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The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR369- larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously.

Melatonin has both neuritogenic and neuroprotective effects in mammalian cell lines such as neuroblastoma cells. The mechanisms of action include receptor-coupled processes, direct binding and modulation of calmodulin and protein kinase C, and direct scavenging of free radicals. While melatonin is produced in invertebrates and has influences on their physiology and behavior, little is known about its mechanisms of action. We studied the influence of melatonin on neuritogenesis in well-differentiated, extensively-arborized crustacean x-organ neurosecretory neurons. Melatonin significantly increased neurite area in the first 24h of culture. The more physiological concentrations, 1 nM and 1 pM, increased area at 48 h also, whereas the pharmacological 1 μM concentration appeared to have desensitizing effects by this time. Luzindole, a vertebrate melatonin receptor antagonist, had surprising and significant agonist-like effects in these invertebrate cells. Melatonin receptors have not yet been studied in invertebrates. However, the presence of membrane-bound receptors in this population of crustacean neurons is indicated by this study. Melatonin also has significant neuroprotective effects, reversing the inhibition of neuritogenesis by 200 and 500 μM hydrogen peroxide. Because this is at least in part a direct action not requiring a receptor, melatonin's protection from oxidative stress is not surprisingly phylogenetically-conserved.

Amines are one class of signaling molecules used by nervous systems. In crustaceans, four amines are recognized: dopamine, histamine, octopamine, and serotonin. While much is known about the physiological actions of amines in crustaceans, little is known about them at the molecular level. Recently, we mined the Daphnia pulex genome for proteins required for histaminergic signaling. Here, we expand this investigation, mining the D. pulex genome for proteins necessary for dopamine, octopamine and serotonin signaling. Using known Drosophila protein sequences, the D. pulex database was queried for genes encoding homologs of amine biosynthetic enzymes, receptors and transporters. Among the proteins identified were the biosynthetic enzymes tryptophan-phenylalanine hydroxylase (dopamine, octopamine and serotonin), tyrosine hydroxylase (dopamine), DOPA decarboxylase (dopamine and serotonin), tyrosine decarboxylase (octopamine), tyramine β-hydroxylase (octopamine) and tryptophan hydroxylase (serotonin), as well as receptors for each amine and several amine transporters (dopamine and serotonin). Comparisons of the Daphnia proteins with their Drosophila queries showed high sequence identity/similarity, particularly in domains required for function. The data presented in this study provide the first molecular descriptions of dopamine, octopamine and serotonin signaling systems in Daphnia, and provide foundations for future molecular, biochemical, anatomical, and physiological investigations of aminergic signaling in this species.

The influence of melatonin on locomotor activity levels was measured in the fiddler crab Uca pugilator. First, activity in untreated, laboratory-acclimated crabs was measured over 48 hours in a 12L:12D photoperiod; this study showed a nocturnal increase in activity. In eyestalk-ablated crabs, overall activity was significantly reduced, and no significant activity pattern occurred. Next, crabs were injected with melatonin or saline (controls) at various times during the 12L:12D photoperiod (0900h, 1200h, and twice at 2100h; each trial was separated by 3-4 days) and monitored for 3 hr post-injection. Control crabs had low activity during early photophase, high at mid-photophase, increasing activity during the first scotophase trial, and decreasing activity during the second scotophase trial. Melatonin had no significant influence on activity when injected during the early-photophase activity trough or early-scotophase activity decline, but significantly increased activity when injected during the mid-photophase activity peak and early-scotophase activity incline. Next, crabs were injected during an early scotophase activity trough and monitored throughout the twelve-hour scotophase. Melatonin did not increase activity until the mid-scotophase activity increase, approximately 6 hours later, showing that the pharmacological dosage persisted in the crabs' systems and had later effects during the incline and peak of activity but not the trough. Eyestalk-ablated crabs were injected with melatonin or saline during early photo- and scotophase. Melatonin significantly increased activity in the photophase but not the scotophase trial, indicating that the responsiveness to melatonin continues following eyestalk removal, but the timing may not match that of intact crabs. Melatonin may be involved in the transmission of environmental timing information from the eyestalks to locomotor centers in U. pugilator.

Melatonin and N-acetyltransferase (NAT) activity were measured in the eyestalks of fiddler crabs acclimated to various photoperiods: constant light, a L:D 12:12 h photoperiod, or constant dark. Following acclimation, eyestalks were collected every 3 h over a 24-h period; they were assayed for melatonin with a radioimmunoassay and for NAT activity with a radioenzymatic assay. In constant light, melatonin levels increased at 1300 h, from 142 to 431 pg x mg(-1) eyestalk; NAT activity increased concurrently, from 97 to 203 pmol x h(-1) x mg(-1) eyestalk, and both remained elevated until 0400 h. In the L:D 12:12 h photoperiod, melatonin levels increased at 1300 h from 28 to 230 pg x mg(-1) eyestalk, and though NAT activity increased significantly, from 80 to 122 pmol x h(-1) x mg(-1) eyestalk, an even greater increase occurred at 0400 h, when melatonin levels were low. In constant dark, melatonin levels increased at 1600 h, from 22 to 196 pg x mg(-1) eyestalk, with a concurrent increase in NAT activity from 93 to 140 pmol x mg(-1) x h(-1) eyestalk. However, the second peak in melatonin (111 pg x mg(-1)), occurring at 0400 h, was out of phase with the second peak of NAT activity (113 pmol x mg(-1) x h(-1) eyestalk) which occurred at 0700 h. NAT may be a rate-limiting step in melatonin synthesis in fiddler crabs under some conditions (constant light and the 1300 h peak in constant dark); however, NAT activity correlates poorly with melatonin levels in a L:D 12:12 h photoperiod and in constant dark relative to the 0400 h melatonin peak.

We measured melatonin (MEL) with RIA in two species of damselfly, Ischnura verticalis and Enallagma civile. I. verticalis were sampled over 24 h in ambient outdoor L13.5:D10.5 photoperiod and thermoperiod (15 to 30.5°C), and we found a significant MEL cycle with a peak during scotophase/cryophase. The range of [MEL] was 11.2 to 20.2 pg mg body mass−1. E. civile were sampled over 24 h at a L12:D12 photoperiod and 25°C and at mid-photophase and mid-scotophase at both 10 and 30°C. Mean [MEL] was 0.957 pg mg body mass−1, and [MEL] did not differ significantly among times of day or temperatures.

Essentially nothing is known about the molecular underpinnings of crustacean circadian clocks. The genome of Daphnia pulex, the only crustacean genome available for public use, provides a unique resource for identifying putative circadian proteins in this species. Here, the Daphnia genome was mined for putative circadian protein genes using Drosophila melanogaster queries. The sequences of core clock (e.g. CLOCK, CYCLE, PERIOD, TIMELESS and CRYPTOCHROME 2), clock input (CRYPTOCHROME 1) and clock output (PIGMENT DISPERSING HORMONE RECEPTOR) proteins were deduced. Structural analyses and alignment of the Daphnia proteins with their Drosophila counterparts revealed extensive sequence conservation, particularly in functional domains. Comparisons of the Daphnia proteins with other sequences showed that they are, in most cases, more similar to homologs from other species, including vertebrates, than they are to those of Drosophila. The presence of both CRYPTOCHROME 1 and 2 in Daphnia suggests the organization of its clock may be more similar to that of the butterfly Danaus plexippus than to that of Drosophila (which possesses CRYPTOCHROME 1 but not CRYPTOCHROME 2). These data represent the first description of a putative circadian system from any crustacean, and provide a foundation for future molecular, anatomical and physiological investigations of circadian signaling in Daphnia.

Serum melatonin concentrations in the diamondback water snake, Nerodia rhombifera, were measured with a radioimmunoassay to determine the influence of photoperiod and temperature on melatonin levels. A diel cycle of serum melatonin, with elevated concentrations during scotophase, occurred in snakes acclimatized for 2 weeks to a 12L:12D photoperiod and 25 degrees. This cycle persisted in snakes acclimatized to a reverse photoperiod, with a longer duration of elevated scotophase levels. In snakes acclimatized to a normal 12L:12D photoperiod and 10 degrees, photophase serum melatonin concentrations were lower than at 25 degrees, and there was no increase during scotophase. Snakes acclimatized to a normal 12L:12D photoperiod and 35 degrees had lower serum melatonin levels than those at 25 degrees, but this difference was not statistically significant. Photoperiod and temperature interact to influence serum melatonin in N. rhombifera, with photoperiod affecting the phase and temperature affecting the amplitude of the diel melatonin cycle.

Melatonin affects a variety of circadian processes such as behavior and neurotransmitter release in vertebrates. Crayfish melatonin production occurs in the eyestalks, and the cycle of production may change seasonally. To date, however, melatonin's roles and mechanisms of action in crustacean physiology are unclear. We injected melatonin or saline into crayfish in scotophase and monitored activity and hemolymph glucose/lactate over 24 h in early spring. Crayfish were significantly more active in photophase versus the expected scotophase, and had concurrent glucose/lactate peaks. Melatonin reversed the activity pattern, causing a scotophase activity peak, but not the glucose/lactate patterns. This study was repeated in late summer, during which control activity and glucose/lactate levels were elevated in scotophase. Melatonin decreased the amplitude of scotophase activity and glucose/lactate, eliminating activity and glucose cycles. We also injected melatonin or saline at various times of day in early summer and monitored locomotor activity for 1 h. Controls had high activity at 1200 (mid-photophase) and 2100 h (early scotophase), and melatonin increased activity at 1200 h but decreased it at 2100 h. Melatonin also increased activity at 1500 h but not 1800 h (late photophase). Next, we examined the influence of melatonin on crayfish neurophysiology. Melatonin (10 microM) enhanced synaptic transmission at the neuromuscular junction (NMJ). The presynaptic action resulted in more vesicles being released during evoked stimulation. Our study indicates that melatonin may have a phylogenetically conserved role in the transduction of circadian information in invertebrates as in vertebrates. Behavioral and physiological effects may be mediated by modulation of central pathways, enhanced at the peripheral level via neuromodulation of the NMJ.

Melatonin was injected into intact and eyestalk-ablated fiddler crabs (Uca pugilator), and its effects on hemolymph glucose and lactate levels were studied. In intact crabs, glucose and lactate levels cycled simultaneously, with peaks occurring during early and late photophase. Melatonin caused a shift in the glucose and lactate cycles, with only one peak occurring closer to mid-photophase. In eyestalk-ablated animals, the glucose rhythmicity was lost; lactate cycled, but levels were significantly lower than in intact animals. Melatonin caused a delayed hyperglycemia in eyestalk-ablated animals, with concurrent but much lower increases in lactate. Overall, melatonin demonstrated delayed hyperglycemic effects that do not appear to be mediated solely via eyestalk factors such as crustacean hyperglycemic hormone (CHH), though involvement of the eyestalks cannot be ruled out. An influence on extra-eyestalk CHH secretion is a potential mechanism of melatonin activity.

Tilden AR, Rasmussen P, Awantang RM, Furlan S, Goldstein J, Palsgrove M, Sauer A. Melatonin cycle in the fiddler crab Uca pugilator and influence of melatonin on limb regeneration. J. Pineal Res. 1997; 23:142–147. © Munksgaard, Copenhagen Abstract Melatonin was measured over 24 hr in the eyestalks of Uca pugilator by means of radioimmunoassay; crabs were acclimatized either to a LD 12:12 photoperiod or constant darkness. A significant peak occurred at 13.00 hr in the LD 12:12 crabs. A photophase peak in melatonin has only been reported in one other species, also a crustacean. In constant darkness, two melatonin peaks occurred, one at 16.00 hr and the other 12 hr later; these results suggest that the melatonin cycle is a true circadian rhythm. HPLC with ultraviolet-visible detection was used to confirm the identity of melatonin immunoactivity. The influence of melatonin on regeneration of the walking legs was also examined: eyestalks were either removed or left intact, and limb bud length was measured every other day for at least 17 days in control and melatonin-treated crabs (60 μg ml-1 seawater). Melatonin significantly increased the rate of limb regeneration in both eyestalk-intact and eyestalk-removed groups; this is contrary to results of regeneration studies in other phyla, in which similar melatonin concentrations inhibited regeneration.

Tilden AR, Becker MA, Amma LL, Arciniega J, McGaw AK. Melatonin production in an aerobic photosynthetic bacterium: An evolutionarily early association with darkness. J. Pineal Res. 1997; 22:102–106. © Munksgaard, Copenhagen Abstract Melatonin was measured in a species of aerobic photosynthetic bacteria, Erythrobacter longus, grown in either constant light or constant dark. A radioimmunoassay was used to quantify melatonin levels and thin-layer chromatography to confirm the identity of melatonin immunoactivity. Melatonin levels were significantly higher (nearly 2.3-fold) in the dark-grown than in the light-grown samples. Also, the homogenates of the dark-grown bacteria retained melatonin-producing enzymatic activity, whereas the light-grown homogenates did not; melatonin levels extracted from the dark-grown homogenates increased with increasing extraction time, reaching as high as 29.2 ng-mg-1 protein at 120 min. Removal of membrane fragments from homogenates did not influence melatonin levels in light-grown homogenate, but this procedure increased melatonin levels in dark-grown homogenate, indicating that at least some of the enzymes in the pathway of melatonin production are not membrane-bound. This study is the second to demonstrate the presence of melatonin at the prokaryotic level, supporting the evidence that melatonin appeared very early in evolution. Its function in prokaryotes has not been determined, but may relate to its antioxidative actions.