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Enzymatic variability in three species of Sitophilus (Coleoptera: Curculionidae)
J. stored Prod. Res. Vol. 30, No. 3, pp. 201-213, 1994 Copyright 0 1994El&ier Science Ltd Printed in Great Britain. All rights reserved 0022474X(94)EOO14-J 0022-474X/94 $7.00 + 0.00
Pergamon
Enzymatic Variability in Three Species of Sitophilus (Coleoptera: Curculionidae) A. M. GRENIER, Laboratoire
B. PINTUREAU
and P. NARDON
de Biologie Appliqute, Bit. 406, I.N.S.A.-I.N.R.A. F-69621 Villeurbanne Cedex, France
20 Au. A. Einstein,
(Received 21 February 1994)
Abstract-Electrophoretic analysis of esterases was carried out on a total of 37 strains of Sitophiius oryzae, S. zeamais and S. granarius. Polymorphism rate was higher in S. zeamais than in the two other species. Esterase characters allow the three species to be distiquikd and confirm that S. oryzue and S. zeumui.9are closer to each other than to S. granarius. They aiso allow differentiation between populations of the same species, but do not indicate the geographical origin of all populations. A high mixture of populations due to the transport of cereals all over the world could explain such a result. Key words-Sitophilus
oryzae, zeamais, granaries,
esterases, diversity, taxonomy.
INTRODUCTION Weevils from the genus Sitophilus (Coleoptera: Curculionidae) are major pests of stored products all over the world. Among the three main species, only S. granarius (L.), the grain weevil, can easily be distinguished from S. oryzae (L.), the rice weevil, and S. zeamais Motsch., the maize weevil, by its morphological characters (bright aspect of the cuticle and rudimentary wings). It is very difficult to discriminate S. oryzae from S. zeamais. One good criterion in identifying these species was to consider the morphology of their symbiotes. Gram-negative bacteria, rod-shaped or flexuous, occur in S. oryzae, while curled or spiralled bacteria occur in S. zeamais (Musgrave and Homan, 1962; Nardon, 1971; Nardon and Grenier, 1989). These are located in a larval bacteriome and at the apex of each ovariole in the female (Mansour, 1930; Nardon, 1971; Nardon and Wicker, 1981). These symbiotes were recently identified as members of the Enterobacteriaceae in the y-3 subgroup of the Proteobacteria (Campbell et al., 1992). However, the isolation of symbiotes is a tedious process. Therefore, there is a need to search for new discriminating characters to distinguish these two sibling species. Our previous study dealt with esterase patterns of one population in each of the three Sitophilus species (Pintureau et al., 1991) and showed a difference between the three species. It also showed that S. oryzae was slightly closer to S. zeamais than to S. granarius. The present study takes into account 37 populations belonging to the three Sitophilus species, in order to investigate the possible discriminating power of esterases to characterize populations from various geographic origins. Such biochemical research for criteria to define geographic origin has been attempted with success in other Curculionidae transported by humans (Hsiao and Stutz, 1985; Unruh and Goeden, 1987). 201
202
A. M. GRENIER et al.
MATERIALS
Culture of Sitophilus
AND
METHODS
strains
The 37 strains of Sitophilus used in this study have been reared at our laboratory for several years and have different origins: crops from known geographic location, French groceries or laboratories (Table 1). They were found on various seeds (cereals or lotus). S. oryzae and S. zeatnais strains originated from different continents, whereas the three S. granarius strains all originated from East France. W AA31 was obtained from W after a selection for 50 generations to a reduced ovariole number. After their arrival at the laboratory, all the strains were maintained on wheat at 27S”C and 70% r.h. in plastic latticed boxes kept in an incubator, according to the method of Laviolette and Nardon (1963).
Table I. Strains of Sitophilus used in this study: names, geographical origins, collection plants and dates Sitophilus swcies
s. oryzae from known geographic origin
from groceries
Name
Origin
Plant
Date
Tunisie 1 Tunisie Nas Maroc Benin Mexique 1 Guad I Guad 111 Haiti Chine
Tunisia, Beja Tunisia, Nasrallah Morocco, Oujda Benin, Paracou Mexico West Indies, Guadeloupe West Indies, Guadeloupe Haiti China, Guangzhou
wheat wheat wheat sorghum maize maize maize maize paddy
1981 1984 1989 1982 1983 1983 1985 198.5 1983
W
France, Lyons (Armenian grocery) Obtained from selection of the W strain
wheat
1978
wheat
1978
France, Lyons (from industrial storage) Australia, Queensland (CSIRO collection 1962)
wheat
1959
wheat
1990
Burkina-Faso, Bobo-Dioulasso Cameroun, Yaoundi Ivory Coast, Bouake Congo, Kiodongala Congo, Nkosso Congo, Loudima Reunion Island Reunion Island Syria, Lattaquie Mexico, Puebla Mexico, Puerto Quintana Mexico, Potzohuacan Peru, Iquitos French Guyana Azores Islands, Santa Maria Azores Islands, Sao Miguel Azores Islands, Sao Miguel
maize
1991
maize maize maize maize paddy rice rice wheat maize maize maize maize sorghum maize maize maize
1982 1981 I984 1984 1984 1979 1987 1985 1984 1984 1984 1990 1984 1990 1990 I 990
Fran=, Lyons (Vietnamese grocery) France, Paris (Vietnamese grocery)
rice
1983
ZMA DAX
English laboratory France, INRA laboratory
maize wheat
1966 1982
AG ChamM MaCon
France, Jura, Courlaoux France, Jura, Passenans France, Sadne & Loire, Macon
wheat wheat barley
1978 1982 1983
W AA31
SFR from laboratories
Austrahe
S. zeamais
Burkina-Faso
known geographic origin
from groceries
Cameroun C&e d’lvoire Congo I Congo 3 Congo 4 FB R&union Gerard R&union Syrie Mexique 2 Mexique 4 Mexique 5 Iquitos Guyane Acores SMI Acores Lagos AFores Sete Cidades Vietnam Lotus
from laboratories
I
lotus seeds 1989
S. granaries
from known geographic orinin
Enzymatic variability in three species of Sitophilus
203
Experimental procedure
Electrophoresis was performed on vertical polyacrylamide slab gels (7%, pH 8.9) as described previously (Pintureau and Babault, 1981; Pintureau, 1987) with some modifications (Pintureau et al., 1991). Samples from each population were prepared from one entire adult (female or male) homogenized in a small vial with 30 ~1 of Trudgill solution containing sucrose (17%) then centrifuged for one min at 9000g and stored at -20°C before use. Supernatant fractions were examined by electrophoresis. Esterase staining was carried out in the dark, at 37°C in a pH 7.2 phosphate buffer, with Fast Blue BB (substrates: a and /3 naphthyl-acetate). Only major bands genetically analyzed (Pintureau et al., 1991) were considered. Statistical analyses
Esterase polymorphism was estimated from three indices: the mean heterozygosity, the mean number of alleles per locus and the polymorphism rate (number of polymorphic loci/number of studied loci). In S. oryzae (13 strains) and S. zeamais (21 strains), mean indices were compared by a t-test, after a transformation arc sin ,,f for mean heterozygosity and polymorphism rate values. No statistics were carried out with S. granarius because only three strains were studied. From the matrix of allele frequencies, we carried out a global study with a Factorial Correspondence Analysis (Lefebvre, 1976), giving the degrees of relative divergence between the three species. In S. oryzae and S. zeamais, where we analyzed a sufficient number of strains, the same analysis was carried out in order to separate the various strains. With the matrix of genetic distances from Cavalli-Sforza chord measure (Cavalli-Sforza and Edwards, 1967) we constructed a dendrogram giving the degree of proximity of strains by using the distance matrix phylogeny program Kitsch (PHYLIP package) (Felsenstein, 1990). Such a method, used by Dainou et al. (1993) with other distances (Nei’s genetic distance and Gregorius’ absolute distance) and algorithms (UPGMA method and minimum-sum-of-squares of Fitch and Margoliash), obtained satisfactory results in differentiating between different populations of Drosophila melanogaster Meigen by using polymorphism of amylases. RESULTS
Electrophoretic patterns of esterases and variability rates in the three species
We found similar patterns to those obtained in previous experiments (Pintureau et al., 1991) but with a high variability between strains. The results require no modification of the genetic interpretation for S. oryzae (Est 2, Est 3, Est 4 and Est 5) or S. zeamais (Est 2, Est 4 and Est S), but a correction must be effected in S. granarius species. Est 2 is unaltered (Est 273in AG strain), but the band which was interpreted as Est 3 belongs in fact to Est 4 (Est 4’j5and Est4” in AG strain instead of Est 396) and the band which was interpreted as Est 4 belongs in fact to Est 5 (Est 576 in AG strain instead of Est 4’O). Tables 2, 3, and 4 show the allelic frequencies in the different strains and the variability rates. The mean heterozygosities vary from 0 to 0.431 in S. oryzae (mean = 0.277 + 0.084; n = 13), from 0.123 to 0.629 in S. zeamais (mean = 0.419 f 0.067; n = 21), and from 0.120 to 0.395 in S. granarius (mean = 0.270; n = 3). Mean values of heterozygosity between S. oryzae and S. zeamais are different, after arc sin ,,f transformation, at the 2% level: t-test = 2.62. Other indices are also different between the 2 species: the mean allele number per locus is 1.90 in S. oryzae vs 2.48 in S. zeamais (t-test = 2.86; 0.01 > P > O.OOl),and the hism rate is 0.62 transformation). in S. oryzae vs 0.89 in S. zeamais (t-test = 3.38; 0.01 > P > 0.001, after arc sin These mean indices are respectively 1.67 and 0.67 in S. granarius. Discrimination of the three species
In the FCA (Factorial Correspondence Analysis) carried out on the three species by integrating data from Tables 2, 3 and 4, the first three axes express 43.4% of the variability (axis 1: 19.2%, axis 2: 15.4%, and axis 3: 8.8%). The first axis completely separates S. granarius strains from the two other species (S. oryzae and S. zeamais), and more weakly strains of these two latter species from each other. The second axis separates S. zeamais strains from other strains (Fig. 1A). The
(n) = number
of individuals
Mean heterozygosity Mean alkk number/locus Polymorphism rate
analysed
92 94 96 100
100 102 I06 115 122
Eat 4
5
0 90 100 I04
F&t 3
I3
0 100 107 (n _05l)
(n =Oll3)
0.338 2 0.75
at each locus in each strain.
0.417 2.25 0.75
0 0.864 0 0.136 (n = II)
(nOzO)
(n =Ol4l)
0.221 0.103 0.272 0.404 (n = 68)
0 0 0.51 0.26
(n _05l)
(n =Oll3)
0.635 0 0.365 0
I
0 0 I 0 0
0.542 0.458
W
I
0.222 I .75 0.50
0 0.307 0.226 0.468 (n = 31)
8 (n-34)
0 0.147 0.853
(n =o36)
: 1
0.388 2.25 I
(n =o28)
0.304 0.178 0.518
(n =o27,
0.370 0 0.630 0
(n : 30)
0.258 0 0.742
0 0.950 0.050 (n = 30)
Mexiaue
I
0.301 2.25 0.50
0.204 0.333 0.426 0.037 (n = 27)
0 0 0.532 0.419 0.049 (n = 31)
(n =o 32)
0 0 I
(n =o 16)
0 I
Nas
for esteram
Tunisie
of the 4 loci coding
8 (n = 36)
1
Tunisie
allele freauencks
0.490 0.510
SFR
Table 2. Ekctrooboretic
Allcks
Est 2
L.nci
0.328 I .75 0.75
(n =o26) (n =o28)
0 I 0
0.346 0 0.654
(n =o29)
0.397 0 0.603 0
(n =o29)
0 0 I
(n =o29)
0.743 0.257
0 I 0
(n =o28)
0 0 0 I
(n :28)
0 0 I
(n =o28)
I 0
Maroc
and uolymor&ism W AA31
0 I 0
0 I 0 0 (n = 26)
0 0 I 0 0 (n = 26)
0 0 I 0 (n = 26)
I 0 0 (n = 26)
tin
0.207 1.75 0.50
0 0.090 0.1% 0.714 (n = 28)
(n =o 30)
0 0 I 0
(n :30)
0 0 I
(n :30)
0.258 0.742
Haiti
0.307 2.25 0.75
0.103 0.259 0.086 0.552 (Ii = 29)
(n :28)
0.143 0 0.857 0
(n : 30)
0 0 I
(n =o30)
0.753 0.247
Australie
I
indices in 13 strains of Sitophihts oryrne
0.339 2 0.75
0.517 0.310 0.173 0 (n = 29)
(n =o 33)
0.833 0 0.167 0
0.378 0 0.622 0 (n = 35)
0 1 0 (n=l8)
ChitlC
0 0 0
I
0.326 2.25 0.75
0.241 0.5% 0.130 0.073 (n = 27)
(n =o 29)
0.569 0 0.431 0
(n : 30)
&4)
0.886 0.114
Goad
III
0.431 2.25 I
(n =“,I)
0.402 0.378 0.220
(n ,041)
0 0.549 0.451 0
(n ,041)
0 0.050 0.950
(” =“22)
0.426 0.574
Guad
Mean heterozygosity Mean allele no./locus Polymorphismrate
(n :22, 0 0 0.125 0 0.359 0 0.516 (n :32)
0.477 3 I
0 0 0.500 0 o.sOO 0 0
(n ,015)
0.525 2.33 1
88
90 92 94 96 99 100 102.5
0.114 0 0.432 0.454 0
0.316 0 0.131 0 0 0.553 (n = 19)
100 102 1% 112 115 II8
Ests
(n ="I,)
(n _039)
Fst4
0.056 0.083 0 0.861 0 0
0
0 84 87 91 94 % 100
F&2
0.387 2 1
0 0 0 0.600 0.400 0 0 0 (n = 30)
0.520 0 0.480 0 0 0 (n =2S)
0 0.100 0.900 0 0 0 0 (n = 45)
C6te d'lvoire Congo I
0.539 0 0.461 0 0
Syrie
Alleles
Loci
0.629 3 I
(n 1030)
0 0 0.350 0 0.2St-J 0.400 0
(n ="3l)
0 0.241 0.323 0.436 0
0 0 0.569 0.209 0 0 0.222 (n = 36)
Congo 3
(n ,027) 0 0 0.147 0.177 0.676 0 0 (n ,017)
(n _02S) 0 0 0.333 0.667 0 0 0 (n _033)
0.375 2.33 0.67
0.577 0 0 0.423 0
0.241 0 0.278 0.481 0
0.268 0 0.732 0 0
0.453 2.33 I
(n :49,
(n : 30)
I
0.502 2.33
(n 227)
0 0 0.352 0 0.444 0.204 0
(n :26)
0 0.745 0 0.255 0 0
Burkina-Faso Cameroun 0 0 0 0 0 0
0 0 0.361 0.583 0 0 0.056 (n = 36)
Congo 4
0.289 2 0.67
0 0 0 0.022 0.664 0.314 0 0 (n =67)
(n :68)
0 0 0.713 0.287 0
(n :70,
0 0 0 0
1
0
Guyane
I
0.516 2.33
0.200 2.33 0.67
(n ,029)
0 0 0.018 0 0.103 0.879 0
0 0 0 0.482 0.321 0.197 0 (n _028)
(n :30,
0.100 0 0.133 0.767 0
(n :22,
0 1 0 0 0
0
Mexique 2
(n ,028)
0.482 0 0.518 0 0
(n _033)
0 0 0.697 0 0.303
0
Iquitos
Table 3. Electroohontic allele freuuencies of the 3 locicodingforestcrases and t~~lymor&ismindices in 21 strains of Sitophilus mm&
0.209 1.67 0.67
0.569 2.67
-continued overleaf
I
(n ,032)
0.156 0 0 0.844 0 0 0
(n : 32)
8
0 0 0
(n ,027)
0.7059 0 0
0 0.241
Mcxique S
(n _03S)
0.229 0.542 0 0.229 0
0
0
0.672 0 0 0 0 0.328 (n = 32)
0 0.323 0 0.354 0 0 0.323 (n = 31)
Mexique 4
0 84 87 91 94 96 100
100 102 106 112 115 118
88 90 92 94 96 99 100 102.5
Est 2
Est 4
Est 5
0.143 I .67 0.67
(n : 19)
0 0 0 0 0.105 0.895 0
(n =o29)
0 0 0.138 0.862 0
(n =o29,
0 0 1 0 0 0
FB RCunion
I
0.452 2.67
0 0 0 0 0.035 0.179 0 0.786 (n = 68)
0 0 0.329 0.671 0 0 (n = 35)
0 0.592 0.204 0.204 0 0 0 (n =22)
GCrard RCunion
(n) = number of individuals analysed at each locus in each strain.
Mean heterozygosity Mean allele no./locus Polvmorohism rate
Alleles
Loci
Table 3-continued
0.475 3
I
I
(n =o27)
0 0 0 0.074 0 0.148 0.788
(n :48)
0 0.094 0 0.375 0.531
(n =o34)
0 0.265 0.662 0 0.073 0
ACOreS SMI
0.457 2.33
(n ,028)
0 0.035 0.304 0 0 0.661 0
(n _030)
0 0.283 0 0.717 0
(n ,021)
0.548 0 0.452 0 0
0
Vietnam
I
0.579 3.67
(n =o 16)
0.312 0 0.156 0 0.094 0 0.438
(n =o29)
0 0.259 0.068 0.259 0.414
(n =o26)
0 0.769 0.193 0 0.038 0
AFOreS Lagos
I
0.533 3.67
(n =“I,,
0.077 0 0 0.230 0.039 0.077 0.577
(n _022)
0 0.159 0.023 0.318 0.500
(n ,012)
0 0 0.750 0 0.250 0
Azores Sete Cidades
1
0.416 3
0 0 0.124 0.697 0.179 0 0 0 (n = 28)
0 0.475 0.250 0 0 0.275 (n = 20)
0.022 0.922 0 0.056 0 0 0 (n = 45)
Lotus
I
0.490 2.33
(n ,033)
0 0 0.409 0.303 0.288 0 0
(n :24,
0.500 0 0.500 0 0
(n =o 18)
0 0.194 0 0.806 0 0
DAX
0.123 I .33 0.33
(n :47,
0 0 I 0 0 0 0
(n =” 50)
0 1 0 0 0
(n _051)
0.243 0 0 0 0.757 0
ZMA
F
?
Enzymatic
variability
in three species of Sirophilus
207
Table 4. Electrophoretic allele frequencies of the 3 loci coding for esterases and polymorphism indices in 3 strains of Sirophilus granorius Loci
alleles
AG
Est 2
0 73
0
91 Est 4
Est 5
Mean heterozygosity Mean allele number/locus Polymorphism rate
Chambl
Macon 0 0
(n _048,
0.646 0 0.;54 (n = 12)
(a 114)
65 71
0.235 0.765 (n = 51)
0.542 0.458 (n = 36)
0.515 0.485 (n = 32)
75
0
76
(n 128)
0. I30 0.870 (n = 23)
0.260 0.740 (n = 25)
0.120 I .33 0.33
0.395 2 I
0.295 I .67 0.67
I
(n) = number of individuals analysed at each locus in each strain.
l-2 plan seems to be sufficient to completely discriminate between the three species. The third axis brings little additional information (Fig. 1B), but indicates that S. oryzae and S. zeamais are closer to each other than to S. granarius. The dendrogram (Fig. 2) shows that the esterase characters completely separate the three Sitophilus species. Moreover, it distinguishes S. granarius from the two other species at the first separation. Diferentiation
of populations
of S. oryzae
The first three axes of FCA express 68.5% of the variability (axis 1: 26.5%, axis 2: 25.2%, and axis 3: 16.8%). Strains were scattered along the first axis, but an apparent structuring of the strains appears according to their geographical origins (W, W AA3 1, SFR and Australie 1 strains, coming from groceries and laboratories, were not taken into account). On this axis, from negative to positive values, the Asiatic strain, American strains and African strains occur successively (Fig. 3A). Axis 2 mainly separates Guad I strain from the other American strains, and weakly the West African strain (Benin) from the North African strains. Axis 3 only discriminates the Asiatic strain from other strains (American and African ones) (Fig. 3B). Nevertheless, there are many overlaps between groupings, making the determination of strain geographic origin by means of esterase characters alone unreliable. W strain came from an Armenian grocery but is similar to African strains. A selection of this strain for a physiological character (reduction in the number of ovarioles) led to the W AA31 strain which deeply diverged from the W strain for esterases. SFR could have originated from America or North Africa, and Australie 1 from North Africa. The dendrogram (Fig. 2) confirms the lack of classification of strains according to their origins (American and North African strains are dispersed). W is close to the African strain (Benin) but W AA31 diverges widely. SFR and Australie 1 appear to be close to the American and North African strains. Dijierentiation
of populations
of S. zeamais
The first three axes of FCA express 46.8% of the variability (axis 1: 18.7%, axis 2: 15.1%, and axis 3: 13.0%). As for S. oryzae, the first axis to a large extent scatters the strains. Only one differentiation based on geographical origin can be discerned (Vietnam, Lotus, ZMA and DAX strains were not taken into account): the three Azorean strains are relatively well separated from the American, Asiatic and African ones (including the Reunion strains) (Fig. 4A). Axis 2 also separates the three Azorean strains from the others. Axis 3 only isolates the Gerard Reunion strain from the other African strains (senso largo) (Fig. 4B). In this species, the determination of geographical origins of the different strains by esterase characters is even less clear than for S. oryzae. Vietnam and Lotus strains found in Vietnamese groceries are close to African and American strains, but only one strain (Syrie) coming from Asia
A. M. GRENIERet al.
208
(Middle East) is taken into account in the analysis. DAX is rather close to Asiatic and American strains, whereas ZMA is very different from all the other strains. The dendrogram (Fig. 2) firstly confirms the separation of ZMA strain and secondly of Azorean strains from all the others. American and African strains are very dispersed and the origins of Lotus, Vietnam and DAX strains are uncertain.
A l-
S. oryzae it
A
S. granarius
•1 0 0
A
t-4
Y
O-
0
4
OO WQ
S. zeamais
00
$
-l0:
0
I
I
I
I
-1
A 2
1
AXIS
I
3
4
1
0
0
1
0
i
1 I
0
0
15
e-4
3 2
0
0 •I B
0
S. oryzae
I
A
0
&OS
. zeamais
S. granarius
000
A
o” 0
A
0 8 0
0
-1
0
B
I
I
I
1
I
-1
0
1
2
3
AXIS
1 4
I
Fig. 1. Factorial Correspondence Analysis (FCA) carried out from allele frequencies of 4 loci coding for esterases (Est 2, Est 3, Est 4 and Est 5): dispersion of the 37 strains belonging to Sitophilus oryzae, S. zeamair and S. granarius. A: axes 1 and 2; B: axes 1 and 3.
Enzymatic
variability
in three species of
Sitophilus
209
1 -A~OIZS
Lagos
S. grunarius
(Azores) (South America) (Indian Ocean) (Central America)
(Middle East) (Africa) (Africa) (Central America) (French grocery) (South America)
S. zeamak
(Indian Ocean) (Africa) FAr;;;;k%Xatory) t (French laboratory) (West Indies) (North Africa) (NormAfrica)
I
S. orjzae
Fig. 2. Classification of the 37 strains belonging to Sitophilus otyae. S. xamais and S. grunarius from allele frequencies of 4 loci coding for esterases (Est 2, Est 3, Est 4 and Est 5). The dendrogram was obtained by the distance matrix phylogeny program Kitsch using Cavalli-Sforza’s chord measure.
DISCUSSION
AND
CONCLUSION
Only aminopeptidases (Baker, 1982), amylases (Baker and Woo, 1985; Baker, 1987; Baker et al., 1989) and esterases (Beiras and Petitpierre, 1981; Pintureau et al., 1991) have been electrophoretitally studied in Sitophilus. Experiments dealing with homogenates of only one individual are still more rare. These studies allow us to analyze the variability rates and to compare the different populations by taking into account their allelic frequencies. Although we only studied a small number of loci in strains that could have been modified by long rearing in the laboratory, the large number of strains of S. oryzae and S. zeamais tested enable the variability of these two species to be compared. It seems that variability is higher in S. zeamais than in S. oryzae, contrary to the preliminary results from Pintureau et al. (1991). The order zeamais > oryzae > granarius was obtained by Beiras and Petitpierre (1981) who analyzed three strains from each Sitophilus species. These authors found lower values than us for genetic variability, because they studied fewer strains and not only analyzed esterases, but also five other enzymatic systems which were monomorphic. Mean heterozygosity values for Sitophilus are in line with those calculated for other Curculionidae. Values obtained for S. zeamais are close to those calculated for three populations of Anthonomus grundis Boheman (Bartlett, 198 l), and values obtained for S. oryzae are close to those calculated for six populations of Hypera postica (Gyllenhal) (Hsiao and Stutz, 1985) and one population of Otiorrhynchus scaber (L.) (Suomalainen and Saura, 1973). Those obtained for S. granarius (only three populations from the same region were tested) are close to those calculated for 17 populations of Pissodes strobi (Peck) (Phillips and Lanier, 1985) six populations of
A. M. GRENIER et al.
210
2q Guad I
l-
q wAA31
q Benin
Maroc Australibl q q W SFRCP Tunisie 1 Guad III
oq Chine
•I Mexique 1
A
%I •1 Tunisie Nas Haiti
1
0
-1
I
I
I
AXIS
2
1
lq W AA31
o Chine
q Tunisic Nas q Mexique 1
[3W 0 z? X a
o-
GuadI
0 •1 Marw SFR 13 0 Guad III
t3 Benin
[3 Australie 1 Haiti 0
0 Tunisie
1 B
-1 -1
I 1
I 0 AXIS
1
Fig. 3. Factorial Correspondence Analysis (FCA) carried out from allele frequencies of 4 loci coding for esterases (Est 2, Est 3, Est 4 and Est 5): dispersion of the 13 strains belonging to Sitophilus oryzae. A: axes 1 and 2;B: axes 1 and 3.
Enzymatic variability in three species of Sitophilus
211
2
A OZMA
1 1 o ApresLagoa
1
Acores SeteCidadcs 8 ACOW.SMI
0 Mexique 5
c-4
VJ
0 Lotus
. d 1. viemam oo Cote lvo~rc
0 Syrie 0
ODE
Cameroun 0 00 Mexique 4 Congo 4 0 Congo 3 0 Guyane o FB Rdunion Congo 1 0 o o Mexique2 Iquitos 0 Gdrard RCunion Burkina-Faso 0
-1 -1
I
I
1
0
1
2
AXIS
1
2-
B
0 GCrard Reunion
l-
I
m v3
a
0 Vietnam
OW
1
Cameroun 0
0
DAX
0 Syrie
0
0
0 Lotus
0 Guyanc 0 C&e d’Ivoire
0 Burkina-Faso 0 Congo 4
Mexique 4
0 Mexique 2
0 Congo3
0 Aqores Lagoa Aqores SM 1
0
0 FB Riunion
8 Congo 1 Iquitos
Apes
0 Sete Cidades
o Mexique 5
- _i
i,
i AXIS
i
1
Fig. 4. Factorial Correspondence Analysis (FCA) carried out from allele frequencies of 3 loci coding for esterases (Est 2, Est 4 and Est 5): dispersion of the 21 strains belonging to Sirophilus zeamais. A: axes 1 and 2; B: axes 1 and 3.
Rhinocylfus conicus Froelich (Unruh and Goeden, 1987) and three populations capita&s De Geer (Suomalainen and Saura, 1973).
of Srrophasomus
This study confirms that esterase patterns can enable distinction of the three Sitophilus species, and that S. oryzue and S. zeamais are closer to each other than to S. granarius. One population
A. M. GRENIER et al.
212
of S. oryzae (Guad I) and one of S. zeamais (FB Reunion) are able to produce a limited number of viable hybrids when crossed together, and notably Guad I is the closest S. oryzae strain to S. zeamais strains in the dendrogram. Esterases also permit some differentiation between populations of a particular species, but the variability is not classifiable according to geographic origin of strains. This situation is probably the result of a high mixture of populations due to trading of cereals all over the world, and perhaps also the result of different pesticide exposures of the strains (esterases are involved in pesticide resistance mechanisms). These results seem to be inconsistent with those obtained by Hsiao and Stutz (1985) and by Unruh and Goeden (1987). In fact, the first authors studying Hypera posticu (from only three geographical origins) by examining 22 loci, found only some of them to differ systematically. The second authors experimenting on seven loci of Rhinocyllus conicus, from only two geographical origins, found allelic frequency differences but no fixed allelic differences. We also found such differences. In S. zeamais, for instance, some alleles characterized certain populations: Est 296in the Iquitos strain, Est 4”’ in Azorean strains, Est 5”*.’ in a Reunion Island strain, and Est 5” in the Vietnam strain. If chemotaxonomic markers do exist among populations of S. oryzae and S. zeamais, it will be necessary to analyze other loci in a large number of populations to find them. are grateful to Yvan Rahbe for his help in computing the dendrogram, and to numerous colleagues who sent us weevils from all over the world. Acknowledgements-We
REFERENCES Baker J. E. (1982) Digestive proteinases of Sitophilus weevils (Coleoptera: Curculionidae) and their response to inhibitors from wheat and corn flour. Can. J. Zool. 60, 32063214. Baker J. E. (1987) Electrophoretic analysis of amylase isozymes in geographical strains of Sitophilus oryzae (L.), S. zeamais Motsch, and S. granarius (L.) (Coleoptera: Curculionidae). J. stored Prod. Res. 23, 125-131. Baker J. E., Lum P. T. M. and Halliday W. R. (1989) Phenotypic variants and total cl-amylase in the maize weevil (Coleoptera: Curculionidae). J. Kansas Ent. Sot. 62, 43&434. Baker J. E. and Woo S. M. (1985) Purification, partial characterization, and postembryonic levels of amylases from Sitophilus oryzae and S. granarius. Arch. Znsect Biochem. Physiol. 2, 415428. Bartlett A. C. (1981) Isozyme polymorphisms in boll weevils and thurberia weevils from Arizona. Ann. En?. Sot. Am. 74, 359-362.
Beiras M. J. and Petitpierre E. (1981) Allozymic variability and genetic differentiation in the three species of Sitophilus L. (Coleoptera, Curculionidae). Egypt. J. Genetic. Cytol. 10, 95-104. Campbell B. C., Bragg T. S. and Turner C. E. (1992) Phylogeny of symbiotic bacteria of four weevil species (Coleoptera: Curculionidae) based on analysis of 16s ribosomal DNA. Insect Eiochem. Molec. Viol. 22, 415421. Cavalli-Sforza L. L. and Edwards A. W. F. (1967) Phylogenetic analysis: models and estimation procedures. &oh&on 32, 55&570. Dainou O., Cariou M. L., Goux J. M. and David J. R. (1993) Amylase polymorphism in Drosophila melanogaster: haplotype frequencies in tropical African and American populations. Genet. Sel. Evol. 25, 133-151. Felsenstein J. (1990) PHYLZP Manual Version 3.3. University Herbarium, University of California, Berkeley, California. Hsiao T. H. and Stutz J. M. (1985) Discrimination of alfalfa weevil strains by allozyme analysis. Ent. exp. appl. 37, 113-121. Laviolette P. and Nardon P. (1963) Action des rayons gamma du cobalt 60 sur la mortalitt et la fertilitt des adultes d’un charan9on du riz. Bull. Biol. Fr. Belg. 97, 305-333. Lefebvre J. (1976) Zntroduction aux analyses statistiques multidimensionnelles. 219 pp. Masson, Paris, N.Y ., Barcelone, Milan. Mansour K. (1930) Preliminary studies on the bacterial cell mass (accessory cell mass) of Calandra oryzae (Linn.): the Rice Weevil. Quart. J. Microsc. Sci. 73, 422435. Musgrave A. J. and Homan R. (1962) Sitophilus sasakii (Tak.) (Coleoptera: Curculionidae) in Canada. Anatomy and mycetomal symbiotes as valid taxonomic characters. Can. Ent. 94, 1196-l 197. Nardon P. (1971) Contribution a l’ttude des symbiotes ovariens de Sitophilus sasakii: localisation, histochimie et ultrastructure chez la femelle ad&e. C. R. Acad. Sci. Paris, Ser D 272, 2975-2978. Nardon P. and Grenier A. M. (1989) Identification, cytology, heredity, culture, metabolism and genetics of Beetle endocytobiotes. In Insect Endocytobiosis: Morphology, Physiology, Genetics and Evolution (Edited by Schwemmler W.) pp. 175-216. CRC Press Inc., Fla. Nardon P. and Wicker C. (1981) La symbiose chez le genre Sitophilus (Coleoptere Curculionidetprincipaux aspects morphologiques, physiologiques et gbnetiques. Ann. Biol. 20, 327-373. Phillips T. W. and Lanier G. N. (1985) Genetic divergence among populations of the white pine weevil, Pissodes strobi (Coleoptera: Curculionidae). Ann. Em. Sot. Am. 78, 744-750. Pintureau B. (1987) Systematique evolutive du genre Trichogramma Westwood (Hym. Trichogrammatidae) en Europe. These de Doctorat d’Etat, Universite Paris VZZ, France, 311 pp. Pintureau B. and Babault M. (1981) Caracterisation enzymatique de Trichogramma evanescens et de T. maidis (Hym.: Trichogrammatidae); Etude des hybrides. Entomophaga 26, 1 l-22.
Enzymatic variability in three species of Sitophilus
213
Pintureau B., Grenier A. M. and Nardon P. (1991) Polymorphism of esterases in three species of Sitophilus (Coleoptera: Curcuhonidae). J. stored Prod. Am. 27, 141-151. Suomalainen E. and Saura A. (1973) Genetic polymorphism and evolution in parthenogenetic animals. 1. Polyploid Curcuhonidae. Generics 74, 4899508. Unruh T. R. and Goeden R. D. (1987) Electrophoresis helps to identify which race of the introduced weevil, Rhinocyllus conicus (Coleoptera: Curcuhonidae), has transferred to two native Southern California thistles. Enciron. En,. 16, 979-983.
Pergamon
Enzymatic Variability in Three Species of Sitophilus (Coleoptera: Curculionidae) A. M. GRENIER, Laboratoire
B. PINTUREAU
and P. NARDON
de Biologie Appliqute, Bit. 406, I.N.S.A.-I.N.R.A. F-69621 Villeurbanne Cedex, France
20 Au. A. Einstein,
(Received 21 February 1994)
Abstract-Electrophoretic analysis of esterases was carried out on a total of 37 strains of Sitophiius oryzae, S. zeamais and S. granarius. Polymorphism rate was higher in S. zeamais than in the two other species. Esterase characters allow the three species to be distiquikd and confirm that S. oryzue and S. zeumui.9are closer to each other than to S. granarius. They aiso allow differentiation between populations of the same species, but do not indicate the geographical origin of all populations. A high mixture of populations due to the transport of cereals all over the world could explain such a result. Key words-Sitophilus
oryzae, zeamais, granaries,
esterases, diversity, taxonomy.
INTRODUCTION Weevils from the genus Sitophilus (Coleoptera: Curculionidae) are major pests of stored products all over the world. Among the three main species, only S. granarius (L.), the grain weevil, can easily be distinguished from S. oryzae (L.), the rice weevil, and S. zeamais Motsch., the maize weevil, by its morphological characters (bright aspect of the cuticle and rudimentary wings). It is very difficult to discriminate S. oryzae from S. zeamais. One good criterion in identifying these species was to consider the morphology of their symbiotes. Gram-negative bacteria, rod-shaped or flexuous, occur in S. oryzae, while curled or spiralled bacteria occur in S. zeamais (Musgrave and Homan, 1962; Nardon, 1971; Nardon and Grenier, 1989). These are located in a larval bacteriome and at the apex of each ovariole in the female (Mansour, 1930; Nardon, 1971; Nardon and Wicker, 1981). These symbiotes were recently identified as members of the Enterobacteriaceae in the y-3 subgroup of the Proteobacteria (Campbell et al., 1992). However, the isolation of symbiotes is a tedious process. Therefore, there is a need to search for new discriminating characters to distinguish these two sibling species. Our previous study dealt with esterase patterns of one population in each of the three Sitophilus species (Pintureau et al., 1991) and showed a difference between the three species. It also showed that S. oryzae was slightly closer to S. zeamais than to S. granarius. The present study takes into account 37 populations belonging to the three Sitophilus species, in order to investigate the possible discriminating power of esterases to characterize populations from various geographic origins. Such biochemical research for criteria to define geographic origin has been attempted with success in other Curculionidae transported by humans (Hsiao and Stutz, 1985; Unruh and Goeden, 1987). 201
202
A. M. GRENIER et al.
MATERIALS
Culture of Sitophilus
AND
METHODS
strains
The 37 strains of Sitophilus used in this study have been reared at our laboratory for several years and have different origins: crops from known geographic location, French groceries or laboratories (Table 1). They were found on various seeds (cereals or lotus). S. oryzae and S. zeatnais strains originated from different continents, whereas the three S. granarius strains all originated from East France. W AA31 was obtained from W after a selection for 50 generations to a reduced ovariole number. After their arrival at the laboratory, all the strains were maintained on wheat at 27S”C and 70% r.h. in plastic latticed boxes kept in an incubator, according to the method of Laviolette and Nardon (1963).
Table I. Strains of Sitophilus used in this study: names, geographical origins, collection plants and dates Sitophilus swcies
s. oryzae from known geographic origin
from groceries
Name
Origin
Plant
Date
Tunisie 1 Tunisie Nas Maroc Benin Mexique 1 Guad I Guad 111 Haiti Chine
Tunisia, Beja Tunisia, Nasrallah Morocco, Oujda Benin, Paracou Mexico West Indies, Guadeloupe West Indies, Guadeloupe Haiti China, Guangzhou
wheat wheat wheat sorghum maize maize maize maize paddy
1981 1984 1989 1982 1983 1983 1985 198.5 1983
W
France, Lyons (Armenian grocery) Obtained from selection of the W strain
wheat
1978
wheat
1978
France, Lyons (from industrial storage) Australia, Queensland (CSIRO collection 1962)
wheat
1959
wheat
1990
Burkina-Faso, Bobo-Dioulasso Cameroun, Yaoundi Ivory Coast, Bouake Congo, Kiodongala Congo, Nkosso Congo, Loudima Reunion Island Reunion Island Syria, Lattaquie Mexico, Puebla Mexico, Puerto Quintana Mexico, Potzohuacan Peru, Iquitos French Guyana Azores Islands, Santa Maria Azores Islands, Sao Miguel Azores Islands, Sao Miguel
maize
1991
maize maize maize maize paddy rice rice wheat maize maize maize maize sorghum maize maize maize
1982 1981 I984 1984 1984 1979 1987 1985 1984 1984 1984 1990 1984 1990 1990 I 990
Fran=, Lyons (Vietnamese grocery) France, Paris (Vietnamese grocery)
rice
1983
ZMA DAX
English laboratory France, INRA laboratory
maize wheat
1966 1982
AG ChamM MaCon
France, Jura, Courlaoux France, Jura, Passenans France, Sadne & Loire, Macon
wheat wheat barley
1978 1982 1983
W AA31
SFR from laboratories
Austrahe
S. zeamais
Burkina-Faso
known geographic origin
from groceries
Cameroun C&e d’lvoire Congo I Congo 3 Congo 4 FB R&union Gerard R&union Syrie Mexique 2 Mexique 4 Mexique 5 Iquitos Guyane Acores SMI Acores Lagos AFores Sete Cidades Vietnam Lotus
from laboratories
I
lotus seeds 1989
S. granaries
from known geographic orinin
Enzymatic variability in three species of Sitophilus
203
Experimental procedure
Electrophoresis was performed on vertical polyacrylamide slab gels (7%, pH 8.9) as described previously (Pintureau and Babault, 1981; Pintureau, 1987) with some modifications (Pintureau et al., 1991). Samples from each population were prepared from one entire adult (female or male) homogenized in a small vial with 30 ~1 of Trudgill solution containing sucrose (17%) then centrifuged for one min at 9000g and stored at -20°C before use. Supernatant fractions were examined by electrophoresis. Esterase staining was carried out in the dark, at 37°C in a pH 7.2 phosphate buffer, with Fast Blue BB (substrates: a and /3 naphthyl-acetate). Only major bands genetically analyzed (Pintureau et al., 1991) were considered. Statistical analyses
Esterase polymorphism was estimated from three indices: the mean heterozygosity, the mean number of alleles per locus and the polymorphism rate (number of polymorphic loci/number of studied loci). In S. oryzae (13 strains) and S. zeamais (21 strains), mean indices were compared by a t-test, after a transformation arc sin ,,f for mean heterozygosity and polymorphism rate values. No statistics were carried out with S. granarius because only three strains were studied. From the matrix of allele frequencies, we carried out a global study with a Factorial Correspondence Analysis (Lefebvre, 1976), giving the degrees of relative divergence between the three species. In S. oryzae and S. zeamais, where we analyzed a sufficient number of strains, the same analysis was carried out in order to separate the various strains. With the matrix of genetic distances from Cavalli-Sforza chord measure (Cavalli-Sforza and Edwards, 1967) we constructed a dendrogram giving the degree of proximity of strains by using the distance matrix phylogeny program Kitsch (PHYLIP package) (Felsenstein, 1990). Such a method, used by Dainou et al. (1993) with other distances (Nei’s genetic distance and Gregorius’ absolute distance) and algorithms (UPGMA method and minimum-sum-of-squares of Fitch and Margoliash), obtained satisfactory results in differentiating between different populations of Drosophila melanogaster Meigen by using polymorphism of amylases. RESULTS
Electrophoretic patterns of esterases and variability rates in the three species
We found similar patterns to those obtained in previous experiments (Pintureau et al., 1991) but with a high variability between strains. The results require no modification of the genetic interpretation for S. oryzae (Est 2, Est 3, Est 4 and Est 5) or S. zeamais (Est 2, Est 4 and Est S), but a correction must be effected in S. granarius species. Est 2 is unaltered (Est 273in AG strain), but the band which was interpreted as Est 3 belongs in fact to Est 4 (Est 4’j5and Est4” in AG strain instead of Est 396) and the band which was interpreted as Est 4 belongs in fact to Est 5 (Est 576 in AG strain instead of Est 4’O). Tables 2, 3, and 4 show the allelic frequencies in the different strains and the variability rates. The mean heterozygosities vary from 0 to 0.431 in S. oryzae (mean = 0.277 + 0.084; n = 13), from 0.123 to 0.629 in S. zeamais (mean = 0.419 f 0.067; n = 21), and from 0.120 to 0.395 in S. granarius (mean = 0.270; n = 3). Mean values of heterozygosity between S. oryzae and S. zeamais are different, after arc sin ,,f transformation, at the 2% level: t-test = 2.62. Other indices are also different between the 2 species: the mean allele number per locus is 1.90 in S. oryzae vs 2.48 in S. zeamais (t-test = 2.86; 0.01 > P > O.OOl),and the hism rate is 0.62 transformation). in S. oryzae vs 0.89 in S. zeamais (t-test = 3.38; 0.01 > P > 0.001, after arc sin These mean indices are respectively 1.67 and 0.67 in S. granarius. Discrimination of the three species
In the FCA (Factorial Correspondence Analysis) carried out on the three species by integrating data from Tables 2, 3 and 4, the first three axes express 43.4% of the variability (axis 1: 19.2%, axis 2: 15.4%, and axis 3: 8.8%). The first axis completely separates S. granarius strains from the two other species (S. oryzae and S. zeamais), and more weakly strains of these two latter species from each other. The second axis separates S. zeamais strains from other strains (Fig. 1A). The
(n) = number
of individuals
Mean heterozygosity Mean alkk number/locus Polymorphism rate
analysed
92 94 96 100
100 102 I06 115 122
Eat 4
5
0 90 100 I04
F&t 3
I3
0 100 107 (n _05l)
(n =Oll3)
0.338 2 0.75
at each locus in each strain.
0.417 2.25 0.75
0 0.864 0 0.136 (n = II)
(nOzO)
(n =Ol4l)
0.221 0.103 0.272 0.404 (n = 68)
0 0 0.51 0.26
(n _05l)
(n =Oll3)
0.635 0 0.365 0
I
0 0 I 0 0
0.542 0.458
W
I
0.222 I .75 0.50
0 0.307 0.226 0.468 (n = 31)
8 (n-34)
0 0.147 0.853
(n =o36)
: 1
0.388 2.25 I
(n =o28)
0.304 0.178 0.518
(n =o27,
0.370 0 0.630 0
(n : 30)
0.258 0 0.742
0 0.950 0.050 (n = 30)
Mexiaue
I
0.301 2.25 0.50
0.204 0.333 0.426 0.037 (n = 27)
0 0 0.532 0.419 0.049 (n = 31)
(n =o 32)
0 0 I
(n =o 16)
0 I
Nas
for esteram
Tunisie
of the 4 loci coding
8 (n = 36)
1
Tunisie
allele freauencks
0.490 0.510
SFR
Table 2. Ekctrooboretic
Allcks
Est 2
L.nci
0.328 I .75 0.75
(n =o26) (n =o28)
0 I 0
0.346 0 0.654
(n =o29)
0.397 0 0.603 0
(n =o29)
0 0 I
(n =o29)
0.743 0.257
0 I 0
(n =o28)
0 0 0 I
(n :28)
0 0 I
(n =o28)
I 0
Maroc
and uolymor&ism W AA31
0 I 0
0 I 0 0 (n = 26)
0 0 I 0 0 (n = 26)
0 0 I 0 (n = 26)
I 0 0 (n = 26)
tin
0.207 1.75 0.50
0 0.090 0.1% 0.714 (n = 28)
(n =o 30)
0 0 I 0
(n :30)
0 0 I
(n :30)
0.258 0.742
Haiti
0.307 2.25 0.75
0.103 0.259 0.086 0.552 (Ii = 29)
(n :28)
0.143 0 0.857 0
(n : 30)
0 0 I
(n =o30)
0.753 0.247
Australie
I
indices in 13 strains of Sitophihts oryrne
0.339 2 0.75
0.517 0.310 0.173 0 (n = 29)
(n =o 33)
0.833 0 0.167 0
0.378 0 0.622 0 (n = 35)
0 1 0 (n=l8)
ChitlC
0 0 0
I
0.326 2.25 0.75
0.241 0.5% 0.130 0.073 (n = 27)
(n =o 29)
0.569 0 0.431 0
(n : 30)
&4)
0.886 0.114
Goad
III
0.431 2.25 I
(n =“,I)
0.402 0.378 0.220
(n ,041)
0 0.549 0.451 0
(n ,041)
0 0.050 0.950
(” =“22)
0.426 0.574
Guad
Mean heterozygosity Mean allele no./locus Polymorphismrate
(n :22, 0 0 0.125 0 0.359 0 0.516 (n :32)
0.477 3 I
0 0 0.500 0 o.sOO 0 0
(n ,015)
0.525 2.33 1
88
90 92 94 96 99 100 102.5
0.114 0 0.432 0.454 0
0.316 0 0.131 0 0 0.553 (n = 19)
100 102 1% 112 115 II8
Ests
(n ="I,)
(n _039)
Fst4
0.056 0.083 0 0.861 0 0
0
0 84 87 91 94 % 100
F&2
0.387 2 1
0 0 0 0.600 0.400 0 0 0 (n = 30)
0.520 0 0.480 0 0 0 (n =2S)
0 0.100 0.900 0 0 0 0 (n = 45)
C6te d'lvoire Congo I
0.539 0 0.461 0 0
Syrie
Alleles
Loci
0.629 3 I
(n 1030)
0 0 0.350 0 0.2St-J 0.400 0
(n ="3l)
0 0.241 0.323 0.436 0
0 0 0.569 0.209 0 0 0.222 (n = 36)
Congo 3
(n ,027) 0 0 0.147 0.177 0.676 0 0 (n ,017)
(n _02S) 0 0 0.333 0.667 0 0 0 (n _033)
0.375 2.33 0.67
0.577 0 0 0.423 0
0.241 0 0.278 0.481 0
0.268 0 0.732 0 0
0.453 2.33 I
(n :49,
(n : 30)
I
0.502 2.33
(n 227)
0 0 0.352 0 0.444 0.204 0
(n :26)
0 0.745 0 0.255 0 0
Burkina-Faso Cameroun 0 0 0 0 0 0
0 0 0.361 0.583 0 0 0.056 (n = 36)
Congo 4
0.289 2 0.67
0 0 0 0.022 0.664 0.314 0 0 (n =67)
(n :68)
0 0 0.713 0.287 0
(n :70,
0 0 0 0
1
0
Guyane
I
0.516 2.33
0.200 2.33 0.67
(n ,029)
0 0 0.018 0 0.103 0.879 0
0 0 0 0.482 0.321 0.197 0 (n _028)
(n :30,
0.100 0 0.133 0.767 0
(n :22,
0 1 0 0 0
0
Mexique 2
(n ,028)
0.482 0 0.518 0 0
(n _033)
0 0 0.697 0 0.303
0
Iquitos
Table 3. Electroohontic allele freuuencies of the 3 locicodingforestcrases and t~~lymor&ismindices in 21 strains of Sitophilus mm&
0.209 1.67 0.67
0.569 2.67
-continued overleaf
I
(n ,032)
0.156 0 0 0.844 0 0 0
(n : 32)
8
0 0 0
(n ,027)
0.7059 0 0
0 0.241
Mcxique S
(n _03S)
0.229 0.542 0 0.229 0
0
0
0.672 0 0 0 0 0.328 (n = 32)
0 0.323 0 0.354 0 0 0.323 (n = 31)
Mexique 4
0 84 87 91 94 96 100
100 102 106 112 115 118
88 90 92 94 96 99 100 102.5
Est 2
Est 4
Est 5
0.143 I .67 0.67
(n : 19)
0 0 0 0 0.105 0.895 0
(n =o29)
0 0 0.138 0.862 0
(n =o29,
0 0 1 0 0 0
FB RCunion
I
0.452 2.67
0 0 0 0 0.035 0.179 0 0.786 (n = 68)
0 0 0.329 0.671 0 0 (n = 35)
0 0.592 0.204 0.204 0 0 0 (n =22)
GCrard RCunion
(n) = number of individuals analysed at each locus in each strain.
Mean heterozygosity Mean allele no./locus Polvmorohism rate
Alleles
Loci
Table 3-continued
0.475 3
I
I
(n =o27)
0 0 0 0.074 0 0.148 0.788
(n :48)
0 0.094 0 0.375 0.531
(n =o34)
0 0.265 0.662 0 0.073 0
ACOreS SMI
0.457 2.33
(n ,028)
0 0.035 0.304 0 0 0.661 0
(n _030)
0 0.283 0 0.717 0
(n ,021)
0.548 0 0.452 0 0
0
Vietnam
I
0.579 3.67
(n =o 16)
0.312 0 0.156 0 0.094 0 0.438
(n =o29)
0 0.259 0.068 0.259 0.414
(n =o26)
0 0.769 0.193 0 0.038 0
AFOreS Lagos
I
0.533 3.67
(n =“I,,
0.077 0 0 0.230 0.039 0.077 0.577
(n _022)
0 0.159 0.023 0.318 0.500
(n ,012)
0 0 0.750 0 0.250 0
Azores Sete Cidades
1
0.416 3
0 0 0.124 0.697 0.179 0 0 0 (n = 28)
0 0.475 0.250 0 0 0.275 (n = 20)
0.022 0.922 0 0.056 0 0 0 (n = 45)
Lotus
I
0.490 2.33
(n ,033)
0 0 0.409 0.303 0.288 0 0
(n :24,
0.500 0 0.500 0 0
(n =o 18)
0 0.194 0 0.806 0 0
DAX
0.123 I .33 0.33
(n :47,
0 0 I 0 0 0 0
(n =” 50)
0 1 0 0 0
(n _051)
0.243 0 0 0 0.757 0
ZMA
F
?
Enzymatic
variability
in three species of Sirophilus
207
Table 4. Electrophoretic allele frequencies of the 3 loci coding for esterases and polymorphism indices in 3 strains of Sirophilus granorius Loci
alleles
AG
Est 2
0 73
0
91 Est 4
Est 5
Mean heterozygosity Mean allele number/locus Polymorphism rate
Chambl
Macon 0 0
(n _048,
0.646 0 0.;54 (n = 12)
(a 114)
65 71
0.235 0.765 (n = 51)
0.542 0.458 (n = 36)
0.515 0.485 (n = 32)
75
0
76
(n 128)
0. I30 0.870 (n = 23)
0.260 0.740 (n = 25)
0.120 I .33 0.33
0.395 2 I
0.295 I .67 0.67
I
(n) = number of individuals analysed at each locus in each strain.
l-2 plan seems to be sufficient to completely discriminate between the three species. The third axis brings little additional information (Fig. 1B), but indicates that S. oryzae and S. zeamais are closer to each other than to S. granarius. The dendrogram (Fig. 2) shows that the esterase characters completely separate the three Sitophilus species. Moreover, it distinguishes S. granarius from the two other species at the first separation. Diferentiation
of populations
of S. oryzae
The first three axes of FCA express 68.5% of the variability (axis 1: 26.5%, axis 2: 25.2%, and axis 3: 16.8%). Strains were scattered along the first axis, but an apparent structuring of the strains appears according to their geographical origins (W, W AA3 1, SFR and Australie 1 strains, coming from groceries and laboratories, were not taken into account). On this axis, from negative to positive values, the Asiatic strain, American strains and African strains occur successively (Fig. 3A). Axis 2 mainly separates Guad I strain from the other American strains, and weakly the West African strain (Benin) from the North African strains. Axis 3 only discriminates the Asiatic strain from other strains (American and African ones) (Fig. 3B). Nevertheless, there are many overlaps between groupings, making the determination of strain geographic origin by means of esterase characters alone unreliable. W strain came from an Armenian grocery but is similar to African strains. A selection of this strain for a physiological character (reduction in the number of ovarioles) led to the W AA31 strain which deeply diverged from the W strain for esterases. SFR could have originated from America or North Africa, and Australie 1 from North Africa. The dendrogram (Fig. 2) confirms the lack of classification of strains according to their origins (American and North African strains are dispersed). W is close to the African strain (Benin) but W AA31 diverges widely. SFR and Australie 1 appear to be close to the American and North African strains. Dijierentiation
of populations
of S. zeamais
The first three axes of FCA express 46.8% of the variability (axis 1: 18.7%, axis 2: 15.1%, and axis 3: 13.0%). As for S. oryzae, the first axis to a large extent scatters the strains. Only one differentiation based on geographical origin can be discerned (Vietnam, Lotus, ZMA and DAX strains were not taken into account): the three Azorean strains are relatively well separated from the American, Asiatic and African ones (including the Reunion strains) (Fig. 4A). Axis 2 also separates the three Azorean strains from the others. Axis 3 only isolates the Gerard Reunion strain from the other African strains (senso largo) (Fig. 4B). In this species, the determination of geographical origins of the different strains by esterase characters is even less clear than for S. oryzae. Vietnam and Lotus strains found in Vietnamese groceries are close to African and American strains, but only one strain (Syrie) coming from Asia
A. M. GRENIERet al.
208
(Middle East) is taken into account in the analysis. DAX is rather close to Asiatic and American strains, whereas ZMA is very different from all the other strains. The dendrogram (Fig. 2) firstly confirms the separation of ZMA strain and secondly of Azorean strains from all the others. American and African strains are very dispersed and the origins of Lotus, Vietnam and DAX strains are uncertain.
A l-
S. oryzae it
A
S. granarius
•1 0 0
A
t-4
Y
O-
0
4
OO WQ
S. zeamais
00
$
-l0:
0
I
I
I
I
-1
A 2
1
AXIS
I
3
4
1
0
0
1
0
i
1 I
0
0
15
e-4
3 2
0
0 •I B
0
S. oryzae
I
A
0
&OS
. zeamais
S. granarius
000
A
o” 0
A
0 8 0
0
-1
0
B
I
I
I
1
I
-1
0
1
2
3
AXIS
1 4
I
Fig. 1. Factorial Correspondence Analysis (FCA) carried out from allele frequencies of 4 loci coding for esterases (Est 2, Est 3, Est 4 and Est 5): dispersion of the 37 strains belonging to Sitophilus oryzae, S. zeamair and S. granarius. A: axes 1 and 2; B: axes 1 and 3.
Enzymatic
variability
in three species of
Sitophilus
209
1 -A~OIZS
Lagos
S. grunarius
(Azores) (South America) (Indian Ocean) (Central America)
(Middle East) (Africa) (Africa) (Central America) (French grocery) (South America)
S. zeamak
(Indian Ocean) (Africa) FAr;;;;k%Xatory) t (French laboratory) (West Indies) (North Africa) (NormAfrica)
I
S. orjzae
Fig. 2. Classification of the 37 strains belonging to Sitophilus otyae. S. xamais and S. grunarius from allele frequencies of 4 loci coding for esterases (Est 2, Est 3, Est 4 and Est 5). The dendrogram was obtained by the distance matrix phylogeny program Kitsch using Cavalli-Sforza’s chord measure.
DISCUSSION
AND
CONCLUSION
Only aminopeptidases (Baker, 1982), amylases (Baker and Woo, 1985; Baker, 1987; Baker et al., 1989) and esterases (Beiras and Petitpierre, 1981; Pintureau et al., 1991) have been electrophoretitally studied in Sitophilus. Experiments dealing with homogenates of only one individual are still more rare. These studies allow us to analyze the variability rates and to compare the different populations by taking into account their allelic frequencies. Although we only studied a small number of loci in strains that could have been modified by long rearing in the laboratory, the large number of strains of S. oryzae and S. zeamais tested enable the variability of these two species to be compared. It seems that variability is higher in S. zeamais than in S. oryzae, contrary to the preliminary results from Pintureau et al. (1991). The order zeamais > oryzae > granarius was obtained by Beiras and Petitpierre (1981) who analyzed three strains from each Sitophilus species. These authors found lower values than us for genetic variability, because they studied fewer strains and not only analyzed esterases, but also five other enzymatic systems which were monomorphic. Mean heterozygosity values for Sitophilus are in line with those calculated for other Curculionidae. Values obtained for S. zeamais are close to those calculated for three populations of Anthonomus grundis Boheman (Bartlett, 198 l), and values obtained for S. oryzae are close to those calculated for six populations of Hypera postica (Gyllenhal) (Hsiao and Stutz, 1985) and one population of Otiorrhynchus scaber (L.) (Suomalainen and Saura, 1973). Those obtained for S. granarius (only three populations from the same region were tested) are close to those calculated for 17 populations of Pissodes strobi (Peck) (Phillips and Lanier, 1985) six populations of
A. M. GRENIER et al.
210
2q Guad I
l-
q wAA31
q Benin
Maroc Australibl q q W SFRCP Tunisie 1 Guad III
oq Chine
•I Mexique 1
A
%I •1 Tunisie Nas Haiti
1
0
-1
I
I
I
AXIS
2
1
lq W AA31
o Chine
q Tunisic Nas q Mexique 1
[3W 0 z? X a
o-
GuadI
0 •1 Marw SFR 13 0 Guad III
t3 Benin
[3 Australie 1 Haiti 0
0 Tunisie
1 B
-1 -1
I 1
I 0 AXIS
1
Fig. 3. Factorial Correspondence Analysis (FCA) carried out from allele frequencies of 4 loci coding for esterases (Est 2, Est 3, Est 4 and Est 5): dispersion of the 13 strains belonging to Sitophilus oryzae. A: axes 1 and 2;B: axes 1 and 3.
Enzymatic variability in three species of Sitophilus
211
2
A OZMA
1 1 o ApresLagoa
1
Acores SeteCidadcs 8 ACOW.SMI
0 Mexique 5
c-4
VJ
0 Lotus
. d 1. viemam oo Cote lvo~rc
0 Syrie 0
ODE
Cameroun 0 00 Mexique 4 Congo 4 0 Congo 3 0 Guyane o FB Rdunion Congo 1 0 o o Mexique2 Iquitos 0 Gdrard RCunion Burkina-Faso 0
-1 -1
I
I
1
0
1
2
AXIS
1
2-
B
0 GCrard Reunion
l-
I
m v3
a
0 Vietnam
OW
1
Cameroun 0
0
DAX
0 Syrie
0
0
0 Lotus
0 Guyanc 0 C&e d’Ivoire
0 Burkina-Faso 0 Congo 4
Mexique 4
0 Mexique 2
0 Congo3
0 Aqores Lagoa Aqores SM 1
0
0 FB Riunion
8 Congo 1 Iquitos
Apes
0 Sete Cidades
o Mexique 5
- _i
i,
i AXIS
i
1
Fig. 4. Factorial Correspondence Analysis (FCA) carried out from allele frequencies of 3 loci coding for esterases (Est 2, Est 4 and Est 5): dispersion of the 21 strains belonging to Sirophilus zeamais. A: axes 1 and 2; B: axes 1 and 3.
Rhinocylfus conicus Froelich (Unruh and Goeden, 1987) and three populations capita&s De Geer (Suomalainen and Saura, 1973).
of Srrophasomus
This study confirms that esterase patterns can enable distinction of the three Sitophilus species, and that S. oryzue and S. zeamais are closer to each other than to S. granarius. One population
A. M. GRENIER et al.
212
of S. oryzae (Guad I) and one of S. zeamais (FB Reunion) are able to produce a limited number of viable hybrids when crossed together, and notably Guad I is the closest S. oryzae strain to S. zeamais strains in the dendrogram. Esterases also permit some differentiation between populations of a particular species, but the variability is not classifiable according to geographic origin of strains. This situation is probably the result of a high mixture of populations due to trading of cereals all over the world, and perhaps also the result of different pesticide exposures of the strains (esterases are involved in pesticide resistance mechanisms). These results seem to be inconsistent with those obtained by Hsiao and Stutz (1985) and by Unruh and Goeden (1987). In fact, the first authors studying Hypera posticu (from only three geographical origins) by examining 22 loci, found only some of them to differ systematically. The second authors experimenting on seven loci of Rhinocyllus conicus, from only two geographical origins, found allelic frequency differences but no fixed allelic differences. We also found such differences. In S. zeamais, for instance, some alleles characterized certain populations: Est 296in the Iquitos strain, Est 4”’ in Azorean strains, Est 5”*.’ in a Reunion Island strain, and Est 5” in the Vietnam strain. If chemotaxonomic markers do exist among populations of S. oryzae and S. zeamais, it will be necessary to analyze other loci in a large number of populations to find them. are grateful to Yvan Rahbe for his help in computing the dendrogram, and to numerous colleagues who sent us weevils from all over the world. Acknowledgements-We
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Pintureau B., Grenier A. M. and Nardon P. (1991) Polymorphism of esterases in three species of Sitophilus (Coleoptera: Curcuhonidae). J. stored Prod. Am. 27, 141-151. Suomalainen E. and Saura A. (1973) Genetic polymorphism and evolution in parthenogenetic animals. 1. Polyploid Curcuhonidae. Generics 74, 4899508. Unruh T. R. and Goeden R. D. (1987) Electrophoresis helps to identify which race of the introduced weevil, Rhinocyllus conicus (Coleoptera: Curcuhonidae), has transferred to two native Southern California thistles. Enciron. En,. 16, 979-983.