Paternal help in larval release of three mudskipper species

Abstract

After the discovery of aerial embryonic incubation in mudskippers, it was found that the male induces larval hatching by removing the air from the egg-chamber so that the eggs immerse in water. Do the adults provide additional support to the newly hatched larvae to help them leave the life-endangering hypoxic water inside the burrow? This paper compares 18 hatching events of 3 species: dusky gilled mudskipper, Pearse´s mudskipper and slender mudskipper. After air removal 3 different burrow-shape dependent strategies of paternal help for larval burrow leaving were observed: (1) very frequent diving toward the burrow shaft or into the egg-chamber to generate turbulences that can help to propel the larvae toward the water surface; (2) tail-undulation to create a water current out of the burrow that would take along the hatched larvae; and (3) intake of larvae into the paternal mouth, subsequently spitting them out to expel them from the burrow.

KEYWORDS:

• Mudskipper
• hatching
• larval burrow leaving
• parental care
• tail-undulation behavior
• larvae-expelling behavior

Mudskippers are tropical and subtropical fish naturally living in the inter-tidal zones of the Indo-West-Pacific and eastern Atlantic. Even though they routinely move about in inter-tidal habitats, they also spend time in burrows filled with hypoxic water. They even spawn inside self-dug burrows and take care of their brood there. Environmental pressure caused them to develop morphological, physiological, and behavioral adaptations to habitats that are unusual for fish (e.g. Clayton 1993; Sayer 2005; Polgar 2014; Parenti and Jaafar 2017; Kumaraguru et al. 2020).

Mudskipper reproduction

Air storage in mudskipper burrows was first described in 1998 (Ishimatsu et al. 1998) and was later confirmed again and again by a series of studies (Hong et al. 2007; Ishimatsu et al. 2007, 2009; Toba and Ishimatsu 2014; Rupp 2021). This allows the development of mudskipper embryos in hypoxic water. Furthermore, it was shown that male removes the air from the egg chamber after the completion of embryonic development. Male transports egg-chamber air in its mouth and expels it into the burrow shaft (Ishimatsu et al. 2007; Rupp 2021). This increases the water level in the chamber and immerses the eggs in water, thus inducing larval hatching. In barred mudskippers (Periophthalmus argentilineatus), the larvae free themselves very quickly, most of them hatching in the first 10 s after immersion (Brillet 1976). This process normally takes place during nocturnal high tides. At that time, the burrow entrances are covered with water, which allows the larvae to exit the burrow. Etou et al. (2007) showed that the survival time of freshly hatched larvae of shuttles hoppfish (Periophthalmus modestus) declines rapidly in hypoxic water. At dissolved oxygen levels of 10% saturation, all larvae died within 1 h. Therefore, it is essential that larvae leave the burrow as quickly as possible. However, when larval hatching is artificially induced in the burrow after removing the male, only a few larvae find their way out of the burrow (Ishimatsu et al. 2007). This raises the question of how freshly hatched larvae find their way out of the burrow as quickly as possible under natural conditions (Ishimatsu et al. 2007; Ishimatsu and Graham 2011). This is all the more significant because, with the exception of the plainfin midshipman (Porichthys notatus) and possibly its relatives, post-hatching care in intertidal fish is not known (Coleman 1999). In 2021, it was shown that the dusky gilled mudskipper (Periophthalmus variabilis) sometimes used tail-undulation to create strong water currents, causing water to flow out of the burrow – which is likely to help the larvae leave the burrow (Rupp 2021). Tail-undulation behavior was only observed during two of several hatching events but also at some high tides before hatching, so that the significance of this behavior remained uncertain. The objective of this study is therefore to answer one of the ‘key questions’ (Ishimatsu et al. 2007; Ishimatsu and Graham 2011; Martin 2015; Martin and Ishimatsu 2017): Are mudskipper parents indeed routinely involved in larval release from the burrow?

Species studied

For this purpose, two additional mudskipper species were studied: the Pearse’s mudskipper (Periophthalmus novemradiatus) and the slender mudskipper (Periophthalmus gracilis). Similar to the dusky gilled mudskipper, on whose brood care behavior video material was already available (Rupp 2021), these two species do not grow too large, which makes them suitable for keeping in aquaria (Mleczko and Rupp 2017). The maximum recorded length of a Pearse’s mudskipper is 51 mm SL. Its habitat is the Bay of Bengal, from East India to southern Thailand (Polgar 2014; Murdy & Jaafar 2017; Parenti and Jaafar 2017). Since their males have a greatly elongated first spine of a more reddish D1 than the female, the two sexes can be easily distinguished. So far, their reproductive cycle has not been described in the literature (Polgar 2014; Martin and Ishimatsu 2017).

The slender mudskipper is even smaller, with a maximum recorded length of 45 mm. Its habitat is the Indo-Pacific region, from Sumatra to the Philippines and Queensland, Australia (Polgar 2014; Murdy & Jaafar 2017; Parenti and Jaafar 2017). In mature males, D1 is medially yellow, and D2 has a yellowish to reddish margin. So far, no study on the reproductive behavior of this species has been published (Polgar 2014; Martin and Ishimatsu 2017).

The previously observed dusky gilled mudskipper is the largest of the three mudskipper species studied, with a maximum length of 65 mm SL. Its habitat is South-East Asia, from the Malacca Straits to the Sulu Sea (Thailand, Malaysia, Vietnam, and Indonesia) (Polgar 2014; Murdy & Jaafar 2017; Parenti and Jaafar 2017). Mature females are stockier and a somewhat paler than their male counterparts (Rupp 2021). Regarding their reproductive behavior, it was observed that spawning took place in an egg chamber filled with water and not with air (Rupp 2021). This is noteworthy in as much as it contradicts speculations by Ishimatsu et al. (2007); Ishimatsu and Graham (2011); and Toba and Ishimatsu (2014), who assumed that spawning takes place in air-filled chambers. Since Ishimatsu et al. (2009), several authors (Ishimatsu et al. 2009, 2018; Martin and Ishimatsu 2017; Mai et al. 2020) had already doubted the reliability of this speculation.

Tide simulation

Two male and eight female dusky gilled mudskippers were purchased from an aquaristics shop in Salzgitter on 25 October 2012. In addition, 12 Pearse’s mudskippers of both sexes from India and three male and five female slender mudskippers from West-Java were provided by a wholesaler in Dietzenbach on 17 February 2021 and 27 March 2021, respectively. The different mudskipper species were kept separately in three experimentation paludaria (80 cm × 50 cm × 40 cm). The tidal setups originally designed for keeping mudskippers or fiddler crabs (Rupp 2013, 2014, 2015a, 2015b; Mleczko and Rupp 2017) were adapted to the objective of this study (Fig. S1, Table 1): Each tank was equipped with a customized tidal system. It provided dusky gilled mudskippers with about 16 h of low tide (LT I and LT II) and about 8 h of high tide (HT I and HT II) while offering both other species about 14 h of low tide (LT I and LT II) and about 10 h of high tide (HT I and HT II). An overflow marked the high-tide level and kept the water in motion, preventing microfilm development on the water surface. Low tide prevailed for 8 and 7 h, respectively, while a second outlet was opened every 12 h, using an electrically powered ball valve connected to an auto timer. Low tide in the experimentation tank thus automatically induced high tide in a basin below. Each tidal change took about 25 min. Before the water flowed into the basin below the tank, it was conducted into a basin with a constant water level where a plankton net trapped any hatched larvae. The number of larvae of the dusky gilled mudskipper was estimated before they were transferred directly into a kreisel-tank for rearing experiments (Rupp 2014, 2015a, 2015b; Mleczko and Rupp 2017). In most cases, the larvae of the slender mudskipper and the Pearse’s mudskipper were first spread out in a Petri dish and photographed to accurately determine their numbers before they were also used for rearing experiments. A mud slope (10–25 cm high) was built inside the tanks using natural mud from a North Sea beach. During low tide, the water level in the tank was 10 cm. During high tide, the water level rose to about 30 cm, covering the entire slope, leaving its peak 5 cm below the surface. Parts of roots and two bamboo sticks (only in the case of dusky gilled mudskippers) offered the mudskippers a place to sit above the water surface during high tide. In dusky gilled mudskippers, neap tides were experimented with during five breeding cycles: 1 week of normal tides (with two low and two high tides per day) alternated with 1 week without high tides. In slender mudskippers, neap and spring tides were experimented with during one cycle. One day with neap tide alternated with 1 day with spring tide.

Table 1. Experimental design.

	P. variabilis	P. novemradiatus	P. gracilis
Tides: LT I	02:00–10:00	04:00–11:00
HT I	10:00–14:00	11:00–16:00
LT II	14:00–22:00	16:00–23:00
HT II	22:00–02:00	23:00–04:00
Salinity	15 ppt
Temperature: daytime At night	26°C 24°C
Light/dark cycle: L	09:15–22:45	08:00–22:00
D	22:45–09:15	22:00–08:00
Food	Wet-frozen bloodworms and Mysis; live brine shrimps and Drosophila flies
Video-recording time span per day	09:15–22:45 (three times: 24 h)	24 h
Total time of observation	17 March 2013 –	1 November 2021 –	4 January 2022 –
	13 December 2015	30 September 2022	30 September 2022

Water parameters

The required brackish water (salinity: 15 ppt, measured with a refractometer; pH 8.4 measured by colorimetric analysis) was prepared by mixing artificial sea salt and tap water. An electrical heater inside the basin below the tank heated the water to 26°C during daytime and 24°C at night. The water was continuously pumped into the tank. The top of the tank was open so as to enable video recording. Therefore, the air temperature corresponded to the room temperature (about 22°C in the winter and up to 25°C in the summer). Water was only sponge-filtered and evaporated water was replaced once a week. When the sponge filter was clogged, the water level rose slower than normal, generating high tide.

Food and feeding

Since all species of the genera Periophthalmus and Periophthalmodon are opportunistic carnivores, although some species also ingest some algae as well (Polgar 2010; Clayton 2017; Hidayat et al. 2022), the mudskippers were fed once a day or every other day with wet-frozen bloodworms and Mysis, also occasionally with live Drosophila flies and live brine shrimps. Feeding time was in the evening during low tide. In some species, and within some populations, seasonal spezialisation may exist presumably a response to changes in prey availability (Polgar 2010). Under aquarium conditions, mudskippers have proven to be very adaptable, although small live foods such as flies and spiders are clearly favored. Once settled, wet-frozen foods and even dry pellets are readily accepted (Schäfer 2005; Mleczko 2006; Polgar 2010).

Lighting conditions and filming

Artificial light/dark cycles equaled 13.5 and 10.5 h, respectively, in dusky gilled mudskippers. During the day, a fluorescent tube (Lumilux 18 W/6500K Cool Daylight) with 1300 lm was used. For the dusky gilled mudskippers, there was no light at night (with only a few exceptions). In Pearse’s mudskippers and slender mudskippers, artificial light/dark cycles equaled 14 and 10 h, respectively. The same type of fluorescent tubes was used during the day. At night, one LED (0.3 W/2700 K) with 12 lm was used to enable filming. All tanks were encased with black cardboard to minimize visual disturbance for the mudskippers. In dusky gilled mudskippers, a Panasonic HC-V510 digital video cam recorder mounted over the tank was used only during daylight times. Three nocturnal high tides were also video-recorded. In Pearse’s mudskippers and in slender mudskippers, the cam recorders were running 24/7 (Table 1). Due to technical problems and excessive turbidity caused by the tail-undulation behavior of the male Pearse’s mudskippers, a small part of the video footage is missing. All film material was sifted at 8× speed. The behavior of the animals during the hatching event at high tide was analyzed in detail. Furthermore, plaster casts were made of some burrows of each mudskipper species.

Mudskippers´contact with their burrow

All 18 hatching events occurred during high tide. Fifteen happened at night, with only three (one in each species) during daylight. Throughout these complete high tide periods, the length of ‘burrow touch’, ‘burrow residence’, and single ‘diving phases’ was determined. In this study, the term ‘diving phases’ describes the incidents of a male being completely submerged inside the burrow entrance or even diving either toward the burrow shaft or deeper into the egg chamber. A ‘burrow residence’ is defined as instances when a mudskipper was either diving or just sitting inside the burrow entrance with its head (or head and pectoral fins) resting on the edge of the entrance opening. A ‘burrow touch’ covers the diving and residence phases and also includes a mudskipper’s physical contact with one of the burrow entrance openings (at least from the outside) or with places very nearby. These differentiations are useful in dusky gilled mudskippers and slender mudskippers, but not in Pearse’s mudskippers due to the special construction of the T-shaped upper part of their burrow: Most of the time the openings were still sealed until hatching occurred. When the entrances were finally opened to prepare air-removal behavior, the male stayed submerged inside the burrow. Sometimes, the male looked out of one opening; very occasionally it left the burrow for a while, but it never stayed exclusively in contact with the burrow entrances from the outside or touched places very nearby to sit.

Air adding and air removal

If air-adding behavior occurred during the hatching event, the length of the period was determined. Especially during low tide, this behavior could be observed in dusky gilled mudskippers and slender mudskippers throughout the entire breeding cycle after the female left the burrow for the last time: The male gulped mouthfuls of air and vigorously dived down into the burrow with a strong flick of the tail (dusky gilled mudskipper) (Video S1). In slender mudskippers, the diameter of the burrow entrance was very small. During high tide, the male therefore gulped a mouthful of air and sank backward down the burrow entrance area towards the burrow shaft. Then, it turned around and vigorously dived down with a strong flick of the tail. If the water level above the entrance was high, the male had to leave the burrow completely, gulp air, and enter the burrow again (Video S1). Air-adding behavior of the male Pearse’s mudskipper (if it happened) could not be filmed as the burrow entrance was closed and the mudskipper stayed inside most of the time.

The length of both, air-removal phases and the complete air-removal periods was determined for all three species.

Tail undulation

Tail-undulation behavior was also observed in all three species: The males dived into one of the two burrow entrances and waved their tails vigorously back and forth. While doing so, they stayed turned upside down in the burrow entrance area holding this position with their pelvic and pectoral fins. This action created a strong water current flowing out of the burrow entrance in which the male was sitting, while at the same time, freshwater streamed into the other burrow entrance (Fig. S3a-c; Video S2-4). At the start of the tail-waving action, the water flowing out of the burrow was very turbid but gradually became clearer in dusky gilled mudskippers and slender mudskippers. In Pearse’s mudskippers, the water remained murky during all tail-undulation phases. In dusky gilled mudskippers, it was often possible to see the caudal fin during this action. In both other species, tail-undulation behavior mostly occurred deeper inside the burrow so that only the water flowing out of one burrow entrance (Pearse’s mudskipper), or sometimes out of both entrances (slender mudskipper) was observed.

Larvae spitting

In slender mudskippers, the intake of the larvae into the paternal mouth took place inside the burrow, but the subsequent larvae expulsion from the burrow could be filmed in two hatching events (Video S5).

The role of females

In all three species, the female was not relevant for hatching or larval release. Only twice a short ‘burrow touch’ of the female was observed during the relevant high tide.

The number of larvae

Similar to the observations of hatching events of barred mudskippers and shuttles hoppfish (Maeda et al. 2017), the number of hatched larvae has a wide range (Table 2), especially in the case of slender mudskippers, and shows no obvious correlation with the type of paternal helping strategy in larval release.

Table 2. Hatching events in three mudskipper species.

Species/ Setting ♂,♀ (Date of hatching)	Burrow touch [% of high-tide time span]	Burrow residence [% of high-tide time span]	Single diving phases		Air-adding period [% of high-tide time span]	Air-removal period [% of high-tide time span]	Tail-undulation period [% of high-tide time span]	Number of the larvae/Larvae expelling
			Number (total/after air removal period)	Ø Duration after air removal period
P. v. /1,1 (08/ 9 February 2015)	96.90	95.24	100/68	0,79 min	10.40	7.04	0	>500/-
P. v. /1,1 (22/ 23 February 2015)	62,26	52,74	70/46	0,59 min	4.87	3.57	0	>500/-
P. v. /2,2 (07/ 8 February 2014)	100	100	9/1	69.03 min	4.36	10.24	84.91	>500/-
P. v. /1,1 (4 July 2014)	100	100	1/1	250.2 min	0	20.10	94.45	>500/-
P. n. /1,1 (14 November 2021)	76.53		23/6	12 min	?	11.05	16.23	?/-
P. n. /1,1 (01/ 2 December 2021)	81.70		5/4	49,54 min	0	10.61	42.84	1379/-
P. n. /1,2 (28/ 29 March 2022)	57.08		1/1	80.67 min	?	5.00	10.09	635/-
P. n. /1,2 (8/ 9 May 2022)	≥45.98		1/1	≥84.93 min	?	5.83	21.45	804/-
P. n. /1,2 (14/ 15 August 2022)	≥57.89		1/1	≥114.68 min	?	9.21	45.17	1667/-
P. n. /1,2 (05/ 6 September 2022)	96.58		1/1	239.02 min	0	4.98	70.70	1088/-
P. n. /1,2 (21/ 22 September 2022)	87.53		3/2	88.56 min	0	22.08	29.27	1607/-
P. g. /2,2 (20/ 21 February 2022)	83.36	35.17	10/7	7.02 min	0	4.13	15.26	164/-
P. g. /2,2 (23/ 24 March 2022)	89.29	82.91	34/1	16.88 min	33.5	6.66 + 3.69	1.35	?/-
P. g. /2,2 (05/ 6 April 2022)	99.58	99.58	5/5	45.57 min	0	3.49	88.96	2940/-
P. g. /2,3 (08/ 9 May 2022)	41.66	35.40	2/2	27.43 min	0	9.64	23.72	?/-
P. g. /2,3 (07/ 8 June 2022)	95.03	95.03	1/1	214.92 min	0	1.00	83.43	2788/-
P. g. /2,2 (26 January 2022)	73.78	50.43	120/60	0,67 min	30.18	1.82 + 2.96	0	292/✓
P. g. /1,2 (24/ 25 July 2022)	98.52	96.93	147/45	1,89 min	56.72	3.17	10.49	3396/✓

Paternal help in dusky gilled mudskipper

Video footage of four hatching events (Figure 1a-d, Table 2, Video S2) addresses the question of whether parent animals help freshly hatched larvae leave the burrow. In two of these recordings, a short air-adding period was observed after the burrow entrances were flooded by the evening high tide. The male then began to remove egg-chamber air interrupting the activity several times to resurface. Large bubbles and extremely turbid water flowed out of the burrow. In the first hatching event (Figure 1a), the first larvae was seen on the water surface 24 min after the male began removing egg-chamber air; in the second (Figure 1b), it took 11 min. After removing the air, the male still undertook very frequent short dives into the burrow. The frequency of diving decreased with time, while the length of each diving phase tended to increase.

Figure 1. Four hatching events in dusky gilled mudskippers.

The third (Figure 1c) and fourth (Figure 1d) hatching events showed another behavior: Between the single air-removal phases the male never resurfaced. Tail-undulation behavior happened before and after (Figure 1c), or before, after, and during the removal of the egg chamber air (Figure 1d). Simultaneously, the number of diving phases after air-removal decreased sharply (1 ± 0) compared to the first and second hatching event (57 ± 11). On the other hand, the total length of the diving phases increased strongly from 0.69 ± 0.1 min to 159.62 ± 90.58 min. Unlike hatching events #1–3, the fourth occurred during the morning high tide after a prolonged period of artificial neap tides during which the burrow entrances were never inundated. In addition, there was no air-adding period. In all four hatching events, the number of larvae was well over 500.

Paternal help in Pearse’s mudskipper

Footage of seven hatching events is available (Figure 2a-g, Table 2, Video S3). In contrast to the dusky gilled mudskippers, these videos showed tail-undulation during all hatching events. Normally, this behavior occurred only after air removal, but in the fifth hatching event (Figure 2e), it was also used prior to air removal. Air-removal phases were usually observed in the first third of the high tide. There was only one exception (Figure 2a): in this case, individual air-removal phases were sometimes interrupted by leaving the burrow. Overall, the number of single diving phases after air removal was minimal (2.29 ± 1.83). The length was 107.61 ± 72.18 min. Sometimes, tail-undulation behavior was so intense that the resulting turbidity of the water made it impossible to observe the male leaving the burrow (Figure 2d-e). The number of larvae per trap ranged from 635 to 1667 (Table 2).

Figure 2. Seven hatching events in Pearse’s mudskippers.

Paternal help in slender mudskipper

Footage of seven hatching events is available (Figure 3a–g, Table 2). The first five show tail-undulation behavior (Video S4). With one exception (Figure 3b), air-removal phases could be observed in the first third of the high tide. This exceptional case is also the only one where air addition prior to the air-removal period was observed. This case featured two more exceptions: two air-removal periods instead of only one, and tail undulation only after air removal. The other four show undulation before and after air removal. The sixth (Figure 3f) hatching event, which occurred by daylight, and the seventh (Figure 3g) showed another behavior: In both cases, air adding was observed – in the first of the two events even twice between two air-removal periods.

Figure 3. Seven hatching events in slender mudskippers.

Particularly, noteworthy is the occurrence of the intake of larvae into the paternal mouth inside the burrow and their subsequent expelling out of the burrow after removing the egg chamber air (Video S5). Simultaneously, there was a steep increase in the number of diving phases after air removal (52.5 ± 7.5) compared to the other five hatching events mentioned above (3.2 ± 2.4). The total length, however, strongly decreased (1.39 ± 0.5 min instead of 67.85 ± 75.63 min). In the last hatching event (Figure 3g), a short tail-undulation behavior was also observed. The number of larvae per trap ranged from 164 to 3396 (Table 2).

Supporting type and burrow shape

The choice of helping strategy after the air-removal period seems – possibly among other factors – to depend on the shape of the burrow and its entrances:

Dusky gilled mudskippers inhabited Y-shaped burrows with two funnel-shaped turreted entrances, often – but not always – featuring very large diameters in comparison to the body size of the male (Fig. S2a). This alone might have facilitated the orientation of the positive phototactic larvae (Brillet 1976; Maeda et al. 2017) toward the exits, especially in moonlight even if the water is turbid. Additionally, in this case, one of the two alternative helping strategies of the male after air removal was observed: very frequent diving toward the burrow shaft or deeper into the egg chamber. These short diving phases, which are similar to those during the air-adding period, generated very strong turbulences that could lift the larvae to the water surface (Video S2). The other strategy found in dusky gilled mudskippers seems to be tail-undulation behavior, which was observed during only two hatching events. In these cases, the diameter of the entrance openings was small (Fig. S2b) so that frequent diving alone would not suffice to lift the larvae to the water surface. According to Brillet (1975), tail-undulation behavior could serve to flush the burrow, a phenomenon that was also observed outside of hatching events (Rupp 2021). In both the flushing and hatching events, water flowed out of the burrow entrance in which the male was sitting, while freshwater streamed into the other burrow entrance (Fig S3a). During the hatching event, the water current out of the burrow entrance in which the male was sitting helped to either wash the larvae out of the burrow passively or make them swim actively against the water current – depending on whether the mudskipper larvae were positive or negative rheotactic (which is not known). Tail undulation occurred not only after but also prior or even during air-removal behavior, so the increase of both oxygen content and water clarity at least in the upper part of the Y-shaped burrow also helped the larvae to find their way out of the burrow (Video S2, S5).

In Pearse’s mudskippers, unlike the dusky gilled mudskippers, tail undulation behavior was observed in all cases (Video S3) as the only helping strategy during hatching events. Tail undulation was never used for other purposes or at other times. Whether there was air-adding behavior prior to air removal cannot be answered for four hatching events (Figure 2a, c-e): Unlike the dusky gilled mudskippers and slender mudskippers, the Pearse’s mudskippers male often enclosed itself in the burrow during high tide and stored air inside the upper T-shaped part of the burrow. It is therefore possible that the male also showed air-adding behavior during high tide, before the hatching occurred using enclosed air, and then starting air removal when the entrance was opened. In three hatching events during high tides, however, at least one entrance was open during the entire period, so no enclosed air could be used by the male. In these cases, air-adding behavior was not observed (Figure 2 b–f, g). The male showed tail-undulation behavior only after both entrances had been opened and mostly only after the subsequent air-removal period was completed (Figure 2 a-d, f, g). Only once this behavior could also be observed before air removal (Figure 2 e). Tail undulation behavior appears to be the most effective method in this mudskipper species to help the larvae exit this particular burrow type. Due to the small diameter of the T-shaped upper part of the burrow, tail undulation created very strong water currents that could carry the larvae along (Fig. S3c). Repeated back-and-forth swimming at high speed within the T-shaped upper part of the burrow also seems to be very effective: Since the diameter of the tubes was very small, this behavior caused water to pulse out of the opening each time in front of the male.

Slender mudskippers built Y-shaped burrows with turreted entrances similar to dusky gilled mudskippers. However, there were two crucial differences: (1) the entrances were not funnel-shaped, and their diameter was always very small (Fig. S2c) which made it energy-intensive for the male to enter the burrow during high tide. The height of the water level above the entrances therefore made all the difference for air adding: either the male could stay inside the burrow, gulping air to bring it into the egg chamber, or it had to leave the burrow completely for air-gulping (Video S1). In the second hatching event (Figure 3b), with water levels high above the burrow entrances, the air-adding period lasted 103.52 min with only 19 diving phases. In the sixth (Figure 3f) and seventh (Figure 3g) event, water levels above the burrow entrances were low. As a result, air-adding took 88.13 min with 48 diving phases and 159.38 min with 98 diving phases, respectively.

(2) The distance between the two openings was very different in the individual burrows which may have had an influence on the type of helping strategy used. Just as in dusky gilled mudskippers, two strategies were observed to help the larvae leave the burrow. Strategy #1: four hatching events in burrows with more widely spaced openings (Figure 3 a, c-e) showed tail-undulation behavior before and after air removal with the implications described above (Video S4). The second hatching event (Figure 3b) with two air-removal periods showed short tail-undulation behavior only after air removal. Unlike the dusky gilled mudskipper, but just like the Pearse’s mudskipper, tail undulation was never observed outside the hatching events. Strategy #2: In two hatching events that occurred in burrows with closely spaced openings (Figure 3f, g), another helping strategy was detected: after air-adding and air-removal behaviors, the male dived very frequently and for a short time toward the burrow shaft or even into the egg chamber, generating turbulences that can lift the larvae into the upper part of the burrow. Due to the small diameter of the entrances, this was, however, not enough to lift the larvae to the water surface as described in the dusky gilled mudskippers. Instead, the male took larvae in its mouth inside the burrow and expelled them out of the burrow (Video S5). In the last hatching event, this behavior was completed with a short tail-undulation behavior. As was to be expected, the number of diving phases in this case was lower and their length somewhat longer. The comparison of 16 hatching events without larvae-expelling behavior shows that the occurrence of tail-undulation behavior is related to a small number (2.43 ± 2.14) of long-duration diving phases (≥53.84 ± 78.12 min). In contrast, the absence of tail-undulation behavior is related to a large number (57 ± 15.56) of short diving phases (0.71 ± 0.74 min) (Table 2). It can be concluded that tail-undulation behavior and frequent diving are two alternative paternal helping strategies for larval release.

The two hatching events with larvae expelling tend to show a similar relationship between the occurrence of tail-undulation behavior and the number and duration of diving phases: with tail-undulation behavior, 45 diving phases of 1.89 ± 3.46 min duration were observed, while 60 diving phases of 0.67 ± 0.53 min duration were seen without tail-undulation behavior. This higher number of relatively short diving phases despite tail-undulation is likely to be due to the fact that, in this particular case, tail undulation occurred only long after hatching (65.75 min) – and this is unlike all other hatching events (12.92 ± 7.54 min) in which it was observed.

Resume and outlook

The question of how freshly hatched larvae find their way out of the burrow as quickly as possible before suffocating in the hypoxic water (Ishimatsu et al. 2007; Ishimatsu and Graham 2011) came up in experiments with artificially induced larval hatching after removing the male in shuttles hoppfish. Only a few larvae found their way out of the burrow (Ishimatsu et al. 2007). The observation of 18 hatching events in three different mudskipper species now shows that this had happened most probably due to the lack of paternal help because in no case did the male leave the burrow immediately after air removal. On the contrary: the male stayed in the burrow for a long time after hatching. Over this time span, at least three strategies helped the larvae to find their way out of the burrow. This is all the more significant because, with the exception of the plainfin midshipman and possibly its relatives, post-hatching care was previously unknown in intertidal fish (Coleman 1999). Apart from the different helping strategies (which are discussed above), the oxygen level of the burrow water seems to play an important role in the process. Air adding – if it occurs – is likely to optimize the oxygen level not only of the egg chamber air but also of the water in the burrow: again and again, air pockets bubbled out of the egg chamber through the burrow shaft (Video S1) when the air bell in the chamber grew too large. In that way, the water inside the burrow was not only swirled but is likely to have been enriched with oxygen at the same time. The air-removal behavior afterward should have the same effect.

Further studies are needed to clarify whether there are additional factors that influence the nature of paternal helping strategies. In dusky gilled mudskippers, the shape of the entrances might determine whether tail undulation or frequent diving will take place. In slender mudskippers, the burrow entrances were always small in diameter. Here, possibly, the distance between the burrow openings and the height of the water surface above the edge of the burrow entrances played a role as it seems to lead to different air-adding intensities before larvae hatching.

Declarations

This study did not require ethics approval. The author has no conflicts of interest to declare that would be relevant to the content of this article. No funds, grants, or other support was received.

Supporting information on supplemental data

Fig S1: Experimentation equipment

Fig S2a-d: The male of dusky gilled mudskipper, Pearse’s mudskipper and slender mudskipper inside an entrance of their burrow

Fig S3a-c: Tail-undulation behavior inside the species-specific shaped burrows of dusky gilled mudskipper, slender mudskipper and Pearse’s mudskipper caused water to flow out of the burrow, which helped larvae to exit the burrow

Video S1: Air-adding behavior of dusky gilled mudskipper and slender mudskipper (high and low water levels above the burrow opening)

Video S2: Dusky gilled mudskipper: Air removal and larval release by frequently diving or tail-undulation behavior

Video S3: Pearse’s mudskipper: Air removal and larval release by tail-undulation behavior

Video S4: Slender mudskipper: Air removal and larval release by tail-undulation behavior

Video S5: Slender mudskipper: Air removal and larval release by larvae expelling out of the burrow

Acknowledgments

I would like to thank Herbert Nigl from Aquarium-Dietzenbach for providing specimen of Pearse’s mudskipper and slender mudskipper as well as Caroline Römer and Christopher David Sloan for revising the English of the first manuscript version and especially Gabriele Schmitt-Bylandt for revising the English of the final text.

Disclosure statement

No potential conflict of interest was reported by the author.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10236244.2023.2211219.