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Haemolymph protein electrophoretic pattern in Periplaneta americana after administration of farnesyl methyl ether
J. InsectPhysiol.,1972, Vol. 18, pp. 1435 to 1440. PergamonPress. Printedin Gteut Britain
HAEMOLYMPH PROTEIN ELECTROPHORETIC PATTERN IN PERIPLANETA AMERICANA AFTER ADMINISTRATION OF FARNESYL METHYL ETHER V. K. K. PRABHU
and K. K. NAYAR
Department of Zoology, University of Kerala, Kariavattom, Trivandrum,
India
(Received 22 November 1971)
Abstract-Administration of a single 4~1 dose of the juvenile hormone analogue farnesyl methyl ether in the male cockroach Periplaneta americana brings about a conspicuous increase in concentration of serum proteins of fractions 4,6, and 7, but not 5, on the subsequent day compared to their controls. The change is reversible. It is fractions 4, 5, and 7 that show pronounced changes during the vitellogenic cycle in the female, whereas in the male they are weaker and do not show any change in concentration.
INTRODUCTION
THE FAT body of the female, but not of the male cockroach Periplaneta americana and of the moth Hyalophora cecropia incorporates, in vitro, labelled amino acids into vitellogenins (PAN et al., 1969). Vitellogenin is not normally found in the male Polyphemus silk moth, but it is found after implantation of female fat body (BLUMENFELDand SCHNEIDERMAN,1968). In Leucophaea, juvenile hormone is necessary for the synthesis of yolk proteins by the female in vivo. This yolk protein is absent in the male (ENGELMANN, 1969). Sex-limited vitellogenins are also detected in P. americana females (BELL, 196913). Ovaries implanted in the adult male cockroach P. awicana continue vitellogenesis though at a slower rate and ultimately change the electrophoretic pattern of serum proteins of the male host to that of the female (PRABHUand HEMA, 1970). The present experiment further supports the hypothesis that the male fat body is capable of secreting those yolk proteins at a higher rate which are normally present in its blood in minute quantities, under the influence of a juvenile hormone analogue. MATERIALS
AND
METHODS
Farnesyl methyl ether (Hoffmann-La Roche), a substance known to mimic juvenile hormone (EMMERICHand BARTH, 1968; BELL, 1969a; BELL and BARTH, 1970), 2, 3,4, 5, and 6 ~1 per animal, was injected by means of a microlitre syringe into batches of 15 adult male cockroaches (Periplaneta americana), a corresponding number of animals receiving Ringer injections being kept as controls. No carrier medium was used as release of the hormonally active analogue from the carrier 1435
1436
V. K. K. PRABHU ANDK. K. NAYAR
medium would introduce uncertainty in the experiment. In an equal number of animals the injection was continued on the second and third days. Some normal adult females on each day of the vitellogenic cycle were studied for comparative purposes. Blood was collected on the subsequent day through a pleural puncture into capillary tubes and centrifuged at low speed to remove haemocytes. Disk electrophoresis of the serum proteins was carried out essentially as described by NIELSEN and MILLS (1968). However, 5 ~1 blood samples were used, as this concentration was found to give optimum separation of the desired proteins. Electrophoresis was carried out for 25 min at room temperature (30°C). RESULTS AND DISCUSSION Animals receiving 5 and 6 ~1 doses of farnesyl methyl ether showed a high rate of mortality (more than 50 per cent), as also did those receiving repeated injections of 4 ~1 and so these animals were discarded. However, those receiving a single 4 ~1 dose of the analogue and also those receiving repeated injections of lower doses apparently were quite normal and were included in the study. Fourteen fractions of proteins numbered from the origin to the front respectively are distinct in the serum of the male and the female cockroach. Taking into consideration the cyclicity of most of the fractions in the female cockroach during vitellogenesis (NIELSENand MILLS, 1968; BELL, 1969b), which we have also observed (Figs. 1,2), the fundamental difference between the electrophoretic patterns of serum proteins of the male and the female cockroach is that in the female fractions 4, 5, and 7 are very much more concentrated during different periods of the vitellogenic cycle than in the male in which these fractions remain uniformly weaker. Administration of a single dose or repeated daily doses of 2 or 3 ~1 of farnesyl methyl ether up to 3 days was without any visible effect on the serum proteins. However, a single injection of 4 ~1 of the analogue brought about an increase in concentration of serum proteins of fractions 4, 6, and 7, but not 5, the next day, whereas in the control there were no changes visible. Fraction 6 in fact appeared more concentrated in the injected male than in the female at any time in the vitellogenie cycle. On the other hand, fraction 12 appeared less concentrated in the experimental male than in the control. Three days after administration of a single 4 ~1 dose of the analogue, reverse changes in the electrophoretic pattern were already apparent. Fraction 6 was already very weak and comparable to that of the control male, whereas fraction 7 was beginning to fall in concentration. Fraction 12 had already started to accumulate. These findings indicate that the juvenile hormone analogue farnesyl methyl ether is capable of stimulating in the male synthesis of those proteins which normally occur at lower concentrations than in the female. Similar changes have been recorded in Peripluneta males after implantation of ovaries (PRABHU and HEMA, 1970). Though these fractions do not appear to be vitellogenins, which form only a very small fraction of the total blood proteins in P. americana (BELL, 1969b), these observations are interesting in the light of the findings of PAN et al. (1969) that unlike the female fat body of the cockroach P. americana and of the
1437
Origin +
FIG. 1. Complete disk electrophorograms of the cockroach. (a), Male cockrc path 1 da:y after a single injection of 4 ~1 farnesyl methyl ether; (b), control male 1 day after injection of 4 fll Ringer solution; (c), normal female carrying fully protru ded o&heca, designated day 4 animal.
1438
Origin +
(d)
W
if)
FIG. 2. Electrophorograms (only the first seven bands shown) of the haemolymph of: (a), male cockroach 1 day after administration of farnesyl methyl ether; (b), control male 1 day after Ringer injection; (c), male 3 days after a single injection of 4 ~1 farnesyl methyl ether; (d), normal female with well-protruded oiitheca (day 4) ; (e), normal female 3 days after deposition of oijtheca; (f), normal female 2 days after deposition of iiotheca.
BLOODPROTEINSIN THE COCKROACH AFTERJH ANALOGUE TREATMENT
1439
moth Hyalophora cecropia, the male fat body was not capable of synthesizing sexlimited vitellogenic blood proteins in &YO, and the in oiwo findings of ENGELMANN (1969) and ENGELMANNand PENNEY(1966) that synthesis and release of proteins (both sex specific and non-specific) essential for egg maturation are stimulated by juvenile hormone in the female Leucophaea. The fact that juvenile hormone stimulates fat body protein synthesis in females differentially is also reported by BLUMENFELDand SCHNEIDERMAN (1968). In the male Periplaneta the differences in response among the five blood proteins to the juvenile hormone analogue observed in the present study indicate that the fat bodies, which are protein synthetic organs in female Periplaneta (PAN et al., 1969), also respond to the juvenile hormone analogue in the male by altering the rate of synthesis of different fractions and releasing them into blood, although not exactly as in the female cockroach. This difference in protein synthetic activity may be due to the differential activity of the fat body of the two sexes (BLUMENFELD and SCHNEIDERMAN, 1968; BUTTERWORTH and BODENSTEIN,1969; PAN et al., 1969). It is interesting that the present work has shown that high doses of the analogue and repeated 4 ~1 doses are toxic to the male cockroach. This may at first appear to be due to the accumulation of proteins in the blood which the male cannot normally use. However, it is unlikely in view of the fact that in the presence of implanted ovary the male can tolerate a very high load of blood proteins for a prolonged period of time (PRABHUand HEMA, 1970). The fact that the male fat body is susceptible to the juvenile hormone analogue and yet it does not normally play this r81e fully may be because of the very low titre of juvenile hormone in the male. Acknowledgements-We thank the Ford Foundation and the Indian Council of Medical Research for a research grant and Professor HOWARDA. BERN for the farnesyl methyl ether. REFERENCES BELL W. J. (1969a) Dual role of juvenile hormone in the control of yolk formation in Periplaneta americana. J. Insect Physiol. 15, 1279-1290. BELL W. J. (1969b) Continuous and rhythmic reproductive cycle observed in Periplaneta americana (L.). Biol. Bull., Woods Hole 137, 239-249. BELL W. J. and BARTHR. H., Jr. (1970) Q uantitative effects of juvenile hormone on reproduction in the cockroach Byrsotria fumigata. J. Insect Physiol. 16, 2303-2313. BLUMENFELDM. and SCHNEIDEWAN H. A. (1968) Effect of juvenile hormone on the synthesis and accumulation of a sex limited protein in the polyphemous silk moth. Biol. Bull., Woods Hole 135, 466475. BUTTERWORTH F. M. and BODENSTEIND. (1969) Adipose tissue of Drosophila melanogaster -IV. The effect of corpus allatum and synthetic juvenile hormone on the tissue of the adult male. Gen. camp. Endocr. 13, 68-74. EMMERICHH. and BARTHR. H., Jr. (1968) Effect of farnesyl methyl ether on reproductive physiology in the cockroach, Byrsotria fumigata (GuCrin). 2. Naturf. 23b, 1019-1020. ENGELMANNF. (1969) Female specific proteins: biosynthesis controlled by corpus allatum in Leucophaea maderae. Science, Wash. 165, 407409. ENGELMANNF. and PENNEY D. (1966) Studies on the endocrine control of metabolism in Leucophaea maderae (Blattaria)-I. The haemolymph proteins during egg maturation. Gen. camp. Endocr. 7, 314-325.
1440
V. K. K. PRABHUANDK. K. NAYAR
NIELSEN D. J. and MILLS R. R. (1968) Changes in electrophoretic properties of haemolymph and terminal oacyte proteins during vitellogenesis in the American cockroach. J. Insect Physiol. 14, 163-170. PAN M. L., BELL W. J., and TJILFER W. H. (1969) Vitellogenic blood protein synthesis by insect fat body. Science, Wash. 165, 393-394. PRABHUV. K. K. and HEMA P. (1970) Effect of implantation of ovaries in the male cockroach Perifilaneta americana. J. Insect Physiol. 16, 147-156.
HAEMOLYMPH PROTEIN ELECTROPHORETIC PATTERN IN PERIPLANETA AMERICANA AFTER ADMINISTRATION OF FARNESYL METHYL ETHER V. K. K. PRABHU
and K. K. NAYAR
Department of Zoology, University of Kerala, Kariavattom, Trivandrum,
India
(Received 22 November 1971)
Abstract-Administration of a single 4~1 dose of the juvenile hormone analogue farnesyl methyl ether in the male cockroach Periplaneta americana brings about a conspicuous increase in concentration of serum proteins of fractions 4,6, and 7, but not 5, on the subsequent day compared to their controls. The change is reversible. It is fractions 4, 5, and 7 that show pronounced changes during the vitellogenic cycle in the female, whereas in the male they are weaker and do not show any change in concentration.
INTRODUCTION
THE FAT body of the female, but not of the male cockroach Periplaneta americana and of the moth Hyalophora cecropia incorporates, in vitro, labelled amino acids into vitellogenins (PAN et al., 1969). Vitellogenin is not normally found in the male Polyphemus silk moth, but it is found after implantation of female fat body (BLUMENFELDand SCHNEIDERMAN,1968). In Leucophaea, juvenile hormone is necessary for the synthesis of yolk proteins by the female in vivo. This yolk protein is absent in the male (ENGELMANN, 1969). Sex-limited vitellogenins are also detected in P. americana females (BELL, 196913). Ovaries implanted in the adult male cockroach P. awicana continue vitellogenesis though at a slower rate and ultimately change the electrophoretic pattern of serum proteins of the male host to that of the female (PRABHUand HEMA, 1970). The present experiment further supports the hypothesis that the male fat body is capable of secreting those yolk proteins at a higher rate which are normally present in its blood in minute quantities, under the influence of a juvenile hormone analogue. MATERIALS
AND
METHODS
Farnesyl methyl ether (Hoffmann-La Roche), a substance known to mimic juvenile hormone (EMMERICHand BARTH, 1968; BELL, 1969a; BELL and BARTH, 1970), 2, 3,4, 5, and 6 ~1 per animal, was injected by means of a microlitre syringe into batches of 15 adult male cockroaches (Periplaneta americana), a corresponding number of animals receiving Ringer injections being kept as controls. No carrier medium was used as release of the hormonally active analogue from the carrier 1435
1436
V. K. K. PRABHU ANDK. K. NAYAR
medium would introduce uncertainty in the experiment. In an equal number of animals the injection was continued on the second and third days. Some normal adult females on each day of the vitellogenic cycle were studied for comparative purposes. Blood was collected on the subsequent day through a pleural puncture into capillary tubes and centrifuged at low speed to remove haemocytes. Disk electrophoresis of the serum proteins was carried out essentially as described by NIELSEN and MILLS (1968). However, 5 ~1 blood samples were used, as this concentration was found to give optimum separation of the desired proteins. Electrophoresis was carried out for 25 min at room temperature (30°C). RESULTS AND DISCUSSION Animals receiving 5 and 6 ~1 doses of farnesyl methyl ether showed a high rate of mortality (more than 50 per cent), as also did those receiving repeated injections of 4 ~1 and so these animals were discarded. However, those receiving a single 4 ~1 dose of the analogue and also those receiving repeated injections of lower doses apparently were quite normal and were included in the study. Fourteen fractions of proteins numbered from the origin to the front respectively are distinct in the serum of the male and the female cockroach. Taking into consideration the cyclicity of most of the fractions in the female cockroach during vitellogenesis (NIELSENand MILLS, 1968; BELL, 1969b), which we have also observed (Figs. 1,2), the fundamental difference between the electrophoretic patterns of serum proteins of the male and the female cockroach is that in the female fractions 4, 5, and 7 are very much more concentrated during different periods of the vitellogenic cycle than in the male in which these fractions remain uniformly weaker. Administration of a single dose or repeated daily doses of 2 or 3 ~1 of farnesyl methyl ether up to 3 days was without any visible effect on the serum proteins. However, a single injection of 4 ~1 of the analogue brought about an increase in concentration of serum proteins of fractions 4, 6, and 7, but not 5, the next day, whereas in the control there were no changes visible. Fraction 6 in fact appeared more concentrated in the injected male than in the female at any time in the vitellogenie cycle. On the other hand, fraction 12 appeared less concentrated in the experimental male than in the control. Three days after administration of a single 4 ~1 dose of the analogue, reverse changes in the electrophoretic pattern were already apparent. Fraction 6 was already very weak and comparable to that of the control male, whereas fraction 7 was beginning to fall in concentration. Fraction 12 had already started to accumulate. These findings indicate that the juvenile hormone analogue farnesyl methyl ether is capable of stimulating in the male synthesis of those proteins which normally occur at lower concentrations than in the female. Similar changes have been recorded in Peripluneta males after implantation of ovaries (PRABHU and HEMA, 1970). Though these fractions do not appear to be vitellogenins, which form only a very small fraction of the total blood proteins in P. americana (BELL, 1969b), these observations are interesting in the light of the findings of PAN et al. (1969) that unlike the female fat body of the cockroach P. americana and of the
1437
Origin +
FIG. 1. Complete disk electrophorograms of the cockroach. (a), Male cockrc path 1 da:y after a single injection of 4 ~1 farnesyl methyl ether; (b), control male 1 day after injection of 4 fll Ringer solution; (c), normal female carrying fully protru ded o&heca, designated day 4 animal.
1438
Origin +
(d)
W
if)
FIG. 2. Electrophorograms (only the first seven bands shown) of the haemolymph of: (a), male cockroach 1 day after administration of farnesyl methyl ether; (b), control male 1 day after Ringer injection; (c), male 3 days after a single injection of 4 ~1 farnesyl methyl ether; (d), normal female with well-protruded oiitheca (day 4) ; (e), normal female 3 days after deposition of oijtheca; (f), normal female 2 days after deposition of iiotheca.
BLOODPROTEINSIN THE COCKROACH AFTERJH ANALOGUE TREATMENT
1439
moth Hyalophora cecropia, the male fat body was not capable of synthesizing sexlimited vitellogenic blood proteins in &YO, and the in oiwo findings of ENGELMANN (1969) and ENGELMANNand PENNEY(1966) that synthesis and release of proteins (both sex specific and non-specific) essential for egg maturation are stimulated by juvenile hormone in the female Leucophaea. The fact that juvenile hormone stimulates fat body protein synthesis in females differentially is also reported by BLUMENFELDand SCHNEIDERMAN (1968). In the male Periplaneta the differences in response among the five blood proteins to the juvenile hormone analogue observed in the present study indicate that the fat bodies, which are protein synthetic organs in female Periplaneta (PAN et al., 1969), also respond to the juvenile hormone analogue in the male by altering the rate of synthesis of different fractions and releasing them into blood, although not exactly as in the female cockroach. This difference in protein synthetic activity may be due to the differential activity of the fat body of the two sexes (BLUMENFELD and SCHNEIDERMAN, 1968; BUTTERWORTH and BODENSTEIN,1969; PAN et al., 1969). It is interesting that the present work has shown that high doses of the analogue and repeated 4 ~1 doses are toxic to the male cockroach. This may at first appear to be due to the accumulation of proteins in the blood which the male cannot normally use. However, it is unlikely in view of the fact that in the presence of implanted ovary the male can tolerate a very high load of blood proteins for a prolonged period of time (PRABHUand HEMA, 1970). The fact that the male fat body is susceptible to the juvenile hormone analogue and yet it does not normally play this r81e fully may be because of the very low titre of juvenile hormone in the male. Acknowledgements-We thank the Ford Foundation and the Indian Council of Medical Research for a research grant and Professor HOWARDA. BERN for the farnesyl methyl ether. REFERENCES BELL W. J. (1969a) Dual role of juvenile hormone in the control of yolk formation in Periplaneta americana. J. Insect Physiol. 15, 1279-1290. BELL W. J. (1969b) Continuous and rhythmic reproductive cycle observed in Periplaneta americana (L.). Biol. Bull., Woods Hole 137, 239-249. BELL W. J. and BARTHR. H., Jr. (1970) Q uantitative effects of juvenile hormone on reproduction in the cockroach Byrsotria fumigata. J. Insect Physiol. 16, 2303-2313. BLUMENFELDM. and SCHNEIDEWAN H. A. (1968) Effect of juvenile hormone on the synthesis and accumulation of a sex limited protein in the polyphemous silk moth. Biol. Bull., Woods Hole 135, 466475. BUTTERWORTH F. M. and BODENSTEIND. (1969) Adipose tissue of Drosophila melanogaster -IV. The effect of corpus allatum and synthetic juvenile hormone on the tissue of the adult male. Gen. camp. Endocr. 13, 68-74. EMMERICHH. and BARTHR. H., Jr. (1968) Effect of farnesyl methyl ether on reproductive physiology in the cockroach, Byrsotria fumigata (GuCrin). 2. Naturf. 23b, 1019-1020. ENGELMANNF. (1969) Female specific proteins: biosynthesis controlled by corpus allatum in Leucophaea maderae. Science, Wash. 165, 407409. ENGELMANNF. and PENNEY D. (1966) Studies on the endocrine control of metabolism in Leucophaea maderae (Blattaria)-I. The haemolymph proteins during egg maturation. Gen. camp. Endocr. 7, 314-325.
1440
V. K. K. PRABHUANDK. K. NAYAR
NIELSEN D. J. and MILLS R. R. (1968) Changes in electrophoretic properties of haemolymph and terminal oacyte proteins during vitellogenesis in the American cockroach. J. Insect Physiol. 14, 163-170. PAN M. L., BELL W. J., and TJILFER W. H. (1969) Vitellogenic blood protein synthesis by insect fat body. Science, Wash. 165, 393-394. PRABHUV. K. K. and HEMA P. (1970) Effect of implantation of ovaries in the male cockroach Perifilaneta americana. J. Insect Physiol. 16, 147-156.