Science, Faculty of
Non UBC
Oceans and Fisheries, Institute for the
DSpace
Diversity 16 (12): 719 (2024)
Project Seahorse
Palma, Jorge
Correia, Miguel J.
Leitão, Francisco
Andrade, José Pedro
This experiment aimed to determine the temperature limits beyond which seahorse growth and reproduction become suboptimal due to climate change. Four temperatures (16, 20, 24, and 28 °C) were tested to evaluate their effect on juvenile (1–56 days post-parturition (DPP)) and adult (one year old) long-snout seahorses, Hippocampus guttulatus. Additionally, the reproductive performance of adults was observed. Another experiment measured oxygen consumption (MO2) in the same age groups and temperatures. Adults showed significantly higher growth rates at 20 and 24 °C compared to 16 and 28 °C. Adult mortality rates were 0%, 0%, 6.2%, and 62.5% at the respective temperatures. Juvenile growth performance was higher at 20 °C and 24 °C but significantly lower at 16 °C and null at 28 °C, with survival rates of 8%, 62%, 10%, and 0%, respectively. Oxygen consumption increased with temperature, ranging from 106.3 ± 3.1 to 203.3 ± 3.1 μmol O2/g BW/h at 16 °C, and from 127.6 ± 3.5 to 273.3 ± 3.1 μmol O2/g BW/h at 28 °C for adults and 1 DPP juveniles, respectively. The study highlights that juvenile and adult H. guttulatus have narrow thermal boundaries, beyond which reproduction, growth, and survival are seriously affected. Under climate change, the species appears unable to cope, potentially leading to their rapid disappearance.
Citation: Palma, J.; Correia, M.; Leitão,F.; Andrade, J.P. Temperature Effectson Growth Performance, Fecundityand Survival of Hippocampus guttulatus.Diversity 2024, 16, 719. https://doi.org/10.3390/d16120719Academic Editor: ElenaKupriyanovaReceived: 5 August 2024Revised: 6 November 2024Accepted: 19 November 2024Published: 25 November 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).ArticleTemperature Effects on Growth Performance, Fecundity andSurvival of Hippocampus guttulatusJorge Palma 1,* , Miguel Correia 2,3 , Francisco Leitão 1 and José Pedro Andrade 11 Centre of Marine Sciences (CCMAR), University of the Algarve, 8005-139 Faro, Portugal;fleitao@ualg.pt (F.L.); jandrade@ualg.pt (J.P.A.)2 Seahorse, Pipefish, and Seadragon Specialist Group, IUCN Species Survival Commission,1196 Gland, Switzerland; m.correia@oceans.ubc.ca3 Project Seahorse, Institute for the Oceans and Fisheries, The University of British Columbia,Vancouver, BC V6T 1Z4, Canada* Correspondence: jpalma@ualg.ptAbstract: This experiment aimed to determine the temperature limits beyond which seahorse growthand reproduction become suboptimal due to climate change. Four temperatures (16, 20, 24, and 28 ◦C)were tested to evaluate their effect on juvenile (1–56 days post-parturition (DPP)) and adult (one yearold) long-snout seahorses, Hippocampus guttulatus. Additionally, the reproductive performance ofadults was observed. Another experiment measured oxygen consumption (MO2) in the same agegroups and temperatures. Adults showed significantly higher growth rates at 20 and 24 ◦C comparedto 16 and 28 ◦C. Adult mortality rates were 0%, 0%, 6.2%, and 62.5% at the respective temperatures.Juvenile growth performance was higher at 20 ◦C and 24 ◦C but significantly lower at 16 ◦C and nullat 28 ◦C, with survival rates of 8%, 62%, 10%, and 0%, respectively. Oxygen consumption increasedwith temperature, ranging from 106.3 ± 3.1 to 203.3 ± 3.1 µmol O2/g BW/h at 16 ◦C, and from127.6 ± 3.5 to 273.3 ± 3.1 µmol O2/g BW/h at 28 ◦C for adults and 1 DPP juveniles, respectively.The study highlights that juvenile and adult H. guttulatus have narrow thermal boundaries, beyondwhich reproduction, growth, and survival are seriously affected. Under climate change, the speciesappears unable to cope, potentially leading to their rapid disappearance.Keywords: seahorses; Hippocampus guttulatus; temperature; growth; reproduction; oxygen consumption;climate change1. IntroductionSeahorses, like other small marine fish species, are an integral part of marine bio-diversity and ecosystem function [1]. They are recognized as flagship and bioindicatorspecies [2,3], making them ideal models for assessing risks from marine contaminantsand environmental stressors [4–7]. Seahorses are considered as such due to their uniquelife history, characterized by sparse distribution, low mobility, site fidelity, small homeranges, low fecundity, mate fidelity, and lengthy parental care for small broods [1,8–10].These traits may render them vulnerable to overfishing and environmental disruptions,including habitat damage and degradation [1]. Additionally, seahorses inhabit shallow,coastal areas worldwide, where anthropogenic disturbances tend to be most frequent andsevere [3]. These constraints help explain why 2 seahorse species are listed as “Endangered”,12 as “Vulnerable”, and 1 as “Near Threatened” on the 2024 IUCN Red List of ThreatenedSpecies, with only 10 listed as “Least Concern” and 17 still as “Data Deficient”. This reflectssubstantial gaps in knowledge, even for a heavily exploited fish group like seahorses.The two European seahorse species, H. guttulatus, and H. hippocampus, which inhabitthe Ria Formosa (southern Portugal), are also exposed to similar environmental stressors.Populations of the two seahorse species experienced a significant decline between 2001 and2009, with a 94% reduction in H. guttulatus and a 73% reduction in H. hippocampus, dueDiversity 2024, 16, 719. https://doi.org/10.3390/d16120719 https://www.mdpi.com/journal/diversityDiversity 2024, 16, 719 2 of 14to unknown causes [4]. These authors did not link the population decline to any of thetested environmental factors. Instead, they suggested it could be related to differences inwater quality across the lagoon, shifts in dominant seagrass species, or natural populationfluctuations. Between 2012 and 2013, a slight population increase was observed [5], indicat-ing that H. guttulatus populations fluctuated and were positively correlated with habitatloss. Previously, ref. [11] already identified human activities (e.g., fishing, illegal fishing,anchoring, and dredging) and natural changes in the lagoon’s dynamics (e.g., silting andshifting currents) as the main causes of habitat loss. Later, in 2018, ref. [12] reported anothersignificant decline in H. guttulatus populations, again linked to the same human activitiesidentified by [11]. This decline continued until 2021, bringing population levels back tothose observed by [4] in the earlier study (Palma, personal observation). The causes ofpopulation decline were directly related to anthropogenic disturbances but do not fullyexplain it, and the effect of climate change should be regarded as an additional stressor.Combined, these drivers of change have a major impact on these seahorse populations,which, due to their sensitive life history, may become powerless to cope with these im-pacts. Therefore, well-designed conservation plans are urgently needed to preserve thebiodiversity of this ecosystem.Sensitivity to regional climatic conditions and anthropogenic disturbances interactto strongly affect habitat loss and fragmentation [6], threatening biodiversity, decreasingdispersal rates, and increasing population mortality of marine species [7]. Climate changenegatively impacts inshore marine habitats and their fauna, including seahorses, throughchanges in temperature, ultraviolet radiation (UVR), rainfall patterns, community com-position, the status of coastal habitats, and storm activity [8]. These changes are likely tobe more severe in enclosed waters, where local warming occurs [9]. According to NOAADaily Sea Surface Temperature Analysis data, over the past 30 years, there has been asignificant increase in coastal sea surface temperatures (◦C/decade) throughout most ofthe Iberian Peninsula and the North African Atlantic coast (avg = 0.214 ◦C). Despite theunpredictability of climate systems, forecasts indicate a global mean temperature rise of2 ◦C by 2100 [10]. Heat waves, which currently last for 1–2 weeks along the Portuguesecoast [13], are expected to become more intense, more frequent, and of longer duration inwarmer climate scenarios [14].The Ria Formosa is a mesotidal, shallow coastal lagoon prone to extreme daily andseasonal temperature variations [15] and UVR exposure [16]. These conditions are ex-pected to become more frequent and severe under the current climate change scenario.Previous data [17] showed a direct relationship between seawater temperature and sea-horse habitat use. In some surveyed locations, seahorse abundance decreased duringhigh temperature peaks (25 ◦C), whereas in locations with lower temperatures (20 ◦C),the abundance remained more stable. It was also observed that in locations with equalhabitat complexity, seahorse abundance was always lower at higher temperatures. Thispopulation shift coincided with the breeding season. Since temperature can influence gonaddevelopment and the survival of larvae, post-settlement juveniles, and adults in manyspecies [4], seahorses may be more susceptible to temperature fluctuations and unableto cope due to their life history and specific habitat requirements. Overall, temperatureplays a critical role in shaping the distribution, life cycle traits, and biological activities ofectothermic animals, including fish. Elevated temperatures resulting from global warmingdisrupt homeostasis in fish, leading to metabolic imbalances and increased vulnerabilityto stress and disease [18]. While some fish species demonstrate resilience to a wide rangeof temperatures [19], others experience temperature fluctuations in their habitats but aremore susceptible to stress, which negatively impacts their health [20]. Temperature hassignificant effects on the performance and biogeography of ectothermic animals, largelydue to its impact on metabolism [21,22]. As temperature increases, there is an exponentialrise in cellular respiration rates, which in turn elevates whole-animal oxygen consumptionrates (i.e., the standard metabolic rate) to meet aerobic energy demands [23]. Measuringaerobic metabolic rate (often equated to oxygen consumption rate, MO2) can help addressDiversity 2024, 16, 719 3 of 14hypotheses like the metabolic theory of ecology [24,25], which suggests that metabolic rateis a fundamental biological factor governing many ecological patterns. This approach isparticularly relevant to fish biology and climate change research, as it helps to explore thehypothesis that aerobic scope is linked to whole-animal performance and fitness [26–28].The theoretical foundation of this hypothesis suggests that the biochemical and physio-logical capacities of aquatic ectotherms have evolved to maximize aerobic scope within acertain temperature range to optimize performance related to fitness (e.g., growth, repro-duction, and locomotion). As aerobic scope decreases at temperatures outside this range,performance declines [29]. The hypothesis implies that critical functions such as growth,locomotion, and reproduction are causally linked to aerobic scope, meaning that animalsshould experience optimal fitness when living within their optimal thermal range [29]. Therate of respiration, commonly measured by oxygen consumption, is often used to indicatethe metabolic rate in aquatic organisms. Fish oxygen consumption can be categorizedbased on their physiological condition [30]. Basal metabolism represents the lowest energyexpenditure required to sustain life [30,31]. However, due to spontaneous activity in fish,measuring basal metabolism is challenging, so the term “resting metabolism” is preferredover the minimum oxygen consumption rate [30,31]. Metabolic scope refers to the rangewithin which an animal’s aerobic metabolic rate can vary [32]. The metabolic scope relatedto feeding is defined as the difference between the maximum oxygen consumption observedduring feeding and the oxygen consumption at a fasted state [33,34]. Thus, analyzing theoxygen consumption of a fish after feeding, when metabolic levels are higher, will providemore contextualized information about the maximum oxygen consumption limit, which iscrucial in situations of rising temperatures and lower oxygen saturation in the water.Metabolism generally scales allometrically with body size, where larger individualshave higher absolute metabolic rates but lower mass-specific metabolic rates [35]. Inectotherms, metabolic rate is primarily governed by temperature, the ‘master variable’,due to its control over enzyme kinetics [36]. However, it is also influenced by endogenousfactors, such as body size [37] and physiological characteristics like thermal history [38].The present study aims to improve our knowledge on this issue by gathering informationto define the temperature effects on the growth, reproduction, and survival of H. guttulatus,as well as the fish metabolic response to different temperature scenarios.2. Materials and Methods2.1. Adult Growth and ReproductionBefore the trial began, H. guttulatus broodstock was kept under the same husbandryconditions, except for the temperature, which was allowed to follow natural wild conditions.The trial lasted for seven months, starting two months prior (March) to the expected start ofthe breeding season and ending in September. At the beginning of the experiment, 64 adultF4 generation H. guttulatus (32 males, 32 females) were selected and stocked in each of4 units of 250 L white plastic flat-bottom rectangular tanks (one tank per treatment) at16 animals (8 males, 8 females) per tank. Nautical rope (1 cm in diameter) resemblingnatural Codium spp. algae was added to the tanks to serve as holdfasts for the seahorsesto attach to. The tanks were assembled in two semi-closed systems, with a constant waterflow (≈50 L h−1) and moderate aeration. The temperature was controlled as follows: onetank system was connected to a chiller, and through different water inflows to the tanks,the water temperature was controlled at 16 ± 1.2 ◦C and 20 ± 1.5 ◦C in each of the twotanks. In the second tank system, water was heated and controlled at 24 ± 1.2 ◦C and28 ± 1 ◦C in each of the two tanks. Salinity was not controlled and followed the naturalseasonal pattern. Tanks were illuminated from above with 2 × 36 W fluorescent tubes, withan intensity of 600 ± 25 lux at the water surface, and a photoperiod controlled by a timer.The photoperiod was adjusted every two weeks to match the natural photoperiod. Waterquality parameters (ammonia, nitrates, and nitrites) were recorded twice a week and keptstable throughout the experiment; ammonia values were always below detectable levels,nitrate < 0.3 mg L−1, and nitrite < 1.25 mg L−1.Diversity 2024, 16, 719 4 of 14During the experiment, seahorses were fed ad libitum once daily at 10:00 a.m. withthe same dietary treatment, composed of two live mysid species, Mesopodopsis slabberiand Diamysis lagunaris, in slightly different proportions depending on the season. As theprey were supplied live, seahorses could feed on demand, which justified the single dailyfeeding. Each day, uneaten food and feces were removed from the tanks by siphoning everymorning before feeding. All adult fish were sampled for length and weight at the start andend of the trial. During the experiment, seahorses were allowed to mate and reproducefreely. When broods occurred, juvenile seahorses from each brood were gently collectedfrom the broodstock tanks with a beaker and counted.2.2. Juvenile GrowthOnce broods began to occur and juveniles were born, 180 juveniles from the samebrood were collected from the 20 ◦C broodstock tank and randomly distributed into therearing tanks. The rearing tanks were initially maintained at 20 ◦C, and only after thejuveniles had been transferred was the temperature gradually increased or decreasedto the target experimental temperatures over a 24 h period. The juvenile growth trialwas conducted according to a completely randomized design, with three replicate tanksassigned per temperature. Fifteen juvenile H. guttulatus were stocked in each of threereplicate 10 L glass rectangular tanks at a density of 1.5 fish per liter. In each tank, thelateral and back walls were covered with black adhesive to improve prey detection [39].The front wall was left uncovered for any necessary observation. Husbandry conditionsand the experimental design used in this experiment were the same as described by [40],except for temperature control. Rearing tanks were assembled in semi-closed systems toallow temperature control for each set of three replicate tanks (Figure 1).Diversity 2024, 16, x FOR PEER REVIEW 4 of 14 stable throughout the experiment; ammonia values were always below detectable levels, nitrate < 0.3 mg L−1, and nitrite < 1.25 mg L−1. During the experiment, seahorses were fed ad libitum once daily at 10:00 a.m. with the same dietary treatment, composed of two live mysid species, Mesopodopsis slabberi and Diamysis lagun is, in slightly different pr portions depen ing on the s ason. A the pr y were supplied live, seahorses could feed o demand, which justified the single daily feed-ing. Each day, un aten food and fe es were remov d from t e tanks by siphoning every morning before feeding. All adult fish were sampled for length and weight at the start and end of the trial. Duri the experiment, s ahorses were allowed to mate and reproduce freely. When broods occurred, juvenile seahorses from ach brood were ge tly collected from the broodstock tanks with a beaker and counted. 2.2. Juvenile Growth Once broods began to occur and juveniles were born, 180 juveniles from the same brood were collected from the 20 °C broodstock tank and randomly distributed into the rearing tanks. The rearing tanks were initially maintained at 20 °C, and only after the ju-veniles had been transferred was the temperature gradually increased or decreased to the target experimental temperatures over a 24 h period. The juvenile growth trial was con-ducted according to a completely randomized design, with three replicate tanks assigned per temperature. Fifteen juvenile H. guttulatus were stocked in each of three replicate 10 L glass rectangular tanks at a density of 1.5 fish per liter. In each tank, the lateral and back walls were covered with black adhesive to improve prey detection [39]. The front wall was left uncovered for any necessary observation. Husbandry conditions and the experimental design used in this experiment were the same as described by [40], except for temperature control. Rearing tanks were assembled in semi-closed systems to allow temperature con-trol for each set of three replicate tanks (Figure 1). Figure 1. Rearing system: 1: heater, 2: water recycling units, 3: cooler, 4, 5: inflow water for temper-ature control. The arrows indicate the direction of water flow. Four different temperatures (the same tested for adult seahorses) were tested: 16 ± 0.8 °C, 20 ± 0.8 °C, 24 ± 1 °C, and 28 ± 0.9 °C. Salinity and dissolved oxygen were kept, respectively, at 37.2 ± 0.2‰ and 7.3 ± 0.2 mg/L. Tanks were illuminated from above with two 36 W fluorescent tubes, with a light intensity of 900 ± 40 lux at the water surface and a photoperiod controlled by a timer at 16L:8D (06:00–22:00 h). Seawater quality data (am-monia, nitrates, and nitrites) were recorded biweekly. Values remained stable throughout Figure 1. Rearing system: 1: heater, 2: water recycling units, 3: cooler, 4, 5: inflow water fortemperature control. The arrows indicate the direction of water flow.Four different temperatures (the same tested for adult seahorses) were tested: 16 ± 0.8 ◦C,20 ± 0.8 ◦C, 24 ± 1 ◦C, and 28 ± 0.9 ◦C. Salinity and dissolved oxygen were kept, respec-tively, at 37.2 ± 0.2‰ and 7.3 ± 0.2 mg/L. Tanks were illuminated from above withtwo 36 W fluorescent tubes, with a light intensity of 900 ± 40 lux at the water surface and aphotoperiod controlled by a timer at 16L:8D (06:00–22:00 h). Seawater quality data (ammo-nia, nitrates, and nitrites) were recorded biweekly. Values remained stable throughout theexperiment; ammonia values were always below detectable levels, nitrate < 0.3 mg/L, andnitrite < 1.25 mg/L.The juvenile grow-out trial lasted for 56 days, from 0 to 56 days post-parturition (DPP),during which juvenile seahorses were fed ad libitum with live copepods (Oithona nana).Juveniles were sampled every 14 days until the end of the experiment (56 DPP). To reduceDiversity 2024, 16, 719 5 of 14stress during sampling, seahorses were measured using a modified approach: rather thanthe three measurements suggested by [41] (head, trunk, and tail lengths combined), thetotal length was calculated by summing the head length (from the tip of the snout to themidpoint of the cleithral ring) and the height of the fish (from the cleithral ring to the tipof the fully extended tail). The data were used to calculate the following: (1) weight gain(WG, mg d−1) = (Wf − Wi)/d, where Wf is the final seahorse wet weight (mg), Wi is the ini-tial wet weight (mg), and d is the number of days; (2) growth rate expressed as thermal-unitgrowth coefficient (TGC) per fish in each tank, TGC = [(Wf(1/3) − Wi(1/3))/Σ(T × d)] × 100,where Wf, Wi, and d, are as described above, and T is the water temperature; and (3) condi-tion factor (CF) = (wet weight (g)/length (cm)3) × 100.2.3. Respirometry—Experimental ProceduresSeahorse oxygen consumption was determined at three different life stages: juvenileswith 1, 14, and 28 DPP, and adults (12 months old). For the 14 and 28 DPP juveniles, asecond batch was raised at 20 ◦C in two replicate tanks, as the initial group experiencedcomplete mortality at 28 ◦C. At 14 DPP, in one tank, the water temperature was graduallyincreased to 28 ◦C over a 48 h period, followed by respirometry analysis. In the secondtank, the same process was repeated, with the analysis conducted at 28 DPP. Oxygenconsumption was measured by flow-through respirometry following an adapted protocoldefined by [42]. In brief, before transferring to the respiratory chambers, fish were fedad libitum; mealtime started at 10:30 h, and fish were fed for 45 min (group n = 12).Measurements started immediately after transfer (11:00 h) and continued for 22 h, with 8 hin the light and 14 h in the dark. To keep the stress level of the fish at a minimum, they werenot disturbed during the respirometry measurements. The wet mass of each individual wasdetermined immediately after removal from the chamber. The flow-through respirometrysystem consisted of six individual metabolic chambers (2.3 L capacity). Each fish wasindividually placed in a metabolic chamber. Since housing in respirometry chambersmay induce stress responses [43,44], measurements taken during the first 3 h of the lightperiod were discarded. The water inlet was always at an oxygen saturation level, while theoxygen concentration at the outlet was measured by a polarographic microelectrode model8–730 (Microelectrodes Inc., Bedford, MA, USA). Measurements were controlled by a PCusing the Oxilogger 2009 software.The oxygen saturation level in the water reservoir was maintained by aeration. Airbubbling was achieved using a 10 cm rectangular air stone diffuser, while a small pump(Eheim® compact+) ensured thorough mixing of the water. A peristaltic pump (ISMATEC,model ISM920A, Switzerland) controlled the water flow in the chambers using Tygon® tub-ing (480-µm inner diameter). Each respirometry run consisted of sequential measurementsfrom all the chambers. The magnetic valves controlled by the Oxilogger 2009 softwaredetermined in which chamber the oxygen consumption was being measured. At the startof each cycle, the oxygen dissolved in water was measured for 30 s to calibrate the software,followed by a 120 s washing step of seawater from the next chamber before starting thenext measurement period (30 s). This step was always performed before and after themeasurement in each chamber. Six oxygen consumption measurements were registeredover a 30 s period (5 s each). A complete measurement cycle was performed in 18 min,comprising the oxygen probe calibration, a washing step, dissolved oxygen measurementin the water chamber, another washing step, and so forth in each of the six chambers insequence. The water temperature in the outlet water of each chamber was measured witha temperature probe, and oxygen calibration was performed each time a measurementrun started.2.4. Statistical AnalysisAfter confirming normality and homoscedasticity, variance analyses were performed.Differences in juvenile seahorse length, wet weight, CF, TGC, and FCR were tested usingnested ANOVA with post hoc Newman–Keuls (NK) multiple comparison test (p = 0.05).Diversity 2024, 16, 719 6 of 14Oxygen uptake rates were determined by calculating the slope for each sealed measurementperiod. The slope from each phase (expressed as µmol O2/g BW/h) was corrected forbackground respiration by using the average of background measurements taken beforeand after the trial. These rates were further adjusted to account for the volume of thechamber and associated tubing, and then converted to a mass-specific rate of oxygenconsumption (MO2, in µmol O2/g BW/h) based on the average of the weights recordedat the beginning and end of the experiment. The metabolic rate (MR) was modeled usinga temperature-dependent mass-scaling equation, MR = aMbecT, where a is the intercept,M represents body mass, b is the mass-scaling coefficient, c is the temperature-scalingcoefficient, and T is temperature. To apply this model, both sides were ln-transformed andanalyzed through linear regression. Data were expressed as mean ± SD of three runs ofrespirometry data, and statistical analysis was performed based on the average of the threeruns per fish. A paired t-test was used to compare the oxygen consumption between thelight and dark phases. Significance levels were set at p < 0.05. Statistical analyses wereperformed using GraphPad Prism 10.0.3. Results3.1. Adult Seahorse Growth and Breeding PerformanceThroughout the experimental trial (seven-month period), adult seahorses increasedin both length and weight (Table 1). Growth parameters were proportionally higher inmales than in females, irrespective of the rearing temperature. Significantly higher growthrates (p < 0.05) were observed in fish reared at 20 and 24 ◦C compared to those reared at16 and 28 ◦C, resulting in a higher WG for fish reared at the intermediate temperatures(Table 1). Conversely, in all temperature treatments, both genders decreased their CF duringthe experiment.Table 1. Growth parameters of adult males and females H. guttulatus at the start and at the end of theseven-month trial (CF—condition factor; WG—weight gain).Growth Parameters16 ◦C 20 ◦C 24 ◦C 28 ◦CFemales Males Females Males Females Males Females MalesInitial length (cm) 16.3 ± 1.1 a 17.5 ± 0.7 a 16.5 ± 0.7 a 17.7 ± 0.4 a 16.3 ± 0.5 a 17.5 ± 0.8 a 16.8 ± 0.8 a 17.6 ± 0.5 aFinal length (cm) 17.7 ± 1.5 a 19.4 ± 1.3 a 18.6 ± 1.2 a 21.1 ± 0.6 a 18 ± 0.6 a 20.1 ± 0.4 a 18.3 ± 0.7 a 20.7 ± 0.8 aInitial weight (cm) 12.3 ± 0.7 a 12.5 ± 1.8 a 12.4 ± 1.3 a 13.2 ± 0.7 a 12.3 ± 0.5 a 12.2 ± 1.6 a 12.4 ± 0.5 a 12.6 ± 0.5 aFinal weight (cm) 14.0 ± 0.2 a 16.9 ± 5.1 a 14.9 ± 3.1 b 20.5 ± 1.4 a 14.2 ± 0.8 b 18.1 ± 1 a 14.8 ± 1.3 b 19.7 ± 4.2 aCF on start ofthe experiment 0.28 ± 0.1a 0.23 ± 0 a 0.28 ± 0.1 a 0.24 ± 0 a 0.28 ± 0 a 0.23 ± 0 a 0.26 ± 0 a 0.23 ± 0 aCF at the end ofthe experiment 0.25 ± 0.1a 0.23 ± 0.1 a 0.23 ± 0 a 0.21 ± 0 a 0.24 ± 0 a 0.22 ± 0 a 0.24 ± 0 a 0.22 ± 0 aWG (g/fish d−1) 0.03 ± 0.01 a 0.08 ± 0.02 a 0.04 ± 0.1 b 0.13 ± 0.07 a 0.03 ± 0.01 b 0.11 ± 0.03 a 0.04 ± 0.01 b 0.13 ± 0.05 aMortality (nº ind.) 0 0 0 0 0 1 3 7% mortality 0 0 6.2 b 62.5 aCF—Condition Factor; WG—Weight Gain. Within gender analysis: Rows with different superscripts letters aresignificantly different (p < 0.05).Adult mortality varied significantly at the four tested temperatures. No mortality wasobserved at 16 and 20 ◦C, but with increasing temperatures, the mortality rate increasedfrom 6.2% (one fish) at 24 ◦C to 62.5% (10 fish) at 28 ◦C. Notably, the mortality rate amongmales was higher than that of females, representing 43.7% of the total mortality at 28 ◦C.Regarding reproductive performance, no reproductive activity (including courtshipbehavior) was observed in seahorses kept at 16 ◦C. At 28 ◦C, only sporadic courtshipbehavior was observed, with no successful broods resulting from it. Conversely, seahorseskept at 20 and 24 ◦C produced 20 and 16 successful broods, respectively (2.5 and 2 successfulbroods per couple). The number of juveniles per brood decreased significantly (p < 0.05),from 410 ± 133 at 20 ◦C to 257 ± 157 at 24 ◦C.3.2. Juvenile GrowthData on length, weight, mean weight gain (WG), condition factor (CF), thermal-unitgrowth coefficient (TGC), and survival are reported in Table 2. At the end of the experimentDiversity 2024, 16, 719 7 of 14(56 DPP), significant differences (p < 0.05) in growth were observed between juvenilesreared at 20 ◦C and 24 ◦C compared to those reared at 16 ◦C. However, no significantdifferences (p > 0.05) were observed between juveniles reared at 20 ◦C and 24 ◦C (Table 2).At 28 ◦C, all fish died before the first sampling at 14 DPP. This differential in the growthperformance of fish reared at the intermediate temperatures was readily observed fromthe first sampling period (Figure 2a,b) until the end of the experiment. CF was the onlyunaffected growth parameter throughout the experiment, as no significant differences in CFwere found among juvenile H. guttulatus reared at each of the four temperatures (Table 1).Survival ranged from 0% in fish reared at 28 ◦C to 62% in fish reared at 20 ◦C, with lowintermediate values for fish reared at 16 ◦C (8%) and 24 ◦C (10%) (Figure 2c).Table 2. Standard length (cm), body weight (g), thermal-unit growth coefficient (TGC), conditionfactor (CF), and feed conversion rate (FCR) (mean ± SD) of juvenile H. guttulatus at the end of the56-day study under the four tested temperatures.Growth Parameter 16 ◦C 20 ◦C 24 ◦C 28 ◦CStandard length (cm) 5.4 ± 0.2 b 7.4 ± 1.3 a 6.7 ± 1.2 a 0Body weight (g) 0.36 ± 0.07 c 0.92 ± 0.4 a 0.71 ± 0.24 b 0WG (g.d−1) 0.006 ± 0.001 b 0.016 ± 0.002 a 0.013 ± 0.002 a 0TGC 0.12 c 0.31 a 0.24 b 0CF 0.22 ± 0.02 a 0.23 ± 0.03 a 0.24 ± 0.03 a 0% survival 8 b 62 a 10 b 0Initial weight = 0.002 ± 0.001 g: Initial length = 1.2 ± 0.2 cm, Initial Condition Factor = 0.12 ± 0.01. Rows withdifferent superscripts are significantly different (p < 0.05).Diversity 2024, 16, x FOR PEER REVIEW 7 of 14 CF on start of the experiment 0.28 ± 0.1a 0.23 ± 0a 0.28 ± 0.1a 0.24 ± 0a 0.28 ± 0a 0.23 ± 0a 0.26 ± 0a 0.23 ± 0a CF at the end of the experiment 0.25 ± 0.1a 0.23 ± 0.1a 0.23 ± 0a 0.21 ± 0a 0.24 ± 0a 0.22 ± 0a 0.24 ± 0a 0.22 ± 0a WG (g/fish d−1) 0.03 ± 0.01a 0.08 ± 0.02a 0.04 ± 0.1b 0.13 ± 0.07a 0.03 ± 0.01b 0.11 ± 0.03a 0.04 ± 0.01b 0.13 ± 0.05a Mortality (nº ind.) 0 0 0 0 0 1 3 7 % mortality 0 0 6.2b 62.5a CF—Condition Factor; WG—Weight Gain. Withi gend r analysis: Rows with different superscripts letters ar significantly different (p < 0.05). 3.2. Juvenile Growth Data on l ngth, weight, m n weight gain (WG), condition factor (CF), thermal-unit growth coefficient (TGC), and survival are reported in Table 2. At the end of the experiment (56 DPP), significant differences (p < 0.05) in growth were observed between juveniles reared at 20 °C and 24 °C compared to those reared at 16 °C. However, no significant differences (p > 0.05) were observed between juveniles reared at 20 °C and 24 °C (Table 2). At 28 °C, all fish died before the first sampling at 14 DPP. This differential in the growth performance of fish reared at the intermediate temperatures was readily observed from the first sampling period (Figure 2a,b) until the end of the experiment. CF was the only unaffected growth parameter throughout the experiment, as no significant differences in CF were found among juvenile H. guttulatus reared at each of the four temperatures (Table 1). Survival ranged from 0% in fish reared at 28 °C to 62% in fish reared at 20 °C, with low intermediate values for fish reared at 16 °C (8%) and 24 °C (10%) (Figure 2c). Length (cm)Weight (mg)0 14 28 42 56020406080100Days16°C20ºC24°C28°C Figure 2. (a) Length increase, (b) weight increase, and (c) percent survival of juvenile H. guttulatus reared at four tested temperatures (16, 20, 24, and 28 °C). Table 2. Standard length (cm), body weight (g), thermal-unit growth coefficient (TGC), condition factor (CF), and feed conversion rate (FCR) (mean ± SD) of juvenile H. guttulatus at the end of the 56-day study under the four tested temperatures. Growth Parameter 16 °C 20 °C 24 °C 28 °C Standard length (cm) 5.4 ± 0.2 b 7.4 ± 1.3 a 6.7 ± 1.2 a 0 Figure 2. (a) Length increase, (b) weight increase, and (c) percent survival of juvenile H. guttulatusreared at four tested temperatures (16, 20, 24, and 28 ◦C).3.3. Respirometry AnalysisA distinct individual variation in oxygen consumption was observed after fish weremoved to the respir metry cha b rs. Oxygen consumption values for each of the testedseahorse groups are detailed in Table 3. It was observed a constant decrease in oxygenconsumption according to the seahorse age, from 203.3 ± 3.1 to 107.5 ± 3.7 µmol O2/gBW/h between 1 DPP juveniles and adults (year old) at 16 ◦C, and from 273.3 ± 3.1 to128.8 ± 3.8 µmol O2/g BW/h between 1 DPP juveniles and adults (year old) at 28 ◦Cduring the light period. Likewise, the same pattern was observed during the dark periodwith a constant decrease in the oxygen consumption according to the seahorse age, fromDiversity 2024, 16, 719 8 of 14198.7 ± 3.5 to 106.3 ± 3.1 µmol O2/g BW/h between 1 DPP juveniles and adults (year old)at 16 ◦C, and from 127.6 ± 3.5 to 269 ± 6.2 µmol O2/g BW/h between 1 DPP juveniles andadults (year old) at 28 ◦C. Regardless of the age, the oxygen consumption was observedto increase linearly with increasing temperatures. No matter the tested temperature, anallometric relation between fish size/age and oxygen consumption was observed, as newlyhatched juveniles (1 DPP) consumed almost two times more oxygen than adult seahorses(one year old) (Figure 3).Table 3. Average oxygen consumption (mean ± s.d. in µmol O2/g BW/h) for each of the four agegroups tested (DPP—days post-parturition).16 ◦C 20 ◦C 24 ◦C 28 ◦C1 DPP juveniles 203.3 ± 3.1 d 227.3 ± 4.9 c 242.3 ± 3.1 b 273.3 ± 3.1 aLight 14 DPP juveniles 123.7 ± 3.7 b 128.4 ± 3.3 b 142.3 ± 3.5 a 156.4 ± 3.5 a28 DPP juveniles 119.4 ± 4.3 b 123.6 ± 3.2 b 132.8 ± 5.6 a 134.3 ± 4.7 aAdults (one year old) 107.5 ± 3.7 c 116.5 ± 4.7 b 126.3 ± 3.5 a 128.8 ± 3.8 a1 DPP juveniles 198.7 ± 3.5 c 213 ± 4.1 b 213 ± 4.1 b 269 ± 6.2 aDark 14 DPP juveniles 112.5 ± 4.8 d 125.2 ± 3.7 c 138.2 ± 3.6 b 151.6 ± 6.4 a28 DPP juveniles 117.7 ± 3.5 b 119.1 ± 4.6 b 127.1 ± 4.4 a 129.7 ± 3.8 aAdults (one year old) 106.3 ± 3.1 c 117.7 ± 3.4 b 131.5 ± 3.8 a 127.6 ± 3.5 aRows with different superscripts are significantly different (p < 0.05).Diversity 2024, 16, x FOR PEER REVIEW 8 of 14 Body weight (g) 0.36 ± 0.07 c 0.92 ± 0.4 a 0.71 ± 0.24 b 0 WG (g.d−1) 0.006 ± 0.001 b 0.016 ± 0.002 a 0.013 ± 0.002 a 0 TGC 0.12 c 0.31 a 0.24 b 0 CF 0.22 ± 0.02 a 0.23 ± 0.03 a 0.24 ± 0.03 a 0 % survival 8 b 62 a 10 b 0 Initial weight = 0.002 ± 0.001g: Initial length = 1.2 ± 0.2cm, Initial Condition Factor = 0.12 ± 0.01. Rows with different superscripts are significantly different (p < 0.05). 3.3. Respirometry Analysis A distinct individual variation in oxygen consumption was observed after fish were moved to the respirometry chambers. Oxygen consumption values for each of the tested seahorse groups are detailed in Table 3. It was observed a constant decrease in oxygen consumption according to the seahorse age, from 203.3 ± 3.1 to 107.5 ± 3.7 µmol O2/g BW/h between 1 DPP juveniles and adults (year old) at 16 °C, and from 273.3 ± 3.1 to 128.8 ± 3.8 µmol O2/g BW/h between 1 DPP juveniles and adults (year old) at 28 °C during the light period. Likewise, the same pattern was observed during the dark period with a constant decrease in the oxygen consumption according to the seahorse age, from 198.7 ± 3.5 to 106.3 ± 3.1 µmol O2/g BW/h between 1 DPP juveniles and adults (year old) at 16 °C, and from 127.6 ± 3.5 to 269 ± 6.2 µmol O2/g BW/h between 1 DPP juveniles and adults (year old) at 28 °C. Regardless of the age, the oxygen consumption was observed to increase linearly with increasing temperatures. No matter the tested temperature, an allometric relation between fish size/age and oxygen consumption was observed, as newly hatched juveniles (1 DPP) consumed almost two times more oxygen than adult seahorses (one year old) (Figure 3). Table 3. Average oxygen consumption (mean ± s.d. in µmol O2/g BW/h) for each of the four age groups tested (DPP—days post-parturition). 16 °C 20 °C 24 °C 28 °C 1 DPP juveniles 203.3 ± 3.1 d 227.3 ± 4.9 c 242.3 ± 3.1 b 273.3 ± 3.1 a Light 14 DPP juveniles 123.7 ± 3.7 b 128.4 ± 3.3 b 142.3 ± 3.5 a 156.4 ± 3.5 a 28 DPP juveniles 119.4 ± 4.3 b 123.6 ± 3.2 b 132.8 ± 5.6 a 134.3 ±4.7 a Adults (one year old) 107.5 ± 3.7 c 116.5 ± 4.7 b 126.3 ± 3.5 a 128.8 ± 3.8 a 1 DPP juveniles 198.7 ± 3.5c 213 ± 4.1 b 213 ± 4.1 b 269 ± 6.2 a Dark 14 DPP juveniles 112.5 ± 4.8 d 125.2 ± 3.7 c 138.2 ± 3.6 b 151.6 ± 6.4 a 28 DPP juveniles 117.7 ± 3.5 b 119.1 ± 4.6 b 127.1 ± 4.4 a 129.7 ± 3.8 a Adults (one year old) 106.3 ± 3.1 c 117.7 ± 3.4 b 131.5 ± 3.8 a 127.6 ± 3.5 a Rows with different superscripts are significantly different (p < 0.05). Oxygen consumption ( mol O2/g BW/h)Oxygen consumption ( mol O2/g BW/h)(a) (b) Figure 3. Metabolic rates (MR) (µmol O2/g BW/h) during (a) light phase, and (b) dark phase of juveniles (1, 14, and 28 DPP) and adults for each of the four tested water temperatures (16, 20, 24 and 28 °C). Figure 3. Metabolic rates (MR) (µmol O2/g BW/h) during (a) light phase, and (b) dark phase ofjuveniles (1, 14, and 28 DPP) and adults for each of the four tested water temperatures (16, 20, 24 and28 ◦C).Regardless of age and tested temperature, with the exception of 1 DPP juveniles at24 ◦C, the oxygen consumption was always higher, but not significantly different (p > 0.05)between the light period vs. the dark period.4. DiscussionFish thermal tolerance can be defined as heat and cold tolerance, which correspond tothe highest summer water temperature and the coldest winter water temperature withintheir ranges [45]. While species can withstand temporary cold and heat peaks, the pro-longed occurrence of these extreme temperatures may cause significant changes in species’growth, reproduction, and survival. The fish thermal environment is thought to have agreater impact on their distribution, overall condition, and survival than any other aquatichabitat characteristic [46]. Therefore, increasing air temperatures resulting from climatechange are expected to increase water temperatures, altering the fish’s thermal environ-ment with direct effects on their growth, reproduction, and survival, as well as that of theirprey [47–49]. Rising temperatures, within certain limits, can be beneficial. For example,ref. [50] observed that adult H. reidi density was positively and significantly correlated withincreased water temperatures during El Niño events. Similarly, in aquaculture, higher tem-peratures can accelerate growth rates and shorten the maturation period of fish. Ref. [51]Diversity 2024, 16, 719 9 of 14reported that juvenile H. barbouri can tolerate temperatures between 25 ◦C and 31 ◦C, butoptimal growth and survival were observed between 25 ◦C and 28 ◦C. However, whentemperatures exceed the optimal limits for a species, it can negatively impact the healthof aquatic animals by causing metabolic stress, increased susceptibility to disease, theproliferation of pathogens, and higher oxygen demand. Ref. [52] found that H. erectus,when exposed to acute thermal stress (30 ◦C for 2–10 h), exhibited a significant increasein basal metabolic rate (breathing rate) and elevated expression of stress response genes(Hsp genes). Chronic exposure to the same temperature (30 ◦C for four weeks) resultedin a persistently elevated basal metabolic rate, increased stress response gene expression,and higher mortality rates. Ref. [53] noticed that even short-term increases in temperaturecaused significant changes in the feeding behavior of juvenile H. kuda. Overall, theseauthors suggested a similar pattern where these species were unable to acclimate to chronicthermal stress, potentially leading to mass mortality due to excessively high metabolic ratesand stress-induced damage.Similarly, in the present study, it was also observed that temperature variation had amajor impact on the reproduction, growth, and survival of both juvenile and adult longsnout seahorses. For adult seahorses, male weight gain was consistently and significantlyhigher (p < 0.05) than that of females, a trait not directly related to the tested temperaturesand previously observed in this species [54]. However, male weight gain was significantlylower at 16 ◦C (the lowest temperature tested) compared to the other three temperatures.This result is expected, as it reflects a decrease in metabolic rate due to the low temperature.Nonetheless, female weight gain did not vary significantly (p > 0.05) across the testedtemperatures, indicating a different growth response between the sexes. However, thismetabolic decrease did not result in any mortality in the tested fish. Conversely, theincreased mortality observed at 28 ◦C is a clear negative effect caused by temperature and astrong indication that this species is unable to cope with prolonged heat periods. At 28 ◦C,reproduction was impaired as no viable broods were produced. Overall, it was observedthat when subjected to a prolonged heat period, the viability of the species is compromiseddue to the cessation of reproductive activity and, in most cases, death. Similar findingswere reported by [55] for the same species, where adult H. guttulatus were exposed totemperatures of 17 ◦C, 20 ◦C, and 24 ◦C. The researchers observed that fish were moreactive and fed more at 24 ◦C, but the increase in energy intake did not result in a highergrowth rate. Ref. [56] made similar observations for the broad-nose pipefish (Syngnathustyphle). When exposed to 24 ◦C, the fish became less active, showed reduced growth, andexperienced significantly lower survival rates compared to those kept at 20 ◦C.Transposing this evidence to natural circumstances implies that if this species’ popula-tions find themselves trapped in prolonged heat periods, their resilience will be affected,leading to a potential reduction in abundance or even the species’ disappearance withina short period of time. For example, in the Ria Formosa, according to [15], water tem-perature varies from 12 ◦C in winter to 27 ◦C in summer. These temperature ranges arequite similar to those tested in this study, indicating that the effects of temperature onwild seahorse populations in the lagoon may already be occurring. In a climate changescenario, even a small increase in temperature will negatively impact the species’ welfareand their ability to remain healthy and stable [57,58]. Moreover, the Ria Formosa lagoonis a highly dynamic system composed of shallow channels where the water temperatureis homogenized and tends to be similar at all depths preferred by this species (3–11 m,personal observation), thus preventing seahorses from moving to find suitable thermalhabitats. Therefore, as mentioned above, heat waves, which currently last for 1–2 weeks onthe Portuguese coast [13], will become more intense, more frequent, and longer-lasting inwarmer climate scenarios [14], leading to extended periods of heat waves with the potentialnegative impacts described here.It was observed that fecundity starts to be impaired at 24 ◦C, as the number of juvenilesproduced per brood was significantly lower (p < 0.05) than at 20 ◦C. Considering that evenunder normal circumstances, seahorse fecundity is much lower compared to other fishDiversity 2024, 16, 719 10 of 14species [1], this constraint may cause an additional impact on the species’ conservation.In this study, this result, combined with the fact that at 24 ◦C only 10% of the producedjuveniles survived to the end of their second month of life—much lower compared tothe 62% survival rate at 20 ◦C—indicates significant thermal sensitivity. Concordantly,refs. [40,59] observed that juvenile H. guttulatus have their optimal growth rate at 21 ◦C,and ref. [60] documented a significant increase in the metabolic rates of newborn juvenilesand adult H. guttulatus with rising temperatures. According to the authors, the juvenilestages display greater thermal sensitivity and may face greater metabolic challenges withpotential cascading consequences for their growth and survival compared to adults. Thiswas confirmed in the present study, where juvenile survival dropped to 10% at 24 ◦C andreached 100% mortality at 28 ◦C. Similarly, juvenile H. reidi mortality rates can reach 100%at temperatures above 30 ◦C [61,62]. Ref. [52] also reported increased mortality in juvenileH. erectus at 32 ◦C in laboratory conditions, temperatures that are considered to be at theupper limit of thermal tolerance for these species.In this study, oxygen consumption was higher during the light phase compared tothe dark phase, regardless of the temperature to which the fish were exposed. Since thefish were fed during the light phase, the increased oxygen consumption may be partlyexplained by the feeding stimulus, which helped to overcome the light–dark cycle. Thehigher metabolism in the light phase may be due to the metabolic processing of food, afinding similar to that of [42], who observed a similar pattern for juvenile Senegalese sole(Solea senegalensis). However, this increase in metabolic activity was also directly related tothe temperature increase; irrespective of the feeding effect, oxygen consumption was am-plified by temperature. As observed in the first part of the experiment, juvenile growth andsurvival and adult reproductive success and survival were severely impacted by tempera-ture. In the second part, an overall increase in oxygen consumption was noted, indicatingthat the observed results are solely a consequence of temperature effects. Metabolic rate iscommonly described as scaling with body size following a power function and increasingexponentially with temperature [63], with the mass-scaling exponent decreasing as tem-perature rises. In the present study, oxygen consumption was significantly higher in thenewborn age group (1 DPP) compared to adult seahorses, aligning with previous studiessuch as [23], who observed a similar trend across 69 other teleost species. It is noteworthythat the oxygen consumption of newly hatched juveniles (1 DPP) was significantly higher(p < 0.05) than that observed in 14 and 18 DPP juveniles and in adults. This indicates greaterthermal sensitivity in the first days of life, which compromises individual survival. Overtime, older juveniles increase their thermal tolerance, reaching levels almost identical tothose of adults. Therefore, a clear relationship between allometric variation and thermalsensitivity was observed, consistent with the findings of [23]. Fish sensitivity to cyclic hightemperatures during the first life stages is not specific to seahorse species. Ref. [64] reportedthat when the mean water temperature was increased by just 0.6 ◦C, there was a reductionin the life history diversity of rainbow trout (Oncorhynchus mykiss), and ref. [65] notedthat high temperatures (31–35 ◦C) decreased growth in juvenile Channel catfish (Ictaluruspunctatus) due to reduced food consumption, feed conversion, and increased activity levels.The prevalence of high-temperature periods above the species’ optimal range can, therefore,imply a long-term negative effect that may impact the species beyond its coping point,impairing long-term population health.In this study, it was observed that adult fish (one year old) had a 34.4% increase inoxygen consumption from 16 ◦C to 28 ◦C. Since male seahorses provide paternal care totheir broods by maintaining the developing embryos in the brood pouch, oxygen require-ment and consumption are hypothesized to be even higher during the breeding season,which occurs during the spring-summer period when longer heat waves also happen. Thisconstraint implies an additional physiological demand on the males and will potentiallyaffect them and their resulting offspring in the same way observed in this study. Environ-mental factors do not play isolated roles in animal physiology; instead, they interact andcombine, causing increased detrimental effects on species’ survival. For aquatic species,Diversity 2024, 16, 719 11 of 14increased water temperatures lead to a decrease in dissolved oxygen, requiring additionalphysiological effort to maintain the necessary oxygen levels in their bodies. Seahorsesare no exception. For pregnant males, who provide parental care to their broods, thisconstraint may represent an additional physiological effort, as they need to supply oxygento the developing embryos in their pouch. This long-term metabolic requirement may helpexplain the increased male mortality compared to females.In conclusion, both juvenile and adult H. guttulatus are affected by long-term exposureto increased temperatures, with direct implications for their growth, survival, and repro-duction. Under natural conditions, this may represent a major constraint for the long-termconservation of this and other seahorse species in coastal areas similar to the Ria Formosa,and ultimately for local aquatic biodiversity as a whole.Author Contributions: Conceptualization, J.P.; methodology, J.P. and M.C.; formal analysis, J.P.;writing/original draft preparation, J.P.; review and editing, J.P.; F.L.; M.C. and J.P.A.; project admin-istration, J.P.; funding acquisition, J.P. and F.L. All authors have read and agreed to the publishedversion of the manuscript.Funding: This study was funded by the project HIPPOCAMPUS_RIA “Avaliação e implementaçãode conservação ecossistémica de cavalos-marinhos” (Fundo Ambiental) and received Portuguese na-tional funds from FCT—Foundation for Science and Technology through projects UIDB/04326/2020,UIDP/04326/2020 and LA/P/0101/2020.Institutional Review Board Statement: CCMAR facilities and the research are certified to house andconduct experiments with live animals (Group-C licenses from the Direção Geral de Alimentação eVeterinária, Ministério da Agricultura, Florestas e Desenvolvimento Rural, Portugal). The captivebreeding program for H. guttulatus (Project HIPPOCAMPUS_RIA) was approved by ICNF—Institutoda Conservação da Natureza e das Florestas under the permit S-009617/2024. Under this approval, thestudy was performed in compliance with the Guidelines of the European Union Council (86/609/EU)and Portuguese legislation for the use of laboratory animals.Data Availability Statement: The data used during the current study are available from the corre-sponding author upon reasonable request.Acknowledgments: The authors would like to acknowledge António Sykes and João Reis for theirvaluable contribution and assistance in the respirometry analyses.Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the designof the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; orin the decision to publish the results.References1. Foster, S.J.; Vincent, A.C.J. Life history and ecology of seahorses: Implications for conservation and management. J. Fish Biol. 2004,65, 1–61. [CrossRef]2. Shokri, M.R.; Gladstone, W.; Jelbart, J. 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Vancouver : University of British Columbia Library
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seahorses
Hippocampus guttulatus
temperature
growth
reproduction
oxygen consumption
climate change
Temperature Effects on Growth Performance, Fecundity and Survival of Hippocampus guttulatus
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