Submitted 17 April 2016Accepted 12 September 2016Published 6 October 2016

Corresponding authorStella Maris Martinsmartinfcnymunlpeduar

Academic editorTim Collins

Additional Information andDeclarations can be found onpage 9

DOI 107717peerj2548

Copyright2016 Martin and Diacuteaz

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Histology and gametogenesis in Heleobiapiscium (Cochliopidae) from the MultipleUse Reserve lsquolsquoIsla Martiacuten Garciacutearsquorsquo BuenosAires ArgentinaStella Maris Martin1 and Ana C Diacuteaz2

1Divisioacuten Zoologiacutea Invertebrados-FCNyM-UNLP-Investigador Independiente CIC Universidad Nacional deLa Plata Buenos Aires Argentina

2Divisioacuten Zoologiacutea Invertebrados-FCNyM-UNLP-Becaria Doctoral CONICET Universidad Nacional de LaPlata Buenos Aires Argentina

ABSTRACTHeleobia piscium (drsquoOrbigny 1835) a member of the Cochliopidae family found onlyin South America is distributed from Entre Riacuteos Delta del Paranaacute and the littoral ofthe Riacuteo de la Plata down as far as to Punta Indio (Buenos Aires) the southernmostlimit of the snailrsquos geographical distribution To date little information is availableregarding the reproductive cycle of species within this family either in Argentina orthroughout SouthAmerica The presentwork analyzed the histology of the reproductivesystem of the gonochoric species H piscium and determined the stages oogenesis andspermatogenesis under natural conditions Specimens of H piscium were collected inthe Multiple-Use Natural Reserve Isla Martiacuten Garciacutea located in the Upper Riacuteo de laPlata estuary to the south of the mouth of the Uruguay River The gametogenic cycle inboth sexes was found to consist of the following stages early maturation maturationand evacuation The maturation period was found to extend from January to Octoberand evacuation of the gametes to start in November and end in February (summer inthe Southern Hemisphere) The results indicated the H piscium exhibit a reproductivecycle without a resting period

Subjects Zoology HistologyKeywords Gametogenesis Histology Heleobia Isla Martiacuten Garciacutea Argentina

INTRODUCTIONHeleobia Stimpson 1865 is a genuswithin the family Cochliopidae Tryon 1866 (Wilke et al2001 Szarowska Falniowski amp Steffek 2011) comprising 101 species of which 90 are foundin South America (Hershler amp Thompson 1992 Pons da Silva amp Veitenheimer-Mendes2004 Cazzaniga 2011 Collado 2015) Rumi et al (2006) and Rumi et al (2008) reported16 Heleobia species for Argentina of which 10 are endemic

Heleobia piscium (drsquoOrbigny 1835) a South American species is distributed from EntreRiacuteos Delta del Paranaacute and the littoral of the Riacuteo de la Plata estuary down to Punta Indio(Buenos Aires) the southernmost limit of the snailrsquos geographical range Research on theCochliopidae to date has tended to focus on the taxonomy morphology and biology ofthe family but little information is available on certain aspects of the anatomy life cycle

How to cite this article Martin and Diacuteaz (2016) Histology and gametogenesis in Heleobia piscium (Cochliopidae) from the Multiple UseReserve lsquolsquoIsla Martiacuten Garciacutearsquorsquo Buenos Aires Argentina PeerJ 4e2548 DOI 107717peerj2548

reproduction dispersal strategies behavior and genetics of its species Accordingly fewstudies have dealt with the reproductive cycle species from Argentina or from South Amer-ica except for those conducted by Cazzaniga (1982) on Littoridina parchappii (drsquo Orbigny1835) in the drainage channels of the lower valley of the Colorado-River province of BuenosAires Argentina Arconada amp Ramos (2002) investigated the pseudohermaphroditism ofSpathogyna fezi (Altimira 1960) a member of the Hydrobiidae family The analysis ofother freshwater gastropods has provided information on the gametogenesis of speciesbelonging to the families Tateidae eg Potamopyrgus antipodorum (Gray 1843) aninvasive species from New Zealand that has colonized Australia Europe and the Americas(Tair-Abbaci amp Garric 2012) and Ampullariidae eg Pomacea canaliculata (Lamarck1801) Pomacea scalaris (DrsquoOrbigny 1835) and Asolene platae (Maton 1809) speciesfrom Argentina (Martin 1986 Martin 1987 Martin 1992) In Meacutexico Carreoacuten-Palau etal (2003) studied the reproductive system of Pomacea padula catemacensis (Baker 1922)in Taiwan Wu et al (2011) studied the reproduction of the invasive the South Americanapple snails Pomacea canaliculata (Lamarck 1822) and Pomacea scalaris (drsquoOrbigny 1835)(Ampullariidae)

Thework reported herewas aimed at contributing essential information on the reproduc-tive process and strategy of a species of the genusHeleobia through histological examinationof the reproductive system of the gonochoric speciesH piscium along with a determinationof the gonadalmaturation stages oogenesis and spermatogenesis under natural conditions

MATERIAL AND METHODSThe specimens were collected in the Multiple-Use Natural Reserve Isla Martiacuten Garciacutea(authorized by the Direccioacuten de Aacutereas Naturales Protegidas-OPDS Provincial Authorityfor Sustainable Development Buenos Aires-La Plata Argentina Authorization grantedby note under docket number 2578-15302005-0) and deposited in the MalacologicalCollection at theMuseo de La Plata Buenos Aires Province Argentina (MLP-Ma)Monthlycollections were made from August 2005 through June 2007 in the eastern part of the islandArena Beach (3411prime09primeprimeS 5815prime09primeprimeW) (Isla Martiacuten Garciacutea in located in the upper Rio dela Plata) to the south of the mouth of the Uruguay river This coastal stretch is delimitedby the Canal del Infierno which is characterized by a remarkable exposure to strong windsfrom the southeast This area constitutes an outcropping of the Brazilian massif ofPrecambrian crystalline basement rock upon which lie sediments of the Holocene andPleistocene epochs of the Quaternary Period (Ravizza 1984)

Samples of H piscium were taken by hand in the coastal drainage channels formed bytidal erosion A total 1306 specimens were collected The ratio of male to female specimenswas 1 to 1

In the histological analysis of gonads 10 individuals being females and 10 males fromdifferent sizes at random for each climatic season (summer autumn winter and spring)for a total of 80 (eighty) were used In the laboratory the specimens were anesthetizedwith menthol crystals for 24 h before being fixed with Bouin or Raillet-Henryrsquos solutionAfter embedding the gonads in Paraplax 10-microm cuts were made with a microtome and the

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 211

resulting slices stained with hematoxylin-eosin (Gabe 1968) Preparations of gonadal tissueat different stages of gametic development were observed under a Leica DM LS microscopeand photomicrographs taken with a Tucsen USB20 camera connected to a microscope at25times and 40timesmagnifications After following the development of the gametogenic cycle inboth sexes (from the gametogenic stages present in each individual) within an annual cyclethroughout the two consecutive years of the study the following stages were determinedearly maturation maturation and evacuation

RESULTSHeleobia piscium is a dioecious oviparous species with internal fertilization Male andfemale gonads form a spiral within the digestive gland and are composed of numerousfollicles surrounded by connective tissue Themale gonad consists of a number of digitiformprojections gathered in small groups The tube that emerges from each of those groups isthe vas deferens that travels from the base of the gonad to the anterior portion of the snailOnce in the ventral region the vas deferens runs toward the stomach but before reachingthe posterior part of the stomach turns back again toward the posterior part finallyreturning to the stomach through the same route In the posterior part of the stomach thevas deferens becomes rolled up with the last section constituting the seminal vesicle Themale gonad is located in a small portion in the pallial cavity that is both the prostate gland

The female reproductive system consists of the ovary composed of follicles the oviductthe seminal receptacle and the albumen gland Like the penis the gonadic follicles aredorsally located in the ventral or dorsolateral region of the digestive or hepatopancreaticgland extending from the posterior portion of the stomach to the posterior end of thebody These follicles cover a smaller portion than that of the penis with respect to size ofthe digestive structure The albumen gland is whitish and is topographically located in theposterior region of the pallial cavity

HistologyWe conducted a histological study of the male and female gonads to ascertain the detailsthe gonadogenesis development and the organization of the internal tissues Therefore tofollow the evolution of the different stages of spermatogenesis and oogenesis we mainlyconsidered the characteristics and morphology of the sexual cells

MalesDuring spermatogenesis in the early maturation of the follicles large numbers ofspermatogonia (364ndash455 microm) and spermatocytes (182ndash273 microm) are present Uponcomplete maturation spermatozoids become abundant in the lumen of the follicles whilethe spermatocytes and spermatids (455 microm) are observed around the border of the acinarwall (Figs 1 2 and 3)

At the evacuation stage the male follicles exhibit a different cytology with the follicularlumen becoming almost empty and amoebocytes appearing that proceed to phagocytize thesexual cells remaining (Fig 4) This process constitutes firm evidence that the evacuationof gametes has occurred The histological cuts observed in the male revealed a tubularstructure resembling tubes separated by connective tissue

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 311

Figure 1 Stages of spermatogenesisMaturation of the germinal sexual cells in the male acini Spermato-zoid sp follicle f hepatopancreatic acini ahp

Figure 2 Stages of spermatogenesisMaturation of the germinal sexual cells in the male acini Spermato-zoid sp follicle f hepatopancreatic acini ahp

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 411

Figure 3 Stages of spermatogenesisMaturation of the germinal sexual cells in the male acini Spermato-zoid sp follicle f hepatopancreatic acini ahp

Figure 4 Stages of evacuation The acinus characterized by the spermatozoids and spermatocytes speNote the phagocytosis of the residual male cells by the amoebocytes a spermatozoid sp follicle f

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 511

Figure 5 Stages of oogenesis Yolk granules surrounded by oocytes The nucleus was prominent duringthe stages of maturation Oocyte oo follicle f hepatopancreatic acini ahp

FemalesDuring oogenesis immature female gonadal follicles are present they measure less than 3mm Only developing oocytes were identified

Follicles measuring less than 3 mm have only immature female gonads lacking vitellusgranules along with developing oocytes In female specimens that have reached a totallength of 3ndash4 mm the vitellogenic oocytes occupy these gonadic follicles This stage infact constitutes the indication that gonadal maturation has been completed (Figs 5 and6) Once reaching complete maturation the female gonads begin evacuating their sexualcells This evacuation is revealed by a spreading out of vitellus granules and a thinning ofthe oocytes those being attached to the now extremely thin follicular walls (Fig 7) Atthis stage cells referred to as amoebocytes with well colored nuclei located at one end of awide granulated cytoplasm appear to phagocytize residual oocytes The histological cutsobserved in females revealed that female follicles are surrounded by connective tissue Thesmall number of oocytes in the follicles attached to these thin follicular walls constitutesthe evidence that sexual-cell evacuation has occurred

DISCUSSIONThe female gonad of H piscium presents a ribbon-like distribution with few follicles Thesize and intrafollicular location of the oocytes agree with reports by Jong-Brink Boer amp Joose(1983) In the present work the female gametogenic cycle involved fully mature oocytesthat were surrounded by abundant yolk granules as observed in marine Caenogastropoda(Covarrubias amp Romero 2009) The presence of vitellus granules revealed a difference from

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 611

Figure 6 Stages of oogenesis Stages of oogenesis Detail of the nucleus and nucleolus was as prominentin the stages of maturation Oocyte oo nucleo n

Figure 7 Stages of oosorption Stages of oosorption and degeneration of the oocytes and yolk granulesOocyte oo yolk granules yk follicle fc

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 711

other Caenogastropoda such as the freshwater species of the Ampullariidae family thatdo not show such cells surrounding oocytes during the maturation stage (Martin 1986Martin 1987Martin 1992)

The maturation stage for both males and females took place from January through Oc-tober DuringMarch April andMay (the Southern-Hemisphere autumn)male and femaleindividualsmeasuring 30ndash45mmwere at the earlymaturation stage Completematurationwas observed during August September and October in specimens measuring 50ndash55mm Tair-Abbaci amp Garric (2012) in the histological study and gonadal development ofPotamopyrgus antipodorum showed early gonadogenesis when they reached 2 mm size inH piscium observed for about the same size

Themale gonad was found to be distributed in a centripetal form similar to that observedin other Caenogastropods of the Littoridinidae family (eg Echinolittorina peruviana(Lamarck 1822) Castillo amp Brown 2008) This characteristic gives a variegated appearanceto the gonadndashdigestive-gland complex a formation quite different from a clearly separatedgonad and digestive gland or from digestion in a topographically separate location inother species Hershler amp Ponder (1998) described the same form of gonadal structure inseveral species of Hydrobiidae and based their description on the genital characteristics ofSpurwinkia salsa (Pilsbry 1905) Carreoacuten-Palau et al (2003) in Pomacea patula catemacen-sis (Baker 1922) and Wu et al (2011) observed that the gonad of Pomacea canaliculataand Pomacea scalaris is in close contact with the digestive gland which constitutes thevisceral coil

The evacuation of the gametes started in November and ended in February (theSouthern-Hemisphere summer) Cazzaniga (1982) had observed in L parchappii amongsouthern snail populations that the maturation stage for bothmales and females took placefrom January to September (springndashsummer) with the evacuation of the gametes startingin September and ending in January for each annual cycle of that species in ArgentinaHeleobia piscium exhibits a reproductive cycle without any resting period a characteristicshared with members of the genera Pomacea (Perry 1811) and Asolene (DrsquoOrbigny 1837)studied byMartin (1986)Martin (1987)Martin (1992)

This present analysis of the population of H piscium constitutes the first study on thegametogenic cycle of a species of Cochliopidae Both the males and the females withinthe population displayed a synchronous gametogenic cycle These results indicate that theH piscium populations exhibit a continuous reproductive cycle in the absence of anyresting period

The present work conducted at the Multiple-Use Natural Reserve Isla Martiacuten Garciacutea(Argentina) provides essential information on the annual reproductive cycle ofH pisciumThis species could be used as lsquolsquotest taxonrsquorsquo in the assessment of the possible long-termnegative effects of herbicides that are used in irrigation canals to control the growth ofsubmerged aquatic vegetation Environmental applications such as these were reportedby Hershler amp Davis (1980) who suggested that the hydrobiid family is one of the mostfundamental groups of invertebrates used as a food source for other organisms in NorthAmerica and Europe

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 811

ACKNOWLEDGEMENTSThe authors wish to thank Dr Donald Haggerty a career investigator and native Englishspeaker for editing the final version of themanuscript andMr JoseacuteMaciel for his hospitalityon the Isla Martiacuten Garciacutea

ADDITIONAL INFORMATION AND DECLARATIONS

FundingFinancial support for this work was provided by an institutional grant from the Comisioacutende Investigaciones Cientiacuteficas de la Provincia de Buenos Aires (CIC) PICT 2042-2008Agencia Ciencia y Teacutecnica and Facultad de Ciencias Naturales y Museo UniversidadNacional de La Plata (Proyect 11N636) The funders had no role in study design datacollection and analysis decision to publish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsComisioacuten de Investigaciones Cientiacuteficas de la Provincia de Buenos Aires (CIC)PICT 2042-2008Agencia Ciencia y Teacutecnica and Facultad de Ciencias Naturales y MuseoUniversidad Nacional de La Plata Proyect 11N636

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull StellaMarisMartin and Ana C Diacuteaz conceived and designed the experiments performedthe experiments analyzed the data contributed reagentsmaterialsanalysis tools wrotethe paper prepared figures andor tables reviewed drafts of the paper

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

Direccioacuten de Aacutereas Naturales Protegidas-OPDS Provincial Authority for SustainableDevelopment Buenos Aires-La Plata Argentina Authorization granted under docketnumber 2578-15302005-0

Data AvailabilityThe following information was supplied regarding data availability

The specimens are deposited at Divisioacuten Zoologiacutea Invertebrados-Seccioacuten Malacologiacutea-Museo de La Plata Facultad Ciencias Naturales y Museo Universidad Nacional de LaPlata DZI-MLP-UNLP Number MLP MA14179-14180-14181-14182

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 911

REFERENCESArconada B RamosMA 2002 Spathogyna a new genus for Valvata (iquest Tropidina)

fezi Altimira 1960 from eastern Spain a second case of pseudohermaphroditismin a Hydrobiidae species (Mollusks Prosobranchia) Journal of Molluscan Studies68319ndash327 DOI 101093mollus684319

Carreoacuten-Palau L Uria-Galicia E Espinosa-Chaacutevez F Martiacutenez-Jeroacutenimo F 2003Desarrollo morfoloacutegico e histoloacutegico del sistema reproductor de Pomacea patulacatemacensis (Baker 1922) (Mollusca Caenogastropoda Ampullariidae) [Morpho-logical and histological development of the reproductive system of Pomacea patulacatemacensis (Baker 1922) (Mollusca Caenogastropoda Ampullariidae)] RevistaChilena De Historia Natural 76665ndash680 DOI 104067S0716-078X2003000400010

Castillo VM Brown DI 2008Microscopic anatomy of the male reproductive systemin Echinolittorina peruviana (Mollusca Caenogastropoda) International JournalMorphology 26(2)423ndash432 DOI 104067S0717-95022008000200030

Cazzaniga NJ 1982 Notas sobre hidroacutebidos argentinos 5 Conquiliometriacutea de Littorid-ina parchappii (DrsquoOrbigny 1835) (Gastropoda Rissoidea) referida a su ciclo de vidaen poblaciones australes Iheringia Zoologiacutea 6197ndash118

Cazzaniga NJ 2011 Heleobia Stimpson 1865 taxonomiacutea In Cazzaniga NJ ed Elgeacutenero heleobia (Caenogastropoda Cochliopidae) Amici Molluscarum NuacutemeroEspecial 12ndash17

Collado G 2015 A new freshwater snail (Caenogastropoda Cochliopidae) from the Ata-cama Desert northern Chile Zootaxa 3925(3)445ndash449DOI 1011646zootaxa392539

Covarrubias AA RomeroMS 2009 Anatomy of the female and male reproductivesystem of Sinum cymba (Menke 1828) (Gastropoda Naticidae) Revista De BiologiacuteaMarina Y Oceanografiacutea 44(3)673ndash683

GabeM 1968 Tecniques histologiques Paris Masson et Cie Editeurs 1113Hershler R Davis GM 1980 The morphology of Hydrobia truncata (Gastropoda

Hydrobiidae) relevance to systematics of Hydrobia Biology Bulletin 158195ndash219Hershler R PonderWF 1998 Areview of morphological characters of Hydrobioid

snails Smithsonian Contributions to Zoology 6001ndash55Hershler R Thompson FG 1992 A review of the aquatic gastropod subfamily

Cochliopinae (ProsobranchiaHydrobiidae)Malacological Review 51ndash140Jong-BrinkM Boer HH Joose J 1983 Mollusca In Adiyodi KJ Adiyodi RG eds

Reproductive biology of invertebrates oogenesis oviposition and oosorption Vol 1New York John Wiley and Sons Ltd 297ndash355

Martin SM 1986 Ciclo reproductivo de Ampullaria canaliculata (Gastropoda Am-pullariidae) en el aacuterea rioplatense Neotropica 32(88)171ndash181

Martin SM 1987 Ciclo reproductivo de Asolene (A)platae Maton (GastropodaAmpullariidae)del arroyo Migueliacuten (Punta Lara Buenos Aires) Neotroacutepica133(89)19ndash28

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 1011

Martin SM 1992 Ciclo anual de la gametogeacutenesis de Ampullaria scalaris drsquoOrbigny(Mollusca Gastropoda Ampullariidae) Anales De La Sociedad CientiacuteFica Argentina2211ndash8

Pons da Silva MC Veitenheimer-Mendes IL 2004 Nova espeacutecie de Heleobia (Ris-soidea Hydrobiidae) da planicie costeira do sul do Brasil Iheringia Seacuterie Zoologia94(1)89ndash94

Ravizza GB 1984 Principales aspectos geoloacutegicos del Cuaternario en la Isla MartiacutenGarciacutea Riacuteo de la Plata superior Revista Asociacioacuten Geoloacutegica Argentina Buenos Aires39(1ndash2)125ndash130

Rumi A Gutierrez Gregoric DE Nuntildeez V Cesar II RocheMA Tassara MP MartinSM Lopez Armengol MF 2006 Freshwater gastropoda from Argentina speciesrichness distribution patterns and an evaluation of endangered speciesMalacologia49(1)189ndash208 DOI 1040021543-8120-491189

Rumi A Gutierrez Gregoric DE Nuntildeez V Darrigran GA 2008Malacologia Lati-noamericana Moluscos de agua dulce de Argentina Revista de Biologiacutea Tropical56(1)77ndash111

SzarowskaM Falniowski A Steffek J 2011 Phylogenetic relationships of Alzoniellaslovenica (Lozek et Bretk 1964) (Caenogastropoda Hydrobiidae) Folia Malacologica19(2)87ndash95 DOI 102478v10125-011-0009-4

Tair-Abbaci K Garric J 2012Histological study of gonadogenesis in Potamopyrgusantipodorum and Valvata piscinalis Journal Cytology and Histology 3(2)1ndash7

Wilke T Davis GF Falniowski AJ Giusti F BodoacutenM SzarowskaM 2001Molecularsystematics of Hydrobiidae (Mollusca Gastropoda Rissooidea) testing monophylyand phylogenetic relationships Proceeding Academic Natural Science Philadelphia1511ndash21 DOI 1016350097-3157(2001)151[0001MSOHMG]20CO2

Wu J-YWu Y-T Li M-C Chiu Y-W LiuM-Y Liu L-L 2011 Reproduction and juvenilegrowth of the invasive apple snails Pomacea canaliculata and Pomacea scalaris(Gastropoda Ampullariidae) in Taiwan Zoological Studies 50(1)61ndash68

Martin and Diacuteaz (2016) PeerJ DOI 107717peerj2548 1111