Lampreys (Petromyzontiformes) are one of the oldest living groups of vertebrates (Docker et al., 2015). Along with hagfishes, lampreys number over 40 species and comprise the agnathan (jawless) vertebrates (Potter et al., 2015) with a lineage dating back 500 million years (Janvier, 2007). The lamprey species Petromyzon marinus, often generically referred to as the sea lamprey (SL), have a complex metamorphic life cycle. Following hatching, SL transition into a microphagous (filter-feeding-like) larval stage that burrow in soft sediment in or near streams. After several years (ranging 2–17) (Dawson et al., 2015), the larvae undergo a dramatic metamorphosis (Manzon et al., 2015), transform into juveniles that increase markedly in size, reaching 30–110 cm, and parasitize fishes in lakes or oceans, primarily feeding on their blood (Potter et al., 2015). Parasitic lampreys ultimately return to streams where they cease feeding, spawn, and die to complete the life cycle (Johnson et al., 2015).

Sea lamprey are endangered in parts of its native range in Europe (Lucas et al., 2020), but are also an invasive pest in the Laurentian Great Lakes (Marsden and Siefkes, 2019). In the Great Lakes, SL feed on the blood of ecologically and economically valued fishes, and because fishes in the Great Lakes are smaller relative to marine fishes, SL often kill their host (Smith and Tibbles, 1980). A binational consortium involving the United States and Canada, which is managed by the Great Lakes Fishery Commission and its partners, has been tasked to develop strategies for controlling SL and reducing their impact on native Great Lakes fish species (Brant, 2019). Barriers are used to block spawning migrations and selective pesticides are used to control larval SL in streams (Siefkes, 2017). In both their native and non-native ranges, improved and more efficient rearing of SL could benefit research and management, and in the Great Lakes, new selective pesticides could improve control (Hume et al., 2020).

Gut microbiota are critical for the overall health of nearly all animals species studied to date (Kostic et al., 2013). Each species has a relatively distinct and coevolved microbiota contributing to host nutrition and health. Diet has a major influence on the diversity and function of the gut microbiota and relationships between diet and microbes have been studied extensively in recent years (David et al., 2014). However, how the gut microbiota of animals change in response to their multistage life histories (i.e., metamorphosis) is relatively understudied (Kohl et al., 2013), especially in hematophagous animals (Tetlock et al., 2012; Maltz et al., 2014; Michel et al., 2018; Zepeda Mendoza et al., 2018). Given this complex physiological life cycle, the gut microbiota of SLs likely significantly changes in concert with changing dietary needs.

The objective of the current study was to identify and characterize the bacterial communities associated with landlocked SL during different life stages using a 16S rRNA gene-based sequencing approach. We hypothesized that bacterial communities would differ among life stages and become less complex and more specialized in parasitic juvenile SL, because they feed exclusively on blood and bodily fluids. A thorough understanding of bacterial communities associated with each SL life stage may help inform restoration of valued populations, as well as control invasive populations.

The gut bacterial communities associated with SL larval, parasitic juvenile, and adult life stages differed significantly (ANOSIM R = 0.881; p-value = 0.001). Notably, the transition from larval to the parasitic juvenile stage was marked with a significant shift in bacterial community structure (Figure 1) and a reduction in alpha diversity indices (Figure 2). Bacterial communities in larvae were significantly more diverse (Shannon) and showed greater species richness (observed OTUs and Chao1), as well as evenness (Simpson’s E) relative to the parasitic and adult SLs.

The most abundant phyla associated with SLs were Proteobacteria, Fusobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria, and Firmicutes (Figure 3). Members of the phyla Fusobacteria, Firmicutes, and Proteobacteria were overrepresented in larval, parasitic juvenile, and adult stages, respectively. At the family level, larval SLs were enriched for Fusobacteriaceae, unclassified Verrucomicrobiales, unclassified Bacteroidales, unclassified Micrococcales, Rhodocyclaceae, and Rubritaleaceae, whereas Streptococcaceae was enriched in parasitic SLs, and Aeromonadaceae, Chitinibacteraceae, and Shewanellaceae in the adult SLs (Figure 4).

Linear discriminant analysis effect size analysis identified significant differences in taxa (at all levels of taxonomic classification) among the larval, parasitic, and adult lamprey stages; only taxa with linear discriminant analysis (LDA) effect sizes ≥3.75 are shown in Table 1. The larval SLs were enriched with members of the unclassified Fusobacteriaceae, unclassified Verrucomicrobiales and Cetobacterium. In contrast, members of the genera Streptococcus, Haemophilus, Cutibacterium, Veillonella, and Massilia were 3–4 orders of magnitude more abundant in the parasitic SLs compared to the larval SLs Other taxa that were dominant, but not significantly enriched during the parasitic stage were: Pseudomonas, unclassified Lactobacillales, and unclassified Enterobacteriaceae. In addition, adult SLs were enriched with Aeromonas, Iodobacter, Shewanella, and Flavobacterium. Furthermore, CCA confirmed the association of these taxa with each life stage (Figure 5).

A total of 116, 2, and 6 OTUs were identified in ≥90% of the larval, parasitic, and adult SLs, respectively, which were defined as members of their core microbiota (Supplementary Table 1). None of the core OTUs were shared between each life stage.

Microbial functional groups in larval, parasitic, and adult SLs were predicted by comparing taxonomic information of OTUs against the FAPROTAX database. This analysis revealed that parasitic SLs showed significantly higher relative abundances of animal parasite- and human-associated taxa, as well as those involved in ureolysis, compared to larval and adult SLs (Figure 6). Relative to larvae, both the parasitic and adult SLs had significant increases in taxonomic groups related to heterotrophy and fermentation. Moreover, the adults had a greater proportion of taxa related to nitrate reduction and respiration than the other stages.

Sea lampreys have a complex metamorphic life cycle consisting of three distinct stages each with a unique feeding behavior: (a) larvae typically feed on detritus and algae; (b) juveniles feed on fish blood; and (c) adults essentially cease feeding, spawn, and then die. We hypothesized that distinct intestinal microbiota in SLs likely correspond with their three different life stages. In this study we show that the lamprey gut microbiota change in concert with its dietary needs. For clarity and ease of discussion, the relationship between lamprey life stages and gut bacterial community composition is presented sequentially.

Larval SL are filter feeders burrowing in sediments and slowly draw water across their pharynx, resulting in the trapping and concentration of suspended particles in mucus (Mallatt, 1981). Suspended particles in stream water are diverse and can consist of diatoms, detritus, and bacteria (Sutton and Bowen, 1994; Mundahl et al., 2005). Larvae appear non-selective in capturing particulate matter (Moore and Beamish, 1973), and therefore their diet is diverse and likely varies by season and among inhabited streams. Given the low nutritional value of most of these food sources and potential competition by other aquatic biota, SL’s successful establishment in certain streams may be attributed to their efficient nutrient assimilation efficiencies and digestion (Moore and Beamish, 1973; Mundahl et al., 2005). The diversity of bacterial communities in the larval stage was noteworthy – with taxa representing as many as eight major phyla (e.g., Actinobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Fusobacteria, Proteobacteria) – relative to the parasitic and adult stages (Supplementary Table 1). Moreover, the increased relative abundance of bacterial taxa representing Verrucomicrobiales and Chitinibacteraceae (Table 1), which are capable of degrading complex carbohydrates, suggests that such complex food sources are available to the larvae while growing in streams (Molinari et al., 2007; Sichert et al., 2020).

Juvenile parasitic lamprey are opportunist (Silva et al., 2014; Happel et al., 2017), feeding on fishes ranging from small Catostomidae (150 mm) in the Great Lakes to Blue-(Prionace glauca; Moyer et al., 2020) and basking-sharks (Cetorhinus maximus; Wilkie et al., 2004) in marine waters. As much as 98% of the parasitic lamprey’s diet is blood, with the remainder being products of tissue cytolysis and other bodily fluids (Farmer, 1980). Conversion efficiency of the blood meal diet is high (nearly 40% at 10°C), with growth rates highest between 15 and 20°C (Farmer, 1980). These drastic changes in the digestive system during metamorphosis, have led to speculation that there is selection and retention of specific microbiota whose functional roles are consistent with lamprey dietary needs (Tetlock et al., 2012). Digestion of blood likely requires a different set of microbial communities than other feeding strategies, given its lack of essential vitamins, high osmolarity, and toxic levels of iron and urea (Song et al., 2019). Predictive functional profiling via FAPROTAX database revealed an enrichment of animal parasite- and human-associated bacterial taxa in parasitic SLs compared to the larval and adult SLs (Figure 6). It should be cautioned, however, that the predicted functions are tentative and somewhat speculative pending further isolation and biochemical characterization of individual taxa is achieved.

We found a significant metamorphosis-induced shift in bacterial community structure (Figure 5), as well as enrichment of certain bacterial genera, such as Streptococcus, Haemophilus, and Cutibacterium (Table 1). These are all fastidious bacteria that have specific nutritional needs and are typically grown (in vitro) in complex media supplemented with blood products. Bacterial genera, such as Streptococcus, and Hemophilus, which are capable of hemolytic activity (Fink and Geme, 2006; Toit et al., 2014), are likely to assist in the digestion of blood and bodily fluids in parasitic lamprey. The increased relative abundance of these taxa could also partly explain the reduction of the alpha diversity indices within parasitic SLs compared to larval SLs (Figure 2). Tetlock et al. (2012) reported the isolation of Aeromonas species capable of hemolysis from the gut of parasitic SL. However, Aeromonas were not detected in juvenile SL. In our study, none of the core OTUs in parasitic stage samples were shared with those from larval stage (Supplementary Table 1), clearly indicating that the transition from larval to the parasitic juvenile stage is accompanied by concurrent selection of bacterial taxa matching their dietary needs.

During the transition from the juvenile to adult stage, the intestine atrophies and the space once occupied by the deteriorated intestine is replaced with gonad (Applegate, 1950) in preparation of spawning. At this point, the SLs stop feeding. This transition was accompanied by significant shift in microbial community structure, as well as the enrichment of certain bacterial genera, including Aeromonas, Iodobacter, Shewanella, and Flavobacterium (Table 1). Drastic changes in gut microbiota during a fasting stage has been observed in many animals such as mice, alligators, and pythons (Costello et al., 2010; Keenan et al., 2013; Zarrinpar et al., 2014). While the lack of nutrient availability during the fasting state has been shown to result in the enrichment of bacterial taxa that can degrade host mucin glycans (Derrien et al., 2004), it is also possible that some residual populations are carried over from the parasitic stage or from stream bacteria in the surrounding environment. Frequent samplings across SL life stages will help better define the core bacterial communities during and between the transitions.

Results of this study show that the gut bacterial communities associated with each SL life stage were significantly different, reflecting their feeding behavior and nutritional requirements. While it is expected that metamorphosis would lead to significant shifts in bacterial community structure, post metamorphic events, such as those present in the transition from parasitic juvenile to spawning adult were equally dramatic. The significantly reduced alpha diversity in gut microbiota observed during parasitic phase appears to be related to the SL’s specialized blood-feeding behavior. The functional composition of the gut microbiota present in the adult is more difficult to reconcile with nutritional and energy requirements needed for reproduction during the non-feeding stage. More work needs to be done to elucidate the functional role of gut microbes during each life stage. This information may reveal new opportunities or targets for developing SL-specific control tools (Thresher et al., 2018), describing differences in growth and survival among lamprey populations (Lucas et al., 2020), or for hatchery rearing protocols for native species requiring restoration (Lampman et al., 2020).

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, PRJNA727788.

Sea lamprey were captured and maintained in conditions as recommended under the American Fisheries Society Guidelines for the Use of Fishes in Research (2014).

PM, MB, NJ, and MS conceived of the studies, wrote, and edited the manuscript. NJ obtained the samples. MB and PM prepared the samples. PM analyzed the data. All authors contributed to the article and approved the submitted version.

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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