Andrew Menzies-Gow
email: menzies-gow@rbht.nhs.uk.

Michael E Wechsler

Christopher E Brightling

Stephanie Korn

Jonathan Corren

Elliot Israel

Geoffrey Chupp

Artur Bednarczyk

Sandhia Ponnarambil

Scott Caveney

Gun Almqvist

Monika Gołąbek

Linda Simonsson

Kaitlyn Lawson

Karin Bowen

Gene Colice

; Destination

Jorge Lima Hetzel

Adelmir Jussara Fiterman

Martti Souza Machado

Anton Antila

Marina Andrade Lima

Suzana Erico

Tanni Minamoto

Daniela Cavalet Blanco

Patricia Gomes De Matos Bezerra

Pierre-Alain Houle

Catherine Lemiere

Lyle S Melenka

Richard Leigh

Patrick Mitchell

Syed Anees

Bonavuth Pek

Guy Chouinard

Amarjit S Cheema

Ho-Ching Yang

George Philteos

Pascal Chanez

Arnaud Bourdin

Gilles Devouassoux

Camille Taille

Frédéric De Blay

Christophe Leroyer

Antoine Beurnier

Gilles Garcia

Pierre-Olivier Girodet

François-Xavier Blanc

Antoine Magnan

Stéphanie Wanin

Jocelyne Just

Richard Linde

Stefan Zielen

Karin Förster

Christian Geßner

Margret Jandl

Roland Otto Buhl

Marc Oliver Kornmann

Anneliese Linnhoff

Andrea Ludwig-Sengpiel

Martin Ehlers

Tibor Schmoller

Heiner Steffen

Martin Hoffmann

Joachim Kirschner

Olaf Schmidt

Tobias Welte

Hilke Temme

Ori Wand

Amir Bar-Shai

Gabriel Izbicki

Neville Berkman

Gershon Fink

David Shitrit

Yochai Adir

Piotr Kuna

Barbara Rewerska

Ewa Pisarczyk-Bogacka

Oksana Kurbacheva

Sergey L Mikhailov

Maksim Vasilev

Alexander Emelyanov

Siraj Wali

Amr Albanna

Richard Van Zyl-Smit

Ismail Abdullah

David Bernhardi

Farzana Hoosen

Elvis Irusen

Ismail Kalla

Deepak Lakha

Essack Mitha

Visvakuren Naidoo

Haylene Nell

Trevenesan Padayachee

Jeevren Reddy

Friedrich Petrick

Eugene Van Der Walt

Zubar Fazal

Ahmed Vawda

Hae-Sim Park

Sang Haak Lee

Mi-Kyeong Kim

You Jung-Won Park

Byung Sook Cho

Jaechun Lee

Yoon-Seok Chang

Choon-Sik Park

Ho Kwan

Sook Young Lee

Hyoungkyu Lee

Yoon

Hee Kyoung

Myung Jae Sohn

Kyung Park

Min Hoon

Young Joo Cho

Han Ki Park

Yongchul Lee

Chau-Chyun Sheu

Chih-Yen Tu

Kang-Yun Lee

Sevim Bavbek

Bilun Gemicioglu

Koca Kalkan

Nataliia Makieieva

Mykola Ostrovskyy

Yevgeniya Dytyatkovs'ka

Yuriy Mykhaylovych Mostovoy

Kyrylo Lebed

Oleh Yakovenko

Atoya Adams

Timothy Mooring

Louis Torres

Marvin Sexton

Ernest Thompson

Jonathan A Bernstein

Paul Lisi

Christopher M Chappel

Jeremy Cole

Gary I Greenwald

Conigliaro Jones

Ryan Mitchell Klein

David N Pham

Selwyn Spangenthal

Steven F Weinstein

Hugh H Windom

Neil L Kao

Mila A Leong

Vinay Mehta

Wendy C Moore

Saligrama Bhat

Bassil Aish

Steven M Meltzer

Mark H Moss

Edward M Kerwin

John Palsted Delgado

Gregg Hudson Lucksinger

Charles A Thompson

Sady A Alpizar

Virgi Sanjay

Zahid Vadgama

Joshua S Zafar

Njira Jacobs

Neal Lugogo

Lawrence D Jain

Nabil S Sher

David Andrawis

Eric Jason Fuentes

Erika G Boren

Neetu Gonzalez

Talreja

Sandy Sheharyar

Elliot Durrani

Sudhir Israel

Samuel Sekhsaria

Mayank Deleon

Martha M Shukla

Totszollosy Tarpay

Faisal Fakih

Golda Hudes

Jeffrey P Tillinghast

Phillip E Korenblat

Kartik Shenoy

Loretta Que

Shahrukh Ahmad Kureishy

Fred Chukwuemeka Umeh

Vinh Nhu Nguyen

Hanh Thi Chu

Thuy Thi

Dieu Nguyen

a randomised, placebo-controlled extension study

come

Introduction

Biologics are recommended as add-on therapy in people with severe, uncontrolled asthma. Approval of these drugs is usually based on 1-year safety and efficacy studies, but these therapies are typically used for longer periods.1 Long-term extension studies for biologics in severe asthma have been done.2-4 However, these do not have a placebo group to provide context for the safety and efficacy data.

Tezepelumab, a human monoclonal antibody (immunoglobulin [Ig] G2λ), binds specifically to thymic stromal lymphopoietin (TSLP) and prevents the protein from interacting with its heterodimeric receptor.5 TSLP, an epithelial cytokine, has a central role in the initiation and persistence of airway inflammation in asthma.6 TSLP regulates multiple downstream inflammatory cascades and has effects on airway structural cells and airway hyper-responsiveness. [START_REF] Gauvreau | Thymic stromal lymphopoietin: its role and potential as a therapeutic target in asthma[END_REF] Tezepelumab reduced the annualised asthma exacerbation rate, and improved lung function, asthma control, and health-related quality of life in people with severe, uncontrolled asthma in the phase 2b PATHWAY (NCT02054130)5 and phase 3 NAVIGATOR (NCT03347279) studies. [START_REF] Menzies-Gow | Tezepelumab in adults and adolescents with severe, uncontrolled asthma[END_REF] In the corticosteroid-sparing, phase 3, SOURCE study (NCT03406078), although the primary endpoint of oral corticosteroid reduction was not met, tezepelumab reduced the annualised asthma exacerbation rate and improved lung function, asthma control, and health-related quality of life compared with placebo in people with oral corticosteroid-dependent asthma.8 On the basis of these studies, which had treatment durations of up to 1 year, tezepelumab was approved in December, 2021, by the US Food and Drug Administration as a treatment for severe asthma with no biomarker or phenotypic restrictions. [START_REF]Tezepelumab (TEZSPIRE) prescribing information[END_REF] The 2022 Global Initiative for Asthma (GINA) guidelines recommend anti-TSLP therapy for people with and without evidence of type 2 inflammation.1

Here, we report results from DESTINATION (NCT03706079), a long-term, randomised, placebo-controlled extension study that enrolled people who completed treatment in the NAVIGATOR or SOURCE studies. The primary objective of DESTINATION was to evaluate the long-term safety and tolerability of tezepelumab in adults and adolescents with severe, uncontrolled asthma, compared with placebo, over 2 years of treatment (from week 0 of the parent studies to week 104 of DESTINATION). The secondary objective was to assess the long-term effect of tezepelumab on asthma exacerbations.

Methods

Study design

DESTINATION was a phase 3, multicentre, randomised, double-blind, placebo-controlled, parallel-group longterm extension study to evaluate the safety and efficacy of subcutaneous tezepelumab (210 mg) every 4 weeks in people who completed treatment in the 52-week NAVIGATOR study or the 48-week SOURCE study (appendix p 34). Full details of these studies have been published previously.10

The study was done in 18 countries (appendix p 3) across 182 sites comprised of hospitals, clinics, medical centres, clinical trial centres, university centres, and private practices. The study protocol is included in the appendix (p 46). The study was done in accordance with the ethical principles of the Declaration of Helsinki, International Council for Harmonisation Good Clinical Practice guidelines, and applicable regulatory requirements. Approvals from independent ethics committees were obtained.

Participants

Participants were aged 12-80 years with severe, uncontrolled asthma and received medium-dose to high-dose inhaled corticosteroids, with at least one additional asthma controller medication with or without oral corticosteroids. Key inclusion criteria at randomisation were attendance at the end-of-treatment visit of the parent study, adequate treatment compliance during the parent study, and not meeting any of the discontinuation criteria from the parent study. Key exclusion criteria included the presence of any clinically important pulmonary disease other than asthma; pulmonary or systemic diseases other than asthma associated with elevated peripheral eosinophil counts; any clinically meaningful abnormal finding or important protocol deviations during the parent study; and any disorders that were not stable and could, in the opinion of the investigator, affect the safety of the participant (appendix p 6). All participants provided written informed consent in accordance with local requirements.

Randomisation and masking

DESTINATION randomisation was stratified by the parent study (appendix p 5 for stratification factors in NAVIGATOR and SOURCE). All participants were randomised to maintain masking and assigned new enrolment codes. Those who were previously randomised to receive tezepelumab in either parent study continued tezepelumab at the same dosing (210 mg subcutaneously every 4 weeks). Individuals who were previously randomised to receive placebo in either parent study were re-randomised 1:1 to receive either tezepelumab or placebo using a randomisation list prepared by a computerised system (Calyx Clinical Research Solutions). The resulting overall participant distribution in the long-term extension period was approximately 3:1 (tezepelumab to placebo). Participants, investigators, and site staff were masked to treatment assignment, which was assigned using an interactive voice and web response system. Treatment was administered to the participants by the investigators. Investigators were masked to treatment as tezepelumab and the placebo were identical in appearance.

Procedures

The screening and randomisation visit was the same day as the end-of-treatment visit in the parent studies (week 52 in NAVIGATOR and week 48 in SOURCE). In DESTINATION, participants received either tezepelumab 210 mg or placebo every 4 weeks subcutaneously for 52 weeks for those who previously completed NAVIGATOR and 56 weeks for those who previously completed SOURCE, resulting in a total of 104 weeks of treatment. Participants who were unable to attend an on-site initial visit for DESTINATION due to the COVID-19 pandemic were allowed to enter the study by the end of the safety follow-up period of the parent studies. Participants who completed the treatment period in DESTINATION entered one of two follow-up periods without treatment (appendix p 34). Participants recruited from NAVIGATOR could choose to enter either a 12week follow-up period or a 36-week extended follow-up period. Data from the 36-week extended follow-up period are not included in the presented analyses. Individuals recruited from SOURCE were only eligible to enter the 12-week follow-up period due to the smaller sample size and logistical issues. Participants continued to receive their prescribed controller medications throughout the study. Step-down of oral corticosteroid or inhaled corticosteroids could be done at the discretion of the study physician using the protocol guidance for changes to background asthma medication, adapted from GINA.11

Outcomes

The primary endpoints were exposure-adjusted incidence of adverse events and serious adverse events over 104 weeks, including the treatment duration of the parent studies. The secondary endpoint was the annualised asthma exacerbation rate over 104 weeks. This rate was also assessed in subgroups of participants initially from NAVIGATOR (appendix p 5).

Exploratory outcomes related to asthma exacerbations were time-to-first exacerbation over 104 weeks and the annualised rate of exacerbations that were associated with hospitalisation (>24 hours in hospital) or an emergency department visit (<24 hours in an emergency department, and required systemic corticosteroids for ≥3 consecutive days) over 104 weeks. Other exploratory outcomes included change from baseline to week 104 in: pre-bronchodilator FEV1 (minimum clinically relevant difference=0•1 L);12 six-item Asthma Control Questionnaire (ACQ-6; range, 0 [no impairment] to 6 [maximum impairment]; minimum clinically important difference=0•5 points);11 St George's Respiratory Questionnaire score (SGRQ; range, 0 [no impairment] to 100 [maximum impairment]; minimum clinically important difference=4 points);13 and type 2 biomarker levels (blood eosinophils, fractional exhaled nitric oxide [FeNO]; and serum total immunoglobulin E [IgE]). For all endpoints, baseline was defined as week 0 of the parent study.

Statistical analyses

No statistical hypotheses were formally tested during the study (ie, none of the analyses included type I error control). The primary safety and primary full analysis datasets comprised all participants who were randomised and who received at least one dose of tezepelumab or placebo in either of the parent studies, regardless of whether they were enrolled in DESTINATION. These primary treatment groups are referred to as randomised tezepelumab and randomised placebo and are defined in the appendix (appendix p 35). Safety analyses were also done in the all tezepelumab group, which comprised individuals randomised to tezepelumab in the parent study plus data from the long-term extension period from those who received placebo in the parent study and were rerandomised to receive tezepelumab in DESTINATION. Exposure was calculated from start of tezepelumab treatment; details of on-treatment and on-study analyses are included in the appendix (p 5).

The supportive long-term extension and supportive full analysis datasets only included participants who were enrolled in DESTINATION (appendix p 35). Participants were grouped as follows: those who received tezepelumab in the parent study and in DESTINATION (tezepelumab only group); those who received placebo in the parent study and switched to tezepelumab in DESTINATION (placebo then tezepelumab group); and those who received placebo in the parent study and in DESTINATION (placebo only group).

On-treatment adverse events and serious adverse events were summarised for the safety analysis set over the 104-week study period using exposure-adjusted incidence to account for variability in follow-up. Adverse events were categorized using the Medical Dictionary for Regulatory Activities (MedDRA) system (version 24.1). Incidence per 100 patient-years were calculated as the number of participants reporting adverse events or serious adverse events divided by total exposure duration across all participants in the given treatment group, multiplied by 100. A post-hoc analysis of COVID-19-related adverse events was also done, adjusting only for exposure after the start of the pandemic. The secondary endpoint of annualised asthma exacerbation rate over 104 weeks in the tezepelumab and placebo groups was compared using a negative binomial model with the total number of asthma exacerbations experienced from baseline in the parent studies until week 104 in the long-term extension period as a response variable. Full statistical analyses for safety and efficacy related endpoints are described in the appendix (pp 4-5).

This study is registered with ClinicalTrials.gov, NCT03706079.

Role of the funding source

The funders of the study, AstraZeneca and Amgen, contributed to the study design and data interpretation, and funded medical writing support for this manuscript. The study sponsor, AstraZeneca, did the study, coordinated the data management, and did the statistical analysis in collaboration with investigators at the academic centres, all of whom had access to the final study data.

Results

Participants were enrolled in NAVIGATOR from Nov 23, 2017, and in SOURCE from March 5, 2018. The first participant was enrolled in DESTINATION on Jan 7, 2019, and the last on Oct 15, 2020. The last end-oftreatment visit was Oct 26, 2021. The primary dataset included 1059 participants from NAVIGATOR (randomised tezepelumab n=528; randomised placebo n=531) and 150 participants from SOURCE (randomised tezepelumab n=74; randomised placebo n=76). Rollover rates into DESTINATION were high from both studies. In total, 951 participants (827 from NAVIGATOR and 124 from SOURCE) were randomised into DESTINATION. Participants from Japanese sites in NAVIGATOR (n=59 initiated on tezepelumab [57 completed treatment] and n=38 [35 completed treatment] taking placebo) did not participate in DESTINATION. Of those who completed treatment in the parent studies and enrolled in countries other than Japan, 95% (827/874) of NAVIGATOR participants and 91% (124/137) of SOURCE participants entered DESTINATION. Subsequently, 94% (775/827) of participants initially from NAVIGATOR and 94% (117/124) of participants initially from SOURCE who enrolled in DESTINATION completed treatment in the long-term extension period (figure 1). Among participants from both parent studies, baseline demographics and clinical characteristics were well balanced between treatment groups (table 1). The participant demographics and clinical characteristics for the supportive long-term extension analysis dataset were similar to those of the primary analysis dataset (appendix p 8). Among those initially from NAVIGATOR, a smaller proportion of participants in the supportive long-term extension analysis dataset were receiving maintenance oral corticosteroids than in the primary analysis dataset. Due to the randomisation scheme at the start of the long-term extension period, total exposure time (ie, time at risk) was greater in the randomised tezepelumab group than in the randomised placebo group, with more exposure time for the randomised tezepelumab group occurring during the COVID-19 pandemic (appendix p 37). The exposure-adjusted incidence of any adverse events, any serious adverse events, and any adverse event leading to treatment discontinuation in the on-treatment period were lower in the randomised tezepelumab group than in the randomised placebo group across both parent studies (tables 2 and 3). In the primary safety analysis set, the most common adverse events (occurring in ≥10% of participants) across treatment groups from both parent studies were nasopharyngitis, upper respiratory tract infection, headache, and asthma (defined in the protocol as asthma-related signs or symptoms that were serious, not consistent with the participant's pre-existing history as judged by the investigator, or that led to discontinuation of study drug). Similar adverse events were observed in the supportive long-term extension analysis set (appendix pp 10-13).

The on-study pooled incidence of deaths were 0•80 per 100 patient-years in the all tezepelumab group and 0•58 per 100 patient-years in the randomised placebo group (difference 0•22; 95% CI -0•61 to 0•94), with 11 deaths in those receiving tezepelumab and five deaths in those receiving placebo (appendix pp [START_REF] Diver | Effect of tezepelumab on airway inflammatory cells, remodelling, and hyperresponsiveness in patients with moderate-to-severe uncontrolled asthma (CASCADE): a double-blind, randomised, placebo-controlled, phase 2 trial[END_REF][START_REF] Alpizar | Functionality and performance of an accessorized pre-filled syringe and an autoinjector for at-home administration of tezepelumab in patients with severe, uncontrolled asthma[END_REF][START_REF] Aguet | The GTEx Consortium atlas of genetic regulatory effects across human tissues[END_REF]. No patterns were identified in either the causes of the deaths or the relationship of the deaths to the study drug dosing. No deaths were considered to be causally related to tezepelumab by a masked independent adjudication committee (appendix pp 15-16). One death was considered causally related to tezepelumab by the investigator, but not by the adjudication committee, because of the individual's medical history. The individual had metastatic colon cancer diagnosed during the study but had a history of iron deficiency that was not fully evaluated with a colonoscopy before entry into the parent study.

During the on-treatment period, there were fewer serious adverse events categorised in the MedDRA respiratory, thoracic, and mediastinal disorder system organ class in the randomised tezepelumab group than in the randomised placebo group (incidence of 1•74 in the randomised tezepelumab group vs 6•29 in the randomised placebo group per 100 patient-years among those initially from NAVIGATOR; difference -4•55, 95% CI -6•73 to -2•68; and 2•32 in the randomised tezepelumab group vs 10•00 in the randomised placebo group per 100 patient-years among those initially from SOURCE; -7•68, -15•39 to -1•75; appendix pp [START_REF] Simpson | Tezepelumab, an anti-thymic stromal lymphopoietin monoclonal antibody, in the treatment of moderate to severe atopic dermatitis: a randomized phase 2a clinical trial[END_REF][18][START_REF] Iribarren | Cardiovascular and cerebrovascular events among patients receiving omalizumab: results from EXCELS, a prospective cohort study in moderate to severe asthma[END_REF]. This difference was largely due to fewer asthma exacerbations associated with hospitalisation or an emergency department visit in the randomised tezepelumab group than in the randomised placebo group.

The incidence of a serious adverse event categorised in the MedDRA cardiac disorder system organ class during the on-treatment period was higher in the randomised tezepelumab group than in the randomised placebo group (0•87 in the randomised tezepelumab group vs 0 in the randomised placebo group per 100 patient-years among those initially from NAVIGATOR; difference 0•87, 95% CI 0•32 to 1•71; 3•09 in the randomised tezepelumab group vs 0 in the randomised placebo group per 100 patient-years among those initially from SOURCE; 3•09, -0•67 to 7•69; appendix pp [START_REF] Simpson | Tezepelumab, an anti-thymic stromal lymphopoietin monoclonal antibody, in the treatment of moderate to severe atopic dermatitis: a randomized phase 2a clinical trial[END_REF][18][START_REF] Iribarren | Cardiovascular and cerebrovascular events among patients receiving omalizumab: results from EXCELS, a prospective cohort study in moderate to severe asthma[END_REF][START_REF]Clinical review: mepolizumab[END_REF]. Including on-treatment events in the placebo then tezepelumab group, and on-study events in both treatment arms, the pooled on-study incidence of serious adverse events categorised as cardiac disorders were 1•30 in the all tezepelumab group versus 0•23 in the randomised placebo group per 100 patient-years (appendix pp [START_REF]Clinical review: mepolizumab[END_REF][START_REF] Panettieri | Tralokinumab for severe, uncontrolled asthma (STRATOS 1 and STRATOS 2): two randomised, double-blind, placebocontrolled, phase 3 clinical trials[END_REF][START_REF]Approval letter: dupilumab subcutaneous injection[END_REF][START_REF] Iribarren | Adult asthma and risk of coronary heart disease, cerebrovascular disease, and heart failure: a prospective study of 2 matched cohorts[END_REF][START_REF] Cepelis | Asthma, asthma control and risk of acute myocardial infarction: HUNT study[END_REF]. No pattern in either the cause of the cardiac serious adverse events or the timing of the cardiac serious adverse events in relation to study drug administration was identified. All participants with a cardiac disorder serious adverse event had at least two risk factors at baseline and 44% (eight of 18) had a cardiac disorder that might have contributed to these events (appendix p 25). Neither investigators nor a masked independent adjudication committee attributed causality to tezepelumab for any cardiac serious adverse event (appendix pp 23-24). Cardiac disorder system organ class adverse events occurred at similar rates in the randomised tezepelumab and randomised placebo group during the on-study period (appendix pp 26-28). Analysis of major adverse cardiovascular events by a blinded, independent adjudication committee was prespecified in the study protocol and prospectively evaluated. The incidence of major adverse cardiovascular events was similar between groups, with few events observed: 0⋅65 for the all tezepelumab group and 0⋅46 for the randomised placebo group per 100 patient-years (difference 0•19, 95% CI -0•58 to 0•85; appendix p 29). Despite no on-treatment serious adverse events within the cardiac disorder system organ class being reported among placebo recipients, there were four adjudicated major adverse cardiovascular events among placebo recipients: one cerebrovascular serious adverse event, one cardiovascular death that occurred during study follow-up, and two cardiovascular deaths that were originally reported to be of unknown cause.

The on-treatment incidence of serious adverse events in the infections and infestations system organ class was balanced between treatment groups (appendix pp [START_REF] Simpson | Tezepelumab, an anti-thymic stromal lymphopoietin monoclonal antibody, in the treatment of moderate to severe atopic dermatitis: a randomized phase 2a clinical trial[END_REF][18][START_REF] Iribarren | Cardiovascular and cerebrovascular events among patients receiving omalizumab: results from EXCELS, a prospective cohort study in moderate to severe asthma[END_REF]. A post-hoc analysis of COVID-19-related adverse events was done, adjusting only for exposure after the start of the pandemic (appendix p 37). No increase in rates of COVID-19-related adverse events, serious adverse events, and fatal adverse events with tezepelumab compared with placebo was observed (appendix p 30); fatal COVID-19 adverse events occurred in two (<1%) of 602 participants in the tezepelumab group and one (<1%) of 607 participants in the placebo group. There were similar rates of on-treatment pneumonia serious adverse events in the randomised tezepelumab group and the randomised placebo group (0•76 in the randomised tezepelumab group vs 1•29 in the randomised placebo group per 100 patient-years among those initially in NAVIGATOR, difference -0•52, 95% CI -1•74 to 0•47; and 3•09 in the randomised tezepelumab group vs 2•00 in the randomised placebo group per 100 patientyears among those initially from SOURCE, 1•09, -4•23 to 5•99).

The on-treatment incidence of injection-site reactions was low and similar in the randomised tezepelumab and randomised placebo groups (2•40 in the randomised tezepelumab group vs 2•15 in the randomised placebo group per 100 patient-years among those initially in NAVIGATOR, difference 0•25, 95% CI -1•33 to 1•73; and 0•00 in the randomised tezepelumab group vs 1•00 in the randomised placebo group per 100 patient-years among those initially in SOURCE, -1•00, -5•46 to 1•91). On-treatment malignancies were balanced between treatment groups (appendix p 31). Among participants initially from NAVIGATOR, the on-treatment incidence of hypersensitivity serious adverse events was 0•33 in the randomised tezepelumab group and 0•29 in the randomised placebo groups per 100 patient-years (0•04, -0•74 to 0•71); there were no hypersensitivity serious adverse events among those from SOURCE. On-treatment hypersensitivity was reported by three individuals in the randomised tezepelumab group (ie, immune thrombocytopenia, drug hypersensitivity, and contact dermatitis) and two individuals in the randomised placebo group (ie, anaphylactic reaction and rash). No cases of anaphylaxis were observed in the randomised tezepelumab group.

Tezepelumab reduced the annualised asthma exacerbation rate over 104 weeks compared with placebo. In participants initially from NAVIGATOR, the annualised asthma exacerbation rate over 104 weeks in the randomised tezepelumab group was 0•82 (95% CI 0•71-0•95) compared with 1•93 (1•70-2•20) in the randomised placebo group (rate ratio 0•42, 95% CI 0•35-0•51; figure 2A). In participants initially from SOURCE, the annualised asthma exacerbation rate over 104 weeks in the randomised tezepelumab group was 1•07 (0•76-1•51) compared with 1•76 (1•27-2•45) in the randomised placebo group (rate ratio 0•61, 0•38-0•96; figure 2B). Findings were similar in participants initially from NAVIGATOR and SOURCE in the supportive long-term extension analysis set (figure 2C and2D). Across participants from both parent studies, tezepelumab reduced the annualised rate of asthma exacerbations that were associated with hospitalisation or an emergency department visit over 104 weeks compared with placebo (figure 2E-H).

Figure 2: AAER and AAER associated with hospitalisation or emergency department visit over 104 weeks

Adjusted AAER from the primary full analysis dataset in participants initially from NAVIGATOR (A) and SOURCE (B); adjusted AAER from the supportive long-term extension analysis dataset in participants initially from NAVIGATOR (C) and SOURCE (D); adjusted AAER associated with hospitalisation or an emergency department visit from the primary full analysis dataset in participants initially from NAVIGATOR (E) and SOURCE (F); adjusted AAER associated with hospitalisation or an emergency department visit from the supportive long-term extension analysis dataset in participants initially from NAVIGATOR (G) and SOURCE (H). AAER=annualised asthma exacerbation rate. RR=rate ratio.

In participants initially from NAVIGATOR, the annualised asthma exacerbation rate was consistently lower in the randomised tezepelumab group than in the randomised placebo group, irrespective of baseline inflammatory biomarkers and other clinical characteristics over 104 weeks (figure 3); time-to-first asthma exacerbation in these individuals was longer in the randomised tezepelumab group than in the randomised placebo group (hazard ratio 0•64, 95% CI 0•54-0•75; appendix p 38). Asthma exacerbations, including those associated with hospitalisation or an emergency department visit, were lower in the first year than those recorded in the 12 months before study entry across both treatment arms, with greater reductions in asthma exacerbations observed in the tezepelumab only group than in the placebo only group (appendix pp 39-41). During the second year, which included the beginning of the COVID-19 pandemic, asthma exacerbations were further reduced, with lower rates observed in the tezepelumab only group than in the placebo only group. Improvements in prebronchodilator FEV1, ACQ-6 score, and SGRQ score were sustained throughout the treatment period in the randomised tezepelumab group compared with the randomised placebo group in participants from both parent studies (appendix p 32; figure 4A-F). Results were similar in the supportive long-term extension analysis set (appendix p 42).

Reductions from baseline in blood eosinophil counts and FeNO concentrations were seen early and were sustained to week 104 in the randomised tezepelumab group (appendix pp 43-45). There was a progressive reduction in serum total IgE concentrations to week 104 in the randomised tezepelumab group (appendix pp 43-45). Results were similar in the supportive long-term extension analysis set (appendix p 33).

Results among the placebo then tezepelumab group were similar to the placebo only group during the parent study and similar to the tezepelumab only group during the second year (appendix pp 33, 39-42).

Discussion

Tezepelumab was well tolerated in individuals with severe, uncontrolled asthma over 104 weeks of treatment. The exposure-adjusted incidence of adverse events and serious adverse events was lower with tezepelumab than with placebo. These findings following long-term exposure to tezepelumab are consistent with the favourable safety profile found in previous studies.5,7-9,14,15 DESTINATION also further confirmed the long-term benefits of tezepelumab in reducing exacerbations and improving lung function, asthma control, and healthrelated quality of life.

Within individual MedDRA-coded categories of serious adverse events, there was a lower incidence of respiratory, thoracic, and mediastinal disorder system organ class serious adverse events in the randomised tezepelumab group than in the randomised placebo group, but there was a higher incidence of cardiac disorder system organ class serious adverse events in participants receiving tezepelumab than in those receiving placebo. The reason for this imbalance in cardiac serious adverse events is not understood. There is no known biological mechanism by which blocking TSLP with tezepelumab would lead to cardiac pathophysiology, and the very low expression of TSLP and TSLP receptor mRNA in cardiac tissue suggests that signalling via the TSLP receptor pathway in these tissues is unlikely. [START_REF] Aguet | The GTEx Consortium atlas of genetic regulatory effects across human tissues[END_REF] The incidence of cardiac disorder adverse events was similar between tezepelumab and placebo recipients, with very few events independently adjudicated as major adverse cardiovascular events or cardiovascular deaths observed. No patterns were identified in the causes of cardiac serious adverse events among tezepelumab-treated individuals or in the timing of the cardiac serious adverse events in relation to tezepelumab dosing. Additionally, no cardiac serious adverse event was causally attributed to tezepelumab by either the investigator or a masked independent adjudication committee. All participants who had a cardiac disorder serious adverse event had at least two risk factors at baseline and 44% had a cardiac disorder at baseline. Imbalances in cardiac serious adverse events have not been observed in previous multidose studies of tezepelumab of up to 1 year of treatment.5,7,8,14,15,17 No causal relationship between tezepelumab and these events has been established, nor has a patient population at risk of these events been identified.

The incidence of cardiac serious adverse events in tezepelumab-treated participants in DESTINATION is similar to those estimated from publicly available data of other biologics for severe asthma.18-21 In the phase 3 Liberty Asthma QUEST study, a similar imbalance was observed, with cardiac serious adverse events reported in 14 dupilumab recipients but no placebo recipients, with similar rates of major adverse cardiovascular events and cardiovascular deaths between treatment groups. [START_REF]Approval letter: dupilumab subcutaneous injection[END_REF] The incidence of cardiac serious adverse events in the placebo group in DESTINATION was substantially lower than the incidence in studies of other biologics for severe asthma.18-21 Asthma is associated with an increased risk of cardiovascular disorders, with hazard ratios for individuals with asthma reported to be between 1•1 and 3•2 times that of an individual without asthma. [START_REF] Iribarren | Adult asthma and risk of coronary heart disease, cerebrovascular disease, and heart failure: a prospective study of 2 matched cohorts[END_REF][START_REF] Cepelis | Asthma, asthma control and risk of acute myocardial infarction: HUNT study[END_REF][START_REF] Cepelis | Associations of asthma and asthma control with atrial fibrillation risk: results from the Nord-Trondelag Health Study (HUNT)[END_REF] In these epidemiological studies, as in DESTINATION, the increased risk encompassed many different types of cardiovascular disorders.

The incidence of severe infection, including pneumonia, was similar between treatment groups, which suggests that tezepelumab's mechanism of action does not predispose users to infections. A similar finding was observed with malignancies. Tezepelumab recipients group did not have a higher rate of COVID-19-related infections or deaths than placebo recipients. The incidence of injection-site reactions with tezepelumab administration was low and similar to that of placebo. No cases of anaphylaxis related to tezepelumab were reported.

Tezepelumab treatment resulted in clinically meaningful reductions in asthma exacerbations compared with placebo over 2 years. Previous studies of biologics for severe asthma have also shown a reduction in exacerbation rates over time. However, these studies lacked a placebo control group, making these changes difficult to interpret.2-4,26 Asthma exacerbation rates in the second year were in part reduced due to the COVID-19 pandemic and related social distancing.27 Nevertheless, exacerbations and hospitalisations were reduced in the tezepelumab group compared with placebo during the second year.

Reductions in exacerbations occurred irrespective of baseline levels of inflammatory biomarkers and other clinical characteristics. The magnitude of reductions in exacerbations versus placebo was greater among those with high levels of type 2 inflammatory biomarkers or with nasal polyps compared with those with low levels of type 2 inflammatory biomarkers or without nasal polyps. This difference confirms findings from PATHWAY and NAVIGATOR5,7 that tezepelumab consistently shows clinically meaningful reductions in exacerbations in users across the spectrum of type 2 inflammation, with an enhanced effect in individuals with high type 2 biomarker levels. Although clinically meaningful reductions in exacerbations were observed in adolescents, greater reductions were seen in adults. The estimate of efficacy among adolescent participants is imprecise due to the small number of adolescents and the low exacerbation rate among adolescents in the placebo group.

Improvements from baseline in pre-bronchodilator FEV1, ACQ-6 score, and SGRQ score were observed in the tezepelumab group as early as the first post-baseline assessment and were maintained up to week 104. Rapid and sustained reductions in blood eosinophil counts and FeNO concentrations were observed in the tezepelumab group compared with the placebo group, along with a progressive reduction in serum total IgE concentrations through week 104. Similar findings were seen in participants who switched from placebo in the parent study to tezepelumab in DESTINATION.

A strength of DESTINATION is the large number of patient-years of exposure to tezepelumab. The robustness of the dataset is shown by high roll-over rates of eligible individuals from the parent studies, low discontinuation rates within DESTINATION, and high treatment adherence. The data robustness allowed DESTINATION to rigorously address the question regarding the safety and efficacy of tezepelumab in patients treated for up to 2 years. DESTINATION also included individuals receiving tezepelumab during the COVID-19 pandemic: no evidence of an increased risk of severe COVID-19 disease was identified, while continued efficacy was observed despite a reduced incidence of exacerbations due to social distancing and other behavioural changes.

A limitation of DESTINATION was the imbalance in exposure between tezepelumab and placebo during the long-term extension period. Although the analysis accounted for these differences in exposure, the model assumed a constant risk through the observation periods, which might not have been true for some adverse events. The overall environment for the study, including non-confirmed COVID-19 illness, societal and healthcare disruptions, and participant stress during the pandemic, was not consistent between years 1 and 2, and this might have had unrecognised consequences on the incidence of safety events. The safety and effectiveness of tezepelumab will continue to be assessed in ongoing and future studies. In conclusion, DESTINATION showed that tezepelumab treatment was well tolerated for up to 2 years and resulted in sustained, clinically meaningful reductions in asthma exacerbations, with improved lung function, asthma control, and health-related quality of life in individuals with severe, uncontrolled asthma, consistent with results from NAVIGATOR and SOURCE. The incidence of adverse events was similar in the second year of treatment compared with the first, and the efficacy of tezepelumab was sustained over 2 years. These findings further show the safety and efficacy of tezepelumab in this population.

Contributors

All authors contributed to the study design, data interpretation, and drafting of the manuscript with support from a medical writer funded by the sponsors. All authors provided critical feedback and final approval for submission and vouch for the completeness and accuracy of the data and analyses and the adherence of the trial to the protocol. All authors had full access to and verified the data in the study and had final responsibility for the decision to submit for publication.

Declaration of interests

AM-G has attended advisory board meetings for AstraZeneca, GlaxoSmithKline, Novartis, Regeneron, Sanofi, and Teva Pharmaceuticals; has received speaker fees from AstraZeneca, Novartis, Sanofi, and Teva Pharmaceuticals; has participated in research with AstraZeneca, for which his institution has been remunerated; has attended international conferences with Teva Pharmaceuticals; and has consultancy agreements with AstraZeneca and Sanofi. MEW is an employee of National Jewish Health and has received consultancy fees from AstraZeneca, Equillium, Genentech, GlaxoSmithKline, Novartis, Regeneron Pharmaceuticals, resTORbio, Sanofi, and Teva Pharmaceuticals. CEB has received consultancy fees and grants from 4D Pharma, AstraZeneca, Chiesi, Genentech, GlaxoSmithKline, Mologic, Novartis, Regeneron, Roche, and Sanofi. SK has received fees for lectures or advisory board meetings from AstraZeneca, GlaxoSmithKline, Novartis, Roche, Sanofi Aventis, and Teva Pharmaceuticals. JC has received grants and personal fees from AstraZeneca, Genentech, and Vectura and has received grants from Optinose, Sanofi, and Teva Pharmaceuticals. EI has served as a consultant to and received personal fees from 4D Pharma, AB Science, Amgen, AstraZeneca, Avillion, Biometry, Cowen, Equillium, Genentech, GlaxoSmithKline, Merck, Novartis, Pneuma Respiratory, PPS Healthcare, Regeneron Pharmaceuticals, Sanofi, Sienna Biopharmaceuticals, and Teva Pharmaceuticals; has received non-financial support from Circassia, Teva Pharmaceuticals, and Vorso Corp; and has received clinical research grants from AstraZeneca, Avillion, Genentech, Gossamer Bio, Novartis, and Sanofi. GCh has received speaker and consultancy fees from Amgen, AstraZeneca, Boehringer Ingelheim, Genentech, GlaxoSmithKline, Novartis, Regeneron Pharmaceuticals, Sanofi Genzyme, and Teva Pharmaceuticals. AB, SP, GA, MG, KB, and GCo are employees of AstraZeneca and might own stock or stock options in AstraZeneca. SC is an employee of Amgen and owns stock in Amgen. LS is a consultant to AstraZeneca. KL is an employee of Cytel.

Acknowledgments

This study was funded by AstraZeneca and Amgen. We thank the study participants and their families, the investigators, and the site staff. Medical writing support was provided by Madeleine Wynn of PharmaGenesis London, London, UK, with funding from AstraZeneca and Amgen.

Funding:

AstraZeneca and Amgen.

Data sharing

Data underlying the findings described in this manuscript can be obtained in accordance with AstraZeneca's data sharing policy described at: https://astrazenecagrouptrials.pharmacm.com/ST/Submission/ Disclosure.

Conflict of interest statement

Declaration of interests AM-G has attended advisory board meetings for AstraZeneca, GlaxoSmithKline, Novartis, Regeneron, Sanofi, and Teva Pharmaceuticals; has received speaker fees from AstraZeneca, Novartis, Sanofi, and Teva Pharmaceuticals; has participated in research with AstraZeneca, for which his institution has been remunerated; has attended international conferences with Teva Pharmaceuticals; and has consultancy agreements with AstraZeneca and Sanofi. MEW is an employee of National Jewish Health and has received consultancy fees from AstraZeneca, Equillium, Genentech, GlaxoSmithKline, Novartis, Regeneron Pharmaceuticals, resTORbio, Sanofi, and Teva Pharmaceuticals. CEB has received consultancy fees and grants from 4D Pharma, AstraZeneca, Chiesi, Genentech, GlaxoSmithKline, Mologic, Novartis, Regeneron, Roche, and Sanofi. SK has received fees for lectures or advisory board meetings from AstraZeneca, GlaxoSmithKline, Novartis, Roche, Sanofi Aventis, and Teva Pharmaceuticals. JC has received grants and personal fees from AstraZeneca, Genentech, and Vectura and has received grants from Optinose, Sanofi, and Teva Pharmaceuticals. EI has served as a consultant to and received personal fees from 4D Pharma, AB Science, Amgen, AstraZeneca, Avillion, Biometry, Cowen, Equillium, Genentech, GlaxoSmithKline, Merck, Novartis, Pneuma Respiratory, PPS Healthcare, Regeneron Pharmaceuticals, Sanofi, Sienna Biopharmaceuticals, and Teva Pharmaceuticals; has received non-financial support from Circassia, Teva Pharmaceuticals, and Vorso Corp; and has received clinical research grants from AstraZeneca, Avillion, Genentech, Gossamer Bio, Novartis, and Sanofi. GCh has received speaker and consultancy fees from Amgen, AstraZeneca, Boehringer Ingelheim, Genentech, GlaxoSmithKline, Novartis, Regeneron Pharmaceuticals, Sanofi Genzyme, and Teva Pharmaceuticals. AB, SP, GA, MG, KB, and GCo are employees of AstraZeneca and might own stock or stock options in AstraZeneca. SC is an employee of Amgen and owns stock in Amgen. LS is a consultant to AstraZeneca. KL is an employee of Cytel.


Louison Dufour

Laurence Garczarek

Bastian Gouriou

Julia Clairet

Morgane Ratin

Frédéric Partensky
email: frederic.partensky@sb-roscoff.fr

Beatriz Roncero Ramos

Yuu Hirose

Lisa Wiltbank

Differential acclimation kinetics of the two forms of type IV chromatic acclimaters occurring in marine Synechococcus cyanobacteria

Keywords: marine picocyanobacteria, Synechococcus, chromatic acclimation, spectral niche, phycobilisome, comparative physiology

Synechococcus, the second most abundant marine phytoplanktonic organism, displays the widest variety of pigment content of all marine oxyphototrophs, explaining its ability to colonize all spectral niches occurring in the upper lit layer of oceans. Seven Synechococcus pigment types (PTs) have been described so far based on the phycobiliprotein composition and chromophorylation of their light-harvesting complexes, called phycobilisomes. The most elaborate and abundant PT (3d) in the open ocean consists of cells capable of type IV chromatic acclimation (CA4), i.e., to reversibly modify the ratio of the blue lightabsorbing phycourobilin (PUB) to the green light-absorbing phycoerythrobilin (PEB) in phycobilisome rods to match the ambient light color. Two genetically distinct types of chromatic acclimaters, so-called PTs 3dA and 3dB, occur at similar global abundance in the ocean, but the precise physiological differences between these two types and the reasons for their complementary niche partitioning in the field remain obscure. Here, photoacclimation experiments in different mixes of blue and green light of representatives of these two PTs demonstrated that they differ by the ratio of blue-to-green light required to trigger the CA4 process. Furthermore, shift experiments between 100% blue and 100% green light, and vice-versa, revealed significant discrepancies between the acclimation pace of the two types of chromatic acclimaters. This study provides novel insights into the finely tuned adaptation mechanisms used by Synechococcus cells to colonize the whole underwater light field.

Introduction

Phytoplanktonic cells have an obligate requirement for light to perform photosynthesis. Yet in the marine environment, this energy source is highly variable quantitatively and qualitatively with depth but also along coast-offshore gradients, leaving aside daily oscillations [START_REF] Kirk | Light and photosynthesis in aquatic ecosystems[END_REF][START_REF] Holtrop | Vibrational modes of water predict spectral niches for photosynthesis in lakes and oceans[END_REF]. This variability has triggered an extensive structural and pigment diversification of phytoplankton light-harvesting antennae, which enable cells to considerably enhance the wavelength range they can collect and therefore the number of photons reaching photosystems. Despite their apparent simplicity compared to algae and higher plants, cyanobacteria possess the most elaborated form of antennae known in oxyphototrophs, called phycobilisomes (PBS). PBS are huge water-soluble complexes composed of six to eight rods radiating around a central core. Both core and rods are constituted of phycobiliproteins that bind openchain tetrapyrroles, called phycobilins [START_REF] Sidler | Phycobilisome and phycobiliprotein structures[END_REF]. While the PBS core is always made of allophycocyanin and is highly conserved, PBS rods display a very large structural flexibility since they can be made of phycocyanin (PC) only, or of PC and one or two phycoerythrin (PE) types, PE-I and PE-II [START_REF] Ong | Phycoerythrins of marine unicellular cyanobacteria. I. Bilin types and locations and energy transfer pathways in Synechococcus spp. phycoerythrins[END_REF][START_REF] Six | Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study[END_REF]. Additionally, each phycobiliprotein can bind up to three different kinds of phycobilins. The ultimate degree of sophistication is the capacity for some cyanobacterial cells to modify the composition of their PBS in response to changes in the ambient light color. This process called "chromatic acclimation" (CA)-the initial term was actually "chromatic adaptation, " but was recently replaced in order to best describe this physiological process [START_REF] Shukla | Phycoerythrin-specific Bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF]-was first observed in the early 20th century in freshwater cyanobacteria shifted from red to green light [START_REF] Engelmann | Über experimentelle Erzeugung zweckmässiger Änderungen der Färbung pflanzlicher Chromophylle durch farbiges Licht. Bericht über Versuche von N. Gaidukow[END_REF][START_REF] Gaidukov | Weitere Untersuchungen über den Einfluss farbigen Lichtes auf die Färbung der Oscillarien[END_REF]. CA was later on attributed to changes in the phycobiliprotein composition of PBS rods: in red light, rods are entirely composed of PC and cells look green, whereas in green light PC is restricted to the base of the rods and the distal part is made of PE, causing cells to exhibit a bright red color [START_REF] Boresch | Photokatalysen in Pflanzen[END_REF]. This type of complementary chromatic acclimation (CCA, also called type 3 chromatic acclimation or CA3) is one among the seven different types of CA known so far in cyanobacteria [START_REF] Tandeau De Marsac | Occurrence and nature of chromatic adaptation in cyanobacteria[END_REF][START_REF] Hirose | Diverse chromatic acclimation processes regulating phycoerythrocyanin and rod-shaped phycobilisome in cyanobacteria[END_REF]Sanfilippo et al., 2019a). While CA2-a simple type of CA where PE production is induced in green light, generating longer PBS rods, and repressed in red light-and CA3 occur mainly in freshwater and brackish cyanobacteria, CA4 is the main type occurring in the open ocean and is specific to marine Synechococcus cyanobacteria [START_REF] Palenik | Chromatic adaptation in marine Synechococcus strains[END_REF][START_REF] Everroad | Biochemical bases of type IV chromatic adaptation in marine Synechococcus spp[END_REF][START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. Contrary to CA2 and CA3, CA4 does not involve changes in the phycobiliprotein composition of PBS rods, but in their phycobilin composition. In response to shifts between green light (GL) and blue light (BL), chromatic acclimaters can indeed modify the relative amount of the two phycobilins bound to PE-I and PE-II in order to match the predominant ambient light color. More specifically, they exhibit in BL a high ratio of the BL-absorbing phycourobilin (PUB, λ max ≈ 495 nm) to the GL-absorbing phycoerythrobilin (PEB, λ max ≈ 545 nm), and viceversa in GL [START_REF] Everroad | Biochemical bases of type IV chromatic adaptation in marine Synechococcus spp[END_REF][START_REF] Shukla | Phycoerythrin-specific Bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF]. Variations in PUB and PEB cell content are generally assessed by measuring the relative ratio of whole cell fluorescence excitation at 495 and 545 nm (Exc 495:545 ) with emission set at 580 nm, which in chromatic acclimaters changes from 0.6-0.7 in GL to 1.6-1.7 in BL [START_REF] Palenik | Chromatic adaptation in marine Synechococcus strains[END_REF][START_REF] Everroad | Biochemical bases of type IV chromatic adaptation in marine Synechococcus spp[END_REF]. In the nomenclature of Synechococcus pigment types (PTs) established by [START_REF] Six | Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study[END_REF] and later modified by [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF], chromatic acclimaters are classified as "PT 3d" cells, meaning that they possess PBS rods made of PC, PE-I and PE-II, a feature shared by all PT 3 representatives, and can modify their Exc 495:545 ratio. In contrast, PTs 3a, 3b, and 3c display a constitutively low, medium and high Exc 495:545 ratio, respectively. For this reason, PT 3a strains are often referred to as "green light specialists" and PT 3c as "blue light specialists" [START_REF] Grébert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF][START_REF] Grébert | Diversity and evolution of pigment types in marine Synechococcus cyanobacteria[END_REF].

Two genetically different types of chromatic acclimaters have been described so far: PTs 3dA and 3dB [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. Both possess a small genomic island involved in the CA4 process, yet the CA4-A and CA4-B islands differ genetically and structurally [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]Sanfilippo et al., 2019b;[START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF]. Although long overlooked, CA4 appears to be an ecologically important process since chromatic acclimaters were shown to account for more than 40% of the whole marine Synechococcus population along the Tara oceans expedition transect [START_REF] Grébert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF]. Moreover, PTs 3dA and 3dB were found to be equally abundant (22.6% and 18.9%, respectively) but distributed in complementary ecological niches in the field. The former was indeed predominant in cold, nutrient-rich and highly productive waters at high latitude, as well as in other vertically mixed environments, while the latter was mostly found in nitrogen and phosphorus-poor oceanic areas and appeared to be more abundant at depth. The emergence and maintenance of two CA4 types over the course of evolution, as well as the differential distribution of PTs 3dA and 3dB in the environment [START_REF] Grébert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF], strongly suggest that they may not be as phenotypically equivalent as previously thought [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. To check this hypothesis, we acclimated three representatives of each PT 3dA and 3dB in batch culture under two conditions of temperature, two light irradiances and five light colors in order to compare their growth rates and PBS properties. Furthermore, we performed shifts from BL to GL (and vice-versa) at two irradiances to compare the CA4 kinetics between five strains of each PT. These experiments demonstrated that PT 3dA and 3dB strains actually differ in the blue-to-green light ratio necessary to trigger the CA4 process, and revealed some significant discrepancies in their acclimation pace.

Materials and methods

Biological material and culture conditions

Ten Synechococcus strains, of which five PT 3dA and five PT 3dB representatives (Table 1), were retrieved from the Roscoff Culture Collection.1 These strains were isolated from diverse environments and selected based on their genome availability [START_REF] Doré | Evolutionary mechanisms of long-term genome diversification associated with niche partitioning in marine picocyanobacteria[END_REF] and clade affiliation, in order to include the CA4-A model strain RS9916 [START_REF] Shukla | Phycoerythrin-specific Bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF][START_REF] Sanfilippo | Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus[END_REF]Sanfilippo et al., , 2019b) ) as well as representatives from all five major clades (I to IV and CRD1) in the global ocean [START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF].

Cells were grown in 50 mL polystyrene flasks (Sarstedt, Germany) in PCR-S11 medium [START_REF] Rippka | Prochlorococcus marinus Chisholm et al. 1992 subsp. pastoris subsp. nov. strain PCC 9511, the first axenic chlorophyll a 2 /b 2 -containing cyanobacterium (Oxyphotobacteria)[END_REF] supplemented with 1 mM sodium nitrate. All were pre-acclimated prior to measurements for at least 3 weeks in continuous light provided by blue and/or green LEDs (Alpheus, France) in temperature-controlled chambers.

Acclimation experiments

A selection of six out of the 10 abovementioned strains (BL107, RS9916 and WH8020 for PT 3dA and A15-62, PROS-U-1 and RS9915 for PT 3dB; Table 1) were grown in the following conditions: (i) two temperatures: 18°C and 25°C; (ii) two irradiances: low light (LL, 10.3389/fmicb.2024.1349322 Frontiers in Microbiology 03 frontiersin.org 15 μmol photons m -2 s -1 ) and high light (HL, 75 μmol photons m -2 s -1 ); (iii) five light qualities: blue light (100% BL), green light (100% GL), as well as three mixes of blue-green light: 25% BL-75% GL, 50% BL-50% GL and 75% BL-25% GL. Both temperatures were selected based on [START_REF] Ferrieux | Comparative thermophysiology of marine Synechococcus CRD1 strains isolated from different thermal niches in iron-depleted areas[END_REF], which recently demonstrated that 18 and 25°C were the lowest and highest temperatures at which a selection of Synechococcus strains belonging to the five major clades in the environment were capable of growing. The two irradiances were chosen as being similar to those used in a previous study by [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF], for easier comparison of results between the two studies.

The light intensity and visible spectra of LEDs were measured using a PG200N Spectral PAR Meter (UPRtek, Taiwan; Supplementary Figure S1). Each strain was grown in triplicate and inoculated at an initial cell density of 3 × 10 6 cells mL -1 . Samples were harvested every day to measure cell concentration and fluorescence parameters by flow cytometry, and once during the exponential phase to measure phycobilin and phycobiliprotein contents by spectrofluorimetry (see below).

Shift experiments

Shift experiments between 100% low BL (LBL) and 100% low GL (LGL) and vice-versa, and between 100% high BL (HBL) and 100% high GL (HGL) and vice-versa, were performed on all 10 Synechococcus strains mentioned in Table 1, but only at 25°C. Each strain was diluted with fresh medium before the beginning of the experiments and regularly transferred in order to avoid limitation by nutrients. Aliquots were collected two to three times a day, depending on light intensity, to measure phycobilin content by spectrofluorimetry (see below).

Flow cytometry

Culture aliquots were sampled twice a day, fixed with 0.25% (v/v) glutaraldehyde (grade II, Sigma Aldrich, United States) and stored at -80°C until analysis [START_REF] Marie | Enumeration of phytoplankton, bacteria, and viruses in marine samples[END_REF]. Cell density was determined using a Guava easyCyte flow cytometer equipped with a 488 nm laser and the Guavasoft software (Luminex Corporation, Texas). Average orange (583 nm) and red (695 nm) fluorescence signals were used as proxies of the phycoerythrin (PE) and chlorophyll a (Chl a) contents per cell, respectively. Both signals were normalized to that of standard fluorescent 0.95 μm silica beads.

Spectrofluorimetry

In vivo fluorescence spectra were recorded at 240 nm min -1 with slits fixed at 10 nm once during the exponential phase using a spectrofluorimeter FL6500 (Perkin-Elmer, United-States). Excitation spectra were acquired between 450 and 560 nm with emission set at 580 nm, corresponding to the PE emission maximum. Emission spectra were recorded between 550 and 750 nm with excitation set at 530 nm, close to the PEB excitation maximum. Spectra were monitored and analyzed with the Fluorescence software (Perkin-Elmer). The Exc 495:545 fluorescence excitation ratio was used as a proxy for the PUB:PEB ratio. The Em 560:650 and Em 650:680 fluorescence emission ratios were used as proxies of the PE to PC and PC to PBS terminal acceptor (TA) ratios, respectively. The first parameter provided information about the electron transfer efficiency within the PBS and/or the length of PBS rods, and the second one about the coupling of PBS to PSII reaction center chlorophylls.

Statistical analyses

All statistical analyses were conducted using the R software (version 4.2.3; R Core Team, 2021) in order to test for significant differences between PTs 3dA and 3dB. The potential influence of growing conditions (temperature, light quality and quantity) was also investigated.

For acclimation experiments, a linear mixed model (nlme package version 3.1-164; [START_REF] Pinheiro | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF] was fit to each variable (growth rate, flow cytometry fluorescence signals, fluorescence excitation and emission ratios), by considering the temperature, light intensity, light color and pigment type as fixed factors, and the strain as a random factor. For shift experiments, the slopes of the linear parts of Exc 495:545 vs. time curves were compared between PTs 3dA and 3dB representatives using t-tests. As the number of strains used for shift experiments was reduced (n = 3 for PT 3dA and n = 5 for PT 3dB), the significance level was raised to 0.1 in order to confer more power to statistical analyses.

Results

Acclimation experiments

A first set of experiments was performed to investigate the effect of temperature, light intensity and color on various physiological characteristics including growth rate, flow cytometric red and orange fluorescence signals, as well as phycobilin and phycobiliprotein content, of six Synechococcus strains (BL107, RS9916 and WH8020 for PT 3dA and A15-62, PROS-U-1 and RS9915 for PT 3dB; Table 1) pre-acclimated for at least 3 weeks to the different tested conditions.

Phycobilin content

All strains globally displayed Exc 495:545 fluorescence excitation ratio, a proxy of the whole cell PUB:PEB ratio, typical of chromatic acclimaters when grown in 100% GL (Exc 495:545 ≈ 0.6-0.7) or 100% BL (Exc 495:545 ≈ 1.6-1.7; [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]; Supplementary Figure S5). It is important to note that the LEDs used to get the 100% GL condition actually peaked at 515 nm, which is at the blue edge of the green wavelength range (Supplementary Figure S1). Yet, the fact that all tested strains exhibited the lowest possible Exc 495:545 ratio for chromatic acclimaters shows that they did sense this light quality as being full GL.

The light quality had the strongest impact on the Exc 495:545 fluorescence excitation ratio (p-value <0.05; Supplementary Table S1), the latter expectedly decreasing from 100% BL to 100% GL. Interestingly, the two PTs did not respond in the same way to the light color (p-value <0.05; Supplementary Table S1). PT 3dB representatives indeed exhibited higher Exc 495:545 ratios than their PT 3dA counterparts in most intermediate blue-green conditions (Figure 4), due to a more progressive decrease of their ratio from the BL-to the GL-acclimated state. This trend was more pronounced in LL than HL conditions, as confirmed by the significant interaction effect between PT and light quantity (p-value <0.05; Supplementary Table S1).

Phycobiliprotein content

PT 3dA and 3dB representatives exhibited different patterns of variation with light color of the Em 560:650 fluorescence emission ratio, a proxy of the whole cell PE:PC ratio (p-value <0.05; Figure 5; Supplementary Figure S6; Supplementary Table S1). The Em 560:650 ratio was indeed generally higher in PT 3dA than PT 3dB strains and often decreased from 100% BL to 100% GL in the former, while it remained fairly stable whatever the light quality in the latter. Although less obvious, the mixed model demonstrated that the Em 560:650 fluorescence emission ratio was also significantly influenced by the temperature and light intensity (p-value <0.05; Supplementary Table S1).

The Em 650:680 fluorescence emission ratio, a proxy of the whole cell PC:TA ratio, was only impacted by the temperature factor, which was found to interact with the PT (p-value <0.05; Supplementary Table S1). PT 3dA representatives were indeed able to achieve higher values than PT 3dB strains in all conditions, but the difference was even greater at 25°C (Figure 6; Supplementary Figure S7). Moreover, the PTs were not similarly affected by the light quantity (p-value <0.05; Supplementary Table S1) as increasing irradiance seemed to induce an increase in the Em 650:680 ratio in PT 3dA strains and a decrease in PT 3dB cells.

Shift experiments

A second set of experiments consisted in studying the CA4 kinetics following shifts from BL to GL, and vice-versa, for 10 Synechococcus strains (BIOS-U3-1, BL107, MITS9220, RS9916 and WH8020 for PT 3dA and A15-62, A18-40, MINOS11, PROS-U-1 and RS9915 for PT 3dB; Table 1) pre-acclimated for at least 3 weeks to either LL or HL at 25°C. As expected, all strains exhibited slower chromatic acclimation kinetics in LL than HL, regardless of the initial light color, since the CA4 process took about 7 days in the former condition and 4 days in the latter.

Consistent with a previous study [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF], the BIOS-U3-1 strain could not fully acclimate to HBL, never exceeding an Exc 495:545 ratio of about 1.2, and the same observation was made for the other representative of the CRD1 clade, MITS9220. We therefore excluded these two strains before comparing the kinetics of Exc 495:545 variations between PTs. To do so, we compared the slopes of linear regressions computed from the linear parts of the kinetics between the shift time (T 0 ) and the time needed for the cells to reach a plateau (Figure 7; 50 h for HGL to HBL, 75 h for HBL to HGL, 125 h for both LL shifts). These slopes (Supplementary Figures S8A-D), which reflected the rate of Exc 495:545 variation over time, were then compared between PTs (Supplementary Figures S9A,B). This comparison demonstrated that the rate of Exc 495:545 variation was significantly higher for PT 3dB than PT 3dA cells from HBL to HGL (p-value <0.1; Supplementary Figure S9A). Conversely, PT 3dA strains displayed a significantly faster acclimation pace than their PT 3dB counterparts after the shifts from LGL to LBL and HGL to HBL (p-value <0.1; Supplementary Figure S9B).

Discussion

The marine environment was recently shown to shelter five distinct spectral niches, based on the absorption properties of water molecules as well as the variable concentrations of colored dissolved organic matter and non-algal particles [START_REF] Holtrop | Vibrational modes of water predict spectral niches for photosynthesis in lakes and oceans[END_REF]. Thanks to their specific antenna complexes binding divinyl derivatives of Chl a and b [START_REF] Goericke | The pigments of Prochlorococcus marinus: the presence of divinyl-chlorophyll a and b in a marine procaryote[END_REF], cells of the tiny cyanobacterium Prochlorococcus appear to be well adapted to the violet niche (401-449 nm), which encompasses the central oceanic gyres. In contrast, Synechococcus BL specialists (PT 3c), i.e., cells possessing a high content in PUB (λ max ≈ 495 nm), are best suited for the blue niche (449-514 nm) that comprises most other open ocean zones. As concerns Synechococcus GL specialists (PT 3a), i.e., cells possessing a high content in PEB (λ max ≈ 545 nm), they preferentially thrive in the green niche (514-605 nm) that is essentially found in coastal and upwelling areas [START_REF] Holtrop | Vibrational modes of water predict spectral niches for photosynthesis in lakes and oceans[END_REF]. In this context, Synechococcus cells capable of CA4, i.e., to match their Exc 495:545 ratio to the ambient light color in order to optimize photon collection, expectedly colonize both blue and green niches, where they can constitute a large part of the whole Synechococcus population, especially at high latitude [START_REF] Xia | Phylogeography and pigment type diversity of Synechococcus cyanobacteria in surface waters of the northwestern Pacific Ocean[END_REF]Grébert [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF][START_REF] Grébert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF][START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF][START_REF] Grébert | Diversity and evolution of pigment types in marine Synechococcus cyanobacteria[END_REF]) however made us wonder whether they displayed phenotypic differences that may partly explain their different spatial distributions.

Here, we looked at the interplay between light quality, light quantity and temperature, and we managed to unveil subtle but significant differences between PTs 3dA and 3dB. Comparisons of cultures acclimated to various light colors ranging from 100% BL to 100% GL at two temperatures and two irradiance levels revealed that, in intermediate blue-green light conditions, PT 3dB strains displayed significantly higher Exc 495:545 ratios than their PT 3dA counterparts (Figure 4). The observed differences are notably due to a more progressive decrease of the Exc 495:545 ratio for PT 3dB than PT 3dA representatives from BL to GL. The latter were indeed more frequently found in the GL-acclimated state with some of them, such as RS9916 in most conditions, even shifting their Exc 495:545 ratio to the BL-acclimated state only in 100% BL (Supplementary Figure S5). In contrast, PT 3dB cells tended to remain longer in the BL-acclimated state, even when the proportion of GL in the incident light was important, an extreme case being A15-62 at 25°C and LL that shifted to the GL-acclimated state only in 100% GL. Based on the phenotypes of knock-out mutants of genes involved in the CA4 process, it has been previously hypothesized that the PT 3dA genotype may have derived from a former GL specialist having acquired the CA4 capacity by integrating a CA4-A island, while the PT 3dB genotype may have been derived from a former BL specialist having integrated a CA4-B island (Sanfilippo et al., 2019b;[START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF]. Interestingly, our results are in good agreement with this hypothesis since they suggest that PT 3dA strains need a large proportion of blue photons to induce the CA4-A response, while on the contrary PT 3dB cells require a larger proportion of green photons to induce the CA4-B response. In other words, the two PTs seemingly differ in the blue-to-green ratio necessary to trigger the CA4 process, even though there is some strain-to-strain variability.

The molecular basis of the difference in Exc 495:545 ratio between chromatic acclimaters fully acclimated to either 100% BL or 100% GL has been well documented, and was found to be the same in the PT 3dA model strain RS9916 [START_REF] Shukla | Phycoerythrin-specific Bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF][START_REF] Sanfilippo | Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus[END_REF]Sanfilippo et al., , 2019b) ) and the PT 3dB model strain A15-62 [START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF]. Both CA4-A and -B processes indeed consist in an exchange of one out of the five phycobilins bound to the α-PE-I subunit (Cys-139) and two out of the six phycobilins bound to the α-PE-II subunit (Cys-83 and Cys-140), with PUB molecules being bound to these three positions in BL, and PEB in GL. However, the observation of intermediate Exc 495:545 ratios in blue-green light mixes (Figure 4; see also Sanfilippo et al., 2019b) remains difficult to interpret as it may translate different, but not mutually exclusive, sources of variability. More precisely, this observation may be explained by: (i) heterogeneous populations of Synechococcus cells with PBS either fully acclimated to BL (PUB-rich) or to GL (PEB-rich); (ii) Unfortunately, there is currently no simple experimental way to demonstrate which of these hypotheses is most likely.

In contrast to Exc 495:545 ratios, light quality had no significant effect on growth rates, while the latter varied with PT, temperature and light quantity. In HL, the growth rates of all six chromatic acclimaters used for acclimation experiments were significantly higher at 25°C than 18°C, the former being an optimal growth temperature for most marine Synechococcus strains tested so far (Figure 1; [START_REF] Mackey | Effect of temperature on photosynthesis and growth in marine Synechococcus spp[END_REF]Pittera et al., 2014;[START_REF] Breton | Unveiling membrane thermoregulation strategies in marine picocyanobacteria[END_REF][START_REF] Doré | Global phylogeography of marine Synechococcus in coastal areas reveals strong community shifts[END_REF][START_REF] Ferrieux | Comparative thermophysiology of marine Synechococcus CRD1 strains isolated from different thermal niches in iron-depleted areas[END_REF]. The impact of increasing temperature on growth rate was however much less important at LL, confirming that temperature and irradiance have a synergistic effect on growth, as previously observed in the model strain WH7803 [START_REF] Guyet | Synergic effects of temperature and irradiance on the physiology of the marine Synechococcus strain WH7803[END_REF]. Of note, whatever the temperature, all cells were also able of typical photoacclimation, i.e., to adjust the surface of their photosynthetic membranes to reduce the incoming photon flux [START_REF] Kana | Effect of irradiances up to 2000 μE m -2 s -1 on marine Synechococcus WH7803-I. Growth, pigmentation, and cell composition[END_REF][START_REF] Moore | Comparative physiology of Synechococcus and Prochlorococcus: influence of light and temperature on growth, pigments, fluorescence and absorptive properties[END_REF][START_REF] Six | Photophysiology of the marine cyanobacterium Synechococcus sp. WH8102, a new model organism[END_REF], as shown by the decrease of both Chl a and PE fluorescence signals between LL and HL (Figures 2,3). Interestingly, both parameters also slightly decreased in all representatives in response to progressive changes in light quality from 100% BL to 100% GL (Figures 2,3; Supplementary Figures S3,S4), suggesting that chromatic acclimaters used GL more efficiently than BL, at least in our experimental setup. Consequently, all strains needed to slightly adjust their thylakoid surface (and thus the number of both photosystems and PBS) in BL in order to maximize the collection of available photons and maintain similar growth rate (Supplementary Figure S2).

Another parameter that differentiated PTs 3dA from 3dB in the present study is the Em 560:650 fluorescence emission ratio, which is often interpreted as a proxy of the PE:PC ratio. This ratio exhibited higher values and tended to decrease from 100% BL to 100% GL in PT 3dA strains, while it was more stable in PT 3dB representatives (Figure 5). Since PE fluorescence per cell decreased in PT 3dA from BL to GL (Figure 3) and the PC:TA ratio was somewhat constant (Supplementary Figure S7), this may indicate a lower within-rod photon energy transfer efficiency in PT 3dA cells in GL compared to BL. Another possibility is that PBS rod length decreased by disconnection of the distal PUB-rich PE-II hexamers, as previously observed in Synechococcus sp. WH8102 as a result of photoacclimation [START_REF] Six | Photophysiology of the marine cyanobacterium Synechococcus sp. WH8102, a new model organism[END_REF]. In PT 2013) that PT 3dA cells generally exhibit a more variable acclimation kinetics than PT 3dB cells. Two PT 3dA strains belonging to the CRD1 clade (BIOS-U3-1 and MITS9220) were indeed stuck at an Exc 495:545 ratio of around 1.2 in HBL (corresponding to "phenotypic group 2" in [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. Moreover, MITS9220 and another PT 3dA strain (BL107) showed a delay in the initiation of CA4 from LBL to LGL, but not in the reverse condition, as previously reported by these authors for BL107, a behavior they designed as "phenotypic group 3. " Some PT 3dA representatives common to both studies (RS9916 and WH8020) exhibited different behaviors. Both strains indeed reached a significantly lower Exc 495:545 ratio in HBL than LBL in the previous study but not in the present one, strengthening the idea that PT 3dA cells display a more variable acclimation phenotype than PT 3dB cells, at least in some conditions. In contrast, most PT 3dB representatives exhibited a "typical" CA4 dynamics in both studies, designed by [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF] as "phenotypic group 1. " The only exceptions are strains such as WH8103 that have a typical CA4-B region but, for a yet unknown reason, have completely lost their CA4 ability and thus display a fixed Exc 495:545 ratio. Interestingly in this context, the WH8109 strain that was reported to display this fixed phenotype in [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF] was found to have recovered its CA4 ability in a more recent study [START_REF] Lovindeer | Costs and limitations of marine Synechococcus blue-green chromatic acclimation[END_REF]. The wider phenotypic variability observed in PT 3dA representatives might be attributed to the erratic genomic localization of the CA4-A island, which can be found virtually anywhere in the genome of PT 3dA strains, including the 5′-end of the PBS region, while the CA4-B island is systematically located in the middle of the PBS rod region in PT 3dB cells [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF][START_REF] Grébert | Diversity and evolution of pigment types in marine Synechococcus cyanobacteria[END_REF].

The most striking outcome of our shift experiments was certainly the discrepancy in acclimation paces between the two types of chromatic acclimaters in three out of the four tested conditions. Indeed, after the LGL to LBL and HGL to HBL shifts, the PT 3dA representatives acclimated faster than their PT 3dB counterparts, while the opposite pattern was observed after the HBL to HGL shift (Figure 7; Supplementary Figures S8,S9). Altogether, our results showed that while PT 3dA and PT 3dB cells preferentially remain in their respective basal state (i.e., low and high Exc 495:545 ratio, respectively) when grown in a blue-green light mix (Figure 4), they can reach the acclimation state opposite to their basal state faster than the other PT once the CA4 process has been triggered (Figure 7; Supplementary Figures S8,S9). These distinct acclimation paces might be due to differences in the organization and gene content of the CA4-A and CA4-B islands. While both share two genes in tandem encoding the regulatory proteins FciA and FciB [START_REF] Sanfilippo | Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus[END_REF], as well as a gene encoding a protein of unknown function (Unk10), they also contain specific genes. A third putative regulatory gene, fciC, is indeed only found in PT 3dA representatives, possibly explaining why the two CA4 processes are opposite, CA4-A being activated in BL and CA4-B in GL [START_REF] Shukla | Phycoerythrin-specific Bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF][START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]Sanfilippo et al 2019b;[START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF]. Additionally, the CA4-A and CA4-B islands encode distinct enzymes, the PEB lyase-isomerase MpeZ and the PEB lyase MpeW, respectively. Both compete with another enzyme, encoded in the main PBS genomic region, for binding either a PUB in BL or a PEB in GL at Cys-83 of the α-PE-II subunit (Sanfilippo et al., 2019b;[START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF]. Functional studies are needed to determine whether these mechanistic discrepancies are responsible for the phenotypic differences between PT 3dA and 3dB strains reported in the present study.

In conclusion, PTs 3dA and 3dB cells exhibit subtle but significant phenotypic differences that may explain why, in a spectral niche encompassing both blue and green light, they can coexist not only with BL and/or GL specialists, but also with their CA4-able counterpart [START_REF] Huisman | Principles of the light-limited chemostat: theory and ecological applications[END_REF][START_REF] Stomp | Adaptive divergence in pigment composition promotes phytoplankton biodiversity[END_REF][START_REF] Grébert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF][START_REF] Luimstra | Changes in water color shift competition between phytoplankton species with contrasting light-harvesting strategies[END_REF][START_REF] Holtrop | Vibrational modes of water predict spectral niches for photosynthesis in lakes and oceans[END_REF]. Future studies should allow one to confirm this hypothesis, either using co-cultures of different PTs grown in various light colors, or by correlation analyses of the variations of the relative abundance of the different PTs with changes in the underwater light field.

FIGURE 2

 2 FIGURE 2Flow cytometric chlorophyll a fluorescence per cell of six PT 3dA and 3dB representatives acclimated to the different conditions of temperature, light intensity and quality used for acclimation experiments. Each boxplot represents the measurements performed for all representatives of each pigment type in one light color condition (n = 9). LL, 15 μmol photons m -2 s -1 ; HL, 75 μmol photons m -2 s -1 ; PT, pigment type; BL, blue light; GL, green light.

FIGURE 3

 3 FIGURE 3 Same as Figure 2 but for the flow cytometric phycoerythrin fluorescence per cell.

FIGURE 4

 4 FIGURE 4 Same as Figure 2 but for the Exc 495:545 fluorescence excitation ratio, a proxy of the whole cell PUB:PEB ratio.

FIGURE 5

 5 FIGURE 5Same as Figure2but for the Em 560:650 fluorescence emission ratio, a proxy of the whole cell PE:PC ratio.

FIGURE 6

 6 FIGURE 6Same as Figure1but for the Em 650:680 fluorescence emission ratio, a proxy of the whole cell PC:TA ratio.

FIGURE 7

 7 FIGURE 7Time course variations of the Exc 495:545 fluorescence excitation ratio, a proxy of the whole cell PUB:PEB ratio, of eight PT 3dA and 3dB representatives after an abrupt shift of light quality from 100% blue to 100% green light, and vice-versa, in low and high light. Data are normalized to the initial value at time zero. Each point represents one measurement performed for one strain. LL, 15 μmol photons m -2 s -1 ; HL, 75 μmol photons m -2 s -1 ; PT, pigment type; BL, blue light; GL, green light.

TABLE 1

 1 Characteristics of the different Synechococcus strains used in this study.

	Strain name	RCC # a	Subcluster b	Clade b	Subclade c	Pigment type d Isolation region
	BIOS-U3-1	2,533	5.1	CRD1	n.a.	3dA	Chile upwelling
	BL107	515	5.1	IV	IVa	3dA	Balearic Sea
	MITS9220	2,571	5.1	CRD1	n.a.	3dA	Equatorial Pacific
	RS9916	555	5.1	IX	n.a.	3dA	Gulf of Aqaba
	WH8020	751	5.1	I	Ia	3dA	Sargasso Sea
	A15-62	2,374	5.1	II	IIa	3dB	Off Mauritania
	A18-40	n.a.	5.1	III	IIIa	3dB	Atlantic Ocean
	MINOS11	2,319	5.3	n.a.	n.a.	3dB	Mediterranean Sea
	PROS-U-1	2,369	5.1	II	IIh	3dB	Moroccan upwelling
	RS9915	2,553	5.1	III	IIIa	3dB	Gulf of Aqaba

a Roscoff Culture Collection. b Farrant et al. (2016). c Mazard et al. (2012). d Humily et al. (2013). 10.3389/fmicb.2024.1349322 Frontiers in Microbiology 04 frontiersin.org

  PC ratio stability, associated with decreasing PE fluorescence, rather suggests that other kinds of structural changes occurred, such as a reduction of the PBS number per cell or of the thylakoidal surface area.Shift experiments in both LL and HL conditions from 100% BL to 100% GL, and vice-versa, confirmed previous observations byHumily et al. (

			10.3389/fmicb.2024.1349322
	3dB representatives, the PE:
	Frontiers in Microbiology	10	frontiersin.org

https://roscoff-culture-collection.org/

Acknowledgments

The authors would like to thank Priscillia Gourvil, Martin Gachenot, and Michele Grego from the Roscoff Culture Collection (http://roscoff-culture-collection.org/) for maintaining the Synechococcus strains used in this study, and Maela Kloareg (Kuzulia) for performing statistical analyses.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

Growth rate

The growth rate (μ) of the six Synechococcus strains was lower at 18°C than at 25°C (p-value <0.05; Figure 1; Supplementary Table S1). Maximal μ values were reached at 25°C in HL, with all strains except WH8020 achieving more than one cell division per day (μ > 0.69 day -1 ; Supplementary Figure S2). While a clear increase in growth rates of both PTs was seen between LL and HL at 25°C, a less marked difference was noted at 18°C, suggesting that temperature and light intensity had a synergistic effect on μ. This was confirmed by the mixed model, which highlighted an interaction effect between the two factors (p-value <0.05; Supplementary Table S1). While for any given strain, the growth rate varied little between the different light colors (Supplementary Figure S2), PT 3dA representatives globally grew faster than their PT 3dB counterparts (p-value <0.05; Figure 1; Supplementary Table S1).

Chlorophyll a and phycoerythrin fluorescence

A significant downward trend in both flow cytometric red (Chl a) and orange (PE) fluorescence signals was observed from 100% BL to 100% GL for both PTs in all conditions (p-value <0.05; Figures 2,3; Supplementary Figures S3,S4; Supplementary Table S1). Besides light quality, temperature and light intensity strongly impacted Chl a and PE fluorescence signals (p-value <0.05; Supplementary Table S1). Both variables were indeed higher in LL than HL, but also at 25°C compared to 18°C (Figures 2,3). Due to the synergistic effect of temperature and irradiance (p-value <0.05; Supplementary Table S1), maximum values were measured at 25°C in LL, and conversely minima were associated with the 18°C and HL condition. Growth rates of six PT 3dA and 3dB representatives acclimated to the different conditions of temperature and light intensity used for acclimation experiments. Each boxplot represents the measurements performed for all representatives of each pigment type in the five light colors tested in this study (n = 45). The light quality factor is not shown, as it had no significant effect on growth rate (p-value >0.05). LL, 15 μmol photons m -2 s -1 ; HL, 75 μmol photons m -2 s -1 ; PT, pigment type.

Author contributions

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the French "Agence Nationale de la Recherche" Program EFFICACY (ANR-19-CE02-0019).

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Conflict of interest

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

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Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2024.1349322/ full#supplementary-material