GENETICA

INTERSPECIFIC DIFFERENCESREVEALED WITH

IN DROSOPHILAPHOTOMETRIC ANALYSIS

R. F. ROENIGSBERG,A. J. CHEJNEand E. BAUTISTA

Instituto de Cenetica, Universidad de los Andes, Bogota, D. E., Colombia

1. INTHODUCTION

The compound eye of Drosophila has been subject to considerablehistological work (Nolte, 1950, 1954; Hertweck, 1931). Hadom and colla-borators (Hadorn and Mitchell, 1951; Radorn and Kurtsteiner, 1955; Ha-dorn and Ziegler.Gunder, 1958) have worked on the chemical basis of thepigmentary system using mutants of Drosophila. Mainx (1938) distinguish-ed two pigments by way of their solubility in water. Later on the work inthis field was greatly simplified because the chromo gens could be extract-ed from the heads of the flies with ethanol acidified with RCI at ph 2.0(Ephrussi and Herold, 1944). The red and the brown pigment, can alsobe separated during the ontogenetic formation of the pupae (Danneel,1941). Another line of research produced results on the action of eye co-lour genes with the spectrophotometer (Nolte, 1952). With this tool, lightextinction differences were soon found between some species (Nolte, 1958).Therefore, it appeared desirable at this point to use the pigment extractionmethod and the spectrophotometric measurement of the pigment in solu-tion in order to analyse the racial affinities among members of the samespecies and the interspecific differences in three important species groupsof the genus Drosophila.

2. MATEHIALS AND METHODS

Several experiments were conducted to study the concentration of thered pigments in the eye of members of the genus Drosophila. The exper i-ments were repeated three times, and the standard errors of the meanswere recorded. Although these methods should be considered satisfactory

116 CALDASIA, VOL. IX, NO 42 DICIEMBRE 21 DE 1964

the results which they procured will be regarded as tentative. The authorshope that other laboratories will repeat these experiments in the hope thatother results will correct errors and vindicate the ones here presented.

The photometric analysis of the various species were carried out witha Beckman DU spectrophotometer (tables 1 and 2) and with a Colemanphotometer Ior the experiments on intraspecific differences within Dro-sophila melanogaster (tables 3 and 4). In the V.V spectrum a H2 lampwas u ed. The visible, from 320 mu to 560 mu, was analysed with a tung-sten lamp.

The red pigments were extracted with an acidified alcohol solution.Concerning the extraction method some relevant facts have to be discussed.The two differential solvents for the red and for the brown pigments arethose first used by Clancy (1942). They were later utilized by Ephrussiand Herold and were named AEA and AMA. The first solvent is preparedas a 3070 ethyl alcohol acidified at ph 2.0 with concentrated HCl. We con-firm the stability of this solvent first realized by Clancy. Regardless of itsstability, AEA was prepared every time an experiment was ready to com-rnence,

The extraction procedure for the red pigment was as follows: the flieswere decapitated with a clean razor blade at room temperature and 5 daysafter emergence. Four milliliters of the solvent were prepared and 20 headswithout proboscis and clypeus were placed in the solvent after they driedfor three minutes. No doubt this method is time consuming, however, itproved to have more merit than the other methods tried in that no turbi-dity formed in the extracts. Besides, our procedure permitted full extrac-tion after 24 Ius. while the other methods recommended by the litera-ture, require 8 or more days. The simplicity of the methods used in manylaboratories consists in simply cutting off heads and placing them in thesolvent. We should like to stress violent shaking during the full 24 Ius. ofextraction at 22°C. All photometric readings were taken in the next 48 hrs,after extraction.

The most practical way to a quantitative determination of pigmentsis by measuring the light absorbed in solutions which contain the pig-ments. This is particularly so in the regions of the maximum absorptionsince there we find the most characteristic wave lengths of the substances.

The Lambert-Beer Law says that light absorption is directly propor-tional to the concentration of the solute and to the depth of the absorbingmedium. However, since in the experiments we used cuvettes of uniformdepth and kind, reason permits to ignore this variable. All our results aregiven in optical density units (in the figures these units are x 102) : Elog 10 / I with 10 being the incident light and 1 the transmitted light.

HOENIGSBERG ET AL.: INTERSPECIFIC DIFF. IN DROSOPHILA 117

Another very relevant variable is temperature. We soon found that itinfluences the amount of red pigments withing the species: it is well knownthat the relative amounts of the two red components in certain mutantsvary in direct relation to temperature (Ephrussi and Herold, 1945). Be-sides, the total amount of pigments depend on the size of the eyes andconsequently on the size of the fly. Furthermore, larvae and pupae deve-lopment depends on temperature for their duration. The size of the flyis affected by the duration of its development. Therefore, it is desirable tobreed all the herds, stocks and species under constant temperatnre: ther-mostatic control of incubators was necessary to maintain the temperatureat 25°C. Moreover, constant size is also influenced by other culturing cha-racteristics: the density of yeast fermentation per individual, adult crowd-ing, density of larval population, humidity, bacterial and fungal infection(Hodson and Chiang, 1948). It was necessary to control humidity to 609'1.-and to achieve optimum size of adults by an adequate supply of food forthe larvae by introducing a sufficient amount of live Fleishman yeast sus-pended in water (Bakker, 1961), with a bacterial and fungal inhibitorcommercially called Tegocept (Goldschmidt Co., New York). The mediapreparation was modified to meet our special requirements: for every1.000 cc. of water, 35 grams of Agar-agar, 16 bananas and 20 cc. of Tego-cept. The Tegocept was a 10% solution of 75% ethyl alcohol. Every halfpound of medium contained 1 cc. of Fleishman yeast which was added10 hrs, after the culture medium was freshly prepared, and 24 hrs. beforethe individuals were introduced in order to have fermentation started.

To have cultures with 200 larvae, 5 females freshly brought from na-ture were placed with 3 males from the same locality, properly labelled,for 48 hrs. to produce 250 eggs during that time. The oviposition is ap-proximately constant and uniform if males and females are placed onfresh media for the first 6 days of imaginal life in the laboratory. Popul-ations developing under these conditions do not exceed 180 individuals perculture. After six generations under such conditions body size and conse-quently eye size show little variation.

Individuals resulting from such cultures were used for the extractionof pigment. Removal of eggs due to excessive laying was necessary in somecases. Usually, however, the number of flies varied from 150 to 180 perculture medium.

3. RESULTS AND DISCUSSION

Brevity advises us to recommend the reader direct examination oftables and figures. Each species presents its own characteristic spectro-

llB CALDASIA, VOL. IX, N° 42 DICIEMBRE 21 DE 1964

graph, although there are intraspecific fluctuations. In some cases the dif-ferences are present in the U.V part of the spectrum only (females of D.melanogaster and of D. ananassae) ; while in other cases the differencesare evident in the visible spectrum as well as in the U. V. Interspecificsimilarities coincide with philo genetic affinities, while racial similaritiesare indicative of the common genetic pool all these populations share,

The authors call attention to the widely different habitats from whichthe races of D. melanogaster came. What affected the survival and the reoproduction of the individuals from the low lands should be totally diffe-rent in intensity, at least, from the circumstances that affected the indi-viduals living in the caribbean island of San Andres: therefore, the tworadically different environments bave almost certainly elicited differentgenetic structures (Dobzhansky, 1951). We are defining environment asthe sum of circumstances that may influence a population in its survivaland in its multiplication (Andrewartha, 1963). In the South Americancontinent a) weather (light, humidity, pressure, temperature) b) foodc) predators d) pathogens and e) peculiar conditions of the terrain aredifferent from the serni-desertic hot northern coast (Soledad, Caracoli-cito and Santa Marta) to the Simi valley (Monteria: hot humid, dense tro-pical forest) that adaptive peaks, genetic-wise, must have produced intime different genotypes within the same Mendelian pool. D. melanogaster,D. aruuuissae, Ii.pseudoobscura, D. repleta, Ir.nebulosa and D.willistoni res-pond so differently in eye pigmentation to these same environments thattheir photometric differences are much greater than those between mem-bers of the same species. The racial dissimilarities are many time smallerthan the interspecific ones. At the same time there are species which differless: melanogaster and ananassae on one hand, and nebulosa and w~llistonion the other hand. These similarities at the interspecific level reveal theirreputed philogenetic kinship.

4. SUMMARY

The above refer to experiments present a new method which permitsthe study of philo genesis in the genus Drosophila. There are several typesof results: a) close kinship among the various geographical races of D.melanogaster in the neo-tropics coincides with their spectrophotometricsimilarities; b) the interspecific differences are also identified with thephotometric analysis; c) finally there are optical density affinities amongthe various species which belong to the same taxonomic groups.

Acknowledgment. The authors want to express their gratitude to Pro-fessor Everet of the physico-chemical laboratory of the National University

HOENIGSBERG ET AL.: INTERSPECIFIC D1FF. IN DROSOPHILA 119

for the use of his Beckman DU spectrophotometer and for his generousadvice. This research is supported by thc American Agricultural ResearchService grant F. G. Co 107. For technical assistance we are indebted toMiss B. I. Cortes and to Mr. L. Castro.

5. REFERENCES

ANDREWARTHA, H. G. 1963. - Introduction to the Study of Animal Populations. TheUniversity of Chicago Press.

BAKKER, K. 1961. - An analysis of factors which determine success in competition forfood among larvae of Drosophila melanogaster. Arch. Neerlandaises de Zoologie.14, 200·281.

CLANCY, C. W. 1942. - The development of eye colors in Drosophila melanogaster,Further studies on the mutant claret. Genetics, 27, 417-440.

DANNEEL, R. 1941. - Die Ausfarhung uberlebender v und en . Drosophila. Augen mitProdukten des Tryptophanstoff wechsels. BioI. Zentr., 61, 388·398.

DOBZHANSKY, T. 1951. - Genetics and the Origin of the Species. 3d. ed. ColumbiaUniversity Press.

Ernaussr, B., and HEROLD, J. L. 1944. - Studies of eye pigments of Drosophila. I. Me-thods of extraction and quantitative estimation of the pigment components. Cene-tics, 29, 148-175.

EPHRUSSI, B., and HEROLD, J. L. 1945. - Studies of eye pigments of Drosophila. II. Ef-feet of temperature on the red and brown pigments in the mutant blood (w b I) •

Genetics, 30, 62-70.HADORN, E., and MITCHELL, H. K. 1951. - Properties of mutants of Drosophila mela-

nogaster and changes during development as revealed by paper chromatography.Proc. Nat. Acad. Sc., 37, 650·665.

HADORN, E., and KURTSTEINER, R. 1955. - Unterschiede in Exkretstoffen bei verschie-denen Genotypen von Drosophila melanogaster. Arch. Julius Klaus Stift-Verer-hungsforsch. Sozialanthropol. u. Rassenhyg., 30, 494·498.

HADORN, E., and ZIEGLER·GUNDER,I. 1958.-Untersuchungen zur Entwicklung, Geschlechts·spezifitiit und phanogenetischen Autonomie der Augen-Pterm verschiedener Ceno-typen. Z. indukt. Ahstamm. u. Vererb.v Lehre, 89, 221·234.

HERTWECK, H. 1931. - Anatomie und Variabilitiit der Nervensystems und der Sinnesor-gane von Drosophila melanogaster (Meigen) . Z. Wiss. ZooI., 139, 559·663.

HODSON, A. C., and CHIANG, H. C. 1948. _. A new method for rearing Drosophila. Sci-ence, 107, 176·177.

MAINX, F. 1938. Analyse der Genwirkung durch Factorenkomhination. Versuche mitden Augenfarhenfaktoren von Drosophila melanogaster, Z. indukt. Ahstamm. u.Vererh .• Lehre, 75, 256·276.

NOLTE, D. J. 1950. - The eye pigmentary system of Drosophila: The pigment cells. J.Genet., 50, 79·99.

NOLTE, D. J. 1952. - The eye pigmentary system of Drosophila: II. Phenotypic effectsof gene comhinations. J. Genet., 51, 130·141.

NOLTE, D. J. 1954. - The eye pigmentary system of Drosophila: IV. The pigments ofthe vermilion group of mutants. J. Cenet., 52, 111·126.

NOLTE, D. J. 1958. - Eye pigment relationships in the species groups of Drosophila.Evolution, 12, 519-531.

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