Use of a pseudomonas alternaria in inhibiting settlement of sea squirts

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to application of a pseudomonas alternaria in inhibition of sea squirt adhesion. The application relates to application of a pseudomonas alternaria in inhibition of sea squirt adhesion. The strain of the application has the function of inhibiting adhesion of fouling sea squirt larvae, can be applied to preparation of marine microbial functional strains, has certain economic benefits and ecological benefits, and therefore has the potential to be applied to prevention and treatment of sea squirt fouling formation.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of a pseudoalternating monocytogenes bacterium in inhibiting sea squirt adhesion. Background Technology

[0002] Marine microbial communities account for over 80% of Earth's biomass and play a vital ecological role (Wang et al., 2022). Furthermore, research indicates that marine bacteria play crucial roles in the life cycles of various invertebrates (McFall-Ngai et al., 2000). *Pseudomonas pseudoalteromonas* (…) Pseudoalteromonas Commonly found in various marine environments (Wietz et al., 2010; Salazar et al., 2016), *Pseudomonas* often live in symbiosis with eukaryotic hosts, including marine phytoplankton, sponges, mussels, scallops, tunicates, and corals (Zeng et al., 2017). Studies have shown that biofilms formed by *Pseudomonas* can inhibit the attachment of various invertebrates, such as *Dobretsov S et al., 2007*, mussels (*Annapoorna et al., 2010; Zeng et al., 2017*), *Hymenia spp.*, and barnacles (*Holmström C et al., 2002*). Furthermore, certain strains of *Pseudomonas* can promote the attachment of sea urchin larvae (Megan J. Huggett et al., 2006).

[0003] In the marine environment, all submerged surfaces (natural or artificial) are rapidly colonized by a series of organisms, resulting in what is known as "biofouling." Organic and inorganic molecules cover the surface, forming a regulatory film. Bacteria adhere to the regulatory film, forming a biofilm. Eventually, invertebrate larvae attach to the biofilm, resulting in biofouling (Mieszkin et al., 2013). Marine biofouling is a global problem that causes huge economic losses to the marine industry and also poses environmental problems (Katherine A et al., 2011).

[0004] However, the inhibitory effect of bacteria on larval attachment is often highly species-specific. Larvae of different fouling species may respond significantly differently to the same bacterial strain or its metabolites, even exhibiting opposite effects (Holmström, C., et al., 1996; Holmström, C., et al., 2002). This means that a microorganism or metabolite that can effectively inhibit one fouling species may not necessarily have the same inhibitory effect on another. Furthermore, existing studies on the inhibitory effects of *Pseudomonas* on attachment have mostly focused on fouling organisms such as barnacles, tubeworms, and mussels, and have largely failed to conduct functional verification on typical tunicate fouling organisms that are increasingly causing harm.

[0005] Marine fouling organisms are diverse, with sea squirts being a typical example. Belonging to the phylum Chordata, subphylum Caudata, they are numerous (Shenkar N et al., 2011). They typically attach themselves to various marine substrate surfaces, including ship hulls, rocks, aquaculture facilities, and other materials, competing with farmed species for oxygen and nutrients, leading to decreased aquaculture yields (Murray CC et al., 2012). This has resulted in numerous biofouling problems and significant economic losses (Aldred N et al., 2014). More seriously, sea squirts are highly mobile, often becoming invasive species as they are transported to new habitats by ships (Cheng et al., 2021). Therefore, there is an urgent need to screen for non-toxic strains that inhibit the attachment and metamorphosis of sea squirt larvae, reducing sea squirt adhesion and providing support for the prevention and control of sea squirt fouling in my country. Summary of the Invention

[0006] The purpose of this invention is to provide an application of *Pseudomonas alterniflora* in inhibiting sea squirt adhesion.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Application of a pseudoalternating monocytogenes in inhibiting sea squirt adhesion.

[0008] The application of the pseudoalternating monocytogenes in inhibiting sea squirt attachment metamorphosis.

[0009] Application of the pseudoalternating monoclonal bacteria in inhibiting attachment metamorphosis in sea squirt larvae.

[0010] The pseudoalternating monoclonal bacteria is pseudoalternating monoclonal bacteria ( Pseudoalteromonas spongiae MP_PS-3.

[0011] The sea squirt larvae mentioned are *Sarcoidea* larvae. Ciona savignyi larva.

[0012] An inhibitory formulation for inhibiting sea squirt adhesion, the formulation containing the aforementioned *Pseudomonas pseudoalteromonas* ( Pseudoalteromonas spongiae MP_PS-3.

[0013] The preparation is the aforementioned *Pseudomonas alterniflora* (…). Pseudoalteromonas spongiae MP_PS-3 fermentation products.

[0014] An inhibitory formulation for inhibiting sea squirt adhesion, and the application of the formulation in inhibiting sea squirt adhesion.

[0015] Advantages of this invention: This invention uses *Pseudomonas aeruginosa* (… Pseudoalteromonas spongiae MP_PS-3 effectively inhibits the attachment of sea squirt larvae, blocking their metamorphic development and preventing the larvae from completing the transition from the planktonic stage to the sessile adult. This significantly inhibits the attachment metamorphosis of sea squirt larvae, thus achieving fundamental control over sea squirt fouling. Furthermore, this strain is safe, non-toxic, and easy to cultivate, making it suitable for further research and development of technologies to control sea squirt fouling. The *Pseudomonas aeruginosa* MP_PS-3 possesses the function of inhibiting the attachment metamorphosis of fouling sea squirt larvae, and can be applied to the preparation of marine microbial functional agents, demonstrating certain economic and ecological benefits and potential for controlling sea squirt fouling. Attached Figure Description

[0016] Figure 1 It is Pseudomonas alterniflora ( Pseudoalteromonas spongiae ) Fluorescence micrograph of biological membrane on MP_PS-3 (1000×).

[0017] Figure 2 The images show the condition of the control and experimental groups of larvae (18 hpf) after 24 h under a dissecting microscope (2×).

[0018] Figure 3 It is Pseudomonas alterniflora ( Pseudoalteromonas spongiae MP_PS-3 against Sassiniformes ( Zion savigny The effect of larval attachment rate.

[0019] Figure 4 It is Pseudomonas alterniflora ( Pseudoalteromonas spongiae MP_PS-3 against Sassiniformes ( Zion savigny The effect of larval metamorphosis rate.

[0020] Figure 5 After placing swimming larvae for different periods of time, *Pseudomonas aeruginosa* (…) Pseudoalteromonas spongiae MP_PS-3 for Sassiniformes ( Ciona savignyi The effect of larval metamorphosis rate. Detailed Implementation

[0021] The following is a further elaboration of the present invention with reference to specific implementation examples, but it is not intended to limit the invention.

[0022] The following examples are of Pseudoalteromonas ( Pseudoalteromonas spongiae MP_PS-3, this strain was isolated by Professor Zhang Weipeng's team, and the related research was published in iMeta in 2024 under the title "Bioprospecting of culturable marine biofilm bacteria for novel antimicrobial peptides"; and was obtained as a gift from Professor Zhang Weipeng's team.

[0023] Example 1: 1. Activation of the strain: The pseudoalternating monoclonal bacteria MP_PS-3, stored in glycerol at -80 ℃, was taken out, thawed at room temperature, mixed by inversion, and 100 µl of bacterial suspension was spread on 2216 E solid medium and placed in an incubator at 25 ℃ for incubation. Single colonies were picked and streaked on plates for later use.

[0024] The components of the 2216E solid culture medium are as follows: The composition of 2216E solid culture medium is as follows: 5 g peptone, 1 g yeast extract, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.98 g magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium carbonate, 0.08 g potassium bromide, 0.034 g strontium chloride, 0.022 g boric acid, 0.004 g sodium silicate, 0.0024 g sodium fluoride, 0.0016 g ammonium nitrate, 0.008 g disodium hydrogen phosphate, 15 g agar, and 1000 mL ddH2O. Mix the above culture medium components, adjust the pH to 7.6 ± 0.2, and sterilize.

[0025] 2. Preparation of Pseudomonas alterniflora MP_PS-3 bacterial suspension Pick a single colony of Pseudomonas alterniflora MP_PS-3 from the plate and inoculate it into 20 ml of 2216E liquid medium. Incubate in a shaker (25℃, 220 rpm) for later use.

[0026] The components of the 2216E liquid culture medium are as follows: The composition of 2216E solid culture medium is as follows: 5 g peptone, 1 g yeast extract, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.98 g magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium carbonate, 0.08 g potassium bromide, 0.034 g strontium chloride, 0.022 g boric acid, 0.004 g sodium silicate, 0.0024 g sodium fluoride, 0.0016 g ammonium nitrate, 0.008 g disodium hydrogen phosphate, and 1000 mL ddH2O. Mix the above components, adjust the pH to 7.6 ± 0.2, and sterilize.

[0027] 3. Determination of MP_PS-3 cell concentration in Pseudomonas pseudoalteromonas The cultured bacterial suspension was centrifuged at 1600 xg for 15 min, the supernatant was discarded, and the suspension was resuspended in sterile filtered seawater (filtered through a 0.2 μm membrane). This process was repeated three times to remove any residual liquid culture medium. Finally, the bacterial precipitate was dissolved in 10 ml of sterile filtered seawater, and 200 μL was transferred to a 96-well plate. The OD value of the bacterial suspension at 610 nm was measured using a microplate reader. The absorbance of the bacterial suspension was 0.6–0.8, and the bacterial cell concentration was 1 × 10⁻⁶. 8 Approximately 1 cell / mL.

[0028] 4. Preparation of Pseudoalteromonas MP_PS-3 biofilm Take 1 mL of the *Pseudomonas aeruginosa* MP_PS-3 bacterial suspension dissolved in sterile filtered seawater from step 3 into a 35 mm sterile petri dish, and add 4 mL of peptone containing 0.5%. Incubate in a sterile incubator for 48 h to obtain a *Pseudomonas aeruginosa* MP_PS-3 biofilm. As shown in Figure 1, the bacteria are densely distributed, indicating that *Pseudomonas aeruginosa* MP_PS-3 can form a biofilm well in the petri dish.

[0029] 5. Pseudorabies MP_PS-3 inhibits larval attachment. The culture dishes with biofilm formation were gently rinsed with sterile filtered seawater, and 5 mL of fresh sterile filtered seawater was added. 200 fertilized sea squirt swimming larvae (18 h old) were then added. A clean sterile culture dish with sterile filtered seawater was used as a control. Each group had three replicates. After 24 h, the attachment and metamorphosis of the sea squirt larvae were recorded.

[0030] The results are as follows Figure 2 As shown, the pseudoalternating monotypic bacteria MP_PS-3 biofilm group showed almost no attachment of sea squirt larvae.

[0031] Simultaneously, the counting results were observed through a stereoscope. Figure 3As shown, in the *Pseudomonas alterniflora* MP_PS-3 biofilm group, the attachment rate of sea squirt larvae was close to 0, significantly lower than that in the control group. Sea squirt larva attachment is crucial for sea squirt growth and development, and the biofilm significantly inhibited larval attachment. Furthermore, observations 24 h after larval exposure to the biofilm revealed that the metamorphosis rate of sea squirt larvae in the *Pseudomonas alterniflora* MP_PS-3 biofilm group (43%) was significantly lower than that in the control group (78%) (see [link to documentation]). Figure 4 ).

[0032] In addition, such as Figure 5 As shown, under continuous observation at different time points, the metamorphosis rate of sea squirt larvae in the biofilm group was significantly lower than that in the control group. In conclusion, the Pseudorabialis MP_PS-3 biofilm inhibits the attachment metamorphosis of sea squirt larvae, with the attachment rate being almost zero.

[0033] The above are merely preferred embodiments of the present invention. It should be noted that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A pseudoalteromonas ( Pseudoalteromonas spongiae Application of MP_PS-3 in inhibiting sea squirt adhesion.

2. The application according to claim 1, characterized in that: The application of the pseudoalternating monocytogenes in inhibiting sea squirt attachment metamorphosis.

3. The application according to claim 2, characterized in that: Application of the pseudoalternating monoclonal bacteria in inhibiting attachment metamorphosis in sea squirt larvae.

4. The application according to claim 3, characterized in that: The sea squirt larvae mentioned are *Sarcoidea* larvae (… Ciona savignyi )larva.

5. A strain containing the pseudoalternating monoclonal bacteria as described in claim 1 ( Pseudoalteromonas spongiae Application of MP_PS-3 formulation in inhibiting sea squirt adhesion.

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