Characteristic profiles of haemolymph proteins during larval development of molting mutants of the silkworm, Bombyx mori

Characteristic profiles of haemolymph proteins during larval development of molting mutants of the silkworm, Bombyx mori

Comp. Biochem. PhysioLVol. 105B,No. 2, pp. 361-367, 1993 Printed in Great Britain 0305-0491/93 $6.00+ 0.00 © 1993Pergamon Press Ltd CHARACTERISTIC P...

1008KB Sizes 0 Downloads 39 Views

Comp. Biochem. PhysioLVol. 105B,No. 2, pp. 361-367, 1993 Printed in Great Britain

0305-0491/93 $6.00+ 0.00 © 1993Pergamon Press Ltd

CHARACTERISTIC PROFILES OF HAEMOLYMPH PROTEINS DURING LARVAL DEVELOPMENT OF MOLTING MUTANTS OF THE SILKWORM, BOMBYX MORI YUTAKA KAWAGUCHI,*t YUTAKABANNO,~"KATSUMIKOGA,~ HIROSHIDOIRA~ and HIROSHI FUJII~ tLaboratory of Sericultural Science; and :~Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka 812, Japan (Tel. 092-641-1101; Fax 092-641-2928) (Received 27 October 1992; accepted 27 November 1992)

Abstract--1. Electrophoresis of the haemolymph from developing larvae of Bombyx mori revealed that the banding patterns of proteins could be grouped into three characteristic types. 2. The young larval type exhibited at the first to the third instars (which are the phase of nutritional growth). 3. The late larval type specific to the fifth instar (which is the phase of reproductive growth). 4. The intermediary type shown at the fourth instar (which is the stage of switchover between the above two phases). 5. The tetramolter mutant M 3 lacked the intermediary type (i.e. the feature of the fourth instar) during its development, whereas the different tetramolter mutant rt had a mixture of the late and the intermediate types at its fourth (last) instar. 6. On the other hand, the mutant M 5, which is a pentamolter, repeated the intermediary type at its fourth and fifth (penultimate) instars.

INTRODUCTION The changes in content of haemolymph proteins during development of insects have been extensively studied using various species (for review see Thomson, 1975; Wyatt and Pan, 1978). Numerous components of insect haemolymph proteins, i.e. storage proteins, vitellogenin, iipophorin, proteinase inhibitors, chromoproteins, some enzymes etc. have been purified and characterized (Kanost et al., 1990). In the domesticated silkworm, Bombyx mori, the fluctuation in content of haemolymph proteins during the larval development has been observed by Wyatt et al. (1956). The protein components in the haemolymph during the larval-pupal and pupal-adult development were detected as about 10-15 bands by polyacrylamide gel electrophoresis (Nakasone and Kobayashi, 1965; Kobara, 1967). Also recognized were two kinds of the female specific proteins designated the larval type (FL) and the pupal type (FP) (Doira, 1968; Kawaguchi and Doira, 1973). The storage proteins 1 and 2 (SP-1 and SP-2) in females, and SP-2 in males, are actively synthesized in the fat body and are continuously released into the haemolymph during the middle stage of the fifth instar (Tojo et aL, 1980). Vitellogenin, recognized as the precursor of the yolk counterpart vitellin, appears in the haemolymph of young pupae (Ono et al., 1975; Ogawa and Tojo, 1981). SP-I and vitellogenin were *To whom correspondence should be addressed. CBPB m05/2--K

361

later found to be identical with FL and FP, respectively. The 30-kDa proteins, a material for the yolk formation, exist in the haemolymph at the mature larval stage as non-sex-limited plasma proteins (Izumi et al., 1981). Protease inhibitors (Eguchi and Kanbe, 1982; Eguchi et al., 1982; Fujii et al., 1989) and a carotenoid-binding protein (CBP) (Fujii et al., 1988a,b) were purified from the haemolymph of the mature larvae and pupae. Much attention has been paid to the purification and characterization of the above proteins, as well as their biosynthesis in the larvae and pupae, by using normal and standard strains of B. mori. Meanwhile, the so-called "molting mutants", in which the process of the post-embryonic development is altered, have been obtained in B. mori (Doira, 1983, 1986). In this paper, we make clear the genetically mediated characteristic profiles of the haemolymph proteins during the larval development of the molting mutants in comparison with a normal strain.

MATERIALSAND METHODS Genetic stocks and rearing o f larvae

The four strains p22, c30, tl01 and t21 stocked in the Institute of Genetic Resources, Kyushu University, were used. Larvae were raised on mulberry leaves at 25°C. The strain p22 is a normal tetramolter (+M), which has five feeding periods and undergoes four molts during the larval development. The strains

362

YUTAKA

et

KAWAGUCHI

c30 and tl01 reach the final larval instar after four feeding periods and three molts (thus these are trimolters) because of the dominant mutation M 3 and the recessive mutation rt, respectively. The t21 strain, containing the recessive mutation M 5, exuviates five times and the final instar is the sixth (pentamolter). M 3, M 5 and + M are alleles of the the M locus, which is mapped at 3.0 on the 6th linkage group, with the dominance relationships of M 3 > + M > M 5. On the other hand, the rt gene lies at the position of 9.0 on the 7th linkage group. All of these genes have arisen as spontaneous mutations (Chikushi, 1972; Doira, 1983).

Quantitative analysis of haemolymph proteins Haemolymph was collected at 24-hr intervals from day 1 of the second instar to the maturation stage of the final instar, 6 days before the larval-pupal ecdysis. Larvae were cut at the caudal horn and clear haemolymph was dropped into a micro test tube containing a few crystals of phenylthiourea. The haemolymph was spun at 5000g for 5 min at 5°C using a Sakuma Microlabofuge and the precipitate (haemocytes) was discarded. The supernatant was subjected to precipitation with trichroloacetic acid (final 5%). The precipitate was washed several times with 5% cold trichroloacetic acid and with ethanol-ether (1:1) to remove free amino acids and lipids, and then was dissolved in 0.1 N N a O H . The solution was measured for the protein concentration by the method of Lowry et al. (1951) with bovine serum albumin as a standard.

Be

A ~" 4o

RESULTS

Changes in concentration of haemolymph proteins The developmental changes in concentration of haemolymph proteins were analysed at each of the whole feeding periods from the second to the final instars of the normal tetramolter strain (+M). As shown in Fig. 1A, the protein concentration was low until day 3 of the fourth instar, with little difference

....

¢.a

,o

,,

0 L I_d.~l

~

I

1231 231 231 234567

*"

II

Ill

IV

V

S

~0

"2

(9

60 v

,~ 4 0 = 20

/

=

0 *-

L-.LJ

231 231 2 3 4 5 II

Ill

IV

~0 "2 "m

I

i

I

/J I:i, i

231231

&

X-..d

x.....J

II

III

5

°

/

i

i

i

i

i

234567 \

lV

/l A

5

6o

v

Polyacrylamide gel electrophoresis Discontinuous polyacrylamide gels for disc electrophoresis were prepared essentially according to Davis (1964) and Ornstein (1964). The gels used here contained 6.5 % acrylamide instead of the standard 7.5 %. The volume of haemolymph applied was 5 #1/column for second- and third-instar larvae and 1 / d / c o l u m n for fourth- and fifth-instar larvae. The gels were stained in 0.1% Coomassie Brilliant Blue R250 solution (ethanol : acetic acid: distilled water = 4:1 : 5) for 1 hr and destained in 7% acetic acid. The gels were subjected to densitometry using a Beckman D U - 8 spectrophotometer, and the FL concentration was calculated from the total protein concentration on the bases of the ratio of F L fraction toward all protein fractions.

al.

20 ~ .o/~.. -~ .-

0

I

I

I

L.J..J

~"l

I

231231

231 23123456

II

IV

eu

STAGES

Ill OF

V

DEYELOPME~T

IV ( DAY )

A

S

Fig. I. Protein concentration of haemolymph in normal and molting mutants during progression of larval stages. A, B, C and D, normal (tetramolter), dominant trimolter, recessive trimolter and pentamolter, respectively; II-VI, the second to the sixth instars; 1~5, day 1 to day 6. Solid line, female; broken line, male; interruption along abscissa indicates larval-larval ecdysis; S, spinning. Each point represents the average value from five measurements. The patterns for II, III of the trimolters and those for II, III and IV of the pentamolter were omitted because these were indistinguishable from those of the tetramolter.

between the female and the male. However, at the fifth instar, i.e. the final instar of the normal strain, the concentration in both sexes was steeply increased from day 1 to day 6. There was a marked sex-dependent difference in value, with the female predominating the male. Similar experiments done in the strain with the dominant trimolter mutant M 3 revealed that the concentration of haemolymph protein was almost the

Haemolymph proteins in molting mutants same as that of the tetramolter from day 1 of the second instar to day 3 of the third instar. Noticeably, the concentration was rapidly augmented in both sexes at the fourth instar, i.e. the final instar of this mutant (Fig. 1B). Moreover, a marked difference was observed between the sexes. This was a situation very similar to that at the fifth instar of the tetramolter. The strain with the recessive trimolter rt exhibited changes of haemolymph protein concentration almost resembling those of the tetramolter at the second and third instars. Even during the early period (from day 1 to day 2 or 3) of the fourth instar, the concentration remained low (Fig. IC) and was reminiscent of the fourth instar of the tetramolter. However, at the late period (from day 3 or 4 to day 6) of the fourth instar, the concentration rose remarkably with a gap between the sexes (Fig. 1C), again giving the pattern comparable to that of the fifth instar of the tetramolter.

363

As shown in Fig. ID, the results for the strain with the pentamolter M s at the second to the fourth instars were similar to those of the tetramolter. However, the concentration at the fifth (penultimate) instar was still low, like that at the fourth instar of the tetramolter. At the sixth (final) instar of the mutant, the concentration rapidly increased showing a clear difference between the sexes, as the normal strain did at the fifth instar.

Changes in component of haemolymph proteins The electrophoretic patterns of haemolymph proteins from day 1 of the second instar to day 6 of the fifth instar in the tetramolter (normal) are shown in Fig. 2. At the second instar, two major components (bands D and E) and 10 or more minor components were observed. At the third instar, bands B and C in addition to D and E became the major components. Bands D and E were weakened gradually with the

-1LM:3rd instar

I-M."2nd instar .... A

--D

II-1

11-2

II-3

f.M: 4th instar

lV-1

|V-2

11I-1

III-2

111-3

it-M: 5th instar

IV-3

IV-/.

V-1

V-2

V-3

V-4

V-5

V-6

Fig. 2. Polyacrylamide gel electrophoresis of haemolymph proteins at the second to the fifth instars in the tetramolter +M. Electrophoresis was done under the conditions without denaturing agent. F and M, female and male, respectively. II-VI, the second to the sixth instars; 1~5, day 1 to day 6. Tentative identification of major bands are as follows: A, carotenoid binding protein (CBP) (Fujii et al., 1988a); B, female specific protein FL (SP-I); C, SP-2; D and E, young larval protein A and B (PYL-1 and PYL-2, respectively); al-a7, other major components at the last (fifth) instar. See text for details.

364

YUTAKAKAWAGUCHIet al.

progress of the larval development. At the fourth instar, the major components were those named A, B and C. Bands D and E only remained as a trace. No marked differences in pattern of the haemolymph proteins between the sexes were exhibited from the second instar to the fourth instar. On days 1 and 2 of the fifth instar, the proteins consisted of A, B and C and about 10 kinds of minor bands. All the bands were weak. However, from day 3 to day 6 of the fifth instar, bands A, B and C became intense, and the bands named al to a7 were also detected more markedly than before. Band B was increased in intensity rapidly in the female at the later period of the fifth instar, but not in the male where this band gradually became weak at the same period. In contrast, band C was observed consistently in both sexes. The differential behaviour of band B makes the electrophoretic patterns of the haemolymph proteins at the final instar markedly different between the sexes. Band B corresponds to the previously named component FL (i.e SP-1), and band C to SP-2 (see Introduction). Bands D and E have completely disap-

peared in the matured larvae. These are the components that have already been named the young larval proteins A and B (abbreviated as PYL-A, PYL-B), respectively, in our previous study (Banno et aL, unpublished). The patterns of haemolymph proteins at the fourth (final) instar of the dominant trimolter (M 3) and the recessive trimolter (rt) are shown in Fig. 3. The protein compositions in M 3 on day 1 of the fourth instar consisted of bands B and C, as well as several minor bands, and no difference was observed between the sexes. After day 2 of the fourth instar, the bands named bl to b6 became intense both in the female and the male. Band B was augmented in intensity in the female haemolymph, while almost disappearing in the male haemolymph. Thus, the protein patterns at the fourth instar of M 3 was similar to that at the fifth instar, but not to that at the fourth instar, of the tetramolter. On the other hand, the protein compositions in rt on days 1, 2 and 3 of the fourth instar were similar to the respective ones at the fourth instar of the

M3:/4th instar

IV-1

IV-2

IV-3

IV-4

rt :~th instar

IV-I

IV-2

IV-3

IV-6

IV-5

IV- 6

Fig. 3. Polyacrylamide gel electrophoresis of haemolymph proteins at the fourth instar in the dominant and recessive trimolter M 3 and rt, respectively. Bands hi-b6 and cl-c?, major components other than A, B and C at the last (fourth) instar. See the legend to Fig. 2 for other comments.

Haemolymph proteins in molting mutants tetramolter. However, after day 4 of the fourth instar, band C and the components named cl to c7 became remarkably strong. Band B was clearly increased in the female haemolymph but gradually disappeared from the male haemolymph. Therefore, the protein constituents of the later period (days 4, 5 and 6) of the fourth instar of rt resembled that at the fifth instar of the tetramolter. The changes of protein compositions in the pentamolter M 5 at the fifth and the sixth instars are illustrated in Fig. 4. At the fifth instar, major bands B and C, as well as about 10 several kinds of minor bands, were seen. No difference was observed between the sexes. After the fifth molt, bands A, C and those named dl to d7 were rapidly increased in strength with the progress of the 6th instar, and band B became profound only in the female, making the striking difference between the sexes. These protein patterns at the fifth and the sixth instars in this pentamolter mutant resembled those at the fourth and the fifth instars, respectively, of the tetramolter.

365

,I 8 IIl-I -2 -3 IV-1 -2 -3 V-2 -~. "6 Ii .5 ~S I B

C

,, ~=31-

ll

~9[ D

~5

M 5: 5 t h instar

~3 8 ¢-1

zn

iv v-1 -2 -3 VH-3-s $l STAGF-S OF DEVELOPMENT Fig. 5. Changes in concentration of female specific protein (FL) in the haemolymph at the third to sixth instars. Shaded column, female;open column, male. II-VI, the second to the sixth instars; 1-6, day 1 to day 6. S, spinning. Each point represents the average value from three measurements. The patterns for III of the trimolters and those for III and IV of the pentamolter were omitted because these were indistinguishable from those of the tetramolter.

V-1

V-2

V-3

Changes in concentration of female specific protein (FL ) in larval haemolymph M5 : 6 t h

instar

VI-1

VI-2

VI-3

V I - Z,

Fig. 4. Polyacrylamide gel electrophoresis of haemolymph proteins at the fifth and sixth instars in the pentamolter M 5. Bands dl~:17, major components other than A, B and C at the last (sixth) instar. See the legend to Fig. 2 for other comments.

The quantitative evaluation of band B, i.e. the female specific protein (FL), was carried out with a Beckman DU-8 spectrophotometer by using the gels shown in Figs 2-4. In the tetramolter (Fig. 5A), the FL concentration at each stage before the fourth instar was quite low, and at the fourth instar it rose gradually in both sexes. With the progress of the 5th instar, the FL concentration was rapidly increased in the female, while that in the male was at low levels. The FL concentration in the dominant trimolter M 3 was elevated in the female, but rather declined in the male at the fourth instar (Fig. 5B). This pattern was similar to that at the fifth instar of the tetramolter. In the recessive trimolter rt, the FL concentration was low in both sexes at the first period (days 1 and 2) of the fourth instar, but thereafter it rose rapidly in the female but still remained low in the male (Fig. 5C). This was taken to be a composite pattern of the fourth and the fifth instars of the tetramolter.

YUTAKAKAWAGUCHIet al.

366

In the pentamolter M 5, the FL concentration rose in both sexes at the fifth instar, but only in the female at the sixth instar (Fig. 5D). These were quite similar to the respective patterns observed at the fourth and the fifth instars in the tetramolter.

DISCUSSION F r o m the viewpoint of quantitative and qualitative changes, the haemolymph proteins were analysed during the feeding periods of the second to the last larval instars of B. mori by using the normal strain and the molting mutants in which the number of ecdysis is changed from the normal. This method revealed that the characteristics of the haemolymph proteins in terms of both concentration and constitution, particularly the concentration of F L or SP-1 (band B), could be divided into the following three stage-dependent types. (1) The young larval type exhibited at the first to the third instars, where the protein concentration of haemolymph was low and no significant differences in concentration and banding pattern were found between both sexes. The most characteristic of this stage was the presence of P Y L - A and PYL-B (bands D and E, respectively). The expression of P Y L - A and PYL-B was known to be mediated by the genes named Pyl-A and Pyl-B, which were located at 11.2 on the 20th linkage group (Banno et al., unpublished). Thus the young larval stage, which is the typical phase of nutritional growth, was characterized by the activation of the Pyl-A and Pyl-B genes. (2) The late larval type characteristic to the last larval stage, where the protein concentration of the haemolymph was rapidly increased and this was more marked in the female than in the male. SP-2 (band C) and the components called al to a7 became strong in intensity in both sexes, but F L rose only in the female. The latter causes the sexual difference in profile of the haemolymph proteins. In previous genetic experiments (Banno et al., 1984, 1987; Shimada et al., 1985; Kawaguchi et al., 1986), the genes controlling the

expression of SP-2 and FL have been identified and named Pst and Pfl, respectively; the former was localized at 16.7 on the 3rd linkage group and the latter at 8.3 on the 23rd linkage group. Thus the last larval stage was characterized by the accelerated expression of the Pst gene in both sexes and of the Pfl gene preferentially in the female. F L may serve as an indispensable resource of yolk formation, and in this respect, the last larval instar is a phase in which the reproductive growth is already taking place. (3) We suggest that the switch-over from the nutritional growth phase (the young larval stage) to the reproductive growth phase (the last larval stage) occurs at the intermediary growth phase, i.e. at the fourth instar in case of the normal tetramolter, where the intermediary types of the haemolymph proteins could be seen. On the basis of the above criterion, the characteristics of the M 3, rt and M 5 mutants could be as summarized in Table 1. M 3 apparently lacked the intermediary phase, i.e. the fourth instar of the normal tetramolter. Thus the mutant can be considered to have the first, second, third and fifth instars. As to the rt mutant, an intermediary phase occurred at the onset of the last instar. Thus the mutant seemed to possess the first, second and third instars followed by a composite fourth plus fifth instar as the final larval stage. On the other hand, M s duplicated the intermediary phase; i.e. it had the first, second, third, fourth, fourth and fifth instars during its larval life. As a whole, the molting mutants analysed in the present study were recognized to have their respective phenotypes of intermediary growth phase. We conclude that the nutritional and reproductive growth phases at the early and final stages, respectively, are scarcely modified, despite the fact that the number of the exuviation or that of the larval instars can be varied by gene mutation.

Acknowledgements--The study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

Table 1. The summarized features of larval haemolymph proteins after quantitative and qualitative analyses Roman numerals: instar no. of larval life Arabic numerals: instars specified by the Characteristic phenotypes Strain Gene features of haemolymph proteins of haemolymph proteins Tetramolter +M I II III IV V Normal type (standard) . . . . (1) 2 3 4 5 Trimolter M3 I II III IV Omission type of (dominant) . . . . the fourth instar (1) 2 3 5 Trimolter rt I II III IV Conjunction type (recessive) . . . . of the fourth and (1) 2 3 4+ 5 fifth instar Pentamolter M5 I II III IV V VI Duplicationtype of (recessive) . . . . . the fourth instar (1) 2 3 4 4 5 The features of the second and third instars (2 and 3, respectively)were almost the same. The first instar was not analysed for proteins but expressed in parentheses.

Haemolymph proteins in molting mutants REFERENCES

Banno Y., Kawaguchi Y., Xu M. K. and Doira H. (1984) Inheritance of female specific protein in larval hemolymph of Bombyx mori. J. Seric. Sci. Jpn 53, 549-550. Banno Y., Kawaguchi Y., Xu M. K. and Doira H. (1987) Linkage studies on the "Larval female protein" gene in Bombyx mori. J. Seric. Sci. Jpn 56, 549-550. Chikushi H. (1972) Genes and Genetical Stocks of the Silkworm. Keigaku, Tokyo. Davis B. J.(1964) Disc electrophoresis. II. Method and application to human serum protein. Ann. N. Y. Acad. Sci. 121, Art2, 404~27. Doira H. (1968) Developmental and sexual differences of blood proteins in the silkworm, Bombyx mori. Sci. Bull. Fac. Agric. Kyushu Univ. 23, 205-214. Doira H. (1983) Linkage map of Bombyx mori: status quo in 1983. Sericologia 23, 245-269. Doira H. (1986) Linkage map of Bombyx mori: revised in 1986. Sericologia 26, 485-488. Eguchi E. and Kanbe M. (1982) Changes in haemolymph protease inhibitors during metamorphosis of the silkworm, Bombyx mori. L. Appl. Ent. Zool. 17, 179-187. Eguchi M., Haneda I. and Iwamoto M. (1982a) Properties of protease inhibitors during the haemolymph of silkworm, Bombyx mori, Antheraea pernyi and Philosamia cynthia ricini. Comp. Biochem. Physiol. 71B, 569-576. Fujii H., Morooka J., Tochihara S., Kawaguchi Y. and Sakaguchi B. (1988a) Existence of carotenoid binding protein in larval hemolymph of the yellow blood strain of Bombyx mori. J. Seric. Sci. Jpn 57, 94-99. Fujii H., Matsui T., Tochihara S. and Kawaguchi Y. (1988b) Purification of carotenoid binding protein from larval hemolymph in the yellow blood strain of Bombyx mori. J. Seric. Sci. Jpn 57, 398-404. Fujii H., Aratake H., Deng L. R., Nakamura M., Kawaguchi Y. and Sakaguchi B. (1989) Purification and characterization of a novel chymotrypsin inhibitor controlled by the chymotrypsin inhibitor a (Ict-A) gene from the larval hemolymph of the silkworm, Bombyx mori. Comp. Biochem. Physiol. 94B, 149-155. Izumi S., Fujie J. and Tomino S. (1981) Molecular properties and biosynthesis of major plasma proteins in Bombyx mori. Biochem. biophys. Acta 670, 222-229.

367

Kanost M. R., Kawooya J. K., Low J. H. Ryan R. O., Van Heusden M. C. and Ziegler R. (1990) Insect haemolymph proteins. Adv. Insect Physiol. 22, 299-396. Kawaguchi Y. and Doira H. (1973) Gene-controlled incorporation of haemolymph protein into the ovaries of Bombyx mori. J. Insect Physiol. 19, 2083-2096. Kawaguchi Y., Banno Y., Shimada T. and Kobayashi M. (1986) Linkage studies on the storage protein-2 gene (Pst) in Bombyx mori. J. Seric. Sci. Jpn 55, 243-245. Kobara R. (1967) Sexual difference in haemolymph protein of several insects. Jap. J. appl. Ent. Zool. 11, 71-75. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Nakasone S. and Kobayashi M. (1965) Acrylamide gel electrophoresis of blood protein during the moulting and the metamorphosis in the silkworm, Bombyx mori. J. Seric. Sci. Jpn 34, 257-262. Ogawa K. and Tojo S. (1981) Quantitative changes of storage proteins and vitellogenin during the pupal-adult development in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Appl. Ent. Zool. 16, 288-296. Ono S., Nagayama H. and Shimura K. (1975) The occurrence and synthesis of female- and egg-specific proteins in the silk-worm, Bombyx mori. Insect Biochem. 5, 319-329. Ornstein L. (1964) Disc electrophoresis I. Background and theory. Ann. N.Y. Acad. Sci. 121, Art2, 321-349. Shimada T., Kobayashi M. and Yoshitake N. (1985) Genetical analysis of storage proteins in the silkworm, Bombyx mori. J. Seric. Sci. Jpn 54. 464--469. Thomson J. A. (1975) Major patterns of gene activity during development in holometabolous insects. Adv. Insect Physiol. 11, 321-398. Tojo S., Nagata M., Kobayashi M. (1980) Storage proteins in the silkworm, Bombyx mori. Insect Biochem. 10, 47-53. Wyatt G. R., Lougheed T. C. and Wyatt S. S. (1956) The chemistry of insect haemolymph. Organic components of the haemolymph of the silkworm, Bombyx mori, and two other species. J. gen. Physiol. 39, 853 868. Wyatt G. R. and Pan M. L. (1978) Insect plasma proteins. Ann. Rev. Biochem. 47, 779-817.