Optimized artificial seed production of Marphysa victori (Annelida, Eunicidae)

Abstract

The genus Marphysa (Annelida, Eunicidae) occurs worldwide in a wide variety of habitats and has an important role of controlling the benthic ecological environments as the second producer. This genus has long been in the spotlight for an important bait for fisheries and sports fishing, and the demand has increased rapidly in recent years since these species are considered an important nutrient source for the reproductive development of fish and shrimp in the aquaculture industry. In Korea, the genus Marphysa is the most valuable in terms of industrial and practical use, so researches on technological development on land-based artificial seedling production for mass production have been continuously conducted at the Fisheries Science and Technology Center since 2003. However, the development of technology that can be contributed to actual industrial production has been delayed due to the extreme drop in survival rate in the early stages of seed production. However, as the possibility of improving the survival rate was confirmed through the accumulated research results over a long period of time, it was possible to elucidate the root cause of this study and to present a simple and practical mass production technology. On the other hand, considering the ecological role of large benthic animals including this species, it is inevitable to think about the possibility of utilization for restoration and to improvement of the coastal benthic environment through mass production in the future. Therefore, the habitat characteristics of regional populations of blood worms distributed on the coast of Korea and their taxonomic review were conducted simultaneously.

1. Habitat ecology and classification of the genus Marphysa Environmental characteristics were surveyed on regional habitat of the high-value polychaete, the genus Marphysa which was sampled at the representative places of the South-eastern, the South-western, the West and Jeju-do coasts in Korea. Habitat ecology was simply described on the general local conditions such as topography, tidal current, substrate composition and so on. In order to classify regional populations of the genus Marphysa regional populations, samples collected from the surveys on habitat ecology were commissioned to specialized organizations for species identification.

1.1. Ecological characteristics of blood worm habitats The population of blood worms was divided according to their habitat regions, in the comparison of ecological characteristics of habitats in the key coastal areas, that is, the southeastern coastal area, the southwestern coastal area, the western coastal area, and the coastal area of Jeju-do, which were well known for the distribution of high-valued blood worms.

▷ Lower intertidal zone habitats Ecological characteristics are similar among the populations distributed in lower intertidal zone habitats in the southeastern coastal area (Deokho-ri, Geoje-si, Gyeongsangnam-do), in the southwestern coastal area (Geumdang-do, Wando-gun, Jeollanam-do), in the western coastal area (Iwon-myeon, Taean-gun, Chungcheongnam-do), and in the coastal area of Jeju-do (Biyang-do, Hallim-eup). In the light of oceanogeography, the characteristics of the surveyed coastal areas are muddy sand formed around neighboring rock beds by good sea flow exchanges and by the development of shielded places, and also an abundance of sediments with lots of organic substances and with seaweeds such as green algae.

▷ Subtidal benthic habitats The benthic water around the southwestern coastal area (Sahu-do, Wando-gun, Jeollanam-do) has a wide tidal flat with a length of about 7 meters. It also has a comparatively smooth physical flows, and has the characteristics of a good screen-shield from surrounding islands and of semi-closed embayment abundant in supplies of nutrients. Different age populations are simultaneously distributed including juveniles in the fine tidal sediment not yet affected by pollution. This area has a role of breeding habitats suitable for the reproduction of the population, and needs to be examined on the ecological characteristics of distribution in the light of survival strategy in comparison with the population inhabiting intertidal zone habitats in its vicinity.

1.2. Classification of blood worm polychaetes from Korean coastal waters Despite the species abundance of the genus Marphysa, taxonomic records from Korean waters had only two species, M. sanguinea and M. tamurai Okuda, 1934 to date (Paik, 1989). Until now, the domestically dominant opinion is that species reported as M. sanguinea has different regional populations in different habitat conditions. Their names are also so diverse that they are called M. sanguinea representing Honmusi, Hongeosi, Galgasi, etc. Therefore, this study described the genus Marphysa species complex from the Korean coastal areas based on both morphological observation and DNA barcode analysis, as the information can be quoted and applied.

v Morphological identification of regional specimens Until now, M. sanguinea complex has also attracted great attention in Korea, as it is a commercially high-valued species. Specimens were selected from three regional samples (Taean-gun, Geoje-si and Jeju-do) collected in lower intertidal zone of Korean waters, fixed with 95% ethyl alcohol for both morphological and genetic analyses. Samples for morphological analysis were sent for an analysis in the National Marine Biodiversity Institute of Korea. In morphological description, it was characterized by having four types of pectinate chaetae, appearance of pectinate chaetae from chaetiger 2, both pectinate chaetae and compound spinigers, and pygidium with only one pair of pygidial cirri. Maxillary apparatus with four pairs and one single of maxillae. The specimens that were newly found in Korean waters showed the several morphological characteristics that generally agreed well with those from its type locality that was originally described as Marphysa victori sp. n. by Lavesque et al., (2017) based on specimens collected from Arcachon Bay in the west of France. However, Korean specimens showed minor differences in morphology from the original description. The subacicular hooks were present in the Korean specimens, while they were absent from the original description. In addition, the Korean specimens differed from the original ones in the Maxillary formula (1+1, 5+5, 5-6+6-7, 3-4+0, 1+1 for French specimen’s vs 1+1. 4+4, 5-6+0, 3-4+6-7, 1+1 for Korean ones). Branchiae pectinate beginning on chaetiger 32 (29–36); number of branchial filaments 1–3 in first chaetigers, maximum number of branchial filaments 6 in mid-body: these branchial characteristics have been presented as a valid key from experts in the classification on the morphological species description, but it was observed that it is difficult to distinguish the differences between species in young individuals of 1-2 years old. Further taxonomic examination is required for these morphological variations.

v Molecular identification of regional specimens A gene analysis was entrusted to a professional company on population samples from different regions surveyed on the ecology of habitats, as there was some confusion over the morphological classification of blood worm polychaete, M. sanguinea, which drew the most attention and reports among commercial species. All specimens of the genus Marphysa which were sampled at the six different places of the South-eastern, the South-western, the West and Jeju-do coasts in Korea, were uesd to obtain cytochrome oxidase I (COI) gene sequences and compared to them to sequences stored in Genbank (NCBI) for the species identification.

▷ Analysis of intertidal habitat specimens COI gene was successfully sequenced and published at NCBI GenBank for the eleven specimens sampled at the intertidal areas of the South-eastern coast (four specimens; A-Geoje-si, S-FSTC), the South-western coast (two specimens; Y-Geumdang-do), the West coast (two specimens; T-Taean-gun), and the Jeju-do coast (three specimens; J-Biyang-do) of Korea near the locality type. Their lengths were identical and 964 bp in total. The result of BLAST searches showed the highest similarity of 98.9~99.7% to M. victori, and a relatively low similarity of 87.2~87.5% to M. sanguinea. The DNA barcode sequences of the mitochondrial COI gene distinguished all specimens M. victori from other species with sequences available in GenBank.

▷ Analysis of subtidal habitat specimens A molecular identification was conducted based on the mitochondrial COI gene for the eleven specimens sampled at the subtidal areas of the South-western coasts neiboring Sahu-do, Wando-gun (six specimens; SH) and the Holtong beach, Hyeonkyong-myeon, Muan-gun (five specimens; SA). The mitochondrial COI DNA sequences of eleven subtidal specimens showed their lengths to be identical and 1,023 bp in total. The genetic similarity of their mitochondrial COI genes was comparatively high at 96.9%~100%. The genetic similarity of SA01~SA05 (Hyeonkyong-myeon, Muan-gun, Jeollanam-do) was 99.8~100%. The genetic similarity of SH01-S~SH03-S, SH04-L~SH06-L (Sahu-do, Wando-gun) was the highest at 99.7~100%. The genetic similarity between specimens from the two regions was the lowest at 96.9~99.4%. The result of BLAST searches in NCBI showed the highest genetic similarity of 96.96~99.90% to M. sanguinea. On the other hand, SA01~SA05 (Holtong beach, Hyeonkyong-myeon, Muan-gun) showed the highest similarity of 99.80~99.90%, and SH04-L~SH06-L (Sahu-do, Wando-gun) showed a relatively low similarity of 96.96~97.06%. Molecular phylogenetic tree based on mitochondrial COI genes of eleven specimens from the subtidal areas of two regions is shown in Fig. 15. These analyzed specimens were supported by bootstrap values of 99%, being branched together with M. sanguinea (NC-023124), and separated molecular phylogenetically from the intertidal specimens. However, a considerable inter-regional phylogenetic difference was found between specimens of Hotong beach (Haeje-myeon, Muan-gun) and Sahu-do (Wando-gun). On the other hand, no intra-regional difference was found between small-sized specimens (SH01-S~SH03-S) and large-sized specimens (SH04-L~SH06-L) from the subtidal area near to Sahu-do, Wando-gun, Jeollanam-do. In conclusion, the specimens collected from the lower intertidal zone in the southern and western coastal area and the coastal area of Jeju-do were identified as M. victori. On the other hand, the specimens distributed in bottom mud flats in the subtidal zone (neighboring Sahu-do, Wando-gun) and in muddy sand flats in lower intertidal to subtidal areas (Holtong beach, Muan-gun) were found to be identical with M. sanguinea. These newly described M. victori and M sanguinea in the Korean coasts may also have chosen and been adapted to ecological environments favorable for its breeding and survival strategy for hundreds of millions of years. Given the spawning and breeding characteristics of marine invertebrates and induction of settlement in meroplankton, their habitats and breeding areas may have been chosen among places favorable for the recruitment and settlement of population in terms of the physicochemical characteristics such as tidal currents, substrates, inflow of food source, etc. rather than in terms of the habitat types.

2. Reproductive Biology for Artificial Seed Production of Marphysa victori In general, the propagation of vertebrates and invertebrates occurs when conditions such as temperature, light, precipitation, and food are favorable for the maximization of the chances of survival of their offspring. The environmental factors for the initial development in the ocean are direct and indirect influential factors that determine the ecology of organisms. Whereas some of the developmental processes show a spatiotemporal variability, the release of most invertebrates shows periodicity resulting from the circadian rhythm, circatidal rhythm, and circasemilunar rhythm that show simultaneity.

2.1. Environmental characteristics on larvae release Most of the existing studies on the correlation between the reproductive cycles of invertebrates and the influential factors were conducted through field collection, and no study has reported the correlation between artificial larval release conditions and tides. Given the process of artificial seedling production in which floating larvae released by mothers (mother worms) are collected, reared, and managed, a lot of more efficient information on larval release is necessary. Therefore, in this study, a terrestrial aquaculture system was used to mainly investigate the larval release rates to determine the artificial spawning and larval release cycles of Marphysa victori, and the circasemilunar rhythm of tidal water levels, sea level pressure, and many other environmental influential factors were considered to analyze the environmental influential factors for the foregoing thereby analyzing the correlation between the foregoing and the environmental influential factors.

2.1.1. Reproductive cycle and fertilized egg care in burrow When monthly changes of reproductive cells of M. victori were observed, the reproductive cycle of this worms was divided into multiplicative stage (December to February), growing stage (January to April), mature stage (March to May), spawning stage (May to August) and degenerative and resting stage (September to November). In order to observe the laying behavior and larval release process of the breeder worms, the eggs scattered in the habitats were identified while breeding and managing in a transparent acrylic tank. The breeder adult showed the behavior of circulating the seawater in the habitat by continuously sweeping and rolling the larvae using brush like branchie through the peristaltic movement. This type of care continued for six days. During this time, mother worms was able to move their bodies within the habitat, and they could move the larvae under management to the desired position. When all the larvae had developed into postlarvae, the mother released them. During the released, the tail with pigidium was used to release the larvae strongly into the outer water column along with the peristaltic movement of the body.

2.1.2. Yearly larvae production and water temperature In the summer natural sea flow through system (NSW) and the winter semi-circulating water temperature control system (TRSW) in Fisheries Science and Technology Center, the larval release of M. victori for 4 years from 2015 to 2018 and corresponding water temperature changes were observed. The water temperature range from spawning to larval release varied depending on the year, but was up to 17~30℃, and the proper water temperature for larval release showed the range of 18.5-21.5℃.

2.1.3. Yearly and annual monthly different larvae production The total larval production by year showed a difference by the biennial pattern between odd years with large numbers (2015, 2017) and even years (2016, 2018) with the lower numbers. This trend has been consistent in the same production site since 2010, and the odd emergent years tended to have an intensive synchronous release (90.01%) early in seedling production (mid-May to early June), whereas even years tended to have a divergent release over the entire period.

2.1.4. Characteristics of larval release by seawater level The amount of larvae collected at the dawn of each day was recorded in the adult tank by the continuous running of natural seawater (NSW) and the semi-recirculating adult tank (TRSW) through artificially winter temperature control. From 2015 to 18, when data was analyzed based on observations of nearby tide levels, larval release was found to be related to the tidal cycle, despite being a terrestrial water tank. There were more larval releases at the mid water level period between the low water period (neap tide) and the high water period (spring tide). The larval release rate showed a tendency to increase remarkably when 3-4 days passed after the high tide period or the low tide period. When the variations of tidal water levels were observed centering on the larval release time (4:00~6:00, 5:00-7:00 am) in order to check the larval release rates by time zone, it was found that the larval release rate showed a tendency to increase around the tide level depending on the phase of tidal time cycle. In other words, larval release occured at the high tide time at the time when the tidal range increased from the low water period (neap tide) to the high water period (spring tide). On the other hand, when the tidal range decreased from spring tide to neap tide, there was a clear tendency to release a large amount of larvae at the time of low water. In particular, it was clearly demonstrated that this pattern appeared throughout the production period by the revised results of reviewing larval release time and tide level since 2010.

2.1.5. Tidal cycle selectivity of larval release according to the semilunar rhythm Rhythmic patterns of larval releases from accumulated relative percentages of larvae released from 2015 to 2018, were appeared to show two clear rhythms that were peaked mostly at 4-6 and 10-13 tides in natural seawater in summer, but not clear pattern in 2016, and at 3-5 and 9-11 tides in semi-recirculation system with TRSW in winter. On reviewing the pattern of changes in the larval release rates during the four to six circasemilunar rhythms (14.8 days) during the larval release period, it could be seen that the larval release with clear synchronized patterns showed at the first and 2nd rhythms. As the temperature increased toward the end of seedling production, not only did the larval release tend to be decrease as the rhythms passed, but also the semilunar rhythm became unclear. In the result of accumulating total larval production over the four years for each tidal time in NSW-summer and TRSW-winter, there was a tendency to show a semilunar rhythmic pattern, in which the first synchronized semilunar rhythm between neap tide and spring tide showed a clear peak at 3-5 tidal time which was exact intermediate tide. However, the second synchronized rhythm, which was proceeded from ST to NT, did not show a distinct peak in natural seawater (NSW), but it spread. Also in the water temperature regulated semi-recirculation system (TRSW), it showed the overall predominance ranges with slightly wider in the mid-term tides of 8-12 tidal time. In conclusion, although environmental factors affecting the spawning of M. victori were diverse, this study found that larval release rates showed differences due to the effects of tides, circasemilunar rhythms, and sea level pressure. This indicates that changes in water levels caused by various influential factors have important effects on larval release rates. These results are important for the establishment of seedling production technology in M. victori aquaculture, and it is judged that predictable and effective seedling production technology can be secured using the findings of this study.

2.2. Productivity enhancement by establishment of convinient technology for artificial seed production of M. victori The polychaete worm culture is divided into seedling, rearing and culture systems. In the field of seedling production and aquaculture technology, development of effective seed production technology is necessary for the settlement of the worm farming industry. Due to a lack of researches on the high-valued polychaete worms including the genus Marphysa in Korea, a research for the seed production of the polychaete worm has been ongoing since 2003 (Pukyong National Fisheries Science and Technology Center) with the aim of its industrializing and utilizing. In the course of this study, the high mortality rate of about two months from the larval stage in seedling production makes it difficult to industrialize worm culture. In particular, the low survival rate in the early stages of larval stage is related to the environmental problems in the feeding tank related to the feeding, and the breeding environment is deteriorated due to the deterioration of the feed source. In order to realize industrialization through the establishment of full farming technology, it is judged that systematic step-by-step technology development through intermediate fostering and nurturing based on stable seed supply is essential in the management of worms.

2.2.1. Productivity enhancement by using artificial enriched seawater (AESW) 1) Improvement of survival rate increase through rearing in AESW In order to find the cause of deterioration of the survival rate of the early life stage of M. victori, the nutritionally enhanced seawater medium ASP2 (NTA) containing similarly various major elements contained in seawater was selected and used by slightly modifying (ASP2m). The growth pattern of the segment showed similar results. After 36 days' rearing, juveniles showed a higher number of segments at ASP2m with 13.88 ± 0.93 than those of NSW with 12.16 ± 1.07. However, there was no significant difference between two groups. On the other hand, ASP2m showed a surprisingly high survival rate (64.7 ± 8.03%) until 1 month passed. In contrast to natural seawater, not only in terms of individual growth, but also in survival rate, it can be judged as a result of fully utilizing many energy sources necessary for growth and survival in artificial seawater.

2) Effect of mineral supplement on growth and survival rate improvement By showing a significantly higher survival rate in artificial nutrient-enriched seawater (ASP2m), in order to find out which elements contained in artificial nutrient-enriched seawater are used as energy sources to increase the survival rate, trace elements were firstly investigated that were likely to be limiting factors in seawater. When a 30-day experiment was performed to confirm the effect of adding trace elements PII metal and FeCl3, the survival rate over 30 days showed a significant difference between the PII metal-added section (38.47±0,29%) and the non-added section (3.14%, 1.68%). In other words, it was judged that the survival rate was not secured unless the macro elements and trace elements were faithfully present. Also experiments of the supplement effect on S2 trace metal separately (ASPp+S2) and mixed with PII metal (ASPp+PII+S2) were conducted for 2 months. Overall survival decreased after 2 months, but ASPn was (59.1±4.1%), ASPp(+PII+S2) was 20.0±1.3%, ASPp and ASPp+S2 were 14.7%, 11.6%. In order of survival rate, ASPn(ASP2m), ASPp+PII+S2, ASPp+PII (~ASPp+S2), ASPp, NSW, and so on. Eventually, it was found that the more insufficient these trace elements were, the more the survival rate decreased over time.

2.2.2. Simple and practical high-efficiency seed production technology development 1) Simple and efficient seedling productivity by adding the salt soup Considering the experimental results so far, it was judged that it would be the most convenient and practical method to dilute and use a solution of natural salt concentrated with natural seawater. Therefore, after high-temperature sterilization of natural seawater, which had the lowest survival rate so far, salt soup was added thereto to conduct an experiment to find an appropriate concentration range. During the period from 2 months to 3 months, the concentration section of 0.01-1 ppm showed faster growth than the other lower sections (0.001 ppm and control) and showed a significant difference (P<0.05). Among the concentration sections, the most effective was the 1 ppm section, which showed a survival rate of 53.48±3.8%, and did not show a significant difference from the ASP2m (60.52±1.3%).

2) Exploring the proper feeding of biofloc as an initial food source Effects of different amounts of biofloc feeding on segment numbers in the result changes in the early juvenile stage of M. victori were observed for 60 days, and the result suggested that the supply of biofloc cultured in salted water as a food source had a significant effect on segment proliferation in the early juvenile stage of seed production. Like segment growth, the results showed that the highest survival rate of 72.3 ± 8.6 % corresponding to 9% of biofloc feeding. Next was 62.56 ± 6.6 % and 46.50 ± 6.2 % of survival rate for 6 and 3% of biofloc feeding, respectively. High growth and survival rates in the high-concentration biofloc supply section indicated that the limiting factors in the nutritional aspects of the initial growth process were hardly affected. It was estimated that the mineral components contained in the medium, various metabolites generated by microorganisms decomposing organic feeds, biofloc itself, etc. were used as metabolic energy sources and food sources for juveniles on settlement process in the early life cycle stage of M. victori.

3) Guide line preparation for intensive seed production based on simple and efficient techniques Separation of harmful or grazing organisms as much as possible after mass collection of larvae is the most important process among biological treatments necessary to secure the initial survival rate. However, in a mass production site where repetitive larvae collection and breeding management are conducted daily, it is difficult for even an expert to perform a perfect process, so a simple and efficient countermeasure is required for intensive seed production.

(1) Effects on segment growth by larval stocking days before injection in the rearing system After the larvae were collected, they were usually inoculated into a breeding tank soon. However, considering that postlarvae does not have a long floating time before settlement and has lethithotrophic characteristics, it is suggested that maintaining a suitable environment for a certain period after settlement will be effective for the overall seed production process. So, an effect of different maintenance (stock) culture days in ASP2m medium before inoculation into breeding tank was observed at first. There was no significant difference in segment growth in all sections until 1 month later, and after 2 months, it was higher in the 3 days’ stock culture section than in other sections.

(2) Effects on survival rate by larval stocking days before injection in the rearing system As a result of culturing in ASP2m for 2 months (60 days), there was no significant difference in all sections, and the average survival rate was 70.93%. After 4 months, it was significantly higher in the 5th and 7th intervals (69.01±2.7%, 66.61±3.8%) and averaged 61.68%. After 6 months, it was the highest at 68.51±2.268.5% in the 5-day section, with an average of 54.02%. Overall, it was confirmed that the low survival rate was shown in the 3rd and 9th sections, while the high survival rate was shown in the 5th and 7th sections. These results are contrary to segment growth, and ultimately, it is most important to ensure their survival rate during the early juvenile stage of inhabiting the substrate surface (for 2 months). After their submerging into the substrate, it can be said that the key to efficient seedling production is to supply the energy sources necessary for growth and development so as not to be insufficient.

(3) Guidelines presentation for simple and efficient artificial seed production of M. victori By combining the techniques obtained through various experiments for artificial seedling production, now it is possible to present guidelines for simple and efficient mass seedling production. In the early life stage of M. victori, young juveniles undergo a process of selective settlement and metamorphosis in the surface matrix for about 2 months corresponding with other predators' grazing, interspecies competition for limited space, and various physical factors such as hydrodynamic, pressure, gravity, light cues, etc. In this process, it was assumed that juveniles evolved in the direction of using more energy to form a tube to defend themselves from attack or exposure rather than preferentially securing a food source for survival. In other words, it was suggested that during this period, energy sources for exercise were absolutely needed much more in quantity rather than in growth. Therefore, by adding natural salt or saline solution containing various minerals contained in seawater, juvenile worms were able to absorb and utilize necessary elements, thereby increasing the initial survival rate. Here is presented as a guideline for simple, efficient and economic artificial seed production of M. victori (Fig. 1).

Fig. 1. Abstract of guidelines showing changes in survival rates for 2 months culture with supply of artificial nutrient-enriched seawater (NESW) and with substitution of natural seawater flow through on the 4th and 6th month.