INTRODUCTIONTop

Estuaries can be areas of rapid environmental change as physico-chemical variables such as salinity and temperature may vary within tidal scales, localized seasonal weather patterns and inter-annual climatic shifts (Wallace et al. 1984Wallace J.H., Kok H.M., Beckley L.E., et al. 1984. South African estuaries and their importance to fishes. S. Afr. J. Sci. 80: 203-207., James et al. 2013James N.C., van Niekerk L., Whitfield A.K., et al. 2013. Effects of climate change on South African estuaries and associated fish species. Clim. Res. 57: 233-248., Potts et al. 2015Potts W.M., Götz A., James N. 2015. Review of the projected impacts of climate change on coastal fishes in southern Africa. Rev. Fish Biol. Fish. 25: 603-630.). This is notwithstanding the further variability caused by anthropogenic drivers such as freshwater abstraction, impoundments, pollution and climate change (Koehn et al. 2011Koehn J.D., Hobday A.J., Pratchett M.S., et al. 2011. Climate change and Australian marine and freshwater environments, fishes and fisheries: synthesis and options for adaptation. Mar. Freshw. Res. 62: 1148-1164., James et al. 2013James N.C., van Niekerk L., Whitfield A.K., et al. 2013. Effects of climate change on South African estuaries and associated fish species. Clim. Res. 57: 233-248., Adams et al. 2016Adams J.B., Cowie M., Van Niekerk L. 2016. Assessment of completed ecological water requirement studies for South African estuaries and responses to changes in freshwater inflow. Water Research Commission, WRC Report No. KV 352/15, Pretoria, 57 pp.). Hypersaline conditions arise when salinity exceeds that of seawater through excess evaporation exceeding freshwater inflow during droughts, or through physical modifications to the estuary, with the biogeochemistry of the catchment area also playing a role (Potter et al. 2010Potter I.C., Chuwen B.M., Hoeksema S.D., et al. 2010. The concept of an estuary: A definition that incorporates systems which can become closed to the ocean and hypersaline. Est. Coast. Shelf Sci. 87: 497-500., Gonzalez 2012Gonzalez R.J. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J. Comp. Physiol. B 182: 321-329., Strydom 2015Strydom N.A. 2015. Patterns in larval fish diversity, abundance, and distribution in temperate South African estuaries. Estuar. Coasts 38: 268-284.). For example, the intermittently open Gqutywa Estuary in the Eastern Cape, South Africa, becomes hypersaline at times due to its closed nature and catchment geochemistry (Teske and Wooldridge 2001Teske P.R., Wooldridge T. 2001. A comparison of the macrobenthic faunas of permanently open and temporarily open/closed South African estuaries. Hydrobiologia 464: 227-243., Strydom et al. 2003Strydom N.A., Whitfield A.K., Wooldridge T.H. 2003. The role of estuarine type in characterizing early stage fish assemblages in warm temperate estuaries, South Africa. Afr. Zool. 38: 29-43.).

Hypersaline conditions may result in several physiological challenges to fish species (Brauner et al. 2012Brauner C.J., Gonzalez R.J., Wilson J.M. 2012. Extreme environments: hypersaline, alkaline and ion-poor waters. In: McCormick S.D., Farrell A.P., Brauner C.J. (eds), Fish physiology: Euryhaline fishes. Academic Press, Cambridge, pp. 435-476.). The difference in osmotic pressure between the internal structures and the external environment of teleost fish, such as the common bream Rhabdosargus holubi (family Sparidae), results in a loss of water, which must be mitigated by drinking (Brauner et al. 2012Brauner C.J., Gonzalez R.J., Wilson J.M. 2012. Extreme environments: hypersaline, alkaline and ion-poor waters. In: McCormick S.D., Farrell A.P., Brauner C.J. (eds), Fish physiology: Euryhaline fishes. Academic Press, Cambridge, pp. 435-476.). This results in salt absorption across the gut, which must be excreted to prevent elevated internal levels and osmotic disturbances (Bœuf and Payan 2001Bœuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 130: 411-423., Brauner et al. 2012Brauner C.J., Gonzalez R.J., Wilson J.M. 2012. Extreme environments: hypersaline, alkaline and ion-poor waters. In: McCormick S.D., Farrell A.P., Brauner C.J. (eds), Fish physiology: Euryhaline fishes. Academic Press, Cambridge, pp. 435-476.). A generalized trend for the impact of osmoregulation on fish respiration and growth is unclear (Bœuf and Payan 2001Bœuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 130: 411-423., Gonzalez 2012Gonzalez R.J. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J. Comp. Physiol. B 182: 321-329.). Previous studies have shown that the impact of osmoregulation on the energy expenditure of fish may be higher than 50% or lower than 10%, varying considerably between species (Bœuf and Payan 2001Bœuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 130: 411-423., Gonzalez 2012Gonzalez R.J. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J. Comp. Physiol. B 182: 321-329., Ern et al. 2014Ern R., Huong D.T.T., Cong N.V., et al. 2014. Effect of salinity on oxygen consumption in fishes: a review. J. Fish Biol. 84: 1210-1220.). Thus, it is possible that changing salinity may have species-specific effects on the physiology of fish (Bœuf and Payan 2001Bœuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 130: 411-423.). This may be especially true for euryhaline species such as R. holubi, which is an estuarine-dependent marine species that is endemic and common in South African estuaries, where salinities may vary considerably (Blaber 1973aBlaber S.J.M. 1973a. The ecology of juvenile Rhabdosargus holubi (Steinachner) (Teleostei: Sparidae). PhD thesis, Rhodes University, Grahamstown, 123 pp., Whitfield 1998Whitfield A.K. 1998. Biology and ecology of fishes in South African estuaries. In: Smith J.L.B. (ed), Ichthyological Monographs 2, Institute for Ichthyology, Grahamstown, 223 pp., Whitfield et al. 2006Whitfield A.K., Taylor R.H., Fox C., et al. 2006. Fishes and salinities in the St Lucia estuarine system—a review. Rev. Fish Biol. Fish. 16: 1-20.).

Hypersalinity can cause osmotic stress in organisms living in estuaries, resulting in potentially lower growth rates, skeletal deformities and mortality at extreme levels (Bœuf and Payan 2001Bœuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 130: 411-423., Gonzalez 2012Gonzalez R.J. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J. Comp. Physiol. B 182: 321-329., Boglione et al. 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.). These kinds of skeletal deformities are prevalent in aquaculture conditions, where even small changes to salinity may impact on the survival and growth of larvae and juvenile fish (Boglione et al. 2013aBoglione C., Gavaia P., Koumoundouros G., et al. 2013a. Skeletal anomalies in reared European fish larvae and juveniles. Part 1: normal and anomalous skeletogenic processes. Rev. Aquacult. 5: S99-S120., bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.). Skeletal anomalies usually occur during skeletogenesis, which persists into the juvenile phase, but skeletal and fin anomalies can also occur in smaller-sized juveniles (Boglione et al. 2009Boglione C., Marino G., Giganti M., et al. 2009. Skeletal anomalies in dusky grouper Epinephelus marginatus (Lowe 1834) juveniles reared with different methodologies and larval densities. Aquaculture 291: 48-60., 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167., 2014Boglione C., Pulcini D., Scardi M., et al. 2014. Skeletal anomaly monitoring in rainbow trout (Oncorhynchus mykiss, Walbaum 1792) reared under different conditions. PLoS ONE 9: e96983.).

Larvae and small juveniles of R. holubi migrate into estuaries at a size of less than 30 mm and grow for about a year and a half to over 140 mm before migrating back to the ocean to spawn (Whitfield 1998Whitfield A.K. 1998. Biology and ecology of fishes in South African estuaries. In: Smith J.L.B. (ed), Ichthyological Monographs 2, Institute for Ichthyology, Grahamstown, 223 pp., Götz and Cowley 2013Götz A., Cowley P.C. 2013. Cape stumpnose (Rhabdosargus holubi). In: Mann (ed) South African Marine Linefish Species Profiles. Oceanogr. Res. Inst. Spec. Publ. 9: 268-269, Durban.). Juvenile R. holubi are usually highly resident, preferring to stay within habitats such as Zostera capensis seagrass beds within the estuary (Edworthy and Strydom 2016Edworthy C., Strydom N.A. 2016. Habitat partitioning by juvenile fishes in a temperate estuarine nursery, South Africa. Sci. Mar. 80: 151-161., Grant et al. 2017Grant G.N., Cowley P.D., Bennett R.H., et al. 2017. Influences of selected geophysical and environmental drivers on the movement patterns of Rhabdosargus holubi in a southern African estuary. Environ. Biol. Fish. 100: 1265-1283., Muller and Strydom 2017Muller C., Strydom N.A. 2017. Evidence for habitat residency and isotopic niche partitioning in a marine-estuarine-dependent species associated with mangrove habitats from the east coast of South Africa. Estuar. Coasts 40: 1642-1652.). R. holubi is a strong osmoregulator able to maintain its internal osmotic environment up to 65 (Blaber 1973aBlaber S.J.M. 1973a. The ecology of juvenile Rhabdosargus holubi (Steinachner) (Teleostei: Sparidae). PhD thesis, Rhodes University, Grahamstown, 123 pp., 1974bBlaber S.J.M. 1974b. Osmoregulation in juvenile Rhabdosargus holubi (Steindachner)(Teleostei: Sparidae). J. Fish Biol. 6: 797-800.). Previous investigations of R. holubi tolerance to high salinity carried out in the laboratory have indicated a maximum threshold of 70 after a 16-hour exposure (Blaber 1973bBlaber S.J.M. 1973b. Temperature and salinity tolerance of juvenile Rhabdosargus holubi [Steindachner (Teleostei: Sparidae)]. J. Fish Biol. 5: 593-598.). However, R. holubi individuals have been recorded alive in the wild at salinity levels up to 90, and it is one of the few species capable of surviving under intense drought conditions (Whitfield 1998Whitfield A.K. 1998. Biology and ecology of fishes in South African estuaries. In: Smith J.L.B. (ed), Ichthyological Monographs 2, Institute for Ichthyology, Grahamstown, 223 pp., Whitfield et al. 2006Whitfield A.K., Taylor R.H., Fox C., et al. 2006. Fishes and salinities in the St Lucia estuarine system—a review. Rev. Fish Biol. Fish. 16: 1-20.). This makes it a euryhaline species able to tolerate a large variation in salinity, as opposed to stenohaline species that can tolerate only minor changes (Whitfield et al. 1981Whitfield A.K., Blaber S.J.M., Cyrus D.P. 1981. Salinity ranges of some southern African fish species occurring in estuaries. Afr. Zool. 16: 151-155., Brauner et al. 2012Brauner C.J., Gonzalez R.J., Wilson J.M. 2012. Extreme environments: hypersaline, alkaline and ion-poor waters. In: McCormick S.D., Farrell A.P., Brauner C.J. (eds), Fish physiology: Euryhaline fishes. Academic Press, Cambridge, pp. 435-476., Kültz 2015Kültz D. 2015. Physiological mechanisms used by fish to cope with salinity stress. J. Exp. Biol. 218: 1907-1914.). Spinal and fin deformities can still be observed in euryhaline fishes such as Atherina lagunae due to the potential combined effects of temperature, pollution, and oxygen deficiency (Ayed et al. 2008Ayed N., Faure E., Quignard J.-P., et al. 2008. Incidence of kyphosis deformities in natural population of Atherina lagunae (Trabelsi et al. 2002) from the Tunis North Lake, Tunisia. Mar. Biol. 153: 319-325.). Despite the high tolerances reported in the literature, the physiological effects of R. holubi living under high salinities for long periods are unknown and there are still potential effects to external structures, such as fin abrasions or swimbladder anomalies due to buoyancy changes and bacterial infections (Latremouille 2003Latremouille D.N. 2003. Fin erosion in aquaculture and natural environments. Rev. Fish. Sci. 11: 315-335., Boglione et al. 2013aBoglione C., Gavaia P., Koumoundouros G., et al. 2013a. Skeletal anomalies in reared European fish larvae and juveniles. Part 1: normal and anomalous skeletogenic processes. Rev. Aquacult. 5: S99-S120., bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.). This study aims to assess the impact of natural in situ and laboratory-manipulated hypersaline conditions on the growth and frequency of skeletal anomalies of juvenile R. holubi.

MATERIALS AND METHODSTop

Field collections

To assess the skeletal descriptors to identify anomalies in R. holubi juveniles growing at different salinities, wild specimens were collected from the lower reaches of the naturally hypersaline Gqutywa (33°21′45′′S; 27°21′31′′E) and the marine-dominated Swartkops (33°51′54′′S, 25°38′00′′E) estuaries during March 2015. At least 50 specimens were collected from each site using a 30×1.5-m seine net of 10 mm bar mesh size. Specimens were fixed in 10% formalin and transported to Nelson Mandela University for skeletal descriptor analysis. A YSI 6600 multiparameter probe was used to measure the environmental variables present at the time of sampling.

The Swartkops Estuary is categorized as permanently open while the Gqutywa Estuary is categorized as temporarily open/closed (Whitfield and Baliwe 2013Whitfield A.K., Baliwe N.G. 2013. A century of science in South African estuaries: Bibliography and review of research trends. SANCOR Occasional Report No. 7, 289 pp.). The Swartkops has been shown to be marine-dominated at the lower reaches with comparatively low freshwater inflow (Baird et al. 1986Baird D., Hannekom N.M., Grindley J.R. 1986. Estuaries of the Cape. Part II: synopsis of available information on individual systems. CSIR Report No. 23, Stellenbosch, 82 pp., Pattrick and Strydom 2014Pattrick P., Strydom N.A. 2014. Recruitment of fish larvae and juveniles into two estuarine nursery areas with evidence of ebb tide use. Est. Coast Shelf. Sci. 149: 120-132., Strydom 2015Strydom N.A. 2015. Patterns in larval fish diversity, abundance, and distribution in temperate South African estuaries. Estuar. Coasts 38: 268-284.), with salinities ranging from 24.61 to 33.44 in the autumn sampling season. At the time of sampling, the following values for the key environmental variables were recorded at the Swartkops Estuary: temperature, 23.42°C; salinity, 31.32; pH, 7.51; turbidity, 13.2 NTU; oxygen saturation, 100%; conductivity, 48.46 µS cm^–1; and total dissolved solids, 31.51 mg l^–1. The Gqutywa Estuary is categorized as hypersaline, with salinities previously ranging from 35.66 to 43.19 during the period 1998-1999 (Strydom et al. 2003Strydom N.A., Whitfield A.K., Wooldridge T.H. 2003. The role of estuarine type in characterizing early stage fish assemblages in warm temperate estuaries, South Africa. Afr. Zool. 38: 29-43.). At the time of sampling, the following values were recorded for the environmental variables at the Gqutywa: temperature, 24.54°C; salinity, 39.55; pH, 7.76; turbidity, 2.4 NTU; oxygen, 100%; conductivity, 58.77 µS cm^–1; and total dissolved solids, 38.77 mg l^–1. The climate in the sampling region is categorized as warm temperate, with rainfall peaking bimodally in winter and spring (Strydom et al. 2003Strydom N.A., Whitfield A.K., Wooldridge T.H. 2003. The role of estuarine type in characterizing early stage fish assemblages in warm temperate estuaries, South Africa. Afr. Zool. 38: 29-43., Pattrick and Strydom 2014Pattrick P., Strydom N.A. 2014. Recruitment of fish larvae and juveniles into two estuarine nursery areas with evidence of ebb tide use. Est. Coast Shelf. Sci. 149: 120-132.).

Aquarium rearing and fish growth

To assess the skeletal descriptors of R. holubi juveniles grown at different salinities in the laboratory, specimens (n=589 total) were collected at the Swartkops Estuary between April and May 2017 using a 30×1.5-m seine net of 10 mm bar mesh size and transported to the aquarium laboratory at Nelson Mandela University. The specimens were then distributed among 12 glass 200-L capacity tanks for laboratory acclimatization. Each tank contained mechanical floss outflow filters, a Bubble Magus protein skimmer and a Matrix Biofilter treated with Seachem Stability. For at least two weeks prior to experiments, the specimens were acclimatized to standard laboratory conditions of 19°C to 21°C (measured with aquarium thermometers) and 29 to 31 salinity (measured with an ATC refractometer) obtained using reverse osmosis water with Blue Treasure Marine Salt. Fish were fed daily to satiation with Omega One marine flakes (43% protein, 11% fat and 2% fibre). A RED SEA Marine Lab aquarium kit was used to test pH (maintained at 8.0-8.4) and concentrations of ammonia (maintained at <0.2 ppm), nitrite (maintained at <0.05 ppm), nitrate (maintained at <20 ppm) and phosphate (maintained at <0.2 ppm) on a weekly basis. Mechanical filters and 10% water were also changed weekly.

For the salinity experiments, six aquaria were used with 20 fish of 46.48±7.2 mm (mean±SD) length (120 total). Three tanks served as controls maintained at standard laboratory conditions which mimicked those of the sampling site (salinity=29.15±0.55, mean±SD) while three tanks were maintained at a higher salinity of 49.9±1.16 (mean±SD) for the hypersaline treatment, which was higher than levels recorded in the Gqutywa. The hypersaline exposure salinity level (~50) was chosen based on a combination of pilot observations in the laboratory and to closer reflect salinities seen in the Gqutywa Estuary, which is generally considered a hypersaline sampling station for ecological studies. Salinity in hypersaline treatment tanks was gradually raised from standard conditions over three weeks via daily ~5% water changes of 50-60 salinity, and was then left at ~50 for the remainder of the study. Fish were measured for total length weekly for nine consecutive weeks using measuring boards, while environmental variables were measured with a YSI 6600 multiparameter probe. After 9 weeks, the fish began exhibiting signs of fin erosion, at which point the growth experiment was stopped. At the end of the exposure, the fish were removed and fixed in 10% formalin and kept until processing for skeletal descriptor analysis.

Skeletal descriptor analysis

To assess skeletal descriptors and identify anomalies, the fish tissues were cleared, and bone and cartilage were stained using methods described by Taylor and Van Dyke (1985)Taylor W.R., Van Dyke G.C. 1985. Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. Cybium 9: 107-109.. In summary, the fish specimens were descaled and deskinned with a scalpel before transfer to 95% ethanol for dehydration for 24 hours. They were then transferred to Alcian Blue cartilage stain for 8 hours and transferred to saturated sodium borate solution for a six-hour neutralization. The cartilage stain solution consisted of 70% of 95% ethanol, 30% acetic acid and ~2 mg Alcian Blue per litre of medium. The bones were also stained using Alizarin Red (mixed until dark purple) in 1% potassium hydroxide solution. Specimens were then transferred to a digestion solution containing diluted saturated sodium borate solution and porcine trypsin powder of roughly five teaspoons per two litres of medium. The fish were left to digest until the tissues were clear enough for analysis. Digestion solutions were changed every week until complete, and digestion times varied between fish sizes. The fish skeletons were observed on both sides under a stereomicroscope (BestScope, ~40-50× magnification depending on size) and analysed for the presence of anomalies considered in Table 1 and adapted from Boglione et al. (2001)Boglione C., Gagliardi F., Scardi M., et al. 2001. Skeletal descriptors and quality assessment in larvae and post-larvae of wild-caught and hatchery-reared gilthead sea bream (Sparus aurata L. 1758). Aquaculture 192: 1-22.. The anomalies considered in this analysis were described in a review by Boglione et al. (2013b)Boglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167., and included severe deformities such as shorter-than-usual fin rays, supernumerary fin rays, lordosis (downward concave curvature), kyphosis (upward concave curvature) and vertebral fusions. The most abundant types of anomalies were reduced fin rays and slight malformations in neural or haemal spines of vertebrae.

Statistical analyses

One-way ANOVA indicated that R. holubi specimen length (mean 46.48 mm ±7.23 SD) did not differ between tanks at the start of the growth experiment (n=20, F=0.204, p=0.96). A repeated-measures ANOVA was used to determine the difference in length between control (salinity ~30) and hypersaline (salinity ~50) treatments over time. Mauchly’s test of sphericity was violated (W=0.03, p<0.05), so the Greenhouse Geisser value was applied (ε=0.52).

The average number of vertebra-related anomalies per fish and the average number of fin ray-related anomalies per fish were compared between salinity treatments for the wild-caught and aquarium-grown fish using a Mann-Whitney U test since parametric test assumptions were violated. Mann-Whitney U tests were also used to compare skeletal anomaly frequencies i.e. the number fish per anomaly type between fish collected in the Swartkops and Gqutywa estuaries, as well as between control and hypersaline treatments. Data were analysed and compared in total and according to two size classes for the field data (38-60 mm and 61-104 mm). Statistics were performed in SPSS V 21.0.

RESULTSTop

Fish growth versus salinity in the aquarium

Repeated-measures ANOVA indicated that R. holubi length increased weekly (F=223.48, p<0.005, Fig. 1) and that the interaction between time and treatment was significant for the within-subject effects (F=2.93, p=0.02). However, overall length did not differ between control and hypersaline treatments (F=0.003, p=0.96).

Skeletal anomalies in wild-caught fish

Standard lengths of wild-caught individuals ranged from 40 to 104 mm. Of the 44 anomalies considered, 12 types were present in wild-caught individuals. Twenty-one of a hundred individuals were malformed, the majority of them from the naturally hypersaline Gqutywa Estuary samples. No severe anomalies such as vertebral kyphosis, lordosis or fusions were detected. The average number of anomalies found was higher in the Gqutywa Estuary samples, especially for fin-related samples, as these were mostly absent in Swartkops Estuary samples (Table 2, Fig. 2). Fin-related anomalies in Gqutywa and Swartkops estuary samples included mostly dorsal and caudal fin breaks and erosions (Fig. 2). Vertebra-related anomalies consisted mainly of minor neural spine anomalies. Total frequency (number of individuals) with each of the 44 anomalies also differed between the Gqutywa and the Swartkops estuaries, with higher incidences of fin-related anomalies in the Gqutywa Estuary (Mann-Whitney U=336, Z=–12.42, p<0.005). Anomalies were only present in the smaller size class in the Swartkops Estuary, while there was no difference between size classes in the Gqutywa Estuary (Mann-Whitney U=906.5, Z =–0.21, p=0.83).

Skeletal anomalies in fish reared at different salinities

Most individuals in both the 30 and 50 salinity treatments grown in aquaria exhibited no skeletal anomalies. Of the 120 individuals surveyed, 18 were anomalous, the majority exhibiting lordosis in the haemal or caudal regions or an eroded caudal fin ray (Fig. 2). There was no significant difference between the number of vertebra-related or fin-related anomalies between the control and hypersaline groups in the laboratory (Table 2). Total frequency of each anomaly tested (n=44) differed between control and hypersaline treatments, with higher incidences of fin-related anomalies being found in the hypersaline treatment (Mann-Whitney U=1294.5, Z=–9.98, p<0.005).

DISCUSSIONTop

Hypersaline conditions may potentially place a significant impact on the physiology of estuarine fish during drought conditions or freshwater impoundments, thus affecting their ecology (Bœuf and Payan 2001Bœuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 130: 411-423., Harrison and Whitfield 2006Harrison T.D., Whitfield A.K. 2006. Temperature and salinity as primary determinants influencing the biogeography of fishes in South African estuaries. Est. Coast Shelf Sci. 66: 335-345., Gonzalez 2012Gonzalez R.J. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J. Comp. Physiol. B 182: 321-329.). This study was aimed at assessing the growth and skeletal anomalies of R. holubi juveniles growing at different salinities in situ and in the laboratory using clearing and staining techniques (Taylor and Van Dyke 1985Taylor W.R., Van Dyke G.C. 1985. Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. Cybium 9: 107-109.) to determine the impact of hypersaline conditions over the long term. It was found that hypersaline conditions with a salinity of 50 for two months did not significantly impact the growth of R. holubi for most of the duration of the experiment, compared with control samples kept at salinity levels around 30, similar to those in their natural habitat. It was found that skeletal anomalies were rare, occurring in less than 20% of all skeletal samples surveyed. The highest incidences of anomalies were found in R. holubi juveniles from the naturally hypersaline Gqutywa Estuary, particularly affecting their fin rays. Conversely, vertebra-related anomalies, i.e. those in vertebra, spines, arches and crania, did not differ significantly between treatments.

The degree of metabolic activity due to osmoregulation varies widely in the literature, ranging from less than 10% to more than 50% of the total energy cost (Bœuf and Payan 2001Bœuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 130: 411-423., Gonzalez 2012Gonzalez R.J. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J. Comp. Physiol. B 182: 321-329., Ern et al. 2014Ern R., Huong D.T.T., Cong N.V., et al. 2014. Effect of salinity on oxygen consumption in fishes: a review. J. Fish Biol. 84: 1210-1220.). Generally, when fish are gradually acclimated, tolerance to hypersaline conditions increases and may also lead to lower metabolic costs (Gonzalez et al. 2005Gonzalez R.J., Cooper J., Head D. 2005. Physiological responses to hyper-saline waters in sailfin mollies (Poecilia latipinna). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 142: 397-403., Whitfield et al. 2006Whitfield A.K., Taylor R.H., Fox C., et al. 2006. Fishes and salinities in the St Lucia estuarine system—a review. Rev. Fish Biol. Fish. 16: 1-20., Gonzalez 2012Gonzalez R.J. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J. Comp. Physiol. B 182: 321-329.). Fish may respond to increased solute concentrations by increasing salt excretion rates and reducing gill epithelial permeability (Gonzalez et al. 2005Gonzalez R.J., Cooper J., Head D. 2005. Physiological responses to hyper-saline waters in sailfin mollies (Poecilia latipinna). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 142: 397-403., Gonzalez 2012Gonzalez R.J. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J. Comp. Physiol. B 182: 321-329.). Growth rates in many fish species, such as the marine estuarine-opportunist Mugil cephalus, are shown to decrease at high salinities due to the compromise of reducing oxygen intake while decreasing gill permeability to prevent the absorption of solutes (Bœuf and Payan 2001Bœuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 130: 411-423.). Additionally, many other metabolic pathways are impacted during changing salinity, including changing gill ventilation, acid-base regulation and hormonal regulation (Claireaux and Lagardère 1999Claireaux G., Lagardère J.P. 1999. Influence of temperature, oxygen and salinity on the metabolism of the European sea bass. J. Sea Res. 42: 157-168., Deane and Woo 2009Deane E.E., Woo N.Y.S. 2009. Modulation of fish growth hormone levels by salinity, temperature, pollutants and aquaculture related stress: a review. Rev. Fish Biol. Fish. 19: 97-120., Ern et al. 2014Ern R., Huong D.T.T., Cong N.V., et al. 2014. Effect of salinity on oxygen consumption in fishes: a review. J. Fish Biol. 84: 1210-1220.). In some species, growth may even increase at high salinities. In the euryhaline milkfish Chanos chanos for example, swimming activity declines at a salinity of 55 compared with 35, leading to a higher growth rate over a period of four weeks (Swanson 1998Swanson C. 1998. Interactive effects of salinity on metabolic rate, activity, growth and osmoregulation in the euryhaline milkfish (Chanos chanos). J. Exp. Biol. 201: 3355-3366.). Thus, the lack of effect of salinity on growth may be due to the offset of osmoregulatory metabolic demands by a reduction in movement. This was seen anecdotally in the laboratory and also evidenced by a decrease in respiration rates at high salinities found in pilot studies (Kisten 2018Kisten Y. 2018. The effects of environmental variability on the physiology and ecology of Cape stumpnose Rhabdosargus holubi (Steindachner, 1881) (Sparidae). PhD thesis, Nelson Mandela University, Port Elizabeth, 115 pp.). In an ecological context, these results show why R. holubi is able to tolerate and proliferate under high-salinity conditions (Whitfield et al. 2006Whitfield A.K., Taylor R.H., Fox C., et al. 2006. Fishes and salinities in the St Lucia estuarine system—a review. Rev. Fish Biol. Fish. 16: 1-20.) and can be the most populous marine estuarine-dependant species in intermittently open estuaries during the closed mouth state (James et al. 2007James N.C., Cowley P.D., Whitfield A.K. 2007. Abundance, recruitment and residency of two sparids in an intermittently open estuary in South Africa. Afr. J. Mar. Sci. 29: 527-538., Whitfield et al. 2017Whitfield A.K., Grant G.N., Bennett R.H., et al. 2017. Causes and consequences of human induced impacts on a ubiquitous estuary-dependent marine fish species. Rev. Fish Biol. Fish. 28: 19-31.).

If fish are able to manage the solute levels in their internal environment, it follows that damage to, or malformations of, internal structures such as the skeleton may be minimized (Gonzalez 2012Gonzalez R.J. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J. Comp. Physiol. B 182: 321-329., Boglione et al. 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.). The ability of the euryhaline R. holubi to maintain its internal osmotic environment near isosmotic levels at salinities up to 65 (Blaber 1974bBlaber S.J.M. 1974b. Osmoregulation in juvenile Rhabdosargus holubi (Steindachner) (Teleostei: Sparidae). J. Fish Biol. 6: 797-800.) may explain the lack of incidence and difference between treatments of vertebral anomalies obtained in this study. Further, skeletal anomalies tend to be more pronounced if they develop during earlier stages in the life cycle (Boglione et al. 2001Boglione C., Gagliardi F., Scardi M., et al. 2001. Skeletal descriptors and quality assessment in larvae and post-larvae of wild-caught and hatchery-reared gilthead sea bream (Sparus aurata L. 1758). Aquaculture 192: 1-22., 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167., Alarape et al. 2015Alarape S.A., Hussein T.O., Adetunji E.V., et al. 2015. Skeletal and other morphological abnormalities in cultured Nigerian African Catfish (Clarias gariepinus, Burchell 1822). Int. J. Fish. Aquat. Stud. 2: 20-25.). This may explain the low incidence of skeletal anomalies in juveniles, especially in the aquarium samples, as the early preflexion larval stages occur in the relatively stable marine environment. This may also explain why wild hypersaline juveniles exhibited anomalies while aquariums samples did not, as individuals in the Gqutywa Estuary may have been exposed to high salinities and other factors at a younger age and for a longer period. However, due to a combination of pollution and other environmental variables, skeletal anomalies such as those observed in Atherina lagunae (Ayed et al. 2008Ayed N., Faure E., Quignard J.-P., et al. 2008. Incidence of kyphosis deformities in natural population of Atherina lagunae (Trabelsi et al. 2002) from the Tunis North Lake, Tunisia. Mar. Biol. 153: 319-325.) can also occur in early juvenile euryhaline fish species. Lordosis (abnormal upward concave curvature) and vertebral spine/arch malformations were some of the most common vertebral-related anomalies present in anomalous samples. Lordosis of the haemal and caudal regions can be attributed to high water current, forced swimming and high temperatures (Divanach et al. 1996Divanach P., Boglione C., Menu B., et al. 1996. Abnormalities in finfish mariculture: an overview of the problem, causes and solutions. International Workshop on Sea Bass and Sea Bream Culture: Problems and Prospects, Verona, pp. 45-66., 1997Divanach P., Papandroulakis N., Anastasiadis P., et al. 1997. Effect of water currents on the development of skeletal deformities in sea bass (Dicentrarchus labrax L.) with functional swimbladder during postlarval and nursery phase. Aquaculture 156: 145-155., Boglione et al. 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.), which may explain why smaller individuals in aquaria exhibited this in both salinity treatments used in this study, as the other variables were kept constant. Anomalies affecting neural and haemal spines and arches are considered less severe and less likely to impact the performance of fish (Boglione et al. 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.), especially at the low levels observed in this study. These anomalies may also be linked to other environmental variables, and particularly a nutrient-deficient diet rather than environmental salinity (Divanach et al. 1996Divanach P., Boglione C., Menu B., et al. 1996. Abnormalities in finfish mariculture: an overview of the problem, causes and solutions. International Workshop on Sea Bass and Sea Bream Culture: Problems and Prospects, Verona, pp. 45-66., Izquierdo et al. 2010Izquierdo M.S., Socorro J., Roo J. 2010. Studies on the appearance of skeletal anomalies in red porgy: effect of culture intensiveness, feeding habits and nutritional quality of live preys. J. Appl. Ichthyol. 26: 320-326., Boglione et al. 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.). As such it is difficult to conclude that a hypersaline environment may have any effect on the internal structures of R. holubi at the salinity levels and exposure durations tested in this study, particularly since other factors such as temperature were kept constant in the laboratory but would vary considerably in the wild.

Fin rays were the most significantly affected structures found in hypersaline conditions both in situ in the Gqutywa Estuary and in the laboratory at a salinity of 50 (Fig. 2). Fin ray anomalies are common in many species in both the wild and in aquaculture conditions (Divanach et al. 1996Divanach P., Boglione C., Menu B., et al. 1996. Abnormalities in finfish mariculture: an overview of the problem, causes and solutions. International Workshop on Sea Bass and Sea Bream Culture: Problems and Prospects, Verona, pp. 45-66., Boglione et al. 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.). However, the impact on fin rays, especially the erosions exhibited in this study, may not be related to physiology, skeletal growth and developmental anomalies (Boglione et al. 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.). Rather, fin erosions may be caused by factors such as abrasions, intraspecific aggression, nutritional deficiencies and bacterial infections (Latremouille 2003Latremouille D.N. 2003. Fin erosion in aquaculture and natural environments. Rev. Fish. Sci. 11: 315-335.). Overall, the stress of unfavourable environments may also lead to fin erosion, as in the case of juvenile cobia Rachycentron canadum reared at suboptimal low salinities of 5 (Denson et al. 2003Denson M.R., Stuart K.R., Smith T.I., et al. 2003. Effects of salinity on growth, survival, and selected hematological parameters of juvenile cobia Rachycentron canadum. J. World Aquac. Soc. 34: 496-504.). The presence of eroded fins does affect mobility and results in changes to swimming patterns that may result in skeletal deformities over the long term (Divanach et al. 1996Divanach P., Boglione C., Menu B., et al. 1996. Abnormalities in finfish mariculture: an overview of the problem, causes and solutions. International Workshop on Sea Bass and Sea Bream Culture: Problems and Prospects, Verona, pp. 45-66., Latremouille 2003Latremouille D.N. 2003. Fin erosion in aquaculture and natural environments. Rev. Fish. Sci. 11: 315-335., Boglione et al. 2013bBoglione C., Gisbert E., Gavaia P., et al. 2013b. Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factors. Rev. Aquac. 5: S121-S167.). Ecologically, fin damage and pathological infections may make fish more vulnerable to predators potentially impacting on population size (Latremouille 2003Latremouille D.N. 2003. Fin erosion in aquaculture and natural environments. Rev. Fish. Sci. 11: 315-335., Hostetter et al. 2012Hostetter N.J., Evans A.F., Roby D.D., et al. 2012. Susceptibility of juvenile steelhead to avian predation: the influence of individual fish characteristics and river conditions. Trans. Am. Fish Soc. 141: 1586-1599.).

In an ecological context, the results of this study indicate that R. holubi juveniles are resilient to hypersaline conditions, enabling them to persist in a saline-tolerant food web. Juvenile R. holubi depend on vegetated refugia for food and protection from piscivores (Blaber 1974aBlaber S.J.M. 1974a. Field studies of the diet of Rhabdosargus holubi (Pisces: Teleostei: Sparidae). J. Zool. 173: 407-417., Edworthy and Strydom 2016Edworthy C., Strydom N.A. 2016. Habitat partitioning by juvenile fishes in a temperate estuarine nursery, South Africa. Sci. Mar. 80: 151-161., Nel et al. 2017Nel L., Strydom N.A., Perissinotto R., et al. 2017. Feeding ecology of Rhabdosargus holubi (family Sparidae) in multiple vegetated refugia of selected warm temperate estuaries in South Africa. Est. Coast. Shelf Sci. 197: 194-204.). This refuge is particularly important if mobility is reduced, due to the potential reduction in activity and fin erosion seen in this study. Conversely, the persistence of R. holubi populations together with other halotolerant species ensures the availability of food for halotolerant piscivores if planktivorous fish prey items decline during hypersaline conditions (Whitfield et al. 2006Whitfield A.K., Taylor R.H., Fox C., et al. 2006. Fishes and salinities in the St Lucia estuarine system—a review. Rev. Fish Biol. Fish. 16: 1-20., Durand 2015Durand J.R. 2015. A conceptual model of the aquatic food web of the upper San Francisco Estuary. San Francisco Estuary Watershed Sci. 13: 1-37.). Thus, both the resilience to, and potential impacts of, hypersaline conditions seen in this study enable R. holubi to buffer food webs during hypersaline conditions. Droughts and hypersaline conditions may become more frequent and intense, especially during the dry phase of ENSO in the future (Gillanders et al. 2011Gillanders B.M., Elsdon T.S., Halliday I.A., et al. 2011. Potential effects of climate change on Australian estuaries and fish utilising estuaries: a review. Mar. Freshw. Res. 62: 1115-1131., Koehn et al. 2011Koehn J.D., Hobday A.J., Pratchett M.S., et al. 2011. Climate change and Australian marine and freshwater environments, fishes and fisheries: synthesis and options for adaptation. Mar. Freshw. Res. 62: 1148-1164., James et al. 2013James N.C., van Niekerk L., Whitfield A.K., et al. 2013. Effects of climate change on South African estuaries and associated fish species. Clim. Res. 57: 233-248.). This is further exacerbated by freshwater reduction practices in rivers and estuaries (Turpie et al. 2002Turpie J.K., Adams J.B., Joubert A. et al. 2002. Assessment of the conservation priority status of South African estuaries for use in management and water allocation. Water SA 28: 191-206., Adams et al. 2016Adams J.B., Cowie M., Van Niekerk L. 2016. Assessment of completed ecological water requirement studies for South African estuaries and responses to changes in freshwater inflow. Water Research Commission, WRC Report No. KV 352/15, Pretoria, 57 pp.). The potential synergistic effect of other factors such as increased temperature, ocean acidification, heavy metal pollution and salinity on the skeletal development of larvae and juveniles is yet unknown and may be an important aspect for study in the future.

ACKNOWLEDGEMENTSTop

Research costs and bursary support was funded by the South African Research Chairs Initiative (SARChI) of the Department of Science and Technology (DST) and National Research Foundation (NRF). Any opinion, finding and conclusion or recommendation expressed in this material is that of the authors and the NRF does not accept any liability in this regard. Much appreciation goes to Mr. Mfundo Mpinga (Nelson Mandela University) for his invaluable assistance in the field and in the laboratory.

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