Adipokinetic activity of shrimp and locust peptide hormones in butterflies

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

43, 256-258 (1981)

NOTES Adipokinetic

Activity of Shrimp and Locust Hormones in Butterflies

Peptide

Synthetic shrimp red-pigment-concentrating hormone (RPCH) and pure locust adipokinethormone (AKH) both cause significant, dose-dependent elevations of hemolymph lipids in the Monarch butterfly, Danaus plexippus, and in the Painted Lady butterfly, Vanessa cardui. Both hormones are effective when administered in picomole amounts; AKH appears to be about 100 times more active than RPCH. These results suggest a highly conservative evolution of some mandibulate arthropod neurohormones. ic

Previous experiments have suggested the existence of an adipokinetic hormone acting to elevate the hemolymph lipid levels in the Monarch butterfly, Danaus plexippus (Dallmann and Herman, 1978), and recent studies suggest the presence of a comparable hormone in the Painted Lady butterfly, Vanessa car&i (Herman and Dallmann, 1980). Earlier research on the Monarch system (Dallmann and Herman, 1978) indicated that pure shrimp red-pigment-concentrating hormone (RPCH), an octapeptide known to be adipokinetic in locusts (Mordue and Stone, 1976, 1977; Herman et al., 1977), was without significant activity when assayed in nondiapause adult Monarchs. Subsequently, we have obtained evidence (Dallmann, unpublished) that the adult Monarch in reproductive diapause is more sensitive to Monarch adipokinetic peptides than is the nondiapause adult. We have also obtained samples of pure locust adipokinetic hormone (AKH) and locust “Compound II” (Carlsen et al., 1979). Compound II is an octapeptide with RPCH-AKH-like structure and biological activity recently isolated from locust corpora cardiaca by Carlsen and co-workers (1979). In view of these developments, we decided to reexamine the effects of RPCH and to test the effects of AKH and Compound II on diapause Monarchs. Due to the very limited supply of Compound II, only RPCH and AKH were tested on diapause

Painted Ladies. Our results demonstrate that low dosages of RPCH and AKH are adipokinetic in both species and provide strong evidence for a close similarity of RPCH, AKH, and putative butterfly adipokinetic hormone. The butterflies used in these studies were collected in the wild in central Minnesota from late August to mid-October; all were in adult reproductive diapause (Herman and Dallmann, 1981; Herman, 1981). They were stored in programmed incubators set at 10” with an 8-hr photophase for up to 1 month, fed 30% honey at room temperature every 5-9 days and 24 hr prior to use in experiments, and placed at room temperature about 1 hr prior to use. This protocol seemed to minimize initial hemolymph lipid levels and to maximize response to treatment. Individual butterflies were never used in more than one experiment. Head extracts and pure peptides were solubilized and injected, and assays in both species were conducted as previously described for Monarchs (Dallmann and Herman, 1978). RPCH was synthesized (Fernlund and Josefsson, 1972) and AKH and compound II purified (Carlsen et al., 1979) in Copenhagen and assays were performed in Minnesota. Figure 1 summarizes the effects of RPCH and AKH in butterflies. A dose-response relationship, at low doses, is evident for both hormones in both species. Owing to a 256

0016-6480/81/020256-03$01.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

NOTES

0.1 Dose

( pmotes)

1. Effect of locust adipokinetic hormone (0) snd shrimp red-pigment-concentrating hormone (A) on hemolymph lipid levels in Monarch (Danaus plexipDUS) and Painted Lady (Vanessa cardui) adult butterflies; peptides injected in 10 pl (Vanessa) or 20 ~1 (Danam) distilled water; n in parentheses; lipids measured before and 1 hr after injection. FIG.

shortage of material, higher doses of AKH were not tested in Vunessa. For comparison, extracts of homospecific butterfly heads, injected at a dose of 1 head equivalent per animal, raised hemolymph lipids in 1 hr by 11.6 r 1.4 pg/pl in 10 Monarchs and 12.8 c 1.0 pg/pl in 9 Painted Ladies (data are mean zt SEM). By contrast, distilledwater injections resulted in changes of - 1.5 + 1.4 ,hg/pl (n = 5) and 2.0 -t 1.0 pg/pl (n = 6), respectively, in the two species. The results obtained with RPCH suggest that our previous failure to demonstrate an adipokinetic action of this hormone in Monarchs (Dallmann and Herman, 1978) may have been due to our use of nondiapause adults. Compound II at a dose of 100 pmol per animal failed to change hemolymph lipid levels in four Monarchs. Additional studies with this peptide were

257

impossible owing to the exhaustion of existing supplies. Existing data allow for quantitative comparisons of the effects of the above hormones in locusts and butterflies. Locust AKH produces a 50% elevation of locust hemolymph lipid at a dose of about 2 pmol (Mordue and Stone, 1977; Stone et ai., 1976), and this value is comparable to that observed in Monarchs and Painted Ladies. In addition, maximal elevations seem to result from injections of about 10 pmol in both locusts (Mordue and Stone, 1977; Stone et al., 1976) and Monarchs. We conclude that locust AKH is about equally effective in either test system. By contrast, approximately 5 pmol RPCH elicits a 50% response, and about 20 pmol is maximally effective in locusts (Mordue and Stone, 1977; Stone et aE., 1976), while both 58% and maximal elevations apparently require substantially more RPCH in butterflies. Similarly, although Compound II equals AKH in lipid-mobilizing ability in locusts and chromatophorotropic activity in shrimp (Carlsen et al., 1979), 100 pmol of this material was ineffective in Monarchs, while maximal effects resulted from comparable doses of AKH. It therefore appears that the mobilization of butterfly lipids is substantially less sensitive to RPCH and Compound II than is the corresponding process in locusts. The above data demonstrate for the first time that two similar neurosecretory peptide hormones (i.e., RPCH and AKH), isolated from arthropods as distantly related as shrimp and locusts, mimic the effect of a putative neurosecretory hormone ‘(Dallmann and Herman, 1978; Hermarrand DallMann, 1981) of two holometabolic insects. Furthermore, the presence of materials with RPCH- AKH-like biological act’ivity in the neuroendocrine systems of many holometabolic insects is well established (Thomsen, 1943; Nat&i and Frontali, 1966; Friedman, 1967; Berman et al., 1977; Ziegler, 1979). Similarly, ongoing attempts

258

NOTES

to chemically characterize such materials from the holometabolic insects Apis mellifera, Tenebrio molitor, and the Monarch butterfly currently indicate substantial chemical similarity between the active substances from these species and RPCHAKH (Dallmann and Herman, 1978; VanNorstrand et al., 1980). We therefore believe that a family of peptide hormones, closely resembling RPCH, AKH, and perhaps also Compound II, may be generally used as hormones in many holometabolic insects. It will thus be of considerable interest to fully characterize such molecules from a greater variety of insects, and to examine the effects of the currently available pure materials in additional species. ACKNOWLEDGMENTS We thank M. and C. Lessman, M. and C. Herman, and J. Sawyer for assistance with capturing butterflies, and M. VanNorstrand for manuscript review. This work was supported by the University of Minnesota Graduate School, USPHS Grant HD-07336, NIH Training Grant 5T32 GM~07323-03, and the Danish Natural Science Research Council (Project 3565).

REFERENCES Carlsen, J., Herman, W. S., Christensen, M., and Josefsson, L. (1979). Characterization of a second peptide with adipokinetic activity and red pigment concentrating activity from the locust corpora cardiaca. Insect Biochem. 9, 497-501. DalImann, S. H., and Herman, W. S. (1978). Hormonal regulation of hemolymph lipid concentration in the Monarch butterfly, Danaus plexippus. Gen. Camp. Endocrinol. 36, 142-150. Fernlund, P., and Jose&son, L. (1972). Crustacean color change hormone: Amino acid sequence and chemical synthesis. Science 177, 173 - 175. Friedman, S. (1967). The control of trehalose synthesis in the blowfly, Phormia regina Meig. J. Insect Physiot. 13, 397-405. Herman, W. S. (1981). Studies on the adult reproductive diapause of the Monarch butterfly. Biol. Bull., in press. Herman, W. S., Carlsen, J. B., Christensen, M., and Josefsson, L. (1977). Evidence for an adipokinetic

function of the RPCH activity present in the desert locust neuroendocrine system. Biof. Bull. 153, 521-539. Herman, W. S., and Dahmann, S. H. (1981). Endocrine biology of the Painted Lady butterfly, Vanessa cardui. J. Insect Physiol., in press. Mordue, W., and Stone, J. V. (1976). Comparison of the biological activities of an insect and crustacean neurohormone that are structurally similar. Nature (London) 264, 287-289. Mordue, W., and Stone, J. V. (1977). Relative potencies of locust adipokinetic hormone and prawn red pigment concentrating hormone in insect and crustacean systems. Gen. Comp. Endocrinol. 33, 103-108. Natal%, G. M., and Frontali, M. (1966). Purification of insect hyperglycemic and heart accelerating hormones. J. Insect Physiot. 12, 1279-1297. Stone, J. V., Mordue, W., Batley, K. E., and Morris, H. R. (1976). Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilization in flight. Nature (London) 263, 207-211. Thomsen, M. (1943). Effect of corpus cardiacum and other insect organs on the color change of the shrimp, Leander adspersus. K. Dan. Vidensk. Selsk. Biol. Medd. 19, l-38. VanNorstrand, M. D., Carlsen, J. B., Josefsson, L., and Herman, W. S. (1980). Studies on a peptide with red pigment-concentration and hyperglycemic activity from the cephalic endocrine system of the honeybee, Apis mellifera. Gen. Comp. Endocrinol. 42, 526-533. Ziegler, R. (1979). Hyperglycaemic factor from the corpora cardiaca of Manduca sexta (L.) (Lepidoptera: Sphingidae). Gen. Comp. Endocrinoi. 39, 350-357.

S. H. DALLMANN W. S. HERMAN Department of Genetics and Cell Biology University of Minnesota St. Paul, Minnesota 55108 J. CARLSEN L.

Biochemistry C, Panum Institute University of Copenhagen Blegdamsvej 3, DK-2200 Copenhagen N, Denmarh Accepted June 21, 1980

JOSEFSSON