Confalonieri, VA

Saidman, BO

Vilardi, JC

English

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VA Confalonieri

JC Vilardi

BO Saidman

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Esterase variation among Argentine populations of Trimerotropis pallidipennis (Orthoptera)

Original article
Esterase variation
among Argentine populations
of Trimerotropis pallidipennis
(Orthoptera)
VA Confalonieri, JC Vilardi, BO Saidman
Universidad de Buenos Aires, Faculdad de Ciencias Exactas y Naturales
Departamento de Ciencias Bioldgicas, I128 Buenos Aires, Argentina
(Received 4 January 1989; accepted 10 March 1990)
Summary - Isozyme studies were carried out in 3 populations of Trimerotropis palli-
dipennis from Tucumin and San Luis Provinces (TU, SLi and SL2) (Argentina). The
purposes of this study were: determination of the numbers of genes and alleles involved
in the production of the isozymes and their frequencies, detection of diagnostic loci for
population identification and measuring the genetic variability and the degree of genetic
differentiation among populations. With this aim, the esterase (EST) isozyme system was
studied and 6 loci were analysed (Est 1 to Est 6). The EST patterns allowed population
samples from different biogeographic provinces to be distinguished. Qualitative differences
between populations were observed in the expression of some loci (Est 1, Est 2 and Est
3) and the presence of additional alleles for other ones (Est 5 and Est 6), characterizing
TU, were also detected. Therefore, this differentiation among populations refers to both
gene expression and allelic frequencies which may represent 2 distinct responses to envi-
ronmental differences. Possible correlations of genetic and chromosomal variation are also
discussed.
orthoptera / isozyme / esterase / Trimerotropis / genetic variation
Résumé - Variabilité des estérases dans des populations argentines de Trimerotropis
pallidipennis (Orthoptères). Trois populations de Trimerotropis pallidipennis situées dans
les provinces de Tucumdn et San Luis (TU, SLl et SL2) (République argentine) ont été
étudiées afin de déterminer le nombre de gènes et d’allèles responsables de la synthèse
des isoenzymes estérasiques, d’identifier les locus pouvant caractériser des populations, et
d’évaluer la variabilité et le degré de différentiation génétique entre les populations. Six
locus probables (Est 1 à Est 6) ont été reconnus. Les enzymogrammes estérasiques ont
permis de distinguer les échantillons des différentes provinces biogéographiques, tandis que
ceux de la même province ne pouvaient pas être distingués. Il existe, entre les populations,
des différences qualitatives dans d’expression des locus Est 1, Est 2 et Est 3 et ainsi que
dans la présence d’allèles différentiels aux locus Est 5 et Est 6. Cette différence entre les
populations se manifeste aussi bien dans l’expression des gènes que dans les fréquences
aldéliques, ce qui pourrait représenter 2 types de réponses adaptatives. Les corrélations
entre la variabilité génétique et chromosomique sont discutées.
orthoptère / isozyme / estérase / Trimerotropis / variabilité génétique
INTRODUCTION
Grasshopper species of the genus Trirn,erotropis are of interest from an evolutionary
genetics standpoint because they show chromosomal variation with respect to
the position of the centromere (White, 1973), usually considered as produced by
pericentric inversions (Hewitt, 1979; John, 1983).
Most of the species of this genus inhabit arid regions from Western North
America; T pallidipennis is one of the few Trimerotropines to have successfully
extended its distribution to Andean South America (White, 1973). Argentine
populations studied are polymorphic for pericentric inversions (Mesa, 1971; Vaio et
al, 1979; Goni et al, 1985; Confalonieri, 1988; Confalonieri and Colombo, 1989).
Goiii et al (1985) observed for this species a geographical pattern of chromoso-
mal polymorphisms associated with neither phytogeographical nor climatic charac-
teristics. Clinal variation along an altitudinal gradient of these polymorphisms was
recently found (Confalonieri and Colombo, 1989). There is, therefore, a need for a
more exhaustive ecological approach. This requires a parallel study of chromosomal,
genetical and ecological variation.
In spite of the known usefulness of isozymal studies to population genetics, only
a few studies (Moran et al, 1980; Gill, 1981; Halliday et al, 1983; Nevo et al, 1984;
Chapco and Bidochka, 1986) have been performed dealing with allozyme variation
in grasshopper species.
In this work, isozyme studies were carried out in T padlidipennis with the pur-
poses of: a) estimating the number of responsible genes and their allelic frequencies;
b) detecting diagnostic loci for populations; c) determining genetic variability and
the degree of differentiation between populations; d) correlating genetic and chro-
mosomal variation.
This paper reports the results obtained from the study of the esterase (EST)
isozyme system in 3 populations of T pallidipennis.
MATERIALS AND METHODS
Samples of adult grasshoppers were collected from TucumAn (TU) and San Luis
(SL, and SL2, Republica Argentina. TU is located at Amaicha del Valle (2 040 m
above sea level); SL, is situated on the National Road No 7, km 792.5, and SL2
on the same road at km 816 (560 and 440 m above sea level respectively). The
populations sampled belong to 2 different biogeographic provinces of the Chaco
Dominion, Neotropical Region (Cabrera and Willink, 1973). TU is in the Prepuna
while both SL populations are situated in the Chaco Biogeographic Province (fig 1).
Twenty-four individuals from TU, 29 from SL1 and 20 from SL2 were collected.
After removal of the testes for chromosomal analysis, the grasshoppers were frozen
and stored in liquid air until enzyme assays were done.
Extracts were obtained from the eviscerated head and thorax. Each individual
was thoroughly homogenised in approximately 0.6 ml of 0.1 mol/1 Tris-HCI, pH 7.1.
The homogenate was centrifuged at 7 000 rpm for 10 min in a refrigerated centrifuge.
The electrophoresis was conducted in 7% polyacrylamide horizontal gels accor-
ding to the technique of Cordeiro (1974). The system of gel and tray buffers was
discontinuous Poulik, pH 7.9, modified by Schaal and Anderson (1974).
Staining techniques are described by Cladera (1981). Different substrates (esters)
were used to characterize the different bands: a-naphthyl acetate (a-NA), ,Q-
naphthyl acetate (/3-NA), a-naphthyl laurate (a-NL), a-naphthyl butyrate (a-
NB), a-naphthyl oleate (a-NO), a-naphthyl propionate (a-NP) and naphthol AS-D
acetate (AS-D).
The interpretation of the genetic variation in electrophoretic patterns is inferen-
tial according to Saidman and Naranjo (1982).
Bands are labelled in capital letters and numbered according to decreasing
electrophoretic mobilities. Loci are named with an initial capital letter followed by
lowercase letters and numbered according to decreasing mobilities of isoenzymes.
Alleles are named with the symbol of their loci and an index according to decreasing
mobilities of allozymes.
RESULTS
Considering the 3 populations, 17 anodic bands were observed but only 12 (EST
1 to EST 12) could be analysed (fig 2).
The results obtained with different substrates are presented in table I. Bands
could be grouped according to their ability to react with different esters: EST 1 -
EST 2 - EST 3; EST 4 - EST 5; EST 6; EST 7 - EST 8; EST 9; EST 10 - EST
11; EST 12. The probable loci and alleles implicated in the genetic determination
of bands were inferred taking into account the combinations of the differential
substrate a!nities.
EST 1 and EST 2 are invariable and may be produced by 2 monomorphic loci:
EST 1 and EST 2 (fig 2). EST 3 and EST 4 show similar affinities and migrate very
close to each other. Three kinds of patterns were observed: EST 3, EST and EST
3 with EST .!. On these grounds the bands were considered as allozymes coded by
1 locus with 2 alleles, EST 31 and EST F respectively.
EST 1 band was never observed in the TU population, and the expression of
bands EST 2 to EST l in this sample was very poor or absent, showing some
degree of individual variation.
EST 5, EST 6 and EST 7 are also considered as allozymes. They appeared in
the following combinations: EST 5; EST 6; EST 7; EST 5 with EST 6; EST 5 with
EST 7; EST 6 with EST 7 (fig 2). Since the EST 7 band is at a low frequency, it is
absent in the individuals depicted in figure 2a; in any case, its relative mobility is
also indicated in 2b. The corresponding alleles were named; EST l!l, EST 42 and
EST 43.
EST 8, EST 9 and EST 10 react similarly to a- and !i-NA. EST 9 and EST
10 also gave similar staining affinities with the rest of the substrates employed.
Because of its low frequency, EST 8 was absent in the individuals assayed with
the latter substrates, and therefore its affinities could not be confirmed. Despite
this fact, these results along with the combinations observed (EST 8; EST 9, EST
10; EST 8 with EST 9; EST 8 with EST 10; EST 9 with EST 10; absence of
bands) may correspond to those expected for a locus with 4 alleles, 3 codominants,
EST 51,EST 5’z, EST 5! and 1 null EST 5°. The latter in a homozygous condition is
characterized by the absence of bands. All phenotypes are shown in figure 2 except
the combination EST 8 - EST 10 which appeared in other gels not depicted in this
figure.
EST 11 and EST 12 varied showing the following combinations: EST 11;
EST 12; EST 11 with EST 12. They were considered as produced by 1 locus with
2 alleles: EST 61 and EST 61 (fig 2). The bands corresponding to EST 6 could not
be properly ascertained in 1 individual from TU and therefore it was excluded from
the analysis of this locus.
Five additional bands, 4 between EST 4 and EST 5 and 1 between EST 10 and
EST 11 were also found. Their respective affinities differentiated them from those of
EST 1 to EST 12, but their patterns were not constant enough to be unequivocally
analysed.
Assuming that this genetic interpretation of bands is correct, phenotype and/or
genotype frequencies for each locus were determined (table II). Then, allelic
frequencies were estimated by counting or, in the case of the EST 5 locus which has
a null allele, by the method of Neimann-Sorensen (1956) (table III). In addition,
mean frequencies of heterozygotes per locus were also estimated and are indicated
in table III.
From the 6 loci analysed, 4 displayed qualitative differences between TU and
both SL populations. Thus, EST 1, EST 2 and EST 3, clearly expressed SL, and
SL2, were either absent or almost non detectable in TU. EST 6 was polymorphic
in the latter and monomorphic in the other 2 populations.
The allelic frequencies at EST 3, EST 4 and EST 5 were compared among
populations by means of contingency tables (table III). Allelic frequencies of
EST 3 from both SL populations were not statistically different and, at the EST
4 locus, differences were also not significant when all populations were considered
(though in this case the X2 for SL1 vs SL2 was significant at the 5% level). However,
EST 5 locus significantly differentiates TU from both SL populations. This is due
to the fact that EST 5°, the most frequent allele in SLI and SL2, was absent in
TU. Also, the EST 52 frequency was high in TU and low in the other samples.
The average expected heterozygosity was also similar for SL, and SL2 (about
0.24) and clearly different from that of TU (0.505) (table III).
In all populations, observed genotypic frequencies were compared with Hardy-
Weinberg expectations by means of chi-square tests for the loci EST 3, EST 4 and
EST 6 (table II). These statistics were further applied only to the data of EST 5
from TU because some classes were too small or absent in the other samples. In
every case differences were not significant.
DISCUSSION
The genetic interpretation of bands studied was inferential, but the hypotheses
put forward agree with the fact that in all cases where statistical comparisons could
be performed, observed genotypic frequencies fit very well with those expected
according to the Hardy-Weinberg law.
Since 5 of the 6 loci analysed showed marked differences in either gene expression
on allelic frequency, esterase zymogrames could be used to identify populations from
different (but not from the same) provinces.
An additional difference between TU and SL populations is the level of variability
of these loci. In the former, H has more than twice the value it has in the latter.
The virtual lack of expression of EST 1 to EST 3 loci in TU may be explained
by different (but not necessarily mutually exclusive) hypotheses:
- 1) Mutations can produce changes in the amino acid sequence of polypeptide
chains affecting the specific activity (null alleles or alleles with a very slight
expression).
- 2) Lower amounts of enzymatic molecules with the same structure are possibly a
consequence of:
a) a regulatory gene;
b) different factors in the environment which produce the activation (or inacti-
vation) of certain regulatory gene(s);
c) the existence of chromosome inversions fixed in this population that silence
these loci by position effects;
The first hypothesis implies that alternative alleles for 3 out of 6 loci analysed
are fixed in different populations. However, the most common situation among
conspecific populations is that 1 allele is the most frequent in almost all of them
(Lewontin, 1979). Besides, this hypothesis seems to be untenable according to the
parsimony principle.
The hypothesis 2a implies a mutation affecting only a single gene (controlling
EST 1 to EST 3 loci) which seems to be a simpler and more likely explanation,
though 2b and 2c are also acceptable.
The hypothesis 2b is plausible because esterases are enzymes frequently acting
on external substrates proceeding from ingestion. The precise plant species that
constitutes this grasshopper’s food in Argentina is not known. Possible candidates
are annual plants associated with species of the genus Larrea (creosote bush) (Otte
and Joern, 1975; Wallner, 1987). As TU and both SL populations are located in
different phytogeographic provinces, it is expected that the food supply exploited
will not be the same. In fact, L divaricata is widely distributed over the arid and
semiarid regions in Argentina, but morphological differences are readily observed
between plants .at different altitudes. This may be a consequence of both genetical
and physiological adaptations (Hunziker, personal communication). The plants
associated with the creosote bush which constitue T paddidipennis food are also
expected to adapt to the corresponding environment. Therefore, distinct enzyme
expression between the orthopterans feeding upon them is not surprising.
The hypothesis 2c is based on the high frequency of inversion polymorphisms and
chromosomal differentiation among T pallidipennis populations (Vaio el al, 1979;
Goni et al, 1985; Confalonieri, 1988; Confalonieri and Colombo, 1989). A simi-
lar kind of differentiation between the populations reported here was also evident
when chromosome investigation was carried out (Confalonieri et al, submitted).
The 3 populations were polymorphic for 3 centric shifts which involved 4 medium
chromosomes; when they were statistically compared, differences with respect to
inversion frequencies were highly significant among all populations except SL ones.
Moreover, some chromosome arrangements were at high frequencies in TU and ab-
sent or at very low frequencies in SLI and SL2, respectively. Therefore, these results
are in agreement with the last hypothesis (2c) because if 2 populations have diffe-
rent fixed inversions, or are of very different frequencies, position effects are not an
unexpected phenomena. Finally, inversion frequencies from 15 populations (inclu-
ding TU, SL, and SL2 from Argentina) were correlated in a very significant fashion
with altitude (Confalonieri and Colombo, 1989; Confalonieri, in preparation), sug-
gesting the action of natural selection in the maintenance of these polymorphisms.
Therefore, as TU and both SL populations have very different inversion frequen-
cies, most probably as a result of living at different altitudes, their chromosomal
variation might also be related to genetical differentiation.
ACKNOWLEDGMENTS .
The authors wish to express their sincere gratitude to H Hunziker for his
guidance, constant encouragement and critical reading of the manuscript and to
PC Colombo for his generousity in providing material from Tacumin. We also
thank the anonymous referees for their valuable suggestions. The financial support
from Consejo Nacional de Investigaciones Cientificas y T6cnicas, Secretaria de
Ciencia y T6cnica (Argentina) through grants to H Hunziker and A Naranjo is
also acknowledged.
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Esterase variation among Argentine populations of  (Orthoptera)

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Esterase variation  among Argentine populations  of Trimerotropis pallidipennis  (Orthoptera)

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Vilardi JC

Confalonieri VA

Saidman BO

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Genetics Selection Evolution

Esterase variation among Argentine populations of <it>Trimerotropis pallidipennis </it>(Orthoptera)

Insect territoriality and its evolution: Population studies of desert grasshoppers on creosote bushes.

The role of chromosome change in the evolution of orthopteroid insects. In: Chromosomes in evolution of eukariotic groups,

Willink A

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Esterase variation among Argentine populations of Trimerotropis pallidipennis (Orthoptera)

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