The Consequences of Daily Cyclic Hypoxia on a European

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The consequence of daily cyclic hypoxia on a European grass shrimp Peruzza, Luca; Gerdol, Marco ; Oliphant, Andrew; Wilcockson, David; Pallavicini, Alberto; Hawkins, Lawrence; Thatje, Sven; Hauton, Chris

Published in: Functional Ecology DOI: 10.1111/1365-2435.13150 Publication date: 2018 Citation for published version (APA): Peruzza, L., Gerdol, M., Oliphant, A., Wilcockson, D., Pallavicini, A., Hawkins, L., Thatje, S., & Hauton, C. (2018). The consequence of daily cyclic hypoxia on a European grass shrimp: From short-term responses to long-term effects. Functional Ecology, 32(10), 2333-2344. https://doi.org/10.1111/1365-2435.13150

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Download date: 09. Jul. 2020 Accepted Article This article copyright.protected is by rights All reserved. 10.1111/1365-2435.13150as doi: leadto differences between this versionand theVersion Please Record. of article this cite beenthrough thecopyediting, typesetting,pagination andproofreadingprocess,which may beenThisandarticle has accepted undergone publication for peer not reviewfull has but Peruzza Luca effects long-term The consequences cyclic of daily hypoxia on aEuropean grass shrimp: from short-term responses to Section:Physiological Animal Ecology Overgaard Johannes Dr Editor: Article :Research type Article DR PERUZZA LUCA (Orcid: ID 0000-0001-5001-8653) reproduction, ammonium excretion excretion ammonium reproduction, transcriptomic shrimp, hypoxia, cyclic Keywords: Aberystwyth, Ceredigion 3DA, SY23 UK; Penglais, University, Aberystwyth Sciences, Rural and Environmental Biological, of Institute 3 2 University ofDepartment Trieste, Lifeof Sciences,Via Giorgieri Trieste, 34127 5, Italy UK; 3ZH, SO14 Southampton, Southampton, 1 Ocean and Earth Science, University of Southampton, of University Science, Earth Oceanand 1 Hawkins 1 , Sven Thatje , Sven 1 , MarcoGerdol 1 , Chris Hauton , Chris 2 , Andrew Oliphant , Andrew Functional Ecology Ecology Functional 1

3 , David Wilcockson David , s, gill morphology, moult cycle, body size, size, body cycle, moult morphology, gill s,

National Oceanography Centre Centre Oceanography National 3 , AlbertoPallavicini 2 , Lawrence Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. ABSTRACT: [email protected] email: Kingdom, 3ZH, United SO14 Southampton, Southampton, Centre Oceanography National Southampton, of University Science, and Earth Ocean address: Peruzza, Luca author: Corresponding Running title: Physiological and molecular effects of cyclic hypoxia hypoxia cyclic of effects molecular and Physiological title: Running 1. 2. 3.

oxygen partial pressure (pO pressure oxygen partial stre hypoxic cyclic daily experience can species where environments variable are marshes Salt crustaceans. and fish estuarine and marine Salt marshes are a keycoastal environment for (7.1±1.8 hours day August in habitat its in experienced currently regime hypoxic cyclic daily the to Europe, the shrimp exposing of consequences physiological the assessed we conditions, laboratory under Here, response to cyclic hypoxia, with the benefit benefit the with hypoxia, cyclic to response in triggered is moulting faster that indicate data morphological and phenotypic Overall, conditions (~3kPa) in ~12-hours. ~12-hours. in (~3kPa) conditions µm with shrimpscyclic in hypoxiahaving a13.6% larger lamellar surface area(measured in detected, were gills the to changes morphological days, 16 After genes. moult stage-related on hypoxia cyclic of effect an indicated cycle moult entire an over proteins cuticular of two expression of pattern the Similarly, normoxia. in animals to compared hypoxia cyclic kept in of acceleration We recorded an for 7-hours each night and in normoxic conditions for the rest of the time. time. ofthe rest the for conditions normoxic in and night each for 7-hours 2 /mg animal) than normoxic animals, which could improvegas exchange capacity. Palaemon varians, Palaemon -1 below 4.0kPa). below In the laboratory adults were kept atwater pO

2 ) from supersaturated conditions (~42kPa) to extremely hypoxic hypoxic extremely to (~42kPa) conditions supersaturated ) from a species commonly found in the salt marshes of northern northern of marshes salt the in found commonly species a P. varians P. ’ moult cycle, which was 15% shorter in animals animals in shorter 15% was which cycle, moult ’ that gill modifications can be prompted more beprompted can modifications gill that ss, characterized by profound variations in in variations byprofound characterized ss, their important role as nursery habitats for for habitats asnursery role their important

2 <4.5kPa

Accepted Article pressure (pO partial oxygen and temperature particularly parameters, physiochemical main the in variability characteristic of salt marshes isthat these habitatscancharacterised be by substantial a diel important Another 2013). (Oliphant UK marshes, salt Lymington ofthe case the in as birds, roosting and feeding breeding, of number a large support Theycan 2006). Hampel & (Cattrijsse nekton role asnursery habitats fringesimportant for their This article is protectedThisarticle is byAll rightsreserved. copyright. INTRODUCTION: normoxic periods) from stressful conditions are given to the exposed organism, under cyclic hypoxic hypoxic cyclic under organism, exposed the to given are conditions stressful from periods) normoxic (i.e. time recovery no hypoxia chronic under Whilst, hypoxia. chronic and cyclic between difference hypoxia. cyclic daily as known is that phenomenon a night, the during kPa) (~1 conditions hypoxic to day the during kPa) (>21 conditions supersaturated 2000; Sokolova rest to possibility the have organisms conditions 4.

Similarly, ammonium excretion was reduced by 66-75% during the 1 the during 66-75% by reduced was excretion ammonium Similarly, (dur rates feeding in decrease a 50% recorded we animals, exposed hypoxic cyclic in day, experimental first On the rapidly in order to meet oxygen requirements of the body. body. the of requirements oxygen meet order to in rapidly Salt marshes are a key habitat of conservation importance along many temperate coastal coastal temperate many along importance conservation of habitat key a are marshes Salt The successionofhypoxic and normoxic periodscharacterises the most fundamental impaired by a cyclic hypoxic regime that is currently found in its natural habitat. habitat. natural its in found currently is that regime hypoxic cyclic a by impaired are parameters physiological many environment, coastal key a in living shrimp decapod in a how, underline results Overall, ~24%. by egg each in yolk of amount the reduced conditions hypoxic cyclic in reproduced that Females days. 28 after ~4% by reduced was size day. Body 2 ).

et al. et Smith and Able (2003) demonstrated that pO that demonstrated (2003) Able and Smith 2012). As a consequence of this, under cyclic hypoxia, different molecular and and molecular different hypoxia, cyclic under this, of consequence a As 2012). ing hypoxic conditions) in comparison to normoxic animals. ore normal homeostasis (Coiro, Poucher & Miller Miller & Poucher (Coiro, homeostasis normal ore for estuarine fish, crustacean species and other crustacean fish, estuarine for 2

could fluctuate every day from st and 21 and st experimental Accepted Article exposedcyclic to hypoxia. will gills larger of development the with coupled cycle moult the of acceleration an that hypothesised be can it Therefore, ecdysis. at replaced periodically is that exoskeleton rigid a by covered are gills and body 2015): Sagi & Abehsera (Roer, stressful conditions. Insects and crustaceans poss to exposure the with associated costs maintenance increased or feeding reduced to due probably hypoxia)coupled is with reductiona in body mass (Greenberg &Ar1996; Callier &Nijhout 2011), to (due frequency moult increased the insects, In 2013). 2011, Nijhout & (Callier body the demand of VandenBrooks frequently and develop a larger respiratory system in demonstrated by Greenberg and (1996), insect Ar larvae reared in hypoxic conditions moult more between oxygen supply and oxygen demandcan develop (Massabuau 2012).Hence, & Abele as does oxygen demand)Lehmann (Kivelä, Gotthard & 2016). In hypoxicconditions, mismatch a (Lundquist exoskeleton rigid habitat. natural their in hypoxia cyclic to subjected are which species those on hypoxia) chronic than (rather cyclic of impact the assess to more appropriate is it why underline hypoxia al. cyc by affected differently be can reproduction) hemocyanin of down-regulation a induced hypoxia chronic to exposure 10-days while yolk), egg of protein hemocyanin of up-regulation an induced days 10 for hypoxia cyclic to continent) American the in found This article is protectedThisarticle is byAll rightsreserved. copyright. demonstrated by Li and Brouwer (2013b), exposing grass shrimp as fact, In hypoxia. chronic to comparison in activated be can mechanisms physiological (2000) and and Brown-Peterson (2000) In arthropods, the respiratory system grows primarily at moults because it is covered by the by the covered is it because moults at primarily grows system respiratory the In arthropods, mRNA (respiratory pigment) and a down-regulation of of down-regulation a and pigment) (respiratory mRNA and no change in etal. order(2017)), in toincrease oxygen supply tothe tissue andsustain oxygen et al. vitellogenin etal. 2017), whereas tissue mass increases between moults (and so (2008). Overall, (2008). different the outcomesofchronic cyclicor . Further, physiological processes (e.g. growth, growth, (e.g. processes physiological . Further, lic and chronic hypoxia as demonstrated by Coiro be observedbe in adult shrimp from salt marshes ess the same generalised structural body plan plan body structural generalised same the ess comparison to larvae reared in normoxia (e.g. (e.g. normoxia in reared larvae to comparison

Palaemon pugio Palaemon vitellogenin mRNA mRNA precursor (the (a species only et Accepted Article morphologicalchanges to gills the within 16 days of cyclic hypoxia. Finally,we demonstrated how activated physiological adaptations resulting inan acceleration ofmoult the and cycle in it Further, days. 7 after organs) major all and hepatopancreas including but and hypodermis in hypoxia)changes induced the transcriptome in entire cephalothorax of the (not limitedcuticle to day per hours seven (of regime hypoxic cyclic a daily to exposure the that demonstrated We 2005). Aguzzi 1975; (Welsh ecosystem the in energy and nutrients of transfer in role fundamental decaying marsh flora are processed bydetritivorous species, such as material is organic of marshes, export European exposure, and affected reproduction in adult of days 28 within excretion ammonium and size body adult reduced regime hypoxic cyclic the maintain O maintain responses and effectson the species (Truebano This article is protectedThisarticle is byAll rightsreserved. copyright. chosen as a model due to its key ecological role in the ecosystem (Aguzzi ecosystem the in role ecological key its to due model a as chosen continent. Within Lymington the marshes, shrimpditch UK, the American the from species to exclusively almost confined is it and rare still is conditions hypoxic cyclic reproducing studies of incidence the however, worldwide; marshes and lagoons estuaries, species &Roy 2008). (Fenberg (Cheek reproduction or 2016) Grecay & Targett (Davidson, growth as such processes physiological on consequences negative long-term, the in can have, hypoxia cyclic to exposure changes, morphological re (e.g. the theenlargementof that mechanisms other exposure, After prolonged expensive processes (e.g. excretion feeding(Wei or adjustments (Brown-Peterson At physiological level, the length of exposure to cyclic hypoxia can trigger different different trigger can hypoxia cyclic to exposure of length the level, At physiological Cyclic hypoxia is currently experienced by a vast number of species from freshwater ponds, ponds, freshwater from species of number vast a by experienced currently is hypoxia Cyclic 2 delivery tothe tissue with molecular, metabolic and/or respiratory/circulatory et al. 2009) with potential repercussions on the biology and ecology of the the of ecology and biology the on repercussions potential with 2009) et al. et spiratory system mentioned above). In addition to these spiratory to system above).In addition mentioned 2008; Truebano P. varians et al. et largely absent (Cattrijsse & Hampel 2006) and and 2006) Hampel & (Cattrijsse absent largely imply functional modifications can be triggered triggered be can modifications functional imply et al. et etal. 2018). In the short-term, responses aim to after 40 days exposure. exposure. days 40 after 2009)) to reduce overall oxygen demand. demand. oxygen overall reduce to 2009)) 2018)and tosuppress aim aerobically

Palaemon varians varians Palaemon P. varians P. et al. et 2005). In fact, in , which plays a Leach was (1814), et al. et

Accepted Article

This article is protectedThisarticle is byAll rightsreserved. copyright. General experimental protocol The population site Sampling of In Information. Supporting in available is methods and materials all of description A detailed all experiments, statisticalsignificance was identified atp MATERIALS AND METHODS: Fig. S1A in Supporting Information). At 22 22 At Information). in Supporting S1A Fig. to June (from summer during marsh the in measured found. frequently more adjacent logger placed oxygen temperature and obtained. obtained. that can occur inditch, the pO net-caught from this site. To assess dailycyclic oxicconditions and describe some of the variability loca provided in Supporting Information). P to Mueller and Seymour (2011) by using “highest the vertical distance method” (further details are – 11 th on: 50 2016; February 16 Hypoxia was defined as pO as defined was Hypoxia

̊44’19.8” N and 50 th –23 Palaemon varians Palaemon 2 rd ̊44’22.2” andW) adult data was continuously measured in three discrete weeks (August 4 (August weeks discrete three in measured continuously was data 2017; May 14 May 2017; 2 < critical oxygen pressure (p pressure oxygen critical < crit was determined at 22 22 at determined was ˚ C a mean p C amean originates from the salt marsh of Lymington (UK, (UK, Lymington of marsh salt the from originates th –22 to the to the bottomchannel, theof where animals are nd September) atnight to2000 (from 0800 BST, P. varians 2017). pO crit of 4.55 ±0.63 of 4.55±0.63 kPa wasTable (n=10, S1) ≤ 0.05.

used in experimental work were all all were work experimental in used ˚ 2 C as this temperature is frequently frequently is temperature C this as crit was measured using a custom custom a using measured was ). P ). crit was determined according according determined was th

Accepted Article excretion and reproduction. reproduction. excretion and cycle moult during according the tothestageof made was selection animal No used. was hours) 12 within moulted all had that animals similar-size of (composed population synchronous a histology, gill and cycle moult entire an throughout genes =12L). (volume tank per animals 18 was density shrimp max experiments Inall S2. Table in is reported field the from collected were animals hypoxia or normoxia). RNA was extracted using a TRI-Reagent a using extracted was RNA normoxia). or hypoxia (cyclic conditions experimental to exposure of days seven after hrs 1030 at sampled were randomly treatment) per (n=8 Animals exposure. hypoxic cyclic of days seven after expression gene in changes N bubbling these systems wasconducted in filtered seawater PSU at and33 22 (one system cyclic hypoxic for treatment, treatment). one fornormoxic All experimental work in This article is protectedThisarticle is byAll rightsreserved. copyright. hypoxia to response Transcriptome cephalothoraxes. Library preparation followed Illumina TruSeq RNA-Library Preparation Kit.v2 Kit.v2 Preparation RNA-Library TruSeq Illumina followed preparation Library cephalothoraxes. mean±SD pO mean±SD the exposure, the of days) of terms (in duration the experiment, each For conditions. hypoxic of development the prevent to bubbled was air system flow-through normoxic the In S1B). day (Fig. (b conditions normoxic in were kept andthey hrs) Microx TX 3 (PreSens) sensors. Water andN Water sensors. (PreSens) 3 TX Microx Animals were subjected daily to hypoxic conditions (pO conditions hypoxic to daily were subjected Animals durati inter-moult investigating In experiments To perform all subsequent experiments, two fl two experiments, subsequent all To perform A pilot RNA-seq experiment was performed on the cephalothorax of animals to identify identify to animals of cephalothorax on the performed was experiment RNA-seq A pilot 2 in the experimental tanks and water pO water and tanks experimental the in 2 level recorded during hypoxic periods in each tank and the sampling date in which which in date sampling the and tank each in periods hypoxic during recorded level 2 flow-rate were adjusted in order to obtain pO obtain to order in adjusted were flow-rate experiments involving RNA-seq, growth, feeding, y bubbling air into the system) for the rest of the the restof the for intothesystem) air bubbling y 2 was continuously measured and loggedwith ow-through experimental systems were built built were systems experimental ow-through on, changes in the expression of cuticular cuticular of expression the in changes on, 2 <4.5kPa) for 7hours to (from 0930 0230

TM (Sigma Aldrich) protocol from whole whole from protocol Aldrich) (Sigma ˚ C. Hypoxia was achieved by 2

2 or <-2, supported by FDR-corrected p-values (Kal perform hypergeometric testsoncategories. to generate an referenceannotated transcriptome, sequencingdata were imported into CLC-Genomic the Workbench v.8.5 Bio, (CLC Aarhus,Denmark) (Illumina, California). Paired-end 115bp reads were sequencedon anIllumina HiSeq-2500. Raw etal. The expression of some up-regulated genes was further confirmed by means of quantitative- of means by confirmed further was genes up-regulated of some The expression Statistically significant gene expression changes were detected by use the of aKal’s Z-test 1999). Contigs considered were differentially as regulated for proportion Fold Change coloured numbered tags (Queen-bees marking kit – – kit marking (Queen-bees numberedtags coloured t duration (ecdysis2_dayN – ecdysis1_day0) was was ecdysis1_day0) – (ecdysis2_dayN duration t ene expressionofcuticular genes” section) and to calculate gene expression levels and to to and levels expression gene calculate to ≤ 0.05.

Accepted Article t-test. using tested and weight wet animal’s using mass body in differences for corrected were parameters All S2). (Fig. and density 201 Eliceiri & Rasband (Schneider, software ImageJ haematoxyl with stained were specimen from each each animal was fixed Bouin’s in solution, dehydrated and embedded.Longitudinal sections gillof from cephalothorax the days, 18 After conditions. experimental in cycle moult entire one completed or normoxic (n=5) treatment and exposed to experimental conditions for days18 tomake sure they values(Sakamoto,Ishiguro 1986). – Kitagawa & Criterion” AIC – Information “Akaike's using compared were models between Differences (GAM). model additive the cyclic hypoxic group was comparedwith the pattern normoxic of the group using general a of expression of pattern overall the genes, selected the of expression the alter to able was hypoxia were reactions transcribed. qPCR and hrs). 1030 RNA was extractedwhole from cephalothorax,DNase treated and reverse 1000 (between frozen flash and werecollected treatment) per (n=6 animals from cephalothoraxes 16), (day event this after days 16 to up 0) (day of ecdysis day the from day, other Every days. 16 to up for conditions experimental to andexposed treatment (n=54) ornormoxic (n=54) This article is protectedThisarticle is byAll rightsreserved. copyright. hypoxia Changes to phenotype in Gene expression of cuticular genes during moult cycle Shrimps from a synchronous population were randomly allocated to the cyclic hypoxic (n=5) (n=5) hypoxic cyclic the to allocated randomly were population synchronous a from Shrimps hypoxic cyclic the to allocated randomly were population synchronous a from Shrimps Palaemon varians performed using primer-sets reported in Table 1. To assess if cyclic cyclic if assess To 1. in Table reported primer-sets using performed : gill modification in response to cyclic cyclic to response in modification : gill 2) to determine lamellar width, length, perimeter perimeter length, width, lamellar determine to 2) in and eosin. Micrographs were analysed using using analysed were Micrographs and eosin. in

Accepted Article water at the same temperature andpO temperature same the at water normoxia. or hypoxia cyclic test. F squares weight regr overtime treatments, linear between usin weretested replicates experimental between differences systematic of Absence microbalance. analytical an using weighed were animals all days, 28 to days seven every then and 0, Onday days. 28 for tank) per shrimps n=17 tanks, experimental compared were time over weight wet in and changes This article is protectedThisarticle is byAll rightsreserved. copyright. excretion: and Feeding Changes in bodysize: used to calculate feed ingestion rate. rate. feedingestion calculate to used its wet weight) withcommercial pellet and, after 150minutes,uneaten feed was dried,weighed and “Ammonia Salicylate Method” (Hach, Colorado USA). USA). Colorado (Hach, Method” Salicylate “Ammonia io andammonium collected were samples water sea-water at the same temperature andpO temperature same the at sea-water for the cyclic hypoxic group; water pO water group; hypoxic cyclic for the kPa for the cyclic hypoxic group; pO water group; hypoxic cyclic kPa the for Feed ingestion was quantified in animals (n=11 shrimps/treatment) placed in artificial sea- artificial in placed shrimps/treatment) (n=11 animals in quantified was ingestion Feed Feedingof excretion and adult days 28 for pertank) shrimps n=17 tanks, experimental (6 hypoxia cyclic in kept were Shrimp Ammonium excretion was quantified in adults (n adults in quantified was excretion Ammonium

2 =21 kPa for the normoxic group). Each adult was fed (~4.5% (~4.5% fed was adult Each group). normoxic the kPa for =21 P. varians P. 2 as their respective experimental aquaria (water pO (water aquaria experimental respective their as 2 =21 kPa for the normoxic group). After 210 minutes, minutes, 210 After group). normoxic the for kPa =21 2 as their respective expe astheir ns were measuredns with a methodHach 8155 were quantified after 1 and 21 days of exposure to to exposure of days 21 and 1 after quantified were ession models were compared using extra sum-of-using extra essionwere compared models g a nested ANOVA. To test for changes in wet wet in changes for test To ANOVA. nested a g with a control population kept in normoxia (6 (6 normoxia in kept population control a with =14-18 adults/treatment) placed in artificial artificial in placed adults/treatment) =14-18

rimental aquaria (water pO (water aquaria rimental 2 =3.0 kPa 2 =3.0 Accepted Article period in August (4 August in period feed ingestion and ammonium excretion, followedTukey’s by multiplecomparisons test. August, where pO August, where This article is protectedThisarticle is byAll rightsreserved. copyright. Environmental variability RESULTS: Reproduction: oscillations seemed “irregular” (with peaks in pO hours. August, In hypoxicconditions below 4.0 kPa lasted, on average, 7.1 hours±1.8 day 12- every detected was troughs and peaks adjacent between difference kPa ~39 a where August to and assessed using Chi-square testand t-test. were dryweight egg and weight) number/body (egg gr between (ratio success reproductive experiment, in hypoxia, 10 pairs innormoxia) were housed in retentionchambers up to 40days.During the temperature variationstemperature ~2 of pO 2 oscillations were less pronounced in February, while they became very marked in May and and inMay marked very became they while February, in pronounced less were oscillations Two-way ANOVAwith‘treatment’ and ‘time’ as factors was used toassess differences in During and2016 oxygen2017, and temperature measurements were takenover one-week with female a and male a of pairs Reproductive

2 could vary from ~40 kPa to ~3.5 kPa within 12-h. In May, the amplitude of the the of amplitude the May, In 12-h. within kPa ~3.5 to kPa ~40 from vary could th –11 th th 2016), February (16 February 2016), ˚ C were recorded in February ~5.5 in to C were increased recorded and th –23 2 that changed considerably every day), in contrast contrast in day), every considerably changed that avid and non-gravid females), relative fecundity fecundity relative females), non-gravid and avid compared with control pairs kept in normoxia normoxia in kept pairs control with compared rd rd 2017) and May (14 ovaries in primary vitellogenesis 12pairs (n=

th –22 nd ˚ 2017) (Fig. 1).Daily C in August.C in Diel -1 . Accepted Article down-regulation of a vitellogenin transcript was observed. observed. was transcript vitellogenin a of down-regulation regulated transcripts, majoritythe were This article is protectedThisarticle is byAll rightsreserved. copyright. peptides ( protein post-moult exoskeleton: formed newly an crustaceans of phase post-moult in the expressed involved cuticlewith synthesis (e.g. cuticular proteins, Table Further, S4). we identified proteins bygenes dominated was transcriptome the overall contigs, up-regulated annotated the Among transcriptome. day 7 the in down-regulated) and187 up-regulated (214 treatment hypoxic cyclic the transcriptome. assembled the of and integrity completeness high the highlighting transcripts, full-length as present ortholog copy single benchmarking ofthe 91% referenc the of and integrity Thecompleteness which revealed only assembledof that 5% the transcripts were absent and 4%were fragmented. GFPG01000000. version, first this isthe paper deposited at DDBJ/EMBL/GenBank under the accessionGFPG00000000. versionThe described in Tr This S3). (Table fragments covered poorly data contained 105,325 contigs, further reduced to59,370 reference sequences upon the of removal PRJNA389547. The complete Sequence Read Archive (accessions SRX2894799, SRX2894801), as apart ofBioProject the and 25,253,636 raw reads,respectively. TheIllumina raw reads weredeposited at the NCBI generated fromcyclic hypoxic and normoxic treatments animals(n=8 each) for 31,461,523returned A pilot RNA-seq experiment was performed toidentify genes putatively involved in of groups treatment in conditions hypoxic cyclic to tolerance hypoxia to response Transcriptome Differential expression analysis DD5 and M28 M28 (Ikeya de novo de et al. et 2001; Inoue assembled transcriptome generated from trimmed sequencing sequencing fromtrimmed generated transcriptome assembled identified a total of 399 contigs expressedof total indifferentially 399 identified a identified as chitinase (Abehsera enzymes identified as anscriptome Shotgun Assembly project has been has project Assembly Shotgun anscriptome ousgenes conserved across all metazoanswere etal. PMP d involved in deposition and calcification of the ), calcification-associated peptide ( 2004; Roer 2004; e transcriptome were evaluated with BUSCO, BUSCO, with wereevaluated e transcriptome

P. varians P. etal. Among2015). down- the . Pooled cDNA libraries Pooled . et al. CaAP 2015) and 2015) a ), Accepted Article of moult the cycle ecdysis,after (just 0, day (Ikeya Fig.4) cyclic expression their selected is because were markers three All 4). (Fig. normoxia or hypoxia cyclic to exposed populations synchronous on 16) selected and theirexpression leve p=0.03, n=54). moult duration (hypoxia times 12andnormoxia 14days) were different (Mann-Whitney, U=239, inter- median In addition, respectively). B, 3A and (Figure normoxia to comparison in (anticipated) W=239, p=0.03), with the frequency distributioncyclic of hypoxic animals being shifted to left the normoxia,whereas fold change calculated RNA-seq from was 189, 5.3 and 4.7, respectively). were all up-regulated according dataqPCR to (~246, 31and 15-fold, respectivelyin comparison to for coding Contigs 2). (Fig. data transcriptome the from genes S5). (Table contigs regulated down the among annotation dominant the was which activity, chitinase with contrast in genes, up-regulated the processes involving cuticle and chitin-binding proc This article is protectedThisarticle is byAll rightsreserved. copyright. 2015). testwhether To the overall pattern of expression between differed treatments, a general Following experiment, resultsfrom RNA-seq cuticular markers ( three Gene expression of cuticular genes during moult cycle Changes in inter-moult duration Similar results were obtained from hypergeometric on tests annotations, as extracellular test rank-sum (Wilcoxon differed treatments among distributions frequency Moulting cuticular post-moult those of up-regulation the confirmed PCR quantitative MIQE-compliant ls were quantified on alternate days (from ecdysis (day 0) to day 0) to (day ecdysis days(from on alternate were quantified ls with a peak in expression during post-moult phase phase post-moult during expression apeak in with esses clearly emerged asover-represented among et al. et

PMP, CaAP PMP, 2001; Inoue PMP, and and et al. DD5

CaAP CaAP in cyclichypoxia in 2004; Roer and DD5 ) were ) were et al.

Accepted Article nested ANOVA (“tank within nested treatment”, F This article is protectedThisarticle is byAll rightsreserved. copyright. Changes in bodysize: density (t=1.49, p=0.17, were observed.n=10) perimeter (t=2.21, p=0.055, n=10),change but no in lamellar width (t=1.82, p=0.12, andn=10) hypoxia Changes to phenotype in points together (without ( treatments) generated the that process described better (i.e. DD5 for that showed AIC of Comparison 2). (Table gene run for each was (GAM) model additive sum-of-squares Ftest, F weight-time relationship cyclic in hypoxia was statistically different from that in normoxia (extra different in comparisonthe normoxic to group. treatment,cyclicin the groupexpression hypoxic overallpattern of the for average ~4% smaller than mean weight of normoxic animals. animals. normoxic of weight mean than smaller ~4% average pugio Brown-Peterson by reported similarly was captivity of consequence 0.6 incyclic the hypoxic exposed animals and -0.26 in normoxic the animals negative (a growth a as the model fitting independently the treatments independently fitting model the for over 30-days in experimental tanks). At day 28, mean weight of hypoxic animals was on on was animals hypoxic of weight mean 28, day At tanks). experimental in over 30-days for The absence of systematic differences between experimental replicates was confirmed by by confirmed was replicates experimental between differences systematic of The absence Cyclic hypoxic shrimps longerhad lamellae (t-test=2.20, p=0.053, n=10,with Fig. a 5) longer 1, 971 1, =7.182, p=0.01, rate Fig. of decrease6). The weight in over - time was Palaemon varians ∆ AIC PMP : 8.6; 10,144 : gill modification in response to cyclic cyclic to response in modification : gill the data) over simplerthe model fitting all data ∆ (cyclic hypoxia and normoxia) was preferred preferred was normoxia) and hypoxia (cyclic =0.71, regression linear p-val=0.71). The the of AIC DD5

: 3.2). Hence, dueto the effect of the et al. et (2008) (2008) whenkeeping PMP and and DD5 PMP P. P. was was and and Accepted Article worldwide. This study assessed the consequences of exposing the shrimp shrimp the of exposing consequences the assessed study This worldwide. hypoxic females compared controls to (unpaired 2.344, t-test= df= 119, p=0.03, Fig. 8C). normalised weight dry Egg 8B). Fig. p=0.72, df=10, for the relative fecundity of females, which did not show statistical difference (unpaired t-test= 0.35, cyclic hypoxia and normoxia (Chi-square: 1.564, df=1,p=0.21, Fig. 8A). Similar results were obtained This article is protectedThisarticle is byAll rightsreserved. copyright. DISCUSSION: Reproduction: excretion: and Feeding Two-way ANOVA revealed an effect of treatment on ammonium excretion excretion (F Two-way ammonium on oftreatment effect an ANOVA revealed 7A). (Fig. detected be could difference no 21 day at but, 1, day at animals normoxic to compared hypoxic cyclic in reduced was while,acceleration long-term, the moul in an of the hypoxia cyclic to exposed inadults detected were excretion ammonium and feeding in reduction a August. theshort-term in In its habitat in experienced currently regime hypoxic cyclic daily and F 7B). (Fig. 21 day and day 1 p<0.0001). Cyclic hypoxic animals had alower excretioncomparison in rate normoxic to animals at 1,40 Cyclic hypoxia is currently experienced by a vast number of species in different ecosystems ecosystems different in species of number vast a by experienced currently is hypoxia Cyclic between different not was females), non-gravid to gravid of (ratio success The reproductive trea of effect significant was a There =6.22, p=0.02, respectively),withinteraction no effect (F tment time ingestionand (F tment onfeed to female body weight was lower (~24%) in cyclic cyclic in (~24%) lower was weight body female to t cycle and an increase in gill surface area were were area surface gill in increase an and cycle t

1,40 = 0.16, p=0.69). Feed ingestion 0.16, p=0.69). Feed = Palaemon varians Palaemon 1,40 = 12.35, p=0.001 1,51 = 77.57, tothe Accepted Article differentially expressed genes we genes expressed differentially of the majority The hypoxia. cyclic to subjected shrimp asynchronously-moulted in contigs expressed This article is protectedThisarticle is byAll rightsreserved. copyright. hypoxia: to response Transcriptome pO in changes rapid study, Inthis Environmental variability: organization. biological of levels many at invertebrate coastal key of a physiology the impact to able is conditions field mimicking regime hypoxic by produced of eggs weight dry the in decrease a and size body adult in decrease ina resulted hypoxia cyclic to exposure long-term a Further, found. temporarilysuitable environmental more relocate to conditions. (frequently summer higher the temperatures to due year, probably of July and Augustwere stressful more in comparis conditions hypoxic cyclic that possible is It obs.). (unpubl. year ofthe rest the during collected easily collect maximum fluctuations kPa of ~42 in August. similar A magnitude ofvariability diel in pO DD5 together with animals, decreases pO decreases animals, with together atmosphere) and by a thick algal coverage (that increases pO increases (that coverage algal thick a andby atmosphere) are characterized stagnantby waters prevent (that mixing and limit gas with the exchange foundLymington in isprobably due to a combinationof abiotic and biotic factors: water channels reported otherfor salt marshes in USA (e.g. Able (Smith 2003;& Cheek ), cuticle re-arrangement (i.e. chitinases) and cuticle calcification (i.e. (i.e. calcification cuticle and chitinases) (i.e. re-arrangement cuticle ), P. varians P. Differential expression analysiswhole from cephalothorax identified 399differentially with hand net at the sampling site during July and August, whereas the species was was species the whereas August, and July during site sampling the at net hand with re involvedcuticle with depositioncuticular (i.e. proteins, 2 during the night via respiration). respiration). via night the during 2 (within 12 hours) were found in all sampling weeks, with with weeks, sampling all in found were hours) 12 (within females. resultsThese emphasize howcyclic a on with conditionson with experienced theinrest of the 2

during the day via photosynthesis and, and, photosynthesis via day the during ≥ 20 Indeed, it proved difficult to to proveddifficult it Indeed, et al. et ˚ C), forcing CaAP, CaAP, 2009)). Diel variability gastrolith proteins). proteins). gastrolith P. varians P. 2 has been has PMP to , Accepted Article hypoxia, undergo prolonged metabolic depression that depression metabolic prolonged undergo hypoxia, chronic from stress continuous to subjected when shrimps, that behypothesised could it process, expensive energetically an is moulting Since hypoxia. cyclic and hypoxia chronic tested: conditions Brown-Peterson by found patterns GE different These proteins. cuticular of up-regulation no and proteins chain electron-transport (similar to this study), whereas chronic hypoxia for pugio Palaemonetes to normoxia. In agreement withour results, Brown-Peterson

(over 16 days) of the post-moult markers markers post-moult of the (over 16days) pattern expression overall The 1). model with (modelled population the of variability natural the differences due pattern overall in to the the treatment (modelledwithmodel were than greater 2) beetle the of cycle inlarvae moult reported accelerated an who (1996) Ar and Greenberg with accordance in animals), normoxic to compared shorter 15% was moult phase in crustaceans (Ikeya crustaceans in moult phase supportedup-regulation the by of insect for similarly thatreported to of cycleeffect moulton the might exposure exert an cyclic hypoxia suggested that This article is protectedThisarticle is byAll rightsreserved. copyright. shrimp:synchronously Changes moulted of gillmoulting morphology cycle and in al. is ofThe expression these genes (2014) and and Abehsera (2014) Gene expression analysis (GE) of synchronously-moulted shrimp showed how the Exposure to cyclic hypoxia accelerated the moult cycle of adult ofadult cycle moult the accelerated hypoxia cyclic to Exposure to 14 days of cyclic hypoxia induced induced hypoxia cyclic of days 14 to et al. et (2015)). The increased expression of cuticular contigs after 7-days 7-days after contigs cuticular of expression increased The (2015)). inevitably related to the phases of the moult cycle seeTom themoult of (e.g. phases the to related inevitably etal. PMP, CaAP etal. larvae reared in hypoxia (Kivelä hypoxia in larvaereared 2001; Inoue 2001; (2008) might experimental explained be different theby (2008) PMP and and DD5 14 days exclusively induced a down-regulation of of down-regulation a induced daysexclusively 14 DD5 et al. et genes, markers highly expressed during post- during expressed highly markers genes, completely inhibits moulting (as reported by reported (as moulting inhibits completely was accelerated in cyclic hypoxia compared an up-regulation of cuticular proteins proteins cuticular of up-regulation an 2004; Roer Tenebrio molitor et al.

(2008)showed that exposing etal. et al. P. varians 2016). This 2016). conclusion was 2015). reared in hypoxia. (inter-moulttime P. varians P. , et Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. laboratory the In hypoxia. cyclic experiencing not site another from population a to comparison afundame considered generally is Bodysize predator-prey relationships and competitionwith other species 2008).(Fenberg &Roy In excretion: size,feeding,and reproduction ammonium body Effectscyclic hypoxiaon of time” to restore homeostasis and stimulates acceleration an of the cycle. moult Wei smaller than averagethe weight of normoxic animals. Li and Brouwer demonstrated(2013a) that a ~4% was weight body average the hypoxia cyclic to exposure 28-days After animals. normoxic to comparison in animals hypoxic cyclic in faster was time over weight wet in decrease of rate the pO lowered the with cope to animals for solution a functional represents area surface in increase ex reportedduring been has lamellar area surface in increase an where species fish several in beendemonstrated has hypoxia chronic of consequence had a13.6% larger lamellar surface area. In agreement with our findings, gill remodelling as a was, on average, 13.6% longer thancontrol shrimp. Animals after moultone cycle in cyclic hypoxia field population of population field (2013). of requirements oxygen meet order to in rapidly be the with hypoxia, cyclic to in response triggered is moulting faster that indicate here presented data Overall, exchange. gas for capacity improve the respiratoryHence,surfacesurface(Massabuau largerareaa 13.6% areawould 2012). Abele & ac movements oxygen fact, In periods. hypoxic during

et al. et The lamellar lengthof (2008)).contrast, In cyclic in hypoxia, the turnover hypoxia-normoxia allows “recovery P. pugio P. from a cyclic hypoxic salt marsh was ~10% shorter and ~22% lighter in in lighter ~22% and shorter ~10% was marsh salt hypoxic cyclic a from P. varians gills (expressed as µm/mg_animal) of cyclic hypoxic animals animals hypoxic cyclic of µm/mg_animal) as (expressed gills the body, in accordance with Callier and Nijhout Nijhout and Callier with accordance in body, the posure hypoxiato (Nilsson 2007). observed The nefit that gill modifications can more can be produced modifications gill that nefit ntal biological trait affecting reproduction, reproduction, affecting trait biological ntal ross the aregills directly proportional tothe

P. varians P. 2

, Accepted Article concentration of O of concentration 21, arguably a as resultof long-term effects such food in difference this However, hypoxia. during provided was food when animals hypoxic-exposed cyclic in reduced significantly was ingestion (Brandt hypoxia by affected be negatively can it therefore and 2009) impairment. physiological hypoxic on stress physiologicalprocesses, while shorter normoxic periods result greater in in normoxia the ones (as in this study) facilitate the recovery hypoxia from by reducing the ofimpact times longer that beargued could it Hence, work). our of normoxia 17-h and hypoxia 7-h to contrast in growth (Coiro continent) This article is protectedThisarticle is byAll rightsreserved. copyright. Davidson some in beendemonstrated has depression growth exposed animals (~66-75%in comparison tonormoxicanimals) during the 1 stress. varians by populated ecosystems on consequences important have could excretion ammonium in reduction substantial excretion was still contrastsuppressed (in to food ingestion). From an ecologicalperspective, the that indicated days 21 after shrimps hypoxic-exposed cyclic in rates excretion reduced exposure. The has been reported to play a key ecological role in macerating detritus and dead marsh plants plants marsh dead and detritus in macerating role ecological key a play to reported been has Feeding is a fundamental biological process that requires aerobic conditions (Wei conditions aerobic requires that process biological fundamental a is Feeding

In etal. P. vulgaris P. P. varians P. P. varians P. and2016) in etal. et al. et 2 -binding proteins (e.g. Truebano (e.g. proteins -binding a significant reduction in ammonium excretion was found in cyclic hypoxic- cyclic in found was excretion ammonium in reduction significant a larvae under a 12-h hypoxia and 12-h 12-h and hypoxia 12-h a under larvae 2009). Indeed, on the first experimental day of cyclic hypoxic exposure, food food exposure, hypoxic cyclic of day experimental first the on Indeed, 2009). 2000). Coiro (i.e. salt marshes), in particular on nutrient cycling and energy fluxes. fluxes. energy and cycling nutrient on particular in marshes), salt (i.e. Palaemon vulgaris etal. (2000)identified between and 15% 70% reduction in larvae(a species only found in the American ingestion during hypoxia was not detectable at day day at detectable not was hypoxia during ingestion as the increased surf as the gill fish species (Stierhoff, Targett & Miller 2006; &Miller Targett species (Stierhoff, fish et al. hypoxia, as documented in relation to chronic chronic to relation in documented as hypoxia, (2018)) that could (2018)) mitigate the hypoxic normoxia cyclic hypoxic regime (in (in regime hypoxic cyclic normoxia

ace area or an increased increased an or area ace st and 21 and st dayofcyclic etal. P.

Accepted Article would not affect the long larval development process. Overall these different results underline a a underline results different these Overall process. development larval long the affect not would for hence, of development the 2013) (Oliphant processes thespecies. of regime) without changingPOI. couldIt be hypo hypoxic cyclic the to according 60%, to 30% (from fecundity its reduced hypoxia cyclic to exposed This article is protectedThisarticle is byAll rightsreserved. copyright. varians Brown-Peterson shrimp sacrifice offspring quality in order tomaintainoffspring number. In contrast to our results, Truebano with accordance In development. larval delaying tran would POI Areduced (Giménez 2004). &Torres Marshall &Uller 2007), as higher POIs are generally associatedwith greater offspring fitness (Oliphant & Thatje 2014).This of istrait fundament the level of maternal resources invested into eggs, alsoknown as per offspring investment (POI) Berrigan with accordance in size, egg and fecundity between a trade-off suggesting conditions, hypoxic cyclic in reproduced that females in found cyclic hypoxia (Li 2009, &Brouwer 2013b). A significant reduction (~24%) in eggdryweightwas (W ofammonia quantities large excreting and in after 7 days of cyclic hypoxia (Table S3), and was reported for reported was and S3), (Table hypoxia cyclic of days 7 after foun was transcript vitellogenin a of down-regulation animals experience stressfulconditions (Petes, Menge &Harris 2008;Sokolova marsh. environmentaffect thecommunitynutrient salt microbial ofthe alter turnover in turn, may and, of microflora (Welsh 1975). Therefore, reduced ex a growth significant supporting and levels trophic of variety a at available energy making thus 2005), and Reproduction is another energetically expensiv energetically another is Reproduction P. pugio, P. pugio P. et al. et lowering fecundity and retaining POI could be functional in cyclic hypoxia as it it as hypoxia cyclic in functional be could POI retaining and fecundity lowering are the result of constraints dictated by the different larval development development larval different the by dictated constraints of result the are (2008) and (2011) demonstrated in the lab and in the field that While the development of P. pugio P. encompasses 11 different larval stages (Broad 1957) thesised that the observed differences between between differences theobserved that thesised elsh 1975; Escaravageelsh 1975; &Castel 1990; Aguzzi al importance within lifehistory biology (J (1991). Egg dry weight is considered a proxy for for aproxy considered is weight dry Egg (1991). cretion of ammonia due tocyclic hypoxia in the slate intoslate smaller a energy reserve, potentially P. varians P. d in the transcriptome of cyclic hypoxic animals animals hypoxic cyclic of transcriptome the d in e process that is frequently affected when when affected is e process that frequently et al. et

comprises only 5 larval stages larval comprises 5 only P. pugio P. (2018), our results (2018), suggest that following 5and 10 daysof etal. 2012). The 2012). P. pugio P. et al. et

P. P.

Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. interest. of conflict no declare Authors interest:Conflict of varians Palaemon shrimp decapod the of physiology the to changes assessed study This Europe). in (especially habitats but basis daily a on hypoxia cyclic by affected be can ecosystems coastal important Many ph on the consequences rather little is the of known CONCLUSIONS: crustacean species. European on hypoxia cyclic of impact the assessed hitherto have that of studies paucity the given study, this of importance the emphasize and hypoxia cyclic to response in variability species-specific influenced the physiology of a decapod crustacean species. species. crustacean decapod of a physiology the influenced and marshes salt Lymington the within conditions environmental mimicked that regime hypoxic ~24%. by egg each in allocated female that longer exposurecyclic to hypoxia reduced adult bo a Finally, cycle. moult one after area surface gill in increase a13.6% and animals) hypoxic cyclic in effects Long-term an involved itstranscriptome. ammonium excretion ~66%by and, after 1week, 399differentially expressed genes were identified 4.0 kPa). day -1 ), mimicking a cyclic hypoxic regime measured in in measured regime hypoxic cyclic a ), mimicking

Overall, data emphasise how different physiologi different how emphasise data Overall, In the adultshort-term induced by a long-term daily cyclic hypoxic regime (water pO (water regime hypoxic cyclic daily long-term bya induced P. varians P. responded by reducing their feeding rate by ~50% and and ~50% by rate feeding their reducing by responded acceleration of the moult cycle (~15% shorter in shorter (~15% cycle moult ofthe acceleration dy size by ~4% and decreased the amount of yolk yolk of amount the decreased and by~4% size dy ysiology and/or ecologyof species living in these P. varians P. cal processes were affected by this cyclic cyclic this by affected were processes cal

’ habitat (7.1 ±1.8 hours day hours ±1.8 (7.1 ’ habitat 2 < 4.5kPa for 7hours -1 below Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. edited themanuscript. bioi the performed AP AOand MG, LP, sequencing. LP, ST and CH conceived the work. LP performed the research. AO andDW performed the Author contributions: https://doi.org/10.5061/dryad.d12635v (Peruzza Repository: Digital Dryad the in deposited was data raw Remaining first version, GFPG01000000. This project isassociated with theBioProject IDPRJNA389547. DDBJ/EMBL/GenBank under accession the GFPG00000000. The version described in this study isthe at deposited been has project Assembly Shotgun Transcriptome This SRX2894801). deposited been have reads RNA-sequencing Raw Data accessibility: paper. the of quality the improved substantially that comments constructive their for reviewers anonymous the Ly in and temperature of the measurements oxygen for Hartman Susan Dr and Campbell Jon Dr Beaton, Alex Dr Martincic, Urska Miss inputs, useful the models, additive general the with help Trueman for the experimental tanks, Mr Robbie Robinson the assistancefor in looking animals, the after Clive Dr Oceanography Centre, Southampton.LP would personally thank MrKelvin forhelpAylett inbuilding National the of School Graduate the from LP to scholarship PhD a by supported was study This Acknowledgements: et al. et on the NCBI Sequence Read Archive (SRX2894799– Archive Read Sequence NCBI the on nformatics.LP wrote the manuscript. All authors mington. Authorsgrateful are tothe editor and Prof PieroGiulianini andDr Alastair Brown for 2018)

Accepted Article Callier, V. & Nijhout, H.F. (2011) Control of body size by oxygen supply reveals size-dependent and size-dependent reveals supply oxygen by size body of Control (2011) H.F. Nijhout, & V. Callier, Davidson, M.I. Davidson, Callier, V.&Nijhout, Body (2013) H.F. size determination in insects: review and a synthesis of size- N.J. Brown-Peterson, N.J. Brown-Peterson, Broad, (1957)Larval A. development of Coiro, L.L. Cheek, A.O. Cattrijsse, & Hampel, (2006) A. EuropeanH. intertidalmarshes:habitat reviewof their functioning A Brandt, S.B. Berrigan, allometry The D. (1991) of egg and size number in insects. Aguzzi, J. This article is protectedThisarticle is byAll rightsreserved. copyright. S. Abehsera, References: Escaravage, impact& Castel, V. (1990)The J. lagoonal of the shrimp Academy of Sciences of the United States of America, of States United the of Sciences of Academy metamorphosis. and molting of mechanisms size-independent grass shrimp, comparison. laboratory versus Field shrimp: grass the in patterns and brain-dependent and and and brain-dependent 141. 144-161. Experimental Marine Biology and Ecology, and Biology Marine Experimental species:: Using constant exposure data toestimate cyclic exposure response. Philosophical Society, fish populations. populations. fish and value for aquaticorganisms. ( from southwestern Europe. Europe. from southwestern 556, survival of juvenile summer flounder metabolism. of Ecology, impoundment. coastal temperate a in on meiofauna Morone saxatilis et al. et al. et et al. et etal. etal. 223-235. etal. (2005) Daily (2005) and seasonal feeding rhythmicityof (2000) Hypoxic effects on growth of of growth on effects Hypoxic (2000) (2009) Diel hypoxia in marsh creeks impairs the reproductive capacity of estuarine estuarine of capacity reproductive the impairs creeks marsh in hypoxia Diel (2009) (2009)Effects of hypoxiaon food consumption and growth of juvenile striped bass (2015) Binary (2015) expression gene patterningof cycle: molt the case of The chitin

11, (2016) Evaluating the effects of diel-cycling hypoxiaonpH and growth and et al. et al. PLoS ONE, Palaemonetes pugio Palaemonetes 409-418. Marine Ecology Progress Series, Progress Ecology Marine ). ). (2008) Effects of cyclic hypoxia on gene expression and reproduction ina (2011)Impacts cyclicof hypoxia onreproductivegeneexpression and Journal of Experimental Marine Biology and Ecology, and Biology Marine Experimental of Journal

88,

10, 944-954. independent mechanisms. mechanisms. independent 4. Marine Biology, Marine Palaemonetes pugio Palaemonetes Marine EcologyProgress Series, . Biological Bulletin, Paralichthys dentatus Paralichthys

247,

Palaemonetes vulgaris Palaemonetes 148, 243-255.

141-147. 392, Holthuis. Acta Oecologica-International Journal Journal Oecologica-International Acta

Biological Reviews of the Cambridge Cambridge the of Reviews Biological 108,

211-221. 214, Palaemonetes varians Palaemonetes 14664-14669. 6-16. . Oikos, Marine Ecology Progress Series, Progress Ecology Marine Palaemonetes varians Palaemonetes The Biological Bulletin, Biological The Proceedings of the National National ofthe Proceedings

324, Aquatic Sciences,

60, larvae and other other and larvae 293-307. 313-321.

381, S143-S149. Journal of (Leach 1814) (Leach) (Leach)

73,

112, 127-

Accepted Article Lundquist, T.A. Lundquist, Li, T. & Brouwer, M. (2013b) Gene expression expression Gene (2013b) M. Brouwer, & Li, T. cyclic of study Field (2013a) M. Brouwer, & T. Li, Li, &Brouwer,T. M.(2009) Bioinformatic analysis of expressed sequence tags from grass shrimp Kivelä, S.M. Kal, A.J. This article is protectedThisarticle is byAll rightsreserved. copyright. regu The (2011) R.S. Seymour, Mueller, C.A.& Massabuau, &Abele,J.C. D. (2012) adaptive? effect maternal a is When (2007) T. Uller, & D. J Marshall, H. Inoue, T. Ikeya, Greenberg, Effects S.&Ar, A. (1996) of chronichypoxia, normoxia and hyperoxiaon larval Giménez, (2004) G.K.A. &Torres, Linking life history in traits successive phasescomplex of a life evolut and Ecological (2008) K. Roy, & P.B. Fenberg, etal. of Insect Physiology hornworm, tobacco the in beta and alpha factors hypoxia-inducible and Proteomics, D:Genomics Part pugio Palaemonetes breathing animals breathing Proteomics, hepatopancreas. Proteomics, and Genomics D: Part Physiology pugio Palaemonetes 3061. level. individual the at test An empirical moulting? biology of the cell, sources. carbon different two on grown yeast from profiles transcript expression Biochemical Zoology, Biochemical oxygen. environmental and consumption oxygen between relationship clarkii Biology, Molecular and Biochemistry B: Part Physiology and Biochemistry structure. repeat tandem novel a with protein exoskeletal putative beetle the in development granulata Chasmagnathus crab, anestuarine in development juvenile early on biomass larval of Effects cycle: know? we do much how etal. et al. et et al. et (1999)Dynamics of gene expressioncomparison revealed by of serial analysis of gene (2001) The DD5 (2001) gene of the decapodcrustacean . (2004) A novel calcium-binding peptide from the cuticle of the crayfish, crayfish, the of cuticle the from peptide calcium-binding A novel (2004) et al. et Biochemical and Biophysical Research Communications, Research Biophysical and Biochemical (2016) Do respiratory Do (2016) limitations affect metabolism of insect larvae before

8, (2017) The effectwithin-instar of development on trachealdiameter and 309-316. Comparative Biochemistry and Physiology Part D: Genomics and Genomics D: Part Physiology and Biochemistry Comparative . Blackwell Science Publ, Oxford. Oxford. Publ, Science Blackwell .

10, . exposed to cyclic hypoxia. hypoxia. cyclic to exposed exposed to environmental stressors. stressors. toenvironmental exposed

84, 1859-1872. 522-532. 522-532. Molecular Ecology, . Tenebrio molitor Principles of oxygen uptake and tissue oxygenation in water- in oxygenation tissue and uptake oxygen of Principles Oikos,

104, 8, 1-10. profile of hepatopancreas from grass shrimp shrimp grass from of hepatopancreas profile lation index: a new method for assessing the the assessing for method new a index: lation 570-580. hypoxic effects on gene expression in grass shrimp shrimp grass in expression gene on effects hypoxic ionary consequences of size-selective harvesting: harvesting: size-selective of consequences ionary .

17, Journal of Insect Physiology, Insect of Journal

4, Comparative Biochemistry and Physiology Physiology and Biochemistry Comparative 187-195. 209-220. The Journal of Experimental Biology, Experimental of Journal The

Penaeus japonicus Penaeus Comparative Biochemistry and Oikos,

318,

116, Comparative 649-654. Manduca sexta Manduca Physiological and 1957-1963.

128, 42, encodes a a encodes 991-996. 379-388. Procambarus Molecular Molecular . Journal Journal

219,

Accepted Article VandenBrooks, J.M. VandenBrooks, Truebano, M. Truebano, Tom, M. Stierhoff, K.L. Sokolova, I.M. Smith, K.J. & Able, K.W.(2003)Dissolved oxygen C.A. Schneider, Sakamoto, Y. Roer, R. Petes, L.E. Petes, L.Peruzza, EnergeticOliphant, adaptations Thatje,brackish-waterwithinA.to (2014) export larval & S. the larval crustacean Decapod (2013) A. Oliphant, This article is protectedThisarticle is byAll rightsreserved. copyright. Nilsson,G.E. (2007)Gill remodelingin fish -anew fashionor an ancient secret? Wei, L.Z. Wei, L.-Z. etal. during development development during affect potential to hypoxia. hypoxia. to potential Biology, Experimental crayfish the of hypodermis the Ecology ProgressSeries, quality. nursery estuarine on effects of analyses modeling and Experimental hypoxia: 1-15. environmental stress toleranc impactson fish assemblages. 671-675. Reidel biochemistry and genetics. genetics. and biochemistry Experimental Biology, Experimental stress resistance. shrimp: from short-term responses to long-term effects. Dryad Digital Repository. palaemonine shrimp, Southampton. of University development. abbreviated and lecithotrophy Physiology hypoxic exposure. exposure. hypoxic etal. et al. et al. et etal. et al. et et al. et (2015) Exoskeletons (2015) across Pancrustacea: the comparative morphology, physiology, et al. et et al. et (2014) Expressioncytoskeletal(2014) molt-related of andis temporally genes scheduledin et al. et . (2009) Effects of limited dissolved oxygen of (2009)Effects oxygen limiteddissolved (2008) Compensatory (2008) growth of Chinese shrimp, etal. (2008) Intertidal (2008) mussels exhibit energetic trade-offs between reproduction and (2018) Data from: The consequences of daily cyclic hypoxia on a European grass grass European a on hypoxia cyclic of daily consequences The from: Data (2018) (1986) Akaike (1986) informationcriterion statistics. . (2006) Ecophysiological responses of juvenile summer and winter flounder to to flounder winter and summer juvenile of responses Ecophysiological (2006) (2018) Short-term acclimation in adults does not predict offspring acclimation acclimation offspring predict not does adults in acclimation Short-term (2018) (2012) Energy (2012) homeostasis as an integrative tool for assessing limits of et al. et (2012) NIH (2012) Image toImageJ: 25years of image analysis. (2017) Supply and demand: How does variation in atmospheric oxygen oxygen atmospheric in variation How does demand: and Supply (2017) Ecological Monographs, Ecological Aquaculture International, Aquaculture Scientific reports, Scientific

Palaemonetes varians Palaemonetes 217, 210,

325, 4193-4202. 2403-2409. insect tracheal and mitochondrial networks? networks? mitochondrial and tracheal insect Integrative and Comparative Biology, Comparative and Integrative 255-266. Marine Ecology Progress Series, Progress Ecology Marine e in aquatic invertebrates. invertebrates. e in aquatic Procambarus clarkii Procambarus developmental plasticity evolution and the of

8, dynamics insalt marsh pools and its potential 3174.

78, . Marine Ecology Progress Series, Progress Ecology Marine

387-402. 16, 455-470. supply on the growth and energy allocation allocation energy growth and the supply on during premolt. premolt. during

Fenneropenaeus chinensis Fenneropenaeus Marine EnvironmentalResearch, Dordrecht, The Netherlands: D. D. Netherlands: The Dordrecht,

258,

55, 223-232. The Journal of of Journal The 771-791. Nature Methods, Nature Journal of of Journal Journal of Insect Insect of Journal

505, 177-191. following Marine

79,

Accepted Article Welsh, The B.L.role (1975) of grass shrimp, This article is protectedThisarticle is byAll rightsreserved. copyright. three discrete weeks: 4 in marsh salt Lymington the from channel one within measured temperature and Oxygen 1: Figure quantitativePCRTable 1:Primers in usedfor and tables: Figures Additional supporting information maybe found in theonlineversion of this article. informationSupporting respectively. Real-time PCR (right) of the same cuticular genes: gene expression (on a log (on a expression gene genes: cuticular same the of (right) PCR Real-time respectively. for change fold Proportional experiment. RNA-seq the Figure 2: concentration 10 concentration undiluted the from started and dilution serial 10-fold by (produced dilutions cDNA of number normalized to multiple reference genes ( genes reference normalized multiple to CaAP by“*”. indicated are treatments between values Significance deviation. ±Standard (CNRQ) quantities : calcification associated peptide; peptide; associated calcification : Society, of juvenile Chinese shrimp, Ecology,

Normalized expression values of

40,

56, 0 cDNA)of for which each primer pair 483-492. 513-530. th – 11 – th August 2016;16 Fenneropenaeus chinensis Fenneropenaeus DD5 EF1-alpha three cuticular contigs (left) differentially expressed from from expressed differentially (left) contigs cuticular three : peptide DD5; DD5; : peptide Palaemonetes pugio Palaemonetes Palaemon varians Palaemon th –23 and rd CaAP showed the reported Efficiency and R and Efficiency reported the showed February; 14 February; RPL8 PMP , ), giving calibrated, normalized, relative relative normalized, calibrated, ), giving DD5

. : post-moult protein. Journal of the World Aquaculture . Linear range is expressed as the the as expressed is range Linear . , in atidal marshecosystem., , PMP th –22 was 4.7 and5.3, 189, nd May. 10 scale) is 2 . Accepted Article different treatments (n=6 for each treatment for each time point). Expression (on a log a time (on each Expression point). treatments for treatment for different (n=6 each Figure 4: values between treatments. indicate significance Different letters treatment. each Horizontal for median the linerepresents This article is protectedThisarticle is byAll rightsreserved. copyright. n=28). normoxia Figure 3: letters indicate significance betweenvalues treatments. Figure Morphological5: on analysis model. inthe parameters of number chosen. df: lower was AIC Akaike's Information values Criterion AIC– betweenmodels– were compared and modelthe with each for (“X”) data expression gene independently data, regardless Formula: of the treatments. Y~smooth(X); Modelfitting GAM model 2: expression gene all fitting model GAM 1: Model used. models GAM of statistics Descriptive 2: Table the formula: Y~smooth(X). For by=treatment). Y~smooth(X, formula: the with cyclic normoxia) dashed: hypoxia, (solid: treatment each for fitted For deviation. ±Standard (CNRQ) quantities ( genes reference two normalized to

Expression of markers at different time points from ecdysis (day 0) to 16 days after in in after days 16 to 0) (day ecdysis from points time different at markers of Expression A) Frequencymoulted of animals overwithin time two treatments (cyclicthe hypoxia n=26; B) Scatter plot showing inter-moult duration the (days) two in treatments. CaAP , dot-dashed lineGAM represents a model without fitted treatments, with P. varians EF1-alpha DD5 gills. For all graphs, mean±SD are plotted. Different are mean±SD allgraphs, gills. For and and and and treatment. Formula: Y~smooth(X,by=treatment). Y~smooth(X,by=treatment). Formula: treatment. RPL8 PMP ), giving calibrated, normalized, relative relative normalized, calibrated, ), giving , plotted lines represent GAM models models GAM represent lines , plotted

10 scale), Target: RPL8 CaAP PMP Accepted alpha EF1- DD5 Article (mean±SD, n=4 hypoxia, n=8 normoxia); normoxia); n=8 hypoxia, n=4 (mean±SD, This article is protectedThisarticle is byAll rightsreserved. copyright. normoxia).Different letters indicate significance values between treatments. on hypoxia cyclic of Impact 8: Figure letters indicate significance betweenvalues treatments. in normoxia or hypoxia cyclic Figure 7: Y=245.7-0.26X). Formula: line. dotted blue regressions fitted for eachtreatment (cyclic hypoxia: red line. Formula: Y=245.7-0.6X; normoxia: linear indicate Lines animals/treatment/day). (n=90-95 ±SEM weight body Mean 6: Figure

contigs: contigs: Reference contig_226 13 contig_104 19 contig_163 contig_41 0.3/0.3 47 contig_235 A) Feed ingestion rates and and rates ingestion Feed (µM): conc. primer R F / 0.3/0.3 0.9/0.9 0.3/0.3 0.3/0.3 sequence: sequence: 5'-3' FOR GG GCCCAAAGT AATTCAGCA TATT TGGTGCACC TCCCGGTCG GA TTCGAGTTG ATCGTGGAC TG ATTCGTGGC ACACTATGC AT AGCCCAAGT ACAGCACTG P. varians varians P. A) B) proportion of reproductive females; females; ofreproductive proportion ammonium excretion after different days of exposure to to exposure of days different after excretion ammonium (mean±SD, n=10-13 animals/treatment/day). Different n=10-13 (mean±SD, C) egg dry weight (means±SEM, n=36 hypoxia, n=88 n=88 hypoxia, n=36 (means±SEM, weight dry egg sequence: sequence: 5'-3' REV CACCAGTCTTT GACGGCCTCGGT CGTCAGGT AATACTCGTTGC AACTCAGC CAGGCAGACATG ATTGGCACA GAAATGGGAAGG GTCCAACA CAGGAACTGGAG

(bases): n size Amplico 179 179 106 113 115 1.88 0.99 5 116 1.91 0.98 4 B) relative fecundity fecundity relative ncy: ncy: Efficie 1.84 1.85 1.86 R 0.95 1 0.99 2 : 5 4 4 range: Linear Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. Model 2: Model 1: fAC df dfAIC df AIC AIC 7.64 267.18 7.10 209.14 7.06 293.97 293.97 7.06 209.14 10.85 11.31 205.85 285.30 269.22 12.26 7.10 267.18 7.64 CaAP

DD5 PMP PMP DD5

Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright.