Introduction

Copepods are prominent members of the mesozooplankton community in diverse aquatic ecosystems, including lakes1, estuaries2, and coastal and pelagic waters3. These microcrustaceans play a critical role in aquatic food webs, connecting primary producers with higher trophic levels and serving as essential prey for fish larvae and other aquatic organisms4. Consequently, copepod abundance is a key factor influencing fishery resources, such as cod5, herring and sprat6, and anchovy7.

A key adaptive strategy of calanoid copepods for surviving adverse environmental conditions is the production of resting eggs8,9,10. These eggs undergo arrested embryonic development, forming a dormant “egg bank” within the sediment that acts as both a genetic reservoir and a biomass pool. When environmental conditions improve, nauplii from these eggs can emerge, replenishing the zooplankton community11. In ecosystems with pronounced seasonal changes, these egg banks are essential for nauplii recruitment and shaping copepod community structure12,13.

Therefore, understanding the dynamics of copepod resting eggs and their interactions with environmental factors is therefore crucial. Seasonal changes, especially in temperature and photoperiod, are key drivers of copepod embryonic dormancy, influencing its initiation, maintenance, and termination11. Additionally, various natural and anthropogenic factors, including salinity14, food availability15, oxygen levels16, and predation17, can affect the production of copepod resting eggs, as well as their viability after burial in the sediment. Research on copepod resting eggs has largely focused on temperate regions, which experience pronounced seasonal changes, particularly in sea surface temperature18. In contrast, studies on tropical and subtropical waters, where stable temperatures likely reduce the necessity for resting egg production, remain comparatively rare19,20,21,22.

Categorizing copepod resting eggs by their embryonic development and physiological processes remains difficult because of the potential for numerous variations. Even within the same species, different populations may exhibit different types of dormancies23. In general, three types of copepod resting eggs, namely quiescent, diapause and delayed-hatching eggs, have been recognized based on their hatching characteristics. Quiescent eggs form when subitaneous eggs, embryos that do not undergo a period of dormancy or delayed development, encounter sudden environmental adversities. These eggs maintain suppressed embryonic development, but can accelerate embryogenesis once favorable conditions return24. Based on current knowledge, the morphology of quiescent eggs does not differ from that of subitaneous eggs9. The hatching rate of copepod quiescent eggs decreases over time, as the embryos gradually consume their energy reserves while maintaining a low metabolic rate24,25. In contrast, diapause eggs can remain viable in sediment for years or even decades due to deep developmental arrest, with extremely low or no energy consumption11,23. The diapause eggs are genetically induced when maternal copepods receive signals of deteriorating conditions, such as unfavorable photoperiods and temperatures. This preparatory process may take some time11. Developmentally arrested diapause eggs require a relatively long recovery period from the refractory phase, even after environmental conditions become favorable9. Additionally, diapause eggs are characterized by the protective mechanisms, such as multilayered chorion and spiny ornamentation26,27, and they typically need specific stimuli to terminate the refractory phase28. The third type of resting egg, delayed-hatching eggs, is considered an intermediate dormant type between diapause and quiescent eggs29,30.

Tapong Bay, a tropical lagoon in southwestern Taiwan with limited water exchange through a single tidal inlet31, provides an ideal setting for investigating copepod egg dynamics. Historically, Tapong Bay supported traditional fishery, and intensive cage or oyster raft aquaculture industries until the early 2000s, when it transitioned to a recreational area. Despite this shift, the bay continues to experience natural environmental fluctuations as well as anthropogenic impacts from nearby aquaculture activities32,33. With its unique environmental characteristics and high primary productivity, Tapong Bay serves as a large mesocosm for marine ecological research. It exhibits significant seasonal and spatial variations in the distribution of the predominate free-spawning copepod species, such as Acartia spp., Bestiolona spp., and Parvocalanus spp., and the egg-bearing copepod species like Oithona spp. and Pseudodiaptomus spp. And the abundances of copepods could be correlated with temperature, chlorophyll, and salinity in the bay34,35. However, copepod egg abundance in the sediment of Tapong Bay has yet to be studied. This study aimed to investigate the temporal and spatial distribution of benthic copepod eggs in the intertidal sediment of Tapong Bay and to assess their embryonic dormancy characteristics.

Materials and methods

Study area and sampling

Tapong Bay, a semi-enclosed tropical lagoon on Taiwan’s southwest coast, covers approximately 5.32 km2 with an average depth of 2.2 m. It connects to the Taiwan Strait via a narrow inlet, leading to limited water exchange driven by semi-diurnal tides. This restricted circulation, combined with water inputs from aquaculture and urban sources, has impacted the distribution of organisms in the bay. Seasonal variations are influenced by the northeastern monsoon (winter dry season, with atmospheric temperatures ranging from approximately 22–25 °C) and the southwestern monsoon (summer wet season, with atmospheric temperatures ranging from approximately 32–36 °C). These monsoons affect freshwater input and water mixing, often resulting in salinity fluctuations between the dry and wet seasons36,37.

Environmental parameters, including water temperature and salinity, were measured using a portable multiparameter device (HI98194, Hanna Instruments). Monthly cumulative precipitation data were obtained from Taiwan’s Central Weather Administration. Copepod and sediment samples were collected from five stations on the tidal mudflat of Tapong Bay (Fig. 1), in December 2020, and March, July, and October 2021. The sampling stations were selected based on their varying hydrodynamic characteristics and spatial distribution. Stations 1, 2, and 5 are located in the inner-bay area, which receives water discharge from aquaculture ponds and experiences limited seawater exchange. In contrast, Stations 3 and 4 are positioned near the interior and exterior sides of the tidal inlet, where the water exchange rate is higher. At each station, four sediment cores were randomly collected within a 1 m2 area using a customized sediment corer (diameter: 9 cm; length: 30 cm) during low tide. The cores were transported under dark conditions and processed in the laboratory within 24 h. The top 6 cm of each core was sectioned at 2 cm intervals, and each layer was divided into four equal subsamples (31.8 cm3 per subsample) designated for different analyses as illustrated in Fig. 2.

Fig. 1
figure 1

Map of Tapong Bay (Pingtung County, Taiwan) showing the locations of the five sampling stations.

Full size image
Fig. 2
figure 2

Experimental protocols employed to evaluate abundances of benthic eggs, ready-to-hatch eggs, and resting eggs in the present study.

Full size image

Abundance of benthic eggs

A subsample (31.8 cm3) from each core (n = 4) was analyzed to determine benthic egg abundance using a modified sucrose flotation method38. This method has been widely used to isolate benthic copepod eggs in previous studies13,39,40. Sediment samples were gently resuspended in freshwater and filtered through an 800 μm sieve to remove large debris, then through a 40 μm sieve to exclude fine particles. This screening process was completed within 5 min to minimize osmotic stress on the eggs.

The filtered sediment was transferred to a 50 mL centrifuge tube, resuspended in saturated sucrose solution, and centrifuged at 2000 rpm for 5 min. The supernatant, containing copepod eggs, was filtered through a 40 μm sieve and rinsed with 1–µm filtered natural seawater. This extraction was repeated three times, after which the eggs were transferred to a Petri dish and counted under a stereomicroscope (Stemi 305, Zeiss). Only morphologically intact calanoid eggs, identified based on the descriptions by Choi et al.3 and Diodato et al.41, were counted. The abundance of benthic eggs per station was calculated by averaging the data from the three sediment layers (0–2 cm, 2–4 cm, and 4–6 cm) across the four replicates. The average seasonal abundance was determined as the mean of the abundances across the five stations.

Sediment incubation: identification of egg types and copepod species

Direct and secondary incubations (ready-to-hatch and resting eggs)

A sediment subsample (31.8 cm3) was incubated immediately under ambient conditions to assess the abundance of ready-to-hatch egg-derived copepods. The sediment was transferred to a 200 mL beaker (n = 4 per station and depth) containing 150 mL of 1–µm filtered seawater adjusted to the salinity levels measured at each sampling station. The incubations were gently aerated and fed every two days with a mixed algal diet (Isochrysis galbana and Proteomonas sulcata in a 1:1 cell density ratio, total concentration of 105 cells mL− 1).

The incubations were conducted in climate-controlled rooms, programmed to match the ambient conditions of each sampling station, for 14 days. Daily observations were performed, and once mature copepods were detected, they were individually sorted, transferred to a cavity slide, photographed alive using a compound microscope (BA210, Motic), and then preserved in 5% buffered formaldehyde for further morphological taxonomy. The immediate removal of adult copepods was crucial to prevent overestimating the number of eggs in the sediment due to potential reproduction. At the end of the incubation period, the entire suspension was filtered through a 38 μm mesh and transferred to a Petri dish. All newly-matured adult copepods were collected, while nauplii and copepodites were individually cultured in 6-well plates until reaching maturity, after which they were analyzed as described above. Only individuals that reached the adult stage were included in the abundance analysis. In addition, a few copepods (20–30 individuals) were maintained in a mono-species culture to collect newly spawned eggs under laboratory conditions. These eggs were used for morphological comparison with field-collected benthic eggs.

The incubation containers containing the pre-incubated sediments were then covered with plastic wrap and stored at 4 °C for one month. This post-incubation cold treatment was applied to simulate an artificial overwintering cue aimed at terminating the refractory phase of the unhatched resting eggs20,42,43,44. After the one-month cold treatment, a secondary incubation was conducted for an additional 30 days following the aforementioned protocol, and individuals emerging from this incubation were classified as resting-egg-derived copepods.

Incubation after cold treatment (resting eggs)

Another sediment subsample (31.8 cm3, n = 4) was immediately stored at 4 °C after the sampling for a period of one month. This pre-incubation cold treatment was applied to simulate a chilling adversity, inducing subitaneous eggs to enter a quiescent resting state and terminating the refractory phase of diapause resting eggs20,42,43,44. After cold treatment, the sediment was incubated according to the previously described protocol, and the abundance and species identification of resting egg-derived copepods were analyzed.

Taxonomic examination of copepods

Adult copepods collected from sediment incubations were examined morphologically. One to three specimens per sample were dissected using stainless steel tweezers under a stereomicroscope (Stemi 305, Zeiss). Morphological characteristics were observed and measured under a compound microscope (BA210, Motic) equipped with a digital camera (ToupCam, E3ISPM) and image analysis software (ToupView, ver. 4.11). Identification to species level was based on keys by Ito45, Chihara46, Shih et al.47 and Ueda48.

Abundance of copepods in pelagic community

Copepod samples were collected using a customized surface-trawling zooplankton net (diameter: 30 cm; length: 60 cm; mesh size: 100 μm) equipped with a flowmeter (2030R, General Oceanics). These samples were immediately preserved in ambient seawater with 5% buffered formaldehyde for further analysis.

Subsequently, copepod samples were split using a Folsom plankton splitter until each aliquot contained 300–500 individuals49. Adult calanoid and cyclopoid copepods were sorted and identified to the genus level based on identification guides by Chihara46, Lian et al.50 and Shih51, except for Acartia copepods, which were identified to the species level as described previously. Harpacticoid copepods, found in small numbers, were excluded from the analysis. The abundance of adult copepods was recorded as individuals per cubic meter (ind. m⁻3), and the relative abundance (RA) was calculated as the percentage of individuals of a specific genus or species relative to the total number of all copepods in the sample.

Relationships between environmental parameters, copepod and egg abundances

To assess the relationships between environmental variables and the abundances of copepods and eggs, pairwise Pearson correlation analyses were conducted. The analyses examined the correlations between temperature, salinity, precipitation and abundance of pelagic A. cf. tsuensis, and abundances of benthic eggs, ready-to-hatch eggs, and resting eggs, respectively. All statistical analyses were executed utilizing SPSS 18.0 (SPSS, Chicago, IL, USA), with the predetermined significant level set at p < 0.05.

Results

Environmental parameters

Tapong Bay exhibited clear seasonal variations across the dry, rainy, and post-rainy seasons (Fig. 3). During the dry season (December 2020 and March 2021), lagoon water showed lower temperatures (25.5–26 °C) and higher salinities (33.8–34.9‰), with monthly precipitation ranging from 0 to 1.5 mm. In the rainy season (July 2021), precipitation increased markedly to 480 mm, with an increase in average temperature to 32.6 °C and a decrease in salinity to 25.5‰. By the post-rainy season (October 2021), cumulative precipitation decreased to 90 mm, with average water temperature at 28.2 °C and salinity at 22.4‰. Notably, lower salinities (13–25‰) were recorded at inner-bay stations (St.1, 2, and 5) during the season.

Fig. 3
figure 3

Environmental parameters at the five sampling stations across seasons: (A) temperature; (B) salinity; (C) precipitation.

Full size image

Abundance of benthic eggs

Abundance of benthic calanoid eggs in sediment showed temporal and spatial variation (Fig. 4A). During the dry seasons, average abundances were 5.68 × 104 eggs m⁻3 in December 2020 and 6.41 × 104 eggs m⁻3 in March 2021. The abundance decreased to 1.11 × 104 eggs m⁻3 in the rainy season (July 2021), while a peak of 11.35 × 104 eggs m⁻3 was observed in the post-rainy season (October 2021). For the spatial distribution, the highest benthic egg abundance was found at St.2 and the lowest was found at St.4 among all stations. For the vertical distribution, higher numbers of the calanoid eggs tended to accumulate in the 0–2 cm upper layer (1.76 to 20.31 × 104 eggs m⁻3) compared to 2–6 cm lower layers (0 to 11.41 × 104 eggs m⁻3).

Fig. 4
figure 4

Abundances of different types of calanoid copepod eggs in the intertidal sediment of the five sampling stations in Tapong Bay across seasons: (A) benthic calanoid eggs in the sediment; (B) ready-to-hatch egg-derived copepods from the sediment; (C) resting egg-derived copepods from the sediment. The left y-axis presents the abundance of eggs per station; and the right y-axis presents the average abundance of eggs per season. Data are presented as the mean of the three layers and four replicates for the abundance per station, and as the mean ± standard error across stations for the abundance per season.

Full size image

Sediment incubation: identification of egg types and copepod species

Direct and secondary incubations (ready-to-hatch and resting eggs)

Abundances of ready-to-hatch egg-derived copepods varied across stations and seasons (Fig. 4B). In the dry seasons, average abundances were 0.26 × 104 ind. m− 3 in December 2020 and 0.42 × 104 ind. m− 3 in March 2021, both lower than the peak of 1.56 × 104 ind. m⁻3 observed during the rainy season (July 2021). No copepod emerged from the sediment samples collected during the post-rainy season (October 2021). In the secondary incubation, no copepod was recovered from the post-incubation cold-treated samples of all seasons. Vertically, ready-to-hatch egg-derived copepods were more abundant in the 2–6 cm lower layers (0 to 0.94 × 104 eggs m⁻3) during the dry seasons and in the 0–2 cm upper layer (2.73 × 104 eggs m⁻3) during the rainy season.

Incubation after cold treatment (resting eggs)

Abundances of resting egg-derived copepods fluctuated temporally and spatially (Fig. 4C). During the dry seasons (December 2020 and March 2021), abundances were generally low across stations (0 to 1.82 × 104 ind. m⁻3), except at St.1, where a higher abundance of 6.51 × 104 ind. m⁻3 was recorded in March 2021. The lowest and highest average abundances were observed in the rainy season (0.39 × 104 ind. m⁻3 in July 2021) and post-rainy season (2.45 × 104 ind. m⁻3 in October 2021), with resting egg-derived copepods found only at St.2 in these seasons. Vertically, higher abundances were found in the 2–6 cm lower layers (0.63 to 6.56 × 104 ind. m⁻3) in December 2020 and October 2021, and in the 0–2 cm upper layer (0.2 to 4.22 × 104 ind. m⁻3) in March 2021 and July 2021.

Taxonomic examination of copepods and benthic eggs

Copepods (Fig. 5A-E) collected from sediment incubations were morphologically similar to Acartia tsuensis as described by Ito45 and Ueda48. For instance, the exopodal segment 2 of right fifth leg with round-cornered rectangular process in male (Fig. 5B), and the female fifth leg with bilobed lobular process and a long terminal spine curved inwards midway (Fig. 5D). However, some differences in morphological (i.e. spinules on urosomite segments) and molecular (i.e. CO1 sequence) features were noted (data not shown), leading to the provisional designation of these specimens as A. cf. tsuensis. The average size (n = 20, each) of adult male was 871.69 μm (Fig. 5A) and female was 1002.96 μm (Fig. 5C), and detailed descriptions and illustrations of A. cf. tsuensis will be provided in a separate report. In addition, the field-collected benthic eggs have similar morphological features to the eggs produced in the mono-species culture of A. cf. tsuensis. The average size (n = 40) of the egg was 82.83 μm, and the eggs have smooth chorion without spiny ornamentation (Fig. 5E).

Fig. 5
figure 5

Photographs of the calanoid copepod Acartia cf. tsuensis collected from the sediment incubations: (A) adult male; (B) fifth leg of the male; (C) adult female; (D) fifth leg of the female (E) egg. Scale bars: (A, C): 250 μm; (B, D, E): 25 μm.

Full size image

Abundance of copepods in pelagic community

Copepod composition varied by station and season (Table 1). The cyclopoid Oithona spp. dominated at all stations and in all seasons, with relative abundances of 66.49–97.84%. Acartia spp., the most prevalent calanoid, constituted 0.83–22.25% of the copepod community, with higher abundance in the dry and rainy seasons compared to the post-rainy season. The Acartia copepods showed the tendency to assemble in the inner-bay area, particularly at St.1 and St.2. Other calanoid copepods, including Pseudodiaptomus spp. and Bestiolina spp. were present at lower relative abundances ranging from 0.48 to 9.08% and 0.85–10.28%, respectively. The Acartia copepod specimens were further examined at the species level, and all collected individuals were identified as A. cf. tsuensis.

Table 1 Abundance of pelagic copepods (ind. m− 3) in the intertidal waters of Tapong Bay.
Full size table

Relationships between environmental parameters, copepod and egg abundances

The Pearson correlation analysis revealed distinct relationships between environmental factors, pelagic A. cf. tsuensis abundance, and egg abundances. The abundance of ready-to-hatch eggs exhibited significant positive correlations with temperature (r = 0.49, p = 0.03), precipitation (r = 0.65, p = 0.003), and the abundance of pelagic A. cf. tsuensis (r = 0.50, p = 0.03), suggesting that higher temperatures, increased rainfall and greater copepod abundance may contribute to a higher number of ready-to-hatch eggs. In contrast, the abundances of benthic eggs and resting eggs showed no significant correlations with temperature, salinity, or pelagic copepod abundance (p > 0.05). Similarly, the abundance of pelagic A. cf. tsuensis was not significantly correlated with either temperature or salinity (p > 0.05).

Discussion

Tapong Bay is a shallow, semi-enclosed lagoon with a single narrow tidal inlet (138 m wide) and receives discharge from two ditches that carry wastewater from surrounding brackish aquaculture ponds. Consequently, the water level, salinity, and nutrient concentrations in the lagoon are influenced by both tidal pumping and aquaculture discharge37,52. Seasonal salinity and temperature fluctuations, consistent with previous studies35, were observed and were closely linked to precipitation. Notably, the inner-bay area (St.1, 2, and 5), which has limited seawater flushing (4–12% per day), exhibited particularly low salinities during the post-rainy season due to the combined effects of rainfall and aquaculture discharge36.

The cyclopoid genus Oithona dominated at all intertidal stations across seasons, consistent with its prevalence in tropical and subtropical waters53,54. In this study, Oithona spp. was found the most dominant copepod in all the intertidal stations across seasons. This contrasts with previous studies in the pelagic zone of Tapong Bay, where calanoids were found to be more dominant34,35. The high abundance of Oithona spp. in the intertidal zone may reflect eutrophic conditions in nearshore waters due to aquaculture discharge, although nutrient and chlorophyll concentrations were not measured here. Oithona species, like other cyclopoids, do not produce resting egg55 explaining their absence in the sediment incubations. Three calanoid genera—Bestiolina, Pseudodiaptomus, and Acartia—were also observed, consistent with previous findings34,35. Bestiolina, a neritic copepod genus typically found in low abundance, was likely transported into Tapong Bay by tidal currents34, thus it was absent in the inner-bay area, where tidal influence is minimal. On the other hand, Pseudodiaptomus and Acartia, both known for their salinity tolerance56,57, were abundant in the inner-bay area, likely due to their affinity for brackish environments. Some Pseudodiaptomus and Acartia species are commonly found in the brackish aquaculture ponds along Tapong Bay34,56,58. In addition to the presence of potential resident populations, these copepods are likely introduced from the aquaculture ponds into the bay via the two ditches. They subsequently aggregate in the inner-bay area due to the poor water exchange and salinity stratification35. Among the three genera, Acartia is characterized by its capacity to produce resting eggs20,59,60, whereas Pseudodiaptomus and Bestiolina do not exhibit such physiology adaptation18,61.

Indeed, morphological comparisons suggest that the benthic calanoid egg abundance in the sediment is exclusively attributable to A. cf. tsuensis, a common species in nearby brackish aquaculture ponds. Copepods and their eggs likely enter Tapong Bay via aquaculture water evacuation34, particularly accumulating in the inner-bay area, and contribute to the benthic egg abundance. Notably, the limited tidal current turbulence at stations 1, 2 and 536 facilities the suitable conditions for copepod egg to sink and accumulate on the bottom. Although sediment grain size and sedimentation rate were not examined in this study, the presence of muddy sediments observed at the inner-bay stations suggests a higher sedimentation rate and lower water turbulence, which may contribute to the greater abundance of benthic eggs in these stations (Table 2).

Table 2 Pearson correlation analyses between temperature, salinity, precipitation, and the abundances of pelagic A. cf. tsuensis, benthic eggs, ready-to-hatch eggs and resting eggs.
Full size table

Benthic egg abundance may reflect the duration of egg retention in the sediment and is associated with reproductive rates and developmental status. These factors are influenced by environmental conditions such as food availability59, salinity62,63, and temperature60,64. The lower benthic egg abundance and higher ratio of ready-to-hatch eggs to resting eggs during the rainy season may be related to accelerated embryonic development under favorable temperature and salinity conditions65,66. Under such conditions, embryos may develop more rapidly and remain in the sediment for only a short period. In contrast, a relatively higher number of benthic eggs were isolated from sediments collected during the dry and post-rainy seasons (Fig. 4A), when the egg population was characterized by a greater accumulation of resting eggs (Fig. 4C). This shift in egg production suggests that A. cf. tsuensis experienced unfavorable environmental conditions during these periods, leading to the production of more resting eggs that accumulated and stayed in the sediment for an extended duration.

The identification of calanoid to species level could be very challenging when examining only their egg morphology under a compound microscope10. While DNA barcoding could aid species identification67,68, it is constrained by database limitations and potential contamination. Incubation-based identification, though time-consuming, provides reliable species information. Our attempts to incubate the isolated benthic eggs were unsuccessful, possibly due to reduced egg viability resulting from the sucrose flotation protocol, despite its widespread use in many studies (Table 3). Additionally, Berasategui et al.30 and Berasategui et al.69 noted that the sucrose flotation method may not recover all types or forms of copepod eggs (e.g., individual eggs or egg sacs). This highlights the need for improved egg isolation techniques in future research. Overall, the benthic egg abundance found in Tapong was relatively lower than previously reported in high-latitude locations (Table 3), aligning with the general understanding of latitude-based copepod resting egg production and temperature-dependent embryonic development18.

Table 3 Comparison of benthic calanoid egg abundances across latitudinal gradients.
Full size table

The sediments were incubated without prior egg isolation and counting, limiting precise hatching rate and egg type percentage estimations. However, trends in reproductive strategy were inferred by comparing benthic egg, ready-to-hatch, and resting egg-derived copepod abundances. All copepods collected from the sediment incubation were morphologically similar to A. tsuensis (Fig. 5), consistent with its known abundance in warm seasons when introduced from aquaculture ponds34. However, some morphological and molecular characteristics of the specimens (e.g. number of spines on the urosomite segments) were found different from the A. tsuensis described in Japan45 and the closely related new species Acartia ohnoi found in the Philippines48, therefore the specimens are referred to as A. cf. tsuensis here.

During the dry seasons (December 2020 and March 2021), higher ratios of benthic eggs to ready-to-hatch eggs, and of resting to ready-to-hatch eggs suggest that lower temperatures (25.5–26 °C) and higher salinity (33.8–34.9‰) may reduce egg viability and increase resting egg production. In contrast, higher ratios of ready-to-hatch eggs to benthic eggs, and to resting eggs indicate a trend of higher egg hatching rate and lower resting egg production in the rainy season. Moreover, the abundance of ready-to-hatch eggs was significantly positively correlated with temperature and precipitation (warm and rainy conditions) and negatively correlated with salinity, although the latter is not statistically significant (Table 2). These findings suggest that the A. cf. tsuensis population in Tapong Bay, is more adapted to warm (32.57 °C) and moderate-salinity (25.5‰) waters, and it coincides with the observation in the previous study34. In addition, similar temperature and salinity preferences have also been reported in the populations of A. tsuensis in Japan and the Philippines45,70,71,72.

In the post-rainy season, a high number of benthic calanoid eggs was isolated from the sediment, but no ready-to-hatch egg was documented. Whereas, a great number of resting eggs emerged at St.2, which was the highest among all seasons. During this period, relatively low salinity levels (13–25 ppt) were observed in the inner-bay area (St.1, 2 and 5). Since salinity levels in aquaculture ponds in the Tungkang area can drop as low as 15–16 ppt during late summer58,73. The salinity reduction in the bay may be attributed to increased freshwater runoff and low-salinity water discharge from nearby aquaculture farms. It appears that A. cf. tsuensis produces more resting eggs when exposed to prolonged and extensive salinity reductions in aquaculture ponds across the rainy and post-rainy seasons. Subsequently, these resting eggs are transported and accumulate at the innermost station (St.2) due to the hydrodynamic conditions in Tapong Bay36.

Temperature has commonly been employed to simulate seasonal variations that induce or terminate copepod embryonic dormancy74,75. The responses of copepod resting eggs to temperature cues can be species- and population-specific8,10,11. Some copepod populations, such as Acartia hudsonica in Narragansett Bay, Rhode Island, USA74, Acartia omorii in Tokyo Bay, Japan60 and Acartia bifilosa in Jiaozhou Bay, China76, could produce over-summering resting eggs. Conversely, the production of over-wintering resting eggs has been reported in Acartia californiensis in Yaquina Bay, Oregon, USA77, Acartia erythraea in Gamak Bay, South Korea59, and Acartia steueri in Okkirai Bay, Japan78. These over-summer and over-winter resting eggs may need to experience either chilling or warming signals to reactivate their embryonic development. Although the population of A. cf. tsuensis can be found year-round, their abundance decreased during the colder seasons in Tapong Bay (Table 1), suggesting that some individuals may enter an overwintering life cycle as resting eggs.

The alternate chilling-warming treatment was therefore applied to induce and terminate embryonic dormancy prior to sediment incubation. Cold simulations have been used in some studies to artificially induce embryonic quiescence of some Acartia species24,25, as well as to trigger the hatching of zooplankton diapause eggs, including those of copepods20, rotifers and daphniids79. The low temperature (4 °C) applied in the present study successfully induced the eggs of A. cf. tsuensis enter resting status, however, such low temperature does not naturally occur in Tapong Bay. The effects of various temperatures on the induction and termination of embryonic dormancy in this tropical species remain to be explored. Our findings suggest that the physiological capability for cold-resistant embryonic dormancy in A. cf. tsuensis may be phylogenetically linked to its congener species inhabiting colder environments80. Future research should investigate the functional phylogeny of embryonic dormancy across Acartia species.

Salinity variation is another environmental parameter know to induce and terminate embryonic dormancy in copepods9,81. In a laboratory experiment, variations in egg hatching rate were documented when the maternal populations of copepod Acartia ohtsukai were exposed to various salinity levels62. The authors noted that the reduced hatching rate might be correlated with a higher proportion of resting egg production under adverse salinity conditions. In addition to maternal control, copepod subitaneous embryos may enter dormancy when they experience abrupt salinity fluctuations14,75. The salinity change (i.e. the high salinity in the dry season and the low salinity in the post-rainy season) seems to be one of the factors inducing the higher resting egg production in Tapong Bay. Further laboratory-based studies should be conducted to verify the combined effects of salinity and temperature variations on the induction, maintenance, and termination of resting egg production in A. cf. tsuensis females, as well as on the post-spawning embryonic dormancy.

Classifying copepod resting egg types from field-collected samples is highly challenging, as multiple egg types at different embryonic stages and resting statuses can coexist in the sediment. In our study, we employed different incubation strategies to assess the abundance of various egg types. In the direct incubation experiment, benthic copepod eggs were not subjected to any additional environmental stimulation. Therefore, the eggs that hatched readily were primarily assumed to be subitaneous. However, due to uncertainty regarding the presence of resting eggs at pre-hatching stages, the copepods that hatched from the direct incubation were inclusively referred to as ready-to-hatch egg-derived copepods. Notably, no copepod hatched from the secondary incubations, suggesting that the applied cold stimulation and ambient incubation conditions failed to terminate the refractory phase of diapause embryos of A. cf. tsuensis. Alternatively, the species may simply lack the physiological capacity to produce true diapause eggs.

In the post-rainy season, copepods did hatch from the sediment stored immediately at 4 °C after sampling. Given the absence of true diapause eggs, the copepods derived from these cold-stored sediments were likely derived from quiescent eggs induced from subitaneous eggs. However, the subitaneous egg-derived copepod was also absent in this season. This finding suggests that the eggs may represent a distinct form of dormancy—delayed-hatching eggs, which exhibit hatching characteristics intermediate between quiescent and diapause eggs. A similar egg type has been reported in a subtropical population of the congeneric species Acartia tonsa29. Overall, the identification of true diapause or delayed-hatching eggs in A. cf. tsuensis remains unresolved and should be further investigated using advanced approaches, such as transcriptome analysis11,82 embryonic staining83,84,85, and with refined induction and incubation protocols.

In our study, no clear pattern was observed in the vertical distribution of different egg types across the sediment layers at the five stations. The vertical distribution of copepod eggs may be influenced by complex sediment dynamics13,44, while embryonic status could be affected by sediment physicochemical properties such as pH, dissolved oxygen, and hydrogen sulfide concentrations3,10,44. Additionally, bioturbation and predatory activities may further contribute to shaping the egg vertical distribution10,13,44. This complex mechanism, involving multiple interacting factors, warrants further investigation in future studies.

In conclusion, this study documents the seasonal and spatial distribution of A. cf. tsuensis eggs in Tapong Bay, suggesting that warm and rainy conditions favor the emergence of ready-to-hatch eggs, while resting egg abundance increases during colder and drier seasons. These findings provide the first insights into the reproductive strategies of A. cf. tsuensis in Tapong Bay and highlight the potential impacts of temperature and salinity on its reproductive patterns. Future studies should further investigate the effects of environmental factors on the dormancy physiology of A. cf. tsuensis.