63
Assessing he uppe he mal limi cons aining he physiological
pe o mance o Callinec es sapidus emb yogenesis unde clima e
wa ming scena ios
Ángela Rod íguez-Ruiz1,2 , Gus a o F. de Ca alho-Souza1, Inma He e a3,
Ignacio González-Go dillo2, En ique González-O egón1
1 Ins i u e o Ma ine Sciences o Andalusia, Spanish Na ional Resea ch Council (ICMAN-CSIC), Campus Uni e si a io Río San Ped o, 11519, Pue o Real, Spain
2 Depa men o Biology, Ma ine Resea ch Ins i u e (INMAR), Uni e si y o Cadiz, Pue o Real Campus, Pue o Real, Spain
3 G upo de In es igación en Biodi e sidad y Conse ación (BIOCON), Ins i u o Uni e si a io ECOAQUA, Uni e sidad de Las Palmas de G an Cana ia (ULPGC),
Telde, Spain
Co esponding au ho s: Ángela Rod íguez-Ruiz (angela. [email p o ec ed]); En ique González-O egón (e.gonzalez.o [email p o ec ed])
Copy igh : © Ángela Rod íguez-Ruiz e al.
This is an open access a icle dis ibu ed unde
e ms o he C ea i e Commons A ibu ion
License (A ibu ion 4.0 In e na ional – CC BY 4.0).
Resea ch A icle
Abs ac
Emb yonic de elopmen ep esen s a ulne able li e s age in ma ine o ganisms, ye i s ole in
shaping he in asion success o non-na i e species unde clima e change emains unde s udied.
In his s udy, we assessed he uppe he mal sensi i i y o emb yogenesis o he blue c ab Call-
inec es sapidus, a globally in asi e species, by quan i ying hei physiological esponses ac oss a
empe a u e g adien ele an o p ojec ed clima e wa ming scena ios. Using Elec on T anspo
Sys em (ETS) ac i i y as a p oxy o ae obic me abolism, we e alua ed espi a ion, egg size,
ha ching ime, and la al mo phology in b ooding eggs incuba ed a 22 °C, 24 °C, 26 °C, and
28 °C. Ele a ed empe a u es induced inc eased ETS ac i i y, indica ing heigh ened me abolic
s ess, and we e associa ed wi h educed egg size and ea lie ha ching o mal o med, non- iable
la ae. Wi hin he Oxygen- and Capaci y-Limi ed The mal Tole ance (OCLTT) amewo k, we
iden i ied a physiological pejus ange (24–26 °C) beyond which emb yonic pe o mance de-
clined. These esul s sugges ha mode a e wa ming may accele a e de elopmen and acili a e
in asion, bu ex eme empe a u es cons ain ae obic capaci y and comp omise la al iabili y.
Ou esul s highligh emb yogenesis as a po en ial bo leneck o blue c ab ec ui men unde
u u e wa ming, wi h implica ions o p edic ing he in asi e po en ial o ma ine species.
Key wo ds: Callinec es sapidus, ecophysiology, ETS, hea wa es, la al ecology, ma e nal e ec s,
he mal s ess
In oduc ion
Phenological windows ha e been sugges ed as a pa o a wa ning sys em enabling
mo e a ge ed p og ams o moni o ing in asi e species (Giménez e al. 2020).
Wa ming coas al wa e s ha e likely con ibu ed o he ec ui men and no hwa d
expansion o he in asi e blue c ab Callinec es sapidus Ra hbun, 1896, beyond i s
his o ical ange a Cape Cod, esul ing in he es ablishmen o pe manen popu-
la ions in new a eas (Johnson 2015; Taylo e al. 2022; C ane e al. 2024). The
Ame ican blue c ab C. sapidus (B achyu a, Po unidae) is na i e o he A lan ic
Academic edi o : Paula Chainho
Recei ed:
31 Janua y 2025
Accep ed:
12 June 2025
Published:
7 Oc obe 2025
Ci a ion: Rod íguez-Ruiz Á, de
Ca alho-Souza GF, He e a I,
González-Go dillo I, González-O egón
E (2025) Assessing he uppe he mal
limi cons aining he physiological
pe o mance o Callinec es sapidus
emb yogenesis unde clima e
wa ming scena ios. In: Anas ácio P,
Ribei o F, Chainho P (Eds) In asions
in Aqua ic Sys ems. NeoBio a 102:
63–79. h ps://doi.o g/10.3897/
neobio a.102.148122
NeoBio a 102: 63–79 (2025)
DOI: 10.3897/neobio a.102.148122
Ad ancing esea ch on alien species and biological in asions
A pee - e iewed open-access jou nal
NeoBio a
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NeoBio a 102: 63–79 (2025), DOI: 10.3897/neobio a.102.148122
Ángela Rod íguez-Ruiz e al.: Tempe a u e e ec s on Callinec es sapidus ea ly de elopmen
coas , anging om No a Sco ia, Canada, o no he n A gen ina, including he
Gul o Mexico (Squi es 1990). Ou side i s o iginal dis ibu ion, he species has
es ablished in asi e popula ions in a ious egions o A ica, Asia and Eu ope
(Neh ing 2011). In he Ibe ian Peninsula, he i s eco d o he species occu ed
in he Tagus Es ua y in 1978 (Gaudencio and Gue a 1979). Since hen, i has
apidly sp ead along he Spanish coas s, eaching he Guadalqui i Es ua y in he
Gul o Cadiz by a ound 2017 (González-O egón e al. 2020).
The p ima y ec o o he in oduc ion o C. sapidus is hough o be bal-
las wa e eleases, which can anspo plank onic la ae du ing up ake (Neh ing
2011). Conside ing ha la al de elopmen in C. sapidus las s 37–69 days, he
ime ame allows o plausible ansoceanic anspo , al hough seconda y dispe -
sal is also possible (Neh ing 2011). The species’ success ul es ablishmen can be
a ibu ed o se e al ecophysiological and de elopmen al ai s, such as he la ae’s
high esis ance o abio ic and bio ic condi ions (Ange 2006; Mo ais e al. 2019),
enabling adap a ion o a wide ange o en i onmen al ac o s.
The la al ecology o C. sapidus is o pa icula in e es , as adul popula ion
ec ui men depends on he su i al o la ae and ju eniles o eplenish he pa-
en al s ock (Sandi e 1975), ac ing like a popula ion bo leneck. La al su i al
is a ulne able li e s age hea ily in luenced by en i onmen al ac o s, pa icula ly
seawa e empe a u e and salini y (Cos low and Bookhou 1959; Cos low 1967;
Rosenbe g and Cos low 1976; Rum ill 1990; Ange 2006). While nume ous
s udies ha e been conduc ed on he biology and ecology o C. sapidus in i s
na i e ange, mos esea ch has ocused on i s comme cial impo ance and he
en i onmen al condi ions in i s o iginal habi a s (Olmi and O h 1995; Daly
e al. 2021). Howe e , he e is a no able lack o s udies add essing he e ec s o
en i onmen al changes in in aded egions such as he Gul o Cadiz.
The Gul o Cadiz is a empe a e and wa m ecosys em en iched by i e dis-
cha ges ha injec nu ien s and ace me als which s imula es p ima y and sec-
onda y p oduc ion (P ie o e al. 2009; González-O egón e al. 2019), hus c e-
a ing a po en ially sui able en i onmen o he de elopmen o in asi e species
like C. sapidus. The Gul o Cadiz connec ed o he Medi e anean Sea h ough
he S ai o Gib al a , acili a es a wo-laye wa e exchange: a su ace in low
om he Eas e n No h A lan ic and deepe ou low o Medi e anean saline
wa e s (Sánchez-Leal e al. 2017). This hyd odynamic egime, along wi h sig-
ni ican an h opogenic ans o ma ion along he coas , including majo po s
like Algeci as and ex ensi e aquacul u e in as uc u e (González-O egón and
Mo eno-And és 2021), may ha e acili a ed he wes wa d expansion o he in-
asi e A lan ic blue c ab Callinec es sapidus om he Medi e anean in o he A -
lan ic. Gene ic e idence suppo s a single in asion e en in o he Medi e anean
ollowed by seconda y sp ead in o adjacen A lan ic wa e s (González-O egón
e al. 2022). The po en ially less ex eme uppe he mal limi s in he A lan ic
egion may o e mo e a ou able condi ions o he species’ es ablishmen and
su i al. This is consis en wi h he in asi e success o C. sapidus, which gene -
ally exhibi s wide he mal ole ance and may possess adap i e ad an ages o e
na i e species such as Ca cinus maenas (Ange 2006; Neh ing 2011).
Labo a o y expe imen s sugges ed ha C. sapidus equi es high empe a u es
(abo e 21 °C) o op imal la al de elopmen (Hill e al. 1989; Bembe e al. 2017).
Combined wi h he wa m sea empe a u es o he Gul o Cadiz, his aises he
ques ion o how hese condi ions will a ec i s de elopmen and expansion in he
65
NeoBio a 102: 63–79 (2025), DOI: 10.3897/neobio a.102.148122
Ángela Rod íguez-Ruiz e al.: Tempe a u e e ec s on Callinec es sapidus ea ly de elopmen
egion. While mos s udies ocus on es ima es o c i ical he mal maxima (CTmax)
o le hal limi s (LT50) when exposed o he mal s ess, insigh s on he in e media e
physiological cons ain s a e mo e ele an in ecological s udies, especially in o -
ganisms wi h c i ical li e s ages as c us aceans. Explo ing he animal Oxygen- and
Capaci y- Limi ed The mal Tole ance (OCLTT) h ough hei physiological pe -
o mance, could p o ide aluable in o ma ion abou hei clima e esponses in a
mo e ealis ic scena io o g adual ising empe a u es (Pö ne e al. 2017).
In his sense, he objec i e o he p esen s udy is o assess he uppe he mal
limi cons aining he emb yogenesis pe o mance o C. sapidus, as he global
wa ming con ex in he Gul o Cadiz could ac as a bo leneck o popula ion
ec ui men . Pe o mance is explo ed h ough in e linked pa ame e s cha -
ac e ising emb yonic he mal sensi i i y and ae obic window, as he Elec on
T anspo Sys em (ETS) ac i i y, egg size de elopmen , ha ching success, and
la al mo phology. ETS ac i i y assays, a po en ial indica o o espi a ion, is a
common p oxy o plank onic espi a ion (Packa d 1971; He e a e al. 2017),
and has p o en e ec i e o es ima ing me abolic a es in c us aceans (Simčič
and B ancelj 2004; Simčič e al. 2014; Ruiz-Delgado e al. 2019; He e a e
al. 2024). Explo ing ETS ac i i y on b ooding eggs o C. sapidus exposed o
di e en empe a u es could p o ide insigh s in o a ia ions on he me abolic
dynamics du ing emb yonic de elopmen unde wa ming condi ions. Egg size
dynamics o e incuba ion ime may be an indica o o de elopmen a es, which
coupled wi h ha ching ime and la al mo phology, p o ides aluable in o ma-
ion abou la al i ness and su i al success unde wa ming condi ions.
Me hods
Egg collec ion and expe imen al design
Eggs masses o Callinec es sapidus we e ea ed in he labo a o y o assess he e -
ec s o inc eased seawa e empe a u e on emb yonic de elopmen p io o la al
ha ching. Eggs we e ob ained om six o ige ous emales ( e e ed as F1, F2, F3,
F4, F5 and F6) a simila s ages o emb yonic de elopmen . These o ige ous e-
males we e manually collec ed by a local ishe man in a shallow inle o he Gua-
dalqui i Es ua y (Sanluca de Ba ameda, Gul o Cadiz, Spain) du ing Augus
2023. Gene ic analysis was pe o med o de e mine he haplo ype o each emale,
in o de o explo e whe he he exis ence o possible ou lie s in he s a is ical anal-
ysis could be a ibu ed o gene ic di e ences be ween hem. Two di e en hap-
lo ypes o C. sapidus coexis in he s udied a ea: CSWM1, p edominan in Span-
ish A lan ic coas ; and CSWM2, p edominan in Spanish Medi e anean coas
(González-O egón e al. 2022). In ou s udy, F1, F2, F3, F5 and F6 belonged o
CSWM1 haplo ype, while F4 belonged o CSWM2.
Each egg mass was emo ed om he emale abdomen and i s olume was
e enly di ided among ou empe a u e ea men s in indi idual con aine s
(1000 ml): 22 °C, 24 °C (de ined as con ol), 26 °C, and 28 °C. These em-
pe a u es we e selec ed based on he (i) a ailable bibliog aphy which se le he
lowe he mal limi o he success ul emb yonic de elopmen (> 21 °C) (Ji o
e al. 2007; Bembe e al. 2017), (ii) he mean summe wa e empe a u e a he
sampling si e o o ige ous emales when b ooding and spawning season occu
(24 °C), and (iii) he IPCC RCP8.5 p ojec ed wa ming scena ios o +2 °C and
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NeoBio a 102: 63–79 (2025), DOI: 10.3897/neobio a.102.148122
Ángela Rod íguez-Ruiz e al.: Tempe a u e e ec s on Callinec es sapidus ea ly de elopmen
+4 °C o he end-o -cen u y (Pö ne e al. 2019). One g oup was kep a a
con olled empe a u e, while he o he s we e g adually exposed o di e en ex-
pe imen al empe a u es (22 °C, 26 °C, and 28 °C). A e 24 hou s a 24 °C
(T0), hey we e g adually ans e ed o 22 °C and 26 °C (T1) o e a 24-hou
pe iod, while ano he g oup, p e iously a 26 °C, was ans e ed o 28 °C one
day la e o e a 24-hou pe iod (T4), allowing he eggs ime o adap o he h ee
new empe a u es (Fig. 1). Fil e ed seawa e (35 o salini y) was enewed daily,
o p e en ungal, bac e ia and o he mic oo ganism’s g ow h in he egg mass.
Emb yonic de elopmen
Du ing he expe imen , a sample o eggs pe ea men and emale was collec -
ed o emb yonic de elopmen al analysis, in o de o es he possible e ec s o
empe a u e on egg size o e ime. Samples we e s o ed in 2 mL Eppendo ubes
con aining seawa e a -20 °C. Fo each sample, he majo diame e s o wen y
andomly selec ed eggs we e measu ed unde a s e eo mic oscope (SMZ25/18,
Nikon Ins umen s Inc.) using he NIS-elemen s Imaging So wa e . 5.21.00.
Thus, he median egg size (µm) and s anda d de ia ion we e calcula ed. Images
we e cap u ed a a scale o 250 µm.
Elec on T anspo Sys em (ETS) assay
Egg samples (~15 mg) om each ea men and emale we e collec ed daily o
measu e espi a o y ETS ac i i y (in µL O2 · h−1 · mg p o −1), e lec ing cellu-
la -le el changes du ing emb yonic de elopmen . Samples we e p ese ed in
Eppendo ubes, ozen in liquid ni ogen (-196 °C), and s o ed a -80 °C p io
o analysis. The ETS assay ollowed he me hod o Packa d (1971), modi ied by
Owens and King (1975), and adap ed o mic opla e eadings by Ruiz-Delga-
do e al. (2019). Eggs we e homogenized in 0.5 mL o ice-cold homogenizing
bu e solu ion (20 mM T izma base, pH 7.8, Sigma-Ald ich) using an ul a-
sonic homogenize (UP2005 Hielsche ) se o 1 cycle a 25% ampli ude o 60
seconds. The homogena e was cen i uged a 3 °C o 10 minu es a 5,000 pm
(Eppendo Cen i uge 5417R), and he supe na an was used o he ETS assay.
In a mic opla e assay, 60 µL o supe na an (in duplica e) was incuba ed wi h
180 µL o subs a e solu ion (0.1 M phospha e bu e , pH 8.5, con aining NA-
DPH 30 mM and NADH 1.76 mM, Sigma-Ald ich) o wi hou subs a e (con-
ol, con aining only 180 µL o phospha e bu e , pH 8.5). Then, 60 µL o INT
solu ion (0.2%, 4 mM, pH 8.5) was added o each sample, and abso bance was
measu ed a 490 nm o e 8 minu es using a mic opla e eade (BioTek Syne gy
H1) and Gen5 3.10 so wa e. ETS ac i i y was co ec ed o in si u empe a u e
using an ac i a ion ene gy o 15 kcal·mol−1 (Packa d 1971) and he A henius
equa ion o calcula e in si u ETS ac i i y (uni s: µL O2 · h−1). To calcula e ETS
ac i i y pe uni biomass, p o ein biomass (mg p o ein) was de e mined using
he bicinchoninic acid (BCA) me hod desc ibed by Smi h e al. (1985). Fo
his, 25 µL o each sample and s anda ds we e incuba ed wi h 200 µL o BCA
wo king solu ion o 30 minu es a 37 °C, and abso bance was ead kine ically
a 562 nm. ETS ac i i y was hen no malized o p o ein con en and exp essed
as ETS ac i i y pe uni p o ein (µL O2 · h−1 · mg p o −1).
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Ángela Rod íguez-Ruiz e al.: Tempe a u e e ec s on Callinec es sapidus ea ly de elopmen
Egg ha ching ime and la al iabili y
Egg mass cul u es we e main ained unde di e en empe a u e ea men s un il
la al ha ching, which ma ked he end o he expe imen o each sample condi-
ion. Ha ching ime was eco ded as he numbe o days om day 0 ( he s a o
incuba ion) un il la ae appea ed.
The app oxima e numbe o newly ha ched la ae was coun ed o each ea -
men and emale. Obse a ions on he mo phological condi ion and mo ili y
o he la ae we e documen ed o assess hei iabili y. This included no ing he
p esence o absence o abe an zoea, and e alua ing hei pho o ac ic swimming
beha io , acco ding o Ji o e al. (2007). In his s udy, abe an zoea is de ined
as an indi idual a a mo phological s age simila o p ezoea bu ha is immobile,
exhibi s sinking beha iou , and subsequen ly dies. In con as , unde op imal con-
di ions, p ezoea mol s in o he i s zoeal s age wi hin he i s 3 minu es o li e
a e hei elease (Cos low and Bookhou 1959; Da is 1965).
S a is ical analysis
The ela ionship be ween p edic o s wi h he ETS ac i i y (µL O2 · h-1 · mg p o -1)
and egg size (µm) o C. sapidus emb yos was examined h ough a Gene alized Ad-
di i e Model (GAM) wi h logno mal and no mal dis ibu ion, espec i ely, based
on he dis ibu ion o he dependen a iables (Zuu e al. 2009). The GAM model
was selec ed as i assumes no unc ional o m be ween dependen and independen
a iables and desc ibes bo h linea and non-linea e ec s, and was pe o med on
RS udio so wa e .2024.12.0. A a iance in la ion ac o (VIF) wi h a h eshold
o 3 was used o iden i y possible collinea i y be ween p edic o s in he da a se ,
be o e i ing models o he da a (Zuu e al. 2010). This analysis indica ed no
mul icollinea i y as all explana o y a iables had VIF < 3 (see Suppl. ma e ial 1:
ables S1, S2), so he gene al o m o he GAM was:
g(y) ~ α + 1(xi) + ε + ε
Figu e 1. Expe imen al design. Schema ic diag am o he empe a u e ea men design and accli-
ma ion p o ocol applied o C. sapidus emb yos ac oss he expe imen al pe iod (T0-T10). Dashed line
indica es con ol a 24 °C.
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NeoBio a 102: 63–79 (2025), DOI: 10.3897/neobio a.102.148122
Ángela Rod íguez-Ruiz e al.: Tempe a u e e ec s on Callinec es sapidus ea ly de elopmen
whe e g() is he link unc ion, y is he esponse a iable (ETS ac i i y o egg size),
1 is he smoo h unc ion o he xi con inuous explana o y a iable (incuba ion
ime), ε is he ca ego ical e ec o he empe a u e ea men s, and ε is he an-
dom e ec o emale o igin o emb yos, cap u ing i s in insic a iabili y. Also,
due o he unbalance ep esen a ion o haplo ypes, his ac o was no included
in he GAM analysis. Es ima ed R2 and explained de iance we e used o e alua e
he p edic i e pe o mance o he model. The esiduals we e g aphically e alua ed
wi h QQ-plo s, his og ams, and plo s o esidual esponse agains i ed alues o
explo e any pa e ns in he esidual e o s (see Suppl. ma e ial 1: igs S2, S3).
Pos -hoc analysis using he Tukey es o a ying amily sizes was pe o med o
iden i ying signi ican di e ences be ween speci ic le els o he ca ego ical ac o
a ec ing emb yo ETS ac i i y and egg size.
Resul s
Tempe a u e-dependen a ia ion in egg size
A gene al pa e n o dec easing egg size o e de elopmen ime was obse ed ac oss
all empe a u e ea men s (see Suppl. ma e ial 1: ig. S1). Unde con ol condi-
ions (24 °C), among all emales, eggs a he e y ea ly s age o emb yonic de el-
opmen had a mean diame e o 273.4 ± 13.8 µm, which dec eased o 241.5 ±
12.8 µm jus be o e ha ching, a educ ion o app oxima ely 11.7%.
The global median egg size esul ed in 247.59 µm. Smalle egg sizes, below 245 µm,
we e ound unde wa me condi ions o 26 °C and 28 °C; while la ge eggs, abo e 245
µm, esul ed in colde empe a u es o 22 °C and 24 °C, eaching he maximum a
con ol condi ions o 24 °C (306.40 µm) (Table 1). These esul s sugges ha ele a ed
empe a u es nega i ely a ec emb yonic de elopmen by educing egg size.
GAM analysis showed signi ican e ec o incuba ion ime, empe a u e ea -
men , and ma e nal o igin on egg size (see Suppl. ma e ial 1: able S1). The model
explained a mode a e po ion o he a iabili y in egg size (R2 = 0.282; de iance ex-
plained = 28.7%), wi h all p edic o s showing signi ican e ec s (p < 0.001). The e -
ec o incuba ion ime on egg size was non-linea and highly signi ican (ed = 7.37,
F = 70.02, p < 0.001). The smoo h unc ion in Fig. 2A indica ed a sha p decline in
egg size du ing he ea ly s ages o emb yo de elopmen , ollowed by a pe iod o s abi-
liza ion and a seconda y decline a ound T8. Con idence in e als a ound he smoo h
e m sugges ed a p ecise es ima ion o his empo al pa e n, wi h wide in e als
in la e s ages, whe e da a densi y is lowe due o di e en la ae ha ching imes.
The andom e ec o ma e nal o igin was signi ican as well (ed = 4.78, F = 20.71,
p < 0.001), indica ing ha indi idual emales con ibu ed o a iabili y in egg size,
jus i ying i s inclusion as a andom smoo he in he model. Al hough he gene ic
backg ound o emales could be a ac o con ibu ing o he a iabili y in egg size, he
p esence o ou lie s was no es ic ed o a single emale (F4 belonged o CSWM2
haplo ype), bu a he dis ibu ed ac oss se e al indi iduals.
Tempe a u e ea men s also signi ican ly a ec ed egg size (F = 39.51, p < 0.001).
Compa ed o he e e ence le el (22 °C), egg size dec eased signi ican ly a highe
empe a u es o 26 °C and 28 °C (es ima es o -6.77 µm and -4.17 µm, espec i e-
ly; p < 0.0001 o bo h cases), and o a lesse ex en a 24 °C (Es ima e = -1.73 µm;
p = 0.016). The Tukey-adjus ed pos hoc compa isons con i med ha all pai wise
di e ences we e signi ican , excep be ween 22 °C and 24 °C empe a u e g oups
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NeoBio a 102: 63–79 (2025), DOI: 10.3897/neobio a.102.148122
Ángela Rod íguez-Ruiz e al.: Tempe a u e e ec s on Callinec es sapidus ea ly de elopmen
(see Suppl. ma e ial 1: able S1). These esul s indica ed ha small inc emen s in
empe a u e beyond he op imal ange can in luence egg de elopmen . The io-
lin plo in Fig. 2B suppo ed hese indings, p o iding a isual summa y o bo h
cen al endency and da a dis ibu ion. The boxplo s nes ed wi hin he iolins
highligh he cen al endency o dec easing median egg size wi h inc easing em-
pe a u e, as well as inc easing ou lie p e alence a 26 °C and 28 °C, sugges ing a
s ess- ela ed de elopmen al cons ain .
Elec on T anspo Sys em (ETS) ac i i y assay
Emb yos ETS ac i i y, a p oxy o po en ial espi a ion a es and exp essed as
log- ans o med speci ic ETS ac i i y, was signi ican ly in luenced by incuba ion
ime, empe a u e ea men , and ma e nal o igin (see Suppl. ma e ial 1: able S2).
The GAM explained 61.4% o he de iance in ETS ac i i y (R2 = 0.584), indica -
ing s ong explana o y powe o hose selec ed a iables and ein o cing he biolog-
ical ele ance o he mal and ma e nal in luences on emb yonic me abolic pe o -
mance. The smoo h e ec o incuba ion ime was signi ican (ed = 2.80, F = 33.14,
p < 0.001), indica ing a non-linea inc ease in ETS ac i i y h oughou he incu-
ba ion pe iod (Fig. 3A). The i ed smoo h e m sugges ed a g adual accele a ion
o ETS ac i i ies o e ime, wi h a p onounced inc ease a ound T4 (4 h day), when
Table 1. Egg size (µm) summa y by empe a u e ea men . Median egg size (µm), s anda d de ia-
ion (SD), and ange (maximum and minimum alues) pe empe a u e ea men .
Tempe a u e (°C) Median ± SD (µm) Maximum (µm) Minimum (µm)
22 248.22 ± 13.21 296.06 215.03
24 250.55 ± 17.34 306.40 211.15
26 244.49 ± 12.64 291.90 218.21
28 244.84 ± 13.24 301.26 220.66
Figu e 2. G aphical ep esen a ion o he e ec s o he incuba ion ime and empe a u e on C. sapidus egg size (µm). Resul s o he GAM
analysis showing A. Pa ial e ec o incuba ion ime (du ing 10 days) on egg size, whe e he black solid line indica es he modelled ela ion-
ship, and he g ey band deno es he 95% con idence in e al abou he es ima ed ela ionship, and B. Violin plo showing he dis ibu ion
o egg size unde es ed empe a u e ea men s (22 °C, 24 °C, 26 °C, and 28 °C), whe e boxplo s embedded wi hin he iolins indica e
he in e qua ile anges and cen al endency.
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Ángela Rod íguez-Ruiz e al.: Tempe a u e e ec s on Callinec es sapidus ea ly de elopmen
he expe imen al inc ease in empe a u e o 28 °C was pe o med. This poin o
in lec ion indica es a me abolic shi , ollowed by con inued inc easing o ETS and
widening con idence in e als owa d he la e s ages o de elopmen . The e ec
o ma e nal o igin was also highly signi ican (ed = 4.75, F = 17.31, p < 0.001),
unde lining indi idual emale a iabili y in he baseline me abolic ac i i y du ing
emb yogenesis. Al hough he gene ic backg ound o emales could be a ac o con-
ibu ing o he a iabili y in ETS ac i i y, he p esence o ou lie s was no es ic -
ed o a single emale (F4), bu a he dis ibu ed ac oss se e al indi iduals.
Tempe a u e ea men s s ongly a ec ed ETS ac i i y o emb yos. Compa ed o he
e e ence le el o 22 °C, ETS ac i i ies we e signi ican ly highe a majo empe a u e
egimes o 24 °C (Es ima e = 0.39, p < 0.001), 26 °C (Es ima e = 0.82, p < 0.0001),
and 28 °C (Es ima e = 0.79, p < 0.0001). Pos hoc Tukey-adjus ed compa isons con-
i med signi ican pai wise di e ences be ween 22 °C and all o he ea men s, as well
as be ween 24 °C and bo h 26 °C and 28 °C (see Suppl. ma e ial 1: able S2). ETS ac-
i i y seemed o become cons an a he wo highes empe a u es, wi h no signi ican
di e ence be ween 26 °C and 28 °C (p = 0.998), sugges ing a h eshold e ec whe e
ETS ac i i y ceases o inc ease despi e addi ional wa ming. The Fig. 3B co obo a ed
his pa e n wi h wide dis ibu ions and highe medians unde wa me condi ions,
consis en wi h he mally enhanced me abolic a es. The shape o he iolins indica es
ela i ely symme ic dis ibu ions, con as ing wi h he igh -skew obse ed in egg size.
Ha ching ime
A gene al pa e n o ea ly la al ha ching a highe empe a u es was obse ed
(Table 2), which was also con i med by he pho og aphic analysis o he mo pholog-
ical cha ac e is ics on egg de elopmen samples (Fig. 4). In all emales, ea ly ha ching
occu ed be ween 6–7 days a he highes empe a u es (26–28 °C), while ha ching
a lowe empe a u es (22–24 °C) was delayed, aking up o a maximum o 11 days.
Figu e 3. G aphical ep esen a ion o he e ec s o he incuba ion ime and empe a u e on ETS ac i i y (µL O2 · h−1 · mg p o −1) o
C. sapidus emb yos. Resul s o he GAM analysis showing A. Pa ial e ec o incuba ion ime (du ing 10 days) on ETS ac i i y, whe e he
black solid line indica es he modelled ela ionship, and he g ey band deno es he 95% con idence in e al abou he es ima ed ela ion-
ship, and B. Violin plo showing he dis ibu ion o ETS ac i i y unde es ed empe a u e ea men s (22 °C, 24 °C, 26 °C, and 28 °C),
whe e boxplo s embedded wi hin he iolins indica e he in e qua ile anges and cen al endency.
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Ángela Rod íguez-Ruiz e al.: Tempe a u e e ec s on Callinec es sapidus ea ly de elopmen
In gene al, lowe empe a u es esul ed in a highe numbe o ha ched la ae,
al hough he e we e di e ences among emales. Only a small numbe o la ae
om emales F2 and F3 ha ched a lowe empe a u es (22–24 °C), bu hese
la ae exhibi ed ac i e and pho o ac ic swimming beha iou , simila o hose om
o he emales unde he same empe a u e ea men s. In con as , a 26–28 °C,
ea ly ha ching o en p oduced > 50 la ae ini ially, bu many we e abe an o
non- iable. Thus, he mal s ess likely induced p ema u e ha ching, leading o
mal o ma ions in la ae mo phology (Fig. 5).
Table 2. Ha ching ime. Eggs ha ching day pe emale and empe a u e ea men . The plus symbol
in b acke s indica es samplings whe e massi e la ae ha ching occu ed (> 50 la ae ha ched o o al
egg olume, simila ac oss all empe a u e ea men s and emales). The as e isk symbol indica es he
p esence o abe an la ae.
Female Tempe a u e (°C)
22 24 26 28
18 (+) 7 (+) 6* 6*
27 (+) 6 6* 6*
37 6 6* 6*
411 (+) 10 (+) 7* 0
511 (+) 10 (+) 7 (+) 7 (+)*
69 (+) 8 (+) 6* 7 (+)*
Figu e 4. Compa a i e mo phological analysis o eggs’ de elopmen be ween empe a u e ea men s. Emb yonic de elopmen o blue
c ab eggs om emale F4 a wo di e en empe a u es A. 22 °C, and B. 28 °C, o e h ee di e en imes: 4-day-old emb yos, 5-day-old
emb yos, and 7-day-old emb yos. A s age 1, egg a achmen s alks a e isible; a s age 2, eye pigmen is de eloping; and a s age 3, abdo-
men and ully o med eyes a e isible, emb yos a e eady o ha ch (Ji o e al. 2007). Scale ba : 250 µm.
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Supplemen a y ma e ial 1
Figu es and ables o he s a is ical analysis o bo h dependen a iables (ETS
ac i i y on emb yos and egg size)
Au ho s: Ángela Rod íguez-Ruiz, Gus a o F. de Ca alho-Souza, Inma He e a, Ignacio
González-Go dillo, En ique González-O egón
Da a ype: pd
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