EXPOSURE OF FIRST-STAGE LARVAE
OF MUELLERIUS CAPILLARIS (NEMATODA) TO DESICCATION:
SMALLER LARVAE AND REDUCED INFECTIVITY
IN THE LAND-SNAIL HOST
CANDIDULAINTERSECTA
CABARET J.*, DIEZ-BAÑOS P.**, LOPEZ-SANDEZ C.** & MORRONDO-PELAYO P.**
S u m m a r y :
First-stage larvae (L1) of the nematode Muellerius capillaris are resistant to dehydratation in desiccated faeces of definitive host.
The L1 were exposed to increasing desiccation with different durations (non desiccated; 70% dry-matter (DM), 0 h; 7 0 % DM, 2 4 0 h; 9 0 % DM, 2 4 0 h; 95% DM, 4 8 h). The larger L3 in snails were recovered from infection using the less desiccated L3 and the infection rates were also higher. The size of L3 could be an indicator of the stress that L1 underwent before infection of the intermediate hosts.
KEY W O R D S : nematode, lungworm, land-snail, Muellerius, desiccation, larval size.
R é s u m é : LA DESSICATION DES LARVES DU PREMIER STADE DE MUELLERIUS CAPILLARIS (NEMATODA): DES LARVES DE PETITE TAILLE ET UNE INFESTIVITÉ RÉDUITE POUR LE MOLLUSQUE TERRESTRE САNDIDULA INTERSECTA
Les larves du premier stade (L1) du nématode Muellerius capillaris sont résistantes à la déshydratation au sein des fèces. Des L1 dans des fèces ont été soumises à des conditions de déshydratation croissantes (non-deshydraté; 70% de matière sèche (MS), 0 h;
70 % MS, 240 h; 90 % MS, 240 h; 95 % MS, 48 h) avant I'infestation. L'infestation des mollusques hôtes intermédiaires est moins importante et les larves L3 sont moins grandes dans les lots infestés par des L1 soumises au préalable à la déshydratation. La taille des L3 pourrait être un indicateur des stress subis par les larves L1 avant l'infestation des hôtes intermédiaires.
MOTS CLÉS : nématode, protostrongle, mollusque terrestre, Muellerius, déshydratation, taille des larves.
INTRODUCTION
T
h e Muellerius capillaris protostrongylid n e m a - tode is a very frequent parasite o f small rumi- nants. It has a two-host life-cycle: small rumi- nants e x c r e t e first-stage larvae ( L 1 ) in their faeces, which infect terrestrial molluscs. T h e L1 d e v e l o p into s e c o n d - s t a g e larvae ( L 2 ) and finally third-stage larvae (L3). T h e L3 are ingested by small ruminants, and develop in the lungs into fourth-stage larvae and finally adults; the eggs s h e d evoluate into L1 in the lungs and are passed in the digestive-tract after regurgitation, and then e x c r e t e d in faeces.Desiccation o f faeces is an important factor in survival o f first-stage larvae (L1) in faecal pellets (Cabaret et al., 1991 ; Morrondo-Pelayo et al., 1 9 9 2 ) . T h e infectivity o f the survivors originating from the d e s i c c a t e d faeces is significantly reduced (Morrondo-Pelayo et al., 1 9 9 2 ) : they penetrated less and d e v e l o p p e d in smaller num- bers into second-stage and third-stage larvae. Mor-
p h o m e t r y variations are often g o o d indicators o f fit- n e s s traits in free-living o r g a n i s m s ( J o n e s , 1 9 8 7 ; McKenzie & Clarke, 1 9 8 8 ) . Desiccation during larval d e v e l o p m e n t induces reduction o f size o f infective larvae o f several species o f trichostrongylid n e m a t o d e s (Rossanigo & Gruner, 1 9 9 2 ) , possibly resulting from a reduced feeding activity in desiccated faeces.
W e will consider morphological c h a n g e s in the larval stages o f the protostrongylid n e m a t o d e M. capillaris in snails after the L1 had b e e n submitted to varied envi- ronments, i.e. several desiccation conditions. T h e snail Candidala intersecta is a particularly convenient host as it is usually found in dry sites, w h e r e rapid desic- cation o f f a e c e s is likely to occur. W e will try to a n s w e r two questions: i) d o e s desiccation o f L1 result in smaller L3 in snails?, ii) and d o e s the size o f L3 reflect infectivity o f the L1 for snails?
MATERIALS AND METHODS
* INRA, Station de Pathologie aviaire et de Parasitologie, 3 7 3 8 0 Nou- zilly, France.
** Universidad Santiago de Compostela, Facultad de Veterinaria, Parasitologia, 2 7 0 8 0 Lugo, Spain.
Correspondence : Jacques Cabaret.
Tél. :
(33) 2 47 42 77 68- Fax :
(33) 2 47 42 77 74.E.mail: cabaret@tours.inra.fr
PARASITE MATERIAL
T
h e L1 w e r e o b t a i n e d from a goat infected with M. capillaris. T h e excretion at the period o f e x p e r i m e n t was approximately 1,200 L l / g o f faeces. This goat w a s reared indoors and fed o n a dietParasite, 1 9 9 7 , 4, 3 0 7 - 3 1 0
Mémoire 307
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1997044307
C A B A R E T J., D I E Z - B A N O S P . . L O P E Z - S A N D E Z С . & M O R R O N D O - P E L A Y O P .
o f hay and c o m m e r c i a l pellets: the range o f faecal dry- matter ( D M ) was 4 2 - 3 6 %. Freshly c o l l e c t e d faeces w e r e dehydrated to obtain 7 0 , 9 0 an 9 5 % o f DM. This w a s performed in a ventilated c h a m b e r at 31 ± 1 °C o n faecal pellets and lasted approximately 3 h to 21 h to obtain 7 0 and 9 5 % DM respectively. After desic
cation p r o c e s s , the faeces w e r e left from 0 to 2 4 0 h according to groups at 2 0 °C before extraction o f L1, in order to reveal the influence o f interaction b e t w e e n desiccation and duration o f stay in d e s i c c a t e d faeces.
T h e L1 w e r e then extracted (Cabaret et al., 1 9 8 0 ) from control ( n o n - d e s i c c a t e d faeces) and from desiccated faeces.
INFECTION OF SNAILS AND MEASURES OF LARVAE T h e adult C. intersecta snails originated from an iso
lated wild c o l o n y in Nouzilly, free o f any protostron- gylid infection. Snails w e r e infected individually a c c o r ding to a m e t h o d modified from Kassai ( 1 9 5 7 ) : e a c h snail was in contact with a larval suspension o f 100 living L1 for 3 h. Six b a t c h e s o f 2 0 snails e a c h w e r e e x p o s e d to M. capillaris L1 that had survived in desic
cated faeces. T h e influence o f desiccation and its dura
tion o n L1 survivals has b e e n previously tested: i) very high survival ( 7 8 - 8 6 % ) was found in control and in desiccated f a e c e s stored for less than 12 h w h e r e a s l o w e r survivals ( a v e r a g e 6 7 % ) w e r e r e c o r d e d in b a t c h e s stored for 2 4 0 h; it) increased desiccation c o m b i n e d with longer storage d e c r e a s e d L1 survival (Morrondo-Pelayo et al. 1 9 9 2 ) . T h e following condi
tions may b e c o n s i d e r e d as a gradient o f increasing stress resulting from desiccation: M l ( c o n t r o l ) , M2 ( 7 0 % DM, 0 h), M3 ( 7 0 % DM, 2 4 0 h ) , M4 ( 9 0 % DM, 240 h), and M5 ( 9 5 % DM, 4 8 h ) . T h e coding for batches is as follows: ( 7 0 % DM, 0 h ) m e a n s that faeces w e r e dehydrated up to 7 0 % o f dry-matter, w h i c h lasted 3 h and the larvae w e r e then extracted w h e r e a s ( 7 0 % DM, 2 4 0 h ) indicated that faeces, dehydrated the s a m e way, w e r e maintained during 2 4 0 h at 2 0 °C before extraction and use for infection o f snails. T h e snails w e r e maintained and fed o n lettuce at 20 °C until they w e r e serially killed from day 4 to 3 0 after infec
tion in order to obtain successively L1, L2 and L3. T h e extraction of larvae from the foot o f snails for the iden
tification o f larval stages was performed as previously described (Morrondo-Pelayo et al, 1 9 8 0 ) . T h e inten
sity o f infection ( n u m b e r o f larvae/infected snail) w a s assessed in e a c h o f the 2 0 snails per batch. T h e fol
l o w i n g m e a s u r e s w e r e p e r f o r m e d b y m e a n s o f a c a m e r a lucida on e a c h larval stage: length o f larvae LL and width o f larvae at the e n d o f o e s o p h a g u s , WL.
PROCESSINO OF DATA
T h e estimation o f arithmetic averages, standard-devia
tion, o n e - w a y analysis o f variance - Anova (using
Newman-Keuhls test o f b e t w e e n b a t c h e s c o m p a r i s o n ) w e r e classical. T h e size and s h a p e o f larvae w e r e esti
mated in a simple m a n n e r as respectively LL x WL and LL / WL, and the difference b e t w e e n batches w e r e eva
luated by m e a n s o f a discriminant analysis. T h e rela
tionships b e t w e e n size (LL χ W L ) o r s h a p e o f L3 and intensity o f infection w e r e established by m e a n s o f l e a s t - s q u a r e r e g r e s s i o n s a n d t h e n v a l i d a t e d using resampling procedures (bootstrap with 5 0 0 repeats).
RESULTS
THE MORPHOLOGICAL CHARACTERISTICS OF LARVAL STAGES
T h e y are r e c o r d e d in table I. T h e L1 w e r e signi
ficantly smaller (Anova, Ρ < 0 . 0 5 ) in the pre- viously most desiccated L1 group ( 9 5 % - 4 8 h ) . T h e L2 and L3 in desiccated groups (M2 to M 5 ) w e r e smaller (Anova, Ρ < 0 . 0 5 ) than in the control g r o u p ( M 1 ) .
T h e size (LL x W L ) and s h a p e (LL/WL) o f larvae L1 and L2 w a s not significantly different b e t w e e n control and d e s i c c a t e d group (data not s h o w n ) . Conversely, differences b e t w e e n size and s h a p e o f L3 w e r e found as s h o w n in table II. T h e size o f L3 w a s significantly larger in the control group c o m p a r e d with the desic
cated groups. T h e s h a p e w a s modified too: the larvae w e r e less slender (ratio LL/WL b e i n g larger) in the control group.
RELATIONSHIP BETWEEN SIZE AND SHAPE AND INTENSITY OF INFECTION
Significant differences (P < 0 . 0 5 ) in intensities w e r e r e c o r d e d b e t w e e n t h e c o n t r o l a n d t h e d e s i c c a t e d groups (Anova with Newman-Keuhls test).
Only size ( S ) c o u l d b e related to intensity o f infection:
S = ( 6 6 5 . 8 χ intensity) + 2 0 , 6 3 5 (R = 0 . 9 6 ; Ρ = 0 . 0 1 ) Confidence intervals at Ρ = 9 0 % b a s e d o n bootstrap procedures w e r e 19,380 to 2 2 , 1 8 0 for the constant and 541 to 910 for the slope o f regression indicating a g o o d fit o f the regression to actual data.
3 0 8 Parasite, 1997, 4, 307-310
DISCUSSION
T
h e lengths o f LÍ w e r e smaller than it has b e e n recorded by M o r r o n d o - P e l a y o et al. ( 1 9 8 0 ) in Spain: 3 9 2 in the control group or even 3 4 9 pm in the most desiccated larvae (batch 9 5 % MS during t w o days) versus 4 3 6 pm. This could b e due to the fact that larvae L Í , prior to infection have different sizes from o n e site to another ( K u l o et ai, 1 9 9 4 ) or thatMémoire
DESICCATION OF FIRST-STAGE LARVAE OF MUELLEMUS САPILLARIS
First-stage larvae maintained during Ü to 240 h in desiccated faeces (70 to 95 % DM) before infection of snails
Non-desiccated 70 % - 0 h 70 % - 240 h 90 % - 240 h 95 % - 48 h
faeces ( M l ) (M2) (МЗ) (M4) (М5)
First-stage larvae 6* 2 18 8 16
Length of larvae 392 (21) 396 (2) 385 (24) 396 (10) 349 (27)
Width of larvae 25 (3) 32 (1) 21 (5) 24 ( 6 ) 23 (5)
Second-stage larvae 46 27 31 35 23
Length of larvae 541 ( 3 8 ) 541 (32) 501 <42) 512 (56) 521 (45)
Width of larvae 40 ( 6 ) 35 (3) 37 ( 6 ) 42 (10) 46 (9)
Third-stage larvae 80 58 24 23 38
Length of larvae 633 (37) 610 (38) 608 (26) 604 (32) 603 (32)
Width of larvae 48 (6) 43 (11) 43 ( 8 ) 41 (6) 42 ( 8 )
• Number of larvae examined.
Table I. — Averages in pm and standard deviations of the first-, second- and third- larval stage measures of Muellerius capillaris in snails (Candidula intersecta) in relation to desiccation of infective first-stage larvae before infection (batches Ml to M5).
First-stage larvae maintained during 0 to 240 h in desiccated faeces
(70 to 95 % DM) before infection Non-desiccated
faeces 70 % - 0 h 70 % - 240 h 90 % - 240 h 95 % - 48 h
Size 30286 26334 26091 24689 25512
(Length χ width in pm) (4824)* (7943) (5382) (3770) (5719)
Shape 13.2 15 14.7 15 14.9
(Length/width) (1.8) (3.2) (2.9) (2.6) (3)
Intensity o f infection 14.9 10.6 8.6 6.9 5.8
(19.4) (15.7) (7.7) (8.5) (8.7)
(20)** (18) (15) ' (19) ( 19)
* Standard-deviation; ** number of infected snails.
Table II. — Size and shape of M. capillaris third-stage larvae in relation to desiccation history of first-stage larvae in faeces and actual infection of snails ( C a n d i d u l a intersecta).
d e v e l o p m e n t differs a c c o r d i n g to the s p e c i e s o f land- snail. Conversely, the s e c o n d larval stage in control g r o u p ( 5 4 1 p m ) w a s larger than that r e c o r d e d b y G e r i c h t e r ( 1 9 5 1 ) , R o s e ( 1 9 5 7 : 5 2 9 µm) o r Morrondo- P e l a y o et al. ( 1 9 8 0 : 5 0 5 µ m ) . T h e third larval stage length ( 6 3 3 p m ) w a s similar in size to that found b y M o r r o n d o - P e l a y o et al. ( 1 9 8 0 : 6 4 1 µm) but w a s m u c h larger than the s p e c i m e n s e x a m i n e d by Rose ( 1 9 5 7 : 5 9 4 p m ) . T h e differences r e c o r d e d in s e c o n d - and third-stage larvae could b e due as a b o v e m e n t i o n e d to origin o f L1, s p e c i e s o f snail o r slug used as inter
mediate host, or fixation and clearing m e t h o d s ( F a g e - rholm, 1 9 7 9 ) .
T h e sizes o f L3 did present a c o h e r e n t pattern b e t w e e n b a t c h e s : they w e r e larger in the heavier infections, i.e.
in control group. W e may h y p o t h e s i s e that larger L1 originating from non-desiccated faeces w e r e more a b l e
to infect snails and that they d e v e l o p e d better than their counterparts extracted from desiccated faeces.
Thus the size o f the o b t a i n e d L3 should reflect the infectivity o f L1. T h e s e findings are in agreement with those o f Rossanigo & G a i n e r ( 1 9 9 2 ) on trichostrongylid n e m a t o d e s (size w a s modified w h e n faeces are desic
c a t e d ) but they also give indications on infectivity o f larvae (this aspect was not evaluated by Rossanigo &
Gruner, 1 9 9 2 ) . Our results could b e c o m p a r e d with t h o s e o b t a i n e d o n asymmetry in insects submitted to stress ( M c K e n z i e & Clarke, 1 9 8 8 ) . In n e m a t o d e larvae, larger size w o u l d b e the positive indicator o f infecti
vity (a putative indicator o f fitness) in contrast with asymmetry in insects wich is a negative indicator o f fitness.
T h e size variations o f larvae are o n e c a s e o f p h e n o - typic plasticity as an adaptation to variable environ-
Mémoire 309 Parasite, 1997, 4, 307-310
C A B A R E T J . , D I E Z - B A Ñ O S P., L O P E Z - S A N D E Z С. & M O R R O N D O - P E L A Y O P .
merits. It could b e an e x a m p l e o f allelic sensitivity, in which the e x p r e s s i o n o f individual g e n e s is altered by c h a n g e s in external conditions (e.g. g e n e s w h o s e pro
ducts are directly responsive to c h a n g e s in desiccation) and the p h e n o t y p i c o u t c o m e is c o n t i n u o u s and pro
portional to fluctuations in environment (Schlichting &
Pigliucci, 1 9 9 5 ) .
T h e results might summarized as follows: desiccation o f L1 in faeces w a s unfavourable for future d e v e l o p ment within the snail host, fewer larvae b e c o m e esta
blished, and the L1 that c o m p l e t e d their d e v e l o p m e n t into L3 r e m a i n e d smaller in desiccated larvae groups.
T h e question is o p e n to w e t h e r these smaller L3 larvae will b e less fit or not to infect small ruminants.
ACKNOWLEDGEMENTS
W
e gratefully a c k n o w l e d g e the funding by the University o f Santiago d e Compostela (Spain) o f short post- (P. Diez-Baños, 1 9 8 9 and P. Morrondo-Pelayo, 1 9 9 1 ) and a pre-doctorate (C. López-Sández, 1 9 9 3 ) grants, and a visit to the Vete- rinary Faculty o f Lugo ( J . Cabaret, 1 9 9 5 ) .MORRONDO-PELAYO P., CORDERO DEL CAMPILLO M., DIEZ-BAÑOS P.
& MANGA-GONZÁLEZ Y . Infestación e x p é r i m e n t a l d e tres Cer- nuella s p p ( M o l l u s c a , S t y l o m m a t o p h o r a ) c o n larvas d e Muellerius capillaris y Neostrongylus capillaris ( N e m a - toda, Protostrongylinae). Anales de la Facultad Veterinaria de León, 1 9 8 0 , 26, 1 0 7 - 1 2 3 .
R O S E J . H . O b s e r v a t i o n s o n t h e larval s t a g e s o f Muellerius capillaris within the intermediate hosts Agriolimax agrestis a n d A. reticulatus. Journal of Helminthology, 1 9 5 7 , 31, 1- 16.
ROSSANIGO C.E. & GRINER L. T h e length o f strongylid n e m a - t o d e infective larvae as a reflection o f d e v e l o p m e n t a l c o n d i t i o n s in f a e c e s a n d c o n s e q u e n c e s o n their viability.
Parasitology Research, 1 9 9 2 , 82, 3 0 4 - 3 1 1 .
SCHLICHTING C D . & PIGLIUCCI M. G e n e regulation, quantita- tive g e n e t i c s a n d e v o l u t i o n o f r e a c t i o n n o r m s . Evolutio- nary Ecology, 1 9 9 5 . 9, 1 5 4 - 1 6 8 .
R e ç u le 17 s e p t e m b r e 1 9 9 6 A c c e p t é le 3 juillet 1 9 9 7
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