CHINESE PHLEBOTOMINE SANDFLIES
OF THE SUBGENUS ADLERIUS NITZULESCU, 1 9 3 1 (DIPTERA: PSYCHODIDAE) AND THE IDENTITY OF P. SICHUANENSIS LENG & YIN, 1 9 8 3
II - GENOTYPING AND NUMERICAL ANALYSIS
Z H A N G LING MIN* & LENG Y A N JIA*
S u m m a r y :
The authors carried out isoenzyme characterization and genotype comparison studies for Phlebotomus chinensis and Phlebotomus sichuanensis imagoes collected from their holotype/paratype original localities: West Mountain, West Suburb, Beijing and Lixian County, Sichuan Province. Numerical analysis was carried out and the dendrogram of four species of Adlerius was drawn up according to morphological characteristics of these four species.
The results showed that P. chinensis and P. sichuanensis can be identified enzymatically in PGM and the MDH-2 allele also differs in P. chinensis from Beijing and. P. sichuanensis. The dendrogram of four Adlerius species showed that P. fengi and P. longiductus are very similar and they clustered first, then P. sichuanensis combine with them to form a combined group.
P. chinensis is a separate group from other three. These two groups come to combine at last. Above results have proved that P. sichuanensis is an isolate species instead of a so-called large type P. chinensis.
KEY W O R D S : Phlebotomus sichuanensis, isoenzyme characterization, genotyping, numerical analysis, taxonomy.
Résumé: LES PHILÉBOTOMES DE CHINE DU SOUS-GENRE ADLERIUS NITZULESCU, 1931 (DIPTERA: PSYCHODIDAE) ET IDENTITÉ DE P. SICHUANENSIS LENG & Y l N , 1983. II - GÉNOTYPE ET ANALYSE NUMERIQUE
les auteurs ont réalisé une comparaison des isoenzymes et des génotypes de Phlebotomus chinensis et Phlebotomus sichuanensis i collectés dans les localités des holotypes et paratypes des
montagnes de l'ouest et de la périphérie ouest de Beijing et Lixian, Province du Sichuan. Une analyse numérique et un dendrogramme de quatre espèces du sous-genre Adlerius ont été faites selon leurs caractéristiques morphologiques. Il résulte que P. chinensis et P. sichuanensis peuvent être identifiés au plan enzymatique pour l'étude de la PCM et également que l'allèle MDH-2 est différent chez P. chinensis provenant de Beijing et P. sichuanensis. Le dendrogramme des quatres espèces d'Adlerius montre que P. fengi et P. longiductus sont très proches. P. chinensis constitue un groupe séparé de celui des trois autres espèces. Ces résultats prouvent que P. sichuanensis est une espèce en soi et non un représentant de grande taille de P. chinensis.
MOTS CLES : Phlebotomus sichuanensis, isoenzyme, génotype, analyse numérique, taxonomie.
INTRODUCTION
F
our s p e c i e s o f Adlerius p h l e b o t o m i n e sandflies h a v e b e e n r e c o r d e d in China, n a m e l y Phlebo- tomus chinensis N e w s t e a d , 1 9 1 6 ; P. fengi Leng &Zhang, 1 9 9 4 ; P. longiductus Parrot, 1 9 2 8 and P. sichua- nensis Leng & Y i n , 1 9 8 3 . Adlerius p h l e b o t o m i n e s are t h e main v e c t o r s o f v i s c e r a l l e i s h m a n i a s i s ( V L ) in China, three o f t h e m are incriminated VL vectors (Leng
& Zhang, 1 9 9 2 ; Leng & Zhang, 1 9 9 4 ; Y i n et al., 1 9 8 5 ;
ABBREVIATIONS:
Pc: Phlebotomus chinensis, P f Phlebotomus fengi, PI: Phlebotomus lougiciuctus. Ps: Phlebotomus sichuanensis. V L : visceral leishma- niasis.
ISS: investigators from Shanghai Institute of Parasitic Diseases, Chi- nese Academy o f Medical Sciences, Shanghai and Shanxi Medical University, Taiyuan, Shanxi Province.
* Department of Medical Parasitology. Faculty of Medicine, Jinan Uni- versity, Guangzhou, 510632 China.
Correspondence: Zhang Ling Min.
Tel.: +86-20-85220255 - Fax: +86-20-85221343.
E-mail: 85220255@l63.net
Zhang & Leng, 1 9 9 7 ) and P. fengi is c o n s i d e r e d a potential VL v e c t o r for the s o u t h w e s t e r n m o u n t a i n o u s region (Leng & Zhang, 1 9 9 4 ) . X i o n g et al. ( 1 9 9 0 ) s y n o - nymized P s to P c and c o n s i d e r e d P s to b e a large type of P c . In the first part o f the present study (Leng &
Zhang, 2 0 0 1 ) , the authors p r o v e d the isolate identity of P s by t a x o n o m i c a l a n d z o o g e o g r a p h i c a l studies. In this report g e n o t y p i n g and n u m e r i c a l analysis are stu- died.
MATERIALS AND METHODS
Studied specimens
P
c a n d P s i m a g o e s w e r e c o l l e c t e d from their holotype/paratype original localities: West Moun- tain, W e s t S u b u r b , Beijing a n d Lixian County, Sichuan P r o v i n c e respectively. Pf s p e c i m e n s m e a s u r e d are the h o l o t y p e and paratypes preserved in J i n a n Uni- versity (Leng & Zhang, 1 9 9 4 ) and the m e a s u r e m e n t s of Pl are q u o t e d from original descriptions a n d well k n o w n p a p e r s (Parrot, 1 9 2 8 ; W a n g et al, 1 9 6 6 ; Arte- miev, 1 9 8 0 ; Lewis, 1 9 8 2 ; F e n g , 1 9 8 3 ) .M i s e au point -287
Parasite, 2002. 9, 287-291
ISOENZYME AND GENOTYPE ANALYSIS
P c and P s imagoes w e r e collected from the places res
pectively m e n t i o n e d a b o v e . After stunning by c h l o r o form the sandflies w e r e individually o n e b y o n e trans
ferred into labeled small plastic tubes for immediate preservation in liquid nitrogen at - 196° C. Before e l e c trophoresis the fly's head w a s r e m o v e d for s p e c i e s identification and the rest o f e a c h fly was h o m o g e n i z e d in 5 μl o f e n z y m e stabilizer ( 2 mM EDTA, 2 mM 6- a m i n o c a p r o i c acid, 2 mM dithiothreitol) in the well o f a plastic titration plate with a glass rod. Each h o m o - g e n a t e w a s a b s o r b e d into a p i e c e o f 4 x 6 m m filter paper b e f o r e it was inserted into o n e e n d o f 12.5 % horizontal starch gel. 0.1M Tri-0.1M Maleic-0.01M EDTA buffer system was used. After electrophoresis, the gel was sliced horizontally into three to four slices 1mm in thickness. Each slice is stained for different enzymes.
Detection o f following enzymes was attempted: alcohol d e h y d r o g e n a s e ( A D H E - E . C . 1 . 1 . 1 . 1 ) ; aldehyde oxidase ( A O - E . C . 1 . 2 . 3 . 1 ) ; aspartate aminotransferase (ASAT or G O T - E . C . 2 . 6 . 1 . 1 ) ; esterase ( E S - E . C 3 . 1 . 1 . 1 ) ; g l u c o s e - p h o s p h a t a t e i s o m e r a s e (PGI-E.C.5.3-1-9); glycerol-3- p h o s p h a t e d e h y d r o g e n a s e (a-GPD-E.C. 1.1.1.8); h e x o - kinase (HK-E.C.2.7.1.1); lactate d e h y d r o g e n a s e (LDH- E.C.1.1.1. 27); malate dehydrogenase (MDH-E.C. 1.1.1.37);
p h o s p h o g l u c o m u t a s e (PGM-E.C.5.4.2.2).
T h e staining t e c h n i q u e used w a s similar to those des
cribed by Siciliano & S h a w ( 1 9 8 0 ) .
NUMERICAL ANALYSIS
17 s e l e c t e d m o r p h o l o g y c h a r a c t e r i s t i c s o f t h e m a l e ( 1 2 ) ( A 3 : a n t e n n a 3; A 3 / L : a n t e n n a 3 / l a b r u m ; A4:
a n t e n n a 4; WL: wing length; AF: a s c o i d formula; HC:
hairs o n i n n e r surface o f c o x i t e ; FHC: farmost hair f r o m b a s e o f c o x i t e ; S T : s u b a p i c a l t u b e r c l e o f a e d e a g u s ; G F : genital filament, Style; Aed: a e d e a g u s ; F / P : genital f i l a m e n t / g e n i t a l p u m p ) a n d f e m a l e ( 5 ) ( A 3 , A3/L, A4, WL, C D S : c o m m o n duct o f s p e r m a - t h e c a ) adult o f t h e four s p e c i e s in s u b g e n u s Adle- rius w e r e u s e d as o p e r a t i o n a l t a x o n o m i c u n i t s , n u m e r i c a l analysis w a s c a r r i e d out.
SPSS 10.0 statistic software (Liu, 2 0 0 0 ) was used to the hierarchical cluster analysis, and the cladistic dendro
gram o f four s p e c i e s o f Adlerius was plotted up.
RESULTS
ENZYME LOCI
Among the 10 enzymes, LDH and ADH showed no acti
vity although treated with several kinds of buffer sys
tems and adjusted the conditions for electrophoresis.
T h e s e two enzymes may b e absent in the sandflies if the technique fails to detect them. HK and ES appeared
very dimly and the diffuse bands w e r e difficult to iden
tify. AO displayed two bands only in the T V B buffer system. T h e activity o f ASAT was very unstable.
MDH with t w o loci migrated a n o d i c a l l y ( M D H - 1 , MDH-2) and o n e l o c u s migrated cathodically (MDH- 3 ) ( T a b l e I, Fig. l a ) . α-GPD with a single locus mani
fested in all p h l e b o t o m i n e s e x a m i n e d a m o n g w h i c h only a few s h o w e d t w o b a n d s ( T a b l e I, Fig. l b ) . P G M w i t h o n e l o c u s c o m p r i s e d four a l l e l e s ( T a b l e I, Fig. l c ) . PGI with two loci, n a m e l y PGI-1 ( e x t r e m e l y active) and PGI-2 (migrates faster than P G I - 1 ) ( T a b l e I, Fig. I d ) .
DIFFERENT GEOGRAPHICAL POPULATION
OF P. CHINENSISAllozyme phenotypes appeared almost the s a m e except in MDH-2 w h i c h revealed slight variations ( R M D ) in the 8 0 o f the alleles in P. chinensis from Beijing but was absent in the P c from Sichuan. ( T a b l e I, Fig. 1).
P. CHINENSIS AND P. SICHUANENSIS
Adlerius p h l e b o t o m i n e s w e r e identified mainly by the male m o r p h o l o g y o f the species. T h o u g h their females r e s e m b l e them morphologically but their enzymic cha
racteristics are different. T h e characteristic e n z y m e loci o f MDH-2 and PGM differ b e t w e e n these two sand
flies. T h e alleles o f locus in MDH-2 situated at 8 0 ( o r a b s e n t ) and 8 4 for P c and P s respectively. T h e locus
Enzyme P. chinensis P. chinensis
phenotypes* (BJ) (SC) P. sichuanensis
MDH-1 100 54 32 30
MDH-2 84 0 0 10
80 24 0 0
α-GPD 100 32 25 13
100/103 0 0 1
97 0 0 1
95 0 1 0
PGM 100 17 12 I
108 0 0 9
100/105 5 2 0
100/108 0 0 1
103/108 0 1 1
PGI-1 100 43 40 18
100/96 5 1 3
100/94 1 0 0
PGI-2 110 6 15 8
110/114 8 4 3
114 0 0 1
* The nomenclature of the enzyme phenotypes was based on an arbitrary designation of "100" for the distance of the most common band at the locus considered in P chinensis. Subsequently desi
gnation were derived from measurements relative to the standard
"100".
(BJ) : specimens from Beijing: (SC) : specimens from Sichuan.
Table I. - Number of allotypes at different loci in P. chinensis and P. sichuanensis.
2 8 8 Mise au point Parasite, 2002, 9. 287-291
Fig. 1. - Composite diagram of enzyme phenotype in starch gel elec
trophoresis for P. chinensis and P. sichuanensis.
Pcb: P. chinensis (Beijing); Pes: P. chinensis (Sichuan); Ps: P. sichua
nensis.
Species GI* GD*
P. chinensis (BJ) - P. chinensis (SC) 0.907 0.098 P. chinensis (BJ) - P. sichuanensis 0.702 0.353 P. chinensis (SC) - P. sichuanensis 0.783 0 . 2 4 5 (BJ) = specimens from Beijing, (SC) = specimens from Sichuan.
* 1 = ΣXiYi/√ΣXi2Yi2; GI = IXY \√IXIY GD = -logeGI (Nei, 1972; Zhen, 1987).
Table II. - Average genetic identity (GI) and genetic distance (GD) between P. chinensis and P. sichuanensis.
in PGM for g e n o t y p e AA(100) is over 77 % and 8.3 % and g e n o t y p e DD( 108) is negative and 7 5 % in Pc and P s respectively (Fig. 2 ) . T h e GI and G D calculated (Nei, 1972; Zhen, 1 9 8 7 ) from allelic frequencies o f six loci for these two flies are listed in T a b l e II.
NUMERICAL ANALYSIS
Rescaled distance cluster c o m b i n e d analysis (hierar
chical cluster method: Liu, 2 0 0 0 ) o f m o r p h o l o g y cha
racters o f four s p e c i e s s h o w e d that P f and P i are very closely related species, they cluster first, then P s c o m bine with them to form a group. P c is another group.
T h e s e two group cluster last. (Fig. 3 ) .
DISCUSSION
P
errotey et al. (2000) separated two sympatric species in the s u b g e n u s Adlerius, P. simici and P. brevis, by m e a n s o f agarose e n z y m e electrophoresis, and found differential patterns at the PGM and HK loci.B e n a b d e n n b i et al. ( 1 9 9 9 ) separated two closely related species, P. longicuspis and P. pemiciosus, by their i s o e n z y m e patterns using isoelectrofocusing o f seven e n z y m e systems. In this study, P c and P s could b e identified enzymatically in PGM, their differences are also d e m o n s t r a t e d in P c Beijing strain a n d in P c Sichuan strain living in the s a m e area as P s . T h e MDH-2 allele is also different in the P c from Beijing
Fig. 2. - A comparison of PGM genotype of P. chinensis and P. si
chuanensis from Bei
jing ( B J ) and Sichuan (SC).
Parasite, 2002. 9, 287-291
Mise au point 289
Rescaled Distance Cluster Combine
0 5 10 15 20 25 Num + + + + + +
2 _+ +
2 _+ + + Fig. 3- - The cladistic dendrogram of numerical analysis of four spe-
4 + I cies of Adlerius.
1 + l = P c ; 2=Pf; 3=PI; 4=Ps
Cluster analysis of 17 morphology characters of four species of Adlerius: SPSS 10.0 statistic software.
and P s , but it is absent in the P c from Sichuan (Fig. l a ) (it may b e a g e n e t i c variety c a u s e d by geographical separation and e c o l o g i c a l environment factors).
From the dendrogram o f morphological numerical ana
lysis o f four s p e c i e s o f Adlerius, it is clear that P f and P i are very c l o s e s p e c i e s and they cluster first, then P s c o m b i n e s with them to form the first group. P c is a n o ther separate group. T h e s e two groups c o m b i n e last.
In the present study, P s s p e c i m e n s are c o l l e c t e d from a b o v e 9 0 0 m altitude, Lixian County, Northwestern Sichuan w h e r e its holotype or paratypes w e r e first col
lected. P c s p e c i m e n s w e r e c o l l e c t e d from: 1) West Mountain, W e s t Suburb, Beijing w h e r e its h o l o t y p e w a s c o l l e c t e d in 1 9 1 5 ; 2 ) Nanping County, North
western Sichuan w h e r e P c and P s are parapatrically distributed. ISS have not c o l l e c t e d s p e c i m e n s from the p l a c e w h e r e the original h o l o t y p e o r paratypes o f P c and P s w e r e formally c o l l e c t e d . T h e i r s p e c i m e n s w e r e collected from the parapatric z o n e for Pc and P s . Data o f t h o s e s p e c i m e n s have not b e e n given for size dis
tinction but just a c c o r d i n g to n a k e d e y e to identify large and small type P c . S o it is hard to avoid incor
rect identification and confusion with the original s p e c i m e n s used in their w o r k ( Q i a o & Lu, 1 9 8 9 ; Z h a o et al., 1997; Z h a o et al, 1 9 9 8 ) .
CONCLUSION
C
o m b i n e d with the m o r p h o l o g i c a l difference, g e o g r a p h i c a l d i s t r i b u t i o n , i s o e n z y m e study, numerical analysis and the development o f their premature stages o f four s p e c i e s o f Adlerius, P s is an isolate s p e c i e s instead o f a large type o f P c .ACKNOWLEDGEMENTS
T
h e authors are indebted to Research Funds Supporting by the Office o f Affairs Concerning Natio
nals Living Abroad o f the State Council. Many- thanks to Professor Stella Ghouti for her great help.
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