MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 95(6) Nov/Dez 2000
PAGES: 893-898 DOI: Full paper
Changes in Nuclear Phenotypes Following Cold Shock in Panstrongylus megistus (Burmeister)

Simone L Garcia, Maria Luiza S Mello +, Nancy L GarciaI, Vera Lúcia CC RodriguesII

Departamentos de Biologia Celular e Parasitologia, Instituto de Biologia
IDepartamento de Estatística, Instituto de Matemática, Estatística e Computação Científica, Universidade Estadual de Campinas (Unicamp), 13083-970 Campinas, SP, Brasil
IISUCEN, Mogi-Guaçu, SP, Brasil

Abstract

The nuclear phenotypes of Malpighian tubule epithelial cells of 5th instar male nymphs of the blood-sucking insect Panstrongylus megistus were studied immediately after a short (1 h) cold shock at 0oC, and 10 and 30 days later. The objective was to compare the responses to a cold shock with those known to occur after hyperthermia in order to provide insight into the cellular effect of cold in this species. Nuclei which usually exhibited a conspicuous Y chromosome chromocenter were the most frequent phenotype in control and treated specimens. Phenotypes in which the heterochromatin was unravelled, or in which there was nuclear fusion or cell death were more abundant in the shocked specimens. Most of the changes detected have also been found in heat-shocked nymphs, except for nuclear fusion which generates giant nuclei and which appeared to be less effective or necessary than that elicited after heat shock. Since other studies showed that a short cold shock does not affect the survival of more than 14% of 5th instar nymphs of P. megistus with domestic habit and can induce tolerance to a prolonged cold shock, heat shock proteins proteins are probably the best candidates for effective protection of the cells and the insects from drastic damage caused by low temperature shocks.

Thermal shocks affect survival, molting incidence and nuclear phenotypes in blood-sucking hemipterans, including the vectors of South American trypanosomiasis. Variations in the responses are a function of the shock duration and of the insect developmental phase, sex and species (Rodrigues et al. 1991, Dantas & Mello 1992, Garcia et al. 1999b).Panstrongylus megistus, for instance, is less resistant than Triatoma infestans to heat and cold shock assays (Rodrigues et al. 1991, Garcia et al. 1999b). P. megistus is a very important disease vector because of its wide distribution in Brazil, its high rate of infection with Trypanosoma cruzi and its adaptability in invading artificial ecotopes (Forattini et al. 1978, Forattini 1980). Knowledge of how this species responds to stress is necessary not only to establish the optimal conditions for rearing specimens in the laboratory but also to evaluate the altered biological characteristics of specimens captured in natural ecotopes.

Exposure for l h at 0°C affects the survival of P. megistus nymphs at various developmental stages and adults, more than does a heat shock at 40°C for the same time, although this difference is relatively less pronounced in 5th instar nymphs (Garcia et al. 1999b). Cold shock also drastically affects the frequency and time of molting, to a greater extent than does heat shock (Garcia et al. 1999b). Even so, more than 50% of the P. megistus nymphs at different developmental phases survive for at least one month after a short cold shock (Garcia et al. 1999b). While cryoprotectants are unknown in these insects, it is hypothesized that heat shock proteins (hsp) confer a certain amount of protection to P. megistus against cold in a similar way to that reported for some other insect species (Chen et al. 1987, Burton et al. 1988, Komatsu et al. 1996). Indeed, cold tolerance may be induced in P. megistus (Garcia et al. 1999a).

Considering that changes in nuclear phenotypes indicative of cell survival and cell death are induced by heat shock in 5th instar nymphs of P. megistus (Garcia et al. 2000), we have examined the cellular effects of cold shock in specimens of the same developmental phase of this species for comparison purposes.

 

MATERIALS AND METHODS

Male 5th instar nymphs, descendants of a domestic population of P. megistus (Burmeister) (Hemiptera, Reduviidae) and reared in the laboratory were used. Control groups were maintained at 28°C and 80% relative humidity whereas the experimental specimens were subjected to cold shock at 0°C for 1 h. The shock temperature conditions were selected based on a previous report on the survival and molting incidence for this species (Garcia et al. 1999b).

The specimens underwent a 15-day moderate fasting period before the shock after which they were fed with hen blood once a week. This nutritional regime was chosen because there was no significant difference between the survival of slightly fasted and fully-nourished 5th instar nymphs of domestic P. megistus (Garcia et al. 1999b) while the moderate fasting facilitates the dissection procedure.

Whole Malpighian tubules were removed from the insects immediately after the shock and also 10 and 30 days later. Organs from at least three specimens under experimental and control groups were used. The control groups were the same as those used for study of the heat shock effects (Garcia et al. 2000), since heat and cold shock experiments were processed simultaneously, although cold shock results were analyzed later. The organs were mounted on histological slides, fixed in absolute ethanol: glacial acetic acid (3:1, v/v) for 1 min, rinsed in 70% ethanol for 5 min and air dried at room temperature. The tubules were then processed for the Feulgen reaction, acid hydrolysis being performed in 4 M HCl at 25°C for 65 min. The stained preparations were rinsed in three washes of sulfurous water and one wash of distilled water, air dried, cleared in xylene, and mounted in Canada balsam.

The total number of Malpighian tubule epithelial cell nuclei and the number of different nuclear phenotypes present in the organ were determined in each specimen by counting with a Zeiss light microscope. Photomicrographs were obtained using a Zeiss Axiophot II microscope. The linear relationship between the shock conditions and the various nuclear phenotypes was assessed statistically with the MinitabTM software.

 

RESULTS

The Feulgen-stained polyploid nuclei of the Malpighian tubules of P. megistus males showed homogeneously distributed fine chromatin granules encircling one small heterochromatic body which contained several copies of the Y chromosome (Mello et al. 1986) (Fig. 1). This phenotype was the most frequent in both, the control and experimental specimens (Table I), although it decreased in relative frequency 30 days after the cold shock (Table II).

Other nuclear types were more prominent in the cold-shocked specimens (Table I) and were characterized by heterochromatin unravelling (Fig. 2), nuclear fusion (giants) (Fig. 3), typical and suspected apoptosis (Figs 4, 5) and necrosis (Fig. 6). Apoptosis and necrosis were defined morphologically and were also seen in some giants.

The absolute, but not relative, frequency of heterochromatin unravelling increased immediately after the shock. With increasing time after the shock, both the absolute and relative frequencies of this nuclear phenotype increased significantly (Tables III).

The absolute frequency of giant nuclei increased slightly just after the shock, but decreased thereafter, whereas the relative frequency of these nuclei was not significantly affected immediately after the shock but decreased moderately throughout the remainder of the experiment. Rare necrotic giant nuclei, and giant nuclei suspected of apoptosis or with heterochromatin unravelling were observed with increasing time after the shock (Tables III).

There was a significant increase in the frequency of apoptotic nuclei but not in that of nuclei suspected of apoptosis following the cold shock. However, the frequency of apoptotic nuclei subsequently decreased whereas that of nuclei suspected of apoptosis was not significantly correlated with increasing time after the shock (Tables III). The frequency of necrotic nuclei did not change immediately after the shock but increased significantly throughout the rest of the experiment (Tables III).

 

DISCUSSION

The control specimens of P. megistus studied here had lower nuclear frequency than that reported for fully-nourished, laboratory-reared specimens (Mello et al. 1986, Garcia et al. 2000). This may reflect nuclear fusion and cell death induced by other stressing agents (such as fasting) prior to the shock. The presence of a certain frequency of nuclear phenotypes different from that of the normal phenotype agrees with this hypothesis. The increased frequencies of altered nuclear phenotypes following cold shock indicate mechanisms involving cell survival (heterochromatin unrav-elling, nuclear fusion) and cell death (apoptosis, necrosis). Most of the nuclear changes observed here have also been reported after hyperthermia in P. megistus and T. infestans (Dantas & Mello 1992, Tavares et al. 1997, Garcia et al. 2000). Heterochromatin unravelling, with its possible role in the activation of silent genes during stress (Simões et al. 1975), is longer-lasting in P. megistus compared with T. infestans, irrespective of the eliciting temperature used (Dantas & Mello 1992).

Giant nuclei generated by fusion in the organs of several blood-sucking hemipteran species have been associated with survival mechanisms under unfavorable conditions (Wigglesworth 1967, Mello & Raymundo 1980, Mello 1989). The frequency of giant nuclei in P. megistus nymphs increased slightly just after the short cold shock but remained very low with increasing time after the shock. This finding contrasts with the situation found after short heat shock when there was an increase in the absolute frequency of giant nuclei 10 days after a 1 h-treatment and a decreased frequency thereafter (Garcia et al. 2000). The activation of nuclear and/or cell fusion which generates giant nuclei (Wigglesworth 1967) was found to be less effective or necessary in response to cold shock than that elicited by heat shock. In addition, some of the giant nuclei showed morphological signs of cell death.

The apoptosis and necrosis phenomena seen in the Malpighian tubules of P. megistus after a cold shock have also been observed after a heat shock (Garcia et al. 2000). In both cases, only the apoptosis program intensified immediately after the shocks; necrosis was a later response.

When stress is enhanced beyond a certain level and hsp are incapable of protecting the cells from the deleterious effects of stress, apoptosis is activated. If the stress damage is very severe, necrosis predominates (Lindquist & Craig 1988, Harmon et al. 1990, Sakaguchi et al. 1995, Samali & Cotter 1996). Although the effects of a short cold shock in P. megistus are not completely controlled by hsp or survival mechanisms involving cell/nuclear fusions and heterochromatin unravelling since cell death mechanisms are elicited, cell death was not grave enough to affect the survival of more than 14% of the 5th instar nymphs of P. megistus after this shock(Garcia et al. 1999b).

Prolonged cold and heat shocks are extremely deleterious to P. megistus nymphs (Garcia et al. 1999b). Indeed, tolerance to prolonged heat and cold shocks develops in P. megistus, if preceded by short heat and cold shocks, respectively (Garcia et al. 1998, 1999a). The development of tolerance after sequential shocks has been attributed to the action of hsp (Lindquist 1986, Burton et al. 1988). Thus, if hsp are activated in P. megistus by a short cold shock, thereby producing tolerance to a prolonged cold shock, hsp would be the best candidates for effectively protecting the cells and insect from drastic damage by low temperature.

 

ACKNOWLEDGMENTS

To Dr Stephen Hynslop for revision of the manuscript and Mr Pedro Ribeiro da Silva for technical help.

 

REFERENCES

Burton V, Mitchell HK, Young P, Petersen NS 1988. Heat shock protein against cold stress of Drosophila melanogasterMol Cell Biol 8: 3550-3552.

Chen C-P, Denlinger DL, Lee RE 1987. Cold shock injury and rapid cold hardening in the flesh fly Sarcophaga crassipalpisPhysiol Zool 60: 297-304.

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

Forattini OP 1980. Biogeografia, origem e distribuição da domiciliação de triatomíneos no Brasil. Rev Saúde Públ 14: 265-299.

Forattini OP, Ferreira OA, Rocha-e-Silva EO, Rabello EX 1978. Aspectos ecológicos da tripanossomíase americana. XII - Variação regional da tendência de Panstrongylus megistus à domiciliação. Rev Saúde Públ 12: 209-233.

Garcia SL, Rodrigues VLCC, Mello MLS 1998. Termotolerância em Panstrongylus megistus Burmeister após choque hipertérmico. Genetics Mol Biol 21 (Suppl.): 81.

Garcia SL, Mello MLS, Garcia NL, Rodrigues VLCC 1999a. Tolerância a choque frio e efeito sobre a taxa de sobrevivência e ocorrência de mudas em Panstrongylus megistus Burmeister. XVI Congresso Brasileiro de Parasitologia, Poços de Caldas, 1998, p. 85.

Garcia SL, Mello MLS, Rodrigues VLCC, Garcia NL 2000. Nuclear phenotype changes after heat shock in Panstrongylus megistus (Burmeister). Mem Inst Oswaldo Cruz 95: 271-277.

Garcia SL, Rodrigues VLCC, Garcia NL, Ferraz-Filho AN, Mello MLS 1999b. 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 42o-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.

Komatsu Y, Kodama O, Fujita K 1996. Heat shock treatment reduces in situ temperature. Cell Mol Biol 42: 839-845.

Lindquist S 1986. The heat shock response. Ann Rev Biochem 55: 1151-1191.

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

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

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 Públ 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 Rhyn-chosciara.Ciênc Cult 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 27 January 2000
Accepted 19 April 2000

This paper received financial support from the State of São Paulo Research Foundation (Fapesp, grants no. 95/1954-8, 95/6629-8, 99/2547-8) and the Brazilian National R&D 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 a Master degree.

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