N E W FEATURES ON THE MOULTS AND MORPHOGENESIS OF THE HUMAN FILARIA LOA LOA B Y USING RODENT HOSTS
CONSEQUENCES
BAIN O.*, WANJI S**, ENYONG P.***, PETIT G.*, NOIREAU F.****, EBERHARD M.I.***** & WAHL G.
S u m m a r y :
The development of the human filaria Loa loa (Dirofilariinae, Onchocercidae), previously studied in monkeys, was studied using the non permissive hosts-mice and jirds. The development proved to be rapid: moult 3 occurred on day 8 post-inoculation, the adult stage was reached on day 25 and measured at that time 3-3.5 mm in length. As in the other filarioids, the female genital apparatus developed during the fourth stage.
A critical analysis of the studies on the development of Onchocercid species was made. The optimal duration of the stages (i.g. the shortest time| was chosen for the comparison. It appeared that the duration of the stage 3 was a constant character in a given species whatever the experimental conditions, whereas moult 4 might be retarded in a non susceptible host.
Comparison between the 1 8 developmental cycles of Onchocercidae in the vertebrate host was made. Two biological types could be distinguished: either the moult 3 occurred on day 2-3 and was followed apparently by a late moult 4 ( ≥ 5 0 days], or the moult 3 occurred after about one week of development and it was associated with a less long stage 4 (20-40 days]. The first group includes Dirofilaria and Onchocerca, the second group brings together mainly Loa and the Onchocercinae of the Dipetalonema line and related genera (Acanthocheilonema, Brugia, Litomosoides, etc.).
The groups thus formed suggest real relationships as they fit with the morphology of the infective stage and the results of a recent molecular analysis of the 5 S DNA.
KEY W O R D S : Loa loa, laboratory mouse, jird, moults, morphogenesis, Onchocercinae, Dirofilariinae, phylogeny.
Résumé : NOUVELLES DONNÉES SUR LES MUES ET LA MORPHOGENÈSE D E LA FILAIRE HUMAINE LOA LOA, EN UTILISANT DES RONGEURS. C O N S É Q U E N C E S
Le développement de la filaire humaine Loa loa (Dirofilariinae, Onchocercidae), antérieurement étudié chez les singes, est précisé chez des souris et des mérions. Il s'avère rapide: la mue 3 s'effectue à J8, le stade adulte est atteint à J25 et mesure alors 3-3,5 mm de long; comme chez les autres filaires, l'appareil génital femelle se développe durant le stade 4.
Une analyse des travaux sur les développements larvaires des Onchocercidés est effectuée. Les durées optimales des stades (c'est-à- dire les plus rapides) sont choisies pour la comparaison. Il apparaît que la durée du stade 3 est un caractère stable indépendant des conditions expérimentales pour une espèce donnée, alors que la durée du stade 4 peut être plus longue chez de mauvais hôtes.
Parmi les 18 cycles d'Onchocercidae réalisés chez l'hôte vertébré, on remarque qu'il existe deux types biologiques: la mue 3
s'effectue soit à J2-J3 et est suivie apparemment d'une mue 4 tardive (≥ 50 purs), soit après une semaine environ de développement et associée à un stade 4 moins long (20-40 jours). Le premier groupe comprend Dirofilaria et Onchocerca; le second rassemble, entre autres, Loa et les Onchocercinae de la lignée Dipetalonema et des genres proches (Acanthocheilonema, Brugia, Litomosoides, etcj.
Les groupes ainsi constitués suggèrent de réelles affinités car ils concordent avec la morphologie du stade infectant et les résultats d'une récente analyse moléculaire sur le 5S DNA.
MOTS CLÉS : Loa loa, souris de laboratoire, mérion, mues, morphogenèse, Onchocercinae, Dirofilariinae, phylogénie.
INTRODUCTION
A
m o n g the 18 d e v e l o p m e n t a l c y c l e s o f O n c h o - c e r c i d filariae e l u c i d a t e d in the definitive host ( A n d e r s o n , 1 9 9 2 a n d T a b l e I ) , that o f t h e h u m a n filaria Loa loa a p p e a r s slow: the fourth moult* Biologie Parasitaire, Protistologie, Helminthologie, CNRS URA 114, Museum National d'Histoire Naturelle, 61, rue Buffon, 75231 Paris Cedex 05. École Pratique des Hautes Etudes.
** University of Buea, Faculty of Science. Department of Life Sciences, PO Box 63, Buea, South West Province, Cameroon.
*** Tropical Medicine Research Institute, Kumba, South West Pro- vince, Cameroon.
**** ORSTOM, Instituto Boliviano de Biología de Altura, La Paz, Bolivia.
*****National Center for Infectious Diseases CDC, Division of Parasitic- Diseases, 4770 Buford Highway NE, Atlanta, GA 30341-3724, USA.
****** CIRMF, Franceville, Gabon; present address: Unter der Birken 21, D-56154 Boppard, Germany.
Correspondence: O. Bain
w a s d e s c r i b e d to o c c u r at a b o u t day 5 0 post inocula- tion ( E b e r h a r d & Orihel, 1 9 8 1 ) , as w a s Dirofilaria immitis ( O r i h e l ,
1961).
This similarity o f the b i o l o g i c a l c h a r a c t e r s s e e m s satisfactory b e c a u s e t h e t w o g e n e r a are p l a c e d in the Dirofilariinae. H o w e v e r , in the inter- pretation o f the results with L. loa obtained in the e x p e - rimentally infected m o n k e y s t h e r e lay a doubt: only o n e o f the t w o moults h a s b e e n o b s e r v e d , at day 1 8 ; it has b e e n identified as the third moult, although the well a d v a n c e d organization o f the genital apparatus at this date c o r r e s p o n d e d to what is generally o b s e r v e d in a late filarial fourth stage.W e h a v e inoculated m i c e a n d jirds with Loa loa larvae ( T a b l e II) a n d treated most o f t h e m with an anti- i n f l a m m a t o r y d r u g ( h y d r o c o r t i s o n e ) . T h e e a r l i e s t n e c r o p s y w a s p e r f o r m e d o n day 8 b e c a u s e , in m a n y filarial s p e c i e s , the moult 3 o c c u r s n e a r the e n d o f the first w e e k f o l l o w i n g larval i n o c u l a t i o n . T h e e i g h t
Parasite, 1998, 5, 37-46
Mémoire 37
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1998051037
G e n u s S p e c i e s D a y M 3 D a y M 4 H o s t R e f e r e n c e s m m M 3 m m M 4
Loa loa 18
8
50 19
monkeys mouse, jird
Eberhard & Orihel, 1981 Present paper
3 2.1
≤ 16.8 3. 1 8
Pelecitus fulicaeatrae ≤ 2 0 * Fulica Bartlett & Anderson, 1989
scapiceps 6 1 2 * Sylvilagus Barrien, 1984 1.4 2.8
Dirofilaria immitis 9
≤ 3
≤ 3
≤ 3 2 2 3
60 58 50
* dog
* dog
* dog rat in vitro in vitro in vitro
Orihel, 1961
Kotani & Powers, 1982 Lichtenfels et al., 1985 Sawyer & Weinstein, 1965 Sawyer & Weinstein, 1965 Lok et al., 1984
Lichtenfels et ai, 1985
1.2 12.8
Litomosoides sigmodontis 8
8
<10 6+3
23
< 28
* Sigmodon
* Sigmodon BALB/c mice
in vitro
Scott et al., 1951 Nelson et ai, 1982 Maréchal et ai, 1996 Nelson et ai, 1982
1.2
< 1.4
> 6 . 4
ßreinlia booliati 6 24 * Rattus Singh et ai, 1976 < 1.5 9.4
Monanema globulosa <10 25 jird Bianco et al., 1983 M 5.98
martini <10 21 * Lemniscomys Wanji et al, 1990 1.4 4.5*
Acanlbocheü. viteae 7?
6
7
6
< 21 20 23
* Meriones hamster
jird jird
Chabaud, 1954 Original
Johnson et al., 1974 Eisenbeiss et al., 1994
1.8 11.9
Molinema arbuta < 20 < 28 * Erethizon Bartlett & Anderson, 1985 2.6 > 8.6
dessetae 20
10
40 34
* Proechimys ' Proechimys
Gayrai et al., 1982
Bain et ai, 1994 1.35 9*
Brugia malayi 7
7
35 29
* cat jird
Edeson & Buckley, 1959 Ash & Risley, 1970a
2.2 1.6
5.8
pahangni 8
6
23 18
* cat jird
Schacher, 1962 Ask & Risley, 1970b
2.1
Wuchereria bancrofti < 14 8
< 42 monkey jird
Cross et ai. 19~9 Ash & Schacher. 1971
2.3 1.5
< 4.7
Elaeopbora schneidert < 14 * Odocoileus Hibler & Metzger, 1974 < 10
Onchocerca lienalis 2
2
<3
* cow in vitro micro C h a m b e r
Bianco & Muller. 1982 Lok et al., 1984
Bianco et al., 1989 0.5
volvulus 5
<3
in vitro micro chamber
Lok et al.. 1984
Bianco et al.. 1989 0.59 Splendidofilaria picacardina < 14 < 21 * Pica Hibler, 1963
Table I. - The 18 cycles o f Onchocercidae in the vertebrate host.
Upper part: Dirofilariinae; middle part: Onchocercinae; lower part: Splendidofilariinae. Day M3: the shortest time for moult 3 in days p.i., according to the different authors (italic numbers correspond to the reliable data); day M4: the shortest time p. i. for the male moult 4.
Host: * means natural host; micro chamber = micropore chamber containing infective larvae and implanted into diverse hosts. References:
a non exhaustive list o f the main works, mm M3: maximum length at moult 3, in millimeters; mm M4: maximum length of male larvae at moult 4, in millimeters; * means original data. Acanthocheil: Acanthocheilonema.
rodents n e c r o p s i e d from that date to day 25 post i n o culation h a r b o u r e d d e v e l o p i n g w o r m s a n d w e r e c o vered s u c c e s s i v e l y larvae u n d e r g o i n g moult 3, moult 4 a n d y o u n g adult w o r m s . T w o m o n k e y s , Cercopithecus leucopbaeus, presenting a patent e x p e r i m e n t a l infec
tion with L. loa, w e r e r e - i n o c u l a t e d with infective larvae, h o w e v e r n o d e v e l o p i n g larva w a s r e c o v e r e d 8 a n d 15 days later, a n d all results p r e s e n t e d h e r e c o m e from the rodent hosts.
MATERIAL
I
nfective larvae for m o r p h o l o g i c a l study. During 1 9 9 6 - 1 9 9 7 , the infective larvae w e r e c o l l e c t e d b y t w o o f us ( S . W . & P . E . ) from Chrysops silacea fed o n a v o l o n t e e r , n e a r K u m b a , in C a m e r o o n ( n ° 2 9 5 SE, MNHN-Paris).Infective larvae for inoculations. T h e y had different ori
gins b e c a u s e the s e a r c h w a s p e r f o r m e d in different
38 Parasite, 1998, 5, 37-46
Mémoire
MOULT AND MORPHOGENESIS OF LOA LOA
Host Inoc. date Larvae inoculated D p.i. N worms recov Stage Hydrocortisone
Mouse 24SE 26.11.1991 13 infective larvae 9 4 M3 25 mg x 2/week
Mouse 31SE 05.12.1991 3 larvae 9 day old from 24SE 23 female 1 L4 25 nig x 2/week
Jird 138SE 22.02.1995 100 infective larvae 19 female 1, male 1 L4, M4 0
Jird 140SE 22.02.1995 200 infective larvae 25 female 1, male 1 Ad 25 mg x 2/week
Mouse 310SE.1 23.11.1996 200 infective larvae 8 female 3, male 5 L3. M3 50 mg x 2/week Mouse 310SE.2 29 11 1996 200 infective larvae 9 female 5, male 7 M3, L4 50 mg X 2/week
Mouse 310SE.3 05.12.1996 200 infective larvae 15 female 4. male 3 L4 50 mg x 2/week
Mouse 310SE.4 15.01.1997 200 infective larvae 22 female 5 L4, M4 50 mg x 2/week
Table II. - l.oa ha in Swiss mice and Meriones unguiculatus: experimental procedures and worms recovered.
Inoc. date: dates of inoculation of larvae into rodents. Larvae inoculated: infective larvae from flies were inoculated, except for 31 SE which was inoculated subcutaneously with the larvae recovered from the mouse 24 SE. D p.i.: time between inoculation of the infective larvae and necropsy (for 34 SE, D 23 - 9 days in 24 SE + 14 days in 31 SE). N worms recov: number of worms recovered. Stage: M, moult;
L, larval stage; Ad, adult immature worm. Hydrocortisone: it was administered intraperitoneally just after the larval inoculation twice a week at a concentration of 25 or 50 mg/kg.
Mice 24 SE and 310 SE came from a colony maintained at the Institut Pasteur, Yaounde; mouse 31 SE and the jirds came from the Bio- logie Parasitaire laboratory, Paris.
p l a c e s and times. In 1 9 9 1 , in C a m e r o o n near C a m p o a n d in 1 9 9 5 , in G a b o n , at the CIRMF in Franceville, t h e i n f e c t i v e l a r v a e w e r e r e c o v e r e d from u n f e d C. silacea and C. dimidiata captured with a net and harbouring wild infections, h o w e v e r it is k n o w n that these diurnal flies transmit essentially the human strain o f L. Ioa ( D u k e & Wijers, 1 9 5 8 ) . T h e flies w e r e dis
s e c t e d individually o r a c c o r d i n g to the t e c h n i q u e o f Wahl et al. ( 1 9 9 5 ) . In
1996-1997,
at K u m b a , the infective larvae originated from flies fed o n a volunteer.
D e v e l o p i n g larvae. T h e rodents, the inoculations, the dates o f n e c r o p s i e s a n d the w o r m s r e c o v e r e d are detailed in T a b l e II. Infective and d e v e l o p i n g larvae w e r e fixed in 5 % formalin for m o r p h o l o g i c a l study.
Adult mature worms. O n e male s p e c i m e n extracted from a h u m a n h y d r o c o e l e , fixed in hot 7 0 % a l c o h o l and sent by Pr C. Ripert (n° 426 H B , collection MNHN- Paris). F o u r m a l e and four female w o r m s r e c o v e r e d from a m o n k e y exprimentally infected with a h u m a n strain o f L. Ioa by Eberhard & Orihel, in 1 9 8 1 , fixed in glacial a c e t i c acid and stored in 7 0 % e t h a n o l plus 5 % glycerine (n° 2 7 5 SE, MNHN-Paris).
RESULTS
T
h e dates o f the moults, the larval growth a n d the m o r p h o g e n e s i s o f L. loa are d e s c r i b e d o n a total o f 26 w o r m s r e c o v e r e d from rodents.MOULTS
O n day 8 p.i., seven o f the eight larvae recovered w e r e u n d e r g o i n g moult 3. and o n e w a s retarded; o n day 9, n i n e larvae w e r e moulting, o f w h i c h three w e r e in the p r o c e s s o f e x s h e a t h m e n t , a n d three larvae w e r e at fourth stage. O n day 15, the s e v e n larvae r e c o v e r e d
w e r e at fourth stage. Moulting 4 o f a male w o r m w a s o b s e r v e d o n day 19 in a jird, and moulting 4 o f a female w o r m o n day 22 in a m o u s e ( T a b l e III, Fig. 1 E, F, K; Fig. 3 ) .
GROWTH
T h e growth w a s slight. At moult 3 the larvae w e r e 1.9- 2.15 m m long, like the infective stage but they w e r e slightly wider ( 3 0 - 3 9 nm). O n day 15 p.i., the male fourth stage larvae w e r e 2.1-2.2 m m long a n d 3 1 - 36 |im wide, the female fourth stage larvae w e r e 2 . 4 - 2.77 m m long and 3 4 - 4 3 μΜ wide; o n day 2 1 - 2 2 p.i.
the five female fourth stage larvae w e r e 3-3-45 m m long and 4 0 - 5 5 μm wide. T h e t w o y o u n g female and male adult w o r m s w e r e respectively 3-65 and 3-3 m m long, 4 2 and 4 5 μm wide.
MORPHOGENESIS
T h e evolution o f the main structures was analyzed from the infective larva to the adult w o r m (Figs. 1 and 2, T a b l e III).
• Caudal lappets. T h r e e c o n s p i c u o u s and r o u n d e d caudal lappets w e r e present during the w h o l e stage 3 (Fig. 1 B , C, F ) ; they persisted during the stage 4 but they w e r e pointed (Fig. 1 I et K ) ; they d e c r e a s e d at the adult stage: the dorsal lappet disappeared and the lateral lappets w e r e vestigial (Figs. 1 N, P, and 2 Q ) .
• Papillae o f the head. In the infective larvae, the externo-labial papillae w e r e pointed and salient; these papillae and the c e p h a l i c o n e s w e r e arranged as rec
tangles slightly stretched in the lateral plane (Fig. 2 A, B , C ) . In the y o u n g and mature adult worm, the t w o groups o f papillae w e r e arranged as rectangles stret
c h e d in the median p l a n e (Fig. 2 F, G, H ) .
• B u c c a l capsule. It w a s 6 Lim high at the third stage, flattened laterally, hardly sclerotized e v e n at its j u n c -
Parasite, 1998, 5, 37-46
Mémoire 39
Fig. 1. - Loa loa morphogenesis in mice and jirds.
A to C: male infective larva from Chrysops. A: anterior region, lateral view (note the post-cephalic constriction); B : tail, ventral view;
C: caudal extremity, left lateral view. D to I, from mice; D to G: female larva, third moult, day 9 p.i.: E: head with the loosened cephalic- cuticle of the third stage and the buccal capsules o f stages 3 and 4 imbricated; F: tail, with caudal lappets o f stage 4 fully developed under the cuticle of stage 3; G: caudal region, lateral view. H & I: fourth stage female larva, day 23 p.i., respectively anterior part, in lateral view and tail, in ventral view. J to P, from jird; J & K: male larva, fourth moult, ventral view, day 19 p.i.; J : posterior part, with spicules; K: caudal papillae under the cuticle of moult 4; L & M: female, day 26 p.i.; L: vulva and vagina, lateral view; M: caudal region, lateral view; N: male, day 26 p.i., tail, ventral view with incipient caudal alae. O & P-. mature adult worm from laboratory monkey;
O: tail, lateral view; P: extremity, right lateral view. Scales: A, G, J , L, M, O, 100 μm B, C, E, F, I, K, N. P, 50 μm ; D, 200 μm.
40 Mémoire Parasite, 1998, 5, 37-46
MOULT AND MORPHOGENESIS OF LOA LOA
Fig. 2. - Morphogenesis of cephalic and female genital structures of Loa ha.
A to D: head, infective stage larva; A & B: lateral and median view; C: apical view; D: optical cross-section at the level of the buccal cavity.
E: head, fourth stage larva from jird; F & G: head, young adult female stage, lateral and median view; H to J: head, mature female adult from monkey; apical view, detail of mouth and base of buccal cavity, optical transversal section of the oesophagus, respectively. L & M:
female fourth stage larva from mice, ovaries and uteri (intestine drawn along a short distance), vulvar region and ovejector, respectively.
N: young adult from jird with open vulva in ventral view. O to Q: mature adult; O: vulva and ovejector; P: initial portion of an ovary;
P: caudal extremity, ventral view. Scales: A to D, 10 μm; E. F. G, M, 20 μm; II, I.. P. 200 μm; I, J , K, N, Q, 100 μm; O, 1000 μm.
Parasite, 1998, 5, 37-46
Mémoire 41
Measurements F e m a l e Male Measurements Measurements
day 9; moult 3 day 22; stage 4 day 19; stage 4 day 25; adult day 25; adult day 19; moult 4
Measurements
Length 2,130 3,280 3,500 3,650 3,300 3,180 Length
Width 35 50 50 42 45 55 Width
Nerve ring 110 1 10 110 115 110 105 Nerve ring
Excretory pore 220 180
-
- - 175 Excretory poreOesophagus 760 730 630 790 630 650 Oesophagus
(muse) (280) (250) (350) (220) (muse)
Vulva-apex 350 340 340 330 145 (60) 160 (50) Left spicule (shaft)
Genital length 45 2,030 1,300 1,740 95 70 Right spicule
(ovej) (90) (210)
Tail 55 62 60 55 50 50 Tail
Table III - Detailed measurements of Loa loa worms recovered from mice, on day 9 and 23, and jirds, on day 19 and 25.
muse: muscular; ovej: ovejector. All measurements are given in μm.
For comparison, mature adult female worms are 50-70 mm long and 500 μm wide and mature adult male worms are 2,7 mm long and 350 urn wide with left and right spicules 180-120 μm long (Eberhard & Orihel, 1981 and present study); spicules are 123-88 um long accor
ding to Brumpt (1949).
Fig. 3 - Third moulting of L. loa, eight days after inoculation. A: general aspect of a larva. B , C: anterior extremity; D, E: posterior extre
mity, lateral and median views (Scale: A, 500 um; B, D, E, 50 am; C, 10 um).
42- Mémoire Parasite, 1998, 5, 37-46
tion with t h e o e s o p h a g u s (Fig. 2 A to D ) . It w a s reduced in the stage 4 (Fig. 2 E ) and absent in the adult stage (Fig. 2 G ) .
• F e m a l e genital apparatus. During the stage 3, the genital anlage hardly e v o l v e d c o m p a r e d to the infec
tive larva (Fig. 1 D ) . T h e differentiation o f the genital apparatus o c c u r r e d during the stage 4 (Fig. 2 L, M ) . T h e vagina remained simple in s h a p e , without any curve or sphincter, but the muscular ovejector b e c a m e very long in the mature adult (Fig. 2 O ) .
• Male genital apparatus. During the stage 3, the genital anlage was at mid b o d y and it increased in size; it was 60-130 L I M long and 7 u.m wide at moult 3 ; its ante
rior extremity was rounded ( 1 0 - 1 2 u.m) and shortly flexed. No c o e l o m o c y t e , as described by Bartlett (1984) with Pelecitus scapiceps, was o b s e r v e d near this extre
mity. T h e spicular primordia o f the fourth stage larvae w e r e 55 μM long; the left spicular p o u c h at moult 4 w a s 2 1 5 μM long.
• Caudal papillae and alae o f the male w o r m (Fig. 1 K, N, O ) . T h e r e w a s n o i m p o r t a n t c h a n g e b e t w e e n moult 4 and maturation, e x c e p t the development o f the lateral alae. H o w e v e r it was n o t e d that the p r e s e n c e o f four p r e c l o a c a l papillae o n the left side o f a y o u n g adult male (Fig. 1 N) suggested a primitive s c h e m e o f ten pairs o f caudal papillae ( C h a b a u d & Petter, 1961), rapidly reduced to nine pairs. T h e papillae had a size a n d w e r e a r r a n g e d a c c o r d i n g to a v e r y c o n s t a n t s c h e m e ( s e e also Fig. 12 in Eberhard & Orihel, 1981).
T h e y formed a subventral set o f three equidistant pairs (the median pair has not b e e n s e e n in the young adult) and a set o f six latero-ventral pairs: three large pre
cloacal pairs, o n e small paracloacal, o n e large and o n e small post-cloacal pairs.
• Cuticular b o s s e s . Consistent with the description o f Eberhard & Orihel ( 1 9 8 1 ) , they w e r e absent in the young adult worm. T h e y a p p e a r e d during the matu
ration and formed o n e o f the anti-sliding apparatus n e c e s s a r y during mating, w h i c h have diverse origins and s h a p e s in filarioids ( B a i n & Chabaud, 1988).
DISCUSSION
N
o mature adult w o r m s o f L. loa w e r e r e c o v e r e d in m i c e a n d jirds, e v e n f o l l o w i n g i m m u n e d e p l e t i o n w i t h h y d r o c o r t i s o n e . H o w e v e r , larval d e v e l o p m e n t until the y o u n g adult stage was o b t a i n e d and the diverse e x p e r i m e n t s performed presented very constant results. C o m p a r e d to the d e v e l o p m e n t in humans o r susceptible simian spe
cies, the growth and c o n s e q u e n t l y the moults might have b e e n e x p e c t e d to b e retarded. T h u s the features o b s e r v e d in r o d e n t s s e e m r e l i a b l e . L. loa m a k e s moults 3 and 4 as early as day 8 and day 19 respecti
vely, its growth is very slight during the stage 4. Stu
dies o f the larval d e v e l o p m e n t o f the O n c h o c e r c i d s have b e e n performed in different experimental condi
tions and a critical analysis o f the results is n e e d e d before comparing the duration o f the stages as between species. T h e moulting is a long process and it is n e c e s sary to agree o n the term moult: it is only w h e n the caudal extremity and the b u c c a l capsule o f the follo
wing stage are formed under the cuticle that the e x u viation is impending; these two last phases are called the moult. In addition, moults are not perfectly syn
c h r o n o u s for a given s p e c i e s , particularly w h e n the host or experimental conditions are not suitable. For e x a m p l e , moult 3 o f O. volvulus b e g a n on day 3 and c o n t i n u e d through day 14, in surrogate primate and r o d e n t h o s t s ( A b r a h a m et al, 1993). H o w e v e r , a reliable datum is the optimal development, that is the m o r e rapid and with the greater growth. Thus w e have b a s e d the c o m p a r i s o n o n the shortest times o f stages ( T a b l e I ) .
T h e d e v e l o p m e n t s to the adult stage have b e e n e x a mined in the natural or surrogate hosts inoculated with infective larvae. T h e stage 3 has b e e n also studied under artificial conditions: infective larvae placed in m i c r o p o r e c h a m b e r s implanted in the sub-cutaneous tissue o f hosts, and in vitro cultures o f infective larvae or o f larvae w h i c h h a v e b e g u n their d e v e l o p m e n t in the vertebrate host.
T h e duration o f the stage 4, w h i c h corresponds to the genital development, a p p e a r e d d e p e n d e n t u p o n the experimental conditions. Moult 4 might b e retarded in resistant hosts. In fact, in many species, the time o f moult 4 is not k n o w n b e c a u s e it did not o c c u r , ' e v e n w h e n the survival o f the larval w o r m s lasted a long time, such as Wuchereria bancrofti in jirds (Ash &
Schacher, 1 9 7 1 ) .
O n the other hand, it appeared that the optimal dura
tion o f the stage 3 was a stable character, as well as its size, not depending upon the experimental conditions.
For e x a m p l e , in the resistant B 1 0 D 2 m i c e , the size o f Litomosoides sigmodontis larvae at moulting 3 and the date o f moulting w e r e similar to those o b t a i n e d in the susceptible B A L B / c m i c e (Maréchal et ai, 1996), in Meriones unguiculatus and in the natural host, Sig-
modon hispidus.
T h e in vitro trials are particularly interesting. T h e first o n e s , performed with Dirofilaria immitis, gave results different to t h o s e in the in vivo observations: in vitro the moult 3 was s e e n at day 3 (Yoeli et al, 1964) w h e n it was said to o c c u r b e t w e e n day 9 and day 12 in the d o g (Orihel, 1 9 6 1 ) . Later, Sawyer & Weinstein (1965) c o m p a r e d the d e v e l o p m e n t o f D. immitis in the dog, the y o u n g rat and in vitro and they noted that, in all cases, the moult o c c u r r e d at days 2-3. T h e s e features w e r e confirmed b y the other authors ( s e e review in
Parasite, 1998, 5, 37-46
Mémoire 43
Lichtenfels et al, 1 9 8 5 and T a b l e I) and the moults o b s e r v e d by Orihel c o r r e s p o n d e d probably to retarded larvae.
W o n g et al. ( 1 9 8 2 ) c o m p a r e d Brugia pahangi with D. immitis in vitro and demonstrated that the first spe
cies m o u l t e d s e v e n days later than the other, that is after nine days o f development, just as it d o e s in the host.
T h e use o f the m i x e d protocol, in vivo followed b y in vitro, performed with B. pahangi b y C h e n & Howells ( 1 9 7 9 ) and L. sigmodontis b y Nelson et al. ( 1 9 8 2 ) s h o w e d that the total in vivo + in vitro duration o f stage 3 was equal to that o b s e r v e d in the natural condi
tions.
Presently the only contradictory c a s e c o n c e r n s Oncho
cerca volvulus which, according to B i a n c o et al. ( 1 9 8 9 ) and A b r a h a m et al. ( 1 9 9 3 ) , did its first moult 3 slightly earlier than day 3 in the m i c r o p o r e c h a m b e r s and, a c c o r d i n g to Lok et al. ( 1 9 8 4 b), at day 5 in vitro. T h e first data is thought to b e the most reliable. It is similar to that o f O. lienalis for w h i c h all the authors agree ( B i a n c o & Muller, 1 9 8 2 ; Lok et al, 1984 b; B i a n c o et al, 1 9 8 9 ) .
T a k i n g into a c c o u n t these remarks, the 18 d e v e l o p mental cycles o f O n c h o c e r c i d a e elucidated in the defi
nitive host w e r e c o m p a r e d . T h e y are distributed in three subfamilies ( T a b l e I ) , t h e Dirofilariinae: t w o g e n e r a and three s p e c i e s ; the O n c h o c e r c i n a e : nine g e n e r a and 13 s p e c i e s ; the Splendidofilariinae: o n e genus, o n e s p e c i e s . H o w e v e r the data are s o m e t i m e s
^insufficient; it is the c a s e for t w o s p e c i e s : Pelecitus fulicae-atrae and Elaeophora schneideri w h i c h are
thus eliminated from the analysis.
O n e feature b e c a m e apparent by consulting the w h o l e data o f the T a b l e I: moult 3 o c c u r r e d either very early, at day 2-3, or after a b o u t a w e e k o f d e v e l o p m e n t . T h e c a s e o f Molinema dessetae n e e d s a c o m m e n t ; moult 3 w a s said to o c c u r o n day 2 0 (Gayral et al., 1 9 8 2 ) h o w e v e r w e have maintained this s p e c i e s o v e r m a n y years and o b s e r v e d the exsheathment o f moult 3 much earlier, o n day 10 ( B a i n et al, 1 9 9 4 ) . In M. arbuta, stu
died by Bartlett & Anderson ( 1 9 8 5 ) , fourth stages w e r e r e c o v e r e d t o g e t h e r with m o u l t i n g third s t a g e s o n day 2 0 , strongly suggesting that in this s p e c i e s t o o the first moults 3 o c c u r r e d earlier.
T h e distribution o f the g e n e r a b a s e d o n the duration o f the stage 3 d o e s not a g r e e with the classification o f Anderson & Bain ( 1 9 7 6 ) . H o w e v e r , the regrouping m a d e according to that character did allow association o f s o m e g e n e r a w h i c h s e e m e d to have other features in c o m m o n , s u c h as the duration o f the stage 4 and the m o r p h o l o g y o f the infective larva. T a k e n together, they c o u l d well illuminate real relationships.
T h e g r o u p presenting an early moult 3 includes two genera, w h i c h are respectively the types o f the Diro
filariinae and O n c h o c e r c i n a e subfamilies: Dirofilaria, o f w h i c h D. immitis is the only cycle elucidated, and Onchocerca, with two elucidated cycles, O. lienalis ( B i a n c o & Muller, 1982; B i a n c o et al, 1 9 8 9 ) and O. vol- vulus ( B i a n c o et al, 1 9 8 9 ) . As far as w e know, moult 4 has not b e e n determined in Onchocerca spp. (Abraham et al, 1 9 9 2 ) although the micropore c h a m b e r technique a l l o w e d r e c o v e r y o f a y o u n g adult m a l e w o r m at day 100 (Strote, 1 9 8 5 ) . Moult 4 o f D. immitis d o e s not o c c u r b e f o r e d a y 5 0 (Orihel, 1 9 6 1 ; Kotani & P o w e r s , 1 9 8 2 ; Lichtenfels et al, 1 9 8 5 ) . T h e infective larvae o f both g e n e r a have a short tail and tiny caudal lappets ( B a i n & Chabaud, 1 9 8 6 ) .
T h e s e c o n d group, with a late moult 3, includes - the seven other genera studied in the O n c h o c e r c i n a e , Lito- mosoides, Breinlia, Monanema, Acanthocheilonema, Molinema, Brugia and Wuchereria; they b e l o n g to the Dipetalonema line or are closely related to it ( C h a b a u d
& Bain, 1 9 7 6 ) , - the t w o other g e n e r a studied in the Dirofilariinae, Loa a n d Pelecitus (sensu Bartlett &
Greiner, 1 9 8 6 ) , - and the Splendidofilariinae Splendi- dofilaria ( T a b l e I ) . In this s e c o n d group, moult 4 occurs well before day 50. H o w e v e r this group appears h e t e r o g e n e o u s as Pelecitus b e c o m e s adult as s o o n as the s e c o n d w e e k following inoculation while the other g e n e r a b e c o m e adult in 2 0 - 4 0 days. T h e infective larvae o f Loa and the Dipetamonema line sensu lato have a long tail with c o n s p i c u o u s caudal lappets. Pele
citus and Splendidofilaria have different morphologies.
T h e affinities w h i c h are suggested b e t w e e n Loa and the Dipetalonema line o f the O n c h o c e r c i n a e o n o n e hand, and b e t w e e n Dirofilaria and Onchocerca o n the other, a g r e e with the results o f an analysis o f m o l e cular phylogeny, b a s e d o n the 5S DNA spacer, per- fomed b y X i e et al. ( 1 9 9 4 ) with s e v e n o f these filarial genera, and Mansonella.
CONCLUSION
T
h e rodent models, e v e n w h e n they are p o o r hosts, a p p e a r interesting for elucidating the m o r p h o g e n e s i s and the larval b i o l o g y o f fila- rioids in the definitive host. T h e k n o w l e d g e o f this p h a s e o f the cycle s o far is p o o r as data have b e e n published for only 13 out o f a b o u t 8 0 g e n e r a w h i c h c o m p o s e the O n c h o c e r c i d s .T h e determinations m a d e o n the dates o f the L. loa moults and the distinction o f t w o biological types o f filariae a c c o r d i n g to the long o r short duration o f stage 3 are o f practical interest for the simian m o d e l s o f loiasis (Pinder et al, 1 9 9 4 ) and other filarial models.
Moult 3 c o r r e s p o n d s to important c h a n g e s in the c o m position o f the cuticle ( r e v i e w e d by B e h n k e et al, 1 9 9 2 ) and s o m e authors give this p h a s e o f the c y c l e
44 Mémoire Parasite, 1998, 5, 37-46
a predominant role in the establishment o f host pro
tection against filarial infection (Eisenbeiss et al, 1 9 9 4 ; Daniel et al, 1 9 9 6 ) .
T h e s e biological features s e e m also to present a phy- logenetic interest. M o r p h o l o g y is p o o r and conver
g e n c e s important in the filarioids, due to their tissue invading habits, and n e w characters are necessary.
T h e g e n u s Loa is presently p l a c e d in the Dirofilariinae on the basis o f adult characters. T h e larval biology, the m o r p h o l o g y o f the infective larva and s o m e s e q u e n c e s o f DNA d o not s e e m to confirm its links with Dirofi- laria, w h i c h itself might b e closely related to Oncho
cerca. T h e features p r e s e n t e d here suggest that Loa should b e brought n e a r e r to the O n c h o c e r c i n a e o f the Dipetalonema line sensu lato. W e d o not suggest h o w e v e r that these hypotheses should bring about any formal systematic modifications as yet.
ACKNOWLEDGEMENTS
T h i s i n v e s t i g a t i o n r e c e i v e d s u p p o r t f r o m Elf A q u i t a i n e ( M i s s i o n R a d e a u d e s C i m e s , C a m e r o o n , 1 9 9 1 ) a n d from t h e E u r o p e a n Union, contract I C 1 8 - C T 9 5 - 0 0 2 6 .
W e are very grateful to Professor R.S. Bray for revising the English version.
REFERENCES
A B R A H A M D., E B E R H A R D M.L., L A N G E A.M., Y A T A N A W I B O O N - C H A I W . , P E R L E R F.B. & L O K J . B . Identification of surrogate hosts for larval Onchocerca lienalis and induction of pro
tective immunity in a model system. Journal of Parasito
logy, 1992, 78, 447-453.
A B R A H A M D., L A N G E A.M., Y A T A N A W I B O O N C H A I W., T R P I S M., D I C - K E R S O N J.W., S W E N S O N B. & E B E R H A R D M.L. Survival and development of larval Onchocerca volvulus in diffusion chambers implanted in primate and rodents hosts. Journal of Parasitology, 1993, 79, 571-582.
A N D E R S O N R.C. Nematode parasites of vertebrates. Their deve
lopment and transmission. CAB International, University Press, Cambridge, 1992, 578 p.
A N D E R S O N R.C. & B A I N O. CIH Keys to the nematode parasites of vertebrates. № 3. Keys to genera of the order Spirurida.
Part 3. Diplotriaenoidea, Aproctoidea and Filarioidea, 1976, 59-116. Ed. R.C. Anderson, A.G. Chabaud & Willmott S., Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, England.
A S H L.R. & R I L E Y J.M. Development of subperiodic Brugia malayi in the jird, Meriones unguiculatus, with notes on infection in other rodents. Journal of Parasitology, 1970a, 56, 969-973.
A S H L.R. & R I L E Y J.M. Development of subperiodic Brugia pahangi in the jird, Meriones unguiculatus, with notes on infection in other rodents. Journal of Parasitology, 1970b, 56, 962-968.
A S H L.R. & S C H A C H E R J.F. Early life cycle and larval morpho
genesis of Wuchereria bancrofti in the jird, Meriones unguiculatus. Journal of Parasitology, 1971, 57, 1043-1051.
B A I N O. & C H A B A U D A.G. Atlas des larves infestantes de Filaires. Tropical Medicine and Parasitology, 1986, 37, 301-340.
B A I N O. ¿4 C H A B A U D A.G. Un appareil favorisant l'accouple- ment des Filaires: les renflements de la région antérieure du corps. Annales de Parasitologic Humaine et Comparée,
1988, 63, 376-379.
B A I N O., W A N J I S., V U O N G P.N., M A R É C H A L P., LE G O F F L. &
P E T I T G . Larval biology of six filariae of the subfamily Onchocercinae in the vertebrate host. Parasite, 1994, 1, 241-254.
B A R T L E T T CM. Development of Dirofilaria scapiceps (Leidy, 1886) (Nematoda: Filarioidea) in lagomorphs. Canadian Journal of Zoology, 1984, 62, 965-979.
B A R T L E T T CM. & A N D E R S O N R.C. The third stage larva of Molinema arbuta (Highby, 1943) (Nematoda) and deve- lopment of the parasite in the porcupine (Erethizon dor- satum). Annales de Parasitologie Humaine et Comparée.
1985, 60, 703-708.
B A R T L E T T CM. & A N D E R S O N R.C. Mallophagan vectors and the avian filarioids: new subspecies of Pelecitus fulicaeatrae (Nematoda: Filarioidea) in sympatric North American hosts, with development, epizootiology, and pathogenesis of the parasite in Fulica americana (Aves). Canadian Journal of Zoology, 1989, 67, 2821-2833.
B A R T L E T T CM. & G R E I N E R E.C. A revision of Pelecitus Railliet and Henry, 1910 (Filarioidea, Dirofilariinae) and evidence for the « capture » by mammals of filarioids from birds. Bul- letin du Muséum National d'Histoire naturelle, 1986, série 4, section A, 8, 47-99-
B E H N K E J.M., B A R N A R D C.J. & W A K E L I N D. Understanding chronic nematode infections: evolutionary considerations, current hypotheses and the way forward. International Journal for Parasitology, 1992, 22, 861-907.
B I A N C O A.E. & MULLF.R R. Experimental transmission of Oncho- cerca lienalis to calves. In: Parasites-Their World and Ours. Proceedings of the Fith International Congress of Parasitology, August 7-14, Toronto, 1982, Molecular and Biochemical Parasitology, Supplement, p. 349.
B I A N C O A.E., M U L L E R R. & N E L S O N G.S. Biology of Monanema globulosa, a rodent filaria with skin-dwelling microfilariae.
Journal of Helmintbology, 1983, 57, 259-278.
B I A N C O A.E., M U S T A P H A M.B. & H A M J.P. Fate of the develo- ping larvae of Onchocerca lienalis and O. volvulus in micropore chambers implanted into laboratory hosts.
Journal of Helmintbology, 1989, 63, 218-226.
B R U M P T E. Précis de Parasitologie, Éd. Masson & Cie, 1949, 1, 1042 p.
C H A B A U D A.G. Sur le cycle évolutif des spirurides et des nema- todes ayant une biologie comparable. Valeur systématique des caractères biologiques (suite). Annales de Parasitologie Humaine et Comparée, 1954, 29, 206-249.
C H A B A U D A.G. & B A I N O. La lignée Dipetamlonema. Nouvel essai de classification. Annales de Parasitologie Humaine et Comparée, 1976, 51, 365-397.
Parasite, 1998, 5, 37-46
Mémoire
M O U L T A N D M O R P H O G E N E S I S O F LOA LOA
- 4 5
CHABAUD A.G. & BAIN O. The evolutionary expansion of the Spirurida. International Journal of Parasitology, 1994, 24, 1179-1201.
CHABAUD A.G. & PETTER A. Remarques sur revolution des papules cloacales chez les Nematodes Phasmidiens para
sites de Vertebres. Parassitologia, 1961, 3, 51-70.
CHEN S.N. & HOWELLS R.E. The in vitro cultivation of the infec
tive larvae and the early mammalian stages of the filarial worm Brugia pahangi. Annals of Tropical Medicine and Parasitology, 1979, 73, 473-486.
CROSS J . H . , PARTONO F., HSU MY.K., ASH L.R. & OEMIJATI S.
Experimental transmission o f Wuchereria bancrofti to monkeys. American Journal of Tropical Medicine and Hygiene, 1979, 28, 56-66.
DANIEL C. & EISENBEISS W.F. Protection against Acanthochei- lonema viteae provided by Caenorhabiditis elegans larval stages in jirds. Spring Meeting of the British Society for Para
sitology, Univ. of Wales, Bangor, April 1-3, 1996, Abstracts.
DUKE B.O.L. & WIJERS D.J.B. Studies on loiasis in monkeys.
I. The relationship between human and simian Loa in the rain forest of the British Cameroon. Annals of Tropical Medicine and Parasitology, 1958, 52, 158-175.
EBERHARD M.I. & ORIHEL T.C. Development and larval mor
phology of Loa loa in experimental primate hosts. Journal of Parasitology, 1981, 67, 557-564.
EDESON J.F.B. & BUCKLEY J J.C. Studies on filariasis of Wuche
reria malayi from man to domestic cat. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1959, 51, 366-370.
EISENBEISS W.F., APFEL H . & MEYER T.F. Recovery, distribution and development of Acanthocheilonema viteae third and early fourth stage larvae in adult jirds. Journal of Parasi
tology, 1991, 52, 580-596.
EISENBEISS W.F., APFEL H . & MEYER T.F. Protective immunity linked with a distinct developmental stage of a filarial para
site. Journal of Immunology, 1994, 152, 735-742.
GAYRAL P., DREYFUS G . & GANTIER J-C. Dipetalonema dessetae chez Proechimys oris. Cycle biologique de la filaire et pro
position d'un modele de filariose experimentale de ron
geur. Cahiers de l'ORSTOM, serie Entomologie medicale et Parasitologic 1982, 20, 81-94.
HIBI.FR C P . Onchocercidae (Nematoda: Filarioidea) of the American magpie, Pica pica hudsonia (Sabine) in northern Colorado. PhD thesis, Colorado State University, Fort Col
lins, Colorado, USA (unpublished).
HIBLER C P . & METZGER C J , Morphology of the larval stages of Elaeophora schneideri in the intermediate and defini
tive hosts with some observations on their pathogenesis in abnormal definitive hosts. Journal of Wildlife Diseases, 1974, 10, 361-369.
JOHNSON M.H., ORIHEL T.C. & BEAVER P.C. Dipealonema viteae in the experimentally infected jird, Meriones unguicu- latus. I. Insemination, development from egg to microfi
laria, reinsemination and longevity of mated and unmated worms. Journal of Parasitology, 1974, 60, 302-309.
KOTANI T. & POWERS K . G . Developmental stages of Dirofilaria immitis in the dog. American Journal of Veterinary Research, 1982, 43, 2199-2206.
LiCHTF.NFELS J.R., PiUTT P.A., KOTANI T. & POWERS K . G . Mor
phogenesis of developmental stages of Dirofilaria immitis (Nematoda) in the dog. Proceedings of The Helmintholo- gical Society of Washington, 1985, 52, 98-113.
LOK J . B . , MIKA-GRIEVE M., GRIEVE R.B. & CHIN T.K. In vitro deve
lopment of third- and fourth-stage larvae of Dirofilaria immitis. comparison of basal culture media, serum levels and possible serum substitutes. Acta Tropica, 1984a, 41, 145-154.
LOK J . B . , POLLACK R.J., CUPP E . W . , BERNARDO M.J., DONNELLY J . J .
& ALBIEZ E . J . Development of Onchocerca lienalis and O. volvulus from the third to fourth larval stage in vitro.
Tropenmedizin und Parasitologic, 1984b, 35, 209-211.
MARÉCHAL P., LE GOFF L., PETIT G . , DIAGNE M., TAYLOR D.W.
& BAIN O. The fate of the filaria Litomosoides sigmodontis in susceptible and naturally resistant mice. Parasite, 1996, 3, 25-31.
NELSON P . B . , WEINER D.J., STROMBERG B . E . & ABRAHAM D.
In vitro cultivation of third-stage larvae of Litomosoides carinii to the fourth stage. Journal of Parasitology, 1982, 68, 971-973.
ORIHEL T.C. Morphology of the larval stages of Dirofilaria immitis in the dog. Journal of Parasitology, 1961, 47, 251- 262.
PINDER M., EVERAERE S. & ROELANTS G . E . Loa loa-. Immunolo
gical responses during experimental infections in mandrills.
Experimental Parasitology, 1994, 79, 126-136.
SAWYER T . K . & WEINSTEIN P.P. Third molt of Dirofilaria immitis in vivo and in vitro. Journal of Parasitology, 1965, 51, sect. 2, 48.
SCHACHER J.F. Developmental stages of Brugia pahangi in the final host. Journal of Parasitology, 1962, 48, 693-706.
SCOTT J.A., MCDONALD E.M., TERMAN B . A description of the stages in the life cycle of the filarial worm Litomosoides carinii. Journal of Parasitology, 1951, 37, 425-432.
SINGH M., YAP E.H., Ho B . C . , KANG K.L., LIM E.P.C. & LIM B . I . Studies on the Malayan forest rat filaria Breinlia booliati (Filarioidea: Onchocercidae): course of development in the rat host. Journal of Helminthology, 1976, 50, 103-110.
STROTE G . Development of infective larvae of Onchocerca vol
vulus in diffusion chambers implanted into Mastomys natalensis. Tropical Medicine and Parasitology, 1985, 36, 120-122.
WAHL G . , MOUKAGNI R., TOURÉ F. & GEORGES A.J. Large scale collection of viable infective larvae o f Loa loa. Tropical Medicine and Parasitology, 1995, 46, 203-204.
WANJI S., CABARET J . , GANTIER J . - C , BONAND N . & BAIN O. The fate of the filaria Monanema martini in two rodent hosts:
recovery rate, migration and localization. Annales de Para
sitologic Humaine et Comparée, 1990, 65, 80-88.
XIE H., BAIN O. & WILLIAMS S. Molecular phylogenetic studies on filarial parasites based on 5S ribosomal spacer sequences. Parasite, 1994, 1, 141-151.
YOELI M., UPMANIS S. R. & MOST H. Studies on filariasis.
III. Partial growth of the mammalian stages of Dirofilaria immitis in vitro. Experimental Parasitology, 1964, 15, 325- 334.
Reçu le 14 mai 1997 Accepté le 15 décembre 1997
Í 6 Mémoire Parasite, 1998, 5, 37-46
