Industrial, agricultural and livestock activities have been identified as drivers of the changes in atmospheric composition, providing evidence supporting the influence of human activities on global warming (IPCC, 2018). The global mean temperature of Earth surface has increased by 1.09°C over the last 150 years, and it is projected to warm by 1.0°C–5.7°C by 2100, in relation to the emissions of greenhouse gases (Chown et al., 2022). Especially, the Antarctic region has been experiencing some of the planet’s strongest climatic changes, including extreme climate and weather events, droughts, floods, accelerated ice loss, increased glacier and ice sheet meltwater, ocean acidification and changes in mean sea level (Golledge et al., 2019; Edwards et al., 2021; Chown et al., 2022; Constable et al., 2022). The land temperature increased to 0.61°C ± 0.34°C per decade between 1989 and 2018 (IPCC, 2018; Robinson, 2022), while the ocean is experiencing the greatest absolute oxygen loss (Bronselaer et al., 2020). Moreover, significant stratospheric ozone depletion persists over Antarctica during the austral spring season, even though evidence suggests that the hole in the ozone layer over Antarctica is showing signs of recovery since the year 2000 (IPCC, 2021). The increased frequency, severity, and duration of extreme events in the short term, will lead many terrestrial, freshwater, coastal, and marine ecosystems to a very high or high risk of losing biodiversity (IPCC, 2022). Antarctic ecosystems have significantly lower biodiversity than the rest of the planet (Convey and Stevens, 2007). Life on the land is mostly restricted to the small ice-free areas of the continent (<0.5% by total area; Chown et al., 2022); mosses and lichens are common, and include different microbial groups (prokaryotes, algae, fungi, and protists), micro-arthropods (mites, springtails, and midges) and micro-invertebrates of the meiofauna (i.e., nematodes, rotifers, and tardigrades; Câmara et al., 2021; Phillips et al., 2022). Nevertheless, switches between dominant and least represented communities for both moss communities and invertebrate species have been recently evidenced (Chown et al., 2022). The primary factors influencing the abundance, composition, and distribution of Antarctic terrestrial biodiversity are temperature, liquid water and nutrient availability (Kennedy, 1993; Convey, 1996; Hogg et al., 2006; Sinclair et al., 2006; Bissett et al., 2014; Chown and Convey, 2016). However, recent evidence indicates an important role of biotic interactions within and between species in shaping Antarctic communities (Potts et al., 2020).

Due to the ongoing trend of increasingly warmer summers and more frequent warming events, the effects of climate change on Antarctic biodiversity are becoming progressively clear in the transformation, damage, and degradation of ecosystems (Andriuzzi et al., 2018; IPCC, 2022). To expand the knowledge about the effects of global warming on Antarctic meiofauna, we investigated the adaptive responses of the Antarctic tardigrade Acutuncus antarcticus (Richters, 1904) to increasing temperature. We analyzed and compared its life history traits at different temperatures (5°C vs. 15°C), and we performed a transcriptome analysis to investigate the molecular mechanisms involved in heat response and tolerance of this species. A. antarcticus is the most abundant and common eutardigrade in Antarctica (Cesari et al., 2016) inhabiting freshwater ecosystems and terrestrial soils, mosses, and lichens (Murray, 1910; Dastych, 1991; Cesari et al., 2016). It is considered to be a parthenogenetic pan-Antarctic species and it is herbivorous and bacteriophagous (Altiero et al., 2015; Cesari et al., 2016). Previous studies demonstrated that it is able to tolerate dehydration, freezing, short exposure (1 h) to high temperatures (up to 37°C), and exposure to UV radiation (Giovannini et al., 2018). Since it colonizes different habitats, it could be considered as a representative animal model for Antarctic terrestrial communities.

The active life span of A. antarcticus reared at 5°C reached a maximum value of 686 days in a specimen of F₂ generation. Adult females laid eggs freely once a fortnight, and they molted before every egg oviposition. Throughout her life span, each female laid up to 93 eggs, with a maximum of 34 ovipositions. Newborns molted once before their first oviposition at the age of about 34 days. The eggs hatched in about 22 days. Detailed collected data and performed analyses of life history traits of P, F₁, and F₂ generations reared at 5°C are presented in Figure 2 (Supplementary Table S2, Supplementary files S1–S3).

The eutardigrade Acutuncus antarcticus was considered an animal model to study the effects of increasing temperature due to global climate change on Antarctic ecosystems. The life cycle and the fitness of this species at different increasing temperatures were investigated to simulate the ongoing global warming. The most surprising result was the maximum life span of 686 days (about 23 months) reached by a specimen of the F₂ generation reared at 5°C, in contrast with the maximum value of 130 days reached by a F₁ specimen reared at 15°C (Altiero et al., 2015). A life span so long has never been recorded before in tardigrade species (Altiero et al., 2018), where the longest record of longevity was 518 days (about 17 months) for a clonal strain of the terrestrial eutardigrade Paramacrobiotus fairbanksi (Altiero et al., 2006), and 18 months for the marine heterotardigrade Halobiotus crispae (Kristensen, 1982). This result was also unexpected, even comparing it with the life cycle in rearing conditions of other extremophile organisms inhabiting Antarctica: the rotifers Adineta grandis and Philodina gregaria lived 40 and 89 days, respectively (Dartnall, 1992); the soil nematode Scottnema lindsayae, reared at 10°C, lived 218 days (Overhoff et al., 1993), and the terrestrial soil nematode Plectus murrayi, reared at 15°C, shows an annual life cycle duration (de Tomasel et al., 2013). Consistent with our results, previous studies showed that temperature has different effects on various aspects of life cycle and development of soil nematodes, especially cold temperature produces an extended adult lifespan (Moorhead et al., 2002).

Acutuncus antarcticus specimens reared at 5°C lived longer, molted more times, and reached sexual maturity later than those reared at 15°C. Moreover, during their life span, animals at 5°C laid more eggs that hatched after a longer time and in lower percentage, and showed a longer interval time between successive ovipositions. Other Polar invertebrates exposed to increasing temperature showed a shorter development (Yoon et al., 2023) and their eggs hatched earlier (Weydmann et al., 2015), even though their hatching success decreased (Birkemoe and Leinaas, 2000). In A. antarcticus, the egg hatching time also decreased when the eggs were exposed to 15°C, although their hatching percentage increased. This increased hatching success could balance the low number of laid eggs when tardigrades were reared at the highest temperature. Overall, this strategy could represent a warm climatic adaptation in response to the unpredictable Antarctic environmental conditions exhibiting temperature fluctuations even within the same day (Altiero et al., 2015).

Differences among generations of A. antarcticus reared at two different temperatures were evidenced in relation to some life history traits. In particular, animals belonging to the first generation lived longer than the second generation. The interval time between successive ovipositions was minimum for the animals of parental generation, and it increased for the filial generations. As a possible adaptive strategy to balance the longer interval time between ovipositions, the eggs laid by the F₂ generation hatched earlier than other eggs. The hatching percentage was higher in eggs laid by the parental generation, and it strongly decreased in eggs laid by filial generations. The unhatched eggs could however be resting eggs, needing a stimulus such as desiccation-rehydration or freezing-thawing to hatch, as observed in other tardigrade species (Altiero et al., 2010; Møbjerg and Neves, 2021). Furthermore, the animals belonging to the first generation, reared at both tested temperatures (5°C and 15°C), showed the highest fitness. Otherwise, the fitness of the F₂ generation at 15°C was significantly lower than the fitness of the other animal groups. These data suggest that the first generation is able to acclimate to the increasing temperature, while the second one is negatively affected. As a whole, the differences among generations in the fitness and in the life history traits, especially the ones associated with reproduction, could be due to genetic and maternal effects, and consequently to phenotypic plasticity (Marshall and Uller, 2007; Yeates et al., 2009; Fusco and Minelli, 2010; Altiero et al., 2015; Pazzaglia et al., 2021).

The first transcriptome of A. antarcticus exposed to increasing temperature was obtained. Upon a first analysis, expression data showed a high degree of inter sample diversity, which could not be ascribable to experimental variables. Such an effect could be the result of an unexpectedly high inter-individual diversity, which in turn could explain the unexpectedly low total number of statistically significant DEGs. The high duplication rate resulting from the BUSCO results could indeed indicate high heterozygosity, but this needs to be supported by genomic data, which are not yet available for this species. After removing the unwanted variability, a set of DEGs for both exposure times could be found. Finally, the reduced availability of high-quality genomes of tardigrade species represents a limitation to the precise annotation of transcripts in our assembled transcriptome, restricting the retrieval of information to databases such as UniProt/SwissProt and PFAM.

When animals were exposed for 1 day to heat (short-term), 67 DEGs were found. The expression patterns of such genes showed a linearity between expression fold change and applied temperature, reinforcing the idea that the observed alterations are a transcriptional response to the temperature increase. While the difference in gene expression is small between 5°C and 10°C, the changes become evident at 15°C and even more dramatic at 20°C. The 23 upregulated genes were significantly enriched in GO terms suggesting alterations of mitochondrial activity and oxido-reductive processes. Among these, NADH-ubiquinone oxidoreductase chain 4 and 5, cytochrome b, and cytochrome c oxidase subunits 1 and 2 are involved in the respiratory chain (Sousa et al., 2018). Since NADH-ubiquinone oxidoreductase is a major source of Reactive Oxygen Species (ROS) in mitochondria (Kusmann and Hirst, 2006; Esterházy et al., 2008), its upregulation, evidenced also in other tardigrade species exposed to heat stress (Neves et al., 2022), induces cellular oxidative stress. Moreover, as evidenced by Oomen et al. (2022), transcripts involved in cellular respiration are upregulated in response to heat, increasing metabolic energy requirements, oxygen consumption, and ROS, damaging complex molecules and cellular structures. The downregulation of two peroxidase (peroxidasin and chorion peroxidase) with the increasing temperature is in line with an oxidative stress scenario, since their functions are related to the protection of animals and eggs from ROS (Benoit et al., 2012; Schokraie et al., 2012).

Nudix hydrolases are widely found in prokaryotes and eukaryotes, and are involved in a variety of cellular processes such as cellular metabolism, homeostasis, and mRNA processing (Yoon et al., 2018; Yang et al., 2022). The observed upregulation of Nudix hydrolases with increasing temperature suggests a role in defense against oxidative stress, as it has already demonstrated in potato (Brouwer et al., 2021).

Disordered proteins help mediate tolerance to different abiotic stresses including freezing, osmotic stress, high temperatures, and desiccation in several organisms (Hesgrove and Boothby, 2020). Recently, three novel families of intrinsically disordered proteins (IDP) were discovered in tardigrades and revealed to contribute to a general stress response (Yamaguchi et al., 2012; Tanaka et al., 2015; Boothby et al., 2017; Hesgrove and Boothby, 2020; Yagi-Utsumi et al., 2021; Neves et al., 2022; Krakowiak et al., 2023). In this study, two intrinsically disordered protein genes, namely, Cytosolic-abundant heat soluble protein (CAHS) and secretory-abundant heat soluble protein (SAHS), were upregulated in response to heating. The aforementioned genes refer to several assembled transcripts (40 and 8 respectively) that were grouped under the same gene by best similarity and, due to the absence of a reference genome, may represent splicing isoforms and/or paralogous genes as well. As previously evidenced in Ramazzottius varieornatus for other IDPs (Neves et al., 2022), these obtained results confirm the role of tardigrade IDPs to cope with heat stress. The putative gene for conserved regulator of innate immunity protein 3 was also upregulated with the increasing temperature. This gene is described in nematodes as homologous to the mammalian C1qbp (Alper et al., 2008), which has several functions in cell activity and a key role in the activation of the complement system, and therefore of innate immunity (Ghebrehiwet and Peerschke, 2004).

Among the 44 DEGs that were downregulated with the increase in temperature, there are genes related to the protein turnover (cathepsin B and UBA domain-like superfamily; Förster et al., 2012), a gene (Multidrug resistance-associated protein 1) involved in dauer larva regulation of the nematode Caenorhabditis elegans (Yabe et al., 2005), and two genes (CREB-binding protein and Homeodomain-interacting protein kinase 2) relevant for the control of cellular proliferation, differentiation and apoptosis (Kovács et al., 2015). The loss of function of a Homeodomain-interacting protein kinase (HPK) shortens lifespan and hastens tissue aging in C. elegans (Berber et al., 2016). Similarly, the downregulation of HPK gene in specimens of A. antarcticus exposed to increasing temperature could have reduced the life span of animals reared at 15°C compared to those reared at 5°C.

Regarding the long-term (15 days) exposure, only two DEGs out of 14 could be annotated. The gene coding for a von Willebrand factor A domain containing protein (VWA) is downregulated. The VWA plays a role in cell adhesion, extracellular matrix proteins, and integrin receptors (Whittaker and Hynes, 2002). However, the oldest known proteins containing the VWA domain are intracellular and involved in functions such as transcription, DNA repair, ribosomal and membrane transport, and the proteasome (Whittaker and Hynes, 2002). The other annotated DEG (i.e., putative conserved regulator of innate immunity protein 3) is upregulated in response to long-term exposure as well as short-term exposure, as described in the previous paragraph. The observed decline in transcriptomic response after a 15-day exposure indicates that the differences observed in the short term are likely due to an acclimation response. It is reasonable to hypothesize that with longer exposure durations, a return to a normal transcriptional landscape would be expected, although further data is needed to confirm this assertion.

Considering that A. antarcticus is a pan-Antarctic species and that the average daily pond temperatures in Victoria Land (Antarctica) range between −5 and +6°C during the summer (Cucini et al., 2022), the exposure to 15°C represents an unusual condition for this species, as well as for other Antarctic organisms. Overall, our findings indicate that the short-term heat exposure leads to significant changes in the transcriptomic landscape of A. antarcticus, as evidenced by the differential expression of 67 genes after 1 day of heat exposure. Notably, several genes involved in oxidative metabolism and oxidative stress response display changes in their expression patterns. Nevertheless, the gene expression alterations observed after long-term exposure are limited to 14 genes, and only the upregulation of a gene (putative conserved regulator of innate immunity protein 3) is evidenced both after 1 and 15 days of heat exposure.

Therefore, according to the results obtained from the life history traits and gene expression this Antarctic tardigrade species could be able to acclimate to higher temperatures over time, including possible future conditions imposed by global warming.