Bonnefoy, S.

Le Pont, François

Tibayrenc, Michel

Name not available

An isozymic study of LUTZOMYIA LONGIPALPIS (DIPTERA, PSYCHODIDAE), the vector of visceral leishmaniasis in the "Yungas" (Bolivia)

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An isozymic study 
of Lutzomyia longipalpis 
(Diptera, Psychodidae), 
the vector of visceral leishmaniasis 
in the “ Yungas ” (Bolivia) (‘) 
S. BONNEFOY r2), Michel TIBAYRENC PJ @), 
François LE PONT f-?), Jean-Pierre DUJARIXN (*), 
Philippe DES~JEUX c5), Francisco J. AY-ALA c6) 
Summary 
Isozyme variation was assayed in a population of Lutzomyia longipalpis, the vector of Leishmania chagasi in the 
” Yungas ” (Bolivia) in order to ascertain its taxonomie homogeneity. Although the specimens sampled exhibited some heteroge- 
ne@ in their size, there was no isozyme indication of taxonomie heterogeneip within the population. The Gpi locus exhibited a 
temporal variation from February to July in the allelic frequencies and the level of heterozygosiv, which may be related to clima.tic 
adaptation. Cenetic variability was low, which could bc explained if t/le present populations of L. longipalpis havc originated 
from a small number of individuals (” Founder EJect ‘9. 
Key words : Speciation - Seasonal variation - Sand flies - Epidemiology - Genetic variation - Bolivia. 
Résumé 
UNE ÉTUDE ISOENZYMATIQUE CHEZ k~7zo,w~z~4 LONGIPALPIS (DIPTERA, PSXHODIME), LE VEC;TEUR DE IA LEISHMA- 
NIOSE VISCÉRALE DANS LES « YUNGAS » (BOLIVIE). Nous avons étudié la variabilité isoenzymatique d’une population de Lut- 
zomyia longipalpis, le vecteur de Leishmania chagasi dans les <( Yungas » (Bolivie) dans le but de tester l’homogénéité taxo- 
nomique de cette population. Quoique les spécimens récoltés aient montré une certaine variabilité quant à leurs dimensions, les don- 
nées isoenzymatiques n’ont fourni aucun indice en faveur d’une hétérogénéité axonomique de la population considkée. le locus Gpi 
a montré une variation temporelle de février à juil& quant aux fréquences alléliques et au niveau d’hékkozygotie. Ces faits peuvent 
(1) This studv was carried mt with support from the French Technical Gmperation and fiwrn the Ministère de l’Industrie et de la 
Recherche ; ext&im of grant nun;her P\‘D/&Jl/L-1423, md with the support of a grant from UNDP/World Bank/WHO Special Program 
for Research and Trainin in Tropical Diseases, and with a financial support from E.E.G. (No-TSD/M-W2/BOL). 
(2) IBBA, Embajuda de Francis, C&illa 824, La PU~, Bolisia (addrm -for rtprint requests). 
(3) Entomolo.~i~rtr: médical ORSTOM, IBBA, and Centre OHSTOMs 70-74 route d’Aulnay, 93140 Bondy, Franre. 
(4) IBBA, nnd Institut de .Wdtrine tropicale P&c Leopold, 155 Nationalestruat, 2 000 Antuwptn, Br&m. 
(5) IBBA, and Insfitut Pasteur, 28 rue du Docteur-Roux, 75015 Paris, France. 
(6) Dcpartment of Ckwtics, Uniami& of Cal$mia, Davis, Ca 95j616, IfsA. 
Coh. ORSTOM, &. Ent. rnid, rt Pamritul., vol. XXIir, nu 3, 1986 : 213-217 
Hre dus à urw adapfation climatique. La variubilife’ gk’tiqw de la population con~idt% e’fait jàibk. LJnr hypothèse pouvant 
e.xjApw 0 &wltat e,rf que les populations actuelles de 1,. longipalpis descendent d’un petit nombre d’individus (CC efft>t fonda- 
Mots-clés : Su&iation - Variation saisonnière - Phlébotomes - 
Bolivie. ’ 
Lutzorr+a longipalpis, Lutz and Neiva 1912, is an 
anthropophilic sand fly which has been shown to be 
the vector of LeLrhmania chagasi, the causative agent of 
visceral leishmaniasis, in the “ Yungas ” region 
(department of La Paz, Bolivia) (Le Pont and 
Desjeux, 1985). A recent morphological study has 
raised the question wether L. longipalpis is taxonomi- 
cal& heterogencous (Lane and Ward, 1984). Indeed 
it was observed within a population of L. longipalpis 
studied in the &’ Yungas ” region (locality of Santa 
Barbara), that there is a certain degree of size hetero- 
genrity among the specimens sampled. We report 
here the results of a study of isozyme variability 
directed towards ascertaining any possible taxonomi- 
cal heterogeneity within this population. 
Material and methods 
The sand flirs were sampled and stored in the 
fïeld in licluicl nitrogen in February and July 1984. 
Each individual was‘identifïed as L. longipalpis in the 
Iabnratory by morphological examination, and then 
immecliately homogenizecl in 10 ~1 of hypotonic 
enzyme stabilizer (Gcrdfrey and Kilgour, 1976). 
The sarryles SC) obtained werr stored at - 70°C until 
use. Bo’h sexes wrre examined in this study. 
Becausr the presrnt work was finished before we had 
rend thr article by Lane and Ward (1984), we were 
unable to take into account the micromorphological 
c,hnrncters used by these authors to suggest the exis- 
tcncr of tasonomicul heterogeneity within the species 
L longi$alpis . 
Elrctrophoresis was carried out on cellulose 
acetate plates (Hrlena laboratories). The follow- 
ing rnzyrrle systems wcre assayed : glucose-6-phos- 
phatr isomernse (E.C.5.3.1.9, GPI) ; CY glycero- 
phosphate dehydrogenase (E.C. 1.1.1.8, 01 GPD) ; 
hexokinasc (E.C.2.7.1.1, HK) ; isocitrate dehyclro- 
grnnse (E.C.1.1.1.42, IDH) ; malate dehydroge- 
nase (E.C. 1.1.1.37, MDH) ; malate dehydrogenase 
(trsalc~ncetatr clecarhox?~lating) (Nadp + ) or malic 
rr~zyne (E.C. 1.1.1.40. ME) ; peptidase 2 (brome- 
luin. fi;.(’ ” . . ..~.4.22.4. formerly E.C.3.4.4.24. PEP 2, 
substrate : L-leucyl-L-alanine) ; phosphoglucomutase 
Épidémiologie - Variation génétique - 
(E.C.5.4.2.2, formerly E.C.2.7.5.1, PGM) and 
6 phosphogluconate clrhydrogenase (E.C. 1.1.1.44, 
GPGD). The assay recipes were taken from the fol- 
lowing authors : Kreutzer et ~6. (1983) (orGPD), 
Lanham rt a/. (1981) (HK) and Tibayrenc and 
Le Ray (1984) (a11 other enzymes), with a few modi- 
fications. Each enzyme system was assayed with 
and without its specifïc substrate. 
Results 
No color bancls appeared on the plates when the 
specific substrate was net added to the staining solu- 
tion, IDH and SPGD gave irregular stainings and 
therefore are not reported in the present study. 
Five of the seven enzyme systems finally consid- 
ered exhibited apparently the activity of only one 
locus each (GPI, ~YGPD, HK, ME and PGM) ; one 
enzyme system showed the activity of two loti 
(‘MDH) ; and one showed the activity of three loti 
(PEP 2). Thus there are a total of ten genetic loti. 
Table 1 gives the number of specimens studied for 
each enzyme system. 
EllZYlW February July Total 
-------_---_____________________________-------- 
11 GDH 12 90 102 
GPI 71 113 184 
HK 37 90 127 
MDH 59 67 126 
ME 59 GO 139 
PEP 70 70 140 
PGM 62 90 152 
---_-___________________________________-------- 
Isozymic study of Lutzomyia longipalpis 215 
Only two of the ten loti (Gfii and ~&Z, the 
locus coding for the fastest migrating MDH), appear 
to be polymorphie. The quaternary structure of 
these enzymes seems to be dimeric, given that hetero- 
zygous patterns exhibit three bands (photo 1). We 
found only two alleles at each of these two loti. 
We have used Chi-square tests to test wether the 
observed genotype numbers agree with the Hardy- 
Weinberg expectations at each of the two loti, Gpi 
and Mdhl. The specimens of February and July are 
considered separately as well as together (table II). 
We have pooled together the two less numerous 
genotypes in every instance, given that the expected 
numbers for at least one of them were too low 
(Schwartz, 1963). We had only females for the 
February sample, whereas both males and females 
were considered together for July. Other results 
show, however, that the genetic parameters for Gpi 
and Mdhl do not differ between males and females 
(Bonnefoy, unpublished data), The genotype num- 
bers did not depart from the Hardy-Weinberg expec- 
tations for either Gpi and Mdhl in February, or in 
July, or for February and July combined. 
We have calculated the allelic frequencies for 
Gpi and Mdhl, and the average observed and 
expected heterozygosity, (H, the probability that one 
individual Will be heterozygous at a given locus) 
(table III). Using an E test (comparison between 
two percentages ; see Schwartz, 1963, p.. 58), we 
have compared the allelic frequencies and the 
observed and expected heterozygosities between the 
specimens caught in February and the specimens 
caught in July (table III). There are signifïcant 
differences between February and July at the Gpi 
locus for the allelic frequencies and for the observed 
and expected heterozygosities. There are no signifi- 
tant differences at the Mdfzl locus. 
Discussion 
The question has been raised whether there is 
taxonomical heterogeneity within the sand fly species 
Lutzomyia longipalpis, which plays an important role in 
the epidemiology of visceral leishmaniasis in South 
America. Our results suggest that the population 
surveyed is taxonomically homogeneous. In another 
taxon of sandfly (Psychodopygus carruai>, we have 
recently evidenced (Le Pont et al., 1985 ; Caillard et 
al., in press) the occurence of two cryptic species, 
based on the presence within the same population of 
alternate alleles without the corresponding heterozy- 
PHOTO 1. - A cellulose acetate plate stained for the enzyme GPI, containing samples of the Phlcbotomine sand fly Lutmmyia fungi~afpi~. 
The fïrst and last samples are from a T~~panosoma cruri laboratory strain (“ Tulahuen “), used for reference. Some individual sand flies 
exhibit 3-banded electrophorograms : this indicates that these individu& are heterozygous for the enzyme system stained, and that the 
enzyme has a dimeric structure. The other individuals, exhibiting 1-banded patterns, are homozygotes. 
Cah. ORSTOM, sér. Ent. mid. et Parasitol., vol. XXIV, n” 3, 1986 : 213-217 
216 A’. Bonn&y, AI. Tibayrenc, FE: Le Pont et al. 
Cihi-square test\ ktu test f~>r Hardy-Weinberg equilibrium in February, July, and Februar~- and July combined for the Gpr and M&l 
grnutype nurrlbcrs. Thv rmmbcrs in parentheses are the exprctcd numbers assuming Hardy-Wrinberg equilibrium. The tests wrre made 
attt:r cmnhining the nurnbrrs for the tw« lrss numerous grnntypes, given that thr expected numbrrs of the rarest one are very low 
(Schmxtz. 1Wl). There is 1 degree of frrcdmn for each chi-square. Al1 genotype numbers arc conktent with HW ~quilibrium 
Genotype February July Feb. + Jul. poolsd 
GJi L/’ 51 (51.5) 102 (102.2) 153 (153.4) 
14 19 (17.9) 11 (10.5) 30 (29.2) 
z4 1 (1.61 0 10.3) 1 Cl.41 
-52 0.02 0.004 0.01 
w A/L 44 (45.0) 55 (54.6) 99 (99.8) 
Le 15 (13.1) 11 (11.7) 26 (24.7) 
u2 0 (1.0) 1 (0.6) 1 (1.5) 
,Y2 0.09 0.01 0.03 
---------_-_____________________________------------------------------ 
TABLE III 
Krsllltu of an t tact (Schwartz, 1963, p. 58) for comparing thc allelic frequencirs, and the obserwd and the expected heterozygosities (H) 
bet\\ern the Fcbrunry and July samples fier the G,+JI’ and Mdhl loti (P is the level of signifïcance ; signifiant values are underlined) 
Allel@S 
Allelic frequencies 
February Julr E P 
bpi L 0.852 0.951 3.28 CO.01 
OP' 2 0.148 0.049 
ohserued H 0.268 0.097 3.06 <o.o 
rxpected H 0.252 0.093 2.87 X0.01 
MI 0.873 0.903 0.76 >0.45 
m2 0.127 0.097 
observed H 0.254 0.164 1.25 >0.22 
@xpected H 0.222 0.175 0.66 >0.51 
--_____-------------____________________--------------------------- 
gotes at each of’ two enzyme loti, and of signifïcant 
deviations from the Hardy-Weinberg expectations at 
three other loti. The present data are quite dif- 
ferent : no alternate alleles without the corresponding 
heterozygotes are observed, and the genotype num- 
bers for the two variable loti, Gpi and Mdhl, do not 
depart signifïcantly from the Hardy-Weinberg expec- 
tations. Hence, we take it as a working hypothesis 
that this population belongs to the only species L. 
lon,ppalpis and this hypothesis will be assumed in 
our epidemiological studies. Wé plan, however, to 
conduct further isozyme studies of other populations 
IsozJjmic studq, of Lutzomyia longipalpis 217 
of L. longipalpis ; and we shall take into account the 
micromorphological characters described by Lane 
and Ward (1984), in order to ascertain whether the 
hypothesis of taxonomical homogeneity of this species 
cari be generalized to other Bolivian regions. 
A possible explanation of the seasonal variation 
observed at the Gpi locus for the allelic frequencies 
and the heterozygosity (both observed and expected) 
is that it results from climatic adaptation ; the adap- 
tation might not directly involve the enzyme gene 
loti themselves but other genes linked to them. 
Clearly, further studies would be required in order to 
decide these issues. 
The observed and expected heterozygosities 
averaged over the ten gene loti studied are, respec- 
tively, 0.037 and 0.036 (February and July samples 
pooled together). These are low compared to the 
average heterozygosities of most insects (Ayala, 
1982). A possible explanation is that the present 
populations of L. longipalpis have originated from a 
small number of individuals and have only recently 
increased greatly in numbers as a consequence of 
their adaptation to human environments. This 
hypothesis of a “ Founder Effect ” has been pro- 
posed to explain the low genetic variability of Tria- 
tona infestans, one of the main vectors of Chagas’ 
disease in Bolivia, and the best adapted one to 
human habitats (Dujardin and Tibayrenc, 1985). It 
is worth noting that the heterozygosity values for the 
sand flics Psychodopygus carrerai and P. yucumensis, 
which are both selvatic species, are higher than the 
values recorded in the present study for the perido- 
mestic species L. longipalpis. For P. carrerai, the 
observed H is 0.099 and the expected H is 0,120 ; 
for P. yucumensis, these values are respectively 0.126 
and 0.140 (Caillard et al., in press). These values 
are within the typical range observed for most insects 
(Ayala, 1982). 
Manuscrit accepte’ par lc Comité de Rédaction le .?l juin 1.986 
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An isozymic study of LUTZOMYIA LONGIPALPIS (DIPTERA, PSYCHODIDAE), the vector of visceral leishmaniasis in the "Yungas" (Bolivia)

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