©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 7 (2): 326-335 (2008) Cytogenetic analysis in the spermatogenesis of Triatoma melanosoma (Reduviidae; Heteroptera) V.B. Bardella, M.T.V. Azeredo-Oliveira and E. Tartarotti Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho”, IBILCE/UNESP, São José do Rio Preto, SP, Brasil Corresponding author: M.T.V. Azeredo-Oliveira E-mail: tercilia@ibilce.unesp.br Genet. Mol. Res. 7 (2): 326-335 (2008) Received October 30, 2007 Accepted January 10, 2008 Published April 15, 2008 ABsTRACT. Triatomines are of great concern in public health because they are vectors of Chagas’ disease. This study presents an analysis of the species Triatoma melanosoma. The cytogenetic characteristics of triatomines include holocentric chromosomes, post-reductional meio- sis in the sex chromosomes and nucleolar fragmentation in the meiotic cycle. The methodology utilized consisted of the techniques of lacto- acetic orcein staining and silver ion impregnation. The organs analyzed were adult testicles. The results enabled to classify the chromosomes by number and size, being three large, eight medium and one small hetero- chromosome. The three largest chromosomes and the heterochromo- somes showed heteropyknotic chromatin in meiosis. The heterochro- mosomes in 8.05% of the cells in metaphase I behaved as pseudobiva- lents, contrasting with 91.95% of the cells with individualized sex chro- mosomes, confirming the achiasmatic nature of these chromosomes. However, the pseudobivalents occurred prominently in metaphase II (78.38%), this fact probably is related to the post-reductional nature of the sex chromosomes. The nucleolus in T. melanosoma persisted until the diplotene phase after which it began to fragment. Nucleolar corpus- cles were observed in metaphases I and II and during anaphases I and II, these characteristics being related to the phenomenon of nucleolar 327 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 7 (2): 326-335 (2008) Cytogenetic analysis in the spermatogenesis of T. melanosoma persistence. In the initial spermatids, peripheral silver ion impregnation occurred, which could be analogous to the pre-nucleolar corpuscles ob- served after fragmentation. Thus, this study extends our knowledge of the characteristics of triatomines, in particular, heteropyknotic degree, kinetic activity, formation of sex chromosome achiasmatic pseudobiva- lency, confirmation of the fragmentation phenomenon, and post-meiotic nucleolar reactivation. Key words: Triatomines; Cytogenetics; Meiosis; Nucleolus; Holocentric chromosomes; Spermatogenesis InTROduCTIOn Chagas’ disease is an endemic parasitosis with a pronounced importance among heart diseases in South America (Tartarotti et al., 2004). This disease affects more than 11 million people and occurs in Mexico and Central and South America (Dias et al., 2002). On the American continent, Chagas’ disease is one of the main endemic diseases after malaria (Tartarotti et al., 2004). The etiologic agent of Chagas’ disease is the protozoan Trypanosoma cruzi, and the transmission of this disease occurs by contact with the feces of the triatomine contaminated with the protozoan (Coll-Cárdenas et al., 2004). However, the disease may also be transmitted congenitally and by blood infection (Tartarotti et al., 2004). The triatomines are divided into six tribes: Alberproseniini, Bolboderini, Cavernicoli, Linshcosteini, Rhodniini and Triatomini, totaling 137 species (Galvão et al., 2003). Within the Triatomini tribe, in the Triatoma genus, there is the Triatoma melanosoma species. This species is distributed throughout the extreme north of Argentina, in the province of Misiones. Initially, it was described by Martinez and collaborators (1987) as a T. infestans subspecies, and as a result it was denominated T. infestans melanosoma (Lent et al., 1994). This classifica- tion originated from the erroneous conception of T. melanosoma as a dark form of T. infestans. T. melanosoma is now recognized to be a species (Lent et al., 1994). In cytogenetic terms, male triatomines have a diploid number of chromosomes varying from 21 to 25, and the typical form of the group is 2n = 22 (Ueshima, 1966). The chromosomes of these insects are holocentric and have an unusual meiotic segregation (De Vaio et al., 1985): the autosomes are chiasmatic and pre-reductional, while the sex chromosomes are achiasmatic and post-reductional (Solari, 1979). The heterochromo- somes, during metaphase II, alternatively behave as pseudobivalents. The chromosomal size is usually homogeneous in the triatomines, but there are some exceptions: T. infes- tans, T. platensis, T. rubrovaria and T. pseudomaculata. These species have from one to three chromosomes larger than the others and are heteropyknotic in conventional staining (De Vaio et al., 1985; Pérez et al., 1992). In triatomines, and in other insects, studies of nucleolar behavior have revealed the number of nucleoli in the spermatocytes and their fragmentation during the meiosis prophase I, as well as nucleolar reactivation at the end of spermatogenesis (Warchalowska- Śliwa and Maryanska-Nadachowska, 1992; González-García et al., 1995; Tavares and Azeredo-Oliveira, 1997; Tartarotti and Azeredo-Oliveira, 1999; Morielle and Azeredo- 328 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 7 (2): 326-335 (2008) V.B. Bardella et al. Oliveira, 2004). Another objective of cytogenetic study is the location of nucleolar or- ganizing regions (NORs), observed in some autosomes and/or heterochromosomes. In triatomines, nucleolar fragmentation occurs during prophase I between diplotene and di- akinesis. Nucleolar corpuscles are also observed in the metaphases, together with the nucleolar reactivation that occurs in anaphase II, which are both phenomena typical of triatomines. Usually, these nucleolar corpuscles become smaller as the spermatids mature (Tavares and Azeredo-Oliveira, 1997; Tartarotti and Azeredo-Oliveira, 1999; Morielle and Azeredo-Oliveira, 2004). The aim of the present study was the characterization of the meiotic cycle with an emphasis on nucleolar activity in T. melanosoma, in order to contribute to a better under- standing of the chromosomal behavior and the phenomenon of nucleolar fragmentation in Triatominae. MATERIAl And METHOds The species analyzed was T. melanosoma (Heteroptera: Reduviidae). For the study, 15 specimens were used. The insects were provided by the Special Health Service of Ara- raquara, State of São Paulo, Brazil, belonging to the Department of Epidemiology of Uni- versity of São Paulo (USP). The seminiferous tubules of adult males were submitted to the cytochemical techniques of lacto-acetic orcein (De Vaio et al., 1985, with modifications) and silver staining (Howell and Black, 1980). The slides were examined with a Zeiss- Jenaval photomicroscope and the photomicrographs were made on a 400-ASA film. REsulTs lacto-acetic orcein The polyploidy nuclei of testicular tubule nutritive cells showed dispersed hetero- pyknotic corpuscles (Figure 1A). The spermatogonial metaphases showed chromatin bridges and heteropyknosis at the chromosome ends (Figure 1B). During the diffuse stage (prophase I), in progressive degrees of chromosome condensation, the heteropyknotic regions showed peripheral or central dispositions (Figure 1C,D). In the diplotene, the presence of terminal and interstitial chiasmata was observed in the bivalent autosomes and achiasmatic sex chromosomes. The heteropyknosis of three autosomes and at least one sexual chromosome was observed (Fig- ure 1F,G). In the diakinesis, there was typical chiasmatic terminalization of the autosomes (Fig- ure 1H). In metaphase I (Figure 1I), the sex chromosomes were visualized alternatively in the pseudobivalent (Figure 1J) or individualized form (Figure 1k). The pseudobivalent presence or absence was analyzed by means of cellular score. In metaphase I, 91.95% of the cells dis- played individualized heterochromosomes, and in metaphase II, 78.38% of the observed cells were in the pseudobivalent configuration (Table 1). In metaphase II, the heterochromosomes were again observed to be either individualized (Figure 1L,M) or pseudobivalent (Figure 1N). During anaphase, parallel migration, typical of holocentric chromosomes, was observed and the late migration of chromosomes was verified (Figure 1O-q). In spermiogenesis, dots were observed on the periphery of early spermatids (Figure 1R,S). During differentiation the dots reduced to the formation of small points at the beginning of the spermatozoids (Figure 1T). 329 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 7 (2): 326-335 (2008) Cytogenetic analysis in the spermatogenesis of T. melanosoma silver ion impregnation The nucleolar cycle was observed in the cells of the seminiferous tubules during spermatogenesis (Figures 2 and 3). The polyploidy nuclei of the nutritive cells of the testicular tubules showed several nucleolar corpuscles; in addition, the polyploidization phase of this cell was observed (Figure 2A-C). The spermatogonial cells displayed intense Figure 1. Seminiferous tubule of Triatoma melanosoma stained by lacto-acetic orcein. A. Polyploidy nucleus with dispersed heteropyknotic corpuscles (arrows). B. Spermatogonial metaphase with chromatin bridges joining the chromosomes (arrow). C,d. Diffuse stage with peripheral chromocenter, see arrows. E. Diffused stage showing chromocenter alternatively in central position (arrow). F. Diplotene, observe heteropyknosis in the three largest autosomes (arrows) and at least one heterochromosome (arrowhead). G. Final diplotene stage, note the typical chiasmatic figures, stressing the achiasmatic behavior of the sex chromosomes. H,I. Diakinesis, note the chiasmatic finalization, and above all pseudobivalent heterochromosome behavior. J,K. Circular metaphase I, note achiasmatic sex chromosomes in the center (arrows). l,M. Metaphase II, note the three largest autosomes with a greater degree of pyknosis (arrowheads) and individualized sex chromosomes (arrow). n. Circular metaphase II with sex pseudobivalent (arrowhead). O. Anaphase I, note late chromosome migration (arrow). P,Q. Anaphase II, note parallel chromosome migration. R,s. Spermatids, note peripheral heteropyknotic corpuscles (arrows). T. Spermatids in final elongation stage. Presence of pseudobivalent Absence of pseudobivalent Metaphase I 7 (8.05%) 80 (91.95%) Metaphase II 58 (78.38%) 16 (21.62%) Table 1. Presence of sex chromosomes as pseudobivalents in meiotic metaphases. 330 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 7 (2): 326-335 (2008) V.B. Bardella et al. nucleolar activity (Figure 2D). The cells in spermatogonial metaphase demonstrated no specific staining but showed chromatin bridges between the chromosomes (Figure 2E-G). The spermatogonial interphasic cells showed an evident nucleolus, reflecting the gene ac- tivity of these nuclei (Figure 2H). In the initial diffuse stage, dispersed nucleolar staining was observed throughout the nucleus, together with the presence of the nucleolus (Fig- ure 2I-k). Still in the diffuse stage, it was possible to observe NORs associated with the nucleolus and the beginning of chromosome condensation (Figure 2L). In the diplotene- diakinesis, nucleolar staining was visualized (Figure 3A), and in the advanced diakinesis stages, it was possible to relate the NORs to three autosomes and one heterochromosome (Figure 3B,C). In diakinesis, nucleolar fragmentation was also observed (Figure 3D). In metaphase, as well as in anaphase, the chromosomes became intensely compacted which made it difficult to analyze the staining. However, it was possible to observe small nucleo- lar fragments inside the cell (Figure 3E-H). Still in metaphase II, the behavior of pseudo- bivalents in the heterochromosomes was observed (Figure 3G). The spermatids, at the beginning of spermiogenesis, contained one corpuscle, suggesting post-meiotic nucleolar activity, possibly related to the differentiation process (Figure 3I). During the differentia- tion process of the spermatids, the corpuscles decrease and fragment (Figure 3J), until the spermatozoids formed (Figure 3k). Figure 2. Seminiferous tubules of Triatoma melanosoma stained by silver ion impregnation. A. Polyploid nuclei in early polyploidization. B,C. Polyploid nuclei in progressive ploidy stages, note the great number of nucleolar corpuscles (arrow). d. Spermatogonial nuclei with nucleolar activity (arrow). E-G. Spermatogonial metaphases with chromatin bridges (arrows). H. Primary spermatocyte with evident nucleolus. I-K. Initial diffused prophase stage, note nucleoli (arrows) and nucleolar corpuscles (arrowheads). l. Final diffused stage, note chromatin associated with nucleolar region (arrow). 331 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 7 (2): 326-335 (2008) Cytogenetic analysis in the spermatogenesis of T. melanosoma dIsCussIOn The study of spermatogenesis in T. melanosoma, in association with the information in the literature, has made it possible to observe chromosome characteristics and meiotic be- havior in Heteroptera. The polyploidy nuclei of T. melanosoma displayed several dispersed heterochromatic corpuscles. The study of polyploidy nuclei in other triatomines has revealed the existence of only one heterochromatic corpuscle as a frequent occurrence in these insects (Tavares and Azeredo-Oliveira, 1997; Tartarotti and Azeredo-Oliveira, 1999; Morielle and Azeredo-Oliveira, 2004). The adult males of T. melanosoma showed a diploid number of chromosomes 2n = 22 (20A, XY), corroborating the study of Panzera and collaborators (1996). In the species analyzed, the chromosomes were classified in the metaphase II according to size; thus, they were denominat- ed as three large, eight medium and one small, the last corresponding to one of the heterochromo- somes. As mentioned above, in male triatomines the diploid number of chromosomes varies from 21 to 25 chromosomes (Ueshima, 1966); however, it is believed that 60% of the species analyzed possess a diploid number of chromosomes 2n = 20A + XY (Tavares and Azeredo-Oliveira, 1997). Figure 3. Seminiferous tubule of Triatoma melanosoma stained by silver ion impregnation. A-C. Diplotene-diakinesis with nucleolar organizing regions evidenced in at least three autosomal bivalents (arrows) and one heterochromosome (arrowhead). d. Diakinesis, note the complete nuclelolar fragmentation. E. Early anaphase I, note the persistence of nucleolar corpuscles (arrows). F,G. Ring metaphase II with sex pseudobivalent at the center (arrow), note the nucleolar persistence (arrowhead). H. Anaphase II, note chromosome showing late migration (arrow). I. Spermatid in early differentiation, notice silver ion impregnation in the periphery (arrow). J. Spermatids in elongation phase, note peripheral nuclear stain in opposition to the area of flagellum formation. K. Spermatozoid agglomeration. 332 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 7 (2): 326-335 (2008) V.B. Bardella et al. In T. melanosoma the three largest autosomes of the diploid complement, together with the heterochromosomes, were heteropyknotic. In the triatomines, little variation in chromosome size is usually observed (De Vaio et al., 1985). However, the species T. infestans, T. platensis, T. rubrovaria, and T. pseudomaculata have between 1 and 3 chromosomes larger than the others, appearing heteropyknotic with conventional staining (De Vaio et al., 1985; Pérez et al., 1992). The chromosomes of T. melanosoma are characterized as holocentric based on migra- tion pattern, late migration and lack of primary constriction. These characteristics are typical of the Heteroptera and of the Homoptera, Lepdoptera and Trichoptera orders (Wolf, 1996). In Hemiptera after high dosages of radiation, a series of chromosome structural rearrangements occur; however, the complete elimination of the chromosomes or fragments, due to a faulty inclusion in the telophase, has rarely been observed (Hughes-Schrader and Schrader, 1961). In holocentric chromosomes, the microtubules of the spindle interact with the whole extent of the chromatin and do not have a localized centromere (Pérez et al., 1997). Atomic force microscopic analyses of insects with holocentric chromosomes have shown, in a 3-D recon- struction, that the junction between the two chromatids is totally homogeneous over the whole chromosome (Mandrioli and Manicardi, 2003). In triatomines, meiosis is inverted for the heterochromosomes. During anaphase I, the sex chromosomes are pre-equational, and in anaphase II they become post-reductional. However, the autosomes follow a standard meiotic segregation (Pérez et al., 2000). In ad- dition, the meiosis of T. melanosoma exhibited patterns usually found in triatomines in metaphases I and II. In metaphase I, the sex chromosomes were positioned in the center of the ring formed by autosomes, as occurs in T. infestans, T. pseudomaculata and Rhodnius pictipes (De Vaio et al., 1985; Pérez et al., 1992). In addition, the heterochromosomes in 8.05% of the cells analyzed in metaphase I behaved as pseudobivalents, in contrast to the remaining cells, in which the sex chromosomes were individualized, confirming the achi- asmatic nature of these chromosomes (Solari, 1979). The heterochromosomes also behaved as pseudobivalents during metaphase II, this being constituted of a chromatid from chro- mosome X and another from chromosome Y. These observations have also been made by other researchers (De Vaio et al., 1985; Tartarotti and Azeredo-Oliveira, 1999; Morielle and Azeredo-Oliveira, 2004). Interestingly, in the present study, the pseudobivalent formation occurs to a large extent in metaphase II (78.38%); this fact may have a possible relationship with the post-reductional nature of the sex chromosomes. With regard to the chiasmatic presence in diplotene stages, four bivalents with two chiasmata, one terminal and the other interstitial were observed in T. melanosoma. Six bi- valents exhibited terminal chiasmata, with a characteristic ring format. The heterochromo- somes in this phase are completely individualized and achiasmatic, although in metaphase I they have a side by side association. Usually, holocentric chromosomes show 1 or 2 chiasms; however, this number may be higher in larger chromosomes. This is also the case with the Homoptera Psylla foersteri, which has three chiasmata in an autosomal pair originating in the fusion between 5 or 6 small chromosomes. However, these chromosomes migrate slowly, and the medium chiasmata are not usually segregated in anaphase I, resulting in cell loss during spermatogenesis (Nokkala et al., 2004). A characteristic phenomenon in T. melanosoma was the anaphase with late migration, suggesting that the chromosomes with late migration may be the sexual ones (Morielle and Azeredo-Oliveira, 2004). The anaphasic figures observed in the present study indicate that the 333 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 7 (2): 326-335 (2008) Cytogenetic analysis in the spermatogenesis of T. melanosoma kinetic activity of the chromosomes is located preferentially in terminal regions and its segre- gation mechanism is entirely equidistant throughout the extent of the chromosome, and that the orientation of the bivalent was uniform and parallel to the polar axis of the spindle. These observations are in agreement with that proposed in the literature regarding the chromosome pair in Hemiptera (Hughes-Schrader and Schrader, 1961). In T. infestans, in anaphases I and II, the kinetic activity was restricted to the chro- mosome end, irrespective of whether this was euchromatic or heterochromatic. It was also observed that the kinetic activity at the euchromatic end was greater than that at the hetero- chromatic end (Pérez et al., 1997). However, studies with specific centromeric probes in ho- locentric chromosomes of Cyperaceae detected the presence of centromeric scattering which, interestingly, was restricted to heterochromatic areas (Guerra et al., 2006). With regard to silver ion impregnation, the testicular tubules of T. melanosoma ana- lyzed in the present study, revealed only one nucleolus in the initial nuclei of prophase I, dif- fering in this way from some heteroptera that possess more than one nucleolus (Fossey and Liebenberg, 1995). However, at the beginning of the diffuse stage, in addition to the nucleo- lus, small arginophilic corpuscles dispersed throughout the nuclear area were observed. This observation suggests a transcriptional activity not restricted to the nucleolar structure at the beginning of the meiotic prophase. The nucleolus in T. melanosoma persisted until prophase I, specifically in the diplotene; after this phase the nucleolar fragmentation phenomenon began to occur. Nucleolar corpuscles were observed in metaphases I and II, as well as during anaphases I and II. These characteris- tics relate to the phenomenon of nucleolar persistence, in which the nucleolus does not disap- pear totaly in triatomines, but is preserved as pre-nucleolar corpuscles until the beginning of the next meiotic cycle (Tartarotti and Azeredo-Oliveira, 1999; Morielle and Azeredo-Oliveira, 2004). These pre-nucleolar corpuscles are composed of proteins involved in pre-RNA process- ing, which leave the nucleolus in the prophase and are mainly located in the periphery of the chromosomes during the cell cycle; these proteins originate from the dense fibrillar center and granular center of the active nucleolus (Hernandez-Verdum, 2006). In T. melanosoma, between the diplotene and diakinetic phases, silver ion impregna- tion was observed in at least three autosomes and one heterochromosome, suggesting the ex- istence of NORs in those chromosomes. Studies of the Panstrongylus genus (P. megistus and P. herreri) have demonstrated the existence of NORs in sexual chromosomes, in prophase I of the meiosis and in chromosomal associations of NOR-carrying autosomes in the nucleolus (Tartarotti and Azeredo-Oliveira, 1999). The association between the nucleolus and the sexual chromosomes and autosomes is a common characteristic in insects observed in Heteroptera and also in Psocoptera (Golub et al., 2004). In the T. melanosoma diplotene-diakinesis stage, the positive Ag-NOR staining on one of the heterochromosomes persists until the meiotic metaphases. However, the silver ion im- pregnations do not persist during the meiotic stage; for example, in the Heteroptera Belostoma oxyurum, the sex chromosomes continue to be Ag-NOR positive until the diffuse stage, after which no staining was observed on these chromosomes (Papeschi, 1995). At the beginning of cell differentiation, that is, in the initial spermatids, a prominent peripheral silver ion staining occurs. This may be analogous to the pre-nucleolar corpuscles observed in the early nucleolar fragmentation. In other words, after the reorganization of the nuclear envelope, silver-positive proteins that were previously small corpuscles in the cell, are 334 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 7 (2): 326-335 (2008) V.B. Bardella et al. destined for the nucleus for future nucleolar reorganization. In conclusion, the present study contributes to our knowledge of the cytogenetics of triatomines through the analysis of heteropyknotic degree in T. melanosoma spermatogenesis, chromosome size arrangement and their correlation at the different pyknotic levels. This study also corroborates the behavior of holocentric chromosomes in Heteroptera, terminal anaphase kinetic activity and the formation of achiasmatic pseudobivalents in the sex chromosomes. Fi- nally, the data of this study confirm the phenomena of nucleolar fragmentation in triatomines and post-meiotic nucleolar reactivation at the beginning of cell differentiation. ACKnOwlEdGMEnTs The authors are thankful to Dr. José Soares Barata, director, and João Luis Molina Gil and Maurício R. da Silva Filho, technicians, of the Insectary of the Araraquara Special Health Service (SESA) (Araraquara, SP), organ of the Department of Epidemiology, São Paulo Pub- lic Health School for providing the specimens studied. Special thanks go to FAPESP and PIBIC/CNPq. REFEREnCEs Coll-Cardenas R, Espinoza-Gomez F, Maldonado-Rodriguez A, Reyes-Lopez PA, et al. (2004). Active transmission of human Chagas disease in Colima Mexico. Mem. Inst. Oswaldo Cruz 99: 363-368. 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