Microbial colonization and assemblage on various niches of hosts is a complex process, which is dependent on genetic as well as environmental background. Many studies have reported the significant role of these microbial communities in humans (Rackaityte and Lynch, 2020), fish (Legrand et al., 2020), and livestock (Cholewińska et al., 2021); they are composed of bacteria, fungi, and archaea (Berg et al., 2020). Different aspects of microbiota have been intensively studied to report valuable information about their influence on human health (Rackaityte and Lynch, 2020). Microbiota supports many functions to satisfy the nutritional needs of the host, mainly due to the ability of the microorganisms to produce vitamins (Nagy-Szakal et al., 2012) and valuable metabolites such as short chain fatty acids (García-Mantrana et al., 2019; Silva et al., 2020). In addition, host-associated microbes train and modulate the immune system to establish tolerance to commensal bacteria (Chai et al., 2014) as well as ward off invasive pathogens (Sylvain and Derome, 2017; Ferretti et al., 2018; Yukgehnaish et al., 2020; Cholewińska et al., 2021). It is noteworthy that early life food components can promote the colonization of specific microbes and their syntrophy with beneficial microbes (Kostopoulos et al., 2020). In fact, microbiome development during the early life of hosts helps in the intrinsic training of the immune functions and shaping of microbiome composition (Sylvain and Derome, 2017; Ferretti et al., 2018). Therefore, in the past years, scientists have been studying the vertical transfer of microbes from mother to infant (Ferretti et al., 2018; Rackaityte and Lynch, 2020) and from parents to progeny in animals (Sylvain and Derome, 2017; McGrath-Blaser et al., 2021; Mika et al., 2021). Microbe transfer in most organisms occurs in three ways: (i) vertical transfer of essential maternal microbes that aid host development at the early stage of life; (ii) horizontal transfer via ingestion of microbes from diet or surrounding environment to which host is exposed to; (iii) environmental transfer between conspecific organisms during social or sexual interaction (Leftwich et al., 2020). Nevertheless, the transfer routes vary across species. For example, in chicken, successive transfer of resident microbes takes place from the oviduct and cloaca to eggshell, egg white, and then the embryo (Lee et al., 2019). In livestock, microbes present in the birth canal colonize newborns (Estellé, 2019). In humans, microbes from the skin and vagina of mothers colonize different body sites of infants, and this type of vertical transmission continues through direct contact (Ferretti et al., 2018).

The mechanism of microbial transmission in aquatic animals differs from those of mammals. In most fish species, the early stage microbiome is shaped by the environment (Llewellyn et al., 2014). As the fish grows, the environmental influence will be overshadowed by other factors (Llewellyn et al., 2014). Atlantic salmon (Salmo salar) embryos were reported to have lower diversity compared to hatchlings, probably indicating the impact of factors other than the original determinant (Lokesh et al., 2019). In zebrafish (Danio rerio) larvae, horizontal transmission of microbial symbionts occurs from the surrounding environment (Stephens et al., 2016). In another fish model, discus (Symphysodon aequifasciata), also the larvae obtain their microbial symbionts via horizontal transmission from the surrounding water (Sylvain and Derome, 2017). However, during the fry stage of discus, vertical transmission prevails because parents feed their skin mucus to their offspring (Sylvain and Derome, 2017). Interestingly, in pipefish (Syngnathus typhle), specific bacteria from both parents shape the microbiota of the embryo because eggs are transferred from mother to paternal pouches (Beemelmanns et al., 2019). The little skate (Leucoraja erinacea) egg capsule holds a variety of bacteria, which will be transferred to offspring (Mika et al., 2021). To our knowledge there are no publications on the microbial transmission from a mouthbrooder to their offspring. Hence, we used Nile tilapia (Oreochromis niloticus) females as a model to understand the bacterial transfer from mother to offspring, and across generations employing the 16S rRNA amplicon sequencing technology, based on the presence of bacteria in different samples.

The constructed amplicon 16S rRNA gene libraries generated 8,323,440 high-quality reads with an average coverage of 138,724 reads per sample. The reads were rarefied to 14,000 reads per sample (without replacement). Out of the 58 samples, one library with a number of reads below the cut-off was discarded. After normalization, we obtained 9535 ASVs, distributed among 27 phyla and 383 genera.

We first determined the relative abundance of the most abundant phyla and genera in the buccal cavity mucus and posterior intestine of the wild fish. Thereafter, to understand the microbial transfer across generations, we describe the differences in composition between two F0 families and the corresponding inbred F2, and between F0 and one outbred family (Figure 1). Later we disclose the differences in the bacteria between fish groups within F2.

Microbial transmission from different body sites of parents to offspring is extensively studied in humans compared to other animals. The gathered data on microbial transmission have provided insights into the beneficial as well as disease-causing microbes that are passed on to generations. A recent study has reported that bacterial transmission is dependent on both relationship and cohabitation (Valles-Colomer et al., 2022). Transfer of bacteria from mother to infant shapes the microbial composition in infants. It should be noted that delivery mode (cesarean section) can disrupt the normal assemblage of microbes in infants, and this issue can make the offspring susceptible to diseases such as celiac disease, asthma, and obesity (Mueller et al., 2015). These facts indicate the importance of normal microbial community at the early stage of organism development. Only a few studies have reported microbe transfer in aquatic animals. These studies showed evidence of vertical microbial transmission (Stephens et al., 2016; Sylvain and Derome, 2017). Here we present the first report of bacterial transmission across generations of Nile tilapia, a mouthbrooder fish species.

Our results revealed the dominance of the phyla, Actinobacteria, Proteobacteria, and Firmicutes in the buccal cavity and posterior intestine of the wild Nile tilapia individuals. These phyla are known to be dominant in the gut of wild Nile tilapia (Bereded et al., 2020; Bereded et al., 2021) and many other fish species (Legrand et al., 2020; Yukgehnaish et al., 2020). On the other hand, the buccal cavity microbiome of wild Nile tilapia has not been previously reported. The microbial composition in the buccal cavity and the posterior intestine in F0 and F2 generations was also dominated by the aforementioned phyla. Furthermore, Actinobacteria, Proteobacteria, and Firmicutes were found as the dominant phyla in breast tissue and human milk (Togo et al., 2019). In our study, at the genus level, the most abundant bacteria were Propionibacterium and Nocardioides; in both the buccal cavity and the posterior intestine. We found that these genera are also dominant in the wild fish samples though their abundance was different across individuals.

Various body sites of fishes harbor microbes and different factors such as diet, environment, and host pressure may help in the establishment of a balanced healthy microbiota which is known as normobiosis (Johny et al., 2021). From an ecological point of view, niche- and neutral- processes lead to well-established host microbial communities (Liao et al., 2016). The niche-based theory indicates the deterministic effects of factors such as environmental conditions, among which rearing systems can influence the gut microbiome assemblage during the development of Nile tilapia larvae. Shared OTUs of the rearing water and gut bacterial communities of Nile tilapia larvae points to the niche selection of the water bacteria (Giatsis et al., 2015). Distinct core gut microbiota in zebrafish was suggested to be due to host selective pressure or a niche selection based on certain bacteria in the rearing water (Roeselers et al., 2011). A study reported differences in the gut of larvae reared in two different rearing systems. However, when they were moved to a common recirculating aquaculture system (RAS), the gut microbial diversity and composition was similar in the individuals (Giatsis et al., 2014; Deng et al., 2021). It is also known that as fishes grow host pressure overtakes the environmental factors in deciding the microbial profile (Talwar et al., 2018). In the present study, we did not find any difference in the microbial richness and evenness in F0 and F2 generations that were reared in a common garden. This may indicate that the oral microbiome is colonized by similar microbial communities. However, in the case of the posterior intestine, we found a statistical trend, probably indicating a difference between the microbial communities in F0 and F2 generations. We speculate that the differences are due to breeding/genetic effects in F059 (F0) and F2C1 (F2) families (Abdelhafiz et al., 2021a). To understand this fact, we analyzed the microbial composition in two families of the F2 generation; these results also did not reveal any differences in the diversities of the microbial communities in F2C1 and F2S2, which are both inbred groups. On the other hand, we found dissimilarities between the microbial community compositions of the buccal cavity and the posterior intestine in F2C1 (inbred) and F2S1 (outbred), based on the weighted UniFrac distance of the inbred and outbred groups. Furthermore, the differential expression analysis of ASVs revealed significant differences between ASVs in both the buccal cavity and the posterior intestine in all the F2 family comparisons. When the buccal cavity communities of the two inbred groups (F2C1 and F2S2) were compared, Kocuria was noted to be the less abundant bacteria in F2S2 compared to F2C1. On the other hand, when we compared the buccal cavity communities of an inbred group with those of an outbred group (F2C1 and F2S1, respectively) we found differences in the microbial taxa. Furthermore, one of the ASVs of the core microbiome belonged to Nocardioides (DENOVO 2), which was not found in the outbred group (F2S1). The differences between the microbial communities, in this case, are likely due to the breeding strategy (Abdelhafiz et al., 2021a).

The core microbiome is known to be present across any population of a particular host organism, and this community plays an essential role in the host biological functions (Risely, 2020). Although it is well known that many factors modulate the microbiome composition in a host, the presence of the core microbial community may not be disrupted (Salonen et al., 2012; Henderson et al., 2015; Deng et al., 2021). In our previous study, we reported the lower microbial inter-individual variability amongst the intestine bacteria of the inbred Nile tilapia (Abdelhafiz et al., 2021a). However, in the present study, our analysis showed inter-individual variation across the buccal cavity and posterior intestine samples of the F2 and F0 generation. In fish, microbial inter-individual variation is common. This was observed even in individuals (cod and bluefin tuna larvae) reared in the same tank (Fjellheim et al., 2012; Gatesoupe et al., 2013). Furthermore, when the microbial interactions between core microbiome members and other microbes are positive, competition between the microbes will be less (Jones et al., 2018), allowing host genetic/selection or ecological pressure to shape the microbial compositional variation (Shan and Cordero, 2020). Moreover, the inter- and intra-individual compositional variations in humans are regarded stable over time (Jones et al., 2018).

In Nile tilapia larvae, the core microbiome was not affected by the early life environment (Deng et al., 2021) and these bacteria had high abundance (Wu et al., 2020; Deng et al., 2021). In the current study, the abundance of the members of the core microbiome was high in the buccal cavity and the intestine of the wild as well as F0 and F2 generations, mostly dominated by Nocardioides, Propionibacterium, Sphingomonas, and Enhydrobacter. However, the core microbiome in the intestine of wild Nile tilapia from Lake Awassa and Chamo in Ethiopia was reported by Bereded et al. (2020). At the phylum level, the core microbiome in the fishes from these two lakes was similar to wild tilapia (in the current study) from the Nile river in Egypt. The core microbiome was mostly dominated by Actinobacteria, Firmicutes, and Proteobacteria. However, at the genus level, the core microbiome in our study and that of Bereded et al. (2020) were different. In Awassa and Chamo lakes, the most abundant genera were Clostridium_XI, GPXI, Cetobacterium, and Turicibacter. In the current study, the core microbiome was mostly dominated by Nocardioides, Propionibacterium, Sphingomonas, and Corynebacterium. In our previous study, we observed Cetobacterium as a core member in the mouth and intestine of Nile tilapia from the F1 generation (Abdelhafiz et al., 2021a). These differences in the core microbiome could be attributed to the microbial functional groups. It was reported that microbes with similar metabolic functions can be combined into functional groups which are controlled by various ecological pressures (Shan and Cordero, 2020). Furthermore, in blue tilapia (Oreochromis aureus) maternal cold-tolerant genetic components were reported to be transferred to offspring (Nitzan et al., 2016). In addition, Kokou et al. (2018) reported host-microbe selection of cold-tolerant microbes in the gut of blue tilapia. Therefore, we also speculate that the difference in the core microbiome between wild Nile tilapia from Egypt and Ethiopia could be due to a genetic pressure directed toward environmental factors.

The core microbiome that is vertically transmitted across generations (Funkhouser and Bordenstein, 2013; Sylvain and Derome, 2017; Lee et al., 2019; Jorge et al., 2020) has conserved functions (Ramos et al., 2021). In the current study, we observed a presumed vertical transmission of the core microbiome from the wild Nile tilapia to the subsequent generations (F0 and F2). The core microbiome in the buccal cavity was mostly dominated by different ASVs of Nocardioides, Propionibacterium, Sphingomonas, and Enhydrobacter. These core members were also abundant in the posterior intestine, the exception was Enhydrobacter. Breast milk microbiome of humans is dominated by nine genera, and among them are Propionibacterium and Sphingomonas (Mueller et al., 2015). In infants, breastfeeding promotes the colonization and maturation of the infant gut microbiome in addition to the vertically transmitted microbes from different body sites of mothers (Mueller et al., 2015). However, microbes transmitted from the mother’s skin and vagina are transient microbes that facilitate the early colonization of other microbes also. Maternal gut microbes that are known to have better ecological adaptation capacity were found to be more persistent in the infant gut (Ferretti et al., 2018). In discus fish, maternal skin microbiome that is vertically transmitted to offspring shapes the gut microbial community of the fry (Sylvain and Derome, 2017). In our study, we found that microbes from both maternal mouth and gut shape the microbiome in offspring. For example, in wild fish, Sphingomonas was a member of the core microbiome of only posterior intestine samples. Nevertheless, we detected bacteria belonging to this genus in the mouth of F0 and F2 generations. The egg capsule of little skate was reported to have a high microbial richness and core microbiome that are essential for embryonic development (Mika et al., 2021). Therefore, we speculate that Nocardioides, Propionibacterium, and Sphingomonas may have a role in facilitating the colonization of other microbes in the buccal cavity and gut of Nile tilapia. Furthermore, the incubation of eggs in the buccal cavity of Nile tilapia could be the route for vertical transmission of microbes to the eggs. Propionibacterium have been found in human skin microbiome, raw milk, soil, silage, and anaerobic digesters (Gautier, 2014). Moreover, members of Propionibacterium were reported to break down urea and release ammonia (Gautier, 2014). It was reported that carp and zebrafish gill nitrogen-cycle microbes can detoxify ammonia (van Kessel et al., 2016). In European seabass, Propionibacterium was noted to be dominant in digesta and mucosa (Serra et al., 2021). Furthermore, Propionibacterium species are known for their unique metabolism to convert lactate to propionic acid and acetic acid by fermentation (Ciani et al., 2013). In addition, Propionibacterium sp. have immunomodulatory effects in the mice intestine. It was reported that propionate, which is produced by Propionibacterium, prevents acute colitis in mice (Plé et al., 2015). It is also known that Propionibacterium surface proteins interact with human epithelial cell surface and improve barrier functions in the intestine, and probably act against inflammatory bowel diseases (do Carmo et al., 2017). Nocardioides, the other abundant member of the core microbiome in Nile tilapia, are known to produce the anti-tumor antibiotics, sandramycin, and they can modify complex compounds chemically and enzymatically. Moreover, Nocardioides spp. have antimicrobial and antifungal activities (Lee et al., 2012), and they belong to healthy microbiota, as reported in the case of feces from healthy cottontail rabbits (Zhang et al., 2015). Furthermore, bacteria from this genus were found enriched in mice intestine after Lactobacillus plantarum administration (Xie et al., 2016). Nocardioides was also reported as gut microbiome member in Malaysian population (Chua et al., 2019). Other articles have also indicated the presence of Nocardioides and Sphingomonas in other fishes and fish rearing facilities; Nocardioides in the intestine of Korean spotted sleeper (Odontobutis interrupta) and leopard mandarin fish (Siniperca scherzeri) (Hyun et al., 2021), and Sphingomonas in fish ponds (Chen et al., 2016) and fish intestine (Hyun et al., 2021).

Sphingomonas species produce poly-β-hydroxybutyrate, in situations where carbon is available, but when there is a limited nutrient source (Dedkova and Blatter, 2014; Chen et al., 2016). Furthermore, Sphingomonas species can grow and survive in a wide range of environments that other bacteria do not tolerate (Kuehn et al., 2013). Sphingomonas was found as a core microbiome member in healthy human milk (Hunt et al., 2011), in Cyprus donkey milk (Papademas et al., 2021) and in bovine milk (Kuehn et al., 2013). In humans, feces microbiome of infants are different between breastfed and formula-fed infants, which indicates the transfer of microbes from milk to infant gut and/or involvement of milk prebiotics in the proliferation of specific microbes (Moossavi et al., 2018). Disturbance of the transfer process of the microbes from human milk to the infant was associated with many diseases (Hunt et al., 2011). Sphingomonas protect maternal breast tissue against breast cancer, and the abundance of Sphingomonadaceae family was higher in the nipple aspirate fluid of healthy women (Xuan et al., 2014; Chan et al., 2016). Thus, Propionibacterium, Nocardioides, and Sphingomonas that are transmitted vertically from the wild fish and possess the aforementioned functional potential are likely beneficial members in the core microbiome of Nile tilapia.

Here we report for the first time a presumed vertical transmission (based on similar ASVs in different generations) of buccal cavity and intestine microbial communities across generations in a mouthbrooder species. To our knowledge, the buccal cavity microbiomes in wild Nile tilapia has not been previously reported. We presume that the buccal cavity and the intestine core microbiome facilitate the colonization of other gut microbiome across generations. Furthermore, we suggest that the route of vertical transmission is through the mouth when eggs are incubated in the buccal cavity of Nile tilapia. Based on the literature, we believe that the core microbiome members that were likely vertically transmitted from the wild tilapia are beneficial bacteria and could play an essential role in the development of the offspring.

The data presented in this study are deposited in the repository, Sequence Read Archive (SRA) with accession number PRJNA808265.

The animal study was reviewed and approved by this study was performed under a license from the Norwegian Animal Research Authority (FOTS ID 10427).

VK, JF, and YA designed the study. YA carried out the sampling, lab work, analyzed the data, and wrote the manuscript. CD also analyzed the data. MP performed sequencing and data generation. YA, JF, CD, and VK interpreted the data. VK, JF, and CD reviewed and edited the manuscript. All authors approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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