MO R P H O G E N E S I S OF CONTRACAECUM RUDOLPHII (N EMATODA: ASCARIDOIDEA), A PARASITE OF FISH-EATING BIRDS,
IN ITS COPEPOD PRECURSOR AND FISH INTERMEDIATE HOSTS
BARTLETT C.M.*
Summary :
Eggs of Controcaecum rudolphii obtained from female worms in double-crested cormorants (Phalacrocorax auritus) in Nova Scotia, Canada, hatched in 9-17 days in sea water at 15-20 °C. The newly-emerged, free-living (and presumably second-stage) larva is described in detail, as are larvae from experimentally-infected copepods (Tigriopus sp.), amphipods (Gammarus sp.), and fish (Lebistes reticulatus, Fundulus heteroclitus). Copepods are considered precursor hosts and morphogenesis of the parasite in them primarily involved body size, the ventricular appendix, and the excretory system. Infection of amphipods and fish was much more successful when invading larvae were from copepods than when they were free living; amphipods served as paratenic hosts and fish as intermediates. In fish, second-stage larvae 2 0 or fewer days postinfection were within the intestinal wall. At 4 4 or more days postinfection, larvae were considered third stage; all were in the abdominal cavity, many within a closely adhering « sleeve » of material resembling cuticle. The longest larvae (3,1-3.9 mm) in fish were associated with the oldest infection (152 d), suggesting that larvae continue to grow for a considerable period of time;
growth was also found to be asynchronous, however.
KEYWORDS : nematode, parasite, Controcaecum spiculigerum, Ascaridoidea, morphogenesis, intermediate, paratenic, cormorant, bird.
Résumé : MORPHOGENÈSE DE CONTRACAECUM RUDOLPHII (NEMATODA : ASCARIDOIDEA), PARASITE DES OISEAUX DE MER, CHEZ SON HÔTE PRÉCURSEUR, LE COPÉPODE, ET SON HÔTE INTERMÉDIAIRE, LE POISSON
La larve de deuxième stade de Contracaecum rudolphii, de connaissance récente et vivant sans hôte, est décrite en détail ainsi que les larves venant de copépodes infectés en laboratoire (Trigriopus sp.), d'amphipodes (Gammarus sp.) et de poissons (Lebistes reticulatus, Fundulus heteroclitus). La morphogenèse du parasite dans le copépode, considéré comme hôte précurseur, inclut la taille, l'appendice ventriculaire et le système excrétoire.
L'infection des amphipodes et des poissons est beaucoup plus importante lorsque les larves proviennent des copépodes que lorsqu'elles vivent sans hôte; les amphipodes ont servi d'hôtes paraténiques et le poisson d'hôte intermédiaire. Chez le poisson, les larves ont atteint le troisième stade 44 jours ou plus après l'infection; toutes se trouvaient dans la cavité abdominale, plusieurs à l'intérieur d'un « sac » étroitement ajusté et d'un matériau évoquant une cuticule. Chez le poisson, les larves les plus longues mesuraient de 3,1 à 3,9 mm (152 d). Il est probable que les larves continuent à grandir chez le poisson pendant une longue période mais la croissance en est cependant asynchrone.
MOTS CLES : nématode, parasite, Contracaecum spiculigerum, Ascaridoidea, morphogenèse, intermédiaire, paraténique, cormoran, oiseau.
I N T R O D U C T I O N
A
scaridoid n e m a t o d e s , c o m m o n parasites o f the s t o m a c h or intestines o f vertebrates, are often rather plastic with respect to transmis- sion (Anderson 1992). Contracaecum rudolphii Hart- wich, 1964, for e x a m p l e , u n d e r g o e s « partial d e v e l o p - ment » in c o p e p o d s followed by later d e v e l o p m e n t in fish to the stage infective to the avian final host (e.g.c o r m o r a n t s , Phalacrocorax s p p . ) ( H u i z i n g a 1966) (note: Hartwich (1964) proposed C. rudolphii as a n e w n a m e for the Contracaecum sp. found mainly in cor- morants; Huizinga (1966) referred to his s p e c i m e n s as Contracaecum spiculigerum). H o w e v e r , fish can also b e infected directly, i.e. without the a g e n c y o f c o p e - p o d s (Huizinga 1966), s u g g e s t i n g d e v e l o p m e n t in
* Biology, University College of Cape Breton, PO Box 5300, Sydney, Nova Scotia, Canada B1P 6L2.
Phone: 902-563-1624 - Fax: 902-562-01 19 - e-mail:
c o p e p o d s may b e trivial. T h e present study sought to clarify our understanding o f these events through a detailed examination o f the parasite's m o r p h o l o g y in c o p e p o d s , amphipods, and fish. It w o r k e d from the premise o f Huizinga ( 1 9 6 6 ) that a second-stage larva e m e r g e s from the egg o f C. rudolphii (although stu- dies o n related s p e c i e s [e.g. Koie and Fagerholm 1 9 9 3 ; Measures and Hong 1 9 9 5 1 suggest this requires re-exa- mination using transmission electron microscopy). Mor- p h o g e n e s i s o c c u r r e d in b o t h c o p e p o d s and fish, enabling their designation as precursor and interme- diate hosts, respectively.
M E T H O D S A N D MATERIALS
L
ive females o f C. rudolphii w e r e r e m o v e d from the proventriculus o f double-crested cormorants (Phalacrocorax auritus) shot o n Delorier Island ( 4 5 ° 31'N, 61° 0 6 ' W ) near Arichat or on Red Islands ( 4 5 ° 4 8 ' N , 6 0 ° 4 6 ' W ) near J o h n s t o w n , Nova Scotia,Parasite, 1996, 4, 367-376
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Mémoire
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1996034367
B A R T L E T T C M .
Canada, in J u n e and July, 1992 and 1 9 9 3 . Eggs w e r e dissected from females and incubated at 10°, 15°. and 20 °C in Syracuse dishes ( 4 x 4 c m ) containing sea water that had b e e n passed through a millepore filter (0.47 pm grid size).
O n c e larvae hatched, various e x p e r i m e n t s (outlined b e l o w ) w e r e c o n d u c t e d . C o p e p o d s (Tigriopus sp.), a m p h i p o d s ( G a m m a r u s s p . ) , and larval fish ( m u m m i - c h o g , Fundulus beteroclitus, 0.75-1,25 cm l o n g ) used in the experiments were collected in a tidal marsh near Port Morien ( 4 6 ° 08'N, 5 9 ° 5 2 ' W ) . Nova Scotia (note:
50 c o p e p o d s , 50 amphipods, and 5 0 larval mummichog w e r e also e x a m i n e d as controls; n e m a t o d e larvae were not present). G u p p i e s (Lekistes reticulatus) w e r e pur
c h a s e d from a local pet store.
Experiments A-C e x p o s e d recently-emerged larvae to c o p e p o d s (A), a m p h i p o d s ( B ) , and fish ( C ) . Hundreds o f larvae were placed in filtered water in each o f nume
rous Syracuse dishes and then d o z e n s o f c o p e p o d s , four amphipods, or o n e fish w e r e added to e a c h dish.
Crustaceans and m u m m i c h o g w e r e e x p o s e d in sea w a t e r : guppies in p o n d water. After 1 d. amphipods and fish w e r e transferred to clean water in crystalliza
tion dishes ( 1 0 0 x 50 m m ) and e x p o s u r e dishes e x a mined to determine if all larvae had b e e n eaten (they had). Crustaceans w e r e maintained at 15° C and fish at 22° C. Amphipods w e r e fed pulverized trout c h o w and fish w e r e fed tropical fish food flakes.
Experiments D and E e x p o s e d infected c o p e p o d s to amphipods ( D ) and fish ( E ) . D o z e n s o f infected c o p e pods (from Experiment A) w e r e placed in filtered sea or pond water (as appropriate) in e a c h o f numerous Syracuse dishes and then four a m p h i p o d s or o n e fish w e r e added to e a c h dish. At 1 d. a m p h i p o d s and fish were transferred to clean water and maintained as pre
viously outlined; e x p o s u r e dishes w e r e e x a m i n e d to determine if all c o p e p o d s had b e e n eaten (they had).
Experiment E involved exposure of fish to infected amphi
pods. Ten amphipods (from Experiment 15. and pre
sumed to contain larvae but not verifiable) were placed in filtered sea water in each o f numerous Syracuse dishes and then one fish was added to each dish. At 1 d, fish were transferred to clean water and maintained as pre
viously outlined; exposure dishes were examined to determine if all amphipods had been eaten (they had).
At various times postexposure, crustaceans and fish were killed (via decapitation) and dissected in saline on micro
scope slides. Larvae from crustaceans and fish 20 or fewer days postinfection were fixed by adding an equal volume of 10 % formalin to the saline; a petroleum-ringed cover- glass was then placed over the mixture and larvae stu
died. Larvae from other fish were fixed in a hot solution of 5 % glycerin/70 % alcohol and studied in glycerin. A m i c r o s c o p e e q u i p p e d with differential interference contrast lighting was used to study larvae.
RESULTS
HATCHING O F EGGS
Larvae first e m e r g e d from eggs at 9 d in water at 2 0 °C and at 11 or 17 d at 15 °C ( e g g s from w o r m s from two c o r m o r a n t s ) . At 10 °C, eggs had not e m b r y o n a t e d by 4 0 d and w e r e discarded. Eggs stored at 5 °C for 10 months did not e m b r y o n a t e after b e i n g m o v e d to 20 °C for 15 d and the e x p e r i m e n t w a s terminated.
EXPERIMENTS A - F
Experiment A: At 1-2 d postexposure, about half o f the h u n d r e d s o f c o p e p o d s e x a m i n e d c o n t a i n e d larvae ( p r e v a l e n c e and intensity not precisely d e t e r m i n e d ) . Larvae were motile and generally free in the abdominal h a e m o c o e l w h e r e they w e r e readily visible. Live, infected c o p e p o d s w e r e noticeably lethargic in c o m parison to c o p e p o d s without larvae. Many infected c o p e p o d s died within 2-3 d postinfection; larvae within them frequently remained motile for an additional 2 d.
Experiment B: O n e larva WAS found in o n e a m p h i p o d and n o n e in 57 others ( T a b l e I ) .
Experiment C: T h r e e o f 6 guppies contained larvae (all motile), n o n e o f 2~ m u m m i c h o g did ( T a b l e I ) . At 2 0 d postinfection, larvae w e r e within the intestinal wall. At 53 and 66 d, they w e r e in the abdominal cavity. S o m e o f t h e latter w e r e in a d e l i c a t e , c l o s e l y a d h e r e n t
« sleeve » o f indeterminate nature resembling s h e d cuticle with a delicate fibrous texture (Table I ) . In addi
tion, n u m e r o u s host cells s o m e t i m e s surrounded these
« sleeves » or larvae by themselves, giving the appea
rance o f a delicate capsule.
E x p e r i m e n t D: Five o f nine a m p h i p o d s c o n t a i n e d larvae (all motile) ( T a b l e I ) .
Experiment E: Four o f four m u m m i c h o g c o n t a i n e d larvae, as did six o f s e v e n guppies ( T a b l e I ) . All larvae w e r e motile. T h o s e in fish e x a m i n e d 10 or fewer days postinfection w e r e in the intestinal wall. At later times ( 4 4 - 1 5 2 d ) , larvae w e r e in the abdominal cavity, s o m e free and others within « sleeves », and s o m e also sur
rounded by host cells ( T a b l e I ) .
Experiment F: T w o o f two m u m m i c h o g c o n t a i n e d larvae, as did two o f seven guppies (Table I ) . All larvae w e r e motile. T h o s e in fish e x a m i n e d six o r fewer days postinfection w e r e in the intestinal wall and those in fish at 96 and 132 d w e r e in the abdominal cavity. As in Experiment E, s o m e larvae w e r e within a « s l e e v e » and s o m e free.
D E S C R I P T I O N O F S E C O N D - S T A G E LARVAE RECENTLY EMERGED FROM E G G S
Larvae (Fig. 1) e m e r g i n g from e g g s with retained, moulted cuticle forming loose, striated sheath slightly
3 6 8 . Parasite. 1996, 4, 367-376
MORPHOGENESIS OF CONTRACAECUM SPICULIGERUM
wider and quarter to half longer than larva within. Ante- rior extremity o f sheath generally with several, irregularly digitiform e x t e n s i o n s and larvae attaching, by way o f anterior extremity, to b o t t o m s o f dishes and waving vigorously in medium. Posterior e n d o f sheath attenuated
and sharply pointed. At 15° C , larvae remaining highly active for approximately 10 d after which b e c o m i n g increasingly less motile, dying b e t w e e n 3 0 and 4 0 d.
Free-living, r e c e n t l y - e m e r g e d ( < 2 4 h r ) a n d 10 cl o l d larvae: Long, s l e n d e r ( T a b l e I I ) , with striated c u t i c l e .
a Experiment B, amphipods exposed directly to recently-emerged, second-stage larvae.
b Experiment C, mummichog and guppies exposed directly to recently-emerged, second-stage larvae.
c Experiment D, amphipods exposed to copepods containing second-stage larvae.
d Fxperiment E, mummichog and guppies exposed to copepods containing second-stage larvae.
e Experiment F, mummichog and guppies exposed to amphipods presumed to contain second-stage larvae.
f « sleeve » = closely adhering, sleeve-like structure resembling cuticle and often with fibrous texture.
Note: Larvae at 20 or fewer days in second stage; larvae at 44 or more days in third stage.
Table I. — Numbers of amphipods (Gammarus sp.) and fish (mummichog, Fundulus heteroclitus; guppies, Lebistes reticulatus) infected with larvae of Contracaecum rudolphii at different times after exposure to larvae in different experiments (B-F); numbers and locations of larvae and condition of fish also indicated.
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D a y s p o s t - e x p o s u r e
N o . i n f e c t e d / N o . e x p o s e d
N o . l a r v a e f o u n d
C o n d i t i o n o f h o s t p r i o r t o d i s s e c t i o n a n d c o m m e n t s o n l o c a t i o n o f l a r v a e E x p e r i m e n t B "
a m p h i p o d 3 1/1 1 a m p h i p o d l i v e ; larva in h a e m o c o e l
a m p h i p o d s 2 - 3 3 0 / 5 7 — a m p h i p o d s live
E x p e r i m e n t C ' '
m u m m i c h o g 1-12 0 / 2 7 — s o m e fish live, s o m e d e a d
g u p p y 2 0 1/1 2 fish d e a d ; l a r v a e in intestinal wall
g u p p y 3 4 0 / 1 — fish d e a d
g u p p y 3 7 0 / 1 — fish d e a d
g u p p i e s 5 3 1/2 2 fish d e a d ; l a r v a e in a b d o m i n a l c a v i t y a d j a c e n t
t o i n t e s t i n e within - s l e e v e J c o v e r e d b y host ,., ,i l.-
g u p p y 6 6 1 1 1
c e n s
fish l i v e ; larva in a b d o m i n a l c a v i t y w i t h a f e w a d h e r e n t h o s t c e l l s b u t n o t w i t h i n a • s l e e v e » E x p e r i m e n t D '
a m p h i p o d s 3 2 / 2 1,6 a m p h i p o d l i v e ; l a r v a e in h a e m o c o e l
a m p h i p o d s 14 1/5 1 a m p h i p o d l i v e ; larva in h a e m o c o e l
a m p h i p o d 2 2 1/1 5 a m p h i p o d l i v e ; l a r v a e in h a e m o c o e l
a m p h i p o d 3 3 1/1 1 a m p h i p o d l i v e ; larva in h a e m o c o e l
E x p e r i m e n t E ' '
m u m m i c h o g 4 1/1 11 fish m o r i b u n d ; l a r v a e in intestinal w a l l
m u m m i c h o g 5 1 l >10() fish d e a d ; l a r v a e in intestinal w a l l
m u m m i c h o g 8 1 l 112 fish d e a d ; l a r v a e in intestinal wall
m u m m i c h o g 1 0 1 1 7 0 fish d e a d ; l a r v a e in intestinal wall
g u p p y 44 1 l 1 0 fish d e a d ; l a r v a e within fat a r o u n d
i n t e s t i n e ( p r e s e n c e o r a b s e n c e o f • s l e e v e - n o t d e t e r m i n e d )
g u p p y 4 5 1/1 5 fish d e a d ; l a r v a e within a b d o m i n a l cavity, o n e
within • s l e e v e • a n d four free
g u p p i e s 8 7 2 / 2 3, i o o n e fish live, o n e d e a d ; larvae e i t h e r c o v e r e d
in host c e l l s o r w i t h i n • s l e e v e •
g u p p y I l i 1/1 ì fish d e a d ; larva free within fat a r o u n d i n t e s t i n e
g u p p i e s 152 1/2 1 9 fish l i v e ; l a r v a e free within fat a r o u n d
i n t e s t i n e a n d free in a b d o m i n a l c a v i t y E x p e r i m e n t F1'
m u m m i c h o g 3 1/1 1 fish d e a d ; larva in intestinal w a l l
m u m m i c h o g 6 1/1 1 6 fish d e a d ; l a r v a e in intestinal wall
g u p p y 5 6 0 / 1 — fish d e a d
g u p p i e s 7 6 0 / 2 — o n e fish live, o n e d e a d
g u p p y 8 6 0 / 1 — fish d e a d
g u p p i e s 9 6 1/2 2 fish l i v e ; l a r v a e within • s l e e v e • with a t t a c h e d
h o s t c e l l s within fat a r o u n d i n t e s t i n e
g u p p y 132 1/1 1 fish l i v e ; larva w i t h i n m e s e n t e r y a n d o b s e r v e d
t o pull itself free o f - s l e e v e • w h e n p l a c e d in s a l i n e
P r o m i n e n t , h y a l i n i z e d , c u t i c u l a r t o o t h p r e s e n t o n m o r e dilated for anteriormost 5 p m than remainder o f c e p h a l i c surface ventral to oral opening. Oral o p e n i n g l e n g t h . Cuticular t u b e c o n t i n u i n g p o s t e r i o r l y a n d leading into tube o f hyalinized cuticle ; latter generally a p p e a r i n g to lack i m m e d i a t e l y a d j a c e n t tissue for
Figs. 1-4. — Contracaecum rudolphii, second-stage larvae (free-living, from copepods (Tigriopus sp.), and from amphipods (Gammarus sp.)). Fig. 1. Larva recently emerged from egg. Fig. 2. Larva from copepod 42 hr postexposure. Figs. 3, 4. Larvae from amphipods exposed to infected copepods 14 and 22 d previously, respectively.
Parasite, 1996, 4, 367-376 370
BARTELETT C.M.
MORPHOGENESIS O F CONTRACAECVM SPICVUGESVM
approximately 15-20 p m b e f o r e b e c o m i n g surrounded by delicate, thin wall; together t h e s e comprising o e s o - p h a g u s . P r e s u m p t i v e ventriculus m a r k e d b y slight increase in width o f wall at posterior e n d o f o e s o - phagus. Ventricular-intestinal junction readily a p p a - rent. Presumptive ventricular a p p e n d i x present as deli- cate c o l u m n o f narrow cells adjacent to ventral side o f anteriormost intestine a n d e x t e n d i n g posteriorly for a v e r a g e o f 7 7 pm at 2 4 hr a n d 8 4 p m at 10 d. Pre- sumptive intestinal c a e c u m not distinguishable. Intes- tine generally containing large clear areas in anterior third a n d small, round, b r o w n particles throughout remainder. Short, narrow constriction joining intestine to rectum. Rectum joined by narrow canal to anus. Ner- v o u s a n d e x c r e t o r y systems not discernable. Posterior e n d o f larva digitiform with bluntly r o u n d e d extremity.
D E S C R I P T I O N S O F S E C O N D - S T A G E LARVAE FROM C R l I S T A C K A N S
Larvae from c o p e p o d s (Fig. 2 ) : Larvae 4 2 hr p o s t e x - posure (Experiment A) wider and longer than recently- e m e r g e d larvae ( T a b l e I I ) . O t h e r characteristics: cuticle delicately striated; e x c r e t o r y pore visible on c e p h a l i c surface ventral to cuticularized t o o t h ; o e s o p h a g e a l wall a n d ventriculus m o r e a p p a r e n t than in recently- e m e r g e d larvae; ventricular a p p e n d i x digitiform, exten- ding posteriorly for an average o f 102 pm from ven- tricular-intestinal junction a n d containing several small, scattered c e l l s ; presumptive intestinal c a e c u m m a r k e d b y small swelling anterior to ventricular a p p e n d i x a n d latter containing few small, scattered c e l l s ; nerve ring
apparent at approximately mid length o f o e s o p h a g u s ; prominent cell with large nucleus present in p s e u d o - c o e l o m slightly posterior to tip o f ventricular a p p e n d i x and not appearing associated with any other structure (although presumably cell o f e x c r e t o r y gland).
Larvae from a m p h i p o d s : Larva 3 d p o s t e x p o s u r e ( T a b l e I, E x p e r i m e n t B ) similar in length a n d width ( T a b l e II) and morphologically to larvae from c o p e - pods. Larvae 14 and 22 d p o s t e x p o s u r e ( T a b l e I, E x p e - riment D ) similar in length a n d width to larvae from c o p e p o d s ( T a b l e II) but differing slightly as follows:
o e s o p h a g u s and ventriculus m o r e apparent; ventricular a p p e n d i x a n d presumptive intestinal c a e c u m slightly larger a n d p a c k e d with small cells with i n c o n s p i c u o u s b o r d e r s ; in larva at 14 d (Fig. 3 ) , cell with large nucleus present in p s e u d o c o e l o m posterior to tip o f ventricular a p p e n d i x but appearing not associated with any o t h e r structure; in larva at 22 d (Fig. 4 ) , this cell associated with e x c r e t o r y gland o f excretory system.
D E S C R I P T I O N S O F LARVAE FROM FISH
At 8 d, larvae (from m u m m i c h o g ; T a b l e I, Experi- ment E ) similar in size a n d m o r p h o l o g y (Fig. 5 ) a n d slightly longer a n d wider than recently-emerged larvae or larvae from crustaceans ( T a b l e I I ) . O t h e r differences including: cuticle thin with delicate transverse striations;
intestinal c a e c u m apparent as tiny anterior projection f r o m v e n t r i c u l a r - i n t e s t i n a l j u n c t i o n ; v e n t r i c u l a r a p p e n d i x slightly longer a n d containing, in posterior third, large cell with prominent n u c l e u s ; intestinal wall containing small round cells.
Second- Second-
stage stage Second Second Second- Second- Third- Third-
larvae" larvae" stage- stage stage stage stage stage Third-stage
24 hr 10 d larvae larvae larvae 14 larva larva larva larva
after after ¡2 hr 3 d & 22 d 8 d 45 d 53 d 66 d
emergence emergence in copepods in amphipod in amphipod in mummichog in guppy in guppy in guppy from eggs from eggs (Experiment A) (Experiment B ) (Experiment D) ( Experiment E ) < Experiment E) (Experiment E) ( E x p e r i m e n t C )
N 10 10 12 1 1. 1 10 1 1 1
Total length 23H 245 365 310 340. 385 407 860 1,600 2,700
(223-255) (215-285) (318-417) (370-470)
Maximum 14 14 17 17 17, 18 19 35 70 110
width (13-15) (13-15) (13-20) (18-22)
Nerve ring — — 52 (32-64) 53 —, 55 54 (45-63) 100 110 140
Length oeso- 77 84 102 100 83. 85 98 (90-110) 142 260 310
phagus r 5 - 8 5 ) (75-90) ( 9 2 - 1 1 8b)
Length — — c 13 15. 15 16 (12-21) 30 20 20
ventriculus
Length ventr. 35 26 (20-33) — 50 53. 60 72 (57-93) 140 240 260
appendix (25-53)
Length intes- — — — — — , — — 40 120 140
tinal caecum
Excretory cell — — — 165 160. 180 198 400 680 1.150
(180-220)
Anus 23 (22-25) — 49 (44-54) 25 40. 47 35 (27-45) 54 70 110
" free-living.
'' includes ventriculus.
( included in length o f oesophagus
T a b l e I I . — M o r p h o m e t r i e s o f l a r v a e o f Contracaecum rudolphii at v a r i o u s t i m e s after e m e r g e n c e from e g g s o r p o s t i n f e c t i o n in c o p e p o d s ( Tigriopus s p . ) , a m p h i p o d s (Gammanis s p . ) , o r fish ( g u p p i e s , Lebistes reticulatus; m u m m i c h o g , Fundulus beteroclitus).
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At 2 0 d, larvae (from g u p p y ; T a b l e I, Experiment C) slightly contracted in a p p e a r a n c e but otherwise similar to larvae at 8 d.
At 4 5 d, larva (from g u p p y ; T a b l e I, Experiment E ; Fig. 6 ) slightly longer and wider than larvae at 8 d (Table II). Other differences including: cuticle with pro-
372 Mémoire Parasite, 1996, 4, 367-376
Figs. 5-7 — Contracaecum rudolphii, larvae from fish (mummichog, Fundulus heteroclitus; guppies, Lebistes reticulatus) Fig- 5 Larva from mummichog 8 d postinfection. Fig. 6. Larva from guppy 45 d postinfection. Fig. 7. Larva from guppy 53 d postinfection.
MORPHOGENESIS O F CONTRACAECUM SPICULEGERUM
minent transverse and delicate longitudinal striations;
c e p h a l i c e x t r e m i t y with four p r o m i n e n t p a p i l l a e ; narrow, non-striated wall o f tissue surrounding tube o f hyalinized cuticle i m m e d i a t e l y p o s t e r i o r to b u c c a l region (an area previously lacking visible tissue w a l l ) ; intestinal c a e c u m extending halfway to nerve ring;
large cell in ventricular appendix containing numerous round b o d i e s (rather than n u c l e u s ) ; nucleus o f cell o f excretory gland with irregular margin and containing two round bodies. Larva not within « sleeve ».
At 53 d, larva (from g u p p y ; T a b l e I, Experiment C;
Fig. 7 ) about twice as long and wide as larva at 4 5 d (Table II). Other differences including: lumen apparent within ventricular a p p e n d i x ; intestinal c a e c u m and intestinal wall containing faintly defined, large cells.
Larva within « sleeve ».
At 6 6 d, larva (from g u p p y ; T a b l e I, Experiment C ) c o n s i d e r a b l y l o n g e r and wider than larva at 53 d ( T a b l e I I ) , but otherwise similar to it. Larva not within
« sleeve ».
At 87 d, larvae (from g u p p y ; T a b l e I, Experiment E ) varying in length and width ( T a b l e III). Genital pri- mordium in longer larvae (> 2 m m ) visible as single cell b e t w e e n ventral hypodermis and excretory gland at approximately mid length o f body. Larvae otherwise similar to larva at 5 3 d. S o m e larvae within « sleeve » and s o m e not.
At 112 d, larva (from g u p p y ; T a b l e I, Experiment E ) 1150 pm long, 4 0 pm wide, and similar to larva at 53 d.
Larva not within « sleeve ».
At 132 d, larva (from g u p p y ; T a b l e I, Experiment F ) 2 7 5 0 pm long, 9 0 pm wide, and similar to long larvae at 8 6 d. Larva within « sleeve ».
At 152 d, larvae (from guppy; T a b l e I, Experiment E ; Figs. 8 - 1 1 ) varying in length and width. Five largest larvae ( T a b l e III) longer and wider than larvae at all previous times but otherwise similar to larvae at 8 6 d.
Larvae not within « sleeve ».
DISCUSSION
C
hanges in C. rudolphii while in the c o p e p o d (Tigriopus sp.) primarily involved b o d y size and d e v e l o p m e n t o f the ventricular appendix (from a delicate c o l u m n o f narrow cells to a longer, digitiform structure containing several small, scattered cells) and the excretory system (from unapparent to a prominent cell with a large n u c l e u s ) . Huizinga ( 1 9 6 6 ) reported a similar slight increase in size o f larvae in the c o p e p o d Tigriopus californicus (as well as unex- plained « partial d e v e l o p m e n t ») but « n o morpholo- gical c h a n g e » in Cyclops vernalis. T h e present study considers the c h a n g e s that occurred in the parasite while in the c o p e p o d to b e developmentally signifi- cant; they w e r e associated with markedly increased s u c c e s s b y the parasite in establishing in its fish host ( 1 0 o f 11 fish e x p o s e d to c o p e p o d s harbouring larvae b e c a m e infected versus n o n e o f 27 m u m m i c h o g and 3 o f 6 guppies e x p o s e d directly to recently-emerged larvae). Huizinga ( 1 9 6 6 ) reported n o ( 0 o f 2 5 ) to limited ( 4 o f 2 5 ) s u c c e s s in infecting guppies withT. californicus and C. vernalis harbouring larvae, res- pectively, and considerable s u c c e s s ( 3 o f 5 ) in infec- ting m u m m i c h o g (= killifish) with T. californicus har- bouring larvae.
A m p h i p o d s , in addition to c o p e p o d s , w e r e found c a p a b l e o f transporting larvae o f C. rudolphii to fish, something not previously reported for the species.
Successful infections in a m p h i p o d s w e r e generally established only w h e n amphipods ate c o p e p o d s contai- ning larvae (5 o f 9 b e c a m e infected), however, and not w h e n amphipods ate free-living, second-stage larvae in water (1 o f 58 b e c a m e infected although all larvae w e r e c o n s u m e d ) . This requirement o f larvae to pass through c o p e p o d s before being infective to amphipods is strikingly similar to that n o t e d for infectivity o f larvae to fish and supports the suggestion that the
L a r v a e 8 7 d L a r v a e
1 5 2 d ( \ = 51
- 1 =2 * 3 # 4 * 5 # 6 # 7 # 8 *9 » 1 0
L a r v a e 1 5 2 d ( \ = 51
T o t a l l e n g t h ( m m ) 0 . 8 1.1 1.5 1.8 1.9 2 . 0 2.2 2.5 2.7 2 . 9 3.5 ( 3 . 1 - 3 . 9 )
M a x i m u m w i d t h 3 0 3 5 5 0 6 0 6 5 7 0 8 5 9 0 9 5 9 0 1 1 4 ( 1 1 0 - 1 2 0 )
N e r v e ring 6 0 — — 1 3 0 1 1 0 — 1 3 0 1 5 0 1 4 0 1 4 0 1 6 7 ( 1 5 0 - 2 0 0 )
L. œ s o p h . 1 6 5 — — 2 3 0 2 0 0 2 1 0 — 3 7 0 3 4 0 3 1 0 3 9 1 ( 3 7 0 - 4 1 5 )
L. v e n t r . 15 — — 2 0 2 5 2 0 — 3 0 2 0 3 0 2 9 ( 2 5 - 3 0 )
L. v e n t r . a p p . 1 3 0 — — 2 1 0 2 2 0 2 2 0 — 3 2 5 2 7 0 3 5 0 3 5 9 ( 3 2 0 - 3 8 0 )
L. int. c a e c u m 4 0 — — 8 0 9 0 8 0 — 1 9 0 1 5 0 1 5 0 1 9 1 ( 1 7 0 - 2 2 5 )
E x c r . c e l l ( m m ) — — — 0 . 6 0 . 7 — — 0 . 9 0 . 9 1.0 1.2 ( 1 . 1 - 1 . 3 )
A n u s — 5 0 6 5 6 0 6 5 6 0 6 5 7 5 8 5 8 5 8 8 ( 8 5 - 9 0 )
Table III. — Morphometries (in pm, unless otherwise specified) of third-stage larvae of Contracaecum rudolpbii in guppies (Lebistes reti- culalus) 87 and 152 d after ingesting copepods (Tigriopus sp.) containing second-stage larvae. Note: Ten larvae of considerable size range were recovered at 86 d and five larvae of more similar size at 152 d.
Parasite, 1996, 4, 367-376 373
B A R T L E T T С . М .
Figs . 8-11. — Contracaecum rudolphii, larva from guppy (Lebistes reticulatus) 152 postinfection. Fig 8. Anterior end, lateral view. Fig.9.
Genital primordium, lateral view. Fig. 10. Tail, laterral view. Figs. 11 A-D. Anterior extremity, lateral, ventral, dorsal, and en face views res- pectively
374
Memoire
Parasite, 1 9 9 6 , 4, 3 6 7 - 3 7 6
MORPHOGENESIS O F CONTRACAECUM SPICULIGERUM
copepod serves a partial developmental role in the transmission of C. rudolphii. Within the genus Contra-
caecum, the copepod is reported to serve differentroles in transmission ranging from that suggested herein for C. rudolpbii (i.e., a « precursor » role, see below) to being the site of a moult (e.g. C. micropa-
pillatum (Stossich, 1850)) (see Anderson 1992).Extreme plasticity also apparently characterizes Pseu-
doterranova decipiens (Krabbe, 1878) where a moulthas been suggested in macrocrustaceans (McClelland, 1990).
The amphipod is considered herein a paratenic host of C. rudolpbii. Amphipods have also been reported to harbour larval C. micropapillatum although, in contrast to the present study, infections were said to have been acquired when amphipods ate free-living larvae (Anderson, 1992). Pseudoterranova decipiens can also pass in the food chain from one crustacean host to a second (« serial transmission ») and acquisi
tion by amphipods is, as in the present study, much more efficient when a copepod is initially involved (McClelland, 1990). Copepods serving this role have previously been termed « precursor » hosts (note: other authors may use the term « metaparatenic host » (see Odening, 1976)) and consumption of them by macroin- vertebrates likely explains the presence, in the latter, of larvae of various species of marine and freshwater ascaridoids (Overstreet, 1983; McClelland, 1990). It would be informative to know whether morphogenesis occurs in these parasites in the copepod, similar to that noted herein.
When copepods are involved in a life cycle, it probably is advantageous for the parasite to use a subsequent paratenic host (macroinvertebrate) or be quickly trans
mitted to fish. Copepods harbouring C. rudolpbii are adversely affected by the presence of larvae, becoming lethargic or moribund or dying within a few days of infection (Huizinga 1966; present study), as do cope
pods harbouring other ascaridoid larvae (McClelland, 1990). Affected copepods in the present study were readily fed upon by amphipods. In nature, transmis
sion likely involves copepods which acquire larvae from the free-living environment and are then fed upon by paratenic hosts or fish. McClelland (1990) reported altered behaviour in amphipods harbouring larvae of P. decipiens; the limited observations herein precluded conclusions.
Huizinga (1966) suggested that at 18 d postinfection in fish, « a moult (of C. rudolpbii) was in progress » although « larvae did not shed the second-stage cuticle while encapsulated, but retained it as a closely adhe
ring layer. » In the present study, larvae at 20 d did not appear to be moulting. However, observations at later times support Huizinga (1966); at 45 d and later times many larvae were within a closely adherent
« sleeve » resembling cuticle. In addition, larvae were sometimes surrounded by host cells, giving the appea
rance of a delicate capsule. Onset of a moult may be associated with migration of larvae from the intestinal wall (their site 20 or fewer days postinfection) to the abdominal cavity (their site 44 or more days postin
fection). Huizinga (1966) recovered larvae in fish at times earlier than 18 d postinfection by using a pepsin digest and thus did not know their specific locations;
his larvae at 18 d were « encapsulated along the intes
tinal mesenteries ».
In the present study, third-stage larvae continued to grow in length, with the longest (3-9 mm) having been obtained from the fish with the oldest (152 d) infection. Different larvae in a given fish were some
times of different lengths, however (e.g. at 87 d, Table III), suggesting that growth is asynchronous. Hui
zinga (1966) also noted the longest (1.4 mm) larva came the fish with the oldest (37 d) infection; Moravec (1994) stated that« advanced »third-stage larvae ranged from 15-24 mm. McClelland and Ronald (1974fo) indi
cated that second-stage C. osculatum Rudolphi, 1802 grew continuously for 32 weeks in cultures at 15 °C but commenced to moult to the subadult (fourth) stage when the temperature was raised to 35 °C.
McClelland and Ronald (1974«, 1974b) found a similar phenomenon, namely development from the second to the fourth stage with only one intervening moult, in cultured P. decipiens.
The detailed description of larvae of C. rudolpbii pro
vided herein will help facilitate identification of anisakid larvae in invertebrates and fish (also, see Moravec 1994). In addition to Huizinga (1966), the life cycle of the Contracaecum sp. in cormorants has previously been studied by Thomas (1937«, b, 1940), Dubinin (1949), and Mozgovoy et al. (1965, 1968). However, within the genus Contracaecum as a whole, only the development of C. osculatum has previously been stu
died in a manner as detailed as that presented herein for C. rudolpbii. The study of C. osculatum was done with cultured specimens; its 450 um long, second stage has a large nucleus in the ventricular appendix, a rea
dily distinguishable excretory system, and a genital pri- mordium (McClelland and Ronald, 1974b, Fig. 3) not seen in second-stage C. rudolpbii from crustaceans.
Cephalic structures of third stage C. rudolpbii from fish at 152 d postinfection were basically similar to those of 6 mm long, cultivated larvae of C. osculatum (see McClelland and Ronald 1974 b; Figs. 1, 2) although the lips were not as prominent and the amphids not visible.
They also resembled the cephalic structures of second- stage larvae of P. decipiens (see McClelland and Ronald, 1974«; Fig. 1), Anisakis simplex (Rudolphi, 1809) (see Smith, 1983; Plate 1, Figs, a, b, e), and Rapbidascaris acus (Bloch, 1779) (see Smith 1984; Fig. 5).
Parasite, 1996, 4, 367-376
Mémoire 375
Anderson a n d Bartlett ( 1 9 9 3 ) suggested that precocity may b e widespread but unrecognized a m o n g many n e m a t o d e s , especially those that exhibit plasticity in transmission. N o n e o f the four ways p r o p o s e d b y Anderson ( 1 9 9 2 ) as manifestations o f precocity w a s o b s e r v e d in C. rudolphii. I n d e e d , its s i n g l e - c e l l e d genital primordium w a s first visible in larvae at 8 6 d in fish a n d it remained u n c h a n g e d in the oldest larvae.
The single-celled genital primordium in C. osculatum only grew after larvae moulted to the fourth stage (McClelland and Ronald, 1974 b). Contracaecum rudol- phii is rather unusual, h o w e v e r , in that growth o f
s o m e larvae apparently c o n t i n u e s while what is pre- s u m e d to b e cuticle from an earlier moult is retained.
M c C l e l l a n d a n d Ronald ( 1 9 7 4 a , 1 9 7 4 ft) s u g g e s t e d s o m e w h a t similar d e v e l o p m e n t s in cultured C. oscu- latum a n d P. decipiens to b e n e o t e n y or artifact.
A C K N O W L E D G E M E N T S
he author gratefully thanks Hasina Kavanagh w h o prepared the illustrations a n d h e l p e d dis- J L s e c t n e m a t o d e s , R o d B e r e s f o r d w h o a l s o helped dissect n e m a t o d e s a n d e x a m i n e cormorants, Dr J o h n Roff (University o f G u e l p h ) w h o identified c o p e p o d s , Dr B e v Scott (Huntsman Marine Lab) w h o identified larval fish, Trevor Wilkie and Dave Harris (Nova Scotia Department o f Natural R e s o u r c e s ) w h o helped collect cormorants, B e r n i e MacLennan (Uni- versity College o f C a p e B r e t o n ) w h o provided consi- derable technical support, and Dr Roy Anderson (Uni- versity o f G u e l p h ) w h o read drafts. This study w a s supported by grants from the Natural S c i e n c e s and Engineering Research Council o f Canada a n d the Uni- versity College o f C a p e Breton.
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Reçu le 7 juin 1996 Accepté le 8 août 1996
376 Mémoire Parasite, 1996, 4, 367-376
B A R T L E T T C M .
