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. 2022 Aug 29;98(10):fiac096. doi: 10.1093/femsec/fiac096

Comparative transmission of bacteria from Artemia salina and Brachionus plicatilis to the cnidarian Nematostella vectensis

Quinton A Krueger 1,✉, Madisun H Shore 2, Adam M Reitzel 3
PMCID: PMC9521339  PMID: 36036952

ABSTRACT

The microbial community associated with animals (microbiome) is essential for development, physiology, and health of host organisms. A critical step to understand the assembly of microbiomes is to determine how effectively bacteria colonize and establish within the host. Bacteria commonly colonize hosts through vertical transmission, passively from the environment, or through food consumption. Using the prey feeding method (PFM), we test transmittance of Bacillus velezensis, Pseudoalteromonas spiralis, and Vibrio alginolyticus to Nematostella vectensis using two prey, Artemia salina and Brachionus plicatilis. We compare PFM to a solution uptake method (SUM) to quantify the concentration of bacteria in these host organisms, with plate counts. Larvae had a similar uptake with SUM at 6 h but had greater concentrations at 48 h versus PFM. Juveniles acquired similar concentrations at 6 h for SUM and PFM using B. plicatilis and A. salina. At 2 days, the quantity of bacteria vectored from PFM increased. After 7 days the CFUs decreased 2-fold with B. plicatilis and A. salina relative to the 2-day concentrations, and further decreased after 14 days. Therefore, prey-mediated methods provide greater microbe transplantation than SUM after 24 h, supporting this approach as a more successful inoculation method of individual bacterial species.

Keywords: bacteria, cnidarian, colonization, marine invertebrates, predator, prey

This study quantified bacterial transmittance from prey to predator in a simple estuarine food web.

Introduction

Bacteria and other microorganisms (microbiome) commonly associate with multicellular organisms (host) (Artamonova and Mushegian 2013, McFall-Ngai 2002, McFall-Ngai et al. 2013, Simon et al. 2019, Zhang et al. 2017). These associations have a broad range of impacts on the physiology and health of the host (Glasl, Herndl and Frade 2016). The host provides surfaces to colonize (Wein et al. 2018), while also supplying nutrients to the microbes for growth. The native microbiome can protect the host from pathogenic invaders (Berg, Zhou and Shapira 2016), assist in the digestion of prey (Herndl and Velimirov 1985, Parker et al. 2018), and increase availability of essential nutrients to the host organism through diverse metabolic pathways (Chan et al. 2019, Heinken and Thiele 2015). The establishment and maintenance of an individual's microbiome results from a combination of processes ranging from initial colonization (Ceh, Van Keulen and Bourne 2013, Nyholm et al. 2000), environmental factors (Har et al. 2015), host immune responses (Augustin, Fraune and Bosch 2010, Parisi et al. 2020), and microbe-microbe interactions (Domin et al. 2018). Thus, the residing microbiome can influence the diversity and concentration of subsequent acquired bacterial species, including those that would be beneficial to the host.

Bacterial species from the genus Bacillus, Pseudoalteromonas, and others have demonstrated positive impacts on host health (Holmström and Kjelleberg 1999, Jamali et al. 2015, Soltani et al. 2019). Developing methods to increase the efficacy of microbial transplantation are necessary to facilitate microbiome manipulations. For example, the addition of probiotic organisms has become a recommendation for the protection and preservation of reef building corals (Assis et al. 2020, Voolstra and Ziegler 2020), fish, and other organisms susceptible to climate change and anthropogenic pollutants (Fragoso ados Santos et al. 2015, Peixoto et al. 2017, Reshef et al. 2006, Rosado et al. 2019). Alternatively, marine pathogens such as Vibiro alginolyticus can also be transmitted to hosts, resulting in disease and potentially death. To facilitate effective delivery of beneficial microorganisms, and quantify transmission of potential pathogens, it is imperative to determine the efficiency of potential methods of colonization to understand how individual bacteria associate with the host. Development of methods for efficient bacterial seeding that could be paired with complementary approaches (i.e. visualization and gene expression) will be important in elucidating functional relationships between bacteria and hosts.

Experimental models for the uptake, retention, and loss of bacteria in aquatic habitats remain rare in comparison to terrestrial species. For example, Caenorhabditis elegans is an insightful experimental organism for quantifying and modeling the acquisition of microbial communities (Dirksen et al. 2016). Caenorhabditis elegans, a bacterivorous organism, has several known bacterial colonization methods including vertical transfer to the offspring (Diaz and Restif 2014), uptake from the surrounding environment, and direct feeding (Samuel et al. 2016). Similar processes have been observed in a vast array of animals including anemones (Baldassarre et al. 2021), corals (Sharp, Distel and Paul 2012), and fish (Sullam et al. 2012). The quantity of bacterial cells introduced into C. elegans is important for successful colonization into the intestine (Vega and Gore 2017). A similar experimental system in a model aquatic invertebrate would increase our knowledge on the mechanisms of bacterial colonization for these organisms living in environments surrounded by water.

Artemia salina and Brachionus plicatilis are small planktonic animals used as prey in aquaculture for carnivorous aquatic animals (Bengtson, Léger and Sorgeloos 1991, Lubzens, Tandler and Minkoff 1989). Artemia salina are utilized in the laboratory setting for their ease of culture and stable long-term storage of cysts (Clegg 1967). Juvenile A. salina are approximately 1 mm long whereas adult B. plicatilis reach 500 μm. This size difference as well as their respective swimming strengths may influence a predator's ability to catch prey, particularly for small juveniles. Both A. salina and B. plicatilis have been shown to transmit bacteria through feeding mechanisms to invertebrates including corals (Assis et al. 2020, Galand et al. 2020). Artemia salina harbors less bacteria compared to B. plicatilis in short-term feeding experiments (Makridis et al. 2000). With respect to the bacterial load of each individual prey item, it is unknown how efficiently the bacteria are transferred and maintained in predatory organisms after consumption. While B. plicatilis are easily caught, A. salina are larger and have a higher nutritional content (Haché and Plante 2011), which may allow for extended retention of bacteria. These differences could influence the successful inoculation of newly introduced bacteria into a predatory organism. Assessing the sustainability of introduced probiotic microorganisms could be essential for effective protection or treatment of diseased animals.

Here, we utilize the sea anemone Nematostella vectensis as an experimental organism for quantification of bacterial transplantation in aquatic invertebrates. In recent years, N. vectensis has become a model organism for cnidarians and other invertebrates due to their ease of culture in a laboratory setting, the ability to recapitulate the life cycle, an extensive set of molecular tools, and their broad distribution in coastal habitats (Al-Shaer, Havrilak and Layden 2021, Darling, Reitzel and Finnerty 2004, Darling et al. 2005). Nematostella vectensis has also grown into a model for microbiome studies where previous studies have described the shifts in bacterial communities in the lab and field (Har et al. 2015), identified robust microbial communities in particular life cycle stages or abiotic conditions (Leach, Carrier and Reitzel 2019, Mortzfeld et al. 2016), and determined the contribution of vertical and horizontal transmission (Baldassarre et al. 2021). Because many organisms are predatory in nature, describing the uptake efficiency of bacteria is a key component to determine how the bacterial community changes over time, especially with feeding organisms.

In this study, we quantify the colonization and establishment of bacteria to this cnidarian host. We utilize two different prey species (Artemia salina and Brachionus plicatilis) to better understand the impact of organismal level interactions with respect to the delivery of bacteria. For this study we selected three bacterial species, Bacillus velezensis, a documented probiotic (Thurlow et al. 2019, Yi et al. 2018), Pseudoalteromonas spiralis, an antagonist to other bacteria (Holmström and Kjelleberg 1999), and Vibrio alginolyticus, a known marine pathogen (Yang et al. 2021, Zhenyu et al. 2013). This approach is compared to the transmission of bacteria through solution to assess how contrasting inoculation approaches impact colonization of these environmental isolates. This method can be extended to other aquatic organisms, particularly predatory species capable of acquiring microbes through their prey.

Materials and methods

Bacterial cultivation

We selected three environmental isolates originally collected from Georgetown, South Carolina, Bacillus velezensis, Pseudoalteromonas spiralis, and Vibrio alginolyticus, for the colonization and growth efficacy trials. Bacillus velezensis and P. spiralis were cultivated on Estuarine Broth agar (EB) (S. Table 1 for recipe), while V. alginolyticus was cultivated on Heart Infusion (HI) broth agar plates (Criterion Cat. no.: C5831) in preparation for seeding in 15 ppt Artificial Seawater (ASW). Artificial Seawater was made with Instant Ocean® Sea Salt dissolved into ultrapure water (PureLab flex) to a concentration 15 ppt or 30 ppt, where applicable. Before transfer to the specific seeding condition, the Optical Density (OD) of the bacterial cultures were determined for a cell concentration of 108 cells mL–1. Growth curves were measured in the BioTek LogPhase 600 for all bacteria (Fig. S1). Samples were serially diluted in 90 μL Phosphate Buffered Saline (PBS), and 10 μL of liquid were drop spotted on the appropriate media. The plates were grown at 25°C overnight, and Colony Forming Units (CFUs) were counted the following day.

Prey cultivation

Artemia salina cultivation

Artemia salina cysts were purchased from Artemia International. One gram of A. salina cysts were cultivated in 250 ml of 30 ppt ASW with bubbling to maintain cysts in suspension while under a light source (NT). For axenic culturing (AB), A. salina were exposed to an antibiotic cocktail (50 μg mL–1 Ampicillin (Acros Organics Cat. No.: 61177), Chloramphenicol (VWR, Cat. No.: 0230), Kanamycin (Fisher BioReagents, Cat. No.: BP906), and Neomycin (Alfa Aesar, Cat. No.: J61499) each) for 24 h under identical cultivation conditions as NT. After 24 h hatched A. salina were subsequently washed three times in sterile 15 ppt ASW two hours before bacterial inoculation. Both ‘No Treatment’ (NT) and ‘Antibiotic’ (AB) conditions utilized this wash step to eliminate either antibiotics or bacteria in solution.

Brachionus plicatilis cultivation

Brachionus plicatilis were purchased from LiveAquaria (#BEH-75641) that were pre-fed with algae. This stock culture was maintained by feeding with Storeatula major cultivated in 15 ppt F/2 media with continuous vigorous aeration. One-quarter volume of the culture was cycled weekly to reduce the accumulation of waste products in solution. Prior to experimentation, B. plicatilis were reared in 15 ppt ASW for 24 h to allow clearing of algae from the digestive system. Antibiotic treated B. plicatilis were exposed to the same antibiotic cocktail as A. salina over 24 h to remove resident bacteria, as described for A. salina. Brachionus plicatilis were rinsed 2 h before application of bacteria used for experiments.

Determination of Artemia salina and Brachionus plicatilis short-term feeding capacity

Two groups of both A. salina and B. plicatilis were grown, one axenically (AB), and the other with no treatment (NT). After the wash step, the prey were exposed to individual or combinations of bacteria. For all conditions, the prey items were exposed to bacteria in a 1 : 1 v/v ratio, at a concentration of 108 CFUs mL–1. After 10 min of exposure, the prey items were rinsed with 30 ml sterile 15 ppt ASW, and groups of 10 were dissociated with sterile pestles in 1.5 mL microcentrifuge tubes, then serial diluted as previously described and drop spotted for CFU counts. Four types of media were utilized in all experiments for CFU calculations, Heart Infusion (HI) agar plates (Criterion Cat. No.: C5831), CHROMagar Vibrio plates (CAV) (CHROMagar Ref. No.: VB912), Phenylethyl Alcohol (PEA) agar (BD Ref. No.: 211539), and Marine Broth (MB) agar plates (Table S1). All media were made using instructions provided by the respective manufacturer.

Nematostella vectensis cultivation

Nematostella vectensis adults were cultured in 15 ppt ASW and spawned weekly, following previously established methods that include feeding adults with mussel tissue once and A. salina three times per week, with weekly water changes (Fritzenwanker and Technau 2002, Hand and Uhlinger 1992, Stefanik, Friedman and Finnerty 2013). Fertilized eggs were separated into new bowls of 15 ppt ASW for development. Recently metamorphosed juveniles were fed Brachionus plicatilis several times until the approximate size reached (>3 mm). To create axenic individuals, the polyps were exposed to the antibiotic cocktail for 24 h. Two hours before treatment, organisms were washed with and transferred to sterile 15 ppt ASW.

Seeding Nematostella vectensis with bacteria

Direct application of bacteria

The three bacterial species were grown to a concentration of 108 cells mL–1 in EB. Nematostella vectensis larvae and young juveniles were exposed to approximately 108 cells in 3 mL of sterile ASW. After 10 min the animals were removed from the bacterial solution, washed over a 40 µm Nylon filter with 30 mL sterile ASW, and aliquoted into flat bottom 96-well plates with 100 µL sterile 15 ppt ASW. At each time point animals (NT, AB, and SUM treated) were dissociated using a sterile pestle in a 1.5 mL microcentrifuge tube, then serially diluted and plated on the applicable media for CFU counts (n = 5). We refer to this approach as the solution uptake method (SUM).

Feeding Nematostella vectensis seeded prey

Artemia salina (PFM-A) and B. plicatilis (PFM-B) were exposed to individual bacterial cultures of approximately 108 cells in 15 ppt ASW. For the combination of B. velezensis and V. alginolyticus, a 50 : 50 mix of both bacteria for a total of 108 cells was exposed to the prey items. After 10 min of exposure prey were washed with 30 mL of sterile ASW and placed into a 6-well plate with N. vectensis. To increase the efficiency of the animals' ability to catch prey, the plates were placed over a light source. As the anemones caught single prey, they were isolated to a single well in a 96 well plate containing with 100 µL of sterile ASW. At 6 h, 48 h, 7 days, and 14 days entire anemones were washed over a 40 µm Nylon Mesh Filter with 30 mL sterile ASW and dissociated in sterile 1.5 mL tubes filled with 100 µL 15 ppt ASW (n = 5). The resulting slurry was serially diluted and 10 µL drop spots were plated on the appropriate media for each individual animal. The total number of CFUs were counted the subsequent day. We refer to this approach as the prey feeding method (PFM). To determine the number of bacteria prey items shed into solution, we serially diluted the previously sterile ASW the prey resided in for 10 min after bacterial exposure. Additionally, to determine the relative transfer of non-treated prey to N. vectensis, juveniles were antibiotically treated and exposed to NT prey and plated on MB, HI, PEA, and CAV, as described above over the time course.

Saturated feeding of adult Nematostella vectensis with Bacillus velezensis

Using fully grown axenic adult anemones, we fed animals approximately 50 A. salina each. Following methods described above (Section 2.3), A. salina were exposed to B. velezensis. Adult anemones were starved for 5 days prior to the exposure of the prey items. Over the same time course as juveniles, adult animals were dissociated, serially diluted, and plated on PEA as previously described.

Results

Prey seeding

Recently hatched A. salina harbored similar bacterial concentrations (Fig. 1A) to those previously reported (Makridis et al. 2000). Our laboratory acclimated B. plicatilis had lower quantities of Vibrio spp. and gram-positive bacteria than has been shown in previous studies (Martínez-Díaz 2003), but this did not appear to influence their ability to uptake additional bacteria (Fig. 1A-B). To test the variance in the differing methods we used two-way ANOVA to determine the difference in associated bacterial concentrations between treatments. When exposing the No Treatment (NT) prey to the bacteria, there was significantly less bacteria when adding V. alginolyticus than in the axenic (AB treated) organisms (two-way ANOVA, P = 0.0067, P < 0.0001, PFM-A, PFM-B, respectively). No statistical difference was detected between the NT and AB treated organisms for B. velezensis and P. spiralis in both prey items (all p-values above 0.977, see Table S2). Additionally, when comparing the bacterial isolates, V. alginolyticus accumulated in untreated organisms with significantly higher numbers than B. velezensis in A. salina (P = 0.0142), and both B. velezensis and P. spiralis in the B. plicatilis (P < 0.0001, both comparisons). In axenic prey, V. alginolyticus seeded with higher numbers than the two other bacteria in both A. salina (P < 0.0001, both comparisons) and B. plicatilis (B. velezensis P = 0.0027, P. spiralis P = 0.0025). We found that short term exposure to dense bacterial culture results in high concentrations of seeded bacteria, which in some cases exceeded normal bacterial concentrations in the prey items (Fig. 1B). When A. salina (Fig. 2A) was exposed to combinations of B. velezensis and V. alginolyticus, B. velezensis seeded similarly to individual inoculations (P > 0.9673), and V. alginolyticus decreased (p < 0.0001) from the concentrations they were exposed to individually (Fig. 2A). For B. plicatilis combination exposures, B. velezensis remained close to individual exposures, while V. alginolyticus also decreased (P < 0.0001) relative to individual exposures (Fig. 2B). Thus, short term exposures with combinations of microbes at equal concentrations in the surrounding media resulted in differential concentrations in the prey item.

Figure 1.

Figure 1.

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Prey individual CFU counts. (A)Artemia salina individual bacterial inoculation plate counts 10-minute after introduction to each bacterial species. Dissociated groups of 10 NT or AB treated prey (n = 15) were serially diluted on non-selective Marine Broth Agar (MB) and Heart Infusion Agar (HI), and selective media ChromAgar Vibrio (CAV), and Phenylethyl Alcohol Agar (PEA) where appropriate, as described in Section 2.3. NT = No Treatment, AB = Antibiotic Treatment. A-B (P = 0.0168) C-D (P = 0.0067). (B)Brachionus plicatilis individual 10-minute bacterial inoculation plate counts. A-B (P < 0.0001). Technical replicates across media types were averaged. Antibiotic treated control animals yielded no culturable growth under the same plating conditions. Separate values are graphed in Fig. S1. Points represent the mean, and the bars indicate the Standard Error of the Mean (SEM).

Figure 2.

Figure 2.

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Counts of bacteria resulting from prey combination exposures. Groups of 10 prey (n = 15) were dissociated and serially diluted on appropriate media types. The first and third columns represent the number of CFUs when the respective bacteria is seeded individually, and the second and fourth bars represent the number of CFUs when both B. velezensis and V. alginolyticus are co-inoculated, respective to the species labeled. (A)Artemia salina combination 10-minute bacterial inoculation plate counts. A-B (P < 0.0001). Combinations containing P. spiralis were uncountable due to the inability to effectively select against the other cohorts. (B)Brachionus plicatilis combination bacterial exposure plate counts. A-B (P < 0.0001).

Predator seeding

Seeding Nematostella vectensis larvae

Under NT conditions, 2-day old larvae harbor between 101 and 103 CFUs per individual, depending on media type. While CAV and PEA selected microbes did not increase in CFUs over the time course, both MB and HI had a 1-fold increase after 2 days (Fig. 3A, all statistical values for Nematostella vectensis time courses can be found in S. Table 2). For the larval stage of N. vectensis, SUM had the greatest retention of microbes (Fig. 3B), as the organisms pre-settlement stages are not capable of predatory feeding (Fig. 3C-D) and are not known to actively consume bacteria (Kehr and Jaeckle 2013). The colonization of B. velezensis, P. spiralis, and V. alginolyticus through the SUM was significantly higher in the short term (2D SUM vs. PFM-A and PFM-B, p < 0.0001, all comparisons). Additionally, there was a significant difference between prey feeding methods for V. alginolyticus (2D PFM-A vs. PFM-B, p = 0.0040). While direct uptake of bacteria from PFM is unlikely in this non-feeding life stage (no prey consumption was observed), bacteria that were present in solution seeded these developmental stages. The concentration of bacteria increases from 6 h to 2 days under all conditions (Fig. 3). Vibrio alginolyticus increased 2-fold over this time course. Both B. velezensis and P. spiralis also increased over this early development phase. We found concentrations ranging from 7.0×103–3.6×105 bacterial CFUs in solution after washing A. salina and B. plicatilis, indicating association of bacteria with the larvae was likely through this indirect mechanism (S. Fig. 3). The concentration of bacteria shed into solution was similar to the SUM concentrations of larvae. At 48 h both SUM and Prey Feeding Methods resulted in similar concentrations that exceed the initial 6-hour larvae measurements.

Figure 3.

Figure 3.

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Nematostella vectensis larvae CFU counts over 2 days (n = 5). Points represent the mean and bars represent the SEM. (A) Untreated control larvae. The number of CFUs associated significantly increased from 6 h to 2 days. (B) SUM treated larvae. All bacterial species increased from 6 h to 2 days. (C)Artemia salina PFM treated larvae. CFUs for all bacterial species trended upward from 6 h to 2 days. (D)Brachionus plicatilis PFM treated larvae. Vibrio alginolyticus significantly increased over the time course (P < 0.0033). All statistical values can be found in Table S2.

Feeding vs. solution uptake by juvenile Nematostella vectensis

When juvenile sea anemones were fed NT prey items, the relative uptake of bacteria from A. salina (Fig. 4A) and B. plicatilis (Fig. 4B) were similar, while trending downward through the 7-day cycle. Juvenile animals over 7 days did not increase in measurable CFUs over the time course (Fig. 5A). SUM transferred microbes resulted in less associated bacteria when compared to the PFM at 2 days and retained less CFUs over time in juvenile organisms. The SUM transplanted microbes increased at 2 days, with general decrease in CFUs at and after 7 days (Fig. 5B). Additionally, both prey vectored methods resulted in a higher concentration of bacteria in the organisms at 2 days than at 6 h, except for P. spiralis in A. salina. When B. velezensis and P. spiralis colonized juveniles through the SUM, the number of CFUs transferred were significantly lower and higher, respectively, at 2 days (2D PFM-A, P < 0.0001, P = 0.0018, and 2D PFM-B, P > 0.9999, P = 0.0010, respectively). Additionally, the SUM resulted in significantly higher accumulation of CFUs at 2 days than both PFMs for V. alginolyticus (6H PFM-A, P < 0.0001, 6H PFM-B, P < 0.0001). Bacteria vectored with A. salina increased up to 2-fold than initial concentration (Fig. 5C), while the B. plicatilis vectored bacteria were up to 1-fold greater than initial concentrations at 2 days (Fig. 5D). We further observed that the highest concentration of vectored bacteria was V. alginolyticus within B. plicatilis, potentially indicating a preference of microbial uptake, or differential microbial retention at the genus level. At 7-days the number of vectored microbes from both prey decreased to the 6-hour concentrations and further decreased at 14 days for most conditions (Fig. 5C and D). For B. velezensis and P. spiralis transferred to N. vectensis, both A. salina and B. plicatilis shared similar short-term capacities (Fig. 5C and D). In a satiated feeding experiment, we observed that adult N. vectensis can consume large volumes of prey and still retain the bacteria through the digestion of the prey species. At 2 days the concentration of bacteria increased 2-fold (Fig. 6), likely due to the availability of freshly digested A. salina. At 7 days the concentration of B. velezensis decreased from the 2-day concentration.

Figure 4.

Figure 4.

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Nematostella vectensis juvenile CFU counts over 7 days. MB = Marine Broth Agar, HI = Heart Infusion Agar. CAV = ChromAgar Vibrio, PEA = Phenylethyl Alcohol Agar. (A) Antibiotic treated juveniles fed Non-Treated Artemia salina. (B) Antibiotic treated juveniles fed Non-Treated Brachinous plicatilis. All statistical values can be found in Table S2.

Figure 5.

Figure 5.

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Juvenile Nematostella vectensis CFU counts. (A)Non-treated juveniles over 7 days. (B) SUM treated juveniles over 14 days. (C)Artemia salina PFM fed juvenile N. vectensis. (D)Brachionus plicatilis PFM fed juveniles.

Figure 6.

Figure 6.

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Adult Nematostella vectensis fed Artemia salina seeded with Bacillus velezensis to satiation, CFUs measured over a 7-day time course.

Discussion

With the pace of climate change likely exceeding the ability for many animals to adapt genetically, the introduction and retention of beneficial microbes represents a promising approach for future conservation efforts (Bourne, Morrow and Webster 2016, Peixoto et al. 2017, Rosado et al. 2019, Ziegler et al. 2017). Methodology for controlled manipulation of the microbiome has extensive application for modifying the health and resilience of aquatic hosts. Approaches for successful, efficient introduction of microbes to aquatic animals remains understudied to inform these interventions (Damjanovic et al. 2019, Doering et al. 2021, Rosado et al. 2019, Santoro et al. 2021, Silva et al. 2021, Welsh et al. 2017, Zhang et al. 2021). The use of axenic prey that can be quickly reseeded with probiotic isolates at or above standard bacterial concentrations could be an efficient alternative to introduction of these bacteria in solution. Brachionus plicatilis has been utilized as a vehicle to potentially transmit probiotic bacteria to cnidarians (Assis et al. 2020). In our study, we build upon this research by showing that multiple species of bacteria are concentrated in B. plicatilis and A. salina, and that bacteria seeded into each prey item are successfully transferred to a predatory cnidarian. The utility of small planktonic organisms for delivery of selected bacteria from different phyla has the potential for wide application for conservation of species under threat. Additionally these methods will allow us to better understand the interactions of cnidarians and their associated microbial communities (Peixoto et al. 2021).

Controlled introduction of specific bacteria can be informative for discerning microbe-microbe interactions and the mechanisms of host-microbe interactions. For example, application of Phaeobacter inhibens in non-axenic A. salina exhibits antagonistic properties against Vibrio anguillarum (Grotkjær et al. 2016). Interactions such as these can be investigated within the context of the host organism. To understand the different interactions between individual bacteria, it is important to determine the stoichiometric ratio present in the host system. Here, we quantified the aggregation of both V. alginolyticus and B. velezensis concurrently and found that both prey items accumulate less V. alginolyticus when both bacteria are presented in equal concentration. When constructing engineered microbiomes, such as for probiotic applications, it is important to consider the ratio of seeded bacteria that aggregate in the terminal animal.

The host microbiome can be a protective mechanism that interferes with the colonization of new bacteria, a factor to be considered when investigating host-bacteria interactions. While this mechanism of exclusion is likely dependent on the bacterial interactions, our results suggest that removal of the native microbial community had limited impact on the ability to successfully seed new bacteria. When the native bacteria are removed, V. alginolyticus had increased aggregation in A. salina, while B. plicatilis had less than control animals. Alternatively, B. velezensis and P. spiralis did not differentially seed either prey, indicating this may be species dependent. Additionally, we found V. alginolyticus seeded B. plicatilis with a 2-fold higher CFUs when compared with B. velezensis and P. spiralis. To effectively study changes in individual or combinations of bacterial interactions with the host it may be beneficial to eliminate the pre-existing microbiome to control the composition for some bacteria. For example, to study the direct interactions between a single bacterial species and its host, it is imperative to eliminate other bacteria to reduce unnecessary interactions. We anticipate that there is a species dependent uptake according to what other microbes are present in solution, and in the host. This result suggests that it may be difficult to predict the microbiome of prey items based on the composition of microbes in solution (Carrier and Reitzel 2019).

Nematostella vectensis larvae acquire their microbiomes at early developmental stages which differs from the microbiome of later life stages (Mortzfeld et al. 2016). For the larval stage, short term uptake of all bacteria at 6 h was equitable across all treatment types. At 48 h, SUM treated larvae had increased acquisition for all bacterial species against both PFMs. While active bactivory has not been documented in N. vectensis, there were still associations of bacteria in the larval stages (Mortzfeld et al. 2016), even when exposed to bacteria shed from seeded prey items. After prey were seeded, we measured different shedding between bacterial species. Both B. velezensis and P. spiralis shed 1-fold more CFUs into solution when associated with A. salina, while V. alginolyticus shed 1-fold more with B. plicatilis (Fig. S3). Despite this differential shedding of bacteria into solution, the number of bacteria measured with the larvae remained similar, indicating there may be other factors such as surface area and binding affinities influencing the quantity of associated bacteria.

Juvenile anemones that consumed seeded prey had an increase in CFUs compared with SUM, likely due to additional nutrients from the metabolism of the prey organisms. With B. plicatilis, the CFUs increased 2-fold at 48 h, decreased 2-fold at 7 days, and maintained these concentrations over 14 days. With A. salina anemones had a greater increase in CFUs but these were also lost through 7 days, returning to the 6-hour concentrations. The retention of high concentrations of introduced bacteria for less than one-week indicates that continuous feeding would likely be required to maintain the seeded bacteria, between 2 and 7 days. The tradeoff of size and nutrient composition between A. salina and B. plicatilis is important for applications comparing species or life stages. Young polyps and other small planktivorous organisms may require the smaller prey for ease of capture and consumption. Adults may benefit from larger nutrient-rich prey to both seed and maintain the transplanted microbes. In adults, a higher initial number of seeded prey does not appear to influence bacterial retention in N. vectensis, and resulted in a decrease to the original CFU load for B. velezensis at 7 days. This indicates repeated inoculations is necessary to maintain high concentrations of probiotic organisms.

Our work targeted the quantification of bacterial inoculation of a model cnidarian species through two approaches: solution and prey items. To determine the impact of probiotics or any bacterial species on stressed or diseased organisms, it is important to understand how bacteria colonize hosts, including how different methods of introduction influence these symbioses. Together, these combinations of methods and comparisons result in a quantitative assessment for how particular microbes can be successfully seeded into focal species to permit introduction of bacteria for future applications. Future studies will help determine the longevity of transplanted bacterial species under different abiotic conditions that more closely resembles natural environments to help inform the field-based application of probiotic supplements.

Supplementary Material

fiac096_Supplemental_Files
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ACKNOWLEDGEMENTS

QAK and AMR were supported through Human Frontiers Research Program award RGY0079/2106. QAK and MHS were supported by NIH award R15GM137253 for a portion of this research.

Contributor Information

Quinton A Krueger, Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Boulevard, Woodward Hall, Charlotte, NC 28223, United States.

Madisun H Shore, Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Boulevard, Woodward Hall, Charlotte, NC 28223, United States.

Adam M Reitzel, Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Boulevard, Woodward Hall, Charlotte, NC 28223, United States.

Authors contributions

QAK and AMR conceived the ideas and designed methodology; QAK and MHS collected the data; QAK analyzed the data; QAK and AMR led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.

Conflicts of interest

The authors declare no conflicts of interest.

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