Fabrice Carrat

Jean-Claude Desenclos

Xavier De Lamballerie

Marie Zins

Orcid F Carrat

In reply to Samuel A. Concerns about "Evidence of early circulation of SARS-CoV-2 in France: findings from the population-based "

We thank Dr Samuel for the comments [START_REF] Samuel | Concerns about "Evidence of early circulation of SARS-CoV-2 in France: findings from the population-based "CONSTANCES" cohort[END_REF], which gives us the opportunity to clarify. Dr Samuel first mentions the lack of consideration in our work of the uncertainty about the specificity of the serum neutralization (SN) test. He estimated a lower bound of the specificity of the SN test, and used it to infer that our SN positive findings were likely false positive. The SN test has indeed been validated on 2387 blood donor samples (between 2015 and 2018, none tested positive, and these data have not been fully published) but it is true that the cited reference (2) reports validation on 464 samples from 2017 and 2018. However, Dr Samuel's conclusion that our findings could easily be explained by a lack of specificity of the neutralization test is not correct. First, there is no valid statistical argument for choosing the lower bound of the 95% confidence interval as the reference value for estimating the probability of false positive results in our sample. Indeed, a value as low as (or lower than) the lower bound had an a priori probability of 2.5%. Second, in our study, 6,020 samples were collected between November 2019 and January 2020, 176 had a positive Elisa-S of which 13 were also positive for SN. Assuming that all 176 Elisa-S samples were false positive (i.e. none of the 176 subjects were "truly infected"), and taking the lower bound of the 95% confidence interval for SN specificity estimated from the 464 published samples (lower bound Specificity= 99.2%, 95% Clopper-Pearson exact Confidence Interval), the probability of observing 5 or more false positive SN tests out of 176 samples was 1.4%, and 13 or more false positive tests out of 176 was 2.6 10 -9 . Using the lower bound of specificity estimated from the 2387 samples (99.85%), the probability of 5 or 13 false positive would dramatically decrease. Third, it can be recalled that SN is frequently used as a gold standard to identify false positive serological tests in many diseases (dengue fever, Japanese encephalitis etc...), and in our study, SN tests were replicated 6 times to limit the risk of misclassification by measurement error.

In a second part, Dr Samuel raises an interesting point that cross-reactive immunity with seasonal human coronaviruses (HCoV) might have led to a selection bias when restricting our SN tests to sample with Elisa-S ≥ 0.7. In other words, all Elisa-S ≥0.7 that were also SN positives were false positive by cross-reaction, and none of the Elisa-S <0.7 remaining samples would have been SN positive, if they had been tested. This assumption seems hardly realistic. In reference with our response to the first comment, all 2387 samples that were tested negative by SN were not selected according to a prior history of HCoV infection or given their Elisa-S test valuetherefore, our initial estimate of 100% SN specificity applied to people with or without prior history of HCoV infection and irrespective of their unknown Elisa-S value. Second, we performed additional analyses on 4704 samples collected in September and October 2019 (the biological collections in CONSTANCES started in 2018 and additional analyses are underway). Preliminary findings suggest positivity to Elisa-S and other SARS-CoV-2 serological tests (IgG against RBD or Spike evaluated with high-throughput multiplex technology on a Luminex® platform) in some participants, while only 1 participant with other positive tests had SN antibodies at 40 -this participant was sampled on Oct 10, 2019. We also found some evidence of crossreactivity between seasonal HCoV and SARS-CoV-2 with higher levels of IgG anti-RBD against HCoV (229E, KHU1, NL63) in anti-SARS-COV-2 Elisa-S positive samples than in negative ones, but we did not identify association between serological tests results of HCoV and anti-SARS-CoV-2 SN. Although, as already discussed in our paper, we cannot totally exclude that part of our SN positive were false positive results, we do not believe that all our SN positive samples identified between November 2019 to January 2020 were false positive. Importantly, detailed questioning of the exposure circumstances of some participants shows the occurrence of suggestive symptoms in the days preceding the date of sampling or possible exposure in China.

In another comment, Dr Samuel points out the increase in Elisa-S positivity from early January while SN positivity appeared quite stable before and after January 2020. We have no clear explanation for this result. Remind that the increase of antibody titers occurred within 2 weeks after infection, the increase of Elisa-S positivity in early January is unlikely explained by a wide spreading of SARS-CoV-2 from mid-December 2019. Rather, it could correspond to cross reaction with HCoV as we indicated above (HCoV peaks are observed between December and March in Europe (3)) while SN positivity among Elisa-S positive sample could correspond to real SARS-CoV-2 infection.

In a last comment, it is unclear how Dr Samuel came to a "prevalence" of 5%, while we clearly never assumed that all Elisa-S positive samples were true infected persons. If we restricted our selection to the 13 participants with both SN and Elisa-S positivity, the cumulative incidence of SARS-CoV-2 in end-January 2020 would be 0.3%. In addition, if we assumed that 5 to 6 participants among these 13 patients were indeed true positive, it means that fifty thousand people would have been infected with SARS-CoV-2 between November 2019 and January 2020 in France, which based on estimated infection hospitalization or death ratios would have converted into one thousand hospitalizations and approximately two hundred and fifty deaths [START_REF] Lapidus | Do not neglect SARS-CoV-2 hospitalization and fatality risks in the middle-aged adult population[END_REF]. It is however possible that such events if they were caused by SARS-CoV-2 infection, may have been missed as the average yearly number of hospitalization or deaths for influenza is >10 times larger and there was a concurrent influenza epidemic at this period in France [START_REF] Boelle | Excess cases of influenzalike illnesses synchronous with coronavirus disease (COVID-19) epidemic, France[END_REF].

Finally, although a recent report clearly shows that the SARS-CoV-2 outbreak was originated from the Huanan market (6), our findings do not contradict with this hypothesis. It is indeed possible that a progenitor of the virus has been circulating worldwide giving low-level infections and not inducing severe respiratory disease to the same extent as the Wuhan virus. A recent study reports early identification of SARS-CoV-2 RNA in a patient with measles-like syndrome in September 2019 in Italy [START_REF] Amendola | Molecular evidence for SARS-CoV-2 in samples collected from patients with morbilliform eruptions since late 2019 in Lombardy, northern Italy[END_REF], and indicated other SARS-CoV-2 RNA sequences obtained from samples collected in Brazil in November 2019. They concluded that a potential progenitor of the B.1 strain may have circulated worldwide since June-July 2019, before the Wuhan outbreak. We agree with these conclusions.

For all these reasons, our conclusion remains unchanged and suggests an early circulation of SARS-CoV-2 in France.

Conflict of interest

The authors have no conflicts of interest to declare that are relevant to the content of this article


Elsa Gadoin

Christelle Desnues

Thierry Bouvier

Emmanuelle Roque D'orbcastel

Jean- Christophe Auguet

Sandrine Crochemore

Adingra Antoinette

Yvan Bettarel

Tracking spoilage bacteria in the tuna necrobiome

Keywords: necrobiome, tuna, histamine, Photobacterium, microbiome, spoilage microorganisms

Like other seafood products, tuna is highly perishable and sensitive to microbial spoilage. Its consumption, whether fresh or canned, can lead to severe food poisoning due to the presence of histamine-producing bacteria and other specific spoilage organisms (SSOs) found in the tuna microbiome. Such bacteria generally develop in dead fish after their capture if conservation conditions are deficient. However, many grey areas persist regarding their ecology, their conditions of emergence and proliferation, and their distribution within different organs. In this study, we used 16S rDNA barcoding to investigate post-mortem changes in the tuna necrobiome until the advanced stages of decomposition (i.e. 120 h). The analyses were performed on fresh and brine-frozen yellowfin tuna (Thunnus albacares) captured in the tropical Atlantic Ocean. The results revealed that despite standard refrigeration storage conditions (i.e. 4°C), a diverse and complex spoilage bacteriome continued to develop in the gut and liver. In general, the relative abundance of SSOs increased rapidly in both organs, representing 82% of the bacterial communities in fresh yellowfin tuna, and less than 30% in brine-frozen ones. Interestingly, Photobacterium was identified as a major bacterial genus, and its temporal dynamics were positively correlated with histamine concentrations, which ultimately, in fresh tunas, exceeded the recommended sanitary level of 50 ppm established by the United States Food and Drug Administration. Finally, the study of the tuna necrobiome shows that the sanitary risks associated with the consumption of this widely eaten fish is strongly influenced by the post-capture storage conditions.

Introduction

Like other living organisms, fish live in close association with a diverse assemblage of microorganisms, including bacteria, viruses, archaea and microeukaryotes, which constitute their microbiome. Increasing attention has been paid to the fish microbiome in recent years, and we now know that it ensures a number of essential functions for the health and fitness of the host [START_REF] Egerton | The Gut Microbiota of Marine Fish[END_REF][START_REF] Sehnal | Microbiome composition and function in aquatic vertebrates : Small organisms making big impacts on aquatic animal health[END_REF]). It has also been shown to be highly heterogeneous in the body, with specific microbial signatures in different fish organs, including the gut, gills, skin, liver, etc. [START_REF] Apprill | Marine Animal Microbiomes : Toward Understanding Host-Microbiome Interactions in a Changing Ocean[END_REF][START_REF] Egerton | The Gut Microbiota of Marine Fish[END_REF][START_REF] Ross | The skin microbiome of vertebrates[END_REF]Gadoin et al. 2021). Numerous studies have reported that the composition of the fish microbiome depends on various factors such as species [START_REF] Chiarello | High diversity of skinassociated bacterial communities of marine fishes is promoted by their high variability among body parts, individuals and species[END_REF][START_REF] Chiarello | Skin microbiome of coral reef fish is highly variable and driven by host phylogeny and diet[END_REF][START_REF] Givens | Comparison of the gut microbiomes of 12 bony fish and 3 shark species[END_REF][START_REF] Larsen | Diversity of the skin microbiota of fishes : Evidence for host species specificity[END_REF], stage of development [START_REF] Hansen | Bacterial Interactions in Early Life Stages of Marine Cold Water Fish[END_REF], sex [START_REF] Dhanasiri | Changes Electronic copy available at: https://ssrn.com/abstract=3985243 in the Intestinal Microbiota of Wild Atlantic cod Gadus morhua L. Upon Captive Rearing[END_REF], diet [START_REF] Cordero | Modulation of immunity and gut microbiota after dietary administration of alginate encapsulated Shewanella putrefaciens Pdp11 to gilthead seabream (Sparus aurata L.)[END_REF][START_REF] Parata | Age, gut location and diet impact the gut microbiome of a tropical herbivorous surgeonfish[END_REF], geographical location [START_REF] Chiarello | Environmental conditions and neutral processes shape the skin microbiome of European catfish ( Silurus glanis ) populations of Southwestern France[END_REF][START_REF] Xavier | The effects of environment and ontogeny on the skin microbiome of two Stegastes damselfishes (Pomacentridae) from the eastern Caribbean Sea[END_REF] or captive state [START_REF] Dhanasiri | Changes Electronic copy available at: https://ssrn.com/abstract=3985243 in the Intestinal Microbiota of Wild Atlantic cod Gadus morhua L. Upon Captive Rearing[END_REF][START_REF] Parata | Age, gut location and diet impact the gut microbiome of a tropical herbivorous surgeonfish[END_REF]. However, little is known about the evolution of this microbiome in the different organs after the death of the fish, which nevertheless partly conditions its sanitary quality for consumption. After a fish dies, numerous physical and chemical alterations take place in the body (i.e. decrease in pH, cellular lysis), inducing taxonomic and functional shifts in the bacterial community initially present in the organism [START_REF] Boziaris | Specific Spoilage Organisms (SSOs) in Fish[END_REF][START_REF] Duarte | Quality Assessment of Chilled and Frozen Fish-Mini Review[END_REF][START_REF] Gram | Microbiological spoilage of fish and fish products[END_REF]. The microbial assemblage that grows in a dead fish and leads to its decomposition is known as the necrobiome, from the Greek word nekrós for 'death'.

In the last three decades, numerous studies have analysed the diversity and activity of spoilage microorganisms in many seafood products, mainly using a culture-based approach (reviewed in [START_REF] Boziaris | Specific Spoilage Organisms (SSOs) in Fish[END_REF][START_REF] Gram | Microbiological spoilage of fish and fish products[END_REF][START_REF] Gram | Fish spoilage bacteria -problems and solutions[END_REF]. These microorganisms, referred to as specific spoilage organisms (SSOs), typically belong to the bacterial genera Aeromonas, Vibrio, Photobacterium, Shewanella or Enterobacteriaceae, to cite a few, and they are commonly found in the flesh of fish and seafood products [START_REF] Boziaris | Specific Spoilage Organisms (SSOs) in Fish[END_REF][START_REF] Gram | Fish spoilage bacteria -problems and solutions[END_REF]. In general, most SSOs are known to produce specific metabolites (trimethylamine oxide, ammonia, biogenic amines, organic acids, acetate and sulphur) leading to the organoleptic rejection of the seafood product during quality control checks [START_REF] Boziaris | Specific Spoilage Organisms (SSOs) in Fish[END_REF][START_REF] Gram | Fish spoilage bacteria -problems and solutions[END_REF]). Among SSOs, several species such as Shewanella spp., Vibrio spp., Salmonella and Listeria monocytogenes Electronic copy available at: https://ssrn.com/abstract=3985243 are also human pathogens [START_REF] Parlapani | Microbial diversity of seafood[END_REF]. The levels of these SSOs in the host organism are mainly dependent on storage conditions and the previous fish evisceration [START_REF] Huss | Quality and quality changes in fresh fish[END_REF].

Chilling, freezing and vacuum storage can reduce the production of degrading metabolites by SSOs and thus increase the shelf-life of seafood products [START_REF] Dawson | Effect of Freezing on the Shelf Life of Salmon[END_REF][START_REF] Ghaly | Fish Spoilage Mechanisms and Preservation Techniques : Review[END_REF][START_REF] Sivertsvik | A review of modified atmosphere packaging of fish and fishery products-Significance of microbial growth, activities and safety[END_REF]. The diversity of SSOs also varies according to the fish species [START_REF] Parlapani | Indigenous and spoilage microbiota of farmed sea bream stored in ice identified by phenotypic and 16S rRNA gene analysis[END_REF]Parlapani et al. 2018;[START_REF] Reynisson | Effects of different cooling techniques on bacterial succession and other spoilage indicators during storage of whole, gutted haddock (Melanogrammus aeglefinus)[END_REF], the geographical location (Parlapani et al., 2018), and the composition of the initial microbiome [START_REF] Boziaris | Specific Spoilage Organisms (SSOs) in Fish[END_REF][START_REF] Gram | Fish spoilage bacteria -problems and solutions[END_REF].

The majority of studies on the spoilage microbiome in fish have been conducted on flesh [START_REF] Antunes-Rohling | Characterization of the Spoilage Microbiota of Hake Fillets Packaged Under a Modified Atmosphere (MAP) Rich in CO2 (50% CO2/50% N2) and Stored at Different Temperatures[END_REF][START_REF] Chytiri | Microbiological, chemical and sensory assessment of iced whole and filleted aquacultured rainbow trout[END_REF][START_REF] Eliasson | The effects of superchilling on shelf-life and quality indicators of whole Atlantic cod and fillets[END_REF][START_REF] Taliadourou | Microbiological, chemical and sensory changes of whole and filleted Mediterranean aquacultured sea bass (Dicentrarchus labrax) stored in ice[END_REF][START_REF] Wang | Spoilage potential of three different bacteria isolated from spoiled grass carp (Ctenopharyngodon idellus) fillets during storage at 4 °C[END_REF][START_REF] Zotta | Dynamics of bacterial communities and interaction networks in thawed fish fillets during chilled storage in air[END_REF], while the viscera such as the gut and liver have received less attention. Yet the latter are recognized as important microbial reservoirs: the digestive tract of fish is known to host specific bacterial taxa that play key roles in the digestion, immunity and fitness of the host [START_REF] Egerton | The Gut Microbiota of Marine Fish[END_REF][START_REF] Ghanbari | A new view of the fish gut microbiome : Advances from next-generation sequencing[END_REF].

More recently, diverse microbial communities have also been discovered in the liver of several fish species, including tuna, mullet, sardinella and Randall's threadfin bream (Gadoin et al. in rev.;Meron et al. 2020), showing the importance of including this organ in microbiome studies on marine organisms.

Of the main fish species consumed worldwide, tuna show one of the highest risks of food poisoning [START_REF] Hungerford | Scombroid poisoning : A review[END_REF][START_REF] Tortorella | Histamine Poisoning from Ingestion of Fish or Scombroid Syndrome[END_REF]. From a microbiological perspective, tuna, like other members of the Scombridae family, is an interesting study model, as the consumption of this species can lead to histamine poisoning [START_REF] Hungerford | Scombroid poisoning : A review[END_REF][START_REF] Hungerford | Histamine and Scombrotoxins[END_REF].

Histamine is produced by specific SSOs [START_REF] Gram | Fish spoilage bacteria -problems and solutions[END_REF][START_REF] Jørgensen | The effect of biogenic amine production by single bacterial cultures and metabiosis on cold-smoked salmon[END_REF] called histamine-producing bacteria (HPB), from a precursor amino acid (histidine) present in high concentrations in Scombridae, that HPB catalyse with the enzyme histidine decarboxylase (HDC) [START_REF] Prester | Biogenic amines in fish, fish products and shellfish : A review[END_REF]. It has been clearly established that storage temperature is a major factor influencing the production of histamine by HPB [START_REF] Economou | Changes in histamine and microbiological analyses in fresh and frozen tuna muscle during temperature abuse[END_REF][START_REF] Guizani | The effect of storage temperature on histamine production and the freshness of yellowfin tuna (Thunnus albacares)[END_REF][START_REF] Hungerford | Histamine and Scombrotoxins[END_REF][START_REF] Mahusain | Changes of histamine levels and bcterial growth in longtail tuna, Thunnus tongsol stored at different temperature[END_REF][START_REF] Silva | Storage Temperature Effect on Histamine Formation in Big Eye Tuna and Skipjack[END_REF]). Yet, while these histamineproducing bacteria have been identified and the production mechanisms of this biogenic amine are relatively well known, the ecology and development of HPB within the postmortem microbiome of tuna remain poorly understood.

Electronic copy available at: https://ssrn.com/abstract=3985243

In this study, we chose to conduct our investigations on a particular species: the yellowfin tuna (Thunnus albacares), which is found in tropical waters worldwide and is the second most consumed tuna species in the world (FAO 2020). Our objective was to understand how the necrobiome of this key species evolves after fish capture/death by examining two major bacterial reservoirs: the gut and the liver. We used a metabarcoding approach depict the dynamics of the whole bacterial community as well as the emergence of more specific SSOs and HPBs. The results are discussed in the light of fish conditioning process by comparing the development of the necrobiome in fresh and brine-frozen tuna fished by artisanal and industrial techniques, respectively.

Material and methods

Sampling

The yellowfin tuna (Thunnus albacares) were captured using two different fishing techniques and post-capture storage conditions: (1) artisanal fishing with immediate storage on ice of fresh individuals, and (2) industrial fishing followed by immediate brine-freezing treatment.

For freshly caught yellowfin, 12 individuals were captured around fish-aggregating devices (FADs) located in the Gulf of Guinea (Ivory Coast, N04°55'00", W03°42'19.97'') on 20-21 November 2019. The capture and euthanasia of the fish were performed by professional fishermen. The tuna were individually placed in plastic bags and kept on ice until they reached the laboratory, less than 5 h after death. The mean fork length of the individuals was 49.5 cm (min 45.7 cm -max 52.3 cm) and the average weight was 2.1 kg (min 1.7 kgmax 2.6 kg).

For the brine-frozen yellowfin tuna, 12 individuals were collected at the Abidjan tuna port Ocean (Gulf of Guinea and off the coast of Senegal) and immediately chilled brine to lower their temperature to around -15°C. The fish remained frozen in the tanks until their landing in the Port of Abidjan and were then thawed at 4°C in the laboratory, 24 hours before the Electronic copy available at: https://ssrn.com/abstract=3985243 beginning of the experiment (Fig. 1). The mean fork length of these individuals was 63.4 cm (min 58.0 cm -max 70.0 cm) and the average weight was 4.4 kg (min 3.1 kg -max 5.9 kg).

Experimental design

For each fresh and brine frozen lots, three yellowfin individuals were dissected and sampled at the beginning of the experiment (T 0 ) to analyse their liver and gut microbiota, as well as the histamine concentration (see sampling procedure below) (Fig. 1). For brine-frozen tuna, T 0 corresponded to 24 h after thawing at 4°C, which is considered as the standard temperature for home-storage. For fresh tuna, T 0 corresponded to the time of death of the fish since they were dissected directly onboard. The 12 remaining fish in each batch were kept at 4°C in temperature-controlled refrigerators. Every 48 h until the end of the 120-h experiment (i.e. T 120 ), three individuals from each batch were randomly selected to sample their hepatic and intestinal microbiota (Fig. 1).

Sampling the gut and liver microbiota

Gut

The tuna were dissected, extracting the gastrointestinal tract after cutting from below the stomach to the rectum using sterile tools. Each gut was opened, squeezed, and its inner surface entirely rubbed to expel the contents (minimum volume of 5 mL) on a sterile surface. The contents were homogenized before sampling (Gadoin et al. 2021).

Liver

A 2 x 0.2 x 2 cm (L x W x H) piece was trimmed from the right lobe of each tuna liver using sterile tools. Liver samples were rinsed with distilled water filtered through a 0.2 μm filter to avoid any contamination from other internal organs or fluids.

All the gut and liver samples were placed in 5-mL sterile cryovials, frozen in liquid nitrogen and stored at -80°C in the laboratory until the extraction of bacterial nucleic acid.

Bacterial DNA extraction, amplification and sequencing

The bacterial DNA was extracted from 250 ± 0.5 mg of the gut (n= 30) and liver (n= 30) samples. All extractions were performed with the PowerSoil DNA Isolation Kit (Qiagen ® , Hilden, Germany) following the manufacturer's instructions. DNA quality and quantity were assessed by spectrophotometry (NanoDrop ® , Wilmington, DE, USA). The V3-V4 region of the 16S rDNA gene was amplified using universal bacterial primers modified for Illumina sequencing: 343F (5'-ACGGRAGGCAGCAG) [START_REF] Economou | Changes in histamine and microbiological analyses in fresh and frozen tuna muscle during temperature abuse[END_REF][START_REF] Guizani | The effect of storage temperature on histamine production and the freshness of yellowfin tuna (Thunnus albacares)[END_REF][START_REF] Hungerford | Histamine and Scombrotoxins[END_REF][START_REF] Mahusain | Changes of histamine levels and bcterial growth in longtail tuna, Thunnus tongsol stored at different temperature[END_REF]) and 784R (5'-TACCAGGGTATCTAATCCT) [START_REF] Andersson | Comparative analysis of korean human gut microbiota by barcoded pyrosequencing[END_REF]. The reaction mixture consisted of 12.5 μL of 2X Phusion Mix (New England Biolabs ® , Ipswich, MA, USA), 1 μL of each primer at 10 μM (Eurofin ® , Luxembourg), 10 ng of DNA template and enough molecular-grade H 2 O (Qiagen ® ) to reach a final volume of 25 μL.

All samples were amplified in triplicate to avoid PCR bias in the taxonomic diversity of the community [START_REF] Perreault | Characterization of the prokaryotic diversity in cold saline perennial springs of the Canadian high arctic[END_REF]). Successfully amplified samples (n= 30) were sequenced on the Illumina platform using 2x250 bp MiSeq chemistry.

Bacterial sequence processing and analysis

A total of 16,277,785 reads were obtained. Raw reads were processed with RStudio (R version 3.5.3) using the DADA2 package (v1.10.1) [START_REF] Callahan | DADA2 : High-resolution sample inference from Illumina amplicon data[END_REF]) following the authors' tutorial (https://benjjneb.github.io/dada2/tutorial.html). The quality of forward and reverse reads was analysed before removing adaptors and primers based on their length. Using the DADA2 tutorial with default parameters, reads were then filtered, trimmed and merged into 8312 amplicon sequence variants (ASVs). Chimaeras were removed, and sequences were aligned to the SILVA 123 database [START_REF] Quast | The SILVA ribosomal RNA gene database project : Improved data processing and webbased tools[END_REF]

Histamine concentration

In all the gut and liver samples, histamine concentration was assessed by enzyme-linked immunosorbent assays (ELISA) using the Veratox® kit for tuna histamine (Neogen®, Lansing, MI, USA) following the manufacturer's instructions. Samples were suspended in distilled water, filtered and diluted 10X prior to the ELISA tests. Assays were performed under sterile conditions and the optical density was measured at 650 nm using a TECAN Infinite M200 Pro (Tecan®, Männedorf, Switzerland). The optical densities of the six standards available in the kit allowed us to trace the standard curve against which the optical density of a sample was plotted to calculate its histamine concentration in parts per million (ppm).

Statistical analysis

All statistical analyses were performed with RStudio. The effect of time and post-catch storage conditions on the composition of hepatic and intestinal bacterial communities was determined by single-factor and multiple-factor PERMANOVA with 999 permutations on the Bray-Curtis dissimilarity matrix, using the "adonis" function of the vegan package [START_REF] Dixon | VEGAN, a package of R functions for community ecology[END_REF]. Correlations between histamine concentration and the relative abundance of potential HPB were evaluated using a Spearman correlation test performed in RStudio.

Results

Short-term dynamics of the tuna necrobiome

The results revealed that the composition of the tuna necrobiome changed significantly over time in both the gut and liver (Tab. 1).

The gut microbiome

In fresh tuna, the initial enteric microbiome was comprised of numerous taxa that included the genera Cutibacterium, Enhydrobacter, BD1-7 clade and Neorickettsia, as well as several potential SSO genera such as Photobacterium, Shewanella, Pseudomonas, Novosphingobium and Vibrio. Over the 120-h period, the abundance of Photobacterium then rapidly increased to reach almost 90% of the total abundance of bacteria, while most of the other genera decreased (Fig. 2).

In brine-frozen yellowfin, significant changes in the composition of the gut necrobiome were also observed during the experiment (Fig. 2). In addition, in these fish, the presence of several potential SSOs that were not found in fresh tuna were identified (Lactococcus, Lactobacillus, Psychrobacter, Psychrilyobacter and Proteus). The occurrence of certain SSOs such as Psychrobacter, Lactococcus and Shewanella increased throughout the experiment.

At T 96 , Photobacterium, Lactobacillus, BD1-7 clade and Mycoplasma were the most abundant bacterial genera, but taxa with a relative abundance of less than 2% represented more than 25% of the community. Their proportion increased at T 120 , when the potential SSO genera Shewanella, Psychrobacter, Proteus, Pseudomonas, Photobacterium, Lactobacillus and Psychrilyobacter were detected and together represented 22.6% of the bacterial community.

The liver microbiome

The composition of the bacterial community in the liver was highly diverse and was significantly different from that of the intestine (Fig. 2, Permanova, p = 0.003). At T 0 , the microbiome in fresh tuna was mostly comprised of Enhydrobacter, Micrococcus, Neorickettsia and Massilia. In contrast with gut samples, the liver of fresh yellowfin initially hosted few SSOs, but these proliferated rapidly over time. The only SSO genus observed in liver samples at the beginning of the experiment was Pseudomonas, but at T 48 the relative abundance of other SSO genera such as Photobacterium, Shewanella, Psychrobacter and Vibrio began to increase. By T 96 , SSO genera were dominant within the liver necrobiome, representing 76% of the bacterial community. They remained the major component of the liver microbiota until the end of the experiment, when other genera such as Salegentibacter, Sporosarcina, Enhydrobacter and Cutibacterium were also detected.

The storage conditions greatly impacted the composition of the necrobiome in this organ.

The liver-associated bacteria in brine-frozen tuna evolved in a different way than in fresh tuna (Tab. 1, Fig. 2). For example, the genus Photobacterium, which was highly dominant in the liver microbiome of fresh fish, was much less abundant in brine-frozen fish. Generally, although the relative abundance of SSO genera increased over time, their occurrence remained lower in brine-frozen than in fresh samples. At the beginning of the experiment, hepatic bacterial communities were composed of Enhydrobacter, Cutibacterium, Brachybacterium, Macrococcus, Halomonas, Acinetobacter and Methylobacterium, as well as two main SSO genera (Photobacterium and Pseudomonas), and potential pathogens such as Electronic copy available at: https://ssrn.com/abstract=3985243

Staphylococcus and Corynebacterium. At the end of the experiment (T 120 ), the liver microbiome hosted several other potential SSO genera including Proteus, Psychrobacter, Photobacterium, Shewanella and Psychrilyobacter, which together represented 29% of the bacterial community.

Diversity of histamine-producing bacteria and histamine concentrations

In general, the relative abundance of HPB was much higher in fresh than in brine-frozen tuna. Photobacterium ASVs were generally dominant in gut samples, while other HPB genera (Pseudomonas and Acinetobacter) were also present in the liver in equivalent proportions (Fig. 3). Interestingly, the genus Proteus was only detected at the late stage of fish decomposition (T 120 ) and exclusively in brine-frozen samples.

In fresh yellowfin, the temporal dynamics of Photobacterium ASVs were significantly correlated with histamine concentration in both gut and liver samples (Pearson, p <0.05).

Other potential HPB genera such as Pseudomonas, Vibrio, Acinetobacter and Enterobacter were also detected, but at low levels (Fig. 3).

Fresh and brine-frozen tuna exhibited contrasting patterns of histamine concentration. In fresh fish, histamine concentration increased abruptly after T 48 to reach a maximum at T 96 in the gut (mean = 676 ppm) and at T 120 in the liver (mean = 59 ppm), thus exceeding the 50 ppm sanitary threshold established by the United States Food and Drug Administration (FDA 2021)(Fig. 3). Conversely, in brine-frozen fish, histamine concentrations remained below that threshold throughout the experiment.

Discussion

Modifications in animal's microbiome composition are normal phenomena following their death, resulting from physical and chemical changes, as well as the loss of immune response [START_REF] Benbow | Death and Decomposition in Aquatic Ecosystems[END_REF]. In fish, however, the evolution of the post-mortem bacteriome over time has been poorly studied. In this study, we explored the tuna necrobiome by examining the dynamics of the main SSOs together with the production of histamine in two major bacterial reservoirs: the gut and the liver. We compared the incidence of post-capture storage conditions (fresh and brine-frozen individuals) on the development of these spoilage bacteria.

Occurrence and diversity of SSOs

As expected, the proportion of potential specific spoilage organisms (SSOs) increased significantly throughout the experiment in the gut and the liver (Fig. 2). This trend was particularly marked in fresh tunas. In both organs, we observed an increase in abundance of Psychrobacter, Pseudomonas, Proteus, Aeromonas, Lactobacillus, Shewanella and Photobacterium, which have all been previously detected in the flesh of various fish species such as haddock, Atlantic salmon, gilthead sea bream, European sea bass and yellowfin tuna [START_REF] Dalgaard | Biogenic amine formation and microbial spoilage in chilled garfish (Belone belone belone)-Effect of modified atmosphere packaging and previous frozen storage[END_REF][START_REF] Fogarty | Spoilage indicator bacteria in farmed Atlantic salmon (Salmo salar) stored on ice for 10 days[END_REF][START_REF] Jääskeläinen | Metabolomics and bacterial diversity of packaged yellowfin tuna (Thunnus albacares) and salmon (Salmo salar) show fish species-specific spoilage development during chilled storage[END_REF]Parlapani et al. 2018;[START_REF] Reynisson | Effects of different cooling techniques on bacterial succession and other spoilage indicators during storage of whole, gutted haddock (Melanogrammus aeglefinus)[END_REF][START_REF] Syropoulou | HRM and 16S rRNA gene sequencing reveal the cultivable microbiota of the European sea bass during ice storage[END_REF]) (Fig. 2). The development of SSOs in fish and seafood products is well documented, and their proliferation typically depends on conservation conditions [START_REF] Boziaris | Specific Spoilage Organisms (SSOs) in Fish[END_REF]. Several studies have demonstrated that SSO diversity in the flesh of different fish species varies between chilling, vacuum packaging or thawing temperature [START_REF] Antunes-Rohling | Characterization of the Spoilage Microbiota of Hake Fillets Packaged Under a Modified Atmosphere (MAP) Rich in CO2 (50% CO2/50% N2) and Stored at Different Temperatures[END_REF][START_REF] Odeyemi | Seafood spoilage microbiota and associated volatile organic compounds at different storage temperatures and packaging conditions[END_REF][START_REF] Reynisson | Effects of different cooling techniques on bacterial succession and other spoilage indicators during storage of whole, gutted haddock (Melanogrammus aeglefinus)[END_REF][START_REF] Syropoulou | Primary Processing and Storage Affect the Dominant Microbiota of Fresh and Chill-Stored Sea Bass Products[END_REF]. Bacteria from the Shewanella, Photobacterium and Pseudomonas genera are known for their ability to produce high quantities of H 2 S, trimethylamine and volatile nitrogenous compounds respectively [START_REF] Boziaris | Specific Spoilage Organisms (SSOs) in Fish[END_REF][START_REF] Carrascosa | Predictive models for bacterial growth in sea bass ( Dicentrarchus labrax ) stored in ice[END_REF][START_REF] Chinivasagam | Volatile components associated with bacterial spoilage of tropical prawns[END_REF], while species such as S. putrefaciens, Proteus mirabilis and P. damselae are bacteria potentially pathogenic to humans [START_REF] Gennari | The microflora of fresh and spoiled sardines (Sardina pilchardus) caught in Adriatic (Mediterranean) Sea and stored in ice[END_REF][START_REF] Ozogul | Antimicrobial activity of thyme essential oil nanoemulsions on spoilage bacteria of fish and foodborne pathogens[END_REF][START_REF] Speranza | Use of Desirability Approach to Predict the Inhibition of Pseudomonas fluorescens, Shewanella putrefaciens and Photobacterium phosphoreum in Fish Fillets Through Natural Antimicrobials and Modified Atmosphere Packaging[END_REF]).

It should be noted that the spoilage activity of SSOs is a relatively complex mechanism that has multiple determinants. The production of spoiling metabolites is clearly speciesdependent and varies according to the storage conditions, such as temperature [START_REF] Antunes-Rohling | Characterization of the Spoilage Microbiota of Hake Fillets Packaged Under a Modified Atmosphere (MAP) Rich in CO2 (50% CO2/50% N2) and Stored at Different Temperatures[END_REF][START_REF] Parlapani | Monitoring of spoilage and determination of microbial communities based on 16S rRNA gene sequence analysis of whole sea bream stored at various temperatures[END_REF], atmosphere conditions [START_REF] Emborg | Significant histamine formation in tuna (Thunnus albacares) at 2 °C-effect of vacuum-and modified atmosphere-packaging on psychrotolerant bacteria[END_REF][START_REF] Silbande | Quality assessment of ice-stored tropical yellowfin tuna (Thunnus albacares) and influence of vacuum and modified atmosphere packaging[END_REF][START_REF] Sivertsvik | A review of modified atmosphere packaging of fish and fishery products-Significance of microbial growth, activities and safety[END_REF], as well as microbial interactions between communities [START_REF] Joffraud | Effect of bacterial interactions on the spoilage of cold-smoked salmon[END_REF][START_REF] Zotta | Dynamics of bacterial communities and interaction networks in thawed fish fillets during chilled storage in air[END_REF]. Various analyses such as the detection of spoilage genes, the growth of SSOs on selective media and the quantification of spoilage metabolites are usually required to assess the spoilage potential of SSOs [START_REF] Fu | Quorum sensing system-regulated genes affect the spoilage potential of Shewanella baltica[END_REF][START_REF] Syropoulou | HRM and 16S rRNA gene sequencing reveal the cultivable microbiota of the European sea bass during ice storage[END_REF][START_REF] Tang | Characterization of LuxI/LuxR and their regulation involved in biofilm formation and stress resistance in fish spoilers Pseudomonas fluorescens[END_REF]. Although these analyses were not performed in this study, the taxonomic identification of SSO genera in the two digestive organs raises questions about their dispersion from the viscera to the flesh after fish death (Shen & Wang 2020).

The effect of storage conditions on the tuna necrobiome

One of the main findings of this study was that the relative abundance and dynamics of SSOs greatly varied according to the initial storage conditions (Fig. 2). At the end of the experiment, they represented on average (for the two organs) 82% of the bacterial community in fresh tuna, in contrast to less than 30% in brine-frozen samples (Fig. 2). The influence of storage conditions on the composition of the fish microbiome has long been investigated [START_REF] Ghaly | Fish Spoilage Mechanisms and Preservation Techniques : Review[END_REF][START_REF] Zhuang | Spoilage-related microbiota in fish and crustaceans during storage : Research progress and future trends[END_REF]. For example, a delayed development of SSOs was reported in frozen fillets of Atlantic cod, mackerel and salmon compared to fresh samples [START_REF] Fagan | Effect of freeze-chilling, in comparison with fresh, chilling and freezing, on some quality parameters of raw whiting, mackerel and salmon portions[END_REF][START_REF] Sørensen | Thawed and chilled Atlantic cod (Gadus morhua L.) from Greenland-Options for improved distribution[END_REF]. While lowtemperature chilling is known to decrease the growth of microorganisms, freezing between -18 and -30°C kills between 10% and 60% of viable bacteria [START_REF] Berkel | Preservation of Fish and Meat[END_REF][START_REF] Rahman | Food Preservation by Freezing[END_REF]). In addition, the presence of sodium chloride is also known to inactivate autolytic enzymes in fish, as well as to negatively impact the growth of several spoilage bacteria [START_REF] Ghaly | Fish Spoilage Mechanisms and Preservation Techniques : Review[END_REF][START_REF] Henney | Preservation and physical property roles of sodium in foods[END_REF][START_REF] Mejlholm | Effect of brine marination on survival and growth of spoilage and pathogenic bacteria during processing and subsequent storage of ready-to-eat Electronic copy available at: https://ssrn.com/abstract=3985243 shrimp (Pandalus borealis)[END_REF][START_REF] Turan | Salting technology in fish processing[END_REF]. This may partially explain why brine-frozen yellowfin exhibited a limited abundance of SSOs in the gut and liver microbiota compared to fresh tuna.

SSOs and histamine production in tuna

Among the potential SSO genera detected in the tuna necrobiome, histamine-producing bacteria (HPB) are of particular interest, as they have been implicated in cases of food poisoning worldwide [START_REF] Hungerford | Scombroid poisoning : A review[END_REF][START_REF] Hungerford | Histamine and Scombrotoxins[END_REF]. We identified several HPB genera in the gut and liver samples, including Acinetobacter, Enterobacter, Morganella, Proteus, Pseudomonas and Vibrio, but Photobacterium was the most abundant, especially in fresh fish, where it rapidly dominated the bacterial community in both organs (Fig. 3). The genus Photobacterium is ubiquitous in marine environments and is composed of several species [START_REF] Thyssen | Genus II. Photobacterium. In Bergey's manual of systematic bacteriology[END_REF]. It has been described as commensal in various fish species [START_REF] Egerton | The Gut Microbiota of Marine Fish[END_REF][START_REF] Estruch | Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene[END_REF][START_REF] Givens | Comparison of the gut microbiomes of 12 bony fish and 3 shark species[END_REF], but some Photobacterium species such as P. damselae and P. piscicida are known as fish and human pathogens [START_REF] Rivas | Photobacterium damselae subsp. Damselae, a bacterium pathogenic for marine animals and humans[END_REF][START_REF] Romalde | Photobacterium damselae subsp. piscicida : An integrated view of a bacterial fish pathogen[END_REF]. Photobacterium has also been identified as an SSO in Atlantic cod [START_REF] Kuuliala | Microbiological, chemical and sensory spoilage analysis of raw Atlantic cod (Gadus morhua) stored under modified atmospheres[END_REF], haddock [START_REF] Reynisson | Effects of different cooling techniques on bacterial succession and other spoilage indicators during storage of whole, gutted haddock (Melanogrammus aeglefinus)[END_REF]) and Atlantic salmon [START_REF] Jääskeläinen | Metabolomics and bacterial diversity of packaged yellowfin tuna (Thunnus albacares) and salmon (Salmo salar) show fish species-specific spoilage development during chilled storage[END_REF]. Indeed, several Photobacterium species are able to synthetize histamine, including P. angustum, P. aquimaris, P. kishitanii, P. damselae and P. phosphoreum, which are designated as high histamine producers (> 200ppm) [START_REF] Bjornsdottir-Butler | Biogenic Amine Production by and Phylogenetic Analysis of 23 Photobacterium Species[END_REF]. While histamine-production capacity has been demonstrated to vary across different Photobacterium species, this capacity is also influenced by temperature [START_REF] Bjornsdottir-Butler | Biogenic Amine Production by and Phylogenetic Analysis of 23 Photobacterium Species[END_REF][START_REF] Morii | Activity of Two Histidine Decarboxylases from Photobacterium phosphoreum at Different Temperatures, pHs, and NaCl Concentrations[END_REF][START_REF] Takahashi | Effects of environmental factors on histamine production in the psychrophilic histamine-producing bacterium Photobacterium iliopiscarium[END_REF]. Insufficiently cold temperatures are known to favour the production of histamine and maintaining the cold chain is essential to prevent its formation [START_REF] Hungerford | Scombroid poisoning : A review[END_REF][START_REF] Hungerford | Histamine and Scombrotoxins[END_REF]. Some psychrotrophic HPB, such as P. phosphoreum and Morganella psychrotolerans, are able to synthetize histamine at temperatures between 0° and 5°C [START_REF] Bjornsdottir-Butler | Biogenic Amine Production by and Phylogenetic Analysis of 23 Photobacterium Species[END_REF][START_REF] Emborg | Significant histamine formation in tuna (Thunnus albacares) at 2 °C-effect of vacuum-and modified atmosphere-packaging on psychrotolerant bacteria[END_REF][START_REF] Kanki | Photobacterium phosphoreum caused a histamine fish poisoning incident[END_REF][START_REF] Wang | Histamine Production Behaviors of a Psychrotolerant Histamine-Producer, Morganella psychrotolerans, in Various Environmental Conditions[END_REF]. Although our data did not allow us to identify the potential HPB down to the species level, we can consider that the Photobacterium taxa observed in both the gut and liver of fresh yellowfin tuna were HPB, as their temporal dynamics were positively correlated with the increase in histamine concentration in the different incubations (Fig. 3). As early as 96h after their capture, histamine concentration in the gut and liver of fresh yellowfin exceeded the United States Food and Drug Administration (FDA 2021) recommendations of 50 ppm.

The vast majority of studies investigating Scombroid (histamine) poisoning have been conducted on tuna flesh or in processed products such as filets or canned tuna [START_REF] Emborg | Significant histamine formation in tuna (Thunnus albacares) at 2 °C-effect of vacuum-and modified atmosphere-packaging on psychrotolerant bacteria[END_REF][START_REF] Guizani | The effect of storage temperature on histamine production and the freshness of yellowfin tuna (Thunnus albacares)[END_REF][START_REF] Kim | Histamine Production by Morganella morganii in Mackerel, Albacore, Mahi-mahi, and Salmon at Various Storage Temperatures[END_REF][START_REF] Kung | Isolation and identification of histamine-forming bacteria in tuna sandwiches[END_REF][START_REF] Silva | Occurrence of histamine in Brazilian fresh and canned tuna[END_REF]. Our study extends this by revealing the presence of histamine and HPB in both gut and liver samples. These organs have been previously identified as important reservoirs of HPB in tuna (Bjornsdottir-Butler et al. 2015;Taylor & Speckhard 1983;Gadoin et al. 2021), but few studies have considered the liver and gut in their investigations on histamine formation in scombroid fish. [START_REF] Glória | Histamine and Other Biogenic Amines in Albacore Tuna[END_REF] observed that the intestinal wall of yellowfin tuna contained a substantial concentration of histamine. Similarly, Fernández-Salguero & Mackie (1979) reported significant histamine concentration in the liver of mackerel, in an even greater proportion than in muscles.

Another key finding was that histamine was not detected in the gut or liver samples of brinefrozen tuna, despite the presence of potential HPB genera (Fig. 3). This suggests that the brine-freezing treatment may alter the capacity of HPB to produce histamine in these two organs. Freezing has been previously observed to limit the production of this biogenic amine Electronic copy available at: https://ssrn.com/abstract=3985243 in tuna fillets [START_REF] Tahmouzi | Histamine formation and bacteriological quality in Skipjack tuna (Katsuwonus pelamis): effect of defrosting temperature[END_REF]). In addition, brine immersion is known to inhibit the activity of the histidine decarboxylase enzyme in HPB, and therefore to limit the synthesis of histamine from its precursor histidine [START_REF] Hwang | Effect of Brine Concentrations on the Bacteriological and Chemical Quality and Histamine Content of Brined and Dried Milkfish[END_REF][START_REF] Morii | Activity of Two Histidine Decarboxylases from Photobacterium phosphoreum at Different Temperatures, pHs, and NaCl Concentrations[END_REF][START_REF] Tabanelli | Effect of Chemico-Physical Parameters on the Histidine Decarboxylase (HdcA) Enzymatic Activity in Streptococcus thermophilus PRI60[END_REF]. Overall, in line with previous studies, our results confirm the usefulness of applying a brine-freezing treatment to tuna to prevent the formation of histamine, and thus reduce the health risk associated with their consumption [START_REF] Hungerford | Histamine and Scombrotoxins[END_REF].

Conclusion

Our results highlight the sanitary risks associated with the development of SSOs and of histamine concentrations in tuna's digestive organs, reminding the importance of removing their viscera prior to consumption. Such sanitary risks were much more elevated with fresh than brine-frozen fishes, confirming the incidence of the storage conditions on the evolution of the tuna necrobiome. Finally, this study confirms the need to take into account the gut and liver in further investigations on the ecology of HPB in scombroid fish. Electronic copy available at: https://ssrn.com/abstract=3985243 x 3

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Figure 2

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Figure 3

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Community dissimilarity

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(

  Ivory Coast) by the Exploited Tropical Pelagic Ecosystem Observatory (IRD, Ob7, certified ISO 9001:2015) within the framework of multiannual European fishery data collection (DCF, financed by the European Maritime and Fisheries Fund, Article 77). All individuals were caught by purse seine vessels between May and December 2019 in the Eastern Atlantic

  to access their taxonomy. Analyses were performed on a random subsample of 2337 sequences per sample, corresponding to the sample with the smallest number of sequences after trimming and quality processing. Using the phyloseq package (McMurdie & Holmes 2013), final taxonomic and ASV tables were linked to sample metadata (including biological compartment, sampling time and conservation conditions). The relative abundance of ASVs in each sample were assessed by phyloseq, and ASVs assigned to non-prokaryotes, archaea, chloroplasts and mitochondria were removed. Using the phyloseq and ggplot2 packages, the composition and diversity of bacterial communities were then represented at the class level, based on the relative abundance of ASVs in each sample. Referring to the literature, a list of putative histamine-producing bacteria (HPB) genera was established, and their presence/absence in our samples was assessed by comparing the list of HPB to our taxonomy table.

Figure 1 .

 1 Figure 1. Experimental design to study the post-mortem microbiome of yellowfin tuna stored at 4°C. Gut and liver samples were collected in triplicate at the beginning of the experiment (T 0 ) and after 48, 96 and 120 hours, on fresh (A) and brine-frozen (B) individuals.

Figure 2 .

 2 Figure 2. Temporal variation in the relative abundance of the main bacterial genera in the gut and liver samples of fresh and brine-frozen yellowfin tuna. The size of the dot is proportional to the relative abundance of each bacterial genus from T 0h to T 120h . Genera identified as potential specific spoilage organisms (SSOs) are coloured in red. Arrows represent the overall development of each bacterial genus during the experiment. Bacterial genera with a relative abundance inferior to 2% were grouped and designated as < 2% abund.

Figure 3 .

 3 Figure 3. Dynamics of histamine concentration (ppm) (right abscissa) and relative abundance (left abscissa) of the main putative histamine-producing bacteria (HPB) found in the gut (A,B) and liver (C,D) of fresh (B,D) and brine-frozen (A,C) yellowfin tuna. The red horizontal bar represents the sanitary threshold of 50 ppm established by the United States Food and Drug Administration (FDA 2021).

  Figure1

Table 1 :

 1 Results of permutational ANOVAS (PERMANOVA, 999 permutations) performed on Bray-Curtis dissimilarities matrices to test the variation of bacterial community composition with time and post-capture conservation conditions in gut and liver samples. Bold values indicate a significant effect of the tested factor (p < 0.05).

			Time			Conservation
		p value	r²	df	p value	r²	df
	Gut	0.001	0.21	2	0.255	0.05	1
	Liver	0.023	0.13	2	0.003	0.09	1

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Acknowledgements

We thank the Montpellier University of Excellence (I-site MUSE; Project The MOME) and the Insitute of Research for Development (JEAI; Project MOSANE) for their financial support. he data used here was collected within the framework of the Data Collection Framework program co-financed by the IRD and Measure 77 of the European Maritime and Fisheries Fund (EMFF). We are grateful to Justin Aurélie Guillou for their assistance during sampling.

We would like to thank the members of the IRD's Exploited Tropical Pelagic Ecosystems Observatory in Abidjan (ie, Aurélie, Guillou, Pascal Bach) for providing the brine frozen tunas. We also grateful to the CRO staff for providing access to their laboratory and facilities.