Internationat Journatjor Parasitology Vol. 13, No.1. pp. 19-43, 1983.Printed in Great Britain.
0020-7519/83/010019-25$03 .00/0Pergamon Press Ltd.
1983 Australian Society for Parasilology
ULTRASTRUCTURE OF LOA LOA MICROFILARIA
WIESLAW J. KOZEK and THOMAS C. ORIHEL
The International Collaboration in Infectious Diseases Research Program, Centro Internacional deInvestigaciones Medicas, Tulane University-COLCIENCIAS, Apartado Aereo 5390, Cali, Colombia,
S.A. and Tulane University Medical Center, New Orleans, LA 70112, U.S.A.
(Received 11 Seplember 1981)
Abstract-KoZEK W. J. and ORIHEL T. C. 1983. Ultrastructure of Loa loa microfilaria. InternationalJournal for Parasitology 13: 19-43. The ultrastructure of Loa loa microfilaria was elucidated by theusc of scanning and transmission electron microscopy during the examination of micro filariae for thepresence of intracellular bacteria. In general, the structure of the sheath, body wall, nerve ring, amphids,phasmids, excretory and anal vesicles, central body, and the R cells of L. loa closely resembles that ofother species of microfilaria. Characteristics which appear to be unique to L. loa include the externallateral cuticular ridges which extend throughout most of the body length, four sensory papillae aroundthe buccal orifice and dense cytoplasmic inclusions in the intestinal cells which envelop the central body.A small cephalic hook is located near the amphids. Buccal orifice and capsule are present; primordialesophagus extends from the buccal capsule to the central body. These resul\s support previous sug-gestions that the microfilariae of different species have not attained the same degree of maturity. Theinternal development of L. loa microfilariae is comparable to, or may be more advanced, than that ofBrugia spp. The population of L. loa studied was probably free of intracellular bacteria since none weredetected in any of the sections examined.
INDEX KEY WORDS: Nematoda; Filarioidea; Loa loa; microfilaria; microanatomy; ultrastructure;scanning electron microscopy; transmission electron microscopy; intracellular bacteria.
INTRODUCTIONTHE PRESENCE of intracellular bacteria in Brugiapahangi and Dirofilar1a immitis, as reported, respec-tively, by Vincent, Portaro & Ash (1975) andMcLaren, Worms, Laurence & Simpson (1975) andour observations of similar bacteriae in Onchocercavolvulus adults and larval stages (Kozek & Figueroa,1977) prompted us to examine other filariae,especially those which infect man, to determine theprevalence of intracellular microorganisms amongthe species of the Filarioidea. Subsequent studieshave demonstrated that both adult and larval stagesof many filariae harbored similar microorganisms(Kozek, 1977; and unpublished observations) andthat these microorganisms could also be aetected inmicrofilariae, if sufficient numbers of microfilariaewere examined. We were especially interested to deter-mine whether Wuchereria bancrofti and Loa loa, twofilariae of considerable medical importance andinterest, also harbored intracellular bacteria, butunfortunately our access was limited to the micro-filaria of L. loa. Since a careful examination of themicrofilariae with an electron microscope was neces-sary to demonstrate conclusively the presence of themicroorganisms, we have conducted, concurrentlywith this search, a morphological study to elucidatethe microanatomy of this microfilaria. The resultsobtained, which complement those of McLaren(1969, 1972), are presented in this report.
MATERIALS AND METHODS
The microfilariae examined were obtained from anexperimentally infected patas monkey at the Delta RegionalPrimate Research Center, Covington, Louisiana, U.S.A.This monkey was infected with larvae harvested fromChrysops spp, which had fed on another patas monkey(No. 3657) which was inoculated with infective larvaeobtained from flies fed on a human volunteer in Cameroon,West Africa. The blood was withdrawn from the infectedmonkey at the Delta Regional Primate Center, diluted 3-4times its vol. with phosphate-buffered saline and filteredthrough a Millipore@ filter membrane with 14 /-lrn poresize. After filtration, the membrane with the filtered micro-filariae was removed from the filter assembly and fixed atambient temperature in 4070 glutaraldehyde in Milonigsbuffer, pH 75. Numerous membranes and all the micro-filariae collected in this manner were mailed in the originalfixative to the California Primate Center, University ofCalifornii\, Davis, California, U.S.A., where they weresubsequently. processed, in essentially the same mannerused to study nodules containing O. volvulus adults (Kozek& Figueroa, 1977), for examination with both transmissionand scanning electron microscopes.
RESULTSThe following morphological features can be
routinely identified in the microfilaria by lightmicroscopy: cephalic space, nerve ring, nuclearcolumn, excretory vesicle, excretory cell, R cells,anal vesicle and a finely striated cuticle (Fig. 1).
10 11m s
WIESLAW 1. KOZEK and THOMAS C. ORIHEL
I.J.P. VOL. 13. 1983
FlOS. 1-43 depict L. loa microfilariae. Abbreviations: AV, anal vesicle; BC, buccal capsule; BO, buccalorifice; CB, central body; CR, cuticular ridges; CS, cephalic space; DM, dense material; DNT, dorsalnerve trunk; EC, excretory cell; ET, esophageal tube; EV, excretory vesicle; G, Golgi apparatus; GC,ganglionic cell; L, lysosome-like body; M, muscle cell; m, mitochondrion; MV, microvilli; N, nucleus; n,nucleolus; NCC, nuclear column cell; NR, nerve ring; P, phasmids; RI-4, R cells; RER, rough endo-
plasmic reticulum; S, sheath; VNT, ventral nerve trunk.
FIG. I. Montage of L. loa microfilaria, Knott's preparation, hematoxylin stain. Arrows indicatetransverse cuticular striations. The numbers refer to the figures which illustrate the areas indicated by thevertical lines. Figures 1 a-c. L. loa (Knotts) stained with PAS, bis benzimide and pyronin. Figure lao
Amphids (arrows) in the cephalic space. Figure Ib. Central body (arrows). Figure Ie. Phasmids.FIG. 2. En face view of the cephalic end. Buccal orifice is surrounded by 4 sensory papillae (small arrows).
The hook (H) covers the aperture of the second amphid (A).FlO. 3. Lateral view of the cephalic end of exsheathed microfilaria depicting an amphid (A) and the origin
of the lateral cuticular ridge (arrow).
I.I.P. VOL. 13. 1983 Ultrastructure of Loa loa microfilaria 21
Special staining also reveals the amphids within thecephalic space (Fig. la), the central body (Fig. Ib) thephasmids in the tail region (Fig. 1c) and the esophagealtube in the anterior portion of the microfilaria(Ftilleborn, 1913; Laurence & Simpson 1968, 1969;Simpson & Laurence, 1972).
Although the microftlariae of L. loa are ensheathed,only a few among those examined retained theirsheaths (Figs. 4, 12). Almost all of the others losttheir sheath during filtration. The sheath ranges from30 to 80 nm in thickness and seems to be comprisedof two layers. The outer, 5-50 nm thick, appearsrough due to the presence of dense, granular depositson its surface (Figs. 6, 12, 13). The inner, approxi-mately 30 nm thick layer, is of more uniform thick-ness and appears to be homogeneous (Fig. 12).
The body wall consists of a multilayered cuticle,hypodermis, muscle cells and nerve trunks. Thecuticle bears fine transverse striations discerniblewith the light microscope (Fig. 1); interstrial distanceranges from 03 to 05 J..lm. It is from 60 to 70 nmthick and consists of at least 4 sublayers. The outer-most sublayer (Fig. 13, No. I), approximately 13 nmthick, is trilaminate; its two electron-dense sublayersare separated by an electron-lucent sublayer. Thesecond sublayer (Fig. 13, No.2), approximately 6 nmthick, is more electron-lucent than the first. The thirdsublayer (Fig. 13, No.3), is thin, electron-dense andapproximately 3 nm thick. The fourth and thickestsublayer of the cuticle (Fig. 13, No.4), ranging from25 to 50 nm, appears to be homogeneous, but someelectromicrographs suggest that it may be comprisedof three layers: an outer, more electron-dense layer(Fig. 19, 4a) and an inner, more electron-lucent layer(Fig. 19, 4c) separated by what appears to be a finefibrillar middle layer (Fig. 19, 4b).
A unique external feature of L. loa microfilaria is acuticular ridge, extending on each side from approxi-mately the 10th anterior annulation to about the 73rdannulation from the tip of the tail (Figs. 3, 5, 6).These ridges, obvious in most crosS and obliquesections (Figs. 12, 17, 23,24, 26, 34, 36), are mostprominent in Figs. 16, 19 and 31. In cross sectionthey appear as cuticular corrugations measuring ap-proximately 190 nm in height and from 300 to 560 nmin width at the base. They are covered by the outer 3(Nos. 1, 2, 3) sublayers of the cuticle, but the princi-pal component of each ridge is a homogenous, electron-lucid material similar to that which constitutes theouter layer (b) of the 4th cuticular sublayer. Theridges either originate from, or rest upon, the innerlayer (c) of the 4th cuticular sublayer (Fig. 19).
The musculature of the body wall consists of adorsal and ventral muscle band. Cross sectionsthrough different levels of the microfilaria reveal thateach muscle band consists of numerous pairs of cellsarranged in tandem (Figs. 15, 17,24,31,34,36,37).The dorsal and the ventral nerve trunk is locatedbetween the two cells of each respective muscle band(Fig. 31). The platymyarian muscle cells are charac-teristically divided into an outer contractile and an
inner, noncontractile portion. The contractileportion consists of two bundles of myofilaments,each containing an aggregation of thick filaments,thick and thin filaments (Fig. 20). The ratio of thick:thin filaments is I: 10-12. The muscle cell mito-chondria are usually situated between the bundles ofmyofibrils and the nucleus (Fig. 20), or beneath themyofibrils within the noncontractile portion of thecell (Figs. 17, 34). They are electron-dense, long butof small diameter and contain few cristae (Figs. 20,30). Muscle cell nuclei, located in the noncontractileportion of the cell, are similar in diameter andappearance, but are longer than the nuclei of nuclearcolumn cells (Figs. 21, 27). They have moderate amountsof peripheral chomatin and irregular strands of chro-matin extending into the center of the nucleus (Fig. 20).
The hypodermal cell nuclei and most of the hypo-dermal cytoplasm are located within the lateral trunks.The nuclei are surrounded by a large volume ofcytoplasm which contains ribosomes, small mito-chondria, lysosome-like bodies and centrioles (Figs.17-19). The hypodermal nuclei are usuallyspheroidal with small amounts of peripheralchromatin and a prominent, centrally located nucleo-lus (Fig. 19). A thin, sleeve-like lateral extension ofhypodermal cytoplasm lies sandwiched between thecuticle and the muscle cells (Fig. 20).
The nuclear column is comprised of the esophagealcells which occupy the region from the cephalic spaceto the central body; the ganglionic cells of the nervering and peripheral nerves; central body cells whichsurround the central body; and other undifferentiatedcells which do not appear to be associated with anyparticular organ primordium. These 4 groups of cellsare similar in appearance but differ in size. Theirnuclei are spheroidal with moderate amounts of peri-pheral chromatin and some strands of centralchromatin. A thin sheath of cytoplasm, containingfree ribosomes and small mitochondria, envelops thenuclei (Figs. 12, 17,31).
Scanning electron micrographs revealed a slender,1 J.lm long hook and a primordial buccal orifice,approximately 120 nm in diameter, on the anteriortip of the microfilaria. A circlet of 4 papillae, eachapproximately 05 J.lm in diameter, surrounds thebuccal orifice and the apertures of the two amphidialchannels (Fig. 2).
The cephalic space (Figs. 7-11) is essentially amuscular tube, formed by 8 muscle cells, ensheathedby the hypodermis and the cuticle (Fig. 11). Thebuccal capsule, flanked by the amphids, is located inthe center of the cephalic space; papillary axons fillthe space between these structures and the musclecells (Fig. 10). The amphids, a pair of cuticularizedchannels approximately 36 Iffi1 long, contain somefine filamentous material and 6-8 cilia which aremodified terminations ofaxons originating in thenerve ring. A plug-like accumulation of an amorphouselectron-dense material is located at the aperture oteach amphid (Figs. 8-10). The papillary axons alsoappear to be modified cilia which, however, do not
22 WlESLAW J. KOZEK and THOMAS C. OalREL I.J.P. VOL. 13. 1983
FIG. 4. Anterior portion of an ensheathed microfilaria.FIG. 5. Tail region of exsheathed microfilaria. Arrow indicates termination of the lateral cuticular ridge.FIG. 6. Partially exsheathed microfilaria. Large arrow indicates the outer, granular layer of the sheath,small arrow the inner> homogenous layer. Lateral cuticular ridge originates near the anterior end of
J.P. VOL. 13. 1983 Ultrastructure of Loa loa microfilaria 23
FIG. 7. Oblique section through the cephalic space. Axons (A) proceeding from the nerve ring terminateas modified cilia (C) within the amphidial channel. Sensory papilla is indicated by large arrow, sections of
the buccal capsule by small arrows.FIG. 8. Longitudinal section through the cephalic space depicting a complete amphidial channel con-taining modified cilia (C), obliquely sectioned buccal capsule and 2 papillary axons (small arrows). Large
arrows indicate the cuticular lining of the amphidial channel and of the buccal capsule.FIG. 9. Higher magnification of the opening of the amphidial channel, in which terminations of cilia (C)
are visible. Large arrow indicates a sensory papilla, small arrows papillary axons.
24 WIESLAW J. KOZEK and THOMAS C. ORIHEL I.J.P. VOL. 13. 1983
terminate freely as do the cilia within the amphidialchannels but end either within some papillae or sub-cuticularly. Sections through the tip of the cephalicspace show 4 small papillary axons located aroundthe buccal capsule and a circlet of 4 larger papillaryaxons surrounding the smaller ones (Fig. 10). Thelarger papillary axons may terminate within the 4papillae shown in Fig. 2.
The buccal capsule extends from the buccal orificeto the esophageal tube. In cross sections it appears asa round, cuticularized tube, approximately 015 }lmin diameter, surrounded by numerous semicirculardesmosomes (Fig. 10); in oblique sections it appearsas a long, cuticularized slit (Fig. 8). The esophagealtube extends from the base of the cephalic space tothe central body. It is similar in cross section to thebuccal capsule, but is surrounded by a cartwheel-likepattern of desmosomes formed by the modifiedcytoplasmic membranes of the adjacent esophagealcells. At some levels, up to 10 of these junctionscontact the esophageal tube (Fig. IS). Figure 12depicts the contact of the cytoplasmic membranes ofthe esophageal cells with the esophageal tube.
The nerve ring represents a local accumulation ofnerves a...