Jean-Baptiste Baudet

Benoît Xuereb

Aurélie Duflot

Agnès Poret

Emeline Maniez

Frank Le Foll

Romain Coulaud
email: romain.coulaud@univ-lehavre.fr

Temperature-mediated developmental plasticity in winter and summer larvae of Palaemon serratus

Keywords: Larval ecology, Plasticity, Developmental pathways, Larval instars, Larval 26 stages, Palaemon serratus. 27

In a seasonal environment, variation in larval phenotype and developmental plasticity allow crustacean larvae to maximise survival by lengthening or shortening their development. The aim of this study is to investigate the effects of temperature, laying season and their interaction on larval developmental pathways (larval instars and larval stages). We monitored the different larval stages and calculated the number of larval instars reached during the development of winter and summer larvae of Palaemon serratus incubated at 12, 16 or 20°C. We observed a great variability in the larval development (6 to 13 larval instars and 6 to 11 larval stages). A higher temperature decreases the development time and the number of larval instars. At a given temperature, the development time of winter and summer larvae was not different. Two larval stages were considered supernumerary (zoea 4 and 6), as they were more frequent at low temperatures. At higher temperatures, some larvae started to develop pleopods as early as the third instar, larval stage which had never been described (named here zoea 3'). This phenomenon was more common in winter larvae than in summer larvae. These results provide new insights into the expression of developmental plasticity in decapod larvae.

Introduction

Ecological Developmental Biology (Eco-Devo) approaches focus on the interactions between organisms and their environment, and their ability to optimise their growth and development [START_REF] Gilbert | Ecological Developmental Biology: Developmental Biology Meets the Real World[END_REF]. For Eco-Devo studies, marine decapod crustaceans represent interesting biological models because their complex life cycle is generally biphasic (for a review see Anger 2006), divided between a pelagic larval phase and a benthic adult life. Given the seasonal or spatial variability of the pelagic environment (e.g. food availability and quality, temperature), decapods may therefore exhibit a high degree of developmental plasticity (i.e. the capacity of a genotype to produce alternative developmental pathways; West-Eberhard 2003), allowing their larvae to cope with variable or even sub-optimal conditions [START_REF] Anger | The Biology of Decapod Crustacean Larvae[END_REF]. Several authors have studied the effects of environmental parameters on the larval development of marine decapods.

Among abiotic parameters, temperature affects intermoult time, growth rate and number of larval moults achieved [START_REF] Linck | Einfluß von Temperatur und Salzgehalt auf die Larven der Nordseegarnele Crangon crangon[END_REF], read in Giménez 2006;[START_REF] O'connor | Temperature control of larval dispersal and the implications for marine ecology, evolution, and conservation[END_REF]Oliphant et al. 2013). The amount of food ingested by the larvae determines the number of larval moult cycles and the rate of morphogenesis between them [START_REF] Anger | The D0 threshold: a critical point in the larval development of decapod crustaceans[END_REF][START_REF] González-Ortegón | Environmentally mediated phenotypic links and performance in larvae of a marine invertebrate[END_REF]. Thence, prolonged development has frequently been observed under poor zootechnical conditions [START_REF] Críales | Experimental studies on the larval development of the shrimps Crangon crangon and C. allmanni[END_REF][START_REF] Giménez | Larval performance in an estuarine crab, Chasmagnathus granulata, is a consequence of both larval and embryonic experience[END_REF][START_REF] González-Ortegón | A multiple stressor approach to study the toxicity and sub-lethal effects of pharmaceutical compounds on the larval development of a marine invertebrate[END_REF][START_REF] González-Ortegón | Environmentally mediated phenotypic links and performance in larvae of a marine invertebrate[END_REF][START_REF] Knowlton | Larval developmental processes and controlling factors in decapod Crustacea, with emphasis on Caridea[END_REF]Oliphant et al., 2013;[START_REF] Ostrensky | Effects of different diets on the larval development and ammonia excretion rates of the crab Chasmagnathus granulata Dana, 1851, under laboratory conditions[END_REF]. Conversely, abbreviated developments have been encountered under optimal conditions such as the thermal preference [START_REF] Críales | Experimental studies on the larval development of the shrimps Crangon crangon and C. allmanni[END_REF]Oliphant et al., 2013;Oliphant and Thatje, 2013). It is these different plastic responses to environmental conditions that lead to different developmental pathways [START_REF] Críales | Experimental studies on the larval development of the shrimps Crangon crangon and C. allmanni[END_REF].

In other way, few authors have studied the interactions of environmental conditions and the offspring phenotype on the developmental plasticity of decapods [START_REF] Linck | Einfluß von Temperatur und Salzgehalt auf die Larven der Nordseegarnele Crangon crangon[END_REF], read in Giménez 2006;[START_REF] Hancock | The relationship between egg size and embryonic and larval development in the freshwater shrimp Paratya australiensis Kemp (Decapoda: Atyidae)[END_REF]Oliphant & Thatje 2013;[START_REF] González-Ortegón | Environmentally mediated phenotypic links and performance in larvae of a marine invertebrate[END_REF]; Oliphant J o u r n a l P r e -p r o o f et al. 2014). The larval size is highly correlated with the nutritional reserves transferred per egg by the females during oogenesis [START_REF] Giménez | Relationships among salinity, egg size, embryonic development, and larval biomass in the estuarine crab Chasmagnathus granulata Dana, 1851[END_REF][START_REF] Giménez | Larval growth in the estuarine crab Chasmagnathus granulata: the importance of salinity experienced during embryonic development, and the initial larval biomass[END_REF][START_REF] Hancock | The relationship between egg size and embryonic and larval development in the freshwater shrimp Paratya australiensis Kemp (Decapoda: Atyidae)[END_REF][START_REF] Moland | Maternal influences on offspring size variation and viability in wild European lobster Homarus gammarus[END_REF]. This increases their fitness by allowing them to better resist starvation and achieve a shorter development [START_REF] Giménez | Linking life history traits in successive phases of a complex life cycle: effects of larval biomass on early juvenile development in an estuarine crab, Chasmagnathus granulata[END_REF][START_REF] Giménez | Larval performance in an estuarine crab, Chasmagnathus granulata, is a consequence of both larval and embryonic experience[END_REF][START_REF] González-Ortegón | Environmentally mediated phenotypic links and performance in larvae of a marine invertebrate[END_REF][START_REF] Hancock | The relationship between egg size and embryonic and larval development in the freshwater shrimp Paratya australiensis Kemp (Decapoda: Atyidae)[END_REF]Oliphant et al., 2014;Oliphant and Thatje, 2013). The larval phenotype is therefore determined by maternal investment, in line with environmental variations. The largest larvae correspond to the most difficult environmental conditions, such as the beginning of the season [START_REF] González-Ortegón | Maternal Trophic Status and Offpsring Phenotype in a Marine Invertebrate[END_REF][START_REF] Paschke | Untersuchungen zum Energiestoffwechsel der Nordsee-Garnele Crangon crangon (Linneaeus (1758) (Decapoda : Caridea)[END_REF][START_REF] Paschke | Seasonal variation in starvation resistance of early larval North Sea shrimp Crangon crangon (Decapoda: Crangonidae)[END_REF][START_REF] Urzúa | Seasonal variations in larval biomass and biochemical composition of brown shrimp, Crangon crangon (Decapoda, Caridea), at hatching[END_REF]. Thus, decapods living in seasonally variable environments and reproducing several times during the year show a seasonal pattern in the "fertility x offspring size trade-off" [START_REF] Bas | Seasonal and interpopulational variability in fecundity, egg size, and elemental composition (CHN) of eggs and larvae in a grapsoid crab, Chasmagnathus granulatus[END_REF][START_REF] Boddeke | The occurrence of winter and summer eggs in the brown shrimp (Crangon crangon) and the pattern of recruitment[END_REF][START_REF] Vázquez | Seasonal variation in reproductive traits of the oriental shrimp Palaemon macrodactylus (Crustacea: Caridea: Palaemonidae) in a nonnative population[END_REF]. The developmental pathways of the larvae of these species should be seasonally different, linked to the difference in larval size between the seasons.

The larval developmental pathway is therefore the result of the interaction between larval genotype, environmental conditions during development and the environment or phenotype of the mother (i.e. maternal effects, [START_REF] Lubzens | Potential advantages of frozen algae (Nannochloropsis sp.) for rotifer (Brachionus plicatilis) culture[END_REF]. In order to study the developmental plasticity response to this interaction, it is necessary to define the developmental pathways taken. Decapod larvae develop through a succession of moults with morphological, ecological and behavioural changes, which are generally called "Larval Stages" [START_REF] Anger | The Biology of Decapod Crustacean Larvae[END_REF]. However, in caridean shrimps that develop anamorphically in particular (i.e. through a gradual pattern of development, [START_REF] Møller | Patterns of larval development[END_REF], the degree of morphological changes between moults is variable [START_REF] Anger | The Biology of Decapod Crustacean Larvae[END_REF]; for a review see [START_REF] Quinn | Extra and "intermediate" larval stages in decapod crustacea: a review of physiological causes and ecological implications, with emphasis on lobsters, Homarus sp[END_REF]. Additional larval stages may be "intercalated" into the developmental sequence, often called "supernumerary stages", displaying intermediate morphology [START_REF] Knowlton | Larval developmental processes and controlling factors in decapod Crustacea, with emphasis on Caridea[END_REF][START_REF] Little | The Larval Development of the Shrimp, Palaemon macrodactylus Rathbun, Reared in the Laboratory, and the Effect of Eyestalk Extirpation on Development[END_REF]. Each larval stage can also be "repeated", without showing any significant change (Rothlisberg, 1980;[START_REF] Walsh | Larval Development of Paratya australiensis Kemp, 1917 (Decapoda: Caridea: Atyidae), Reared in the Laboratory, With Comparisons of Fecundity and Egg and Larval Size Between Estuarine and Riverine Environments[END_REF] J o u r n a l P r e -p r o o f [START_REF] Wehrtmann | Larval development of Nauticaris magellanica (A. Milne Edwards, 1891) (Decapoda: Caridea: Hippolytidae), reared under laboratory conditions[END_REF]. We thus explain that the larvae of a species do not necessarily develop through a fixed number of moults. Therefore, the neutral term "Larval Instar" is preferred to refer to the successive moult cycles during larval development, without describing them [START_REF] Martin | Atlas of Crustacean Larvae[END_REF][START_REF] Phillips | Larval ecology[END_REF]. (2013) monitored both the larval stages and larval instars of P. varians during development at 3 temperatures. [START_REF] Críales | Experimental studies on the larval development of the shrimps Crangon crangon and C. allmanni[END_REF] described the developmental pathways (larval stages and instars) of Crangon crangon and C. allmanni according to rearing temperature. [START_REF] Linck | Einfluß von Temperatur und Salzgehalt auf die Larven der Nordseegarnele Crangon crangon[END_REF], read in Giménez, 2006) is the only author to have studied the effects of the interaction between temperature and laying season on larval development pathways (C. crangon).

To investigate the effects of the interaction between temperature and seasonal variations in larval size on the variability of larval developmental pathways, we chose to work with the caridean shrimp, Palaemon serratus (Pennant, 1777). This species is abundant in European coastal environments and has a life span of 3 to 5 years [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF]. Its reproductive cycle consists of two annual clutches (commonly called "winter laying" and "summer laying"), characterised by larger eggs, and thus larvae, in the first clutch [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF][START_REF] González-Ortegón | Maternal Trophic Status and Offpsring Phenotype in a Marine Invertebrate[END_REF]. This species is known to carry out larval development with a variable number of instars; mostly 6 to 9 but ranging from 6 to 22 in the laboratory [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF] J o u r n a l P r e -p r o o f [START_REF] Fincham | Larval keys and diagnoses for the subfamily Palaemoninae (Crustacea: Decapoda: Palaemonidae) in the north-east Atlantic and aspects of functional morphology[END_REF][START_REF] Kelly | Effects of temperature and salinity on the survival and development of larval and juvenile Palaemon serratus (Decapoda: Palaemonidae) from Irish waters[END_REF]Reeve, 1969a). This developmental plasticity is mainly temperature-mediated, with the larvae of this species performing fewer instars in warm water than in cold water [START_REF] González-Ortegón | Environmentally mediated phenotypic links and performance in larvae of a marine invertebrate[END_REF]. However, variations in developmental pathways due to temperature-mediated plasticity have not been investigated in P. serratus (larval stages and instars). The effects of seasonal variation in the size of the larvae of this species on their development have also not been studied, although this should also influence their developmental pathways [START_REF] González-Ortegón | Maternal Trophic Status and Offpsring Phenotype in a Marine Invertebrate[END_REF]. We hypothesise the following two scenarios. Large winter larvae develop faster than small summer larvae at the same temperature, completing fewer larval instars. Furthermore, the interaction between temperature and laying season has synergistic effects on developmental pathways. To test these hypotheses, we monitor the different stages and calculate the number of instars reached during the development of larvae from winter and summer females of P. serratus incubated at 3 temperatures, 12, 16 and 20°C.

Material and methods

Collection and maintenance of females

To study the larval development of the winter and summer laying, ovigerous P. serratus females (6 per seasons ; Fig. 1) were respectively sampled between 29/04/2021 and 12/05/2021, and between 23/06/2021 and 28/06/2021 by hand-netting on Saint-Jouin-Bruneval foreshore (Seine-Maritime, France). Only the specimens carrying eggs at the end of development were selected for the study. The length of the cephalothorax was constant for both seasons, measured respectively 40.8 mm (± 1) for winter females and 39.8 mm (± 1.1) for summer females, in mean (± S.E.). The shrimps were transferred to the laboratory of the University of Le Havre (Seine-Maritime, France), and then maintained under photoperiod 12: 12 (L: D) in 100 L tank J o u r n a l P r e -p r o o f temperature-controlled at 16°C, filled with filtered aerated natural seawater at 30 ppt (± 4) of salinity (Fig. 1). Seawater was renewed by half 3 times per week to avoid nitrate increase. Each specimen was isolated in wire baskets covered with fine mesh and fed individually twice a week ad libitum with pieces of squid. Larval hatching was checked daily.

Larval maintenance

The objective was to incubate larvae from 12 clutches (6 winter and 6 summer) at three temperature treatments of 12, 16 and 20°C, as shown in Figure 1. On hatching, actively swimming larvae were separated from females using a pipette. The rostro-telson length of 30 newly hatched larvae from each clutch was measured [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF]. For each clutch, 3 groups of 104 larvae were distributed in three conical vessels, with filtered seawater at 30 ppt of salinity (± 4), at a density of 100 larvae per L [START_REF] Reeve | Laboratory culture of the prawn Palaemon serratus[END_REF][START_REF] Torres | Methods to study organogenesis in decapod crustacean larvae. I. larval rearing, preparation, and fixation[END_REF]. The water was initially at the same temperature as for the maintenance of the females, and then the conical vessels were distributed to 3 incubators (temperature ± 0.3°C, regulated by a TK700 cooling unit by TECO), with photoperiod 12: 12 (L: D) and constant thin aeration. This resulted in a gradual change which allowed time for the larvae to acclimate to the new temperatures [START_REF] Richard | Tolérance aux températures extrêmes de Palaemon serratus (Pennant): Influence de la taille et de l'acclimatation[END_REF]. Larvae were fed daily with freshly-hatched Artemia sp. nauplii at a density of 10 nauplii per mL [START_REF] Reeve | Laboratory culture of the prawn Palaemon serratus[END_REF]. The water was changed (100 %), and larval mortality and development, assessed by identification of stages (see section 2.4), were monitored 3 times a week. During development, the oxygen saturation was 103 % (± 4), the pH was 8 (± 0.1) and the ammonium concentration was 1 mg per L (± 0.3), in mean (± S.E.). On moulting to the juvenile stage, shrimps were removed.

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

Intermoult time and number of instars

As several successive stages can occur without morphological change, it is necessary to determine the number of larval instars. Because the larvae are mass-reared, the larval average intermoult time cannot be individually measured and then need to be calculated. The average intermoult time of P. serratus larvae is known to be constant in good zootechnical conditions [START_REF] González-Ortegón | Environmentally mediated phenotypic links and performance in larvae of a marine invertebrate[END_REF]Reeve, 1969a). We compiled data on the duration of larval development of P. serratus under controlled temperature conditions available in the literature. We did not use data concerning larvae exposed to other stresses (salinity, contaminant, ...). This allows the construction of a function expressing the theoretical intermoult time of larval as a function of temperature. For each juvenile obtained in the experiment, its number of instars realised is estimated by dividing its developmental time by the corresponding theoretical intermoult time.

Identification of larval stages

The different larval stages are identified using morphological criteria. In the larvae of European palaemonidae, the morphological criteria corresponding to the rostrum, caudal region and pleopods are commonly used to identify the different larval stages [START_REF] Fincham | Larval keys and diagnoses for the subfamily Palaemoninae (Crustacea: Decapoda: Palaemonidae) in the north-east Atlantic and aspects of functional morphology[END_REF]. [START_REF] Fincham | Larval keys and diagnoses for the subfamily Palaemoninae (Crustacea: Decapoda: Palaemonidae) in the north-east Atlantic and aspects of functional morphology[END_REF] described 9 larval stages for P. serratus, while Campillo (1979) described 6. Description and terminology for the naming of larval stages differ between these authors, but it is possible to do correspondences and characterise 10 different larval stages (Table 1; according to Duflot et al., unpublished). According to these authors, these larval stages are potentially all observable, but are not obligatory stages through which each larvae must pass to develop. On the recent recommendations of [START_REF] Møller | Patterns of larval development[END_REF] regarding the terminology of caridean shrimp larvae, we call these stages zoea 1 to zoea 10 in chronological order of morphogenesis.

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

Table 1: Correspondences between the nomenclatures of the larval stages of Palaemon serratus 176 described [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF], [START_REF] Fincham | Larval keys and diagnoses for the subfamily Palaemoninae (Crustacea: Decapoda: Palaemonidae) in the north-east Atlantic and aspects of functional morphology[END_REF]

Statistical analysis

Statistical procedures were implemented with R software (R Core Team, 2020). The relationship between larval intermoult time and temperature (Fig. 3; Eq. 1) was obtained using the nls function. The difference in larval size between winter and summer laying was tested using a nonparametric Wilcoxon Rank Sum test. To test the effects of temperature and laying season on the rate of larval metamorphosis and the development time of the metamorphosed larvae, we used mixed-effects models to consider the fact that for each of the seasons, the larvae used in the different temperatures came from the same females. For the rate of larval metamorphosis, we fitted generalised linear mixed-effects model with a logit link function and a binomial error distribution (glmer function of the package lme4) and performed deviance analysis procedure including interaction terms (using the package car). For the development time of the metamorphosed larvae, we fitted linear mixed-effects model (using the function lmer of the package lme4) and performed deviance analysis procedure including interaction terms (using the package car).

Results

Zootechnical performances during larval development.

The average total length of hatching larvae was significantly higher in winter (3.68 ± 0.07 mm), than in summer (3.18 ± 0.05 mm) in mean ± S.E. (Wilcoxon Rank Sum test; p < 0.05).

While all females produced juveniles at 16 and 20°C, only 5 winter and 3 summer females produced juveniles at 12°C. At 12°C, only 18 of the 624 winter larvae and 5 of the 624 summer larvae metamorphosed (Fig. 2a). At 16 and 20°C respectively, 30 % (± 5) and 41 % (± 7) of the winter larvae metamorphosed, compared to 43 % (± 7) and 55 % (± 7) of the summer larvae, in mean (± S.E.). The rate of larval metamorphosis varied significantly with temperature and

Number of instars achieved by larvae.

The compilation of literature data on the larval development of P. serratus under controlled temperature conditions allows us to construct a function expressing the theoretical larval intermoult time versus temperature (Fig. 3; Eq. 1): 𝐿𝐼𝑇 = 147.96 𝑒 (-0.25 𝑡𝑒𝑚𝑝) + 2.45 Eq. 1

where LIT is the theoretical larval intermoult time and temp the incubation temperature.

This function allows us to calculate the theoretical intermoult times at 12°C (9.8 days), 16°C

(5.2 days) and 20°C (3.4 days) as well as the number of instars achieved by each juvenile obtained in this experiment. In all experimental conditions, the larvae that metamorphosed did so after 6 to 13 larval instars (Fig. 4). Only about 3.7 % of them developed for more than 9

instars, and about 1 % for 6 instars. While about 33 and 45 % of the larvae performed 7 instars at 16 and 20°C, about 43 and 31 % of them performed 8 instars. At 20°C, larvae metamorphosing after 7 instars represented about 40 % of the total in winter and 50 % in J o u r n a l P r e -p r o o f summer. At 16°C, 3 larvae metamorphosed after 6 instars in summer, while none did so in winter. than 50 % mortality from the zoea 1 stage (Fig. 5).

Succession of larval stages.

All 10 larval stages described were observed at each temperature. However, we observed differences in the proportions of the different larval stages. At 12, 16 and 20°C, respectively, at least 15.3, 5.9 and 0.4 % of the larvae passed through the zoea 4 stage, and at least 9.1, 0.6 and 0.3 % of the larvae passed through the zoea 6 stage.

Furthermore, during the monitoring of the larval stages presented here, we frequently observed larvae with 2 rostral spines (criteria for zoea 3) and pleopod buds (criteria for zoea 5). This larval stage seems to take place instead of the zoea 3 stage, so we called it zoea 3'. This never described larval stage was not observed at 12°C. At 16 and 20°C, respectively, at least 58.1 and 67.8 % of the winter larvae passed through the zoea 3' stage, compared to at least 20.8 and 19.9 % of summer larvae.

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

Discussion

Regardless of the incubation temperature and the laying season from which the larvae hatched, we observed a great variability in the larval development of P. serratus (6 to 13 larval instars and 6 to 11 larval stages). The different developmental pathways observed underline the developmental plasticity of this species.

In contrast to several authors studying the number of larval instars of decapods during development (Reeve, 1969a;[START_REF] Dalley | The survival and development of the shrimp Crangon crangon (L.), reared in the laboratory under non-circadian light-dark cycles[END_REF][START_REF] Linck | Einfluß von Temperatur und Salzgehalt auf die Larven der Nordseegarnele Crangon crangon[END_REF], read in Giménez, 2006), we did not rear the larvae individually. This choice was made in view of the survival rates of P. serratus larvae in the laboratory available in the literature. While González-Ortegón and Giménez (2014) and [START_REF] Lassus | Etude de la sensibilité thermique des larves du crustacé décapode: Palaemon serratus (Pennant)[END_REF] achieved respectively 40 and 45-70% metamorphosis when rearing larvae individually, [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF] and [START_REF] Reeve | Laboratory culture of the prawn Palaemon serratus[END_REF] achieved respectively 76 and over 90% metamorphosis with mass-rearing. Furthermore, Reeve (1969a) observed longer larval development with lower survival when rearing P. serratus individually than when massrearing.

We calculated the number of instars made by the larvae from their average intermoult time according to bibliographic data. Intermoult times obtained for the same temperature may vary slightly between the 6 sources used (Fig. 3). This can be explained by the differences in experimental protocol and by maternal effects. The limited information available on this second factor complicates discussions on this subject. The females used in these 6 experiments came from populations in Ireland, Wales, Brittany and the Mediterranean (the latter being phylogenetically distant from the Atlantic populations; [START_REF] Weiss | Genetic differentiation between Mediterranean and Atlantic populations of the common prawn Palaemon serratus (Crustacea: Palaemonidae) reveals uncommon phylogeographic break[END_REF]. Only González-Ortegón and Giménez (2014) specify the period of collection of females (autumn) as well as their size (between 17 and 24 mm from the postorbital margin to the end of carapace, between 18 and 22 mm in our study). According to Reeve (1969a) and González-Ortegón and Giménez (2014), the intermoult time of P. serratus is constant during larval development under good J o u r n a l P r e -p r o o f zootechnical conditions. Nevertheless, these authors reared the larvae of this species between 18 and 24°C. It is possible that the intermoult time of P. serratus larvae increases as development proceeds at lower temperatures, as reported by [START_REF] Kelly | Effects of temperature and salinity on the survival and development of larval and juvenile Palaemon serratus (Decapoda: Palaemonidae) from Irish waters[END_REF] and [START_REF] Lassus | Etude de la sensibilité thermique des larves du crustacé décapode: Palaemon serratus (Pennant)[END_REF] between 10 and 13°C. However, the few individuals that metamorphosed at 12°C leads us to put the interpretation of the number of instars carried out at this temperature into perspective.

Temperature-mediated developmental plasticity

The larval development of P. serratus is temperature-driven. Here, metamorphosis rates were temperature dependent (about 2, 36 and 49 % at 12, 16 and 20°C respectively). [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF] experienced the highest rates of metamorphosis (85 %) by incubating P. serratus larvae from Roscoff (France) at 27°C, a temperature above the local maximum. Other authors who studied the larval development of P. serratus at temperatures between 10 and 13°C observed a mortality of all individuals between the third and fourth larval instar [START_REF] Kelly | Effects of temperature and salinity on the survival and development of larval and juvenile Palaemon serratus (Decapoda: Palaemonidae) from Irish waters[END_REF][START_REF] Lassus | Etude de la sensibilité thermique des larves du crustacé décapode: Palaemon serratus (Pennant)[END_REF][START_REF] Yagi | The role of temperature and salinity on moult, metamorphosis and growth at each larval stage of Palaemon serratus (Pennant) (Crustacea, Decapoda, Palaemonidae)[END_REF]. We also observed very low survival rates at 12°C, but we managed to get some individuals to metamorphosis (< 2%) after 7-13 instars. We chose to incubate larvae at 12°C because this is the temperature of the sea water in the Bay of Seine (Normandy) at the beginning of May [START_REF] Claquin | MAREL SMILE buoy data and metadata from coriolis Data Centre[END_REF], the date at which the larvae from the winter spawning are released by females of the same size as those used for this study (personal observations). Mortality at 12°C was constant during development in the laboratory.

At this temperature, only the larvae from 3 of the 12 females suffered mortality rates above 50 % by the end of the first instar. The mortality rate of the larvae was approximately 52 % after 30 days of incubation, when most of them were in their fourth instar. The thermal optimum of crustacean larvae experiences an ontogenic shift [START_REF] Sastry | Diversity in metabolic adaptation of pelagic larval stages of two sympatric species of brachyuran crabs[END_REF]. These variations between larval stages are due to temperature-induced changes in metabolic rates J o u r n a l P r e -p r o o f [START_REF] Small | Stage-Specific Changes in Physiological and Life-History Responses to Elevated Temperature and Pco2 during the Larval Development of the European Lobster Homarus gammarus (L.)[END_REF]. During the month of May, the temperature of the sea water in the Bay of Seine increases by approximately 3°C [START_REF] Claquin | MAREL SMILE buoy data and metadata from coriolis Data Centre[END_REF]. A constant temperature of 12°C in the laboratory negatively affects the survival rates of P. serratus larvae. These effects probably increase during ontogeny.

In addition to decreasing survival, colder water increases the duration of development. First of all, as we know from the literature, the intermoult time increases as the temperature decreases following a negative exponential relationship (Fig. 2). Secondly, larvae tend to go through a greater number of instars in colder waters (Fig. 4). Although the duration of development is variable between individuals incubated at the same temperature, this is not so with the value of the average duration per family (Fig. S1, Supplement). The plastic response to temperature therefore appears to be the same for larvae produced by different females. However, differences in survival rates appear between larvae of different families (Fig. S1, Supplement).

Concerning the larval stages, zoea 4 and zoea 6 are more frequent in colder water (Fig. 5 to 7).

These larval stages can be interpreted as supernumerary, or intercalated, stages occurring under unfavourable conditions such as low temperature. This would explain why [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF] did not describe them at 27°C (Table 1). Conversely, at 16 and 20°C, many larvae with zoea 3 criteria (2 rostral spines) and zoea 5 criteria (pleopod buds) were observed (about 39 and 44 % at 16 and 20°C respectively). This never described stage, which we have called zoea 3', seems to replace the zoea 3 stage. This larval stage can be interpreted as a sign of abbreviated As stated in the introduction, this temperature-mediated developmental plasticity has already been demonstrated in caridean shrimps. The larvae follow shorter developmental pathways in temperatures closer to their thermal optimum [START_REF] Críales | Experimental studies on the larval development of the shrimps Crangon crangon and C. allmanni[END_REF][START_REF] Linck | Einfluß von Temperatur und Salzgehalt auf die Larven der Nordseegarnele Crangon crangon[END_REF], read in Giménez, 2006;Oliphant et al., 2013). Generally, at a given temperature, the more instars the larvae develop through, the larger the juveniles. This trend has been demonstrated in the larvae of several decapods including Chasmagnathus granulata [START_REF] Giménez | Larval growth in the estuarine crab Chasmagnathus granulata: the importance of salinity experienced during embryonic development, and the initial larval biomass[END_REF] and P. varians (Oliphant et al., 2013). Therefore, it would be interesting to compare the consequences of temperature-mediated developmental plasticity of P. serratus larvae on the phenotype of juveniles.

These differences in survival between thermal conditions can also be explained by differences in nutritional requirements. Crustacean larvae require long-chain polyunsaturated fatty acids (PUFAs) for development, such as eicosapentaenoic acid, or EPA, and docosahexaenoic acid, or DHA [START_REF] Anger | Patterns of growth and chemical composition in decapod crustacean larvae[END_REF][START_REF] Bergé | Fatty Acids from Lipids of Marine Organisms: Molecular Biodiversity, Rolesas Biomarkers, Biologically Active Compounds, and Economical Aspects[END_REF]. These are generally synthesised by phytoplankton and transferred into the food chain [START_REF] Lubzens | Potential advantages of frozen algae (Nannochloropsis sp.) for rotifer (Brachionus plicatilis) culture[END_REF]. PUFAs, DHA in particular, maintain membrane integrity and viscosity [START_REF] Estévez | Fatty Acid Composition of Neural Tissues of Normally Pigmented and Unpigmented Juveniles of Japanese Flounder Using Rotifer and Artemia Enriched in n-3 HUFA[END_REF][START_REF] Los | Membrane fluidity and its roles in the perception of environmental signals[END_REF][START_REF] Pagliarani | Effect of diets containing different oils on brain fatty acid composition in sea bass (Dicentrarchus labrax L.)[END_REF], especially in cold waters [START_REF] Harwood | Fatty Acid Metabolism[END_REF][START_REF] Munro | The role of polyunsaturated fatty acids in the expression of torpor by mammals: a review[END_REF]. Like many authors, we fed P. serratus larvae with artemia nauplii [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF][START_REF] Reeve | Laboratory culture of the prawn Palaemon serratus[END_REF][START_REF] Wickins | The food value of Brine Shrimp, Artemia salina L., to larvae of the prawn, Palaemon serratus Pennant[END_REF][START_REF] Yúfera | Effect of prey density on feeding rates during larval rearing of Palaemon serratus Pennant (Crustacea: Palaemonidae)[END_REF]. Although these are traditionally used to feed decapod larvae, they are known to be deficient in PUFAs [START_REF] Narciso | Fatty Acid Profile of Palaemon Serratus (Palaemonidae) Eggs and Larvae During Embryonic and Larval Development Using Different Live Diets[END_REF][START_REF] Navarro | Lipid conversions during enrichment of Artemia[END_REF]. These deficiencies may explain the very low survival of P. serratus larvae in cold water observed here and by the authors mentioned in the paragraph above. PUFAs also influence growth, stress and disease resistance in decapods [START_REF] Anger | Patterns of growth and chemical composition in decapod crustacean larvae[END_REF][START_REF] Kanazawa | Effects of dietary lipids, fatty acids, and phospholipids on growth and survival of prawn (Penaeus japonicus) larvae[END_REF][START_REF] Mourente | Effects of salinity and dietary DHA (22:6n -3) content on lipid composition and performance of Penaeus kerathurus postlarvae[END_REF]. It would be interesting to study the J o u r n a l P r e -p r o o f effects of the interaction between temperature and diet quality on the developmental plasticity of P. serratus.

Developmental plasticity induced by laying season and its interaction with temperature

The newly hatched larvae used in this study were larger in winter than in summer. These results are consistent with González-Ortegón et al. (2018) who observed difference in P. serratus larval biomass between laying seasons. Because of this difference in phenotype, we hypothesised that P. serratus larvae from winter females would develop faster than those from summer females. These effects were not significant in our data analyses. At a given temperature, summer larvae even appeared to perform fewer larval instars than winter larvae.

These results are in contradiction with those obtained on another caridean shrimp species [START_REF] Linck | Einfluß von Temperatur und Salzgehalt auf die Larven der Nordseegarnele Crangon crangon[END_REF], read in Giménez, 2006). The author incubated larvae of C. crangon, from large winter eggs and small summer eggs, at two relatively cold and warm temperatures (12 and 18°C). Regardless of the temperature, summer larvae metamorphosed after a greater number of larval instars than winter larvae. We need to further investigate the effects of the interaction between the laying season and environmental conditions on the developmental plasticity of P. serratus.

The winter larvae were larger than the summer ones. Few authors have studied the effects of larval size or larval biomass on developmental plasticity of decapod larvae. The number of larval instars made by larvae decreases with increasing larval size or larval biomass in C. crangon [START_REF] Paschke | Seasonal variation in starvation resistance of early larval North Sea shrimp Crangon crangon (Decapoda: Crangonidae)[END_REF], C. granulata [START_REF] Giménez | Linking life history traits in successive phases of a complex life cycle: effects of larval biomass on early juvenile development in an estuarine crab, Chasmagnathus granulata[END_REF], Homarus gammarus [START_REF] Moland | Maternal influences on offspring size variation and viability in wild European lobster Homarus gammarus[END_REF], P. varians (Oliphant et al., 2014;Oliphant and Thatje, 2013) and P. australiensis [START_REF] Hancock | The relationship between egg size and embryonic and larval development in the freshwater shrimp Paratya australiensis Kemp (Decapoda: Atyidae)[END_REF], a phenomenon that is accentuated under adverse conditions.

Although they highlighted the decrease in the number of larval instars with increasing initial J o u r n a l P r e -p r o o f larval biomass in P. serratus larvae under starvation conditions, González-Ortegón & Giménez (2014) did not confirm this trend under favourable feeding conditions. This result is consistent with those presented here.

Although the development time of the larvae in the two laying seasons did not differ, at 16 and 20°C, the zoea 3' stage was observed more often in winter larvae than in summer larvae (Fig. 5 to 7). The morphogenesis of the winter larvae (pleopod buds) was earlier than that of the summer larvae, at least in the early larval stages. The winter larvae may have received more available energy resources (transmitted by the mother) than summer larvae and used them during these early instars. This phenomenon may have been favoured by the choice of females used. The cephalothorax of the winter females measured on average about 40 mm. These would be more than two years old, which corresponds to less than 2 % of the female population of P. serratus [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF]. The limited information available on the size of the females and the date of their sampling in previous publications does not allow comparison of our results.

The lack of description of the zoea 3' stage in previous studies can be explained by the choice of females used. We need to investigate the effects of female age on maternal investment and developmental plasticity in P. serratus.

When they hatch, decapod larvae have visible vitelline reserves in the cephalothorax in the form of yolk droplets [START_REF] Anger | Physiological and biochemical changes during lecithotrophic larval development and early juvenile growth in the northern stone crab, Lithodes maja (Decapoda: Anomura)[END_REF]Oliphant and Thatje, 2014). These can be used to cope with starvation conditions during the first larval instars (Anger, 1995a(Anger, , 1995b;;Thessalou-Legaki et al., 1999), e.g. when larvae are exported from a brackish environment to the marine environment [START_REF] Anger | From lecithotrophy to planktotrophy: ontogeny of larval feeding in the Amazon River prawn Macrobrachium amazonicum[END_REF]Oliphant and Thatje, 2014). Such yolk droplets are observed in hatched larvae of P. serratus (personal observation). [START_REF] Narciso | Fatty Acid Profile of Palaemon Serratus (Palaemonidae) Eggs and Larvae During Embryonic and Larval Development Using Different Live Diets[END_REF] [START_REF] Oliphant | Decapod crustacean larval developmental plasticity and the evolution of lecithotrophy and abbreviated development[END_REF], as a result of the laying season and their effects on larval development will provide a better understanding of the developmental plasticity in P. serratus larvae.

In P. serratus, winter larvae are larger than summer larvae. We expected that this phenotypic difference would allow winter larvae to develop faster than summer larvae at the same temperature. Although we did not observe this result, we did observe earlier morphogenesis in winter larvae at the warmest temperatures. As explained in the previous paragraphs (see section 4.2), we believe that this phenomenon is related to the amount of energy allocated per offspring.

This pattern of maternal investment buffers winter larvae from variable early season conditions (low temperature, Torres et al., 2020; less abundant planktonic food resources, [START_REF] Paschke | Seasonal variation in starvation resistance of early larval North Sea shrimp Crangon crangon (Decapoda: Crangonidae)[END_REF]. Furthermore, the negative effects of food limitation on larval survival are exacerbated by unfavourable temperatures (Torres and Giménez, 2020). The few months separating the hatching of the larvae from the two laying periods of P. serratus result in two cohorts of juveniles [START_REF] Campillo | Contribution à l'étude de la crevette rose Palaemon serratus (Pennant) : Exploitation, Biologie, Elevage, Epidémiologie[END_REF]. As these cohorts share the same habitats, they compete for resources or even cannibalise [START_REF] Moksnes | Predation on postlarvae and juveniles of the shore crab Carcinus maenas: importance of shelter, size and cannibalism[END_REF]. The shift in the recruitment periods of these two cohorts results in a difference in the size of the individuals (P. serratus juveniles of 0.5g which double or triple their weight in 2 months depending on their sex at temperatures between 18 and 29°C; [START_REF] Rodriguez | Growth and sexual maturation of Penaeus kerathurus (Forskäl, 1775) and Palaemon serratus (Pennant) in salt ponds[END_REF]. These traits probably favour juveniles from winter laying over those from summer laying in years when early season conditions are favourable [START_REF] Temming | Facultative lecithotrophy during larval development of the burrowing shrimp Callianassa tyrrhena (Decapoda: Callianassidae)[END_REF]. The population structure and dynamics of P. serratus should be studied to assess the settlement success of winter and summer cohort. This would facilitate understanding the effects of juvenile recruitment phenology on their interactions.

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

Conclusion

Although larval development of P. serratus is plastic in response to temperature, the interaction between temperature and laying season does not seem to influence its developmental pathways as expected. As the development of some groups of decapods is gradual, understanding their developmental pathways will require consideration of the number of instars and the succession of larval stages.

Vitae

Jean-Baptiste Baudet is working on crustacean ecology, developmental plasticity, phenotypic based on data from bibliography (Reeve, 1969a, squares;Campillo, 1979, filled circles;Lassus & Maggi, 1980, cross;Yagi & Ceccaldi, 1985, stars;Kelly et al., 2012, filled squares;González-Ortegón & Giménez, 2014, filled triangle). J o u r n a l P r e -p r o o f • Higher temperature decreased the development time and the number of instars.

Figure captions

• The seasonal phenotype had no effect on the development time.

• Supernumerary stages were observed at low temperatures.

• Early morphogenesis was observed at high temperatures.

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

  It seems necessary to describe the number of larval instars reached and the different successive larval stages to accurately characterise the larval developmental pathway. Some authors have study the developmental plasticity of caridean shrimp larvae in response to environmental factors by focusing on larval stages, larval instars or both. Hancock (1998) described the larval stages of Paratya australiensis encountered during development at 2 temperatures. Oliphant et al. (2014) counted the number of larval instars achieved by Palaemonetes varians in relation to food availability. González-Ortegón and Giménez (2014) carried out the same type of experiment on P. serratus larvae adding the effects of temperature and salinity. Oliphant et al.

  [START_REF] Fincham | Larval keys and diagnoses for the subfamily Palaemoninae (Crustacea: Decapoda: Palaemonidae) in the north-east Atlantic and aspects of functional morphology[END_REF]

  J o u r n a l P r e -p r o o f laying season without interaction of these two factors (Deviance analysis, interaction term: Chisq = 3.18, p = 0.07; temperature: Chisq = 16.19, p < 10 -5 ; season: Chisq = 26.6, p < 10 -7 ; n = 6 per temperature and season).The metamorphoses took place between days 65 and 111 at 12°C, between days 30 and 56 at 16°C and between days 19 and 42 at 20°C (Fig.2b). The development time of the metamorphosed larvae varied significantly between the three incubation temperatures, but neither as a function of the laying season nor as a result of the interaction of these two factors (Deviance analysis, interaction term: Chisq = 0.76, p = 0.4; temperature: Chisq = 138.73, p < 10 -16 ; season: Chisq = 0.62, p = 0.43; n = 6 per temperature and season).

Figures 5

 5 Figures 5 to 7 show the number of individuals observed for each larval stage during

  measured a decrease in fatty acid content during the first 48 hours of development of P. serratus larvae under starvation conditions. Their results and ours seem to confirm the use of vitelline reserves during the first larval instars in P. serratus. Future analyses to compare the quantity J o u r n a l P r e -p r o o f and quality of resources allocated per offspring, called POI (i.e. per offspring investment;

  links and larval ecology during his PhD project started in December 2020. His previous experience was in penaeid shrimp research and development. Benoît Xuereb is a researcher at the research unit for Environmental Stresses and Biomonitoring of Aquatic Ecosystems laboratory (SEBIO), Le Havre -Normandy University. His research interest focuses on the description of ecophysiology and life traits in the palaemonid shrimps, with a perspective to propose reliable effect-based methods for ecotoxicological applications.J o u r n a l P r e -p r o o f Aurélie Duflot works in the SEBIO laboratory on various fields related to the physiology of marine organisms and is an expert on the embryo-larval development of Palaemon serratus. Agnès Poret is an engineer at the SEBIO laboratory working on marine invertebrates, especially on reproduction and immunology. Emeline Maniez has completed a master's degree in marine sciences. She specialises in marine and fisheries ecology, as well as in the functioning and management of marine ecosystems. She is interested in shrimp and plankton, for example, in relation to global changes and/or fisheries. Frank Le Foll is a professor of organismal biology at the University of Le Havre Normandie and currently the head of the Environmental Stresses and Biomonitoring of Aquatic Ecosystems laboratory (SEBIO). SEBIO carries out basic and translational research in bioenvironmental sciences and ecotoxicology. Frank research interest focuses on invertebrate immune cell activity, host-microbe-environment interactions, multixenobiotic resistance phenotype, immunomarker research and innovative cage design for biological monitoring of polluted water bodies. Romain Coulaud is a researcher at the research unit for Environmental Stresses and Biomonitoring of Aquatic Ecosystems laboratory (SEBIO), Le Havre -Normandy University. His research interest focuses on the ecology, ecophysiology and ecotoxicology of macroinvertebrates (particularly crustaceans), using multi-scale approaches ranging from sub-J o u r n a l P r e -p r o o f individual biomarkers to community functioning and combining laboratory and field experiments and modelling.

Figure 1 :

 1 Figure 1: Experimental design to study the effects of temperature and laying season on larval

Figure 2 :

 2 Figure 2: (a) Metamorphosis rate (%) and (b) development time (days) of Palaemon serratus

Figure 3 :

 3 Figure 3: Theoretical larval intermoult time of Palaemon serratus versus rearing temperature,

Figure 4 :

 4 Figure 4: The relative frequency (%) of the total number of larval instars reached by

Figure 5 :

 5 Figure 5: Number of Palaemon serratus larvae observed for each larval stage during their

Figure 6 :

 6 Figure 6: Number of Palaemon serratus larvae observed for each larval stage during their

Figure 7 :Figure 2 Figure 3 J

 723 Figure 7: Number of Palaemon serratus larvae observed for each larval stage during their

Figure 5 J

 5 Figure 5

Figure 6 J

 6 Figure 6

  Figure 7 771

Acknowledgments

We would like to thank the Thalasso of Deauville for the supply of natural sea water and the University of Le Havre for the facilities. This study was supported by the Normandie Region and the European Maritime and Fisheries Fund (Action 28 "Partnerships between scientists and fishermen"; GeDuBouq project).

Funding

This work was supported by the EMFF Gedubouq program of the European Union.

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