A COMPARISON OF CHORIONIC SCULPTURING OF FOUR INDIAN PHLEBOTOMINE SANDFLIES
(DIPTERA: PSYCHODIDAE) BY SCANNING ELECTRON MICROSCOPY
GHOSH K.N.* & MUKHOPADHYAY J.*
Summary:
The chorionic sculpturing of four Indian sandfly species:
Phiebotomus argentipes Annandale & Brunetti, P. papatasi (Scopoli), P. major major Annandale and Sergentomyia zeylanica (Annandale] was investigated with the help of scanning electron microscopy. It was found that P. argentipes and P. papatasi show similar type of chorionic sculpturing i.e., parallel connected ridges and ridge column palisade consisting of several small granules spaced in a double series, arranged longitudinally. However, morphology of each ridge is very different, particularly near the two ends of the egg and also where there is a formation of hexagonal areas. The inter- ridge area and basal layer of P. papatasi egg show multiple granular elevation which look like multiple minute microvilli like projections whereas in case of P. argentipes, there is reticular structure formation by fibrous material. In P. m. major and S . zeylanica, the egg surface pattern is found to be polygonal but in case of S. zeylanica almost all the polygons are of equal sizes and each ridge consists of flattened cells of different sizes arranged in a palisade. On the other hand, in P. m. major, there is no uniformity on the size and shape of the polygons and they look like that the ridges are formed by the fusion of the flattened cells. These species specific characters will be useful in identifying different sandfly species on the basis of their chorionic sculpturing pattern and consequently it will help to indicate the relatedness of sandfly species i.e., sandfly taxonomy.
KEY WORDS : sandflies, Phiebotomus argentipes, P. papatasi, P. m. major, Sergentomyia zeylanica, eggs, chorionic sculpturing, SEM.
MOTS CLES: phlébotomes, Phiebotomus argentipes, P. papatasi, P.m. major, Sergentomyia zeylanica, œufs, ornementation chorionique, SEM.
Résumé : COMPARAISON PAR MICROSCOPIE ÉLECTRONIQUE À BALAYAGE DES ORNEMENTATIONS CHORIONIQUES DE QUATRE ESPÈCES INDIENNES DE PHLÉBOTOMES (DIPTERA: PSYCHODIDAE)
Les ornementations chorioniques de quatre espèces indiennes de phlébotomes (Phlebotomus argentipes Annandale et Brunetti, P. papatasi (Scopoli), P. major major Annandale et Sergentomyia zeylanica Annandale) sont décrites par analyse en microscopie électronique à balayage. P. argentipes et P. papatasi possèdent des ornementations chorioniques similaires, telles que les rainures parallèles connectées et les rainures en colonnes palisadées formées de plusieurs petits granules espacés en séries doubles et orientés
Jongitudinalement. Cependant, la morphologie de chaque rainure diffère, en particulier aux deux extrémités de l'œuf ainsi qu'au niveau des zones de formation des aires
hexagonales. La région entre les rainures ainsi que la couche basale des œufs de P. papatasi se caractérisent par de multiples protubérances granuleuses ressemblant à des projections microvillositaires multiples. Chez P. argentipes, cette zone se présente sous forme d'une formation réticulaire constituée de matériel fibreux. Le motif de surface des œufs de P. m. major et S . zeylanica esf polygonale. Quant à celui de S . zeylanica, la quasi totalité des polygones sont de taille égale et chaque rainure est formée de cellules aplaties de différentes tailles organisées en palisade. Par contre, chez P.
m. major, il n'y a aucune uniformité de taille et de forme des polygones et il semblerait que les rainures sont issues de la fusion de cellules aplaties. La spécificité des motifs
chorioniques s'avère être un bon critère d'identification des différentes espèces de phlebotomes et devrait en conséquence être utile à définir plus clairement la position taxonomique des diverses espèces de phlébotomes.
Department of Zoology, University o f Calcutta, 35 Ballygunge Cir- cular Road, Calcutta 700 019, India.
* Present address: Yale Arbovirus Research Unit, Department of Epi- demiology and Public Health, School of Medicine, 60 College street, PO Box 208034, New Haven, Connecticut 06520-8034, USA.
Correspondence: K.N. Ghosh.
INTRODUCTION
F
ollicular d e v e l o p m e n t o f sandflies like other h a e - m a t o p h a g o u s i n s e c t s is d e p e n d e n t o n b l o o d m e a l . H o w e v e r , s o m e o f t h e t h e m h a v e b e e n r e p o r t e d t o b e a u t o g e n o u s in nature ( J o h n s o n , 1 9 6 1 ; Killick-Kendrick, 1 9 8 7 ) . T h e time o f e g g laying varies in different i n s e c t s a n d e a c h e g g reflects characteris- tics o f the s p e c i e s , b o t h b i o c h e m i c a l l y a n d m o r p h o l o - gically. P r o b a b l y , it is i m p o s s i b l e to differentiate thevery tiny e g g s o f the closely related s p e c i e s with s o m e morphological parameters. Therefore, attempts have b e e n m a d e to differentiate eggs o f related s p e c i e s b y analysing the e x o c h o r i o n o f the laid eggs. T h e struc- ture o f the e x o c h o r i o n reflects the s h a p e o f the folli- cular cells in the ovariole and thus, they are the mani- festation o f a character o f the adult. T o an e v e n larger extent, the s a m e thing is true for the tertiary e g g shells, w h i c h are formed b y secretions o f the female genitalia in combination with action b a s e d o n inherited instincts o f the adult (Emden, 1957). This egg shell pro- tects the d e v e l o p i n g e m b r y o from the external envi- ronment. It also helps in g a s e o u s e x c h a n g e a n d pre- v e n t s d e s s i c a t i o n a n d t h e r e b y h e l p in t h e p r o p e r d e v e l o p m e n t o f the e m b r y o .
Studying egg surface o f the phlebotomine sandflies had b e e n initiated l o n g a g o with light m i c r o s c o p y o f P. ARGENTIPES (Howlett, 1 9 0 9 ) . Later, similar attempts have b e e n c o n t i n u e d to study the N e w W o r l d sandfly eggs (Linguist, 1936; Barretto, 1 9 4 1 ; Addis, 1 9 4 5 ; Sher- l o c k 1 9 5 7 ; S h e r l o c k & Caneiro, 1 9 6 3 ; Chaniotis &
Anderson, 1 9 6 4 ) and also the O l d World sandfly eggs ( S a c c a , 1 9 5 0 , 1 9 5 2 ; Perfil'ev, 1 9 6 6 ; A b o n n e n c , 1 9 7 2 ; Trouillet, 1977, 1 9 7 9 ) . H o w e v e r , most o f the authors faced difficulty in separating the eggs o f the closely related s p e c i e s with light m i c r o s c o p y .
Scanning Electron M i c r o s c o p y ( S E M ) has b e e n intro- d u c e d later to s e e the finer structure o f the c h o r i o n i c sculpturing o f insect eggs with the help o f its three dimensional view. Initially m o r e w o r k has b e e n d o n e with SEM o f m o s q u i t o eggs (Hinton, 1 9 6 8 , Hinton &
Service, 1 9 6 9 , Matsuo ET AL, 1 9 7 4 ) and later Ward &
Ready ( 1 9 7 5 ) first introduced SEM to s e e the outer c h o - rionic sculpturing o f 13 s p e c i e s o f N e w W o r l d sand- flies from Brazil. O t h e r w o r k e r s ( Z i m m e r m a n ET AL,
1 9 7 7 ; Endris ET AL, 1987 & Feliciangeli ET AL, 1 9 9 3 ) continued similar study o n other s p e c i e s o f N e w World sandflies and contributed altogether egg sculpturing o f 4 9 s p e c i e s . O n the other hand, very little attempt has b e e n m a d e with the O l d W o r l d sandflies. O f the ten s p e c i e s s o far described, Irungu ET AL. ( 1 9 8 6 ) , Lane &
El-Sawaf ( 1 9 8 7 ) , Gebre-Michael & Lane ( 1 9 9 1 ) & Fausto ET AL. ( 1 9 9 2 ) have e x p l o r e d the egg sculpturing pattern o f four, o n e , t w o a n d t h r e e s p e c i e s r e s p e c t i v e l y ( T a b l e I ) . T h e i r observations clearly indicate that the Old W o r l d sandflies are less studied. Here, w e report the c h o r i o n i c sculpturing o f four Indian p h l e b o t o m i n e sandflies with the help o f SEM.
MATERIALS AND METHODS
L
a b o r a t o r y c o l o n i e s o f PHLEBOTOMUS ARGENTIPES Annandale & Brunetti a n d P. PAPATASI ( S c o p o l i ) w e r e maintained following the m e t h o d o f G h o s h ET AL. ( 1 9 9 1 ) . T h e e g g s o f the two s p e c i e s w e r e taken from the respective c o l o n i e s . F e m a l e s o f P. M. MAJOR Annandale a n d S. ZEYLANICA ( A n n a n d a l e ) w e r e c o l - lected from Dankuni and Salt Lake area o f West B e n g a l respectively. T h e eggs w e r e c o l l e c t e d by feeding the wild caught females o n h o u s e lizard in the laboratory following the m e t h o d o f G h o s h & Bhattacharya ( 1 9 8 9 ) - T h e s p e c i e s w e r e identified b y the keys provided b y T h e o d o r ( 1 9 3 8 ) as modified b y Lewis ( 1 9 7 8 ) .Species Origin
P PR(C)
Pattern
PR(UC) Vol Network
Reference
P. argentipes India X Ghosh ¿4 Bhattacharya, 1993
X This paper
P. celiac Ethiopia X Gebre-Michael & Lane, 1991
P. duboscqi Kenya X Fausto et al, 1992
P. langeront Egypt X Lane & El-Sawaf, 1986
P. martini Kenya X Irungu et al., 1986
Ethiopia X Gebre-Michael tk Lane, 1991
P. major India X This paper
P. papatasi Italy X Fausto et al, 1991
Italy X Fausto et al, 1992
India X This paper
P. perfiliewi Italy X Fausto et al., 1992
P. perniciosus Italy X Fausto et al, 1992
S. bedfordi Kenya X Irungu et al, 1980
S. garnhami Kenya X Irungu et al, 1980
S. kirki Kenya X Irungu et al, 1980
S. zeylanica India X This paper
Table I. — Chorionic sculpturing of Old World sandflies as observed by SEM.
P: polygonal, PR(C) : parallel ridge (connected), PR(UC): parallel ridge (unconnected), Vol: volcanic and Network (synonymous to reticular).
62 Parasite, 1996, 3, 61-67
For SEM examination, the laid eggs w e r e allowed to embryonate for 2-3 days. T h e n , the eggs were collected with the help o f a fine brush and w a s h e d with P B S to clear the egg surface. T h e n , they w e r e fixed in 1 % gluteraldehyde solution in c o l d for 2-3 hrs. T h e solu
tion was r e p l a c e d by 3 % gluteraldehyde and left overnight at 4 °C. O n the following day, the eggs w e r e w a s h e d with PBS (pH 7 . 2 ) to r e m o v e e x c e s s gluteral
dehyde from the samples and subsequently p a s s e d through upward grades o f ethanol k e e p i n g 10-15 min in e a c h grade. Lastly, absolute ethanol was r e p l a c e d by isoamyl acetate. T h e samples w e r e then dried in a critical point drying apparatus ( P o l a r o n E 5 0 0 ) and mounted on a metal stub with the help o f double sided adhesive tape. T h e eggs w e r e then coated with an alloy o f Au-Pd o f 2 0 0 - 3 0 0 A0 thickness in a sputter c o a t e r (Edwards S 1 5 0 ) . T h e c o a t e d samples w e r e v i e w e d and p h o t o g r a p h e d under a Philips SEM (PSEM 5 0 0 ) at varying b e a m o f current, tilt angles.
RESULTS
F
igs. 1-3 indicate the ultrastructure o f e x o c h o r i o n o f the eggs o f the following four Indian phle- b o t o m i n e s .P . ARGENTIPES
Egg length 3 2 0 um; breadth (middle) 72 um. T h e eggs are almost spindle s h a p e d with o n e e n d o f the spindle broader than the other. T h e b a s i c pattern o f chorionic sculpturing s h o w s parallel interconnecting ridges. In s o m e areas, the bifurcating b r a n c h e s o f the adjacent parallel longitudinal ridges unite o n e another e n c r o a c h i n g few h e x a g o n a l s t r u c t u r e s ( F i g . 2 K ) . T h e s e b r a n c h e s act as interconnecting ridges which are very short in length. Closer observation reveals that e a c h parallel ridge is actually c o m p o s e d o f two very close rows o f small granular protuberances with intermediate
Fig. 1. — SEM micrographs showing the characteristics ridge pattern of the outer chorionic sculpturing of four Indian sandfly eggs. P. argen- tipes (A); P. papatasi (B); P. m. major (C); S. zeylanica (D). (Bar = 30 um).
s p a c e s . T h e individual granules o f the two rows are alternately arranged. At e v e n higher magnification, e a c h granule appears to b e triangular in s h a p e with central elevation. T h e basal egg surface (inter-ridge areas) is actually a rough area having intercalated net
w o r k s w h i c h e x t e n d up to the b a s e o f the granules o f the ridges (Fig. 3A).
P. PAPATASI
Egg length 3 1 7 Um; breadth (middle) 106 um. T h e two e n d s o f the eggs are almost blunt, thus giving a thick, short, rod s h a p e d a p p e a r a n c e . Here, the b a s i c pattern o f c h o r i o n i c sculpturing is also parallel i n t e r c o n n e c t e d ridges but the longitudinal ridges are not always uni
form. In areas, w h e r e the ridges are thicker, particu
larly closer to the two ends, they form quadrangular gaps o f different sizes (Fig. 2 B ) . It may b e the fact that the ridges are actually c o m p o s e d o f t w o individual rows w h i c h in s o m e areas have fused and in o t h e r areas exist separately thereby e n c r o a c h i n g an area in b e t w e e n the two rows. T h e pattern o f distribution o f ridges nearer the e n d s o f the e g g s h o w s n e t w o r k like structure w h i c h is formed by the b r a n c h i n g o f the lon
gitudinal ridges (Fig. 2 B ) . T h e interconnecting ridges are thinner in c o m p a r i s o n to the longitudinal ridges and l o o k s like a very small side b r a n c h o f the longi
tudinal ridges (Fig. 2 B ) . T h e basal e g g surface (inter- ridge a r e a s ) is rough, having uniform small granular p r o t u b e r a n c e s w h i c h l o o k like multiple microvilli.
Fig. 2. — SEM micrographs of the selected region of egg surface of the four sandflies showing ridge arrangement, interconnection and polygon formation.
P. argentipes (A); P. papatasi (B) (Bar = 8 u:m). P. m. major (C); S. zeylanica (D) (Bar - 4 urn).
64 Parasite, 1996, 3, 61-67
P. M. MAJOR
Egg length 2 0 0 um and breadth ( m i d d l e ) 7 4 um. Eggs are spindle s h a p e d with polygonal structures on their surface area. T h e pattern o f p o l y g o n formation is not uniform having quadrangles, p e n t a g o n s or h e x a g o n s (Fig. 1C). Each ridge o f the chorion consists o f columns o f granular cells arranged to form a palisade. In s o m e areas, the ridge s h o w s single row o f quadrangular gra
nular cells w h e r e a s in other areas, as in the corners, there is n o separate e x i s t e n c e o f individual cells a n d the cells a p p e a r to b e fused and the ridges are also wider (Fig. 2 C ) . At higher magnification, the ridge sur
face shows multiple small depressions which m a k e s the surface rough (Fig. 3 G ) .
S. ZEYLANICA
Egg length 2 6 6 um and breadth (middle) 6 4 um. Each egg is a narrow, rod like structure having two blunt ends
Fig. 3. — SEM micrographs showing the ridge morphology of the four Indian sandflies.
P. argentipcs (A); P. papatasi ( B ) ; P. m. major(C); S. zeylanica (D). (Bar = 1 |im).
(Fig. I D ) . Here, the egg surface consists o f symmetrical h e x a g o n s which are uniformly distributed throughout the egg surface (Fig. I D ) . In lower magnification, the ridges appear to b e wide and solid, uniform cord like stmctures. However, at higher magnification, the surface of the ridge shows unevenness with the existence o f dif
ferent solid granules o f various sizes, arranged in a column to form a palisade (Fig. 3 D ) .
DISCUSSION
M orphological
characters o f the adult insects are always t a k e n into a c c o u n t in the way o f describing a s p e c i e s o r for t a x o n o m i c identification. C h o r i o n i c sculpturing o f insect eggs has b e e n a c c e p t e d as a basis o f o o t a x o n o m y which is suc
cessfully analysed with the help o f SEM (Hinton, 1981).
Parasite, 1996, 3, 61-67
From the result it is clear that the pattern o f chorionic sculpturing h a s significant variation a m o n g the Indian p h l e b o t o m i n e s . However, in the s a m e genus o r clo
sely related s p e c i e s , the patterns a r e almost similar e x c e p t t h e differences at the ultrastructural level. T h e c h o r i o n i c sculpturing o f P . ARGENTIPES eggs h a s b e e n g r o u p e d under " c o n n e c t e d parallel ridge". Although, P . PAPATASI egg falls under this category o n the basis o f distribution o f ridges, there is a clear difference in the m o r p h o l o g y o f the ridges and the pattern b y w h i c h they m a k e i n t e r c o n n e c t i o n a n d form s o m e polygonal areas o n the surface o f the eggs. T h e two terminal ends o f a P . PAPATASI egg are very diagnostic b e c a u s e the distribution o f the ridges bears a very unique, dia
gnostic feature w h i c h is quite different from the rest o f the e g g surface (Fig. 2 B ) .
In c a s e o f S. ZEYLANICA, o n the surface o f the egg, t h e ridges form several, almost equal h e x a g o n a l areas but the pattern o f formation o f t h o s e h e x a g o n a l areas a r e quite different. T h e ridges never run in parallel and the six arms o f t h e h e x a g o n s are almost o f equal size unlike the hexagonal areas o f PHLEBOTOMUS eggs, where t w o arms o f the h e x a g o n (in most c a s e s w h i c h a r e parallel) are always longer than the other four arms ( w h i c h are formed b y small interconnecting ridges). In SERGENTOMYIA group, the h e x a g o n a l areas are distri
buted throughout the e g g surface w h e r e a s in c a s e o f PHLEBOTOMUS they are very scanty.
T h e sculpturing pattern o f parallel c o n n e c t e d ridges, o b s e r v e d in P . ARGENTIPES and P . PAPATASI in this pre
sent investigation h a s also b e e n reported in P . PERNI- CIOSUS b y F a u s t o ET AL. ( 1 9 9 2 ) a n d in LUTZOMYIA CRU- CIATA a n d LU. ANTHOPHORA b y Endris ET AL. ( 1 9 8 7 ) . T h e o t h e r type o f parallel ridges i.e., n o t transversely c o n n e c t e d ( o r in other word, u n c o n n e c t e d ) h a s b e e n found in P . LANGERONI (Lane & El-Sawaf, 1986), P . MAR
TINI ( K e n y a ) a n d S. GARNHAMI (Irungu ET AL, 1 9 8 6 ) . H o w e v e r , P . MARTINI from Ethiopia s h o w e d different type o f sculpturing pattern i.e., network type ( G e b r e - Michael & Lane, 1991). This u n c o n n e c t e d parallel ridge pattern has also b e e n reported in P . PERFILIEWI (Fausto ET AL, 1 9 9 2 ) a n d in LU. DIABOLICA (Endris ET AL, 1 9 8 7 ) The p o l y g o n sculpturing pattern as o b s e r v e d here in c a s e o f P . M. MAJOR and S. ZEYLANICA is most c o m m o n in N e w World sandflies. Zimmerman ET AL. ( 1 9 7 7 ) , Endris ET AL. ( 1 9 8 7 ) a n d Feliciangeli ET AL. ( 1 9 9 3 ) des
cribed polygon sculpturing pattern in four, two and six New World sandflies out o f six, five a n d eight s p e c i e s studied b y them respectively.
From the present investigation it appears that parallel ridge formation is t h e b a s i c pattern in P . ARGENTIPES (subgenus PHLEBOTOMUS Rondani & B e n e ) and P . PAPA
TASI ( s u b g e n u s EUPHLEBOTOMUS) w h e r e a s in c a s e o f S. ZEYLANICA ( s u b g e n u s NEOPHLEBOTOMUS) a n d P . M. MAJOR ( s u b g e n u s LARROUSSIUS), the basic pattern
is polygonal. However, the finer differences b e t w e e n the e g g s o f P. ARGENTIPES a n d P. PAPATASI m a y b e d u e to the microhabitat they use for their breeding and also for the structure o f the follicular cells in t h e ovariole w h i c h is t h e manifestation o f t h e c h a r a c t e r o f the adult. Ward & Ready ( 1 9 7 5 ) stated that t h e form o f sculpturing is b e l i e v e d to b e influenced b y environ
mental conditions o f the breeding places. T h e y also noted that, in the s p e c i e s o f the genus PSYCHODOPYGUS, there w e r e s o m e differences in t h e c h o r i o n i c sculptu
ring although they are most abundant in the forest (Ward & Killick-Kendrick, 1 9 7 4 ) . T h e y indicated that the differences in microhabitat might b e the c a u s e b e h i n d t h e c h o r i o n i c sculpturing variations a m o n g them. H o w e v e r , t h e c h o r i o n i c sculpturing o f Indian sandflies has b e e n least studied and m o r e informations are n e e d e d o n t h e c h o r i o n i c sculpturing o f o t h e r Indian sandflies to have a better understanding o n the relation o f e c o l o g i c a l habitat a n d t h e sculpturing pat
tern. However, it will b e interesting to s e e w h e t h e r genetic similarities o r breeding habitat o r a c o m b i n a tion o f both have s o m e role in determining t h e c h o rionic sculpturing pattern o f a s p e c i e s which, in turn, may provide the possible relationship a m o n g c h o rionic sculpturing, breeding habitat a n d genetics o f p h l e b o t o m i n e sandflies. This might also help to have a solid understanding o n sandfly t a x o n o m y o n the basis o f c h o r i o n i c topography.
ACKNOWLEDGEMENTS
W
e are grateful to t h e S c a n n i n g Electron M i c r o s c o p e Unit, Regional Sophisticated Instrumentation Centre (RSIC), B o s e Institute, Calcutta for providing SEM facility. T h e authors are thankful to Dr. François Palluault & Dr. Martine Armstrong, Y a l e University for their h e l p in translating the title, summary a n d k e y words into French.
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