Changes in dry weight, protein, and nucleic acid content during diapause and normal development of the blowfly, Lucilia sericata

9. Insect Physiol., 1973,VoI. 19,pp. 481to 494.Pergamon

Press. Printed in Great Britain

CHANGES IN DRY WEIGHT, PROTEIN, AND NUCLEIC ACID CONTENT DURING DIAPAUSE AND NORMAL DEVELOPMENT OF THE BLOWFLY, LUCILIA SERICATA R. A. RING Department

of Biology, University of Victoria, Victoria, B.C., Canada

(Received 1 January

1972; revised 24 August 1972)

Abstract-Dry weight (D.W.), protein, RNA, and DNA have been determined in the blowfly, Luciliu sericata, throughout all stages of normal development in long and short photoperiod regimes and during larval diapause. During normal development protein/D.W. levels fluctuate markedly during the larval and puparial stages, increased levels being correlated with the synthesis of new cuticle, etc. prior to ecdysis and the histogenesis of adult tissues prior to emergence. Protein levels remain relatively high and constant during adult life to senescence. RNA/D.W. levels are highest in first instar larvae but decline rapidly during larval development until just before puparium formation. Sharp increases are found prior to pupation and then again prior to adult emergence. In the adult stage, the levels decline steadily throughout the life span. DNA/D.W. levels are very low in the egg but reach their highest levels in early first instar larvae. They then decline during larval development, with small increases being found prior to puparium formation and adult emergence. Adult levels remain relatively constant throughout the life span. The RNA/DNA ratio has extremely high values in the egg, indicating the high degree of synthetic activity that takes place during embryogenesis. There is a steady decline in RNA/DNA values during adult life to senescence in both sexes, suggesting that physiological ageing in L. sericata is accompanied by a decrease in protein synthesis potential. During larval diapause all parameters, with the exception of the RNA/DNA ratio, are maintained at low and constant levels, reflecting the fact that diapause is a period when synthetic and mitotic activity are minimal. The great variations in RNA/DNA levels, however, indicate that individuals within a group of larvae can terminate diapause spontaneously at 24°C and return to the normal processes of morphogenesis. INTRODUCTION MANY of the changes taking place during insect morphogenesis and the induction and termination of larval diapause are under the control of hormones. The manner in which these hormones produce their effects is not well understood, but in recent years evidence has been growing which indicates that the moulting hormone, at least, may be acting directly on the chromosomes (WIGGLESWORTH, 1970). It would seem that the effect of the hormone is to free some latent component of the gene system from repression so that specific messenger RNA can be produced. This passes into the cytoplasm and protein synthesis follows. The study of nucleic 15

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acid and protein metabolism, therefore, is of obvious interest in the study of diapause physiology. There is little information available concerning the events surrounding the resumption of normal morphogenesis in diapause larvae of Luciliu spp. By convention the termination of diapause in Lucilia larvae is ‘pre-dated’ by the appearance of the puparium. The study of protein, RNA, and DNA levels would appear to be a more accurate way of defining diapause in biochemical terms and of determining the exact timing of diapause termination and the resumption of growth. In the present experiment dry weight, protein, RNA, and DNA measurements have been determined in the blowfly, Lucilia sericuta, throughout all stages of normal development and during larval diapause. MATERIALS

AND METHODS

C%emicals

Yeast RNA (Type XI, Sigma Chemical Co.) was used to determine the standard curve for RNA from 3 to 30 pg RNA/ml O-2 N perchloric acid (PCA). Highly polymerized calf thymus DNA (Type I, Sigma Chemical Co.) was used to prepare the DNA standard curve for concentrations from 1.46 to 14.64 pg DNA/ ml O-3 N KOH. Casein (J. T. Baker Co.) was used to prepare the protein standard curve for use with the Lowry reagents. A standard curve was determined for concentrations ranging from 10 to 400 pg protein/ml 0.15 N KOH. Blowjies

Blowflies were obtained from a colony which had been reared in the laboratory for 23 generations. Adults and larvae were reared at 24°C according to standard procedures already outlined (RING, 1967) in both long (LPR = 18 hr light; 6 hr darkness) and short (SPR = 12 hr light; 12 hr darkness) photoperiod conditions. Eggs were taken within 1 hr of oviposition. Larval samples (5 larvae per sample) were taken on the day of hatching and at daily intervals thereafter until puparium formation. Larvae entering diapause were sampled at weekly intervals up to a maximum of 12 weeks, all diapause larvae coming from the SPR. Puparia were sampled on their first day and then at 48 hr intervals thereafter until adult emergence. Adults were taken on the day of emergence and sexed, the sexes being kept separate. Experimental animals were maintained on a diet of sugar and water and samples (3 adults per sample) were taken at weekly intervals until death of the remaining individuals of the colony. Each sample was washed with distilled water, weighed for wet weight, and then freeze-dried. Samples were stored in the deep-freeze at -20°C until used in the experiment. Preparation

of samples

When the desired range of samples had been accumulated, each vial, containing one sample, was placed on the freeze-drier for 30 min to remove any residual moisture. Dried samples were then weighed on a Cahn G2 electrobalance for dry

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weight content. Four millilitres of ice-cold distilled water were added to each sample (10-20 mg of sample proved best) and the samples homogenized in a ground-glass tissue grinder. During homogenation the tubes were kept cold in an ice jacket. The homogenate was then poured into a centrifuge tube and the homogenizer rinsed with 1 ml cold distilled water which was added to the homogenate. From the total 5 ml sample, a 1 ml aliquot was removed for protein analysis, the remaining 4 ml being employed in nucleic acid analyses. Ten to twelve samples were analysed concurrently. The analytical methods of DAGG (1969) were used whereby protein, RNA, and DNA could be determined from the same homogenated extract. Protein determination Protein determinations were carried out using a technique modified after LOWRY et al. (1951). T o each protein aliquot was added 1 ml 0.3 N KOH and the tubes left for at least 30 min so that the protein could dissolve. The tubes were then cooled and centrifuged at 0°C for 5 min at 10,000 revlmin. Two hundred microlitres of supernatant were extracted and mixed with 2 ml of the alkaline copper reagent (50 ml 2% Na,CO, in O-1 N NaOH+ 1 ml 05% CuSO, in 1% Na citrate). The tubes were allowed to sit for 15 min before adding 200 ,uI of Folin’s reagent (50% 2 N Phenol reagent supplied by Fisher Scientific Co.). The contents of each tube were thoroughly agitated as the Folin reagent was being added. The tubes were then allowed to sit for 1 hr before being read at 750 nm on a Beckman DB-G spectrophotometer. The absorbance was read and plotted on the standard curve to give protein content. Nucleic acid determination Accurate determination of nucleic acid content in biological material poses many problems which have been reviewed by HUTCHINSONand MUNRO(1961) and MUNROand FLECK(1966). Essentially, the indole method (CERIOTTI, 1952) slightly modified by HUTCHINSONand MUNRO(1961) was adopted for DNA determination and the recommendations of MUNRO and FLECK (1966) for the SCHMIDT and THANNHAUSER (1945) technique were adopted for RNA determination. Freshly prepared homogenates were fractionated as quickly as possible and kept at 0°C to minimize any nuclease activity. To the 4 ml homogenate of each sample was added 2 ml ice-cold 0.6 N perchloric acid. At this point a blank containing 4 ml distilled water and 2 ml 0.6 N PCA was prepared and treated similarly to all other samples. Homogenates were placed in a refrigerated centrifuge precooled at 0°C and allowed to stand for 10 min before centrifugation for 10 min at 10,000 rev/min. The supernatant was discarded and the precipitate washed twice with 2 ml cold O-2 N PCA. Four millilitres of O-3 N KOH were added to the washed precipitate and the tubes were placed in a hot water-bath at 37°C for 1 hr. After incubation the tubes were replaced in the ice jacket and 2.5 ml 1.2 N cold PCA added to

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each tube. Again they were allowed to stand for 10 min at 0°C before being centrifuged at 10,000 rev/min for 10 min. The supernatant contained the RNA and the precipitate the DNA. The precipitate was washed with 4 ml cold O-2 N PCA, centrifuged, and the supernatant pooled with that from the previous centrifugation. The pooled supernatants were then ready to be read at 260 nm for RNA content on a Beckman DB-G spectrophotometer. LINZENand WYATT (1964) and Wyatt (personal communication) reported complications in RNA measurements of the tissues of Cecropia silk moth pupae due to the interference of uric acid. A preliminary experiment was therefore carried out to determine the extent to which uric acid contaminated the RNA extracts of L.. sericata. A scanning absorption spectrum, from 190 to 450 nm, was conducted on the RNA extracts prepared by the method above. Two peaks occurred, at 205 nm and at 260 nm, with only minimal absorbance at 290 nm, the peak absorption wavelength of uric acid. It was concluded from this and from subsequent uricase tests, that the readings for RNA concentration in L. sericata were prejudiced little by the presence of uric acid when using the extraction procedure described above. The precipitate containing DNA was mixed with 4 ml 0.3 N KOH and allowed to stand for 30 min. Two millilitres of the mixture were then taken and to these samples were added 1 ml 0.04% indole solution and 1 ml concentrated HCl. The tubes were placed in boiling water for 10 min, removed, and then placed in an ice-bath for a few minutes until cool. Each sample was then shaken vigorously for approximately 1 min after the addition of 5 ml of chloroform. After centrifugation for 5 min at 8000 rev/min the top aqueous phase was withdrawn and the extraction with chloroform done twice more. The aqueous phase was read at 490 nm for DNA content. RESULTS

Since Lucilia larvae are variable in size and weight depending on their nutritional state, results are presented in ratios to dry weight (D.W.). As HINTON (1968) points out, in many studies on the biochemistry of the pupal stage the biochemistry of the pharate adult is often attributed to the pupa and the early part of the pupal stage, the pharate pupa, is usually ignored. Since the precise timing of events during the pupal and pharate adult stages of L. sericata is not known with any degree of accuracy, the whole period has been designated the puparial period and is dated from the formation of the white puparium. Protein

Protein/D.W. levels fluctuate during larval life, showing three important peaks at 2, 4, and 7 days during the course of larval development (Fig. 1). First instar larvae have slightly higher protein contents than their corresponding egg stages, but thereafter these levels decrease during each instar with a sharp increase occurring prior to ecdysis when the larvae are synthesizing new cuticle, etc. The increase in protein content at 7 days can be closely correlated with the proliferation of imaginal disks as well as the synthesis of new cuticle. In the LPR, protein/D.W.

CHANGES

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NORMALLY

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BLOWFLY

levels return to approximately the same values after each moult, but in the SPR the levels fall during larval life, becoming progressively lower in the second and third instars. Protein levels immediately prior to puparium formation are, however, comparable in both photoperiod regimes, and fall to approximately the same level during the puparial period.

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FIG. 1. Protein1D.W. ratios during development of L. sericata in long and short photoperiod conditions. Immature stages in short photoperiod conditions, O-O-O ; immature stages in long photoperiod conditions, 0 . . . . . . . 0 . . . . . . .O ; adult males in short photoperiod conditions, O-O-O ; adult females in short photoperiod conditions, O- * - * -O- * - -0 ; adult males in long photoperiod conditions, 0 . . . . . . . 0 . . . . . . .O ; adult females in long photoperiod conditions, O- - - - -O- - - - -0. l

In the puparial stage, protein/D.W. levels drop for the first few days during the period of histolysis of larval tissues, but increase again rapidly to reach immediate prepupal levels before adult emergence, i.e. during the period of histogenesis and differentiation of adult tissues. The protein levels then remain relatively high throughout adult life with only minor fluctuations occurring. Males and females have similar protein/D.W. levels. These adults were maintained on sugar and water with the absence of protein in their diet, hence the lack of fluctuation in the females’ protein content. Females fed on protein and going through normal cycles of oijgenesis would be expected to show correlated fluctuations in protein content. Adult protein content in the SPR, although as constant as that in the LPR, is at a generally lower level (0.36-0.72 compared to 044-0~73). It should be noted that adult longevity is significantly greater in the SPR than in the LPR, a phenomenon which has been noticed consistently during the cultural history of this species in the laboratory. Furthermore, the development of the immature stages also takes longer in the SPR, being 7 days in the larval stage, compared to 6 days in the LPR, and 8 days in the puparium, compared to 6 days in the LPR. During larval diapause the protein/D.W. ratio remains relatively constant throughout the 12 week test period (Fig. 7A). There is, however, a slight increase in this ratio in 12 week diapause larvae perhaps indicating some prepupal metabolic activity. It has already been determined that most larvae break diapause spontaneously within 2 to 12 weeks when maintained at a constant temperature of

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24°C (Ring, unpublished). The level of this ratio which is maintained during diapause is approximately the same as that of 6-day-old non-diapause larvae maintained under SPR, i.e. a point which corresponds to an intermoult period when there is little mitotic activity. RNA RNA content can be considered an index of the capacity of an organism for protein synthesis. Total RNA is, therefore, a measure of the potential rate of protein synthesis. Although RNA/D.W. levels in the eggs are already high, they are more than doubled in the first instar larvae in both photoperiod regimes (Fig. 2). These levels rapidly decrease by 90 to 95 per cent during larval develop-

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FIG. 2. RNA/D.W.

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ratios during development of L. sericutu in long and short photoperiod conditions. Legend as in Fig. 1.

ment, reaching their lowest level in 6-day-old third instar larvae. There follows a sharp increase in RNA levels in the 7- to S-day-old larvae prior to puparium formation, but a further decline occurs in early puparial life, during histolysis, before increasing once again during the pharate adult stage. Newly emerged adults have RNA levels equivalent to that of 3- to 4-day old larvae, but these levels drop steadily throughout the adult life span until they are approximately one-half of the original values. During diapause the RNA/D.W. levels fluctuate very slightly within a narrow range of values (Fig. 7D) equivalent to S-day-old non-diapause larvae under short photoperiod conditions. There is, however, once again the indication of increased RNA activity (2.7 fold increase) at the lZweek-old diapause larva stage reminiscent of the activity seen just prior to puparium formation in non-diapause larvae. This probably indicates the termination of diapause and a resumption of normal

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morphogenesis, although this has to remain speculative since there is no accurate way of determining the precise point of diapause termination in an individual.

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FIG. 3. RNA/protein ratios during development of L. sericata in long and short photoperiod conditions. Legend as in Fig. 1.

DNA DNA/D.W. levels are very low in the egg during the early stages of embryogenesis, but reach their highest levels during development in the first instar larvae, sampled within 12 hr of eclosion (Fig. 4). During that period there are thirty-fourand fifteenfold increases in the DNA levels in the LPR and SPR respectively.

FIG. 4. DNA/D.W.

ratios during development of L. sericata in long and short photoperiod conditions. Legend as in Fig. 1.

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There follows a rapid decline in DNA/D.W. during larval life reaching low levels at 3 to 5 days in the LPR and 4 to 7 days in the SPR. A slight increase in this ratio occurs immediately prior to puparium formation, dropping-off during histolysis in the pupa, with a resurgence during the pharate adult stage. The adult values are equivalent to Z- to 6-day-old larvae, or early and late puparium levels, and are maintained at relatively constant levels throughout the life span. No significant differences in this respect can be detected between males and females, and the ratios are similar in corresponding life stages in both photoperiod regimes except, perhaps, in the early puparium where it is higher in LPR than in SPR. In diapausing larvae, the DNA/D.W. ratio remains remarkably constant throughout the diapause period of a population (Fig. 7B). The onefold increase at the 12 week period reflects the increased DNA activity which is also seen in the prepupal activity of non-diapause individuals. Similarly, those peaks after 2 and 4 weeks in the diapause state could represent individuals in the sample that had completed their diapause development and had resumed the normal morphogenesis pre-requisite to puparium formation. Otherwise DNA/D.W. levels of diapausing larvae remain low, comparable with 6-day-old non-diapause larvae or with early puparial stages in the SPR.

FIG. 5. DNA/protein ratios during development of L. sericata in long and short photoperiod conditions. Legend as in Fig. 1.

RNA/DNA The per cell. increase increase capacity first-day

RNA/DNA ratio can be regarded as an index of protein synthesis capacity Since cyclorrhaphan larvae grow by increase in cell size and not by in cell number (although AGRELL (1964) points out that cell nuclei can in size), fluctuations in this ratio represent variations in protein synthesis per whole animal. Peaks in these curves in first-day third instar larvae, puparia (not apparent in SPR), pre-emergence pharate adults, and

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newly emerged adults (Fig. 6), are reflections of the increased protein synthesis needed for new cuticle formation and formation of new tissues. Extremely high values of the RNA/DNA ratio in the egg stage indicate the high degree of synthetic activity that takes place during embryogenesis. In the adult stage the levels of this ratio decline slowly throughout the life span of both males and females in both photoperiod regimes.

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FIG. 6. RNA/DNA ratios during development of L. sericata in long and short photoperiod conditions. Legend as in Fig. 1.

During diapause there are great variations in RNA/DNA levels in the larval population (Fig. 7F). This is probably due to individuals within a group which terminate diapause spontaneously at any time during the range in diapause intensity in this species (2-12 weeks), and in which the normal processes of morphogenesis have been, or are about to be, restored. The RNA/DNA levels in diapause larvae are relatively high, being equivalent to those of second instar or early third instar larvae, indicating that the ability to synthesize proteins is retained by all larvae during diapause. DISCUSSION There is a variety of ways for measuring growth in insects based on physical, physiological, and biochemical parameters. In this study the biochemical parameters of protein, RNA, and DNA levels in whole insects have been used to study the growth of the sheep blowfly, L. sericata, throughout normal growth and during larval diapause, a period during which normal growth and morphogenesis cease. Relationships between growth in insects and RNA content have already been reported by a number of authors. RNA concentration has been used in growth

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c.

mg DNA/ mg Protein

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3

4

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FIG. 7. Biochemical changes during the refractory period of diapause development in a population of L. sericata larvae maintained under short photoperiod conditions. A, Protein/D.W.; B, DNA/D.W.; C, DNA/protein; D, RNA/D.W.; E, RNA/ protein; F, RNA/DNA.

studies of Aedes aegypti L. (LANG et al., 1965), Anthonomus grandis Boheman (VICKERS and MITLIN, 1964), D rosaphiIa melanogaster (CHURCHand ROBERTSON, z ora erythrocephala Mg. (LEVENBOOK, 1953; 1966; BURRand HUNTER, 1969), Ca ZZ’ph PRICE,1965; SEKERI et al., 1968), L. cuprina (LENNIEet al., 1967), GryZZusbimaculatus (KRISHNAKUMARAN, 1961), and also in the crustaceansArtemia salina and Euchaeta japonica (Dagg, 1969). However, in most studies specific tissues or specific life stages only were selected for analysis. In this study, the various parameters have been determined in whole insects throughout the complete life span

CHANGES IN DIAPAUSING AND NORMALLY

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in an effort to define growth and diapause in biochemical terms, and to determine the relationships between diapause and the known hormonal activities of this species. RNA content can be considered an index of the capacity of the organism for protein synthesis, and DNA content an estimate of cell number. The RNA/DNA ratio is, therefore, an index of the protein synthesis capacity per cell (LANG et al., 1965). The use of DNA as an index of cell number is complicated in cyclorrhaphan Diptera where larval growth in most tissues is restricted to an increase in cell size and not to an increase in cell number. As AGRELL(1964)points out, however, the growth in cell size in many species is not restricted to the cytoplasm but also involves growth of the nucleus resulting in polyploidy and polyteny. The decreasing DNA/D.W. values found during the larval development of L. sericata, therefore, do not accurately predict increasing dry weight content of the larvae since decreasing DNA values and increasing dry weight content may not be linearly related. They do, however, reflect the increase in cell size that occurs during larval development. The greatest fluctuations in concentration of protein, RNA, and DNA per organism occurred during the maturation stages of development, i.e. the egg, nondiapause larval, and puparial stages. It is obvious that the moulting cycle had important effects on protein and RNA synthesis. These effects were particularly apparent when the protein1D.W. curve was examined, the cyclic secretion of new cuticle, development of imaginal disks, etc. being indicated by sharp fluctuations in protein synthesis activity. The highest RNA/DNA ratios were present in the egg stage in both photoperiod regimes. Such high values indicate the extremely rapid rate of protein synthesis that takes place during the early embryonic development of the insect egg. In first instar larvae twenty- and sevenfold decreases occurred in the RNA/DNA ratio in the LPR and SPR respectively. However, sharp increases were found in samples taken on the first day of the third instar larval stage (day 3), early puparial, and early adult stages, indicating increased protein synthesis at these moults. There was a steady decline in RNA/DNA values throughout adult life to senescence in both sexes. Contrary to the results of LANG et al. (1965) with the mosquito, these results suggest that physiological ageing in L. sericatu is accompanied by a decrease in protein synthesis potential. This appears to be valid for both sexes. In females undergoing normal cycles of oiigenesis, cyclic changes in protein synthesis would be anticipated. However, in this experiment females were maintained on a sugar and water diet only, so that the normal sequence of oijgenesis was prevented. This explains the lack of cyclic changes in protein synthesis during their life span. The DNA/protein ratio was highest in newly hatched larvae and fell to approximately 25 per cent of that value by the second day of larval life, whereafter it remained relatively constant except for a decrease during the third larval instar prior to puparium formation (see Fig. 5). The RNA/protein ratio (Fig. 3) followed a similar curve but with its fluctuations lagging slightly behind those of the DNA/ protein curve. The RNA/protein ratio was also highest in the early larval stages and showed a steady decline to 10 per cent of its original value at the third day before

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puparium formation (day 5 in LPR; day 6 in SPR) when there followed a slight increase. During the puparial period this ratio remained relatively constant in the SPR but decreased in the LPR until a few days before adult emergence when another peak, equivalent to 25 per cent of the value for newly hatched larvae, was reached. A slow but steady decline followed during adult life to senescence. LANG et al. (1965) suggest that such decreasing curves are due to the fact that protein is increasing more rapidly than nucleic acids. No sexual differences were found in either ratio. During the puparial stage, which is essentially a closed system, all parameters showed decreasing values during the early part of the period, coinciding with the histolysis of larval tissues, and increasing values toward the end of the period when histogenesis and differentiation of adult tissues occurred. The changes in RNA values during the puparial period were similar to those found in C. erythrocephala (AGRELL, 1964). No significant differences in this respect were found between insects reared in the LPR and SPR. During larval diapause when mitotic activity has ceased (AGRELL, 1964) there The DNA/D.W. ratio, and all other parameters was no new DNA synthesis. except the RNA/DNA ratio, were maintained at relatively low and constant levels. Comparison with SPR larvae undergoing normal development showed that the low values for all ratios in diapause larvae were similar to those of late third instar larvae undergoing normal development. This is the prepupal period at which diapause is induced in Lucilia spp. and the period at which these parameters have their lowest values of any stage in life cycle. RNA/protein and DNA/protein curves were remarkably constant throughout the diapause period (see Fig. 7E, C), indicating that if any changes in these parameters occurred then they did so at equal rates. The fluctuations that occurred in the RNA/DNA curve, however, suggest that protein synthesis capacity per indiviThis could be credited to the fact that individuals dual varies during diapause. within a group can terminate diapause spontaneously at any time between 2 and 12 weeks, and that normal morphogenesis had been initiated. Furthermore, the irregularities that occurred in the RNA/D.W. curve, and to a lesser extent in the DNA/D.W. curve, may denote stages in diapause development (‘refractory phase’ of MANSINGH, 1971) of varying protein synthesis capacity. Low and constant levels of RNA/D.W. and RNA/DNA during diapause indicate that this stage is a period of synthetic rest. Cell division does not occur and there is little enzymatic activity, although some of the brain neurosecretory cell groups remain functioning and continue to elaborate and secrete their products (FRASER, 1959). Increased RNA activity, as reflected in the RNA/DNA ratio, would, therefore, appear to be the most reliable biochemical indicator of the termination of diapause. Such increases among diapause larvae indicate a resumption of potentially intensive hormonal and enzymatic activity in larvae which have newly completed diapause development, or about to complete it, and have returned to normal morphogenesis. It was noted with interest that larval and pupal plus pharate adult development was protracted by about 25 per cent under short photoperiod conditions, and that

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adult longevity was much greater, being 49 days for females and 42 days for males in the SPR as opposed to 21 days for females and males in the LPR. The reasons for this are as yet unknown. Acknowledgements-1 am grateful to Mr. M. J. DAGG for the analytical techniques employed in this experiment, and to KATHY RAUCHERTand BARBARARICK~ONfor technical assistance. This work was financed by a grant from the National Research Council of Canada.

REFERENCES AGRELL I. (1964) Physiological and biochemical changes during insect development. In Physiology of Insecta (Ed. by ROCK~TEINM.), 1, 91-149. Academic Press, New York. BURR M. J. and HUNTERA. S. (1969) Effects of temperature on Drosophila--V. Weight and water, protein and RNA content. Comp. Biochem. Physiol. 29,647-652. CERIOTTI G. (1952) A microchemical determination of desoxyribonucleic acid. r. biol. Chem. 198, 297-303. CHURCHR. B. and ROBERTSONF. W. (1966) A biochemical study of the growth of Drosophila melanogaster. J. exp. Zool. 162, 337-352. DAGG M. J. (1969) Relationships between growth rate and RNA, DNA, protein and dry weight in Artemia salina and Euchaeta japonica. MSc. Thesis, University of Victoria, Victoria, B.C. FRASER A. (1959) Neurosecretion in the brain of the larva of the sheep blowfly, Lucilia Caesar L. Q2uart.J. micr. Sa’. 100, 377-394. HINTON H. E. (1968) Spiracular gills. Adv. Insect Physiol. 5, 65-162. HUTCHINSONW. D. and MLINROH. N. (1961) The determination of nucleic acids in biological materials. A review. Analyst 86, 768813. KRISHNAKUMARAN A. (1961) Ribonucleic acid in the moult cycle of an insect. Nature, Lond. 189,243-245. LANG C. A., LAU H. Y. and JEFFER.SOND. J. (1965) Protein and nucleic acid changes during growth and aging in the mosquito. Biochem. J. 95, 372-377. LENNIE R. W., GREGORYD. W., and BIRT L. M. (1967) Changes in the nucleic acid content and structure of thoracic mitochondria during development of the blowfly, Lucilia cuprina. J. Insect Physiol. 13, 1745-1756. LEVENBOOKL. (1953) The variation in phosphorus compounds during metamorphosis of the blowfly, Calliphora erythrocephala Meig. J. cell camp. Physiol. 41, 313-334. LINZFN B. and WYATT G. R. (1964) The nucleic acid content of tissues of cecropia silkmoth pupae. Relations to body size and development. Biochem. Biophys. Acta 87, 188-198. LOWRY 0. H., ROSEBROUGHN. J., FARR A. L., and RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. r. biol. Chem. 193,265-275. MANSINGHA. (1971) Physiological classification of dormancies in insects. Can. Ent. 103, 983-1009. MUNRO H. N. and FLECK A. (1966) Recent developments in the measurement of nucleic acids in biological materials. A supplementary review. Analyst 91, 78-88. PRICE G. M. (1965) Nucleic acids in the larva of the blowfly, CuZZiphora erythrocephala. J. Insect Physiol. 11, 869-878. RING R. A. (1967) Photoperiodic control of diapause induction in the larva of Lucilia Caesar L. (Diptera, Calliphoridae). J. exp. Biol. 46, 117-122. SCHMIDT G. and THANNHAUSERS. J. (1945) A method for the determination of deoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. biol. Chem. 161,

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SEKERIK. E., SEKERIS C. E., and KARLSONP. (1968) Protein synthesis in subcellular fractions of the blowfly during different developmental stages. J. Insect Physiol. 14, 425-431. VICKERSD. H., and MITLIN N. (1965) Changes in nucleic acid content of the boll weevil, Anthonomus grandis Boheman during its development. Physiol. Zoiil. 39, 70-76. WIGGLESWORTH V. B. (1970) Insect Hormones. Oliver & Boyd, Edinburgh.