Biochemical changes in tissue composition of Periplaneta americana (Linn.) acclimated to high and low temperatures

d. therra, Biol. VoL 5, pp. 211 to 218 0306-4565/80/1 O01-0211 $02.00/0 ¢) Pergamon Press Ltd 1980. Printed in Great Britain

BIOCHEMICAL CHANGES IN TISSUE COMPOSITION OF PERIPLANETA AMERICANA (LINN.) ACCLIMATED

TO HIGH AND LOW TEMPERATURES

S. P. Sr~OH

Postgraduate Department of Zoology, Udai Pratap College, Varanasi, India

and

A.B. Dm /

Department of Zoology, Viswa-Bharati University, Santiniketan, India

(Received 16 January 1980; accepted 10 March 1980)

Abstract--I. Cold acclimation apparently favours an increase of water content in fat body, but not in coxal muscle, of cockroaches.

2. A remarkable enhancement in the accumulation of total protein in fat body characterizes the cold acclimation of cockroaches, particularly adult males (175% increase in protein/DNA ratio)~ The increase in protein content of coxal muscle during acclimation to 15°C, observed in nymphs (67%) and males (16%) but not in females, is less pronounced than that of fat body.

3. A diminution (28-32~'0) in the free amino acid/DNA ratio due to cold acclimation has been recorded in both coxal muscle and fat body of nymphs and females, but not in males.

4. No qualitative change occurs in the free amino acid spectrum of haemolymph and tissues of this insect during acclimation to 15 and 35°C.

5. An augmentation (15--30%) of the RNA/DNA ratio occurs in fat body and coxal muscle of nymphs and males but in fat body alone of females following cold acclimation.

6. The glycogen reserve has been shown to increase by up to 30% in fat body and coxal muscle of cold acclimated cockroaches compared to warm acclimated ones.

INTRODUCTION A WID~ variety of poildlotherms demonstrate thermal acclimation at different levels of biological organization, as reviewed by Precht et al. (1973) and Prosser (1962, 1967, 1973). The potential molecular mechanisms underlying the metabolic rate compensation in ectotherms against environmental thermal fluctuation have been reviewed and classified excellently in recent years by Hockachka (1967), Rao (1967), Hochachka & Somero (1973), Hazel & Prosser (1974) and Somero & Hochachka (1976). The apparent variations in a largi~ number of enzymatic concentrations which occur during thermal acclimation of different poikilotbermic organisms (Wilson, 1973; Hazel & Prosser, 1974; Shaklee et ai., 1977) are mainly due to specific changes in protein synthesis and gene regulation (Somero & Hochachka, 1976). The molecular mechanisms, like augmentation of the net synthesis of proteins and nucleic acids during cold acclimation and the reverse process during warm acclimation, seem to have evolved independently in widely separated taxo- nomic categories of ectotherms like fish (Smith & Morris, 1966; Das, 1967; Das & Prosser, 1967; Morris & Smith, 1967; Haschemeyer, 1968, 1969a, b; Das & Krishnamoorthy, 1969; Haschemcyer & Per- sell, 1973; Somero & Doyle, 1973; Nielsen et al., 1977), amphibia (Mews, 1957; Jankowsky, 1960), crustacea (McCarthy et al., 1976), the black locust tree (Siminovitch et al., 1967) and the frost-resistant "winter wheat" (Devay & Paldi, 1977). On the whole, the cold acclimation of poikilothermic organisms

seems to involve a "biosynthetically directed metabolic reorganization", as concluded by Hochachka (1967) and Somero & Hochachka (1976). Even in homeotherms, like laboratory rats and meadow voles, cold acclimation or winter acclimatization seems to favour an increase in the turnover rate of tissue proteins (Yousef & Chaffee, 1970; Chauhan et al., 1971 ; Singh & Alexsink, 1972). The above-mentioned metabolic reorganization during thermal acclimation is obviously reflected in the alteration of biochemical tissue composition (cellular concentrations of total protein, free amino adds, RNA, glycogen and lipids etc.) of fish (Das, 1967; Prosser, 1967), tropical earth- worms and freshwater mussel (Rao, 1967), crustacea (McWhinnie, 1967), the bed bug (Okasha, 1964) and even a plant (Siminovitch et al., 1967). as reviewed by Precht et al. (1973).

Although thermal acclimation of biosynthetic processes and the concomitant compensatory changes in cellular concentrations of macromolecules (even some substances of low molecular weight), which are of structural and functional significance in the organism seem to be fairly widespread phenomena in poikilo- t h e m , the information is quite fragmentary and debatable in insect& Certain authors such as Edwards (1953), Bullock (1955) and Keister & Buck (1965) have concluded that there is a general inability for insects to compensate metabolically for thermal fluctuations. On the other hand, a few cases of capacity acclimation to temperature, even at the enzymatic level, have been reported for a variety of insects by different

211

212 S.P. SINGH and A. B. DAS

workers such as Mutchmor (1967), Precht (1967), Somme (1968), Burr & Hunter (1970), Hunter & Cediel (1970), Anderson & Mutchmor (1971), Davison (1971), Das & Singh (1972) etc. Acclimation to high and low temperatures has been already demonstrated for nymphs and the two sexes of adult Periplaneta americana, with respect to the rate of respiratory metabolism of whole insects (Dehnel & Segal, 1956; Singh & Das, 1977a), the rate of endogenous oxygen consumption of tissues (Das & Singh, 1974), the cata- lytic efficiency of certain enzymes (Mutchmor, 1967; Thiessen & Mutchmor, 1967; Singh & Das, 1977b), cytochemical reorganization and morphological transformation of the fat body tissue (Singh & Das, 1978). The present investigation throws light on the quantitative strategy of compensatory alterations in the biochemical composition of a storage tissue like fat body, a highly active tissue like coxal muscle and circulatory medium (haemolymph) of this insect due to acclimation to 35 and 15°C.

M A T E R I A L S A N D M E T H O D S

Nymphs of the penultimate instar, males and females of Periplaneta americana (Linn.), collected from local grocery stores, were acclimated to 15 + I°C and 35 + I°C in B.O.D. incubators (Munson, 1953; Dehnel & Segal, 1956; Richards, 1963; Mutchmor, 1967; Singh & Das, 1977a). The coxai muscles were dissected out from meso- and recta-thoracic legs. Besides the regular yellowish-white "foliate" fat body, the transluscent, shining-white "capsulated" type tissues (Singh & Das, 1978) were removed separately. Samples of haemolymph were obtained through punctures made at the sternal joints of thorax of the insects.

The water contents of coxal muscle and "foliate" fat body of 15 and 35*C-acclimated nymph, male and female cockroaches were calculated from the records of wet weight and dry weight of tissues (dried at 110 + I°C for 2448 h until there was no further change in weight).

The total protein, free amino adds, RNA and DNA were isolated from each tissue following the methods of Das (1967).

The protein concentration was measured using the Folin--Ciocaltcau reagent as suggested by Lowry et aL (1951), using bovine serum albumin as the standard. The quantitative assay of amino acids was conducted by ninhydrin reaction according to the method described by Moore & Stein (1948), using glycine as the standard amino acid. Yeast RNA-hydrolysate was used as the RNA standard for the determination of RNA concentration using the orcinol reagent according to the procedure of Mejbaum (1959). The determination of DNA concentration was made following Dische's (1930) method based on diphen- ylamine reaction and calf thymus DNA standard was employed for this purpose. Glycogen was isolated from the tissues following the method of Good et aL (1933).

For the separation and identification of free amino acids of the coxal muscle and the "foliate" fat body of cold and warm adapted roaches, each tissue was homogenized in 1 ml of 96~ ethyl alcohol. The supematant of this extract, obtained after centrifugation at 10009 for 15 rain and the known amino acid solutions (0.1%) were spotted on What- man No. 1 chromatographic paper. The solvent employed (Cramer, 1955} was a mixture of butanol-glacial acetic acid-water (4:1:5). The chromatograms were developed by spraying 0.2% ninhydrin solution in acetone and drying at 800C.

The data on the biochemical tissue compositions, except for glycogen, were expressed per unit DNA content instead

of the usual percentage yield values in order to avoid any possible error due to change in water content of the tissues. The glycogen concentration was. however, expressed as g~% value. In haemolymph the protein and amino acid levels were represented as g/100 ml. The significance of the data was statistically tested by the standard t-test and its modifi- cation for unknown variance in the two samples (as revealed by F-test). All the formulae for the tests of significance were used as given by Jacob & Self (1964). P < 0.05 was accepted as statistically significant level.

R E S U L T S

Tissue composition of water, protein, amino acids, RN A and alycogen in cold and warm acclimated cockroaches

Figures 1 and 2 illustrate the following alterations in biochemical composition of the tissues of cockroach due to acclimation to low and high temperatures:

1. A significant increase (P < 0.001) is evident in the water content of the "foliate" fat body of nymphs (101.2~), adult males (81.2~o) and females (71.3%) due to cold accfimation. The value is 25-35% in warm acclimated insect tissue, which becomes 58-65~ during cold acclimation. However, the coxal muscle does not demonstrate a statistically significant change in its water content (70-78~) during thermal acclimation of either nymphal or adult cockroaches.

2. Cold acclimated nymphal, female and male insects exhibit an augmentation (P < 0.01) of the protein content (protein/DNA ratio) of their "foliate" fat body up to 54.0, 60.2 and 174.7~ respectively over warm acclimated individuals. The value of protein/ DNA in the coxal muscle also increases by 67.2~ in nymphs (P < 0.01) and marginally (16.1~) in males (P < 0.05), but not in females, due to cold acclimation. The concentration of total protein in "capsu-

"o * o

140

60 4O 20

+l -

~ 40

i 020

Woter Protein/ Amino RNAI Glycogen content DNA aciO/ DNA ( q% )

(g%) DN A

Fig. 1. Percentage changes in the biochemical composition of fat body of 15°C-acclimated cockroaches (nymph, male

and female) over 35°C-acclimated insects.

Biochemical changes in Periplaneta americana (Linn.) 213

8O 6O

"o w 40 o 20 .E -Pi 000

• o " 4 0

~ 4 C

- - ' - IC

.~- ~

c o ~ 6C

~ . , -2C -4C

Nymph

Male

Fem01e

Water Protein / Amino RNA / Glycogen content" DNA acid/ DNA ( g %) (9%) DNA

Fig. 2. Percentage changes in the biochemical composition of coxal muscle of 15°C-acclimated cockroaches (nymph,

male and female) over 35°C-acclimated insectg

lated" fat body (protein/DNA value) and haemolymph (g%) were found to remain 62-71 and 3-4 respectively in nymphal as well as adult roaches, irrespective of their thermal history.

It is noteworthy that the highest level of total protein is found in the "capsulated" fat body of cockroach, whose protein/DNA ratio is almost 100°/0 more than that of the "foliate" tissue in cold acclimated insects (22-35, equivalent to 3-5g% protein) and about 200% more than that of the same tissue in warm acclimated insects (10-22, equivalent to 2.6-3.6 g% protein). The protein contents of coxal muscle in nymphal (3.0-5.5g%) and adult male (5.7-5.9 g%) roaches are not significantly different than the values of fat body Cfoliate" type) in the same insects. However, the female cockroach seems to pos- sess a slightly lower concentration of protein in its leg muscle (4.9-5.4 g%) and a slightly higher protein yield of its "foliate" fat body 0.6-4.8 g%) than the male insect (5.7-5.9 g% in leg muscle and 2.6-3.3 g% in "foliate" fat body).

3. The free amino acid pool size, as measured by amino acid/DNA ratio, in the "foliate" fat body of 15°C-acclimated cockroaches is reduced (P < 0.02) in nymphs (32_2%) and in females (28.2%), but shows an increase (P < 0.01) of 46.7% in males compared to that of 35°C-acclimated insects. The coxal muscle of cold acclimated nymph and female roaches, unlike the males, exhibits a diminution (P < 0.05) in the amino acid concentration (30.1 and 27.5% respectively) as compared to that of warm acclimated insects. The free amino acid pool size, measured as amino acid/DNA ratio in "capsulated" fat body (3--4) and gO/0 value in haemolymph (0.5--0.6), do not change due to thermal acclimation, in either adult or nymphal cockroaches.

It is also interesting to note that the largest free amino acid pool size is present in the coxal muscle

(1.0-1.5 g% or 3--7 in amino acid/DNA ratiog This value is marginally higher than that of the "capsulated" fat body, but is significantly greater than that of the ~foliate" fat body (0.3-0.5 g% or 2-3 in amino acid/DNA ratio) of nymphal and adult male cockroaches. The level is remarkably lower in this tissue of female insects (0.2-0.3 g% or 0.7-1.0 in amino acid/ DNA ratio). However, there is an apparent lack of sexual dimorphism in the free amino acid content of the leg muscle in roaches.

4. The RNA/DNA ratio of the "foliate" fat body demonstrates similar increase (20-35%) in nymphs and adults of both the sexes due to cold acclimation and this is statistically significant (P < 0.05). An augmentation (P < 0.05) of a lesser degree (13-18%) is observed in RNA/DNA ratio of the coxal muscle of 15°C-acclimated nymph and male over 35°C-accli - mated insects, while the females fail to demonstrate any alteration in the RNA concentration of this tissue due to thermal acclimation. The "capsulated" fat body presents a constant value in this biochemical parameter (1.4-1.5) in nymphal and adult roaches, irrespective of the temperature of acclimation.

It may be observed that the RNA/DNA ratio of coxal muscle (0.6-0.8 in nymphs and males, while 0.4 in females) is almost 50% of the value in "foliate" fat body (1.0-1.9 in nymphs and males, but 1.4--1.7 in females). Thus the male roach has a higher (75-85%) RNA concentration in its coxal muscle but a lower (40-45%) level in its fat body as compared to the other sex.

5. Cold acclimation increases the glycogen content of"foliate" fat body of nymphal (16%), male (9%) and female (25%) cockroaches. However, the "capsulated" fat body does not show any variation in its glycogen yield (0.4--0.5 g%) due to thermal acclimation of either nymphal or adult insect. This value is almost I0 times less than the level in the "foliate" tissue of cold acclimated insects. The extent of the increase in glycogen content of coxal muscle due to acclimation to low temperature seems to be almost identical (27-30%) in nymphal, adult male and female roaches.

It is also noteworthy that the glycogen content of coxal muscle of male cockroach (2.7-3.5 g%) is four to six times higher than that of the same tissue of female (0.6-0.8g%) and nymphal (0.4-0.6g%) cockroach irrespective of the acclimation temperature. However, the female insect exhibits a higher value (about 30°/0) of glycogen yield of its "foliate" fat body (4.0-4.8 g%) compared to that in adult male (3.3-3.6 g%) or nymph 0.3-3.9 g%).

Qualitative analysis of free amino acid pool in the tissues of cold and warm acclimated cockroaches

Thirteen amino acids could be resolved in the haemolymph of male, female and nymphal roaches and two of these could not be identified. The identified ones were leucine, phenylalanine, methionine, tyro- sine, proline, alanine, glutamic acid, aspartic acid, arginine/glycine, histidine and cystine. Phenylalanine could not be traced in muscle, although present in fat body. Two additional amino acids, hydroxyproline and aspargine could be identified in both the tissues of the insect. However, it is interesting to note that no alteration in the number and types of amino acids occurs in a tissue due to thermal acclimation.


DISCUSSION

Alteration in water content of tissues

Fat body of cold acclimated cockroach seems to accumulate a higher (70--100%) water content compared to that of warm acclimated insect, although this is not true for coxal muscle. However, it is interesting to note that coxal muscle has a higher water content than the normal "foliate" fat body in this insect, irrespective of its thermal history. Kanungo & Prosser (t959) reported a diminution of water content in goldfish muscle and liver during cold acclimation, but Murphy (1961), Heinicke & Houston (1965) and Das (1967) could not confirm this. Burr & Hunter (1969) did not observe any difference in water content of four species of Drosophila due to acclimation to high and low temperatures. Although Precht et al. (1973) have suggested an association of cold acclimation with diminution of free water content of tissues in ectotherms, an increase in water content, rather than a decrease, seems to characterize the acclimation of insects to low temperature (Baldwin & House, 1954; Edwards, 1958). As a matter of fact the decrease in osmotic concentration (Atwal, 1960) and specific gravity (Baldwin & House, 1954) of haemolymph in certain insects during cold acclimation can be explained on the basis of an increase in free water content. In particular in a storage tissue like fat body of cockroach, which exhibits an accumulation of macromolecules like protein and RNA due to acclimation to 15°C, an osmotic increase of water content is nor- mally expected, as actually observed in the present investigation. It is noteworthy that the variations of total protein and RNA concentrations in the coxal muscle due to thermal acclimation are much less when compared to those in fat body.

Alteration in protein content of tissues

While in the case of nymphal cockroach both fat body ("foliate") and coxal muscle exhibit a significant increase (55-65%) of total protein content, the augmentation of protein accumulation is more pronounced in fat body (60--175~o) than in coxal muscle (0-15%) of adult insects due to cold acclimation. The "foliate" fat body of 15°C-acclimated male roaches in particular demonstrates a 175~o increase in the steady state concentration of protein over 35°C-acclimated insects. This situation is interestingly analogous to the observation of Das (1967) regarding a similar greater accumulation of total protein in liver than in muscle of goldfish during cold acclimation. Fat body of an insect and liver of a fish are both examples of storage tissue and are also the chief regulatory sites of inter- mediary metabolism in these animals.

Dean & Vernberg (1965) reported an "inverse compensation" (diminution) of protein synthesis during cold acclimation of Uca. Burr & Hunter (1969) failed to observe any difference of protein nitrogen content between fruit flies (Drosophila species) grown at 15 and 25°C. On the contrary, Rhodnius prolixus exhi- bited an augmentation of protein concentrations in abdominal fat body and haemolymph in association with cold acclimation (Okasha, 1964). The fact that an increase in protein level of tissues characterizes the phenomenon of cold acclimation of ectotherms (Precht et al., 1973) is confirmed by the data of the

present investigation. Hence, it would be very interesting to explore further the molecular mechanisms underlying the phenomenon of increased net accumulation of protein in cockroach tissues (particularly fat body) during cold acclimation, as analysed in terms of the kinetics of absolute synthesis and degradation in fishes like Carassius auratus (Smith & Morris, 1966; Morris & Smith, 1967; Das & Prosser, 1967), Opsanus tau (Haschemeyer, 1968, 1969a, b; Hasehemeyer & Persell, 1973; Nielson et al., 1977), Salmo gairdneri (Dean & Berlin, 1969) and Gillichthys mirabilis (Somero & Doyle, 1973). McCarthy et al. (1976) have already reported a compensatory translation (Prosser, 1973) of the rate of incorporation of [3H]-leucine into the acid soluble fraction of lobster (Homarus ameri. canus) tissues during acclimation to 5 and 20°C.

Alteration in free amino acid pool size of tissues

The extent of diminution (28-32~o) in amino acid/ DNA ratio in "foliate" fat body and coxal muscle of nymphs and female cockroaches during acclimation to 15°C, when compared to the 35°C-acclimated insects, cannot explain the much greater increase in protein/DNA ratio recorded in both of these tissues in nymphs (54 and 67%) and in fat body only of females (60%) due to cold acclimation. In male fat body, on the contrary, a significant increase (47%) has been noticed in amino acid/DNA ratio, in conjunc- tion with a 175% augmentation of protein accumulation, during acclimation to low temperature. The coxal muscle of male insects, exhibiting only a margi- nal (16%) accumulation of protein, shows no change in free amino acid concentration during cold acclimation. Therefore, the role of the availability of free amino acids as a limiting factor in the thermal acclimation of net synthesis and accumulation of total protein in cockroach tissues cannot be determined on the basis of the inconsistent changes noticed in the two sexes of the adult insect. This can only be finally decided through kinetic analysis of the incorporation of labelled amino acids into the free amino acid pools of the insect tissues, as was done in goldfish by Das & Prosser (1967). The previously reported close association of the enhancement in protein accumulation and diminution in free amino acid level in tissues of tropical invertebrates during cold acclimation (Rag- hupathiramireddy & Rao, 1963; Saroja & Rao, 1965; Rao, 1967) cannot be confirmed in case of Periplaneta americana on the basis of the present investigation.

No qualitative change in the free amino acid pool has been detected in the tissues and haemolymph of cockroach due to thermal acclimation, although Anders et al. (1964) found a diminution of glutamic acid content (in spite of an increase of the total free amino acid level) of Drosophila melanogaster due to a decrease in the rearing temperature from 30 to 15°C, and Hansen & Viik (1975) reported an increase of alanine concentration in the diapausing third and fourth instar larvae of Arctia caja due to its maintenance at 0 to - 5 ° C for more than 2 weeks. Barlow (cited by Prosser, 1967) observed an increase in concentrations of some amino acids, a decrease of some and no alteration of others in fish brain during warm acclimation. The significance of change in the concentration of specific amino acids in thermal acclimation


and extreme temperature tolerance of ectotherms is still unresolved.

Protein and free amino acid levels of haemolymph Although Okasha (1964) observed a 1000/0 increase

in the amino acid level and a remarkable decrease in the protein content of haemolymph in Rhodnius prolixus at 36.5°C over the insects maintained at 28°C, this is not true for another insect, Periplaneta americana acclimated to 35 and 15°C as found by the present investigators. A strict regulation of the protein and amino acid concentrations of the haemolymph, in spite of the significant changes in the tissues, apparently characterizes the thermal acclimation of this insect.

Alteration in RN A concentrations of tissues An increase in the RNA/DNA ratio induced by

cold acclimation can be correlated with increased accumulation of protein in the same tissue of cockroach. For example, the higher protein concentration due to cold acclimation is more pronounced in fat body than in coxal muscle of the male insect, which also exhibits a greater increase in RNA concentration in fat body (35%) than in coxal muscle (13%). On the other hand, an enhanced accumulation of protein due to low temperature of acclimation is noticeable only in fat body of the female insect, which also demonstrates a significant increase in RNA/DNA ratio of its fat body (20~) but not of coxal muscle. The degree of augmentation of RNA content in both the tissues of cold adapted nymphs over warm acclimated ones is almost identical (18-20%), which corroborates with a similar accumulation of protein (55-65%) in these tissues induced by cold acclimation. Such alterations in RNA/DNA ratio accompanying the changes in protein concentration may be indicative of a compensatory augmentation of net protein synthesis during cold acclimation. Similar changes in RNA content of tissues were reported earlier in thermal acclimation of Indian freshwater mussel (Rao, 1963) and earthworm (Saroja &Rao, 1965) and also in the insect, Droso- phila viracochi (Burr & Hunter, 1969). However, a kinetic analysis of the turnover rates of the different fractions of RNA, for determining the compensatory alteration of net synthesis of RNA during thermal acclimation, has been attempted only in goldfish tissues (Das, 1967) and plant cells (Siminovitch et aL, 1967; Devay & Paldi, 1977).

It is noteworthy that the RNA/DNA ratio of leg muscle of cockroach (nymphs or adults) is significantly lower (50°/0) than that of fat body, irrespective of the temperature of acclimation although the level of total protein of both these tissues is almost of the same order. This is similar to the situation in rat, reported by Florini (1962), and Florini & Breuer 0965) or in goldfish, reported by Das (1967), regarding the lower level of protein synthesis and the lower level of ribosomal RNA in muscle than in liver of the animal.

Alteration in glycogen yield of tissues The carbohydrate nature of the respiratory sub-

strate is indicated for leg muscle of the American cockroach by the value of its respiratory quotient being close to unity (Cornwell, 1968). Although a

strict correlation between glycogen availability in a tissue and the rate of glycogenolysis associated with the respiratory metabolism is not possible, the earlier findings of Bah'on & Tahmisian (1948), confirmed by the present investigators, are strongly suggestive of an intimate association between the 4=6 fold difference in glycogen level of leg muscle of male and female Peri- planeta americana and a similar sexual d/morphism of the respiratory rate (Das & Singh, 1974).

McWhinnie (1967) reported in the crayfish, Orco- nectes virilis, a reduction of glycogen level in muscle but no change in hepatopancreas during acclimation to 9°C when compared to 24°C. The present investigation on female cockroaches reveals a significant (25%) enhancement of glycogen accumulation in fat body ("foliate"), correlated with the compensatory increase of in vitro oxygen consumption (without any exogenously added sub strate) of this tissue (Das & Sing, h, 1974), due to cold acclimation. The narrow margin of increase in the glycogen yield of fat body during acclimation to 15°C of male (9%) and nymphs (16%), over 35°C-acclimated insects, may be corrobor- ated with the lack of thermal compensation of endogenous respiratory rate of this tissue in nymphal and male roaches (Das & Singh, 1974). An almost identical increase (27-30%) in glycogen reserve of coxal muscle of nymphs and adult insects of both the sexes during adaptation to 15°C is strikingly similar to an almost translational compensatory pattern of respiratory rate of this tissue against thermal fluctuation (Das & Singh, 1974).

Biochemical alteration underlying the cytomorphologi- cal transformation of fat body

The most important biochemical alteration in the transformation of "foliate" fat body into its "capsulated" form, accompanying cold acclimation of the insect (Singh & Das, 1978), is the remarkably high degree (200%) of protein (but not RNA) accumulation. The glycogen yield, however, is significantly less in "capsulated" fat body compared to the "foliate" tissue in roaches maintained at 15°C. These changes seem to be chiefly related to the storage func- tion of the "capsulated" fat body in cold acclimated roaches, because this tissue has been reported as being metabolically inert (Das & Singh, 1974).

Sexual dimorphism in cellular chemistry and degree of thermal acclimation

A sex-correlated difference in biochemical tissue composition and the extent of compensatory changes during acclimation to high and low temperatures is evident from the data recorded in the present investigation. A male cockroach possesses a greater (12%) protein content, a much greater (80%) RNA concentration and glycogen reserve (4--6 fold) of its coxal muscle than a female insect. On the other hand, a female insect has higher protein yield (400/0), RNA/ DNA ratio (60%) and glycogen level 00%) of its "foliate" fat body than a male. Even in the degree of compensatory alterations of protein and RNA accumulation in tissues due to thermal acclimation there is a sex-specific difference. The females, in con- trast to males, do not exhibit any compensation in their leg muscle although the acclimation changes are evident in their fat body. Such a sexual dimorphism in

216 S. P. SINGH and A. B. DAS

the pattern of thermal response of this insect may be due to hormonal changes associated with reproduc- tion in the two sexes. The role of endocrines in metabolic compensation to thermal changes in poikiioth- erms is not yet finally resolved (Prosser, 1967; Precht et al., 1973). The only documented case in insect is the report by Clarke (1966) on the plausible stimulation of protein synthesis at low temperature (15°C) in Locusta mioratoria through neurosecretion, as evi- denced by the exhaustion of corpora cardiaca.

The observations presented in this paper are strongly suggestive, in case of an insect for the first time, of the validity of the generalization made by Hochachka (1967) and Somero & Hochachka 0976) regarding "a biosynthetically directed metabolic reorganization" and the accompanying changes in cellular metabolism of ectotherms during cold acclimation. A poikilotherm, like the cockroach, can afford to chan- nel a larger proportion of its additional free energy into processes like net synthesis and accumulation of protein, RNA and glycogen in its tissues at low temperature of acclimation, perhaps due to the reduction of the maintenance metabolism. Such a quantitative strategy employed for cold acclimation is not a rate- compensatory mechanism per se. However, it would be extremely interesting and challenging to explore the underlying qualitative strategy of compensatory regulation of the rates of absolute synthesis and degradation of the gene products (protein and RNA) in the tissues of cockroach during thermal acclimation, which has been successfully attempted in case of a number of fish, a frog, a lobster, the black locust tree and the frost-resistant "winter wheat", as dis- cussed already.

Acknowledgements--The authors are indebted to Pro- fessor H. S. Chaudhary, Head of the Department of Zoo- fogy, University of Gorakhpur, for providing necessary facilities (where this work was conducted) and to the State Council of Scientific and Industrial Research, U.P. (India) for financial support during the work. Gratitude to Pro- fessor C. L. Prosser, Department of Physiology and Bio- physics, University of Illinois, is also expressed herewith for many helpful suggestions and criticisms.

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Key Word Index--Insect; Periplaneta americana; cockroach; acclimation; biochemical composition of fat body and muscle.

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Biochemical changes in tissue composition of Periplaneta americana (Linn.) acclimated to high and low temperatures