On the role of the proventricle region in reproduction and regeneration in Typosyllis antoni

16 Pages·2016·5.76 MB·English

by  Michael Weidhase

Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196
DOI10.1186/s12862-016-0770-5
RESEARCH ARTICLE Open Access
On the role of the proventricle region in
reproduction and regeneration in Typosyllis
antoni (Annelida: Syllidae)
Michael Weidhase1, Patrick Beckers2, Christoph Bleidorn1,3* and M. Teresa Aguado4
Abstract
Background: Syllids are a species rich annelid family possessing remarkable regenerative ability, which is not only
theresponse after traumatic injury, but also a key step during thelife cycle of several syllid taxa. Inthese animals
theposterior part of thebody becomes an epitoke and is later detached as a distinct unit named stolon. Such a
sexual reproductive mode is named schizogamy or stolonization. The prostomium and the proventricle, a modified
foregutstructure,havebeenproposedtohaveacontrolfunctionduringthisprocess,thoughtheconcretemechanisms
behindithaveneverbeenelucidated.
Results:Byusingdifferentexperimentalset-ups,histologyandimmunohistochemistrycombinedwithsubsequentcLSM
analyzes,weinvestigateanddocumenttheregenerationandstolonizationinspecimensofTyposyllisantonithatwere
amputatedatdifferentlevelsthroughouttheantero-posteriorbodyaxis.Theremovaloftheanteriorendincludingthe
proventricleimpliesanincompleteanteriorregenerationaswellasseveredeviationsfromtheusualreproductivepattern,
i.e.acceleratedstolonization,masculinizationandtheoccurrenceofaberrantstolons.Thedetailedanatomyofaberrant
stolonsisdescribed.Ahistologicalstudyoftheproventriclerevealednosignsofglandularorsecretorystructures.The
ventricleandthecaecaarecomposedofglandulartissuebuttheyarenotinvolvedinthereproductiveandregenerative
processes.
Conclusions:As in other investigated syllids, the proventricle region has a significant role during stolonization
and reproduction processes in Typosyllis antoni. When the proventricle region is absent, anterior and posterior
regeneration are considerably deviated from the general patterns. However, proventricle ultrastructure does not
show any glandular component, thereby questioning a direct involvement of this organ itself in the control of
reproduction and regeneration. Our findings offer a comprehensive starting point for further studies of
regeneration and reproductive control in syllids as well as annelids in general.
Keywords: Confocal laser scanning microscopy (cLSM), Proventricle, Schizogamy, Scissiparity, Stolon
Background developmental biology in annelids and their regeneration
Annelids provideexcellent model organismsfor investi- patterns have been possible because the life-cycles ofsome
gating regenerative processes. Their regenerative cap- ofthemarewell-knownandhavebeenreproducedsuccess-
abilities are remarkable. They are able to renew worn fully under laboratory conditions. Such is the case of spe-
structures such as chaetae or opercula [1, 2]. Further- cies of the clitellates Enchytraeus, Helobdella and Pristina,
more,someannelidsredevelopacompletebodyoriginated and polychaetes Alitta, Capitella, Hydroides, Platynereis,
only in one or a few segments [3–5]. Studies concerning amongothers[6–15].
However, this group of model organisms represents
very few of the numerous annelid lineages. Recently,
*Correspondence:[email protected]
1MolecularEvolution&AnimalSystematics,InstituteofBiology,Universityof members of other groups such as Syllidae, have been
Leipzig,Talstraße33,D-04103Leipzig,Germany proposed as possible model species [16]. Syllids are in-
3MuseoNacionaldeCienciasNaturales,SpanishResearchCouncil(CSIC),José
teresting for developmental studies due to their complex
GutiérrezAbascal2,28006Madrid,Spain
Fulllistofauthorinformationisavailableattheendofthearticle life cycles and reproductive modes, named epigamy and
©2016TheAuthor(s).OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196 Page2of16
schizogamy or stolonization, the latter include steps of continue during recent decades, and therefore
segment regeneration [17–19]. During reproduction in new microscopic techniques could not be used. Some of
severalannelidfamilies,thecompletebodyistransformed the most relevant results suggested an endocrine func-
intoanepitoke,aprocesscalledepigamy.Incontrast,dur- tion of the proventricle (a specific structure in the di-
ing schizogamy only the posterior part becomes an epi- gestive tube) during the cyclic reproductive activity of
toke, which is later detached as a distinct reproductive Typosyllisspp.[38,40–42,44,48,68].
unit called stolon, while the posterior end of the stock is The proventricle is a muscular structure with radially
regenerated(Fig.1). arrangedstriatedmusclecellssurroundingthegut[74–77].
The regeneration patterns of syllids and their repro- These cells consist of usually only one or two sarcomeres
ductive methods were studied during the 20th century with up to 100 μm length, being the longest known sarco-
by Abeloos [20], Albert [21], Allen [22, 23], Berrill [24], meres within the Metazoa [78, 79]. Within the muscular
Boilly [25–32], Boilly & Thibaut [33], Caullery [34], fiberstherearemembrane-boundgranulescontaininghigh
Deyle [35], Durchon [36–38], Durchon & Wissocq [39], amounts of calcium and phosphorus, contributing to the
Franke [18, 40–45], Gidholm [46], Hauenschild [47], calcium metabolism of the muscle cells [79]. Several
Heacox & Schroeder [48], Izuka [49], Langhammer [50], authors [38, 40, 69] proposed that the proventricle
Malaquin [51], Mesnil [52], Mesnil & Caullery [53], produces a hormone that, in high concentrations,
Michel [54], Müller & Kreischer [55], Okada [56–62], attenuates the stolonization while promoting the regen-
Pruvot [63], Schiedges [64], Verger-Bocquet [65–67], eration of the posterior end. In later studies, Franke
and Wissocq [68–73]. However, these studies did not [41,42,44]aswellasHeacox&Schroeder[48]suggested
Fig.1Specialmodesofsexualreproductioninannelids.aEpigamy.Thespecimenbecomesanepitokeandproducesgametes.Aftermating,the
individualoftendies.bSchizogamy.Onlytheposteriorpartoftheindividualistransformedintoaseparateepitokousunitcalledstolon.Gametes
couldbealsoproducedinnon-stolonsegmentsandlatertransferredintothelatter.Aftermaturation,thestolondetaches,matesanddies,
whereasthestockregeneratesitsposteriorend
Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196 Page3of16
the prostomium as the main control core that manages was set up with three untreated individuals as a con-
theproventricleendocrineactivity.However,thesestudies trol. For more detailed information on animal main-
did not specify the hormone’s nature and the exact loca- tenance please check reference [16]. A comparable
tion for its production. Recently, Aguado et al. [16] sug- setup for amputation in site 3 was conducted also in
gested that the hormones might be produced by adjacent August and September 2014. This third experiment
digestive structures that follow the proventricle, the ven- was performed to study the regenerating body ends
tricle and caeca, which are supposed to have glandular during a longer time in order to check their ability to
functions. However, a detailed histological study of these produce stolons. Two sets with six specimens each
structureshasnotbeenperformedandthespecificcontrol (numbers 1–1 to 1–6 and 2–1 to 2–6, respectively)
mechanismsofstolonizationandregenerationcontinueto were set up. In this experiment every specimen (anterior
beunknown. and posterior end) was placed in a distinct plastic bowl
In this study, we investigate the anterior and posterior with the same conditions as described above. Over a
regeneration and the removal effect of the proventricle period of 25 days (specimens 1–1 to 1–6) and 23 days
region in Typosyllis antoni (Syllinae) involving recent (specimens 2–1 to 2–6) specimens were examined every
microscopicaltechniques.Weusedavarietyofexperimen- day. Therefore, anteriorand posterior endswereremoved
talset-ups,histologyandimmunohistochemistrycombined from their boxes, anesthetized in 3.5 % MgCl for 5–
2
with subsequent confocal laser scanning microscopy 10min,thenumberofsegmentswascountedandmodifi-
(cLSM) in order to investigate the relationships between cations (e.g., stolon development) were documented.
regeneration,reproductionandtheproventriclefunction. Afterwards,specimenswereplacedbackintheirbox.
Methods Histology
Specimensandexperimentalsetup For histology, specimens were treated as described previ-
Specimens of Typosyllis antoni used in this study were ously [80], with the following modifications. Specimens
taken from aquaria located at the University of Leipzig were anesthetized in 7 % MgCl dissolved in artificial sea-
2
(Germany). In total, three different amputation sites water,fixedovernightinBouin'sfixative(saturatedaqueous
were used in the regeneration experiments (Fig. 2): (1) picricacid,37%formaldehyde,glacialaceticacid;15:5:1by
amputation directly in front of the proventricle, (2) volume), washed in 70 % ethanol and dehydrated in an
amputation at the border between proventricle and ascending ethanol series. Azan stains the neuropil of the
ventricle, and (3) amputation between chaetigers 35 nervoussystemgray,theextracellularmatrixblue,andthe
and 36. Regeneration experiments 1 and 2 were per- musculature orange. The color of nuclei is variable based
formed during August and September 2014 with 18 ontheircontent.
specimens each. The specimens were anesthetized in
3.5 % MgCl dissolved in artificial seawater and ampu- Immunohistochemistry
2
tated with a scalpel. Afterwards, anterior and posterior Immunohistochemistry was performed as described pre-
ends were placed separately in plastic bowls and kept viously [5]. For this study, we used combined stainings
at 25 °C with water changes every third day. Each ex- of anti-α-tubulin or anti-serotonin with phalloidin-
periment (amputation sites 1 and 2, respectively) was rhodamine. For anti-α-tubulin staining,amixtureofanti-
split into two sets with a delay of two days (set 1: days acetylated α-tubulin (monoclonal anti-tubulin, acetylated
2, 8, 10, 16; set 2: days 4, 6, 12, 14, 18). For each men- antibody, produced in mouse, ascites fluid, Sigma-Aldrich,
tioned day, two specimens were removed, anesthetized St. Louis, MO, USA; dilution 1:500 in PBST-NGS) and
and fixed in 4 % paraformaldehyde. An additional box anti-tyrosinated α-tubulin (monoclonal anti-tubulin,
Fig.2SchematicdrawingofTyposyllisantoniillustratingthedifferentcuttingsitesforeachregenerationexperiments:1.Infrontoftheproventricle,2.
Betweenproventricleandventricle,3.Betweenchaetigers35and36
Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196 Page4of16
tyrosineantibody, produced inmouse, ascites fluid, Sigma- by a thin epithelium, which is covered by a prominent
Aldrich,St.Louis,MO,USA;dilution1:250inPBST-NGS) cuticle (Fig. 4b). At the transition from the pharynx to
was used. The labeled α-tubulin is a structural component theproventricle,thecuticlebecomesweaker,theepithelium
of microtubules, which are amongst others present in isbroadenedandthelumendecreases(Fig.4c).Incontrast,
axons, while serotonin (=5-HT) is a neurotransmitter. themusclelayerenlarges,startingtoshowthetypicalradial
Phalloidin-rhodamine labels filamentous muscular actin (f- arrangementofthesarcomeres(Fig.3b).Thesesarcomeres
actin)[5]. are extremely long, thus only two sarcomeres per radius
occur,asvisiblebythez-discs(connectionsiteofactinfila-
Neutralredstaining,lightmicroscopyandimage ments), on half cross-section of the proventricle muscula-
processing ture(Fig.4c,d).Thesarcoplasmislimitedtotaperedareas
Forneutral red staining, a specimen wasplacedfor 2min betweenthesarcomeres;thenucleiarepresentinsidethese
inneutralredsolution(2mg/mlinartificialseawater)and areas,neartheoutermargin.Throughouttheposteriorend
afterwards anesthetized in 3.5 % MgCl dissolved in oftheproventricle,theinnerepitheliumbecomesgradually
2
artificial seawater. Light microscopic pictures of neutral thinner, the musculature broadens and the interior lumen
red staining and regenerating specimens were taken diminishes (Fig. 4d). Possible secretory structures are cer-
using a Leica (Leica Microsystems, Wetzlar, Germany) tainly not detectable within the proventricle. Instead, the
DM1000microscopewithattachedLeicaDFC295camera ventricle has an abundance of glandular epithelium pro-
and the Leica LAS v3.6 software. All drawings, images vided by cells with large vacuoles, while it almost lacks
and final image plates were processed and compiled musculature(Fig.4e). Inthe caecaregion,themusculature
using Adobe (San Jose, CA, USA) Photoshop CS6 and starts to increase again and the large glandular cells get
Illustrator CS6. scarcerandsmallerinsize(Fig.4f).Thetwocaecashowan
arrangementoftissuescomparabletothefollowingmidgut
Results (Fig. 4g), with a voluminous epithelium and a thin outer
Proventriclemorphologyandfunction musclelayer(Fig.4f,g).
TheforegutofTyposyllisantoniiscomposedofapharynx
armedwithoneanteriorpharyngealtooth,followedbythe Regenerationdiffersacrossamputationsites
proventricle, the ventricle and two caeca (Figs. 3a, 4a). In order to investigate if anterior regeneration varies de-
Thepharynxconsistsofanoutermuscularlayer,followed pending on the presence of the proventricle region and
Fig.3LightmicroscopicimagesofTyposyllisantoni,neutralredstaining.Anteriorendupperleft,allimagesaredorsalviews.aModifiedforegut
withproventricle(p),ventricle(v)andcaeca(ca).bHighermagnificationofproventriclesurface.cHighermagnificationofleftcaecum(c).
Abbreviations:ca,caecum;g,gut;p,proventricle;v,ventricle.Scalebar=100μm
Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196 Page5of16
Fig.4(Seelegendonnextpage.)
Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196 Page6of16
(Seefigureonpreviouspage.)
Fig.4HistologicalcrosssectionsofanteriorsegmentsinTyposyllisantoni(twodifferentspecimens,b-drespectivelye-g),Azanstaining.aSchematic
representationofT.antoniwiththelocationsofeachcrosssection.bPharynx.cAnteriorendofproventricleandschematicrepresentationoftheupper
leftquarteroftheproventricle(downright,withcolorsaccordingtolabelcolors).dProventricle.eVentricle.fCaecaregion.gMidgut.Abbreviations:
ca,caeca;cu,pharyngealcuticle;dbv,dorsalbloodvessel;dci,dorsalcirrus;dlm,dorsallongitudinalmusculature;ec,gutepitheliumofthecaecaregion;
eca,caecaepithelium;ecm,extracellularmatrix;eg,midgutepithelium;ep,epitheliumoftheproventricle;eph,pharyngealepithelium;ev,epitheliumof
theventricle;mc,gutmusculatureofthecaecaregion;mca,musculatureofcaeca;mg,midgutmusculature;mph,pharyngealmusculature;
nc,neurochaetae;nu,nucleus;sa,sarcomere;sp,sarcoplasm;vlm,ventrallongitudinalmusculature;vn,ventralnervecord;zd,z-disc
to exclude an involvement of the subsequent structures specimen at 18 dpa. However, not all the individuals
(ventricle+caeca), we tested three cutting sites (1, 2 and regenerated at the same rate and some remained in the
3). Amputation site 1 (Fig. 2) was located directly anter- invagination stage for more than one week without any
ior to the proventricle, so that the posterior body part signs of blastema formation. In addition, during the
kept the latter feature, but no additional parts of the experiment, a redevelopment of the ventricle and caeca
pharynx. Amputation site 2 (Fig. 2) was at the border wasnotobservable.
between the proventricle and the ventricle plus caeca, Amputation site 3 - anterior regeneration: In this case,
thus the posterior part lacks the proventricle, but kept T. antoni usually regenerates two or sometimes three
theotherstructures.Amputationsite3(Fig.2)wasbetween segments visible at once at 4 dpa. Afterwards the pro-
chaetiger35and36,clearlybehindthemodifiedforegut. stomium and the segments grow, but no more seg-
Amputation site 1 - anterior regeneration: When am- ments were added within one month after (Table 1,
putating specimens directly in front of the proventricle, for more details and pictures please refer to [16]).
anterior regeneration starts with invagination followed Amputation site 3 - posterior regeneration: The posterior
by blastema formation and blastema patterning. This regenerationinspecimensamputatedbetweenchaetiger35
leadstothedevelopmentoftheprostomiumwithantenna and36iscomparabletoamputationsite2.
and palps after 4 days post amputation (dpa) (Fig. 5a).
Afterwards, new segments are regenerated consecutively
directly anterior to the amputation site (re-segmentation) Stolonizationandstolonmorphology
(Fig. 5b-d). The maximum number of observed segments Stolons of T. antoni are dicerous, possessing two anterior
was six after two weeks (Fig. 5d). Regeneration speed, lobes, two pairs of eyes and two anterior antenna [16].
represented by the number of new segments, varies be- Whilefemalestolonsarefullofgrayoocytes,malescontain
tweendifferentspecimens.Allthespecimensregenerateda two packages of yellow sperm per segment. T. antoni can
pharynxbutanewpharyngealtoothwasneverobserved. pass through several successive stolonization events. Sto-
Amputation site 1 - posterior regeneration. The anterior lons in T. antoni are not generated by segment addition,
ends of specimens dissected in front of the proventricle but arise from a transformation of the posterior segments.
regenerate slowly and are incomplete over the period of After detachment of a stolon, the posterior end is regener-
observation: After 14 dpa, about one third of the anterior ated before new stolons are developed. The number of
bodyendsremainedintheinvaginationstagewithoutany transformed segments varies between specimens, and pre-
signs of posterior regeneration (e.g., blastema formation). sumably also between each stolonization event in a single
The remaining ones regenerated some few segments, but specimen, from about 5 to 18 segments. The anteriormost
less than specimens amputated at sites 2 or 3. The max- stolon segment bearing the eyes and antenna underwent
imum number of regenerated segments was four, first extensive morphological changes and became the stolon
seeninaspecimenattendpa. “head”. Additionally, the nervous system and the muscula-
Amputation site 2 - anterior regeneration: The anterior ture passed through a remodeling process. Already in the
regenerationinspecimensamputatedbetweenproventricle stillattachedstolon,adorsalorientatedringofneuritesoc-
and ventricle bears striking differences in comparison with curredattheanteriormarginofthestolonhead(Fig.6a,b).
the other two sites. At first, two or usually three segments Having its origin in the ventral nerve cord, this structure
were visible at once (first seen 6 dpa) and no more seg- representsthestolon brain. Nevertheless,the ventral nerve
ments were added until 16 dpa. At 18 dpa, one specimen cord was not interrupted between the remaining body and
showedintotalfourandtheotheronefiveregeneratedseg- the stolon at this stage. Moreover, the body wall muscula-
ments.Thus,additionalsegmentswereaddedsequentially. ture is reduced at the border between the remaining body
Amputation site 2 - posterior regeneration: Posterior (stock) and the stolon (Fig. 6c, d). Inside the stolon head,
regeneration in specimens amputated between proven- the transverse muscle fibers contract to rings which later
tricle andventricleisfasterthaninsite1.Themaximum becomeasphinctermusclepriortodetachment(Fig.6e,f).
number of regenerated segments observed was 16 in a Theanteriorthirdofthestolonheadisfreeofmusculature.
Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196 Page7of16
Fig.5AnteriorregenerationinTyposyllisantonispecimensamputateddirectlyinfrontoftheproventricle(Fig.2,site1),lightmicroscopicpictures.All
imagesaredorsalviewsoftheanteriorendexcepta(dorso-lateral),anteriorisleftinallimages.Thedottedlineindicatesthesiteofamputation.a4dpa.
Theprostomium(pr)anditsappendages(lat,ps)areredevelopingbutsegmentsarenotvisible.b10dpa.Twomoresegments(I.-II.)havedeveloped.c12
dpaandd14dpa.Segments(III.-VI.)wereaddedsequentiallydirectlyanteriortotheamputationsite.Abbreviations:I.-VI.,regeneratedsegments;dci,dosal
cirrus;dtc,dorsaltentacularcirrus;mat,medianantenna;lat,lateralantenna;pr,prostomium;ps,palpus;vtc,ventraltentacularcirrus.Scalebars=100μm
Allfurtherstolonsegmentsexhibitedanervoussystemand (latter ones without a proventricle) were monitored over a
musculaturecomparabletostocksegments. longerperiodtoinvestigatetheproductionofstolons.
Posterior regeneration - During posterior regeneration
(anterior body parts), all 12 specimens showed an invagin-
Theeffectofprostomiumandproventricleregion ation at 1 dpa. One specimen was not found after 2 dpa
removalduringstolonization and another specimen remained at this stage until the end
Inordertogetabetterunderstandingofpotentialrelation- of the experiment (25 dpa) without any signs of regener-
ships between regeneration and stolon development, 12 ation. The other ten regenerated their posterior ends, but
additional individuals were amputated between chaetiger inthreespecimens,aftertwoweeks,theposteriormostpart
35 and 36 (site 3, Fig. 2). The anterior and posterior ends was lost and regenerated again. The maximum number of
Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196 Page8of16
Table1SummaryofregenerationinTyposyllisantoniafter developmentofthestolon.Thenumberofsegmentswithin
dissectingatsite2and3 each single stolon was between ten and 17 segments.
dpa regeneration Four specimens (1–5, 2–1, 2–4, 2–6, Fig. 7d, e, f, h)
amputationsite2–anteriorregeneration(observationonlyevery2ndday) produced aberrant male stolons (described below) in
series of two or three, which were detached as a whole.
0 invaginationof1stremainingsegmentatwoundsite
Only in three specimens (1–2, 1–5, 1–6, Fig. 7b, d, Add-
2 blastemaformation
itional file 1: Figure S1b) female stolons occurred. In some
4 prostomiumwithdevelopingantenna,palpsandeyes individuals (e.g., 2–1, Fig. 7e) the stolon wasalready devel-
6–16 twoorthreesegments opedwithinlessthanoneweekafteramputation,inothers
18 sequentialadditionoffurthersegments (e.g.,1–4;Fig.7c)thislastsnearlythreeweeks.Duringthis
amputationssite2–posteriorregeneration(observationonlyevery2ndday) time, the former went through their second stolonization
event (e.g., 1–1, Fig. 7a). Neither the number of sequential
0+2 invaginationof1stremainingsegmentatwoundsite
stolons nor the relative position of the first stolon head
4 pygidiumwithdevelopinganalcirriandmedianpapilla
within the aberrant stolons showed a regular pattern.
6onwards sequentialadditionofsegments
Accelerated stolonization, masculinisation and the oc-
amputationsite3–anteriorregeneration currence of aberrant stolons has been also observed in
0–1 invaginationof1stremainingsegmentatwoundsite the specimens amputated at site 2. In contrast, none of
2 blastemaformation these changes was observable in specimens amputated
at site1.
3 blastemawell-developed
4 prostomiumwithdevelopingantenna,palpsandeyes;
twoorthreesegmentswithtentacularcirriregenerating Morphologyofaberrantstolons
in1stsegment Aberrantstolonsshowedagreatvarietyofsegmentnumber
6 pharynxpresent and order (Fig. 8a, c-e). Some of them were composed of
two (Fig. 8c, e), others of three (Fig. 8a) sequential stolons
7onwards growthofsegmentsandstructures
at different developmental stages. In one case, the anterior
amputationsite3–posteriorregeneration
end appeared to be a composition of two segments with a
0–2 invaginationof1stremainingsegmentatwoundsite
total of 11 eyes, three antennae but only one parapodium
3 blastemawell-developed (Fig. 8d). In this case, the stolon head was induced three
4 pygidiumwithdevelopinganalcirriandmedianpapilla times through two subsequent segments. The musculature
5 additionoffirstnewsegment oftheaberrantstolonsalsoshowsdeviationsfromtheregu-
lar pattern (Fig. 8b).Thesphinctermuscles of thefirstand
6onwards additionoffurthernewsegmentsfollowedbytheir
development the third stolon are well developed, but there are few
musclefibersleftbetweenthestolontwoandthree,indicat-
segmentsregeneratedwas24(withoutlossofsegments;25 ing that this stolonization event is younger. This is espe-
dpa),theminimum11(23dpa).Inthosespecimens,which cially true for the second stolon, indicating that this
lost their posterior end, a total of 30 segments were re- stolonizationinitiationistheyoungest.Additionally,within
generated. None of the examined specimens produced the first stolon, musculaturestartsfading at two segmental
stolons. The lost posterior ends are certainly traumatic borders (Fig. 8b, asterisks), maybe representing the
losses, as we did not observe gametes, attached stolons, initiation of two more stolon heads.
or detached stolons in those specimens and their bowls.
Anterior regeneration - During anterior regeneration Discussion
(posterior body parts without proventricle and prosto- Removal of the proventricle region has strong effects on
mium) all 12 specimens regenerated a new prostomium the processes of stolonization and posterior regener-
and usually two new segments (Fig. 7, Additional file 1: ation. Ourresultsareinagreementwithpreviousstudies
Figure S1). Only in specimen number 1–2 (Fig. 7b) and a [37,40,48,69]andclearlysupportanadditionalregulatory
midbody fragment of specimen 2–3 (Additional file 1: function of this region. The presence of a proventricle has
Figure S1d) three regenerated segments were observed. been proposed as a synapomorphy for Syllidae, and its an-
All specimens developed stolons; seven specimens went cestral function is seen in the role of a sucking pump [81,
through only one stolonization event, while five experi- 82]. Comparable structures have not been found in the di-
enced two successive stolonizations. The detachment gestive tract of any other group of annelids [83]. The pro-
was produced several segments anterior to the stolon in posed sucking function may be particularly useful in many
specimens 1–4 and 2–5 (Fig. 7c, g), while in specimens syllids that feed on hydroids, bryozoans or other inver-
1–5,2–6,and2–3(Fig.7d,h,Additionalfile1:FigureS1d) tebrates [75, 84]. However, many other syllids show a
the anterior body was fragmented previously to the different kind of feeding preferences [85–87], e.g., algae
Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196 Page9of16
Fig.6(Seelegendonnextpage.)
Weidhaseetal.BMCEvolutionaryBiology (2016) 16:196 Page10of16
(Seefigureonpreviouspage.)
Fig.6Attached(a-d)anddetached(e-f)stolonsofTyposyllisantoni.Anti-α-tubulin(glowmode,a-b)andanti-f-actin(depthcoded,legend
insertedinc,c-f)staining,confocalmaximumprojections.Anteriorisleft,allimagesareventralviews.Arrowsinbindicatetheborderbetween
stockandstolon,asterisksdesignateeyes,reddottedlinesindindicateedgesofstock(anterior)andattachedstolon(posterior)whilewhite
dottedlinesineandfindicatetheanteriormarginofthestolon.aOverviewandbdetailedimageofmarkedareawithreducedstacknumber.Nearthe
detachmentsiteandbetweentheeyes,adorsalorientatedloopcomingfromtheventralnervecordappeared,representingthestolonbrain(sbr).The
ventralnervecords(vn;arrowheads)ofstockandstolonwerestillconnected.cOverviewandddetailedimageofmarkedarea.Themusculatureisonly
weaklydevelopedatthetransitionbetweentheremainingbodyandthestolon.Attheanteriormarginofthestolon,thetransversemusculature(tm)
appearscontracted.eMalestolonandfstolonofunknownsex.Infrontofalevelbetweentheparapodiaofthestolonhead,nomusculatureisvisible.
Atthislevelthetransversemusculatureformedasphinctermuscle(ssm).Abbreviations:ga,gamete;sat,stolonantenna;sbr,stolonbrain;ssm,stolon
sphinctermuscle;tm,transversemusculature;vlm,ventrallongitudinalmusculature;vn,ventralnervecord.Scalebars=100μm(a,c,e),50μm(b,d,f)
as in the T. antoni diet. These different feeding strat- hormone would not be exclusively inhibiting stolonization,
egies are widely spread especially within Syllinae, where butalowconcentrationmightbealsonecessaryfortiming
theindividualsreproducebyschizogamy.Thus,theproven- reproductiveprocesses.Aswell,theprostomialsignalmight
triclemighthavebeencooptedfromaplesiomorphicsuck- not only target the proventricle, but also other structures
ing pump function into an additional stolonization control involvedinreproduction[41].Amajorreproductivecontrol
purpose. This might explain its presence in a group of an- functionoriginatingintheprostomiumisalsodescribedin
nelids that show a high diversity in reproductive modes, other Phyllodocida such as Eulalia viridis, Harmothoe
but do not seem to primarily rely on it for feeding [17, imbricata, or Hediste diversicolor [88–90]. A recent study
81]. Alternatively, structures located in the proventricle on Nereidae revealed a high complexity of reproduction
region (e.g., nervous system) might be responsible control,describedbyamulti-hormonemodel[91].
for the regulation of reproductive and regenerative However, morphology does not support an endocrine
processes. function of the proventricle itself. The proventricle of
According to our results, the effects of the removal of syllids is basically a muscle structure. It is composed of
the proventricle region in the stolonization-posterior large sarcomeres [74–76, 78, 92, 93] and granules with
regenerationprocessesare:1)Thestolonizationisacceler- high amounts of phosphorus and especially calcium ions
ated; 2) only three segments and the pygidium are regen- [79]. Our histological survey has revealed that there is
eratedafter stolonization;3)usuallyonly male stolons are no other feature associated in the proventricular region,
produced; and 4) appearance of aberrant stolons (Fig. 9). and there are no signs of glandular tissue through the
H-D Franke [40] also found that the removal of the pro- whole proventricular structure. However, the proventricle
ventricle caused acceleration in the production of stolons muscle cells contain large plasmatic areas and remarkably
inS.prolifera.Inthisspecies,only3–4posteriorsegments hugenuclei(Fig.4c,d),suggestingahighmetabolicactivity.
plus the pygidium are regenerated after proventricle re- It has been suggested that the muscle cells of the pro-
moval [40]. An accelerated stolonization also occurs in ventricle in Syllis spongiphilia have a myoepithelial origin
proventricle-less T. pulchra but to a minor degree [48]. [76–78]. Myoepithelial cells have been described with
Additionally, in S. prolifera as well as in T. pulchra the secretory functions in other groups of organisms [94].
male sex of stolons might be linked to the absence of the However, our results and that of other previous authors
proventricle signalling, which seems necessary for female contradict this possible myoepithelial origin. The muscle
determination[40,48].Inourresults,afewfemalestolons cells have no epithelial part and are demarcated with an
(specimens 1–2, 1–5, and 1–6; Fig. 7, Additional file 1: extracellular matrix (presumably basal lamina) on both
FigureS1)occurinthefirststolonization;howeverthesex sides,closetothecoelomaswellastotheepitheliumthat
of these stolons had probably been determined prior to surrounds the gut lumen (Fig. 4d). Additionally, the pres-
amputation. Aberrant stolons were also found in the re- ence of an inner and an outer epithelium encircling the
generating Procerastea halleziana, a member of Autolyti- proventricle musculature in Syllis gracilis has been dem-
nae,aftertheamputationoftheproventricle[50]. onstrated using transmission electron microscopy [33].
Therefore, a relationship between the proventricle and Obviously, the use of the term “myoepithelial” was either
stolonization-regeneration can be assumed. However, a misinterpretation or a misuse. The proventricle muscle
thespecificwaytheproventricle controlstheseprocesses cells are characteristically striated muscle cells with no
is not clearly established. Franke [41, 42] proposed an secretoryfunction.
endocrine function of the proventricle itself, controlled The proventricle muscle cells contain high amounts of
by the prostomium. Two factors might be involved: the calciumions.Recently,ithasbeendemonstratedthatintra-
"stolonization-inhibiting hormone" released by the pro- cellular calcium ion signalling is essential in the regulation
ventricle; and the "stolonization-promoting hormone" of cell activity in intestinal stem cells of Drosophila
mediatedbytheprostomium.Additionally,theproventricle [95]. However, it is likely that the calcium in the syllid