Possible Influences of Circadian Melatonin on the Function of Neurosecretory Neurons and Serotonin-Modulated Behavior on Crayfish.

Crustaceans have frequently been used to study the neuroethology of diverse function as agonistic behavior and circadian rhythms. Whether the highly stereotyped and quantifiable agonistic activity in crustaceans is controlled by circadian pacemakers has so far not been investigated. In order to address this question, I introduce the marbled crayfish (Procambarus spec.), an easy to breed parthenogenetic decapod crustacean of unknown regional origin, as a new preparation for neuroethological studies. Isolated marbled crayfish displayed rhythmic locomotor activity under 12 hours light-12 hours darkness (LD12:12) and rhythmicity persisted after switching to constant darkness (DD) for nine days, suggesting the presence of endogenous circadian pacemakers. Isogenetic females of parthenogenetic marbled crayfish displayed all behavioral elements known from agonistic interactions of previously studied decapod species including the formation of hierarchies. Previously isolated marbled crayfish initially displayed high numbers of agonistic encounters during the first hour of their co-habitation in groups of six animals. With the formation of hierarchies, the frequency of agonistic encounters was reduced to low levels. Group agonistic activity was entrained to periods of exactly 24 hours under LD12:12 and peaks of agonistic activity coincided with light-to-dark and dark-to-light transitions. After switching to DD, enhanced agonistic activity was only observed at times corresponding with light to dark transitions during the preceding three days in LD 12:12. During four days under DD, agonistic activity remained rhythmic with an average circadian period of 24.83 ± 1.22 h in all crayfish groups tested. Analysis of individual agonistic activities revealed that the most dominant crayfish participated in more than half of all agonistic encounters within the group and initiated most of them. Only the most dominant crayfish revealed clear endogenous rhythmicity in their agonistic behavior. Subordinate individuals, depending on their social rank, initiated only between 19.4 and 0.03 % of all encounters in constant darkness and displayed no statistically significant rhythmicity. The results indicate that both, locomotion and agonistic social interactions are rhythmic behaviors of marbled crayfish, which are controlled by light-entrained endogenous pacemakers. The availability of isogenetic populations of marbled crayfish may foster the future analysis of these pacemakers in crustaceans. Crustaceans and insects appear to be sister groups, but their circadian systems seem to differ in degrees of centralization of pacemaker centers in their brains. However, also in Crustacea at least part of the circadian control resides in the optic lobes. In insects, optic lobe pacemaker neurons reside in a specialized neuropil, the accessory medulla (AMe). The insect AMe is innervated by neurons expressing the neuropeptide pigment-dispersing factor (PDF), and because of that it is known as PDFMe. PDF is homologous to the crustacean pigment-dispersing hormone (PDH), which in crustacea is expressed in a variety of brain neurons. With combined immunocytochemical and backfill studies, I identified neural structures in the marbled crayfish that probably correspond to the insect optic lobe circadian pacemaker. In this work, the PDH-expressing neuron system of the crayfish was studied in eyestalks and brain, and results show a tripartite PDH-ir neuron cluster that appeared to be homologous to the insect PDFMe according to the homology criteria of similar position and neurochemical quality. However, I did not find a structure comparable to the insect AMe, thus leading to the assumption, that the insect AMe is a newly attained structure in the evolution of the insect optic lobe, which was not present in the common ancestor of crustaceans and insects. This work opens first insights into the evolution of the arthropod circadian system and presents new valuable information about the location of circadian pacemakers in crustaceans. Melatonin, the classical time messenger of vertebrates, is present in most organisms studied so far. In the melatonin biosynthetic pathway, the indoleamines serotonin (5-HT) and N-acetylserotonin (NAS) are formed from 5-hydroxytryptophan. Serotonin is important for a variety of functions, in both vertebrate and invertebrate animals. In contrast, the knowledge about potential functions of N-acetylserotonin and melatonin in invertebrates is relatively limited. In this work, I adapted an ELISA procedure for the analyses of crustacean tissues, in order to measure serotonin, N-acetylserotonin and melatonin in the brain and eyestalks of the marbled crayfish Procambarus spec. over a complete daily cycle (LD12:12). Serotonin showed mean levels in the range of 21.05 ± 0.93 ng/ml in brains and 6.75 ± 0.36 (mean ± SEM) ng/ml in eyestalks. NAS concentrations were slightly lower than those of serotonin with 5.65 ± 0.29 ng/ml in brains, and 3.12 ± 0.23 ng/ml in eyestalks. Strikingly, melatonin showed levels 1000 times lower than NAS, in brains 30.82 ± 1,61 and in eyestalks 21.66 ± 1.54 pg/ml. Significant changes between minima and maxima over one day cycle were found for serotonin and melatonin levels in both central nervous structures. NAS differences were not significant in both structures, but in eyestalks they were very close to significance. Cosinor analysis did not show a significant (p ≤ 0.05) fit to a sinusoid with a period of 24 h, but significance was close to be reached in some cases. These results enable some assumptions about the biosynthesis of melatonin in the nervous system of arthropods, specifically in the crayfish Procambarus espec. This is the first time that NAS is measured in arthropods and provides new insights into metabolic pathways that form melatonin in invertebrates.