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. 2026 Apr 8;345(6):545–553. doi: 10.1002/jez.70088

Ovulating Female of Nine‐Spined Sticklebacks (Pungitius sinensis) Are Attracted to Substances Derived From Male Kidneys

Miki Nagaya 1, Hidenobu Yambe 2, Yasunori Koya 3,✉
PMCID: PMC13255013  PMID: 41952285

ABSTRACT

Male sticklebacks (Gasterosteidae) produce an adhesive protein called “spiggin” in the tubular epithelial cells of their kidneys, which they use to attach plants or plant debris together and build nesting sites. Spiggin is a glycoprotein excreted in urine. A similar substance has been reported in freshwater sculpins (Cottidae), and it is thought that male sculpins that nest under stones use this glycoprotein in their enlarged kidneys to attract females to their nests. Based on the phylogenetic closeness of sticklebacks and sculpins and the physiological similarities in their kidneys, we hypothesized that spiggin may play a role in attracting females to nesting sites in sticklebacks. We tested this hypothesis using a Y‐maze with the nine‐spined stickleback (Pungitius sinensis), a member of the Gasterosteidae. Ovulated females were more consistently attracted to male kidney extracts than female kidney extracts. The high‐molecular‐weight fraction (molecular weight > 3000) separated from the male kidney extract using a dialysis membrane had an attractive effect on ovulating females, while the low‐molecular‐weight fraction (molecular weight < 3000) had a repellent effect. The water in the aquarium containing nests built by males significantly attracted ovulating females. These results suggest that spiggin or an unknown component associated with spiggin functions to attract ovulating females. In sticklebacks, which evolved from territorial breeding to building elaborate nests, it is possible that the substance that attracts females produced in the male kidney has evolved into an adhesive component that solidifies the nest.

Keywords: female attractant, Gasterosteidae, nesting behavior, olfactory cue, releaser pheromone, spiggin

Summary

  • Ovulating female sticklebacks were attracted to extracts from the male's kidneys, and the attractant component had a molecular weight of over 3000.

  • Ovulating females were attracted to the scent of nests made by the male, which were made from plant debris.

  • The component (spiggin) that glues together nest material, produced in the kidneys of male sticklebacks, may have evolved functionally from a substance that attracts females.

1. Introduction

Males of Gasterosteidae, including Gasterosteus aculeatus, are known to build elaborate nests by gluing together pieces of plant material during the breeding season to protect the eggs laid by females (van Iersel 1953; Baggerman 1966; Morris 1958; Reisman and Cade 1967; Rowland 1974; Östlund‐Nilsson 2001). Studies using G. aculeatus, Spinachia spinachia, and Pungitius pungitius have shown that the adhesive substance used in this nesting material, called “spiggin,” is produced and secreted in the renal tubules of the male's kidneys (Rinkel and Hirsch 1940; Hentschel 1979; Jakobsson et al. 1999). Spiggin is a glycoprotein with a molecular weight of 203 kDa (Jakobsson et al. 1999), and during the breeding season, male kidneys increase in weight by approximately 2.5–3 times their normal weight due to spiggin production (Baggerman 1989).

The phenomenon of male kidney enlargement during the breeding season is not limited to Gasterosteidae, but has been reported in several species of the genus Cottus (Goto 1974; Goto et al. 1979; Hentschel 1982; Bucher and Hofer 1993; Koya et al. 2016a), a family closely related to Gasterosteidae (see Kawahara et al. 2008). Adult Cottus species typically hide in crevices under stones and, during the spawning season, males use the spaces under the stones as a spawning nest to attract females to spawn there (Koyama 1950; Savage 1963; Goto 1982). In Cottus pollux, the renal tubular epithelium of males thickens during the breeding season, causing the kidneys to approximately double in size, and the tubular epithelium produces glycoprotein (Koya et al. 2016a). Males of this species are known to store large amounts of viscous urine in their bladders when nesting during the breeding season (Koya et al. 2016b). Field observations and behavioral experiments with this and other Cottus species suggest that ovulating females are attracted to nesting males (Goto 1994; Koya unpublished data). Based on this, it is possible that the males' kidneys enlarge during the breeding season because the males, who claim the nest as their territory, secrete olfactory stimulants to attract females from afar.

Considering the evolution of the unique nest‐building trait of Gasterosteidae, could kidney secretions have originally been a substance used to attract females? It is possible that the viscous substance used by males to attract females evolved functionally into an adhesive substance for building elaborate nests from plant material. To test this hypothesis, we conducted behavioral experiments to examine whether kidney extracts from breeding males have a female‐attracting effect. We also investigated whether the nests built by males using spiggins themselves are capable of attracting females. To conduct these experiments, we used the Gasterosteidae genus Pungitius. It has been reported that male kidneys in Pungitius exhibit significant enlargement during nest‐building activity (Fukui 1962), and that renal tubules thicken during the breeding season (van Oordt 1924; Rinkel and Hirsch 1940). It is also known that Pungitius have a gene homologous to spiggin in G. aculeatus (Kawahara and Nishida 2007; Seear et al. 2015). Since the genus Pungitius uses emergent plants as a substrate and builds nests made of rolled‐up plant fragments high up on the bottom (Morris 1958), we considered this fish species to be an ideal species for experiments examining female attraction, as nests built by males can be easily retrieved.

There are two cryptic species of Pungitius in Japan: a freshwater species and a brackish‐water species (referred to as Pungitius sp. 1 and Pungitius sp. 2, respectively) (Takata et al. 1987). These are now considered to be distinct species morphologically and genetically, and Takahashi et al. (2016) tentatively named the freshwater species Pungitius sinensis (Guichenot 1869) and the brackish‐water species P. pungitius (Linnaeus 1758). In the present study, we focused on the freshwater species P. sinensis. P. sinensis is widely distributed throughout Hokkaido, inhabiting the middle and lower reaches of rivers (Ishikawa et al. 2013; Takahashi et al. 2003; Tsuruta et al. 2008), and generally nests only in freshwater (Machida et al. 2023).

2. Methods

2.1. Fish Collecting and Rearing

The experiment was conducted over 4 years from 2020 to 2023. The P. sinensis used in this study were collected with dip nets and cage nets from several rivers in Hokkaido (Abashiri River System in 2020 and 2021, Shibetsu River System in 2022, and Nishitappu, Tarumae, and Orito River systems in 2023) in May–June in each year. Collections were conducted immediately prior to the breeding season in each year. The collected fish were transported to an experimental facility (Tsukiyodani Fureai‐no‐Sato) in the mountainous region of Ibigawa Town, Gifu Prefecture. Male (min. and max. standard body length: 37.7–51.7 mm) and female (min. and max. standard body length: 44.7–54.6 mm) fish were housed in a mixed‐gender rearing tank (50 cm wide, 85 cm long, 56 cm high; 150 L capacity; FRP). The breeding tanks were filled with river water (no Pungitius species inhabiting them). Terrestrial grass plants (Poaceae) that grow near the water was planted in the aquarium as a nesting substrate, and the dead grass leaves (approximately 1 cm wide) cut into 2–3 cm long pieces were placed in the aquarium as nesting material. The fish were fed frozen chironomid larvae (Kyorin Co. Ltd.) once or twice daily. The river water temperature during breeding was approximately 14°C in early June, when breeding began, and reached 18°C by late July, when breeding ended. Experiments were conducted from mid‐June to mid‐July each year.

The presence or absence of ovulation was estimated almost daily during the experimental period by checking whether the female's abdomen was distended, and females that were presumed to be ovulating were used for the experiment on the same day. Females used in the experiments were brought back to the laboratory on the same day, anesthetized (Eugenol; approximately 5–10 μL/L; FUJIFILM Wako Pure Chemical Corporation), dissected, and their ovaries were examined to confirm whether ovulation had actually occurred. If no presumed ovulating females were found, non‐ovulating females were used in the experiment.

All animal experiments were conducted in accordance with the regulations of the Animal Experimentation Committee of Gifu University.

2.2. Experimental Equipment

A Y‐maze (made of 5‐mm‐thick vinyl chloride boards) with two separate upstream sections was used for the experiment. The dimensions of the Y‐maze waterway are shown in Figure 1. The channel was infused with river water at 0.11 L per second. The test solution was introduced into the upstream sections on both sides of the Y‐maze, and observations were made to see which section the test females placed in the downstream section would enter. Silicone tubing (4 × 6; As One; https://www.as-1.co.jp/en/) and a peristaltic pump (PST‐100N; IWAKI; https://iwaki.atgc.co.jp/) were used to introduce the test solution into the upstream section. The introduction point was 50 cm downstream from the river water inlet (approximately halfway up the upstream section), on the outer side. The pump flow rate was set to 0.8 mL/s. Methylene blue (Waldeck GmbH & Co. KG; https://www.waldeck-ms.de/en/) was used to observe the spread of the test solution throughout the tank, and it took approximately 60 s for the methylene blue to reach the top of the downstream area (movable gate) after the pump was turned on.

Figure 1.

Figure 1

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Schematic diagram of the Y‐maze used in the behavioral experiments. Above is the top view, and below is the side view.

2.3. Behavioral Experiments

Three types of choice experiments using Y‐maze were conducted: a “kidney extract experiment” in 2020, 2021, and 2023, an “ultrafiltration fractionation experiment” in 2021, and a “nest water experiment” in 2021 and 2022. After separating the upstream and downstream sections of the Y‐maze with a wire mesh partition to prevent test fish from moving between them, test females were placed in the downstream section and allowed to acclimate for 10 min. The pump was then turned on to begin introducing the test solution, the wire mesh partition was removed by pulling it straight up with a rope, and the female's behavior in the tank was filmed for 15 min with a video camera (HDR‐CX700, Sony Corporation, Tokyo, Japan) placed directly above the tank. This procedure constituted one trial. After each trial, the Y‐maze was drained, washed with water using a sponge, rinsed, and then washed with ethanol and rinsed again. The side into which the test solution was injected was alternated between trials.

2.3.1. Kidney Extract Experiment

As test solutions, the control solution (saline: 0.8% sodium chloride solution), male kidney extract, and female kidney extract were used. To prepare kidney extracts, fish were anesthetized (Eugenol; approximately 5–10 μL/L; FUJIFILM Wako Pure Chemical Corporation), then the head and tail were cut off, and the cut surface was immersed in saline for a while to remove the blood. The abdomen was then opened, the kidneys were removed, weighed, and placed in micro test tubes. An appropriate amount of saline was added and homogenized using a plastic homogenizer pestle. The mixture was centrifuged (12,100×g; 15 min; 4°C), and the supernatant was used as kidney extract. The amount of saline was adjusted to a final kidney component concentration of 0.04 g kidney/mL saline. The tweezers and scissors used were washed and wiped with ethanol after each test solution was prepared for each fish. One mL of the prepared kidney extract (or control) was placed in a small cube water tank (15 cm × 15 cm, approximately 3.4 L capacity) and diluted to a final kidney concentration of 0.119 µg/mL.

This experiment provided data for both ovulated and non‐ovulated females. Experiments were performed on one ovulating female in the following order: control versus control, male kidney versus control, male kidney versus female kidney, and female kidney versus control. For this experiment, each experiment was repeated three times for each female, and the average value of the three experiments was used as the data for that female. Because female kidneys are smaller than males, there were cases where the test solution for female kidneys was insufficient. In such cases, only experiments using male kidneys were conducted.

2.3.2. Ultrafiltration Fractionation Experiment

Kidney extracts were prepared from the kidneys of several males using the method described above (0.04 g kidney/mL 0.85% saline). Five hundred µL of this extract was taken and centrifuged at 4000g at 4°C for 90 min using an ultrafiltration membrane with a molecular weight of 3000 (Microcon Centrifugal Filter, Ultracel YM‐3, Millipore Co. Ltd.), separating the supernatant (high‐molecular‐weight fraction with a molecular weight of 3000 or more) from the filtered fraction (low‐molecular‐weight fraction with a molecular weight of 3000 or less). Physiological saline was added to the high‐molecular‐weight fraction to make it equal to the low‐molecular‐weight fraction. One mL of each fraction was placed in a small aquarium (15 cm × 15 cm, 3.4 L capacity) and used as a test solution.

Experiments were performed on one ovulating female in the following order: high‐molecular‐weight fraction versus control, low‐molecular‐weight fraction versus control, and high‐molecular‐weight fraction versus low‐molecular‐weight fraction. Data from non‐ovulating females were not used. In cases where test solution was insufficient, only experiments for which test solution was available were performed. For this experiment, one trial was conducted with one female in each experimental group. The number of female individuals (trials) used was 19 for the high‐molecular‐weight fraction versus control, 19 for the low‐molecular‐weight fraction versus control, and 17 for the high‐molecular‐weight fraction versus low‐molecular‐weight fraction.

2.3.3. Nest Water Experiment

The rearing tank was observed daily, and any nests built by males were found and collected. If an ovulating female was present on the day the nest was discovered, an experiment was conducted. The nest was placed in a small tank (15 cm on each side, 3.4 L capacity), and the water was used as the test solution. A control group was used, in which only the same plant fragments as those placed in the rearing tank were placed in a small tank.

One trial was conducted for one ovulating female, in the order of control versus control and nest water versus control. Experiments were conducted on 19 ovulating females (19 trials). Data from non‐ovulating females were not used.

2.4. Video Analysis and Statistical Analysis

Using the video footage, the upstream branch point of the Y‐maze was used as the boundary, and the time spent in both upstream areas (staying time) was measured for 10 min, from 1 to 11 min after the pump was turned on. Mean values (±standard error) were calculated for each choice condition. Mean staying time was compared using the Wilcoxon signed‐rank test. A significance level of p < 0.05 indicated a significant difference.

3. Results

3.1. Kidney Extract Experiment (Figure 2)

Figure 2.

Figure 2

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Staying time of ovulating females (a) and non‐ovulating females (b) in the upstream area in an experiment using kidney extract as the test solution. Cont. indicates the control group (saline was used). N indicates the number of females used. ** indicates a significant difference (p < 0.05) in the Wilcoxon signed‐rank test.

In the experiments using ovulating females (Figure 2a), when the control solution was injected from both the left and right sides, the average staying time was 143.3 ± 36.2 s on the right and 196.1 ± 41.3 s on the left, with no significant difference observed (p = 0.980). In experiments using male kidney extract and control solution, the average staying time was 85.5 ± 23.2 s on the control solution side, and 320.0 ± 39.3 s on the male kidney side, with male kidney side significantly longer (p = 0.0005). In experiments using female kidney extract and male kidney extract, the average staying time was 121.8 ± 39.4 s on the female kidney side and 312.4 ± 49.5 s on the male kidney side, with the male kidney side being significantly longer (p = 0.044). In experiments using female kidney extract and control solution, the average staying time was 277.4 ± 60.0 s on the control side and 192.6 ± 57.1 s on the female kidney solution side, with no significant difference observed (p = 0.304).

In the experiments using non‐ovulating females (Figure 2b), when the control solution was injected from both the left and right sides, the average staying time was 244.9 ± 39.9 s on the right and 195.5 ± 44.5 s on the left, with no significant difference observed (p = 0.757). In experiments using control solution and male kidney extract, the average staying time was 211.3 ± 36.3 s on the control solution and 241.6 ± 34.3 s on the male kidney side, with no significant difference observed (p = 0.589). In experiments using female kidney extract and male kidney extract, the average staying time was 189.0 ± 37.2 s on the female kidney side and 260.3 ± 41.6 s on the male kidney side, with no significant difference observed (p = 0.339). In experiments using the control solution and female kidney extract, the average staying time was 244.7 ± 34.7 s on the control solution side and 217.8 ± 38.7 s on the female kidney side, with no significant difference observed (p = 0.765).

3.2. Ultrafiltration Fractionation Experiment (Figure 3)

Figure 3.

Figure 3

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Staying time of ovulating females in the upstream area in an experiment using a kidney extract fractionated by ultrafiltration as the test solution. Cont. indicates the control group (saline was used). N indicates the number of females used. ** indicates a significant difference (p < 0.05) in the Wilcoxon signed‐rank test.

In experiments using high‐molecular‐weight fractions and control solutions, the average staying time was 86.5 ± 29.4 s on the control solution side and 207.0 ± 50.1 s on the high‐molecular‐weight fraction side, which was significantly longer on the high‐molecular‐weight fraction side (p = 0.016). In experiments using low‐molecular‐weight fractions and control solutions, the average staying time was 102.7 ± 32.4 s on the control solution side and 48.7 ± 19.3 s on the low‐molecular‐weight fraction side, which was significantly shorter on the low‐molecular‐weight fraction side (p = 0.034). In experiments using high‐molecular‐weight fractions and low‐molecular‐weight fractions, the average staying time was 65.0 ± 27.5 s on the low‐molecular‐weight fraction side and 136.1 ± 41.9 s on the high‐molecular‐weight fraction side, which was not significantly different (p = 0.225), but the high‐molecular‐weight fraction side showed an average value twice as long.

3.3. Nest Water Experiment (Figure 4)

Figure 4.

Figure 4

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Staying time of ovulating females in the upstream area in an experiment using nest‐soaked water as the test solution. Cont. indicates the control group (saline was used). N indicates the number of females used. ** indicates a significant difference (p < 0.05) in the Wilcoxon signed‐rank test.

In experiments where a control solution was used on both the left and right sides, the average staying time was 52.2 ± 16.13 s on the right and 54.4 ± 14.48 s on the left, with no significant difference being observed. In experiments where nest water and control solution were used, the average staying time was 93.2 ± 19.15 s on the control solution side and 146.9 ± 23.73 s on the nest water side, with the nest water side being significantly longer (p = 0.029).

4. Discussion

In a Y‐maze choice experiment using kidney extract, ovulating females significantly spend longer time in the compartment containing male kidney extract than in the control or female kidney extract compartments. However, the female kidney extract had no effect on attracting ovulating females. This suggests that ovulating females are attracted not simply to the kidney odor of conspecifics, but to the kidney odor of males. The male kidney extract is thought to contain a large amount of spiggin, a component of nest material adhesion. Although blood components were washed away as much as possible when preparing the kidney extract, some blood‐derived components may still be present. It is known that male G. aculeatus have high levels of androgens in their blood during this period (Mayer et al. 1990; Jakobsson et al. 1999), and P. sinensis also has high levels of 11‐KT in its serum during the breeding season (our unpublished data). These androgens and other steroid hormones, prostaglandins, and their precursors and metabolites have been reported to act as pheromones to the opposite sex and affect reproductive behavior and physiology in over 10 orders of bony fish (see Stacey 2024). In P. sinensis, the possibility cannot be ruled out that blood‐borne steroids and prostaglandins may also have attracted females. Therefore, we conducted female choice experiments using the ultrafiltrate fraction of male kidney extract as the test solution.

A choice experiment was conducted using high‐molecular‐weight and low‐molecular‐weight fractions separated by ultrafiltration at a molecular weight of 3000. The high‐molecular‐weight fraction was more effective at attracting ovulating females than the control group. On the other hand, the low‐molecular‐weight fraction showed a repellent effect compared to the control group. G. aculeatus is known to be sensitive to chemical alarm signals contained in skin extracts of the same or closely related species (Brown and Godin 1997). The existence of low‐molecular‐weight alarm substances has been reported in zebrafish, Danio rerio (Masuda et al. 2024). It is possible that Pungitius also reacted to some alarm substance present in trace amounts in the low‐molecular‐weight fraction, which repelled them. Furthermore, when comparing the high‐molecular‐weight fraction with the low‐molecular‐weight fraction, the high‐molecular‐weight fraction showed an average residence time twice as long, but this difference was not statistically significant. This suggests that the component that attracts females is not a single substance, but rather a combination of multiple substances contained in both the high‐molecular‐weight and low‐molecular‐weight fractions. In other words, the different attractant components flowing separately from the upstream area to the left and right are mixed in the downstream area, exerting a strong attraction, stimulating females to stay in the downstream area, but then escaping to the upstream area with high‐molecular‐weight components in response to trace amounts of repellent components contained in the low‐molecular‐weight fraction. It cannot be denied that steroid hormones and their precursors/metabolites (molecular weight of about 500) that have pheromone activity in some fish, as well as eicosanoids such as prostaglandins (molecular weight of less than 400), may act as female attractants in their low‐molecular‐weight fractions. Meanwhile, proteins and peptides are likely to be the main female‐attracting components contained in the high‐molecular‐weight fraction.

Do the males attach female‐attracting components to their nests? Therefore, in this study, we collected nests built by males in stock tanks during the breeding season and conducted a choice experiment in which water from the tank containing only the nests was introduced into a Y‐maze. The results showed that females spent significantly longer in the water compartment containing the nests than in the water compartment containing only plant fragments. These results suggest that the attractant is applied to the nest, effectively luring females back to it. So, is the attractant the spiggin itself that binds the nesting material together? Spiggin is a glycoprotein derived from the mucin gene family (Kawahara and Nishida 2007). In Salaria pavo, a species of the Blenniidae, the anal gland and testicular glands of males develop in response to androgen, and it has been suggested that the neutral mucins and sialomucins secreted from them function as olfactory stimuli to attract females (Serrano et al. 2008). It is quite possible that mucin‐derived spiggins have also evolved to attract females in the Gastrosteidae. While the possibility that the attractant is a substance other than spiggin cannot be ruled out (Figure 5; hypothesis‐1), in that case, it is thought that the attractant is coated on the nest along with spiggin and gradually dissolves from the nest to exert its attractive effect. The attractive effect of attractants applied to nests may weaken over time. In the present study, we were able to demonstrate the attractiveness of nests to females by using them in attraction experiments as soon as we found them. In preliminary experiments, we collected and froze the nests, then thawed them and performed the same experiment, but no female attraction was observed (data not shown). The frequent nest‐strengthening behavior of males during the breeding season (Morris 1958; Fukui 1962) may not only be for structural reinforcement but also serve to sustain the attractive effect. This is consistent with the possibility that the attractant is spiggin itself.

Figure 5.

Figure 5

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Hypothesis regarding the functional evolution of female attractants and nesting material adhesives (spiggin) based on knowledge of the phylogenetic relationships of Gasterosteoidei (modified from Kawahara et al. 2009) and the reproductive habits of each genus (see main text). Illustrations of spawning sites are from: Hypoptychus, Akagawa and Okiyama 1993; Aulichthys, Akagawa et al. 2008; Aulorhynchus, Limbaugh 1962; Spinachia, Östlund‐Nilsson 2000; Apeltes, Rowland 1974; Culaea, McKenzie 1969; Pungitius, Morris 1958; Gasterosteus, Tinbergen 1952.

Males of Pungitius have been observed fanning toward the nest after building it, even though they have not laid any eggs (Morris 1958), which is likely a way of dispersing attractants attached to the nests. Pungitius and Gasterosteus live in flowing rivers (Wootton 1976), and in the case of Gasterosteus, males in good condition settle and nest further upstream and have higher reproductive success (Kraak 1999), suggesting that upstream nests chemically attract downstream females.

This is the first study to directly demonstrate that male secretions attract females in the Gasterosteidae family, but several previous studies have indirectly demonstrated this. It has been shown that females of S. spinachia prefer males with nests heavily smeared with spiggin (Östlund‐Nilsson 2001), and that under visual deprivation, females choose water containing males and nests over unconditioned water (Östlund 1995). These findings can be interpreted as females of S. spinachia also responding to the scent of nest‐attached substance. It has been reported that females of G. aculeatus and Culaea inconstans recognize each other's male odors and preferentially identify their own males (Mclennan 2003). Furthermore, attraction experiments with C. inconstans showed that ovulating females were strongly attracted to the odor of water that had been conditioned by multiple males for 24 h (Mclennan 2005). This result makes sense if we consider that the substances that attract females, produced in the male′s kidneys, are released in the urine. From the above, it can be said that the property of males producing olfactory stimulants in the kidney that attract females is common among Gasterosteidae.

The present study suggests that in P. sinensis, non‐ovulating females do not respond to the attractant cues emitted by males. Previously, it was shown that the attraction of females to male odors in C. inconstans increases with ovulation (Mclennan 2005). Considering that females are attracted to male nests to spawn eggs, the difference in responsiveness between those who are ovulating and those who are not is reasonable, suggesting that endocrine changes that occur during final oocyte maturation and ovulation in females play an important role in their detection of attractants released by males, or in the behavioral changes that occur after reception. In many teleost species, final oocyte maturation is triggered by a surge of luteinizing hormone secreted from the pituitary and the subsequent secretion of maturation‐inducing hormone (17α,20β‐dihydroxy‐4‐pregnen‐3‐one; 17α,20β‐DP) from follicle cells in the ovary (Nagahama and Yamashita 2008). In breeding female G. aculeatus, 17α,20β‐DP has been detected in the blood at very low concentrations of 1‐2 ng/ml (Borg and Mayer 1995). However, it is not always present at high concentrations during the breeding season, and it is reasonable to assume that the blood concentration of 17α,20β‐DP rises sharply only at the time of final oocyte maturation and ovulation, followed by a rapid decrease (e.g., Stacey et al. 1989). It is quite possible that the secretion of 17α,20β‐DP during final oocyte maturation may directly or indirectly affect the receptivity to attractants released by males.

“Nest” is defined as a breeding style in which males modify habitats by constructing new structures or modifying existing structures to establish breeding territories and attract females to lay eggs in order to increase their reproductive fitness (Bessa et al. 2021). This type of nest‐based breeding has been observed in several species across 10 families, including Gasterosteidae (Bessa et al. 2021) (11 families including Cottidae), and is thought to have evolved polyphyletically. Among these, the possibility that males attract females to the nest by olfactory stimulation has been suggested in Gobiidae (Colombo et al. 1980; Murphy et al. 2001), Blenniidae (Serrano et al. 2008), and Cottidae (Katsel et al. 1992; Goto 1994; Koya et al. 2016b). In nesting species, males have territories for breeding, and territorial behavior is thought to have two aspects: attachment to the nest and aggression against intruders (Wootton 1976). In other words, because males cannot leave the nest to protect it, it is thought that a method of attracting females to the nest using olfactory stimuli has evolved. In Gasterosteidae, did adhesive substances come to function as female attractants as nest‐based reproduction evolved? Or did substances that originally functioned as attractants evolve into adhesive substances?

Figure 5 shows the phylogenetic relationships of Gasterosteoidei (Kawahara et al. 2009) and the reproductive ecology (breeding territories and nest building) of each species. Hypoptychus, which is thought to have branched off from other groups early within Gasterosteoidei, does not build nests but engages in territorial breeding (Akagawa and Okiyama 1993), suggesting that this genera may have already employed chemical attractants for females. Aulorhynchus, which is a sister to Gasterosteidae, is known to not only establish territorial boundaries for breeding but also build very simple nests made of seaweed bound together with mucus threads excreted from its urogenital opening (Limbaugh 1962). It is thought that the Gasterosteidae, which build more elaborate nests using more adhesive substances, branched off from this ancestral species. This suggests that Gasterosteoidei is a territorial breeding species, and that males likely possessed the ability to chemically attract females before acquiring the ability to build sticky nests.

Spiggin belongs to the MUC19 group of secreted mucins (Kawahara and Nishida 2007). MUC19 is present in vertebrates other than fish, and its gene expression has been confirmed in the kidneys of zebrafish (Lang et al. 2016), suggesting that it is a glycoprotein that is universally produced in the kidneys of many fish species. Mucin secreted by the kidneys may function as a female attractant during reproduction in some territorial fish species (Figure 5). In Cottus sp. SE, it is believed that during the breeding season, the male kidneys produce a polysaccharide‐containing compound, which causes a large amount of viscous urine to accumulate in the bladder, and that this substance attracts females (Koya et al. 2016a, 2016b). In Gasterosteidae, five spiggin genes exist due to gene duplication, and they show diversity in length and similarity (Kawahara and Nishida 2007). This suggests that mucins with enhanced nest‐material adhesion capabilities may have evolved from kidney mucin with female‐attracting properties (Figure 5; hypothesis 2). To prove this hypothesis, it is necessary to investigate the expression of specific mucin genes in the kidneys of male Hypoptychus, which does not build nests, during the breeding season, as well as the female‐attracting properties of renal secretions.

5. Conclusion

The present study, for the first time in the Gasterosteidae, demonstrated through behavioral experiments the existence of olfactory stimuli for mating. Ovulating females of P. sinensis are chemically attracted to a high‐molecular‐weight substance produced by male's kidney. This substance was also found to be applied to nests made by gluing together plant fragments, suggesting that the nests themselves attract females. This chemical attraction of females by males may be a common phenomenon among territorial breeding males. At least in the Gasterosteidae, female attraction may have occurred before the evolution of nest‐building habits using plant fragments, suggesting that the attractant component may be a type of mucin that is the precursor to spiggin, or it may be spiggin itself. In other words, spiggin is thought to be a substance that evolved functionally from a female‐attracting substance.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

This study was financially supported by JSPS KAKENHI (grant numbers 17K07906 and 21K05780).

Data Availability Statement

Data will be made available on request.

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