Cytochemical study of Rhodnius neglectus and Rhodnius prolixus salivary gland cells ( Hemiptera , Triatominae )

Triatomines are hematophagous bugs of medical interest in South and Central America, where they may act as invertebrate hosts of the hemoflagellate protozoa Trypanosoma cruzi (the causative of Chagas’ disease) and Trypanosoma rangeli (Tejera, 1920). Triatomines of Rhodnius genus have salivary gland formed by two close and independent units: the principal and the accessory. This gland secretes saliva that abounds in substances that facilitate and permit feeding. Despite this importance, there are few reports on its cytochemistry. In purpose of amplifying this understanding, in this work it was investigated the nuclear structures (chromatin and nucleolar corpuscles) of salivary gland cells of Rhodnius neglectus (Lent, 1954) and Rhodnius prolixus (Stål, 1859). The salivary glands were removed from adult insects, fixed and submitted to different cytochemical methods: lacto-acetic orcein, silver ion impregnation, Feulgen reaction, Toluidine Blue, Variant method of critical electrolyte concentration and C-banding. The results evidenced predominance of binucleated cells, with bulky and polyploid nucleus, decondensed chromatin and a large nucleolar area. In addition, cytoplasmic metachromasy and a clear association between nucleolar and heterochromatic corpuscles were observed. Such characteristics were associated with intense synthesis activity to produce saliva. Besides, the heterochromatic corpuscles observed with C Banding permitted the differentiation of sexes and species.

Triatomines are vector of Trypanosoma cruzi (Chagas, 1909), the etiological agent of Chagas disease.In Latin America, it has been estimated that there are 15-17 million people infected and 90-100 million are exposed to infection (Coura & Borges-Pereira, 2010).Rhodnius genus is also vector of Trypanosoma rangeli (Tejera, 1920), a protozoon transmitted by the bite and apparently harmless to humans.It is pathogenic to the insect vector (HeCker et al., 1990).
Triatomines are hematophagous insects throughout the lifecycle.During the blood feeding (probing and engorgement phases), saliva is released (soares et al., 2006).It is enriched with molecules that antagonize host's hemostatic, inflammatory and immunological responses, facilitating feeding (riBeiro & FranCisCHetti, 2003).This saliva, in association with cryptic behavior, nocturnal habits of triatomines and mechanical adaptations of mouthparts, allows few perceivable bites thus enabling a successful blood meal.
Saliva is produced by a salivary gland formed by two close and independent units: the larger is reddish and elongated (principal unit) and the smaller is round and translucent (accessory unit) (BaPtist, 1941;reis et al., 2003).Ultrastructural analysis showed abundant endoplasmic reticulum, mitochondria and microvilli in the apical plasma membrane domain, thus enlarging the membrane area available for saliva secretion (Meirelles et al., 2003;reis et al., 2003).In addition, an intense acid phosphatase activity was found in nucleolus, suggesting the activity of this enzyme during rRNA molecule transcription (anHê et al., 2007).Recently, roCHa et al. (2010) developed a primary culture of Rhodnius prolixus (Stål, 1859) salivary gland cells.Three types of cells were identified, mostly binucleated.In addition, some secretion vesicles were observed budding from duct cells, suggesting a possible secretory role of this structure.
Despite its importance, the cytochemical characteristics of this gland are little known.So, the purpose of this work is to describe nuclear structures (chromatin and nucleolus) of salivary gland cells of two species of Rhodnius: Rhodnius neglectus (Lent, 1954) and Rhodnius prolixus.Besides, some aspects that allow comparing and differentiating sexes and species are described.

MATERIAL AND METHODS
Salivary glands of R. neglectus and R. prolixus of both sexes were used in this study.They were from Frutal (state of Minas Gerais, Brazil) (R. neglectus) and Colombia (R. prolixus) and were obtained from the Insectary of the Araraquara Special Health Service, organ of the São Paulo University (Araraquara, state of São Paulo, Brazil).Forty newly-emerged adults (twenty males and twenty females) of each species were analyzed.They fed on mice and were dissected seven days after feeding.

RESULTS
The techniques described nuclear structures of salivary gland cells of both sexes of R. neglectus (Figs 1-12) and R. prolixus .Bulky and polyploid nuclei were observed with a predominance of binucleated cells.
In addition, techniques permitted the differentiation of sexes, since just on males a unique heterochromatic corpuscle was observed.Panzera et al. (1992) proposed a way of naming the sex chromosomes according to their C-heterochromatin: the Y is the heterochromatic sex chromosome.Thus, the heterochromatic corpuscle observed just on males, probably, corresponds to the Y chromosome and, so, justify its absence in females.Similar results were found in other species of triatomines.In T. infestans, a heterochromatic body called chromocenter (Mello, 1971(Mello, , 1975) ) is characteristic of males (anHê & azereDo-oliVeira, 2008) and corresponds to the packed three largest autosomes and the sex chromosome (solari, 1979;Panzera et al., 1992;Perez et al., 1997).In P. megistus, just males presented a heterochromatic corpuscle (anHê & azereDo-oliVeira, 2008), which is characteristic of Y chromosome and can also be present in X1 and X2 chromosomes (tartarotti & azereDo-oliVeira, 1999).In addition, C-Banding permitted the differentiation of the species of Rhodnius genus, since just R. prolixus showed a lot of heterochromatic corpuscles.
For both nucleolar and chromatic staining, the technique of Variant of CEC was used (Mello et al., 1993).In this technique, slides were fi rst treated with a Toluidine Blue solution, which stains DNA and RNA in blue (control technique).After, the material was submitted to a magnesium chloride 0.05M solution until the metachromasy of DNA be abolished and it stained in green, while RNA continues bluish.So, the results showed small nucleolar corpuscles (bluish) surrounded by halos of heterochromatin (greenish), which confi rmed association between DNA and RNA, proposed by other techniques utilized.This very close association, classically observed in other tissues and organisms, evidences the functional relationship between these structures, very important to the maintenance of nucleolar architecture (CarMo-FonseCa et al., 2000).Staining with just Toluidine Blue was employed as control  (13,15,16,18) and some on females (l).Magnifi cations: 1344x.
for the technique, when DNA and RNA showed the same metachromatic blue coloration.
Besides, different forms and sizes of nuclei were observed, which could be related to the saliva secretion.According to BartH (1954), there are four to six phases, including transport of substances, secretion of the saliva and cell regeneration.BartH (1954) and laCoMBe (1999) also observed that the insect nutrition changes the cell morphology.To avoid this variable, in this study the insects were dissected seven days after feeding.
Cytoplasmic staining evidenced by the techniques of silver ion impregnation, Toluidine blue and variant of CEC indicated the presence of cytoplasmic RNA (mRNA, tRNA and rRNA).This suggested the high protein synthesis in the gland, necessary to produce saliva.
Thus, these results clearly suggest that polyploid binucleated cells with bulky nuclei, large nucleoli and the high metachromasy in cytoplasm were related to the intense activity of salivary gland cells.These characteristics reflect the high protein synthesis, necessary to the composition of saliva and contribute to a successful blood feeding.In addition, chromatin staining techniques enable to differentiate the sexes and the species.