Background

Polychaetes with a relatively short life cycle and a strong reproductivity not only play a role of secondary consumers in the ocean but also sometimes purify deposit by changing the organic ingredients through feeding behavior (Clark 1977; Paik 1989; Heo 2011). Polychaetes are considered as an indicator organism of marine pollution (Belan 2003; Giangrande et al. 2005; Samuelson 2001), as the major prey of benthic fish and as bait for angling becoming a target species of fishermen’s sideline (Gambi et al. 1994; Olive 1994, 1999; Younsi et al. 2010). Among the polychaete species, especially, the rockworm Marphysa sanguinea (Montagu, 1813) (Eunicidae), is a commercially important species for aquaculture.

M. sanguinea lives in a rock block or between gravels mixed in tender deposit of upper and low intertidal region in the whole coast of South Korea and is well distributed around the world (Glasby and Hutchings 2010; Hutchings et al. 2012). The studies on the breeding of M. sanguinea were done by Imai (Imai 1975, 1976, 1981). Besides, the studies on feeding habit and inhabiting environment of adults (Prevedelli et al. 2007), on the early larval development (Imai 1982; Prevedelli et al. 2007), and on the salinity tolerance of juvenile (Garcês and Pereira 2011) were reported. However, the study on early nursery-stock cultivation for mass production of M. sanguinea was meager.

The aquaculture of M. sanguinea required the definite technologies for mass production and by supply of appropriated feed in each production stage. Unfortunately, no feed is placed on the market and only a pellet type of shrimp or finfish feed is used in the culture of M. sanguinea. It is not clear that efficiency of such feed is proper as a food source. It is uncertain how such feed affects growth and survival of M. sanguinea. Especially, survival of larva-juvenile until the third month is crucial to determine a seed production of M. sanguinea, and it is necessary to establish a reasonable food source and feeding rate to prevent a high mortality in early life stage. Hence, the purpose of this study was to examine survival rate and growth rate by testing various feeds to know appropriate feed, feeding rate, and supply rate in an early stage of nursery-stock production, the biggest fatal stage of M. sanguinea aquaculture.

Methods

To investigate an appropriate feed for early life stage of M. sanguinea, three different experiments were performed.

Experiment 1: preliminary survey with eight different feeds (20 days)

To investigate a suitable food source in early life stage of M. sanguinea, 15 ml of seawater filtered by membrane filter (GF/C) was put in 6-hole well plate (SPL Life Sciences Inc.), and 100 individuals of larvae were introduced in each hole. The larvae were produced in Fisheries Science and Technology Center of Pukyong National University. One microliter of diluted solution of 0.1 g of mud with 10 ml of filtered seawater was put in each hole as substrate to induce the planktonic larvae to do metamorphosis. Water temperature was maintained as 20 °C, and water was changed in 10 ml every 2 days. Without food source (control), Chaetoceros sp. (1 × 104 cells/ml), benthic diatom sp. (1 × 104 cells/ml), Chlorella powder (1 mg), Tetraselmis suecica (1 × 10 cells/ml), sea mustard (1 ml), decapsulated Artemia (1 mg), and extruded pellet (EP) for shrimp (1 mg) were used as food sources. Among them, decapsulated Artemia and EP for shrimp were started to supply after 6 days of introduction of larvae when they formed (Kim 2015). Feeds were supplied in every 2 days after renewal of seawater. The feeding tests were triplicated, and the experimental period was 20 days. The survival and growth rate were calculated at the end of the experiment. The living worms were counted, and the length was measured by taking 10 juveniles randomly in each experimental group for calculating the growth rate. The Dixi image program (Ver 2.89, Dixi optics) was used to measure the length of juveniles.

Experiment 2: investigation on the source and the amount of feed for early juvenile culture (for 2 months)

Five feed sources with four quantities in each (total 20 conditions) were tested. Decapsulated Artemia and shrimp feed showing the positive results in experiment 1 were also used in addition to eel feed. For microalgae, mixed microalgae solution sold on the market and benthic diatom species generally used for larva of invertebrate as a feed were selected (Table 1). For shrimp feed, eel feed, and decapsulated Artemia, four different quantities such as 5 mg/3000 inds, 10 mg/3000 inds, 50 mg/3000 inds, and 100 mg/3000 inds were supplied. For mixed microalgae and benthic diatom, 1 × 104 cells/3000 inds, 1 × 105 cells/3000 inds, 1 × 106 cells/3000 inds, and 1 × 107 cells/3000 inds were supplied. Nutritional contents of used feed are presented in Table 1. The feeding was done for every 2 days after 7 days from installation of experiment. Filtered seawater was renewed constantly, and remained food was not removed. The experimental periods were 60 days by reflecting the high mortality in early juvenile period. Number of setigers and survival rate of worms were examined at the end of experiment. A rearing tank (Fig. 1) was constituted with three reserves of 45 cm × 9 cm × 5 cm which allowed three replicates of experiments. Mud was used as a substrate with 1-cm depth. Experimental installations are shown in Fig. 2. The conditions of seawater were maintained at 20 ± 2 °C, 32–33 psu, pH 8.0, and 6–8 ppm of DO during the experimental period.

Table 1 Nutritional contents of feed used in this experiment and their ingredients
Full size table
Fig. 1
figure 1

Schematic diagram of rearing tank of M. sanguinea juveniles. Arrows indicate the flow of direction of running seawater

Full size image
Fig. 2
figure 2

Schematic diagram of experimental setup for experiments 2 and 3. Arrows indicate flow of direction of running seawater

Full size image

Experiment 3: more detailed investigation on the source and the amount of feed for juvenile cultivation (3 months of experiment)

Decapsulated Artemia and shrimp feed could be considered as a good efficient feed through the results of experiment 2; hence, a closer feeding amount ranges were investigated in the third experiment for 3 months. Three quantities such as 25 mg/3000 inds, 50 mg/3000 inds, and 75 mg/3000 inds of two feeds were reinstalled. The experimental conditions were similar to experiment 2.

Statistical analysis

Growth rate (GR) was measured with growth of body length in experiment 1.

GR (growth rate) = [(final body length − initial body length)/initial body length] × 100

For experiments 2 and 3, GR was measured with number of setigers.

Survival rate was calculated as

$$ \mathrm{SR}\ \left(\mathrm{survival}\ \mathrm{rate}\right)=\left(\mathrm{final}\ \mathrm{population}\ \mathrm{number}/\mathrm{initial}\ \mathrm{population}\ \mathrm{number}\right)\times 100 $$

The validation of each study section was made by two-way ANOVA test with squared transformed data, and the significant test was conducted with a p value of 0.05. The statistical analysis was performed with the Systat v.9 package.

Results

Experiment 1: a preliminary survey about appropriate feed source of an early larval/juvenile cultivation for nursery-stock cultivation

The decapsulated Artemia and shrimp feed showed the high growth (Fig. 3) and survival rate (Fig. 4). The growth rate (GR) of shrimp feed group was 451.5 ± 13.29% which was the highest, and decapsulated Artemia was 411.3 ± 6.94% at the end of 20-day experiment. The other experimental groups were showed similar or lower growth rate than the control group (without feed).

Fig. 3
figure 3

Growth rate (GR) of early larval culture with eight different food sources after 20 days. A, without food source (control); B, Chaetoceros sp. (1 × 104 cells/ml); C, benthic diatom sp. (1 × 104 cells/ml), D, Chlorella powder (1 mg); E, Tetraselmis suecica (1 × 10 cells/ml); F, sea mustard (1 ml); G, decapsulated Artemia (1 mg); and H, extruded pellet (EP) for shrimp (1 mg)

Full size image
Fig. 4
figure 4

Survival rates of early larval culture with eight different food sources after 20 days. A, without food source (control); B, Chaetoceros sp. (1 × 104 cells/1 ml); C, benthic diatom sp. (1 × 104 cells/1 ml); D, Chlorella powder (1 mg); E, Tetraselmis suecica (1 × 10 cells/1 ml); F, wakame (1 ml); G, decapsulated Artemia (1 mg); and H, extruded pellet (EP) for shrimp (1 mg)

Full size image

The survival rate (SR) was the highest in decapsulated Artemia with 61 ± 3.2%. Shrimp feed was followed with 51 ± 3%. The control group was 35 ± 4.6%. The lowest survival rate was 28 ± 3% of sea mustard which is lower than control (Fig. 4). The results of GR and SR were shown that decapsulated Artemia and shrimp feed were good candidate for feed of early juvenile stage.

Experiment 2: first survey for appropriate feed source of an early juvenile cultivation for nursery-stock cultivation (2 months of experiment)

Growth and survival rates of juveniles at the end of the experiment according to feeding amount with different feed types are shown in Table 2 and Figs. 5 and 6. In the survival rate of different feed sources, decapsulated Artemia showed the highest survival rate, followed by shrimp feed and eel feed. They showed higher SR than other two feed sources such as benthic diatom and mixed microalgae. Fifty milligrams of decapsulated Artemia showed the highest average survival rate with 33.88 ± 2.54%. Followed by 50 mg of eel feed 11.92 ± 3.28% and 50 mg of shrimp feed 11.81 ± 1.77%, 100 mg of decapsulated Artemia showed 11.62 ± 2.98%.

Table 2 Survival rate (SR) and number of setigers of M. sanguinea juvenile by feeds (2 months)
Full size table
Fig. 5
figure 5

Total survival rate of M. sanguinea juvenile according to the different feeds for 2 months

Full size image
Fig. 6
figure 6

Number of setigers of M. sanguinea juvenile according to the different feeds for 2 months

Full size image

In feeding amount, 50 mg showed the highest survival rate in general. Benthic diatom and mixed microalgae did not show survival individuals except in 1 × 107 cells (Fig. 5). Two-way ANOVA test showed the significant effect (p < 0.05) of feed and quantity of feed for survival rate of juvenile (Table 3). In growth presented with number of setigers, decapsulated Artemia and shrimp feed were higher than eel feed. Benthic diatom and mixed microalgae were showed 22.00 ± 3.07 setigers and 18.67 ± 3.06 setigers, respectively; however, they could not survive until 20 experimental days (Fig. 6). In decapsulated Artemia section with the highest growth rate, 100 mg/3000 inds section was 33.43 ± 3.2 setigers, and 5 mg/3000 inds section was 29.66 ± 3.65 setigers, but 50 mg/3000 inds section with the highest survival rate showed a low growth as 25.03 ± 0.32 setigers. In all sections, 100 mg/3000 inds section showed the highest growth and 50 mg/3000 inds section showed the highest survival rate (Table 2). Considering survival and growth, decapsulated Artemia 50 mg/3000 inds section showed good result.

Table 3 Two-way ANOVA test results of survival rate (SR) cultured in different feeds for M. sanguinea juveniles
Full size table

Two-way ANOVA test showed the non-significant effect (p < 0.05) of feed and quantity of feed for increase of setiger numbers of juvenile (Table 4). So the effects of feed and feed quantity were significant for survival rate, not for juvenile’s growth. Two-way ANOVA test was performed only with shrimp feed, eel feed, and decapsulated Artemia which give statistically enough numbers at 20 experimental days.

Table 4 Two-way ANOVA test results of setiger numbers cultured in different feeds for Marphysa sanguinea juveniles
Full size table

Experiment 3: final survey for appropriate feed source of an early juvenile cultivation for nursery-stock cultivation (3 months of experiment)

Decapsulated Artemia group showed higher survival rate than shrimp feed group on average (Table 5); 75 mg/3000 inds of decapsulated Artemia section showed 7.91 ± 1.28% of survival rate. Next, decapsulated Artemia 50 mg/3000 inds showed 4.36 ± 0.81%, and shrimp feed 75 mg/3000 inds section showed 3.99 ± 1.19% (Fig. 7). In feeding amount, 75 mg/3000 inds showed the highest survival rate in general. Two-way ANOVA test showed the non-significant effect (p > 0.05) of feed, significant effect (p < 0.05) of quantity of feed, and significant effect (p < 0.05) of interaction of two factors for survival rate of juvenile (Table 6).

Table 5 Survival rate (SR) and number of setigers of M. sanguinea juvenile after 3 months
Full size table
Fig. 7
figure 7

Total survival rate of M. sanguinea juvenile according to the different feeds for 3 months

Full size image
Table 6 Two-way ANOVA test results of survival rate (SR) cultured in different feeds for M. sanguinea juveniles
Full size table

In growth presented with number of setigers, decapsulated Artemia had more setigers than shrimp feed. Decapsulated Artemia showed the highest growth in 75 mg/3000 inds with 42.2 ± 8.31 setigers, followed by shrimp feed 75 mg/3000 inds section with 40.33 ± 5.94 setigers. In low amount of feed, growth was similar to shrimp feed and decapsulated Artemia. With high amount of feed, growth was higher in decapsulated Artemia than in shrimp feed (Fig. 8). Two-way ANOVA test showed that the non-significant effect (p > 0.05) of feed and significative effect (p < 0.05) of quantity of feed and significant effect (p < 0.05) of interaction of two factors for setiger numbers of juvenile (Table 7).

Fig. 8
figure 8

Number of setigers of M. sanguinea juvenile according to the feeds for 3 months

Full size image
Table 7 Two-way ANOVA test results of setiger numbers cultured in different feeds for Marphysa sanguinea juveniles
Full size table

Discussion and conclusions

Imai (1975) reported that mucilage secreted after 10–13 days from fertilization, later it was well agreed to the report of Kim and Jang (2008). In this experiment, we made an attempt of food supply from the seventh day of larval release, as per the report of Kim (2015), and observed that larvae ate organic matter with jaw plate from the seventh day. In the larval stage, like other lecitotrophic larvae, the worm cannot feed and only looking for appropriate settling place (Thorson 1950; Jablonski and Lutz 1983). The other polychaetes Nereis pelagica and Nereis grubei showed polyphagia, Perinereis cultrifera shows phytophagy feeding seaweed, and Perinereis nuntia showed polyphagia placing too much emphasizing on creophagy. Pereneris nuntia showed more than 80% of feeding efficiency with mixed feed of eel feed as powder in the Japanese aquaculture farm (Yoshida 1976). But only few reports are available on the early juvenile stage of M. sanguinea (Kim 2015).

In the pilot survey for 20 days, creophagy- and phytophagy-type feeds were tested. It was very clear that decapsulated Artemia and shrimp feed were reasonably good for early life stage of M. sanguinea culture and the other feed were shown similar level to the control group (without food) (Fig. 3). Similar result was reported by Nielsen et al. (1995), filter-feeding Nereis diversicolor grew on a diet of suspended algal cells, but the maximum specific growth rate was lower than the feeding experiments with shrimp meat. In the case of experiment 1, growth and survival rate of shrimp feed and decapsulated Artemia were higher than other feeding sources. Generally, 50 mg/3000 inds sections showed good survival rate in feeding amount of shrimp, eel, and decapsulated Artemia feeds. Decapsulated Artemia showed the highest growth rate. Studies performed by Vedel and Riisgård (1993) showed that N. diversicolor fed algal cells (Rhodomonas sp.) obtained specific growth rates of 3.1% comparable to 3.9% measured in worms in glass tubes placed 15 cm above the bottom in the eutrophicated Odense Fjord. The maximum specific growth rate of the facultative N. diversicolor fed algae is lower than 9% found for the obligate suspension-feeding blue mussel Mytilus edulis grown in nature in net bags (Riisgård and Poulsen 1981).

However, 50 mg/3000 inds section with the highest survival rate showed lower growth than other feeding amount sections. Both 5 mg/3000 inds and 10 mg/3000 inds sections were showed very low survival rate by insufficient food sources. It seems that there was a cannibalization in the low feeding amount sections. The other section 100 mg/3000 inds was also found with relatively low survival rate due to the degradation of water quality by the accumulation of unused feeds. In addition, there was already no survival being in adhesive diatom and microalgae sections, and it showed a very low survival rate below 0.2% of survival rate; thus, they could not play a role of food source for M. sanguinea. Hence, 50 mg/3000 inds of decapsulated Artemia was the best food and feeding amount for the first 2 months of nursery-stock cultivation.

Two-way ANOVA test showed significant effect of feed and feed quantity for survival rate. However, there was no significant difference in growth rate. The good survival rate was showed in decapsulated Artemia. Eel feed showed slightly higher survival rate than shrimp feed for M. sanguinea larvae. But growth rate was higher in shrimp feed and easier to control in water quality than in eel feed (Kim 2015). This is similar to the result of N. diversicolor, where the maximum specific growth rate was higher in shrimp feed (Nielsen et al. 1995). Hence, decapsulated Artemia and shrimp feed were chosen for experiment 3. The quantity of feed supplied was differentiated more and less 25 mg/3000 inds in the base of 50 mg/3000 inds. In the experiment 3, our results showed that enough amount of feed can help survival and growth rates, no matter what kind of feed is provided. However, decapsulated Artemia showed high survival rate and 75 mg/3000 inds section provided the best quantity of feed in the earlier life stage culture of M. sanguinea. Since there were no much studies on the larvae of M. sanguinea in Korea and other countries, our results were not extensively compared. However, further comparative studies are necessary to clarify the extent of feeding in the larvae of M. sanguinea.