versión On-line ISSN 0717-7712
Parasitol. latinoam. v.58 n.1-2 Santiago ene. 2003
Parasitol Latinoam 58: 49 - 53, 2003 FLAP
Effects of blood ingestion on patterns on the chorion of
eggs of Lutzomyia ovallesi (Diptera: Psychodidae)
Chorion patterns on eggs of Lutzomyia ovallesi fed on seven types of blood were studied using scanning microscopy. The results showed that the different blood sources produced no differences in the patterning. The eggs had a chorion with sinuous longitudinal bridges and a few transverse bridges that formed rectangular shapes. These results confirm that chorion patterns on the eggs of sand flies are a useful taxonomic parameter.
Key words: Lutzomyia ovallesi, blood ingestion, egg chorion, scanning microscopy.
Chorion patterns on sand flies eggs are significantly different between species and have been proposed as a taxonomic tool for species identification and classification, since this feature makes it possible to distinguish between species that are morphologically very similar.1-8 On the other hand, have been found intraspecies differences in the exochorion of L. gomezi which were explained as the result of geographical differences.9 Also exists intraspecies variations in the exochorion of Phlebotomus pedifer, P. aculeatus and P. martin which made it difficult to use this feature for species identification.10 The same phenomenon was recorded in females of L. gomezi in Colombia and Brazil.11 These differences were produced by microenviron-mental factors and require further study to determine precisely what factors produced them.10 The morphological differences in the chorion between species are due to specific adaptation to the environmental conditions of sites where eggs were laid and the small intraspecies differences in L. verrucarum are associated with the geographical isolation of colonies in different Andean valleys.8 The intraspecies differences between colonies of L. gomezi in Panama-Colombia and Venezuela-Brazil and colonies of L. verrucarum result from isolation due to geographical barriers and separation.5
Since the factors producing variations in chorion patterns in the same species of sand flies are still unknown, and since blood is a determining factor in egg production and maturation, it seemed possible that these variations might be due to different types of blood. Thus the present work is a scanning microscopy study of the chorion patterns on eggs of Lutzomyia ovallesi fed on seven different types of blood.
MATERIALS AND METHODS
Females of L. ovallesi were used. These came from a laboratory colony established with specimens collected in El Arenal, Ejido, in the state of Merida, Venezuela at 1 360 mts. a.m.s.l. Following the techniques described previously,12 the colony is kept in an incubator at 25°C ± 1° with a relative humidity of 80% ± 10% in the Parasitology Laboratory, "José F. Torrealba", in the University of Los Andes, Mérida, Venezuela.
Blood from healthy animals was collected in citrate tubes, with 0.2 ml of citrate per ml of blood. Blood sources were horse (Equus caballus), chicken (Gallus domesticus), pig (Sus scrofa domestica), cow (Bos taurus) and goat (Capra hircus). Human (Homo sapiens sapiens) and dog (Canis familiaris) blood was also used.
Two-day-old females were used, artificially fed to repletion using chicken membrane. They were kept in an incubator at 25°C ± 1° with a relative humidity of 80% ± 10%, with 12 hours of light and 12 hours of darkness. As a dietary supplement they were given a 50% fresh saccharose solution, renewed daily.
Chorion patterns on the eggs
Three-day-old L. ovallesi eggs were used. These came from different females of each group fed on the blood of 7 different hosts. The eggs were washed for 2 minutes in 7.2 pH phosphate buffer at 4° C. They were then fixed in glutaral-dehyde following the method described pre-viously.5
After washing, the eggs were fixed for 2 hours at 4°C in glutaraldehyde in 0.1M 7.2 pH phosphate buffer. They were then washed in 7.2 pH phosphate buffer at 4° C after fixing, for 1 hour in a 1% osmium tetroxide solution in a 7.2 pH phosphate buffer at 4° C. They were washed in a phosphate buffer and dehydrated in an alcoholic series (30%, 50%, 70%, 80%, 90%, 95% and 100%), and then exposed to the air to dry overnight. They were coated with gold (Au) in an SPI ion coater for 90 seconds at 18 mAmp and at a pressure of 0.3 mbar.
Observation and photography
The chorion of the eggs was viewed and photographed using a Hitachi scanning electron microscope (S-2500). Observations were made to determine the values for the sides of the polygonal cells from the exochorion of eggs of L. ovallesi fed on the blood of 7 vertebrate hosts and one-way variation analysis (ANOVA) was used.
The chorion had sinuous longitudinal bridges and a few transverse bridges that formed rectangular shapes. This pattern was the same for each one of the eggs of L. ovallesi fed on the different types of blood in the study.
Table 1 shows the average values for the sides of the polygonal cells from the exochorion of eggs of L. ovallesi fed on the blood of 7 vertebrate hosts. Variation analysis (g.1 6 and 37) no shows significant differences (p = 0.316).
The chorion of insect eggs functions as protection for the embryo from the environment, preventing desiccation and regulating gaseous interchanges. These are some of the functions associated with the structure of the chorion, which may also have a taxonomic value.4 Chorion patterns in sand flies have only been studied in a limited number of species due to the difficulty of finding eggs in the natural state and to the difficulty of establishing laboratory colonies.5,7,10
Chorion patterns on eggs of L. ovallesi fed on blood from 7 different hosts were within the parameters described for this species, with sinuous longitudinal bridges and a few transverse bridges that formed rectangular shapes.5,7
There were no significant differences in the average values of the polygonal cell sides of the exochorion of L. ovallesi.
These results would seem to indicate that blood source does not modify chorion patterns in L. ovallesi; the experiment should be repeated for those species were differences have been observed between different colonies.
The chorion is secreted by cells in the follicular epithelium when eggs are laid; it is viscous, allowing the egg to adhere to the substrate. When dry, the chorion assumes its characteristic patterns.4 8 The genital atrium of phlebotomines change shape as eggs passed through.13 On the other hand, the shape and size of the genital atrium in 17 species of sand flies found in Venezuela is a constant feature of individuals from the same species and diffe-rentiates them from individuals of other species.14 In the same way, spines on the genital armature can be used as a classification feature. This suggests that the parameters of chorion patterns are mainly the result of the shape and size of the genital atrium and spines, features dependent on genetic information in sand flies. For Lutzomyia spp. chorion patterns are a useful taxonomic tool.
Se estudio la escultura del corion de los huevos de Lutzomyia ovallesi alimentadas con siete tipos de sangre utilizando microscopia de barrido, los resultados no muestran diferencias estructuras del corion de los huevos de L. ovallesi alimentadas con las distintas fuentes sanguíneas estudiadas, presentando los huevos un corion con puentes longitudinales sinuosos con pocos puentes que se cruzan formando áreas rectan-gulares, estos resultados confirman el uso de la escultura del corion de los huevos de los flebotominos como un buen parámetro taxonómico.
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Acknowledgements: This work was supported by CDCHT (the Scientific, Humanistic and Technological Development Council) (C-1082-01-03-C), and to CONICIT (the National Scientific and Technological Research Council) (S1-2000000818). Our thanks to: Maritza Rondón and Carlos Araque, the personnel of Chemical Analysis and Material Structure Laboratory); Profs. Efrain Entralgo, Guillermo Bianchi, Paolo Ramoni and Prof. Leida intero.
* Laboratorio de Investigaciones Parasitológicas "J.F. Torrealba", Departamento de Biología, Facultad de Ciencias, Universidad de Los Andes. La Hechicera, Mérida - Venezuela. 5101.
** Laboratorio de Parasitología. Departamento de Biología. Facultad de Ciencias Universidad de Los Andes. La Hechicera, Mérida- EDO-Mérida, 5101. Venezuela. Fone: 74 2 712565 - FAX: 74 2 401286, e-mail:email@example.com