PERIODIC INFECTIVITY OF PLASMODIUM GAMETOCYTES TO THE VECTOR.
A REVIEW
GAUTRET P.* & MOTARD A.**
S u m m a r y :
Frank Hawking, in 1 9 6 6 postulated that in synchronous malaria infections, the brief period of infectivity of gametocytes was timed to occur when the vector bites. Since this early work, numerous studies had contributed to confirm and explain this phenomenon with bird, rodent and primate Plasmodium. Data on the periodic production of gametocytes, the duration of their maturation, the effect of the schizogony on the infectivity and the circadian bioavailability of gametocytes provide some more informations on the periodic Plasmodium gametocyte infectivity to the vector. This paper is intended to be a review of contributions on the "Hawking phenomenon" and to summarize the principal causal hypotheses.
The conclusion stresses the practical consequences for experimental studies and epidemiological surveys.
KEY WORDS : Plasmodium, Leucocytozoon, gametocytes, infectivity, transmission, sequestration, periodicity.
Resume : INFECTIVITÉ PÉRIODIQUE DES GAMÉTOCYTES DE PLASMODIUM POUR LE VECTEUR. MISE AU POINT
Franck Hawking en 1966 énonce et confirme l'hypothèse suivante : dans les infections palustres synchrones, la période d'infectivité maximale des gamétocytes coincide avec le pic d'activité des moustiques vecteurs. Depuis, de nombreux travaux ont confirmé ses observations et expliqué ce phénomène pour
certains Plasmodium d'oiseaux, de Rongeurs et de Primates. Les observations sur la production périodique des gamétocytes, la durée de leur maturation, l'effet de la rupture des schizontes sur leur infectivité et les variations circadiennes de leur biodisponibilité pour le moustique ont permis des avancées dans la
compréhension du mécanisme de périodicité d'infectivité des gamétocytes de Plasmodium. Cet article a pour objectif de présenter une compilation des données disponibles sur le
"phénomène de Hawking" et d'en résumer les principales hypothèses explicatives. La conclusion indique les conséquences pratiques pour les travaux expérimentaux et les études
épidémiologiques.
MOTS CLÉS : Plasmodium, Leucocytozoon, gamétocytes, infectivité, transmission, séquestration, périodicité.
A
remarkable feature o f most o f bird, rodent and primate malaria is the p r e c i s e timing o f its recurrent attacks w h i c h are generally at s o m e multiple o f 24 hours. This implies that the duration o f the erythrocytic asexual cycle is stable and that all t h e parasites b e h a v e synchronously, reaching s c h i z o - g o n y at the s a m e time. Cell division takes p l a c e at an hour o f the day constant for e a c h s p e c i e s o f malaria parasite w h i c h d e p e n d s o n the location o f the host or for s o m e s p e c i e s mostly on the time o f inoculation o f the parasite. T h e biological p u r p o s e o f such an a c c u r a t e timing in the c y c l e o f Plasmodium w a s s h o w n b y Hawking ( 1 9 7 0 ) to assist them to present infective g a m e t o c y t e s at t h e time m o s q u i t o e s bite and* Laboratoire de Parasitologie et de Mycologie Médicale, CHU La Milétrie, BP 577, 86021 Poitiers Cedex, France.
** 32 Lot. Alvina, 97354 Remire, Guyane Française.
Correspondence: Dr. Philippe Gautret.
Tel: 0033(0)549443959 - Fax: 0033(0)549443908.
E-mail: parasitologie@chu.univ-poitiers.fr
s o rendering the transmission m o r e efficient. In other terms, Hawking s h o w e d that the production o f short- lived g a m e t o c y t e s was cyclic, leading to circadian variations in the ability o f Plasmodium to infect the m o s q u i t o v e c t o r and it was called by G a r n h a m &
P o w e r s ( 1 9 7 4 ) , "the Hawking p h e n o m e n o n " . Since Hawking's work, n e w data o n periodic infectivity o f Plasmodium and o t h e r Haemosporidia to the v e c t o r have b e e n published and experimental work has b e e n d o n e in order to determine its m e c h a n i s m . This per- iodic infectivity d e p e n d s o n the duration o f g a m e t o - cyte maturation, periodic sequestration o f g a m e t o - cytes and periodic release o f parasite and/or vertebrate host c o m p o u n d s that regulate the g a m e t o c y t e infecti- vity. This p a p e r is intended to b e a review o f contri- butions o n the periodic infectivity o f the g a m e t o c y t e s o f the Haemosporidia to their vectors and to sum- marize the principal causal h y p o t h e s e s . T h e c o n c l u - sion stresses the practical c o n s e q u e n c e s o f the Haw- k i n g p h e n o m e n o n for e x p e r i m e n t a l s t u d i e s a n d e p i d e m i o l o g i c a l surveys.
Parasite, 1999, 6, 103-111
Mise au point 103
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1999062103
PERIODIC PRODUCTION OF GAMETOCYTES
T
h e periodicity o f the production o f g a m e t o c y t e s can b e assessed in different ways ( T a b l e I ) . Total g a m e t o c y t e counts can b e m a d e at short timed intervals over a period o f several days, exfla- gellation rate can b e o b s e r v e d at regular intervals and m o r p h o l o g i c a l analysis o f the g a m e t o c y t e s c a n b e made over time. G a m e t o c y t e m o r p h o l o g y u nde r goe s age-related transformations so that different morphological stages can b e defined, although all intermediates can b e observed. During the course o f normal ageing, the gametocyte diameter increases at first and b e c o m e s then smaller while its chromophilic character increases.
T h e nucleus is at firts relatively small, then enlarges before b e c o m i n g c o n d e n s e d and progressively smaller.
T h e nucleus is granular at the begining o f its evolu
tion and colloidal at the e n d (Landau et al, 1 9 7 9 ) .
AVIAN PLASMODIUM
Periodic production o f sexual stages was first d e m o n s - traded with avian Plasmodium. In 1934, Shah claimed that the n u m b e r o f g a m e t o c y t e s o f P. cathemerium ( 2 4 hour c y c l e ) in the peripheral circulation o f Serinus canarias varied at different periods during the day, and s h o w e d a daily increase around 6 p. m. This was the first report o f a periodicity in the production o f Plas
modium sexual stages. In a continuation o f that ear
lier work, Gambrell ( 1 9 3 7 ) also observed a p e a k in the n u m b e r o f g a m e t o c y t e s at 6 p. m. Hawking et al.
( 1 9 6 8 a ) c o n f i r m e d the periodical c h a r a c t e r o f the g a m e t o c y t o g e n e s i s o f P. cathemerium and s h o w e d a
nocturnal p e a k o f exflagellating m i c r o g a m e t o c y t e s at 4-6 a. m. A corresponding cycle was demonstrated in the morphology o f the gametocytes w h i c h undergo six maturation stages.
P. relictum matutinum was studied by Gambrel ( 1 9 3 7 ) and w a s s h o w n to present a definite increase in num
bers o f g a m e t o c y t e s around the time o f segmentation which takes place daily at 8 a.m. P. gallinaceum game
tocytes w e r e s h o w n by Lumsden & Bertram ( 1 9 4 0 ) to b e p r o d u c e d in b r o o d s and to p e a k at about the s a m e time as the p e r c e n t a g e o f schizonts increased (i. e.
alternatively at midnight and at midday in relation to the 3 6 hour cycle). Similarly, Hawking et al. ( 1 9 7 2 ) des
c r i b e d a p e a k o f exflagellation o f P. gallinaceum m i c r o g a m e t o c y t e s w h i c h d e v e l o p p e d in four matura
tion stages. Roller & D e s s e r ( 1 9 7 3 ) demonstrated that the numbers o f Leucocytozoon simondi gametocytes in ducks p e a k e d during day time w h e n the activity o f the vector Simulium rugglesi was maximum. Similar fluc
tuations o f the numbers o f g a m e t o c y t e s in the per
ipheral b l o o d were seen in L. smithi infected fowls. T h e diurnal p e a k s o c c u r e d at the time w h e n S. slossonae, the vector, bites (Noblet & Noblet, 1 9 7 6 ) . T h e inver
sion o f the photo-period shifts the p e a k s by 12 hours (Noblet & Noblet, 1 9 7 7 ) and c o n t i n u e d e x p o s u r e o f fowls to light suppressed the periodicity o f gametocytes in the peripheral b l o o d ( G o r e & Noblet, 1 9 7 7 ) . Pinea- lectomy, h o w e v e r did not have any effects on this per
iodicity ( G o r e et al., 1 9 8 2 ) .
RODENT PLASMODIUM
With rodent malaria, the first observation o f a rhythm in the production o f g a m e t o c y t e s was that o f Hawking et al. ( 1 9 7 2 ) w h o s h o w e d that the exflagellating P. cha-
Time o f
Number Interval between
Total g a m e t o c y t e Exflagellation o f g a m e t o c y t e two g a m e t o c y t e s
peak peak stages described b r o o d s ( h o u r s )
P. catbemerium 6 p.m. 4-6 p.m. 6 stages 24
P. relictum 8 a.m. NS NS 24
P. gallinaceum noon/midnight noon/midnight 4 stages 36
L. simondi day time NS NS 24
L. smithi day time NS NS 24
P. chabaudi NS midnight-4 p.m. 4 stages 24
P. vinckei NS NS 4 stages 24
P. bergbei * NS NS NS
P. yoelii NS 4 stages 24
P. cynomolgi NS midnight (Langur)
6 p.m. (Nilgiri)
8 stages 48
P. knowlesi midnight midnight 4 stages 24
P. falciparum NS night time 5 stages 48
P. vivax λ S NS 7 stages 48
NS = not studied; * = Depending to the time of parasite inoculation.
Table I. - Periodic production of Haemosporidia gametocytes.
104 Mise au point Parasite, 1999, 6. 103-111
PERIODIC INFECTIVITY O F PLASMODIUM GAMETOCYTES
baudi g a m e t o c y t e s w e r e in great n u m b e r at 0-4 a. m at the time o f schizogony. Later, a 24 hour rhythm o f production o f g a m e t o c y t e s was confirmed by Gautret et al. ( 1 9 9 6 c ) with P. chabaudi as w e l l as with P. vinckei vinckei and P. v. petteri (Gautret et al., 1995;
1996α). Landau et al. ( 1 9 7 9 ) described four evolutio
nary stages (0, I, II and III) in rodent Plasmodium gametocytes. In parasites like P. berghei and P. yoelii, a periodic production o f g a m e t o c y t e s can also b e s h o w n after synchronization. P. berghei w a s synchro
nized b y Hawking et al. ( 1 9 7 2 ) in Thamnomys sur- daster by repetitive p a s s a g e s from Thamnomys to Jbamnomys and s h o w e d a p e a k o f exflagellation o f m i c r o g a m e t o c y t e s at a time depending o f the time o f inoculation o f the parasite to the rodent. P. y. yoelii and P. y. nigeriensis w e r e synchronized by Percoll-glu- c o s e concentration o f y o u n g stages for inoculation ( D e h a r o et al, 1994) and a 24 hour rhythm o f pro
duction o f g a m e t o c y t e s was demonstrated (Gautret et al. 1995; 1996b.).
PRIMATE PLASMODIUM
W o r k was also performed with primate Plasmodium.
Light stages o f maturation were identified with P. cyno- molgi ceylonensis, Langur strain (C strain) gametocytes in rhesus m o n k e y s (Hawking et al., 1968α) with a peak o f exflagellation occuring at midnight every 4 8 hours.
Rao et al ( 1 9 7 1 ) also o b s e r v e d a p e a k o f exflagella- tion but it o c c u r e d at 6 a. m. probably b e c a u s e they used the Nilgiri strain and w o r k e d in Delhi w h e r e a s Hawking w o r k e d in London. Similarly, five stages o f maturation w e r e d e m o n s t r a t e d with P. knowlesi in rhesus m o n k e y s and the total numbers o f gametocytes increased at midnight every 24 hours as well as the numbers o f exflagellating gametocytes (Hawking et al, 1968a). P. vivax has b e e n less studied. B o y d (1935) estimated the duration o f the maturation o f m o r p h o logically infective gametocytes to b e 4 8 hours and des
cribed s e v e n evolutive stages.
P. FALCIPARUM
P. falciparum g a m e t o c y t e s undergo five maturation stages in man (Field & Shute, 1956), in splenectomized Aotus ( H a w k i n g et al, 1 9 7 1 ) and in vitro (Smalley, 1976). Stage V n e e d s nine days to b e p r o d u c e d but three m o r e days are required for the g a m e t o c y t e s to be able to exflagellate (Jeffery & Eyles, 1 9 5 5 ) . O b s e r vation with P. falciparum in man s h o w e d m o r e exfla- gellations at night than during day time and, exflagel
lation p e a k s w e r e o b s e r v e d at 4 8 h o u r intervals ( H a w k i n g et al, 1 9 7 1 ) . T h e authors c o n s i d e r e d this observation to b e related to a circadian t e n d e n c y in the production o f infective sexual stages. However, the difficulty to e v i d e n c e this periodicity, due to both the
approximate 4 8 hour rhythm o f P. falciparum asexual stage d e v e l o p m e n t and the long time o f gametocyte maturation led Hawking to c o n c l u d e that his results required futher investigations to b e confirmed.
Thus, in all the s p e c i e s studied, n e w g a m e t o c y t e s are p r o d u c e d f o l l o w i n g s u c c e s s i v e s c h i z o g o n i e s a n d undergo morphological maturation stages leading to the formation o f g a m e t o c y t e s infective to the vector at a precise timing. T h e life cycle o f the parasites is carefully timed s o that they are most ready for transfer from the vertebrate host to m o s q u i t o during the hours w h e n the m o s q u i t o e s are most likely to b e feeding (Hawking, 1 9 7 5 ) . T h e c a s e o f P. gallinaceum with an alternative production o f infective gametocytes at mid
night and at n o o n , in relation with its 36 hour schi- zogonic rhythm could b e the c o n s e q u e n c e o f an adap
tation to the bitting habits o f Aedes or Culex vectors in Asia.
INFECTIVE STAGES OF GAMETOCYTES AND DURATION OF THEIR MATURATIONS
The chronology o f gametocyte development (i. e.
the time required by a merozoite to undergo the different evolutionary stages o f g a m e t o c y t o g e - nesis) can b e assessed by evaluating the relative pro
portion o f e a c h stages at short timed interval during the c o u r s e o f g a m e t o c y t a e m i a ( T a b l e II). T h e scatte
ring o f the pigment or the exflagellation ability o f male g a m e t o c y t e s are used by several authors as an indi
cator o f the maturity/infectivity o f sexual stages. W e consider, however, correlation analysis b e t w e e n the proportion o f e a c h stage at the time o f v e c t o r feeding and the n u m b e r o f oocyst in insect midgut to b e a much m o r e accurate and reliable parameter for the identification o f the infective g a m e t o c y t e stage.
Duration Mean life Infective o f maturation span o f morphological from merozoite infective
stage to infective stage stage
P. cathemerium III (F)-IV (M) 24 h/30-36 h/26-29 h P. gallinaceum IV 60/64 h 17 h P. chabaudi II 48 h 3-6 h P. vinckei II 36 h 3-6 h P. berghei NS 26 h 7 h P. yoelii 0 24 h S h P. cynomolgi IV/V (F)-VII (M) 58 h 12 h P. knowlesi IV 31 h 5 h P. falciparum V 12 days 2.5 days
NS = not studied; h = hours; F = female; M = male.
Table II. - Infective stage of Plasmodium gametocytes and duration of their maturation.
Parasite, 1999, 6, 103-111
M ¡se au point 105
B I R D PLASMODIUM
Shah by studing the morphology o f P. cathemerium gametocytes c o n c l u d e d that they r e a c h e d their matu
rity after about 24 hours, at the time o f schizogony.
Gambrel ( 1 9 3 7 ) , by contrast, considered the lenght o f the maturation o f a merozoite into mature gametocyte to b e 30-36 hours, and the gametocytes ageing 24 hours to b e premature gametocytes. Both authors c o n s i d e r e d the scattering o f the pigment as a criterion for matu
rity. Hawking et al. (19638a), identified type III and type IV g a m e t o c y t e s to b e the mature stages respecti
vely for m a c r o g a m e t o c y t e s and m i c r o g a m e t o c y t e s and the time required was 2 6 - 2 9 hours. T h e y c o n s i d e r e d the exflagellation to b e the indication o f the full matu
rity o f the m i cr ogam et oc yt es . In this latter work, the authors u s e d an Italian strain isolated from a sparrow near R o m e in 1966 and the time o f s c h i z o g o n y w a s 11 p. m. Shah used a strain from the University o f Syra
c u s e w h o s e origin w a s not m e n t i o n e d and Gambrell used the D strain from R o m e . B o t h strains underwent s c h i z o g o n y at 6 p. m. T h e discrepancies in the dura
tion o f the g a m e t o c y t e maturation and in the time o f s c h i z o g o n y may b e due to the use o f different strains called P. cathemerium. P. gallinaceum g a m e t o c y t e maturity was c o n s i d e r e d by Lumsden and Bertram ( 1 9 4 0 ) to b e a c h i e v e d in 6 0 hours. This w a s confirmed by Hawking et al. ( 1 9 6 9 ; 1 9 7 2 ) w h o demonstrated a relatively brief period ( 1 7 hours) o f exflagellation o f male gametocytes and evaluated the duration o f the maturation to reach type IV gametocytes to b e 64 hours.
RODENT PLASMODIUM
With P. chabaudi, P. v. vinckei and P. v. petteri, the time o f maturation o f merozoites to type II infective g a m e t o c y t e s is 4 8 , 4 8 and 3 6 hours and they d e g e n e rate in six to three hours (Gautret et al. 1 9 9 5 ; 1 9 9 6 a ; 1 9 9 6 c ) . P. chabaudi microgametocytes w e r e s h o w n by Hawking et al. ( 1 9 7 2 ) to b e able to exflagellate during seven hours. Mons et al. ( 1 9 8 5 ) synchronized the deve
l o p m e n t o f P. berghei g a m e t o c y t e s in vitro and in vivo in rats and estimated the time o f maturation to b e 26 hours (confirming work by Hawking et al. ( 1 9 7 2 ) in Thamnomys) and the m e a n duration o f infectivity was s h o w n to b e about 13 hours. P. y. yoelii and P. y. nigeriensis, w h e n synchronized by the Percoll-glu- c o s e technic, d e v e l o p their merozoites into mature type 0 gametocytes in 24 hours and degenerate in three h o u r s . T h i s is c o n s i s t e n t with the o b s e r v a t i o n b y Killick-Kendrick & Warren ( 1 9 6 8 ) w h o s h o w e d that the first infective g a m e t o c y t e s appeared 24 hours after the rupture o f hepatic meronts. T h e discrepancy b e t w e e n the infective stages o f the different rodent Plasmodium (stage II for P. vinckei and P. chabaudi and stage 0 or P. yoelii) is surprising but correlates with a similar
discrepancy in the sequestration o f g a m e t o c y t e s in capillaries ( s e e b e l o w ) .
PRIMATE PLASMODIUM
P. cynomolgi ceylonensis (Langur С strain) merozoites transform into mature female (type IV and IV) and male (type VII) gametocytes in 5 8 hours and degenerate in about 12 hours (Hawking et al, 1 9 6 8 a ) . P. knowlesi mature type IV male and female g a m e t o c y t e s n e e d 31 hours to b e produced and degenerate in n o m o r e than five hours (Hawking et al, 1 9 6 8 a ; 1 9 6 8 b ) .
P. FALCIPARUM
T h e life span o f P. falciparum mature stage V g a m e tocyte is longer than that o f other m a mma li a n Plas
modium but relatively short. Smalley & Sinden ( 1 9 7 7 ) o b s e r v e d the p r e s e n c e o f exflagellating stage V g a m e tocytes in the b l o o d smears obtained from patients fol
lowing radical c h l o r o q u i n e treatment during a m e a n time o f 2.4 days. P. falciparum gametocytes can remain infective to the v e c t o r for m a n y days as demonstrated by Jeffery, Y o u n g & Eyles ( 1 9 5 6 ) and Smalley & Sinden ( 1 9 7 7 ) w h o successfully infected m o s q u i t o e s from g a m e t o c y t e carriers up to 11-12 days following radical chloroquine treatment. It must b e pointed out h o w e v e r that these infections led to considerably reduced oocyst burden per infected mosquito.
T h u s , with the e x c e p t i o n o f P. falciparum, mature infective g a m e t o c y t e s o f the different s p e c i e s o f Plas
modium have a short life span w h i c h d o e s not e x c e e d 17 hours and is most o f the time, only b e t w e e n three and seven hours.
TEMPORARY LOSS OF GAMETOCYTE INFECTIVITY DURING SCHIZOGONY
I
ndependently o f cyclical maturation o f infective g a m e t o c y t e s , several authors recently d e s c r i b e d m e c h a n i s m s o f periodic production o f factors inhibiting g a m e t o c y t e infectivity.
RODENT PLASMODIUM
Motard et al. ( 1 9 9 0 ) s h o w e d that g a m e t o c y t e s in m i c e infected with P. v. petteri l o o s e temporarily their infec
tivity for Anopheles stephensi at the time o f schizogony.
This t e m p o r a r y loss o f g a m e t o c y t e infectivity w a s demonstrated to b e linked with a p e a k o f production o f nitric o x i d e during the s c h i z o g o n y (Motard et al, 1993). T h e responsability o f antibodies in this p h e n o m e n o n is very unlikely as g a m e t o c y t e infectivity inhi
bition o c c u r s in P. berghei infected SCID mice for A. stephensi (Sinden et al., 1 9 9 3 ; Fleck et al., 1 9 9 4 ) .
106 Mise au point Parasite, 1999, 6, 103-111
PERIODIC INFECTIVITY O F PLASMODIUM GAMETOCYTES
PRIMATE PLASMODIUM
A transcient inhibition o f P. vivax g a m e t o c y t e infecti
vity w a s o b s e r v e d in m a n at t h e time o f clinical paroxisms (Mendis et al. 1 9 9 0 ) . T u m o u r necrosis factor a n d g a m m a interferon l e v e l s are i n c r e a s e d w h e n P. vivax and P. cynomolgi g a m e t o c y t e s infectivity for A. tessellatus is reduced during clinical paroxisms (Karu- naweera et al., 1 9 9 2 ; Naotune et al, 1991). White b l o o d cells and reactive nitrogen intermediates are necessary to obtain an inhibition o f gametocyte infectivity by the supernatant o f cultured peripheral b l o o d mononuclear cells stimulated by frozen-thawed b l o o d stages o f Plas
modium. This is the c a s e for P. vivax/A. tessellatus in man and for P. falciparum ( 3 D 7 ) / A . stephensi, in vitro (Naotunne et al, 1993).
In conclusion, inhibition o f Plasmodium g a m e t o c y t e infectivity at the time o f s c h i z o g o n y may b e the c o n s e q u e n c e o f the activation o f cellular immunity effectors via the secretion o f cytokines and production o f nitric- oxides. It is very likely that parasite origin factors are released at the time o f s c h i z o g o n y also and play a role o f initial activators o f the white cells.
SEQUESTRATION AND ORCADIAN BIOAVAILABILITY OF GAMETOCYTES
A
n alternative explanation to the Hawking phen o m e n o n is the e x i s t e n c e o f a circadian prefe
rential distribution (sequestration, retention or r e l e a s e ) o f infective g a m e t o c y t e s in the capillaries, r e n d e r i n g t h e m a v a i l a b l e to t h e m o s q u i t o v e c t o r . Sequestration o f asexual b l o o d stages o f Plasmodium in d e e p capillaries is very well k n o w n and hundreds o f publications have b e e n p r o d u c e d since the first o b s e r v a t i o n with P. falciparum b y Marchiafava &
Bignami ( 1 8 9 4 ) . Sequestration o f other Haemosporidia g a m e t o c y t e s b y contrast have b e e n less studied.
B I R D PLASMODIUM
Missiroli ( 1 9 3 9 ) m a d e the first report o f sequestration o f P. praecox g a m e t o c y t e s in sparrows by demonstra
ting an increase o f their n u m b e r s in the b l o o d o f birds submited to m o s q u i t o Culex bites. Roller & D e s s e r ( 1 9 7 3 ) s h o w e d that g a m e t o c y t e s o f L. simondi were in greater numbers in hepatic b l o o d than in cardiac b l o o d o f d u c k s w h e n the peripheral parasitemia w a s low at night.
RODENT PLASMODIUM
Experiments with rodent Plasmodium are very n u m e rous and signifiant. Comparison b e t w e e n g a m e t o c y t e c o m p o s i t i o n o f b l o o d from the tail vein o f P. y. yoelii
infected mice and b l o o d from mosquito midgut i m m e diately after e n g o r g e m e n t s h o w e d an enrichment o f type 0 and I m i c r o g a m e t o c y t e s in the latter w h e n the infectivity to the v e c t o r is maximal (Landau et al, 1979; Gautret et al., 1996b). This p h e n o m e n o n was nei
ther s e e n b y J a n s e et al. ( 1 9 8 5 ) w h e n c o m p a r i n g P. berghei gametocyte composition o f b l o o d from mice tail vein, mice heart b l o o d and mosquito b l o o d meal nor by Mons et al. ( 1 9 8 5 ) w h o observed the same num
bers o f exflagellating gametocytes in vitro and in vivo.
This is probably b e c a u s e it only occurs at the time o f m a x i m u m infectivity and affects the infective stage only. With P. v. petteri and P. chabaudi, stage II infec
tive gametocytes are found in greater numbers in mos
quito b l o o d meal than in mice tail vein b l o o d (Gautret et al, 1 9 9 6 a ; Gautret et al., 1 9 9 6 c ) . W h e n experiments w e r e performed with P. chabaudi at the time type 0 gametocytes peak in m i c e blood, n o sequestration was observed indicating that the retention o f gametocytes in d e e p capillaries affects only the infective stage and therefore occurs at a precise time in the cycle o f matu
ration.
PRIMATE PLASMODIUM
With primates, Dei-Cas et al. ( 1 9 8 0 a ; 1 9 8 0 b ) also o b s e r v e d a difference in the counts o f various stages o f P. inui m a c r o g a m e t o c y t e s w h e n comparing per
i p h e r a l m o n k e y b l o o d a n d m o s q u i t o m e a l s a n d c o n c l u d e d that a sequestration o f certain types o f gametocytes in m o n k e y capillaries occurs.
P. FALCIPARUM
P. falciparum mature g a m e t o c y t e s are detectable in h u m a n peripheral b l o o d around 10 days after asexual stages (Ross & T h o m s o n , 1 9 1 1 ) , c o r r e s p o n d i n g to 10 days after the first p e a k o f fever producing schi- zonts (Bray, personal communication). Immature game
tocytes are s e q u e s t e r e d in spleen capillaries and in b o n e marrow ( D e Beaurepaire Arago, 1 9 3 0 ; Garnham, 1 9 3 1 ; T h o m s o n & Robertson, 1 9 3 5 ; Smalley et al, 1980) but not in placental vessels (Blacklock & Gordon, 1 9 2 4 ; Bray & Sinden, 1 9 7 9 ; Desowitz & B u c h b i n d e r , 1 9 9 2 ) . In s p l e n e c t o m i z e d Aotus y o u n g P. falciparum g a m e t o c y t e s are not s e q u e s t e r e d anymore, indicating that the spleen plays a major role in this p h e n o m e n o n ( H a w k i n g et al, 1 9 7 1 ; W a r d et al, 1 9 7 2 ) . Similar results w e r e obtained by Bray ( 1 9 5 8 ) in c h i m p a n z e e s . Van den B e r g h e ík C h a r d o m e ( 1 9 5 1 ) s h o w e d that P. falciparum gametocytes in man w e r e m o r e n u m e rous in smears m a d e o f c u t a n e o u s scarifications than in peripheral b l o o d smears. T h e c h a n g e s o f shape o f erythrocytes parasitized b y P. falciparum gametocytes is associated with the d e v e l o p m e n t o f microtubules under the parasite m e m b r a n e (Sinden et al, 1 9 7 8 )
Parasite, 1999. 6, 103-111
Mise au point 107
which may m a k e the infected cell m o r e rigid, leading to a m e c h a n i c a l s e q u e s t r a t i o n in t h e c a p i l l a r i e s . Recently, specific c y t o a d h e r e n c e to C32 cells o f the red b l o o d cells parasitized in vitro by stages I and II P. fal- ciparum gametocytes was demonstrated (Rogers et al., 1996α). This adherence was inhibited by anti-CD36 anti
bodies and by anti-ICAM I antibodies. Infected red cells use the modified b a n d 3 to adhere to C32 and CD36- transfected CHO cells (Rogers et al.,
1996b).
This adherence to C D 3 6 was confirmed by Day et al. ( 1 9 9 8 ) and demonstrated to b e linked with k n o b p r e s e n c e and HRP1 expression in parasitized red b l o o d cells.
In conclusion, p h e n o m e n o n o f sequestration/release o f infective g a m e t o c y t e s in the fine capillaries is cyclical and p r o b a b l y results from the cyclical production o f mature g a m e t o c y t e s . B y favoring the absorption o f the gametocytes b y the vector it facilitates the transmission o f the sexual stages from the mamalian to the insect.
PERIODIC TRANSMISSION
OF PLASMODIUM TO THE VECTOR
T
he logical c o n s e q u e n c e o f the a b o v e reported observations should b e a circadian cycle in the infectivity o f Plasmodium to t h e m o s q u i t o . T r a n s m i s s i o n e x p e r i m e n t s h a v e b e e n t h e r e f o r e c o n d u c t e d by several authors in order to investigate variations in the production o f oocysts in insects according to the time o f feeding.
BIRD PLASMODIUM
A periodical variation in the n u m b e r s o f avian Plas
modium oocysts o b t a i n e d in m o s q u i t o e s fed o n the v e r t e b r a t e host at different times o f the day w a s demonstrated with P. catbemerium (Shah et al. 1 9 3 4 ; Hawking et al, 1 9 6 8 a ) , P. gallinaceum (Lumsden &
Bertram, 1 9 4 0 ) , P. praecox (Missiroli, 1 9 3 7 ) . In all ins
tances, the p e a k o f oocysts w a s found to b e b e t w e e n s c h i z o g o n i c peaks.
RODENT PLASMODIUM
With rodent malaria, it w a s demonstrated for P. v. pet- teri only (Motard et al 1 9 9 0 ; Gautret et al, 1 9 9 6 α ) . B y contrast, P. cbabaudi d o e s not s h o w any variation in its ability to infect A. stephensi m o s q u i t o e s throughout the day despite a circadian variation in the numbers o f type II infective g a m e t o c y t e s . This w a s c o n s i d e r e d to b e due to the c o e x i s t e n c e o f the p e a k o f type II g a m e t o c y t e s and o f the s c h i z o g o n y in this particular s p e c i e s (Gautret et al., 1 9 9 6 c ) . A circadian t e n d e n c y in the infectivity o f P. y. nigeriensis g a m e t o c y t e s to the m o s q u i t o was o b s e r v e d b y Motard during asynchro
nous infections; the infectivity b e e i n g greater w h e n the
proportion o f mature s c h i z o n t s and rings w a s t h e lowest (unpublished data). No variation h o w e v e r , w a s o b s e r v e d over time, in the infectivity o f P. y. yoelii g a m e t o c y t e s b e c a u s e it d e v e l o p a s y n c h r o n o u s l y with different g a m e t o c y t e stages present at all the times (Gautret et al., 1 9 9 6 b ) .
PRIMATE PLASMODIUM
With P. cynomolgy ceylonensis (Langur С strain), it was found b y Hawking et al. (1966-1968α) that infectivity o f gametocytes to A. stephensi increased at midnight e v e r y 4 8 hours. This results w e r e c o n f i r m e d with P. cynomolgi cynomolgi (M strain) and A. maculatus (Coatney et al, 1971) and with P. cynomolgi ceylonensis (Langur С strain) and A. stephensi ( G a r n h a m & Powers,
1974). With P. knowlesi, oocyst numbers in A. ste
phensi midguts w e r e higher w h e n feeding at midnight than at mid-day (Hawking et al. 1968b). P. coatneyi was s h o w n to have a 48-hour rhythm o f infectivity for A.
freebomi (Coatney et al, 1 9 7 1 ) . Y a n g et al. (1984) and Y a n g (1996) demonstrated that P. vivax g a m e t o c y t e infectivity to A. sinensis s h o w e d a 4 8 hour pattern in patients naturally infected with malaria.
P. FALCIPARUM
With P. falciparum, neither e x p e r i m e n t s by Bray et al.
(1976), or by G i t h e k o et al. (1993) s h o w e d a greater infectivity o f young g a m e t o c y t e carriers to m o s q u i t o e s during night-time. H o w e v e r , in Bray's e x p e r i m e n t , m o s q u i t o e s w e r e successively fed at 10 a. m. and 10 p. m. and in G i t h e k o ' s study, at 4 p. m. and 7 p. m.
Therefore, these results d o not e x c l u d e a variation o f P. falciparum g a m e t o c y t e infectivity during the day or over a 48-hour cycle as transmission experiments w e r e performed at 12 and s e v e n hour intervals. T h e 2.4 day life span o f P. falciparum infective gametocytes cannot a c c o u n t h o w e v e r , for an impairment o f periodical infectivity in fully synchronous infections. Mature game
tocytes are p r o d u c e d at e a c h s c h i z o g o n y (i. e. at 4 8 hourly intervals) and released in the peripheral circu
lation s o m e nine days after. If the great majority o f g a m e t o c y t e s remain infective for 2.4 days, infective g a m e t o c y t e s from t w o s u c c e s s i v e b r o o d s s h o u l d overlap during a brief period o f 0.4 days ( 1 0 hours).
B e t w e e n these overlaping period, the remaining infec
tive p o p u l a t i o n s h o u l d b e t h e r e f o r e s i g n i f i c a n t l y r e d u c e d . In natural i n f e c t i o n s . P. falciparum c a n d e v e l o p synchronously in relatively n o n - i m m u n e chil
dren in e n d e m i c areas (Bray, personal c o m m u n i c a t i o n ) but this is not the c o m m o n feature, particularly during t h e primary a t t a c k and in s e m i - i m m u n e p a t i e n t s , contrary to the other human plasmodia. Furthermore, in most natural P. falciparum infections, t w o b r o o d s o f parasites are present, cycling 2 4 hours out o f p h a s e (White et al., 1992). It is probable that variations in the
1 0 8 Mise au point Parasite, 1999, 6, 103-111
PERIODIC INFECTIVITY OF PLASMODIUM GAMETOCYTES
infectivity o f P. falciparum g a m e t o c y t e s has not b e e n shown, partly b y reason o f the w e a k synchronicity o f its s c h i z o g o n i c d e v e l o p m e n t . It should b e o f interest to study during t w o 4 8 hour cycles, the effect o f schi
z o g o n y o n transmission in this particular species, in relation with the d e g r e e o f synchronicity o f the para
sitemia a n d gametocytemia a n d the circadian bioavai
lability o f the g a m e t o c y t e s for the mosquito.
In conclusion, in all fully synchronous Plasmodium spe
cies, (with the e x c e p t i o n o f P. chabaudi) a periodicity o f infectivity o f gametocytes to mosquitoes c a n b e evi
d e n c e d with an inter-schizogonic p e a k o f oocysts.
GENERAL CONCLUSION
Τ
η all the Plasmodium s p e c i e s , a periodic production o f mature g a m e t o c y t e s c a n b e demonstrated, frequently associated with a sequestration/release p h e n o m e n o n o f infective gam e t o cy t e s. In most o f the cases, the kinetic o f morphologically mature g a m e t o cytes in the vertebrate host a n d / o r the resulting infec
tivity to the vector s h o w s an accurate circadian rhythm adapted to the bitting habits o f t h e insects. It has b e e n demonstrated for Leucocytozoon in ducks (Roller &
Desser, 1 9 7 3 ) a n d for t h e plasmodia o f Thamnomys rutilam (Gautret et al., 1 9 9 8 ) . T h r e e parasites fail to s h o w circadian variations in their infectivity to Ano
pheles. P. chabaudi b e c a u s e s c h i z o g o n y a n d produc
tion o f mature g a m e t o c y t e s c o i n c i d e , d o e s not s h o w any circadian variation o f gametocyte infectivity. P. fal
ciparum g a m e t o c y t e s w e r e n o t demonstrated to s h o w a circadian rhythm in their ability to infect m o s q u i t o e s b e c a u s e infections in h u m a n are frequently asynchro
nous. P. berghei b e c a u s e o f its s p o n t a n e o u s asyn
c h r o n o u s development, produces mature g a m e t o c y t e s independently o f the time. With s y n c h r o n o u s s p e c i e s o f Plasmodium, w h e n experimental or natural infecti
vity is studied, it is strongly r e c o m m e n d e d that the e x p e r i m e n t s are performed at the s a m e time o f the day if a valuable comparison is to b e made. It is impor
tant to perform the experiment at the time o f maximum infectivity if o n e wants to study t h e influence o f the i m m u n e system or o f drugs o n transmission. During epidemiological records, the identification o f g a m e t o cyte carriers should c o n s i d e r infective g a m e t o c y t e s while taking t h e morphological a n d c h r o n o b i o l o g i c a l data into account.
ACKNOWLEDGEMENTS
W
e are very grateful to I. Landau a n d A. G.Chabaud for c o m m e n t s a n d suggestions a n d to R. S. Bray for pertinently reviewing this manuscript.
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Reçu le 24 novembre 1998 Accepté le 25 mars 1999
Mise au D O i n t 111
