Cuticle Formation in Hemicycliophora Arenaria, Aphelenchus Avenue and Hirschmannieua Gracilis 1
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Cuticle Formation in Hemicycliophora arenaria, Aphelenchus avenue and HirschmannieUa gracilis 1 P. W. JOHNSON, S. D. VAN GUNDY AND W. W. THOMSON 2 Abstract: The onset of molting in all stages of Hemicycliophora arenaria was preceded by the appear- ance of numerous, discrete globular structures which were termed "molting bodies" because they were present in the hypodermis only during the production of the new cuticle. In all parasitic stages the molt commenced with the separation of the cuticle from the hypodermis from which the new sheath and cuticle were differentiated. Following completion of the new sheath and cuticle most of the old outer covering was apparently absorbed before ecdysis. Electronmicrographs of body wall cross sections in molt- ing 1,4 male specimens revealed the final molt to be a double molt in which an additional sixth cuticle was produced. Since both a new sheath and cuticle were produced during the molt of each stage, the sheath must be considered as an integral part of the cuticle and not as a residual cuticle or the result of an incomplete additional molt. Molting in Aphelenchus avenae and Hirschmanniella gracilis was less complex and "molting bodies" were not observed. After cuticle separation the hypodermis gave rise to a new trilaminate zone, the future cortex, and (later) the matrix and striated basal layers. Key Words: Cuticle, Molting, Ultrastructure, Hemicycliophora arenaria, Aphelenchus avenae, Hirschmanniella gracilis. The generalized life cycle of free-living or study was to clarify this situation. Com- parasitic nematodes consists of six stages, parative ultrastructural studies of cuticle egg, four larval stages and adult. Develop- formation in H. arenaria, Aphelenchus ment beyond the first larval stage depends avenue and Hirschmaniella gracilis were upon successful molting; a process about undertaken with these objectives in mind. which very little is known, particularly at the ultrastructure level. The electron micro- MATERIALS AND METHODS scope studies by Bird and Rogers (1), Lee All nematodes were maintained and ex- (9) Samoiloff and Pasternak ( 13 ) and Wat- tracted as previously reported (7). The son (18) are the only ones dealing with molting sequence in H. arenaria was studied nematode molting. in active, handpicked, fourth-stage (L4) The molting nematode may be at one of male and female larvae held in tap water in the weakest points in its life cycle for with- a moist chamber at 26.5 C. When nematodes standing periods of stress (17). The main became quiescent and relaxed, it was as- purpose of the present ultrastructural studies sumed the molting process had been initiated and this was arbitrarily called "time zero." of molting was to gain a better understand- Molting specimens were selected after various ing of both the molting process and cuticle time intervals, depending on the develop- structure. Considerable controversy exists mental stage of the nematode, from time concerning the origin of the sheath in Hemi- zero to exsheathment. Second and third- cycliophora arenaria in particular and Hemi- stage (L2, L3) molting larvae were visually cycliophora spp. and Hemicriconemoides selected in various stages of the molt, without spp. in general. A second purpose of this regard to time, directly from the washings from the 325-mesh screen. Molting speci- Received for publication 5 June 1969. mens of A. avenae and H. gracilis were 1 Supported in part by National Science Foundation Grants GB-6569, GB-4513, and GB-8199. selected directly from the washings without Department of Nematology and Department of Life Sciences, University of California, Riverside, California regard to elapsed time in the molt. 92502. Present address of senior author: Canada Depart- All histological techniques were the same ment of Agriculture, Research Station, Harrow, Ontario, Canada. as those previously described (7). 59 60 Journal of Nematology, Vol. 2, No. 1, January 1970 RESULTS concurrent with its separation from the old HEMICYCLIOPHORA ARENARIA: The onset cuticle (Fig. 2-A, B, arrows). An electron of molting in all stages was preceded by the dense (osmiophilic), multilayered region appearance of numerous, peculiar, globular then formed at the outer edge of the hypo- structures, termed "molting bodies" (Fig. dermis as the fibrillar matrix and striated l-A, D): intimately associated with the basal layer began to differentiate (Fig. 3-A, production of the new cuticular outer cover- B, C). At this time many folds began to ing appearing just prior to molting and dis- form in the new cuticle (Fig. 3-B, C, arrows, appearing shortly after its completion. They Fig. 4-D) and the fibrillar structure of the varied considerably in size (0.2-1.2 g in matrix was easily distinguished (Fig. 4-A, diameter) and were confined to the hypo- B). The matrix also increased in thickness dermal chords (Fig. l-A). Internally the as the striated basal layer became further molting bodies exhibited three distinctive differentiated (Fig. 4-A, C). types of substructure: crystalline areas, dis- Approximately 50 hr after initiation of coid structures and amorphous areas (Fig. the molt, the trilaminate cortex of the new l-B). In some sections they appeared to be cuticle became visible (Fig. 5-A, arrow) in releasing material into the hypodermis (Fig. the trailing edge of the multilayered region, l-C, arrow). In later stages of the molt, resulting in a decrease in its total thickness molting bodies were frequently observed to (Fig. 5-A). Continued differentiation of be deteriorating (Fig. l-D). Just prior to electron-dense layers (Fig. 5-B, arrows) oc- and during the molt, the hypodermis stained curred in the multilayered region as the new more intensely than during intermolt periods sheath developed (Fig. 5-B, C.$5). The (Fig. l-C). outer osmiophilic layers of the sheath were In L4 female specimens (at 26.5 C the probably derived from what remained of the molting and exsheathing processes required multilayered region after the other osmio- approximately 72 hr) the first ultrastructural philic layers of the cuticle and sheath had been sign of molting was an increase in thickness differentiated from it. The new cuticle and of the interchordal hypodermis (Fig. 2-B) sheath were essentially complete and the old FIG. 1. Electron micrographs of molting bodies in H. arenaria. A. Longitudinal section just prior to molt with numerous molting bodies in chordal area. X 53,000; B. Transverse section of large molting body showing three types of substructure. X 47,000; C. Transverse section showing molting body apparently releasing (arrow) material into hypodermis. X 78,000; D. Transverse section show- ing molting body in apparent state of breakdown in late stages of the molt. X 78,000. KEY TO LETTERING OF FIGURES A. amorphous area in molting body order bl. basal lamella H. hypodermis C. cortex M. matrix Cr. crystalline area in molting body Mb. molting body Cu. cuticle Ms. modified sheath of male H. arenaria Cu2, Cu3, etc. cuticle of stage designated by nu- Mu. somatic musculature meral R. multilayered region Cu6. sixth cuticle of male H. arenaria S. sheath Di. discoid shaped structure in molting body $2, $3, etc. sheath of stage designated by numeral F. fibrillar layer St. striated layer FI, F2, etc. fibrillar layers signified in centripetal T. trilaminate zone CUTICLE FORMATION " Johnson, Van Gundy, Thomson 61 62 Journal o/ Nematology, Vol. 2, No. 1, January 1970 striated layer was no longer visible (Fig. breaking down (Fig. 9-A, B). Some micro- 5-C) after approximately 60 hr. graphs (Fig. 9-C, arrows) suggest that this During the next 10-12 hr much of the old breakdown material was probably reabsorbed cuticle and sheath degenerated and was either but, as with the L4 females, absolute proof reabsorbed or simply broken down. Obser- of this was not obtained. The cuticle break- vations of micrographs indicate material from down may or may not be completed before the old cuticle and sheath may have been the next stage of the molt, 30-40 hr after reabsorbed (Fig. 5-D, arrows) although con- molt initiation. clusive proof has not been obtained. After The hypodermis separated from the newly- approximately 72 hr, only remnants of the completed cuticle (Fig. 10-A, Cu5) and a old cuticle and sheath remained (Fig. 6-A, densely staining (osmiophilic) multilayered B). The exsheathment process in water region, similar to that formed in the other required as little as 15 min to occasionally stages, was formed at its outer edge (Fig. more than 24 hr and resembled the molt of 10-A, arrows). A new striated layer formed Caenorhabditis briggsae (6). inside this region (Fig. 10-B). The multi- Molting of L2 larvae (Fig. 7-A, B) and layered region and striated layer became L3 larvae (Fig. 7-C, D) were comparable further separated as the fibrillar matrix layer to molting of the L4 females; in each case a continued to increase in thickness (Fig. new sheath and cuticle were produced. There 10-C) and the trilaminate cortical zone and was no evidence of retention of the sheath modified four-layered sheath became dif- and/or cuticle of the previous stage. ferentiated (Figs. 10-C, D; ll-A). In the L4 male specimens, also required approxi- region of the lateral field, the striated layer mately 72 hr for molting but actually under- was forked (Fig. 10-D, arrows) and replaced went a double molt; two new cuticles, only by a layer of fibrillar material which ap- the second of which included a sheath, were peared as two layers in mature male speci- produced. The first of these new cuticles mens. Dense-staining material accumulated was shed at ecdysis. at the base of the matrix adjacent to the Initially, as in the other stages, the hypo- striated layer (Fig.