Cellular and volumetric changes in relation to the activity cycle in the corpora allata of Diploptera punctata

CELLULAR AND VOLUMETRIC CHANGES IN RELATION TO THE ACTIVITY CYCLE IN THE CORPORA ALLATA OF DIPLOPTERA PUNCTA TA CATHARINE M. SZIBBO* and STEPHEN S. TOBE

L>rpartment of Zoology. l_Jniversity of Toronto, 25 Harbord

(Rrcrivrcl I fk~r,&r

Street. Toronto

M5S IAI. Ontario.

Canada

1980: rc~i,~d 6 March I98 I )

Abstract

In the corpora allata (CA) of the viviparous cockroach. Diploprera puncrata, a cycle of juvenile during ovarian maturation can be correlated with cyclical changes in CA volumes and cell numbers. llptake of [JH]-thymidine occurs in nuclei of CA cells during periods of increase in cell number. Both members of a pair of CA maintain symmetry of volume, cell number and rate of JH synthesis. After a cycle of CA activity. the CA can be transplanted to a young. allatectomized female, where they

hormone

support

(JH) synthesis

a second

wave of oiicyte development.

INTRODUCTION THY MLATION between corpus allatum (CA) volume and ovarian development in Diploptera punctata Eschscholtz was first described by ENGELMANN(1959), who observed a peak in the volume of CA per nucleus midway through the first gonotrophic cycle. Using an irl vitro radiochemical assay for the biosynthesis juvenile hormone (JH), TOBE and STAY (1977) showed that during this period the rate of synthesis of JH increases and decreases by more than IO-fold, with peak synthesis occurring at day 5. Thus in D. punctata, structural changes in the CA appear to be correlated with changes in JH synthesis. A parallel between rates of JH synthesis and CA volume during ovarian development is found in Nauphoeta cinerea (LANZREIN ~‘t al.. 1978) and in Leptinotarsa decernlineata (SCHOOWVELD et a/., 1977; KRAMER. 1978). but not in S~~listo~rr-c,aRre~aria (TOBE and PRATT, 1975; INJEYAN and TOW 1981). The significance of volumetric changes in the CA and the manner in which such changes reflect or underly changes in hormone synthesis has not been resolved (ENGELMANN, 1970). Following the studies of ENGELMANN (1957) on changes in CA volume and ‘cytoplasmic-nuclear ratio’ during ovarian development in Leucophaea n~adercte. SCHARRER and VON HARNAC.K (1958) determined that increases in CA volume were due not only to increases in cytoplasmic volume, but also to an increase in the number of nuclei. It has been suggested that cell division plays a role in volumetric increases in CA of adults of Oncopeltus jhsciatus (JOHANSSON, lY5X) and in N. cinrrea (BARTH and SROKA, 1975). On the other hand, increases in volume are thought to occur without cell division in several other species, e.g. L. ~l~~linrcrtrr~u~u( SWWNEVELD, 1970) and Choleva trygrcsltrltr ( DELI L!RAN(‘Eand CHARPIN. lY75). Our work represents the first study since that of SC‘HARKERand VON HARNACK (1958) to investigate in detail changes in cell number occurring during a cycle * To whom all correspondence should be addressed.

in CA activity. This also represents the tirst study to demonstrate DNA synthesis as manifested by the incorporation of [3H]-thymidine in CA of an adult insect. Because of the recent developments enabling the direct measurement of rates of both spontaneous and farnesoic acid-stimulated synthesis in D. punctatu (TOBE and STAY, 1977; FEYEREISENer ul.. 1981), it is particularly important at this time to establish the underlying cellular changes in the CA of D. punctata. Furthermore, it is possible that manipulations which result in altered CA activity are modulated through changes in CA structure (STAY and TOBE. 1978).

MATERIALS

AND METHODS

The D. punctata colony was maintained at 27°C with a 12 : 12 hr. light : dark photoperiod. Under these conditions, mating occurs on day 0, immediately after imaginal emergence. whereas oviposition occurs at day 8 or 9. Newly-emerged adults were isolated from the colony O-7 hr after ecdysis. Only mated females were used. All operations were performed within 8 hr of ecdysis: animals were maintained in a 28’C incubator without a light/dark cycle thereafter. Implantation operations were performed as described by STAY and TOBE (1977), except that the citratefortified bathing medium (PRATT and TOBE. 1974) did not contain penicillin or streptomycin. The synthesis of JH by isolated CA was determined in vitro by a radiochemical assay based on the incorporation of the methyl moiety of [methyl-‘“Clmethionine (Amersham-Searle. Arlington Heights. Illinois, tinal specifc activity I .3-l .4 GBq;mmol: 35-3Y mCi!‘mmol) into C,,JH (JH III) after a 3 hr incubation period (PRATTand ToB~. I Y74: Tout and PRATT, lY74). C,,JH is the only juvenile hormone synthesized by CA of adult D. punctala in virro (TORIand STAY. 19771. Measurement of the ovaries at the time of assay provides a bioassay for the cumulative effects of JH titres (TOBE and STAY, 1977). For histological studies, CA were removed and tixed

655

CATHAKINE

h5h

M.

SZIBBO AND STEPHEN S. TOBE

in Bouin’s tixative (HUMASON. 1979). They were embedded in paraffin (m.p. 56_57”C), serially sectioned at 5 pm with random planes of section and stained in Mallory-Heidenhain (CASON, 1950).

Initial measurements of CA volume were taken immediately after removal. The CA were placed in a small volume of bathing medium (TOBE and PRATT. 1974) fortified with 20 mg/ml Ficoll (Sigma, St. Louis. Missouri) and three axes measured with an eyepiece micrometer under a dissecting microscope. The volume ot’the CA was assumed to be that of an ablate spheroid and calculated using the formula C’=4/3 U/X, where U. h and (’ represent the radii of the three axes. after the method of TUBE and PRATT (1975). Histological sections of CA were used to obtain CA volume using the method of SCHARRER and VON HAKNAC‘K(1958). With the aid of an eyepiece grid the area of every 5th, 7th or 9th section was meask!red and the section volume determined by multiplylng the value by section thickness. The volume ofintermediate sections was estimated from the mean of the two measured consecutive sections. Figure 1 shows the relationship between the volume of fresh CA and that of the same CA after sectioning. Volumes obtained from paraffin sections were less than one half that calculated before fixation. This is probably due to tissue shrinkage that occurs during values from histological sections dehydration: probably underestimate volume. However, the linear relationship demonstrated between the two methods suggests that either is satisfactory to illustrate relative volume differences between CA. Histological sections were used to determine the CA volume in this study because corresponding studies of the CA cell number required fixation of the CA. It should be noted that these types of volume measurement do not take into account extracellular spaces within the CA, nor the volume of CA nuclei; thus we refer to the volume of CA rather than the volume of cytoplasm.

Nuclear

numbers

in serial sections

Nuclear

under the compound microscope were counted using an eyepiece micrometer grid. Since the nuclear diameter was estimated to be just over 5 pm, it was thought that by counting all possible nuclear profiles in alternate 5-pm-thick CA sections. each nucleus would be counted only once. Those nuclei which fall exactly between the sections would not be counted because the portion of the nucleus falling in the counted section is too small. After observation of serial sections of over 100 CA, nuclear size was compared in one representative section through the middle of the CA from a day-l animal (inactive CA) and from a day-5 animal (active CA). From photographs of the light microscope sections, the diameters of all nuclear profiles in each section were determined and a frequency distribution prepared (Fig. 2). Smaller nuclear profiles probably represented glancing sections of nuclei. When the

of CA viewed

% . :.. I .

.

.

15

3

.

9

1

2

0

.

I

.

t

.

.

.

4

HISTOLOGICAL

l

l

.

.

o9

0. .

VOLUME

6 (106

8

volume).

,

10

)mA

I, Volume ofCA estimated before fixation (fresh volume) compared sections (histological

.

.

.

I

.

.

52

paraffin

(pm)

Fig. 2. Frequency distribution of diameter of all detectable nuclear protiles from one 5 pm section (A) from a day-5 CA or (B) from a day-l CA.

o^ 25 E 7 t

Fig.

Diameter

to estimates

of volume from serial

Each point represents the volume for a single CA.

657

c

.”’

50

pm

Fig. 3. Paraffin sections through CA centre. Material was stained with Mallory-Heidenhain. (A) Day-O CA showing smaller diameter of nuclei in CA interior (B) Day-5 CA (C) Higher maginification view of extracellular spaces in day-5 CA (D) Autoradiogram showing localization of 13H]-thymidine label over CA nuclei of gland exterior.

Corpora allata of Diploptera frequency distributions from active and inactive CA are compared, it is apparent that of day-5 CA is shifted towards slightly larger diameters, the modal nuclear diameter being 6.5 pm compared to 6.0 pm for day-l CA. Differences in nuclear diameter can alter estimates of the number of nuclei per unit volume (ABERCROMBIE. 1946) but a 0.5 pm difference in nuclear diameter between active and inactive CA could contribute to an overestimation of the difference between the total number of nuclei per CA at these ages of, at most, 34; (see the formula Of ABERCROMBIE, 1946). The method of calculation of the total number of nuclei per CA used by SCHARRERand VON HARNACK (1958) relied on thecounting of only 3 sections per CA, but changes in nuclear size were not taken into account. Because of crowding of nuclei in the CA, in distribution of nuclei, slight heterogeneity differences in nuclear size in different areas of the CA and the impossibility of detecting small fragments of nuclei caused by near tangential section, we believe that the counting of nuclei in alternate CA sections provides a valid estimate of the total nuclear number per CA. After counting the nuclei in every alternate section for over 75 CA, it was found that it was only necessary to count the nuclei in every 5th or 7th CA section and estimate those of omitted sections from the means of two adjacent counted sections to obtain the nuclear number to within 50, of the above method. Because at least 7 sections per CA were counted, slight heterogeneities in nuclear distribution and nuclear size would not critically affect the total estimated number of nuclei. There is no evidence for multinucleate cells in CA of D. punctatu; the nuclear number can be assumed to be an estimate of the cell number. The total CA volume was divided by the total nuclear number to give the volume of CA per one million nuclei. Autoradiograph.) For autoradiographic measurement of the incorporation of [3H]-thymidine, D. punctafa of various ages were injected with 1 pl(37 kBq; 1 @i) of [3H]-thymidine (specific activity 74 GBq/mmol; 2 Ci, mmol, Amersham-Searle, Arlington Heights, Illinois.) through the membrane at the coxatrochanter junction of a metathoracic leg after the insects had been chilled on ice. The animals were incubated at 28 C for 4 hr. then killed and the CA dissected out. After tixation for at least 48 hr in Bouin‘s tixative. CA were embedded and sectioned as above. Following hydration, mounted sections were coated with Kodak NTB-2 nuclear emulsion after the method of KOPRIWA and LE BLOND(1962). Slides were e.\posed for 12-14 Jays at 4°C. then developed and stained with Metanil yellow-haematoxylin (SIMMELet a/.. 195 I ). For quantifcation of results, the number of labelled nuclei were counted in alternate CA sections. the total number of nuclei per CA determined and the percentage of labelled cells calculated. Label was localized almost entirely over nuclei, the background being less than 2 grains per nucleus. Where there were fewer than 4 cells labelled per CA. the percentage was expressed as 0. Nuclei with fewer than 7 grains were not consrdered to be labelled: however most labelled nuclei were head\ labelled.

659

RESULTS Structure

of the CA of D. punctata

The histological appearance of CA of D. punctatu changed during the cycle of JH synthesis of the first gonotrophic period (Fig. 3). As previously reported by ENGELMANN (1959), there was an increase in internuclear distance and in the total volume of the CA (compare Fig. 3A and 3B). Nuclei were relatively spherical (Fig. 3). In CA of all ages, interior cells often stained more darkly with Mallory-Heidenhain than peripheral cells. In inactive day-0 CA (Fig. 3A) the central nuclei were slightly smaller than those of the CA periphery, but there was much less difference between central and peripheral nuclei in active CA (Fig. 3B). Occasionally, extracellular spaces were visible between exterior cells of active CA (Figs. 3B, C). Such spaces are also apparent at the EM level (JOHNSONand STAY, unpublished observations) as well unfixed unpublished as in tissue (Lococo, observations). Symmetry

of the CA

Histological CA volumes and nuclear numbers were recorded in both members of 18 CA pairs from animals of different ages. For each pair, the smaller CA volume or nuclear number was divided by the larger (Table 1). In terms of both volume and nuclear number, the CA of D. punctata were highly symmetrical. Seventeen out of the eighteen pairs (94”“) had individuals whose volumes and nuclear numbers fell within a ratio of 0.8-1.0. Thus the volume or cell number of one member of a pair can be judged from the other member with at least 201, accuracy. Subsequent measurements were done on only one member of a pair. To determine if the CA of D. punctata are functionall\ as well as structurali! symmetric. JH synthesis by the right and left members of pairs of CA was determined. Figure 4 shows the sum of rates of JH synthesis by right and left members of a CA pair vs. age. Figure 5 shows the ratio of JH synthesis by’ the right CA over that of the left CA vs the total rate of JH synthesis for the gland pair for these same CA. Figure 5 also indicates that the synthesis of one member of a pair was less than one half or greater than twice that of its partner for only 10% of the pairs. Analysis of variance showed that over the total range of CA activities the variability in rates of JH synthesis between members of the same pair was less than that between different pairs (P
Table 1. Symmetry of CA volumes and cell numbers Ratio of smaller/larger

0.9 -1.0 0.8-0.9 0.7-0.8

Parameter measured (I’,,) CA volume Cell number 83 11 6

50 44 6

For 18 pairs of CA of different age during the tirst gonotrophic cycle, volume and cell number was determined for both members of the pair and the smaller value in each pair divided by the larger. The percentage of CA pairs failing in each ratio category is shown for both number of nuclei and CA volume.

M. SZIBBO

C‘ATHAKIN~

6

4

2

8

Age (days) IFig. 4. Rate of JH synthesis per paw ah a functton of age during the lir\t oviposition cycle. Sample six i\ given beside the verttcal bars which represent standard errs of the mean

(S.E.M.). r

AND

STEPHEN

S.

TOBE

Figure 6 shows that there were major changes in CA volume and cell number (Figs. 6B, C) that occurred during oiicyte growth (Fig. 6AL The changes in C.4 volume and cell number paralleled changes in rates ot JH synthesis (Fig. 4). The CA used for Fig. 6 were different from those used for Fig. 4, although the rate of oiicyte development was similar in each case and was similar to that reported by TOBE and STAY (1077). According to Fig. 6. CA volume increased to more than 200”,, of its original value during the gonotrophic cycle and the maximum volume was observed on day 5, the time of maximum hormone synthesis (Fig. 4: TOBE and STAY, 1977). The number of nuclei per CA mcreascd from 6000 at da! 0 IO approi. WOO h! da\ 5 (a ?3”,, increaser. Thereat&r. the number ~~fnuclci tic, CA decreased to its original value. Mitotic tigures were observed between 2 and 4 days after adult emergence. hut only in a small percentage ofcells. No attempt was made to quantify pycnotic nuclei or lysosomal activit) during the time of apparent cellular necrosis. Because at times of peak synthesis the increases m CA i olume surpass those of nuclear number. there is ;i marked increase in the volume of CA per nucleus (Fiy. 6D). This contirms the results of EW~.L.MANN ( 1959). although he arrived at larger absolute values of the ‘cytopiasmic-nuclear ratio’. ENWL MANN ( 1959) also stated that CA volume peaks at the mid-ovarian cycle stage. hut Fig. hC extends his finding to
X=L Y=R

A.

0

._..: :.~L...’

.9.

.L..

100 C,SJH

SYNTHESIS

200

(pmol

h-l

perpa~r)

l:is. 5. The ratw ofC‘,,JH hynthe\ls by I& (LI and riyhr (R) C‘A of adult female D. l~trr~lcrrcr as a function of total JH s~nrhes~stleft plus righl CA). A value of I .Oindicates that the left and right CA have synthesized the same quantity of JH. For values above the broken line. L > R and for values below. R > I.. Fach pomt ~c~~~-cwntsa single determination on an

3

Age

‘days:

I-lg h. C’hanges in CA paramctels
Corpora

I

1

2

3

4

661

I”H]-thymidine between ages 3 to 5 Jays and verb few at other times. As might be expected. the period of peak DNA synthesis preceded the time at which the maximal cell number was observed; thus it is probable that DNA synthesis followed by cell division occurred prior to the time of maximal cell number. In some autoradiographic CA sections with a high number of labelled cells. it was clear that labelled cells were localized in the gland periphery (Fig. 3D). Because changes in CA volume and cell number occurred at the same time as changes in rates of JH synthesis, it is useful to express the changing rate of JH synthesis on a per unit tissue basis (Fig. 8), following the practice of TOBE and PRATT (1975). This value was calculated by dividing the values for mean rates of JH synthesis from Fig. 4 bl, either CA volume or cell number (Figs. 6B. C) at each age. Since these measurements were not performed on the same CA, this ratio must be regarded as only an estimate of the actual rate of JH synthesis per unit tissue. Figure 8 shows that, due to the large changes in rates of JH synthesis in comparison to changes in CA volume and cell number, there were major changes in the rate ofJH synthesis per unit CA volume. This calculated ratio was low in the first few days and the final stages in the gonotrophic cycle, but increased more than 5-fold between days 3 and 4.

‘k. 5

0

allata of Diploptera

5

'0

6

11

c

7

Age fdaysl Fig. 7. Mean percentage of CA cells labelled with [3H]thymidine, 4 hr after injection, as a function of age during the first reproductive cycle. Sample size is presented beside the vertical bars which represent S.E.M.

part of the volumetric increase coincides with an increase in cell number. The number of cells labelled 4 hr after injection of [-‘HI-thymidine was determined at each day between 0 and 7 days after emergence. Figure 7 shows that onI) a small percentage of cells became labelled: however. there were detinitely some cells incorporating

The ability qf day-8 CA to support successsive cycles qf’ oiicvte development

To determine whether day-8 CA of D. punctata were

0 8 Age of CA at

1

,_

t

1.6

f : f

1.4

: E U-l 6 1.2 B ‘.O

0

2

4

6

,

a

Age (dars) Fig. 8. Estimated values for rates of JH synthesis: (A) per lotal CA volume, (B) per total number of CA cells. as a function of age during the first reproductive cycle. Values were calculated from Fig. 4 and 6B or C as described in text. Vertical bars represent the S.E.M. calculated by pooling the variance about both means used to calculate the ratio.

immediatel~~

1

time

0 8 of implantation

ldwl

B

0 Age of CA at

8 time

16 of

implantation

khsl

Fig. 9. (A) Age at spermatophore release (Sp-.) or oviposition (0~‘) when allatectomized day-0 hosts were implanted with a pair of day-0 CA (n= 5) or day-8 CA (n = IO). (B) Oiicyte length at day 5 when day-0 hosts were implanted with a pair ofdayCA (age of CA at operation = 0 days; n = 61. day-8 CA (age of CA at operation = 8 days. n = IO). or-day-8 CA that had been implanted previously into dav-0 animals for two cycles of oiicyte development in succession (age of CA at operation = 16 days, n = 51. Narrow vertical bars represent S.E.M.

able to support another wave of oiicyte development immediately following a cycle of activity. CA from day-8 animals that oviposited on day 8 were implanted into allatectomized day-0 animals. Controls were dayO-CA implanted in allatectomized da>-0 females. Figure 9A shows the time required for spermatophore release and oviposition for implants of CA aged 0 or 8 days. These times were similar in both cases. To determine whether day-l 6 CA could support yet another cycle of egg growth. CA were transplanted twice: once from 8-day females to O-day females and again to O-day females when the tirst host was 8 days old. The ability of these glands to support growth is shown by the length of oiicytes of the host 5 days after receiving CA implants. This is shown in Fig. 9B in comparison with CA that had supported I or 2 cycles of ovarian development.

DISCUSSION

Both CA volume and number of nuclei per CA are similar in both members of a CA pair in D. punctuta (Table I ). In contrast, S~HOONEVELD(1970) observed large differences in volume between right and left CA in L. decernlineutu which he presumed were due to differences in cell number per CA. l-or all levels of actlvith from ages tJ to 7 da!s. lett and right CA of a pair exhibit similar rates of JH s> nthesis I Fig. 51. S> mmetrl in rates of JH s!,nthesis within pairs of CA has been observed pre\iousl\ for the CA of Pcriplruwtu umw(~mu. where the difference in 5) nthetlc rates is less than Z-fold for 70”,, of the cases examined (Wt:.\vt~. 1979). In 11. purrc~tu/u the difference ih less than 2-t’oltl for 90”,, of the CA pairx. Contrq to the hndings in P. tuwrkmu (WEAVIK. 19791. in /). prtr?c’ftrluthere is no indicallon in 4exuall\ Immature co&roaches da hr~el‘postecd~sal period 01 as\ mmetr! m CA 1unctwn. Kate5 01. J H 5) n thesis at-c less symmetric than either CA Lolumcs or cell

numbers. The symmetry in rates ofJH synthesis in D. punctatu and P. clnlrricana contrast with those for CA of S. greguriu. where asymmetries of IOO-fold are not uncommon (TORE. 1977). WEAVEK (1979) proposed that the CA a.,ymmetry of S. greguriu occurs because JH biosynthetic activity is controlled primarily by nervous connectives (TORE et u/., 1977). but in P. anzrric~unu the CA are controlled predominantly via chemicals released into the haemolymph. Nonetheless. nervous connectives between right and left CA in cockroaches (ADIY~DI. 1974: FRASEK and PIPA. 1977) may play an important part in maintaining symmetric synthesis. An experimental manipulation such as unilateral allatectomy or nerve section disturbs this normal CA symmetry. Clrung~~s in the CA during the gonotrophic

cycle

For D. punctota, changes in JH synthesis, CA volume and CA cell number are clearly parallel (Figs. 4.6). Minimum CA volume, nuclear number and rate of JH synthesis are observed in newly-ecdysed and pregnant females, whereas maximum values are found at the mid-gonotrophic cycle stage. These changes in CA volume and cell number are similar to those which

occur during the ovarian cycle in L. muderue, where a cycle in number of nuclei per CA occurs during both the tirst and second pre-ovulation periods. Similar parameters must be studied in detail in insects other than cockroaches to determine if. as a general rule. changes in CA cell number accompany changes in \olttmc and acti\ll!. The 33”,, increase observed in cell number by day 5 is accompanied by an increase in the percentage of cells incorporating [“HI-thymidine. The low percentage of cell\ Incorporating 1‘l-1 I-lh!mldine (Jab-5 mean approximately 100 cells per CA) could be because rapid clearance of labelled thymidine and failure of a large proportion of labelled precursors to reach tissues far from the site of injection (SELMAN and KAFATOS. 1974) reduces the effective time of exposure to radioactive precursor. Circadian rhythms in the cell cycle of the CA could also introduce variation in the uptake of [jH]-thymidine by CA cells; daily rhythms in the nuclei of CA of Drosophila nzelunogaster were observed by RENSING et a/. (1965). Nevertheless. the mcorpot-allon of ( ‘H l-th> midme and mitotlc tigures preceding peak cell numbers in the CA the conclusion b[rengthens that increases in cell number before day 5 are due to cell division. The corresponding decrease in cell numbers occurring in the CA during the la!ter half of the gonotrophic cycle presumably involves cellular destruction. In the CA of L. nzuderue. SCHARRERand VON HARNAC~ (1958) also observed some mitotic figures in the CA of L. muderur prior to the attainment of maximum cell number and found that pycnotic nuclei appeared more often in CA returning to the inactive state. Changes in nuclear number in the CA. they postulated, are due to changes in the rate of cell turnover. DNA synthesis in CA has not been recorded previously in an adult insect: KRISHNAKUMARANet al. C1967) ohserved uptake of (JH]-thymidine by less than

I I I’: 01 ( :I cell\ iti 1;1r\ ;IC 01 .Ir~/lr~wc~~ p~~lypiwnus. but I~IILOLICgruuth durmg ~CL~II~~II~CIIL&SO occurs in many other somatic tissues. In D. punctata the incorporation of [“HI-thymidine is most pronounced Ill the C‘A periphery (Fig. 3,. SimilarI\ WIGGLESWORTH(1934) observed that in the CA of Rhodnius prolixus only peripheral cells grow and divide. In the CA of D. punctuta, there is some heterogeneity in nuclear diameters within the CA, with nuclei at the CA centre being slightly smaller than those at the periphery in inactive CA (Fig. 3A). There is much less central/peripheral variation in active CA (Fig. 3B). SCHARRERand VON HARNACK (1958) failed to establish any relationship between nuclear diameter and <‘A volume in L. mukruc. However. SCMAKKLK (1964) stated that nuclear size does fluctuate somewhat during an activity cycle. In the CA of D.punc/utu it is possible that the slight increase in modal nuclear diameter occurring during a gonotrophic cycle (Fig. 2) could be primarily due to the enlargement of interior nuclei. Nuclear enlargement has been associated with increased CA activity elsewhere (e.g. SCHARRER. 1964; TAREDA. 1977: JOL’I Cl Nl.. 1968; PAL~V~DY and GRIMAL. 1976; ScHooNEvELr), 1970), and in mammalian cells nuclear enlargement and chromatin dispersion are associated with the activation of nuclei (Lew~u. 1974).

663

Corpora allata of Diploptera The ability, o/‘CA to support successive waves ofocicyte development The CA of D. punctata undergo extensive structural and synthetic changes during the course of the gonotrophic cycle (Figs. 4,6,7; TOBE and STAY, 1977). It was not known if inactive CA from day-8 animals immediately following a cycle in CA activity could support a second successive cycle of oiicyte development, or whether a quiescent period was required before CA again acquired the ability to respond to the signals causing cell proliferation and increased JH synthesis. That this ability is acquired by day 18is indicated by the observation that inactive CA of day-18 females can achieve high rates of JH synthesis after transplantation into allatectomized day-0 animals (STAY and TOBE. 1977). However, LANZREIN rt al. (1978) found that in N. cinerea the CA of ovulating females seemed incapable of stimulating oiicyte growth after transplantation into young females. Figure 9 shows that inactive day-8 CA are capable of supporting immediately a second cycle of oijcyte maturation when transplanted into allatectomized day-0 females. In fact. the rate of oiicyte growth is accelerated in comparison to that in the presence of day-0 CA f Fig. 9). although O-day and 8-day CA have similar numbers of cells (Fig. 6C). The same CA seem less able to support three cycles in succession, possibly due to damage in handling. Therefore, the CA of D. punctuta appear to be able to respond to the activating stimulus in allatectomized day-0 animals despite the fact that CA of this age have recently undergone a decrease in volume and cell number. Whether cycles in volume and cell number occur in the implanted CA during the second induced gonotrophic cycle is presently under investigation. The possible importance 41 cellular changes in the CA to changes in rutes o/ JH synthesis

It is clear from Fig. 6 that increases in CA volume and cell number are small when compared with the more than IO-fold increase in JH synthesis (Fig. 4). This is reflected in a change in the calculated rates of JH synthesis per unit CA volume and per cell (Fig. 8). which are low in young animals and in animals nearing oviposition but which rise sharply between days 3 and 4. Changes in rates ofJH synthesis per cell and per unit CA volume parallel changes in the spontaneous rate of JH synthesis divided by the farnesoic acid-stimulated rate [the I’ractional endocrine activit! ratio (FEAR): (TOBf. and PKA 1.I 1976) 1In !j punc~turo ( Ff-\‘tKl-ISFrl et d., 1981).The increase in rate of JH synthesis per unit CA volume or per cell and in FEAR between days 3 and 4 indicates that at this age there is an increase in the spontaneous rate ofJH synthesis by all CA cells. an increase in the fraction of spontaneously synthesizing CA cells. or both (see TOBE and PRATT. 1975; 1976). For the CA of D. punctata we have no information concerning the synthetic rates of individual cells and whether the proportion of active cells increases at times of peak synthesis. However, the observations of morphological and proliferative differences between peripheral and central CA cells suggest that not all CA cells function similarly. Such features as the smaller diameter of nuclei and the smaller internuclear

distances in the centre of CA, the difference in staining intensity of interior and exterior cells and the presence of extracellular spaces between exterior cells (Fig. 3) have been observed in the CA of L. maderae (SCHARRER and VON HARNACK, 1958; SCHARRER, 1964), as well as in other species [see NAYAR (1956): TAKEDA (1977); MENDES (1948); WIGGLESWORTH (IY34): SCHOONEVELI) (lY70): DAY t 1Y43): Snu (1965); YIN and CHIPPENDALE(1979)]. Dividing cells. which are localized at the CA periphery, and nondividing cells may be in a different physiological state with regard to JH synthesis. SCHARRER and VON HARNACK (1958) postulated that in the CA of L. maderae, mitosis replaces ‘exhausted’ JH-synthesizing cells with younger cells. In the phase of activation more secretory cells are added to the CA, whereas in the declining phase necrosis of CA cells accounts for the decline in CA activity. Mass necrosis of cells within the CA interior has also been proposed by JOLY (1967) to alter output of the CA of Locusta migratoria after the first gonotrophic cycle. Thus. increases and decreases in cell number in the CA could be directly responsible for alterations in the rate of JH synthesis, despite the fact that cell number changes are small in comparison, provided that proliferation and necrosis modify a JH-synthesizing subpopulation of CA cells. The determination of whether previously inactive cells are recruited, perhaps from the CA interior, during increases in CA activit! and whether thts recruitment involves mitosis in the CA awaits further study on the rate of JH synthesis by individual cells of the CA (TOBE and PRATT, 1976; TOBE and SALEUDDIN, 1977). In vertebrate endocrine glands cell proliferation is often provoked by the same trophic factors that stimulate hormone synthesis (GARREN et al., 1971: ERMANSet al.. 1972). In the case of the adrenal cortex, ACTH-stimulated DNA synthesis occurs in the peripheral zona glomerulosa ( IDELMAN.1970) whereas ACTH-stimulated steroidogenesis occurs in the interior zona reticularis and zona fasciculata (ICHIKAWA ef al., 1978). In an analagous manner. cell proliferation in the CA of D. punctata could be merely a secondary response to humoral factors (STAY and TOBE, 1977; 1978) causing the stimulation of JH synthesis. .4cXnowlrd~m~m/,s~~ -We

are

indebted

to

Professor

BARBARA STAY for

many helpful discussions and critic,d comments. We would also like to thank Dr. R. FEYEREIS~V. Dr. G. JOHNSON. JOHN DALE and DONALI) LOCYXY~for critically reading the manuscript. This work was supported by grants from the National Sciences and Engineeiing Research Council of Canada. CMS acknowledges receipt of an NSERC postgraduate fellowship.

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664

CATHAKINE M. SZIBBO AND STEPHEN S. TOBE

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TOBE

of Diploptera

(l(Ji

WIGGLESWORTH V. B. (1934) The physiology of ecdysis m Rhodnius prolixus (Hemiptera). II. Factors conrrolhng moulting and metamorphosis. Q. JI micro.x SC,/. 77, 191-222. YIN C. M. and CHIPPENDALE G. M. (1979) Ultrastructural characteristics of insect corpora allata in relation to larval diapause. Cull Tissue Res. 197. 45346 I.