1.2 Sample collection

Sampling was carried out at MG, SG, and SG+CR in March 2015 and 2016 (Figure 1A). Two bait traps (10 m long and 6 mm mesh size) were set overnight in each area to collect fishes and macro-invertebrates. The muscle tissues of fishes and macro-invertebrates were used for stable isotope analysis, because the constant isotopic value of this tissue reflects the isotopic value of food sources utilized over long periods (i.e., several weeks to months) (Herzka, 2005).

The leaves of mangrove species (Rhizophora stylosa) and seagrass species (Thalassia hemprichii and Halodule pinifolia) were collected by hand in the mangrove and seagrass areas, respectively. Mangrove and seagrass leaves were washed with MQ pure water (Arium^® 611VF, Sartorius Stedim Biotech GmbH, Germany) to remove all detritus before it was stored at -20°C until further treatment. Phytoplankton samples were collected using a plankton net. In both habitats, 100 L of seawater was pre-filtered using a mesh with a pore size of 73 mm to remove large material (e.g., detritus, zooplankton). Phytoplankton was captured using a mesh size of 10 mm. Retained phytoplankton were filtered onto pre-combusted Whatman GF/F filters (~0.7 mm) and were stored at -20°C. Aliquots of the surface sediment (upper 2 cm) were sampled to study primary microbial producers. These aliquots were homogenized and stored in Corning tubes (50 mL) at -20°C.

DZ were collected with a modified “emergence trap” of conical shape, according to Hobson and Chess (1979), using 2 chambered traps with a mesh size of 73 µm (Figure 1B). Three demersal traps were set-up at MG, SG, and SG+CR. The mouth of each trap was fixed at 5 cm under the sediment surface using soil anchors, and was set up from 18:00 to 07:00 of the next day. DZ samples were separated into fractions using sieves; these fractions were: 73 µm (73 to 100 µm), 100 µm (100 to 250 µm), 250 µm (250 to 500 µm), 500 µm (500 to 1000 µm), 1000 µm (1000 to 2000 µm), and greater than 2000 µm (>2000 µm). Then the fractions were divided into 2 sub-samples: one for identification, for which zooplankton was fixed in 5% formalin, and one for stable isotope analysis (SIA). The SIA samples of demersal zooplankton were examined under the stereomicroscope (SMZ 1000, Nikon Inc., Tokyo, Japan) to remove detritus to avoid contaminating the isotopic signal. The SIA samples of demersal zooplankton were then stored at -20°C until isotopic analysis.

1.3 Determining the abundance and biomass of DZ

DZ was identified and counted under a stereomicroscope (SMZ 1000, Nikon Inc.). The abundance of DZ taxa was calculated as the number of individual m^-2 (ind.m^-2) on the bottom surface area, based on the mouth area of the “emergence trap” (Figure 1B). The carbon biomass of DZ was calculated as the carbon content in µg C ind^-1, based on the taxonomic level and the average size class, using the regression equation given by Heidelberg et al. (2010): LN (Copepod biomass) = 1.82 × log (L) + 1.28 (r² = 0.893, df = 16, F = 125; sig. = 1.12 × 10^-8) and LN (Other taxa biomass) = 1.46 × LN (L) + 1.03 (r² = 0.733, df = 16, F = 80.7; sig. = 3.47 × 10^-7), where LN is the natural logarithm and L is the average size (length) in mm.

1.4 Analysis of fish stomach content

The stomach and gut were dissected from the fish body and preserved with 90% ethanol. The stomach content was observed under a stereomicroscope (SMZ 1000, Nikon Inc.), and the percentage of items present was estimated. The food sources were identified to the lowest possible taxon.

1.5 Sample treatment

Sediment containing bacteria, small primary producers, and fine detritus was dried at 60°C in an oven to a constant weight (around 20 h), and was then passed through a sieve with a pore size of 63 mm to separate out large particles (coarse gravel, mangrove detritus, seagrass roots, and mollusk shells). Sediment powder was homogenized for sediment SIA. The litter that remained on the sieve (< 200 mm) that was derived from mangrove and seagrass was ground to a fine powder for detritus stable isotope analysis. The other SIA samples were dried at 60°C until a constant weight was reached. Then, the samples were ground to a fine powder.

The homogeneous powder of the SIA samples was divided into 2 fractions for δ¹⁵N and δ¹³C analysis. The samples for δ¹⁵N were stored in a dry box until analysis without acid treatment to prevent acidification affecting the δ¹⁵N values (Bunn et al., 1995; Mateo et al., 2008). The samples for δ¹³C analysis were further treated to remove the lipid content and carbonates. In brief, the lipid content in animal tissue can alter the results and conclusions of δ¹³C analysis in the aquatic food web and migration studies (Bunn et al., 1995; Focken and Becker, 1998). Thus, in the present study, fishes and macroinvertebrate samples were treated to remove lipids following a method modified from Logan et al. (2008). Specifically, dried powder samples were placed in centrifuge tubes to which a solvent with a 2:1 ratio of chloroform:methanol was added, with a volume 3 to 5 times larger than the sample size. Samples were mixed for 30 s in an MS1 Minishaker (IKA^® Works (Asia) Sdn. Bhd., Malaysia) at 1000 rpm. The samples were then left undisturbed for about 20 min, after which they were centrifuged at 2500 × g for 10 min. The supernatant containing the solvent and lipids was discarded. This process was repeated until there was an entirely clear supernatant. After removing the lipids, the animal tissues were subjected to an acidification procedure to remove carbonates. The samples (animal tissues, sediment, and seagrass leaves) were acidified by dropping a solution of 1 N HCl onto the sample until bubbling ceased (Jacob et al., 2005). Then, the samples were washed 3 times with Milli-Q water before being dried at 60°C to a constant weight and ground to a fine powder. DZ samples and phytoplankton trapped on GF/F were fumed with concentrated 12 N HCl in a glass desiccator for 12 h to remove carbonates, and were then dried at 60°C to a constant weight and ground to a fine powder. All powder samples were stored in Eppendorf tubes inside a dry box until analysis.

1.6 Stable carbon and nitrogen isotope analysis

Stable carbon and nitrogen isotopes were analyzed at the Laboratory of Aquatic Animal Ecology, School of Marine Biosciences, Kitasato University, Japan. The samples were dried in an electric oven at 60°C for 6 h before analysis. Subsamples of 0.5 ± 0.07 mg (Mean ± SD) dry weight for fishes and macroinvertebrates tissues and 2.0 ± 0.08 mg (Mean ± SD) dry weight for DZ were placed in ultra-pure tin capsules, and the samples were burned in an elemental analyzer (Flash EA, Thermo Fisher Scientific, Waltham, MA, USA). Combustion gasses continuously moved through a flow controller (ConFlo, Thermo Fisher), and then the stable carbon and nitrogen isotope compositions were detected with a mass spectrometer (Delta^plusXP, Thermo Fisher). L-alanine was used as the working standard. Repeated measurements of the standard showed a standard deviation of 0.2‰ or less. Stable isotope ratios were expressed in δ notation (part per thousand, ‰) as deviations from international standards according to the following equation:

δX = (R_sample/R_standard – 1) × 1000

Where X represents ¹³C or ¹⁵N, and R represents isotope ratios ¹³C/¹²C or ¹⁵N/¹⁴N, respectively. R_sample and R_standard are the isotope ratio of the sample and working standard, respectively.

1.7 Estimation of proportional contribution from food sources

The SIAR package (Parnell and Jackson, 2013) of R software (R Core Team, 2015) was used to calculate the proportion of total diet that was contributed by food sources, based on the stable isotope values of consumers, the mean and standard deviation (SD) stable isotope values of food sources, and the trophic enrichment factor (TEF) of food sources. To compare the percentage of DZ with other food sources at each of the selected habitats, we estimated the contribution of food sources to the diets of consumers (fishes, macroinvertebrates) in each habitat using the mean of the TEF (± SD) for δ¹³C and δ¹⁵N as 0.4 ± 1.3‰ and 3.4 ± 1.0‰, respectively (Post, 2002). On the basis of the results of the stomach contents analysis, we compared DZ against other food sources (including crabs, shrimps, mollusks and, detritus) in the diet of fishes. In the diet of macro-invertebrates, we compared DZ against the plant (mangrove or seagrass) leaves, phytoplankton, detritus, and sediment.

1.8 Statistical analysis

The Shannon diversity index (Hʹ) was calculated to estimate the diversity of the DZ community. One-way ANOVA was performed to test the differences (p < 0.05) in the isotopic values of DZ and their proportion in the diets of consumers.

2 Results

2.1 Composition of DZ communities

In the present study, 18 DZ taxa were identified (Table 1). The species groups that had high-frequency distributions and higher abundance in all 3 habitats were Harpacticoida (copepodite), Foraminifera, and Gammaridea (Table 1). In the MG habitat, Harpacticoida (copepodite) contributed 43.2%, followed by Nematoda (24.3%) and the nauplii of Copepoda (10.8%). In the SG habitat, Foraminifera contributed 27.8%, followed by Isopoda (22.2%). In the SG+CR habitat, Oithona rigida had the highest abundance (25.7%), followed by the nauplii of Copepoda (22.9%) and Harpacticoida (copepodite) (12.9%).

2.2 Stomach contents of fishes

Table 2 shows the stomach contents of 11 fish species collected in Fukido Estuary. The preferred foods of fishes in MG were zooplankton, which are components of DZ (Table 1). Fishes from MG primarily consumed copepods (5-15%) and polychetes (5-25%), followed by amphipods (3-10%), nematodes (5-10%), and foraminifers (2-5%). Some fishes, such as Y. cringer (2%), C. punctata (5%), F. amboinensis (7%), and Lutjanus sp. (20%) consumed crabs. Shrimp and mollusks were consumed by larger fishes, such as Lutjanus sp. (10%) and A. semipunctata (10%), respectively (Table 2). However, the main stomach content of some fish specimens in MG was detritus (G. oyena [80%] and A. semipunctata [50%]) and fine soil and sand grains (F. amboinensis [40%] and Z. dunckeri [30%]) (Table 2). Fish specimens collected in the LG habitat mainly consumed crabs (10-15%), mollusks (10-30%), and shrimp (10%). Moreover, some fishes consumed DZ species, such as amphipods (20-30%), copepods (10%), and polychetes (5%), in the lagoon habitat (Table 2).

2.3 Isotopic signature of consumers, DZ, and other food sources

Figure 3 shows the stable carbon and nitrogen isotope signatures of DZ, other food sources (mangrove leave, seagrass leave, detritus, sediment, and phytoplankton), and their consumers (fishes, crabs, shrimps and mollusks) in the aquatic food web of Fukido Estuary. The δ¹³C values of DZ ranged from -24.9 to -20.4‰ and -20.5 to -15.4‰ in the MG and LG areas, respectively. The δ¹⁵N signatures of DZ in both habitats were not significantly different (ANOVA, p = 0.479), ranging from 2.7 to 4.6‰ (Figure 3).

The δ¹³C and δ¹⁵N values of other food sources (mangrove and seagrass leaves, phytoplankton, detritus, and sediment) ranged from -29.4 to -25.9‰ and 0.6 to 2.1‰, respectively, in MG; and from -21.3 to -10.3‰ (δ¹³C) and 0.7 to 1.8‰ (δ¹⁵N) in the LG habitat. The δ¹³C and δ¹⁵N values of consumers ranged from -26.7 to -18.8‰ and 4.3 to 9.3‰ in MG, respectively. In comparison, these signatures ranged from -14.2 to -11.0‰ (δ¹³C) and from 4.2 to 9.7‰ (δ¹⁵N) in the lagoon habitat (Figure 3).

2.4 Proportional distribution of DZ in the diets of higher trophic levels

The contributions of the potential food sources of the fishes and macro-invertebrates in Fukido Estuary are presented in Table 3 and Table 4, respectively. Table 3 shows that DZ of larger size classes (500 to 2000 mm) contributed more to the diet of MG fishes than the DZ of smaller size classes (73 to 250 mm). In the diet of some mangrove fish species, the proportional contribution of DZ from the larger size classes (500 to 2000 mm) was higher than that of other food sources (e.g., crabs, shrimp, mollusks, and detritus). A. semipunctata consumed large quantities of DZ large than 2000 mm (15.0%), followed by 500–1000 mm DZ (13.4%) and 1000-2000 mm DZ (12.9%). DZ contributed 11.1-12.5% to the diet of Y. criniger. DZ large than 2000 mm contributed the highest proportion (12.9%) to the diet of Z. dunckeri, followed by 1000-2000 mm DZ (12.6%) and 500-1000 mm DZ (12.1%). Other food sources contributed 4.3-11.7%, 7.1-10.5%, and 7.3-9.7% to the diet of A. semipunctata, Y. criniger, and Z. dunckeri, respectively. In comparison, some mangrove fish species consumed more crabs and shrimps than DZ. For instance, F. amboinensis consumed the highest proportion of crabs (19.0%), followed by shrimps (15.8%). The diet of C. punctate contained 13.1% crabs and 11.7% shrimps. DZ contributed just 6.2-7.8% (73-250 mm size classes) and 8.7-13.6% (500-2000 mm) to the diet of F. amboinensis, and 8.8-9.6% (73-250 mm) and 10.1-11.6% (500-2000 mm) to the diet of C. punctata.

Lutjanus sp. and G. oyena consumed similar quantities of DZ (500-2000 mm) and other crustaceans (such as crabs and shrimps) in MG habitat; however, Lutjanus sp. collected in the LG habitat consumed more crabs (12.2%) and shrimps (11.2%) than DZ (9.8-10.1%). Other lagoon fishes also consumed more on crabs, shrimps, and mollusks than DZ (Table 3).

The results of the mixing model of the macro-invertebrates diet items and their proportions in Fukido Estuary are presented in Table 4. Mangrove crabs (Scylla serrata and Charybdis sp.) consumed more DZ of bigger size classes (500 to 2000 mm) than smaller size classes (73-250 mm) or other food sources (i.e., those derived from mangrove leaves, detritus, sediment, and phytoplankton). In comparison, some macro-invertebrates (such as E. japonica and C. septemspinosa) consumed more DZ of smaller size classes (73-250 mm) than larger size classes (500-2000 mm). Almost all macro-invertebrates (except Charybdis sp. and S. serrata) from both habitats preferentially consumed food sources derived from detritus, sediment, plants, and phytoplankton compared to DZ.

3 Discussion

3.1 Role of DZ as a potential food source in an estuarine food web

Mangroves, seagrasses, and coral reefs support and provide shelter for a large number and high diversity of fish and invertebrates (Zieman et al., 1984; Larkum et al., 2007). The average size ratio of predator to prey is usually around 10 to 1, while the abundance ratio is typically the inverse (Litchman et al., 2013; Chen and Terry, 2014). Therefore, DZ must contribute substantially to the system, as a large reserve of highly abundant food source with a wide range of body dimensions. Previous studies reported that DZ are highly abundant in shallow habitats (Melo et al., 2010). DZ usually reside in the top 3 cm of the sediment, emerging in large numbers at night and occupying the water column up to 30 cm from the bottom (Alldredge and King, 1985). They only remain in the water column for a short time to avoid predators that use vision to locate their prey (Alldredge and King, 1985). In the present study, DZ abundance ranged from 4.0 to 11.3 × 10⁴ ind.m^-2 (Figure 2A), with a wide range of body sizes being detected (75 to 9100 µm) (Table 1). At Fukido Estuary, the abundance of DZ was higher than that previously reported in other geographical areas. For instance, Melo et al. (2010) reported comparatively lower DZ abundance (5 × 10³ ind.m^-2) in a seagrass area in the southwestern Atlantic Ocean. In comparison, the abundance of DZ in Onslow Bay (North Carolina, USA) ranged from 1 to 6 × 10⁴ ind.m^-2 depending on substrate structure and season (Cahoon and Tronzo, 1992).

The biomass of DZ in the 1000-2000 µm and > 2000 µm size classes were lower than those smaller size classes in MG compared to that in the other habitats (Figure 2B). Compared to that in the other habitats in our study, the abundance of DZ in MG was intermediate, with smaller biomass. Sultana et al. (2016) reported that the sediment in mangrove habitat supports high primary production. Thus, DZ might be present in higher abundance and biomass than that detected in our study. However, our results showed that smaller DZ contributed the most to DZ biomass, indicating that higher trophic level organisms preferentially consume larger DZ (Figure 2). Therefore, DZ with lower biomass and of smaller size might remain in the mangrove habitat. This suggestion is supported by the result of the mixing model used to estimate the proportion of DZ in the diet of fishes (Table 3). This model showed that the proportional contribution of larger DZ (500-2000 mm) were higher than that of smaller DZ (73-250 mm) in the diet of almost all mangrove fishes. Thus, consumers in the mangrove area might actively feed on more DZ than previously thought (Nakamura et al., 2008; Tue et al., 2013). This hypothesis is consistent with the stomach contents of fishes, which showed that the mangrove fishes primarily fed on DZ compared to other food sources (Table 2).

3.2 Proportional contribution of DZ in the diet of consumers

Estimation of the proportional contributions of prey in the diet of consumers in coastal ecosystems is traditionally performed by analyzing stomach contents. This method is convenient because it can be carried out rapidly and economically, providing a direct estimation of prey contents (Hyslop, 1980; Pasquaud et al., 2010; Winemiller et al., 2011). However, stomach content analysis has some limitations, because living organisms digest food more rapidly than non-living organic matter (Fry and Ewel, 2003). The SIAR mixing model is based on the isotopic signatures of prey and consumers, and has been increasingly used to determine the proportional contributions of food sources (Parnell et al., 2010). In this study, we combined both methods (stomach content and mixing model) to clarify and highlight the contributions of DZ to the diet of fishes and macroinvertebrates at Fukido Estuary.

The mixing model showed that DZ contributed more to the diet of mangrove fishes than other food source in the MG habitat, particularly for A. semipunctata, Y. criniger, and Z. dunckeri (Table 3). In particular, these fish species consumed larger DZ (of 500 to 2000 mm), which were dominated by nematodes, polychetes (larvae), copepods, amphipods, and gammarids (Table 1). The results of the stomach content analysis supported this observation (Table 2). Our results were also consistent with those of previous reports (Masuda et al., 1984; Rainboth, 1996; Myers, 1999). In comparison, crabs and shrimp contributed to the diet of F. amboinensis and C. punctata more than DZ. Other studies also reported that F. amboinensis primarily consumes small crustaceans (Masuda et al., 1984), whereas C. punctata preferentially feeds on small benthic invertebrates and small crabs (Knapp, 1999). However, our stomach content analyses of these fishes showed that they preferentially fed on nematodes and copepods compared to crabs and shrimps (Table 2). This discrepancy might be explained by the fact that stomach contents only reflect feeding events, rather than assimilated material; consequently, this type of analysis is biased by the differences in digestibility of prey items (Polunin and Pinnegar, 2002). Thus, the proportions of food sources based on isotopic analysis probably provides a more accurate picture of feeding behavior than stomach contents. Our results showed that mangrove fishes predominantly fed on DZ compared to other food sources. In comparison, lagoon fishes consumed more crabs, shrimps, and mollusks than DZ. These differences were supported by both the results of the mixing model (Table 3) and the stomach contents analysis (Table 2). Lagoon fishes of large body size might preferentially feed on larger food sources. This suggestion was supported by the results of the feeding preferences of Lutjanus sp., which is a migratory fish that frequents Fukido Estuary (Nakamura et al., 2008). Juvenile stage Lutjanus sp. are relatively small (around 30-125 cm) (Nakamura et al., 2008; Shibuno et al., 2008), and they inhabit MG as a nursery. Juvenile Lutjanus sp. consumed similar proportions of DZ (500-2000 mm) as crabs and shrimps in MG (Table 3), perhaps because the Lutjanus sp. might prefer DZ than crustaceans that have hard exoskeletons. As adults, when their body size becomes greater than 150 cm, Lutjanus sp. migrate to the lagoon area (Nakamura et al., 2008), where they switch their feeding preference to crustaceans (crabs and shrimps) during ontogenetic migration (Table 2; Table 3). Thus, DZ might be an important food source for fishes in mangrove habitats compared to other studied habitats.

Benthic macroinvertebrates are distributed in all coastal ecosystems (mangrove, seagrass and coral reef, etc.), and inhabit areas on or near the seabed (Attrill, 1998; Castro et al., 2008; Barnes, 2013; Kumar and Khan, 2013). This group exhibits diverse ecological niches and invests in a variety of feeding behaviors. For instance, there are deposit feeders (mainly polychetes and some mollusks), detritivores (some echinoderms and crustaceans), predators (echinoderms and crustaceans), filter feeders (mainly bivalves and crustaceans), among others. Our dataset showed that DZ contributed more than other food sources to the diet of 2 crab species (Scylla serrata and Charybdis sp.) (Table 4). Other studies have also reported that these 2 crab species preferentially feed on fishes, crustaceans, mollusks, and polychetes in mangrove habitats (Sara et al., 2007; Wikipedia, n.d.). The stable carbon and nitrogen isotope signature of S. serrata are comparable to that of some mangrove fishes (Z. dunckeri, A. semipunctata, F. amboinensis) (Figure 3); thus, these crabs probably feed on food sources similar to fishes, preferentially selecting large-sized DZ. This suggestion was supported by the results of the mixing model on the diet of S. serrata, which showed that they feed on larger rather than smaller DZ (Table 4). In contrast, some species (such as E. japonicas and C. septemspinosa) primarily consumed food sources that were derived from detritus, sediment, and plant debris rather than DZ (Table 4). This result supports that obtained by previous report (Kolpakov et al., 2012). Other macroinvertebrates (except Scylla serrata and Charybdis sp.) appeared to consume similar quantities of small (73-250 mm) and large DZ in the lagoon habitat, but preferentially fed on smaller DZ in mangrove habitat (Table 4).

4 Conclusions

This study demonstrated that DZ are an important food source in Fukido Estuary. The mangrove habitat supported the lowest DZ biomass, and was mostly made up of small-sized DZ. Thus, higher trophic level organisms might consume large-sized DZ, resulting in the smaller sizes remaining (unconsumed) in the mangrove habitat. This suggestion supports the proportions of DZ size classes detected in the stomach contents of mangrove fishes. Large percentages of DZ were detected in the stomachs of mangrove fishes, confirming their importance as a food source. In contrast, DZ did not have a significant role in the diet of fishes in lagoon habitats compared to other food sources. The combined results of abundance and biomass with the proportions of DZ in the diet of consumers highlight the important role of DZ in the diet of active feeders in mangrove habitats compared to that in other habitats. To our knowledge, this study is the first attempt to elucidate the role of DZ within the food web of an estuary, using the stable isotope mixing model of size class and biomass combined with direct observations of stomach contents. In conclusion, we showed that the suggested approach (combining stable isotope mixed models with stomach content observations) was reliable, demonstrating that DZ of different body sizes serve as food sources for different consumers in different habitats.

Authors’ contributions

HMV, BEC, LS, and YS designed the study, conducted the experiments, and collected the samples. HMV, BEC, and KT identified demersal zooplankton and stomach contents. HMV and KH completed the stable isotope analysis. All authors read and approved the final manuscript.

Acknowledgments

We thank Mr. S. Tamada of the Laboratory of Aquatic Animal Ecology, School of Marine Biosciences, Kitasato University for the supporting the stable isotope analysis. We also thank Mr. S. Uehara (Growing Coral Co.) for providing support during the fieldwork, and we thank the International Coral Reef Research & Monitoring Center, Ministry of Environment, Ishigaki, Japan for providing laboratory facilities. The Environmental Leaders Program of Shizuoka University Corporation (ELSU) and the Global Coral Reef Conservation Project (GCRC) of Mitsubishi Corporation supported this study. This study was conducted under the umbrella of Core-to-Core Program and Asian CORE Program of the Japan Society for the Promotion of Science.

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