Molina, M. C.

Reyes, A.

Revistes Catalanes amb Accés Obert

Miscel.lania Zooloaica 20.1 (1997) . 19 
Allozyme variation in a natural 
population of the horse fly 
Haematopota italica Meigen 
(Diptera, Tabanidae) 
A. Reyes & M. C. Molina 
Reyes, A. & Molina, M. C., 1997. Allozyme variation in a natural population of the horse fly 
Haematopota italica Meigen (Diptera, Tabanidae). Misc. Zool., 20.1 : 19-25. 
Allozyme variation in  a natural population o f  the horse fly Haematopota italica Meigen (Diptera, 
Tabanidae).- The species has traditionally been studied morphologically, but no genetic studies 
have been carried out so far. A sample of 24 individuals of a natural population of Haematopota 
italica was analysed for six allozymic loci (Aph-1, Aph-2, Est-1, Est-2, Pgm, Sod). Four out of six loci 
were polymorphic (Aph-2, Est-2, Pgm, Sod). The estimates of genetic variation (heterozygosity, 
polymorphism and mean number of alleles per locus) were among the highest values described for 
hematophagous insect species. The loci Est-2 and Sod presented an excess of heterozygotes, 
possibly due to  selection or to  its association with chromosome inversions. 
Key words: Haematopota italica, Tabanidae, Horseflies, Allozymic polymorphism. 
(Rebut: 24 V 96; Acceptació condicional: 2 6  IX 96; Acc. definitiva: 4 IV 97) 
A. Reyes, Centro Studi sui Mitochondri e Metabolismo Energetico-CNR, Univ. degli Studi di Bari, Via 
Amendola 165/A, 70126 Bari, Italia (Italy).- M.  C. Molina, Dept. de Fisiología Vegetal, Univ. 
Complutense, 28040 Madrid, España (Spain). 
Introduction 
The evolutionary potent ial  o f  a populat ion 
is highly correlated w i t h  its genetic vari- 
ability. The analysis o f  genetic variation can 
therefore be useful i n  determining aspects 
related t o  t h e  structure and dynarnics o f  a 
populat ion ( A Y A ~ ,  1976; DOWNHOWER e t al., 
1987). 
O f  al1 t h e  existing rnethods fo r  studying 
genetic variation, gel electrophoresis has 
been t h e  rnost cornrnonly used. Since its 
introduct ion i n  t h e  sixties (HUBBY & LEWON- 
TIN, 1966; LEWONTIN & HUBBY, 1966). it has 
provided a great deal o f  inforrnat ion about  
the  genetic variability. 
Nevertheless, unt i l  t h e  beginning o f  t h e  
last decade this technique was n o t  applied 
ISSN: 021 1-6529 O 1997 Museu de Zoologia 
Reyes & Molina 
to  those insects considered as agricultura1 
pests (MENKEN & ULENBERG, 1987; LOXDALE & 
HOLLANDER, 1989; REYES & OCHANDO, 1994; 
OCHANDO et al., 1994; REYES, 1995) or hema- 
tophagous insects which affect man and 
domestic animals (MCLAIN et al., 1985; BON- 
NEFOY et al., 1986; TABACHNICK, 1992; HARRY et
al., 1992; FRENCH et al., 1995). 
Little genetic attention has been paid to  
biting hematophagous species, included in 
the family Tabanidae, even though they 
can transmit a great deal of infectious dis- 
eases, including those involving viruses. Few 
surveys have been published to  date based 
on morphology (CHVA'LA et al., 1972; ELGER 
et al., 1980; PORTILLO, 1984). ecology 
(IASAKOVA, 1974; FOMINYKH & EREMINA, 1984) 
or cytogenetics (GRINCHUK, 1969; BOYES &
WILKES, 1972). 
In this paper, starch gel electrophoresis 
was used to  analyse allozyme variation in a 
natural populat ion o f  the  horse f ly  
Haematopota italica, a hematophagous spe- 
cies that bites both man and domestic ani- 
mals. The aim of the present work was to 
obtain the first estimates of genetic vari- 
ability in horse flies and compare these 
with the values reported for other insect 
species. 
Material and methods 
A sample of 24 females of Haematopota 
italica Meigen (Diptera, Tabanidae) was 
used. The capture was carried out at feed- 
ing time in the late summer in 1992 in 
Atajate (Málaga-Spain), situated at 1700 m 
above sea level and with a typical Mediter- 
ranean climate. 
Horseflies were captured alive, classified 
and immediately frozen and stored at - 20°C 
until analysis. ' 
Protein extraction of each fly was car- 
ried out in 50 p1 Tris-HCI 0.1M pH 7.1, 0.1 % 
Triton X-100, and the electrophoretic con- 
ditions for the 12% starch gel were accord- 
ing to  the methodology currently used for 
insect species (AYALA et al., 1972; REYES, 
1995). Eight randomly chosen allozymic sys- 
tems, were assayed: APH. Alkaline phos- 
phatase; EST. Esterase; HBDH. Hydroxibuty- 
rate dehydrogenase; HK. Hexoquinase; IDH. 
lsocitrate dehydrogenase; MDH. Malate 
dehydrogenase; PGM. Phosphoglucomutase; 
SOD. Superoxide dismutase. Four of the 
eight systems showed a good resolution 
with our running conditions (APH, EST, PGM 
and SOD) and were retained for further 
analysis. 
Genotypic and allelic frequencies, such 
as mean heterozygosity (H), polymorphism 
(P) and mean number of alleles per locus 
(A), as well as deviations from Hardy-Wein- 
berg equilibrium expectations, were calcu- 
lated based on the banding patterns. 
Results 
After running the electrophoresis, one 
horsefly was found to  present a very differ- 
ent banding pattern. It was assumed to 
belong to  a different species and was ex- 
cluded from the analysis. 
The schemes of the zymograms with the 
different banding patterns found in the 
remaining 23 horse flies, and for the four 
retained enzyme systems, are shown in fig- 
ure 1. It can be seen that there is a single 
Pgm locus, as well as one Sod locus. How- 
ever, for Aph and Est there are two differ- 
ent loci; one of which is monomorphic and 
the other is polymorphic. 
The enzymes of the Aph-2 , Est-2, Pgm 
and Sod loci could have a monomeric struc- 
ture as shown in figure 1. The different 
genotypes can thus be simply identified by 
the nurnber and running position of differ- 
ent bands (alleles). However, PGM could 
have postranslational modifications or non- 
charged aminoacid variation within each 
electromorph as more than one band i s  
observed in the homozygotes. The Aph-1 
and Est-1 loci do not provide information 
about the structure of the enzyme as they 
are monomorphic. 
The information about the genotypes of 
each fly for the four polymorphic loci is 
presented in table 1. Genotypic and allelic 
frequencies are shown in table 2. The loci 
Aph-2 and Sod presented four genotypes 
and three alleles, while in Est-2 and Pgm 
three genotypes and two alleles were found. 
The estimates of genetic variation in this 
population for the four analysed loci were: 
mean heterozygosity, H = 0.3696, propor- 
tion of polymorphic loci, P = 0.6667, and 
Miscel.lania Zooloqica 20.1 (1997) . 21 
APH EST 
PGM SOD 
Fig 1. Different banding patterns found for  each enzyme in the 23 analyzed horse 
flies o f  Haematopota Italica. Different alleles and genotypes for  each polymorphic 
locus are shown. 
Patrones de bandas encontrados para cada enzima en las 23 moscas de caballo 
analizadas, correspondientes a Haematopota italica. Se muestran los diferentes 
alelos y genotipos para cada locus polimórfico. 
mean number o f  alleles per locus, A = 2. 
Deviations from the expectations under 
Hardy-Weinberg equilibrium for  hetero- 
zygosity values are presented in table 3. 
Two loci are in equilibrium (Aph-2 and Pgm). 
In the other two, Est-2 and Sod, the ob- 
served heterozygosities are significatively 
higher than expected under equiiibrium 
conditions. 
Discussion 
The first estimates o f  genetic variation in  
the horse f ly Haematopota italica are pre- 
sented. 
The genotypic and allelic variability 
found in H. italica, referred as the number 
of different genotypes or alleles in the poly- 
morphic loci (table 1). is comparable t o  
that  found in other hematophagous spe- 
cies such as Aedes aegypti (TABACHNICK & 
POWELL, 1978), Lutzomyia longipalpis (Bo- 
NNEFOY e t  al., 1986), different species of 
Rhodnius (HARRY et  al., 1992), Cuclioides 
variipennis (TABACHNICK, 1992) or  Lucila 
caprinus (FRENCH et  al., 1995). Moreover, 
when compared wi th non-hematophagous 
species from the same geographical area 
(Atajate-Málaga) such as Dacus oleae (OCHAN- 
DO et al., 1994) or Ceratitis capitata (REYES, 
1995), no  important differences were found, 
except in the case o f  the Sod locus, which 
presented three alleles in  H. italica while 
only one was detected in C. capitata. 
Regarding the estimates o f  genetic vari- 
ation, the values o f  mean heterozygosity, 
degree o f  polymorphism and mean number 
Reyes & Molina 
Table 1. Observed genotypes for the 
four polymorphic loci in the 23 horse 
flies belonging t o  the Haematopota 
italica species. 
Genotipos observados para los cuatro 
loci polimórficos en las 23 moscas del 
caballo pertenecientes a la especie 
Haematopota italica. 
Genotypes 
Indiv. Aph-2 Est-2 Pgm Sod 
1 BB BB BB AB 
o f  alleles per locus in  this population of H. 
italica were H = 0.3696, P = 0.6667 and 
A = 2, respectively. In other hematophagous 
species, these parameters range from the 
low values found in Rhodnius, H = 0.0600, 
P = 0.2200, A = 1.2 (HARRY et al., 1992) and 
the high values o f  A. aegypti, H = 0.3524, 
P = 0.75, A = 3.0 (TABACHNICK & POWELL, 1978). 
Thus, the values obtained for H. italica are, 
wi th those described for A. aegypti, the 
highest values reported for hematophago.~~ 
species. When compared wi th non hema- 
tophagous species f rom the same geo- 
graphical area, it has been shown that ge- 
netic variability is higher than that found 
in C. capitata, H = 0.0966, P = 0.4000, 
-A = 1.74 (REYES, 1995) while in comparison 
wi th D. oleae only heterozygosity was found 
t o  be higher, H = 0.1322, P = 0.7727, A = 2.6 
(OCHANDO et al., 1994). 
The high value o f  mean heterozygosity 
found in  this population of H. italica, as 
well as that described for A. aegypti (based 
on four polymorphic loci), is probably an 
overestimate o f  the real value, as it has 
been shown that the higher the number of 
loci under consideration, the lower the value 
of this parameter, approaching i t s  real value 
(SINGH & RHOM.BERG, 1987). Thus many more 
loci must be analysed in order t o  obtain a 
more  re l iable estimate o f  t h e  mean 
heterozygosity in this species. 
The analysis of the single locus hetero- 
zygosities in each o f  the four polymorphic 
loci is presented in table 3. Two loci, Aph-2 
and Pgm, did not show statistically signifi- 
cant deviations from the Hardy-Weinberg 
equilibrium. This could mean that such loci 
are not  under selection pressure or that 
there are mutually compensed forces, for 
example, selection versus migration, selec- 
t ion versus mutation, or even antagonic 
selective effects (MARINKOVIC & AYALA, 1975a, 
1975b). In other Diptera species, these loci 
have shown t o  be related t o  fitness under 
similar circumstances, and are conse- 
quently subject t o  selection (MARINKOVIC & 
AYALA, 1975a, 1975b). In contrast, Est-2 and 
Sod loci showed significant deviations from 
the Hardy-Weinberg equilibrium, caused by 
an excess o f  heterozygotes. This also occurs 
in other species, such as Drosophila, due t o  
a developmental homeostatic effect or t o  
overdominance (DANZMANN et al., 1985). I t  i s  
also possible that these loci are located 
within a chromosomic inversion. This i s  a 
common phenomenon in  Dipteran species, 
and leads t o  deviations from the Hardy- 
Miscel.lania Zoologica 20.1 (1997) 2 3 
Table 2. Observed genotypic and allelic frequencies in the four polymorphic loci o f  
Haematopota italica. Designation o f  genotypes and alleles are shown in figure 1 and 
table 1. 
Frecuencias genotípicas y alélicas observadas en los cuatro loci polimórficos de 
Haematopota italica. La designación de los genotipos y alelos se realizó en base a la 
figura 7 y la tabla 7. 
Genotypes Alleles 
Locus AA AB B B B C A B C 
Aph-2 0.1739 0.1739 0.6087 0.0435 0.2609 0.7174 0.021 7 
Est-2 0.0435 0.6956 0.2609 0.3913 0.6087 
Pgm 0.2609 0.4348 0.3043 0.4783 0.5217 
Sod 0.0435 0.7827 0.0869 0.0869 0.4348 0.521 7 0.0435 
Weinberg equilibrium as well as t o  higher 
values o f  heterozygosity than those ob- 
tained for  loci outside the inversions (SINGH 
& RHOMBERG, 1987) (table 3). Nevertheless, 
as no cytogenetic study has yet been car- 
Table 3. Chi-square comparisons between 
observed and expected heterozygosities 
under Hardy-Weinberg equilibrium for 
the four polymorphic loci: 0. Observed; 
E. Expected; * p<0.05; *** p<0.01. 
Comparación mediante la prueba 
del Ji-cuadrado entre la heteroigosidad 
observada y la esperada en condiciones 
de equilibrio de Hardy- Weinberg en 
los cuatro loci polimórficos analizados: 
O. Observados; E. Esperados; * p <  0,05; 
*** p < 0,Ol. 
Locus O E Statistic value 
ried out  in H. italica it is not  possible t o  say 
whether inversions are common in  this spe- 
cies or not. 
In conclusion, the analysis o f  six allozymic 
loci in H. italica has revealed high values o f  
genetic variability, w i t h  an excess of 
heterozygotes in  t w o  loci. This i s  pos- 
sibly explained by selection or association 
wi th chromosome inversions. 
Acknowledgements 
We are grateful t o  Prof. Pilar Arana, Prof. 
María Dolores Ochando and Prof. Vicente 
Córdoba for valuable comments on the 
manuscript, Prof. Francisca Salom for classi- 
fying the species and t o  Jonathan for revi- 
sion of the English text. 
Resumen 
Variabilidad aloenzimática en una población 
natural de la mosca del caballo Haematopota 
italica Meigen (Diptera, Tabanidae) 
Aph-2 0.2174 0.4168 3.7833 n.s. Los estudios llevados a cabo hasta la fecha 
Est-2 0.6957 0.4764 4.4295 * en las diferentes especies de la familia 
Tabanidae se han centrado básicamente en 
Pgm aspectos morfológicos, siendo escasos o 
Sod 0.8696 0.5369 10.2337 *** inexistentes los enfoques de t ipo genético. 
En el presente trabajo se ha analizado la 
Reyes & Molina 
variabilidad genética de una población na- 
tural  de Haematopota italica mediante 
electroforesis en gel. A partir de los patrones 
de bandas de cuatro sistemas aloenzimá- 
ticos: APH, EST, PGM y SOD (fig. 1) en cada 
individuo de la población (tabla 1) se calcu- 
laron las frecuencias genotípicas y génicas 
para cuatro loci polimórficos (tabla 2), se 
estimaron parámetros de variabilidad genéti- 
ca como hetericigosidad, polimorfismo y nú- 
mero medio de alelos por locus, y se compro- 
bó la existencia de equilibrio de Hardy- 
Weinberg en los loci polimórficos (tabla 3). 
Los resultados muestran que los valores 
de variabilidad genética obtenidos para esta 
especie se encuentran entre los más altos 
descritos para especies de insectos hema- 
tófagos, especialmente la heterocigosidad; 
observándose un exceso de heterocigotos 
en los loci Est-2 y Sod que podría ser expli- 
cado por selección o por su asociación con 
inversiones cromosómicas. 
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Allozyme variation in a natural population of the horse fly Haematopota italica Meigen (Diptera, Tabanidae)

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