Transcript
Page 1: Ultrastructure of Loa loa microfilaria

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 sug­gestions 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, respec­tively, 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 deter­mine whether Wuchereria bancrofti and Loa loa, twofilariae of considerable medical importance andinterest, also harbored intracellular bacteria, butunfortunately our access was limited to the micro­filaria of L. loa. Since a careful examination of themicrofilariae with an electron microscope was neces­sary 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.

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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 micro­filariae was removed from the filter assembly and fixed atambient temperature in 4070 glutaraldehyde in Milonigsbuffer, pH 7·5. Numerous membranes and all the micro­filariae 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).

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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).

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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, approxi­mately 30 nm thick layer, is of more uniform thick­ness 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 0·3 to 0·5 J..lm. It is from 60 to 70 nmthick and consists of at least 4 sublayers. The outer­most 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 approxi­mately 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 ap­proximately 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 princi­pal component of each ridge is a homogenous, electron­lucid 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 charac­teristically 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 mito­chondria 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 chro­matin extending into the center of the nucleus (Fig. 20).

The hypodermal cell nuclei and most of the hypo­dermal cytoplasm are located within the lateral trunks.The nuclei are surrounded by a large volume ofcytoplasm which contains ribosomes, small mito­chondria, lysosome-like bodies and centrioles (Figs.17-19). The hypodermal nuclei are usuallyspheroidal with small amounts of peripheralchromatin and a prominent, centrally located nucleo­lus (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 peri­pheral 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 0·5 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 3·6 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

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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

microfilaria.

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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 con­taining 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.

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terminate freely as do the cilia within the amphidialchannels but end either within some papillae or sub­cuticularly. 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 0·15 }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 around the esophageal tube. Many axons aretwisted about each other, others are dilated andcontain neurosecretory-like granules (Figs. 14, IS).Nerve trunks which originate from the nerve ringextend anteriad or posteriad (Fig. 14). The individualaxons are thin, contain some microtubules, and verysmall, dense mitochondria (Figs. 14, 17).

The excretory complex is a unicellular structure whichcan be divided into three anatomic components: (a)the excretory vesicle and the specialized cytoplasmicterminations within it, (b) the cytoplasmic tube whichconnects the vesicle to the excretory cell, and (c) theexcretory cell and its nucleus (Figs. 21-26). Theexcretory vesicle is a spherical cavity. Its ventral halfis cuticularized, contains the pore and is filled withfine, filamentous dense material which extends intothe pore (Figs. 21, 23). The dorsal half of the vesicleconsists of numerous membranous terminations ofthe cytoplasmic arm which converge towards thepore from all directions (Figs. 21-23). Theesophageal tube lies dorsal to the cytoplasm of theexcretory cell at the level of the pore (Fig. 23). Thenucleus of the excretory cell is approximately 2 Iill1 indiameter and characterized by a prominent dense,eccentric nucleolus (Fig. 24). The perinuclear cyto­plasm contains free ribosomes or glycogen particles,some strands of rough endoplasmic reticulum, Golgiapparatus and a few small mitochondria (Fig. 24)which are less electron-dense than the mitochondriaof the muscle cells. Rough endoplasmic reticulum isconcentrated in the region posterior to the nucleus;adjacent areas contain numerous Golgi apparatusand lysosome-like bodies (Figs. 25, 26). Some of thenuclear column cells located near the excretory cellextend thin, long cytoplasmic axon-like projectionstowards the nerve ring (Fig. 25).

The central body is a 30-50 /lm long cylindroid of

uneven diameter, located anterior to the R-I cell(Figs 1, 1b, 27, 28). It consists of electrondense,homogenous material surrounded by a cytoplasmiccylinder formed by thin, epithelioid cells which willbe referred to as the central body cells.

Sections through any level of the central bodyreveal that it is enveloped by three central body cells(Figs. 31, 32); cytoplasmic membranes of theadjacent cells are joined by septate desmosomes(Figs. 31-33). Their nuclei are smaller than the nucleiof the nuclear column cells, and have less peripheraland central chromatin material (Fig. 33). Theunusual feature of these cells is the apparent lack of acell membrane at the interface with the central body,although normal cell membranes are present wherethe cells are in contact with either the cells of thenuclear column or of the body wall (Figs. 31, 32).Spherical inclusions, up to 0-5 /lm in diameter,which are either homogeneous and electron-dense, orwith r;entral vacuoles, were commonly observed in thecytoplasm of the central body cells (Figs. 28, 31-33).

The R-l cell, approximately 15 jAm long, is thelargest cell within the microfilaria. Its nucleus iselongated, approximately three times as long as it iswide; the nucleolus is located near the anterior poleof the nucleus. Its cytoplasm contains ribosomes,small electron-lucid mitochondria, few Golgi appar­atus, and lysosome-like bodies (Figs. 29, 30).

The R-2 and R-3 cells are much smaller than theR-l cell. Their nuclei resemble that of the excretorycell by virtue of the centrally located nucleolus. Theirperinuclear cytoplasm filled with free ribosomes isnot as abundant as that of R-l (Fig. 35). Most of themitochondria and Golgi apparatus are located withinthe cytoplasmic trunks which extend posteriorly(Figs. 34, 35) and terminate as microvilli within theanal vesicle (Fig. 39).

The general structure of the anal vesicle resemblesthat of the excretory vesicle, but differs from it intwo respects: it is not a unicellular unit-it is formedby cytoplasmic extensions of at least 3 cells, theseextensions terminate as microvilli. Cell walls of theadjacent cytoplasmic extensions are modified intothick junctions (Figs. 39, 40). Numerous mitochon­dria, of the same electron opacity as the mitochondriaof the excretory vesicles, are located in the cytoplasmat the base of the microvilli (Figs. 39, 40). A finelygranular electron-dense material fills the ventral partof the vesicle and extends through the pore to theoutside of the microfilaria (Fig. 39). A muscle cell islocated on each side of the anal pore (Fig. 39).

The two phasmids are situated in the tail area,approximately 28 jAm posterior to the anal vesicle.Each phasmid, a 4 lillI-long, cuticularized channellocated within the hypodermis, opens to the outsidethrough a small pore in the cuticle. Each containsone cilium, approximately 3·5 jAm long and 0·4 jAm indiameter and finely granular filamentous material(Figs. 41-43). The cuticular lining, similar to thelining of the amphids and of the two vesicles, extendsas far as the basal portion of the cilium (Fig. 42).

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FIG. 10. Oblique section near the tip of the cephalic space. Buccal orifice is flanked by 2 amphidialchannels (A) and surrounded by 8 papillary axons grouped as an inner circlet of 4 small axons (smallarrows) and the outer circlet of 4 larger axons (large arrows). Crescent-shaped desmosomes are visible

around the buccal capsule.FIG. 11. Oblique section through the cephalic space near the base of the amphidial channels. Eachchannel contains 5-8 cilia (C). Eight muscle cells (small arrows) surround the amphids and the buccal

capsule (large arrow).FIG. 12. Oblique sectIOn of ensheathed microfilaria near the level of the nerve ring. Three esophageal cells(E I-E3) surround the cuticularized esophageal tube. Small arrows indicate the lateral cuticular ridges;

large arrows, the sheath.FIG. 13. Higher magnifications of the sheath and of the cuticle; sublayers of the cuticle are numbered (seetext). Arrows indicate granular deposits on the external surface of the sheath. C, cuticlej H, hypodermis.

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FIG. 14. Longitudinal section through the nerve ring. Dilated axons contain neurosecretory granule.(large arrows), bundles ofaxons (small arrow) extend anteriorly and posteriorly from the nerve ring.FIG. 15. Oblique section through the nerve ring. Numerous axons contain neurosccretions (small arrows),

large arrow indicates the esophageal tube.

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FlO. 16. Lateral cuticular ridge (arrow) at midbody level.

Flo. 17. Oblique section near the level of the nerve ring depicting typical internal morphology of themicrofilaria. Muscle bands consist of two muscle cells. Nerves and axons lie near some of the muscle cells.

Large arrow indicates a mitochondrion within an axon, small arrows the lateral cuticular ridges.FlO. 18. Highly magnified portion of the hypodermis of Fig. 17. depicting two centrioles (large arrows).

Lateral cuticular ridge (small arrow).

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FIG. 19. Hypodermal cell in the lateral field, note prominent nucleolus within the nucleus. The lateralcuticular ridge (small arrow) is covered by the 3 cuticular sublayers. The 3 layers (4a, 4b, 4c) of the fourth

cuticular layer can be discerned.FIG. 20. Muscle cell of the microfilaria. The nucleus is located in the metabolic portion of the cell belowthe mitochondria and myofilaments. Large arrow indicates aggregation of thick filaments, small arrowsindicates aggregation of thin filaments, intermediate zone contains both thick and thin filaments. Thin

hypodermal sheath (H) extends between the cuticle (C) and the muscle cell.

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FIG. 21. Oblique section through the excretory vesicle. Specialized membrane projections converge ontothe pore. Dense filamentous material rills the cavity of the vesicle. Small arrow indicates a muscle cell

nucleus, large arrow the esophageal tube.

FIG. 22. Lateral section through the excretory vesicle depicting the converging membranous projectionsonto the region or the pore.

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FIG. 23. Oblique section through the excretory vesicle and excretory pore, showing the relationship of theesophageal tube to the vesicle. Large arrow indicates the cuticular lining of the vesicle, small arrows the

lateral cuticular ridge.

FIG. 24. Oblique section through the excretory cell nucleus. Cytoplasm of the ccll fills the central portionof the microfilaria. Arrow indicates the esophageal tube. Dorsal and ventral muscle bands are prominent.

Lysosome-like body is present in a muscle cell. Small arrows indicate the lateral cuticular ridges.

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fto. 2). Longitudinal section through the cytoplasmic portion of the excretory ceIl. One portion isoccupied by the nucleus, the second by rough endoplasmic reticulum and the third by Golgi bodies and

lysosome-like inclusions. Arrows indicate 2 ganglionic cells and their axons (small arrows).

FlO. 26. Oblique section through the excretory cell posterior to the nucleus, depicting the rough endo­plasmic reticulum, Golgi apparatus, esophageal tube (large arrow). and the lateral cuticular ridge (small

arrows).

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FIGS. 27-30 depict segments of one longitudinal section through the central body and the R-l cell.Horizontal lines identify the common reference level in successive figures; the numbers refer to the figures

which depict areas that precede or follow the reference line.

l"IG. 27. The central body surrounded by central body cells (small arrows), large arrow indicates a musclecell nucleus.

FIG. 28. Cytoplasmic inclusions (small arrows) in the central body cells. Lysosome-like bOdy (large arrow)and long mitochondria present in a muscle cell.

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FIG. 29. Nucleus of the R-I cell with its eccentric nucleolus. Axons (large arrows) with small densemitochondria (small arrows) are visible on one side of the microfilaria.

FlO. 30. Cytoplasm of R-l cell containing lysosome-like bodies (small arrows). Large arrOw indicates along mitochondrion within a muscle cell.

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FIGS. 31-33. Oblique and transverse sections depicting the central body enveloped by the cytoplasm of atleast three central body cells. Large arrows indicate tight junctions between the cytoplasmic membranes

of adjacent central body cells. I, cytoplasmic inclusions.FIG. 31. Cross section through a narrow portion of the central body. A dorsal and a ventral nerve trunk is

prominent within the respective muscle band. Small arrows indicate lateral cuticular ridges.FIG. 32. Oblique section through a wide portion of the central body. Note the absence of a cytoplasmic

membrane between the central body and the medial wall of the central body cells (small arrows).

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®FIG. 33. Oblique section through the central body, a nucleus of one (arrow) and the cytoplasm of several

central body cells. Inclusions within these cells vary in size and density.FIG. 34. Cross section through the microfilaria anterior to the anal vesicle. Cytoplasmic processes (P I' P2)

of the R-2 and R-3 cells.

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FlO. 35. An oblique section through R-2 and R-3 cells. Cytoplasmic process (delineated) extends pos­teriorly from R-3 towards the anal vesicle.

FIGS. 36-38. Transverse section through tail region of the microfilaria.FIG. 36. Section near the termination of the lateral cuticular ridges (arrows).

FlO. 37. Section posterior to that shown in Fig. 36, showing the terminal portion of one cuticular ridge(arrow).

FIG. 38. Section near the tip of the tail which consists of muscle cells (arrows) and hypodermalcomponents.

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FIGS. 39-40. Oblique and longitudinal sections through the anal vesicle. Arrows indicate tight junctionsbetween the cytoplasm of the cells which form the vesicle.

FIG. 39. Oblique section through the anal vesicle. Microvilli and dense, fine filamentous material fill thecavity.

FIG. 40. Longitudinal section through the anal vesicle depicting microvilli cut transversely. Adjacenthypodermal cell contains a lysosome-like body.

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FIGs. 41-43. Sections through the phasmids.

FIG. 41. Longitudinal section through a phasmid. The channel colltains one cilium (C); dense material islocated near its opening. Arrow indicates the location of the opening of the second phasmidial channel.

FIG. 42. A phasmid highly magnified. Cuticular lining (arrows) of phasmidia! channel extends up to thebase of the cilium (BC).

FIG. 43. An oblique section depicting a cilium (arrows) in each phasmidiaJ channel.

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An accumulation of dense material is located at eachpore (Figs. 42, 43).

The tail region posterior to the phasmids containssome nuclei, muscle cells, and nerves (Figs. 36-38).The lateral cuticular ridges terminate at this level(Figs. 36, 37). Prominent muscle bandscan be seen inthe successive sections towards the end of tail; thelateral trunks graduaJly diminish in size until only themuscle cells and some hypodermal tissue can bedemonstrated near the tip of the tail (Fig. 38).

Intracellular bacteriae were not detected in any ofthe many thousands of sections examined during thisstudy.

DISCUSSION

Our previous experience indicated that the intra­cytoplasmic bacteria could be easily detected in themicrofilariae jf the adult worms also harbored them.Since none of the sections of L. loa microfilariaeexamined during this study contained any micro­organisms, it may be concluded that this and possiblyother human strains of L. loa are probably free ofthese micro-organisms. Although an extremely lowinfection rate of the microfilariae examined cannotbe ruled out, the examination of adult wormsnecessary to confirm or refute these observationscould not be performed at the time.

The basic microanatomy of L. loa microfilariaconforms closely to that of other species ofmicrofilariae (Kozek, 1971; McLaren, 1972; Tongu,1974; Laurence & Simpson, 1974; Kanagasuntheram,Singh, Ho and Chan, 1974; Singh, Kanagasuntheram,Ho, Yap and Chan, 1974; Kanagasuntheram, Singh,Ho, Yap and Chan, 1974; Suguri, 1977; Martinez­Palomo & Martinez-Baez, 1977). The structure andcomposition of the body wall, nuclear column cells,nerve ring, amphidial and phasmidial channels,excretory complex, R cells, and the anal vesicle, arevery similar to the corresponding structures of othermicrofilariae. The combined use of scanning andtransmission electron microscope, however, revealedsome additional, hitherto undescribed morphologicalfeatures of L. loa microfilariae, including the buccalorifice, a small cephalic hook, sensory papillae at thetip of the cephalic space, the number and arrangementof cephalic papillary axons and intracytoplasmicinclusions within the central body cells.

The sheath of L. loa microfilariae depicted byMcLaren (1972) was covered with a thick, filamen­tous mat. The few sheaths observed during thepresent study were covered with coarse granulardeposits of dense material similar to those shown byLaurence & Simpson (1974) and Suguri (1977) on thesheath of B. pahangi. This minor difference maymerely reflect the different techniques used toprocess the microfilariae for each study. The lateralcuticular ridges, first observed on the L. loa micro­filariae by McLaren (1972) can be helpful in dis­tinguishing L. loa from other microfilariae in trans­mission electron micrographs. Although similar

ridges have not been detected to date on other micro­filariae, this feature might not be unique to L. loa;similar structures will undoubtedly be detected onother species of microfilariae as more are examinedwith the electron microscope. It remains to bedetermined whether these ridges serve some usefulpurpose when the microfilaria is either in thevertebrate host or in the vector.

Some microfilariae, e.g. those of Dipetalonemareconditum, Brugia pahangi, possess a cephalic hooklarge enough to be observed by light microscopy(Esslinger, 1962; Sawyer, Rubin & Jackson, 1965).The cephalic hook of others is too small to bedetected with a light microscope, consequently, itspresence has to be verified either by scanning ortransmission electron microscopy. This study hasdemonstrated that L. loa microfilaria has a shortcephalic hook. A small pouch to house the hook isprobably also present, although we were unable todemonstrate it. Cephalic hooks were demonstratedto date by transmission electron microscopy on themicrofilariae of Dirofilaria immitis andDipetalonema witei by Kozek (1971) and McLaren(1972), respectively, and both the hook and thepouch have been demonstrated by scanning electronmicroscopy on the microfilariae of Onchocercavolvulus from Liberia (Franz & Schulz-Key, 1981)and from Guatemala (Franz, 1979; Kozek, unpub.lished observations).

Microfilariae of L. loa possess 8 cephalic axonsarranged in 2 concentric rings around the primordialbuccal capsule; each ring consists of 4 axons. Four ofthese axons appear to terminate within the 4 cephalicpapillae observed with the scanning electronmicroscope. These terminations are usually difficultto demonstrate by transmission electron microscopy,although an axon within one such papilla can be seenin Fig. 4 of McLaren (1969) and another 2 in Plate 4,Fig. 1 of McLaren (1972). Eight papillary axons havealso been demonstratednear the tip of the cephalicspace of B. pahangi by Suguri (1977), and in Breinliasergenti by Kanagasuntheram, Singh, Ho, Yap &Chan (1974). These axons probably represent the in­nervation and the future site of the cephalic papillaewhich, in the adult and in the more advanced larvalstages of many filariae, are arranged in 2 rings eachconsisting of 4 or more papillae (Anderson, 1968;Wong & Brummer, 1978; Franz, 1979; 1980). Itappears, therefore, that the blueprint for theinnervation of the cephalic end is already determinedin the microfilarial stage and the number of papillaryaxons in each species of microfilaria corresponds tothe number of cephalic papillae, a characteristic for aspecies, present in the adults. This characteristicnumber and distribution of papillary axons are rarelyobserved during ultrastructural studies since it can beseen clearly only in cross sections through a shortsegment of the cephalic space. The random orienta­tion of the microfilariae in the embedding mediumrenders it difficult to obtain such a section.

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40 WIESLA W J. KOZEK and THOMAS C. ORIHEL I.J.P. VOL. 13. 1983

The number of cephalic papillae present on eachspecies of microfilariae remains to be elucidated. Wehave demonstrated 4 on L. loa. However, similarpapillae were not detected with the scanning electronmicroscope on the microfilariae of O. volvulus ofMexican origin by Martinez-Palomo & Martinez­Baez (1977), nor on microfilariae of D. immitis byAoki & Katamine (1975), although some micro­graphs suggest that similar papillae may be presenton the cephalic tip of O. volvulus microfilariae ofAfrican and Guatemalan origin (Franz, 1979, Fig.28D; Franz & Schulz-Key, 1981, Fig. 2; Kozek, un­published observations).

Histochemical studies conducted on variousspecies of microfilariae by Laurence & Simpson(1968, 1969) and Simpson & Laurence (1972)disclosed stained areas in forms of hooks, spines andrings on the anterior tip of the most of themicrofilariae studied, including those of L. loa. Ourscanning electron micrographs could onlydemonstrate a small cephalic hook, a buccal 'orifice,and the cephalic papillae. The stained areas depictedby these investigators might represent a coating, oraccumulations of the dense material present in theapertures and within the amphidial channels, on thesurface of the cuticle and the structures adjacent tothe amphidial pores. Since the samples used for thesehistochemical tests consisted of microfilariae in thickblood smears, some of these "cephalic spines" couldhave been produced by the shrinkage of the cuticlewhen the microfilariae dried. Additional defor­mation of the cuticle might have been produced bypre-mortem constriction of the muscle cells whichinsert near the tip of the cephalic space or near thehook. However, some microfilariae may havecephalic spines; a structure resembling a spine hasbeen demonstrated by Tongu (1974) on microfilariaof B. malayi.

The L. loa microfilaria has a primordial digestivesystem which consists of a buccal orifice, a buccalcapsule located within the cephalic space, a pri­mordial esophagus which consists of the esophagealtube with its esophageal cells and the central bodywith the central body cells which envelop it. We wereunable to obtain during this study sections whichwould have demonstrated the junction of the buccalcapsule with the esophageal tube. However, thecontinuity of the buccal capsule with the esophagealtube was clearly demonstrated by histochemicalmethods at light microscope level in the microfilariaeof L. loa, B. malayi, B. pahangi, W. bancrofti,Cardioji/aria ni/esi and M. ozzardi, by Laurence &Simpson (1967), although their study did not demon­strate the esophageal tube in the micro filariae of O.volvulus, Litomosoides carin ii, or Dipetalonemaperstans.

The buccal orifice and the esophageal tube appearpatent, but only physiological studies can demonstratewhether they are functional or not. The buccalcapsule is similar in appearance to the esophageal

tube but lacks the desmosomal attachments whichgive the tube the characteristic cartwheel appearance.The termination of the esophageal tube at the centralbody of L. loa was not observed in this study, but it isprobably similar to that shown in B. malayi byTongu (1974), and in Breinlia sergenti by Singh,Kanagasuntheram, Ho, Yap & Chan (1974).

The nuclear column cells which lie between thecephalic space and the central body can be dividedinto at least two groups: the esophageal cells andganglionic cells of the nerve ring and the peripheralganglia. The esophageal cells surround the eso­phageal tube to which their cytoplasmic membranesare attached by means of thick junctions. Since someof the esophageal cells are displayed by the nerve ringand the excretory complex, their cytoplasmicextensions are longer than those of others.

The identity of the cells which will form theintestine in the more advanced larval stage remainscontroversial. According to some investigators(Feng, 1936; Laurence & Simpson, 1971), theintestine is derived from the nuclear column cellslocated near the central body. McLaren (1972), whowas the first to demonstrate with the aid of a trans­mission electron microscope the cells enveloping thecentral body in a microfilaria, proposed that thesecells form the intestine (McLaren, unpublishedPh.D. thesis, BruneI University, 1971, cited byMcLaren, 1972). Laurence & Simpson (1971, 1974)agree with this observation and refer to theanalogous cells in Brugia spp. as the "intestinalcells" (Laurence & Simpson, 1974). Although wehave not studied at the ultrastructural level thedevelopment of L. loa in its vector, the appearanceand the relationship of the epithelioid central bodycells of L. loa to the rest of the primordial digestivetract strongly suggest that the central body cells arethe precursors of the intestinal cells. The cytoplasmicinclusions of the central body cells have been observedto date only in L. loa microfilariae. It is not knownwhether they are storage granules or accumulationsof metabolic waste products. However, their presenceonly in the central body cells constitutes a potentialintracellular marker which could be used to trace thedevelopment and the fate of these cells in subsequentlarval stages.

The structure and organization of the nerve ringand the presence of neurosecretory-like granules insome axons closely resemble the appearance of thenerve ring of other microfilariae (Kozek, 1971;McLaren, 1972; O'Leary, Bemrick & Johnson, 1973;Laurence & Simpson, 1974; Kanagasuntheram,Singh, Ho, Yap & Chan, 1974; Suguri, 1977). Someof the ganglionic cells from which the axons originateare probably located close to the nerve ring, asdiagrammed by McLaren (1972, Text Fig. 1), andsuggested by some electron micrographs (Kozek,1971; McLaren, 1972; O'Leary et al., 1973).However, such a relationship is difficult to confirmwithout a complete set of serial sections through the

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I.J.P. VOL. 13. 1983 Ultrastructure of Loa loa microfilaria 41

nerve ring and its adjacent regions. Since the smalldiameter of the microfilaria limits the number ofnuclei which can be located at the same level withinthe body cavity to about 5, only a few ganglionic cellscan be present immediately next to the nerve ring.The remaining cells of the nerve ring have to belocated at various distances in front and behind thenerve ring and some are apparently located evenbeyond the excretory cell.

A well-developed peripheral nervous system of themicrofilaria is manifested by the nerve trunks whichare located between the two cells comprising eachmuscle band. In adult nematodes the dorsal nervetrunk is usually larger than the ventral trunk. Thesame relationship is observed in L. loa, and probablyalso in other microfilariae, at all levels except in thevicinity of the nerve ring where axons are located nearthe lateral trunks and on the lateral aspects of themuscle bands. The location and the size of the nervetrunks constitute, therefore, 2 useful landmarkswhich can be used for dorso-ventral orientation ofcross sections through most levels of the microfilaria.

The muscle cells of L. loa are of the meromyarian,platymyarian type, being divided into contractile andnoncontractile portions. The thick: thin myofilamentratio (1: 10-12) is the same as in the microfilariae ofD. immitis (Kozek, 1971), and similar to the 1: 8-12ratio reported by Suguri (1977) for B. pahangi and1 : 12 for the microfilariae examined by McLaren(1972). The distribution of thick and thin myofila­ments within the contractile portion indicates thatthe muscle cells of L. loa are obliquely striated. Theventral muscle band separates at the level of theexcretory and anal pore, so that one muscle cell islocated on each side of each pore. Contraction ofthese cells during the undulatory movement of themicrofilaria could change both the diameter of thepore, the volume of the vesicle and the pressurewithin them, thus possibly assisting in the inter­change of substances between the microfilaria and itsmillieu.

The excretory cell of L. loa is larger than that of D.immitis and Brugia spp. The abundance of itsorganelles indicate a highly active metabolic unit withthe capability of synthesis. The latter function issuggested by the presence of rough endoplasmic reti­culum and adjacent Golgi bodies in the posteroventralportion of the cell. Accumulation of lysosome-likeinclusions and vacuoles suggest that the cell also hasthe capacity for metabolic degradation.

The R-l cell is the largest cell of the microfilaria.Although it resembles the excretory cell, the R-l canbe differentiated from the latter by the lack of roughendoplasmic reticulum, fewer Golgi apparatus andlysome-like bodies and by the ellipsoidal nucleus withan eccentric nucleolus. Cross or oblique sections atthe level of the R-l can be distinguished from similarsections through the excretory cell by the absence ofthe esophageal tube which terminates at the centralbody. The rather simple appearance of the R-1 cell

does not offer any clues to indicate which organ willbe formed by its progeny. Some investigatorspostulated that the R-l formed the genitalprimordium (Rodenwald, 1908; Iyengar, 1956), theintestine (Taylor, 1960; Schacher, 1962), or the mus­culature of the adult worm (Bain, 1970). Perhaps astudy of the fate of unique inclusions observedrecently in the R-1 of other species of microfilariae(Kozek, unpublished observations) could indicatewhich organ the R-l is destined to form.

The R-2 and R-3 cells are similar to R-l, but aresmaller and their nuclei are more spheroidal. A cyto­plasmic extension from each of these cells continuesposteriorly to end as microvillar surface within theanal vesicle. Although we did not obtain any sectionsthrough the R-4 cell, it can be postulated that itsmorphology is probably very similar to that of R-2and R-3 and that it also forms a part of the analvesicle, as it does in other species of microfilariae(Kozek, 1971; McLaren, 1972).

Kanagasuntheram, Singh, Ho & Chan (1974)suggested that the excretory vesicle, anal vesicle andthe R cells may constitute a part of the nervoussystem of microfilaria. However, the excretory andthe anal vesicles depicted by their electron­micrographs (Figs. 4, 5, 8, 10, 11) bear onlysuperficial resemblance to nerve tissue. We haveobserved similar appearance of the two vesicles indead microfilarae, or in those improperly fixed inhypotonic fixatives. Since these authors used distilledwater to lyse the erythrocytes and isolate the micro­filariae from blood, the collected microfilariae weresubject to osmotic damage which probably causedthese morphological artifacts.

Although numerous ultrastructural studies havebeen conducted by many investigators, only fewreports note the presence of centrioles in nematodecells. Most of those which have been described weredetected in maturing germinal cells (Jamuar, 1966;Foor, 1970; Neill & Wright, 1973). Wright (1976)found six centrioles in somatic cells of Capillariahepatica: two in non-glandular hypoderman cells, adiplosome in a hypodermal cell, one in a myoepi­thelial cell ensheathing the muscular esophagus andone in an epithelial cell of the vas deferens. Thecentrioles observed in L. loa were also located in ahypodermal cell, but they were slightly smaller thanthe somatic centrioles of Capillaria, were orientedparallel and not at right angles to one another, andeach consisted of nine singlets. Similar centrioleswere detected by Foor (1970) in a spermatid ofAncylostoma caninum, and by Neill & Wright (1973)in a spermatid of C. hepatica. Centrioles areprobably more common in microfilariae than ourobservations suggest, but are difficult to find due totheir small size and the relatively few cells availablefor examination in cross or oblique sections of amicrofilaria.

In routinely prepared and stained blood smearsmicrofilariae appear as cuticular sleeves filled with

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42 WIESLAW J. KOZliK and THOMAS C. ORIHEL l.J.P. VOL. 13. 1983

embryonic nuclei. Even the internal morphologicalfeatures revealed by special staining methods hardlysuggest the high degree of internal organization andthe remarkable development revealed by electronmicroscopy. The primordial digestive tract, theperipheral nervous system, the number and thearrangement of the cephalic papillary axons andpapillae of L. loa microfilaria reflect the patternpresent in the adult stage. Laurence & Simpson(1971) consider the microfilaria of Brugia spp. a verymuch modified first-stage larva. Our study hasshown that the internal development of L. loa micro­filaria is comparable to, or may be even moreadvanced, than that of Brugia spp., suggesting thatnot all species of microfilariae have attained the samelevel of internal development. This range of thedegree of internal maturity in different microfilariaewill become more apparent when additional speciesare critically examined with the electron microscopein the future. Nevertheless, considering thecontinuous progress in the preparative methods,reagents and electron microscopes, extreme cautionis required in interpreting the results obtained toensure that the differences observed in the internalanatomy of different microfilariae are real and donot represent the products to improved technology.

Our results suggest that a microfilaria is a remark­ably adapted embryonic form which has developedthose organ pirmordia, or their analogs, essential forits survival in the vertebrate host and transfer to thevector. The body wall is necessary for protection(cuticle), structural support (cuticle and muscles) andmovement (muscles); the nervous system for orienta­tion and movement. Excretion, ion and osomoregu­lation, are probably the most important postulatedfunctions of the excretory vesicle and possibly of theanal vesicle. It is not known how the microfilariafeeds, but some of its nutrients could be obtainedthrough its rudimentary digestive tract, by transcu­ticular absorption, or by intake through the analvesicle. The extent to which these modes may befunctional, whether alone or in combination with theother two, needs to be elucidated by physiologicalstudies. However, the primordial state of thedigestive tract suggest that its function is minimal, ifit is functional at all. Since the microvilli of the analvesicle most closely resemble the intestinal absorptivesurface of vertebrates and invetebrates, it seemsreasonable to infer that the anal vesicle may serve asan intestinal analog until the intestine proper is fullydeveloped and functional in the late second larvalstage. The hyperthrophy of the anal vesicle (rectalplug) during the early development of the larva in thevector may, therefore, merely reflect the response ofthis organ to the great demand for nutrients neededby the rapidly growing larva.

Acknowledgements-Wc thank Dr. Mark L. Eberhard ofthe Delta Regional Primate Research Center in Covington,Louisiana, for his assistance in collecting and handling ofmicrofilariae and the expert and dedicatcd assistance of Ms.

Mary Ann Brayton, Paul Lee and John Schmidt of TheDavis Primate facility. Thanks are also due to GloriaAmparo Chamorro D. and Esmeralda Caicedo R., andJesus Abelardo Granja in Cali, Colombia, for theirassistance during this study.

Supported, in part, by Grant RR00169, and a ProgramProject Grant Al 16315-01 from the National Institute ofAllergy and Infectious Diseases, National Institute ofHealth, Bethesda, Maryland.

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