Low-diversity bacterial microbiota in Southern Ocean representatives of lanternfish genera Electrona, Protomyctophum and Gymnoscopelus (family Myctophidae)

RESEARCH ARTICLE Low-diversity bacterial microbiota in Southern Ocean representatives of lanternfish genera Electrona,Protomyctophum and Gymnoscopelus (family Myctophidae) Alison Gallet 1 , Philippe Koubbi 2,3 , Nelly Le ´ger 4 , Mathilde ScheiflerID 5 , Magdalena Ruiz- Rodriguez 5 , Marcelino T. Suzuki 6 , Yves Desdevises 5 , Se ´bastien DuperronID 1,7 * 1Muse ´um National d’Histoire Naturelle, CNRS, Mole ´cules de Communication et Adaptation des Microorganismes, MCAM, Muse ´um national d’Histoire naturelle, Paris, France, 2IFREMER, Channel and North Sea Fisheries Research Unit, Boulogne-sur-Mer, France, 3UFR 918 « Terre, Environnement, Biodiversite ´», Sorbonne Universite ´, place Jussieu, Paris, France, 4Sorbonne Universite ´, Biologie des Organismes et Ecosystèmes Aquatiques BOREA, Paris, France, 5Sorbonne Universite ´, CNRS, Biologie Inte ´grative des Organismes Marins, BIOM, Observatoire Oce ´anologique, Banyuls/Mer, France, 6Sorbonne Universite ´, CNRS, Laboratoire de Biodiversite ´et Biotechnologies Microbiennes, LBBM Observatoire Oce ´anologique, Banyuls/Mer, France, 7Institut Universitaire de France, Paris, France *[email protected] Abstract Myctophids are among the most abundant mesopelagic teleost fishes worldwide. They are dominant in the Southern Ocean, an extreme environment where they are important both as consumers of zooplankton as well as food items for larger predators. Various studies have investigated myctophids diet, but no data is yet available regarding their associated microbiota, despite that the significance of bacterial communities to fish health and adaptation is increasingly acknowledged. In order to document microbiota in key fish groups from the Southern Ocean, the bacterial communities associated with the gut, fin, gills and light organs of members of six species within the three myctophid genera Electrona,Protomyctophum and Gymnoscopelus were characterized using a 16S rRNA-based metabarcoding approach. Gut communities display limited diversity of mostly fish-specific lineages likely involved in food processing. Fin and skin communities display diversity levels and compositions resembling more those found in surrounding seawater. Community compositions are similar between genera Electrona and Protomyctophum, that differ from those found in Gymnoscopelus and in water. Low abundances of potentially light-emitting bacteria in light organs support the hypothesis of host production of light. This first description of myctophidassociated microbiota, and among the first on fish from the Southern Ocean, emphasizes the need to extend microbiome research beyond economically-important species, and start addressing ecologically-relevant species. PLOS ONE | https://doi.org/10.1371/journal.pone.0226159 December 11, 2019 1 / 17 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Gallet A, Koubbi P, Le ´ger N, Scheifler M, Ruiz-Rodriguez M, Suzuki MT, et al. (2019) Lowdiversity bacterial microbiota in Southern Ocean representatives of lanternfish genera Electrona, Protomyctophum and Gymnoscopelus (family Myctophidae). PLoS ONE 14(12): e0226159. https://doi.org/10.1371/journal.pone.0226159 Editor: Varenyam Achal, Guangdong Technion Israel Institute of Technology, CHINA Received: July 21, 2019 Accepted: November 20, 2019 Published: December 11, 2019 Copyright: ©2019 Gallet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Raw reads were deposited into the GENBANK Sequence Read Archive (SRA) database under accession number SAMN12077264 to SAMN12077346, belonging to the BioProject PRJNA531247. Funding: The cruise was operated under the auspices of IPEV and MESOPP (H2020) awarded to PK. Lab work and post-doctoral fellowship (MRR) were funded through a Sorbonne Universite ´s EMERGENCE program MICROFISH Introduction Microbiota plays multiple fundamental roles in animal biology, including nutrition, immunity, protection and behavior [1]. The study of microbiota in teleost fish is an emerging research topic, initially owing to its relevance to aquaculture and fisheries research [2,3]. Teleosts have also emerged as good models to investigate vertebrate host-symbiont relationships because they are easy to rear, and display relatively limited bacterial diversity compared to other vertebrates, in particular endotherms. Representing half of the vertebrate species, teleosts as a group experience a broad diversity of environmental conditions and life histories, and are thus good candidates to study how microbiota may contribute to host adaptation and resilience [4]. Myctophids are among the most dominant mesopelagic teleost fishes worldwide and are the most dominant in the Southern Ocean [5–7]. An estimated 24 species of Myctophidae strictly occur in the Southern Ocean while 44 more species are occasionally recorded south of the Subtropical Front [6]. They feed on crustaceans, mostly copepods, amphipods and euphausids [8,9], and are important prey items for larger fauna, in particular mammals (seals) and birds (penguins). The Southern Ocean is one of the most extreme marine environments, most notably due to low temperature and isolation from other water masses, resulting in a low diversity of teleosts [6,10]. Teleost adaptation to cold waters has been studied in particular for the endemic Southern Ocean Notothenioidei [10], but very little is known regarding fish-associated microbiota in this environment despite their significance to fish physiology and ecology is well-estab- lished [2,3]. To our knowledge, two studies have investigated intestine-associated bacteria using culture-independent methods in four nothothenioid species, in Chionodraco hamatus and in Gymnodraco acuticeps [11,12]. No study to date has investigated the microbiota associated with the Myctophidae family, and generally very little is known regarding their nutrition. Their diet implies an ability to degrade large amounts of arthropod cuticle, and high levels of chitinolytic activities were indeed measured in the gut from several species, but these originated from the Monterey Bay, and not the Southern Ocean [13]. Interestingly, Myctophidae are referred to as ‘lanternfish’ owing to their production of light. Light emission in metazoans can be of either animal or bacterial origin, the latter through symbiotic interactions [14]. While early works found positive response to bacterial luminescence gene probes, supporting a bacterial origin for this emission, following work invalidated these results and suggested the absence of bacteriarelated luciferase genes and activity in Myctophidae [15]. This supports a metazoan origin of light emission, yet the mechanism has not been clearly elucidated and a molecular investigation of bacteria potentially associated to light organs is still lacking. Given the ecological importance of Myctophidae in the Southern Ocean and general lack of data, this family is a good target group to investigate the composition, organ-specificity and variability of microbiota associated with Southern Ocean fish. In this study, the microbiota associated with species belonging to three genera, namely Electrona,Protomyctophum and Gymnoscopelus, was characterized. The genus Electrona is the most numerically abundant [16], and E.antarctica is the most abundant mesopelagic species endemic to the Southern Ocean [6,17]. Fish were sampled in the region between Crozet and Kerguelen islands where the three genera co-occur. The area is under the influence of three major fronts, the subtropical front, the subantarctic front and the Antarctic polar fronts which all influence myctophid assemblages from the subtropical zone to the subantarctic one and the Antarctic waters [6,18]. A 16S rRNA-based metabarcoding approach was used to identify and compare the bacterial taxa occurring on the gills, the fins, in the luminous organ and in the intestine of fishes. Bacterial communities present in the surrounding water were analyzed and compared with fish microbiota. Altogether, this study provides the first assessment of microbiota composition in Myctophidae species, and one of the first investigation of Antarctic fish microbiota. Microbiota in Myctophidae from the Southern Ocean PLOS ONE | https://doi.org/10.1371/journal.pone.0226159 December 11, 2019 2 / 17 (SU-16-R-EMR-22-MICROFISH) awarded to SD and YD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Material and methods Sampling Samples were acquired within the program VT155 REPCCOAI conducted during cruise MD206/ObsAustral aboard the RV “Marion Dufresne”, from 8 stations in the area between Crozet islands and Kerguelen (Table 1 and Fig 1) [19]. Individuals of Electrona antarctica (10 specimens), Protomyctophum bolini and P.tenisoni (7 and 4 specimens, respectively), Gymnoscopelus bolini and G.braueri (3 specimens each) were sampled using an IKMT (Isaacs Kidd Midwater Trawl) trawled from the surface to different depths at a speed between 2 to 3 knots (Table 1). The net was 17 m long with a mesh size decreasing from its mouth (4cm) to the cod end where the mesh was 0.5 cm. Upon recovery, fish were immediately measured, photographed and dissected using sterile scalpels and tweezers. Caudal fins were sampled, light organs as well as two branchial arcs were dissected. The full intestine (without stomach) was sampled, its content was removed with sterile water pouring to focus on gut-associated communities and avoid bias due to the transient community occurring in the gut contents of different specimens. Samples were frozen immediately in liquid nitrogen then stored at -80˚C. Water was sampled from 3 stations, including two where fish were also sampled (IK2017-14 and -18), using Niskin bottles at three depths (125, 600 and 1000 m). Upon recovery, 1L water was filtered on a 0.22 μm nitrocellulose filter and filters were frozen. No endangered species were harvested for this study. All necessary authorizations and approval of the study protocol were obtained from the “Re ´serve naturelle des Terres Australes Franc¸aises” (TAAF) prior to the cruise. Re ´serve nationale naturelle des Terres Australes Franc¸aises” and TAAF administration agreed on the project. The natural reserve is a government board having a committee for environmental protection and a scientific council. PK is a member of the TAAF Scientific Committee. DNA extraction and 16S rRNA-based metabarcoding of bacterial communities DNA was extracted using the QIAGEN Blood and Tissue Kit according to the manufacturer’s instructions (Qiagen, CA), and visualized on an agarose gel. A fragment of the 16S rRNA- encoding gene corresponding to the V4-V5 variable region of Escherichia coli was amplified using primers 341F (5’- CCTACGGGNGGCWGCAG-3’) and 805R (5’- GACTACH VGGGTATCTAATCC-3’) [20,21] with Illumina adapters and 8-bp barcodes. The PCR mix contained 1X KAPA2G Fast Ready Mix (Sigma-Aldrich, France), 0.2 μl of each primer (concentration of 0.2 μM), 3.6 μl of ultrapure water and 1 μl of DNA in a final volume of 10 μl. After 3 min of initial denaturation at 95˚C, the PCR was run for 22 cycles (95˚C for 45s, 50˚C for 45s, and 68˚C for 90s), with a final extension step (68˚C for 5 min). Three parallel PCR reactions were run on each sample and then pooled together. PCR products were purified (USB ExoSAP-IT PCR Product Cleanup Kit from Thermofisher, France) and the DNA from different reactions was normalized with the SequalPrep Normalization Plate Kit (96 well, Thermofisher, France), and amplicons were pooled and concentrated by using the Wizard SV Gel and PCR Clean up Kit (Promega, France). Amplicons were sequenced on an Illumina1 HiSeq 2500 platform (2×300 paired-end) by FASTERIS SA, Switzerland, in parallel with other projects. Raw reads were deposited into the GENBANK Sequence Read Archive (SRA) database under accession number SAMN12077264 to SAMN12077346, belonging to the BioProject PRJNA531247. Microbiota in Myctophidae from the Southern Ocean PLOS ONE | https://doi.org/10.1371/journal.pone.0226159 December 11, 2019 3 / 17 Table 1. Samples from this study including sample site, coordinates, depth, species, body length and part. Date Site Lat. S Long. E Depth (m) Species Fish number ID Shannon index Length (cm) Organ Raw reads QF, non chimeric reads Ratio QF/ raw (%) Observed ASVs Accession Sample ID 1/14/ 2017 IK2017- 8 54˚ 58.85 51˚ 59.66 60 Electrona antarctica 7 –P#11 4.44 4.3 Fin 55886 32354 57.89 56 SAMN12077264 Eant_F1 Electrona antarctica 7 –P#11 4.05 4.3 Gill 40151 24948 62.14 40 SAMN12077265 Eant_Gi1 Electrona antarctica 7 –P#11 1.26 4.3 Gut 75283 38638 51.32 20 SAMN12077266 Eant_Gut1 Electrona antarctica 7 –P#11 2.34 4.3 LO 64024 40648 63.49 31 SAMN12077267 Eant_OL1 Electrona antarctica 8 –P#8 3.82 3.7 Fin 55981 33342 59.56 45 SAMN12077268 Eant_F2 Electrona antarctica 8 –P#8 3.53 3.7 Gill 58216 13669 23.48 36 SAMN12077269 Eant_Gi2 Electrona antarctica 8 –P#8 1.62 3.7 Gut 52072 35848 68.84 7 SAMN12077270 Eant_Gut2 Electrona antarctica 9 –P#10 3.76 4.3 Fin 45203 25126 55.58 41 SAMN12077271 Eant_F3 Electrona antarctica 9 –P#10 3.53 4.3 Gill 36139 18166 50.27 33 SAMN12077272 Eant_Gi3 Electrona antarctica 9 –P#10 1.39 4.3 Gut 39628 6250 15.77 10 SAMN12077273 Eant_Gut3 1/17/ 2017 IK2017- 10 56˚ 27.68 62˚ 58.66 70 Electrona antarctica 12 –P#2 3.98 5.6 Fin 28453 2974 10.45 42 SAMN12077274 Eant_F4 Electrona antarctica 13 –P#1 4.67 6.2 Fin 22431 6133 27.34 94 SAMN12077275 Eant_F5 Electrona antarctica 13 –P#1 1.93 6.2 Gill 35501 1749 4.93 15 SAMN12077276 Eant_Gi5 Electrona antarctica 14 –P#4 5.08 4.6 Fin 33881 4917 14.51 81 SAMN12077277 Eant_F6 Electrona antarctica 14 –P#4 0.91 4.6 Gut 39536 24474 61.90 4 SAMN12077278 Eant_Gut6 Protomyctophum bolini 15 –P#51 1.45 5.5 Fin 44925 25090 55.85 42 SAMN12077279 Pbol_F1 Protomyctophum bolini 15 –P#51 0.74 5.5 Gill 60853 45371 74.56 12 SAMN12077280 Pbol_Gi1 Protomyctophum bolini 15 –P#51 0.85 5.5 Gut 50667 35213 69.50 8 SAMN12077281 Pbol_Gut1 Protomyctophum bolini 16 –P#53 1.95 4.9 Fin 34004 19184 56.42 48 SAMN12077283 Pbol_F2 Protomyctophum bolini 16 –P#53 0.78 4.9 Gill 59004 46011 77.98 9 SAMN12077284 Pbol_Gi2 Protomyctophum bolini 16 –P#53 3.41 4.9 LO 85291 1930 2.26 33 SAMN12077285 Pbol_LO2 (Continued) Microbiota in Myctophidae from the Southern Ocean PLOS ONE | https://doi.org/10.1371/journal.pone.0226159 December 11, 2019 4 / 17 Table 1. (Continued) Date Site Lat. S Long. E Depth (m) Species Fish number ID Shannon index Length (cm) Organ Raw reads QF, non chimeric reads Ratio QF/ raw (%) Observed ASVs Accession Sample ID Protomyctophum bolini 17 –P#50 2.78 5.1 Fin 40350 20005 49.58 65 SAMN12077286 Pbol_F3 Protomyctophum bolini 17 –P#50 0.92 5.1 Gill 58934 43180 73.27 19 SAMN12077287 Pbol_Gi3 Protomyctophum bolini 17 –P#50 1.34 5.1 Gut 53496 38654 72.26 8 SAMN12077288 Pbol_Gut3 Protomyctophum bolini 17 –P#50 2.30 5.1 LO 31776 2338 7.36 19 SAMN12077289 Pbol_LO3 1/19/ 2017 IK2017- 12 47˚ 48.64 58˚ 58.54 530 Gymnoscopelus braueri 26 –P#4 0.01 4.7 Fin 58073 33902 58.38 2 SAMN12077320 GGia_F1 Gymnoscopelus braueri 27 –P#5 2.82 4.3 Fin 14028 6676 47.59 22 SAMN12077335 GGia_F2 Gymnoscopelus braueri 27 –P#5 2.99 4.3 Gill 36224 15454 42.66 83 SAMN12077330 GGia_Gi2 Electrona antarctica 28 –P#7 0.01 3.2 Gut 52380 29998 57.27 2 SAMN12077294 Eant_Gut7 Protomyctophum tenisoni 29 –P#32 2.44 4 Fin 4833 1609 33.29 14 SAMN12077314 Pten_F1 Protomyctophum tenisoni 29 –P#32 2.58 4 Gut 34357 8798 25.61 38 SAMN12077315 Pten_Gut1 Protomyctophum tenisoni 29 –P#32 0.90 4 Gill 33862 14430 42.61 9 SAMN12077316 Pten_Gi1 Protomyctophum tenisoni 30 –P#33 1.95 3.2 Fin 42584 11119 26.11 33 SAMN12077317 Pten_F2 Protomyctophum tenisoni 30 –P#33 3.44 3.2 Gill 23270 7994 34.35 36 SAMN12077332 Pten_Gi2 Electrona antarctica 31 –P#8 3.29 3 Fin 32937 5789 17.58 32 SAMN12077302 Eant_F9 Electrona antarctica 31 –P#8 0.48 3 Gut 65710 16983 25.85 3 SAMN12077304 Eant_Gut10 Protomyctophum tenisoni 32 –P#33 0.19 3.1 Fin 45016 31508 69.99 8 SAMN12077334 Pten_F3 Protomyctophum tenisoni 32 –P#33 3.80 3.1 Gill 37021 6989 18.88 69 SAMN12077326 Pten_Gi3 1/19/ 2017 OISO7 47˚ 40.00 58˚ 00.00 125 Water 4.60 41978 28972 69.02 94 SAMN12077336 Water1 Water 6.29 99492 61543 61.86 184 SAMN12077337 Water2 1/19/ 2017 OISO7 47˚ 40.00 58˚ 00.00 600 Water 3.56 43616 28490 65.32 51 SAMN12077338 Water3 Water 6.32 59862 38712 64.67 205 SAMN12077339 Water4 1/19/ 2017 OISO7 47˚ 40.00 58˚ 00.00 1000 Water 5.77 63868 41660 65.23 174 SAMN12077340 Water5 (Continued) Microbiota in Myctophidae from the Southern Ocean PLOS ONE | https://doi.org/10.1371/journal.pone.0226159 December 11, 2019 5 / 17 Table 1. (Continued) Date Site Lat. S Long. E Depth (m) Species Fish number ID Shannon index Length (cm) Organ Raw reads QF, non chimeric reads Ratio QF/ raw (%) Observed ASVs Accession Sample ID 1/21/ 2017 IK2017- 14 48˚ 25.22 64˚ 52.26 650 Protomyctophum bolini 39 –P#31 4.76 6.3 Fin 26654 5381 20.19 77 SAMN12077296 Pbol_F4 Protomyctophum bolini 39 –P#31 1.65 6.3 Gill 46885 29498 62.92 22 SAMN12077297 Pbol_Gi4 Protomyctophum bolini 40 –P#33 1.09 5.4 Fin 51723 28032 54.20 7 SAMN12077298 Pbol_F5 Protomyctophum bolini 40 –P#33 3.52 5.4 Gill 49545 9151 18.47 47 SAMN12077299 Pbol_Gi5 Electrona antarctica 41 –P#22 3.32 4.9 Fin 25710 7624 29.65 48 SAMN12077312 Eant_F8 Electrona antarctica 41 –P#22 0.05 4.9 Gut 23944 15360 64.15 4 SAMN12077313 Eant_Gut8 Protomyctophum bolini 42 –P#34 2.06 6 Fin 41607 3111 7.48 18 SAMN12077301 Pbol_F6 Protomyctophum bolini 42 –P#34 3.43 6 Gill 42034 7124 16.95 44 SAMN12077305 Pbol_Gi6 Protomyctophum bolini 42 –P#34 2.13 6 LO 8494 2656 31.27 15 SAMN12077307 Pbol_LO6 Gymnoscopelus braueri 43 –P#36 3.60 11.1 Fin 5237 1850 35.33 20 SAMN12077331 GGia_F3 Gymnoscopelus braueri 43 –P#36 5.16 11.1 Gill 41642 7933 19.05 136 SAMN12077324 GGia_Gi3 Electrona antarctica 44 –P#25 2.31 4.5 Fin 53227 17487 32.85 44 SAMN12077321 Eant_F8 Electrona antarctica 44 –P#25 0.40 4.5 Gut 38896 15426 39.66 3 SAMN12077322 Eant_Gut9 1/21/ 2017 IK2017- 14 48˚ 25.22 64˚ 52.26 125 Water 5.52 12669 6673 52.67 93 SAMN12077341 Water6 1/21/ 2017 IK2017- 14 48˚ 25.22 64˚ 52.26 600 Water 6.27 55772 31757 56.94 184 SAMN12077342 Water7 1/21/ 2017 IK2017- 14 48˚ 25.22 64˚ 52.26 1 000 Water 3.66 46454 25059 53.94 56 SAMN12077343 Water8 Water 5.86 61181 37545 61.37 171 SAMN12077344 Water9 1/25/ 2017 IK2017- 18 48˚ 48.11 72˚ 19.23 900 Gymnoscopelus bolini 48 –P#1 3.64 17 Fin 22662 11889 52.46 49 SAMN12077290 Gbol_F1 Gymnoscopelus bolini 48 –P#1 3.61 17 LO 8800 2239 25.44 29 SAMN12077291 Gbol_LO1 Protomyctophum tenisoni 50 –P#9 1.28 3.4 Gut 19765 3622 18.33 15 SAMN12077328 Pten_Gut4 Protomyctophum tenisoni 50 –P#9 1.69 3.4 Gill 42116 16903 40.13 14 SAMN12077329 Pten_Gi4 Protomyctophum bolini 53 –P#10 2.56 3.6 Fin 38022 15931 41.90 44 SAMN12077309 Pbol_F8 (Continued) Microbiota in Myctophidae from the Southern Ocean PLOS ONE | https://doi.org/10.1371/journal.pone.0226159 December 11, 2019 6 / 17 Table 1. (Continued) Date Site Lat. S Long. E Depth (m) Species Fish number ID Shannon index Length (cm) Organ Raw reads QF, non chimeric reads Ratio QF/ raw (%) Observed ASVs Accession Sample ID Protomyctophum bolini 53 –P#10 2.39 3.6 Gut 36954 19592 53.02 14 SAMN12077310 Pbol_Gut8 Protomyctophum bolini 53 –P#10 0.62 3.6 Gill 37768 18229 48.27 9 SAMN12077311 Pbol_Gi8 1/25/ 2017 IK2017- 18 48˚ 48.11 72˚ 19.23 600 Water 6.09 44181 26783 60.62 181 SAMN12077345 Water10 1/25/ 2017 IK2017- 18 48˚ 48.11 72˚ 19.23 1000 Water 4.68 60145 37872 62.97 116 SAMN12077346 Water11 1/25/ 2017 IK2017- 19 48˚ 46.61 72˚ 11.09 590 Gymnoscopelus bolini 52 -P#1 3.92 19.1 Fin 18013 1648 9.15 31 SAMN12077292 Gbol_F2 Gymnoscopelus bolini 52 –P#1 2.33 19.1 Gill 7521 3661 48.68 20 SAMN12077318 Gbol_Gi2 1/26/ 2017 IK2017- 20 48˚ 43.73 72˚ 05.19 190 Gymnoscopelus bolini 55 –P#1 3.89 23.6 Gill 24564 8093 32.95 60 SAMN12077323 Gbol_Gi3 F: fin; Gi: Gill; Gut: gut; LO: light organ. Number of raw and quality-filtered (QF) reads are provided. Shannon index and observed ASVs are rarefied to 1,100 reads. https://doi.org/10.1371/journal.pone.0226159.t001 Microbiota in Myctophidae from the Southern Ocean PLOS ONE | https://doi.org/10.1371/journal.pone.0226159 December 11, 2019 7 / 17 Sequence analysis Analysis were performed using the QIIME2 software [22]. Raw reads were demultiplexed, quality checked and trimmed to remove primer regions, paired ends were assembled, chimeric sequences were discarded, and reads were denoised using DADA2 resulting in a list of Amplicon Sequence Variants (ASVs) [23]. Taxonomic affiliations were obtained by the sklearnbased classifier using the SILVA_132_QIIME_release distributed by the Silva project [24]. Sequences matching “Archaea”, “Eukaryota”, “Unassigned”, “Chloroplast” and “Mitochondria”, representing 2.8% of raw reads, were discarded. Rarefaction curves, alpha and beta diversity indexes were generated using a sampling depth of 1,100 corresponding to the lowest number of quality-filtered reads obtained in a sample. A guide phylogenetic tree was produced to compute UniFrac distances and a principal-coordi- nates analysis (PCoA) plot based on Weighted UniFrac (WU) dissimilarities was generated [25]. Community richness estimated by Faith’s Phylogenetic Diversity (PD) were compared using Kruskal Wallis tests, and compositions were compared using PERMANOVA. Venn diagrams were drawn using the web-based software available at http://bioinformatics.psb.ugent. be/webtools/Venn/. Results A total of 1,258,379 assembled paired-end bacterial reads were obtained from 61 fish samples (intestine, light organ, fin and gill) and 11 filtered seawater samples (Table 1). These represented 1,683 distinct ASVs. Individual samples yielded between 1,609 and 61,543 reads (mean 18,505). Numbers of reads were sometimes low, leading us to choose a minimal 1,100 reads level for rarefaction-based analyses. At this level, rarefaction curves reached saturation for animal samples at this level, while water samples did not (S1 Fig). It is thus likely that the majority of animal-associated bacterial diversity was successfully captured. At this level, between 2 and 205 (mean 48) ASVs were observed in individual samples (Table 1). Diversity and composition of bacterial communities Water samples displayed highest ASVs diversity with average 137.2±56.3 ASVs, followed by fin and gill samples (mean 40.1±23.6 (24 samples) and 37.5±32.0 (19 samples), respectively; see Table 2 for mean values according to host genus). Light organs displayed on average 25.4±7.9 ASVs (5 samples). Intestine samples displayed markedly lower diversity, with average 10.5±9.9 ASVs (13 samples). Faith’s PD, which accounts for the phylogenetic distance among observed ASVs, was significantly higher in water compared to all fish samples (p<0.001, S1 Table). Gymnoscopelus samples displayed a PD comparable to that of water (p = 0.78), while both Electrona and Protomyctophum samples displayed markedly lower PDs (both p-values versus water <0.001), comparable between them (Electrona versus Protomyctophum samples, p = 0.62). Among fish organs, light organs were excluded from alpha diversity comparisons because only 5 samples were analyzed. Fin and gill samples were found to display comparable PDs (p = 0.85), both well above those in intestine samples (both p-values versus intestine <0.001). Sampling date did not affect Faith PD (p = 0.72). The two most abundant bacterial groups in all fish samples were Gammaproteobacteria (notably Alteromonadales, Pseudomonadales, Thiomicrospirales and, to a lesser extent, Vibrionales) and Mollicutes (Mycoplasmatales, Fig 2). In the light organ, dominant ASVs belonged to the Mycoplasmatales (41.3±37.4%, notably genus Mycoplasma), Alteromonadales (22.2±29.5%, genus Pseudoalteromonas), and Pseudomonadales (22.3±32.8%, genus Acinetobacter), and abundances were variable across samples. In intestine samples, Mollicutes were consistently dominant in all samples of Electrona antarctica (97.9±4.1% of reads). Mollicutes Microbiota in Myctophidae from the Southern Ocean PLOS ONE | https://doi.org/10.1371/journal.pone.0226159 December 11, 2019 8 / 17 were also abundant in intestine samples of Protomyctophum (mean 29.2±43.6%) but Thiomicrospirales (27.6±40.9%), including two ASVs belonging to the clade SUP-05 of sulfur-oxidiz- ing bacteria, and Vibrionales (13.7±23.1%) represented by two ASVs within genus Vibrio present in one sample each, were also abundant in some samples. Despite that several ASVs belonging to the Mollicutes and Gammaproteobacteria were present, a single ASV sometimes represented up to 99% of reads, emphasizing the overall low bacterial diversity in intestine samples (S2 Table). Fin and gill samples displayed dominance of Gammaproteobacteria (68.6±6.2% and 81.6±5.9%, respectively) and Mollicutes (19.1±33.2% and 13.0±27.7%, respectively), with a greater diversity of ASVs compared to other animal sample types. Water samples were dominated by Proteobacteria of the Gamma, Alpha and Delta groups, and Mollicutes represented less than 0.1% of reads in any of the water sample. Beta diversity Among abundant ASVs, i.e. the 217 representing at least 1% of reads in at least one sample, 53 were shared between at least two genera, or a genus and water. Ten out of the 67 abundant Fig 1. Sampling sites near Kerguelen. Crozet islands are located on the west, not visible. Map obtained using Google Earth. https://doi.org/10.1371/journal.pone.0226159.g001 Table 2. Mean values for Shannon index, and mean observed ASVs and associated standard deviation in each body part (for which sample number exceeds 1) of each genus and in water. Genus Organ Shannon index Observed ASVs SD Electrona Fin 3.85 53.7 20.5 Gill 3.26 31.0 11.0 Gut 0.77 6.6 6.8 Gymnoscopelus Fin 2.80 24.8 17.1 Gill 3.59 74.8 46.7 Protomyctophum Fin 2.12 35.6 24.1 Gill 1.95 26.4 20.0 Gut 1.69 16.6 12.4 LO 2.61 22.3 9.5 Water 5.33 137.2 56.3 https://doi.org/10.1371/journal.pone.0226159.t002 Microbiota in Myctophidae from the Southern Ocean PLOS ONE | https://doi.org/10.1371/journal.pone.0226159 December 11, 2019 9 / 17 8. Pakhomov EA, Perissinotto R, McQuaid CD. Prey composition and daily rations of myctophid fishes in the Southern Ocean. Mar Ecol Prog Ser. 1996; 134: 1–14. https://doi.org/10.3354/meps134001 9. Saunders RA, Collins MA, Ward P, Stowasser G, Hill SL, Shreeve R, et al. 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