Eye Pigments of the Blood-sucking Insect, Triatoma Infestans Klug (hemiptera, Reduviidae)

The pigmentation of black (wild) and red (mutant) eyes of Triatoma infestans was studied spectro-photometrically and compared with red-eyed (wild) and white-eyed (mutant) forms of Drosophila melanogaster. The spectral absorption profiles of the black and red eye pigments of T. infestans were similar to each other and to that of the wild-type eyes of D. melanogaster. The similarity to the wild form of D. melanogaster indicated that both eye forms of T. infestans contained ommochromes of the xanthommatin type, a finding confirmed by ascending paper chromatography. Pteridines, mela-nins, and ommins were not detected as eye pigments in T. infestans. The eye color difference in T. infestans was assumed to be a function of the xanthommatin concentration, with a smaller content of ommochrome in red eyes, although this probably did not affect the insect's visual acuity. These data support other findings regarding the similarities between black-and red-eyed specimens of T. infestans for other characteristics. A pigmentação de olhos pretos (forma selvagem) e vermelhos (forma mutante) de Triatoma infestans foi estudada por espectrofotometria e comparada à de olhos vermelhos (selvagem) e brancos (mutante) de Drosophila melanogaster. Os perfis do espectro de absorção dos pigmentos de olho preto e vermelho de


INTRODUCTION
Eye color in Triatoma infestans is genetically controlled at a single autosomal locus in such a way that black eyes are produced by the dominant gene (wild) and red eyes by the homozygous recessive gene (mutant) (Wygodzinsky & Briones, 1954;Dujardin & Bermúdez, 1986).No significant differences in oviposition or egg hatching rates have been associated with the parental eye-color phenotypes in this species (Pires et al., 2002).Although no impairment of mating attractiveness or mating success has been related to the red-eyed color, it has been suggested that sperm cells from red-eyed males have a lower viability compared to those of black-eyed specimens, thus explaining why red-eyed mutants of T. infestans are uncommon in natural populations (Pires et al., 2002).
At the cellular level, nuclear phenotypes in the Malpighian tubules of red-eyed T. infestans do not differ from those of black-eyed individuals, at least under full nourishment conditions (Gonçalves et al., 1997).
Although investigations have dealt with redeyed mutants of T. infestans, no information has up to now been available on the nature of the pigments in the red and black eyes of this species.The only reduviid hemipteran in which the eye pigments have so far been studied is Rhodnius prolixus for which, based on paper chromatography, the presence of ommins and xanthommatin has been suggested (Butenandt et al., 1960;Linzen, 1974).On the other hand, there are numerous studies of eye pigmentation in dipterans that have demonstrated the occurrence of ommins, ommatins, and pteridines, and their relevance in mutations, insect physiology, and evolution, particularly in Drosophila species (Ephrussi & Herold, 1944;Cordeiro, 1959;Rasmuson et al., 1960;Fuge, 1967;Connolly et al., 1969;Stark & Wasserman, 1974;Nash & Henderson, 1982;Pfeiderer, 1992;Lewin, 2000).The typical ommatin in this case is xanthommatin (Linzen, 1974).
In this work, we used spectrophotometry to study the pigmentation of black and red eyes of T. infestans.Since fluorescence characteristics of the eye pigmentation was not the primary focus of the present study, they were were not assessed.

MATERIAL AND METHODS
Black-eyed (wild) and red-eyed (mutant) adult males of Triatoma infestans Klug (1834) (Hemiptera, Reduviidae) were used.The insects were reared at 30ºC and 80% RH in a laboratory at Superintendência de Controle de Endemias (Sucen), Mogi-Guaçu (São Paulo State).Adults were used because the pigment content of their eyes was greater.
The black-eyed specimens, which were from colonies reared at Sucen beginning in 1975, originated from domestic insects collected in the states of São Paulo (north) and Minas Gerais (south), Brazil.The colonies have periodically been crossed with specimens collected in southern Minas Gerais.The red-eyed colony originated from specimens collected in Argentina and was homozygous recessive for eye color mutation.
Red-eyed wild forms and white-eyed mutant adults of Drosophila melanogaster, strain Canton S, kindly provided by Dr. Louis Bernard Klaczko (Unicamp, Campinas) were used as standards.In the white mutant of D. melanogaster, the synthesis of ommochrome and pteridines is affected (Linzen, 1974).
Forty eyes of T. infestans (20 wild and 20 mutant specimens) and 200 eyes of D. melanogaster (100 wild and 100 mutant specimens) were used for each assay.The eyes were initially treated, overnight at 25 o C, with 5 ml of 30% ethanol acidified to pH 2.0 with concentrated HCl (AEA).This procedure extracts pteridines.The eyes were then removed and placed in a second solution consisting of 5 ml of absolute methyl alcohol acidified with concentrated HCl to 1% (v/v) (AMA).This procedure, based on Ochando's (1981) modification of the method described by Ephrussi & Herold (1944), extracts ommochrome pigments after 24 h at 25 o C. All extractions were done in a dark room to avoid changes in the spectral properties of the extracted pigments.
Spectral absorption curves were obtained for the eye pigment solutions using an HP8452A diode array spectrophotometer (Roseville, CA, USA) over the wavelength range of 200 through 700 nm.The spectral profiles were compared with each other and with those reported by Ephrussi & Herold (1944) and Ochando (1981) for D. melanogaster.
Since treatment with acidified methanol extracts ommochromes and melanins, and since the latter are not soluble in concentrated sulfuric acid and HCl (Ajami & Riddiford, 1971), a pigment extract was obtained with these acids in order to assess whether melanins were present in T. infestans eyes.
To confirm the conclusions resulting from the analysis of the spectral absorption profiles, eye pigment solutions were also subjected to ascending chromatography on Whatman n. 1 paper, using npropanol-ammonia (5%) (2:1, v/v) as the solvent.The chromatograms were run at room temperature in the dark (Brown & Nickla, 1977) until the solvent front was approximately 15 cm from the point of sample application.The spots were detected using a short wavelength (254 nm) UV lamp (LF215S, UVItec Limited, Cambridge, UK) and their R f were calculated.

RESULTS
Pigments were not extracted from T. infestans eyes when ethanol was used as the solvent, thus excluding the presence of drosopterins (Ephrussi & Herold, 1944).However, when pigments were obtained with acidified methanol, which extracts ommochromes and melanins (Ephrussi & Herold, 1944), pigments were revealed.The treatment of T. infestans eyes with concentrated sulfuric acid resulted in total extraction of eye pigments.Since such a procedure does not extract melanins (Ajami & Riddiford, 1971), these results indicate that ommochromes are the pigments present in both black and red eyes of T. infestans.
The absorption spectra of the eye pigments extracted from T. infestans and D. melanogaster with acidified methanol are shown in Fig. 1.Comparison of the absorption profiles of the ommochromes present in the eyes of both species revealed similarities.As expected, based on wellcharacterized ommochrome absorption profiles (Schäfer & Geyer, 1972;Linzen, 1974 -review), four peaks were evident: A (λ = 210-225 nm), B (λ = 265-275 nm), C (λ = 350-360 nm), and D (λ = 440-450 nm).These peaks were not well defined in the curves for black-eyed T. infestans, but they were for red-eyed D. melanogaster, whose ommochromes belong to the xanthommatin family (Linzen, 1974), and for red-eyed T. infestans.Indeed, the black eye pigmentation of T. infestans is characterized by decreased absorption at region D and enhanced absorption in region C, a typical response of concentrated ommochromes to the H 2 O 2 present in the AMA extractive solution (Ochando, 1981) (Fig. 1).
Chromatography of the pigments extracted from black and red eyes of T. infestans and from red eyes of D. melanogaster showed a main spot (R f = 0.15) close to the point of sample application.Weakly absorbing spots with higher R f values were observed in the material from the three sources and were assumed to be pigment precursors or metabolic by-products.

DISCUSSION
The extraction properties of the T. infestans black and red eye pigments and the similarity of their spectral absorption profiles with those of eyes from wild D. melanogaster under identical experimental conditions indicated the presence of ommochromes and absence of melanins in T. infestans.Since xanthommatin is the ommochrome occurring in wild D. melanogaster eyes, we concluded that xanthommatin also occurs in both black and red eyes of T. infestans.The chromatographic data supported this hypothesis (Brown & Nickla, 1977).
The inability of acidified ethanol to extract eye pigments excluded pteridines as a component of the eye pigments in T. infestans, unlike what happened in the case of D. melanogaster (Cordeiro, 1959;Rasmuson et al., 1960;Fuje, 1967;Connolly, 1969;Pfeiderer, 1992).
The lack of an absorption peak at λ = 520 nm or 547 nm in the spectral absorption profile of the eye pigments of T. infestans indicated that ommins may not contribute to the ommochromes involved (Linzen, 1974).This conclusion differs from the paper chromatography results for Rhodnius prolixus, which suggested that ommins were the main ommochrome in the eyes of this species (Butenandt et al., 1960;Linzen, 1974).
Assuming that black and red eye pigments in T. infestans are the same, the red color may be caused by the smaller pigment amount.Indeed, the eye pigment of wild T. infestans changes from blackbrown to red and then to yellow as the pigment extract is diluted.The lower absorption in the λ = 440-450 nm region (peak D), concomitant with higher absorption in the λ = 350-360 nm region (peak C) only in the spectral profile of pigments from black T. infestans eyes, is typical of redox properties of the ommochrome pigment (Schäfer & Geyer, 1972).Assuming that in T. infestans the ommochrome concentration in black eyes is higher than in red eyes, the H 2 O 2 concentration required to completely stabilize the redox state should not be the same for both eye types.
The reaction generates molecules with peculiar solubilities, redox properties, and spectral characteristics (Linzen, 1974).Ommochrome pigments, including xanthommatin, arise from kynurenine precursors synthesized in organs such as Malpighian tubules and fat bodies during insect development.Kynurenine has been detected microspectrofluorimetrically in the Malpighian tubules of T. infestans (Mello & Vidal, 1985).With the development of the insect optical system, the precursors of eye pigments discharged from the insect's hemolymph are captured by the developing eyes and transformed and deposited in pigment granules (Shoup, 1966;Colombo et al., 1973;Wigglesworth, 1984).Xanthommatin and other ommochromes are synthesized only in the eyes and in epidermal cells (Colombo et al., 1973).
Mutations leading to less eye pigmentation reflect changes in the metabolism of tryptophan or  in the transit of ommochrome pigment precursors to the eye (Wigglesworth, 1984; http:// flybase.bio.indiana.edu).However, in the case of red-eyed T. infestans, this may be not disadvantageous since ommochromes are still present although at a much lower level.The visual acuity of the compound eye has been reported to be reduced when ommochromes are lacking (Linzen, 1974).
In summary, these findings further support the similarities between black-and red-eyed specimens of T. infestans already demonstrated for other characteristics (Gonçalves al., 1997;Pires et al., 2002).

Fig. 1 -
Fig. 1 -Spectral absorption profiles of pigments extracted from T. infestans and D. melanogaster eyes.White mutant and wildtype D. melanogaster were used as negative and positive controls, respectively, for xanthommatin.