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The effects of starvation and subsequent feeding on juvenile hormone synthesis and oöcyte growth in Schistocerca americana gregaria
0022-1910/79/09014701
J. Insect Physiol., Vol. 25, pp. 701 to 708. 0 Pergamon Press Ltd. 1979. Printed in Great Britain.
$02.00/O
THE EFFECTS OF STARVATION AND SUBSEQUENT FEEDING ON JUVENILE HORMONE SYNTHESIS AND 0i)CYTE GROWTH IN SCHZSTOCERCA
AMERZCANA
GREGARZA
STEPHEN S. TOBE*and CANDICE S. CHAPMAN Department of Zoology, University of Toronto, Toronto, Ontario. MSS lA1, Canada (Received 16 March 1979; revised 9 May 1979)
Abstract-Theeffect of starvation on the synthesis of C,, juvenile hormone (JH) and the growth of terminal oijcytes was assessed in Schistocerca americana gregaria at two times during adult life: before activation of the corpora allata and during the first gonotrophic cycle. In both groups, starvation resulted in a decline in JH synthesis within 2-3 days and rates of synthesis remained low throughout the experimental period. The growth rate of oiicytes which were not vitellogenic at the time of starvation was depressed whereas the percentage of resorption of vitellogenic oiicytes increased dramatically with starvation. Although the percentage of resorption increased in animals with vitellogenic oiicytes, some mature otiytes were produced, particularly in animals in which the ohcytes were greater than 5 mm in length at the time of starvation. This suggests that oiicyte maturation can be divided into two distinct phases-an early phase of vitellogenesis associated with high rates of JH synthesis and a late phase, in oiicytes greater than 5 mm, associated with much lower rates of JH synthesis. Stimulation of JH synthesis by farnesenic acid in S-day starved animals resulted in high rates of JH synthesis, indicating that starvation did not appreciably alter the enzymic activities of the final two stages in JH synthesis. Thus rate limitation did not occur at these stages. Feeding of S-day starved animals resulted in a transient increase in the rate of JH synthesis. However, rates of JH synthesis and oacyte growth remained subnormal throughout the observation period, suggesting that the effects of starvation cannot be entirely reversed by feeding. Thus starvation may decrease the reproductive potential of the females. Key Word Index: Starvation, iuvenile hormone synthesis, corpus allaturn, oiicyte growth, resorption,
INTRODUCTION
The cerebral neurosecretory cells (NSC) and/or the neurosecretory tracts retain neurosecretory material in starved adults of many species including Bombyx (BAWJRMANOVAand PANOV, 1967), Periplaneta (QAYYUM and VIDYASAGAR, 1977), Dysdercus (AWASTHI, 1972), Locusta (MCCAFFERY and HIGHNAM, 1975) and Schistocerca (HIGHNAMet al., 1966). This accumulation of neurosecretion may be causally related to the lack of release of neurosecretion from the NSS: and to a decline in the synthesis or release of JH by the CA. For example, MORDUE (1967)
STARVATION of female insects which must feed as adults in order to produce mature eggs suppresses the accumulation of yolk during vitellogenesis (see ENGELMANN, 1970). However, this inability to produce mature eggs may not be the direct result of a nutritional deficiency (which prevents the synthesis of a sufficient amount of vitellogenin and/or other constituents of yolk), but rather the result of suppression of the neuro-endocrine system. Either reduced release of neurosecretory substances which associated the inactivity of the NSC with reduced stimulate the corpora allata or suppression of the release of JH from the CA of starved Tenebrio molitor. corpora allata directly could result in an inhibition of Oiicyte growth in these animals is reduced in oijcyte growth and maturation (see ENGELMANN,comparison not only to control animals but also to 1970). For example, RANKINand RIDDIFORD (1977) starved animals given access to water. Accordingly, using the Manduca pigmentation bioassay for juvenile MORDUE (1967) concluded that the lack of oiicyte hormone (JH), have showa recently that withdrawal development is not the result of lack of nutrients but of food from Oncopeltus fasciatus causes a gradual rather the result of inactivity of the NSC. Starvation decline in the activity of the corpora allata (CA) over a may therefore cause a decline in the activity of the CA 4-6 day period. This is the first conclusive but indirect via the NSC. demonstration of the effect of starvation on CA It has been previously observed in Schistocerca activity and confirms the suggestion that starvation americana gregaria that starvation of sexually inactivates the CA (JOHANSSON, 1958). However, the immature adults prevents or severely depresses oiicyte mechanisms whereby the CA are inactivated remain growth and this is associated with reduced CA volume unknown. and a build-up of neurosecretion in the neurosecretory tracts (HIGHNAM et al., 1966). Starvation of older *To whom all correspondence should be sent animals does not prevent oiicyte growth completely 701
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STEPHENS. TOBE AND CANDICES. CHAPMAN
but the percentage of oiicytes undergoing resorption increases dramatically (HIGHNAMet al., 1966). This evidence suggests that starvation suppresses the activity of the CA rather than directly inhibiting the growth of the oijcytes themselves (as a consequence of reduced nutrients for example). We have investigated these phenomena quantitatively by employing a direct radiochemical assay (PRATTand TOBE,1974; TOBEand PRATT, 1974a, 1975a) to monitor the synthesis of JH by the CA of S. americunn greguriu after periods of starvation. We have also observed the effects of feeding after a period of starvation on JH synthesis and oiicyte growth in order to assess the ability of the animals to recover reproductive competence after feeding.
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MATERIALS
AND METHODS
Locusts were reared by the methods described by TOBE and PRATT (1975a). The temperature of the rearing room cycled between 24. and 40°C in synchrony with a photoperiod of 12 hr L:l2 hr D. Synthesis of C,,JH (JH III; Methyl (2E, 6E)-(lOR)IO,1 l-epoxy-3,7,1 I-trimethyl-2,6_dodecadienoate) by individual pairs of CA was determined by the method of PRATT and TOBE (1974) and TOBE and PRATT (1974a, 1975a) utilizing the incorporation of the methyl moiety of [methyl-14C] methionine into C,,JH. In those experiments noted, farnesenic acid was added to the incubation medium (final concentration, 20 PM) to stimulate the synthesis of C,,JH (PRATT and TOBE, 1974; TOBE and PRATT, 1974a, 1975a). The lengths of the terminal oiicytes of all experimental animals were measured on fresh ovarioles. At least 5 ovarioles from each ovary of each animal were measured. The number of ovarioles and the number of resorptive oiicytes were determined and the number of resorptive oiicytes expressed as a percentage of the total ovariole number. Food was withdrawn from experimental animals for periods of up to 8 days. JH synthesis by the CA and o6cyte lengths were determined for experimental and control groups for each day during the period of starvation. Starved animals were not provided with water and cages were checked for dead animals twice each day to prevent cannabalism. Control and experimental animals were obtained from the same cages.
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Fig. I. The effect of varying periods of starvation on JH synthesis by CA of S. a. gregaria after withdrawal of food on day 6 after fledging Each point represents a single determination of a single pair of glands. The dotted line joins the median values for starved animals and the solid line joins the median values for control animals. m-Control animals; &Starved animals. The numbers adjacent to the median values represent the total number of determinations on any given day. Growth of the terminal oijcytes reflected the decline in JH synthesis by starved animals. Figure 2 shows
that in control animals, oiicytes increased rapidly in length after day 1 whereas in starved animals, the growth of oiicytes was suppressed. It is known that the oijcytes of S. a. greguriu become vitellogenic after attaining a length of 1.8-2.0 mm (see TOBEand PRATT, 8
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RESULTS JH synthesis and oticyte growth in starved animals
In one experimental series, female locusts were deprived of food on day 6. Thus day 6 represents day 0 of starvation and accordingly the duration of starvation for animals on any given experimental day represents the minimal period of starvation. Figure 1 shows that JH synthesis by CA from starved animals rapidly declined, reaching very low values by day 4. The effect of starvation on JH synthesis became apparent after 2-3 days. CA from control animals synthesized JH at rates similar to those observed previously, reaching maximal values between days 2 and 4 (TOBEand PRATT, 1975a).
05 l-me stffved,
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Fig. 2. The effect of varying periods of starvation on growth of terminal oiicytes in S. a. gregaria after withdrawal of food on day 6 after adult emergence. Each point represents the mean &S.E. of measurements from 5 to 15 animals and corresponds to the sample size given in Fig. 1. Symbols and lines as for Fig. 1.
JH synthesis and oocyte growth in
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Schistocerca
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Time starved,
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Fig. 3. The effect of varying periods of starvation on JH synthesis by CA of S. a. gregaria after withdrawal of food on day 10 after adult emergence. Each point represents a single determination on a single pair of glands. Symbols and lines as for Fig. 1.
1975a) and an examination of Fig. 2 reveals that terminal oiicytes of starved animals did not exceed this critical length (except for day 2). In a second series of experiments, animals were deprived of food on day 10 after emergence. Thus day 0 is equivalent to day 10 for control animals (Fig. 3). Animals were observed for a period of 7 days after withdrawal of food. After this time, the experiment was discontinued because of high mortality. Figure 3 shows that the rates of JH synthesis declined rapidly
2nd cycle
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Fig. 4. The effect of varying periods of starvation on growth of terminal oocytes in S. a. gregaria after withdrawal of food on day IO after adult emergence. Each point represents the mean *SE. of measurements from 5 to 30 animals and corresponds to the sample size given in Fig. 3. Symbols and lines as for Fig. 1.
after the withdrawal of food and were obvious after 2-3 days of starvation. Median values for rates of JH synthesis generally remained at or below 1 pmole hr- l pair - L for the entire experimental period after day 1. On the other hand, rates of JH synthesis by CA from control animals reached minimal values on days 12-l 3 (end of the first gonotrophic cycle-see TOBE and PRATT, 1975a) and increased thereafter, in agreement with previous work (TOBE and PRATT, 1975a). Growth of terminal oiicytes in starved animals continued at rates similar to those observed in control animals (Fig. 4), although an increasing number of terminal oiicytes were observed to undergo resorption as the duration of starvation increased (see Fig. 5 and below). Nonetheless, a number of terminal oocytes in starved animals completed growth and chorion formation [oocytes must attain a length of at least 6.9 mm for chorion formation to occur (TOBE and PRATT, 1975a)]. The effect of starvation on oiicyte growth became particularly obvious in the second cycle of oocytes. These were the penultimate oijcytes (T-l oocytes) which became the terminal (T) olicytes after ovulation of the oiicytes of the first cycle. Growth of these oiicytes is normally inhibited until the T oiicytes have been ovulated (TOBE and PRATT, 1975a; TOBE, 1977). Figure 4 shows that although the obcytes of the second cycle apparently entered vitellogenesis, as indicated by their lengths, their growth was depressed when compared to control animals. Thus the decline in JH synthesis as a result of starvation coincided with a depression in the growth of oiicytes of the second gonotrophic cycle. Although the low levels of JH synthesis appear to coincide with depression of growth of second cycle oiicytes, the effect of starvation was particularly apparent on the percentage resorption of hoth Srst
STEPHEN
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CANDICE S. CHAPMAN
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Fig 5. Effect of starvation on percentage of oocytes undergoing resorption in S. II. gregaria after withdrawal of food on day IO after adult emergence. Each point represents the mean +S.E. and the numbers adjacent to the means represent the sample sizes for any given day. Arrow represents the time of oviposition of first cycle oiicytes and accordingly, values for days 3-7 represent resorption percentage during the second gonotrophic cycle. Symbols and lines as for Fig. 1.Standard errors were calculated using the arc-sine transformation for percentages.
second cycle oocytes. Figure 5 shows that, in starved animals, the percentage resorption reached 50% by the time the first cycle oocytes were mature (i.e. 12-l 3 days after emergence or 2-3 days of starvation). In the second wave, the percentage resorption continued to increase during the experimental period, reaching a maximum of 90% by 7 days after withdrawal of food. Control values did not exceed 33% at any time during the experimental period. Thus, although starved animals were able to complete maturation of oiicytes after short periods of prolonged starvation resulted in a starvation, dramatic increase in resorption and an apparent arrest in oocyte growth. and
JH synthesis and oiicyte growth after feeding
In order to investigate the possible effects of a period of starvation on subsequent JH synthesis and oocyte growth when feeding was resumed, groups of animals were starved for a period of 5 days, an interval known to result in a significant depression in JH synthesis (Figs. 1 and 3) and oiicyte growth (Figs. 2 and 4). The animals were then fed and JH synthesis and oiicyte growth monitored. In animals which had been deprived of food on day 6, starved for 5 days and subsequently fed, rates of JH synthesis were elevated by 24 hr, but declined thereafter (Fig. 6B). By 5 days after feeding, not ah the animals appeared to have recovered from the trauma of starvation-in more than half the animals, rates of JH synthesis by the CA remained below 1.0 pmole hr-i pair-’ (Fig. 6B). However, growth of the
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Time since refeeding,
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Fig. 6. The effect of feeding on growth of oiicytes (A) and JH synthesis by the CA (B) of S. a. gregbria after 5 days of starvation. Animals were starved from day 6 after emergence and were fed on day Il. Each point represents the mean f S.E. (A) or a single determination on a single pair of CA (B). In Fig. 6B, the dotted line joins the median values for animals which were not fed and the solid linejoins the median values for fed animals. A-Starved animals; A-fed animals. The numbers adjacent to the median values represent the total number of determinations on any given day and correspond to the sample sizes for mean oiicyte lengths in Fig. 6A.
terminal oocytes was apparent although vitellogenesis had not begun in 3 of the 9 animals (Fig. 6A). These results suggest that a period of starvation can affect subsequent JH synthesis and oocyte growth in at least some animals. In a parallel series of experiments, day 10 animals were deprived of food for a 5 day period and subsequently fed. It can be seen from Fig. 7B that once again, feeding stimulated the rate of JH synthesis by up to one order of magnitude. The median rate of JH synthesis remained between 3 and 9 pmoles hr- i pair-i for the entire experimental period, with the exception of day 9. These rates of JH synthesis are lower than those observed in normal fed animals (compare for example with Fig, 3) but are apparently sufficient for oocyte maturation (Fig. 7A). Oocyte growth did not resume immediately after feeding and in fact, because the percentage of T oiicytes undergoing resorption continued to increase in the early part of the experimental period, mean oocyte length actually appeared to decrease. However by day 3 after feeding, the oijcytes have resumed growth and
705
synthesis and oijcyte growth rn Schistocerca
JH
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Fig. 7. The effect of feeding on growth of oiicytes (A) and JH synthesis by the CA (B) of S. a. gregaria after 5 days of starvation. Animals were starved from day 10 after emergence and were fed on day 15. Each point represents the mean +S.E. of oiicyte lengths (A) or a single determination on a single pair of CA (B). Symbols and lines as for Fig. 6.
by day 6-7, maturation of the second wave of oiicytes was complete. It should be noted that this group of oiicytes represented the second cycle, the first cycle having completed maturation or resorption during the early portion of the starvation period (see Fig. 4). The interval between the onset of vitellogenesis and completion of chorionation in this wave of oiicytes was 4-5 days, an interval similar to or slightly longer than that observed in normal animals (see Fig. 4 and TOBEand PICA-IT, 1975a). However, growth of the third cycle of oikytes appears slower. Thus, in the 10 day interval following the resumption of feeding, experimental animals matured only one batch of oiicytes whereas normal animals would mature two batches during this time. Effect of starvation on stimulated synthesis of JH
had impaired the ability of the CA to carry out these final two stages in JH synthesis, farnesenic acid was added to the incubation medium and the rates of JH synthesis determined after a 3 hr incubation. The results are shown in Table 1. It can be seen that rates of JH synthesis by CA from animals starved for 5 days were considerably higher than the spontaneous rates (compare with Fig. 1 and 3). Thus, although spontaneous JH synthesis (TOBE and PRAIT, 1974b, 1976) was significantly depressed by starvation, JH synthesis in the presence of farnesenic acid proceeded at a high rate, indicating that in starved animals, the final two stages in JH synthesis were not significantly impaired.
DISCUSSION
Addition of the exogenous precursor farnesenic acid to the incubation medium of the CA has been Starvation and JH synthesis demonstrated previously to stimulate the synthesis of The results presented here show clearly that JH C,,JH as a result of rapid esterification and synthesis is depressed by starvation (Figs. 1 and 3). epoxidation (PRATT and TOBE,1974; TOBEand PRATT, This depression becomes obvious 2-3 days after the 1974a; PRATT et al., 1975). To determine if starvation onset of starvation and remains throughout the period of starvation. The decline in JH synthesis is accompanied by a cessation in terminal oiicyte growth Table 1. C, ,JH synthesis in the presence of farnesenic acid 5 days after starvation and/or by an increase in the number of terminal oikytes undergoing resorption. Age at withdrawal of food JH synthesis Two different times were chosen for the withdrawal (pmoles/hr/pair) + S.E. (days) of food-6 days and 10 days after emergence. By 6 days, female S. a. gregaria have undergone substantial 6 62.14k7.14 somatic growth (HILL et al., 1968) but the terminal 36.44* 3.58 10 oocytes have not yet entered vitellogenesis (TOREand
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STEPHENS. TOBE AND CANDICE S. CHAPMAN
PRATT, 1975a). In addition, under the rearing conditions used by TOBEand PRATT(1975a) and in the present study, the CA do not undergo an increase in JH synthesis until day 8. Thus in animals starved from day 6, the CA have not yet been activated at the time of food withdrawal and accordingly, the effect of starvation on activation of the CA can be observed. In animals in which food was withdrawn from day IO, somatic growth was virtually complete (HILL et al.. 1968) and the terminal oiicytes were undergoing rapid vitellogenesis (TOBEand PRATT, 1975a). Rates of JH synthesis by the CA are high at this time (TOBEand PRATT, 1975a) and accordingly, in animals starved from day 10, the effect of starvation on the continued functioning of the CA after activation can be observed. Figure 1 shows that in animals starved from day 6, rates of JH synthesis do not increase and in fact, decrease during much of the experimental period. Starvation therefore appears to prevent the activation of the CA. Similarly Fig. 3 shows that in animals in which the CA have been activated, there is a rapid decline in JH synthesis after withdrawal of food. HIGHNAMet al. (1966), on the basis of CA volume, suggested that starvation of female S. a. gregaria prevented the activation of the CA of young animals and reduced the activity of CA from egg-maturing animals. Although CA volume has been shown not to correlate well with JH synthesis in this species (TOBE and PRATT. 1975b), the present results confirm the suggestions of HIGHNAMet al. (1966). These authors also observed an accumulation of stainable material in the nervi corporis cardiaca I (NCC I) and in the neurosecretory cells of the pars intercerebralis after starvation. A similar accumulation in the cerebral neurosecretory system has been observed in Locusta migratoria after starvation (HIGHNAMand WEST,197 I; MCCAFFERYand HIGHNAM,1975). In S. a. gregnria, this accumulation occurred both in animals starved from early in adult life and in those starved during the gonotrophic cycle. Allatotrophic factors necessary for the maintenance of JH synthesis have been shown to reach the CA by nervous tracts in S. a. gregariu (TOBE er al.. 1977) and because many of these axons originate in the brain and corpus cardiacum (MASON, 1973), it can be suggested that the accumulation of neurosecretion in the brain and NCC I during starvation is associated with the failure of allatotropin to reach the CA (see also SLAMA,1978). HIGHNAMet al. (1966) observed that starvation has a less dramatic effect on oiicyte maturation and resorption than cautery of the NSC of the pars intercerebralis or allatectomy. inferring that synthesis of neurosecretory material and JH is not completely suppressed by starvation. However, cautery of the NSC of the pars intercerebralis may not directly affect the CA because the lateral NSC, rather than the medial cells, directly innervate CA in Schistocerca (STRONG, 1965; MASON, 1973) and accordingly, accumulation of neurosecretory material in the medial NSC may not be directly relevant. The present results demonstrate that there is some synthesis of JH, albeit at low rates, during starvation and in a small number of insects, the rates of JH synthesis may be appreciable (see Figs. 1 and 3), thus confirming the suggestion of HIGHNAMet al. (1966).
OBcyte growth and resorption
It seems likely that the suppression of oiicyte maturation and the increase in resorption during starvation can be associated, at least in part, with the low rates of JH synthesis. JOHANSSON (1955) observed that oiicyte growth in starved Leucophaea muderae resumed after implantation of ‘active’ CA. Although similar experiments cannot be performed on starved S. u. gregaria because denvervated CA rapidly lose their ability to synthesize JH (TOBEet nl., 1977). the results of MCCAFFERYand HIGHNAM(1975) (using a juvenile hormone analog) and MCCAFFERY(1976) (using CA implanted into isolated abdomens) suggest that oijcyte growth can be stimulated by JH in the absence of food in L. migratoria. Thus, the cessation of oijcyte growth occurs as a result of suppression of the CA rather than as a direct result of nutritional deficiency (see also MORDUE.1967; RANKINand RIDDIFORD,1977). It is significant that maturation of a percentage of oiicytes occurs even in starved animals, provided that they have attained a length of 5 mm (Fig. 4). It should be noted that this length approximates that at which resorption percentage no longer increases (TOBEand PRATT, 1975a). This correlation suggests that high rates of JH synthesis may not be necessary for growth of oiicytes greater than 5 mm in length-thus, the JHdependent phase of oiicyte growth in S. a. gregaria may be restricted to oiicytes between 1.8 and 5 mm in length (vitellogenic oiicytes). It can be suggested that vitellogenesis ceases in oiicytes greater than 5 mm in length, thus accounting for the potential JH-independent phase of growth. Hydration of the oiicytes may occur during this phase. However, it is unlikely that a JHindependent phase of oiicyte growth in S. a. gregariucan beexplained on this basis because: (a) the follicular epithelium of oiicytes greater than 5 mm remains patent (TOBEand PRATT, 1975a); (b) the dry weight of terminal oiicytes increases dramatically between 5 and 8 mm (HILL et al., 1968). Thus it appears that vitellogenesis continues in oijcytes above 5 mm in length, even though the CA are synthesizing JH at low rates. This later phase of oiicyte growth may simply require less JH than the early growth phase. In fact, the results of HIGHNAMet al. (1963) suggest that this may be the case, since oiicyte growth ceases completely in allatectomized animals. irrespective of oijcyte length. JH synthesis and feeding after starvation
The present study reveals that feeding of S. a. results in transient elevated rates of JH synthesis, in the case of females starved from day 6 or in elevated but subnormal rates of JH synthesis in females starved from day IO. Although these rates of JH synthesis may be subnormal, when compared to control values (TOBE and PRATT, 1975a), they are nonetheless sufficient to support oiicyte maturation albeit at a reduced rate. Starvation thus appears to suppress the ability of CA to synthesize JH and feeding does not completely reverse this effect within the experimental period. HIGHNAMet al. (1966) reported a rapid reduction in stainable material in the NCC 1 following feeding in females starved for 7 days and a complete depletion by 4 hr after feeding. The relationship between release of
greguria after a 5 day starvation
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JH synthesis and oijcyte growth in Schistocerca
this material and CA activity is uncertain although it can be suggested that the increase in rates of JH synthesis by 24 hr after feeding occurs as a consequence of the release of neurosecretion. However, as noted above, neurosecretory axons in the NCC I do not enter CA directly (STRONG, 1965; MASON, 1973) and therefore, observations on the NCC I may not be directly relevant. Thus, it cannot be ascertained whether starvation causes a direct impairment of the CA or a reduction in synthesis or release of the allatotropin which stimulates the CA. Starvation and subsequent feeding of female Leucophaea maderae does not result in a significant change in the number of nuclei in the CA but does result in large changes in nucleo-cytoplasmic ratios (VON HARNACK, 1958). In this insect, starvation does not appear to cause a decrease in the-number of CA cells and feeding does not cause an increase in cell number. If a similar situation occurs in S. a. gregaria, the changes in rates of JH synthesis during starvation and feeding are not the result of changes in cell number of the CA but rather the result of changes in the levels of activity of rate-limiting steps in JH synthesis and/or in the percentage of cells of the CA synthesizing JH (see also TOBEand PRATT, 1976). Stimulated
JH synthesis
It is possible to conclude that starvation does not impair the final two enzymic stages in JH synthesis, on the basis of the results obtained with farnesenic acid stimulation (Table l), and it is reasonable to assume that feeding does not alter the enzymic activities of these two final stages. In addition, rate limitation does not occur at this stage of JH synthesis, in normal animals or in starved animals. Thus, the decline in JH synthesis during starvation is not the result of rate limitation of these final stages. Starvation does not appear to alter the activities of the two terminal enzymic stages in JH synthesis and accordingly, rate limitation must occur earlier in the biosynthetic pathway. The fact that rate limitation does not occur at the final two enzymic stages in JH synthesis may also be demonstrated through examination of the fractional endocrine activity ratio (FEAR-see TOBE and PRATT, 1976). Using the median values for spontaneous JH synthesis on day 5 after withdrawal of food (from Figs. 1 and 3), the FEAR for animals deprived of food from day 6 was 0.005 and for those deprived of food from day 10 was 0.012. Thus, stimulated JH synthesis (TOBE and PRATT, 1974b, 1976) is approximately two orders of magnitude greater than spontaneous synthesis. Clearly, rate limitation in JH synthesis by starved animals occurs prior to the final two enzymic stages. Acknowledgements-This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. We thank Drs. T. FRIEDEL,H. INJEYAN and E. MUNDALLfor critically reading the manuscript.
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BASSURMANOVA 0. K. and PANOVA. A. (1967) Structure of the neurosecretory system in Lepidoptera. Light and electron microscopy of type A-neurosecretory cells in the brain of normal and starved larvae of the silkworm Bombyx mot-i. Gen. camp. Endocr. 9, 245-262.
ENGELMANN F. (1970) The Physiology of Insect Reproduclion. pp. 143-189, Pergamon, Oxford. HIGHNAMK. C. and WENTM. W. (1971) The neuropilar reservoir of Locusta migrutoria neurosecretory migrarorioides R & F. Gen. camp. Endocr. 16, 574-585. HIGHNAMK. C., LUSIS0. and HILL L. (1963) The role of the corpora allata during oocyte growth in the desert locust, Schistocerca gregaria Forsk. J. Insect Physiol. 9, 587-596. HIGHNAMK. C., HILL L. and MORDUEW. (1966) The endocrine system and oocyte growth in Schistocerca in relation to starvation and frontal ganglionectomy. J. Insect Physiol. 12, 977-994.
HILL L., LUNTZ A. J. and STEELEP. A. (1968) The relationships between somatic growth, ovarian growth, and feeding activity in the adult desert locust. J. Insecl Physiol. 14, I-20.
JOHANSSON A. S. (1955) The relationship between corpora allata and reproductive organs in starved female Leucophaea maderae (Blattaria). Biol. Bull. mar. hiol. Lab., Woods Hole 108, 4@44.
JOHANSSON A. S. (1958) Relation of nutrition to endocrinereproductive functions in the milkweed bug Oncopelrus fusciafus (Dallas) (Heteroptera: Lygaeidae). NYU. Mug. 2001. 1, 1-I 32. MASONC. A. (1973) New features of the brain-retrocerebral neuroendocrine complex of the locust Schistocerca saga (Scudder). 2. Zellforsch. mikrosk. Anat. 141, 19-32. MCCAFWRYA. R. (1976) Effects of electrocoagulat!on of cerebral neurosecretory cells and implantation of corpora allata on oocyte development in Locusta migratoria. J. Insect Physiol. 22, 1081-1092.
MCCAFFERYA. R. and HIGHNAMK. C. (1975) Effects of corpus allatum hormone and its mimics on the activity of the cerebral neurosecretory system of Locusta migraroria migratorioides R. & F. Gen. camp. Endocr. 25, 373-386. MORDUEW. (1967) The influence of feeding upon the activity of the neuroendocrine system during oocyte growth in Tenebrio molitor. Gen. romp. Endocr. 9, 406-415.
PRATT G. E. and TOBE S. S. (1974) Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in vitro. Lz$z S&. 14,575-586. PRATTG. E., TOBE S. S.. WEAVERR. J. and FINNEYJ. R. (1975) Spontaneous synthesis and release of C,, juvenile hormone by isolated corpora allata of female locust Schistocerca gregariu and female cockroach Periplaneta americana. Gen. romp:Endocr.
26, 478-484.
QAYYUMM. A. and VIDYASAGAR P. S. P. V. (1977) Effect of certain environmental factors on the cerebral neurosecretory cells of American cockroach Periplaneta americana. Experientia 33, 1622-1623. RANKIN M. A. and RIDDIFORDL. M. (1977) Hormonal control of migratory flight in Oncopeltus fasciatus: the effects of the corpus cardiacum, corpus allaturn, and starvation on migration and reproduction. Gen. camp. Endocr. 33, 309-32 1. SLAMAK. (1978) The principles of antihormone action in insects. Acta ent. bohem. 75, 65-82. STRONGL. (1965) The relationships between the brain, corpora allata, and oocyte growth in the Central American locust, Schistocerca sp. II. The innervation of the corpora allata, the lateral neurosecretory complex, and oocyte growth. J. Insect Physiol. 11, 271-280. TOBES. S. (1977) Inhibition of growth in developing oocytes of the desert locust. Experientia 33, 343-345. TOBES.S. and PRATTG. E. (1974a)Theinfluenceofsubstrate concentrations on the rate of insect juvenile hormone biosynthesis by corpora allata of the desert locust ,n vitro. Biochem. J. 144, 107-l 13.
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TOBES. S. and PRATTG. E. (1974b) Dependence of juvenile hormone release from corpus allatum on intraglandular content. Nature, Lond. 252, 474-476. TOBES. S. and PRATTG. E. (1975a) Corpus allatum activity in vitro during ovarian maturation in the desert locust, Schistocerca gregaria. J. exp. Biol. 62, 611-627.
TOBES. S. and PRATTG. E. (1975b) The synthetic activity and glandular volumd of the corpus allatum during ovarian maturation in the desert locust Schistocerca gregaria. Life SC;. 17, 417-422.
TOBE S. S. and PRATT G. E. (1976) Farnesenic
acid
stimulation of juvenile hormone biosynthesis as an experimental prohe in corpus allatum physiology. In The Juvenile Hormones (Ed. by GILBERTL. I.) pp. 147-163. Plenum Press, New York. TOBES. S., CHAPMANC. S. and PRATTG. E. (1977) Decay in juvenile hormone biosynthesis by insect corpus allatum after nerve transection. Nature, Lond. 268, 728-730. VONHARNACKM. (1958) Histophysiological studies on the corpus allatum of Leucophaea maderae. II. The effect of starvation. Biol. Bull. mar. biol. Lub.. Woods Hole 115, 521-529.
J. Insect Physiol., Vol. 25, pp. 701 to 708. 0 Pergamon Press Ltd. 1979. Printed in Great Britain.
$02.00/O
THE EFFECTS OF STARVATION AND SUBSEQUENT FEEDING ON JUVENILE HORMONE SYNTHESIS AND 0i)CYTE GROWTH IN SCHZSTOCERCA
AMERZCANA
GREGARZA
STEPHEN S. TOBE*and CANDICE S. CHAPMAN Department of Zoology, University of Toronto, Toronto, Ontario. MSS lA1, Canada (Received 16 March 1979; revised 9 May 1979)
Abstract-Theeffect of starvation on the synthesis of C,, juvenile hormone (JH) and the growth of terminal oijcytes was assessed in Schistocerca americana gregaria at two times during adult life: before activation of the corpora allata and during the first gonotrophic cycle. In both groups, starvation resulted in a decline in JH synthesis within 2-3 days and rates of synthesis remained low throughout the experimental period. The growth rate of oiicytes which were not vitellogenic at the time of starvation was depressed whereas the percentage of resorption of vitellogenic oiicytes increased dramatically with starvation. Although the percentage of resorption increased in animals with vitellogenic oiicytes, some mature otiytes were produced, particularly in animals in which the ohcytes were greater than 5 mm in length at the time of starvation. This suggests that oiicyte maturation can be divided into two distinct phases-an early phase of vitellogenesis associated with high rates of JH synthesis and a late phase, in oiicytes greater than 5 mm, associated with much lower rates of JH synthesis. Stimulation of JH synthesis by farnesenic acid in S-day starved animals resulted in high rates of JH synthesis, indicating that starvation did not appreciably alter the enzymic activities of the final two stages in JH synthesis. Thus rate limitation did not occur at these stages. Feeding of S-day starved animals resulted in a transient increase in the rate of JH synthesis. However, rates of JH synthesis and oacyte growth remained subnormal throughout the observation period, suggesting that the effects of starvation cannot be entirely reversed by feeding. Thus starvation may decrease the reproductive potential of the females. Key Word Index: Starvation, iuvenile hormone synthesis, corpus allaturn, oiicyte growth, resorption,
INTRODUCTION
The cerebral neurosecretory cells (NSC) and/or the neurosecretory tracts retain neurosecretory material in starved adults of many species including Bombyx (BAWJRMANOVAand PANOV, 1967), Periplaneta (QAYYUM and VIDYASAGAR, 1977), Dysdercus (AWASTHI, 1972), Locusta (MCCAFFERY and HIGHNAM, 1975) and Schistocerca (HIGHNAMet al., 1966). This accumulation of neurosecretion may be causally related to the lack of release of neurosecretion from the NSS: and to a decline in the synthesis or release of JH by the CA. For example, MORDUE (1967)
STARVATION of female insects which must feed as adults in order to produce mature eggs suppresses the accumulation of yolk during vitellogenesis (see ENGELMANN, 1970). However, this inability to produce mature eggs may not be the direct result of a nutritional deficiency (which prevents the synthesis of a sufficient amount of vitellogenin and/or other constituents of yolk), but rather the result of suppression of the neuro-endocrine system. Either reduced release of neurosecretory substances which associated the inactivity of the NSC with reduced stimulate the corpora allata or suppression of the release of JH from the CA of starved Tenebrio molitor. corpora allata directly could result in an inhibition of Oiicyte growth in these animals is reduced in oijcyte growth and maturation (see ENGELMANN,comparison not only to control animals but also to 1970). For example, RANKINand RIDDIFORD (1977) starved animals given access to water. Accordingly, using the Manduca pigmentation bioassay for juvenile MORDUE (1967) concluded that the lack of oiicyte hormone (JH), have showa recently that withdrawal development is not the result of lack of nutrients but of food from Oncopeltus fasciatus causes a gradual rather the result of inactivity of the NSC. Starvation decline in the activity of the corpora allata (CA) over a may therefore cause a decline in the activity of the CA 4-6 day period. This is the first conclusive but indirect via the NSC. demonstration of the effect of starvation on CA It has been previously observed in Schistocerca activity and confirms the suggestion that starvation americana gregaria that starvation of sexually inactivates the CA (JOHANSSON, 1958). However, the immature adults prevents or severely depresses oiicyte mechanisms whereby the CA are inactivated remain growth and this is associated with reduced CA volume unknown. and a build-up of neurosecretion in the neurosecretory tracts (HIGHNAM et al., 1966). Starvation of older *To whom all correspondence should be sent animals does not prevent oiicyte growth completely 701
702
STEPHENS. TOBE AND CANDICES. CHAPMAN
but the percentage of oiicytes undergoing resorption increases dramatically (HIGHNAMet al., 1966). This evidence suggests that starvation suppresses the activity of the CA rather than directly inhibiting the growth of the oijcytes themselves (as a consequence of reduced nutrients for example). We have investigated these phenomena quantitatively by employing a direct radiochemical assay (PRATTand TOBE,1974; TOBEand PRATT, 1974a, 1975a) to monitor the synthesis of JH by the CA of S. americunn greguriu after periods of starvation. We have also observed the effects of feeding after a period of starvation on JH synthesis and oiicyte growth in order to assess the ability of the animals to recover reproductive competence after feeding.
.
x
P l
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l
.
. .
MATERIALS
AND METHODS
Locusts were reared by the methods described by TOBE and PRATT (1975a). The temperature of the rearing room cycled between 24. and 40°C in synchrony with a photoperiod of 12 hr L:l2 hr D. Synthesis of C,,JH (JH III; Methyl (2E, 6E)-(lOR)IO,1 l-epoxy-3,7,1 I-trimethyl-2,6_dodecadienoate) by individual pairs of CA was determined by the method of PRATT and TOBE (1974) and TOBE and PRATT (1974a, 1975a) utilizing the incorporation of the methyl moiety of [methyl-14C] methionine into C,,JH. In those experiments noted, farnesenic acid was added to the incubation medium (final concentration, 20 PM) to stimulate the synthesis of C,,JH (PRATT and TOBE, 1974; TOBE and PRATT, 1974a, 1975a). The lengths of the terminal oiicytes of all experimental animals were measured on fresh ovarioles. At least 5 ovarioles from each ovary of each animal were measured. The number of ovarioles and the number of resorptive oiicytes were determined and the number of resorptive oiicytes expressed as a percentage of the total ovariole number. Food was withdrawn from experimental animals for periods of up to 8 days. JH synthesis by the CA and o6cyte lengths were determined for experimental and control groups for each day during the period of starvation. Starved animals were not provided with water and cages were checked for dead animals twice each day to prevent cannabalism. Control and experimental animals were obtained from the same cages.
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Fig. I. The effect of varying periods of starvation on JH synthesis by CA of S. a. gregaria after withdrawal of food on day 6 after fledging Each point represents a single determination of a single pair of glands. The dotted line joins the median values for starved animals and the solid line joins the median values for control animals. m-Control animals; &Starved animals. The numbers adjacent to the median values represent the total number of determinations on any given day. Growth of the terminal oijcytes reflected the decline in JH synthesis by starved animals. Figure 2 shows
that in control animals, oiicytes increased rapidly in length after day 1 whereas in starved animals, the growth of oiicytes was suppressed. It is known that the oijcytes of S. a. greguriu become vitellogenic after attaining a length of 1.8-2.0 mm (see TOBEand PRATT, 8
r t
RESULTS JH synthesis and oticyte growth in starved animals
In one experimental series, female locusts were deprived of food on day 6. Thus day 6 represents day 0 of starvation and accordingly the duration of starvation for animals on any given experimental day represents the minimal period of starvation. Figure 1 shows that JH synthesis by CA from starved animals rapidly declined, reaching very low values by day 4. The effect of starvation on JH synthesis became apparent after 2-3 days. CA from control animals synthesized JH at rates similar to those observed previously, reaching maximal values between days 2 and 4 (TOBEand PRATT, 1975a).
05 l-me stffved,
days
Fig. 2. The effect of varying periods of starvation on growth of terminal oiicytes in S. a. gregaria after withdrawal of food on day 6 after adult emergence. Each point represents the mean &S.E. of measurements from 5 to 15 animals and corresponds to the sample size given in Fig. 1. Symbols and lines as for Fig. 1.
JH synthesis and oocyte growth in
I 0
I
.
I 2
703
Schistocerca
L
1
I 3
I
I
I
4
5
6
Time starved,
l
1 7
days
Fig. 3. The effect of varying periods of starvation on JH synthesis by CA of S. a. gregaria after withdrawal of food on day 10 after adult emergence. Each point represents a single determination on a single pair of glands. Symbols and lines as for Fig. 1.
1975a) and an examination of Fig. 2 reveals that terminal oiicytes of starved animals did not exceed this critical length (except for day 2). In a second series of experiments, animals were deprived of food on day 10 after emergence. Thus day 0 is equivalent to day 10 for control animals (Fig. 3). Animals were observed for a period of 7 days after withdrawal of food. After this time, the experiment was discontinued because of high mortality. Figure 3 shows that the rates of JH synthesis declined rapidly
2nd cycle
0
I 2
I
I 6
4
The sbved,
I a
days
Fig. 4. The effect of varying periods of starvation on growth of terminal oocytes in S. a. gregaria after withdrawal of food on day IO after adult emergence. Each point represents the mean *SE. of measurements from 5 to 30 animals and corresponds to the sample size given in Fig. 3. Symbols and lines as for Fig. 1.
after the withdrawal of food and were obvious after 2-3 days of starvation. Median values for rates of JH synthesis generally remained at or below 1 pmole hr- l pair - L for the entire experimental period after day 1. On the other hand, rates of JH synthesis by CA from control animals reached minimal values on days 12-l 3 (end of the first gonotrophic cycle-see TOBE and PRATT, 1975a) and increased thereafter, in agreement with previous work (TOBE and PRATT, 1975a). Growth of terminal oiicytes in starved animals continued at rates similar to those observed in control animals (Fig. 4), although an increasing number of terminal oiicytes were observed to undergo resorption as the duration of starvation increased (see Fig. 5 and below). Nonetheless, a number of terminal oocytes in starved animals completed growth and chorion formation [oocytes must attain a length of at least 6.9 mm for chorion formation to occur (TOBE and PRATT, 1975a)]. The effect of starvation on oiicyte growth became particularly obvious in the second cycle of oocytes. These were the penultimate oijcytes (T-l oocytes) which became the terminal (T) olicytes after ovulation of the oiicytes of the first cycle. Growth of these oiicytes is normally inhibited until the T oiicytes have been ovulated (TOBE and PRATT, 1975a; TOBE, 1977). Figure 4 shows that although the obcytes of the second cycle apparently entered vitellogenesis, as indicated by their lengths, their growth was depressed when compared to control animals. Thus the decline in JH synthesis as a result of starvation coincided with a depression in the growth of oiicytes of the second gonotrophic cycle. Although the low levels of JH synthesis appear to coincide with depression of growth of second cycle oiicytes, the effect of starvation was particularly apparent on the percentage resorption of hoth Srst
STEPHEN
S. TOBE
AND
CANDICE S. CHAPMAN
(0)
51
1
I
I
I
I
I (b) .
1 9 5
20
4
:
/-7
Fig 5. Effect of starvation on percentage of oocytes undergoing resorption in S. II. gregaria after withdrawal of food on day IO after adult emergence. Each point represents the mean +S.E. and the numbers adjacent to the means represent the sample sizes for any given day. Arrow represents the time of oviposition of first cycle oiicytes and accordingly, values for days 3-7 represent resorption percentage during the second gonotrophic cycle. Symbols and lines as for Fig. 1.Standard errors were calculated using the arc-sine transformation for percentages.
second cycle oocytes. Figure 5 shows that, in starved animals, the percentage resorption reached 50% by the time the first cycle oocytes were mature (i.e. 12-l 3 days after emergence or 2-3 days of starvation). In the second wave, the percentage resorption continued to increase during the experimental period, reaching a maximum of 90% by 7 days after withdrawal of food. Control values did not exceed 33% at any time during the experimental period. Thus, although starved animals were able to complete maturation of oiicytes after short periods of prolonged starvation resulted in a starvation, dramatic increase in resorption and an apparent arrest in oocyte growth. and
JH synthesis and oiicyte growth after feeding
In order to investigate the possible effects of a period of starvation on subsequent JH synthesis and oocyte growth when feeding was resumed, groups of animals were starved for a period of 5 days, an interval known to result in a significant depression in JH synthesis (Figs. 1 and 3) and oiicyte growth (Figs. 2 and 4). The animals were then fed and JH synthesis and oiicyte growth monitored. In animals which had been deprived of food on day 6, starved for 5 days and subsequently fed, rates of JH synthesis were elevated by 24 hr, but declined thereafter (Fig. 6B). By 5 days after feeding, not ah the animals appeared to have recovered from the trauma of starvation-in more than half the animals, rates of JH synthesis by the CA remained below 1.0 pmole hr-i pair-’ (Fig. 6B). However, growth of the
L
2
3
Time since refeeding,
4
5
days
Fig. 6. The effect of feeding on growth of oiicytes (A) and JH synthesis by the CA (B) of S. a. gregbria after 5 days of starvation. Animals were starved from day 6 after emergence and were fed on day Il. Each point represents the mean f S.E. (A) or a single determination on a single pair of CA (B). In Fig. 6B, the dotted line joins the median values for animals which were not fed and the solid linejoins the median values for fed animals. A-Starved animals; A-fed animals. The numbers adjacent to the median values represent the total number of determinations on any given day and correspond to the sample sizes for mean oiicyte lengths in Fig. 6A.
terminal oocytes was apparent although vitellogenesis had not begun in 3 of the 9 animals (Fig. 6A). These results suggest that a period of starvation can affect subsequent JH synthesis and oocyte growth in at least some animals. In a parallel series of experiments, day 10 animals were deprived of food for a 5 day period and subsequently fed. It can be seen from Fig. 7B that once again, feeding stimulated the rate of JH synthesis by up to one order of magnitude. The median rate of JH synthesis remained between 3 and 9 pmoles hr- i pair-i for the entire experimental period, with the exception of day 9. These rates of JH synthesis are lower than those observed in normal fed animals (compare for example with Fig, 3) but are apparently sufficient for oocyte maturation (Fig. 7A). Oocyte growth did not resume immediately after feeding and in fact, because the percentage of T oiicytes undergoing resorption continued to increase in the early part of the experimental period, mean oocyte length actually appeared to decrease. However by day 3 after feeding, the oijcytes have resumed growth and
705
synthesis and oijcyte growth rn Schistocerca
JH
(b)
. : .
0
I
I
I
2
I
I
I
I
,
I
3
4
5
6
7
8
Time since refeMng,
9
I
IO
days
Fig. 7. The effect of feeding on growth of oiicytes (A) and JH synthesis by the CA (B) of S. a. gregaria after 5 days of starvation. Animals were starved from day 10 after emergence and were fed on day 15. Each point represents the mean +S.E. of oiicyte lengths (A) or a single determination on a single pair of CA (B). Symbols and lines as for Fig. 6.
by day 6-7, maturation of the second wave of oiicytes was complete. It should be noted that this group of oiicytes represented the second cycle, the first cycle having completed maturation or resorption during the early portion of the starvation period (see Fig. 4). The interval between the onset of vitellogenesis and completion of chorionation in this wave of oiicytes was 4-5 days, an interval similar to or slightly longer than that observed in normal animals (see Fig. 4 and TOBEand PICA-IT, 1975a). However, growth of the third cycle of oikytes appears slower. Thus, in the 10 day interval following the resumption of feeding, experimental animals matured only one batch of oiicytes whereas normal animals would mature two batches during this time. Effect of starvation on stimulated synthesis of JH
had impaired the ability of the CA to carry out these final two stages in JH synthesis, farnesenic acid was added to the incubation medium and the rates of JH synthesis determined after a 3 hr incubation. The results are shown in Table 1. It can be seen that rates of JH synthesis by CA from animals starved for 5 days were considerably higher than the spontaneous rates (compare with Fig. 1 and 3). Thus, although spontaneous JH synthesis (TOBE and PRAIT, 1974b, 1976) was significantly depressed by starvation, JH synthesis in the presence of farnesenic acid proceeded at a high rate, indicating that in starved animals, the final two stages in JH synthesis were not significantly impaired.
DISCUSSION
Addition of the exogenous precursor farnesenic acid to the incubation medium of the CA has been Starvation and JH synthesis demonstrated previously to stimulate the synthesis of The results presented here show clearly that JH C,,JH as a result of rapid esterification and synthesis is depressed by starvation (Figs. 1 and 3). epoxidation (PRATT and TOBE,1974; TOBEand PRATT, This depression becomes obvious 2-3 days after the 1974a; PRATT et al., 1975). To determine if starvation onset of starvation and remains throughout the period of starvation. The decline in JH synthesis is accompanied by a cessation in terminal oiicyte growth Table 1. C, ,JH synthesis in the presence of farnesenic acid 5 days after starvation and/or by an increase in the number of terminal oikytes undergoing resorption. Age at withdrawal of food JH synthesis Two different times were chosen for the withdrawal (pmoles/hr/pair) + S.E. (days) of food-6 days and 10 days after emergence. By 6 days, female S. a. gregaria have undergone substantial 6 62.14k7.14 somatic growth (HILL et al., 1968) but the terminal 36.44* 3.58 10 oocytes have not yet entered vitellogenesis (TOREand
706
STEPHENS. TOBE AND CANDICE S. CHAPMAN
PRATT, 1975a). In addition, under the rearing conditions used by TOBEand PRATT(1975a) and in the present study, the CA do not undergo an increase in JH synthesis until day 8. Thus in animals starved from day 6, the CA have not yet been activated at the time of food withdrawal and accordingly, the effect of starvation on activation of the CA can be observed. In animals in which food was withdrawn from day IO, somatic growth was virtually complete (HILL et al.. 1968) and the terminal oiicytes were undergoing rapid vitellogenesis (TOBEand PRATT, 1975a). Rates of JH synthesis by the CA are high at this time (TOBEand PRATT, 1975a) and accordingly, in animals starved from day 10, the effect of starvation on the continued functioning of the CA after activation can be observed. Figure 1 shows that in animals starved from day 6, rates of JH synthesis do not increase and in fact, decrease during much of the experimental period. Starvation therefore appears to prevent the activation of the CA. Similarly Fig. 3 shows that in animals in which the CA have been activated, there is a rapid decline in JH synthesis after withdrawal of food. HIGHNAMet al. (1966), on the basis of CA volume, suggested that starvation of female S. a. gregaria prevented the activation of the CA of young animals and reduced the activity of CA from egg-maturing animals. Although CA volume has been shown not to correlate well with JH synthesis in this species (TOBE and PRATT. 1975b), the present results confirm the suggestions of HIGHNAMet al. (1966). These authors also observed an accumulation of stainable material in the nervi corporis cardiaca I (NCC I) and in the neurosecretory cells of the pars intercerebralis after starvation. A similar accumulation in the cerebral neurosecretory system has been observed in Locusta migratoria after starvation (HIGHNAMand WEST,197 I; MCCAFFERYand HIGHNAM,1975). In S. a. gregnria, this accumulation occurred both in animals starved from early in adult life and in those starved during the gonotrophic cycle. Allatotrophic factors necessary for the maintenance of JH synthesis have been shown to reach the CA by nervous tracts in S. a. gregariu (TOBE er al.. 1977) and because many of these axons originate in the brain and corpus cardiacum (MASON, 1973), it can be suggested that the accumulation of neurosecretion in the brain and NCC I during starvation is associated with the failure of allatotropin to reach the CA (see also SLAMA,1978). HIGHNAMet al. (1966) observed that starvation has a less dramatic effect on oiicyte maturation and resorption than cautery of the NSC of the pars intercerebralis or allatectomy. inferring that synthesis of neurosecretory material and JH is not completely suppressed by starvation. However, cautery of the NSC of the pars intercerebralis may not directly affect the CA because the lateral NSC, rather than the medial cells, directly innervate CA in Schistocerca (STRONG, 1965; MASON, 1973) and accordingly, accumulation of neurosecretory material in the medial NSC may not be directly relevant. The present results demonstrate that there is some synthesis of JH, albeit at low rates, during starvation and in a small number of insects, the rates of JH synthesis may be appreciable (see Figs. 1 and 3), thus confirming the suggestion of HIGHNAMet al. (1966).
OBcyte growth and resorption
It seems likely that the suppression of oiicyte maturation and the increase in resorption during starvation can be associated, at least in part, with the low rates of JH synthesis. JOHANSSON (1955) observed that oiicyte growth in starved Leucophaea muderae resumed after implantation of ‘active’ CA. Although similar experiments cannot be performed on starved S. u. gregaria because denvervated CA rapidly lose their ability to synthesize JH (TOBEet nl., 1977). the results of MCCAFFERYand HIGHNAM(1975) (using a juvenile hormone analog) and MCCAFFERY(1976) (using CA implanted into isolated abdomens) suggest that oijcyte growth can be stimulated by JH in the absence of food in L. migratoria. Thus, the cessation of oijcyte growth occurs as a result of suppression of the CA rather than as a direct result of nutritional deficiency (see also MORDUE.1967; RANKINand RIDDIFORD,1977). It is significant that maturation of a percentage of oiicytes occurs even in starved animals, provided that they have attained a length of 5 mm (Fig. 4). It should be noted that this length approximates that at which resorption percentage no longer increases (TOBEand PRATT, 1975a). This correlation suggests that high rates of JH synthesis may not be necessary for growth of oiicytes greater than 5 mm in length-thus, the JHdependent phase of oiicyte growth in S. a. gregaria may be restricted to oiicytes between 1.8 and 5 mm in length (vitellogenic oiicytes). It can be suggested that vitellogenesis ceases in oiicytes greater than 5 mm in length, thus accounting for the potential JH-independent phase of growth. Hydration of the oiicytes may occur during this phase. However, it is unlikely that a JHindependent phase of oiicyte growth in S. a. gregariucan beexplained on this basis because: (a) the follicular epithelium of oiicytes greater than 5 mm remains patent (TOBEand PRATT, 1975a); (b) the dry weight of terminal oiicytes increases dramatically between 5 and 8 mm (HILL et al., 1968). Thus it appears that vitellogenesis continues in oijcytes above 5 mm in length, even though the CA are synthesizing JH at low rates. This later phase of oiicyte growth may simply require less JH than the early growth phase. In fact, the results of HIGHNAMet al. (1963) suggest that this may be the case, since oiicyte growth ceases completely in allatectomized animals. irrespective of oijcyte length. JH synthesis and feeding after starvation
The present study reveals that feeding of S. a. results in transient elevated rates of JH synthesis, in the case of females starved from day 6 or in elevated but subnormal rates of JH synthesis in females starved from day IO. Although these rates of JH synthesis may be subnormal, when compared to control values (TOBE and PRATT, 1975a), they are nonetheless sufficient to support oiicyte maturation albeit at a reduced rate. Starvation thus appears to suppress the ability of CA to synthesize JH and feeding does not completely reverse this effect within the experimental period. HIGHNAMet al. (1966) reported a rapid reduction in stainable material in the NCC 1 following feeding in females starved for 7 days and a complete depletion by 4 hr after feeding. The relationship between release of
greguria after a 5 day starvation
707
JH synthesis and oijcyte growth in Schistocerca
this material and CA activity is uncertain although it can be suggested that the increase in rates of JH synthesis by 24 hr after feeding occurs as a consequence of the release of neurosecretion. However, as noted above, neurosecretory axons in the NCC I do not enter CA directly (STRONG, 1965; MASON, 1973) and therefore, observations on the NCC I may not be directly relevant. Thus, it cannot be ascertained whether starvation causes a direct impairment of the CA or a reduction in synthesis or release of the allatotropin which stimulates the CA. Starvation and subsequent feeding of female Leucophaea maderae does not result in a significant change in the number of nuclei in the CA but does result in large changes in nucleo-cytoplasmic ratios (VON HARNACK, 1958). In this insect, starvation does not appear to cause a decrease in the-number of CA cells and feeding does not cause an increase in cell number. If a similar situation occurs in S. a. gregaria, the changes in rates of JH synthesis during starvation and feeding are not the result of changes in cell number of the CA but rather the result of changes in the levels of activity of rate-limiting steps in JH synthesis and/or in the percentage of cells of the CA synthesizing JH (see also TOBEand PRATT, 1976). Stimulated
JH synthesis
It is possible to conclude that starvation does not impair the final two enzymic stages in JH synthesis, on the basis of the results obtained with farnesenic acid stimulation (Table l), and it is reasonable to assume that feeding does not alter the enzymic activities of these two final stages. In addition, rate limitation does not occur at this stage of JH synthesis, in normal animals or in starved animals. Thus, the decline in JH synthesis during starvation is not the result of rate limitation of these final stages. Starvation does not appear to alter the activities of the two terminal enzymic stages in JH synthesis and accordingly, rate limitation must occur earlier in the biosynthetic pathway. The fact that rate limitation does not occur at the final two enzymic stages in JH synthesis may also be demonstrated through examination of the fractional endocrine activity ratio (FEAR-see TOBE and PRATT, 1976). Using the median values for spontaneous JH synthesis on day 5 after withdrawal of food (from Figs. 1 and 3), the FEAR for animals deprived of food from day 6 was 0.005 and for those deprived of food from day 10 was 0.012. Thus, stimulated JH synthesis (TOBE and PRATT, 1974b, 1976) is approximately two orders of magnitude greater than spontaneous synthesis. Clearly, rate limitation in JH synthesis by starved animals occurs prior to the final two enzymic stages. Acknowledgements-This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. We thank Drs. T. FRIEDEL,H. INJEYAN and E. MUNDALLfor critically reading the manuscript.
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BASSURMANOVA 0. K. and PANOVA. A. (1967) Structure of the neurosecretory system in Lepidoptera. Light and electron microscopy of type A-neurosecretory cells in the brain of normal and starved larvae of the silkworm Bombyx mot-i. Gen. camp. Endocr. 9, 245-262.
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