MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 95(2) Mar/Apr 2000
PAGES: 271-277 DOI: Full paper
Nuclear Phenotype Changes after Heat Shock in Panstrongylus megistus (Burmeister)

Simone L Garcia, Maria Luiza S Mello +, Vera Lúcia CC RodriguesI, Nancy L GarciaII

Departamento de Biologia Celular e Parasitologia, Instituto de Biologia
ISucen, Mogi-Guaçu, SP, Brasil
IIDepartamento de Estatística, Instituto de Matemática, Estatística e Computação Científica, Unicamp, 13083-970 Campinas, SP, Brasil

Abstract

The nuclear phenotypes of Malpighian tubule epithelial cells of male nymphs of the blood-sucking insect, Panstrongylus megistus, subjected to short- and long-duration heat shocks at 40oC were analyzed immediately after the shock and 10 and 30 days later. Normal nuclei with a usual heterochromatic body as well as phenotypes indicative of survival (unravelled heterochromatin, giants) and death (apoptosis, necrosis) responses were observed in control and treated specimens. However, all nuclear phenotypes, except the normal ones, were more frequent in shocked specimens. Similarly altered phenotypes have also been reported in Triatoma infestans following heat shock, although at different frequencies. The frequency of the various nuclear phenotypes observed in this study suggests that the forms of cell survival observed were not sufficient or efficient enough to protect all of the Malpighian tubule cells from the deleterious effects of stress. In agreement with studies on P. megistus survival following heat shock, only long-duration shock produced strongly deleterious effects.

The effect of heat shock on survival and molting incidence in Panstrongylus megistus varies with the duration of the shock, the developmental stage and sex of the specimens, and in certain cases, the insect's habits and nutritional states (Garcia et al. 1999).

P. megistus is less resistant to heat shock than Triatoma infestans, indicating that no generalization can be made about the responses of different reduviid species to temperature shocks (Rodrigues et al. 1991, Garcia et al. 1999).

As with other stress factors, heat shocks induce cytological changes in T. infestans, including nuclear fusion, heterochromatin unravelling, and cell necrosis and apoptosis, as part of the mechanisms of cell survival and cell death, respectively (Mello 1989, Dantas & Mello 1992, Mello et al. 1995, Tavares et al. 1997).

The Malpighian tubule cells in late nymphs of P. megistus are highly polyploid (Mello 1975). In males, the most usual nuclear phenotype of this organ has a homogeneous distribution of granulous chromatin and a small but conspicuous heterochromatic body formed by several copies of the Y chromosome (Mello et al. 1986).

Since P. megistus and T. infestans differ in their normal nuclear phenotypic characteristics (Mello 1971, 1975, Mello et al. 1986), these nuclear phenotypes may be affected differently by heat shock treatment.

In the present study, the nuclear phenotypes of P. megistus nymphs were determined after heat shock and the changes compared with those for T. infestans under similar temperature conditions (Dantas & Mello 1992).

 

MATERIALS AND METHODS

Fifth instar male nymphs of a domestic population of P. megistus (Hemiptera, Reduviidae), descended from insects obtained in Fazendas Pedra Balão and Pedra Branca in São João da Boa Vista (State of São Paulo) and reared in the laboratory at Sucen (Mogi Guaçu, SP), were used. The control nymphs were maintained at 28oC and 80% relative humidity, conditions which have traditionally been used for rearing this species in the laboratory at Sucen since 1980.

The treated specimens underwent heat shock at 40oC for 1 h and 12 h. The choice of this temperature was based on a previous study of survival and molting incidence in P. megistus after heat shock (Garcia et al. 1999). The specimens were fasted for 15 days before the shock and after treatment were returned to a normal diet of hen blood once a week.

Malpighian tubule preparations were obtained immediately after heat shock and 10 and 30 days later. The organs from at least three specimens were used for each experimental condition and the corresponding control.

Whole Malpighian tubules were mounted on glass slides, immediately fixed in acetic ethanol for 1 min, rinsed in 70% ethanol for 5 min and air dried at room temperature. The material was then subjected to the Feulgen reaction, with hydrolysis in 4 M HCl at 25oC for 1 h and 5 min. The Feulgen-stained material was rinsed in sulfurous and distilled water, air dried, cleared in xylene and mounted in Canada balsam.

A Zeiss light microscope was used to count the total number of Malpighian tubule epithelial cell nuclei per specimen, to identify the different nuclear phenotypes and to evaluate their frequencies in each specimen. Photomicrographs were obtained with a Zeiss Axiophot II microscope.

A linear correlation was used to evaluate the relationship between the stress conditions and the various nuclear phenotypes.

 

RESULTS

The most frequent phenotype in the control and heat shocked specimens consisted of nuclei with a small heterochromatic body containing copies of the Y chromosome (Mello et al. 1986) in the middle of evenly distribution of lightly stained chromatin (Fig. 1Table I).

Other nuclear types observed in all the experimental conditions were generally more frequent after heat shock (Table I). These included nuclei with heterochromatin unravelling (Fig. 2), apoptotic nuclei (Fig. 3), nuclei suspected of apoptosis (Fig. 4), necrotic nuclei (Fig. 5) and giant nuclei (Fig. 6). In some cases giant nuclei exhibited signs of necrosis or were suspected of apoptosis (Fig. 6). Some giant nuclei were also observed in which heterochromatin decondensation was suspected. Apoptosis and necrosis were defined here in terms of their classic morphological characteristics (Kerr 1971, Kerr et al. 1972). There was a significant decrease in the relative frequency of normal nuclei with heat shock and its duration (Fig. 7aTable II). The absolute frequency of nuclei with heterochromatin unravelling increased just after the short and long shocks, and 10 and 30 days after the short shock (Table I). However, in terms of relative frequencies, the increase in nuclei with heterochromatin unravelling was significant only immediately after short and long shocks (Fig. 7b,Table II); the frequency of this phenotype decreased after long shocks compared to short shocks (Fig. 7b). There was no significant correlation with the time after the shock (Table II). The appearance of heterochromatin decondensation was moderately correlated with that of apoptotic nuclei (Table II).

A few giant nuclei were detected (Table I). The relative frequency of this nuclear phenotype decreased immediately after long shocks when compared to short shocks (Fig. 7c), i.e. there was a slight negative correlation between shock duration and phenotype frequency (Table II). There was no correlation between the relative frequency of morphologically normal giant nuclei and the time after the shock or the shock temperature, but a moderate correlation was observed between giant nuclei with heterochromatin decondensation and the time after the shock (negative), between the occurrence of necrotic giants and shock temperature (positive) and between giants suspected of apoptosis and the shock temperature and duration (positive) (Table II).

There was a high correlation between giant nuclei with normal morphological characteristics and necrotic giant nuclei as well as between necrotic giants and giants suspected of apoptosis (Table II).

The absolute and relative frequencies of apoptotic nuclei increased immediately after heat shock (Fig. 8a, Tables III), but there was no significant correlation between these frequencies and the time after shock or the duration of the shock (Table II).

The relative frequency of nuclei suspected of apoptosis did not increase with the heat shock temperature or duration, but there was a moderately significant correlation between the increase in this frequency and the time after shock (Fig. 8b,Table II). The absolute and relative frequencies of necrotic nuclei increased significantly 10 and 30 days after the heat shock (Tables IIIFig. 8c).

 

DISCUSSION

The number of nuclei in the Malpighian tubules of fully-nourished laboratory-reared P. megistus isabout 18,000 (Mello et al. 1986). In the present study, control specimens had a much smaller nuclear frequency, indicating that nuclear fusion and cell death induced by other stressors may have occurred under laboratory conditions. Indeed, the specimens used in this investigation had been moderately fasted prior to the heat shock. Fasting is a stress agent in blood-sucking insects (Andrade & Mello 1987, Mello 1989). The choice of a slight fasting condition for the present study was based on the finding that only after it some 5th instar specimens are capable of surviving a long heat shock (Garcia et al. 1999). The observation that some altered nuclear phenotypes also occurred in the control specimens supports the idea that some stressing effect other than heat shock was involved. Maybe an occasional and unexplained refusal of some specimens to feed a blood meal added some fasting period to that intentionally provoked.

Nuclear phenotypes differing from the normal phenotype seen in P. megistus have also been reported for T. infestanssubjected to heat shock or other stressing agents (Álvares-Garcia 1988, Mello 1989, Dantas & Mello 1992, Mello et al. 1995). Such phenotypes may reflect mechanisms of cell survival (heterochromatin unravelling, nuclear fusion) or cell death (apoptosis, necrosis) in response to stress (Dantas & Mello 1992, Tavares et al. 1997).

Heterochromatin unravelling occurred even long after heat shock in P. megistus. This situation differs from that for T. infestans in which such unravelling is most frequent 10-120 min after the shock (Dantas & Mello 1992). If heterochromatin unravelling leads to the activation of silent genes during stress (Simões et al. 1975), its effects may be longer-lasting in P. megistus than in T. infestans. It is not known whether heterochromatic zones contain genes for heat shock proteins (hsp).

In various reduviid species, giant nuclei are produced by nuclear and/or cell fusion (Wigglesworth 1967, Mello & Raymundo 1980, Mello 1989, Dantas & Mello 1992). These nuclei have been suggested to be involved in cell or organ survival mechanisms under unfavorable conditions. In this regard, the high mortality rates seen in P. megistus subjected to long heat shock (Garcia et al. 1999) suggest that giant nuclei were not present in sufficient numbers and/or were not efficient enough to protect the insects from heat shock-induced damage. Indeed, the relative frequency of giant nuclei decreased significantly with the shock duration whereas the number of giants suspected of apoptosis was weakly correlated with this factor.

P. megistus survivors of heat shock showed a significant decrease in the frequency of giant nuclei 10 to 30 days after the shock. In T. infestans, the highest frequency of giants occurs 30 days after the shock (Dantas & Mello 1992). In addition to a decrease in new fusions, the fact that some giant nuclei in P. megistus exhibited necrosis or were suspected of apoptosis probably also contributed to the reduction in their frequency. It is possible that in P. megistuscells and nuclei resulting from fusion may be more susceptible to metabolic failure than in T. infestans.

When the stress is enhanced beyond a certain level, the presence of hsp is incapable of protecting the cells and apoptosis program is initiated (Lindquist & Craig 1988, Maihos et al. 1993, Samali & Cotter 1996). If the stress exceeds this level, cell death by necrosis predominates (Samali & Cotter 1996). Thus, murine mastocytoma cells subjected to a temperature of 43-44oC show an increase in apoptotic index. At a temperature of 45oC, both apoptosis and necrosis are observed in these cells while at 46-47oC only necrosis is found (Harmon et al. 1990). Similar responses have been described in other cell types (Sakaguchi et al. 1995).

In the Malpighian tubules of P. megistus, apoptosis and necrosis occurred simultaneously, especially after hyperthermia. Only the apoptosis program intensified immediately after the heat shock. The frequency of the apoptotic nuclei then remained unchanged whereas necrosis, which was not significantly affected immediately after heat shock, intensified with the shock duration and time after shock.

Hsp expression, apoptosis, heterochromatin unravelling and nuclear fusion were thus not apparently sufficient to protect all P. megistus Malpighian tubule cells from the deleterious effects of heat shock. In terms of insect survival, only long shocks proved to be strongly deleterious (Garcia et al. 1999). In the few survivors of long shocks, there was probably no additional degeneration since no significant difference in the frequency of necrotic nuclei was observed compared to insects subjected to short shocks.

The individual variations in response to hyperthermia were similar to those seen in T. infestans after other stressing agents and suggest that specimens of P. megistus may also vary in their resistance to different stressors, including heat shock.

 

ACKNOWLEDGMENTS

To Mr Pedro Ribeiro da Silva for his technical support.

 

REFERENCES

Álvares-Garcia RS 1988. Efeitos da Radiação Gama sobre os Fenótipos Nucleares de Alguns Tipos Celulares deTriatoma infestans Klug (Hemiptera, Reduviidae),MSc Thesis, Unicamp, Campinas, 104 pp.

Andrade CGTJ, Mello MLS 1987. Phenotypes and number of Malpighian tubule nuclei in Triatoma infestans Klug along development and starvation. Rev Bras Genet 10: 449-457.

Dantas MM, Mello MLS 1992. Changes in the nuclear phenotypes of Triatoma infestans Klug, induced by thermal shocks. Rev Bras Genet 15: 509-519.

Garcia SL, Rodrigues VLCC, Garcia NL, Ferraz-Fillho AN, Mello MLS 1999. Survival and molting incidence after heat and cold shocks in Panstrongylus megistus Burmeister. Mem Inst Oswaldo Cruz 94: 131-137.

Harmon BV, Corder AM, Collins RJ, Gobé GC, Allan DJ 1990. Cell death induced in a murine mastocytoma by 42-47oC heating in vitro: evidence that the form of death changes from apoptosis to necrosis above a critical heat load. Int J Radiat Biol 58: 854-858.

Kerr JFR 1971. Shrinkage necrosis: a distinct mode of cellular death. J Pathol 105: 13-20.

Kerr JFR, Wyllie AH, Currie AR 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257.

Lindquist S, Craig EA 1988. The heat shock proteins. Ann Rev Genet 22: 631-677.

Mailhos C, Howard MK, Latchman DS 1993. Heat shock protects neuronal cells from programmed cell death by apoptosis. Neuroscience 55: 621-627.

Mello MLS 1971. Nuclear behaviour in the Malpighian tubes of Triatoma infestans (Reduv., Hemiptera). Cytologia 36: 42-49.

Mello MLS 1975. Feulgen-DNA values and ploidy degrees in the Malpighian tubes of some triatomids. Rev Bras Pesq Med Biol 8: 101-107.

Mello MLS 1989. Nuclear fusion and change in chromatin packing state in response to starvation in Triatoma infestansRev Bras Genet 12: 485-498.

Mello MLS, Raymundo HH 1980. Nuclear fusion in the Malpighian tubes of a blood-sucking hemipteran. Cytologia 45: 203-209.

Mello MLS, Randi MA, Giorgio S, Ferraz-Filho AN, Rodrigues VLCC, Rocha-e-Silva EO, Cordeiro JA 1986. Number of chromosomes, Feulgen-DNA content and nuclear phenotypes in domestic and wild specimens ofPanstrongylus megistusAnn Trop Med Parasitol 80: 641-648.

Mello MLS, Kubrusly FS, Randi MA, Rodrigues VLCC, Ferraz Filho AN 1995. Effects of heavy metals on chromatin supraorganization, nuclear phenotypes, and survival of Triatoma infestans. Entom Exp Appl 74: 209-21.

Rodrigues VLCC, Mello MLS, Ferraz Filho AN, Dantas MM 1991. Sobrevivência e ocorrência de muda emTriatoma infestans Klug (Hemiptera, Reduviidae) após choque de temperatura. Rev Saúde Pública (São Paulo)25: 461-467.

Sakaguchi Y, Stephens LC, Makino M, Kaneko T, Strebel FR, Danhauser LL, Jenkins GN, Bull JMC 1995. Apoptosis in tumors and normal tissues induced by whole body hyperthermia. Cancer Res 55: 5459-5464.

Samali A, Cotter TG 1996. Heat shock proteins increase resistance to apoptosis. Exp Cell Res 223: 163-170.

Simões LCG, Amabis JM, Cestari NA 1975. Puffs in the heterochromatin in chromosomes of Rhynchosciara.Ciênc Cult São Paulo 27: 159-161.

Tavares MCH, Dantas MM, Rodrigues VLCC, Mello MLS 1997. Alterações em fenótipos nucleares, com ênfase na apoptose, em túbulos de Malpighi de Triatoma infestans Klug após choque hipertérmico, 43o Congresso Nacional de Genética, Goiânia, p. 65.

Wigglesworth VB 1967. Polyploidy and nuclear fusion in the fat body of Rhodnius (Hemiptera). J Cell Sci 2: 603-616.

+Corresponding author. Fax: + 55-19-788.7821. E-mail:  This e-mail address is being protected from spambots. You need JavaScript enabled to view it.
Received 30 July 1999
Accepted 21 December 1999

This work was supported by the State of São Paulo Research Foundation (Fapesp, grants 95/1954-8, 95/6629-8 and 99/02547-8) and the Brazilian National Research and Development Council (CNPq).
This study was part of a thesis presented by SLG to the Instituto de Biologia, Unicamp, in partial fulfillment of the requirements for the Masters degree.

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