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Parasitology Research

, Volume 116, Issue 10, pp 2813–2819 | Cite as

Three-dimensional morphology of rigid structures as a tool for taxonomic studies of Dactylogyridae (Monogenea)

  • María A. RossinEmail author
  • Pablo N. De Francesco
  • Juan T. Timi
Original Paper
  • 254 Downloads

Abstract

Dactylogyridae is overwhelmingly the most abundant and diverse taxon among monogeneans in continental waters of South America. Their small body size requires considerable sampling effort and training for collecting and identifying the worms from the gills, skin, nasal cavities, and other microhabitats. Indeed, diagnostic characteristics as sclerites and male copulatory complex are generally less than 100-μm long and are essential for taxonomic description and identification of species. Here, a combination of simple and routine methods for three-dimensional morphological studies on hard structures is proposed for dactylogirids: SDS treatment for clarification of specimens and enzymatic digestion with proteinase K for freeing sclerotized structures, followed by laser confocal microscopy. This method is applicable to fresh or fixed specimens and does not require staining or dehydration. Indeed, stable autofluorescence emission is detectable at 500–530 nm for bars, anchors, and male copulatory complex when excited by argon laser. Advantages of this protocol over previous methodologies for taking laser confocal images are discussed. Open access software for image processing was used for three-dimensional reconstruction of sclerotized structures generating models and full 360° rotation videos.

Keywords

Monogenea SDS treatment Proteinase K treatment Laser confocal images Autofluorescence Three-dimensional reconstruction 

Notes

Acknowledgements

The authors wish to thank to Dr. Pablo Scarabotti, Instituto Nacional de Limnologia (INALI), and Lic. Manuel Irigoitia, Instituto de Investigaciones Marinas y Costeras (IIMyC), for providing fish samples of Pygocentrus nattereri from Paraná River. We also like to thank Dra. M. Daniela Villamonte and Lic. Viviana Daniel for the support obtaining images from the Nikon C1 SiR confocal microscope and the anonymous reviewer for your suggestions that improved the quality of our work. Finding: this work was supported by the Agencia Nacional de Promoción Científica y Tecnológica (grant number ANPCYT-PICT 2015 N° 2013); CONICET (grant number CONICET-PIP N° 112-20150100973); and Universidad Nacional de Mar del Plata, Argentina (grant number UNMdP-EXA 869/16).

Compliance with ethical standards

The research has been conducted according to Argentine laws. All applicable institutional, national, and international guidelines for the care and use of animals were followed. Permit for fishing was provided by the Ministerio de Asuntos Agrarios de la Provincia de Buenos Aires, Argentina (Disposición 164, August 23, 2012).

Supplementary material

436_2017_5591_Fig4_ESM.gif (263 kb)
Supplementary Fig. 1

LSCFM images. a Rhinoxenus piranhus Kritsky, Boeger and Thatcher, 1988 male copulatory complex (ventral view). b R. piranhus anchor. c Characithecium chascomusensis (Suriano, 1981) male copulatory complex (ventral view). d C. chascomusensis ventral anchor (ventral view). e C. chascomusensis ventral bar (ventral view). (GIF 262 kb)

436_2017_5591_MOESM1_ESM.tif (44.3 mb)
High Resolution Image (TIFF 45313 kb)
436_2017_5591_Fig5_ESM.gif (176 kb)
Supplementary Fig. 2

Maximum intensity Z projection. a Rhinoxenus piranhus Kritsky, Boeger and Thatcher, 1988 male copulatory complex (ventral view). b R. piranhus anchor. c Characithecium chascomusensis (Suriano, 1981) male copulatory complex (ventral view). d C. chascomusensis ventral anchor (ventral view). e Ventral bar (ventral view). (GIF 175 kb)

436_2017_5591_MOESM2_ESM.tif (44.3 mb)
High Resolution Image (TIFF 45313 kb)
436_2017_5591_MOESM3_ESM.avi (4.4 mb)
Supplementary Movie 1 Male copulatory complex of Rhinoxenus piranhus Kritsky, Boeger and Thatcher, 1988. (AVI 4467 kb)
436_2017_5591_MOESM4_ESM.avi (2.3 mb)
Supplementary Movie 2 Ventral anchor of Rhinoxenus piranhus Kritsky, Boeger & Thatcher, 1988. (AVI 2314 kb)
436_2017_5591_MOESM5_ESM.avi (4.1 mb)
Supplementary Movie 3 Male copulatory complex of Characitecium chascomusensis (Suriano, 1981). (AVI 4203 kb)
436_2017_5591_MOESM6_ESM.avi (2 mb)
Supplementary Movie 4 Ventral bar of Characitecium chascomusensis (Suriano, 1981). (AVI 2055 kb)
436_2017_5591_MOESM7_ESM.avi (3.3 mb)
Supplementary Movie 5 Ventral anchor of Characitecium chascomusensis (Suriano, 1981). (AVI 3346 kb)

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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • María A. Rossin
    • 1
    Email author
  • Pablo N. De Francesco
    • 2
  • Juan T. Timi
    • 1
  1. 1.Laboratorio de Ictioparasitología, Instituto de Investigaciones Marinas y Costeras (IIMIyC), Facultad de Ciencias Exactas y NaturalesUniversidad Nacional de Mar del Plata-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Funes 3350Mar del PlataArgentina
  2. 2.Laboratorio de Neurofisiología, Instituto Multidisciplinario de Biología Celular (IMBICE)Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)—Universidad Nacional de La Plata—Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC-PBA)La PlataArgentina

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