specific discrepancies, it is thus imperative to elucidate how heatwaves affect mussel byssus production.

Virtually unaffected activity of the enzyme PPO observed in heatwaves-stressed byssal glands demonstrates that M. strigata is likely able to implement compensatory mechanisms to cope with heatwaves. PPO plays a fundamental role in the biosynthesis of byssal thread and adhesion of adhesive plaque, by cross-linking the foot protein Mfp-1 within byssal threads and enhancing the conversion of tyrosine residues into dihydroxyphenylalanine required for adhesion (Silverman and Roberto, 2007). When exposed to heatwaves, consequently, highly invasive mussels such as M. strigata might give priority to prevent the thermal degradation of PPO. This assumption is supported by the maintenance of energy budget for essential physiological processes (Xu et al., 2023b). In the extensively-cultured mussel Mytilus coruscus in the East China Sea, thermal stress manipulated at 4°C above mean seawater temperature during summer seasons significantly depressed the PPO activity, and weakened byssus production (Li et al., 2020). Yet, given the unaffected activity of PPO seen in M. strigata subjected to heatwaves, it is reasonable to assume that other physiological processes may be responsible for heatwaves-induced fewer, thinner, and shorter byssal threads and smaller adhesive plaques.

Mussel byssal glands have the ability to absorb metal ions from the surrounding environment through the formation of metal complexes among specific amino acids of the foot protein and heavy metals. In particular, metal ions can participate in the cross-linking of foot protein by interacting with amino acids (cysteine, histidine, etc.), and play an important role in regulating the strength and ductility of byssal threads (Krogsgaard et al., 2013; Reinecke et al., 2017). In mussel byssal threads, Ca²⁺, Zn²⁺, Fe³⁺ and Al³⁺ have been identified as the most abundant elements (Li et al., 2018). The mechanical property and structural integrity of the byssus are also constrained by the balance between concentrations of foot proteins and metal ions (Seguin-Heine et al., 2014). In M. strigata subjected to heatwaves, concentrations of Zn²⁺ and Fe³⁺ in mussel byssal threads significantly increased and Ca²⁺ and Al³⁺ significantly decreased compared with those grown at ambient conditions. Previous studies found that the equilibriums of metal ions in pearl oyster byssal threads were disturbed by ocean acidification, leading to a decrease in byssal thread mechanical properties (Li et al., 2017). Therefore, these findings indicate that heatwaves may also alter the homeostasis of metal ions in byssal threads, likely affecting the adhesion of byssal threads.

Specifically, Ca²⁺ can mediate the coupling of the histidine-rich structural domain of Mfp-4 to the histidine-rich terminus of PreCol, thereby allowing Mfp-4 to function as a macromolecular bifunctional junction (Zhao and Waite, 2006). Due to the down-regulation of Mfp-4 and PreCol gene expression levels, the content of Mfp-4 and PreCol in byssus decreased, so the Ca²⁺ concentration decreased accordingly. Fe³⁺ is the main element of byssus adhesion properties (Yap and Tan, 2007), and mussels pump Fe³⁺ through the byssus to enhance the adhesion strength (Sever et al., 2004). As demonstrated by Hwang et al. (2010) and Lee et al. (2011), Fe³⁺ could significantly affect the interaction of Mfp-2 and Mfp-3 by binding to specific amino acid residues and maintaining the core function of the adhesive plaque. Zn²⁺ is mainly cross-linked to Mfp-1. In addition, mussels can opportunistically replace Al³⁺ with Fe³⁺ in the byssal threads (Schmitt et al., 2015b), and Fe³⁺ can replace Al³⁺ to maintain the stiffness and hardness of the byssus. These observations may lend further support to significant changes of Ca²⁺, Zn²⁺, Fe³⁺ and Al³⁺ concentrations in the byssal threads.

The expressions of genes involved in byssus production provide further insights into impacts of heatwaves at lowered levels of biological organization. Expressions of four categories of genes (foot protein gene, cell apoptosis gene, extracellular matrix-receptor interaction gene, and neuroactive ligand-receptor interaction gene) in byssal glands demonstrate that changes in the quantity and quality of byssus may be caused by changes in the physiology of byssal glands. Caspases play a key role in the apoptotic pathway (Creagh et al., 2003). Caspase-3 and Caspase-9 genes are activated in oysters subjected to ocean acidification thus triggering apoptosis (Kvitt et al., 2015), and these studies suggest a close link between apoptosis and environmental stress. Following exposure to heatwaves, the expression levels of Caspase-3 and Caspase-7 genes were significantly increased, indicating that byssal glands of M. strigata may be affected by heatwaves by triggering the apoptosis process. In addition, the response of byssus production to heatwaves can be explained in energetic terms. Adaptation and stress tolerance in bivalves are limited by energy balance (Sokolova et al., 2012). Byssus production is extremely energy intensive, accounting for 2% to 10% to nearly 50% of the daily energy budget (Roberts et al., 2021). Therefore, when apoptosis occurs in the organism, more energy may be preferentially allocated to maintain fitness-related functions (Leung et al., 2020), leading to a decrease in byssus production. The findings are consistent with Sokolova et al. (2012)’s metabolic principles of stress response.

The impact of heatwaves on the byssus production of M. strigata was also reflected in the down-regulated expression of extracellular matrix-receptor interaction genes and neuroactive ligand-receptor interaction genes. The level of expression of extracellular matrix-receptor interaction genes can affect muscle performance of byssal glands (Li et al., 2017). The extracellular matrix, a mixture of structural and functional macromolecules such as tenascin, laminin and collagen (Humphrey et al., 2014), is essential for tissue structural integrity and plays a critical role in controlling adhesion. The down-regulation of collagen, tenascin and laminin gene expression levels reflects the lower expression of genes encoding these macromolecules in mussels, which may affect the motility of the byssal gland and thus lead to reduced byssus production, as byssal gland extension is a prerequisite for byssus production. (Yu et al., 2011; Andrade et al., 2015). Behavioral changes in organisms are regulated by the nervous system, which has recently received more attention (Wang and Wang, 2020). Expression levels of multiple nerve-related genes in byssal glands of M. strigata were down-regulated following exposure to heatwaves, suggesting that heatwaves can induce mussel nervous system dysfunction, which may in turn change the behavior of byssal glands. However, the specific mechanisms involved are still poorly understood and deserve further investigation.

The present study demonstrates that heatwaves can have negative impacts on byssus production of highly invasive mussels M. strigata. These impacts can be manifested at various biological levels of organization. From the individual appearance, the heatwaves led to the decline of the byssus production and quality (length, diameter and volume of byssal threads); from the physiological level, the heatwaves disturbed the metal ion content of the byssal threads, affecting the cross-linking interaction between the byssus protein and the metal ion, reducing movement and impairing physiology of byssal glands, thereby affecting energy allocation of the body; at the molecular level, the heatwaves caused changes in the expression of multiple genes in the byssal glands. However, given the survival and physiological tolerance of M. strigata to heatwaves, it is imperative to elucidate how mussels may likely allocate the energy budget to essential physiological processes and fitness-related functions when heatwaves attacked. As we enter an era of unprecedented climate change, such information is crucial for linking extreme weather events and biological invasions.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

XX: methodology, investigation & writing-Original draft preparation. KY: methodology & investigation. YL: methodology. YD: investigation. LZ: conceptualization, methodology, investigation & writing-original draft preparation. All authors contributed to the article and approved the submitted version.