A marine benthic diatom amphiprora sp. and its application in inducing mytilid adhesion

Abstract

The application belongs to the technical field of biology, and specifically discloses a marine benthic diatom Amphora sp. ECSMBd2408, which is classified as Amphora sp., is preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M2025972 and a preservation time of May 6, 2025. The application also discloses an application of the marine benthic diatom Amphora sp. ECSMBd2408 in inducing Mytilus coruscus attachment, and a biofilm formed by the Amphora sp. ECSMBd2408 has the activity of inducing Mytilus coruscus larvae and juvenile attachment, thereby providing a new scheme for improving the Mytilus coruscus attachment rate and the Mytilus coruscus yield.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of a marine benthic diatom in inducing attachment of juvenile and larval thick-shelled mussels. Specifically, it relates to the isolation, purification, identification and preservation of the marine benthic diatom ECSMBd2408 (Amphora sp.), and the inducing effect of the biofilm formed by it at different ages on the attachment of juvenile and larval thick-shelled mussels. Background Technology

[0002] Thick-shelled mussels (Mytilus coruscus), belonging to the phylum Mollusca, class Bivalvia, order Mytilodia, and family Mytilidae, are an important aquaculture species in my country, distributed along the coasts of the Yellow Sea, Bohai Sea, and East China Sea. Rich in nutrients, thick-shelled mussels possess significant economic value, promising aquaculture prospects, and market development potential. The life cycle of thick-shelled mussels includes two stages: planktonic and benthic. During the later stages of planktonic life, the eyespot larvae undergo irreversible attachment metamorphosis to transform into benthic juveniles, eventually developing into adults. During this developmental process and subsequent growth, if environmental conditions are unsuitable, the mussels can sever their byssal threads to select a suitable substrate for "secondary attachment." In this life cycle, metamorphosis requires successful attachment to a suitable substrate. During aquaculture, the metamorphosed juveniles need to be transferred to the sea area for cultivation, at which point secondary attachment is necessary. All these processes depend on the mussel's attachment ability. Therefore, the attachment success rate determines the metamorphosis success rate, which is the primary bottleneck in natural population breeding and artificial seedling production. Attachment behavior also directly affects the survival rate, growth performance, and final product specifications and quality of individuals during the breeding process.

[0003] The mussel farming industry is currently facing dual pressures: rising global sea temperatures and overexploitation of near-shore fishery resources. These factors not only directly threaten the physiological state of mussels but also, by altering the microenvironment of the attachment substrate, restrict their initial attachment and subsequent reattachment behaviors, becoming a bottleneck for the industry's sustainable development. Therefore, developing an environmentally friendly and efficient attachment induction technology is particularly urgent. Summary of the Invention

[0004] Benthic diatoms are an important component of the marine environment, possessing high temperature tolerance and an intrinsic connection with the feeding needs of mussel larvae. They hold promise for fundamentally improving attachment rates, thereby enhancing metamorphosis rates and seedling quality, and providing innovative technical support for addressing environmental challenges and steadily improving the thick-shelled mussel aquaculture industry.

[0005] The purpose of this invention is to provide a marine benthic diatom, *Diatomum bisporum*, and its application in inducing attachment in thick-shelled mussels, thus providing an environmentally friendly and efficient attachment induction technology for the artificial cultivation of thick-shelled mussels.

[0006] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a marine benthic diatom, Amphora sp. ECSMBd2408, which is taxonomically named Amphora sp. and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M2025972, deposited on May 6, 2025, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Secondly, the present invention provides the application of the above-mentioned marine benthic diatom Amphora sp. ECSMBd2408 in inducing attachment of thick-shelled mussels.

[0009] Furthermore, the biofilm formed by the marine benthic diatom Amphora sp. ECSMBd2408 has the activity of inducing attachment of thick-shelled mussel larvae and juvenile mussels.

[0010] Further, the specific induction method is as follows: Amphora sp. ECSMBd2408 is inoculated into f / 2 liquid medium and cultured to the exponential growth phase. After centrifugation, algal pellets are obtained, washed with sterile filtered seawater and made into a suspension. The suspension is diluted and inoculated onto the surface of an attachment substrate (such as a glass slide) to form a biofilm. The biofilm is then used to induce attachment of thick-shelled mussel larvae and juvenile mussels.

[0011] Furthermore, the biofilm culture conditions were as follows: water temperature range of 22±1℃, light range of 3000±500lx, photoperiod of 12L / 12D (12 hours of light, 12 hours of darkness), salinity of 30±1 (PSU), pH of 8.0~8.2, and culture time of 7~28 days.

[0012] Furthermore, when inducing attachment of thick-shelled mussel larvae, a biofilm cultured for 21 days was used. The induction time was 3–72 hours. Compared with the control group, the sedimentation and attachment effects were significantly improved. The induction effect was more significant starting at 48 hours, at which time the larval sedimentation rate reached 44.62%, which was significantly improved compared with 23.27% in the control group.

[0013] Furthermore, when inducing attachment of juvenile thick-shelled mussels, biofilms cultured for 7–28 days were used, and a certain attachment effect was obtained after 48 hours of induction. The induction activity of biofilms of different ages for juvenile mussels also varied. The biofilms formed after 21 days of culture had an attachment induction rate of 62.2% for juvenile mussels, which is a high induction activity and a significant improvement compared to the 24.4% of the control group.

[0014] The present invention has the following beneficial effects:

[0015] This invention marks the first application of the marine benthic diatom *Amphora sp.* ECSMBd2408 to induce attachment in thick-shelled mussels. The biofilm formed by this diatom exhibits a significant attachment-inducing effect on both larvae and juvenile mussels, providing a new solution for improving attachment rates and increasing mussel production. Furthermore, benthic diatoms, as a natural marine component, possess environmentally friendly characteristics, can adapt to changes in the aquaculture environment, and have significant application value and promising prospects for industrial promotion. Attached Figure Description

[0016] Figure 1 Comparison of the attachment rate of thick-shelled mussel larvae between the blank group and the *E. coli* ECSMBd2408 biofilm group.

[0017] Figure 2 The attachment rate of the biofilm of *Echinopsis lanceolata* ECSMBd2408 to juvenile thick-shelled mussels at different ages. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents used in the following embodiments are all commercially available conventional reagents, and the experimental procedures involved are all conventional procedures in the art unless otherwise specified.

[0019] Example 1

[0020] I. Isolation, purification and preservation of Amphora sp. ECSMBd2408

[0021] The Amphora sp. ECSMBd2408 involved in this invention is derived from a biofilm formed in the waters at a depth of 1-2 m in the thick-shelled mussel farming area of ​​Gouqi Island, Shengsi County, Zhejiang Province (122°46′E; 30°43′N). Pure algal colonies were isolated and purified using an f / 2 solid culture medium containing antibiotics and Na2SiO3: The obtained natural biofilm slides were placed in an f / 2 culture medium containing antibiotics (penicillin: 10 kU / ml, streptomycin: 10 mg / ml, gentamicin: 5 mg / ml) and Na2SiO3 and grown for one week. An appropriate amount of algal solution was then dropped onto a solid culture medium (1% agar) and evenly spread, and the culture was sealed until algal colonies appeared. The pure algal strain was then obtained through repeated purification and separation under a microscope. The obtained algal strain was then expanded using a culture medium containing antibiotics. After 18S rDNA gene sequencing of the pure bacterial colony and comparison with nucleic acid data in GENEBANK (http: / / www.ncbi.nlm.nih.gov / ), the data was uploaded to the NCBI database. It was finally determined that this diatom is a *Diatomia spp.*, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025972, deposit date May 6, 2025, and address 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0022] II. Induction experiment of Amphora sp. ECSMBd2408 on attachment of thick-shelled mussel larvae and juveniles

[0023] Biofilm preparation: Seawater was filtered through a 0.22 μm microporous membrane and autoclaved before use. Algal inoculum was inoculated into 500 mL Erlenmeyer flasks containing culture medium and cultured until the exponential growth phase (3–4 days). After culture, the diatoms were centrifuged at 3500 rpm for 15 min, the supernatant was discarded, and the precipitate was resuspended in autoclaved filtered seawater (AFSW) and centrifuged again, repeating this process three times. 50 mL of fresh AFSW was added to the algal precipitate after complete removal of the culture medium, and the diatom density was calculated using a hemocytometer. A sterile glass slide was placed in each culture dish, and AFSW and algal solution were added to the dish to achieve an initial diatom density of 1.0 × 10⁻⁶. 5 Cells / ml were cultured in a light incubator with a water temperature range of (22±1℃), a light range of (3000±500lx), a photoperiod of 12L / 12D, a salinity of 30±1, and a pH of 8.0~8.2 for 7 d, 14 d, 21 d, and 28 d to obtain biofilms of different ages.

[0024] Mussel attachment includes larval attachment and juvenile attachment. The following experiments demonstrate the induction of biofilm attachment to juvenile and larval attachment in thick-shelled mussels:

[0025] Twenty thick-shelled mussel larvae were placed on a glass slide with a 21-day-old biofilm in a petri dish containing sterilized filtered seawater. Larval behavior, specifically planktonic activity and attachment, was recorded at 3 h, 24 h, 48 h, and 72 h. A glass slide without a biofilm served as a blank control, and the same thick-shelled mussel larval attachment experiment was performed. The results showed that the biofilm formed by *Amphora* sp. ECSMBd2408 stimulated the settling and attachment of thick-shelled mussel larvae. Figure 1 As shown, compared with the control group, the biofilm of Amphora sp. ECSMBd2408 was able to settle significantly, with a settling rate of 44.62% after 48 hours, while the control group only had 23.27%.

[0026] Glass slides with biofilms of different ages (7 d, 14 d, 21 d, and 28 d) and 10 juvenile thick-shelled mussels (shell length 1.31 ± 0.02 mm; shell height 0.89 ± 0.02 mm) were placed in a petri dish containing sterile filtered seawater. The attachment rate of the juveniles was recorded at 48 h, i.e., the percentage of juveniles attached to the glass slide relative to the total number of juveniles in the petri dish. A glass slide without biofilm was used as a blank control, and the same thick-shelled mussel attachment experiment was performed. The results showed that the biofilm formed by *Amphora sp.* ECSMBd2408 had the activity of inducing the attachment of juvenile thick-shelled mussels. Figure 2 As shown, the biofilm formed at different ages exhibited varying induction activities for juvenile mussels. At 21 days of culture, the biofilm showed a high induction rate of 62.2% for juvenile attachment; at 28 days, the biofilm showed an induction rate of 58.8%, while the control group showed only 24.4%. Therefore, the biofilm formed by *Amphora sp. ECSMBd2408* has the effect of inducing juvenile attachment. These findings are significant for elucidating the attachment mechanism of juvenile thick-shelled mussels and for the artificial cultivation of these mussels.

[0027] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. A marine benthic diatom, *Diomysoma bipinnata*, characterized in that, The specimen, named Amphora sp. ECSMBd2408 and classified as Amphora sp., is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025972 on May 6, 2025.

2. The application of the marine benthic diatom Dipterocarpus as described in claim 1 in inducing attachment of thick-shelled mussels.

3. The application according to claim 2, characterized in that, The biofilm formed by the marine benthic diatom Amphora sp. ECSMBd2408 has the activity of inducing attachment of thick-shelled mussel larvae and juvenile mussels.

4. The application according to claim 3, characterized in that, The specific induction method is as follows: Amphora sp. ECSMBd2408 is inoculated into f / 2 liquid medium and cultured to the exponential growth phase. After centrifugation, algal pellets are obtained, washed with sterile filtered seawater and made into a suspension. The suspension is diluted and inoculated onto the surface of the attachment substrate to form a biofilm. The biofilm is then used to induce attachment of thick-shelled mussel larvae and juvenile mussels.

5. The application according to claim 4, characterized in that, The biofilm culture conditions were as follows: water temperature range of 22±1℃, light intensity range of 3000±500lx, photoperiod of 12L / 12D, salinity of 30±1, and pH of 8.0~8.

2.

6. The application according to claim 5, characterized in that, When inducing attachment of thick-shelled mussel larvae, a biofilm cultured for 21 days was used for 3–72 hours.

7. The application according to claim 6, characterized in that, When inducing attachment of thick-shelled mussel larvae, a biofilm cultured for 21 days was used for 48 hours.

8. The application according to claim 5, characterized in that, When inducing attachment of juvenile thick-shelled mussels, a biofilm induced by culturing for 7–28 days was used for 48 hours.

9. The application according to claim 8, characterized in that, When inducing attachment of juvenile thick-shelled mussels, a biofilm was induced by culturing for 21 days for 48 hours.

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