The European common cuttlefish, Sepia officinalis, is used extensively in biological and 24
biomedical research yet its microbiome remains poorly characterized. We analyzed the 25 microbiota of the digestive tract, gills, and skin in mariculture-raised S. officinalis using a 26 combination of 16S rRNA amplicon sequencing, qPCR and fluorescence spectral 27
imaging. Sequencing revealed a highly simplified microbiota consisting largely of two 28 single bacterial amplicon sequence variants (ASVs) of Vibrionaceae and Piscirickettsiaceae. The 29 esophagus was dominated by a single ASV of the genus Vibrio. Imaging revealed bacteria in the 30 family Vibrionaceae distributed in a discrete layer that lines the esophagus. This Vibrio was also 31 the primary ASV found in the microbiota of the stomach, cecum, and intestine, but occurred at 32 lower abundance as determined by qPCR and was found only scattered in the lumen rather than 33 in a discrete layer via imaging analysis. Treatment of animals with the commonly-used antibiotic 34 enrofloxacin led to a nearly 80% reduction of the dominant Vibrio ASV in the esophagus but did 35 not significantly alter the relative abundance of bacteria overall between treated versus control 36 animals. Data from the gills was dominated by a single ASV in the family Piscirickettsiaceae, 37 which imaging visualized as small clusters of cells. We conclude that bacteria belonging to the 38 Gammaproteobacteria are the major symbionts of the cuttlefish Sepia officinalis cultured from 39 eggs in captivity, and that the esophagus and gills are major colonization sites. Symbiotic associations between invertebrates and bacteria are common. Among cephalopods, the 61 most intensely studied association is the colonization of the light organ of the bobtail squid 62
Euprymna scolopes by the bioluminescent bacterium Vibrio (Aliivibrio) fischeri in a highly 63 specific symbiosis (1). A more diverse but still characteristic set of bacteria colonize the 64 accessory nidamental gland from which they are secreted into the egg jelly coat and likely 65 protect the eggs from fungal and bacterial attack (2). The accessory nidamental gland and egg 66 cases of the squid Doryteuthis (Loligo) pealeii and the Chilean octopus (Octopus mimus) have 67 also been reported to contain Alphaproteobacteria and Gammaproteobacteria (3, 4) . These 68 associations indicate that bacteria can play a key role in the physiology of cephalopods. In particular, results showed a consistent and highly simplified microbiota in the 103 esophagus ( Figure 1 ; Table 1 ). A single ASV in the genus Vibrio (referred to as ASV1 in 104 subsequent figures and tables) made up the majority of the 16S rRNA sequence data from the 105 esophagus of the three pilot investigation individuals (mean 92% ± 10%) and of 24 individuals 106 sampled four months later (control group mean 100% ± 1%; treatment group mean 94% ± 10%). 107
Thus, this ASV represents a dominant constituent of the esophagus microbiota stably over two 108 time periods in the study and after exposure to antibiotic treatment with enrofloxacin. Another 109 ASV of the related genus Photobacterium (Vibrionaceae) (referred to as ASV2 in Table 1 ) was 110 present in the esophagus community in the pilot investigation animals (mean 7% ± 10%). 111
Combined, the two Vibrionaceae ASVs (ASV1 and ASV2) in the three pilot animals constituted 112 >99% of the esophagus community. 113
The major Vibrio ASV1 was also a major constituent of downstream sites in the GI tract, 114 although present at lower abundance measured both as relative abundance in 16S sequencing 115 data and by quantitative PCR (qPCR) (Figure 1 ; Table 1 ). qPCR revealed a high abundance of 116
Vibrio cell copies in the esophagus (average 520.4 ± 410 copies/ng of total DNA including host 117 DNA) relative to more distal portions of the GI tract that included stomach, cecum, and intestine 118 (combined average 25.6 ± 77.8 copies/ng) (p < 0.005, χ 2 = 16.5, df =3 Kruskal-Wallis) (Fig. 1B) . 119
Comparison of qPCR measures of ASVs in the genus Vibrio between the esophagus of treatment 120 and control animals revealed a striking and significant decrease of nearly 80% in the quantity (p 121 < 0.02, df = 10.8, Welch two sample t-test). We did not observe a significant difference in Vibrio 122 ASV1 quantity between other organs of the digestive tract with antibiotic treatment (Fig. 1B) . 123
Relative abundance of Vibrio ASV1 in the esophagus and stomach also did not differ 124 significantly between antibiotic and control groups (Welch two sample t-test, p > 0.10), but did 125 differ significantly among the cecum (Welch two sample t-test, p = 0.00235) and intestine 126 (Welch two sample t-test, p = 1.99e-05) of treatment versus control groups. Analysis of GI tracts 127 (esophagus, stomach, cecum, and intestine samples combined) between treated versus control 128 animals from the second period of study revealed significant differences in weighted UniFrac β-129 diversity between the two groups (PERMANOVA Pr(>F) = 0.006, F = 5.63, df = 1), despite the 130 Vibrio ASV1 remaining dominant in most organs. These differences in measured relative 131 abundance and β-diversity may result from stochastic variation in low-abundance sequences, as 132 the non-Vibrio portion of the 16S rRNA sequence data from the GI tract consisted of an 133 assortment of taxa that varied between individuals or between the two time points of the study 134 and thus suggested transient organisms rather than stable microbial colonization. 135
Samples from gills were dominated by a single highly abundant ASV in the family 136
Piscirickettsiaceae (referred to as ASV3 in subsequent figures and tables), which made up an 137 average relative abundance of 96.9% ± 2.5% in the gills. In samples from other body sites this 138 ASV3 was detected only sporadically and at low relative abundance (mean 0.2%, range 0 to 139 5.8%) (Fig. 1A ). An additional Vibrio ASV, ASV4, was a major constituent of the microbiota of 140 the shrimp used as food for the cuttlefish and was also detectable in some samples from stomach, 141 cecum, and intestine ( Figure 1A ). Skin samples did not exhibit much similarity to GI tract or 142 gills with respect to microbiome composition, with most common ASVs found in other 143 anatomical sites comprising < 20% (mean 17.2% ± 2.9% ) relative abundance of the 144 microbiome (Table S1) . 145
In addition to surveying internal organs, we collected fecal samples from the 24 animals 146 from the second time period of our study. These samples were collected daily for each 147 individual throughout the course of the antibiotic treatment experiment (see methods). 148
Comparison of weighted UniFrac dissimilarity of fecal samples from experimental animals 149 revealed significant differences in beta-diversity between cuttlefish fecal samples, seawater, and 150 shrimp (PERMANOVA Pr(>F) = 0.001, F = 5.26, df = 2) upon which the animals were fed. 151
These results, paired with the differences we observed in compositional relative abundance 152 between organs, seawater, and shrimp provide additional support for our finding that bacterial 153 communities associated with cuttlefish differ from those found in their seawater environment and 154 food source (Fig. 2) . Hybridization with the near-universal Eub338 probe showed bacteria in high density in a layer 163 ~20-40 μm thick at the border between host tissue and lumen. Staining with fluorophore-164 conjugated wheat germ agglutinin revealed a mucus layer that covered the epithelium and 165 generally enclosed the bacteria (Fig. 3) . To verify the identity of these bacteria we employed a 166 nested probe set including Eub338 as well as probes for Alphaproteobacteria, 167
Gammaproteobacteria, and probes we designed specifically for Vibrionaceae (Vib1749 and 168
Vib2300, Table 2 ). Bacterial cells imaged in the esophagus showed signal from all probes 169 expected to hybridize with Vibrionaceae, suggesting that the bacteria observed in this organ are a 170 near-monoculture of this taxon ( Fig. 4B-E) . A probe targeted to Alphaproteobacteria was 171 included in the FISH as a negative control and, as expected, did not hybridize with the cells (Fig.  172 4F). As an additional control to detect non-specific binding of probes, we performed an 173 independent FISH with a set of probes labeled with the same fluorophores as the experimental 174 probe set but conjugated to oligonucleotides not expected to hybridize with the cuttlefish 175 microbiota (Table 2) In other parts of the digestive tract we observed a sparser distribution of bacteria without 179 obvious spatial organization. Bacteria in the intestine were present not in a layer but scattered 180 throughout the lumen and mixed with the luminal contents (Fig. 5) . Similarly, in the cecum, we 181 observed bacteria in low abundance in the lumen (Fig. 6) . FISH was also applied to the stomach, 182 posterior salivary gland (poison gland) and duct of the salivary gland, but no bacteria were 183 detected (not shown). 184
Fluorescence in situ hybridization to cross-sections of the gills revealed clusters of 185 bacteria at or near the surface of the tissue (Fig. 7) . These bacteria hybridized with the Eub338 186 near-universal probe and a probe for Gammaproteobacteria ( Fig. 7B , C, Table 2) with a single morphotype that hybridized to probes targeting Vibrionaceae. In the remainder of 197 the GI tract, both imaging and 16S rRNA sequencing indicated a less consistent microbiota. 198
Sequencing also showed lower relative abundance of the dominant Vibrio ASV, and qPCR 199 confirmed a significantly lower total abundance of Vibrio cell copies in the distal GI tract. 200
Imaging revealed sparse and sporadic colonization in the intestine and cecum, with scattered 201 cells in the lumen and no clear colonization of the epithelium. In light of these results, we 202 conclude that the GI tract of laboratory cultured Sepia officinalis has a highly simplified 203 microbiome dominated by the genus Vibrio. 204
Diverse associations with Vibrio and the Vibrionaceae are known from cephalopods. 205
Among the most extensively investigated is the mutualistic association of the bioluminescent 206 Vibrio (Aliivibrio) fischeri with the light organ of the bobtail squid Euprymna scolopes (1, 13). 207
Other well-known symbioses include the colonization of the cephalopod accessory nidamental 208 gland with Alpha-and Gammaproteobacteria, which enables the host to secrete a layer of 209 bacteria into the protective coating of the egg capsule (3, 14-18). Thus, colonization by 210 Gammaproteobacteria and specifically by Vibrionaceae is common in cephalopods, yet 211 colonization of the GI tract, and particularly the esophagus, was unexpected. Vibrio and Photobacterium ASVs serve this function, their localization in high density in the 218 esophagus, near the beginning of the digestive tract, may serve to seed the distal gut; 219 colonization of the lining of the esophagus may provide a reservoir that permits the microbes to 220 avoid washout from the gut by continually re-populating the lumen of downstream gut chambers. 221
An alternative explanation is that the colonization of the esophagus, and the rest of the 222 gut, is pathogenic or opportunistic. Various Vibrio species are known pathogens of cephalopods, 223 causing skin lesions and sometimes mortality in squids and octopuses (22) (23) (24) (27). A previous study of the microbiota of 228
Octopus vulgaris paralarvae found that recently hatched paralarvae had a high-diversity 229 microbiome that changed, in captivity, to a lower-diversity microbiome with abundant 230
Vibrionaceae (21). Whether the Vibrionaceae are an integral part of cuttlefish physiology in 231 nature or whether they represent opportunistic colonists of these laboratory-reared organisms is a 232 question for future research. 233
Our sequence data from gills were dominated by a single ASV classified as 234
Piscirickettsiaceae that was in low abundance at other body sites. The Piscirickettsiaceae are a 235 family within the Gammaproteobacteria (28) that includes the salmon pathogen P. salmonis. 236
Rickettsia-like organisms have been described from the gills of clams and oysters (29, 30) as 237 well as associated with the copepod Calanus finmarchicus (31). In recent years 238
Piscirickettsiaceae have been identified in high-throughput sequencing datasets from seawater 239 and sediment as a taxon that may be involved in biodegradation of oil and other compounds (32-240 38). Whether taxa in this family colonize the gills of cuttlefish and other organisms as symbionts 241 or as opportunistic pathogens is a subject for future investigation. 242
Studies of wild S. officinalis microbiota will be informative for understanding natural 243 host-symbiont associations under natural conditions, as compared to the mariculture-reared 244 animals in the present study. S. officinalis in the eastern Atlantic and Mediterranean are known to 245 prey on small mysids (crustaceans) in their first few weeks post-hatching; then as juveniles and 246 adults they prey mainly on marine fishes and crabs. The sparseness and simplicity of the gut 247 microbiota observed in our study may have been in part a result of the monodiet of grass shrimp 248 (Palaemonetes) we employed. It remains to be seen whether differences in diet and natural 249 variation in environmental conditions influence the association of microbial symbionts with S. 250 officinalis in the wild. 251
Because cuttlefish behavior is well-studied and there exist standardized methods for 252 documenting multiple behaviors (8), we hypothesized that these animals may provide a unique 253 opportunity to study microbes and the gut-brain axisthe effect of gut microbiota on behavior 254 (39)in an invertebrate system. Therefore, in parallel with our study of the microbiome of 255 various organ systems, we conducted extensive preliminary experiments to study the effect of 256 antibiotic treatment on the behavior of S. officinalis. These results were largely negative, perhaps 257 due to the highly simplified microbiota we observed and its resilience to the antibiotic employed. To assess the extent to which cuttlefish microbial symbionts were shared with their 293 environment and food sources, 1L water samples were taken from each of three water tables in 294 which animals were held on the day of euthanasia and filtered using a 0.22 micron Sterivex filter 295 for DNA extraction; grass shrimp used as the food source throughout the duration of the 296 experiment were collected in 1.8ml sterile cryotubes on the same day that water was sampled, 297
and were frozen at -20˚C until prepared for DNA extraction. Carlsbad, CA, USA). We used the standard 515f and 806r primers (49-51) to amplify the V4 305 region of the 16S rRNA gene, using mitochondrial blockers to reduce amplification of host 306 mitochondrial DNA. Sequencing was performed using paired-end 150 base reads on an 307
Illumina HiSeq sequencing platform. Following standard demultiplexing and quality filtering 308 using the Quantitative Insights Into Microbial Ecology pipeline (QIIME2) (52) and vsearch8.1 309 (53), ASVs were identified using the Deblur method (19) and taxonomy was assigned using the 310 Greengenes Database (May 2013 release; http://greengenes.lbl.gov). Libraries containing fewer 311 than 1000 reads were removed from further analyses. All 16S rRNA sequence data and metadata 312 will be made available via the QIITA platform prior to publication. Statistical analyses and 313 figure production were produced with the aid of several R packages including vegan (40) were incubated with wheat germ agglutinin (20 ug ml -1 ) conjugated with Alexa Fluor 488 and 341 DAPI (1 ug ml -1 ) at room temperature for 30 minutes after FISH hybridization to label mucus 342 and host nuclei, respectively. Slides were dipped in ice-cold deionized water, air dried, mounted 343 in ProLong Gold antifade reagent (Invitrogen) with a #1.5 coverslip, and cured overnight in the 344 dark at room temperature before imaging. Probes used in this study are listed in Table 2 Black software (Carl Zeiss) using reference spectra acquired from cultured cells hybridized with 352
Eub338 probe labeled with the appropriate fluorophore and imaged as above. Unmixed images 353 were assembled and false-colored using FIJI software (Schindelin et al., 2012) . showing hybridization with near-universal, and Gammaproteobacteria probes, respectively. (D) Merged image of (B), and (C) showing an exact match of the signal from those two probes. Display in (B, C, and D) was adjusted for clarity. Scale bar=20 µm (A); 5 µm (B-D).
1 Table 1 . Relative abundance of three most abundant ASVs found among two sampling periods of the European common cuttlefish. Averages correspond to relative abundance of individual ASVs from each anatomical site and period. Period 1 consisted of three individuals, Period 2 consisted of eight individuals. 