Jeremy Dalos
email: bjeremy.dalos@ndsu.edu

Raphael Royauté
email: draphael.royaute@gmail.com

Ann Hedrick
email: favhedrick@ucdavis.edu

Ned A Dochtermann
email: ned.dochtermann@gmail.com

Bolley Drive

Stevens Hall

Jonathan Albers

Monica Berdal

Katelyn Cannon

Sarah Felde

Brady Klock

Ishan Joshi

Hannah Lambert

Jenna Lacoursiere

Alondra Neunsinger

Katie Pnewski

Maddi Rick

Teresa Tesarek

Amanda Wilson

David Lightfoot

Scott Bundy

Nico Franz

Sangmi Lee

Cameron Jones

Kenny Chapin

Ti Eriksson

Meranda Feagins

Charlotte Mulloney

Melody Martinez

Allyson Richins

Mauriel Rodriguez

Helen Vessels

David Wikman

Phylogenetic conservation of behavioral variation and behavioral syndromes

Keywords: behavioral syndrome, personality, field crickets, comparative method

Individuals frequently differ consistently from one another in their average behaviors (i.e.

"animal personality") and in correlated suites of consistent behavioral responses (i.e. "behavioral syndromes"). However, understanding the evolutionary basis of this (co)variation has lagged behind demonstrations of its presence. This lag partially stems from comparative methods rarely being used in the field. Consequently, much of the research on animal personality has relied on "adaptive stories" focused on single species and populations. Here we used a comparative approach to examine the role of phylogeny in shaping patterns of average behaviors, behavioral variation, and behavioral correlations. In comparing the behaviors and behavioral variation for five species of Gryllid crickets we found that phylogeny shaped average behaviors and behavioral (co)variation. Variation in average exploratory behavior and response to cues of predator presence attributable to phylogeny was greater or comparable to the magnitude of "personality variation".

Likewise, magnitudes of variation were concordant with evolutionary relationships and behavioral correlations were consistent across species. These results suggest that phylogenetic constraints play an important role in the expression of animal personalities and behavioral syndromes and emphasize the importance of examining evolutionary explanations within a comparative framework.

Introduction

Behavioral syndromes, correlations between behaviors at the among-individual level [START_REF] Dingemanse | Defining behavioural syndromes and the role of "syndrome" deviation in understanding their evolution[END_REF], have been documented across taxa [START_REF] Brommer | Phenotypic correlations capture between-individual correlations underlying behavioral syndromes[END_REF]. Behavioral syndromes can conceptually be thought of as correlations between individual averages and stem from underlying genetic correlations and correlations due to developmental plasticity and other sources of permanent environmental covariance [START_REF] Dingemanse | Defining behavioural syndromes and the role of "syndrome" deviation in understanding their evolution[END_REF][START_REF] Dingemanse | Individual behaviour: behavioural ecology meets quantitative genetics[END_REF]). Among-individual variation in behavior, often referred to as "personality variation", has been found to be similarly ubiquitous [START_REF] Bell | The repeatability of behaviour: a metaanalysis[END_REF]. Similar to behavioral syndromes, this personality variation can be thought of as variation across individuals in their average behaviors and likewise stem from genetic and permanent environmental variation (Dingemanse andDochtermann 2013, Dochtermann et al. 2015). Attempts to infer whether general taxonomic patterns exist for both personality variation and behavioral syndromes have generally been conducted via literature reviews and meta-analyses [START_REF] Bell | The repeatability of behaviour: a metaanalysis[END_REF][START_REF] Dochtermann | Testing Cheverud's conjecture for behavioral correlations and behavioral syndromes[END_REF][START_REF] Garamszegi | A meta-analysis of correlated behaviours with implications for behavioural syndromes: mean effect size, publication bias, phylogenetic effects and the role of mediator variables[END_REF], 2013, Dochtermann et al. 2015[START_REF] Brommer | Phenotypic correlations capture between-individual correlations underlying behavioral syndromes[END_REF]. These synthesis efforts have shown that among-individual variation is common (average repeatability ∼ 0.37, [START_REF] Bell | The repeatability of behaviour: a metaanalysis[END_REF], that the magnitude of behavioral syndromes is generally weak (average r ∼ 0.19, [START_REF] Garamszegi | A meta-analysis of correlated behaviours with implications for behavioural syndromes: mean effect size, publication bias, phylogenetic effects and the role of mediator variables[END_REF][START_REF] Garamszegi | A meta-analysis of correlated behaviors with implications for behavioral syndromes: relationships between particular behavioral traits[END_REF], and that there is general alignment between nested patterns of correlations at the phenotypic, among-individual, within-individual, and genetic levels (Dochtermann 2011, Brommer and[START_REF] Brommer | Phenotypic correlations capture between-individual correlations underlying behavioral syndromes[END_REF].

Despite the observation that both among-individual variation and behavioral syndromes are common, we have a poor understanding of the evolution of either. This gap in our understanding is partly because comparative approaches have rarely been used in studies of among-individual behavioral variation and behavioral syndromes [START_REF] White | Towards a comparative approach to the structure of animal personality variation[END_REF], despite having been the backbone of studies of morphological evolution. Such approaches allow for direct comparison across species of behavioral (co)variation and are necessary for a proper understanding of the importance of phylogeny in shaping "personality" and behavioral syndromes (Royauté et al. 2020[START_REF] White | Towards a comparative approach to the structure of animal personality variation[END_REF].

Direct assessment of evolutionary hypotheses can also be extended to the study of personality and behavioral syndromes: both among-individual variation and behavioral syndromes have clear connections to quantitative genetic parameters; specifically, additive genetic variation, and additive genetic covariances [START_REF] Dochtermann | Testing Cheverud's conjecture for behavioral correlations and behavioral syndromes[END_REF]Roff 2010, Dingemanse and[START_REF] Dingemanse | Individual behaviour: behavioural ecology meets quantitative genetics[END_REF]. The mathematical relationships between amongindividual (co)variances and additive genetic (co)variances [START_REF] Boake | Repeatability -Its Role in Evolutionary Studies of Mating-Behavior[END_REF][START_REF] Dingemanse | Individual behaviour: behavioural ecology meets quantitative genetics[END_REF], Dochtermann et al. 2015) allows the extension of predictions from quantitative genetics to among-individual variation and behavioral syndromes.

One such prediction is that differences in the magnitude of variation present for a trait might be attributable to differences in selection between populations or species. Specifically, [START_REF] Mousseau | Natural selection and the heritability of fitness components[END_REF] argued that traits with low heritability might be indicative of strong selection having eroded genetic variation. Likewise, because amongindividual variation represents the sum of additive genetic variation, dominance (and other epistatic) genetic variation, and permanent environmental variation (e.g. irreversible and developmental plasticity), selection is expected to deplete this variation. Note, however, that drift is often also expected to reduce genetic and, therefore, among-individual variation.

Selection is likewise expected to shape additive genetic covariances and correlations, both by the loss of variation in single traits and changes to the magnitude and directions of covariances [START_REF] Roff | Evolutionary Quantitative Genetics[END_REF]. For example, correlational selection is expected to produce genetic correlations [START_REF] Phillips | Visualizing multivariate selection[END_REF]Arnold 1989, Armbruster and[START_REF] Armbruster | Causes of covariation of phenotypic traits among populations[END_REF]. As in the case of among-individual variances, these effects on genetic correlations are expected to carry over to behavioral syndromes. In other words, behavioral syndromes are expected to reflect the effects of selection on genetic correlations. Therefore, if behavioral syndromes differ across populations, species, or other groupings, then this suggests differences in genetic correlations and correlational selection (i.e. the "adaptive" hypothesis, [START_REF] Bell | Behavioural differences between individuals and two populations of stickleback (Gasterosteus aculeatus)[END_REF]. In contrast, if behavioral syndromes are conserved across groups then this would suggest that either behavioral syndromes stem from pleiotropic effects (i.e.

the "constraints" hypothesis, [START_REF] Bell | Behavioural differences between individuals and two populations of stickleback (Gasterosteus aculeatus)[END_REF] or that selection is similar across groups.

While these topics have been addressed for other types of traits, particularly morphological and chemical characteristics [START_REF] Aguirre | Comparing G: multivariate analysis of genetic variation in multiple populations[END_REF], Hine et al. 2014[START_REF] Mcglothlin | Adaptive radiation along a deeply conserved genetic line of least resistance in Anolis lizards[END_REF], addressing them for behavior remains important for several reasons. First, considerable behavioral research assumes an adaptive framework for both among-individual variation and behavioral syndromes, thereby minimizing the importance of phylogeny and minimizing the potential role of phylogenetic constraints. Second, behaviors, life-history, and physiological traits exhibit substantially lower heritabilities than do morphological traits [START_REF] Mousseau | Natural selection and the heritability of fitness components[END_REF][START_REF] Stirling | Selection, structure and the heritability of behaviour[END_REF], Dochtermann et al. 2019). Consequently, the role of phylogeny and selection in constraining and shaping morphology may not generalize to traits with lower heritabilities and thus greater plasticity.

Here, we compared the behavior of five closely related cricket species: Gryllus integer, Gryllus assimilis, Gryllus lineaticeps, Gryllodes sigillatus, and Acheta domesticus. For each species we measured exploratory behavior and response to cues of predator presence. By working with the same behavioral assays in five closely related species we were able to assess the importance of phylogeny for average behaviors and to evaluate predictions about trait (co)variation. Specifically, we addressed the following questions:

1. Does the average expression of behavior differ among species?

We predicted that species would differ but do so in a manner constrained by phylogeny. Put another way, more closely related species will have more similar average behaviors.

Do among-individual variances differ among species?

We did not have species level predictions but, because selection and drift should both reduce among-individual variance, we predicted that among-individual variation would differ across species independent of phylogeny.

3. Do within-individual variances differ among species? Within-individual variation, typically disregarded as residual variation, includes phenotypic plasticityspecifically reversible plasticity or "phenotypic flexibility" not captured by factors and covariates of a statistical model [START_REF] Piersma | Phenotypic flexibility and the evolution of organismal design[END_REF][START_REF] Whitman | What is phenotypic plasticity and why is it important? Phenotypic plasticity of insects: mechanisms and consequences[END_REF][START_REF] Piersma | The flexible phenotype: a body-centred integration of ecology, physiology, and behaviour[END_REF][START_REF] Westneat | The biology hidden inside residual within-individual phenotypic variation[END_REF], Berdal and Dochtermann 2019). Differences across groups in the magnitude of withinindividual variation therefore are, in part, differences in the magnitude of plasticity.

We did not have a priori expectations as to species differences or phylogenetic signal for within-individual variances.

Do behavioral syndromes differ among species?

Because behavioral syndrome structure has been conserved at the genetic level across cricket populations of G. integer (Royauté et al. 2020), we predicted that syndromes would similarly be phylogenetically conserved and shared across species.

Methods

Cricket Acquisition, Housing, and Rearing Conditions

Data used in this study were originally collected for various studies investigating the effects of development on behavioral variation and the presence of behavioral constraints and behavioral syndromes [START_REF] Royauté | Current energy state interacts with the developmental environment to influence behavioural plasticity[END_REF], Royauté et al. 2020). A. domesticus males and females were obtained as nymphs (~ 1 mm in size) from a commercial supplier (Fluker's Cricket Farm, Port Allen, LA, U.S.A.) and were measured once mature. G. integer females were captured in Aguila, AZ, G. lineaticeps males and females were caught in Dunnigan, CA, and the G. assimilis males and females were caught in Maricopa County, AZ.

These species were all captured during the summer of 2017. G. sigillatus individuals were taken from an outbred population established by S. Sakaluk with crickets collected from California and currently maintained in Fargo, ND. For G. lineaticeps and G. assimilis, the same individuals that were caught in the field were measured, while lab reared offspring of G. integer were measured. All species were reared under a 12:12 light: dark photoperiod at a temperature of 25-28˚C. All individuals were housed in 0.71-liter containers with transparent covers that included food, shelter, and water filled glass vials plugged with cotton balls. A. domesticus were exposed to a mixture of high and low quality diets described in [START_REF] Royauté | Current energy state interacts with the developmental environment to influence behavioural plasticity[END_REF], while all other species included in this study were fed ad libitum food (commercially purchased chicken feed).

Behavior Trials

To measure exploratory behavior and anti-predator responses we repeatedly recorded individuals' activity levels in an open field arena, followed by their responses to cues of predator presence created from diluted Eublepharis macularius excreta (see details below). A. domesticus were measured between March 2015 and October 2016, G. lineaticeps were measured from August 2017 to September 2017, G. assimilis were measured between September 2017 and October 2017, G. integer were measured between May 2018 and June 2018, and G. sigillatus were measured in May 2019. All trials were conducted in a plastic arena (60 cm x 60 cm and 15 cm high) with a Plexiglas lid. The arena was split into four 30 cm × 30 cm arenas separated by a divider, allowing up to four crickets to be tested at one time. Open field trials were always conducted first followed by antipredator response trials either immediately after or on another day to minimized potential carryover effects from exposure to cues of predator presence. After each behavioral assay, arenas were thoroughly cleaned with 70% ethanol wipes to avoid accumulation of any chemical traces of conspecifics. Mass at the time of behavioral trials was recorded to the nearest 1 mg. All individuals were measured in each assay for a maximum of three repetitions, with some individuals measured fewer times due to escape or natural mortality (Table 1). In total, we conducted 2478 behavioral assays across a total of 460 individuals (Table 1).

Open field behavior

Individual crickets were left to rest for 30 seconds under a 5 cm diameter cup after being introduced into the lower right section of the arena (Figure S1). After these 30 seconds we allowed the individuals to move freely through the arena for 220 seconds. We measured each individual's exploratory propensity by calculating the number of unique zones visited (UZ) by the cricket with Ethovision X (Noldus Information Technology, Wageningen, The Netherlands). This behavioral protocol has previously been used with A.

domesticus and G. integer to evaluate genetic and individual differences in activity and exploratory behaviors [START_REF] Royauté | Behaviour, metabolism and size: phenotypic modularity or integration in Acheta domesticus?[END_REF][START_REF] Royauté | When the mean no longer matters: developmental diet affects behavioral variation but not population averages in the house cricket (Acheta domesticus)[END_REF][START_REF] Royauté | Current energy state interacts with the developmental environment to influence behavioural plasticity[END_REF], Royauté et al. 2020).

Predator cue response

To measure responses to cues of potential predator presence, we collected excreta from three adult leopard geckos, Eublepharis macularius, that were fed a mixed diet of A. Behavior Society (2020). Collected excreta was frozen and then finely ground and diluted with deionized water (1 ml H2O: 5 mg of excreta). This solution was then applied to 15 cm diameter filter paper disks with a 5 cm diameter central cutout that allows crickets to be left to rest unexposed to the predator cues [START_REF] Royauté | When the mean no longer matters: developmental diet affects behavioral variation but not population averages in the house cricket (Acheta domesticus)[END_REF][START_REF] Royauté | Current energy state interacts with the developmental environment to influence behavioural plasticity[END_REF], Royauté et al. 2020). Each predator cue disk was left to dry for a minimum of 2 hours then stored at -23˚C until needed for trials. Predator cue disks were allowed to warm to room temperature before use in antipredator trials and discarded after a single use.

Between each trial, cue disks were stored at 4˚C for a maximum of 14 days.

We placed the predator cue disk at the bottom of a 15 cm diameter arena and left the cricket to rest for a minimum of 30 seconds under a 5 cm diameter cup in the nontreated central cutout. We then removed the cup and allowed the cricket to move freely for 220 seconds and estimated the distance travelled in cm (AP distance) using Ethovision X (Figure S1). Previous studies with this protocol show that crickets had heightened activity levels in the presence of this diluted gecko excreta compared to water controls [START_REF] Royauté | When the mean no longer matters: developmental diet affects behavioral variation but not population averages in the house cricket (Acheta domesticus)[END_REF]. Consistent with this, G. sigillatus crickets have been found to increase their activity after direct exposure to predators [START_REF] Bucklaew | The effects of exposure to predators on personality and plasticity[END_REF]. Greater activity during these antipredator response assays, i.e.

greater AP distance, was therefore interpreted as a greater responsiveness to predator cues.

Data Analysis: Univariate Models

To assess differences in behavioral responses between species for means and variances we analyzed behavioral data using separate univariate mixed-effects models for unique zones visited and AP distance (square root transformed). We included species, temperature (Celsius, mean centered), mass (using among-and within-individual centering (Van de Pol and Wright 2009)), and sex as fixed effects. Individual ID was included as a random effect. We compared the fit of four univariate mixed models structured as follows: To determine whether species differed in average behavior, Models 1 and 4 were run with and without species as a fixed effect and compared based on deviance information criterion (DIC) values. If species differ in average behavior, models with species included as a fixed effect would be expected to have lower (DIC) values. Average behavioral differences among species reported in the Results section were then qualitatively assessed using posterior-modal estimates for each species [START_REF] Congdon | Bayesian model choice based on Monte Carlo estimates of posterior model probabilities[END_REF].

We then compared DIC values among models 1 through 4 to determine whether either among-or within-individual variances differed among species following Royauté et al.

(2019) and Royauté and Dochtermann (2020). The model with the lowest DIC value was considered the best model and models with Δ DIC>5 were considered to have a substantively poorer fit [START_REF] Barnett | Using information criteria to select the correct variance-covariance structure for longitudinal data in ecology[END_REF]. Models with ΔDIC<5 were considered as having comparable support relative to the best model [START_REF] Barnett | Using information criteria to select the correct variance-covariance structure for longitudinal data in ecology[END_REF]. All models were specified with the same fixed effect structure as specified above to prevent biased estimates of variance components and repeatability [START_REF] Spiegelhalter | WinBUGS user manual[END_REF], Nakagawa and Schielzeth 2010b[START_REF] Westneat | Individual variation in parental care reaction norms: integration of personality and plasticity[END_REF].

Data Analysis: Phylogenetic Signal

As our primary questions were about differences in behavioral averages and variances, our results and discussion focus on the above model comparisons. However, we also calculated the variation in behavior directly attributable to phylogeny. To do so, we fit mixed effects models with the same fixed effects, prior structure, and chain parameters as above but omitting Species as a fixed effect. Species was instead incorporated as a random effect, along with individual ID, with the relationship among species modeled according to the current phylogeny (Figure 1, [START_REF] Weissman | Crickets of the genus Gryllus in the United States (Orthoptera: Gryllidae: Gryllinae)[END_REF]). From these models we then estimated the strength of phylogenetic signal as the proportion of variation attributable to the hierarchical pattern of relatedness among species (i.e. λ, Pagel 1999, Hadfield and Nakagawa 2010, Nakagawa and Santos 2012). From the same models we also estimated the proportion of variation attributable to among-individual differences (i.e. τ, repetability, Dingemanse and Dochtermann 2013). We estimated both phylogenetic signal and repeatability as unadjusted values; that is, we included the variation attributable to fixed effects in the ratio denominator (Nakagawa and Schielzeth 2010a).

Data Analysis: Bivariate Models

Behavioral syndromes were estimated using bivariate mixed-effects models with unique zones traveled and AP distance as response variables, also using the MCMCglmm library [START_REF] Hadfield | MCMC methods for multi-response generalized linear mixed models: the MCMCglmm R package[END_REF], and analyzed separately for each individual species. We fit models using temperature (Celsius, mean centered), mass (using among-and within-individual centering on subjects (Van de Pol and Wright 2009)), and sex as fixed effects and individual ID was fit as a random effect. These models were fit with 2.6 million iterations, a 600,000 burn-in period, a thinning interval of 2000, and a prior that was flat for correlations.

Among-individual correlations were estimated for all species, while within-individual correlations were only assessed when individuals were measured for unique zones traveled and antipredator activity during the same testing period [START_REF] Dingemanse | Quantifying individual variation in behaviour: mixed-effect modelling approaches[END_REF]. Consequently, we were unable to assess within-individual covariation of G. lineaticeps and G. assimilis due to the fact that these species were not measured for each behavior in immediate succession. Because model comparisons as used above for single traits could not be conducted for correlations (due to software imposed model limitations), differences in behavioral correlations across species were assessed based on whether 95% HPD intervals overlapped. Overlap of 95% intervals is an over-conservative comparison metric (Royauté and Dochtermann 2020), but this did not affect our species comparison results here. All analyses were conducted in R 3.4.4 [START_REF] Team | R: A language and environment for statistical computing[END_REF].

Results

Differences in average behavior among species

Species differed in average behaviors: the inclusion of species as a fixed effect substantially improved model fit for both behaviors (Table 2, Table S1). The monophyletic group of G. assimilis, G. integer, and G. lineaticeps exhibited the lowest number of unique zones visited (Figure 1a) but differences in AP distance were less obviously associated with phylogenetic structure (Figure 1b). Consistent with this, phylogenetic signal was stronger for unique zones visited (λ : 0.27) than for AP distance (λ : 0.16; Table S2).

Table 2. DIC values for statistical models with and without the inclusion of species as a fixed effect. The effect of species was evaluated in a model where variances did not (Model 1) or did (Model 4) differ by species. For both behaviors and both models, the inclusion of species substantially improved model fit, as indicated by the lower DIC values for models with species included as a fixed effect.

Behavior

Differences in variances among species

The best fit model for unique zones visited was Model 4, which allowed both among and within-individual variances to vary across species. All other models were poorly supported (ΔDIC>8; Table 3). This indicates that both among-and within-individual variances differed among species in open field trials. For AP distance, Models 3 and 4 fit comparably well (Table 3). Both of these models support differences among species in within-individual variances for AP distance. The difference between the models therefore suggests mixed support for species differences in among-individual variances for AP distance.

The monophyletic group of G. assimilis, G. integer, and G. lineaticeps exhibited higher among-individual variation for unique zones visited and lower among-individual variation for AP distance (Figure 1c & d). This monophyletic group also exhibited higher withinindividual variation for both unique zones visited and AP distance than observed for A.

domesticus and G. sigillatus (Figure 1e & f).

Differences in behavioral correlations among species

Among-individual behavioral correlations were of similar magnitude for A.

domesticus, G. assimilis, G. lineaticeps, and G. sigillatus (0.3 : 0.5, Figure 2a, Table S3) while the correlation for G. integer was estimated to be slightly higher (0.66, Figure 2a, Table S3).

Importantly, the lower bounds of the HPD intervals for G. assimilis, G. integer, G. lineaticeps, and G. sigillatus also overlapped with 0 (Figure 2, Table S3). This is perhaps unsurprising given the small sample sizes for G. assimilis and G. lineaticeps.

Behavioral correlations at the within-individual level ranged from 0.1 to 0.35 for A.

domesticus, G. sigillatus, and G. integer, with G. integer having the lower bound of its HPD interval overlapping with 0 (Figure 2b). The overlapping of 0 indicates that behavioral plasticity might not be integrated in this species. Behavioral correlations at either level did not show obvious patterns relative to phylogeny and were not significantly different across species (Figure 2). Within-individual correlations for G. assimilis and G. lineaticeps were not calculated as behavior trials were not performed in close succession.

Discussion

Our results demonstrate that species differed in their exploratory behavior and response to cues of predator presence at all levels of variation but that behavioral syndromes were conserved across species. These results suggest an important influence of phylogenetic constraints on how behaviors evolve.

Species differed from one another in their average behaviors (Table 2), in a manner consistent with phylogenetic relationships. Specifically, the monophyletic group of G. assimilis, G. integer, and G. lineaticeps were generally similar in average unique zones visited (Figure 1a). In contrast, while average AP distance differed by species, it did not do so in a manner clearly concordant with phylogeny (Figure 1b). Indeed, phylogenetic signal, the proportion of variation attributable to the hierarchical pattern of relatedness among species, was higher for unique zones visited than for AP distance (Table S2). Interestingly, and relevant for future research, phylogeny explained considerably more variation in our measure of exploratory behavior-unique zones visited-than did among-individual variation, i.e. "animal personality" (τ = 0.15 versus λ = 0.27; Table S2).

The species we examined also differed in among-individual variation in exploratory (unique zones visited) and predator response (AP distance) behaviors, again in a manner consistent with phylogenetic relationships (Figures 1c,d). Unfortunately, phylogenetic methods have been developed primarily with the goal of understanding differences in trait averages rather than trait (co)variances. Our comparisons of "personality" variation and syndromes among species are therefore based on the model comparison methods identifying the presence of species differences and subsequent qualitative comparisons of species level estimates. Nonetheless, the concordance between patterns of the magnitude of among-individual variation and the currently described phylogeny suggests phylogenetic constraints on the magnitude of "personality" variation. Of the five species, the monophyly of G. assimilis, G. integer, and G. lineaticeps exhibited the highest amongindividual variation in unique zones visited and the lowest among-individual variation in AP distance (Figure 1c andd). While the expression of average behaviors and behavioral syndromes might be expected to exhibit phylogenetic signal, we did not expect this to be the case for among-individual variances. One possible explanation would be bottlenecks at more basal phylogenetic nodes leading to reduced genetic variation present in subsequent groupings. While this could explain the lower among-individual variation in AP distance for G. assimilis, G. integer, and G. lineaticeps, it does not explain that those same species exhibit higher within-individual variation for unique zones visited.

Differences observed in among-individual variation could also be attributable to selection differentially acting upon these species by reducing the additive genetic variation present in a population or species [START_REF] Mousseau | Natural selection and the heritability of fitness components[END_REF]. Our results therefore suggest the possibility that exploratory behavior, for which unique zones visited is a proxy, has been under stronger selection for A. domesticus and G. sigillatus than for the other species.

Importantly, because we do not know the strength and direction of selection acting on these phenotypes, the data presented here only suggests this possibility and cannot be used to distinguish between the effects of selection and drift for either behavior.

Alternative explanations for the observed differences in among-individual variances stem from differences across source populations and sampling of these populations. For example, individual A. domesticus used in this study were from a captive population where inbreeding could have reduced genetic variation over generations. This potentially explains the low among-individual variation the species shows for unique zones visited (Figure 1c) but is contradicted by the high among-individual variation in AP distance (Figure 1d). In contrast, G. assimilis and G. lineaticeps behavior was measured for field-caught individuals.

If individuals of these species experienced different developmental environments from one another, we would predict higher among-individual variation in behavior because permanent environmental variation contributes, on average, 50% of the observed amongindividual variation in behavior present in populations [START_REF] Dochtermann | The contribution of additive genetic variation to personality variation: heritability of personality[END_REF]). This explanation is not, however, supported: while G. assimilis and G. lineaticeps indeed showed high relative among-individual variation in unique zones visited, the same was not the case for AP distance (Figure 1c andd). Moreover, for both behaviors, G. assimilis and G.

lineaticeps were very similar to G. integer, for which lab reared individuals were measured.

To summarize, the conflicting patterns of among-individual variation observed between AP distance and unique zones visited prevents clear interpretation.

Estimated within-individual variances include variation from a variety of sources, including plasticity in response to short-term environmental variation and measurement error (Dingemanse et al. 2012, Berdal andDochtermann 2019). In comparing species, if we assume measurement error is similar among species, differences in within-individual variation will primarily represent differences in plasticity. This short-term plasticity, also referred to as phenotypic flexibility [START_REF] Piersma | The flexible phenotype: a body-centred integration of ecology, physiology, and behaviour[END_REF], allows individuals to respond flexibly to an environment [START_REF] Westneat | The biology hidden inside residual within-individual phenotypic variation[END_REF]. As was the case for amongindividual variation and average unique zones visited, G. assimilis, G. integer, and G. lineaticeps were grouped together and exhibited similar magnitudes of within-individual variation (Figure 1e andf). For both behaviors, this group exhibited considerably higher within-individual variation than observed for A. domesticus and G. sigillatus, differences supported by our model comparison results (Table 3). In other words, the Gryllus genus exhibited greater behavioral plasticity.

One possible explanation for this pattern is that our sample of G. assimilis, G. integer, and G. lineaticeps were of individuals either caught from the field or the direct offspring of field inseminated and subsequently captured individuals. In contrast, the population of G.

sigillatus we sampled had been in captivity for around 75 generations and the population of A. domesticus was reared for production purposes for some undetermined but large number of generations. Consequently the differences in within-individual variation could be attributable to exposure to a frequently changing environment [START_REF] Relyea | Morphological and behavioral plasticity of larval anurans in response to different predators[END_REF] in the case of G. assimilis, G. integer, and G. lineaticeps and the loss of plasticity in A. domesticus and G.

sigillatus. This possibility could be assessed for crickets via experimental evolution with populations experiencing different levels of environmental heterogeneity.

With regard to behavioral correlations, [START_REF] Bell | Behavioural differences between individuals and two populations of stickleback (Gasterosteus aculeatus)[END_REF] proposed two hypotheses for the expression of behavioral syndromes within a population relevant to the species level comparisons we performed. The first of these, the constraints hypothesis, chiefly attributes behavioral syndromes to the presence of pleiotropy, with the expression of genes affecting multiple behaviors. This hypothesis can be extended to other mechanistic connections constraining independent trait expression. Second, the adaptive hypothesis states that behavioral syndromes are the adaptive outcome of correlated selection. While pleiotropy and other mechanistic connections can evolve and be adaptive, syndromes attributable to the adaptive hypothesis are expected to respond more quickly to changes in selection [START_REF] Roff | Evolutionary Quantitative Genetics[END_REF]. Consequently, phylogenetic similarity in behavioral syndromes provides indirect support for the constraints hypothesis. Due to among-individual correlations not substantively differing among species (Figure 2), our results therefore support the constraints hypothesis, despite species differing in variances and average expressions of behaviors (Figure 1).

While a comparative approach has only rarely been used for examining behavioral variation, three particular studies are relevant to the interpretation of our results here.

an alternative to the dominant adaptive explanations commonly employed when discussing animal personality and behavioral syndromes.

Table S2. Variance estimates (posterior modes with 95% credibility intervals) for models including phylogenetic structure as a random effect. Models were fit with temperature (centered), sex, and mass (within and between individual centered) as fixed effects.

Phylogeny was modeled according to the trees shown in Figure 1 and with uniform branch lengths. Subject was also included as a random effect. Variance ratios are presented as unadjusted ratios; that is, variance due to fixed effects is included in the denominator. Ratios for unique zones include the distribution specific variance (DSV) in the denominator. λ and τ correspond to unadjusted phylogenetic signal and unadjusted repeatabilities respectively.

Variance NA * while the ratios for any single MCMC estimate will sum to 1, the posterior modes can sum to other values due to uncertainty across the MCMC chain ** estimated as 𝑙𝑛 ( 1 𝑒𝑥𝑝(𝛽 0 ) + 1) following Nakagawa & Schielzeth (2010) Table S3. Among-and within-individual correlations by species. Correlation estimates are posterior modes and are presented along with 95% credibility intervals (CrI).

Species

Among The open field arenas were subdivided into 36 unique "zones" during video processing. For the antipredator response trials the cricket was introduced, under a container, to the center point. This cricket release zone did not have predator cues present.

1) Model 1 :

 1 Vi = & Vw = A null model where the among-(Vi) and within-individual (Vw) variances were kept constant between species. 2) Model 2: Vi ≠ & Vw = A model where the among-individual variance differed between species, but the within-individual variance was kept constant. 3) Model 3: Vi = & Vw ≠ The within-individual variance differs between species, but the among-individual variance was kept constant. 4) Model 4: Vi ≠ & Vw ≠ Both the among and within-individual variances were allowed to vary between species.These models were specified using the MCMCglmm package for Bayesian mixed models[START_REF] Hadfield | MCMC methods for multi-response generalized linear mixed models: the MCMCglmm R package[END_REF]) using Markov-chain Monte Carlo (MCMC) with 1.3 million iterations, 300,000 iteration burn-in, a thinning interval of 1000, and an inverse-Wishart prior. AP distance and unique zone models were fit with Gaussian and Poisson error distributions, respectively.

Figure 1 .

 1 Figure 1. Species posterior-modal values with 95% HPD credibility intervals. (a) Average unique zones visited. (b) Average AP distance in centimeters. (c) Among-individual variances in unique zones traveled. (d) Among-individual variances in AP distance. (e) Within-individual variances in unique zones traveled. (f) Within-individual variances in AP distance.

Figure 2 .

 2 Figure 2. Species posterior-modal values with 95% HPD credibility intervals. (a) Amongindividual behavioral correlations of unique zones visited and AP distance. (b) Withinindividual differences of behavioral correlations of unique zones traveled and AP distance. Within-individual correlations for G. assimilis and G. lineaticeps were not calculated as behavior trials were not performed in close succession.

  Figure S1. Schematics of the open field (left) and predator cue arenas (right).The open field arenas were subdivided into 36 unique "zones" during video processing. For the antipredator response trials the cricket was introduced, under a container, to the center point. This cricket release zone did not have predator cues present.

Table 1 .

 1 Number of individuals, by species, for which behavior was assayed in a first, second, and third repetition.

	Species	Behavioral Assay	Repetition 1	Repetition 2	Repetition 3	Total Trials
	Acheta domesticus	Open field Antipredator	281 262	263 235	225 220	769 717
	Gryllus assimilis	Open field Antipredator	16 16	16 16	16 15	48 47
	Gryllus integer	Open field Antipredator	92 88	91 88	74 72	257 248
	Gryllus lineaticeps	Open field Antipredator	21 21	17 13	11 11	49 45
	Gryllodes sigillatus	Open field Antipredator	50 50	50 50	49 49	149 149
	Total		896	837	743	2478

Table 3 .

 3 DIC and ΔDIC values of model fit for AP distance and unique zones visited.

	Model (variance constraints)	Behavior	DIC	ΔDIC
	Model 1 (Vi = & Vw =)	Unique Zones Visited	8982.97	644.76
	Model 2 (Vi ≠ & Vw =)	Unique Zones Visited	8420.69	82.48
	Model 3 (Vi = & Vw ≠)	Unique Zones Visited	8346.44	8.23
	Model 4 (Vi ≠ & Vw ≠)	Unique Zones Visited	8338.21	0
	Model 1 (Vi = & Vw =)	AP Distance	8025.51	263.31
	Model 2 (Vi ≠ & Vw =)	AP Distance	8010.04	247.84
	Model 3 (Vi = & Vw ≠)	AP Distance	7762.20	0
	Model 4 (Vi ≠ & Vw ≠)	AP Distance	7763.82	1.62

This work was supported by US NSF IOS grants 1557951 and 1558069 to N.A.D. and A.H. respectively.

First, [START_REF] Blankers | Multivariate phenotypic evolution: Divergent acoustic signals and sexual selection in Gryllus field crickets[END_REF] compared the phenotypic variances and (co)variances of seven calling traits of multiple cricket species (including G. lineaticeps, which was included in our study). These authors found that the phenotypic covariance matrices differed among cricket species. One of the major differences among species was in the magnitude of variation present in single traits [START_REF] Blankers | Multivariate phenotypic evolution: Divergent acoustic signals and sexual selection in Gryllus field crickets[END_REF]. This is consistent with our findings that variances of behaviors differed across species (Figure 1c-f). Unfortunately, these authors compared phenotypic (co)variances, which conflate among-and within-individual (co)variation [START_REF] Dingemanse | Defining behavioural syndromes and the role of "syndrome" deviation in understanding their evolution[END_REF]. Second, [START_REF] White | Towards a comparative approach to the structure of animal personality variation[END_REF] compared the amongindividual covariance matrices of seven species of fish. Comparable to our results, these authors detected differences in the magnitude of among-individual behavioral variability and also found overall phylogenetic signal and similarity in how variation was expressed across multiple behaviors [START_REF] White | Towards a comparative approach to the structure of animal personality variation[END_REF] More generally, our findings here suggest that behavioral correlations are phylogenetically conserved. Conserved trait correlations like those observed here constrain the divergence of populations and species [START_REF] Schluter | Adaptive radiation along genetic lines of least resistance[END_REF]. While the potential for such constraints has been speculated about for behaviors ( Jointly, our approach allowed us to determine whether there were differences in average behavior, "personality", behavioral plasticity, and behavioral syndromes among species. Our results demonstrate phylogenetic conservation of behavioral averages, behavioral variation, and behavioral syndromes. This finding is potentially surprising given that behavior is often assumed to be more flexible and labile than other types of traits (but see [START_REF] Zuk | Killing the Behavioral Zombie: Genes, Evolution, and Why Behavior Isn't Special[END_REF] and suggests an important role for phylogenetic constraints as Supplemental Materials Table S1. Fixed effects coefficients for Model 4 (Table 3). The intercept estimate is for Acheta domesticus females (fixed effect coefficients are contrasts versus these values).

AP


David Luque-Paz

Pierre Sesques

Florent Wallet

Emmanuel Bachy

M.D Florence Ader
email: florence.ader@chu-lyon.fr

B-cell malignancies and COVID-19: a narrative review

Keywords: B-cell malignancies, B-cell depletion, COVID-19, SARS-CoV-2, mRNA vaccine, neutralizing monoclonal antibody, convalescent plasma therapy

Background. COVID-19 has been extensively characterized in immunocompetent hosts and to a lesser extent in immunocompromised populations. Among the latter, patients treated for Bcell malignancies have immunosuppression generated by B-cell lymphodepletion/aplasia resulting in a higher susceptibility to respiratory virus infections and poor response to vaccination. The consequence is that these patients are likely to develop severe or critical COVID-19. Objectives. To examine the overall impact of COVID-19 in patients treated for a B-cell malignancy or receiving chimeric antigen receptor T (CAR-T) immunotherapy administered in case of relapsed or refractory disease. Sources. We searched in the Medline database to identify relevant studies, trials, reviews, or meta-analyses focusing on SARS-CoV-2 vaccination or COVID-19 management in patients treated for a B-cell malignancy or recipients of CAR-T cell therapy up to July 8 th , 2022. Content. The epidemiology and the outcomes of COVID-19 in B-cell malignancy patients and CAR-T cell recipients are summarized. Vaccine efficacy in these subgroups is compiled. Considering the successive surges of variants of concern, we propose a critical appraisal of treatment strategies by discussing the use of neutralizing monoclonal antibodies, convalescent plasma therapy, direct-acting antiviral drugs, corticosteroids and immunomodulators. Implications. For B-cell malignancy patients, preventive vaccination against SARS-CoV-2 remain essential and management of COVID-19 includes the control of viral replication due to protracted SARS-CoV-2 shedding. Passive immunotherapy (monoclonal neutralizing antibody therapy, convalescent plasma therapy) and direct-active antivirals such as remdesivir and nirmatrelvir/ritonavir are the best currently available treatments. Real-world data and sub-group analyses in larger trials are warranted to assess COVID-19 therapeutics in B-cell depleted populations.

Introduction

The Coronavirus disease 2019 (COVID-19) pandemic started more than two years ago and its characteristics and outcomes in immunocompetent individuals have been largely described [START_REF] Robinson | COVID-19 therapeutics: Challenges and directions for the future[END_REF].

Although several treatments have been successively approved for the treatment of COVID-19, we still do not have solid evidence-based data regarding the optimal strategy to treat immunocompromised patients. Most treatment guidelines address COVID-19 through disease status (mild, moderate, or severe) and not sufficiently according to host immune status [START_REF] Agarwal | A living WHO guideline on drugs for covid-19[END_REF]. This gap is a consequence of very few inclusions of immunocompromised patients in registered clinical trials [START_REF] Trøseid | Immunocompromised patients have been neglected in COVID-19 trials: a call for action[END_REF]. SARS-CoV-2 variants of concern (VOCs) have caused physicians to constantly revisit management strategies according to retained efficacy of treatments. To date, the VOC Omicron and its sub-lineages may cause less severe disease in the general population, but there is uncertainty regarding their impact in individuals with immune deficiency. Recent data suggest escape of these sub-lineages to vaccine-induced serum neutralizing activity suggesting a gain in neutralization resistance over time, of further concern for immunocompromised populations less likely to be vaccine responders [START_REF] Ulloa | Estimates of SARS-CoV-2 Omicron Variant Severity in Ontario, Canada[END_REF]5]. Thus, it is important to examine the impact of COVID-19 in specific subgroups of immunocompromised populations to improve their practical management throughout the enduring pandemic. Profound humoral deficiency is a risk factor for severe to critical COVID-19 and B-cell malignancies (BCMs) are primary providers of this immune dysregulation.

In this narrative review, we intend to provide an overview of the burden of COVID-19 in patients treated for BCMs. Disease-specific series addressing the clinical outcome and the effect of prevention and treatment strategies are important to adapt the management. We searched in the MEDLINE database to identify the most relevant studies, trials, reviews, or meta-analyses until July 8 th , 2022.

Immunopathology of COVID-19 in B-cell malignancies

J o u r n a l P r e -p r o o f

Recent studies have provided insights regarding immune response to SARS-CoV-2 infection or after vaccination in population having BCMs or receiving B-cell depleting therapy. They have impaired humoral response after vaccination proven by quantitatively and qualitatively lower antibody levels against the SARS-CoV-2 spike protein compared to healthy individuals [START_REF] Haggenburg | Quantitative analysis of mRNA-1273 COVID-19 vaccination response in immunocompromised adult hematology patients[END_REF]7]. This is of importance considering the link between the kinetics of production of neutralizing antibodies and clinical disease outcome. A study conducted in immune competent individuals has shown that those deceased from COVID-19 had delayed neutralizing antibody release in comparison to discharged COVID-19 patients and ambulatory high neutralizers [START_REF] Lucas | Delayed production of neutralizing antibodies correlates with fatal COVID-19[END_REF].

A high viral load (> log10 5.6 per mL) has been found significantly associated with increased risk of mortality, and this lethal risk increased by 7% for each log10 increment in a large general population cohort, underscoring the critical role of neutralizing antibodies [START_REF] Pujadas | SARS-CoV-2 viral load predicts COVID-19 mortality[END_REF].

Active BCM therapies may also impair specific T-cell response [7]. Nevertheless, T-cell immunity, which is highly correlated to antiviral activity [START_REF] Monette | T Lymphocytes as Measurable Targets of Protection and Vaccination Against Viral Disorders[END_REF], still generates a detectable specific response after vaccination or infection in nearly three quarters of the cases [START_REF] Bange | CD8+ T cells contribute to survival in patients with COVID-19 and hematologic cancer[END_REF]. This is indirectly confirmed by the fact that patients with a greater number of CD8+ T cells have an improved survival, regardless of prior anti-CD20+ therapy [START_REF] Bange | CD8+ T cells contribute to survival in patients with COVID-19 and hematologic cancer[END_REF].

COVID-19 outcomes in B-cell malignancies

Summary of overall clinical outcomes and level of immune responses to SARS-CoV-2 mRNA vaccines in BCM patients are provided in Table 1. Most large multicentre COVID-19 studies have pooled all haematological malignancies together (Table S1). Although there are intrinsic differences, common features exist: i) an increased risk of severe or fatal COVID-19; ii) risk factors such as age and aggressive or progressive disease requiring intensive treatment worsen the prognosis [START_REF] Lee | COVID-19 prevalence and mortality in patients with cancer and the effect of primary tumour subtype and patient demographics: a prospective cohort study[END_REF], iii) protracted SARS-CoV-2 shedding [START_REF] Hao | Decreased B Cells on Admission Associated With Prolonged Viral RNA Shedding From the Respiratory Tract in Coronavirus Disease 2019: A Case-Control Study[END_REF], iv) an increased risk of thrombosis in comparison to the general population [START_REF] Cook | Thrombotic and Clinical Outcomes in Patients with Hematologic Malignancy and COVID-19[END_REF].

Lymphoma

J o u r n a l P r e -p r o o f

Lymphoma patients were identified early in the pandemic to be at higher risk of COVID-19, especially those with non-Hodgkin lymphoma (NHL) [START_REF] Wang | When hematologic malignancies meet COVID-19 in the United States: Infections, death and disparities[END_REF]. Clinical outcomes of patients with NHL and Hodgkin lymphoma (HL) infected with SARS-CoV-2 differ considerably to the advantage of HL [START_REF] Visco | A prognostic model for patients with lymphoma and COVID-19: a multicentre cohort study[END_REF]. A prognostic model based on a large prospective cohort of 856 lymphoma patients identified that HL was associated with the best survival, partly explained by younger ages [START_REF] Visco | A prognostic model for patients with lymphoma and COVID-19: a multicentre cohort study[END_REF]. In NHL, an overall mortality rate ranging from 31% to 35% was reported [START_REF] Regalado-Artamendi | Risk Factors and Mortality of COVID-19 in Patients With Lymphoma: A Multicenter Study[END_REF][START_REF] Duléry | Prolonged inhospital stay and higher mortality after Covid-19 among patients with non-Hodgkin lymphoma treated with B-cell depleting immunotherapy[END_REF]. Among risk factors of mortality, the two prominent were age > 70 years and relapsed/refractory lymphoma [START_REF] Regalado-Artamendi | Risk Factors and Mortality of COVID-19 in Patients With Lymphoma: A Multicenter Study[END_REF][START_REF] Duléry | Prolonged inhospital stay and higher mortality after Covid-19 among patients with non-Hodgkin lymphoma treated with B-cell depleting immunotherapy[END_REF]. Several cohorts of lymphoma patients found that persistent viral infection > 6 weeks was associated with mortality [START_REF] Regalado-Artamendi | Risk Factors and Mortality of COVID-19 in Patients With Lymphoma: A Multicenter Study[END_REF][START_REF] Hueso | Convalescent plasma therapy for B-cell-depleted patients with protracted COVID-19[END_REF].

Vaccine response in lymphoma patients depends on the timing of treatment (Table S2). In offtreatment period, a seroconversion rate as high as 89% has been reported [START_REF] Perry | Efficacy of the BNT162b2 mRNA COVID-19 vaccine in patients with B-cell non-Hodgkin lymphoma[END_REF]. Seroconversion is found in less than 10% in case of anti-CD-20+ therapies (rituximab, obinutuzumab) [START_REF] Perry | Efficacy of the BNT162b2 mRNA COVID-19 vaccine in patients with B-cell non-Hodgkin lymphoma[END_REF]. A time > 6 months after the last anti-CD-20+ administration is associated with a significantly enhanced seroconversion rate [START_REF] Avivi | BNT162b2 mRNA COVID-19 vaccine booster induces seroconversion in patients with B-cell non-Hodgkin lymphoma who failed to respond to two prior vaccine doses[END_REF]. The immunogenicity induced by mRNA vaccines can also be blunted by other chemotherapies, explaining why NHL had poorer response to vaccines than HL [START_REF] Lim | Antibody responses after SARS-CoV-2 vaccination in patients with lymphoma[END_REF]. Importantly, COVID-19 vaccine booster doses in lymphoma patients have been reported to partly increase the ability to seroconvert (up to 18-50%) [START_REF] Avivi | BNT162b2 mRNA COVID-19 vaccine booster induces seroconversion in patients with B-cell non-Hodgkin lymphoma who failed to respond to two prior vaccine doses[END_REF], and 69% of patients treated with rituximab who lack antibody response had a mRNA vaccine-induced T-cell response, arguing for maintaining vaccination in these patients [START_REF] Riise | Rituximab-treated patients with lymphoma develop strong CD8 T-cell responses following COVID-19 vaccination[END_REF].

Chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is the most frequent leukaemia and usually affects the elderly with underlying comorbidities. This is probably why the proportion of severe COVID-19 was dramatically high in the first reported cohorts (up to 80%) [START_REF] Scarfò | COVID-19 severity and mortality in patients with chronic lymphocytic leukemia: a joint study by ERIC, the European Research Initiative on CLL, and CLL Campus[END_REF]. CLL is responsible for a profound immune dysregulation affecting both cellular and humoral functions as peripheral healthy B cells are significantly decreased and 85% of CLL patients have J o u r n a l P r e -p r o o f hypogammaglobulinemia [START_REF] Langerbeins | Immune Dysfunction in Patients with Chronic J o u r n a l P r e -p r o o f Lymphocytic Leukemia and Challenges during COVID-19 Pandemic[END_REF]. In two multicentre cohort studies including 198 and 190 CLL patients, the mortality rates of hospitalized patients were 37% and 36.4%, respectively [START_REF] Scarfò | COVID-19 severity and mortality in patients with chronic lymphocytic leukemia: a joint study by ERIC, the European Research Initiative on CLL, and CLL Campus[END_REF][START_REF] Mato | Outcomes of COVID-19 in patients with CLL: a multicenter international experience[END_REF].

Of note, Bruton tyrosine kinase inhibitor (BTKi) had no detrimental impact on mortality due to COVID-19 in comparison to the watch-and-wait strategy in the management of CLL [START_REF] Mato | Outcomes of COVID-19 in patients with CLL: a multicenter international experience[END_REF].

Overall, these mortality rates exceeding 30% decreased during the pandemic to 18% in the most recent data [START_REF] Blixt | Covid-19 in patients with chronic lymphocytic leukemia: clinical outcome and B-and T-cell immunity during 13 months in consecutive patients[END_REF]. This reduction is explained by an optimized management of severe COVID-19 and specific T-cell and B-cell immunity in response to vaccination and prior COVID-19 in treatment-naïve CLL patients [START_REF] Blixt | Covid-19 in patients with chronic lymphocytic leukemia: clinical outcome and B-and T-cell immunity during 13 months in consecutive patients[END_REF]. Vaccine response in CLL has been shown to be highly variable, ranging from 73% of seroconversion in treatment-naïve patients to 29% and 4% in patients receiving BTKi and anti-CD-20+ therapy respectively [START_REF] Molica | mRNA COVID-19 vaccines in patients with chronic lymphocytic leukemia: A systematic review and meta-analysis[END_REF]. Overall, T-cell response was detectable in more than 60% of patients [START_REF] Blixt | Covid-19 in patients with chronic lymphocytic leukemia: clinical outcome and B-and T-cell immunity during 13 months in consecutive patients[END_REF][START_REF] Lyski | Cellular and humoral immune response to mRNA COVID-19 vaccination in subjects with chronic lymphocytic leukemia[END_REF].

Multiple myeloma (MM)

Early in the pandemics, studies focusing on patients with multiple myeloma (MM) infected by SARS-CoV-2 found an in-hospital mortality rates were over 30% [START_REF] Martínez-López | Multiple myeloma and SARS-CoV-2 infection: clinical characteristics and prognostic factors of inpatient mortality[END_REF][START_REF] Chari | Clinical features associated with COVID-19 outcome in multiple myeloma: first results from the International Myeloma Society data set[END_REF]. In a large prospective cohort study which took place during the first year, the highest mortality rate among BCMs was found in MM [START_REF] Booth | Key findings from the UKCCMP cohort of 877 patients with haematological malignancy and COVID-19: disease control as an important factor relative to recent chemotherapy or anti-CD20 therapy[END_REF]. In another study carried out later in the pandemic, the highest occurrence of progression toward severe or critical COVID-19 was found in MM patients, despite the use of neutralizing monoclonal antibodies (NmAb) [START_REF] Weinbergerová | Successful Early Use of Anti-SARS-CoV-2 Monoclonal Neutralizing Antibodies in SARS-CoV-2 Infected Hematological Patients -A Czech Multicenter Experience[END_REF].

Response to SARS-CoV-2 vaccine is elevated in MM patients, with seroconversion reported in 93% of cases and a T-cell response rate of 61 % [START_REF] Ramasamy | Immune response to COVID-19 vaccination is attenuated by poor disease control and antimyeloma therapy with vaccine driven divergent T cell response[END_REF]. Yet, effective mRNA vaccine-induced neutralizing antibodies have been found to be lower than the overall seroconversion rates (one third of vaccinated MM patients lacked detectable serum neutralizing activity) [START_REF] Nooka | Determinants of Neutralizing Antibody Response After SARS CoV-2 Vaccination in Patients With Myeloma[END_REF]. Reduced vaccine efficacy has been observed in male, older age, advanced disease, ongoing MM treatments (especially anti-CD38 and anti-BCMA therapies), and hypogammaglobulinemia [START_REF] Ramasamy | Immune response to COVID-19 vaccination is attenuated by poor disease control and antimyeloma therapy with vaccine driven divergent T cell response[END_REF][START_REF] Avivi | Humoral response rate and predictors of response to BNT162b2 mRNA COVID19 vaccine in patients with multiple myeloma[END_REF].

J o u r n a l P r e -p r o o f

Chimeric antigen receptor T cell (CAR-T) immunotherapy

CAR-T cell immunotherapy is administered after lymphodepleting conditioning, which generates long-term B-cell aplasia and hypogammaglobulinemia [START_REF] Fried | Early and late hematologic toxicity following CD19 CAR-T cells[END_REF]. In the two largest studies reporting clinical outcomes, ICU admission rates were close to 40% while mortality was 43% (n=13/30) and 41% (n= 23/56) respectively [START_REF] Busca | COVID-19 and CAR-T cells: current challenges and future directions-a report from the EPICOVIDEHA survey by EHA-IDWP[END_REF][START_REF] Spanjaart | Poor outcome of patients with COVID-19 after CAR T-cell therapy for B-cell malignancies: results of a multicenter study on behalf of the European Society for Blood and Marrow Transplantation (EBMT) Infectious Diseases Working Party and the European Hematology Association (EHA) Lymphoma Group[END_REF]. Most patients had underlying relapsed/refractory NHL (86%, n= 74/86). Response to mRNA SARS-CoV-2 vaccines has been assessed in small observational studies of CAR-T cell recipients showing a seroconversion rates ranging from 6 to 30% [START_REF] Dahiya | Impaired immune response to COVID-19 vaccination in patients with B-cell malignancies after CD19 CAR T-cell therapy[END_REF][START_REF] Gastinne | Safety and antibody response after one and/or two doses of BNT162b2 Anti-SARS-CoV-2 mRNA vaccine in patients treated by CAR T cells therapy[END_REF][START_REF] Sesques | Immune response to three doses of mRNA SARS-CoV-2 vaccines in CD19-targeted chimeric antigen receptor T cell immunotherapy recipients[END_REF]. The largest cohort (n=33) published to date found that T-cell response (42%) was higher than humoral response (18%), and no clear benefit in vaccine response was found after administering a second booster dose [START_REF] Sesques | Immune response to three doses of mRNA SARS-CoV-2 vaccines in CD19-targeted chimeric antigen receptor T cell immunotherapy recipients[END_REF].

Treatment strategies

Given that most randomized controlled trials (RCTs) have included few immunocompromised patients and mostly took place during the circulation of SARS-CoV-2 ancestral strain or pre-Omicron variants, the actual management of COVID-19 in the setting of BCMs may be challenging in certain aspects.

Corticosteroids

Dexamethasone was the first treatment that showed a reduction of 36% mortality in hospitalized COVID-19 patients [START_REF] Recovery Collaborative Group | Dexamethasone in Hospitalized Patients with Covid-19[END_REF]. The rationale was easily understandable as the hyper-inflammatory response in lungs triggered by viral infection potentially leads to acute respiratory distress syndrome. But corticosteroids impair innate immune pathways, which may add up to the issue of protracted SARS-CoV-2 shedding due to B-cell depletion and T-cell impairment [START_REF] Cour | Effects of dexamethasone on immune dysfunction and ventilator-associated pneumonia in COVID-19 acute respiratory distress syndrome: an observational study[END_REF]. In BCMs, the benefit of corticosteroids has not been demonstrated. In several observational studies of B-cell depleted patients, corticosteroids had no substantial impact on the outcomes, including survival [START_REF] Hueso | Convalescent plasma therapy for B-cell-depleted patients with protracted COVID-19[END_REF][START_REF] Hueso | Convalescent plasma improves overall survival in patients with B-cell lymphoid malignancy and COVID-19: a longitudinal cohort and propensity score analysis[END_REF][START_REF] Roeker | COVID-19 in patients with CLL: improved survival outcomes and update on management strategies[END_REF]. Whether corticosteroids participate in viral persistence in B-cell depleted J o u r n a l P r e -p r o o f patients is a key issue, unanswered so far, leading to likely consider their use in combination with an antiviral therapy.

Other Immunomodulators

Interleukin (IL)-6 inhibitors (tocilizumab and sarilumab) and oral Janus kinase (JAK) inhibitor baricitinib have not been assessed in the immunocompromised settings although there is a strong recommendation by the World Health Organization guidelines in favour of their use in severe or critical COVID-19 based on platform trial results [START_REF] Agarwal | A living WHO guideline on drugs for covid-19[END_REF]. In BCMs, we suggest that their use with corticosteroids should be considered individually only after assessment of the benefitrisk balance.

Convalescent plasma therapy

There is a rationale for the use of high-titre polyclonal convalescent plasma therapy (CPT) to palliate B-cell defect and to provide plasma collected during the circulation of the then dominant VOC. The first published series of 17 B-cell malignancy patients previously treated by anti-CD20+ therapy who had protracted COVID-19 has shown a resolution of symptoms and decrease of SARS-CoV-2 RNAemia in most patients [START_REF] Hueso | Convalescent plasma therapy for B-cell-depleted patients with protracted COVID-19[END_REF]. Then, two retrospective cohort studies of BCM patients hospitalised with COVID-19 confirmed that CPT was efficient in treating patients and showed a significant reduction in mortality at 30 days and 16 days respectively using propensity score analyses [START_REF] Hueso | Convalescent plasma improves overall survival in patients with B-cell lymphoid malignancy and COVID-19: a longitudinal cohort and propensity score analysis[END_REF][START_REF] Thompson | Association of Convalescent Plasma Therapy With Survival in Patients With Hematologic Cancers and COVID-19[END_REF] . Of note, the large REMAP-CAP trial, evaluating the impact of CPT on organ support-free days in over more than 2000 critically ill COVID-19 patients, although stopped for futility, found a non-significant trend for the use of CPT in the subgroup of immunocompromised patients [START_REF] Investigators | Effect of Convalescent Plasma on Organ Support-Free Days in Critically Ill Patients With COVID-19: A Randomized Clinical Trial[END_REF]. CPT has been authorized by the U.S. Food and Drug Administration (FDA) under an emergency use authorization for the treatment of COVID-19 in patients with immunosuppressive disease or receiving immunosuppressive treatment [START_REF]Recommendations for Investigational COVID-19 Convalescent Plasma. FDA[END_REF]. Overall, in BCM patients hospitalised with COVID-19 presenting clinically and virologically persistent infection, the use of high-titre CPT from J o u r n a l P r e -p r o o f donors having recently recovered from COVID-19 is among the therapeutic available options.

Physicians should be aware of the possibility of an antibody-dependent enhancement defined by enhanced antibody-mediated viral uptake into phagocytic cells causing immune complexdependent inflammatory syndrome [START_REF] Ricke | Two Different Antibody-Dependent Enhancement (ADE) Risks for SARS-CoV-2 Antibodies[END_REF]. Other adverse events, namely transfusion-associated circulatory overload, transfusion-related acute lung injury or allergic reaction are very uncommon [START_REF]Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial[END_REF].

Neutralizing monoclonal antibodies

The use of NmAbs is a relevant approach for immunocompromised patients non responders to vaccines. A major issue has been the emergence of VOCs requiring constant adjustment according to retainment of efficacy of the available NmAbs. Multiple RCTs evaluating the efficacy of NmAbs at various stages of SARS-CoV-2 infection have confirmed that the best window of opportunity for these treatments to improve patients' overall outcome was the preexposure or the early post-exposure phases in patients at high risk for progression to severe COVID-19 [START_REF] Dougan | Bamlanivimab plus Etesevimab in Mild or Moderate Covid-19[END_REF][START_REF] Weinreich | REGEN-COV Antibody Combination and Outcomes in Outpatients with Covid-19[END_REF][START_REF] Gupta | Early Treatment for Covid-19 with SARS-CoV-2 Neutralizing Antibody Sotrovimab[END_REF]. Chronologically, the combination of bamlanivimab and etesevimab was the first to induce a reduction in hospitalization and 29-day mortality versus placebo in the outpatient setting, but was proven to be inactive on Beta and Delta variants [START_REF] Dougan | Bamlanivimab plus Etesevimab in Mild or Moderate Covid-19[END_REF]. The combination casirivimab and imdevimab was also found to significantly prevent hospitalization and reduce 28-day mortality vs. placebo for outpatients [START_REF] Weinreich | REGEN-COV Antibody Combination and Outcomes in Outpatients with Covid-19[END_REF] and reduced 28-day mortality among hospitalized COVID-19 patients who were seronegative at baseline vs. standard of care [START_REF]Casirivimab and imdevimab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial[END_REF]. This combination did not retain efficacy on the Omicron variant [START_REF] Bruel | Serum neutralization of SARS-CoV-2 Omicron sublineages BA.1 and BA.2 in patients receiving monoclonal antibodies[END_REF]. The combination tixagevimab and cilgavimab was reformatted with amino acid substitutions in the Fc regions to extend their serum half-lives and reduce Fcγ receptor and complement binding [START_REF] Loo | The SARS-CoV-2 monoclonal antibody combination, AZD7442, is protective in nonhuman primates and has an extended half-life in humans[END_REF].

Preliminary results of a phase III trial designed to evaluate tixagevimab and cilgavimab as pre-exposure prophylaxis, which included high-risk and immunocompromised participants, have showed a reduced risk of developing symptomatic COVID-19 by 83% J o u r n a l P r e -p r o o f vs. placebo [START_REF]Tixagevimab and Cilgavimab (Evusheld) for Pre-Exposure Prophylaxis of COVID-19[END_REF]. Another RCT has shown that the administration of tixagevimab and cilgavimab in the early outpatient setting significantly reduced the occurrence of severe COVID-19 or death [START_REF] Montgomery | Efficacy and safety of intramuscular administration of tixagevimab-cilgavimab for early outpatient treatment of COVID-19 (TACKLE): a phase 3, randomised, double-blind, placebocontrolled trial[END_REF]. Finally, in the setting of hospitalized COVID-19 patients, tixagevimab and cilgavimab reduced by 30% the 90-day mortality vs. placebo (secondary outcome), although the RCT was conducted before the Omicron era and failed to achieve sustained clinical recovery (primary outcome) [START_REF]Tixagevimab-cilgavimab for treatment of patients hospitalised with COVID-19: a randomised, double-blind, phase 3 trial[END_REF]. Sotrovimab has been assessed in the treatment of early stages of infection (symptomatic outpatients ≤ 5 days) showing a risk reduction of hospitalization or 29-day mortality by 85% vs. placebo [START_REF] Gupta | Early Treatment for Covid-19 with SARS-CoV-2 Neutralizing Antibody Sotrovimab[END_REF]. Bebtelovimab, a new Omicron-active NmAb, has received emergency use authorisation by the FDA for the treatment of mild to moderate COVID-19 in outpatients who are at high risk for progression to severe COVID-19, but is not currently available in Europe [START_REF]New Monoclonal Antibody for Treatment of COVID-19 that Retains Activity Against Omicron Variant[END_REF]. Results of neutralizing assays on Omicron sub-lineages influence treatment strategies. Assays performed on live BA.1 and BA.2 showed differences in activity: sotrovimab and bebtelovimab retained activity against BA.1, whereas tixagevimab and cilgavimab and bebtelovimab retained activity against BA.2 [START_REF] Bruel | Serum neutralization of SARS-CoV-2 Omicron sublineages BA.1 and BA.2 in patients receiving monoclonal antibodies[END_REF][START_REF] Iketani | Antibody evasion properties of SARS-CoV-2 Omicron sublineages[END_REF].

As of today, data of neutralizing assays on BA.2.12.1 and BA.4/BA5 revealed that only bebtelovimab and cilgavimab retained activity [START_REF] Takashita | Efficacy of Antibodies and Antiviral Drugs against Omicron BA[END_REF][START_REF] Cao | 2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection[END_REF].

Direct-acting antiviral agents

Direct-acting antivirals (DAAs) are important assets in the management of COVID-19 as they all retain in vitro neutralizing activity against VOCs [START_REF] Takashita | Efficacy of Antibodies and Antiviral Drugs against Omicron BA[END_REF]. In patients hospitalized with COVID-19, the use of remdesivir showed no difference in all-cause mortality and on SARS-CoV-2 viral kinetics in comparison to standard of care [START_REF] Ader | Remdesivir plus standard of care versus standard of care alone for the treatment of patients admitted to hospital with COVID-19 (DisCoVeRy): a phase 3, randomised, controlled, openlabel trial[END_REF]. However, remdesivir was associated with a shortened time to recovery in patients with mild-to-moderate COVID-19 in two trials mostly including immunocompetent patients [START_REF] Beigel | Remdesivir for the Treatment of Covid-19 -Final Report[END_REF][START_REF] Spinner | Effect of Remdesivir vs Standard Care on Clinical Status at 11 Days in Patients With Moderate COVID-19: A Randomized Clinical Trial[END_REF]. In the outpatient setting, a 3-day course of remdesivir resulted in an 87% decrease in the risk of COVID-19 progression, although only 4.1% of patients were immunocompromised [START_REF] Gottlieb | Early Remdesivir to Prevent Progression to Severe Covid-19 in Outpatients[END_REF]. Despite encouraging preliminary data, J o u r n a l P r e -p r o o f disappointing results were obtained with molnupiravir in symptomatic outpatients [START_REF] Extance | Covid-19: What is the evidence for the antiviral molnupiravir?[END_REF]. The nirmatrelvir/ritonavir, an oral protease inhibitor combination, has shown a reduction of 30% in hospitalisation and 89% in mortality leading to authorization of use for mild to moderate COVID-19 in outpatients at high risk for progression to severe COVID-19 [START_REF] Bernal | Molnupiravir for Oral Treatment of Covid-19 in Nonhospitalized Patients[END_REF][START_REF]Pfizer's Novel COVID-19 Oral Antiviral Treatment Candidate Reduced Risk of Hospitalization or Death by 89% in Interim Analysis of Phase 2/3 EPIC-HR Study | Pfizer n[END_REF]. In addition, a large retrospective cohort study in the Omicron era identified immunosuppressed patients as one of the subgroups for whom nirmatrelvir/ritonavir use is most effective to prevent severe COVID-19 or death [START_REF] Najjar-Debbiny | Effectiveness of Paxlovid in Reducing Severe Coronavirus Disease 2019 and Mortality in High-Risk Patients[END_REF]. Drug-drug interactions are the major limit to its wide-spread use, especially in patients with BCMs who frequently have co-medications. There is a growing interest for combined antiviral strategies including NmAbs and DAAs in the immunocompromised populations, which warrants further validation by RCTs.

Conclusion

BCMs carry a high burden in COVID-19 characterized by high case fatality and poor humoral vaccine-induced response. Nevertheless, vaccinating these patients remains an essential measure of prevention. In these populations at high risk for progression to severe COVID-19, pre-exposure prophylaxis using NmAbs must be proposed when feasible. The cornerstone of curative treatment is DAAs or variant-active NmAbs for outpatients with mild to moderate COVID-19 and NmAbs or high-titre polyclonal CPT in hospitalized patients. Combining antiviral approach may gain interest in the future.

Authorship statements

DLP and FA were the project initiators and drafted the manuscript and approved the final version. FW, PS and EB contributed to the revision of the manuscript for important intellectual content and approved the final version.

Acknowledgements

The authors gratefully acknowledge the Haematology physicians of the Lyon HEMINF study group: V. Alcazer, E. Bachy, M. Balsat, F. Barraco, F. Bouafia-Sauvy, S. Ducastelle-Leprêtre, J o u r n a l P r e -p r o o f

Funding

No external funding was received for this review.

E. Ferrant, H. Ghesquières, C. Golfier, M. Heiblig, L. Karlin, H. Labussière-Wallet, A.

Lazareth, M.V. Larcher, H. Lequeu, A. Mareek, V. Safar, F. Wallet.

Competing interests

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest or financial conflict with the subject matter or materials discussed in the manuscript. Production of this manuscript did not require writing assistance. J o u r n a l P r e -p r o o f

Table 1. Clinical outcomes and immune responses to SARS-CoV