Neotrop. Helminthol., 8(1), 2014

2014 Asociación Peruana de Helmintología e Invertebrados Afines (APHIA)

ISSN: 2218-6425 impreso / ISSN: 1995-1043 on line

ORIGINAL ARTICLE / ARTÍCULO ORIGINAL

FIRST REPORT OF TEMNOCEPHALA PEREIRAI (PLATYHELMINTHES,

TEMNOCEPHALIDAE) ON TRACHEMYS DORBIGNI (EMYDIDAE) FROM SOUTHERN

BRAZIL –A COMPLETE MORPHOLOGICAL STUDY

PRIMER REGISTRO DE TEMNOCEPHALA PEREIRAI (PLATYHELMINTHES,

TEMNOCEPHALIDA) SOBRE TRACHEMYS DORBIGNI (EMYDIDAE) EN EL SUR DE

BRASIL – ESTUDIO MORFOLÓGICO COMPLETO

1* 2 1 3*

Samantha A. Seixas , José F. R. Amato , Suzana B. Amato & Carolina S. Mascarenhas

Abstract

Specimens and eggs of Temnocephala pereirai Volonterio, 2010 found on Trachemys dorbigni

(Duméril & Bibron, 1835), were reported for the first time extending the known geographical

distribution of T. pereirai from Uruguay to southern Brazil. Sixteen turtles were collected from a

dam at 'Centro Agropecuário da Palma', Universidade Federal de Pelotas (UFPel), Municipality

of Capão do Leão, State of Rio Grande do Sul, Brazil. The helminthes were fixed according to our

standard protocols. Juvenile and adult temnocephalans were translucent and without the red-eye

pigmentation. The worms were living in the inner corners of the plastron, near the legs, together

with the eggs, and in the center of the plastron. Eggs were elongated, thick-shelled, with a small

and subpolar filament. The most distinctive features of this species were: 1. cirrus's introvert

proximal portion with spines from top to mid-level, followed by a smooth region, extending to the

introvert proximal limit; 2. vagina with strong muscular walls and a single, symmetrical sphincter;

3. vesicula intermedia unusually long; 4. small dorsolateral 'excretory' syncytial epidermal plates

larger in the anterior portion; and 5. excretory pore in the anterior portion of the plate, near the

internal limit. The specimens in the present work had larger body length and base diameter of the

shaft than the Uruguayan specimens.

Keywords: freshwater turtles - Neotropic Region – taxonomy – temnocephalans - Trachemys dorbigni.

Suggested citation: Seixas, SA, Amato, JFR, Amato, SB & Mascarenhas, CS. 2014. First report of Temnocephala pereirai

(Platyhelminthes, Temnocephalida) on Trachemys dorbigni (Emydidae) from Southern Brazil –complete morphological study.

Neotropical Helminthology, vol. 8, n°1, jan-jun, pp. 23 - 35.

1 Laboratório de Helmintologia, Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul. Caixa Postal 15014, 91501-

970 Porto Alegre, Rio Grande do Sul, Brasil. E-mail: seixas.sa@gmail.com; sbamato@ufrgs.br

2 Rua Cabral, 875/301, 90420-121 Porto Alegre, Rio Grande do Sul, Brasil. E-mail: josefelipeamato@gmail.com

3 Laboratório de Parasitologia de Animais Silvestres, Instituto de Biologia, Universidade Federal de Pelotas (UFPel). Caixa Postal 354, 96010-900 Pelotas, Rio

Grande do Sul, Brasil. E-mail: phrybio@hotmail.com

*CAPES Doctoral Fellow. Part of the Doctoral Dissertation of S. A. Seixas.

Seixas et al.

Temnocephala pereirai on Trachemys dorbigni

Resumen

Palabras clave: Región Neotropical- taxonomía – temnocéfalos – tortugas.

Especimenes de Temnocephala pereirai Volonterio, 2010 ectosimbiontes en Trachemys dorbigni

(Duméril & Bibron, 1835) se describen del Municipio de Capão do Leão, Estado de Rio Grande do

Sul, Brasil. Este registro permite la extensión de la distribución geográfica conocida de T. pereirai

desde Uruguay hacia el sur de Brasil. Dieciséis tortugas se recogieron en una represa del 'Centro

Agropecuário da Palma', Universidade Federal de Pelotas (UFPel), Municipio de Capão do Leão,

Estado de Rio Grande do Sul, Brasil. La totalidad de la muestra se fijó de acuerdo a nuestros

protocolos. Temnocéfalos juveniles y adultos eran translúcidos y la pigmentación de ojos rojos

estaba ausente. Se localizan en las esquinas internas del plastrón, cerca de las patas, junto con los

huevos, y en el centro del plastrón. Los huevos eran alargados con cáscara espesa y con un

pequeño filamento desplazado hacia un lado. Las características más distintivas de esta especie

fueron las siguientes: 1. cirro con la porción proximal del introverto con espinas desde la

extremidad distal hasta la mitad, seguido por una región desnuda, que se extiende hasta el límite

proximal del introverto; 2. vagina con fuertes paredes musculares y esfínter vaginal simétrico; 3.

vesícula intermedia inusualmente larga; 4. placas dorsolaterales, sincitiales 'excretoras' pequeñas,

más anchas en su parte anterior; y 5. poro excretor en la porción anterior de la placa, cerca del

límite interno. Los especímenes del presente trabajo tenían una mayor longitud del cuerpo y

diámetro de la base del 'shaft' en comparación con los especímenes uruguayos.

type series deposited by Monticelli in the

'Museum für Naturkunde Zentralinstitut der

Humboldt-Universität zu Berlin, Institut für

Systematische Zoologie', thus observing that his

specimens as well as those studied by

Monticelli, did not have body and ocellar

pigmentation. Based on this information Baer

did not find valid the variety described by

Merton in 1922 (Temnocephala brevicornis var.

intermedia) which could be differentiated from

T. brevicornis by the presence of body and

ocellar pigmentation and insignificant details in

the disposition of the reproductive system

(Cordero, 1946). Pereira & Cuocolo (1940,

1941) conducted an extensive work on the

morphology, bionomy, and ecology of

specimens identified as T. brevicornis. They

studied ectosymbiont specimens from H.

tectifera collected in the State of São Paulo and

from H. maximiliani collected in the States of

Rio de Janeiro and Minas Gerais.

Caballero & Zerecero (1951) recorded

specimens they identified as T. brevicornis on

brachyuran crustaceans of the genus

Temnocephala brevicornis Monticelli, 1889 was

the second species of the genus to be described

and for 121 years was the only species recorded

from chelonians. Monticelli (1889) studied

temnocephalans from Hydromedusa

maximiliani (Mikan 1820) and Hydraspis gibba

(= Mesoclemmys gibba (Schweigger, 1812)

collected in Brazil and deposited in the

helminthological collection of the Zoological

Museum of Copenhagen.

The description of T. brevicornis was made in a

short note, limited to the external characters,

allegedly because there were not enough

specimens in good condition to be studied.

Monticelli (1898) published a detailed study of

the internal anatomy based on specimens

ectosymbiont on Hydromedusa tectifera Cope,

1869, collected in 1894 in the South of Brazil.

Baer (1931) studied specimens identified as T.

brevicornis from H. maximiliani, Hydromedusa

platanensis (= H. tectifera), and M. gibba. The

author studied specimens which included the

INTRODUCTION

Neotrop. Helminthol., 8(1), 2014

Turtles were captured manually (with the

UFPel

with dip nets, and were taken

in plastic boxes to the 'Laboratório de

Parasitologia de Animais Silvestres da UFPel',

to be examined for temnocephalans.

Temnocephalans were fixed with 10%

phosphate-buffered formalin 90ºC or with AFA,

under slight cover slip pressure, following the

protocols established by Amato et al. (2007) and

Seixas et al. (2010). Some specimens were

stained in Delafield's hematoxylin or aceto-

carmine /fast green, cleared in cedar oil, and

mounted as permanent slides in Canada balsam.

Other specimens were prepared for Scanning

Electron Microscopy (SEM) at the 'Centro de

Microscopia Eletrônica da UFRGS (CME)'. The

temnocephalans from T. dorbigni were studied

through a series of techniques focusing

especially on the: 1. morphology of the cirrus

structure; 2. morphology of the vagina and other

female reproductive organs; 3. distribution of

the rhabditogenic glands in juveniles; and, 4.

paired, dorsolateral, post-tentacular 'excretory'

syncytial plates (DLSPs). Photomicrographs

were taken with the microscopes Zeiss Axiolab

and Leica DMR Hc equipped with Nomarski´s

differential interference contrast (DIC) prisms.

The photographic images and line drawings

were scanned and prepared using CorelDraw X5

and Adobe's Photoshop CS6, respectively.

Measurements are in micrometers (µm) unless

otherwise indicated and were taken from

specimens killed under slight cover slip pressure

and mounted in Canada balsam; ranges are

followed (between parentheses) by the mean, the

standard deviation values, and the number of

specimens measured for a given character (when

different than 27). Cirrus measurements were

taken from extracted cirri mounted in Faure´s

mounting medium (F), while the terminology

used to describe the male reproductive structures

followed Seixas et al. (2010). The whole mounts

of adult and juvenile specimens, as well as slides

containing individual cirri mounted in (F) were

deposited in the following scientific collections:

1. 'Coleção Helmintológica do Instituto

Oswaldo Cruz (CHIOC)', Rio de Janeiro, RJ,

Brazil; 2. 'Coleção de Invertebrados do Instituto

permission of the Ethics and Animal

Experimentation Committee of the (nº

3026 CEEA) and

Pseudothelphusa De Saussure, 1857, collected

in Sabana de Piedra, Venezuela. Although these

authors have provided a detailed description of

the specimens, it is probable that the specimens

belong to a different species, as T. brevicornis is

not known to occur on crustaceans.

The first record of specimens identified as T.

brevicornis outside Brazil was published by

Dioni (1967). This author studied ectosymbionts

from H. tectifera collected in Tacuarembó,

Uruguay, as well as four other species of

temnocephalans, publishing a dichotomic key

for these species.

Yuki et al. (1993) recorded T. brevicornis in two

new hosts: Acanthochelys spixii (Duméril &

Bibron, 1835), and Trachemys dorbigni

(Duméril & Bibron, 1835) collected in the

Municipality of Viamão, State of Rio Grande do

Sul. The authors found that the cirrus

morphology (shape) was similar to that

described by Pereira & Cuocolo (1940),

although other characters presented significant

differences. Novelli et al. (2009) recorded

specimens they identified as T. brevicornis on H.

maximiliani collected in the State of Minas

Gerais.

Brusa & Damborenea (2000) published the first

record for Argentina of specimens identified as

T. brevicornis collected from H. tectifera in

Magdalena, Buenos Aires. Volonterio (2010)

described two new species (Temnocephala

pereirai Volonterio 2010 and Temnocephala

cuocoloi Volonterio 2010) ectosymbiont on H.

tectifera collected from a road-killed turtle in

Canelones, Uruguay. The present work is the

second record of T. pereirai and the first record

of the species in Brazil.

Sixteen turtles, T. dorbigni, were collected

between July and December of 2010 in a dam

(31º48'01.10”S, 52º30'48.60”W) at the 'Centro

Agropecuário da Palma', Universidade Federal

de Pelotas (UFPel), located in the rural area of

the Municipality of Capão do Leão, RS, Brazil.

MATERIAL AND METHODS

Seixas et al.

Temnocephala pereirai on Trachemys dorbigni

including pair, of large, round, more central cells

(paranephrocytes?), 77.5–127.5 (101 ± 17; 7)

long (Fig. 11).

Reproductive system. Female. Ovary 85–287.5

(170 ± 41; 26) long, 60–150 (109 ± 22; 26) wide,

located between the vagina and the vesicula

resorbens (Figs 15 and 26-27). Vitellarium

arborescent, thick, almost covering the entire

intestine dorsally (Fig. 6); vagina 112.5–200

(173 ± 24; 26) long, 105-215 (172 ± 33; 27)

wide, muscular walls strong, 25-62.5 (42 ± 11;

26) (Figs 15 and 26-27); single vaginal sphincter

symmetrical, 90-180 (139 ± 23; 25) in total

diameter (Figs 15 and 26-27); vesicula

intermedia unusually long, 62.5-127.5 (92 ± 17;

16) long (Figs 15 and 27); vesicula resorbens

usually full of sperm (Fig. 15), 80–247.5 (156 ±

47; 13) long, 127.5–275 (203 ± 47; 13) wide.

Male. Four testes usually rounded, slightly

oblique; deferent vessels (Figs 14 and 25) unite

in large, seminal vesicle pyriform (Fig. 14 and

25), 140–337.5 (240 ± 42) long, 45–100 (74 ±

17) wide; wall thickness 2.5–15 (8 ± 3); prostatic

bulb short, muscular walls thick (Figs 14 and

25), 67.5–195 (117 ± 30) long, 65–122.5 (96 ±

16) wide, wall thickness 2.5–5 (4 ± 1). Cirrus

302.5–352.5 (322 ± 17; 12) long, introvert

proximal portion with spines from tip to mid-

level, followed by a smooth region, extending to

the introvert proximal limit (Figs 13, 18, and 21).

Shaft 272.5–312.5 (288 ± 14; 12) long, shaft

maximum width at base 77.5–110 (91 ± 9; 12)

(Figs 13 and 18); introvert 30-42.5 (34 ± 4; 12)

long; introvert width at base 15-27.5 (21 ± 4, 12);

maximum introvert width at level of swelling

15-20 (17 ± 2, 12). Introvert´s swelling with

approximately 21 spines per longitudinal row

(Figs 20 and 21). Ratio between total body

length (without tentacles)/ total length of cirrus

9.2: 1; ratio between total length of cirrus/

maximum width of shaft´s base 3.5: 1; ratio

between total length of cirrus/ total length of

introvert 9.5: 1.

Taxonomic summary.

Type host: Hydromedusa tectifera Cope, 1869

(Chelidae).

Type locality: Barra do Carrasco, Ruta 10, km 15

Nacional de Pesquisas da Amazônia (INPA)',

Manaus, AM, Brazil; and 3. 'Coleção

Helmintológica do Laboratório de

Helmintologia, Departamento de Zoologia,

Universidade Federal do Rio Grande do Sul',

Porto Alegre, RS.

Temnocephala pereirai Volonterio, 2010

(Figs 3 - 27)

Description. Based on 391 specimens collected:

27 whole mounted adults and 1 juvenile; 3

specimens mounted on stubs for SEM; 12

dissected cirri mounted in (F); 27 specimens

measured.

External characteristics. Body (without

tentacles) (Figs 6 and 12) 2.11–4.03 mm (2.97

mm ± 451) long, 1.48–2.84 mm (2.23 mm ± 345)

wide; adhesive disk ventral, subterminal,

completely covered by body (Figs 6 and 7)

434–968 (750 ± 137) long, 632–1027 (860 ±

117) wide; disc peduncle 237–612 (411 ± 91)

wide. Red pigmentation of the eyes absent. Two

DLSPs, small and kidney-shaped, larger in the

anterior portion (Figs 16 and 17), left plate

217.5–253.4 (230.5 ± 20; 3) long, 111.7–130.85

(121.3 ± 13; 3) wide; right plate 213.65–250

(226.6 ± 20; 3) long, 76.9–113.85 (94.5 ± 18; 3)

wide; length ratio of DLSPs /total body length,

without tentacles, 13: 1. Excretory pore in the

anterior portion of the plate, near the internal

limit (Fig. 17 - ).

Glands. Rhabditogenic glands forming bunches

extending from posterior level of pharynx to

mid-level of posterior testes, bunches much

wider anteriorly (average 41 cells), in lateral

fields of body (Figs 7-8 and 10), 40-87.5 (62 ±

12) in diameter, ducts conspicuous (Figs 7 and

10). Two groups of two Haswell glands (Fig. 24 -

asterisks), showing little affinity with

hematoxylin, in front of the brain transverse

band (Fig. 24), diameter of largest cell 60–140

(93 ± 23; 26). Disc glands between adhesive disc

and genital complex (Figs 7-9 and 11), 47.5–105

(71 ± 17) in diameter, forming two, lateral

bunches extending from mid-level of posterior

testes to anterior margin of adhesive disc,

head arrow

RESULTS

Neotrop. Helminthol., 8(1), 2014

Figures 1–3. Trachemys dorbigni. 1. Female specimen of Trachemys dorbigni. 2. Ventral view of male specimen of T. dorbigni

showing the temnocephalans concentrated in the femoral and anal areas of the plastron. Scale bar = 1.5 cm. 3. Live eggs deposited

in the inner marginal area of the carapace. Scale bar = 0.5 cm. Figures 4–6. Temnocephala pereirai. 4. Live specimens in the

femoral area of the plastron. Scale bar = 3 mm. 5. Unhatched eggs cleared in cedar oil, showing shape, peduncles, and filament.

Scale bar = 250 µm. 6. Adult specimen. Scale bar = 500 µm.

Seixas et al.

Temnocephala pereirai on Trachemys dorbigni

Figures 7–9. Temnocephala pereirai juveniles cleared in lactophenol. 7. Juvenile showing rhabditogenic gland ducts entering

tentacles (head arrow); rhabditogenic glands (arrow); esophageal glands (eg); and disc glands (asterisk). Scale bar = 250 µm. 8.

Magnification of the left side showing the limit between the anterior rhabditogenic glands and the posterior disk glands (arrow);

pharynx (p); and cirrus (c). Scale bar = 100 µm. 9. Juvenile; posterior portion showing the disk glands; posterior testes (pt); and

cirrus (c). Scale bar = 100 µm.

Neotrop. Helminthol., 8(1), 2014

Figures 10–15. Temnocephala pereirai. 10. Incomplete diagram of a juvenile specimen, showing rhabditogenic gland ducts

entering tentacles (rgd); and the rhabditogenic glands (rg). Scale bar = 250 µm. 11. Juvenile, posterior portion showing disk

glands (dg) and the pair of paranephrocytes (head arrow). Scale bar = 150 µm. 12. Incomplete diagram of adult specimen showing

adhesive disk (ad), anterior testis (at), excretory vesicle (ev), Haswell glands (hg), intestine (i), mouth (m), posterior testis (pt),

pharynx (p), tentacles (t), and vitellarium (v). Scale bar = 500 µm. 13. Cirrus showing the proximal limit of the introvert (head

arrows). Scale bar = 25 µm. 14. Male reproductive complex, showing cirrus (c), prostatic bulb (pb), prostatic cells (pc), prostatic

secretion (ps), seminal vesicle (sv), and vasa deferentia (vd). Scale bar = 50µm. 15. Female reproductive complex, showing

genital atrium (ga), ovary (ov), vagina (va), vaginal sphincter (vs), vesicula resorbens (vr), vesicula intermedia (vi), and vitelline

gland duct (vgd). Scale bar = 100 µm.

Seixas et al.

Temnocephala pereirai on Trachemys dorbigni

size, and the introvert spines present in larger

number although being more delicate. Most of

these differences were attributed by Volonterio

(2010) to the species T. pereirai.

We tried (unsuccessfully) to collect turtles in the

same locality [ULNA (Uma Luz No Amanhã),

Águas Claras, RS, Brasil] where Yuki et al.

(1993) collected their specimens, although, the

examination of the specimens that these authors

deposited at the “Museo de La Plata (MLP)”

confirmed that they belonged to T. pereirai.

Brusa & Damborenea (2000) also reported that

the Argentinean specimens showed smaller

body size, and smaller and thinner cirri than

those described by Pereira & Cuocolo (1940).

The examination of Argentinean specimens of T.

brevicornis deposited at the “Museo de La Plata

(MLP)” also showed that those specimens

belong to T. pereirai.

The specimens of the present work show larger

body length (average length of 2.96 mm) when

compared to the specimens described by

Volonterio (2010) (average length of 2 mm).

Although the average body length of the

specimens identified as T. brevicornis studied by

Pereira & Cuocolo (1940) is even larger

(average length of 3.76 mm), these authors left a

doubt if this measure included or not the

tentacles. Even with this discrepancy in body

size other important characters are similar to T.

pereirai. According to Volonterio (2010), the

vagina has two sphincters, one proximal,

smaller, well developed, and another distal,

larger and less developed.

The specimens of the present work have a well-

developed proximal sphincter (average diameter

of 139 µm – Fig. 26), with almost identical

values of the Uruguayan specimens (average

diameter of 134 µm), however, what the author

called distal sphincter, we believe to be the

vagina wall, which in this species is highly

muscular (average thickness of 42 µm). When

the measurements of the vagina length between

the Uruguayan specimens (average length of

177 µm) and the specimens of the present work

(average length of 172.6 µm) are compared it is

possible to observe that the structure is the same.

o o

(34 52'S, 56 02'W), Departamento de

Canelones, Uruguay.

Other host (present work): Trachemys dorbigni

(Duméril & Bibron, 1835) (Emydidae).

Other locality (present work): Centro

Agropecuário da Palma, Universidade Federal

de Pelotas (UFPel), Capão do Leão, RS, Brazil.

Site of infestation: inner corners of the plastron,

near the legs, together with the eggs, and in the

center of the plastron.

Intensity of infestation: 6-169.

Average Intensity of infestation: 43.4.

Prevalence: 56.25%.

Helminth specimens deposited: 'Coleção

Helmintológica do Instituto Oswaldo Cruz':

CHIOC 37929a - JFA 3222-1-4; CHIOC 37929b

- JFA 3222-1-1; CHIOC 37931 – JFA 3223-1-6

(juvenile); CHIOC 37930 - JFA 3137-1-19

(cirrus); CHIOC 37929c - JFA 3222-1-20

(cirrus). 'Coleção de Invertebrados do Instituto

Nacional de Pesquisas da Amazônia': INPA 636 -

JFA 3137-1-1; INPA 635 - JFA 3223-1-2A

(cirrus).

Other helminth specimens examined:

'Colección de Invertebrados, División Zoología

Invertebrados, Museo de La Plata' (MLP): MLP

3155 (194; 195) – Temnocephala brevicornis

from A. spixii. MLP 4585 (162; 167; 168) –

Temnocephala brevicornis from H. tectifera.

Volonterio (2010) when describing T. pereirai

called attention to some important differences

observed between this species and T.

brevicornis, mainly related to the specimens

studied by Pereira & Cuocolo (1940). Yuki et al.

(1993) also described important differences

between their specimens and those of Pereira &

Cuocolo (1940) stating that even so they were

going to identify their specimens as T.

brevicornis. These differences were in shape and

size of the vagina, the presence of a vesicula

intermedia (described as a duct linking the

ootype to the vesicula resorbens), smaller body

DISCUSSION

Neotrop. Helminthol., 8(1), 2014

Figures 16–17. Temnocephala pereirai observed with SEM. 16. Entire specimen, showing the dorsolateral 'excretory' syncytial

plates (DLSP) (head arrows) and position of excretory pore (ep). Scale bar = 100 µm. 17. Right DLSP and position of excretory

pore (ep). Scale bar = 100 µm.

Seixas et al.

Temnocephala pereirai on Trachemys dorbigni

Figures 18-22. Temnocephala pereirai cirrus mounted in (F) and observed with DIC. 18. Cirrus, showing the proximal limit of

the introvert (arrow). Scale bar = 25 µm. 19-22. Cirrus introvert observed in different focusing planes. 21. The proximal limit of

the introvert (black arrow) and the beginning of the spines row (white arrow). Scale bar = 10 µm.

Neotrop. Helminthol., 8(1), 2014

Figures 23-27. Temnocephala pereirai. 23. Juvenile specimen. Scale bar = 500 µm. 24. Brain transverse band (b) and Haswell

glands (*). Scale bar = 50 µm. 25. Male reproductive system: prostatic bulb (pb), cirrus (c), vasa deferentia (vd), and seminal

vesicle (sv). Scale bar = 50 µm. 26-27. Female reproductive system. 26. Anterior portion of the vaginal sphincter (avs), genital

atrium (ga), ovary (ov), posterior portion of the vaginal sphincter (pvs), vagina (va), and vaginal muscular walls (vmw). Scale bar

= 50µm. 27. Anterior portion of the vaginal sphincter (avs), ovary (ov), posterior portion of the vaginal sphincter (pvs), vagina

(va), and vesicula intermedia (vi). Scale bar = 50 µm.

Volonterio (2010) drew and commented in the

species description of T. pereirai about the

“expansion of duct wall connecting the vesicula

resorbens and the ootype”. This expansion, the

vesicula intermedia, is evident, having an

average length of 92 µm, and probably

substitutes the function of the seminal

receptacles (Damborenea, 1994; Amato et al.,

2005). Differentiating the cirrus of T.

brevicornis described as straight, Volonterio

(2010) described the cirrus of T. pereirai as:

“stylet is quite straight, but the distal portion of

the shaft is slightly sinuous”, exactly as the

cirrus of the specimens studied in the present

work (Fig. 18). The number of spines per

longitudinal row in the introvert (16 in

Uruguayan and 21 in Brazilian specimens), the

length of the shaft (average length of 324 µm in

Uruguayan and 322 µm in Brazilian specimens),

and the length of the introvert (average length of

37 µm in Uruguayan and 34 µm in Brazilian

specimens) are very close. The base diameter of

the shaft in our specimens is larger (average 91

µm) than the values presented by Volonterio

(2010) (average 74 µm). It is known that the base

of the shaft gets thicker until it forms a rim in

adult individuals. All cirri measured in the

present work were micro dissected from adult

individuals, with the copulatory organ well

formed. Some young adults in the sample

measured by Volonterio (2010) could explain the

smaller values found in base diameter of the

shaft in Uruguayan specimens. Although the

proportion shaft length/ introvert length (one of

the cirrus characters with less variation) is,

practically equal, being 8.8 µm in Volonterio's

specimens and 8.5 µm in the specimens of the

present work.

The specimens of the present work also have

very similar DLSPs when compared to those

described by Volonterio (2010). The insertion of

the seminal vesicle into the prostatic bulb in the

Uruguayan specimens was described as

subpolar or equatorial, although in T.

brevicornis, the insertion is subpolar or polar. In

the specimens of the present work, the insertion

is always subpolar (Fig. 25).

ACKNOWLEDGEMENTS

Special thanks are due to Jorge Ernesto de

Araújo Mariath and Rinaldo Pires dos Santos,

Laboratório de Anatomia Vegetal (LaVeg),

Instituto de Biociências, UFRGS, for the

permission to use the Leica DMR Hc

microscope to make the DIC photomicrographs.

Special thanks are also due to the staff of the

'Centro de Microscopia Eletrônica', UFRGS for

the SEM operation; to CAPES (Coordenação de

Aperfeiçoamento do Pessoal de Nível Superior);

to 'Instituto Chico Mendes de Conservação da

Biodiversidade' for the permission (Nº 23196-1)

to capture the turtles; to Marco Antonio Afonso

Coimbra for his help in the field;

and, especially, to Philip J. Scholl, for kindly

reviewing the English of the several versions of

the manuscript.

to Marta Fabián

for her critical review of the Spanish summary;

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