Emergence, Development, and Maturity of the Gonad of Two Species of Chitons

12 Pages·2013·3.28 MB·English

Emergence, Development, and Maturity of the Gonad of
Two Species of Chitons ‘‘Sea Cockroach’’ (Mollusca:
Polyplacophora) through the Early Life Stages
Omar Hernando Avila-Poveda1*, Quetzalli Yasu´ Abadia-Chanona2
1Divisio´ndeestudiosdeposgrado,UniversidaddelMar,(UMAR),PuertoA´ngel,Oaxaca,Me´xico,2Laboratoriodehistolog´ıa,UniversidaddelMar(UMAR),PuertoA´ngel,
Oaxaca,Me´xico
Abstract
Thisstudydescribesandrecognises,usinghistologicalandmicroscopicalexaminationsonamorphometricalbasis,several
gonadtraitsthroughtheearlylifestagesofChitonarticulatusandC.albolineatus.Gonadalontogenesis,gonaddevelopment
stages, sexual differentiation, onset of the first sexual maturity, and growth sequences or ‘‘early life stages’’ were
determined.Inaddition,allometrybetweenlengthsandbodyweightpooledforbothsexespereachchitonwerecalculated
using equation Y=aXb. A total of 125 chitons (4#TL#40mm, in total length ‘‘TL’’) were used. All allometric relations
showed a strong positive correlation (r), close to 1, with b-values above three, indicating an isometric growth. Gonadal
ontogenesis and gonad development stages were categorised into three periods (‘‘Pw’’ without gonad, ‘‘Pe’’ gonad
emergence,and‘‘Pf’’gonadalsacformed)andfourstages(‘‘S0’’gametocytogenesis,‘‘S1’’gametogenesis,‘‘S2’’mature,and
‘‘S3’’spawning),respectively.Compounddigitalimageswereattainedforeachprocess.Periodsandstagesareoverlapped
among them and between species, with the following overall confidence intervals in TL: Pw 6.13–14.32 mm, Pe 10.32–
16.93mm, Pf 12.99–25.01mm, S0 16.08–24.34mm (females) and 19.51–26.60mm (males), S1 27.15–35.63mm (females)
and 23.45–32.27mm (males), S2 24.48–40.24 mm (females) and 25.45–32.87mm (males). Sexual differentiation (in S0) of
bothchitonsoccursfirstasafemalethenasamale;although,malesreachtheonsetofthefirstsexualmaturityearlierthan
females,thusforC.articulatusmalesat17mmandfemalesat32mm,andforC.albolineatusmalesat23.5 mmandfemales
at 28mm, all in TL. Four early life stages (i.e., subjuvenile, juvenile, subadult, and adult) are described and proposed to
distinguish growth sequences. Our results may be useful to diverse disciplines, from developmental biology to fisheries
management.
Citation: Avila-Poveda OH, Abadia-Chanona QY (2013) Emergence, Development, and Maturity of the Gonad of Two Species of Chitons ‘‘Sea Cockroach’’
(Mollusca:Polyplacophora)throughtheEarlyLifeStages.PLoSONE8(8):e69785.doi:10.1371/journal.pone.0069785
Editor:VincentLaudet,EcoleNormaleSupe´rieuredeLyon,France
ReceivedOctober11,2012;AcceptedJune12,2013;PublishedAugust2,2013
Copyright:(cid:2)2013Avila-PovedaandAbadia-Chanona.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:Fundingforthisresearchwassupportedinmultipleways:securingthesamplingswaspersonallydonebyO.H.Avila-PovedawiththeSNI-CONACYT
stimulus(number44175,fromJanuarytoJuly2012).FacilitiesfortheuseofhistologyequipmentweresupportedbyUMARprojectnumber2II1108toO.H.Avila-
Poveda,andbySAGARPA-CONACyTprojectnumber2003-C01-125acquiredbyS.J.Serrano-Guzma´n.Allofthehistologicalmaterialsweregenerouslydonatedby
S.J.Serrano-Guzma´n(UMARProfessor-Researcher;SAGARPA-CONACyTProjectNo.2003-C01-125).Thestayinthelocalityduringallcollections,thehistological
processingandwritingofthisresearchwassponsoredbyagrantfamilial(SERVITORNO-AVILA)toO.H.Avila-Poveda.ExceptforO.H.Avila-Poveda,theother
fundershadnoroleinstudydesign,collectionsanddataanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction In contrast, the origin and/or the development and/or
maturationofthegonadsofchitonsthroughearlylifestageshave
In invertebrates, the origin and development of the gonad is
been described to a much lesser extent, these studies being from
complex and varied, and it is scarcely described in the literature
the early 20th century (i.e., 1898–1912). Plate [30–32] published
eventhoughitisafundamental aspectofdevelopmental biology.
several treatises on the general anatomy of Chilean chitons,
The reproductive anatomical features (gonad and gonoducts) of
including microscopic traverse sections showing the origin and
polyplacophoranshavebeenstudiedinawidespectrumofspecies
development of the gonad. Haller [33] described the coelom of
around the world because of their relevance to phylogeny. In
chitons including an ovary and a testis, each beginning the
essence, these studies have targeted the morphography by light
emergenceofgametogonia(i.e.,spermatogoniaandoogonia),that
and/or electron microscopy of gonad structure and the shape of
is, sexual differentiation. Heath [34] indicated that sexual
gametesbutalwaysofadultchitons[1–8],withspecialregardto:
differentiation occurs first as a female then as a male during the
accumulation of substances such as nucleic acids, proteins, and
early stages of the chiton Trachydermon raymondi, when the ova
mucopolysaccharides duringoocytedifferentiation [9–13];follicle
appear in a typical fashion, but as the chiton grows, some of the
cellsassociatedwiththeoocytesurface[14–18];thechorion(also
primitive sex cells on the wall of the gonad commence to divide
called hull) of the mature ovum surface [19–24]; spermiogenesis
rapidly and ultimately form smallclusters of spermatozoa among
and acrosome formation [25–27]; and even the general aspect of
theneighbouringova.Higley[35]describedthegrossanatomyof
both female and male gametes in the same gonad of a
hermaphrodite chiton[28–29],among others.
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gonad development during the early life of chitons Trachydermon collectedspecieshavenocurrentregulatorybodyconcernedwith
raymondi andNuttallina thomasi. theprotection ofwildlife orfishery management.
Almost a century has elapsed (until 1974–1999) and similar
studies were published, such as Richter [36], who described Collections
histologically the onset of the first sexual maturity in Lepidochitona DuringthecollectionofChitonarticulatusadultsinOaxaca[43],
cinerea (as L. cinereus). Ball [37] identified the smallest male and Chiton articulatus and Chiton albolineatus of different sizes (i.e.,
female chiton, as well as the onset of the first sexual maturity of subjuvenile‘‘post-metamorphosed’’,juvenile,subadult,andadult)
Stenoplax conspicua. Otway [38] indicated that the chitons weredetectednearhightideandonexposedrocks.Hence,along
Onithochiton quercinus and Plaxiphora albida reached the onset of the therockyintertidalshoresofthetownofPuertoAngel(15u399N–
first reproductive maturity at 2 years of age, i.e., when they still 96u299W), C. articulatus (n=72 sampling size, with total length
have a mediumsize as comparedtoadult size. ‘‘TL’’of5#TL#40mm)werecollectedduringAprilandMayof
Even though there is an extensive knowledge of the gonad of 2011, and C. albolineatus (n=53, 4#TL#40mm) was collected
chitons, in both adults and non-adults, this knowledge is not during March of 2012 (Fig. 1). The smallest adult size for both
integrated making it difficult to determine the gonadal ontogeny collected chitons was established at 40mm TL, based on the
(origin, development and maturation) of chitons through their modal size-class from other Mexican populations of C. articulatus
early life stages, which is one of the fundamental aspects of [44–46] andC.albolineatus [47].
developmental biology. However, some generalizations can be
drawn. According to previous authors, the gonadal ontogenesis, Histological procedure and measurements
thesexualdifferentiationandtheonsetofthefirstsexualmaturity, Chitons were relaxed and allowed to extend for 1 h, with
apparentlyhappenstooearlyinthelifecyclealmostimmediately gradual additions of tap water to the seawater, until the salinity
after the metamorphic stage. However, information from other reached half the salinity of the seawater of the sampling site (i.e.,
mollusks has indicated that they undergo many physiological, 50:50 in volume); and this relaxation process was accelerated by
behavioural and digestive changes during this post-embryonic adding a few milliliters of 10% ethanol [43,48]. Later, chitons
stage and/or post-hatching stage (post-metamorphic stage in werefixedin10%neutralformalin-salinesolutioninseawaterfor
chitons) before gonadal morphogenesis. So, the present study two weeks. Subsequently, they were preserved in 70% ethanol
hypothesises that the origin, development and maturation of with 0.1% glycerin until histological processing [49–50]. After
chitons gonads really happens just after the post-metamorphic preservation,allchitonscollectedwereweighedwithananalytical
stage(i.e.,subjuvenilestage)perhapsduringthejuvenilestage;soit balance (60.001 g, body weight ‘‘BW’’) and measured with a
isalsoimportanttodistinguishgrowthsequences(earlylifestages) verniercaliper(60.1 mm,totallength‘‘TL’’includingthemantle
girdle,andtotalwidth‘‘TW’’includingthemantlegirdle).Weight
based on a consistent terminology: subjuvenile ‘‘post-metamor-
lost at the end of preservation was estimated at a 2% loss of live
phosed’’, juvenile, subadult, andadult.
weight [43]. Smaller chitons (TL#15mm) were decalcified for
The aim of our investigation is to provide, on the basis of
oneweek,immersedin5%commercialvinegar(glacialaceticacid
histological and microscopic examinations, descriptions on the
in water), and the others were disarticulated, valve by valve,
emergence, development, and maturity of thegonad through the
keeping thegross softbody intactandcomplete.
early life stages (i.e., from subjuvenile and along the juvenile and
Chitons were haphazardly cut along the total length (TL) in
subadult stages) of two wild chitons of the Chitonidae family,
sagittal (dorsoventral orientation) and frontal (lateral orientation)
Chiton articulatus and Chiton albolineatus. Likewise, we aimed to
planes,aswellasalongthetotalwidth(TW)inseveraltransverse
distinguish qualitatively and quantitatively these early life stages
planes(Fig.2).Eachportionwasdehydratedinanethanolseries,
based on morphometry, as well as recognise the size range at
cleared in CitrisolvH, and infiltrated and embedded in paraffin
whichchitonsdisplaydiscerniblegonads(gonadaldifferentiation),
[50–51].Serialsectionswerecutat5-mmthicknessusingamanual
andtheonsetofthefirstsexualmaturity(andnotreferringtothe
rotary microtome (LEICAH RM2145) and mounted on glass
sizeatwhich50%ofapopulationissexualmaturegiventhatwe
slides.Groat’shematoxylinanderythrosinewasusedasbasicstain
are not evaluating an adult population). In addition, allometric
since it gives better contrast [52–53]. The modified Crossmon’s
relationships between the lengths and body weight of chitons
trichrome method (i.e., Groat’s haematoxylin, erythrosine B-
throughout early life stages are evaluated, because chitons subtly
OrangeG,andtrypanblueand/orlightgreen[54–56])wasused
change their body shape as they develop into adulthood but
tocontrastconnectivetissue,aswellastoreveal vitellinereserves
particularly evolve in response to gonad maturation. This basic
(i.e., yolk granules)andvacuolar processes.
information may be useful to diverse disciplines, from develop-
mental biology to fisheries management, since recently these two
Microscopic examination
chitons [but particularly Chiton articulatus (locally known as ‘‘dog
Here,twolevelsofformationanddevelopmentofgonadswere
tongue’’ or ‘‘sea cockroach’’)] have gained regional importance
followed: 1) emergence and formation of the gonadal sac (i.e.,
and economic interest in the southern Mexican Pacific, where
gonadal ontogenesis) and, later, 2) the gonad development stages
some restaurants have begun to offer the ‘‘sea cockroach’’ to
(i.e., changes in the ovary and/or testis during growth and
touristsasagourmetandaphrodisiacfood([39–42],Avila-Poveda
maturation), respectively: 1) periods and 2) stages. Periods (P) of
and Abadia-Chanona, pers.obs.).
gonadal sac formation were assigned based on descriptions given
forotherspeciesofchitons[6,32–35],usingtheletterinlowercase
Materials and Methods
of the representative process to give the period’s name: Pw:
withoutgonad,Pe:gonademergence,andPf:gonadalsacformed.
Ethics statement
Stages (S) of gonad development were classified based on the
Nospecificlegalpermitswererequiredforthecollectionofthe
sequenceofthedevelopmentprocessofsexualcells(i.e.,gametes)
specimens in the sampling location, because the location is not
of female chitons [1,2,4,14–17,36] and male chitons [25–27];
privately owned or a protected area in any way. Besides, the
thereby, for both sexes: S0: gametocytogenesis (i.e., immature
collections did not involve endangered or protected species. Both
gametes), S1: gametogenesis, S2: mature, and S3 spawning.
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Figure1.ChitonarticulatusandChitonalbolineatuspreserved.Chitonarticulatus(left)andChitonalbolineatus(right)preserved,showingsome
oftheseveralsizesherestudied.
doi:10.1371/journal.pone.0069785.g001
Nonetheless, the S3 spawning (an adult stage) was not observed
and is only marked in some graphs to understand better the
developmentalprocessofsexualcells(i.e.,gametes)inthecontext
of early lifestages.
The photomicrographs were obtained with a digital camera
(Sony Cybershot DSC-W520, 14.1 Mpixels) mounted on a
microscope(OlympusCX21-FS1),andwerestoredinacomputer
ingraphicformat.AdobePhotoshopHCS2,version9.0,wasused
toadjustthecontrastandleveloftheimages,forthephotographic
assembly process, and to produce composite images with
numerous microscopic fields of 1006and 4006visual magnifi-
cations that show the gross microscopic features of chitons.
GeneralmorphologicalterminologyfollowsRichter[4],Plate[30–
32],Haller[33],HigleyandHeath[35],andRichter[36],among
others.Eachoocytealongitsdevelopmentwasmeasuredthrough
sections containing the nucleus using Carl Zeiss Microimaging H
AxioVisionRelease, Version4.8.2.0.
Allometry and confidence intervals
In addition, the allometric relationships between the lengths
(total length ‘‘TL’’, total width ‘‘TW’’) and body weight (BW),
pooled for both sexes per chiton species were determined with
referencetotheequationY=aXb[43,57–58],whereb(exponent)is
ofinterestasitrepresentsthegrowthtype[59].Thelevels(periods
and stages) here described were also analysed using 95%
confidence limits for the mean of the total length ‘‘TL’’ in order
toestimatetheintervalsamongtheselevels[60].Eachconfidence
interval was expressed as P (value mm#m#value mm) [60].
Statistical analyses were carried out using the computer program
STATISTICAH6 andSigmaStatH3.5.
Figure2.DiagramofChitonarticulatusillustratingthelocation
Results
andorientationofthecuttingplanes.(A)Dorsoventralorientation
to permit the cutting of sagittal sections. (B) Lateral orientation to
Allometry through the early life stages
permit the cutting of frontal sections. (C) Dorsoventral orientation to
permitthecuttingofseveraltransversesections. Therelationbetweenthelengths(totallength‘‘TL’’,totalwidth
doi:10.1371/journal.pone.0069785.g002 ‘‘TW’’) and body weight (BW), pooled for both sexes per each
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Figure3.AllometricrelationshipsduringtheearlylifeofChiton
articulatusandChitonalbolineatus.Relationshipsbetweenmeasure-
ments(totalweight‘‘TW’’,andtotallength‘‘TL’’)andbodyweight(BW)
pooledforbothsexesofChitonarticulatus(0.007to4.400gBW)and
Chitonalbolineatus(0.004to3.632gBW)duringtherangeofearlylife
herestudied.Continuouslineindicatesequationtendency.
doi:10.1371/journal.pone.0069785.g003
chitonspeciesduringtheearlylifestages(0.007to4.400gBWin
Chiton articulatus, and 0.004 to 3.632 g BW in Chiton albolineatus),
showedanequation(Y=aXb)ofstrongpositivecorrelation(r)close
to0.99.Theb-valuerangedfrom3.02to3.06inChitonarticulatus Figure 4. Ontogenesis and maturity of gonads through the
early life of Chitonarticulatusand Chitonalbolineatus. Relation
and from3.00to3.16in C.albolineatus (Fig.3).
amongtotallength(mm)andtheperiodsofthegonadalsacformation
(Pw:withoutgonad,Pe:gonadalemergence,Pf:gonadalsacformed),
Gonadal ontogenesis and the stages of gonad development (S0: gametocytogenesis, S1:
The gonadal sac of chitons is a nearly round structure, but its gametogenesis, and S2: mature) in males and females from Chiton
shape and absolute size vary depending on the number and articulatusandChitonalbolineatus.Thearrowheadindicatesthesexual
differentiation, and the arrow indicates the onset of the first sexual
diameter of gametes enclosed. The formation of the gonadal sac
maturity.Numberofdata(n).
takesplaceinthreeperiods,asfollows:withoutgonad(Pw),gonad
doi:10.1371/journal.pone.0069785.g004
emergence(Pe),andgonadalsacformed(Pf),andgenerallyevolves
with increasing total length (TL) of chitons, but each period is
overlapped with Pe, i.e., with confidence interval of P
represented by a narrow range of total lengths with considerable
(15.21 mm#m#21.39 mm), while in C. albolineatus it occurred
overlap among periods. Thereby, the confidence interval in total
withoutoverlappingpreviousperiods,i.e.,withconfidenceinterval
length‘‘TL’’fortheperiodwithoutgonad(Pw)inChitonarticulatus
of P (12.99 mm#m#25.01 mm) (Fig. 4, Pf). Thus, the first
was P (8.12mm#m#14.32mm) and in Chiton albolineatus was P
appearance of the gonad evolves as a projection of squamous
(6.13 mm#m#10.20 mm) (Fig. 4, Pw), and qualitatively showed
epithelium that extends from the wall of the dorsal aorta and is
only the dorsal aorta covered by muscle tissue and connective
thrown toward the visceral cavity; this could be termed the
fibres,withoutprojectionsofbloodsinusandbloodvesselsinward
‘‘gonadal ridge’’ or the precursor to the gonad (Fig. 6). This
of thevisceral cavity (Fig.5).
squamous epithelium continues projecting until closing very
The period of gonad emergence (Pe) in C. articulatus started
narrowly against the dorsal aorta forming twooval lobes (Fig.7).
overlappingtotallywithPw,i.e.,withconfidenceintervalinTLof
Later, thesquamous epithelium fromthewall of thedorsal aorta
P(10.32 mm#m#16.93 mm);whilstinC.albolineatusthisperiodof
becomes ciliated cuboidal epithelial cells, and the two oval lobes,
gonademergence(Pe)wasnotobserved(Fig.4,Pe).Theperiodof
inturn,mergeanddevelopintothegonadsac(Fig.8).Finally,the
gonadal sac formed (Pf) in C. articulatus started very close to and
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Figure 5.Chitonthroughthe periodwithoutgonad.Chitonarticulatus (0.074g BW, 11mmTL, and 7mmTW) during the period without
gonads.(A)Compositedigitalimageintransversesectionoftheentirespecimenshowingthegeneralhistologicalanatomy.(B)Close-upofthe
dorsalpartofawholespecimen.Abbreviations:a,articulamentum;af,accessoryfold;c,cuticle;ct, ctenidium;da,dorsalaorta;f,foot;go,girdle
ornamentations;k,kidney;lm,longitudinalmuscle;ln,lateral‘‘visceral’’nervecord;mg,mantlegirdle;oe,oesophagus;pg,pharyngealgland;pn,
pedalnervecord;r,radula;tm,transversalmuscle;v,valve;vc,visceralcavity‘‘hemocoel’’.
doi:10.1371/journal.pone.0069785.g005
built gonad sac starts to expand to form a hollow space (i.e., Stage 1. Gametogenesis, i.e., period of formation of second-
gonadal lumen), and in turn, the ventral wall of the gonad sac ary oocytes or secondary spermatocytes and spermatids. Females
invaginates, forming folds inward of the lumen (i.e., tissue plates) of C. articulatus showed asynchronous groups of oocytes, i.e.,
(Fig.9). primaryoocytes,secondaryoocytes,andmatureovawerepresent
withoutadominantgroup(Fig.12),whereasC.albolineatusfemales
Gonad development stages showed only a synchronous group of secondary oocytes without
replenishment by earlier oocytes (Fig. 13). This occurred around
Stage0. Gametocytogenesis(i.e.,periodofformationofearly
35mm in total length ‘‘TL’’ (n=2) for C. articulatus and with a
oocytesorearlyspermatocytes,i.e.,immaturegametes)takesplace
confidence interval of P (27.15mm#m#35.63mm) for C.
overlapping the periods of gonad emergence (Pe) and of gonadal
albolineatus (Fig. 4, S1-females). The secondary oocytes measured
sac formation (Pf) in both chiton species (Fig. 4). In females, the
from 46 to 162mm in diameter, and were characterised by a
firstsexualcells(i.e.,oogoniaandearlyoocytes)ariseexceptionally
cytoplasmsomewhatdiminishedofbasophilia,nonvacuolatedbut
early during the end of the gonad emergence period (Pe, Fig. 4),
wholly granular, with relatively smaller, spherical nucleus than
when the cuboidal epithelial cells of the gonadal sac are created
early oocytes. Some secondary oocytes displayed or not follicle
and the two oval lobes have merged, but still without tissue
cells surrounding them (Fig. 12 and 13). Males in gametogenesis
plates (Fig. 8B); i.e., with a confidence interval of P
had a confidence interval in TL of P (23.45mm#m#32.27mm)
(18.83 mm#m#24.34 mm) for C. articulatus and of P
for C. articulatus and of P (23.89mm#m#31.38mm) for C.
(16.08 mm#m#23.92 mm) for C. albolineatus (Fig. 4, S0-females).
albolineatus (Fig. 4, S1-males); showing a testis with spermatocytes
The primary oocytes measured from 15 to 62.5mm in diameter
and spermatids occurring around the tissue plates and forming
andwerecharacterisedbyahyalinecytoplasmthatwasvacuolated
groups,andsomespermatozoabeginningtoreplenishthegonadal
andheavilybasophilic,whereasthenucleus(germinalvesicle)was
lumen (Fig.14).
translucent with one basophilic spherical nucleolus (Fig. 10). In
Stage 2. Mature, i.e., period of formation of mature ova or
males,thefirstsexualcells(i.e.,spermatogoniaandearlyspermato-
sperm.FemalesofbothChitonspp.showedonlyasynchronousgroup
cytes) arose later than in females during the end of the period of
of mature ova without replenishment by earlier oocytes (Fig. 15);
gonad sac formation (Pf, Fig. 4), when the gonad sac is wholly
withaconfidenceintervalinTLofP(31.09mm#m#40.24mm)for
expandedformingagonadallumen,andseveraltissueplatesoccur
C.articulatus and of P (24.48mm#m#38.85mm) for C.albolineatus
abovetheventralwallcoveredbyearlyspermatocytes(Fig.11);i.e.,
(Fig. 4, S2-females). The mature ova, excluding the chorion,
with confidence interval of P (20.07mm#m#26.60mm) for C.
measuredfrom134to216mmindiameter,andwerecharacterised
articulatus and of P (19.51mm#m#25.49mm) for C. albolineatus
byanacidophiliccytoplasmfilledwithuniformproteinyolkgranules
(Fig.4,S0-males). (vitellum) and a fully developed chorionic process with projections
(i.e.,chorionorhull)coveringthematureovaand/orshedintothe
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Figure6.Chitonthroughthestartingperiodofgonademergence.Chitonarticulatus(0.053gBW,9mmTL,and6mmTW)throughthe
startingperiodofgonademergence,showingtheprojectionofthesquamousepitheliumthatextendsalongthewallofthedorsalaortaandis
thrown toward the visceral cavity. (A) Composite digital image in transverse section of the whole specimen showing the general histological
anatomy.(B)Close-upofthedorsalpartfromthewholespecimen.Abbreviations:a,articulamentum;af,accessoryfold;c,cuticle;ct,ctenidium;da,
dorsalaorta;dg,digestivegland;ds,dorsalaortasinus;f,foot;go,girdleornamentations;gr,gonadalridge(i.e.,theprecursortothegonad);gs,
gonadalsinus(i.e.,cavityformedbyabendingorcurvingfromthegonadalridgeandcontainingchieflyblood);i,intestine;k,kidney;lm,longitudinal
muscle;ln,lateral‘‘visceral’’nervecord;mg,mantlegirdle;ms,musclesinus;pn,pedalnervecord;st,stomach;tm,transversalmuscle;v,valve;vc,
visceralcavity‘‘hemocoel’’.
doi:10.1371/journal.pone.0069785.g006
gonadal lumen (Fig. 15). Males reached the mature stage at Sexualdifferentiation(dimorphism)andonsetofthefirst
a smaller size than females, with a confidence interval in TL sexual maturity
of P (25.45mm#m#31.75mm) for C. articulatus and of P
The aforementioned histological examinations, as well as the
(26.05mm#m#32.87mm) for C. albolineatus (Fig. 4, S2-males),
ranges and confidence intervals among stages, indicated that the
showingthegonadallumenfullyreplenishedwithsperm(Fig.16).
sexual differentiation (i.e., dimorphism, from the viewpoint of
Figure7.Chitonthroughtheformationofthegonadallobes.Chitonarticulatus(0.382gBW,21mmTL,and12.5mmTW)throughthemiddle
periodofgonademergence,showingtheformationoftwoovallobes.(A)Compositedigitalimageintransversesectionofthewholespecimen
showingthegeneralhistologicalanatomy.(B)Close-upofthedorsalpartofthewholespecimen.Abbreviations:a,articulamentum;af,accessoryfold;
c,cuticle;ct,ctenidium;da,dorsalaorta;dg,digestivegland;f,foot;go,girdleornamentation;gs,gonadalsinus(i.e.,cavityformedbyabendingor
curvingfromthegonadalridgeandcontainingchieflyblood);k,kidney;lm,longitudinalmuscle;ln,lateral‘‘visceral’’nervecord;mg,mantlegirdle;
pn,pedalnervecord;st,stomach;tm,transversalmuscle.
doi:10.1371/journal.pone.0069785.g007
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Figure8.Chitonthroughthemergingofthegonadallobes.Chitonarticulatus(0.185gBW,16mmTL,andmm9TW)throughthelastperiod
ofgonademergence,showingtheciliatedcuboidalepithelialcellsandthebeginningofthemergingofthelobesofthegonadsac.(A)Composite
digitalimageintransversesectionofthedorsalpartofthewholespecimenshowingthegeneralhistologicalanatomy.(B)Close-upoftheventral
rightsideofthegonadsacwithouttissueplatesbutshowingtheriseoftheearlyoogoniawithbasophilicgranularcytoplasmandonenucleolus,as
wellasthedevelopmentoftheearlycellsthatconstitutethetissueplatesontheventralwallofthegonadsac.Abbreviations:da,dorsalaorta;ds,
dorsalaortasinus;gl,gonadallumen(i.e.,aninneropenspaceorcavityofthegonad),lm,longitudinalmuscle;ms,musclesinus;v,valve;vc,visceral
cavity‘‘hemocoel’’;vw,ventralwallofgonadsac.
doi:10.1371/journal.pone.0069785.g008
Figure9.Chitonformingthegonadsac.Chitonarticulatus(0.347gBW,21mmTL,and12mmTW)throughthelastperiodofgonademergence,
showingtheciliatedcuboidalepithelialcells,themergedlobes,theriseofthetissueplatesand,inturn,developmenttowardthegonadsacformed.
(A)Compositedigitalimageintransversesectionofthewholespecimenshowingthegeneralhistologicalanatomy.(B)Close-upofthedorsalpartof
thewholespecimen.Abbreviations:a,articulamentum;af,accessoryfold;ct,ctenidium;da,dorsalaorta;dg,digestivegland;ds,dorsalaortasinus;f,
foot;go,girdleornamentation;i,intestine;k,kidney;lm,longitudinalmuscle;mg,mantlegirdle;ms,musclesinus;pn,pedalnervecord;rs,residual
gonadalsinus;v,valve;vc,visceralcavity‘‘hemocoel’’.
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GonadalOntogenesisandMaturationofChitons
Figure 10. Gametocytogenesis in a Chitonfemale. Composite
digitalimagein frontalsectionoftheanteriordorsal partofa Chiton
articulatusfemale(1.161gBW,29mmTL,and16.5mmTW)withthe
ovary in the gametocytogenesis stage (S0) showing the formation of
theprimaryoocytes.
doi:10.1371/journal.pone.0069785.g010
Figure11.GametocytogenesisinaChitonmale.Compositedigital
microscopic gonadal differentiation, since differentiable external
imageintransversesectionofaChitonalbolineatusmale(0.596gBW,
traits are not present at this time, either in the gonad or in the 27.5mm TL, and 13mm TW) through the gametocytogenesis stage
anatomyoftheorganism)ofChitonarticulatusandChitonalbolineatus (S0),showingthegeneralhistologicalanatomyofthegonadsacwholly
occursfirstasafemalethenasamale;although,malesreachthe expanded forming a gonadal lumen and with several tissue plates
mature stage earlier than females (Fig. 4). Thus, it would be occurringabovetheventralwall.Earlyspermatocytesoccuraroundthe
tissue plates. Abbreviations: da, dorsal aorta; gl, gonadal lumen, lm,
possible to designate a sexual differentiation (i.e., gametogonia
longitudinalmuscle;st,stomach;vc,visceralcavity‘‘hemocoel’’.
differentiation, stage 0: gametocytogenesis) at around doi:10.1371/journal.pone.0069785.g011
21.93 mm61.16 mm (mean 6 S.E.) in total length ‘‘TL’’ for C.
articulatus,andat21.79mm61.23mm(mean6S.E.)inTLforC. changes in body shape are given by a direct relation with
albolineatus. development and the physiological adjustment of the internal
The onset of the first sexual maturity (i.e., first time to a given organs, but particularly evolve in response to gonad maturation.
sizethat eachsexshowedmature gametes(S2), suchasovafilled Therefore, gonads near maturity grow considerably, forcing the
with yolk granules and/or spermatozoa) for C. articulatus was valvestobulge,makingthemmoreconvexandleadingtoamore
around 32mm in total length ‘‘TL’’ for females, and around oval body shape. This also is supported by observations during
17mmTLformales;whileforC.albolineatusitwasaround28mm dissections of mature gonads, since the gonad compresses the
TL for females and 23.5 mm TL for males. Finally, males of C. ventral organs (i.e., visceral mass) toward the foot to the point of
articulatusmaturebeforethoseofC.albolineatus;incontrast,females almost merging the gonad with the visceral mass, making them
of C.albolineatus mature before those ofC.articulatus (Fig.4). difficult to separate [Abadia-Chanona and Avila-Poveda, pers.
obs.].
Discussion Connorsetal.[64]indicatedthatbodyshapeisabiomechanical
consequenceinresponsetoapassiveanddefensive conformation
Allometry of the shell plate assembly, and a geometric enhancement of the
The b-values obtained here for Chiton articulatus and Chiton structural assembly of the chitons to give mechanical robustness,
albolineatuswereabovethree,demonstratingthatthesespecieshave whichfinallyresultsinanapproximatelycontinuouscurvatureand
a standard isometric growth (i.e., growth that occurs at the same a constant armor thickness that leads to a balance between
rate for all parts of an organism so that its shape is consistent mobility and protection. Therefore, the results of any allometric
throughout development) in total length ‘‘TL’’ and total width analysesinpolyplacophoransarepotentiallysubjectedtoincreased
‘‘TW’’duringtheirearlylifestages,i.e.,fromsubjuvenile,through variability due to differential growth rates of the different
juvenileuntilsubadult(4#TL,40mm).Butjusttowardtheadult components during reproduction and to the flexibility of the
stage(TL.40mm),theb-valuesmovedownwardstobetween2.9 mantle girdle [57,58], so it would be essential in this type of
and2.2forC.articulatus[43–45],andaround2.7forC.albolineatus analysis to perform them according to the early life stages (e.g.,
[47].Therefore,thisindicatesthatthesespecieshavefirst,during subjuvenile ‘‘post-metamorphosed’’, juvenile,subadult, adult).
the early life stages, an isometric growth and subsequently an
allometricgrowth(i.e.,growthwherebypartsofthesameorganism Early life stages
growatdifferentrates)astheydevelopintoadulthood.Indeed,the Conspicuously, much of the literature on reproductive issues
allometricrelationshipwithanotherchiton,Lepidochitonacinerea,has does not indicate a life stage for the organisms investigated, and
beenvariablethroughafullsizerangeandhasshownachangein instead they have beenassumed to beadults and/or islimitedto
thegrowth type,exhibiting b-values from2.5to3.4[61,62]. summarise that specimens were collected mainly during their
Avila-Poveda [43] indicated that these changes in growth type reproductivelyactiveperiod,butthequestioniswhendotheystart
occur because chitons subtly change their body shape, which is to be adults? This matter, in chitons, lacks consensus in the
duetochangesinthedorsalelevationofbodyheight[63].These literatureontheappropriateterminologyandsizetodesignatethe
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GonadalOntogenesisandMaturationofChitons
Figure12.GametogenesisinaChitonarticulatusfemale.CompositedigitalimageinfrontalsectionoftheanteriordorsalpartofaChiton
articulatusfemale(2.469gBW,35mmTL,and22mmTW)withtheovaryinthegametogenesisstage(S1)showingasynchronousgroupsofoocytes,
i.e.,primaryoocytes,secondaryoocytes,andmatureovawithoutadominantgroup.Abbreviations:po,primaryoocytes;so,secondaryoocytes;mo,
matureova.
doi:10.1371/journal.pone.0069785.g012
‘‘early lifestages’’forpolyplacophorans. Hence,somesizeranges genesis) and is defined as the stage between the subjuvenile and
areproposedtodistinguishgrowthsequencesor‘‘earlylifestages’’ subadult stages. Thus, it covers from the formation of oocytes or
in these chitons, Chiton articulatus and Chiton albolineatus, according spermatocytes, both primary (i.e., diploid gametes) until the
to gonadal ontogenesis, gonad development stages, sexual formation of oocytes or spermatocytes, both secondary, which
differentiation, and onset of the first sexual maturity here includespermatids(i.e.,firsthaploidgametes),butwithoutactually
evaluated. forming mature gametes (i.e., ova and spermatozoa). For C.
The term subjuvenile or post-metamorphosed defines the first articulatus and C. albolineatus, this stage was established based on
growth stage that covers the criteria across all periods of gonad females between 14#TL#28mm, i.e., between the lowest value
formation until a gonadal sac isbuilt andexpanded (i.e., Pw, Pe, ofstage-0andthelowestvalueofstage-2,intotallength(Fig.17).
Pf), without actually forming gametogonia or gametocytes. The term subadult introduces chitons that, besides meeting
Particularly for C. articulatus and C. albolineatus, this stage was criteriafromStage0andStage1(particularlyinmales),alsomeet
established at TL#14mm, i.e., until just before the lowest total criteriafromStage2(mature),andisdefinedasthestagebetween
length ‘‘TL’’ of the subsequent stage-0 female was observed the juvenile and the adult stages. The subadult stage commences
(Fig.17). with the full attainment of mature gametes (i.e., ova or
The term juvenile introduces young chitons that meet the spermatozoa), the so-called onset of first sexual maturity. For C.
criteria from Stage 0 (gametocytogenesis) and Stage 1 (gameto-
Figure 13. Gametogenesis in a Chitonalbolineatus female. Figure 14. Gametogenesis in a Chitonmale. Composite digital
Composite digital image in transverse section of the dorsal part of a imageintransversesectionofaChitonalbolineatusmale(0.434gBW,
Chiton albolineatus female (0.953g BW, 28mm TL, and 14mm TW) 21mmTL,and11mmTW)withtestisinthegametogenesisstage(S1)
with the ovary in the gametogenesis stage (S1) showing only a showing spermatocytes and spermatids occurring around the tissue
synchronous group of secondary oocytes without replenishment by plates and forming groups, and some spermatozoa beginning to
earlier oocytes. Abbreviations: da, dorsal aorta; k, kidney; gl, gonadal replenish the gonadal lumen. Abbreviations: da, dorsal aorta; gl,
lumen;lm,longitudinalmuscle;st,stomach;tm,transversalmuscle;vc, gonadallumen,lm,longitudinalmuscle;st,stomach;vc,visceralcavity
visceralcavity‘‘hemocoel’’. ‘‘hemocoel’’.
doi:10.1371/journal.pone.0069785.g013 doi:10.1371/journal.pone.0069785.g014
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Figure 15. Mature Chiton female. Composite digital image in
transversesectionofanovaryofChitonarticulatus(2.228gBW,32mm
TL,and22mmTW)inmaturestage(S2)showingonlyasynchronous
groupofmatureovawithoutreplenishmentbyearlieroocytes.
doi:10.1371/journal.pone.0069785.g015
articulatus and C. albolineatus, this stage is very close and wholly
superimposed(particularlyinmales)tothejuvenilestage;thus,itis
established based on females between 28#TL#40mm, i.e.,
between the lowest value of stage-2 and the lowest value of
stage-3, in total length (Fig.17).
Finally, the adult stage commences after the subadult stage
finalizes, then, for practical purposes, in C. articulatus and C.
albolineatus, this stage was established at TL$40mm, and it
included thefuture spawning stage-3 (Fig.17).
Figure 16. Mature Chiton males. Composite digital images in
Perhapsthesestagesarenotadequateforallpolyplacophorans, transversesection(A)oftheposteriordorsalpartand(B)oftheanterior
but they are at least for the family Chitonidae, which generally dorsal part from mature Chiton albolineatus males (A: 0.648g BW,
exhibits a common ‘‘adult’’-size above 40mm, including the 25mmTL,and13mmTW,andB:1.365gBW,29.5mmTL,and16mm
TW) showing the gonadal lumen fully replenished with sperm.
chitons here studied [65], and also shows concordance with the
Abbreviations: da, dorsal aorta; dg, digestive gland; i, intestine; k,
modal size-class exhibited by other Mexican populations of both
kidney;lm,longitudinalmuscle;s,spermatozoa;tm,transversalmuscle;
chitons [44–47]. vc,visceralcavity‘‘hemocoel’’.
doi:10.1371/journal.pone.0069785.g016
Sexual differentiation of the gonad
For molluscs [66,67] and, particularly, from studies on from Oaxaca, Mexico is ongoing, and histological observations
Aplacophora [68], Polyplacophora [32], Bivalvia [69], Gastro- reveal not hermaphroditism for specimens of total length ‘‘TL’’
poda [70], and Cephalopoda [49,71–75], it has been indicated greater than 40mm, i.e., adult stage [Abadia-Chanona, pers.
thatthegonadremainsundifferentiatedthroughoutthelarvallife obs.].
and their predetermined sex differentiation only appears during In this sense, results obtained in the present study indicate
the post-metamorphic life. In effect, Chiton articulatus and Chiton the practical possibility of recognising early, between
albolineatus are invariably of separate sexes with a well-defined 13#TL#20mm, the sex of the chitons Chiton articulatus and
sexualdifferentiationthathappensfirstasafemalethenasamale, Chitonalbolineatus,i.e.,betweenthelowestvaluesofS0-bothsexes,
during the beginning of the juvenile stage. Similar, to the intotallength,andduringthestartofthejuvenilestage,whichfor
polyplacophora [32–34,37], as well as other mollusks, such as practicalpurposesalsocorrespondstoamediumsize(i.e.,20mm
Aplacophora [68], Bivalvia [69], Gastropoda [70], and Cepha- TL) in regard to the smallest adult size (i.e., 40mm TL). Other
lopoda[49,71–75],ithasbeenindicatedthatthesexualcellsarise
from specialised cells called protogonia, and emerge during the
paired gonad primordium, but first as an ovary that can develop
andsuddenlyasatestis.Infact,thisisthetimingwithinwhichthe
anatomicalsexualphaseisdefinedasambisexual‘‘hermaphrodit-
ism’’or unisexual‘‘gonochorism’’ [34,66,67].
Recently, it has been indicated that Chiton articulatus reveals
anunusuallyhighincidenceofhermaphroditism,commonlybeing
the female tissue which occupies the greatest part of the gonad
sections respecttomale tissue [76].However, thisstudy doesnot
indicate the total length, ‘‘TL’’, of the studied specimens.
Therefore, it is difficult to integrate the information to recognize
whether C.articulatus is: 1) a hermaphrodite species inthe adult
Figure17.EarlylifestagesforChitonspp.Rangesintotallength
stage,and/or2)whetherthesearejustjuvenilespecimenswhoare proposedtodistinguishgrowthsequencesor‘‘earlylifestages’’inthe
in a process of sexual differentiation, which is more likely. studiedchitons,ChitonarticulatusandChitonalbolineatus.
Furthermore, a study of reproductive biology of C. articulatus doi:10.1371/journal.pone.0069785.g017
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