The structure of the shell of the egg of Trichuris trichiura was examined using light microscopy as well as scanning and transmission electron microscopy. The results confirmed its three-layered structure and provided evidence that the cores of the polar plugs, which could be seen to be extensions of the shell's middle layer, could be lost en bloc, either mechanically or chemically, and in this way probably provided an exit for the first stage larva at hatching.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.lmchunu2014mn201

Appleton, C.C.

White, Belinda J.

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.The structure of the shell of the egg of Trichuris trichiura was examined using light microscopy as well as scanning and transmission electron microscopy. The results confirmed its three-layered structure and provided evidence that the cores of the polar plugs, which could be seen to be extensions of the shell's middle layer, could be lost en bloc, either mechanically or chemically, and in this way probably provided an exit for the first stage larva at hatching.lmchunu2014mn201

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Onderstepoort J. vet. Res., 56, 219-221 (1989) THESTRUCTUREOFTHESHELLANDPOLARPLUGSOFTHEEGGOFTHE WHIPWORM, TRICHURIS TRICmURA (NEMATODA: TRICHURIDAE) FROM THE SAMANGO MONKEY (CERCOPITHECUS ALBOGULARIS) C. C. APPLETON<1) and BELINDA J. WHITE<2) ABSTRACf APPLETON, C. C. & WHITE, BELINDA J., 1989. The structure of the shell and polar plugs of the egg of the whipworm, Trichuris trichiura (Nematoda: Trichuridae) from the Samango monkey (Cercopithecus albogularis). Onderstepoort Journal of Veterinary Research, 56,219-221 (1989) The structure· of the shell of the egg of Trichuris trichiura was examined using light microscopy as well as sc~nning ~nd transmission electron microscopy. The r~sults confirmed its three-layered structure and rro~!ded evidence that the cores of the polar plugs, which could be seen to be extensions of the shells m1ddle layer, could be lost en bloc, either mechanically or chemically, and in this way probably provided an exit for the first stage larva at hatching. INTRODUCTION Eggs of the-- nematodes belonging to the whip-w<;mn familylfrichuridae are characterized by being thick-walled, tanned, devoid of sculpture and having bip.olar . "plugs", "protuberances" or "opercula" which give the eggs a barrel shape. This thick egg-~hell undoubtedly plays an important role in promot-mg the survival, development and infectivity of the enclosed embryo (Wharton, 1980). The nature and purpose of the polar "I?lugs" are less clear and although accounts of their structure vary, they are presumably involved in the hatching process. The-stru~ture of the shell and polar "plugs" has been descnbed for 3 species of Trichuris, viz. T. vulpis from the dog (Inatomi, 1960), T. suis from the do~estic pig (Whart~n & ~enkins, 1978) and T. tri-chzura from man (Xianqum, Songshan, Wenquin Boxia, Wensu & Li, 1986). The methodology em: rloyed by the above authors has differed; Inatomi 1960) used light microscopy, Wharton & Jenkins 1978) tr~nsmission electron microscopy (TEM) and several histochemical techniques while Xianquin et al. (1986) used scanning electron microscopy (SEM). In the present study, the e~gs ofT. trichiura were also examined but from a dtfferent host, the Samango monkey (Cercopithecus albogularis) using all 3 forms of microscopy. ' Whipworms live in the caeca of their hosts and hav~ a dire?t life cycle in which the eg¥s, containing undifferentiated embryos, are voided m the faeces. Over the subsequent period of approximately 3 weeks, depending on the prevailing temperature and moisture levels in the soil, the embryo develops to a 1st ~tage larya within the egg. It hatches only after . havmg been mgested by another host. MATERIALS AND METHODS Samango monkeys (Cercopithecus albogularis) harbouring T. trichiura mfections were collected at Karkloof, Natal (30° 24' S; 30° 17' E) in No-vember 1987. Light microscopy Trichuris trichiura e~gs were recovered from fresh C. albogularis scats usmg the formol-ether sedimen-tation technique of Allen & Ridley (1970). They )V~re then measu~ed to the nearest 0,1 J.lm with a miCrometer eyeptece and photographed using a U> Department of Zoology and Entomology, University of Natal P. 0. Box 375, Pietermaritzburg 3200 ' <2> E_lectron Microscope Unit, University of Natal, P.O. Box 375, P1etermaritzburg 3200 Received 29 June 1989-Editor 219 Leitz Laborlux 12 photomicroscope. Measurements were expressed as means ± standard error. Scanning electron microscopy Eggs were collected as described above and pre-served in 4 % formalin until they were processed as follows for SEM: after collecting on 5 J.lm Millipore filter paper, they were rinsed in distilled water, placed in 2 % osmium tetroxide for 2 h, rinsed again, dehydrated through a graded alcohol series and crit-ical point dried ( cpd) in a Hitachi HCP-2 critical point drier. Others were air-dried. With one exception, female worms dissected from C. _ albogularis were fixed in 4 % formalin and thereafter fractured under liquid nitrogen using a razor blade also cooled in liquid nitrogen. Pieces containing uterus were then dehydrated and critical point dried. A single specimen was fixed in 3 % glutaraldehyde buffered in 0,05 M phosphate buffer and critical point dried as described above, except that this specimen was rinsed in 0,05 M phosphate buffer and post-fixed in 2% osmium tetroxide m the same buffer. Examples of these uterus-containing pieces were then mounted on brass stubs before being coated with gold palladium using a Polaron E500 sputter coating unit and viewed on a Hitachi S-570SEM. Transmission electron microscopy Pieces of 4 % formalin-fixed female worm con-taining uterus were rinsed in distilled water ,lost-fixed for 2 h in 2 % osmium tetroxide, rinse and dehydrated through a graded alcohol series before being embedded in Spurr's resin. Ultrathin gold sec-tions were cut, collected on copper grids, stained with uranyl acetate, followed by lead citrate and FIG. 1 Light-mi<:roscope photograph of the egg ofT. trichiura from a faecal sample STRUCfURE OF THE SHELL AND POLAR PLUGS OF THE EGG OF THE WHIPWORM FIG. 2 TEM transverse section through the egg.,shell of an intra-uterine egg, showing the vitelline layer (V), the chitinous layer (C), the lipid layer (L) and the embryo {E). Tlte lamellae of the chitinous layer are clearly visible FIG. 3 SEM micrograph of an intra-uterine egg, showing the collapsed polar plug FIG. 4 TEM section of the polar plug of an intra-uterine egg, showing the expanded lamellae of the chitinous layer (arrow) and the core area (ca) FIG. 5 SEM micrograph showing the empty core area of the plug (arrow) of an intra-uterine egg. The lamellae are also visible viewed at 80 kV using a Jeol 100CX TEM. Mea-surements of egg-shell layers were made to either the nearest 0,1 nm or J.lm and expressed as means ± standard error. RESULTS The dimensions of in utero T. trichiura eggs were 62,4 ± 0,3 x 38,4 ± 0,4 J.lm (n=30) while those recovered from scats were slightly smaller, measuring 60,1 ± 0,4 x 26,9 ± 0,2 J.lm (n=30). Light microsco.ry showed the egg-shell to be multi-layered (Fig. l) but the number of these constituent layers was not clear. TEM sections how-ever, showed a shell comprising 3 layers (Fig. 2). 220 The innermost layer varied considerably in thickness from 180 nm to 2,1 J.lm (mean 834,7 ± 157,1 nm; n=15). The thicker middle layer, which in the cen-tral part of the egg measured 1,7 to 3,8 J.lm (mean 2,3 ± 0,2 J.lm; n=15), consisted of 10 bands of elec-tron-dense material alternating with less dense ones. The outermost layer, which could be seen to cover the entire egg, was the thinnest (30,4 to 144,0 nm; mean 61 ,4 ± 6,6 nm; n=20). Layering was in-distinct when viewed using SEM. Under the light microscope the outer surface of the polar plugs was seen to be convex in both fresh and preserved eggs (Fig. 1), but had collapsed dur-ing preparation for SEM (Fig. 3) and was recessed into the collars of the egg-shell. TEM sections re-vealed the cores of the plugs to be extensions of the shell's middle layer (Fig. 4). The concentric arcs visi-ble on one side of the plug can be seen to be conti-nuations of the bands which comprise this layer but clearly do not traverse the core itself. The electron density of the plug cores was not uniform, being less dense in the peripheral areas. Using SEMon a frac-tured specimen, the contents of these cores could not be seen at all (Fig. 5). While the outer surface of the in utero eggs had a wrinkled appearance, that of the voided egg was smooth and devoid of any decoration. No pores could be seen. DISCUSSION The shell of the trichurid egg has been variously reported to comprise 1, 2 and 3 layers by Inatomi (1960), Xianquin et al. (1986) and Wharton & Jen-kins (1978) respectively. The three-layered structure observed in the present material (Fig. 2) therefore confirms the findings of the latter authors. The in-nermost layer conforms to their lipid layer, the mid-dle one to their chitinous layer and its bands to their "lamellae". The outermost layer equates with their vitelline layer. The structure of the polar plugs, like that of the shell itself, has been the subject of debate. While the collapsed nature of the outer surface might have been an artefact of the preparation techniques, the fact that it had not happened in fresh or preserved eggs suggests that it could well occur in the natural environment after the eggs have been voided from the host. The variation of the electron density of the plug's core so clearly seen in Fig. 4, supports the contention of Wharton & Jenkins (1978) that its composition may differ from that of the rest of the shell and may perhaps be more susceptible to enzy-matic action during the hatching process in the host's caecum. Failure to see any substance to the cores of fractured specimens under the SEM indicates that these contents were lost, either mechanically or che-micall_Y., during preparation. This loss led Xianquin et al. ( 1986) to conclude, mistakenly, that the "plug" 221 C. C. APPLETON & BELINDA J. WHITE was an empty cavity. The lack of sculpture is not surprising because trichurid egg-shells lack a 4th layer, the uterine layer, which forms the proturber-ances and spines that decorate eggs such as those of Ascaris spp. CONCLUSIONS Using T. trichiura from C. albogularis, this study has confirmed the 3-layered structure of the trichu-rid egg-shell and polar "plug" as proposed by Whar-ton & Jenkins (1978). It has afso provided visual demonstration that, while the cores of the "plugs" are indeed continuations of the chitinous layer of the shell, they differ in composition from that in the adjacent shell wall. The absence of this core material in air-dried and critical point dried eggs suggests that it is a "weak-spot" which can be lost during the hatching process and that the term "plug" is an ap-propriate one. ACKNOWLEDGEMENTS We are grateful to M. Bruorton (Department of Zoology and Entomology, University of Natal) for making the gastro-intestinal tracts of C. albogularis available for parasitological study and to A. G. Bruton, V. H. Bandu and P. E. Donnelly (Electron Microscope Unit, University of Natal, Pietermaritz-burg) for their ready help with the preparation of the material. REFERENCES ALLEN, A. V. H. & RIDLEY, D. S. , 1970. Further observations on the formal-ether concentration technique for faecal para-sites. Journal of Clinical Pathology, 23, 545-546. INATOMI, S., 1960. Submicroscopic structure of the egg shell of helminth. II. A study on Trichuris vulpis. Acta Medicinae C>koyarna, 14,257-260. WHARTON, D . A. & JENKINS, T., 1978. Structure and chemistry of the egg-shell of a nematode (Trichuris suis). Tissue & Cell, 10, 427-440. WHARTON, D., 1980. Nematode egg-shells. Parasitology, 81, 447-463. X:iANQUIN, M. , SONGSHAN, W., WENQUIN, Z., BOXIA, W., WENSU, H. & LI, W., 1986. The operculum-plug area and membranous structure of the eggs of Trichuris trichiura. Scan-ning Electron Microscopy, 111, 1015-1018. 

The structure of the shell and polar plugs of the egg of the whipworm, Trichuris trichiura (Nematoda : Trichuridae) from the Samango monkey (Cercopithecus albogularis)

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The structure of the shell and polar plugs of the egg of the whipworm, Trichuris trichiura (Nematoda : Trichuridae) from the Samango monkey (Cercopithecus albogularis)

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