Genetica 109: 211–218, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Chromosomal responses of blowflyLucilia cuprina to heat and heavymetal stress

Archana Joshi & P.K. Tiwari∗School of Studies in Zoology, Jiwaji University, Gwalior (M.P.) 474 011, India;∗Author for correspondence

Received 12 January 1999 Accepted 23 November 2000

Key words:heavy metal stress, heat shock puffs, heat shock response,Lucilia cuprina

Abstract

Chromosomal responses to heat and heavy metal shocks were studied in the pupal trichogen polytene chromosomesof Lucilia cuprina. Heat shock induced seven distinct puffs on different chromosomes at the following loci: 1C2,21A1, 21C, 23B, 24B, 42A and 95B3. Arsenate and mercury, two of the most common toxic environmentalchemical pollutants also, induced almost the same set of puffs except 20B2, which appeared to be induced bymetals only and 95B3, which was not induced by arsenate. The findings suggest that a common set of gene lociencoding the heat shock proteins is responsive to these diverse environmental stresses.

Introduction

The cell’s response to environmental insults was firstobserved by Ritossa (1962, 1964) in salivary glandpolytene chromosomes ofDrosophilaas induction ofa novel set of chromosomal puffs when larval salivaryglands were exposedin vitro to elevated temperature(37◦C) or 2,4-Dinitrophenol. Later studies revealedthat these puffs were the sites of intense transcrip-tional activity (Pelling, 1964; Ashburner & Bonner,1979) and responsive to a variety of other stressors(Ashburner & Bonner, 1979; Ashburner, 1980, 1982;Lakhotia & Singh, 1985). Tissieres, Mitchell andTracy (1974) and Lewis, Helmsing and Ashburner(1975) demonstrated a correlation between the heatshock puffs and the heat induced polypeptides, theheat shock proteins (HSPs), (Spradling, Pardue &Penman, 1977). The induction of heat shock re-sponse was found to be evoked by a wide range ofconditions including elevated heat, heavy metals ortransition series metals, amino acid analogs, oxidants,etc. (for a list, see Nover, 1984; Nover & Scharf,1997). The phenomenon is widely defined as the ‘heatshock response’/or ‘stress response’, and is conservedthroughout the living world; it is suggested to be cyto-protective (for reviews, see Schlesinger, Ashburner &

Tissieres, 1982; Nover, 1984; Lindquist, 1986; Nover& Scharf, 1997; Forrieter & Nover, 1998).

Cytogenetic studies on the heat shock gene locihave so far remained limited to only a few of dip-terans, viz.,Drosophila (Ritossa, 1964; Ashburner,1970; Ashburner & Bonner, 1979; Lakhotia & Singh,1982, 1985),Chironomus(Yamamoto, 1970; Vincent& Tanguay, 1979; Lezzi, 1984; Lezzi, Meyer & Mahr,1981; Lezzi, Gatzka & Meyer, 1984; Nath & Lak-hotia, 1989a),Sarcophaga(Bultmann, 1986 a,b) andmosquito (Nath & Lakhotia, 1989b). The blowflyLu-cilia cuprina (Diptera, Calliphoridae), one of the mostserious pests of farm animals, is highly adapted toa wide variety of extreme environmental conditions,including high tropical temperatures, anoxia, varyingpH conditions and other toxic pollutants common totheir habitat. It, therefore, provides an opportunityto examine a greater scope of conditions relating tothe mechanisms of the stress responses and the func-tions of the heat shock genes in thermotolerance andecological adaptation (Tiwari, Mohan & Joshi, 1995).Further, cytologically analysable polytene chromo-somes are found in both larval and pupal tissues. Inthe present study, a cytological analysis of the heatand metal induced chromosomal puffs was made toidentify the genetic loci responsive to these stress

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agents. Interestingly, the chromosomal loci responsiveto higher temperature and metals, were found to beapparently the same or overlapping.

Materials and methods

The laboratory mass culture (about 11 years old) ofa tropical species of blowfly,Lucilia cuprina cuprina(Phaenicia cuprina; Joshi, 1999), Wiedemann (Dip-tera, Calliphoridae) used in the current study, wasreared at 25–28◦C on raw goat meat and sugar meal(Whitten et al., 1975). In our culture conditions thetotal life cycle (embryonic, larval, pupal to adultemergence) takes about 13–14 days.

Treatments

Relatively good quality cytologically analysablepolytene chromosomes from pupal thoracic trichogencells ofL. cuprinawere used for the present study. Forthe analysis of heat shock puffs, treatments were givento the posterior half of the excised thorax where thelarger scutellar bristles are found.

Heat shockFor heat shock treatment, the posterior scutellar sec-tion of the thorax from 4 to 5 days-old pupae in whichbristle pigmentation had just began, was dissected.Tissues were incubated at different temperatures, suchas 43◦C, 44◦C, 45◦C, or 46◦C for 30 min or 60 min in acavity block containing preheated Poels’ salt solution(Lakhotia & Mukherjee, 1980). In parallel, the controlthoraces were incubated for 30 min or 60 min at roomtemperature (25±1◦C). Heat-shocked and control tis-sues were immediately fixed in aceto-methanol (1 partglacial acetic acid: 3 parts methanol) for 1 h and storedat 4◦C overnight in fresh fixative.

Chemical treatment and induction of chromosomalpuffsTwo of the most potent toxic heavy metals Hg+2 (Mer-curic chloride, HgCl2, Sigma) and As+5 (Sodiumarsenate, Na2HAsO4, Sigma) were used to study thepattern of stress-induced puffing. Various concentra-tions of mercuric chloride (0.01–0.05mM) and so-dium arsenate (0.12, 0.16, 0.2, 0.3, and 0.5 mM) wereprepared in Poels’salt solution. These concentrationsoverlapped those found to induce heat shock proteinsin Drosophilaembryonic cells (Bournias-Vardiabasis,Buzin & Josephine, 1990). For each treatment, thor-aces from pupae were incubated in the test solution

in cavity blocks for 60 min at room temperature. Con-trol tissue was incubated only in Poels’ salt solution.Following treatment, tissues were fixed overnight withaceto-methanol as described above.

Preparation of polytene chromosome spreads

The method of squash preparation of polytene chro-mosomes followed in this study was essentially thesame as that described in Foster et al. (1980). Thescutellar part was dissected out from the fixed controlor treated thoracic tissue in 70% alcohol. After re-moving associated cuticular appendages, the scutelumwas stained with 2% lacto-aceto-orcein for 15–20 min.Following a brief wash in 45% acetic acid, the stainedtissue was squashed in a fresh drop of stain under aclean coverslip. Coverslip was sealed from the sideswith DPX (Distrene-Plastisizer-Xylene) mountant toprevent from drying and stored at 4◦C.

For a detailed analysis of heat shock induced puff-ing a cytological map of trichogen polytene chromo-somes of our laboratory (Indian) stock ofL. c. cuprinawas also prepared (Joshi, 1999) using the map pre-pared for the Australian population ofL. c. dorsalisR.-D. (Foster et al., 1980) as a reference.

Puff size measurement

To assess the relative activities of the heat inducedpuffs (Pelling, 1964; Myohara & Okada, 1988a,b),puff sizes were measured with the help of occulo-micrometer scale using a reference band on the samechromosome arm. Similar measurements were alsodone in control preparations. In each case a minimumof 15 nuclei with well-spread chromosomes were se-lected for the measurement of puff sizes. Due to diffi-culty in obtaining sufficient suitable nuclei in the caseof metal treatments, no puff size measurements werecarried out, and the positions of the metal respons-ive loci were only compared to those induced by heatshock. Photographs of the chromosomes were takenusing a Nikon (Japan) Labophot-2 Photomicroscope.

Results and discussion

Induction of puffs by heat has been correlated withthe concomittant synthesis of HSPs which apparentlyassist in the development of thermotolerance inDro-sophila (Ritossa, 1964; Tissieres, Mitchell & Tracy,1974; Spradling, Pardue & Penman, 1977; Lindquist,1986; Vincent & Tanguay, 1979; Petersen, 1990) and

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Chironomus(Lezzi, Meyer & Mahr, 1981; Nath &Lakhotia, 1989a). In continuation to our earlier studyon the induction of heat shock proteins inL. cuprina(Tiwari, Mohan & Joshi, 1995), in this study, wehave analysed the stress inducible chromosomal lociin order to develop a possible correlation betweenthe two and for further cytogenetic and molecularanalyses.

For the study of stress-induced puffs a cytologicalmap of trichogen polytene chromosomes was prepared(see Joshi, 1999). The banding pattern of the Indianpopulation ofL. c. cuprina(also refered asPhaeniciacuprina; Joshi, 1999) was found, in general, compar-able to that of the Australian population (Foster et al.,1980). However, a separate map for the Indian pop-ulation was prepared to avoid any confusion whichmay result from variations in the general pattern ofband-interband in these two geographic populations(for details of map, see Joshi, 1999).

Heat shock induced puffing

There were no differences in the chromosomal puffingpatterns at 43◦C, 44◦C or 45◦C after 30 or 60 min.Even a heat shock at 46◦C for up to 30 min didnot show any significant effect. However, when thethoracic tissues were exposed to 46◦C for 60 min,several puffs on different chromosome arms were in-duced. When carefully compared with the correspond-ing chromosomes from control tissues, seven distinctheat shock puffs were identifiable (Figure 1). Only onepuff was found induced on the left arm of chromo-some 2 at locus 1C-2. There were four puffs (21A,21C, 23B, and 24B) on the left arm of chromosome3. On chromosome 4 only one heat shock puff waspresent at 42A on the left arm. No heat shock puffswere observed on chromosome 5. On chromosome 6,locus 95B-3 on the right arm was found to be induced.

Relative activities of heat inducible puffs

The extent of gene activity of a given genetic locusis suggested to be directly correlated with its size inthe polytene chromosomes (Pelling, 1964; Myohara& Okada, 1988a,b). Hence, in order to compare therelative activities (in arbitary units) of different tem-perature inducible loci (puffs) their relative sizes incontrol and treated preparations were measured on anocculometer scale along with a reference band on thecorresponding chromosomes (Table 1).

Fifteen chromosomes were measured for eachtreated (heat shock) and control conditions. Not all of

the nuclei exhibited similar polyteny. The relative puffsizes in control and treated chromosomes, when meas-ured in reference to a constitutive (unpuffed) band(Table 1 and Figure 3), showed that the puff at 21Aon chromosome 3 was the largest (2.48±0.18) while95B3 on chromosome 6 was the smallest (1.84±0.04).The increase in the puff sizes in treated chromosomeswere found significant as revealed by Student’s ‘t’-testanalysis (P < 0.001; Table 1). The relative activitiesof different puffed loci were found in the followingorder: 21A>42A>23B>1C-2>24B>21C>95B-3.

For the first time a study on the cytological analysisof the heat shock gene activity in a tropical popula-tion of Lucilia cuprina, a member of Calliphoridae,has been carried out. In contrast toSarcophaga, aclose relative of Calliphora,Lucilia appeared to bemore comparable toDrosophila in its responses toheat stress, except for the level of temperature toler-ance, which generally depends on the thermotolerantability of a given species (Tiwari, Mohan & Joshi,1995; Tiwari, Joshi & Mohan, 1997). Similarity inthe responses ofDrosophilaandLucilia to heat stressspecifically appears in respect to a comparable numberof heat inducible chromosomal loci and the heat shockproteins (HSPs) induced.

Interestingly, in contrast to the observations madein Drosophila(Ashburner, 1970), inLucilia cuprina,heat shock does not appear to affect ongoing puff-ing activity significantly, as also observed in Sarco-phaga (Bultmann, 1986a). For example, locus 25Bon chromosome 3L was found induced in most ofthe control as well as treated chromosomes (see Fig-ure 1). This observation is consistent with our resultson heat induced proteins, both inL. cuprina andM.domestica, since general protein synthesis is not signi-ficantly affected even when the HSPs are maximallyinduced, until the temperature reached to very highlevels (46◦C or 48◦C; see Tiwari, Mohan & Joshi,1995; Tiwari, Joshi & Mohan, 1997). A similar obser-vation was also made inS. bullata(Bultmann, 1986a, b). It is surprising that while multiple cytologicalloci that are distinctly induced upon heat stress inDrosophila, Chironomusand Lucilia, only one ge-netic locus showed significant inducibility in pupalfoot-pad polytene chromosomes ofS. bullata uponexposure to both heat or hypoxia (Bultmann, 1986a& b). The other heat shock puffs, if induced, werenot distinct enough to be clearly recognized. The poorvisibility of smaller puffs may possibly account for re-latively lower polyteny in foot-pad or even trichogencell polytene nuclei.

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Figure 1. Heat induced puffs on pupal trichogen cell polytene chromosomes (nos. 2, 3, 4 & 6) ofL. c. cuprina. Arrows indicate the positionsof reference bands. Locus 25B is shown as puff both in control and treated nuclei. (Magnification in this and Figure 2 is 1100X).

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Table 1. Puff size (puff activity) measurements on the polytene chromosomes ofL.cuprina following heat stress treatment

Chromosome Puff site Reference Mean puff size

number band (N = 15)

Control Heat shock ∗t-value

(±S.E.) (±S.E.)

Chromosome 2L 1C2 4B2 0.94± 0.06 2.04±0.46 9.45

Chromosome 3L 21A 25A3 1.30± 0.01 2.48±0.18 4.68

21C 25A3 1.00± 0.00 1.95±0.03 8.69

23B 25A3 1.02± 0.02 2.04±0.15 5.72

24B 25A3 0.98± 0.01 1.97±0.11 5.23

Chromosome 4L 42A 45C 1.11± 0.04 2.27±0.07 9.28

Chromosome 6R 95B3 99B3 0.87± 0.02 1.84±0.04 9.88

∗(Student’s ‘t’ test:P < 0.001).

Chemical induced puffing

To our knowledge, this is the first report on the effectsof mercury and arsenate on chromosomal puff induc-tion. Various concentrations (mM) of the metal saltswere tested for their effects. Chemical treatments werefound to adversally affect the general chromosomemorphology, particularly at higher concentrations. Asa result, fewer analysable nuclei were found.

Mercury induced puffsTreatment of pupal thoracic tissues with varyingconcentrations (0.01–0.05mM) of mercuric chloride(HgCl2) for 1 h induced eight puffs. While lower con-centrations (0.01–0.03mM) were found to induce thepuffs, higher concentrations (0.04–0.05mM) appearedtoxic or inhibitory and even caused chromosomal dis-tortions and clumping, obliterating clear distinctionof banding pattern. In addition to a puff at 20B2in the right arm of chromosome 2 (Figure 2), allseven heat shock puffs were found induced by thistreatment (same as in Figure 1). InLucilia cuprinamercury, therefore, has induced eight puffs includ-ing those seven induced by heat shock. Although the20B2 was not a prominent puff, it was clearly inducedby this treatment (Figure 2). Mercury is a transitionseries metal and it is known to induce oxidative stressin both insects and vertebrates. InDrosophila em-bryonic cells, it induces low levels of all the majorHSPs and significantly reduces the number of termin-ally differentiated muscle and neuronal cells in culture(Bournias-Vardiabasis, Buzin & Josephine, 1990).The induction of heat inducible chromosomal loci by

Figure 2. Metal (mercury and arsenate) induced puff (20B2) onchromosome 2R.

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Figure 3. Relative puff activities (puff sizes) of different heat shock loci on trichogen cell polytene chromosomes ofL. c. cuprina.

mercury, therefore, appears in accordance to the aboveobservations.

Arsenate induced puffingSimilar to heat shock or mercury, arsenate also in-duced seven new puffs corresponding to those inducedby heat. Lower concentrations (0.1, 0.15 and 0.2 mM)of sodium arsenate induced six heat shock puffs (95B3was not induced; same as in Figure 1), as well as20B2 (Figure 2). At 0.3 mM, however, all the inducedpuffs either regressed or were inhibited, while 0.5 mMsodium arsenate severely affected chromosomal mor-phology. Interestingly, the puff 95B-3 which wasinducible by both heat and mercury, did not showany inducibility by arsenate, while 20B-2 appearedinduced as in mercury treated cells.

Though it may not be appropriate to conclude that20B-2 is a metal inducible locus specifically, its ab-sence in heat shocked chromosomes indicate that thismay be the one. Further testing with other metals is,

therefore, needed to ascertain the generality of thisobservation. Similarly, the absence of 95B-3 puff inarsenate-treated chromosomes suggest some commonmechanism in the regulation of mercury and heat in-duced puffing, which is not shared by arsenate. It wasalso noted that mercury- and arsenate-induced puffswere relatively smaller than the heat induced puffs.Similar to heat shock, mercury or arsenate also did notappear to alter the normal puffing pattern significantly,as some of the puffs on different chromosomes wereobserved common to both control as well as treatednuclei (not presented). The heat or metal induced puffsin Lucilia, therefore, appear to be new puffs with noconcomitant effect on general puffing pattern.

Though our observations on the induction of HSPsby arsenate or mercury are preliminary, low levelinduction of polypeptides of 79, 69, and 20 kD mo-lecular weight (unpublished), corresponding to someof the prominent HSPs induced by heat shock inL.cuprina was noted (Tiwari, Mohan & Joshi, 1995).

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Mercury, however, appeared a relatively weak inducerof these polypeptides.

In our earlier study we identified nine heat shockproteins induced upon increasing environmental tem-perature inLucilia, HSP70 being the major heat in-ducible polypeptide (Tiwari, Mohan & Joshi, 1995).The present study, however, does not allow us to makeany correlation between the heat induced species ofHSPs and the heat shock puffs observed in the presentstudy. Use of heat shock induced mRNAs or clonedheat shock gene cDNAs as molecular probes, fromDrosophilaor other related species, may possibly behelpful to provide the answer.

Acknowledgements

This work was supported by a grant-in-aid from DAE,Govt. of India, Mumbai, India to PKT.

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