Abstract

Melatonin is a hormone synthesized and secreted by the pineal gland. Its production is regulated by daylength with increasing amounts of the hormone being produced in the autumn and winter, as daylight hours decrease. Melatonin has become the newest craze on the U.S. health supplement market and has been described as a panacea for many human ailments. Research in the last decade has elucidated immunoregulatory properties of this hormone in human and rodent studies. In human medicine, melatonin has several clinical applications for enhancing the immune response against certain types of cancer. Field and laboratory studies of mammals have shown that seasonal changes can affect various immune parameters. It has been suggested that melatonin serves to up-regulate the immune system in temperate climates as a natural defense against higher energetic demands and seasonal hardships associated with the winter months. There has been no research to date investigating whether melatonin could alter immune parameters of fish, although there are a few reports indicating that seasonal changes can affect fish immune responsiveness. This study showed increased innate immune responses for extracellular O₂ and lysozyme and near significant increases in phagocytosis, survival, and antibody titers in melatonin implanted fish compared to control fish. If melatonin is shown to enhance the immune response of fish, this hormone may also prove beneficial for the aquaculture industry. Some practical applications would be to prevent disease related losses, either as an immunomodulator in commercial fish feeds and/or as a vaccine adjuvant, boosting overall immune responses against specific aquatic pathogens.

Introduction

Zapata¹³ reviewed seasonal variation of immune function in several poikilotherm animals, failing to find any consistent trends linking season with immunity for ectotherms. Steroid hormones in poikilotherms down regulated immune function similar to observations in homeotherms. There has been no specific investigation to date examining the effects of photoperiod or melatonin on the immune response of these animals. Reviewing an entire group as diverse as the poikilotherms, does not account for the myriad of differences between and within classes. Phylogenetic differences between cyprinids and salmonids are greater than differences between rodents and primates.

Seasonal trends in immune function have been reported in some fish. Earlier records of disease occurrence and mortalities in wild fisheries have been based on an individual fish species or pathogen.^11,12 More recent work has elaborated on the general function of the immune system with regard to seasonal changes. Collazos et al.^1-4 reported seasonal variations in hematologic parameters, immune parameters, and immune function in the tench (Tinca tinca), and Hutchinson and Manning⁵ observed seasonal trends in lysozyme activity in dab (Limanda limanda) from Lyme Bay, U.K. Research investigating seasonal effects on basic immune function may ultimately serve to advance our understanding of the epidemiology of specific fish pathogens.

Methods and Results

Three hundred female rainbow trout were divided into two experimental groups; each consisting of three replicates (ca. 50 fish/replicate). The fish were maintained at 12°C in an outdoor recirculating system under ambient photoperiod. One group was implanted with constant-release melatonin implants (Regulin; Hoechst UK Ltd.), while the other group received a sham-operation but no implant. Fish were sampled for immunologic analysis at 3 and 6 wk post melatonin implantation and subjected to bacterial challenge 10 wk after implantation.

Melatonin Levels

A radioimmunoassay was used to assess the level of melatonin in the plasma of implanted fish and control fish. As measured post implantation, the treated group had levels of melatonin 3-4 times typical of night-time values compared with the normal daytime values observed in the control group at week three and week six.

Cellular Components of the Innate Immune Response

Detection of the Superoxide Anion

The following two assays were used to quantify the intensity of macrophage respiratory burst activity.¹⁰

Intracellular O₂ was detected by dissolving insoluble formazan that was produced by the reduction of the dye nitroblue tetrazolium (NBT). Although the group receiving melatonin implants exhibited a slightly higher production of intracellular O₂, the differences were not significant.

Extracellular O₂ was measured using a solution of ferricytochrome c. The difference, between phorbol myristate acetate (PMA) stimulated cells and unstimulated cells, was significantly higher (P < 0.05) for the melatonin-implanted group compared with the control group at 3 wk post-implant (Table 1). There was no significant difference between the melatonin implanted and control fish at 6 wk post-implantation, although a slight increase in extracellular O₂ was observed in the control group.

Phagocytosis Assay

Phagocytosis of yeast by head kidney macrophages isolated from experimental fish was assessed 6 wk after melatonin implantation. Yeast were used for phagocytosis at 6 wk and media alone was used as a negative control. The melatonin-implanted group had both a higher phagocytic ratio and index (Table 2), indicating, respectively, that there were a higher percentage of phagocytosing macrophages and a larger number of phagocytosed yeast particles. However, neither of these differences was statistically significant. The control group had a standard deviation twice that of the treatment group for the phagocytic ratio.

Lysozyme

The ability of plasma lysozyme to degrade Micrococcus lysodeikticus was measured using spectrophotometry at both 3 and 6 wk post implantation. The data were statistically analyzed and results indicated in Table 3. At 3 wk, post-implantation lysozyme values for melatonin-implanted and control fish appeared nearly identical, but lysozyme was significantly higher (P < 0.05) in the treated group compared with the control group at 6 wk post melatonin implant (Table 3).

Bacterial Challenge and ELISA

The fish were artificially challenged with Vibrio anguillarum and percent survival determined. These were calculated from pooled replicates (note that only two replicates were pooled for the melatonin-implanted group, because the third replicate was lost due to a system malfunction just prior to bacterial challenge):

 Control: Pooled % survival = (27+26+34)/(34+36+39)= 87/109 = 79.82%; SE = 0.0386

 95% CL = 0.0757; Upper 95% CL = 87.39%; Lower 95% CL = 72.25%

 Melatonin Treated: Pooled % Survival = (32 + 28)/(36+32) = 60/68 = 88.24 %; SE = 0.0394

 95% CL = 0.0771; Upper 95% CL = 95.56%; Lower 95% CL = 80.53%

Levels of antibodies against Vibrio anguillarum, which were elicited by fish 3 wk post-challenge, were determined using ELISA (Table 4).

Discussion

This study provides the first reported data on the effects of melatonin on immunoregulation in fish. In the last decade, literature relating to the effects of melatonin on the immune response of humans has expanded greatly. Numerous articles report the immunoregulatory effects of melatonin, from triggering novel opioid peptides that activate T-cells,⁶ to enhancing the production of tumor necrosis factor.⁹ Although fish are poikilotherms they share many of the same cellular and humoral immune components seen in mammals. Melatonin is a widely conserved molecule found in single alga, edible plants, invertebrates and vertebrates.⁸ If ontogeny recapitulates phylogeny, than certainly melatonin may arbitrate similar mechanisms across vertebrate phyla.

This study attempted to elucidate the effects of melatonin on the immune response of rainbow trout (Oncorhynchus mykiss). Several parameters of both cell-mediated and humoral components of the non-specific immune system, as well as humoral components of the specific immune response, were assayed, comparing melatonin-implanted fish with control fish. In comparison with control fish, melatonin-implanted fish had significantly higher values for both the cytochrome c assay at 3 wk post-implant and lysozyme activity at 6 wk post-implant. There was a near-significant increase in phagocytic ratio at 6 wk post-implant, and a tendency towards increased survival and enhanced humoral antibody responses post Vibrio challenge.

In conclusion, these findings provide the first evidence that melatonin may enhance the immune system of rainbow trout, acting to stimulate bactericidal activity and eventually leading to stronger adaptive responses. These results and their significance provide a foundation for further investigations designed to explore neuroendocrine immunoregulation in fish.

Table 1. Statistical data for quantifying extracellular O₂ released by macrophage monolayers from control and melatonin-implanted rainbow trout 3 wk after melatonin implantation.

T-test (1) P = 0.0537
T-test (2) P = 0.8731
T-test (3)* P = 0.0260*
* Denotes significant difference in values
** Results are expressed as OD _{550 nm} for 2 x 10⁵ cells well ^-1.

Table 2. Statistical analysis for the phagocytosis of yeast particles by head kidney macrophages from melatonin-implanted or control rainbow trout at 6 wk post-implant.

1. Mann-Whitney Between both Treatment T = 134.5 P = 0.091
2. T-Test and Control Groups t = 1.10 P = 0.2870

Table 3. Lysozyme values (OD _540nm) measured at 3 and 6 wk post-implant in control and melatonin-implanted fish.

* Denotes a statistical difference between treatment and control groups.
1. Mann-Whitney Week 3 Between both Treatment T=145 P = 0.795
2. T-Test Week 6* and Control Groups t = 2.34 P = 0.0289*

Table 4. Antibody titers produced by fish challenged with Vibrio anguillarum were determined by ELISA at 3 wk post-challenge.

Acknowledgments

This research was supported by a grant (GR3/R927) from the Natural Environment Research Council of the United Kingdom and a Thouron Fellowship from the University of Pennsylvania.

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