Life history and development of Pneumon ema tiliquae (nematoda: Rhabdiasidae)

Inrenmionnl Journal for Parasirology Vol. 2 I, No. 5, pp 52 I-533, I991 Printed in Great Brrtain 0

LIFE HISTORY

002~7519/91 $3.00 + 0.00 PergMton Press plc 1991 Ausrralion Societyfor Parasitology

AND DEVELOPMENT OF PNEUMONEMA (NEMATODA: RHABDIASIDAE)

TLUQUAE

ROBERT J. BALLANTYNE School of Science and Technology,

Charles Sturt University,

P.O. Box 588, Wagga Wagga,

New South Wales 2650, Australia

(Received 22 August 1990; accepted 28 February 199 1)

AIrstraCt-BALLANTYNa R. J. 1991. Life history and development of Pneumonemo tiliquue (Nematoda: Rhabdiasidae). International Journalfor Parasitology 21: 521-533. Pneumonema tiliquae besides its unusual adult morphology has a very precise life cycle. In nature, parasites appear to be restricted to Tiliqua scincoides (blue-tongued lizard); the parasitic generation in the form of a female alternates with a free-living generation of males and females. The development during the alternation is very precise; sperm and ova are alternately produced in the gonad of the parasitic form; eggs are retained in utero until they contain fully formed first-stage larvae, then the uteri are emptied and another cycle ofegg development occurs. Eggs hatch in the large intestine or faeces; if hatching occurs in the large intestine, larvae die unless faeces are deposited before the onset of division of the genital primordium. The free-living generation undergoes four moults to produce adult males and females in 24-36 h with the male inseminating the female immediately after the final moult. The development and hatching of eggs in utero is obligatory and larvae eat out the female body; infective third-stage larvae are ensheathed and are found in cultures 8496 h post-hatching. Third-stage larvae exsheath and are capable of entering molluscan paratenic hosts. Lizards are infected orally; thirdstage larvae enter the body cavity and develop to adults; the third- and fourth-stage sheaths are retained, then shed 7-8 days post-infection; immature adults enter the lungs only if the lungs are free of worms. Once in the lungs, 12-14 days post-infection, the worms continue to grow, developing large body spines and associated body muscles and produce large numbers of eggs. The free-living generation resembles other Rhabdiasidae in the presence of nine pairs of genital papillae in the male and can be differentiated from the Strongyloididae on the size of the vulva, number of genital papillae and the absence of an undifferentiated buccal collar in the pharynx. INDEX

KEY WORDS:

Nematoda;

Rhabdiasidae;

Pneumonema

; life history; development.

INTRODUCTION

MATERIALS AND METHODS

THE Rhabdiasidae is an unusual family of nematodes found in the lungs of amphibians and reptiles. Unusual features of the family are the absence of parasitic males and the presence of an alternation of parasitic and free-living adult generations. The family has received sporadic attention and the literature contains many brief reports on morphology and portions of life histories, except for the detailed studies by Baker (I 978, 1979). Morphological features are ill-defined, and specimens are difficult to fix, hence variations exist in descriptions of many of the important taxonomic features. The family is well represented in Australia and a study using fresh material was undertaken (196% 1970) to produce a comprehensive understanding of the family. Ballantyne (1986) reviewed the morphology of the parasitic adult of Pneumonema tiliquae and described certain aspects of its life history. This present paper covers the complete life history and development of P. tiliqune. Details of the post-embryonic development of the reproductive system are included in a separate report (Ballantyne, 1991).

Animals. All large lizard hosts were kept in wire-mesh bottomed cages. In an endeavour to produce regular defaecation, lizards were fed on a variety of foodstuffs including different meats, fruit and grain, without success. Finally they fed on Whiskas* Jellymeat for cats which produced healthy lizards, but irregular defaecation. Parasitefree scincoides (blue-tongued lizards) for Tiliqua experimental infections were obtained from adult female lizards, captured between August and December. These adult lizards produced young viviparously between January and March. Young lizards were kept in small cages and fed egg yolk and Whiskas. Lizards were housed either in the laboratory at room temperature, in an air conditioned room (25°C) with a 12-12 light-dark regime, or outdoors with shade and water provided; under all conditions lizards became inactive in winter. Cultures. Cultures of free-living stages were obtained from faeces, the contents of lizard’s large intestine, eggs taken from the lungs and eggs removed from parasitic adults. Cultures were grown at room temperature (15-31°C) and water was kept to a minimum because excess moisture was detrimental to development, often causing death. Eggs from the lungs and parasitic females were grown in either rat or guinea pig *Registered 521

trade mark of Uncle Ben’s of Australia.

R.J.

522

BALLANTYNE

TABLE ~-FREE-LIVINGGENERATION.SPECIFICWORMS(MEASUREMENTSIN

pm)

Stage

BL

PhL

CL

PNR

Wst

stw

WC

cw

Iw

Wph

Phw

Wa

RL

Ll

340 380 505 650 903 880 1140 380 640 756 700

100 110 105 110 132 143 170 94 120 130 210

50 50 55 55 66 77 90 40 60 66 95

77 77 80 77 99 99 125 70 92 99 95

13 15 14 14 12 16 16 11 13 12 15

9 10 11 11 10 12 14 7 7 7 5

22 24 21 20 21 24 28 16 20 22 20

12 13 15 15 16 21 24 10 11 11 7

6 6 6 7 7 10 12 4 6 5 5

25 27 25 24 26 37 40 22 23 28 24

14 16 16 16 18 21 24 12 13 13 12

14 16 24 24 26 16 17 12 14 18 16

22 22 33 30 38 33 38 20 22 22 27

900

240

114

115

15

5

25

7

5

28

12

16

27

Adults

Adult? Ll* Ml L2t L3$

TL 45 50 30 30 30 66 83 60 88 94 90 65 110 75

L3: TL 90 and 110 = length of L2 sheath. *In utero; t emerging from female; $ ensheathed infective. Abbreviations: BL, total body length; CL, corpus (pharynx) length; Cw, pharynx width at metacarpus; Iw, isthmus (pharynx) width; PhL, total length of pharynx; Phw, pharynx width at end bulb; pNR, distance of nerve ring from anterior end; RL, rectum length; Stw, pharynx width at anterior end of procorpus; TL, tail length from anus to posterior end; Wa, body width at anus; WC, body width at metacarpus; Wph, body width at pharyngo-intestinal junction; Wst, body width at base of stoma.

Stage

BL

PhL

pNR

Wst

stw

Wph

Phw

WV

Wa

RL

TL

pV%

gpL

L3*

1200 1600 1900 1800 2000 2000 2300 2600 5000 8000 12,000

280 285 360 370 4.00 400 400 440 500 530 650

140 145 165 150 150 150 150 150 150 150 175

17 20 22 25 25 26 28 31 33 33 33

11 13 15 18 18 19 22 24 25 25 25

35 50 55 58 60 60 60 65 90 100 110

20 30 40 40 45 50 45 50 55 55 55

20 50 50 50 55 55 55 55 100 160 200

35 25 30 35 35 35 35 40 60 75 75

28 35 35 35 38 38 38 45 80 120 120

100 110 130 116 130 130 115 150 250 425 530

67 73 73 62 62 62 62 60 57 50 44

45 75 200 365 365 400 550 740 1510 4190 7290

M3 L4t M4f Adultg Adult Adult Adult Adult

1 2 3 4

*In body cavity; t ovary reflexing; $ adult buccal capsule forming; 4 after L3 and L4 sheaths shed; 1, in body cavity; 2, when first eggs produced; 3, standard form; 4, unusually large form. For abbreviations see Table 1. Additional abbreviations: gpL, genital primordium length (anterior reflexion to posterior reflexion); pV%, position of vulva expressed as a percentage of the total body length; WV, body width at vulva.

faeces (laboratory-raised animals) with or without 1% nutrient agar added. These cultures were used to check for direct and repeated free-living development. Cultures were examined at regular intervals to follow their development. The complete development from egg to infective L3 was followed at hourly intervals in two separate cultures to ensure all development was observed. Examination and recording techniques. The various stages were studied alive, heat killed, and heat killed and stained with or without fixation. Camoy’s fixative gave the best results and the stain used was 1% acetic orcein (Hirschmann, 1962). Lugol’s iodine, bismark brown and methylene blue were tried but produced poor results. Glycerine alcohol fixation or any form of chemical fixation followed by evaporation to glycerine from glycerine alcohol produced specimens with little resemblance to the living material. All material was examined using a Leitz Laborlux II microscope

with a 95 x oil objective, drawing tube and camera system. Coverslips were always sealed on the underside with petroleum jelly to ensure that the weight of the coverslip did not alter the arrangement of body parts. Fixed and stained specimens, prepared in this manner, were usable for 3-6 months. Measurements of heat-killed worms were made using an eyepiece micrometer. Tables 1 and 2 give the measurements of individual worms and the text contains a variety of measurements from several hundred worms as a descriptive guide. Collection andpreparation ofparasitic worms. Lizards were killed with chloroform or by injecting Lethabarb (Pento barbitone sodium solution). A ventral incision was made taking care to avoid major blood vessels; the body cavity was irrigated several times with saline, the fluid collected in a tall cylinder and the sediment examined for worms. The lungs, liver, alimentary canal and adjacent mesenteries were then

P. tikguae: life history and development removed and placed in saline; the external surfaces were freed of larvae and subsequently each organ opened. Free worms were removed before each organ was cut into pieces. Each piece was pressed between the inverted lid and bottom of a Petri dish and examined for worms using a stereomicroscope and transmitted light. After the initial examination all organs and the carcass were subjected to either pepsin digestion or placed in saline at room temperature for 6-8 h. The tissue and fluid were examined, then placed in the refrigerator overnight and re-examined the next day for worms. Parasitic material was washed in saline and examined as above. For staining without fixation worms needed to be transferred to water then into stain; saline and acetic orcein do not mix. Stages from the body cavity appeared to be easily stressed in 0.7 and 0.9% saline and often died in a few hours. Material for sectioning was fixed in 5% formalin or Carnoy’s, embedded in paraffin wax and sectioned at S-10 pm. Sections were stained with Mayer’s haemalum and eosin. Enface specimens were prepared after heat fixation, and evaporation to glycerine from 5% glycerine in 70% alcohol. Worms were cut and mounted in glycerine jelly on a coverslip. The coverslip was inverted onto plasticine supports on a slide, pressed gently to ensure the coverslip was level, then the preparation was made permanent by adding Canada balsam. Collection and preservation of infective larvae. Infective larvae were collected by pipette after water had been added to the culture surrounds. Larvae were then stored in a thin film of water in a ctean Petri dish until used. Moarhing.Moulting periods were signified by the division of lateral cord nuclei and commenced with the retraction of the protoplasm from the cuticle in the cephalic region. The moult was completed when the cuticle was loosened over the whole body. Care was required when fixing, staining and mounting material not to artificially loosen the cuticle or alter cellular arrangement. RESULTS Hosts

Forty-eight of the 50 Tiliqua scincoides examined from south-east Queensland had adults in the lungs. The two lizards not showing adults in the lungs had immature stages in the body cavity. Usually two to five adult worms were found in one or both lungs, but some lizards harboured more than 50 adults. The worms had their anterior ends in the alveolar sacs and their intestines contained blood. There was no evidence, either from sections or whole mounts of worms in situ, that the spines were in contact with the alveolar wall. P. tiliquae was not found in six Tiliqua gerrardi (pink-tongued lizard) autopsied from the Brisbane area, but was found in one T. nigrolutea (southern or blotched blue-tongue). An experimental infection of P. tiliquae was established in an adult male Tiiiqua rugosa (shingle-back lizard) from Adavale (south-west Queensland). Development from parasitic generation

At any one time, all eggs in the uteri of parasitic adults were at approximately the same stage of development. When all eggs contained fully formed first-stage larvae (Ll) the uteri were emptied and another cycle of egg development began in the uteri. Eggs and faeces from worms were removed from the

523

lungs and encased in mucus by the normal ciliary current. This mucus encasement remained intact and was easily traced through the alimentary canal of the lizard. Eggs hatched in either the large intestine or the faeces. If hatching occurred in the large intestine, most of the Ll died, unless defaecation occurred before the onset of division of the genital primordium, i.e. prior to the first moult (Ml). If suitable conditions prevailed after hatching, the larvae fed and developed through four moults (MlM4) to free-living adult males and females in 24-36 h. The moults occurred between the following hours: M 1 6-8; M2 9-12; M3 16-24 M4 20-32. Males developed slightly faster than females and were ready to inseminate the female as soon as the latter had undergone M4. Males were short lived and began to die between 48 and 60 h. Fertilized ova developed and hatched in utero. Ll appeared between 36 and 48 h and M 1 occurred between 48 and 60 h. Second-stage larvae (L2) had completed eating out the female after 60-72 h and infective third-stage larvae (L3) were fully formed after 84-96 h. All times were from initial hatching of eggs from the parasitic adult, not from collection of faeces. Each stage could be determined by the development of the nuclei in the reproductive system, whereas the size of the reproductive system varied with body length. Body length and rate of development depended on culture conditions. These conditions were not precisely defined but high and low temperatures, excess moisture, overcrowding and lack of food produced adverse effects. The effect was noticeable after MI and females were affected more than males. There was no direct development to L3 or repeated free-living generations. Eggs. Eggs (Fig. 1) from parasitic adults were thin shelled, 85-110 pm long, 45-65 pm wide, and contained fully formed Ll when laid. First-stage larvae. Recently hatched Ll (Fig. 2), 340-380 pm long; 25-27 pm wide at pharyngointestinal junction and genital p~mordi~; tail 45-55 pm long, tapering to a point; post-anal lip small. Mouth opening 2-3 pm long, 1 pm wide, leading into an undifferentiated buccal tube 9-10 pm long, 1.5 pm wide; buccal tube cuticle thick and dark. Pharynx rhabditiform, 100-110 ,um long, corpus slightly differentiated into pro- and metacarpus; procorpus extending 4-5 pm round base of buccal tube to form an undifferentiated buccal collar; pharyngeal radii of procorpus tuboid, remainder convergent; radii arch anteriorly in procorpus, then taper towards metacorpus (Figs. 2,4); radii of metacarpus expanded but not formed into a valve; valve of end bulb with eight or nine ridges on each sector. Nerve ring in mid-isthmus region of pharynx. Pharyngo-intestinal junction a cuticle-lined extension of the pharynx containing five to seven nuclei, forming part of the alimentary canal wall, i.e. not projecting into the intestinal lumen. Intestine with 20 large (5-8 pm) nuclei; anterior ring of four, remainder in two rows (dorsal and ventral);

R. J BALLANTYNE

524

50w

@\ a

7 I i

I CC

1

,

/

I

I \

5

Ftos. 1-6. Free-living

FIG. I. Egg containing

generation.

L 1 male: cc, coelomocyte;

bcl, buccal collar; pr, pharyngeal precursor.

es, egg shell.

FIG. 2. Ll female recently

hatched:

FIG. 3. Ll male posterior

end: B, spicule/gubexnaculum precursor; F, spicule precursor; junction; U, as in Fig. 2. FIG. 4. Adult anterior

end; a, amphidiat

ray; U, precursor

duct; cp, cephalic

of cloaca development;

Y, ganglion

rc, rectal cell; rj, recta-intestinal

papilla.

FIG. 5. Renette cells, adult. FIG. 6. Male posterior

end: ed, ejaculatory

duct; edv, ejaculatory

duct diverticulum;

ph, phasmid;

sp, sperm; spd, sperm duct.

P. tiliquae: life history and development

constriction present anterior to posterior four nuclei. Recta-intestinal junction poorly developed, with two nuclei. Rectum a cuticle-lined tube 20 pm long, surrounded anteriorly by one dorsal uninucleate and two ventro-lateral binucleate rectal cells and dorsally by four nuclei (accessory genital primordium); in male larvae, two of the four nuclei (B, F) enlarged (Fig. 3). Ventral cord immediately anterior to anus in female with two large (U, Y) and six small nuclei, in the male with one large (U) and six small nuclei; remainder of the ventral cord with about 18 prominent nuclei. Genital primordium prominent, lying ventrally in mid-body region, with six large nuclei in females (Fig. 2) and 10 large nuclei in males. Two coelomocytes laterally, in female small and posterior to the genital primordium, in male large and adjacent to the posterior end of the genital primordium. Amphidial ducts prominent, opening laterally 5 pm from anterior end. Head papillae small and difficult to count, cervical papillae not observed. Phasmids lateral in midtail region. Excretory pore and duct variable in position between nerve ring and end bulb of pharynx; renette cells not visible. Lateral cords with prominent nuclei in a single row. Development to free-living adult. Small bristle-like structures appeared in the base of the buccal tube of male and female Ll and remained in all stages including adults (Fig. 4). These structures were the ends of the t&radiate pharyngeal cuticle projecting into the cylindrical buccal tube. In all larvae the dorsal rectal cell became bi-nucleate at Ml. In some female larvae, and at any stage of development, some of the intestinal nuclei divided or became elongate. The accessory genital primordium in the male formed the spicules and gubernaculum during the late fourth stage (L4), whereas in the female, one or two of the small dorsal nuclei divided. The U nucleus in the ventral cord divided in the male but not in the female. The amphidial ducts and openings remained postlabial, and the glands became prominent dorsal and ventral to the pharyngeal isthmus prior to each moult; between moults the glands were difficult to distinguish. Each amphidial gland contained four nuclei and the dorsally positioned gland opened on the right side. The head papillae remained small and were difficult to study. Cervical papillae were not observed during development. The male genital papillae formed during M4. Paired renette cells (Fig. 5) became prominent during L4, but prior to this, the renette-cell nuclei could not be distinguished from nerve nuclei. The excretory duct entered the renette cells dorsally, often a considerable distance from the anterior end of each cell. The actual connection to each cell was difficult to determine and had one or two nuclei associated with it. Each renette cell was partially surrounded laterally by a group of four nuclei. These four nuclei stained intensely and may have belonged to the nervous system. The lateral cords showed a similar development to that seen in the parasitic generation except gland cells and ducts to the surface were not formed

525

during the L4. The male and female reproductive systems developed through characteristic stages with the vulva in the female failing to increase in size during the late L4 and early adult. Adult free-living male. Body length 0.5-0.9 mm. Body wall, cuticle, head, stoma, pharynx, intestine (nuclei usually not divided) and associated junctions, rectum and excretory system as described for female. Rectal cells obscured by spicule development. Reproductive system single, testis reflexed, containing large spermatocytes, ejaculatory duct prominent with two anterolateral diverticula; reflexed end of testis shortens after sperm ejaculation. Two darkened, equal, slightly curved spicules, 2840 pm long, with pointed distal tips and pincer-like proximal ends for muscle attachment, Gubernaculum darkened, 15-17 pm long, wedge-shaped in cross section, with thin edge (ventral) between the spicules, dorsal side curves and narrows towards anterior end (distal to cloaca), lateral margins with supports for the spicules-supports well developed towards cloaca1 end. Anterior ventral margin (dotted in Fig. 6) often neither thickened nor darkened. Tail short, about 30 pm long excluding terminal taper; appearance variable as the taper may or may not form at M4. Nine pairs of genital papillae (Fig. 6) as follows: two subventral pre-anal, one subventral adanal, four subventral post-anal with the last two close together at the posterior end, one lateral post-anal between 4th and 5th subventrals, one subdorsal at posterior end. Phasmids subventral between 5th and 6th subventral papillae. Small nerve ending present in midline on anterior lip of cloaca. Adult free-living female. Body length 0.58-2 mm (most worms less than 1 mm); basic morphology similar to Ll. Body wall thin, cuticle with fine longitudinal striations; head structures small and ill-defined, true lips absent, four cephalic and six labial papillae present (best seen in stained worms). Amphidial glands not differentiated, duct and opening lateral as in larvae. Cervical papillae (derids) minute near level of nerve ring. Mouth opening 2-3 pm long, buccal tube 910 pm long with bristle-like structures at the base. Pharynx rhabditiform, muscular, glands poorly developed, buccal collar present but not differentiated from remainder of pharynx. Pharyngo-intestinal junction small, with five to seven nuclei. Intestinal cells granular, constriction and nuclei (20 present) as in Ll or with some divided or elongate. Recta-intestinal junction poorly developed with two small lateral nuclei. Rectum tubular, 3&40 pm long. Three binucleate rectal cells present; accessory primordium prominent containing five to seven nuclei. Tail 60-90 pm long, often tapered to a long point. Phasmids small in mid-tail region. Lateral cords broad and flat, nuclei poorly developed, cuticlelined ducts absent. Paired renette cells granular, 3&40 pm long, lateral excretory canals absent. Two complete reproductive systems present with uteri opposed at vulva, oviduct not clearly differentiated from uterus, ovaries reflexed containing large oocytes, vulva minute in mid-body region.

R. J. BALLANTYNE

526

8

Pf

13

14

16

I

I

17

\ 18

FIGS.7-19. Parasitic generation.

P. tiiiquae: life history and development Development from free-living fernate

Fertilized ova from the free-living female developed and hatched in utero, eating oocytes but never fertilized ova. Neither eggs nor larvae were able to escape through the vulva and development within the female appeared to be obligatory because Ll and L2 died if they were artificially released into cultures. Eggs with a morula 70-80 pm long, 40-S5 pm wide. Recently hatched Ll similar to Ll from the parasitic generation, 380-400 l.trn long with tail SO-60 ,um long; genital primordium small; lateral cord nuclei difficult to stain. Larvae pre-Ml SO&680 pm long with tail 90110 pm long ending in a long delicate point; genital primordium 22-26 pm long with six nuclei; some intestinal nuclei divided in a few specimens; lateral cord nuclei clearly defined and beginning to divide. At M 1 which occurred in the uterine-oviduct area, genital primordium with 10 nuclei and longitudinal striations under the Ll cuticle; striations broken 25-35 pm from the posterior end; this break corresponds to the characteristic ringed tail of the sheath of L3 (Fig. IS). L2 increased in length as they ate first the ovaries, then the remainder of the body and escaped when only the parent cuticle remained. The larvae actively destroyed the parent body during feeding, using both ends of their body to break up the body contents as they moved back and forth inside each system. The female was active until the nerve ring was broken. Recently escaped L2 50&800 pm long, pharynx rhabditiform 125-140 pm long; lateral cord nuclei well developed; genital primordium with 14 nuclei. One to eight larvae developed depending on the rate of production and fertilization of ova. Ova fertilized late did not develop to L3 as there was not enough food. Small adult females (580 pm long) produced small L3 (500 pm) but excessively large females (1 S-2 mm) did not produce correspondingly large L3. After escape, L2 became quiescent as M2 occurred. During M2 the cuticle lining the rhabditiform pharynx loosened and the pharynx lengthened and lost the valve from the end bulb; the intestinal constriction disappeared; the posterior extremity of the L3 tail formed level with the ring in the L2 cuticle. L3 remained ensheathed in the L2 cuticle and were usually found in the film of moisture at the edge of cultures, where they lay quiescently with their bodies

527

straight. The free-living generation was more successful in developing if moisture was kept to a minimum; under minimal water conditions there was no migration of larvae from the cultures. If water was added to a culture as soon as infective L3 became quiescent, they would migrate to the lid of the Petri dish. If 24 h or more elapsed before water was added, the larvae would not migrate. Once ensheathed, L3 were infective and remained alive for up to 3 months in a thin film of water. It was not determined whether they remained infective for the whole of this period. Larvae 37 days old produced an infection, but most larvae used for experimental infections were aged between I and 21 days. Females that did not produce fertilized eggs were common in cultures. These females were similar morphologically to larvae-producing females and lived for 12-15 days. Sperm was not found in the reproductive tract and during their increased life span all oocytes enlarged packing the uteri, oviducts and ovaries. The germinal-cell line was depleted and the distal end of the ovaries emptied. Each system contained 25-30 oocytes which was a similar number to that found in larvae-producing females. Infective third-stage larvae L3 were ensheathed in L2 cuticle, length 500-900

pm; width 22-28 pm at pharyngo-intestinal junction and genital primordium; tail of sheath 90-l 10 pm long with characteristic ring (Fig. 15); L3 tail 65-75 pm long ending abruptly with small ridges on the posterior extremity; longitudinal striations on cuticular sheath (L2 cuticle) consist of a series of separate blocks; L3 cuticle with fine lon~tudinal striations and a small longitudinal ala (ridge) on each lateral surface. Head papillae small, the four cephalic papillae barely visible; amphidial glands not differentiated, ducts and openings post-labial as in Ll. Buccal tube 9-10 pm long, buccal collar present (Fig. 7). Pharynx 21@240 pm long, elongate rhabditiform without a valve in the end bulb (Fig. 13). Nerve ring 95-115 pm from anterior end. Pharyngo-intestinal junction small, with five to seven nuclei. Intestinal nuclei variable in number, either 20 or 30-36, posterior constriction as seen in free-living generation absent. Recta-intestinal junction poorly developed, with two small lateral nuclei;

FIGS.7-13. Anterior end. 7. Infective L3. 8. M3: L4 buccal capsule forming, ventral. 9. L4 ensheathed: buccal

capsule

lateral. 10. M4: adult forming, lateral. 11. Immature adult from body cavity: lateral; cp, cephalic papilla; lp, labial papilla; pf, pharyngeal funnel. 12. As 11 enface; ap, amphidial pore. 13. Infective L3 pharynx. FIG. 14. Genital

primordium

infective

FIG. 15. Tail of ensheathed

L3; vc, ventral cord. infective L3.

FIGS. 16-18. Rectal area: rc, rectal cell; rch, rectal chamber. 16. Late L3. 17. M3 (one subventral Immature adult. FIG. 19. Anterior

end, immature

adult from body cavity: ec, renette cell.

rectal cell not shown).

18.

528

R.J.BALLANTYNE

rectum tubular 26-28 pm long. Phasmids small in midtail region. Rectal cells, accessory primordium, renette cells, coelomocytes and amphidial glands not clearly differentiated. Genital primordium 24-30 pm long with 14 nuclei (Fig. 14) located at 64% of the body length. Differentiation of lateral cord nuclei depended on the age of the larvae. Some larvae exsheathed in culture without any apparent stimulation. Exsheathment was stimulated by mucus from snails (Lymnaea lessoni, Physastra sp., Helix aspersa), slugs (Deroceras Zaeve) and from the alimentary tract of blue-tongued lizards. During exsheathment splits 55-65 pm long occurred in the sheath anterolaterally on each side; the anterior extremity remained intact and the L3 escaped through either split. Entry of L3 into snails was observed through the transparent shell of laboratory-raised L. Zessoni under a stereomicroscope. L3 entered the mantle cavity and then moved up the kidney duct to the pericardial cavity where they became inactive. While in the snail, the renette cells, pharyngeal glands and intestinal cells enlarged. If the snail died, larvae did not escape but died in the pericardial cavity. L3 kept in a snail for 64 days were infective to a lizard. Entry of L3 into the small slug D. Zaeve was studied by dissection and was similar to entry into L. Zessoni. L3 also entered other snails, Physastra sp. and H. aspersa, but appeared to show a preference for L. Iessoni. L3 from all these hosts were infective to blue-tongued lizards. Several D. laeve and H. aspersa from an area known to contain a blue-tongued lizard infected with P. tikquae were negative for P. tiliquae larvae. Method of infection The method of infection in the field was not determined. Lizards caught in the field had adults in the lungs and usually immature adults in the body cavity. The number of immature adults in the body cavity varied from 10 to 70. In one lizard autopsied 6 months after capture there were four adults in the lungs and three dead immature adults in the body cavity. Immature adults in the body cavity and adults in the lungs of wild caught lizards did not prevent experimental infections developing to immature adults in the body cavity. These new immature adults did not enter the lungs. Pre-natal infection did not occur in blue-tongued lizards born in captivity. The faeces of all laboratoryraised lizards were negative and one lizard kept for 4 months and then autopsied was negative. Two lizards strangled at birth by their yolk-sac cord were also negative. Experimental infections in both laboratory-raised lizards 3-9 months of age, and field caught lizards containing adult worms in the lungs and immature adults in the body cavity were established: (i) by feeding laboratory-raised slugs and snails containing L3 to lizards; (ii) orally, by injecting ensheathed L3 down the oesophagus of lizards using a hypodermic

syringe with a blunt needle and minimal fluid; and (iii) by placing lizards in a shallow layer of water containing ensheathed L3 for 12-24 h then examining the fluid for L3. Lizards placed in water presumably became infected orally, not by skin penetration because: lizards were observed licking the water, and L3 were found in the stomach of two lizards autopsied 17 h after licking the water; L3 pipetted on to the dorsal and ventral surfaces of a lizard moved under the scales but did not establish an infection; L3 were not stimulated into activity by the presence of lizards. Development in the lizard Ensheathed L3 exsheathed in the stomach and the body cells increased in size and assumed an appearance similar to that of L3 from paratenic hosts. The actual site and method of entry into the body cavity were not determined, but entry occurred between 24 and 48 h after infection. At this time L3 were found in the stomach, small intestine, stomach lymphatic vessels and free in the body cavity. Larvae were not found in the blood system, liver or lungs. Dead L3 were recovered from faeces following experimental infections. It would appear that L3 entered the body cavity through the stomach wall and that the majority of L3 entering the small intestine passed out with the faeces. Larvae fed on coelomic fluid and were free in the body cavity as they were easily washed out at autopsy. From days 3 to 5 the following changes occurred in L3: the body increased in length (1 to 1.6 mm) and width (30 to 50 pm); the pharynx increased in length and width and the rhabditiform shape became less noticeable; the renette cells enlarged and extended posterior to the level of the pharyngo-intestinal junction; the intestinal nuclei divided several times; and the rectal cells and accessory genital primordium enlarged (Fig. 16). On day 5, the renette cells and pharyngeal glands enlarged, the lateral cord nuclei divided, and the genital primordium divided to form into gonads and gonoducts; at this stage M3 began. The L4 buccal capsule formed outside the L3 buccal tube and became clearly differentiated as the cuticle at the base of the new capsule darkened (Fig. 8). When fully formed, the buccal capsule was 4 pm wide and 6 pm deep, with the basal 4 pm darkened (Fig. 9). The mouth opening was about 2 pm deep. The increased width of the pharynx, particularly in the buccal collar region, and the formation of the L4 buccal capsule outside the L3 buccal tube (but still with pharyngeal tissue surrounding the base of the L4 capsule) produced a large, irregularly shaped opening to the pharyngeal radii. The rectum invaginated into each rectal cell, increasing the size and altering the shape of the rectum (Fig. 17). A lumen formed in the accessory primordium and appeared to open into the rectum. The genital primordium was 180-200 pm long and had differentiated into ovaries, separated by a gonoduct area 65-75 pm long. The posterior extremity of the L4

P. tiIiq~~e:life history and development

tail was pointed and formed internally to the abruptly rounded L3 tail. The L3 cuticle was retained as a sheath by the L4, which showed indistinct longitudinal lateral alae. The L4 lasted about 24 h while the following development occurred: the intestinal nuclei continued to divide; the reproductive system developed rapidly forming distinct areas and the distai end reflexed and grew in the opposite direction; lateral cord nuclei began to differentiate; the pharynx increased in length and width; renette cells remained large and prominent, and the accessory genital primordium decreased in size and at M4 showed no signs of a lumen or opening. During M4 the adult buccal capsule became apparent as a clear globular space around the L4 buccal capsule (Fig. 10). There was no buccal collar, but the pharyngeal entrance enlarged and became funnelshaped (Fig. 11) and the anterior extremity of the pharynx was wider than the buccal capsule. The fully formed buccal capsule was 15 pm wide and 10 pm deep and surrounded by thick body tissue. The cuticle of the buccal capsule wall and pharyngeal entrance was only slightly darkened. Four cephalic and six labial papillae became clearly differentiated on the rim of the buccal capsule (Fig. 12). The canals of the cephalic papillae and amphidial glands were large and formed slight swellings lateral to each group of papillae. The cervical papillae became clearly differentiated laterally, near the level of the nerve ring. The tail was short and near the posterior extremity abruptly tapered to a point. The rectum enlarged and formed into a large flask-shaped chamber (Fig. 18). The L4 cuticle was retained as a very thin sheath, so that two sheaths were present for a short period. The lateral surface of young adults contained a smail (l1.5 pm) longitudinal ala (ridge), which was not apparent in fully developed adults. The reproductive system continued to develop to form two complete systems with opposed uteri, and large gland-like cells became apparent in the lateral cords. The sheaths were shed from days 7 to 8 when the body length was about 2.3 mm. Subsequent development depended on whether adult worms were present in the lungs. If adults were present in the lungs, development ceased when the germinal zone of the reproductive system contained 28-30 germinal nuclei and the immature worms remained in the body cavity. At this stage the body length was about 2.5 mm. If adults were not present in the lungs, division continued in the germinal zone of the reproductive system producing sperm before the worms entered the lungs, between days 12 and 14. ~~Fph~~ogy of i~~~tur~

adults in the body cavity

Body slender 2.3-2.8 mm long, width at pharyngointestinal junction 60-65 pm, tail 115-l 50 ,um long tapering to a point; body cuticle not inflated, lateral cords well developed with small cuticle-lined ducts to the surface; six labial and four cephalic papillae on rim

529

of well developed buccal capsule; pharynx clavate 43@470 pm long; renette cells large (Fig. 19); body muscles evenly developed throughout length of body; two opposed reproductive systems, ovary with 20-30 germinal cells. ~eveIopme~t

of adults in lungs

The route of entry into the lungs was not determined, but worms in the lungs fed on blood. Once in the lungs growth continued with nuclear division occurring only in the intestine and in the germinal cells of the ovaries. The intestinal nuclei divided several times and in the mature adult the intestine could be divided into several regions by variation in thickness and in nuclear number per cross section, The spines and lateral expansions were fully formed 2 days after entry into the lungs when the body length was 4-S mm and ova were ready for fertilization. The anterior body muscles developed in association with the filling and darkening of the spines. The number of large spines was constant, whereas there was a variation in number of small spines. The gonads alternatively produced sperm and ova; sperm were stored in the folded section of the oviduct and ova were fertilized as they passed through the oviduct. All the eggs were retained in utero until they all contained fully formed Ll, and were then laid en masse, when another cycle of development began. Egg-producing worms continued to grow and 17-20 days post-infection were 6.5-8 mm long. The lateral expansions, spines and body muscles when formed restricted the growth of the anterior body, and growth occurred mainly in the posterior body as shown by the position of the vulva changing from 57 to 44% of the body length as worms increased in length from 5 to 12 mm. The renette cells apparently atrophied as the adults developed and were not seen after the spines and expansions formed in either whole worms or sections. Lateral cord development

The lateral cords were not easy to study because the activity pattern of the nuclei produced variable staining reactions. In Ll the cord appeared to originate as a single row of nuclei (Fig. 20). After division of the nuclei at Ml (Fig. 21) thecord was 12-15pm wideand made up of a pattern of recurring nuclei as follows: (i) distinct cells 15-17 pm long by 7-8 pm wide with round nuclei, every second nucleus was preparing to divide; (ii) large (8-9 pm) nuclei 70-80 pm apart and separated from each other by four nuclei from (i); (iii) indistinct dorsal nuclei 60-80 pm apart alternating with (ii); (iv) scattered densely staining nuclei (scn) deep in the cord. During L2 the cord remained clearly defined (Fig. 22) and was similar to that seen at M 1 except that: (a) every second nucleus from(i) above had divided giving a distinct pair of nuclei (pn); (b) the indistinct dorsal nuclei from (iii) above became more distinct; (c) there appeared to be more scattered nuclei (scn) from (iv)

R. J. BALLANTYNE

530

c CJ 0 0

--\_ scn

‘-\

20

21

23

24

FIGS. 20-24. Lateral cord, parasitic generation. 20. Ll recently hatched. 21. Ml: i, ii, iii see text; scn, scattered nuclei. 22. M2: pn, paired nuclei. 23. Mid L4. 24. Immature adult from body cavity: gc, gland cell; gn, gland nucleus; gv, gland vacuole; mc, mid-cord nuclei; p. cuticle-lined opening; scn, scattered nuclei; sn, surface nuclei.

above; (d) the pattern of the nuclei had altered in relation to the indistinct dorsal nuclei (iii) above, suggesting the indistinct dorsal nuclei were fixed in position. In infective L3 the cord was similar to that of L2 except it was 4.5 pm wide and the nuclei were more elongate. As L3 aged it became progressively harder to distinguish a nuclear pattern. During the parasitic L3 there was a gradual increase in the size of the cord and nuclei. As M3 approached, nuclei changed rapidly in position, size and staining intensity. It was not possible to interpret the resultant patterns in the material available, so the destination of nuclei could not be determined. At M3 the cord was about 20 pm wide and probably all nuclei had divided or were dividing. During the early L4, and without any increase in the size of the cord, the nuclei differentiated into the final pattern (Fig. 23). All nuclei, except those deep in the cord, which belonged to the future gland cells, stained intensely. The nuclei were characteristically arranged in recurring groups of four nuclei with a vacuole (gv) anterior to the gland nucleus (gn) in each group. Each group was the opposed mirror image of the group at either side. Each group consisted of one gland cell (gn), one mid-cord nucleus (mc) and two surface nuclei (sn). During L4 the cord widened to 30 pm with a slight increase in nuclear size. The gland vacuole developed

into a duct to the surface and became cuticle-lined at M4. While the immature adult remainedensheathed the gland cells became prominent especially in the anterior half of the body (Fig. 24). As the immature adults grew in the body cavity the surface nuclei lost their pattern and the scattered dorsal and ventral nuclei became apparent as cells rather than nuclei. In the adults, the shape of the cord was altered by the development of the anterior body muscles and the reproductive system. Some gland cells near spines appeared to be empty whereas those of the posterior body were flattened by the reproductive system and it was difficult to ascertain their contents. DISCUSSION This study used a simple staining technique to produce extensive morphological detail in animals that are often regarded by zoologists as having limited taxomonic characters. The technique has the disadvantage of being best suited to live or recently fixed material, although secondary fixation should enhance the staining properties of old and/or poorly fixed material. The use of Carnoy’s fixative and acetic orcein staining allowed the morphology of the papillae, amphidial glands, renette cells and the reproductive system to be studied during all stages of development; the contrast between the nuclei and the surrounding tissue was excellent.

P. tiliquae:l&ehistory and development

The morphology of the free-living generation of P. although Chu (1936), Chabaud, Brygoo & Petter (1961), Yuen (1965) and Baker (1979) considered Rhubdius spp. to have 10 pairs of genital papillae in the freeliving male. The extra pair of papillae described by these authors is in all probability the phasmids which appear papillae-like and occur laterally between the 5th and 6th pair of papillae. The above authors do not mention the presence ofphasmids in males. Free-living Rhabdiasidae can be readily differentiated from freeliving Strongyloididae by the presence of the welldeveloped pharyngeal buccal collar in strongyloidids and by the male genital papillae (Little, 1966); strongyloidids have six pairs plus a median domeshaped papilla anterior to the cloaca, whereas rhabdiasids have nine pairs. The renette cells became very prominent when L3 entered snails and slugs and during development to adults in the body cavity of lizards, and they then atrophied during adult life in the lungs. This development may have been associated with osmoregulation and exsheathment in the developing worms environment. Once adults entered the lung environment the activity was not required. The amphidial glands became prominent and glandular in all stages prior to each moult, suggesting a secretory involvement in moulting. The pharyngeal glands may have been associated with moulting as suggested by Singh & Sulston (1978) but they were also active during feeding, especially in parasitic stages. Luc, Taylor & Netscher (1979) reviewed the terms used for in utero development in nematodes and recommended that endotokia matricida should be used for female encystment of larvae and matricidal hatching be used for intrauterine hatching leading to the destruction of the female by the larvae. In their discussion they did not address the situation of obligatory intrauterine development as in P. tiliquue where larvae cannot escape and must remain in the body for food, as they die if removed to the surrounding culture. The concept of intrauterine and intrabody development is interesting as Duggal (1978) suggested that in PanagrelIus redivivus the hatched Ll had to produce a stimulus that induced the uterus to contract and expel the larvae. In P. tiliquae it would appear that the evolution of intraparent development has progressed through several steps: reduction in size of the vulva making expulsion of eggs and larvae difficult; loss of the stimulus that causes expulsion of Ll from the uterus; and dependence on parent tissue for food as larvae do not survive if released into the culture environment. The alternation of parasitic and free-living generations is an unusual animal characteristic. Although superficially the generations appear different, closer examination shows that the parasitic form can be viewed as a modified version of the free-living. The pattern of head papillae, and the structure of the amphidial glands, excretory system and pharyngotiliquae is similar to that of other rhabdiasids,

531

intestinal junction are essentially similar in both generations. The stoma is similar in both generations until M3, when in the parasitic form the globular buccal capsule begins to form. The parasitic form loses its rhabditiform pharynx at M2 and the intestine becomes multinucl~te during development in the lizard. In the parasitic form the rectum invaginates into the three rectal glands at M3, and at M4 the rectum changes from cylindrical to flask-shaped. The pseudo-development of the accessory primordium during M3 in the parasitic generation may be a sign of maleness or confusion in the hermaphroditic form with the lumen representing the spicule chamber. The reproductive systems differ in that there are separate sexes in the free-living form; the vulva is fully functional in the parasitic female; and most areas of the system in the parasitic form are larger than their freeliving counterpart. The major difference is that development in the lateral cord area stops in the freeliving genemtion prior to M4, which is similar to that found in Cuenorhabditis eleguns by Sulston & Horvitz (1977), whereas in the parasitic generation the cells continue to develop during M4 and the early adult stage forming large gland cells and cuticle-lined ducts to the surface. The gland cells appear homologous with the seam cells of C. eieguns as described by Sulston & Horvitz (1977), and appear to be associated with the production of the spines and enlarged muscles of the anterior body. The function of the cuticle-lined ducts to the surface is unknown but they occur in all rhabdiasids. Singh & Sulston (1978) have demonstrated the need for seam cells for lateral alae production in C. eleguns. In P. tiliquue the lateral alae became smalier as worms develop from L3 to adults and in fully formed adults they are absent. The overall life cycle pattern (Fig. 25) shown by P. tiliquue is similar to that observed in other rhabdiasids (Baker, 1979) although skin penetration appears to be absent and paratenic hosts may play an important role in the life cycle. P. tiliquue appears to be the only species where immature adults are suppressed in the body cavity of the definitive host. The study has shown that P. tiliquae has a very successful life cycle with all hosts examined containing worms. This success appears to stem from several factors. The organization of the reproductive system of the parasitic female is such that eggs are passed in batches and if lizard faeces were deposited in a moist environment a large number of eggs would develop. The free-living female has evolved so that eggs hatch and larvae develop in utero; in this way only a small number of larvae are produced per female but they all reach the infective stage. Infective larvae may then enter a paratenic host, thus increasing their survival potential and their chances of entering a lizard when feeding. The above development from eggs to infective L3 may explain why direct and repeated free-living development do not occur in species with obligatory matricidal hatching. The retention of immature adults in the body cavity of lizards if adult worms are present

532

R.J.

Exsheath

BALLANTYNE

in oesophagus

ar stomach

--.

\

I

Paratenic host

/

Exsheath

L3 (ensheathed 84-96hI t 12

I t2

Ew Adult

1 Faeces Eggs

0

I_I-2e3-e4-Adult

Li

8

Ll (24-36

2 -3

-

/

4-c-

t Fertilization Aduit

h)

FIG. 25. Life cycle: diagrammatic.

P. riliquue: life histor ‘y and development

in the lungs is an interesting development. These worms have undergone their full complement of moults but do not begin sperm production as they would if they were entering the lungs. It is not clear why development is arrested and it is not known if the worms have the ability to resume development and enter the lungs. The arrested development may be a mechanism to minimize host immunity and ensure the lizard is always infected with worms that enter the lungs as appropriate and begin egg production. The controlled entry of adults to the lungs where worms feed on blood may ensure that parasite stimulation of the host remains low and reasonably constant, thereby producing a low immunity threshold. Acknowledgements1 am indebted to many people and institutions for their assistance with this study. The bench work was carried out in the Parasitology Department of the University of Queensland with the aid of Department funds and a Commonwealth Postgraduate Award (1968-1970). The Zoology Department of the Australian National University iiovid& office space and library facilities during mv tenure of a Visiting Fellowship in 1989 and Charles Sturt University supported-my PEP Lkave in 1989. I thank John Pearson, Warwick Nicholas and Lesley Ballantyne for their assistance with various drafts of the manuscript. REFERENCES BAKER M. R. 1978. Morphology and taxonomy of Rhabdias spp. (Nematoda: Rhabdiasidae) from reptiles and amphibians of southern Ontario. Canadian Journal of Zoology 56: 2127-2141. BAKERM. R. 1979. The free-living and parasitic development of Rhabdius spp. (Nematoda: Rhabdiasidae) in amphibians. Canadian Journal of Zoology 57: 161-178. BALLANTYNER. J. 1986. Pneurnonema tiliquae (Nematoda:

533

Rhabdiasidae): a reappraisal. In: Parasite LivesPapers on Parasites, Their Hosts and Their Associations: To Honour J. F. A. Sprent (Edited by CREMIN M., DOBSONC. & MOOREHOUSED. E.), pp. 41-55. University of Queensland Press, St Lucia. BALLANTYNER. J. 1991. Post-embryonic development of the reproductive system of Pneumonema tiliquae (Nematoda: Rhabdiasidae). International Journal for Parasitology 21: 535-547. CHABAUD A. G., BRYGOO E. R. & PETTER A. J. 1961. Description et caracters biologiques de deux nouveaux Rhabdias malagaches. Annales de Parasifologie Humaine et Cornparke 36: 752-763. CHU T. 1936. A review of the status of the reptilian nematodes of the genus Rhabdias with a redescription of Rhabdias ficovenosa var. catanensis (Rizzo, 1902) new rank. Journal of Parasitology 22: 13G139. DUGGAL C. L. 1978. Initiation of copulation and its effect on oocyte production and life span of adult female Panagrellus redivivus. i%??IQtO~OgiCQ 24: 266-276. HIRSCHMANNH. 1962. The life cycle of Ditylenchus triformis (Nematoda: Tylenchida) with emphasis on postembryonic development. Proceedings of the Helminthological Society of Washington 29: 30-43. LITTLE M. D. 1966. Comparative morphology of six species of Strongyloides (Nematoda) and redefinition of the genus. Journal of Parasitology 52: 6S84. Luc M., TAYLOR D. P. & NETSCHERC. 1979. On endotokia matricida and intrauterine development and hatching in nematodes. Nematoiogica 25: 268-274. SINGH R. N. & SULSTONJ. E. 1978. Some observations on moulting in Caenorhabditis elegans. Nematologica 24: 63-71. SULSTONJ. E. & HORVITZ H. R. 1977. Postembryonic cell lineage of the nematode, Caenorhabditis e/e&s. Developmental Biology 56: 1 l&156. YUEN P. H. 1965. Some studies on the taxonomy and development of some rhabdiasoid and cosmocercoid nematodes from Malayan amphibians. Zoologischer Anzeiger 174: 275-298.