Resumen

Esta investigación fue realizada para incrementar el conocimiento actual de los datos morfológicos y morfométricos de

los ganchos rostelares de la forma larvaria (metacestode) de Echinococcus granulosus. Los protoescoÛlices fueron

aislados de hígados y pulmones de ovinos y vacunos infectados naturalmente obtenidos de los mataderos en los

departamentos del Perú (Arequipa, Cuzco, Puno, Huancavelica y Junín). Se utilizó la microscopia de campo claro, la

microscopia confocal, la microscopia electrónica de barrido y contraste de interferencia diferencial. La morfometría se

realizó mediante el análisis de imagen computacional. La aplicación de estos ensayos indican que los ganchos grandes

con frecuencia presentaban finos protectores y de superficie irregular entre el protector y el mango. Los datos también

indicaron que los ganchos pequeños presentaron proctectores redondeados y robustos. La hoja no mostroÛ ningún aspecto

relevante. Se observoÛ que no existe distinción morfológica clara entre los ganchos grandes y pequeños. Fue evidente

diferencias fenotípicas en la forma y el tamaño de los ganchos. En conclusión, la comparación de la morfología de los

ganchos rostelares grandes y pequeños mostró ciertas diferencias. Nuestro estudio demostró la utilidad de la

combinación de herramientas tradicionales y nuevas para los estudios morfológicos y ayudar a resolver las cuestiones

pendientes con respecto a la morfología de los ganchos rostelares.

Palabras claves: Echinococcus granulosus – Técnicas microscopicas – morfología –

morfometría - Perú- ganchos rostellares.

Suggested citation: Almeida, BF., Rodrigues-Silva, R., Neves, H.R., Gonçalves, M.M.L., Romani, E.L.S., Machado - Silva, J.R.

Morphological and morphometric studies on protoscoleces rostellar hooks of Echinococcus granulosus

from Peru visualized by several microscopic techniques Neotropical Helminthology, vol. 3, nº 2, pp. 65-72.

1 1,* 1 1

Fernanda Almeida B , Rosângela Rodrigues-Silva , Renata Neves H , Margareth Gonçalves Ml ,

2 3

Elizabeth Romani L S , José Roberto Machado-Silva

MORPHOLOGICAL AND MORPHOMETRIC STUDIES ON PROTOSCOLECES

ROSTELLAR HOOKS OF ECHINOCOCCUS GRANULOSUS

FROM PERU VISUALIZED BY SEVERAL MICROSCOPIC TECHNIQUES

ESTUDIOS MORFOLÓGICOS Y MORFOMÉTRICOS EN GANCHOS

ROSTELLARES DEL PROTOESCOLICES DE ECHINOCOCCUS GRANULOSUS

DEL PERÚ VISUALIZADA POR VARIAS TÉNICAS MICROSCÓPICAS

Neotrop. Helminthol., 3(2), 2009

2009 Asociación Peruana de Helmintología e Invertebrados Afines (APHIA)

Key words: Echinococcus granulosus – microscopycal techniques – morphology –

morphometry - Peru- rostellar hooks.

Abstract

This study was undertaken to expand the current knowledge of the morphology and morphometry of rostellar hooks of

protoscoleces from the metacestode E. granulosus. Protoscoleces were isolated from livers and lungs of naturally infected

ovines and bovines obtained from abattoirs in Peruvian provinces (Arequipa, Cuzco, Puno, Huancavelica and Junin). Bright-

field microscopy, confocal laser scanning microscopy, differential interference contrast and variable pressure scanning electron

microscopy were used. Morphometry was made using computer image analysis. The application of these assay indicated that

the large hooks frequently had thin guards and an irregular surface between the guard and handle. Data also showed that the

small hooks presented rounded and stout guards. The blade did not show any relevant feature. No clear morphological

distinction was observed between large and small hooks. Phenotypical polymorphism was evident in the shape and size of

hooks. In conclusion, the current data show that large and small rostellar hooks have morphological polymorphism. Because the

application of this knowledge for taxonomic study is limited, for this end morphometry techniques are required. Our study

demonstrated the usefulness of combining conventional and new morphological tools to help to solve unresolved matters with

regards to rostellar hooks features.

Laboratório de Helmintos Parasitos de Vertebrados, Departamento de Helmintologia, Instituto Oswaldo Cruz, Avenida Brasil 4365, Manguinhos, Rio de Janeiro,

21040-900, Rio de Janeiro, Brasil.

Departamento de Parasitologia, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Peru.

Laboratório Romero Lascasas Porto, Departamento de Microbiologia, Imunologia e Parasitologia, Faculdade de Ciências Médicas, Universidade do Estado do Rio de

Janeiro, Rio de Janeiro, Rua Prof. Manoel de Abreu 444/5 andar, Vila Isabel, Rio de Janeiro, 20511-070, Brasil.

ORIGINAL ARTICLES/ ARTICULOS ORIGINALES

INTRODUCTION

Echinococcus granulosus is a common small

tapeworm living in the small intestine of

domesticated and wild carnivores (Ingold et al.,

2001). The larval stage (metacestode) causes cystic

echinococcosis (CE) that remains one the most

health problem worldwide, especially in the sheep-

rearing rural areas (Eckert & Deplazes, 2004; Moro

et al., 2004; Ahmadi & Dalimi, 2006). The

metacestode are found in liver and lungs of sheep,

cattle, camels, goats, pigs, buffalo and horses

(Thompson & McManus, 2002).

The rostellar hooks are distributed in 2 rows, each

have a blade, guard and handle region (Rogan &

Richards, 1987). Scanning electron studies revealed

that hooks of upper row (large hooks) are longer,

more pointed, slightly curved and less robust

compared with hooks of the lower row (small

hooks). In addition, large hooks had a projecting part

with a smooth surface, whereas small hooks had a

serrated edge (Antoniou & Tselentis, 1993).

Based on the metacestode rostellar hooks

morphology, phenotypic variations have largely

been observed in E. granulosus from different

species of intermediate hosts (Ponce-Gordo &

Cuesta-Bandera, 1997; Tashani et al., 2002;

Turčeková et al., 2003; Ahmadi, 2004; Almeida et

al., 2007). Evidences also showed that

protoscoleces differed markedly in both the number

and size of hooks, which are host-induced (Hobbs et

al., 1990).

There is often a need to collect images for recording

metacestode rostellar hooks for both taxonomic and

morphological studies in E. granulosus. Usually,

unstained mounts of hydatid fluid sediment are

examined. Diverse techniques as Ryan and modified

Baxby stains were recommended for visualization

under transmitted-light microscopy, whereas Ziehl-

Neelsen stain under green excitation light was very

useful for fluorescence microscopy (Clavel et al.,

1999). However, the potential value of new

techniques does not seem to have been deeply

realized for E. granulosus studies. Because no

specific sample preparation protocols are required,

variable pressure scanning electron microscopy

now is widely used for morphological studies

(Muscariello et al., 2005). In this paper, new data on

the morphology and morphometry of rostellar hooks

are presented on the basis of bright-field

microscopy, confocal laser scanning microscopy,

Normaski differential interference contrast light

microscopy and variable pressure scanning electron

microscopy.

The E. granulosus protoscoleces used in this study

were obtained from livers and lungs cysts of ovines

and bovines origin from abattoirs in Peruvian

o o

provinces Arequipa (16 20' S and 71 30' W), Cuzco

o o o o

(13 32' S and 72 00' W), Puno (15 55' S and 70 03'

o o

W), Huancavelica (12 50' S and 75 05' W) and

o o

Junin (11 24' S and 76 00'W) (Almeida et al.,

2007).

Protoscoleces rostellar hooks were aspirated under

sterile conditions together with the hydatid fluid

from each cyst and rinsed twice in a 0.85% NaCl

solution. Each suspension of protoscoleces was

sieved to remove the larger debris (Ahmadi, 2004).

Then, the material was transferred and stored in

small tubes with 10% buffered formalin at room

temperature.

Protoscoleces for bright-field microscopy (BM)

were squashed under coverslip in polyvinyl

lactophenol on microscope glass slides. Acquired

images were obtained using Image Pro-Plus

software (Media Cybernetics, Inc., Silver Spring,

MD, USA). For confocal laser scanning microscopy

(CLSM) whole-mounts were examined under a

confocal microscopy (LSM 510 – ZETA, Zeiss)

(Neves et al., 2004). All of these slides were also

examined under phase contrast by adding a prism

(Zoom 3.8) to the scanning confocal microscope.

Nomarski differential interference contrast light

microscope images of rostellar hooks were obtained

from a Zeiss bright-field microscope in a differential

interference contrast (DIC) apparatus.

For variable pressure scanning electron microscopy

(VPSEM), hooks were rinsed in Milli-Q water

followed by repeated centrifugation at 1000g for 2

min Samples were placed on glass slides, and

inspected under a LEO 435 VP SEM operating at

15000 V and 150 Pa. Several linear measurements

and area of both large and small hook were

analyzed: total area (TA), total perimeter (TP), total

length (TL), total width (TW) and distance between

blade and guard (BGD).

For the statistical analysis, all procedures were

MATERIAL AND METHODS

Almeida et al.Rostellar hooks from Echinococcus

carried out using Statistical Package for Social

Sciences (SPSS version 9.0, Chicago). Statistical

analysis included Analysis of variance (ANOVA)

followed by the post-hoc Tukey-test. The statistical

significance was assessed at p<0.05.

All microscopic techniques utilized evidenced

morphologic variations in both large and small

rostellar hooks, even though all of them presented

handle, blade and guard (Fig. 1a). Large and small

hooks presented a central amorphous pulp region

(Fig. 1b). Large hooks (Figs. 1b-d) frequently had

thin guards and an irregular surface between the

guard and handle (Fig. 1c). There are some hooks

with stout guards and smooth surface between the

guard and handle (Figs. 1b-d). Small hooks (Figs.

1e-f) presented rounded and stout guards (Figs. 1e-

f). Usually, the surface between the guard and

handle was irregular (Fig. 1a). The blade did not

show any relevant feature.

We obtained good results with differential

interference contrast microscopy (DIC). Some

protoscoleces settled in the cyst fluid (hydatid sand)

were joined by a slender stalk (Fig. 2a). They

possess a variable number of rostellar hooks

arranged in two rows (Fig. 2 b), alternating between

large (upper row) and small hooks (lower row).

It was possible to remove a small number of hooks

from the rostellar pad and analyze the marks they

left on the rostellum. Variable pressure scanning

electron microscopy (VPSEM) imaging revealed

that the marks left by the missing upper row hooks

were larger than lower row ones (Fig. 2c). The larger

hooks are located in the upper row (Fig. 2d).

Confocal laser scanning microscopy (CLSM)

images confirmed that large hooks had thin guards

with an irregular surface between the guard and

handle (Fig. 3a). Hooks with stout guards and a

smooth surface between the guard and handle was

found (Fig. 3b). Small hooks also presented rounded

and stout guards (Fig. 3c), and usually the surface

between the guard and handle was irregular (Fig.

3d).

The morphometric approach allowed discriminating

differences between large and small hooks. The

hooks of the upper row were found to be

Neotrop. Helminthol., 3(2), 2009

RESULTS

DISCUSSION

significantly longer than those of the lower row. The

average measurements of large hooks were 101µ m

(TA), 61µm (TP), 25µm (TL), 9µm (TW) and 10µm

(BGD). Regarding the small hooks, the average

measurements of were 79µm (TA), 52µm (TP),

22µm (TL), 8µm (TW) and 10µm (BGD).

Many issues concerning the taxonomy of E.

granulosus have been resolved on the basis of

morphological and morphometric techniques. For

these purposes, there is often a need to collect

images for recording metacestode rostellar hooks.

To date, only conventional morphological

techniques as bright-field microscopy, scanning

electron microscopy and transmission electron

microscopy were used (Kumaratilake et al., 1986;

Smith & Richards, 1991; Antoniou & Tselentis,

1993; Dubinský et al., 1998; Xiao et al., 2005). The

potential value of new techniques does not seem to

have been deeply realized for E. granulosus studies.

Usually, for bright-field analysis unstained mounts

of hydatid fluid sediment are examined due to

difficulty of staining rostellar hooks (Clavel et al.,

1999). In this work, the features and distribution of

unstained rostellar hooks from Peruvian samples

under several morphological techniques were

examined. This study and others (Said et al., 1988;

Hobbs et al., 1990; Ponce-Gordo & Cuesta-

Bandera, 1997; Dubinský et al., 1998; Turcekov? et

al., 2003; Ahmadi, 2004; Thompson et al., 2006)

evidenced a high degree of polymorphism within

hooks. We observed that large hooks had a slender

guard, whereas the guard of the small hooks was

more robust and rounded. The region between the

guard and the handle was serrated. In contrast,

scanning electron studies revealed that large hooks

were longer, more pointed, slightly curved and less

robust in comparison with small hooks. The large

hooks had a projecting region with a smooth surface,

whereas small hooks had a serrated edge (Antoniou

& Tselentis, 1993).

During protoscolex development this parasitic form

remains attached to the germinative layer through a

stalk. When fully differentiated, the stalk is cut off

and the infective protoscolex is now free in the

hydatid fluid (Galindo et al., 2002). On observing

brood capsules under bright-field microscopy, our

data confirm that developing stages already harbor

rostellar hooks, which are the first fully,

differentiated structures formed at the apical region

of the nascent scolex (Galindo et al., 2002).

The major question arising from these findings is

that large and small hooks are not accurately

discriminated on the basis of morphological tool, as

recently observed for Echinococcus shiquicus (Xiao

et al., 2005). From a taxonomic viewpoint, the

larval-hook morphometry is a valid method for

identifying E. granulosus (Ahmadi, 2004). The

results obtained here indicate that large and small

hooks can be reliably distinguished on the basis of

morphometric differences between them. This result

agrees with previous studies (Ponce-Gordo &

Cuesta-Bandera, 1997; Ahmadi, 2004; Almeida et

al., 2007).

An important finding in relation to previous studies

(Antoniou & Tselentis, 1993; Xiao et al., 2005)

refers to the two rows of rostellar hooks: large

(upper row) and small hooks (lower row). Because

no specific sample preparation protocols are

required, variable pressure scanning electron

microscopy (VPSEM) now is widely used for

morphological studies (Griffin, 2007). This

technique was chosen because does not requires

some proceeding that makes possible damages to

the preparation. This approach combines the

advantages of light microscopy and resolution of

electron microscopy (Muscariello et al., 2005).

Variable pressure scanning electron microscopy

imaging revealed that the marks left by the missing

upper row hooks were larger than lower row ones. In

addition, larger hooks are located in the upper row.

Records exist indicating that these differences in the

morphology of the two types of hooks are related to

the differences in their function (Antoniou &

Tselentis, 1993).

Confocal imaging has been demonstrated both

theoretically and practically to give improved

visibility of optical sections and improved

resolution. Since the images produced by the

confocal system are held in a digital frame store they

are readily available for computer analysis (White et

al., 1987). Another advantage is that a same whole-

unt can be observed either by BM or CLSM. Our

previous studies evidenced that CLSM allows the

study of gross anatomy in chloride carmine-stained

helminthes (Machado-Silva et al., 1998; Neves et

al., 2004). However, in this study the rostellar hooks

failed to stain with chloride carmine (data not

shown). In this respect, CLSM images of unstained

hooks were obtained with a prism coupled for the

phase contrast technique. Apart from their low

degree of fluorescence, the protocol followed in this

study allowed us to support the notion that rostellar

hooks show morphological polymorphism, as above

described for bright-field analysis. Consequently, it

is still difficult to accurately discriminate between

these two rostellar hooks on because morphological

variations were evidenced. Finally, future studies

based on stained rostellar hooks are needed (Clavel

et al., 1999).

In conclusion, the current data show that large and

small rostellar hooks have morphological

polymorphism. Because the application of this

knowledge for taxonomic study is limited, for this

end morphometry techniques are required. Our

study demonstrated the usefulness of combining

conventional and new morphological tools to help to

solve unresolved matters with regards to rostellar

hooks features.

This work was supported by a fellowship from

Conselho Nacional de Desenvolvimento Cient?fico

e Tecnológico (JRMS). We thank Dr. Roberto

Magalhães Pinto (Laboratório de Helmintos

Parasitos de Vertebrados, Instituto Oswaldo Cruz),

Mr. Pedro Paulo de Abreu Manso (Departamento de

Patologia, Instituto Oswaldo Cruz), Mr. Jonas Brito

(Universidade do Estado do Rio de Janeiro), for his

assistance with the VPSEM, and Mr. Bruno

Eschinazi Vieira (Laboratório de Produção e

Processamento de Imagem Científica, IOC) for

technical assistance with the figures.

ACKNOWLEDGEMENTS

Almeida et al.Rostellar hooks from Echinococcus

Neotrop. Helminthol., 3(2), 2009

Figure 1. Bright-field micrographs of

Echinococcus granulosus large and small

rostellar hooks. a Large (left) and small (right)

rostellar hooks showing the guard (arrows) and

the surface between the guard and handle

(?*, ), b large hooks showing the central

amorphous pulp (arrow), c - f rostellar hooks

showing the guard (arrows) and the surface

between the guard and handle (*?): b, blade; g,

guard; h, handle. Scale bar, 9µm.

Figure 3. Confocal laser scanning images of

Echinococcus granulosus showing rostellar hooks. a - b

Large rostellar hooks showing the guard (arrows), c - d

Small rostellar hooks showing the guard (arrows) and

the surface between the guard and handle (?*). Scale bar,

7µm.

Figure 2. Photomicrographies of

Echinococcus granulosus metacestodes. a

Bright-field images in a differential

interference contrast of brood capsules

containing protoscoleces joined by a slender

stalk (arrow), bar 26µm; b Bright-field images

in a differential interference contrast of large

(à) and small (5) rostellar hooks, bar 26µm; c

Variable pressure scanning electron

microscopy micrographs showing marks left

by the missing large (à) and small hooks (5),

bar 5µm; d Variable pressure scanning electron

microscopy micrographs showing rostellar

hooks showing large hooks in the upper row

(à) and small hooks in lower row (5), bar

5µm.

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