Introduction
Marine fish farming was the vanguard activity in Brazilian aquaculture (Cavalli and Hamilton, Reference Cavalli and Hamilton2007). Despite this, until now, marine fish farming has not yet evolved into an economically important activity in Brazil (Valenti et al., Reference Valenti, Barros, Moraes-Valenti, Bueno and Cavalli2021), unlike some countries in Asia and Europe, where marine fish production is consolidated as a perennial activity (Neu et al., Reference Neu, Honorato and Lewandowski2019). Among the most produced diadromous species are the Atlantic salmon Salmo salar, the milkfish Chanos chanos, the Japanese eel Anguilla japonica and mullets of the genus Mugil (FAO, 2022).
Over the years, technological advances, combined with scientific developments on the biological needs of species, have contributed to increased productivity with lower production costs, making aquaculture an economically competitive source of animal protein worldwide (Asche, Reference Asche2008; Silva et al., Reference Silva, Sterzelecki, Alves Musialak, Sugai, Castro, Pedrotti, Magnotti, Cipriano and Cerqueira2020; Føre et al., Reference Føre, Thorvaldsen, Osmundsen, Asche, Tveterås, Fagertun and Bjelland2022; Owatari et al., Reference Owatari, Magnotti, Vargas, de Carvalho, Sterzelecki and Cerqueira2023; Kaneko et al., Reference Kaneko, Ishikawa and Nomura2023).
Currently in Brazil, the Lebranche mullet Mugil liza has stood out in experimental cultivations supported by scientific initiatives such as those by Angelo et al. (Reference Angelo, Lisboa, Magnotti, Pilotto, Mattos and Cerqueira2021a) who verified that the temperature influenced the final phase of embryogenesis, initial development and survival of M. liza larvae, while Silva et al. (Reference Silva, Sterzelecki, Alves Musialak, Sugai, Castro, Pedrotti, Magnotti, Cipriano and Cerqueira2020) verified the effect of feeding frequency on growth performance, blood metabolites, proximal composition and digestive enzymes of juvenile M. liza and recommend feeding juveniles three to five times per day. Castro et al. (Reference Castro, Magnotti, Angelo, Sterzelecki, Pedrotti, Oliveira, Soligo, Fracalossi and Cerqueira2019) verified that the supplementation of dietary ascorbic acid between 107 and 216 mg kg–1 optimizes the spermatic quality in males of Lebranche mullet. Lisboa et al. (Reference Lisboa, Barcarolli, Sampaio and Bianchini2015) tested different salinities and defined 24‰ as the best growth in the cultivation of mullet juveniles in relation to salinities of 0.6 and 12‰. Carvalho et al. (Reference Carvalho, Bianchini, Tesser and Sampaio2010) determined that a diet containing 35% crude protein for juveniles resulted in greater weight gain, feed intake and higher growth rate, while Okamoto et al. (Reference Okamoto, Sampaio and Maçada2006) reported that the temperature of 30 ºC is the most suitable for the growth and survival of juveniles.
The Lebranche mullet M. liza is a fish of the Mugilidae family that inhabits tropical and subtropical regions and is geographically distributed from Florida (USA) to Argentina (Lemos et al., Reference Lemos, Varela, Schwingel, Muelbert and Vieira2014; Mai et al., Reference Mai, Mino, Marins, Monteiro-Neto, Miranda, Schwingel, Lemos, Gonzalez-Castro, Castello and Vieira2014). In Brazil, the species has great economic importance, mainly for artisanal fisheries (Morado et al., Reference Morado, de Andrade-Tubino and Araújo2021). The populations are genetically distinct into two groups. One population is located to the north, above the State of Rio de Janeiro in warmer waters, while another population is located to the south, acclimatized to colder waters, even so, during the winter they migrate northwards in search of warmer waters to reproduce (Lemos et al., Reference Lemos, Varela, Schwingel, Muelbert and Vieira2014; Mai et al., Reference Mai, Mino, Marins, Monteiro-Neto, Miranda, Schwingel, Lemos, Gonzalez-Castro, Castello and Vieira2014). This behaviour of southern populations suggests a great relevance of temperature on reproduction and on the initial development of the species.
Among the various abiotic factors that affect animal physiology, temperature has a great influence, especially in ectothermic animals (Abram et al., Reference Abram, Boivin, Moiroux and Brodeur2017). Fish, for example, are not able to maintain a constant body temperature like endothermic animals, which can affect ontogenetic development, especially in the early stages of embryogenesis (Jonsson and Jonsson, Reference Jonsson and Jonsson2019).
After fertilisation of the oocytes, embryonic development begins, a sensitive phase in which successive meroblastic cell divisions occur, which in a few hours or days results in a newly hatched larva (Kucharczyk et al., Reference Kucharczyk, Luczynski, Kujawa and Czerkies1997). However, under adverse environmental conditions, where temperatures remain outside the thermal comfort range, embryonic deformation and/or infeasibility of hatching may occur, as well as larval anomaly; as observed for other marine fish species (Herbing, Reference Herbing2002; Donaldson et al., Reference Donaldson, Cooke, Patterson and Macdonald2008; Mendonça et al., Reference Mendonça, Ikebata, Araújo-Silva, Manhães and Tsuzuki2020; Angelo et al., Reference Angelo, Lisboa, Magnotti, Pilotto, Mattos and Cerqueira2021a, 2021b; Clarkson et al., Reference Clarkson, Taylor, McStay, Palmer, Clokie and Migaud2021).
Considering that the beginning of ontogenetic development is more sensitive to abiotic conditions such as temperature (Martell et al., Reference Martell, Kieffer and Trippel2005; Angelo et al., Reference Angelo, Lisboa, Magnotti, Pilotto, Mattos and Cerqueira2021b), We herein investigated the influence of this parameter on the entire embryonic development of Lebranche mullet M. liza, from egg fertilisation to larval hatching.
Material and methods
Breeders and experimental site
The study was carried out at the facilities of the laboratory of marine fish culture (LAPMAR) of the Federal University of Santa Catarina (UFSC), located in Barra da Lagoa in Florianópolis-SC. The Lebranche mullet breeders used are part of the first Generation (F1) fish batch produced in captivity. All experiments were carried out with authorisation from the Ethics and Animal Use Committee CEUA – UFSC No. 7385251119.
Obtaining the eggs
For reproduction, the methodology described by Cerqueira et al. (Reference Cerqueira, Carvalho, Sanches, Passini, Baloi and Rodrigues2017) adapted to the current laboratory model, in which two male breeders were selected according to size and a female was selected according to oocyte diameter (>600 μm). Breeders were allocated in a 1000 L circular tank, with continuous water flow (salinity 33‰) from the ocean, collected at Mozambique beach Florianopolis, Brazil (27°34′02″S, 48°25′44″W), with thermostat for temperature maintenance (24.5 ± 0.7 ºC).
For female specimens (length and weight of 48.0 cm and 1270.0 g), hormonal control of spawning occurred with an intramuscular application of carp pituitary extract (CPE) hormone and another application of Luteinizing Hormone-Releasing Hormone analogues (LH-RHa) (des-Gly10, D-Ala6 LH-RH ethylamid salt acetate hydrate, L4513, Sigma-Aldrich, St. Louis, USA), being a first dose with 20 mg CPE kg fish–1, and after 24 h a second dose with 200 µg LH-RHa kg fish–1. For the male specimens (length and weight of 28.0 cm and 270.0 g), a single dose of 100 µg kg fish–1 of LH-RHa was also administered intramuscularly, concomitantly with the second dose of the females (Magnotti et al., Reference Magnotti, Cipriano, Pedrotti and Cerqueira2020).
At 333.7 degree-hours, spawning and natural fertilisation occurred at a temperature of 23.7 ºC. The eggs were collected automatically by a fish egg collection and harvester system for aquaculture coupled to the tank with lentic water flow, and transferred to two cylinder-conical tanks (incubators) with a useful volume of 34 L. The incubators were checked periodically (every 15 min), 3 h before the expected spawning time. As soon as the eggs appeared in the incubators, they were distributed to the experimental units.
Experimental design
To monitor the embryonic development of M. liza at different temperatures, 12 cylindrical experimental units with a useful volume of 400 mL were used. Each set with three experimental units was placed in a water bath inside a polyethylene tank with water at a constant temperature of 23 °C. The systems were maintained at a temperature of 23 ºC until eggs were included. Then the heating thermostats were activated, and the desired temperatures were established. Hundred eggs were distributed in each experimental unit and the treatments were established in triplicate, with four temperatures, 18, 22, 26 and 30 ºC.
The experiment was carried out in an acclimatized room with a constant temperature of 18 ºC. In each water bath box, a heating thermostat maintained the temperature corresponding to the treatment, and an air diffuser homogenized the water, maintaining the same temperature throughout the water bath. Each of the experimental units was equipped with mild aeration to ensure oxygenation and smooth egg movement. Salinity was maintained at 33‰, pH, oxygen and temperature were measured twice daily. It is noteworthy that pH, oxygen and salinity values followed the comfort range for the species (Okamoto et al., Reference Okamoto, Sampaio and Maçada2006).
Every 15 min until hatching, about 10 eggs were randomly sampled in each treatment. The eggs were visualized and photographed in a stereoscopic microscope (EZ4HD, Leica, Germany), coupled with a camera and software (LAZ EZ 2.1, Leica, Germany). The classification of embryonic stages was performed according to the methodology proposed by Fujimoto et al. (Reference Fujimoto, Kataoka, Sakao, Saito, Yamaha and Arai2006).
Statistical analyses
The Levene test was used to verify homoscedasticity and the Shapiro–Wilk test to verify the normality of obtained data. The data were submitted to ANOVA and Tukey’s test. All tests were performed at a significance level of 5% using the STATISTICA 10.0 software.
Results
Regarding water quality, significant differences (p < 0.05) were observed between the treatments. The concentration of dissolved oxygen was significantly lower (p < 0.05) in treatments with higher temperatures (Table 1).
Values for water quality variables (mean ± standard deviation) during incubation of Lebranche mullet Mugil liza eggs at different temperatures. Different letters indicate significant differences between treatments by Tukey’s test
The temperature significantly influenced (p < 0.05) the events of the embryonic development of Lebranche mullet M. liza analyzed in the present study. A more accelerated development was observed according to the increase in temperature until the gastrula phase (Table 2).
Embryonic development of Lebranche mullet Mugil liza subjected to different incubation temperatures. Every 15 min until hatching, about 10 eggs were randomly sampled in each treatment, visualized and photographed under a stereoscopic microscope (EZ4HD, Leica, Germany), coupled with a camera and software (LAZ EZ 2.1, Leica, Germany). Data are related to the time (in hours) for observation of events that occur during embryonic development. (NO) Non-occurrence
At temperatures of 22 and 26 °C, embryonic development occurred from fertilisation to hatching of the larvae (Figures 1–3). In the treatment with a temperature of 18 °C, a stopping embryo development was observed during the final phase of cleavage and the beginning of blastula formation. However, in the embryos that remained in development in this treatment, an anomaly in the growth pattern was observed, presenting body impairments (Figure 4). Finally, there was total mortality of the embryos, without hatching of the eggs.
Eggs of Lebranche mullet Mugil liza during embryonic development at different temperatures of 18, 22, 26 and 30 ºC. In (a), egg in early stages collected from the incubator at 22 ºC. In (b), first cleavage. First two cell divisions under treatment at 26°C after 0.5 h. In (c), cleavage phase with four cells under treatment at 22 ºC after 1.5 h. In (d), cleavage phase with eight cells under treatment at 26 ºC after 2 h; Gastrulation occurs. Every 15 min until hatching, about 10 eggs were randomly sampled in each treatment, visualized and photographed under a stereoscopic microscope (EZ4HD, Leica, Germany), coupled with a camera and software (LAZ EZ 2.1, Leica, Germany).
Eggs of Lebranche mullet Mugil liza during embryonic development at different temperatures of 18, 22, 26 and 30 ºC. In (a), gastrulation phase occurring with cellular movements that lead to a rearrangement of the blastula cells, resulting in the formation of an embryo, under treatment at 26°C after 8 h. In (b), blastopore formation under treatment at 26°C after 8 h. In (c), blastopore closure phase starting the organogenesis phase (differentiation of head and tail) under treatment at 22°C after 14 h. In (d), emergence of optic primordia under treatment at 26°C after 14 h. Every 15 min until hatching, about 10 eggs were randomly sampled in each treatment, visualized and photographed under a stereoscopic microscope (EZ4HD, Leica, Germany), coupled with a camera and software (LAZ EZ 2.1, Leica, Germany).
Eggs of Lebranche mullet Mugil liza during embryonic development at different temperatures of 18, 22, 26 and 30 ºC. In (a), stage of organogenesis characterized by the formed somite and the presence of otolith in the treatment of 22 °C after 21 h. In (b), presence of melanophores, which are cells with a characteristic branched shape, containing melanin granules observed in the treatment at 26 °C after 15.5 h. In (c), pre-hatching larvae at 26 °C treatment. In (d), newly hatched larvae in the treatment of 26 °C after 36 h. Every 15 min until hatching, about 10 eggs were randomly sampled in each treatment, visualized and photographed under a stereoscopic microscope (EZ4HD, Leica, Germany), coupled with a camera and software (LAZ EZ 2.1, Leica, Germany).
Eggs of Lebranche mullet Mugil liza during embryonic development at different temperatures of 18, 22, 26 and 30 ºC. Malformation in fish embryonic development at incubation temperatures of 18 and 30 °C. In (a), non-viable embryo during the blastula phase at 18 °C. In (b), malformed embryos at a temperature of 30 °C, during the initial cleavage phase. In (c) non-viable embryos during the blastula phase at 30 °C. In (d), malformed embryos during the organogenesis phase at 18 °C. Every 15 min until hatching, about 10 eggs were randomly sampled in each treatment, visualized and photographed under a stereoscopic microscope (EZ4HD, Leica, Germany), coupled with a camera and software (LAZ EZ 2.1, Leica, Germany).
In the treatment with temperature at 30 °C, patterns of malformation of the embryos were also verified, with erratic divisions of the blastomeres, resulting in irregular cells (Figure 4). Unlike what was observed at a temperature of 18 °C, none of the embryos incubated at 30 °C reached the blastopore closure phase, stopping at the gastrula (Table 2). The larvae hatched in the treatments at 22 and 26 °C were viable and with intense swimming, with a large amount of reserve material (yolk) and an evident drop of oil. The optical system was still underdeveloped and poorly pigmented. The mouth was closed, as well as the anus.
Discussion
Environmental thermodynamics is a fundamental variable that can interfere with the chemical reactions of metabolism by increasing or reducing molecular kinetic energy in the body of ectothermic species. The consequences of temperature variation include molecular impact changes that alter the stability of chemical bonds, affecting enzymatic activity, as well as macro-level changes that can cause behavioural disturbances (Baldisserotto and Val, Reference Baldisserotto and Val2002; Motta et al., Reference Motta, Glória, Radael, Mattos, Cardoso and Vidal-Júnior2023). During ontogenetic development, temperature is an abiotic factor that affects several fish species (Alfonso et al., Reference Alfonso, Gesto and Sadoul2021). Indeed, in the present study, we verified that the different temperatures were fundamental for the impairments or normal embryonic development of M. liza. Thus, it is important that during the embryonic development of altricial larvae such as those of M. liza (Whitfield, Reference Whitfield1990; Rønnestad et al., Reference Rønnestad, Yufera, Ueberschär, Ribeiro, Sæle and Boglione2013), the culture environment presents ideal conditions for the development of the embryo, since at early stages of life these larvae are planktonic, with low functional capacity and unable to ingest food after hatching (Koumoundouros, Reference Koumoundouros1996; Gluckmann et al., Reference Gluckmann, Huriaux, Focant and Vandewalle1999; Bone and Moore, Reference Bone and Moore2008).
Several ecological, behavioural and physiological characteristics that emerge later in other stages of development can be influenced by temperature during embryonic development within the egg, including the rate of embryonic development, growth, reproductive distribution and migration and sex determination. Such temperature responses are probably regulated by epigenetic mechanisms, such as Deoxyribonucleic Acid (DNA) methylation, histone modification and microRNAs (Jonsson and Jonsson, Reference Jonsson and Jonsson2019).
Results similar to those observed in the present study were described by Mendonça et al. (Reference Mendonça, Ikebata, Araújo-Silva, Manhães and Tsuzuki2020) in the pygmy angelfish Centropyge aurantonotus, where the incubation time was inversely proportional to the temperature, i.e., in treatments at temperatures at 22°C, egg hatching occurred in a period twice as long as at a temperature at 30°C. The best incubation temperature range was established from 24 to 28 °C to incubate C. aurantonotus eggs. Likewise, Petereit et al. (Reference Petereit, Haslob, Kraus and Clemmesen2008) when verifying the influence of temperature on the development of Baltic Sea sprat (Sprattus sprattus) eggs and yolk sac larvae found that egg development and hatching showed exponential temperature dependence. The authors observed that above 14.7 °C no hatching occurred and hatching success was significantly reduced below 3.4 °C. Furthermore, the time until eye pigmentation, as a proxy for mouth opening, decreased with increasing temperature from 17 days after hatching at 3.4°C to seven days at 13°C, while the larval yolk sac phase was shortened from 20 to 10 days at 3.8 and 10 °C, respectively.
When comparing the same species, Angelo et al. (Reference Angelo, Lisboa, Magnotti, Pilotto, Mattos and Cerqueira2021a) verified after the blastopore enclosure, that temperatures between 23.2 and 23.9°C resulted in better hatching rate and larval survival of M. liza, with more efficient consumption of the yolk sac. However, the researchers did not assess the entire stage of embryonic development. Thus, we can infer that eggs incubated at higher temperatures can accelerate biological processes resulting in shorter incubation and hatching times. According to Boltaña et al. (Reference Boltaña, Sanhueza, Aguilar, Gallardo-Escarate, Arriagada, Valdes, Soto and Quiñones2017), temperature variation, even when small, can generate embryos with malformations.
Several studies have reported the occurrence of malformations in embryos due to the incubation of eggs at temperatures outside the comfort range, both in marine and freshwater species. Okamoto (Reference Okamoto2004) observed some deformities in newly hatched larvae of Brazilian flounder Paralichthys orbignyanus when incubated at temperatures close to 23 °C, outside their comfort zone. The author reported that the time for hatching was inversely proportional to temperature, but the percentage of malformed larvae was higher at extreme temperatures, decreasing at temperatures 20, 23 and 26 °C.
On the other hand, Yang et al. (Reference Yang, Ma, Zheng, Jiang, Qin and Zhang2016) verified the presence of adverse impacts in pompano Trachinotus ovatus embryos incubated at temperatures above 33 °C and below 23 °C. According to the authors, the growth of fish kept at 29 and 33 °C was significantly faster than those kept at 23 and 26 °C, while survival at 26 and 29 °C was greater than those kept at 23 and 33 °C. Likewise, the Ribonucleic Acid (RNA)/DNA ratio was higher at 26 and 29 °C than at 33 °C. Furthermore, jaw deformities increased significantly when the rearing temperature exceeded 29 °C, while vertebral deformities were found more frequently at 33 °C than at lower temperatures.
Herein, the malformations that were observed in the 18 °C and 30 °C treatments can be attributed to the tolerance of the species to a specific temperature range, since the temperature in the spawning incubator was set at 23.7 °C, and the most affected treatments were evidenced at 5 °C and +7 °C from this spawning temperature, negatively influencing metabolism and embryonic development.
Indeed, fish are more vulnerable to the variables of the environment in which they live during the initial stages when compared to adults (Rijnsdorp et al., Reference Rijnsdorp, Peck, Engelhard, Möllmann and Pinnegar2009; Ciannelli et al., Reference Ciannelli, Smith, Kearney, Hunsicker and McGilliard2022). According to Jonsson and Jonsson (Reference Jonsson and Jonsson2014), the initial environment influences the later performance of the fish, i.e., the conditions that the fish face during embryogenesis can cause long-lasting deleterious effects, which can be transmitted to the offspring by the parents, mainly by the mother.
The Mugil species naturally spawns in specific periods, when an ideal water temperature is reached in a certain region, acting as an environmental stimulus for reproduction events (Lemos et al., Reference Lemos, Monteiro-Neto, Cabral and Vieira2017). Thus, it was expected that thermal conditions similar to natural conditions would guarantee the best embryonic development (Karås and Klingsheim Reference Karås and Klingsheim1997). However, the planet has experienced the effects of climate change on the oceans, directly affecting fish. Ongoing climate change is estimated to directly affect aquatic organisms during all life stages, contributing to changes in aquatic populations and ecosystem functioning (Pörtner and Peck, Reference Pörtner and Peck2010).
In conclusion, the temperature influenced chronologically and morphologically the embryonic development of the Mugil liza mullet, causing malformations that resulted in mortalities at temperatures of 18 and 30 °C. According to the results obtained, we recommend that the incubation of M. liza eggs be carried out at temperatures between 22 and 26 °C. However, at 26 °C embryogenesis was significantly faster.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0967199424000285
Acknowledgements
The authors thank the National Council for Scientific and Technological Development CNPq and the Coordination of Higher Education Personnel – CAPES.
Author contributions
João Vitor de Azevedo Manhães: Conceptualization, Experimental run, Methodology, Formal analysis, Investigation, Writing – original draft. Douglas da Cruz Mattos: Formal analysis, Writing – original draft. Rômulo Alves Strassburguer: Experimental run. Ulysses da Silva Palma: Investigation, Formal analysis. Fabio Carneiro Sterzelecki: Formal analysis, Investigation. Marco Shizuo Owatari: Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Caio Magnotti: Investigation, Writing – original draft. Vinicius Ronzani Cerqueira: Supervision, Writing – review & editing.
Funding
The study was partially funded by the National Council for Scientific and Technological Development CNPq 422460/2016-8; Universal CNPq 430770/2018-9; The Coordination of Higher Education Personnel – Brazil (CAPES) and the first author (J.V.A.M.) received scholarships from the Federal Agency for the Support and Evaluation of Graduate Studies (CAPES).
Competing interests
The authors declare no conflict of interest.
