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. 2026 Mar 25;109(1):348–361. doi: 10.1111/jfb.70418

Calcium and magnesium ions are the main intrinsic factors that induce the activation of stickiness in the eggs of the fat greenling ( Hexagrammos otakii )

Zhuang Xue 1,2,3,✉, Yuqing Yan 1,2, Chenghao Qiu 4, Juan Chang 4, Wei Wang 1,2,✉, Hongyu Pu 2,✉
PMCID: PMC13397299  PMID: 41879661

Abstract

In fish artificial breeding, adhesive eggs present a significant challenge for fertilization and reduce hatching success rates. Understanding the specific factors and mechanisms that influence egg adhesiveness, as well as developing effective de‐adhesion methods, is crucial for improving fish reproduction efficiency. Fat greenling (Hexagrammos otakii), a typical marine teleost species that produces adhesive eggs, is widely distributed in the northwest Pacific Ocean and is considered a promising candidate for aquaculture. In this study, H. otakii eggs were used as a model to qualitatively analyse the effects of different pH levels (6.0, 7.0, 8.0 and 9.0), osmotic pressures (100, 400 and 1000 mOsm kg−1) and metal ions (Na+, K+, Ca2+, Mg2+, Mn2+ and Cu2+) on egg adhesiveness. In addition, scanning electron microscopy was employed to examine the ultrastructural changes on the egg surface under different conditions. The results indicated that Ca2+ and Mg2+ are the primary factors in seawater that induce egg adhesiveness in H. otakii, while changes in pH and osmotic pressure neither triggered adhesiveness nor altered the underlying adhesive mechanism. Furthermore, the study evaluated the effectiveness of protease treatment for chemical de‐adhesion. The results showed that treating the eggs with a 10 mg/L protease solution for 2 min successfully removed their adhesiveness, confirming the feasibility of chemical de‐adhesion. This research provides significant theoretical insights for optimizing the artificial breeding techniques of H. otakii.

Keywords: adhesive eggs, artificial propagation, chemical de‐adhesion, Hexagrammos otakii, ultrastructure

1. INTRODUCTION

The structure and type of fish eggs play a vital role in their survival, hatching and reproductive success during fish reproduction (Bobe, 2015). The adhesive property represents a crucial reproductive strategy evolved in teleosts, enabling their eggs to resist dispersal by water currents and thereby preventing deposition in unsuitable habitats (Riehl & Patzner, 1998). This adaptation enhances survival under variable environmental conditions (Mansour et al., 2009b). According to the different characteristics of fish eggs, they can be categorized into floating, sinking and adhesive egg types (Chen & Liu, 2022). These egg types have evolved different attachment mechanisms, incubation strategies and adaptations according to environmental and population needs to cope with variable habitat conditions in marine or freshwater. Based on the strength of their surface adhesiveness, fish eggs can be clearly classified into three distinct types: strongly adhesive eggs, weakly adhesive eggs and non‐adhesive eggs (Rizzo et al., 2002). Among these types, adhesive fish eggs are particularly specialized in that they have remarkable adhesive properties. Adhesive eggs are eggs with a strongly sticky surface, which can quickly and firmly attach to substrates, such as rocks, seagrasses, rocky reefs or aquatic plants, after being laid, to avoid drifting away with the current or drifting to unfavourable environments (Mansour et al., 2009a; Murray et al., 2013). These eggs are widely distributed in marine, estuarine and freshwater environments, and are commonly found in species such as Thamnaconus modestus (Gwak & Roy, 2021) and members of the carp family (Cyprinidae) (Chen et al., 2022). The adhesive properties of adhesive fish eggs are ecologically significant in natural environments, yet they may pose certain challenges during artificial propagation. Adhesion activation of adhesive fish eggs is a complex process involving interactions between adhesive substances on the surface of the egg membrane and water, ions and substrates in the environment (Chen et al., 2023). Through water contact, protein or sugar‐based adhesion molecules are rapidly activated to form physical and chemical bonds with environmental substrates (Łuczyński et al., 2022), creating the adhesive capacity to ensure stable egg attachment to the appropriate incubation environment.

To figure out the mechanism of adhesion production, it is necessary to understand the structural characteristics of the eggs specifically. The peritoneum of scleractinian fish usually consists of two structures, the zona radiata interna (ZRI) and the zona radiata externa (ZRE). The composition of the ZRI is mainly proteins, which are responsible for the protection of the embryo, and the ZRE is mainly composed of neutral and acidic mucopolysaccharides, which are responsible for the interactions with water (Huysentruyt & Adriaens, 2005). Non‐adhesive eggs typically have a thinner and relatively simpler ZRE, whereas adhesive eggs are usually surrounded by a thicker and more complex ZRE (Riehl & Patzner, 1998; Rizzo et al., 1998). The ZRE of the adhesive eggs undergoes different physicochemical changes on the egg surface when it is exposed to water, making it sticky and thus adhering to different substrates. The morphology of the ZRE of bony fishes is variable. The surface of the radial band of non‐adhesive eggs of lipozoans is a pore or a network of cilia, the surface of the radial band of weakly adhesive eggs is only a pore and the surface of the radial band of highly adhesive eggs is a pore in the form of a globular, filamentous, fluffy or honeycomb‐like pore (Rizzo et al., 2002). Understanding this mechanism is an important guide for adhesion regulation and de‐adhesion treatment in artificial reproduction.

The fat greenling (Hexagrammos otakii Jordan & Starks, 1895), also known as yellow fish and yellow stick in northern China, belongs to the family Hexagrammidae and the genus Hexagrammos, and is mainly distributed in the northwestern Pacific Ocean, especially in the offshore waters of China, Japan and North Korea (Nishikawa, 1982; Ren et al., 2013). The H. otakii typically inhabits nearshore rocky reefs, with clear water and strong currents being the most common habitat conditions. They can adapt to a range of depths, from shallow waters to deeper regions, usually found near rocky reefs, seaweed or seagrass beds at depths of 20–100 m (Habib et al., 2011). As a marine fish with high economic value, the H. otakii is popular in the market due to its tender flesh and rich nutritional content (Gao et al., 2022). It also plays an important role in recreational fisheries. With the decline of marine fisheries resources, research on artificial breeding and aquaculture technologies has become crucial for the sustainable use and resource restoration of H. otakii. H. otakii eggs are typically strong adhesive eggs, and because the eggs are immediately bonded into clumps after being produced, the operation of single‐layer planar fertilization needs to be very rapid and technically difficult, and the efficiency of large‐scale production is not high. In spawning or incubation ponds, the adhesive eggs tend to accumulate debris and organic detritus on their surface, resulting in excessive bacterial and fungal loads that impair subsequent egg development (Sahu et al., 2023). Furthermore, these adhesive eggs frequently agglomerate into clusters, which impedes proper gas exchange and ultimately leads to hypoxic egg mortality (Siddique et al., 2016). Further improvement and upgrading of the technology requires a more in‐depth understanding of the mechanism of the formation of adhesion of the eggs of H. otakii and the factors affecting it.

Currently, the methods for removing adhesiveness from fish eggs primarily include physical, chemical and mechanical treatments (Kalvani Neitali et al., 2012). Traditional approaches, such as the clay slurry method, involve stirring the eggs in a yellow mud suspension to disperse them, followed by rinsing. The talc powder method utilizes a mixed suspension of talc powder and salt to treat the eggs, effectively reducing fungal infections (Gela et al., 2003). The urea method employs a stepwise treatment with urea solution, achieving thorough adhesion removal but requiring a longer processing time (Pšenička, 2016). The mechanical water‐stirring method relies on water flow agitation and friction to eliminate adhesiveness. Although mechanical treatment is cost‐effective, excessive stirring intensity or prolonged processing may damage the egg membrane, exposing the embryo to the external environment and leading to mortality. Additionally, mechanical friction can compromise the protective gelatinous layer on the egg surface, increasing the risk of pathogen infections, such as Saprolegnia (Thoen et al., 2011). Therefore, selecting an appropriate adhesion removal method based on the specific characteristics of fish eggs is crucial. This study aimed to systematically investigate the effects of environmental factors, such as pH, osmotic pressure and metal ions, on the adhesion of H. otakii eggs, and to observe the changes in the surface ultrastructure of the eggs under different treatments by scanning electron microscopy (SEM) to gain a deeper understanding of the mechanism of egg adhesion formation. In addition, the study also attempted to achieve the de‐adhesion of the eggs by protease treatment to evaluate its effectiveness in reducing the adhesion of the eggs and to assess its effect on the structure and development of the egg membrane, with the aim of providing theoretical basis and technical support for optimizing the artificial propagation technology of the H. otakii.

2. MATERIALS AND METHODS

2.1. Experimental fish and conditions

The H. otakii used in this study were wild individuals from Lushunkou District and Changhai County, Dalian, with body lengths 263.5 ± 21.3 mm and body weights 243.9 ± 34.7 g. The fish were temporarily reared in a pond in the workshop of Dalian Bay Nursery Plant of Dalian Hazelwood Fisheries Ltd at a water temperature of 12–14°C, pH 8.3 ± 0.2, salinity of 29 ± 1 ppt and sufficient dissolved oxygen. After 1 week of temporary rearing, individuals with significantly enlarged and distended abdomens and distinctly protruding genital pores were selected for egg collection. Mature eggs were obtained by gently pressing the abdomen from the anterior to the posterior direction without causing harm to the fish. Following egg collection, the fish were returned to temporary rearing for another week to allow recovery before being released back into their natural habitat. The extruded mature eggs exhibited colours ranging from dark grey, light yellow, brownish‐brown to dark purple, with an average diameter of 1.83 ± 0.1 mm. The eggs were coated with ovarian fluid, presenting a gelatinous state. Experiments were conducted immediately after egg collection. Each treatment group consisted of three replicates (n = 3), with each replicate containing 5 g of eggs.

2.2. State of the egg after exposure to water

First, 5 g of fish eggs exhibiting normal colour and morphology was weighed as observation samples. Dead eggs exhibit a milky white appearance, while eggs of poor quality display a shrunken morphology. Such eggs are excluded from experimental consideration. Healthy eggs, in contrast, present a smooth, plump and fully rounded shape. The collected eggs were placed in a pre‐prepared Petri dish and an appropriate amount of seawater (temperature 12–14°C, pH 8.3 ± 0.2, salinity 29 ± 1 ppt) was added to maintain the activity of the eggs. Using plastic tweezers, five or six fish eggs were tweezed and placed on a slide with a small amount of seawater as the observation medium. The objective and eyepiece of the microscope were adjusted to the intended magnification (40×) and the light source was adjusted to obtain a clear image. The morphology and structure of the fish eggs were photographed using the camera system that came with the microscope. The size, surface structure and other characteristics of the fish eggs were recorded.

2.3. pH treatment

Based on purified water, a HEPES buffer system (final concentration 20 mM) was used as a pH stabilizer. The initial pH of the solution was calibrated with a pH meter, and target pH values (6.0, 7.0, 8.0 and 9.0) were precisely adjusted by dropwise addition of dilute hydrochloric acid (0.1 M or 0.01 M) or sodium hydroxide solution (0.1 M). The same concentration of HEPES buffer was applied across all pH treatment groups to ensure comparability among experimental conditions.

The pH‐adjusted solutions were then poured into four separate Petri dishes, with 25 mL of each solution quantitatively transferred into each dish. Freshly collected fish eggs were randomly divided into four groups. From each group, 5 g of eggs was evenly distributed into the solution corresponding to the respective pH condition, ensuring complete submersion and a monolayer arrangement. Throughout the experiment, the water temperature was maintained at 12–14°C. The status of the fish eggs, including colour, morphology and adhesiveness, was observed under a microscope.

2.4. Osmotic pressure treatment

To simulate the effects of osmotic pressure fluctuations in the marine environment on fish eggs, four types of aqueous solution (AS; sucrose solution) with different osmolalities were prepared using sucrose solution: AS‐0 (distilled water, serving as a low‐osmotic‐pressure reference), AS‐100 (100 mOsm kg−1, corresponding to the osmotic pressure range in coastal low‐salinity zones), AS‐400 (400 mOsm kg−1, approximating isotonic conditions of standard seawater) and AS‐1000 (1000 mOsm kg−1, simulating intertidal evaporation or high‐salinity stress environments). The osmotic pressure gradient was designed to cover typical salinity fluctuation ranges that fish may encounter in natural reproductive environments (from freshwater input to high‐salt evaporation conditions), aiming to systematically evaluate the tolerance thresholds of fish eggs to osmotic pressure changes.

Fish eggs were divided into samples of equal weight (5 g) in 90‐mm glass Petri dishes. Then, 25 mL of AS solution was added to each dish, and the dishes were shaken at 100 rpm for 15–20 s to ensure full contact between the AS solution and the eggs, followed by a 60‐s standing period. The number of floating non‐adhesive fish eggs was counted and recorded. A small number of samples were taken for microscopic examination immediately after counting, and the same procedure was repeated and recorded again after 10 min.

2.5. Metal ion interventions

Based on the major inorganic salt composition of seawater, this study selected four primary metal ions (Na+, K+, Ca2+ and Mg2+) as the core test subjects. Single‐ion exposure experiments were conducted using their corresponding salt solutions (NaCl, KCl, CaCl2 and MgCl2). Additionally, to evaluate the potential effects of trace metal ions, Mn2+ and Cu2+ were included as supplementary test ions in the experimental system, with MnCl2 and CuCl2 used for solution preparation, respectively. All salt solutions were diluted to the target concentrations (Table 1). After preparation, fish eggs were divided into equal‐mass samples (5 g) in 90 mm glass Petri dishes, and 25 mL of salt solutions with different ion concentrations was added to each dish. The dishes were shaken at 100 rpm for 15–20 s to ensure full contact between the solution and the fish eggs, followed by a 60 s standing period before samples were collected for microscopic observation.

TABLE 1.

Metal ion concentrations.

Ion Concentration (mmol L−1)
Na+ 10, 30, 50, 150, 500, 700
K+ 10, 30, 50
Ca2+ 10, 30, 50
Mg2+ 10, 30, 50, 150, 500, 700
Mn2+ 10, 30, 50, 150
Cu2+ 10, 30, 50, 150
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2.6. Protease de‐sticking

The protease (from Bacillus subtilis; CAS: 9001‐92‐7) was dissolved in isotonic solution and configured into 10 mg L−1 protease solution, the fish eggs were mixed thoroughly with the protease solution and after standing for 2 min, the enzyme solution was filtered off, and the eggs were rinsed with the isotonic solution for three times, and then the seawater was added and the Petri dish was shaken after standing for 60 s. Statistical records of the phenomena were made. Then 5 g of fish eggs was placed in a Petri dish and seawater was added to promote adhesion into firm egg masses. The egg masses were thoroughly mixed with the protease solution and shaken at 200 rpm. After standing for 5 min, the eggs were rinsed three times with isotonic solution, then observed and recorded.

2.7. Preparation of scanning electron microscope samples

The treated fish egg samples were fixed with 2.5% glutaraldehyde prepared in 0.1 M phosphate‐buffered saline (PBS, pH 7.4) at 4°C for 4–12 h (overnight) to preserve their morphological structure. After fixation, the samples were transferred to glass vials containing 0.1 M PBS (pH 7.4) and rinsed three times for 15 min each on a horizontal shaker. Gradient dehydration was then performed using tertiary butanol solutions at concentrations of 50%, 70%, 80%, 90% and 95% (v/v), with each step carried out on a shaker at 4°C for 15 min. This was followed by three dehydration steps with 100% pure tertiary butanol under the same conditions, each lasting 10 min, to ensure complete removal of water. The dehydrated samples were placed in a −20°C freezer to solidify the tertiary butanol, after which the solidified samples were transferred to a critical point dryer. Critical point drying was performed using liquid carbon dioxide as the transition medium. The dried samples were mounted on metal stubs with conductive adhesive and coated with an approximately 10 nm‐thick layer of gold–palladium alloy using an ion sputter coater under vacuum. After coating, the samples were ready for observation under a scanning electron microscope.

2.8. Statistical analysis

In this study, data were processed and analysed using SPSS 22.0 statistical software. The data are presented as mean ± standard deviation (SD). In order to assess the differences between groups between time points, one‐way analysis of variance (ANOVA) combined with Duncan's multiple range test was employed to compare the significance of differences among different treatment groups. The results of the experiments are presented graphically, where significant differences are labeled as *p < 0.05, **p < 0.01, and ***p < 0.001.

3. RESULTS

3.1. Changes in eggs before and after exposure to water

As shown in Figure 1a, the eggs of H. otakii are attached to the red alga Mazzaella japonica in their natural marine environment. Before the eggs came into contact with seawater, microscopic observation showed that the surface of the eggs was smooth, light brown, slightly transparent, the oil globules were clearly visible and there were gaps between the eggs with only a little contact. The eggs were gently squeezed with a dissecting needle to bring them together, and the eggs did not show any adhesion (Figure 1b). After the eggs were exposed to seawater (pH 8.3 ± 0.2), the brightness and transparency of the surface of the eggs were obviously strengthened under microscopic observation, the eggs became semi‐transparent at about 8 s, a circular transparent layer appeared on the surface of the eggs at about 15 s, the eggs were adhered to each other after contacting with each other and could not be separated by shaking the slide, the eggs were gently squeezed by a dissecting needle, the contact area among the fish and eggs increased under the action of the external force and the adhesion became stronger. The contact area between the eggs increased and the adhesion became stronger under the external force, and the eggs were tightly combined together to form irregular egg masses in about 40 s, which were not easy to be separated (Figure 1c).

FIGURE 1.

FIGURE 1

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Changes before and after seawater activation in the eggs of H. otakii. (a) Eggs of H. otakii naturally adhered to the red alga Mazzaella japonica, photographed in the coastal waters of Dalian Ocean University. (b) The natural initial state of the egg not activated by seawater. (c) The state of the egg after it has been activated by seawater. Magnification is 40×. Solid arrows indicate eggs that remain aggregated through ovarian fluid cohesion without adhesion, whereas dashed arrows denote significantly adhesive eggs forming tightly bound clusters.

3.2. Effects of pH exposure on egg adhesion

It was observed that after the eggs were treated with the test solutions at pH 6, 7, 8 and 9 for 60 s and 10 min (Table 2), the eggs did not show any adhesion, there was no adhesion between the eggs and the surface of the eggs did not show any obvious change compared with that of the eggs in the natural state. Microscopic examination revealed that after treatment with a pH 6 aqueous solution, the outer layer of the eggs formed a transparent ring and no significant adhesion was observed, the eggs were merely in contact with each other (Figure 2a). Similarly, after treatment with a pH 7 aqueous solution, a transparent ring also formed around the outer layer of the eggs, with no noticeable adhesion (Figure 2b). When the eggs were treated with pH 8 and pH 9 aqueous solutions, no significant adhesion was observed and the transparent outer ring layer became smaller (Figure 2c,d). The proportion of unadhered eggs reached 100% following 10‐min exposure to all tested pH conditions (p > 0.05) (Figure 2e).

TABLE 2.

Changes in egg adhesion after pH exposure.

pH Adhesion (60 s) Proportion of free eggs (60 s) Adhesion (10 min) Proportion of free eggs (10 min)
6 − 100% − 100%
7 − 100% − 100%
8 − 100% − 100%
9 − 100% − 100%
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Note: +, yes; −, clogged. Percentage of free‐living eggs = free‐living eggs/total number of eggs × 100%.

FIGURE 2.

FIGURE 2

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The variation in adhesive properties of H. otakii eggs under different pH treatments. Microscopic observations of H. otakii eggs following treatment with solutions at pH 6 (a), 7 (b), 8 (c) and 9 (d). Proportion of unadhered H. otakii eggs after 10‐min exposure to different pH treatments (e). The symbol ‘→’ indicates non‐adhesive eggs that remain aggregated via ovarian fluid cohesion. ns, not significant.

3.3. Effects of different osmotic pressures on egg adhesion

None of the eggs produced significant adhesion in AS treatments with four different osmotic pressures, AS‐0, AS‐100 (100 mOsm kg−1), AS‐400 (400 mOsm kg−1) and AS‐1000 (1000 mOsm kg−1) (Table 3). Microscopic observation revealed that after immersion in sucrose solutions of AS‐0 (Figure 3a), AS‐100 (Figure 3b), AS‐400 (Figure 3c) and AS‐1000 (Figure 3d), all samples in each group showed no significant differences in morphology or size. The perivitelline space at the outermost layer of the eggs appeared as a distinct transparent ring. No adhesion was observed in eggs treated with different osmotic pressures, with the proportion of unadhered eggs reaching 100% (p > 0.05) (Figure 3e).

TABLE 3.

Changes in egg adhesion after exposure to different osmotic pressures.

AS Adhesion (60 s) Proportion of free eggs (60 s) Adhesion (10 min) Proportion of free eggs (10 min)
AS‐0 − 100% − 100%
AS‐100 − 100% − 100%
AS‐400 − 100% − 100%
AS‐1000 − 100% − 100%
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Note: +, yes; −, clogged. Percentage of free‐living eggs = free‐living eggs/total number of eggs × 100%.

FIGURE 3.

FIGURE 3

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Effects of osmotic pressure treatments on the adhesion of H. otakii eggs. Microscopic observations of H. otakii eggs following treatment with osmotic gradients AS‐0 (a), AS‐100 (b), AS‐400 (c) and AS‐1000 (d). Proportion of unadhered H. otakii eggs after 10‐min exposure to different osmotic pressures (e). The symbol ‘→’ indicates non‐adhesive eggs that remain aggregated via ovarian fluid cohesion. ns, not significant.

3.4. Effects of metal ions on egg adhesion

Natural seawater contains various metal ions, which are key factors in stimulating adhesion in adhesive fish eggs. Experimental results indicated that after treatment with Na+ solutions at concentrations of 10, 30, 50 and 150 mmol L−1 for 60 s and 10 min, the eggs did not exhibit adhesion and the proportion of free eggs remained at 100%. When the Na+ concentration was increased to 500 and 700 mmol L−1, the proportion of free eggs after 60 s of treatment was 84.84% and 48.57%, respectively, while after 10 min of treatment, the proportion was 75.75% and 0%, respectively. Similarly, after treatment with K+ solutions at concentrations of 10, 30, 50, 150, 500 and 700 mmol L−1 for 60 s and 10 min, no adhesion was observed and the proportion of free eggs remained at 100%. In contrast, when the eggs were treated with Ca2+ at a concentration of 10 mmol L−1 for 60 s, the proportion of free eggs was 6.45%. At higher concentrations of 30 and 50 mmol L−1, the eggs exhibited strong adhesion, clustering tightly in irregular aggregates. For Mg2+ treatments, the proportion of free eggs was 11.53% and 3.84% after 60 s of exposure to 10 and 30 mmol L−1 solutions, respectively. At higher concentrations, all treated eggs exhibited adhesion. Furthermore, after 60‐s exposure to Mn2+ at concentrations of 10 and 30 mmol L−1, the proportions of unadhered eggs were 38.19% and 17.62%, respectively, whereas Cu2+ treatment failed to induce egg adhesion (Table 4).

TABLE 4.

Changes in egg adhesion at different metal ion concentrations.

Ions Concentration (mmol L−1) Adhesion (60 s) Proportion of free eggs (60 s) Adhesion (10 min) Proportion of free eggs (10 min)
Na+ 10 − 100% − 100%
30 − 100% − 100%
50 − 100% − 100%
150 − 100% − 100%
500 + 84.84% + 75.75%
700 + 48.57% + 0%
K+ 10 − 100% − 100%
30 − 100% − 100%
50 − 100% − 100%
150 − 100% − 100%
500 − 100% − 100%
700 − 100% − 100%
Ca2+ 10 + 6.45% + 0%
30 + 0% + 0%
50 + 0% + 0%
Mg2+ 10 + 11.53% + 0%
30 + 3.84% + 0%
50 + 0% + 0%
Mn2+ 10 + 38.19% + 0%
30 + 17.62% + 0%
50 + 6.85% + 0%
Cu2+ 10 − 100% − 100%
30 − 100% − 100%
50 − 100% − 100%
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Note: +, yes; −, clogged. Percentage of free‐living eggs = free‐living eggs/total number of eggs × 100%.

Microscopic observation revealed that H. otakii eggs treated with NaCl solutions at different concentrations did not exhibit adhesion. Their appearance was similar to that of eggs in their natural state before activation by seawater, with the eggs merely in contact with each other without adhering (Figure 4a). Similarly, eggs treated with KCl solutions at various concentrations showed the same results as those treated with NaCl, with no activation of adhesion (Figure 4b). In contrast, eggs treated with CaCl2 (Figure 4c), MgCl2 (Figure 4d) and MnCl2 (Figure 4e) solutions for 60‐s exhibited adhesion. Neither Na+ nor K+ served as key ions for egg adhesion induction, with all concentration treatments resulting in 100% unadhered eggs (p > 0.05) (Figure 4f). Moreover, as the ion concentration increased, the adhesive strength of the eggs became more pronounced, leading to irregular aggregation and making separation more difficult. To eliminate the interference of Cl−, H. otakii eggs were treated with gradient concentrations of MgSO4, MnSO4 and CuSO4 solutions. The results showed that egg adhesion was activated in both MgSO4 (Figure 5a) and MnSO4 (Figure 5b) solutions, whereas no adhesion was observed in CuSO4 solutions (Figure 5c).

FIGURE 4.

FIGURE 4

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The changes in adhesiveness of H. otakii eggs under different concentrations of metal ion treatments. (a) Microscopic images of H. otakii eggs treated with NaCl solutions. (b) Microscopic images of H. otakii eggs treated with KCl solutions. (c) Microscopic images of H. otakii eggs treated with CaCl2 solutions. (d) Microscopic images of H. otakii eggs treated with MgCl2 solutions. (e) Microscopic images of H. otakii eggs treated with MnCl2 solutions. (f) Proportion of unadhered H. otakii eggs after treatment with different concentrations of metal ions for 60 s. Solid arrows indicate eggs that remain aggregated through ovarian fluid cohesion without adhesion, whereas dashed arrows denote significantly adhesive eggs forming tightly bound clusters. ns, not significant; **p < 0.01, ***p < 0.001.

FIGURE 5.

FIGURE 5

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Effects of metal ion concentration on the adhesion of H. otakii eggs. (a) Microscopic images of H. otakii eggs treated with MgSO4 solutions. (b) Microscopic images of H. otakii eggs treated with MnSO4 solutions. (c) Microscopic images of H. otakii eggs treated with CuSO4 solutions. Solid arrows indicate eggs that remain aggregated through ovarian fluid cohesion without adhesion, whereas dashed arrows denote significantly adhesive eggs forming tightly bound clusters.

3.5. Protease removes the adhesion of the egg

Microscopic observation demonstrated that seawater‐activated eggs exhibit strong adhesiveness, with individual eggs tightly adhered to each other (Figure 6a). To explore an efficient method for de‐adhesion of H. otakii eggs, we immersed the eggs in protease solutions at concentrations of 10, 20, 30 and 50 mg L−1 (Figure 6b) for 3 min. The results showed that egg adhesion was inhibited and the perivitelline space appeared as a transparent ring. When the protease‐treated eggs were subsequently stimulated with seawater, their ability to develop adhesion was lost (Figure 6c).

FIGURE 6.

FIGURE 6

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Eggs of H. otakii under natural conditions and after protease treatment. (a) Microscopic images of H. otakii eggs after seawater activation. (b) Microscopic images of H. otakii eggs treated with alkaline protease solutions at concentrations of 10, 20, 30 and 50 mg L−1. (c) Microscopic images of H. otakii eggs treated with an alkaline protease solution followed by seawater activation. Solid arrows indicate eggs that remain aggregated through ovarian fluid cohesion without adhesion, whereas dashed arrows denote significantly adhesive eggs forming tightly bound clusters.

3.6. Scanning electron microscopy analysis of eggs

Ultrastructural scanning electron microscopy revealed that the surface of unfertilized and seawater‐unactivated H. otakii eggs is characterized by numerous regularly arranged circular pores with diameters of 0.3–1 μm and inter‐pore spacings of 1.5–4 μm, uniformly distributed across the egg surface. In addition, abundant glycoproteins and fibrous proteins were observed surrounding the egg envelope and the pores of the egg membrane (Figure 7a). These adhesive substances exhibit fibrous or granular structures and constitute the primary factor enabling the eggs to adhere to each other and to submerged surfaces. Upon further magnification to a scale showing only a single oocyte pore (40,000:1), more rounded chorionic projections were visible on the surface of the oocyte theca, arranged in a relatively dense pattern. At this scale, fibrillar fibronectin and punctate glycoproteins could be more clearly observed distributed around the periplasmic pores (Figure 7b). These adhesive substances can rapidly spread or coagulate on exposure to seawater stimulation or fertilization, thereby facilitating the immobilization of the egg in a specific environment.

FIGURE 7.

FIGURE 7

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Electron microscope scanning analysis of H. otakii eggs. The initial state of the egg without seawater activation (a) and a scanning electron micrograph of a single egg membrane pore at a magnification of 40,000:1 (b). (c) Ultrastructure of eggs treated with 700 mmol L−1 Na+. The ultrastructure of the egg envelope after activation by Mg2+ (d) and Ca2+ (e). (f) The ultrastructure of egg treated with 10 mg L−1 of protease for de‐adhesion. Inline graphic, fibrin and glycoproteins; Inline graphic, closed oocyte pore.

To explore which metal ions had a significant effect on the activation pathway of H. otakii eggs, electron microscope ultrastructural scanning analysis was also performed. The results showed that at a Na+ concentration of 700 mmol L−1, the eggs developed significant adhesion. Electron microscope scanning results observed that the fibronectin and glycoprotein attached to the surface of the egg membrane swelled and the adhesion of the egg was activated (Figure 7c). The results of the previous ion concentration experiments have demonstrated that Mg2+ and Ca2+, among these metal ions, are the key ions for the activation of egg adhesion. The electron microscope scanning results here show that the egg envelope of the Mg2+ (Figure 7d) and Ca2+ (Figure 7e) activated eggs became rougher than that of the unactivated eggs, and the villi‐like granules protruded clearly. At this point, the adhesive mechanism of H. otakii eggs was fully stimulated. Artificial de‐adhesion is a commonly used technique for fish breeding with adhesive eggs, and in this study, H. otakii eggs were de‐adhesively treated with a protease solution at a concentration of 10 mg L−1, which had a remarkable effect. The adhesion of the eggs disappeared after 2 min of de‐adhesion treatment. The results of electron microscope scanning showed that the pore of the egg membrane was closed and blocked by some granular materials in the protease‐treated eggs (Figure 7f).

4. DISCUSSION

Fish egg adhesion is a specialized biological phenomenon exhibited by many marine and freshwater fishes during reproduction, especially for benthic spawning species, where adhesive eggs help to immobilize the eggs in suitable spawning environments, preventing them from drifting and being ingested by predators (Ding et al., 2022). This phenomenon of sticky eggs is widespread in many marine fishes, including the H. otakii. It is a typical marine benthic fish that becomes sexually mature at the age of 2 and spawns in the fall and winter, with the peak of spawning occurring from September to mid‐November each year. Spawning occurs in reefs rich in seagrasses or seaweeds, and the fertilized eggs adhere to each other to form large clumps of eggs that are attached to spawning substrates such as reefs, seagrasses or seaweeds. The egg adhesion of the H. otakii plays an important role in the attachment and hatching of its eggs. This study revealed that prior to activation by seawater, the egg membrane surface of H. otakii is relatively smooth and exhibits a honeycomb‐like structure characterized by a regular arrangement of oval pores. On contact with seawater, mucin proteins on the egg surface rapidly undergo conformational changes and hydration‐driven expansion, leading to swift activation of the adhesive mechanism and ultimately resulting in strong egg adhesiveness.

The production of egg adhesion is usually associated with the secretion of glycoproteins or mucins on the surface of the eggshell, which are able to adsorb water and form a sticky matrix in water (Debus et al., 2008; Mansour et al., 2009a; Murray et al., 2013). In addition, egg adhesion is regulated by a number of external environmental factors, such as pH, salinity, temperature and metal ion concentration. These external environmental factors not only affect the efficiency of egg adhesion formation, but also may influence the structure and function of adhesive molecules (Wu et al., 2022). The pH range of the ovarian fluid of scleractinian fishes has been reported to be 6.2–8.8, whereas the pH of surface seawater is generally in the range of 8.0–8.1 (Zadmajid et al., 2019). The results of this study indicate that the gradient change in pH does not activate the adhesiveness of H. otakii eggs, thus proving that pH variation is not the fundamental cause of egg adhesiveness on contact with seawater. Riehl and Patzner found that the eggs of Cyprinodontiformes are non‐adhesive in the ovarian fluid, but on contact with water, they adhere to each other on the aquatic substrate (Riehl & Patzner, 1998). In this study, the eggs of H. otakii failed to exhibit adhesive activation in AS‐0 (distilled water). Furthermore, under the three osmotic pressure conditions tested—AS‐100 (100 mOsm kg−1), AS‐400 (400 mOsm kg−1) and AS‐1000 (1000 mOsm kg−1)—no adhesive response was induced in the eggs. However, the eggs appeared shaded after 10 min of AS‐1000 treatment, which was different from the circular transparent layer that appeared when encountering seawater. In conclusion, neither pH nor osmotic pressure (as manipulated via sucrose in this study) exhibited a positive correlation with the development of egg adhesion in H. otakii. Thus, under the present experimental conditions, they are classified as cofactors. It is worth noting that the osmotic pressure in this study was achieved using only organic osmolytes (sucrose); in natural environments where osmotic pressure results from both organic and inorganic osmolytes, osmolality may influence egg adhesion.

In this study, it was found that the adhesion of the eggs of the H. otakii was mainly regulated by Na+, Mg2+ and Ca2+. Analysis of the results of the study concluded that Na+ mainly acts as a co‐regulator of the production of adhesion in H. otakii eggs, mainly through the regulation of osmotic pressure, hydration, charge balance and synergistic effects with other metal ions. A moderate amount of Na+ ions can help the proteins and polysaccharides on the surface of the egg membrane to undergo moderate hydration and swelling, and enhance adhesion (El‐Gamal & El‐Greisy, 2008). At the same time, Na+ stabilizes the structure of the egg membrane by neutralizing the negative charge on the membrane and acts together with Mg2+ and Ca2+ ions to further promote cross‐linking and cohesion of the adhesive substances, ensuring the stability and adhesion capacity of the egg membrane. In this study, a moderate increase in Na+ concentration (700 mmol/L) significantly enhanced egg adhesiveness. However, this phenomenon does not imply that Na+ is the direct metal ion responsible for inducing egg adhesion; rather, it primarily functions through synergistic interactions with other ions. This is important for the fact that in practical applications, an increase in the appropriate Na+ concentration can improve the adhesion of the eggs, ensure their successful attachment to the substrate during reproduction and create a stable environment for embryonic development.

The adhesive eggs of H. otakii often attach to seaweeds, reefs or other underwater objects, relying on natural water flow and substrates to maintain an optimal hatching environment. Under artificial conditions, the absence of these natural supporting factors necessitates the use of artificial de‐adhesion techniques to compensate for the limitations of the artificial environment, thereby optimizing the hatching conditions and improving the survival and hatching rates of the eggs (Siddique et al., 2016). Nowadays, there are two main types of de‐adhesion methods used in production, namely physical de‐adhesion and chemical de‐adhesion. Physical de‐adhesion, which utilizes de‐adhesion agents such as yellow mud slurry and talcum powder to adhere to the surface of the eggs and thus cover up the adhesion of the eggs (Mizuno et al., 2004; Pšenička & Franěk, 2021), is a commonly used de‐adhesion method in the production process, with the advantages of low cost and simple method, and is currently more commonly used in carp, crucian carp, catfish and sturgeon fish (Chebanov & Galich, 2013; Siddique et al., 2016). Chemical de‐adhesion is a method of destroying or inhibiting egg adhesion by utilizing substances capable of reacting with the egg membrane such as proteases, sodium hypochlorite and tannins (Żarski et al., 2015). Compared with physical de‐adhesion, chemical de‐adhesion is more thorough and efficient. Bian et al. (2022) investigated the effects of different concentrations of tannic acid (0.5%, 0.2% and 0.1%), sodium hypochlorite (0.05%, 0.025% and 0.01%) and alcalase (4%, 2%, 1% and 0.5%) on the removal of egg adhesiveness in the greenfin horse‐faced filefish (Thamnaconus septentrionalis). They found that only the alcalase enzyme was effective in removing egg adhesiveness, and it had no significant impact on hatching rates (Bian et al., 2022). Bouchard et al. (2002) treated Acipenser fulvescens eggs with 50 mg L−1 buffered iodine solution for 30 min, and then used urea, sodium chloride‐tannic acid solution and Fuller's earth to de‐adhesion, which resulted in 99.6% and 97.7% hatching rate after disinfection and then de‐adhesion, respectively (Bouchard III & Aloisi, 2002). The chemical de‐adhesion method has also been successfully applied to the eggs of Sander lucioperca (Kristan et al., 2016), Silurus glanis (Pšenička & Franěk, 2021), Tinea tinea (Linhart et al., 2003) and Clarias gariepinus (Asraf et al., 2013). In this study, the eggs of the H. otakii were successfully de‐adhesive after treatment with a protease solution at a concentration of 10 mg L−1 for 60 s. Electron microscope ultrastructural scans showed that the pores on the egg surface disappeared after protease treatment. The main function of the pores of the egg membrane is to provide a passage for spermatozoa to enter the egg (Yanagimachi et al., 2017). In the egg of most fish species, the egg membrane has a hard protective structure called the eggshell or egg membrane, and the otic pore is the only opening in this layer. When a sperm swims towards the egg, it can only enter the egg through the otic pore to unite with the egg's nucleus and complete fertilization. Therefore, the protease de‐adhesion method needs to follow the sequence of fertilization followed by de‐adhesion. It was also found that the proteins in the adhesive layer on the surface of the protease‐treated eggs disappeared and were decomposed, and the eggs were no longer adhesive when exposed to seawater. However, the adhesive eggs that had been activated by the seawater and adhered together could not be separated after being immersed in the protease solution, so it was evident that the adhesion of the eggs of the H. otakii was a unidirectional reaction and was not reversible. During incubation, the eggs need to obtain oxygen from the surrounding environment and excrete metabolic wastes. Excessively adherent fish eggs can block the flow of water and reduce the exchange of oxygen and nutrients (Siddique et al., 2016). This may result in poor embryonic development or death. Therefore, the application of de‐adhesion technology can positively affect egg hatchability by preventing the formation of egg masses, facilitating water flow, and improving oxygen supply and waste removal.

5. CONCLUSION

In fish artificial breeding, adhesive eggs present a challenge due to their negative impact on fertilization and hatching rates. This study, using H. otakii as a model, found that Ca2+ and Mg2+ are key factors influencing egg adhesiveness, while changes in pH and osmotic pressure had no significant effect. On release, mature eggs absorb ovarian fluid and expand as the oocytes detach from the ovarian wall. The ovarian fluid dissolves in seawater and the mucoproteins on the egg membrane are activated by Ca2+ and Mg2+, resulting in a rough egg surface covered with adhesive macromolecules. The adhesiveness is fully activated during this process, with fertilization occurring simultaneously, ultimately leading to the formation of egg masses attached to the substrate. The study also demonstrated that a 10 mg L−1 protease solution can effectively remove egg adhesiveness without causing damage. This research provides important insights for optimizing the breeding techniques of H. otakii. Future studies should thoroughly evaluate whether protease treatment affects the subsequent growth of larvae hatched from treated eggs.

AUTHOR CONTRIBUTIONS

Yuqing Yan: Data curation, formal analysis, investigation, software, visualization, writing – original draft. Chenghao Qiu and Juan Chang: Resources. Hongyu Pu, Wei Wang, and Zhuang Xue: Conceptualization, investigation, methodology, project administration, validation, writing – review and editing.

CONFLICT OF INTEREST STATEMENT

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

ACKNOWLEDGEMENTS

This work was supported by the Liaoning Province Science and Technology Joint Plan Project (2024‐MSLH‐047), the Liaoning Province Major Science and Technology Special Project (2024JH1/11700010, for Zhuang Xue and Wei Wang), the Basic Research Projects of Higher Education Institutions by Liaoning Provincial Department of Education (JYTZD2023038; JYTMS20230499), and Open Fund of Key Laboratory of Biotechnology and Bioresources Utilization (Dalian Minzu University), Ministry of Education (No. KF2024010), China.

Contributor Information

Zhuang Xue, Email: xg-dzyx@163.com.

Wei Wang, Email: wangwei@dlou.edu.cn.

Hongyu Pu, Email: puhongyu@dlou.edu.cn.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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