GENERAL AND COMPARATIVE ENDOCRINOLOGY 51, 378-383 (1983) Methionine Enkephalin-Induced Changes in Pigmentation of Zebrafish (Cyprinidae, Brachydanio rerio) and Related Species and Varieties, Measured Videodensitometrically II. Pearl and Gold Danios RICHARD GORDON AND SOPHIE LEVINA Department of Pathology, Medical Faculty, University of Manitoba, Winnipeg R3E OW3, Canada Accepted September 22, 1982 In the fish Brachydanio albolineatus (pearl danio) MSH-induced darkening was similar to that of the B. rerio (zebrafish). However, they showed a greatly reduced response to methionine enkephalin (met-E) though these fish apparently have normal dermal melano- phores. This suggests a central nature of met-E-induced darkening and on the other hand at least a partial lack of MSH production and/or release in response to met-E in these fish. Therefore this reduction in the response to met-E may involve a partial lack of opiate receptors in the cells that provide or stimulate MSH production in the pearl danio. In the B. rerio gold mutant met-E appeared to be able to induce production and/or release of MSH, but these fish have almost no dermal melanophores (except a few in the dorsal and tail tins) and therefore show no darkening in response to either MSH or met-E. The Brachydanio genus has the advan- tage of having a number of pigmentation and pattern varieties, which are bred by aquarists. Since methionine enkephalin (met-E) was found to be able to induce dis- persion of pigment granules in the melano- phores of zebrafish (Levina and Gordon, 1982, 1983) it was of interest to examine some other varieties of the Brachydanio genus that could elucidate the mechanism of this unexpected biological activity of met-E. Since we have evidence of a central mechanism for met-E-induced changes of coloration in zebrafish (Levina & Gordon, 1983), we selected for further examination two varieties of the Brachydanio genus: B. albolineatus (called “pearl danio”) and a B. rerio mutant (called “gold danio”). The reasons for our choice are the following: the pearl danio provides some evidence, in its altered ability to adapt to the background color (see below), that it is partially lacking in MSH secretion compared to the ze- brafish. The gold danios appear to have no dermal melanophores over their body. Therefore these two varieties give us an op- portunity to test if met-E acts centrally or peripherally in inducing color changes in these fish. The direct effect of endorphins on iso- lated skin melanophores, in particular the effect of met-E, has been studied in several papers. The effect varies from species to species. Darkening is induced in Ram es- culenta skin in vitro (Medzihradsky and Medzihradsky-Schweiger, 1976), but not in the skin of Rana pipiens (Plotnikoff et al., 1976). Met-E potentiates alpha-MSH ac- tivity in Anolis carolinensis (Carter and Shuster, 1978), while it subsensitizes skin melanophores of Rana berlandieri and Xen- opus Zaevis (Novales and Novales, 1979). The same species specificity had been described earlier in terms of morphine-in- duced darkening of isolated amphibian skin. Humoral and nervous regulation of fish coloration and pigment pattern formation is now under active investigation. However, few observations have yet been carried out on fish to investigate either central or pe- ripheral alteration of MSH activity by en- 378 00164480183 $1.50 Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
Transcript
GENERAL AND COMPARATIVE ENDOCRINOLOGY 51, 378-383 (1983)
Methionine Enkephalin-Induced Changes in Pigmentation of Zebrafish (Cyprinidae, Brachydanio rerio) and Related Species and
Varieties, Measured Videodensitometrically II. Pearl and Gold Danios
RICHARD GORDON AND SOPHIE LEVINA
Department of Pathology, Medical Faculty, University of Manitoba, Winnipeg R3E OW3, Canada
Accepted September 22, 1982
In the fish Brachydanio albolineatus (pearl danio) MSH-induced darkening was similar to that of the B. rerio (zebrafish). However, they showed a greatly reduced response to methionine enkephalin (met-E) though these fish apparently have normal dermal melano- phores. This suggests a central nature of met-E-induced darkening and on the other hand at least a partial lack of MSH production and/or release in response to met-E in these fish. Therefore this reduction in the response to met-E may involve a partial lack of opiate receptors in the cells that provide or stimulate MSH production in the pearl danio. In the B. rerio gold mutant met-E appeared to be able to induce production and/or release of MSH, but these fish have almost no dermal melanophores (except a few in the dorsal and tail tins) and therefore show no darkening in response to either MSH or met-E.
The Brachydanio genus has the advan- tage of having a number of pigmentation and pattern varieties, which are bred by aquarists. Since methionine enkephalin (met-E) was found to be able to induce dis- persion of pigment granules in the melano- phores of zebrafish (Levina and Gordon, 1982, 1983) it was of interest to examine some other varieties of the Brachydanio genus that could elucidate the mechanism of this unexpected biological activity of met-E.
Since we have evidence of a central mechanism for met-E-induced changes of coloration in zebrafish (Levina & Gordon, 1983), we selected for further examination two varieties of the Brachydanio genus: B. albolineatus (called “pearl danio”) and a B. rerio mutant (called “gold danio”). The reasons for our choice are the following: the pearl danio provides some evidence, in its altered ability to adapt to the background color (see below), that it is partially lacking in MSH secretion compared to the ze- brafish. The gold danios appear to have no dermal melanophores over their body.
Therefore these two varieties give us an op- portunity to test if met-E acts centrally or peripherally in inducing color changes in these fish.
The direct effect of endorphins on iso- lated skin melanophores, in particular the effect of met-E, has been studied in several papers. The effect varies from species to species. Darkening is induced in Ram es- culenta skin in vitro (Medzihradsky and Medzihradsky-Schweiger, 1976), but not in the skin of Rana pipiens (Plotnikoff et al., 1976). Met-E potentiates alpha-MSH ac- tivity in Anolis carolinensis (Carter and Shuster, 1978), while it subsensitizes skin melanophores of Rana berlandieri and Xen- opus Zaevis (Novales and Novales, 1979).
The same species specificity had been described earlier in terms of morphine-in- duced darkening of isolated amphibian skin.
Humoral and nervous regulation of fish coloration and pigment pattern formation is now under active investigation. However, few observations have yet been carried out on fish to investigate either central or pe- ripheral alteration of MSH activity by en-
378 00164480183 $1.50 Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENKEPHALIN AND R’SH PIGMENTATION 379
dogenous opiates: Satake (1980) has shown in goldfish that met-E interferes with xan- thophore aggregation, induced by naloxone or melatonin.
MATERIALS AND METHODS All our fish were about 25-35 mm in length and 400-
800 mg body wt at maturity. The fish were maintained and treated as described earlier, with exactly the same experimental design (Levina and Gordon, 1983). All fish were divided into four groups: those injected with (1) saline; (2) MSH solutions (100, 1000, or 10,000 ngi g fish body wt) either alone or with a preceding nal- oxone injection (20 pg/g) given 20 min before; (3) met- E solutions (IO, 100, or 1000 rig/g) either alone or 20 min after naloxone injection (20 l&g); (4) only nal- oxone (20 u&g). In groups 2 and 3 time was measured from the last injection.
The ability of met-E to induce color changes com- pared with that of MSH was examined videodensito- metrically and with the help of melanophore indices (MI). For details see Levina and Gordon (1983).
RESULTS
Even though scale and skin pigmentation vary greatly among these three varieties of the Brachydanio genus, they have similar pigmentation in their pectoral fins, The gold danio pectoral fins have fainter pigmenta- tion than B. rerio (zebrafish) and B. albol- Czeatus (pearl danio).
As in our previous experiments (Levina and Gordon, 1983) separate groups of both kinds of the fish examined (the gold and pearl danios) were maintained for 10 days on a black gravel background with no other treatment, and with the same diurnal light cycle. While the gold danios did not darken, the pearl danios darkened on the dorsal parts of the body. They differed considerably compared to the control group of pearl danios maintained on our usual grey gravel. However, their darkening was far less than the deep darkening of zebrafish under the same conditions.
B. albolineatus. Saline- or naloxone-in- jetted fish (groups 1 and 4, respectively), as well as intact fish, have silver coloration with one visible dark stripe on each of the lateral surfaces near the tail. These stripes are short and stop anteriorly at the level of the anal fin. The dorsal scales of these fish contain both dermal and epidermal melano- phores distributed similarly to those ob- served in zebrafish scales. Their fins also closely resemble zebrafish fins in terms of melanophore population but have almost no xanthophores, Melanophores in the ptri- toneum are rather numerous and moder- ately contracted.
PIG. 1. (A) Changes in the melanophore indices (MI) of light-adapted pearl danios, treated with either fish saline, MSH, or met-E alone or with either one out of the last two substances with naloxone pretreatment. Each point represents the mean index obtained from six to seven fish 2 hr after a single intramuscular (im) injection. (B) Changes in the mean body darkness of the same fish measured by videodensitometry.
380 GORDON AND LEVINA
The darkening that occurred in the second group is again similar to that in zebrafish after MSH treatment, though slightly less intense. Dermal-like scale melanophores were expanded while epidermal melano- phores remained unchanged. MSH-induced darkening is dose related (Fig. 1) and nal- oxone does not interfere with it. The re- sponses of the melanophores of the pec- toral fins (Fig. 2A,B) and of the peritoneum (Fig. 3A,B) are close to that observed in zebrafish (Levina and Gordon, 1983). How- ever, no color changes occurred in the third group and fin melanophores expressed no reaction to the injections of met-E, inde- pendent of naloxone pretreatment (Fig. 1).
B. rerio mutant (gold danio). Intact, sa- line- or naloxone-injected fish (groups 1 and 4) have the same light yellow appearance because they all have many xanthophores. Their epidermal melanophores are small and punctate. There are no dermal-like melano- phores either at the edges of the scales or in the peritoneum. The only location of the scarce, dermal-like melanophores in these fish is in their fins. These melanophores are slightly more numerous in the dorsal and tail fins than in the other fins.
Neither in the second nor in the third group did any treatment induce body dark- ening of the gold danio, though in both groups one could observe dose-related ex- pansion of xanthophores. However, fin me- lanophores proved to be responsive to the treatment in both groups (2 and 3) revealing some dose-related expansion, but less than the response of zebrafish fin melanophores. Within the second hour after injections the dorsal tin darkening was visible to the naked eye, and the tail fin had a typical, though ghost-like, stripe pattern of darkening. The response of the fin xanthophores is more obvious. Naloxone does not alter their MSH-induced changes, but abolishes their met-E-induced responses.
Histological examination confirms the absence of dermal melanophores in the skin and in the peritoneum of the gold danio.
Epidermal melanophores look unchanged (Fig. 4).
Our data on the mean body darkness ob- tained by videodensitometry are similar to that obtained using the MI (Fig. lA,B).
DISCUSSION
The results obtained on B. albolineatus suggest that these fish share with B. rerio a
FIG. 2. (A) Pectoral fin of a pearl danio, treated with saline. Melanophores along bony rays are contracted. (B) The same fin of the same fish, treated with MSH (2 hr after a single im injection of 10,000 rig/g body weight). Melanophores look branched and stellate with dispersed pigment granules (X 10).
ENKEPHALIN AND FISH PIGMENTATION 381
FIG. 3. (A) Peritoneal melanophores of a pearl danio, treated with saline, appear contracted, (B) Peritoneal melanophores of the pearl danio 2 hr after a single im injection of 10,000 rig/g MSH lo& expanded with dispersed granules (hematoxylin and eosin, x 63).
similar set of both dermal and epidermal melanophores as well as the usual respon- siveness to MSH. However, B. alboli- neatus, unlike B. rerio, exhibits a substan- tial lack of endogenous MSH production and/or release in response to met-E. Their prominent darkening in response to a black background on the other hand suggests that this failure to darken in response to met-E (unlike zebrafish) does not depend on a low level of the secretion of MSH in these fish, but rather reflects low levels of opiate re- ceptors on the cells that stimulate MSH production. It is of interest to mention also that the lack of darkening after met-E in- jection again confirms the failure of any di- rect met-E influence on dermal melano- phores in our fish.
On the other hand, the gold B. rerio mu- tant fails to develop dermal melanophores except for a few cells in the fins. However, the changes in these’cells after met-E treat-
ment provide some evidence of MS,H-re- leasing activity of met-E similar to that ob- served in B. rerio. The same activity seems to be made even more evident by the ob- vious expansion of xanthophores in these fish (group 3). The lack of dermal melano- phores may be the result of the death of pigment cells in the fish embryo in the gold danio (Gordon and King, 1982).
The capability of morphine (Kastin et al., 1969) or of endogenous opiates to induce alpha-MSH release has been shown in rats (van Wimersma Greidanus et al., 1979; Celis, 1980) and suggested in goldfish, since met-E could prevent naloxone-induced ag- gregation of xanthophores in these fish (Sa- take, 1980). In rats it has been found that 20 min after a single injection of endorphins the pituitary content of MSH decreased. mln vitro the same response was dose depen- dent (Celis, 1980).
Some other indirect data are avaiiable for
382 GORDON AND LEVINA
FZG. 4. The gold danios carry no melanophores ei- ther in the dermis or in the peritoneum (hematoxylin and eosin, x40).
poikilotherms too. The paper of Mennin and Saland (1980) on Amphibia is particularly relevant. They provided some evidence that in vivo and in vitro naloxone did not affect melanophores when applied directly to the skin and did not prevent darkening in hy- pophysectomized frogs after injections of MSH-containing medium. Therefore they suggested that the effect of naloxone was “mediated via central nervous system con- nections to the pituitary gland.”
Our results also provide evidence for a central MSH-involving mechanism of met- E darkening. Otherwise met-E would be able to induce darkening in B. albolineatus as well, since dermal melanophores of these fish had a rather prompt response to MSH in our experiments on the black gravel background (10 days).
Our videodensitometry method proved especially useful in measuring mean dark- ness. For instance, in our experiments on
the gold danio, while there are no dermal melanophores over its body at all, some persist in its fins and contribute to a mea- surable overall darkening.
The prompt and reproducible darkening of zebra&h in response to both MSH and met-E in conjunction with the response of pearl danio only to MSH, may be used as an easy and inexpensive semiquantitative bioassay for endorphins. The dose-related responses can be evaluated by the naked eye. The MI or videodensitometry are only needed for quantitation. Though radioim- munoassays are now available for endor- phins it is still necessary to check the bioac- tivity since it has been shown that bioac- tivity and immunoactivity do not necessarily coincide (Li et al., 1979).
ACKNOWLEDGMENTS We express our gratitude to Mr. A. Stein for his
valuable assistance in videodensitometry of our fish. Supported in part by the Children Hospital of Win- nipeg Research Foundation, the Sellers Research Foundation, and the Control Data Corporation.
REFERENCES Carter, R. J., and Shuster, S. (1978). Beta-endorphin
potentiates pigmentary activity. J. Invest. Der- matol. 70, 228-229.
Celis, M. E. (1980). Effects of different opiate agonists on MSH release: In viva and in virro studies. Canad. J. Physiol. Pharmacol. 58, 326-329.
Gordon, R., and King, G. M. (1982). Stripe copying in the zebrafish embryo (Brachydanio rerio Ham- ilton). Submitted for publication.
Kastin, A. J., Schally, A. V., Viosca, S., and Miller M. C. (1969). MSH activity in plasma and pitui- taries of rats after various treatments. Endocri- nology 84, 20-28.
Levina, S. E., and Gordon, R. (1982). Met-enkephalin induced changes in pigmentation of zebrafish (Brachydanio reuiu) and reiated species and vati- eties. Proceedings, Western Regional Conference on Comparative Endocrinology, p, 29.
Levina, S., and Gordon, R. (1983). Methionine-enke- phalin induced changes in pigmentation of ze- brafish (cyprinidae, Brachydanio rerio) and re- lated species and varieties, measured videoden- sitometrically. I. Zebrafish. J. Camp. Endocrinol., 51, 370-377.
Li, C. H., Chang, W. C., Yamashiro, D., and Tseng, L. E (1979). Beta-endorphin: Deletion of a single
Medzihradsky, K., and Medzihradsky-Schweiger, H. (1976). Small peptides with MSH-activity. FE&S Lett. 67, 45-47.
Mennin, S. P., and Saland, L. C. (1980). Natoxone prevents dark-background adaptation in amphib- ians. Neuroendocrinology 31, 385-389.
Novales, R. R., and Novales, B. J. (1979). Endorphins supersensitize frog skin melanophores to isopro- terenol, but subsensitize them to alpha-MSH. Gen. Camp. Endocrinol. 39,481-489.
Plotnikoff, N. P., Kastin, A. J., Coy, ID. H., Chris- tensen, C. W., Schally, A. V., and Spirtes, M. A. (1976). Neuropharmacological actions of enke- phalin after systemic administration. Life Sci. 19, 1283-1288.
Satake, N. (1980). Effect of methionine-enkephalin on xanthophore aggregation. Peptides l(l), 73-75.
van Wiiersma Greidanus, T. B., Thody, T. J., Ver- spaget, H., de Rotte, G. A., Goedemans, H. J. H., Croiset, G., and van Ree, J. M. (1979), Effects of morphine and beta-endorphin on basal and ele- vated plasma levels of alpha-MSH and vaso- pressin. Life Sci. 24, 579-583.
