The fluorescent detection of Paradiplozoon sp . ( Monogenea : Diplozoidae ) attachment clamps ’ sclerites and integumental proteins

MILNE, S.J. & AVENANT-OLDEWAGE, A. 2006. The fluorescent detection of Paradiplozoon sp. (Monogenea: Diplozoidae) attachment clamps’ sclerites and integumental proteins. Onderstepoort Jour nal of Veterinary Research, 73:149–152 In the genus Paradiplozoon, few hard structures are present therefore most of the taxonomic studies have focused on the unique ultrastructure of the sclerites. Alcohol-fixed specimens were transferred to BSA-saline for 5 min. before staining indefinitely with 5 μl WGA-TRX, 5 μl SYTO 9 and 5 μl of Cal co fluor White M2R. Rhodamine, Fluorescein and DAPI bandpass filters on the microscope enabled selective light wavelength illumination of the three flourochromes by a mercury light source. This method provided an easy and rapid methodology to show the internal sclerites of attachment clamps. It is suitable for alcohol preserved specimens and may have additional applications in other helminth organisms.

Paradiplozoon (Monogenea: Diplozoidae) is an ectoparasitic helminth of yellowfish, Labeobarbus aeneus within the Vaal Dam that is situated in the Gauteng province of South Africa (26°52' S; 28°10' E).The monogenean gill parasite occurs as two hermaphroditic individuals fused in permanent cross copula.Le Brun, Renaud & Lambert (1988) pointed out that there is a lack of valid morpho-anatomic criteria for determining species of the closely related genus Diplo zoon as they lack sclerified genitalia which are less subjective to adaptive pressures than the haptor.They also found that four species of Diplozoon in Southern France could be separated by differing allo zyme electrophoretic mobilities.
Most of the taxonomic studies on the Diplozoidae have focused on the unique ultrastructure of the sclerites situated in the attachment clamps that are positioned in two rows of four each on opposing sides of the organism's opisthaptor.The usage of standard techniques, such as trichrome stains and scanning electron microscopy (SEM) have limitations when observing the sclerites.In scanning electron micrographs the bivalve-like structure of the attachment clamps is clearly visible.However, due to the apical lamina of the tegument covering the clamps, there is limited visibility of the chitinous scle rites.Therefore, fluorochromy was employed to improve the vis- Direct fluorochromy was also conducted on the organ ism as a nuclear counter stain to ascertain the general morphology of the organism.
Yellowfishes were collected from the Vaal Dam using 90 mm mesh size gill nets.The fish were killed by severing the spinal cord.The gills were subsequently removed, placed in dam water and scrutinized utilizing a dissection microscope.The 27 parasites collected were individually removed from the gill filaments and transferred to a drop of dam water on a glass slide.A cover slip, separated from the slide by petroleum jelly, was then placed onto the first to ensure that each parasite was fixed in a flat position.The two cohesive slides impounding the parasite were transferred to Petridishes containing 70 % ethanol for 10 min, thereby killing the organism.Thereafter each parasite was preserved in 70 % ethanol.
The "Cell Culture Determination Kit" protocol (Molecular Probes, Eugene, Oregon.) was followed as the fluorochrome kit was designed to test for gram positive bacteria and the technique was applied for helminths for the first time.Specimens stained with Horen's trichrome (Fig. 1A) show that the general morphology of the parasite is highlighted but that the visibility of the sclerites in the clamps is limited.It is evident that each clamp consists of a pair of hinged jaws supported by median and lateral sclerites (Ramsamy, Brennan & Halton 1995).At a high magnification the hamuli, which are residual larval hooks, can be observed (Fig. 1B).
The Paradiplozoon specimens were observed to autofluoresce when excited with UV light (365 nm) produced by an Hg lamp source and DAPI bandpass filter.The chemical composition of monogenean attachment clamps is ambiguous and it is unknown whether they are made up of keratin, chitin, quinine-tanned protein or collagen (Roberts & Janovy 2005).However, a strong blue autofluorescence was observed under UV irradiation in hookworm hook lets, which are known to be made of chitin (Rost 1995) indicating that the sclerites in Paradiplozoon probably also contain chitin; which is in contrast to the finding of Lyons (1966), who stated that the sclerites of monogeneans are composed of scleroproteins and not the glycoprotein; chitin.Comparing the sclerites of the clamps as shown in Fig. 1E, 1F and 1G it is observed that much of the detail of the clamp morphology occurs on the posterior side of the clamp.In Fig. 1E and 1F the chitinised components show up in three dimensions, highlighting the Median sclerite of the posterior jaw 6 Lateral sclerite of the posterior jaw 7 Spiky spur 8 anterior jaw BJ Ball joint CT Chitinous thickening SP Serrated median plate hinges and joints in the sclerites which are not visible in trichrome staining (Fig. 1A and 1 B).The chitinised thickening, the ball joint of the lateral sclerite attached to the anterior jaw and the three chitinised joints in the anterioir part of the median plate becomes visible with this technique (Fig. 1F and 1G).
The direct localization of the nucleic acids (antigen) of the Paradiplozoon was done utilizing the fluorescent SYTO 9 green nucleic acid stain (antibody).The SYTO 9 fluorochrome penetrates the compromised plasma membranes of eukaryotic cells labelling the DNA and RNA with a ~1 000-fold fluorescence enhancement upon nucleic acid binding (Haug land 2002).This is observed in Fig. 1D-F.It provides a counterstain to the WGA-Texas Red-X, Calcofluor White M2R providing a point of reference for the general morphology of the organism.WGA-Texas Red is specific for N-acetyl-ß-D-glucosamine, a constituent glucosaminyl residue of chitin as well as N-acetylneuraminic acid (sialic acid) and N-acetyl muraminic acid which are component carbohydrates of the organisms glycocalyx (John sen, Hasner, Schnell & Wuertz 2000).To support this further it was found that two mannose-rich glycoproteins similar to those previously mentioned, are found in the glycocalyx of Gyrodactylus derjavini (Monogenea) (Buchmann & Lindenstrøm 2002).These mannoserich glycoproteins are important antigens and stimulants of the host's alternative complement pathway (Whittington, Cribb, Hamwood & Halliday 2000).
The glycocalyx of the anucleate tegument fluoresced red as shown in Fig. 1C.WGA-Texas Red is a lectin bound fluorochrome that binds to the sialic acid compounds present on the apical lamina of the para dip lozoon's tegument, demonstrating the specific binding of antibodies to antigens.This proves that the glycocalyx of this Paradiplozoon is made up of N-acetylneuraminic acid and N-acetyl muraminic acid, contrasting Lyons ' (1966) finding that it consists of scleroproteins.
This technique provides an easy and rapid methodology to show the internal sclerites and the anatomy of attachment clamps.It is suitable for alcohol preserved specimens and may have additional applications for other helminth organisms.

FIG. 1
FIG. 1 Photomicrographs of Paradiplozoon morphology.Scale bars = 100 μm A Dorsal view of opisthaptor, showing clamp morphology.Hörens trichrome stain B Dorsal view of opisthaptor, arrow indicates position of hamilus.Hörens trichrome stain C Photomicrograph of fluorescing glycocalyx after staining with WGA-1 Texas red D Photomicrograph following staining with composite fluorochromes E Dorsal view showing detail morphology of sclerites following staining with fluorochromes F Ventral view showing detail morphology of sclerites following staining with fluorochromes, arrows indicate position of thickening in the anterior jaw Working solutions were prepared according to the instructions and stored at -20 °C.The preserved Paradiplozoon were transferred onto glass slides and covered by 10 μl of BSAsaline for 5 min before staining with the addition of 5 μl WGA-TRX, 5 μl SYTO 9 and 5 μl of Cal co fluor White M2R indefinitely.A cover slip was placed over each specimen and sealed with nail varnish.The spec imens were subsequently viewed and photographed with a Zeiss Axioplan 2 epi-illumination fluorescence microscope, operated by Axiovision 4.3 software.Rhodamine, Fluorescein and DAPI bandpass filters on the microscope enabled selective light wavelength illumination of the three flourochromes by a mercury light source.
FIG. 1 Photomicrographs of Paradiplozoon morphology (cont.)G Diagram of the second clamp on the left, showing the anatomy of the clamp scler ites following fluorochrome stain ing 1 Median plate 2 Trapeze spur 3 Joining sclerites of the median plate 4 Sclerites of the distal tip of the median plate 5 Median sclerite of the posterior jaw 6 Lateral sclerite of the posterior jaw 7 Spiky spur 8 anterior jaw BJ Ball joint CT Chitinous thickening SP Serrated median plate