ORIGINAL ARTICLE /ARTÍCULO ORIGINAL

A NEW SPECIES OF TEMNOCEPHALA BLANCHARD (PLATYHELMINTHES,

TEMNOCEPHALIDA) ECTOSYMBIONT OF KEMPNYIA RETICULATA (KLAPÁLEK)

(INSECTA, PLECOPTERA) IMMATURES FROM BRAZIL

UMA NOVA ESPÉCIE DE TEMNOCEPHALA (PLATYHELMINTHES,

TEMNOCEPHALIDA) ECTOSIMBIONTE SOBRE IMATUROS DE KEMPNYIA

RETICULATA (KLAPÁLEK) (INSECTA, PLECOPTERA) DO BRASIL

UNA NUEVA ESPECIE DE TEMNOCEPHALA (PLATYHELMINTHES,

TEMNOCEPHALIDA) ECTOSIMBIONTE SOBRE INMATUDOS DE KEMPNYIA

RETICULATA (KLAPÁLEK) (INSECTA, PLECOPTERA) DE BRASIL

1 2 3

Samantha A. Seixas ; José F. R. Amato & Suzana B. Amato

1Setor de Evolução e Ecologia, Programa de Pós-Graduação em Ambiente e Desenvolvimento, Centro Universitário

UNIVATES, Rua Avelino Tallini 171, Prédio 8, salas:102/104, Bairro Universitário, CEP 95900-000, Lajeado, RS, Brasil.

PNPD/CAPES Post-Doctoral Fellow. E-mail: seixas.sa@gmail.com

2Rua Cabral, 875/301, Bairro Rio Branco, CEP 90420-121, Porto Alegre, RS, Brasil. E-mail: josefelipeamato@gmail.com

3Laboratório de Helmintologia, Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio Grande do

Sul, Av. Bento Gonçalves 9500, Prédio 43435, sala 208, Bairro Agronomia, CEP 91501-970, Porto Alegre, RS, Brasil. E-

mail: sbamato@ufrgs.br

Neotropical Helminthology, 2015, 9(2), jul-dec: 277-284.

ABSTRACT

Keywords: aquatic insects - Neotropic Region - plecopterans - taxonomy - Temnocephalidae.

Temnocephalans were found on immature Kempnyia reticulata (Klapálek) collected in a first

order section of Córrego Bonito (19°58'28,4”S, 40°31'54,4”W), in the Estação Biológica de

Santa Lúcia (EBSL), Santa Teresa, State of Espírito Santo, Brazil. Of 29 immature specimens

examined, 5 (17.24%) were positive for specimens of Temnocephala Blanchard, 1849. Juveniles

and adult parasites were found on the head, mesonotum and, median legs of the hosts. Eggs were

found in larger numbers in the intrathoracic gills. The most distinctive characters of the

Temnocephala stoneflyi n. sp. were in the cirrus and the dorsolateral 'excretory' syncytial

epidermal plates (EPs). The comparison of the general anatomy and, in particular, the

morphology of the cirrus and EPs with those of Temnocephala curvicirri Amato & Amato, 2005

described from aquatic heteropterans and Temnocephala caddisflyi Amato, Amato & Seixas,

2011 described from trichopterans, showed that although these characters are similar, they are not

equal, differing primarily in size and morphology of the cirrus, that has an intermediary size

between the three species and smaller EPs, which also have a different format. The egg deposition

sites are different and this is the third species with the reproductive system characterized as being

'complex'.

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ISSN Versión impresa 2218-6425 ISSN Versión Electrónica 1995-1043

INTRODUCTION

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Neotropical Helminthology. Vol. 9, Nº2, jul-dec 2015

Temnocephala decarloi Moretto, 1978 was the

first species described as epibiont on insects. It

was found on Belostoma cummingsi De Carlo,

1935 collected in Otamendi, Delta del Paraná,

Província de Buenos Aires, Argentina

(Moretto, 1978). The species was recorded

again in Argentina by Damborenea & Cannon

(2001). Vianna & De Melo (2002) recorded

temnocephalans on belostomatids and

naucorids in the State of Minas Gerais, Brazil.

Three species have been described as epibionts

on insects in Brazil. Amato & Amato (2005)

described Temnocephala curvicirri Amato &

Amato, 2005 on two species of Belostoma

Latreille, 1807; and Amato et al. (2007)

described Temnocephala minutocirrus Amato,

Seixas & Amato, 2007 on Cryphocricos

granulosus De Carlo, 1967, a naucorid

hemipteran. Both records were made in the

State of Rio Grande do Sul, Brazil.

Temnocephala caddisflyi Amato, Amato &

Seixas, 2011 was described as epibiont on

'caddisfly' larvae (Trichoptera) in the State of

Minas Gerais, Brazil. Amato et al. (2011) made

an extensive study of the peculiar reproductive

system of the species T. curvicirri and T.

caddisflyi, which, most probably, is shared by

T. decarloi.

Species of Kempnyia Klapálek, 1914 are

endemic to the southern half and central

regions of Brazil. The immatures are usually

found in the litter retained by currents,

although they can also be found in puddles

with low current and accumulated litter, which

are formed during periods of drought (Bispo &

Froehlich, 2004). Avelino-Capistrano et al.

(2 01 3 ) r e co r de d t h e p r es e n c e o f

temnocephalans epibiont on immatures of

Kempnyia reticulata (Klapálek, 1916)

collected in the State of Espírito Santo, without

describing the species.

The present study describes a new species of

Temnocephala epibiont on K. reticulata found

in the 'Reserva Biológica de Santa Lúcia',

Santa Teresa, State of Espírito Santo, Brazil.

RESUMO

Palavras-chave: insetos aquáticos - plecópteros - Região Neotropical - taxonomia - Temnocephalidae.

Temnocefalídeos foram encontrados sobre imaturos de Kempnyia reticulata (Klapálek, 1916)

coletados no Córrego Bonito, em uma seção de primeira ordem (19°58'28,4”S, 40°31'54,4”W),

na Estação Biológica de Santa Lúcia (EBSL), Santa Teresa, Espírito Santo, Brasil. Vinte e nove

imaturos foram examinados, cinco (17.24%) estavam positivos para espécimes de

Temnocephala Blanchard, 1849. Juvenis e adultos foram encontrados sobre a cabeça, o mesonoto

e as pernas médias de seus hospedeiros. Ovos foram encontrados em grande número sobre as

brânquias intratorácicas. Os caracteres mais distintivos de Temnocephala stoneflyi n. sp foram o

cirro e o par de sincícios pós-tentaculares, ou placas excretoras (PEs). A comparação da anatomia

geral e, em particular, da morfologia do cirro e das PEs com Temnocephala curvicirri Amato &

Amato, 2005 epibionte sobre heterópteros aquáticos e Temnocephala caddisflyi Amato, Amato

& Seixas, 2011 epibionte sobre tricópteros, mostrou que, embora sejam de mesmo tipo e

natureza, estes caracteres não são iguais, diferindo principalmente em tamanho e morfologia do

cirro, que possui um tamanho intermediário entre as três espécies, e PEs menores e de formato

diferente. Os locais de postura dos ovos diferem e esta é a terceira espécie com o sistema

reprodutor caracterizado como 'complexo'.

Seixas et al.

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Neotropical Helminthology. Vol. 9, Nº2, jul-dec 2015

as slides containing individual cirri mounted in

Faure´s mounting medium, were deposited in

the following scientific collections: 1.

'Coleção Helmintológica do Instituto Oswaldo

Cruz (CHIOC)', Rio de Janeiro, RJ, Brazil; and

2. 'Coleção Helmintológica do Laboratório de

Helmintologia', Departamento de Zoologia,

Universidade Federal do Rio Grande do Sul

(UFRGS), Porto Alegre, RS. The remainder

host specimens were deposited in the 'Coleção

Helmintológica do Laboratório de

Helmintologia', UFRGS, Porto Alegre, RS.

Temnocephala stoneflyi n. sp.

(Figs 3 - 11)

Description. Based on 4 specimens collected: 2

whole mounted adults; 1 specimen mounted on

a stub for SEM; 2 dissected cirri mounted in

(F).

External characteristics. Red pigmentation of

the eyes absent. Two small EPs, rounded

slightly r than wide (Fig. 6), right plate longe

166.34 (n = 1) long, 122 (n = 1) wide;

Excretory pore in the anterior portion of the

plate (Fig. 6 - ep).

Female reproductive system not observed.

Male reproductive system. Cirrus classified as

'complex', composed of long shaft and long

introvert, both curved (lateral view), directed

ventrally, 697.5 (n = 1) long, shaft curved

(approximately 169º) 340 (n = 1) long, shaft

maximum width at base 30 (n = 1) (Fig. 18);

introvert curved (approximately 133º) at mid-

region, introvert total length (ventral) 157.5 (n

= 2), introvert total length (dorsal) 200-357.5

(279, n = 2, 111), 130 (n = 2), ventral portion

without spines, 155-160 (157.5, n = 2, 3) long,

introvert´s swelling portion length (ventral

side) 27.5 (n = 2), introvert´s swelling portion

length (dorsal side) 40-47.5 (44, n = 2, 5). Ratio

MATERIAL AND METHODS

Twenty-nine immature K. reticulata were

collected from July 2008 to February 2009 in a

first order section of Córrego Bonito

(19°58'28,4”S, 40°31'54,4”W), in the 'Estação

Biológica de Santa Lúcia (EBSL)', Santa

Teresa, State of Espírito Santo, Brazil. The

insects were collected by Avelino-Capistrano,

examined for temnocephalans and deposited in

the Coleção do Departamento de Entomologia,

Museu Nacional, Universidade Federal do Rio

d e J a n e i r o ( M N R J ) . H o s t s w i t h

temnocephalans were sent to the Laboratório

de Helmintologia, Universidade Federal do

Rio Grande do Sul. The temnocephalans from

plecopterans were studied through a series of

techniques fully described by Amato et al.

(2007) and Seixas et al. (2010), focusing

especially on the: 1. morphology of the cirrus

structure; 2. shape of individual eggs as well as

the areas of egg deposition on the host; and, 3.

paired, dorsolateral, post-tentacular 'excretory'

syncytial plates (EPs). Photomicrographs were

taken with a Zeiss Axiolab microscope, a Leica

DMR Hc Microscope, and Nomarski

interference contrast prisms. The photographic

images and line drawings were scanned and

prepared using Adobe's Photoshop CC and

CorelDraw X5 , respectively. The image of the

cirrus with total focus was obtained with

Hel i c o n Focu s 5.3 . 7 . 2 sof t w a re.

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 2).

Measurements of cirrus were taken from

extracted cirri mounted in Faure´s mounting

medium (F), and follow Amato et al. (2011).

Measurements of the introvert and shaft

curvature angles were made with Zeiss

Axiovision 4.8 software. Specimens, as well

A new species of Temnocephala from Brazil

RESULTS

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Neotropical Helminthology. Vol. 9, Nº2, jul-dec 2015 Seixas et al.

Figures 1-2. Immature of Kempnyia reticulata. (1) Specimen infested with temnocephalids, dorsal view. Bar = 5 mm. (2) Detail of

the temnocephalid (arrow) and of the eggs (arrow head) fixed to the host. Bar = 2 mm. Figures 3-5. Temnocephala stonefly n. sp. i

(3) Pedunculated eggs. Bar = 250 μm. (4) Detail of a pedunculated egg and filament (arrow). Bar = 250 μm. (5) Adult specimen

mounted in toto, stained with acetic carmine/fast green. Bar = 250 μm.

Site of infestation: Juveniles and adults of T.

stoneflyi n. sp. were living on the head,

mesonotum and, median legs of the

immatures.

Prevalence: 17.2%.

Helminth specimens deposited: 'Coleção

Helmintológica do Instituto Oswaldo Cruz':

CHIOC 38203 (SBA 3113-1-1holotype);

CHIOC 38204 (SBA3112-1-1- cirrus).

between total length of cirrus/ maximum width

of shaft´s base 23: 1; ratio between total length

of cirrus/ total length of introvert 2.5: 1.

Taxonomic summary.

Type host: Immature Kempnyia reticulata

(Klapálek, 1916) (Insecta, Plecoptera,

Perlidae).

Type localit y: Córre go da Div isa

(19°58'28,4”S, 40°31'54,4”W), 'Reserva

Biológica de Santa Lúcia', Santa Teresa, State

of Espírito Santo, Brazil.

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Neotropical Helminthology. Vol. 9, Nº2, jul-dec 2015 A new species of Temnocephala from Brazil

Figure 6. Scanning Electron Microscopy (SEM) of the right post-tentacular 'excretory' syncytial plate of Temnocephala stonefly i

n. sp. showing the excretory pore (ep). Bar = 20 µm.

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Figures 7-11. Cirrus of Temnocephala stonefly n. sp. seen with DIC. (7) Cirrus 'complex' showing the retractor muscle which i

projects toward the front of the cirrus (arrow). Bar = 50 µm. (8) Limit of introvert-shaft (arrow head), portion of the retractor

muscle which projects toward the front of the cirrus (asterisk) and the introvert inflation. Bar = 50 µm. (9-10) Sequence of

different focus views of the distal portion of the cirrus, showing the introvert inflation and the small spines; long spines which are

in the internal wall of the structure. Bar = 20 μm. (11) Image created by the Helicon Focus software 5.3.7.2 made from the

sequence of picture stacking of the distal portion of the cirrus. Bar = 20 μm.

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133º – T. stonefly n. sp.). The epibionts of the i

K. reticulata immatures have the largest

introvert curvature among the three species.

The shaft is smaller in the specimens of T.

stonefly n. sp. and the relationship between the i

lengths of the introvert/length of shaft (2.5:1)

is smaller than that found in T. caddisflyi

(2.8:1) and in T. curvicirri (4.5:1).

Temnocephala curvicirri and T. caddisflyi

have EPs with a similar shape, but different

size, which go beyond the equatorial line of the

body in T. caddisflyi (Amato & Amato 2005;

Amato et al. 2011). Comparing the EPs of the

two species with those of T. stonefly n. sp., the i

new species has the excretory plates of

different shape and size (Fig. 6), they are

rounded while the excretory pore is in the

anterior half of the plate. The excretory plates

are smaller in length (166 µm) than those of T.

caddisflyi (368 µm in average) and T.

curvicirri (492 µm in average).

Just a few specimens of T. stoneflyi n. sp. were

sent to the 'Laboratório de Helmintologia,

UFRGS' and the majority were not correctly

fixed. Given the small number of samples, the

female reproductive system was not described.

To Jorge Ernesto de Araújo Mariath and

Rinaldo Pires dos Santos, Laboratório de

Anatomia Vegetal, Instituto de Biociências,

UFRGS, for the permission to use the Leica

DMR HC microscope to make the DIC

photomicrographs; to Fernanda Avelino-

Capistrano at the 'Departamento de

Entomol o g i a , M useu Nacion a l d a

Universidade Federal do Rio de Janeiro' for the

collection of the insect hosts; to Adriano

Cavalleri for the help with the Helicon Focus

5.3.7.2 software; to the staff of the 'Centro de

Microscopia Eletrônica', UFRGS for the SEM

operation; to CAPES (Coordenação de

Aperfeiçoamento do Pessoal de Nível

DISCUSSION

As observed in T. caddisflyi and T. curvicirri,

the species epibiont on immatures of K.

reticulata has a cirrus classified as 'complex',

and, most probably, it must also have a

'complex' vagina. The feminine reproductive

system of T. stonefly n. sp. could not be clearly i

seen, although the 'complex' vaginas, as

described for the epibionts found on

belostomatids and trichopterans are quite

similar (Amato & Amato, 2005; Amato et al.,

2011). When the 'complex' cirrus of the two

species of epibionts found on insects are

compared with that of T. stonefly n. sp., we i

observed that the new species has the total size

of the cirrus of an intermediary size among the

three (1003 µm in average - T. curvicirri; 560

µm in average - T. caddisflyi, and 697 µm in

average - T. stonefly n. sp.) and the largest total i

length of the introvert (155 µm in average - T.

curvicirri; 140 µm in average - T. caddisflyi

and 157 µm in average - T. stonefly n. sp.).i

Amato et al. (2011) mentioned this species (as

unpublished data) when they compared the

shape of the inflation of the introvert among

the species with cirrus classified as 'complex'.

Temnocephala caddisflyi and T. curvicirri

have approximately the same number of spine

rows and the number of spines per row, as well

as a similar shape (low cylinder in T. caddisflyi

and tall cylinder in T. curvicirri). The inflation

of the introvert of T. stonefly n. sp. has size and i

number of spine rows similar to those two

species mentioned above, although the shape is

globular (Figs 9-11). When curvature angles of

the shaft are compared (158º – T. caddisflyi;

129º – T. curvicirri, and 169º – T. stonefly n. i

sp.), it is possible to see that the epibionts

found on plecopterans have the shaft with the

smallest curvature (almost straight), while T.

curvicirri shows the largest curvature among

the three species. The inverse occurs when the

curvature angle of the introvert is analyzed

(135º – T. caddisflyi; 145º – T. curvicirri, and

ACKNOWLEDGMENTS

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Neotropical Helminthology. Vol. 9, Nº2, jul-dec 2015 Seixas et al.

Temnocephala (Platyhelminthes:

Temnocephalida) in immatures of

Kempnyia reticulata (Enderlein)

(Plecoptera: Perlidae). EntomoBrasilis,

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Bispo, PC & Froehlich, CG. 2004. Perlidae

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São Paulo State, Southeastern Brazil.

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34, pp. 95-99.

Seixas, SA, Amato, JFR & Amato, SB. 2010.

Redescription of Temnocephala iheringi

(Platyhelminthes: Temnocephalida)

based on specimens from Pomacea

canaliculata (Mollusca: Ampullariidae)

of the State of Rio Grande do Sul: the

possible type host and type locality.

Zoologia, vol. 27, pp. 245-257.

Vianna, GJC & De Melo, AL. 2002. Aquatic

heteroptera as host of Temnocephala

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Superior) for the Doctoral Scholarship

awarded to SAS (2008-2013); to Débora N.

Souza and Lucas Casagrande for their help in

the laboratory; and, especially, to Philip J.

Scholl, for kindly reviewing the English in the

several versions of the manuscript.

Amato, JFR & Amato, SB. 2005. New species

o f Te m n o c e p h a l a B l a n c h a r d

(Platyhelminthes, Temnocephalida)

ectosymbiont on giant water bugs,

B e l o s t o m a s p p . ( H e m i p t e r a ,

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pp.107-118.

Amato, JFR, Seixas, SA & Amato, SB. 2007. A

new species of Temnocephala Blanchard

(Platyhelminthes, Temnocephalida)

ectosymbiont on creeping water bugs,

Cryphocricos granulosus De Carlo

(Hemiptera, Naucoridae) from southern

Brazil. Revista Brasileira de Zoologia,

vol. 24, pp. 1043–1051.

Amato, JFR, Amato, SB, Seixas, SA, Vidigal,

THDA & Andrade, CP. 2011.

Trichoptera — the newest insect order

h o s t o f t e m n o c e p h a l a n s

(Platyhelminthes, Temnocephalida) and

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Received June 23, 2015.

Accepted September 25, 2015.