Effects of acclimation temperature and exposure time on the scope for growth of the blackfoot Pāua (Haliotis iris)

Animal collection and acclimation

Pāua of both sexes (80–100 mm in shell length, 124.1 ± 19.9 g) were collected by snorkelling in Little Port Cooper (Lyttelton), Christchurch (S43°613′ E172°816′). The average maximal shell length for this population between 2018 and 2020 was 112.4 mm. Pāua were collected for specific acclimation temperatures close to seasonal values from Lyttelton Harbour (Table 1). They were then transported in a chilly bin in aerated seawater to the aquarium at the University of Canterbury where they were tagged and held in recirculating natural seawater (salinity ~33‰) system for at least 3 days to reduce possible stress. Temperatures during transportation and in the aquarium were maintained similar at temperatures to the original ambient temperature where Pāua were caught (Table 1). After that, Pāua were transferred to control rooms with smaller recirculating seawater systems. Temperatures were raised ~2°C day⁻¹ by adjusting constant temperature rooms until they reached the target water temperatures (i.e. 12, 15, 18 or 21°C). For each acclimation temperature, there were four tank replicates. Each replicate was a 60-L tank (with Pāua) connected to another 60-L tank (without Pāua) by a submersible pump (Moray 1300, 1100 L h⁻¹, China) to maintain nature sea water recirculation. Water from the upper tanks flowed through 50-μm polishing filter pads to trap particles in the water before going to the lower tanks. Air stones were put in the lower tanks to oxygenate the water. The light-dark cycle was 12–12 h. Pāua were fed seaweed (Undaria pinnatifida or Macrocystis pyrifera) at 15% body weight every other day. Salinity, dissolved oxygen, pH and ammonia concentration were monitored and maintained at ~33‰ ± 2, 95% ± 5, 8.2 ± 4 and under 0.25 mg L⁻¹. Water was exchanged for 50% every second day. They were stocked at a rate of 8–10 Pāua per tank.

Experimental design

The experiment was designed to estimate the effects of acclimation at four constant temperatures (12, 15, 18 or 21°C) recorded at four different periods (Table 1) and acclimation times up to 28 days (measured on Days 1, 14, 21 and 28) on physiological responses (absorption efficiency, absorbed energy, respiration energy, ammonia excretion energy and SFG) of Pāua and their haemolymph parameters (haemocyanin, protein, sodium and potassium). A group of 32 Pāua was used for each temperature treatment (except for 18°C where 24 Pāua were used). Eight replicate Pāua were measured for each time interval within each temperature treatment. At 18°C, physiological responses were not measured for the Pāua acclimated on Day 1 because the time and facilities were limited.

Statistical analysis

The R statistical program was used for all analyses (ver. 2021.09.0, R Foundation for Statistical Computing, Vienna, Austria, see https://www.r-project.org/). Two-way ANOVA was used to compare SFG, physiological responses and haemolymph parameters of Pāua between different acclimation temperatures and exposure times. The assumptions of normality and homogeneity of variances were tested for all variables by viewing quantile-quantile normality plots and histograms of the model residuals. If the main effects (acclimation temperature and exposure time) were significant (P ≤ 0.05), then Tukey’s Honest Significant Difference (HSD) post hoc test was applied to detect significant differences between treatment means. When a significant interaction between main effects was observed, plots were used to interpret the interaction effects.

Absorption efficiency

Acclimation temperature and exposure time affected absorption efficiency (Fig. 1a). Values were generally above 70%, with the exception being for Pāua 3 days after they had been collected from the field in summer (February). There was significant variation at the start of the acclimation period when the absorption efficiency in the 15°C acclimation being remarkably high (96.4%) and that for the 21°C acclimation was very low (53.4%). From day 14 onwards, there were not many differences in the absorption efficiency of Pāua at different acclimation temperatures. Both acclimation temperature and exposure time affected absorption efficiency, and there was a significant interaction between these factors (P < 0.001, Table 2).

Fig. 1.

Mean physiological responses and SFG of Pāua acclimated at 12, 15, 18 and 21°C, measured multiple times over an acclimation period of 28 days for (a) absorption efficiencies, (b) absorbed energy, (c) excretion energy, (d) respiration energy, (e) scope for growth, and (f) N:O ratio. Bars depict the mean and error bars are ±s.e. from eight replicates per treatment.

Energy absorbed from food

The energy absorbed from food (J g⁻¹ DW h⁻¹) varied widely on Day 1, with values in the 15°C acclimation 3.7 and 9.6 times higher than that for the 12 and 21°C acclimation treatments respectively. After 2 weeks of acclimation, the absorbed energy was more stable (Fig. 1b). The energy absorption response depended on both acclimation temperature and exposure time, and there was a significant interaction effect between the two factors (P < 0.001, Table 2).

Respiration energy

The energy required for respiration (J g⁻¹ DW h⁻¹) increased with higher temperatures and changed over exposure time. Pāua acclimated to 21°C consumed the highest amount of oxygen at 6.22 ± 0.90 J g⁻¹ DW h⁻¹, whereas that of 12°C was lowest, at 2.77 ± 0.67 J g⁻¹ DW h⁻¹ (Fig. 1d). The values accounted for the highest proportion of energy absorbed, increasing with rising acclimation temperatures of ~36, ~40, ~49 and ~69% at 12, 15, 18 and 21°C respectively. The respiration energy was significantly affected by acclimation temperature and exposure time (P < 0.001, Table 2).

Ammonia excretion energy

The ammonia excretion energy (J g⁻¹ DW h⁻¹) of Pāua increased from Days 1 to 14 before remaining fairly stable until Day 28 (Fig. 2c). During 4 weeks of acclimation, both temperature (P < 0.001, Table 2) and acclimation time (P < 0.001) influenced the ammonia excretion energy significantly, but there was no interaction between them (P < 0.203). However, these values only accounted for a small amount of the absorbed energy, ranging from 2.9 to 5.6%.

Fig. 2.

Haemolymph responses of Pāua acclimated at 12, 15 and 21°C, measured multiple times over an acclimation period of 28 days for (a) haemocyanin concentration, (b) haemolymph protein, (c) haemolymph sodium and (d) haemolymph potassium. Bars depict the mean and error bars are ±s.e. from eight replicates per treatment.

Scope for growth

The SFG (J g⁻¹ DW h⁻¹) of Pāua varied for the different acclimation temperatures and exposure times (Fig. 1e). On Day 1 after collection from the field in spring, the SFG in the 15°C acclimation was very high (18.76 J g⁻¹ DW h⁻¹) and Day 1 values for those collected in summer for the 21°C was negative (−4.10 J g⁻¹ DW h⁻¹). After a 2-week acclimation period, SFG became more stable and the positive SFG values were obtained for Pāua at all acclimation temperatures. The SFG of Pāua acclimated at 21°C was lowest on Day 28. For the 18°C acclimation, SFG was highest on Day 14 and reduced until Day 28. By contrast, the values for 12°C increased during the acclimation period and became highest in all groups on Day 28. The SFG of Pāua acclimated at 15°C remained almost unchanged after 2 weeks’ acclimation. During the 4 weeks, the SFG of Pāua was significantly affected by an interaction between acclimation temperature and exposure time (P < 0.001, Table 2). The significant interaction was due to considerable variation on Day 1.

O:N ratio

After the 2-week acclimation period, the ratio ranged from 18.7 to 32.5, with the highest value at 18°C. This ratio was generally similar between 21 and 28 days (Fig. 1f). Time but not acclimation temperature significantly affected the O:N ratio (P < 0.001, Table 2). The values on Day 1 were significantly higher than those for Days 14, 21 and 28 (Tukey’s HSD test, P < 0.001). All other groups were similar (Tukey’s HSD test, P > 0.05).

Haemocyanin concentration and haemolymph protein

Acclimation temperature and exposure time had similar effects on haemocyanin concentration (mmol L⁻¹) and haemolymph protein (g L⁻¹). They all increased with rising acclimation temperatures, with the highest at 21°C and the lowest at 12°C (Fig. 2a, b). They were affected significantly by acclimation temperature (P < 0.001, Table 2) but not by exposure time (P > 0.05). Also, there was no interaction between the two factors. Haemocyanin concentration at 12°C was significantly lower than 21°C (Tukey’s HSD test, P < 0.001). By contrast, haemolymph protein at 12°C was significantly lower than at 15 and 21°C (Tukey’s HSD test, P < 0.05).

Haemolymph sodium and potassium

Both haemolymph sodium (mmol L⁻¹) and potassium (mmol L⁻¹) were highest at 12°C (Fig. 2c, d). Temperature had a significant effect on the haemolymph sodium (P < 0.001, Table 1), but acclimation time did not (P > 0.001). There was no interaction between the two factors. The levels for Pāua acclimated at 12°C were significantly higher than at other temperatures (Tukey’s HSD test, P < 0.001). However, neither temperature nor time affected haemolymph potassium (P > 0.05, Table 2).

Seasonal physiological responses of field-collected Pāua

It is well known that the physiology of molluscs can show adaptations to seasonal changes in their environment and these results in seasonal differences in reproduction, fecundity and growth (Vilchis et al. 2005; Shin et al. 2020; Oliveira et al. 2021). Such seasonal adjustments have been shown in metabolism, oxygen uptake, ammonia excretion and digestive enzymes (Bayne and Newell 1983). In such studies, the main factors thought to affect these changes include fluctuating or constant exposure temperature, high temperatures or food availability or quality. In the present study, seasonally collected Pāua maintained for 1 day at a constant temperature close to their field temperature had variable feeding, respiration and excretion rates. Pāua collected in spring had the highest energy intake and those collected in summer had the highest energy loss due to respiration. Calculations for the SFG of these Pāua were low for winter collected individuals, highest for spring collected individuals and negative for summer collected individuals. Negative values for SFG have been recorded for other haliotids in other studies (Vosloo and Vosloo 2010). For Pāua these values most likely reflect the field conditions where, during spring, high food availability and temperature conditions close to 15°C are most likely optimal for metabolism, gonad development and spawning. High temperatures over summer are consistent with higher oxygen demands and limited food intake most likely resulting in impaired SFG. If summer collected individuals are not in good physiological health then they may not adapt quickly to laboratory conditions that are two degrees above the seawater temperature at the time of collection.

Absorbed energy and absorption efficiency

For field collected haliotids, absorption efficiencies usually exceed 50% depending on food types (Peck et al. 1987) but in laboratory experiments or in aquaculture, where a single favoured algal food is supplied, the expectation is that absorption efficiency would be higher (Bansemer et al. 2016). In the present study after 2 weeks of acclimation, absorption efficiencies for Pāua were similar to other studies. For example, values for the European abalone H. tuberculata were between 78 and 81% (Peck et al. 1987), for the green abalone, H. fulgens 84.9% (Farías et al. 2003) and for Thai abalone H. asinina, 22–42 mm in length, the average absorption efficiency was 81.04% (Ganmanee et al. 2010).

The energy obtained by grazers from their algal food depends upon its availability, its nutritional value, the feeding rate and the ability of the animal to digest it (Barkai and Griffiths 1987). All of these variables showed variability in the field and linked directly or indirectly with temperature change. The grazing rate of molluscs generally increases with exposure temperature up to a certain limit that is regarded as the optimal temperature. It then declines as in H. midae (Britz et al. 1997). The optimum temperature differs amongst haliotid species depending on species and habitat characteristics. In summer, for H. tuberulata, the amount of energy absorbed from food was greater than H. fulgens but the opposite was found in winter. In the present study, after 2 weeks of acclimation to constant temperatures absorbed energy increased with exposure temperature up to 18°C but decreased in the 21°C acclimation. Following a further 2 weeks of acclimation, the values were broadly similar for all acclimation temperatures. Over the 28-day time period, Pāua acclimated to 12°C had the most stable energy uptake generally increasing over time.

Respiration and ammonia excretion energies

Consistent with numerous other studies on molluscs, the energy consumed in respiration and ammonia excretion energies of Pāua increased markedly with acclimation temperature (McBride et al. 2001; Kang et al. 2019) and also varied over time. The two factors acted independently and values in the current study were generally higher than those from other haliotids. These differences may be due to body size or the exposure temperatures investigated. For H. tuberculata, Lopez and Tyler (2006) found that energy lost due to respiration and ammonia excretion increased greatly with the temperature from 15 to 22°C. They also found that respiration energy depended on size, accounting for 31% of the energy intake for the 10–20-mm length group, and 50.7% for the 50–60-mm group. By contrast, ammonia excretion comprised 0.9% of energy intake for the small up to 3.5% for the larger group.

Barkai and Griffiths (1988) reported that less than 1% of the energy uptake of the South African haliotid (H. midae) was lost by ammonia excretion and ~32% of energy was spent on respiration. Similarly, for Thai abalone H. asinina between 22 and 42 mm, more than 30% of the energy obtained was used for metabolism and from 0.8 to 1.8% spent for ammonia excretion (Ganmanee et al. 2010). In the present study, energy lost by respiration was 36% of intake for animals kept at 12°C, and this increased to over 40% at the higher temperatures of 15 and 18°C. At the highest temperature (21°C) energy lost through respiration was 69%, indicating that this temperature is well above optimal for this species.

For adults of the South African H.midae, Vosloo and Vosloo (2010), acclimation of cold-acclimated individuals to 16, 19 and 22°C for 1 month decreased both their specific oxygen consumption and nitrogen excretion at higher temperatures. This ability to adapt was not the same response recorded for oxygen uptake in Pāua acclimated between 12 and 21°C over a period of 28 days. By contrast, the oxygen uptake of Pāua acclimated to 21°C remained high throughout the acclimation period, confirming the limited ability to adapt to this higher temperature regime.

The O:N ratio is useful in determining the energy sources used in the metabolism of marine ectotherms. In the present study the O:N ratio for field-collected Pāua was between 33 and 41, values similar to H. fulgens, with an average value of 21.6 and 47.0 for large individuals and smaller individuals respectively (Farías et al. 2003). For Pāua, values decreased after 2 weeks of acclimation then remained at levels close to 20 for the following 14 days. Generally, the O:N ratio of Pāua was lower than other haliotids. The O:N ratio of H. discus hannai juveniles was higher than H. iris, ranging from 32.1 ± 1.4 to 38.1 ± 1.8, with a mean of 34.3 ± 0.3 (González et al. 2010). These findings suggest that, in the field, Pāua use both proteins and lipids to produce energy and that during the stable 28-day acclimation period, there was a shift towards utilisation of protein.

Scope for growth

Scope for growth (SFG), the difference between energy gains and losses and the energy available for shell, somatic and reproductive growth, has been used as a predictor of actual growth in many molluscs including bivalves (Widdows and Johnson 1988) but the relationship is less well known for haliotids. For Pāua both acclimation temperature and exposure time had interactive effects on the SFG.

Although Pāua acclimated at 12, 15, 18 and 21°C all had a positive SFG following 2 weeks of acclimation to constant temperatures, the value decreased considerably following 28 days of acclimation at 21°C. This is most likely due to the increased demands of oxygen to maintain metabolism at this temperature (Guppy and Withers 1999; Shao et al. 2015). In the 21°C acclimation, there was a steady decline over the acclimation period indicating that this acclimation temperature is above the optimal level. The positive values for SFG for the 12, 15 and 18°C acclimations suggest these haliotids could grow and mature at these lower seawater temperatures in New Zealand.

SFG values recorded previously for abalone are both higher and lower than those recorded here for Pāua (Ganmanee et al. 2010; González et al. 2010). Some of the differences can be explained by exposure temperature and others on body size; for example for H. asinina, feeding on an artificial diet at 28°C, the SFG increased with size for three size classes between 22- and 42-mm length (approximately 2–25 g) giving SFG estimates between 2.9 and 5.7 J g⁻¹ DW h⁻¹. SFG dependence on size was also found in juvenile green abalone H. fulgens, ranging from 99.8 to 173.7 J abalone⁻¹ day⁻¹ for abalone between 10 and 50 mm at 16°C, using a diet formulation (Farías et al. 2003). Also, the SFG of juvenile European abalone H. tuberculata was 33.9 cal g⁻¹ DW day⁻¹ (~5.91 J g⁻¹ DW h⁻¹) at 15°C when fed on Ulva lactuca (Peck et al. 1987) was a little lower than was found in the current research. The effects of temperature on the SFG have also been investigated in some other species including other molluscs and crustaceans (Yurista 1999; Navarro et al. 2002). These results were consistent with those for Pāua, where, within the optimal temperature range, SFG reached the highest values.

Haemolymph properties

Haemolymph is the circulatory fluid in molluscs transporting oxygen, nutrients, hormones, proteins and immune cells. It contains haemocyanin that accounts for a large proportion of haemolymph proteins (Fredrick and Ravichandran 2012). Both haemolymph haemocyanin and protein levels of Pāua showed a similar response after 14 days of acclimation, with levels increasing with higher acclimation temperatures from 12 to 21°C. The elevation of haemocyanin levels is consistent with the ability of Pāua to maintain high levels of oxygen uptake at temperatures above 20°C. This ability, however, may not be present in juveniles as Wells et al. (1998) reported that aerobic metabolism is limited above 20°C owing to a limitation of their ability for oxygen storage in haemocyanin. These results contrast with research on the marsh isopod Saduria entomon where the haemocyanin concentration remained almost unchanged with increasing temperatures (Borecka et al. 2016).

Total blood protein has been applied as a health status indicator in marine invertebrates (Paschke et al. 2010) and when haemocyanin levels increase due to increased temperatures, haemolymph proteins are also expected to increase. Studies on the effects of heat stress on juvenile haliotids (hybrid Haliotis laevigata × Haliotis rubra) found that haemolymph protein was not significantly affected by increasing temperature from 16 to 26°C after 1 week of acclimation (Hooper et al. 2014). These results suggest that protein regulation in haliotids may differ from other invertebrates.

For Pāua, haemolymph sodium was affected by temperature with the highest values at the lowest acclimation temperature of 12°C. However, haemolymph potassium was similar for Pāua acclimated between 12 and 21°C. Day et al. (2010) found that both haemolymph sodium and potassium of an Australian haliotid increased as exposure temperature decreased. In contrast, in another study on farmed Australian haliotids (hybrid Haliotis laevigata × H. rubra), Hooper et al. (2014), found that haemolymph protein and haemolymph sodium were unaffected by increasing temperature from 16 to 26°C after 1 week of acclimation. The results of that study are similar to the results on the oyster drill (Thais haenzastonza) where Hildreth and Stickle (1980) reported that temperature did not affect haemolymph sodium and potassium of this species.

Ecological context

Like all molluscs, Pāua show seasonal adaptations in their physiology as demonstrated by their patterns of growth and reproduction (Hooker et al. 1997). This is consistent with other haliotids, including those in aquaculture that also show adaptations to multiple stressors including hypoxia, air exposure and elevated temperatures (Rogers-Bennett et al. 2010; Ragg and Watts 2015). Previous research on oxygen uptake in haliotids suggests that the long and short-term responses to elevated temperatures are both species and location specific (Hooper et al. 2014). It also depends on the exposure temperature and whether this is within its normal environmental range.

The laboratory acclimation responses of Pāua are consistent with their seasonal responses and geographical distribution. Because Paūa cannot significantly reduce their energy expenditure due to respiration with increasing temperature they are therefore vulnerable to long periods of seawater temperatures above 18°C. This could induce an energy imbalance at high temperatures, in which the metabolic energy expenses of Pāua exceed their energy gain. In turn, this could affect their growth and reproduction. As a result, Pāua will be likely vulnerable to increasing global warming and marine heatwaves (Morash and Alter 2016; Markle and Kozak 2018).

Some haliotid species appear better able than others to adapt to changing environmental temperatures (Kang et al. 2019). For example, Alter et al. (2017) found that H. laevigata and H. laevigata × H. rubra hybrids have thermal acclimation capacity, but H. rubra do not. In other studies, Barkai and Griffiths (1987), investigated H. midae in South Africa and found that the respiration rate of abalone in the field was highly affected by temperature and did not have an acclimatory adjustment of its metabolism, which rose significantly during the summer. However, studies on the same haliotid suggested this species reduced the specific oxygen consumption and nitrogen excretion as temperature increased (Vosloo and Vosloo 2010). In a later study, Vosloo et al. (2013) suggest that 19°C is the temperature acclimation limit for winter-acclimated H. midae and that above this temperature energy is reallocated to survival mechanisms instead of growth. It follows then that adult Pāua show similar thermal acclimatory responses to juvenile H. midae from South Africa.

The inability of Pāua to reduce energy expenditure at seawater temperatures of 21°C suggests that, in the event of future seawater warming, there is likely to be a southern shift in the distributions to more favourable temperatures. Throughout New Zealand, however, Pāua growth differs with seawater temperatures and on small scales with higher growth on promontories than within bays (Naylor and Andrew 2004; Haist 2018). In addition, ocean warming and marine heatwaves are major threats to marine biodiversity, including effects on primary productivity and kelp forest, which provide food for Pāua (Arafeh-Dalmau et al. 2019; Rogers-Bennett and Catton 2019; Thomsen et al. 2019; Rogers-Bennett et al. 2021). Thus, there may be direct and indirect effects of climate change. The present study highlighted the likely inability of adult Paūa to survive long-term exposure to increased seawater temperatures ~21°C and this vulnerability confirms research by Aalto et al. (2020), which has suggested that, in haliotids, adults are the life stage that is most sensitive to environmental stress. In the current study, if Pāua return back to lower temperatures after a short exposure to 21°C, they may get to recover quickly. This is because they are acclimatised at lower temperature. Therefore, in aquaculture systems for Pāua, adults most likely should managed below 21°C. Also, using high quality foods or varied food sources may help Pāua to survive and grow at elevated temperatures.

Pāua in this study were maintained under the same laboratory conditions with high water quality and enough food. However, space may have been limited in the experimental tanks. Handling could also have resulted in variations of in the physiology of Pāua in this study. Future studies should explore these factors in combination with temperature effects affecting the SFG of Pāua.

In conclusion, the findings from the present study suggest that in the future, if warming seawater temperatures due to climate change result in exceeding 21°C for a long period, this could act as a severe stress to adult Pāua. The results confirm that energy relationships are affected by acclimation temperature and time. At 21°C, energy expenditure exceeded energy uptake suggesting that there would be insufficient energy for growth. Because growth is affected by other factors, then similar studies need to be undertaken using combinations of stressors including reduced oxygen levels and salinity. It would also be useful to examine the responses of juvenile Pāua living in low intertidal habitats with more variable temperature extremes and different food characteristics.