Anthropogenic influence on the production and release of greenhouse gases into the atmosphere has rapidly increased over the last century (Rahman, 2013; Fawzy et al., 2020). Since the mid of the 20^th century, the ocean absorbed more than 90% of the excess heat in the atmosphere (Jewett and Romanou, 2017) and more than 25% of the atmospheric CO₂ (Sabine et al., 2004). According to the Intergovernmental Panel on Climate Change (IPCC) models, the temperature is projected to increase 1.4 to 4.4°C (more conservative and more extreme scenarios, respectively) (IPCC, 2021), until the end of the century. Ocean warming is expected to affect all levels of the structure and function of marine ecosystems and organisms (Parmesan, 1996; McCarthy et al., 2001; Walther et al., 2002. For ectotherms, such as fish, the temperature is a determinant factor for several behavioral (Wong and Candolin, 2015), physiological (Pörtner and Farrell, 2008), and metabolic processes (Clarke and Fraser, 2004). Warming has been shown to lead to increased metabolic rates (Roessig et al., 2004), interfere with growth (Boltaña et al., 2017), reproduction (Lopes et al., 2020; Servili et al., 2020), digestion processes and feeding rates (Rangel and Johnson, 2018; Pilakouta et al., 2020). It has also the potential to induce behavioral deviations, such as changes in swimming performance (Simon et al., 2017), predator-prey interactions (Domenici et al., 2019), and social interactions (Colchen et al., 2017).

In addition to ocean warming and other climate change-related stressors (e.g. acidification, hypoxia) (Sampaio et al., 2021), marine species are also being impacted by other human-related pressures, such as overexploitation of resources (Jackson et al., 2001), poaching (Correia, 2022), habitat degradation and fragmentation (Airoldi et al., 2008) and pollution (Shahidul Islam and Tanaka, 2004). When combined, these pressures are particularly severe for species with unique life history traits, such as seahorses. Seahorses (Hippocampus spp.) are benthic species, with low mobility, small home ranges, low fecundity, sparse distribution, mate fidelity, and lengthy parental care (Foster and Vincent, 2004). These traits, combined with illegal trade for Traditional Chinese Medicine and unsustainable aquarium display (Foster and Vincent, 2004), and habitat loss (Harasti, 2016; Correia, 2022), render them extremely vulnerable to population declines and local extinctions (Foster and Vincent, 2004; Chong et al., 2010; Macusi et al., 2011; Vincent et al., 2011). Hence, for better control over seahorse trade, all species in the genus have been included in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES Convention) (Foster and Vincent, 2005), and in the International Union for Conservation of Nature (IUCN) Red List of Threatened Species (IUCN, 2020). More recently, IUCN World Conservation Congress approved a resolution (WCC-2020-Res-095) that urges IUCN members to take action toward ensuring the status assessment of all syngnathids (seahorses, pipefish, and seadragons), and their inclusion in national/regional Red Lists, while promoting the protection and restoration of their habitats.

Experimental Studies on the effects of global warming on the behavior and physiology of seahorses are still scarce and limited to just a few species. The temperate long-snouted seahorse, Hippocampus guttulatus, distributes across most of Europe and North Africa, including the Atlantic Ocean, Mediterranean Sea, and Black Sea (Woodall et al., 2015; Lourie et al., 2016). Concurrent with this wide geographic range, the species can also experience a wide range of temperatures throughout the year (Planas et al., 2012), which will vary depending on the latitude. Temperatures in the South of the Iberian Peninsula can range from 10 to 28 °C (Ria Formosa lagoon, Curtis and Vincent, 2006), which clearly differs from temperatures experienced in Northern France or the British Isles, where temperatures can range from 4 to 20°C (Garrick-Maidment et al., 2014). To date, two studies looked into the effects of prolonged exposure (1 month) to increasingly warmer water conditions (18°C – 26°C – 30°C) on H. guttulatus (Aurélio et al., 2013; Faleiro et al., 2015). Authors report an increase in ventilation and metabolic rates of adult seahorses with warming, but fail to detect any further physiological and behavioral impairments, which led the authors to conclude that adults of the species are thermally resilient to heat stress. A similar study on a congener sub-tropical species, H. erectus, which occurs from the Gulf of Mexico to Nova Scotia in water temperatures ranging from 5 to 28°C (Teixeira and Musick, 2001; Lourie et al., 2016), exposed fish for 1 month to increasingly warmer water conditions (22°C – 26°C – 30°C) (Qin et al., 2018). Authors found evidence of persistently higher basal metabolic rate at 30°C, higher stress response on gene expression, and higher mortality rates. Further, a transcriptomic analysis suggests an arrest of muscle development processes, activation of heat shock proteins, and a switch to anaerobic metabolism from the first moments of exposure to high temperatures (30 or 33 °C) (del Vecchio et al., 2022).

The extensive latitudinal range these species inhabit might contribute to differences in thermal tolerance across populations (Helmuth et al., 2002; Pereira et al., 2017), with population-specific thermal limits being set by physiological limitations in aerobic performance (Eliason et al., 2011). Therefore, when assessing the potential impact of climate change it is important to use scenarios that reflect temperatures within and outside a population’s natural variability, and not only scenarios that reflect the variability across the species geographical distribution.

‘The present study aimed to assess the effects of chronic exposure to increasing temperature scenarios on growth, feed intake, and behavioral patterns of adults of H. guttulatus. Individuals were collected in the Sado estuary (Portugal), where temperature can annually range from average 15°C to 19°C, but minimum and maximum temperatures range from 13.8°C (January) and 20.5°C (September) (European Centre for Medium-Range Weather Forecasts, ECMWF, 2022) ( Table S1 ). Fish were exposed for a 2-month period to three different temperature scenarios: ambient temperature (17°C); high temperature (20°C); and an extreme temperature scenario (24°C), which is likely to occur by the end of the century (IPCC, 2021). Furthermore, as the study took place during the species’ breeding season, we provide a descriptive discussion of temperature effects on reproduction based on anecdotal observations.

Results from the present study reflect differences in behavioral responses in seahorses exposed to different elevated temperatures for a long-term period (2 months). Fish were significantly more active under extreme temperature conditions (24°C) (less time resting, more time swimming and unattached), and were also seen feeding more under both high and extreme temperatures (20°C and 24°C). Higher food ingestion did not translate into an increased body condition, though, as the weight gain of the fish did not differ across treatments. Temperature can impact differently fish locomotion and activity. While some studies suggest that individual activity increases with temperature (Ojanguren and Brañta, 2000; Biro et al., 2010), others conclude that swimming ability decreases under warming conditions (Johansen and Jones, 2011). The elevated temperature was shown to increase swimming activity in Atlantic cod, Gadus morhua (Claireaux et al., 2000), but impact negatively swimming activity in juvenile brown trout, Salmo trutta (Ojanguren and Brañta, 2000). In the current study, we found a significant temperature effect on swimming activity, with fish under extreme temperature treatment (24°C) being more active (more time spent swimming) and spending less time resting. Similarly, a study on the activity of adult lined seahorses showed that when they were exposed to higher temperatures close to the species’ thermal limit (30°C), they increased their swimming as opposed to seahorses maintained at lower temperatures (22 and 26°C) (Qin et al., 2018). Interestingly, two other studies conducted on the long-snouted seahorse, from the same collection site, exposed to much more extreme temperature conditions (26°C, 28°C, 30°C), did not find an effect of temperature on frequency of swimming or periods of inactivity when compared to fish from control temperatures (18°C) (Aurélio et al., 2013; Faleiro et al., 2015). Some studies in other fish species have demonstrated that swimming activity decreases at low temperatures, increase to a peak and then decrease as the temperature approaches the upper thermal limit (Myrick and Cech, 2000; Ojanguren and Brañta, 2000). This could explain the results from those authors’ studies – the extreme temperatures the seahorses were exposed to might already be close to their thermal limit. Another possible explanation for the observed differences might relate to methodological aspects. In the current study, we run behavioral observations three times a week, for 2 months, and observations were performed early in the morning before feeding. Aurélio et al. (2013) and Faleiro et al. (2015) had shorter periods of behavioral observations and run observations 30 min after feeding. This might have influenced the time spent swimming/inactive as fish might still be under post-prandial effects.

In response to a warmer environment, fish will increase their energetic demands and consequently will need to ingest more food (Sswat et al., 2018) , to maintain core activities. As expected, in our study, fish under the extreme temperature treatment (24°C) significantly ingested more food than fish under ambient and high-temperature treatments (17°C and 20°C). Contrasting results were reported by Aurélio et al. (2013) and Faleiro et al. (2015), with no changes in feed intake rates of H. guttulatus under extreme water temperatures (26°C, 28°C, 30°C). Also, Qin et al. (2018) fail to detect an influence of temperature on the feeding frequency of the H. erectus after 1 week at 30°C, which is close to the species’ thermal limit. However, in these 3 studies, authors observed an increase in metabolic rates and ventilation rates (which we did not measure in the current study), supporting an increased metabolic activity under higher temperatures. The increase in metabolic rates but the absence of a temperature effect on feeding rates might indicate that the species was already outside their optimum temperature limit, as feed intake might decrease if the temperature surpasses the species’ optimum limits (Volkoff and Rønnestad, 2020). Therefore, individuals might have adopted a saving-energy strategy by not spending energy on feeding but making digestion more efficient.

Temperature increases within an appropriate range usually promote fish growth (Green and Fisher, 2004), and therefore we were expecting to have fish under the better condition at higher temperatures. In our study, fish did not show any significant change in their body weight among the different temperatures they were exposed to. Similarly, Qin et al. (2018) did not find differences in weight or body height of adult H. erectus exposed to different temperature scenarios (22, 26, and 30 °C) for a 4-week period. However, the seahorse hepatosomatic index (HSI) was lower under 30 °C, while metabolic rates were higher at this temperature. Authors argue that thermal stress inhibited hepatic energy reserves and leads to lower HSI by increasing energy consumption. In our study the lack of temperature-induces effects in fish growth was possible due to fish being more active under extreme temperatures (24°C), thus spending more energy. Nonetheless, we can also not rule out the possibility that seahorses were already under thermal stress at these extreme temperatures. Mascaró et al. (Mascaró et al., 2016, Mascaró et al., 2019), for example, report that chronic exposure (1 month) to the extreme temperature of 30°C significantly reduced juvenile H. erectus growth. Using transcriptome data, del Vecchio et al. (2022) provide evidence of repression of the MEF2A gene within the first 0.5 h of exposure of the H. erectus to target temperatures (30 and 33 °C). This gene is implicated in the regulation of growth and differentiation of muscle fibers, and its repression might justify changes in growth under temperatures close to the s’ecies’ thermal limits. Despite the relevance of this study, the rate of thermal increase untill reaching the target temperatures (abrupt: < 5 min; gradual, 1-1.5 °C every 3 h) and the fact that the authors only collected data after 0.5 h of exposure prevents us from making further conclusions on the effects of prolonged warming on growth.

A word of caution is needed when interpreting results of the current study, as it took place during the breeding season of the species, and reproduction changes activity rate (which likely justifies the observed significant effect of Day of Observation) and physiological condition (weight gain) of individuals. Therefore, its influence on the response variables under measure is difficult to control. Nevertheless, courtship behaviors and daily greetings (Vincent, 1995) were regularly and evenly recorded across the three temperature treatments during the 8-week exposure period (19 courtship events under ambient temperature; 21 events under high temperature; 21 events under extreme temperatures). Moreover, all males across treatments were seen to become pregnant at a certain point of the experimental period, even though the number of successful pregnancies differed across treatments. Therefore, the observed changes at the behavioral level and feeding rate are likely to truly reflect a temperature effect, rather than just an influence of reproductive activity. A total of 7 successful breeding events were registered, being 4 events (in 3 out of the 4 males) in couples from the extreme temperature (24°C), 2 events (in 2 out of the 4 males) in couples from the high temperature (20°C), and 1 event (in 1 out of the 3 males) in ambient temperature (17°C). As reproduction is considered to be sensitive to thermal stress (Pörtner and Farrell, 2008) our results, then, might indicate that the range of tested temperatures was not enough to negatively impact reproduction, and it could even suggest that breeding is stimulated at higher temperatures. Qin et al. (2018) found no significant negative effects on gonad development or reproductive endocrine regulation genes in response to chronic thermal stress (1 month, at 30 °C) in the H. erectus. The authors argue that the species’ reproductive behavior is adapted to higher-temperature conditions, as H. erectus seasonally migrate into warm waters for breeding and return to offshore areas when the water cools (Boehm et al., 2015). Nevertheless, in the present study, and despite the suggestion of increased reproductive activity under high and extreme temperature conditions (20 and 24°C), episodes of unsuccessful egg transfer from the female to the male were recorded only under these temperature conditions and were frequently observed in couples where males were not responsive to females approach and courtship. Moreover, abortion was also registered at high temperatures (20°C), with the male releasing embryos of several developmental stages. Altogether, these observations might suggest that there is a high but unsuccessful investment in reproduction under warming conditions. Consequences at the offspring level were not possible to infer, as we were unable to maintain the newborns. Future studies should take a closer look at the impacts of high temperatures on the reproductive success of these species.

Overall, and despite the limited number of individuals available to run this experimental study, our results points to a negative consequence of ocean warming on behavior and feeding rate of adult H. guttulatus. To truly understand if these individuals were already under thermal stress at the extreme temperature of 24°C, additional physiological and biochemical endpoints would be needed. Determination of metabolic rates, such as minimum and maximum metabolic rate, is a highly useful physiological tool that allows a better understanding of fish metabolic performance under different environmental conditions, and, as it involves minimum manipulation of the individual, there is a low risk of death. The set of physiological responses to warming is the reflection of a prompt cellular stress reaction triggered to maintain the organism’s homeostasis, and it involves biochemical adjustments of metabolic pathways and the regulatory activity of the antioxidant system (Sopinka et al., 2016). These indicators provide highly relevant information for evaluating stress response levels, but the downside is that they require the euthanasia of individuals, for tissue and blood sampling. To avoid invasive and lethal practices with a species that holds a special conservation status, we decided not to evaluate these physiological responses.

Seahorses are a great model to study the effects of climate change on the fitness and behavior of sedentary and low dispersal species, acting as an indicator of ecosystem health. These species can be used as flagship species for conservation issues and can be found worldwide in some of the most vulnerable marine ecosystems in shallow waters (Vincent et al., 2011). The population of the H. guttulatus from the Sado estuary is subject to intense pressure from other anthropogenic activities, such as overexploitation, pollution, and habitat destruction. All these pressures reduce the likelihood of these species to acclimatize and hinder the opportunity to persist in a changing environment. If protective measures are not taken, seahorse populations might suffer a sharp decline in numbers, and in long term affect this population’s fitness and survival.

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

This study was carried out under the approval of Direção-Geral de Alimentação e Veterinária (DGAV, Portuguese Authority for Animal Health, permit 0421/000/000/2020) and according to the ISPA University’s animal ethics guidelines.

AC: methodology, data collection, data analysis, writing- original draft preparation, writing - review and editing; MC: conceptualization, methodology, writing- original draft preparation, writing - review and editing; GS: methodology, writing - review and editing; AL: data analysis, writing - review and editing; AF: funding acquisition, project administration, conceptualization, methodology, writing- original draft preparation. All authors contributed to the article and approved the submitted version.