*Review* **Methods to Evaluate Bacterial Motility and Its Role in Bacterial–Host Interactions**

**Victoria Palma <sup>1</sup> , María Soledad Gutiérrez 1,2 , Orlando Vargas <sup>1</sup> , Raghuveer Parthasarathy 3,4 and Paola Navarrete 1,2,\***


**Abstract:** Bacterial motility is a widespread characteristic that can provide several advantages for the cell, allowing it to move towards more favorable conditions and enabling host-associated processes such as colonization. There are different bacterial motility types, and their expression is highly regulated by the environmental conditions. Because of this, methods for studying motility under realistic experimental conditions are required. A wide variety of approaches have been developed to study bacterial motility. Here, we present the most common techniques and recent advances and discuss their strengths as well as their limitations. We classify them as macroscopic or microscopic and highlight the advantages of three-dimensional imaging in microscopic approaches. Lastly, we discuss methods suited for studying motility in bacterial–host interactions, including the use of the zebrafish model.

**Keywords:** bacterial motility; motility methods; bacteria; flagella; bacterial–host interaction; microscopy

#### **1. Introduction**

Motility is defined as the movement of cells by some form of self-propulsion [1]. Many bacterial cells are motile as it allows them, for example, to escape from unfavorable conditions and to exploit new resources or opportunities. Combined with chemotaxis, the ability to sense a chemical gradient and direct movement accordingly, it enables bacteria to pursue nutrients and to reach specific niches. In this sense, motility is also involved in the interaction between microorganisms and their host, specifically in colonization or infectious pathogenic processes. Indeed, non-motile mutants are either impaired or completely disabled to colonize and/or cause disease [2].

There are different types of motility, often classified as swimming, swarming, twitching, gliding, and sliding [3,4]. Swimming consists of movement in a liquid environment typically by using flagella, long, thin appendages attached to the cell [1]. Swarming is a coordinated movement of cells that are propelled by flagella through thin liquid films on surfaces and can involve cellular differentiation into a longer and hyper-flagellated phenotype [5]. Other structural molecules can be involved in bacterial movement such as twitching and gliding, both being active ways of moving over a surface. In twitching, type IV pili extend and attach to a solid surface, then retract to allow movement [6]. While twitching is described as intermittent and uneven, gliding is a more organized and smoother cell movement that comprises evolutionarily unrelated machineries which include the use of adhesins that attach to a substratum and either move across the cell or use surface proteins to perform a back-and-forth motion [4,7,8]. Sliding is a passive movement that, instead of

**Citation:** Palma, V.; Gutiérrez, M.S.; Vargas, O.; Parthasarathy, R.; Navarrete, P. Methods to Evaluate Bacterial Motility and Its Role in Bacterial–Host Interactions. *Microorganisms* **2022**, *10*, 563. https://doi.org/10.3390/ microorganisms10030563

Academic Editor: Konstantinos Ar. Kormas

Received: 30 December 2021 Accepted: 6 February 2022 Published: 4 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

requiring an appendage, occurs by bacteria's surfactants (i.e., rhamnolipids) [3]. While dividing, cells are pushed outwards by the growing colony, and surfactants reduce the surface tension decreasing the friction between the surface and bacterial cells, accelerating their spreading [9]. Alternatively, sliding can be attributed to osmolytes (i.e., glycine betaine) secreted by bacteria that draw water to the media surface [9].

Other types of motion are possible [4,10–14]. *Spiroplasma* propagates kinks along its helical body to swim [10], while it is believed that cyanobacteria of the genus *Synechococcus* does so by propagating spicule-like surface extensions along the cell [11,12]. Another example is *Acinetobacter baumannii* 17978, whose type I pili confer surface motility modulated by light [13]. Moreover, some parasitic bacteria can induce actin polymerization to form a tail and move inside the host cell. These motility types and others are included in a recent re-classification based on the structure of the force-producing motor [14].

Different motility types are not mutually exclusive. It has been shown that besides swimming, swarming, and twitching, *Pseudomonas aeruginosa* can also display sliding motility [15], and a recent review discusses different forms of movement observed in *Staphylococci*, including gliding and sliding in *Staphylococcus aureus* [16]. Motility also shows great variability among species and even strains. For example, strains from different serovars of *Salmonella enterica* showed differences up to a factor of 2.7 in swimming speed [17].

Although motility can provide fitness advantages, it also has considerable drawbacks, such as high energetic and metabolic cost [18], and the presence of antigenic structures such as flagella [19]. These costs are a function of the biological context, and therefore realistic assessment of motility requires setting experimental conditions to be as close as possible to the actual environment of interest. We will discuss here common and recent methodological approaches that have been used to study bacterial motility and its role in bacteria–host interactions.

#### **2. Macroscopic Techniques**

We will distinguish between macroscopic and microscopic methods for studying bacterial motility. The former does not resolve the motions of individual bacteria but rather the spread of a population through some medium. Qualitatively, the link between macroscopic spreading and microscopic motility makes sense—a non-motile species, for example, will have little dispersal, and a vigorously moving species may travel far. Quantitatively, the relationship between macroscopic dispersal and the motility of individual cells is more subtle because the spread of a population is driven by growth (cell division) as well as motility. For example, a bacterium *Escherichia coli* that travels in fairly straight "runs" of a constant speed, *νbacteria* that persist on average for time *τ* before the organism "tumbles" and randomizes its direction, executes a random walk through its three-dimensional world with an effective diffusion coefficient *D* proportional to the square of its speed [20,21].

$$D = \frac{1}{3} \, v\_{batteria} \, ^2 \tau \tag{1}$$

If the bacteria are also growing exponentially with growth rate *r*, the population will spread with a velocity:

$$
v = 2\sqrt{rD} \tag{2}$$

as Fisher, Kolmogorov, and others showed nearly a century ago [22,23]. For typical bacterial swimming speeds and growth rates, the macroscopic dispersal speed (perhaps millimeters per hour) will be one or two orders of magnitude lower than the speed of individual bacteria (perhaps tens of microns per second). Besides considering the expansion described by Fisher (Fisher waves), recent work on bacterial range expansion has taken into consideration phenomena such as intraspecific cooperativity [24] and chemotaxis [25].

The most common macroscopic approach to studying macroscopic motility is by examining bacterial spread through semi-solid agar (soft agar) [26]. Starting from an inoculation stab deep inside the agar, non-motile bacteria will remain near the inoculation zone, while motile bacteria will spread and visibly blur the media (Figure 1a). Because of its simplicity, it is particularly well suited to uncover non-motile or hypermotile strains (Table 1). Some bacteria can form, depending on the environmental conditions, characteristic colony patterns in plates, especially during swarming [5]. Spatial patterns seen using the soft agar method are linked to chemotaxis—directed motion induced by chemicals—as chemoattractants present in the agar that are metabolized by bacteria creating radial concentration gradients that boosts outward expansion [27]. Using low concentrations of the metabolizable chemoattractant would accentuate taxis response [28]. Other methods to study chemotaxis have been described, such as the capillary assay, where a capillary tube filled with a chemical is placed in a bacterial suspension and the accumulation of bacteria towards or away from the chemical is assessed visually [27,28].

In soft agar assays, the agar concentration can be adjusted according to the bacterial species and motility type (Table 2). To assess sliding motility, soft agar assays with flagellumand/or type IV pili-deficient strains are usually used to discard swarming and/or twitching, respectively [9]. If the motility zone cannot be visualized because of low cell density, for example, in the case of using agar medium low in nutrients, the bacterial density of an agar plug at a standardized distance can be measured to determine if bacteria has reached this position [29]. Labeling can increase the contrast between the spreading bacteria and the culture media. For example, 2,3,5-triphenyltetrazolium chloride (TTC) can be easily incorporated into the media, coloring bacterial growth [30]. Genetically modified bacteria encoding fluorescent proteins (i.e., GFP) or bioluminescent bacteria can also be used. For example, a fluorescent *Pseudomonas* and a bioluminescent *Salmonella* can both be distinguished in a co-swarming experiment [31]. Staining the biosurfactant rhamnolipids produced by bacteria, by adding Red Nile in the medium, showed that its production on agar surfaces was associated with bacterial swarming motility [31]. *Microorganisms* **2022**, *10*, x FOR PEER REVIEW 4 of 15

**Figure 1.** Some examples of methods to study bacterial motility and motility tracking. (**a**) Soft agar assay is the most common macroscopic method used to study motility. After inoculating 1–6 µL or **Figure 1.** Some examples of methods to study bacterial motility and motility tracking. (**a**) Soft agar

a stab of bacterial culture in soft agar, motile bacteria will spread and blur the media. (**b**) Assessing

trajectories can be obtained by (i) stacks of 2D slices along the z-axis (z-stacking) or by (ii) projecting the 2D image in the z-axis according to certain parameters such as depth-dependent shape in the case of defocused imaging methods. (**d**) Intravital microscopy (IVM) aims to visualize phenomena occurring inside live animals. For example, exposing the tissue of an anesthetized mouse by doing small incisions while carefully preserving its physiological conditions, a glass coverslip can be placed in the knee joint [32] or an intestinal loop [33] to visualize the movement of fluorescently labeled bacteria. Bacteria (green) are shown inside knee joint microvasculature. (**e**) The optical transparency of zebrafish larvae allows non-invasive visualization of the in vivo motility of fluorescent bacteria with light-sheet fluorescence microscopy (LSFM) in which a focal plane is illuminated, ex-

citing all points in the plane simultaneously.

assay is the most common macroscopic method used to study motility. After inoculating 1–6 µL or a stab of bacterial culture in soft agar, motile bacteria will spread and blur the media. (**b**) Assessing motility using some common microscopic methods is based on tracking individual bacteria to obtain their 2D trajectories. If a cell leaves the focal plane (orange cell) the track ends. (**c**) Three-dimensional trajectories can be obtained by (**i**) stacks of 2D slices along the z-axis (z-stacking) or by (**ii**) projecting the 2D image in the z-axis according to certain parameters such as depth-dependent shape in the case of defocused imaging methods. (**d**) Intravital microscopy (IVM) aims to visualize phenomena occurring inside live animals. For example, exposing the tissue of an anesthetized mouse by doing small incisions while carefully preserving its physiological conditions, a glass coverslip can be placed in the knee joint [32] or an intestinal loop [33] to visualize the movement of fluorescently labeled bacteria. Bacteria (green) are shown inside knee joint microvasculature. (**e**) The optical transparency of zebrafish larvae allows non-invasive visualization of the in vivo motility of fluorescent bacteria with light-sheet fluorescence microscopy (LSFM) in which a focal plane is illuminated, exciting all points in the plane simultaneously.

**Table 1.** Macroscopic assays to study bacterial motility.


**Table 2.** Agar concentration in media according to the type of motility type to assess in a semi-solid (soft) agar assay.


<sup>1</sup> The plate is inoculated at the bottom of the media instead of the top.

Environmental factors can also affect motility in agar. Tremblay and Déziel [40] proved that incubation temperature, pH, and drying time of soft agar under laminar flow affected swarming. In fact, even the location of the plates within the laminar flow causes significant differences in the swarming speed. These factors can affect media wetness that causes differences in the thickness of the liquid layer. The wetter the surface, the easier it is for bacteria to overcome frictional forces and move. This makes the reproducibility of these methods difficult to achieve.

#### **3. Microscopic Techniques**

Direct observation of motile bacterial cells provides the clearest insights into their motility but is challenging due to the length and time scales involved, as well as the potential complexity of the microbe's environments. Bacteria are typically around a micron in size, with speeds up to tens of microns per second for flagella-mediated swimming. Video capture rates of at least 10 frames per second (fps) are therefore needed if cellular positions in adjacent images are to be no more than a body-length apart, facilitating reconstruction of trajectories. Slower rates could capture transitions between straight runs and tumbles, but only rates of 10 fps or higher can capture information about instantaneous speed and angle changes [41]. Moreover, if the bacterial density is too high, bacteria will traverse each other constantly, making the reconstruction process difficult.

Even though bacteria can be tracked using simple bright-field imaging, its discerning from the background can be enhanced by techniques such as dark-field microscopy, differential interference contrast microscopy (DIC), and phase-contrast microscopy (Table 3). In dark-field microscopy, illumination comes from the side so that only light scattered by objects such as bacteria is detected, providing a bright signal on a dark background. This enables, for example, visualization of flagella in addition to bacterial cell bodies when using a high light intensity [1,42]. One-sided dark-field illumination variant is useful to simultaneously determine cell rotation and swimming speed in spirochetes [43]. In DIC microscopy and phase-contrast microscopy, the index of refraction gradients and phase shifts, respectively, are mapped onto intensity differences, enhancing the contrast of relatively transparent objects, making these methods suitable for assessing bacterial movement and orientation [44,45]. Recently, Smith et al. [46] were able to quantify twitching throughout a dense bacterial colony where individual cell tracking was not feasible using DIC microscopy. Substantially, the edge of the colony was observed by microscope and light changes over time were mapped and associated with areas with low and high motility within the field of view, where a higher modulation of light implies higher bacterial motility.

Fluorescent microscopy enables clear identification of labeled cells or even specific bacterial components such as flagella [47] (Table 3). Genetically encoded fluorescent proteins are routinely used in model bacterial strains, such as *E. coli* K12 or *P. aeruginosa* PAO1, and increasingly in non-conventional microbes, such as some *Aeromonas* and *Pleisomonas* isolates from the zebrafish intestinal microbiota [48]. Exogenous labels, such as fluorescent probes, can be simpler to apply but will be diluted as bacteria divide, and one must be aware that they can potentially alter bacterial function. Staining with DAPI, for example, halves the swimming speed of *Pseudomonas* species [49], and fusions of fluorescent proteins to components of the bacterial flagellar motor can alter its dynamics [50].

Microscopy in its forms mentioned so far provides views of a two-dimensional image. The truncated fragments of trajectories as bacteria move in and out of the focal plane still allow measurement of swimming speeds, durations of runs, and other characteristics (Figure 1b). Nonetheless, three-dimensional trajectories obtained through stacks of 2D slices (z-stacking) can be worthwhile, giving a more accurate characterization of motility patterns (Figure 1c). The main disadvantages are the requirement of rapid stack acquisition and the high amount of computational resources needed to process large stacks. On the other hand, methods based on 2D projection allow observing a larger volume in exchange for providing less exact measurements [51]. Berg's classic identifications of *E. coli*'s runs and tumbles tracked a microbe in three dimensions through a feedback loop linking image intensities and stage positions [52]. This is a very precise approach but can only track a single cell.

More recent techniques allow three-dimensional imaging of many bacteria within a field of view. In defocused imaging methods, depth-dependent image shape allows localization along the axis perpendicular to the focal plane ("z") (Figure 1c). This approach has long been used for non-bacterial imaging, e.g., nanoparticles [53], and has been applied to bacteria using fluorescence [54] as well as phase contrast [51] microscopy, with a z-range limit of 200 µm in the latter. Gray values can also be used to determine z-distance in cells close to the focal plane [55].


**Table 3.** Microscopic techniques to study bacterial motility and their main applications.

Another technique for three-dimensional reconstruction that has been applied to bacterial systems is digital holographic microscopy (DHM) [56] (Table 3). DHM reconstructs an image from the interference pattern produced by the specimen, illuminated by a coherent light source, although it does not support three-dimensional fluorescence imaging. While a low scattering efficiency of bacteria is a disadvantage, DHM has high imaging speed and, with recent improvements, a lateral resolution of less than 0.5 µm has been achieved [57,58]. Acres and Nadeau [59] described that DHM 2D projections generally suffice for calculating free-swimming bacteria speeds, but z-stacking is more accurate to study motility near a solid surface.

In light-field microscopy (LFM) a whole volume is illuminated and sampled in one snapshot, instead of using a bidimensional image as an input [60] (Table 3). Then, a microlens array translates depth information into a two-dimensional light field image, which can be computationally transformed back into a three-dimensional image. While LFM employs wide-field illumination, selective volume illumination microscopy (SVIM) is a variant that illuminates only the volume of interest, reducing the background noise and increasing the contrast, allowing a lateral resolution of 3 µm [61] (Table 3). Considering the high number of optimizations available, SVIM has a great potential for visualizing dynamic and complex interactions such as the bacterial flow of *Vibrio fischeri* within the seawater surrounding the light organ of its host, the Hawaiian bobtail squid (*Euprymna scolopes*), as well as the selective colonization of that organ by individual bacteria [61].

Differential dynamic microscopy (DDM) [62,63] relies on light scattering caused by a suspension of particles, instead of tracking (Table 3). The scattering forms a speckle pattern whose intensity will vary at a rate depending on the speed of the particles movement. These fluctuations lead to the differential intensity correlation function from which parameters such as speed and motile fraction can be extracted. While the great number of bacteria that can be processed simultaneously is a considerable advantage, this method is unsuited for obtaining more specific motility parameters. DDM is convenient to quickly evaluate motility responses at a whole-population level, such as the speed recovery after osmotic shocks of different magnitudes [64] and local speed changes caused by a light pattern projection in photokinetic *E. coli* genetically modified to swim smoothly with a light controllable speed [65].

All these techniques and more, under the appropriate conditions, are precise enough to reveal strategies for swimming, chemotaxis, and other behaviors. Lastly, new methods for extracting and assessing image-derived trajectories can be used to produce more accurate characterizations of the bacteria's movement. Accordingly, Liang et al. [66] implemented an unsupervised cluster analysis to fractionate the swimming trajectories of *Azotobacter vinelandii* into run and tumble segments, and then extracted the motility parameters distribution for each segment by fitting mathematical distributions. Other examples are the algorithms developed by Vissers et al. [67] (available on GitLab) to determine the positions, and orientations of individual rod-shaped bacteria, and track and analyze their surface dynamics, discerning between adhering, diffusing, and swimming cells.

Several techniques are available to study the role that bacterial appendages play in motility. However, as they are not in the scope of this review, they will be only briefly presented. Common techniques for visualizing nanomachineries include electron microscopy (EM) and its variations: transmission EM, scanning EM, and cryo-EM [68] are used to observe and study the structure of these bacterial components. Specifically, cryo-EM has recently provided 3D structural models of motility- [4] and chemotaxis-related [69] components with high resolution. However, freezing the cell makes capturing the dynamics of the machinery unachievable. Recent advances in fluorescence microscopy have allowed studying the functionality of these bacterial components. The substitution of amino acid residues of flagellin for cysteines or pilin subunits and subsequent labeling them with maleimide fluorescent dyes has allowed the study of flagellar [70] and pili [68] dynamics in real time. Moreover, a label-free technique, interferometric scattering microscopy (iSCAT), has recently been used to study type IV pili motor dynamics three-dimensionally [71]. These advances are vastly improving our knowledge of how the molecular machinery of bacterial motility operates.

#### **4. Study of Bacterial Motility in Bacterial–Host Interactions**

The study of bacterial motility inside a host is a more complex affair, which is why many studies simulate host conditions in vitro. Soft agar can, up to some extent, mimic physical, chemical, and nutritional conditions inside and outside the host [29,72,73]. Furthermore, chambers can mimic environments such as xylem vessels [74], enabling the discovery that *Xylella fastidiosa* migrates against the flow via twitching motility, and anaerobiosis, allowing researchers to prove that *Clostridioides difficile* modulates its swimming speed in the presence of a metabolite related to its host colonization [75]. Likewise, vertical diffusion chambers (VDC) were used to study the role of motility in *Campylobacter jejuni*

invasion of epithelial cells [76]. An alternative closer to in vivo conditions is tissue culture, which allows investigation of motility behavior in processes such as cell invasion and tumor colonization [77–79]. Lastly, artificial systems that reproduce the successive environmental niches of the human gastrointestinal tract can be used to simulate the host's dynamic conditions [80]. A metagenomic analysis of a gastrointestinal model of the colon developed by The Netherlands Organization for Applied Scientific Research (TIM-2) inoculated with human gut microbes showed that higher iron availability resulted in an enrichment of motility and chemotaxis functions [81]. Meanwhile, an early ex vivo approach in infant mice includes the labeling of motile and non-motile strains of *Vibrio cholerae* with fluorescent antibodies to visualize and compare its distribution in the extracted infected tissue [82].

In vivo real-time imaging is crucial to understand the colonization dynamics of bacteria. Intravital microscopy (IVM) consists of imaging inside live animals and often relies on fluorescence microscopy (Figure 1d; Table 3). The main problem is the thickness of the tissue samples, as off-focus blur and light scattering limit the depth of imaging [83]. Confocal microscopy can suffice; Moriarty et al. [84] reported high-resolution multidimensional visualization of bacterial dissemination inside a living mammal using spinning disk confocal IVM, revealing that dissemination of *Borrelia burgdorferi* in microvasculature of mice is a multi-stage process. Nonetheless, the scattered fluorescence limits the imaging depth of confocal microscopy to tens of microns. On the contrary, with multiphoton fluorescence, which is based on the simultaneous absorption of two or more infrared or near-infrared photons, imaging can be deeper than 100 µm in tissue. This is possible because longer wavelengths can penetrate at higher depths, besides lowering endogenous autofluorescence. Moreover, as excitation occurs only in the focal plane, there is minimal bleaching in the rest of the tissue [85,86]. Because of its advantages, IVM has been widely applied to visualize bacterial motility in colonized organs, such as *B. burgdorferi* in the skin [86] and *V. cholerae* in the intestine [33].

Zebrafish (*Danio rerio*) is a particularly advantageous vertebrate animal model for studying host–bacterial interactions due to their optical transparency at the larval stage, allowing for non-invasive examination of bacterial movement inside a living vertebrate host (Figure 1e). There are considerable similarities between zebrafish and mammals [87]. The gut is anatomically organized in separate sections and the intestinal epithelium is constantly renewing its cells. There is a high degree of orthologue genes [88] and their regulation within the gut is similar. The immune system of teleost fish species shares several traits with the system of mammals including the presence of lymphoid tissues, cell-mediated responses, and mucosal immunity [89].

Another advantage of zebrafish is that larvae hatch at 2–3 days post-fertilization (dpf) and open their mouths at 3 dpf, facilitating the production of germ-free or axenic individuals, great tools to study bacterial–host interactions. Fluorescently labeled bacteria can be inoculated via immersion at this developmental stage and visualized both at a whole population and at a single-cell level [90,91]. Germ-free zebrafish larvae colonized with fluorescent bacteria proved to be useful to examine the relationship between bacterial motility and symbiosis within the intestine [92,93]. In the last few years, the use of the zebrafish model coupled with light-sheet fluorescence microscopy (LSFM, also known as selective plane illumination microscopy) has provided new insights into the field of bacterial dynamics within a living host [94–98]. In this technique, only the focal plane is illuminated, exciting all points in the plane simultaneously, while out-of-focus points are not excited, minimizing photodamage and photobleaching and increasing imaging speed compared to point scanning methods, while achieving much higher resolution than wild field microscopy [96,99]. These characteristics make LSFM very suitable to follow bacterial dynamics inside the whole intestine of zebrafish for several hours. Nevertheless, because of light diffraction, generating a thin plane of excitation light is difficult, causing a loss in resolution compared to confocal and multiphoton imaging.

Combining LSFM, larval zebrafish, and bacteria engineered with inducible switches for a flagellar motor component revealed that the swimming motility of a zebrafish-native *Vibrio* species was necessary for its persistence inside the host and avoidance of expulsion with intestinal flow [98]. In a separate study, live imaging revealed that sub-lethal doses of the broad-spectrum antibiotic ciprofloxacin promoted its bacterial aggregation and expulsion from the intestine [100].

Finally, transcriptomic approaches can be used to investigate the effect of host or environmental factors [101–103] and phenomena such as macrophage internalization [104] and host cell infection [105] in the transcriptional regulation of genes related to bacterial motility. Employing microarrays, Snyder et al. [106] first assessed an *E. coli* pathotype's transcriptome in vivo from bacteria extracted from infected mice, showing that flagellar genes were downregulated compared to in vitro conditions. Interestingly, this transcriptome was performed from different urine samples taken across 10 days of the infection period. A similar experiment using an *E. coli* expressing a luminescent reporter for the flagellar gene *fliC* showed that its expression was upregulated during the pathogen's ascension through the upper urinary tract, suggesting a major contribution of motility in the colonization of the urinary system [107]. Recently, a comparison by RNA-seq between *Pseudomonas plecoglossicida* infecting spleens of the fish *Larimichthys crocea* and those cultivated in vitro revealed an up-regulation of motility-related and flagellum-related genes during the fish infection [108].

It is important to consider that, as single-cell transcriptomic approaches are difficult to achieve in prokaryotes [109], only homogenized output from a population is usually obtained for bacteria, impeding the study of phenotypically distinct subpopulations that could be present in the sample. Recent works have focused on overcoming these difficulties with strategies including mRNA enrichment methods. Kuchina et al. (2021) modified SPLiT-seq—a technique that uses combinatorial indexing to label the eukaryotic RNA's cellular origin—to optimize its performance in bacteria. This approach was able to assess the fraction of *Bacillus subtilis* PY79 population that expressed flagellin and surfactin while growing in a rich medium [110].

Lastly, proteomic approaches, particularly those based on mass spectrometry (MS) which measures the mass-to-charge ratio of ionized molecules to identify them—have proven to be a notable tool for assessing abundance changes in bacterial proteins inside a host [111,112]. Proteomic studies using liquid chromatography MS showed that downregulation of *Salmonella enterica* Typhimurium proteins involved in virulence, chemotaxis, and flagellar systems occurs earlier in bacteria inside macrophages compared to bacteria internalized by epithelial cells, suggesting that different host cell types have a different impact on motility adaptations [112].

#### **5. Discussion and Concluding Remarks**

The crucial role of motility in bacterial survival, host colonization, and/or virulence is a fact. This mini review showed that multiple approaches are available to study motility, from soft agar to a wide variety of microscopic techniques. The optimal choice will depend on the specific questions or requirements of the experiment, such as the number of cells or strains to process, z range needed, and growth conditions. In host–bacterial interactions, in vitro set-ups can provide fair approximations to the host environment, whereas intravital microscopy allows in vivo tracking of bacteria within the host tissue. This approach benefits from techniques that allow a greater depth of imaging, namely, confocal, and multiphoton fluorescence microscopy. Alternatively, the zebrafish model allows direct visualization of bacteria inside the host. Assessing the expression level of motility-related genes is also feasible. All these approaches can be combined to have a wider outlook; for example, coupling semi-solid (soft) agar plates with microscopy visualization. Accordingly, Deforet et al. [55] observed that macroscopically, a *P. aeruginosa* hyperswarmer mutant spreads faster, yet does not swim faster than the wild-type at the single-cell level. Further investigation led to realize if this phenomenon is related to wider turns.

Overall, a considerable number of new methods and advances to study bacterial motility have emerged during the last decade, deepening our understanding of bacterial behavior. Nevertheless, there are several issues that still need improvement, such as protocol standardization in soft agar assays; facilitating the implementation of 3D tracking, mostly achieved by microscopy techniques that are technically demanding and/or require complex set-ups and extending the depth of imaging for bacteria within host tissue in in vivo motility studies.

**Author Contributions:** Conceptualization, V.P. and P.N.; writing—original draft preparation, V.P., M.S.G., O.V., R.P. and P.N.; writing—review and editing, R.P. and P.N.; funding acquisition, P.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by FONDECYT/ANID, grant number 1181499.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** MSG acknowledges funding by FONDECYT/ANID, grant number 3200998.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the writing of the manuscript.

#### **References**


## *Review* **Research Progress of the Gut Microbiome in Hybrid Fish**

**Xinyuan Cui <sup>1</sup> , Qinrong Zhang <sup>1</sup> , Qunde Zhang <sup>1</sup> , Yongyong Zhang <sup>1</sup> , Hua Chen <sup>2</sup> , Guoqi Liu <sup>2</sup> and Lifeng Zhu 1,\***

	- liuguoqi@mingkebio.com (G.L.)

**Abstract:** Fish, including hybrid species, are essential components of aquaculture, and the gut microbiome plays a vital role in fish growth, behavior, digestion, and immune health. The gut microbiome can be affected by various internal and/or external factors, such as host development, diet, and environment. We reviewed the effects of diet and dietary supplements on intestinal microorganisms in hybrid fish and the difference in the gut microbiome between the hybrid and their hybrids that originate. Then, we summarized the role of the gut microbiome in the speciation and ecological invasion of hybrid fish. Finally, we discussed possible future studies on the gut microbiome in hybrid fish, including the potential interaction with environmental microbiomes, the effects of the gut microbiome on population expansion, and fish conservation and management.

**Keywords:** hybrid fishes; gut microbiome; community and function; speciation; invasion; fish conservation and management

## **1. Introduction**

The host and its microbiome are regarded as a unique biological entity holobiont, including the genome, which is called the hologenome [1]. The combination of complex microbiota and genes in the intestine are collectively referred to as the gut microbiome [2]. Animal hosts maintain a long, close, and complex relationship with their gut microbiome [3]. The gut microbiome plays a vital role in the nervous system development [4], behavior [5], immunity [6], food digestion, and metabolism [7] of the host. Gut microbiota are highly specialized microbial communities with a complex composition that is affected by many interactions among microorganisms, host, diet, and the environment [8]. Host phylogeny and diet are the two main factors shaping the animal gut microbiome [9–14].

Fish comprise nearly 50% of the total vertebrate diversity, and more than 34,000 species have been described to date, constituting a crucial part of the aquatic ecosystem [15,16]. Microorganisms exist in almost every fish organ, including the skin, digestive tract, internal organs, and luminous organs [17]. The fish gut is a complex ecosystem, composed of highly diverse microbiota. The microbiota is influenced by various factors, such as habitat environmental factors, season, host genetics, developmental stage, nutrition level, and diet composition, with the potential major determinant being the habitat environment [16].

Overall, bacteria are the primary microbial colonizers in the gastrointestinal tract of fish [18–21]. The gastrointestinal microbiota of fish mainly consist of aerobic or facultative anaerobic microorganisms and facultative and obligate anaerobes [20,22–24]. Among them, Proteobacteria, Firmicutes, and Bacteroidetes constitute 90% of the gut microbiome of most fish [15]. In addition, Actinobacteria, Fusobacteria, Bacilli, Clostridia, and Verrucomicrobia are the dominant bacterial phyla in fish gut microorganisms [15,25–29]. The gut microbiota of fish participate in various physiological functions. There are several beneficial effects on the host, such as reproduction, development, nutrition, immunity, and stress responses, and

**Citation:** Cui, X.; Zhang, Q.; Zhang, Q.; Zhang, Y.; Chen, H.; Liu, G.; Zhu, L. Research Progress of the Gut Microbiome in Hybrid Fish. *Microorganisms* **2022**, *10*, 891. https://doi.org/10.3390/ microorganisms10050891

Academic Editor: Konstantinos Ar. Kormas

Received: 4 April 2022 Accepted: 21 April 2022 Published: 24 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the gut microbiota are often referred to as an 'extra organ' [15,30]. Nayak has described the role of fish gastrointestinal microbiota in nutrition, immunity, and health management [20].

Early research on fish gut microflora employed culture-dependent techniques. The emergence of metagenomics and next-generation sequencing techniques has entirely changed fish gut microbiome research by presenting a method that directly analyzes the microbial genome from environmental samples [31,32]. These new research methods have led to a better understanding of the connections between the microorganisms and their respective hosts. The Illumina system, Roche 454 system, and Ion Torrent Personal Genome Machine (PGM) are the primary next-generation sequencing (NGS) platforms used in fish gut microbiome research, and the Illumina system is the most commonly used [15].

The influencing factors and physiological functions of fish intestinal microbiota are two critical issues in NGS analyses [33]. Most studies have explored the effects of various host and environmental factors on the bacterial community composition of gut microbiota. Limited studies have analyzed the beneficial and harmful effects of the gut microbiota on the host [15]. However, there are many valuable bacterial species in the intestines of fish, including *Cetobacterium* spp. and *Lactobacillus* spp. [34]. Hybrid fish are indispensable components of fish species and are essential in aquaculture. We review recent research on the gut microbiome and ecological problems in hybrid fishes and discuss possible future research to improve our understanding of the gut microbiome in fish.

#### **2. The Gut Microbiome in Hybrid Fish**

### *2.1. Effects of Diet and Dietary Supplements on the Gut Microbiome and Immune Health of Hybrid Fish*

Hybridization is a basic step in the long-term evolution of organisms, which may lead to the production of new species. Heterosis is a complex biological phenomenon where the hybrid offspring show superior natural characteristics, when compared with their parents [35,36]. Heterosis occurs in fish, and hybrid fish have advantages of faster growth performance, higher immunity, improved ecological adaptability, and an enhanced tolerance for transportation. Therefore, as wild catch fisheries can no longer support the world consumption of seafood, fish heterosis has been widely assisting aquaculture since the 1980s [37–41]. However, even the improvements made by heterosis may not be enough for the growing world consumption rate of fish [35].

In addition, different fish species inhabiting the same waters may also naturally hybridize in the wild. Hybrid fish may possess improved ecological adaptability compared to their parents and be more widely distributed in the natural environment with heterosis, due to the survival of the fittest theory [42–44]. In reality, the microbiota in hybrids may provide new favorable physiological functions and promote the utilization of new ecological niches, and the hybrid microbiota may also shape reproductive barriers, which may influence the ecological speciation or the expansion of the population range [45–48]. It has been shown that greater than 30,000 variations of hybrid fish species have formed in the wild, and these large fish populations can produce high diversity in the dietary niches. Therefore, exploring the microbiota of wild hybrid fish is of great significance for understanding the basic biological and ecological processes of speciation, population expansion, and invasion ecology [39].

We, firstly, aimed to provide a whole picture of the diet or dietary supplement effects on the fish gut microbiome (Table 1) [15,20,49,50]. Then, we focused on the relationship between the diet and the hybrid fish gut microbiome. We found that many studies have explored changing the diet or dietary additives on the composition and function of the hybrid fish gut microbiome and their promotion of the growth and health of mixed fish (Table 2), but rare in the comparison between the hybrid and their hybrids' origin.



**Table 1.** The application of diet and dietary supplements in fish.




**Table 2.** The studies on the gut microbiome of hybrid fish.





#### 2.1.1. Antibiotics

Infectious diseases caused by various pathogens have severely harmed the health of aquatic organisms around the world [125]. Antibiotics have been widely used as feed supplements to treat intestinal diseases in fish and have become indispensable in human health [33,126,127]. A short-term (6 days) dietary antibiotic mixture (vancomycin, neomycin sulfate, and metronidazole) can improve the lipid metabolism in hybrid groupers (*Epinephelus fuscoguttatus* ♀ × *E. lanceolatus* ♂) fed medium- and high-lipid diets. However, antibiotic treatments can also strongly alter intestinal microbiota by reducing the relative abundance and diversity of hybrid grouper gut microbiota, resulting in a significant increase in the proportion of Bacteroidetes and a decrease in the proportion of Firmicutes [93]. Long-term antibiotic supplementation can cause several side effects on fish health [127–129]. Presently, the pollution and spread of antibiotic-resistant genes caused by the long-term abuse of antibiotics have become a global problem [130]. Recently, probiotics and prebiotics are an emerging strategic approach for sustainable aquaculture, as they do not cause environmental pollution or public health hazards [51,131,132].

#### 2.1.2. Probiotics

Probiotics are beneficial microorganisms that can modulate intestinal microbial composition and improve the host health status [133,134]. Probiotics are commonly used in the aquaculture industry as feed or water additives [20]. The essential probiotic microorganisms employed in aquaculture are lactic acid bacteria (LAB) species [135,136] and *Bacillus spp*. [52,137]. The other general probiotic species used in fish are *Saccharomyces*, *Clostridium*, *Enterococcus*, *Shewanella*, *Leuconostoc*, *Lactococcus*, *Carnobacterium*, and *Aeromonas* [20]. Fish are vulnerable to various pathogenic microorganisms, and innate immunity provides an initial line of defense [138]. The addition of probiotics to the diet plays a vital role in stimulating fish immune responses, and further promotes the innate and adaptive immune system [139]. For an example, *Bacillus subtilis* strain 7k, isolated from the gastrointestinal tract of hybrid hulong grouper (*Epinephelus fuscoguttatus* × *E. lanceolatus*), could be used in grouper culture to stimulate growth, enhance immunity and promote health in the fishes [94]. Studies reveal that *O. mykiss* fed different types of probiotics increased the expression of the TGF-β gene, which regulates fish immunity [140–142]. TGF-β levels increased in juvenile hybrid tilapia (*O. niloticus* ♀ × *Oreochromis aureus* ♂), after consuming a diet supplemented with *Bacillus subtilis* C-3102 [95], and the same occurred in Koi carp (*Cyprinus carpio*) [143]. HWF™ is a paraprobiotic and postbiotic supplementary diet using inactive and beneficial bacteria, and is considered an efficient therapeutic agent in fish. Feeding hybrid sturgeons (*Acipenser baerii* × *Acipensers chrenckii*) with HWF™ improved their growth and immunity by changing the composition and diversity of the gut bacteria, developing their healthy gut microbiota [96].

#### 2.1.3. Prebiotics

Prebiotics are an innovative strategy, providing a dietary supplement to improve growth development and the immune system by regulating gut microbiota [144]. Prebiotics are generally non-digestible oligosaccharides added to fish feed as dietary components to promote the proliferation of specific beneficial microorganisms in the intestine and, thus, enhance host health [145]. Previous research has shown that prebiotics can decrease the adherence and colonization of pathogenic microorganisms in the intestinal tract to improve the general immunity of the host by increasing the number of lactic acid bacteria, especially *Bifidobacterium* [20,146,147]. Fructo-oligosaccharides, galactooligosaccharides, mannanoligosaccharides (MOS), xylooligosaccharides (XOS), inulin, lactulose, and lactosucrose are common prebiotics used in various animals, including humans [20]. The level of gut lactic acid bacteria was significantly increased in hybrid catfish (*Pangasianodon gigas* × *Pangasianodon hypophthalmus*) fed with diets containing 0.6% xylooligosaccharides (XOS) [97]. In addition, several studies have reported that inulin, fructooligosaccharides, xylooligosaccharides, galactooligosaccharides, and arabinoxylan-oligosaccharides can affect growth

development, immune health, and the composition and/or diversity of the gut microbiota in different fish species [53,97,148–151]. Indeed, many researchers have reported the effect of prebiotics on the gut microbiota in fish, such as grass carp [54], Siberian sturgeon [53], Nile tilapia [55], and European sea bass [152].

The prebiotic Grobiotic™AE and dietary brewer's yeast can improve the growth performance, immune response, and resistance to *Streptococcus iniae* infection in hybrid striped bass (*Morone chrysops* <sup>×</sup> *M. saxatilis*) [153]. Dietary supplementation of 4% ESTAQUA® yeast culture (YC) for hybrid grouper (*Epinephelus fuscoguttatus* ♀ × *E. lanceolatus* ♂) could improve the alpha diversity of gut microbiota, growth performance and serum immune responses against *V. harveyi* attacks [98]. N.B.T. is an excellent indicator of the health status and/or immunization effectiveness in fish [56]. Supplementing the diet with raffinose in hybrid sturgeons (*Acipenser baeri* Brandt ♀× *A. schrenckii* Brandt ♂) improved the growth performance and intestinal morphology, modifying the gut microbiota composition and increasing the level of N.B.T. activity [99]. Chitosan oligosaccharide (COS) is a new prebiotic, dietary COS supplementation, which improves the growth performance and health status of *Scopthalmus maximus* [154], *Cyprinus carpio koi* [155], and *Oncorhynchus mykiss* [156]. Dietary COS supplementation improved the intestinal health and immune responses of hybrid groupers (*Epinephelus fuscoguttatus* ♀ × *E. lanceolatus* ♂) when fed a low-fish meal diet [100].

It is worth noting that prebiotic supplementation is only beneficial when a moderate volume is provided; prebiotics at a high concentration can be harmful to the host. Excessive prebiotics may cause an imbalance in the gut microenvironment, which decreases the digestive capacity in fish intestines. A previous study revealed that a high concentration of inulin could damage the enterocytes of *Salvelinus alpinus* [157]. This may explain why 0.4–0.6% COS supplementation was optimum in hybrid groupers [100].

#### 2.1.4. Fishmeal Protein Substitutes

Fishmeal (F.M.) is the most widely utilized high-quality protein source in aquatic feed and has many advantages [158]. However, fishmeal production cannot meet the growing needs of the aquaculture industry due to its rapid development, which is causing a severe impediment to industry development [101,159]. Therefore, using plant proteins is an innovative solution for sustainable aquaculture [160,161].

Cottonseed protein concentrate (CPC) is a new experimental fishmeal (FM) replacement [162]. However, fishmeal replaced with CPC in an inappropriate proportion can have adverse effects on the intestinal health of groupers and leads to intestinal inflammation [163]. A study on pearl gentian groupers (*Epinephelus fuscoguttatus* ♀ × *Epinephelus lanceolate* ♂) revealed that 24% CPC was considered the most appropriate volume for F.M. replacement and growth performance, digestive proteinase activity, intestinal morphology, and intestinal microflora in the pearl gentian grouper reached maximum levels with 24% CPC replacement levels. Subsequently, many physiological parameters are reduced with increasing CPC replacement levels [101]. The substitution of FM with peanut meal (PNM) of up to 50% or CPC up to 60% obviously changed the intestinal microbiota of juvenile hybrid groupers *(E. fuscoguttatus* ♀ × *E. lanceolatus* ♂), which increased intestinal pathogenic bacteria and decreased intestinal beneficial bacteria [102,103]. Similarly, replacing FM with peptides from swine blood (PSB) up to 75% could reduce growth performance for hybrid groupers (*Epinephelus fuscoguttatus* ♀ × *E. lanceolatus* ♂), and increase the abundance of the potentially pathogenic *Pseudomonas* and *Arcobacter* in the gut [104].

Another fishmeal replacement protein is soybean meal (SBM). SBM has been widely considered an inexpensive FM replacement [164]. Nevertheless, anti-nutritional factors in SBM can negatively affect the intestinal morphology of fish [165]. Research reveals that bioprocesses (such as soybean meal ingredients) can reduce the intestinal microorganism diversity in hybrid striped bass (*Morone chrysops* × *M. saxatilis*) [105]. It is challenging to find a suitable fish meal substitute for various fish, and protein substitutes have excellent potential and are important future research topics.

#### *2.2. Hybrid Speciation and Gut Microbiome*

No living organisms exist in isolation from the microbial world, and microbial symbiosis and speciation profoundly shape the biodiversity composition. Animal hosts and microbiomes are closely interconnected and interact over long evolutionary timeframes. They can even be regarded as a unique biological entity-holobiont and include their entire genome, called the hologenome [1]. Diverse and complex interactions exist between hosts and microorganisms. Microorganisms play essential roles in host physiology, health, and survival. Microorganisms can even alter host reproduction [166], resulting in host embryo death [167–170] and affect the host gametic integrity and embryonic viability, which may be closely related to the formation of new species [45,171]. The microorganisms and their interactions with hosts are potentially important factors in stimulating the formation of new species [172].

Species are reproductively isolated groups composed of potentially interbreeding individuals, and hybrids can suffer from post-mating isolation barriers, such as sterility and/or unviability [173]. The composition and functional effects of animal microbiota are closely related to host evolution, and the survival rate and performance of microorganisms can be reduced when interspecific microbiota transplantation occurs between closely related and different host species pairs. The microbiome compositional relationships (i.e., beta diversity) reflect the evolutionary relationships of the host species [173,174]. Thus, natural selection can drive phylosymbiotic changes within the parental species, which may lead to the evolution of deleterious interactions between hybrids and their microbiomes [173].

Based on the holobiont concept, host-genome–microbiome associations and their role in host adaptability demonstrate that microorganisms may participate in the process of speciation, and symbiotic microorganisms may hinder speciation through isolation, including behavioral isolation, geographical isolation, and reproductive isolation [45]. Microbial symbionts can add new functional genes to the host genome, which assists the host in expanding its dietary niche and obtaining new nutritional opportunities. Unfortunately, hybridization can inhibit symbiotic relationships by destroying the vertical transmission of some microorganisms between the host parents and offspring, which are hybridization disadvantages and hinder species formation, as observed in *Acyrthosiphon pisum* [175], *Sitophilus* [176] and the family *Plataspidae* [177]. In hybrid species, microorganisms can hinder speciation by assisting reproductive isolation. *Wolbachia* is a bacterium that widely exists in the reproductive system of arthropods and may cause hybrid male sterility in *Drosophila paulistorum* [178]. In the two-spotted mite (*Tetranychus urticae*), *Wolbachia* can also cause cytoplasmic incompatibility (CI) in the F1 generation and F2 male offspring deaths from the surviving F1 females in the CI cross [178]. Similarly, different CI Wolbachia in *Nasonia* wasp species can cause high levels of F1 hybrid lethality and the reproductive isolation induced by CI has evolutionary potential in the early stages of the speciation process [179,180].

Similarly, a close interaction exists between the gut microbiome and host, and plays an important role in the speciation of hybrid species. For example, the host gut microbiome may hinder the formation of new species by participating in the death of hybrids in *Nasonia* wasp species [181]. Vertebrates are a vital group for interactions in reproductive isolation and speciation research. Alterations in gut microbiota communities and increases in gut pathology exist in hybrid mice *(Mus musculus* × *Mus domesticus*) [46]. The gut microbiome does not always play negative roles in hybrid species. For example, the hybrid offspring of sika deer (*Cervus nippon*) and elk (*Cervus elaphus*) harbor a high abundance of *Acetitomaculum* bacterial species, which may assist in the absorption and metabolism of nutrients [182,183]. A similar phenomenon was identified in the hybrid offspring of ponies and donkeys, which render a completely different gut microbiota from their parents [184].

In the gut microbiome in hybrid fish research, differences in the gut microbiome between hybrid offspring and parents have been observed. In lake whitefish (*Coregonus clupeaformis*), the gut microbiome is significantly different between the F1 hybrids and their parents, especially the abundance difference between Firmicutes and Proteobacteria [106]. The research also found the interactions of the host-microbiota-environment

demonstrated three different evolutionary paths in the gut microbiome [106]. Similarly, the gut characteristics of hybrid fish from herbivorous blunt snout bream (*Megalobrama amblycephala*) and carnivorous topmouth culter (*Culter alburnus*) differ from their parents. The microbial community in the hybrid topmouth culters was markedly distinct from their parents, and varied in the cellulose content in the gut [39]. One study found that the evolutionary characteristics of hybrid fish progeny from *Megalobrama amblycephala* and *Culter alburnus* may be manifested in dietary adaptation and choice; the interactions between gut microbiota and host genetics contributed to hybrid fishes adapting to herbivorous diets more than carnivorous diets [185]. Compared to the parents, the hybrid offspring of two invasive North American carp, *Hypophthalmichthys nobilis* and *Hypophthalmichthys molitrix*, harbor different gut microbiome compositions and display higher alpha diversity than their parents [107].

#### *2.3. The Differences in the Gut Microbiome of the Hybrid Fish and Their Hybrids Origin*

There are still few studies directly comparing gut microbiome between parental and hybrid progeny. However, it has been shown that existing differences in intestinal microbiota between captive parents and hybrid fishes' offspring exist under a controlled environment [106]. There is no doubt that diet will affect the gut microbiome composition and growth performance of the host, and under the same dietary conditions (Artemia and mixed diet), the taxonomic composition of transient gut microbiota between both whitefish (*Coregonus clupeaformis*) parental species and their reciprocal hybrids showed a slight pattern of differentiation, which, within the Artemia diet group, meant a higher abundance for Firmicutes, but lower for Proteobacteria, was observed in hybrids in comparison with their parents' whitefish, while the opposite result was found in the mixed diet group, where there was a higher abundance of Proteobacteria but it was lower for Firmicutes. In addition, in the abundance composition of some specific bacterial genera, the two reciprocal hybrids, and their parents also showed the opposite pattern, that F1 D♀N♂has more specific bacterial genera than its parents, while F1 N♀D♂with fewer specific bacterial genera than its parents. In the hybridization experiment between whitefish and omul (*Coregonus migratorius*), the researchers found that the hybrid progeny had a lower alpha diversity (e.g., Shannon index) in hindgut microbiota than the parents [186].

Host genetics can strongly affect the gut microbial composition of the hybrid offspring [39]. Compared with carnivorous topmouth culter (*Culter alburnus*, TC) parents, the gut microbiome structure of their two-hybrid progenies is more similar to that of herbivorous blunt snout bream (*Megalobrama amblycephala*, BSB) parents, as the alpha diversity of the two types of hybrids and BSB parent is higher than that of a TC parent, as well as beta diversity analysis, which also showed that there was no significant difference between the two hybrids and the BSB parent. Interestingly, in the composition of gut microbiota, Fusobacteria and Proteobacteria are the most abundant intestinal flora in hybrid fishes, and the proportion of Fusobacteria and Proteobacteria in hybrid offspring is similar to the BSB parent but significantly different from the TC parent. Again, the shared bacterial taxa at the phylum level showed different results; the hybrids of the two types share higher proportions of gut bacterial communities with the BSB parent than the TC parent.

Recently, our study reported a direct comparison of the similarities and differences in gut microbiome (composition and potential function) among bighead carps (*Hypophthalmichthys nobilis*, B), silver carps (*Hypophthalmichthys molitr*, S) and their hybrid offspring (SB and BS) in ponding experiments [107]. The hybrid gut microbiome displays the admixed pattern at the community level and harbors the relatively high alpha diversity (e.g., phylogenetic diversity). For example, the hybrid fish had intermediate abundances of Cyanobacteria and Bacteroidetes in the foregut, while Fusobacteria are significantly enriched in parents in the hindgut. Moreover, the hybrid gut microbiome's predicted function shows the enrichment in the genes coding for putative enzymes involved the diet utilization, which suggests the potential benefits to their local adaptation.

#### *2.4. Gut Microbiome Might Promote Ecological Invasion by Hybrid Fish*

Gut microbiota can enhance the adaptability of the host to the environment and improve the successful invasion rate of some invasive species [187]. For invasion success, the species requires a dispersal ability, environmental tolerance, phenotypic plasticity, and associated epigenetics [188,189]. Host shifts can lead to phytophagous insects becoming invasive species [190]. It has already been demonstrated that the gut microbiome plays a vital role in phytophagous insect invasion success [191], and gut bacteria can assist in the successful invasion of insect species by regulating epigenetic factors related to the host [192]. Similarly, some biological mechanisms can enhance the success rate of invasive species, such as genetic diversity [193], reproductive rate [194], food resources [195], and hybridization [44,196].

Therefore, there are complex and close relationships between hybridization, the gut microbiome, and bio-invasion. Bighead carp and silver carp are invasive species, characterized by various hybridization in the Mississippi River Basin [107]. There is higher alpha diversity in the foregut microbiota in the hybrid offspring, and an increasing discrepancy also occurs between the foregut and hindgut. Similarly, the hybrids had a higher proportion of putative genes coding for putative enzymes related to the digestion of filter-feeding phytoplankton (Cyanobacteria, cellulose, and chitin) than their parents. The improved putative enzymes could encourage the utilization of new food resources by the gut microbiota and, therefore, improve survival, environmental adaptation, and invasion by hybrid fish. Therefore, the gut microbiome and host genome may synergistically promote bigheaded carp invasion in the United States [107].

#### **3. The Potential Impact of Environmental Microbiota**

The current research focuses on fish, not hybrid fish. However, environmental microbiota impacts may also occur in hybrid fish.

#### *3.1. Habitat Environmental Microbiome Shapes the Early Gut Microbiome of Juvenile Fish*

The main determinant of fish gut microbiota is the natural environment, and fish intestinal microbiota symbionts are generally obtained from the environment [197] by neutral processes, such as drift and diffusion, which produce most of the microbial diversity [198]. The microorganisms transmitted from the environment to the fish intestine are mainly derived from two paths: the foodborne microorganisms carried by prey and the microorganisms in the water, and most of the environmental microbiota remain temporarily in the fish gut [199]. In most fish species, the ontogeny and colonization of gut microbiota in the early stages of life rely on the horizontal transmission of environmental microbiota [200]. Juvenile zebrafish (*Danio rerio*) acquire gut symbiotic bacteria from the water environment after hatching, which may promote the development and function of their intestines [201]. Similar patterns are observed in wild Atlantic salmon (*Salmo salar*), discus (*Symphysodon aequifasciata*) [200], grass carp (*Ctenopharyngodon idellus*), Mucha perch (*Siniperca chuatsi*), and southern catfish (*Silurus meridionalis*). The composition of the gut microbiota community of juvenile fish was more similar to the habitat water environment than the adults [197,200]. However, fish gut microbiota often differ from their surrounding environment after becoming adults [202]. Therefore, environmental microorganisms play an important role in shaping the gut microbiota in the early juvenile fish stages and, as fish mature, the environmental factors are less influential because the gut microbiota gradually differentiate from the environmental microbiota, showing individual variations [197,203].

#### *3.2. Do Fish Specifically Select Proteus from the Water Environment?*

The gut microbiota of fish are mainly Proteobacteria and Firmicutes, whereas amphibians, reptiles, birds, and mammals contain mainly Firmicutes and Bacteroidetes. The excessive reproduction and presence of *Proteus* may be a sign of ecological imbalance in the gut microbial community of mammals [204], as many symbiotic *Proteus* bacteria can translate into pathogens, and infect and promote inflammation in the host under

**References** 

3229–3236.

*Biol.* **2015**, *13*, e1002226.

specific conditions. Many studies have demonstrated that, regardless of the fish living environment, the gut microbiome is composed of a common core microbiome [205]. Major environmental microorganisms are rarely observed in fish intestines [49]. *Proteus* dominate the gut microbiota of most fish species [206]. The *Proteus* abundance can increase with the growth and nutritional level of the fish (from herbivorous to carnivorous). Conversely, the abundance of Firmicutes usually decreases with increasing nutritional levels [202]. The gut microbiome not only reflects the microorganisms in its surrounding environment but also characterizes the specific selection of the environmental microbiome by the host in grass carp (*Ctenopharyngodon idellus*) [207], silver Prussian carp (*Carassius auratus gibelio*) [208], and zebrafish (*Danio rerio*) [209]. Notably, the higher proportion of *Proteus* in the fish intestines indicates the fish host has specifically selected *Proteus* from the habitat water or *Proteus* has outperformed the other environmental bacterial taxa in the water. This discrepancy is an urgent problem needing to be explored [16].

#### **4. Future Perspectives**

The gut microbiome can promote the successful ecological invasion of hybrid fish, which makes them occupy favorable ecological niches and further improves the potential for population expansion. Following Darwin's theory of evolution, this process greatly improves the potential of hybrid fish to evolve into new species in the future (Figure 1). The gut microbiome plays a role in speciation, but its degree of impact remains unclear. Furthermore, the high genomic similarity between bighead and silver carp, and an over 90% embryonic viability in all crosses, indicate that interspecific hybridization between the carps might have promoted their range expansion [44]. In the future, the role of the gut microbiome in population expansion of hybrid species should not be ignored. It is highly significant for us to better combine the genome and metagenome to improve our understanding of the ecological problems of hybrid fish. The fish gut flora and fecal materials discharged into the water may reflect their diet preferences, physiological behaviors, and presence in the river [210], allowing gut microbiota to potentially monitor fish invasion and population expansion, which is an important research issue in fish conservation and management in the future (Figure 1). water than the adults [197,200]. In addition, different fish tissue types, such as skin, gills, and intestines, may also be the main determinants of microbiota diversity and composition [48]. Successful hybrid fish invasion depends on the relationships and interactions between an individual's characteristics (age and gender), gut microbiome, environmental microbiome, and post-mating reproductive isolation, associated with environmental microbial transmission. Future research is required to assist our understanding of these interactions (Figure 1). In addition, the aquatic environment can become a reservoir of antibiotic-resistant genes (ARGs), providing an ideal path for the acquisition and dissemination of ARGs [211]. Aquatic animals, such as fish, are direct witnesses and victims of ARGwater pollution. Therefore, wild fish can be recipients and disseminators of ARGs in aquatic environments [130]. At present, there are few studies assessing ARG pollution and transmission in wild hybrid fish, providing great research potential in the future (Figure 1).

*Microorganisms* **2022**, *10*, x FOR PEER REVIEW 13 of 22

**Figure 1.** Major research progress and future perspectives on hybrid fish gut microbiome. **Figure 1.** Major research progress and future perspectives on hybrid fish gut microbiome.

**Author Contributions:** L.Z. conceived the ideas and methodology. X.C., Q.Z. (Qinrong Zhang), Q.Z. (Qunde Zhang), Y.Z., H.C. and G.L. collected the data. X.C. and L.Z. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. **Funding:** Financial support was provided by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). For a long time, the source of gut microbiota has been an attractive research topic. Environmental microbiome transmission plays an important role in animal gut microbiota, and the differences between terrestrial and aquatic environments cause the gut of aquatic animals to be very different from that of terrestrial organisms, including fish and aquatic mammals. Research shows 13% of the gut microbiota of threespine stickleback (*Gasterosteus aculeatus*) comes from the surrounding water environment and 73% from prey [199]. In

1. Bordenstein, S.R.; Theis, K.R. Host Biology in Light of the Microbiome: Ten Principles of Holobionts and Hologenomes. *PLoS* 

3. McFall-Ngai, M.; Hadfield, M.G.; Bosch, T.C.G.; Carey, H.V.; Domazet-Lošo, T.; Douglas, A.E.; Dubilier, N.; Eberl, G.; Fukami, T.; Gilbert, S.F.; et al. Animals in a bacterial world, a new imperative for the life sciences. *Proc. Natl. Acad. Sci. USA* **2013**, *110*,

4. Carlson, A.; Xia, K.; Azcarate-Peril, M.A.; Goldman, B.D.; Ahn, M.; Styner, M.A.; Thompson, A.L.; Geng, X.; Gilmore, J.H.; Knickmeyer, R.C*.* Infant Gut Microbiome Associated With Cognitive Development. *Biol. Psychiatry* **2018**, *83*, 148–159.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

2. Rosenberg, E.; Zilber-Rosenberg, I. The hologenome concept of evolution after 10 years. *Microbiome* **2018**, *6*, 78.

6. Levy, M.; Blacher, E.; Elinav, E. Microbiome, metabolites and host immunity. *Curr. Opin. Microbiol.* **2017**, *35*, 8–15.

5. Archie, E A.; Tung, J. Social behavior and the microbiome. *Curr. Opin. Behav. Sci*. **2015**, *6*, 28–34.

addition, in most fish species, the ontogeny and colonization by gut microbiota in the early stages of life mainly occur through the horizontal transmission of environmental microbiota [200]. Juvenile zebrafish (*Danio rerio*) acquire gut symbiotic bacteria from the water environment after hatching, potentially promoting the development and function of the intestines [201]. Similar patterns are observed in wild Atlantic salmon (*Salmo salar*), discus (*Symphysodon aequifasciata*) [200], grass carp (*Ctenopharyngodon idellus*), Mucha perch (*Siniperca chuatsi*), and southern catfish (*Silurus meridionalis*), and the composition of the gut microbiota community of juvenile fish was more similar to the habitat water than the adults [197,200]. In addition, different fish tissue types, such as skin, gills, and intestines, may also be the main determinants of microbiota diversity and composition [48]. Successful hybrid fish invasion depends on the relationships and interactions between an individual's characteristics (age and gender), gut microbiome, environmental microbiome, and post-mating reproductive isolation, associated with environmental microbial transmission. Future research is required to assist our understanding of these interactions (Figure 1). In addition, the aquatic environment can become a reservoir of antibiotic-resistant genes (ARGs), providing an ideal path for the acquisition and dissemination of ARGs [211]. Aquatic animals, such as fish, are direct witnesses and victims of ARG-water pollution. Therefore, wild fish can be recipients and disseminators of ARGs in aquatic environments [130]. At present, there are few studies assessing ARG pollution and transmission in wild hybrid fish, providing great research potential in the future (Figure 1).

**Author Contributions:** L.Z. conceived the ideas and methodology. X.C., Q.Z. (Qinrong Zhang), Q.Z. (Qunde Zhang), Y.Z., H.C. and G.L. collected the data. X.C. and L.Z. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial support was provided by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We thank the team members for the help during literature collection.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

