J. Insect Physiol. Vol. 36, No. 9, PP. 655664, 1990 Printed in Great Britain. All rights reserved

0022-1910/90 $3.00 + 0.00 Copyright 0 1990 Pergamon Press plc

DEVELOPMENTAL CHANGE OF BOMBYXIN CONTENT IN THE BRAIN OF THE SILKMOTH

BOMBYX MORI

A. MIZ~GUCHI, M. HATE-A,’ S. SATO,’ H. NAGASAWA,’ A. SUZUKI*

and H. ISHIZAKI’**

‘Department 01‘ Biology, School of Science, Nagoya University, Chikusa-ku, Nagoya 464 and *Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku,

Tokyo 113, Japan

(Received 28 February 1990)

Abstract-Accumulation of bombyxin in the brain of Bombyx mori was examined throughout post-embryonic development. Immunohistochemically, four pairs of large dorsomedial neurosecretory cells of brain were shown to contain bombyxin throughout the stages from larval hatching to adult eclosion. The bombyxin content in the brain that was estimated by immunohistochemistry, immunoblotting and bioassay remarkably changed during larval-larval and larval-pupal development. The bombyxin level in penultimate-instar larvae was hi8.h in the first half while low in the second half of the stage. It became high again at the time of ecdysis to the final instar, but gradually decreased afterward until the larvae began wandering. After wandering the bombyxin content increased gradually until pupation when a level as high as that in the newly ecdysed, final-instar larvae was regained. Immunoblotting of brain extracts demonstrated that bombyxin consisted of many molecular forms, all of which were present through larval-larval development. Based on the developmen- tal changes in the bombyxin content in the brain, the physiological role of this neuropeptide is discussed.

Key Word Index: Bombyx mori; bombyxin; insulin; neurosecretory cells; immunohisto- chemistry; larval-pupal development

INTRODUCTION

Bombyxin is a brain secretory peptide of the

silkmoth Bombyx mori that was previously called 4K-prothoracicotropic hormone (4K-PITH) (Ishizaki and Suzuki, 1984). Bombyxin activates the prothoracic glands of the saturniid moth Sumiu cynthia ricini to secrete ecdysone in viva (Ishizaki and Ichikawa, 1967) as well as in vitro (Nagasawa et al., 1984). Contrast- ing to this clear-cut prothoracicotropic activity in Sumia, bombyxin is inactive on Bombyx itself as far as the in uivo pupa-adult (Ishizaki et al., 1983) and larva-larva assays (Suzuki and Ishizaki, 1986) are concerned. It is now firmly established that this peptide is a member of the insulin family from the amino acid sequence homology (Nagasawa et al., 1984, 1986) as well as from the cDNA and gene structure (Adachi ei’ al., 1989; Iwami et al., 1989; 1990; Kawakami et al., 1989). Thus, bombyxin is formed of the A- and B-chains which are linked together by disulphide bonds in exactly the same way as in insulin (Nagasawa et al., 1988). Bombyxin consists of highly h[eterogeneous molecular species which differ by some amino acid substitutions. Their

*To whom all correspondence should be addressed.

primary structure has been fully determined for bombyxin-II (Nagasawa et al., 1986, 1988) and -IV (Maruyama et al., 1988) and partially for bombyxin-I, -111 (Nagasawa et al., 1984), and -V (Jhoti et al., 1987).

The function of bombyxin in the Bombyx that produces it is still unknown. Insulin and insulin- related peptides exert a wide variety of physiological effects on growth and metabolism of vertebrates (Froesch et al., 1985). Insulin, when applied to insects, also affects sugar metabolism and in vitro cell growth (Kramer et al., 1985). From these facts, it seems highly probable that bombyxin also plays important roles in regulation of growth and metabolism of Bombyx. The use of antibodies to bombyxin is expected helpful for elucidating the function of this peptide. In a previous report we described the pro- duction and characterization of a monoclonal anti- body against a synthetic bombyxin (l-IO), a peptide corresponding to the amino-terminal lo-amino-acid sequence of the bombyxin-I A-chain (Mizoguchi et al., 1987). By using this antibody, four pairs of large dorsomedial neurosecretory cells of Bombyx brain have been identified immunohistochemically as the bombyxin-producing cells. The axon terminals at the periphery of the corpora allata were also immuno-

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656 A. MIZ~GUCHI et al.

reactive, indicating that bombyxin is transported to, and released from, the corpora allata. These obser- vations were made with O-l-day-old, fifth-instar larvae and freshly ecdysed pupae. We now report on similar studies extended to an entire postembryonic development of Bombyx using the same anti-bombyxin (l-10) antibody. In particular, a detailed study was made for the fourth- to fifth-instar larval devel- opment to examine the fluctuation in bombyxin content in the brain by immunohistochemistry, immunoblotting and bioassay.

MATERIALS AND METHODS

Animals

Larvae of a racial hybrid of Bombyx, Kinshu x Showa (supplied by Kanebo Silk Elegance Co. Ltd, Aichi), were reared on an artificial diet, “Silk Mate” (Nihon Nosan Kogyo Co. Ltd, Yokohama), at 25 + 1°C in a photoperiodic regime of 12-h light and 12-h dark. The animals were restaged on the days of larval ecdysis, wandering, and pupal ecdysis which were designated day 0 of the respective stages. Fourth-instar larvae moulted into the fifth-instar on day 4 or 5 and fifth-instar larvae underwent wandering on day 6 or 7, forming two gates due to the circadian rhythm in development (Fujishita and Ishizaki, 1982). Since the majority of larvae used the second gate, only the second-gate animals were used for experiments. Pupation occurred 4 days after wandering.

Bombyxin preparation

A partially purified bombyxin preparation termed “highly purified bombyxin” (Nagasawa et al., 1979) was prepared from Bombyx heads.

Antibody

A mouse monoclonal antibody generated against a synthetic peptide corresponding to the amino- terminal decapeptide of the bombyxin-I A-chain (Mizoguchi et al., 1987), which we refer to as the anti-bombyxin-I (l-10) antibody hereafter, was used for the immunohistochemical and immunoblotting studies.

Immunohistochemistry

Brain-corpora cardiaca-corpora allata complexes of the third- to fifth-instar larvae, pupae, and pharate adults were dissected out in the Bouin’s fixative and fixed in fresh Bouin’s for 12 h. For first- and second- instar larvae, the whole bodies or heads were fixed by cutting them open in Bouin’s so that the fixative penetrated rapidly into the tissues. Fixation was made 4-8 h after lights-on. Immunohistochemical observation was made with at least five specimens at each stage.

Bombyxin extraction from Bombyx brains

Fifty brains were taken from Bombyx larvae of each developmental stage and day-0 pupae after

anaesthetization by immersing them in an ice bath, and were homogenized with 2% NaCl using a glass-glass homogenizer cooled in an ice-water bath. Following centrifugation at 10,OOOg for Smin, the supernatants were heat-treated in boiling water for 2 min and cooled rapidly. The precipitated protein was removed by centrifugation at 10,000 g for 5 min. The supernatants were used for immunoblotting and bioassay of bombyxin.

Immunoblotting

Twenty-brain equivalent aliquots of the brain extracts were subjected to polyacrylamide gel electro- phoresis (PAGE) without SDS and immunoblotted as described (Mizoguchi et al., 1987), except that the biotin-streptavidin system was applied to sensitize the detection of mouse immunoglobulin G. After blotting, the membrane was successively exposed to a 1:200 dilution of anti-bombyxin-I (l-10) antibody, a 1: 200 dilution of biotinylated anti-mouse immuno- globulin antibody (Amersham), and a 1: 200 dilution of streptavidin-biotinylated peroxidase conjugate (Amersham) for 2, 1 and 1 h respectively. Between respective incubations the membrane was washed three times with Tris-HCl buffered saline containing 0.05% Tween-20. SDS-PAGE was performed using the Laemlli’s system (Laemlli, 1970) without mercap- toethanol on a 15% polyacrylamide slab gel 1 mm thick. In this experiment 10 brain equivalents of the brain extract from day-O, fifth-instar larvae and 40 head equivalents of “highly purified bombyxin” were applied. The procedure for the immunoblotting following SDS-PAGE was the same as that after native PAGE.

Bioassay for bombyxin activity

Ten brain equivalents of the brain extracts were serially diluted with 50 mM phosphate-buffered saline containing 0.04% bovine serum albumin and injected into debrained dormant pupae of Samia Cynthia ricini to assay the bombyxin activity of evoking adult development (Ishizaki and Ichikawa, 1967).

RESULTS

Immunohistochemical assessment of bombyxin content fluctuation in the bombyxin -producing neurosecretory cells

First, we surveyed roughly the immunostaining of the brain and endocrine complex with the bombyxin-I (l-10) antibody throughout the postembryonic devel- opment of Bombyx. Thus, we examined the brain- retrocerebral complex from day-0 larvae of the first- to fifth-instars, day-0 pupae, and pharate adults one day before adult eclosion. Four pairs of large dorso- medial neurosecretory cells of the brain, the same cells as those shown previously to be immunoreactive to bombyxin-I (l-10) antibody (Mizoguchi et al., 1987), were strongly immunoreactive in all the stages

Fig. 1. Bombyxin-producing neurosecretory cells in the brain from various developmental stages of Bowzby.r mori. Medial large neurosecretory cells in the brain of day-0 larva of the first (A). second (B). third (C), fourth (D), and fifth (E) instar, a day-0 pupa (F), and a pharate adult one day before eclosion (G) were immunostained with bombyxin-I (I-10) antibody. Serial sections demonstrated four pairs of

immunoreactive neurosecretory cells in all the stages. Scale bar, 50 pm.

657

Fig. 2. Change of bombyxin-I (I-10) immunoreactivity in the medial neurosecretory cells of Bombyx brain during larval-pupal development. Brains of Bombyx larvae on day-l(A), -3(B), -5(C), -7(D), and -9(E) of the fifth-instar and newly ecdysed pupa (F) were immunostained with bombyxin-I (l-10) antibody.

Note the difference in the intensity of immunostain among the cells. Scale bar, SOpm.

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* L4 W-L - 5

0 1 2 3 4 0 1 2

Fig. 3. Immunoblot of bombyxin-family peptides in the brain of developing B0mby.x larvae. Brain extracts from the larvae on day 04 of the fourth-instar (L4) and on day O-2 of the fifth-instar (L5) were resolved

by native PAGE, and then immunoblotted with bombyxin-I (I-10) antibody.

1 2

kd

17.0 -

14.4 -

8.2 -

6.2 -i

Fig. 4. lmmunoblotting following SDS-PAGE of the brain extract of BombF.y larvae. Ten brain equivalents of day-0 larval brain of the fifth-instar (lane 1) and 40 brain equivalents of “highly purified bombyxin” from Bombys adult heads (lane 2) were resolved by SDS-PAGE and immunoblotted with

bombyxin-I (I-10) antibody.

659

Bombyxin content in the brain of the silkmoth 661

examined (Fig. 1). The nerve fibres at the periphery of the corpora allata were also strongly immuno- reactive (photomicrographs not shown) as have been demonstrated in the previous paper. These results indicate that the synthetic machinery for bombyxin has already been established before larval hatching and bombyxin continues to be produced and released throughout postembryonic development, though the secretory activity may possibly fluctuate as larvae grow.

Next, a detailed examination was made for the fourth- and fifth-instar larvae and freshly ecdysed pupae by sampling every day to detect possible fluctuation in the immunostaining during each moult- ing cycle. During the fourth-instar, four pairs of the brain dorsomedial bombyxin-producing cells were strongly immunostained with no recognizable change in the intensity (photographs not shown). In the fifth-instar, by con’trast, the staining showed a marked change as larvae grew. Thus, during the first 3 days, the neurosecretory cells were heavily loaded with the immunoreactive material [Fig. 2(A)] but thereafter an impairment in the stainability was apparent. Remarkabsly, in most specimens, two of these cells in a hemisphere of the brain were relatively intensely stained whereas the other two cells stained poorly [Fig. 2(B), (C)l. Two strongly stained and the other two weakly smined neurosecretory cells were otherwise indistinguishable from each other, e.g. in their size, shape and position. The immunoreactivity became the weakest on day 7 when larvae began wandering [Fig. 2(D)]. On days 6 and 7, some but not all of the bombyxin cells lost completely the immuno- reactivity. During tb[e prepupal period (days &lo), the cells gradually mgained the immunoreactivity to reach the maximal degree of staining at the time of pupation [Fig. 2(F)], although more or less unequal staining between the 2-cell groups was still recogniz- able. The immunoreactivity of the axon terminals at the corpora allata periphery also tended to change in parallel with the change in somata described above

but the change was far less prominent compared to that of the somata; even on day 7 when the somata stained very poorly, the axon terminals in the corpora allata stained considerably.

Immunoblot analysis to assess the quantitatiue and qualitative developmental changes of bombyxin

When Bombyx brain extracts were immunoblotted after native PAGE, three intense and five weak bands appeared (Fig. 3). When immunoblotting was performed after SDS-PAGE, only a single band appeared which corresponded in position to the similarly treated “highly purified bombyxin” (Fig. 4). Thus, the multiple bands resolved after native PAGE are thought to represent different molecular species of bombyxin.

It is apparent from Fig. 3 that the intensity of the bands are high in the early stages of the fourth-instar (days 0, 1 and 2) while low later (days 3 and 4). This quantititive change contrasts to the results obtained by immunohistochemistry which failed to reveal a visible change throughout the fourth-instar. On day 0 of the fifth-instar, the bands became intense again but thereafter weakened rapidly. The overall band pattern did not change throughout the fourth- and fifth-instars, indicating that no change occurred in the relative amount of bombyxin components during the stages examined.

Bombyxin content in the brain assessed by bioassay

Fluctuation of the bombyxin content in Bombyx brain was assessed by the Samia pupal assay from the day-O, fourth-instar larva to day-0 pupa (Fig. 5). The activity was high (8 Samia units per brain) in the early fourth-instar (day 0, 1 and 2) but decreased later (days 3 and 4) to about a half. When ecdysed to the fifth-instar (day 0), the activity recovered to the level of the early fourth instar and decreased thereafter again. The activity remained low before and after wandering and rose again to reach the initial high level shortly before pupation.

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Fig. 5. Developmental change of bombyxin content in the Eombyx brain assessed by bioassay. Brain extracts from the fourth-instar larvae (day o-4), the fifth-instar larvae (day MO) and pupae (day 0) were

assayed for their prothoracicotropic activity on Samia pupae. W: start of wandering.

662 A. Mtzooucm et al.

DISCUSSION

Critical considerations on the three different methods used to assess the bombyxin content and the conclusion about bombyxin fluctuation in Bombyx brain

First we consider the specificity of the bombyxin-I (l-10) antibody used in this study, with respect to the recognition of the different molecular species of bombyxin. It has been demonstrated that the bombyxin-I (l-10) antibody reacts with bombyxin-II (l-10) and bombyxin-III (l-10) (the synthetic peptides corresponding to the amino-terminal l-10 sequences of the bombyxin-II and -111 A-chains) with a similar affinity to that with bombyxin-I (l-10) (Mizoguchi, 1990). It is worth noting that the amino-terminal l-10 sequence of the A-chain is highly conserved throughout bombyxins so far sequenced (Nagasawa et al., 1986, 1988; Maruyama et al., 1988) and preprobombyxins deduced from the cDNA and gene structure (Adachi et al., 1989; Iwami et al., 1989, 1990; Kawakami et al., 1989); only residues 2, 8 and 9 are variable as are among bombyxin-I, -11 and -111. Therefore, it is highly probable that the bombyxin-I (l-10) antibody recognized all molecular species of bombyxin. Many immunoreactive bands that appeared after native PAGE of the Bombyx brain extract were reduced to a single band after SDS-PAGE. This single band corresponded in position to that of the similarly treated “highly purified bombyxin”. All the native-PAGE bands may therefore presumably represent bombyxins.

A bioassay for bombyxin that relied on the prothoracicotropic activity in Samia pupae might have failed to detect some structurally related but functionaly diverged peptides. It has been argued that C-family bombyxins which were deduced from their gene structure might be functionally different from the conventional bombyxins (Iwami et al., 1990). Thus, the bioassay might have understimated the bombyxin amount.

As to the precision in quantification of bombyxin, immunohistochemistry and bioassay are apparently inferior to immunoblot analysis. The neurosecretory cells containing bombyxin over a certain threshold level would give an equally maximal immunostaining. In fact, during the fourth-instar and the early fifth- instar where immunoblot and bioassay resolved changes in brain bombyxin content, immunohisto- chemistry gave an equally heavy stain. The Samia pupal bioassay is also semiquantitative because this assay is based on the use of 2-fold diluted series of material whose response is evaluated as just positive or negative.

Thus, the three methods employed in this study have their merits and disadvantages. An important fact is that the results obtained by these methods were nevertheless not mutally contradictory though slightly different. After careful comparison of the results, it seems safe to conclude that bombyxin is accumulated in brain in a high amount at the time of

larval ecdysis, diminishes as larva grows, is kept low before and after wandering, and rises again towards the pupal ecdysis.

Speculation about bombyxin secretory activity and function in Bombyx as inferred from the brain content _ktuation

The amount of neurosecretory material contained in a neurosecretory cell at a certain moment results from the balance between the synthesis and release of that material. The bombyxin gene transcript in the Bombyx brain has been shown not to fluctuate appreciably throughout the fifth-instar (Adachi et al., 1989). Bombyxin synthesis seems therefore to occur continually in the fifth-instar, although care should be taken before concluding definitely because the amount of mRNA may not directly be correlated with the rate of synthesis of the final peptidal product, as has been proven for the brain neuro- secretory material of the mealworm Tenebrio molitor (Lack and Happ, 1976). Thus, the diminution of bombyxin in the neurosecretory cell somata may indicate the active release of bombyxin that exceeds its synthesis, based on the assumption of a constant supply of bombyxin. The present data of the im- munoblot, immunohistochemistry, and bioassay analyses then suggest that active release of bombyxin occurs at the feeding stage of the larva where active growth occurs. Insulin and insulin-like growth factors promote growth and differentiation (Froesh et al., 1985), and molluscan insulin-related peptide dictates Limnaea growth (Smit et al., 1988). Bombyxin then might likewise control the growth of various tissues of Bombyx. Another important role of insulin in vertebrates is the control of carbohydrate metabolism (Cahill, 1971). In insects also, the presence of hypo- glycaemic factors in the central nervous system has been reported (Thorpe and Duve, 1984). Since feed- ing causes hyperglycaemia in the larva, the release of bombyxin following ecdysis and its continous release during the feeding stage might be causally related to blood-sugar control. Fluctuation in the haemolymph bombyxin titre as measured by radioimmunoassay, the effect of bombyxin on sugar metabolism, and bombyxin binding to various tissues are now being examined to test the above hypotheses.

DSf)erential immunostaining in two groups of bombyxin -producing neurosecretory ceils

An intriguing fact disclosed by immunohisto- chemistry is the differential immunoreactivity to bombyxin-I (l-10) antibody of two groups of bombyxin-producing neurosecretory cells, each of which consists of two cells within a brain hemisphere. The cells of one group were heavily immunostained whereas in those of the other group the stain was weak; the cells of these 2-cell groups were otherwise indistinguishable. This differential immunostain was observed only during the late fifth-instar and pre- pupal period. It is not known, however, whether this

Bombyxin content in the brain of the silkmoth 663

unequal bombyxin accumulation in two groups of neurosecretory cells is specific to those stages because the immunoreactive material existing in amounts over a certain level and give an equally maximal staining. In fact, at other stages all of the four pairs of bombyxin-neuroscretory cells were equally heavily stained.

The following hypothetical views may account for this differential immunostaining. First, two groups of neuroscretory cells are genetically distinct, being endowed with different developmental programmes for bombyxin gene e:xpression, bombyxin synthesis, or release. Alternalively, two groups of neuro- secretory cells, though they are of the same cell type, are under different physiological regulations, e.g. under different nervous controls from other neurones. Furthermore, it may be speculated that the production and/or release of bombyxin is oscillatory in nature and the bombyxin amount in a 2-cell group and that in the other 2-cell group oscillate with different phases so that the histological profile obtained after fixation at a certain moment differs between the two groups. Pulsatile secretion of pep- tide hormones is a rather general phenomenon (e.g. Rasmussen et al., 1089; Smith et al., 1989). In an insect also, the pulsatile secretion of PTTH has been described (Gilbert ef al., 1981). The present finding of a cell-group-dependent difference in bombyxin accumulation might represent a histological manifestation of such a pulsatile secretion.

Hiruma and Agui (1977) studied the developmental fluctuation of brain neurosecretory material stained with paraldehyde-fuchsin in the armyworm Mamestra

brussicue. These authors named four pairs of medial neurosecretory cells as the group-l neurosecretory cells and classified them into the Type-I and Type-II cell consisting of two pairs of neurosecretory cells each, according to the difference in the amount of the stained material. Th,ese types were classified solely depending on the diff’erence in the amount of paralde- hyde fuchsin-positive material and were indistin- guishable at some sta.ges in which all of the four pairs of neurosecretory cells were equally heavily stained. The paraldehyde fuchsin-positive material was pre- sent abundantly in newly ecdysed final (sixth)- instar larve, decreased as they grew, and increased again after wandering to reach the same high level as in the newly ecdysed sixth-instar. The four pairs of medial neurosecretory cells of Mumestra thus bear a striking resemblance with the four pairs of bombyxin-producing cells of Bombyx, both in the differential staining of 2-cell groups and in the developmental fluctuation, and therefore might be physiologically and evolutionarily homologous to the bombyxin-neuroscretory cells of Bombyx.

The results obtained in the work to identify the PTTH-producing neuroscretory cells of the tobacco hornworm Manduca sexta appear to be interesting in relation to the speculation about the cell-group- dependent differential secretion of neuropeptides.

Agui et al. (1979) once concluded that Manduca

PTTH was produced by only one pair of the brain lateral neurosecretory cells by quantifying mH in the extracts of micro-dissected neurosecretory cells. Later, two pairs of lateral neuroscretory cells have been identified as the PTTH-cells by immunohisto- chemistry using an anti-PTTH antibody (O’Brien et al., 1986). These two different results may be reconciled if we assume that the two pairs of neuro- secretory cells consist of two groups whose PTTH accumulation oscillates with different phases and that the cells of both groups were equally immunostained to the maximal extent. Thus it seems possible that the fluctuation of a neurohormone in the neurosecretory cells with differential timing depending on subgroups is a rather general phenomenon.

Acknowledgements-This work was supported in part by a Grant-in-Aid for Scientific Research (Nos 63740423 and 01060004) from the Ministry of Education, Science and Culture of Japan. We thank Mr K. Soma and Miss I. Kubo for their technical assistance.

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