Gerald A. LeBlanc

Biological rhythms regulate innumerable physiological processes, yet little is known of factors that regulate many of these rhythms. Disruption in the timing of these rhythms can have devastating impacts on population sustainability. We hypothesized that the timing of the molt infradian rhythm in the crustacean Daphnia magna is regulated by the joint action of the protein E75 and nitric oxide. Further, we hypothesized that disruption of the function of E75 would adversely impact several physiological processes related to growth and reproduction. Analysis of mRNA levels of several genes, involved in regulating the molt cycle in insects, revealed the sequential accumulation of E75, its dimer partner HR3, FTZ-F1, and CYP18a1 during the molt cycle. Exposure to the nitric oxide donor sodium nitroprusside early in the molt cycle had no effect on E75 or HR3 mRNA levels, but delayed the peak accumulation of FTZ-F1 and CYP18a1 mRNA. The subsequent exuviation was also delayed consistent with the delay in peak accumulation of FTZ-F1 and CYP18a1. These results supported our assertion that nitric oxide binds E75 rendering it incapable of binding HR3. Excess HR3 protein then enhanced the accumulation of the downstream products FTZ-F1 and CYP18a1. Similarly, suppression of E75 mRNA levels, using siRNA, had no effect on mRNA levels of HR3 but elevated mRNA levels of FTZ-F1. Consistent with these molecular responses, the suppression of E75 using siRNA increased the duration of the molt cycle and reduced the number of offspring produced. We conclude that the molt cycle of daphnids is regulated in a manner similar to insects and disruption of E75 results in a lengthening of the molt cycle and a reduction the release of viable offspring.

Daphnia spp, a keystone genus in freshwater lentic habitats, are subject to environmental sex determination wherein environmental conditions dictate offspring sex and whether they reproduce asexually or sexually. The introduction of males into a population denotes the first step in the switch from asexual parthenogenetic reproduction to sexual reproduction. We tested the hypothesis that photoperiod and temperature co-regulate male sex determination and that these environmental stimuli would activate elements of the male sex determination signaling cascade. Results revealed that photoperiod was a critical cue in creating permissive conditions for male production. Further, under photoperiod-induced permissive conditions, male sex determination was temperature dependent. The two daphnid species evaluated, Daphnia pulex and D. magna, exhibited different temperature dependencies. D. pulex produced fewer males with increasing temperatures between 16-22°C, and D. magna exhibited the opposite trend. We found consistent expression patterns of key genes along the male sex determining signaling pathway in D. pulex independent of environmental stimuli. mRNA levels for the enzyme responsible for synthesis of the male sex determining hormone, methyl farnesoate, were elevated early in the reproductive cycle, followed by increased mRNA levels of the methyl farnesoate receptor subunits, Met and SRC. Environmental conditions that stimulated male offspring production significantly increased Met mRNA levels. Results indicate that male sex determination in daphnids is under the permissive control of photoperiod and the regulatory control of temperature. Further, these environmental cues may stimulate male sex determination by increasing levels of the Met subunit of the methyl farnesoate receptor.

Nitrogenous compounds enter the environment through various anthropogenic sources. Among these are nitrate (NO₃⁻) and nitrite (NO₂⁻) which can oxidize the heme moiety of hemoglobin and reduce the oxygen-carrying capacity of the molecule resulting in toxicity. Of the two anions, nitrite is more toxic. Hemoglobin levels are influenced by environmental conditions; thus, we hypothesized that hemoglobin levels would influence the toxicity of nitrite with low hemoglobin levels resulting in enhanced toxicity and high hemoglobin levels resulting in reduced toxicity. We tested this hypothesis by elevating hemoglobin levels with pyriproxyfen treatment and lowering hemoglobin levels using siRNA in Daphnia magna. Exposure to pyriproxyfen significantly elevated hemoglobin mRNA levels and induced copper coloration of the organisms, indicative of increased hemoglobin protein accumulation. siRNA treatment significantly reduced hemoglobin mRNA levels in both untreated and pyriproxyfen-treated organisms and attenuated copper coloration. Pyriproxyfen treatment increased the tolerance of daphnids to the acute toxicity of nitrite approximately 2-fold while siRNA treatment significantly decreased the tolerance of daphnids to nitrite toxicity. Results indicate that increased hemoglobin levels increase the tolerance of daphnids to nitrite toxicity which may serve to protect daphnids in environments subject to hemoglobin-elevating hypoxia or elevated temperatures.

The high throughput screening of chemicals for interaction with intracellular targets is gaining prominence in the toxicity evaluation of environmental chemicals. We describe ligand-mediated receptor assembly as an early event in receptor signaling and its application to the screening of chemicals for interaction with targeted receptors. We utilized bioluminescence resonance energy transfer (BRET) to detect and quantify assembly of the methyl farnesoate receptor (MfR) in response to various high-production volume and other chemicals. The hormone methyl farnesoate binds to the MfR to regulate various aspects of reproduction and development in crustaceans. The MfR protein subunits Met and SRC, cloned from Daphnia pulex, were fused to the fluorophore, mAmetrine and the photon generator, Rluc2, respectively. Ligand-mediated receptor assembly was measured by photon transfer from the photon donor to the fluorophore resulting in fluorescence emission. Overall, the BRET assay had comparable or greater sensitivity as compared to a traditional reporter gene assay. Further, chemicals that screened positive in the BRET assay also stimulated phenotypic outcomes in daphnids that result from MfR signaling. We concluded the BRET assay is an accurate, sensitive, and cost/time efficient alternative to traditional screening assays.

The methyl farnesoate receptor (MfR) orchestrates aspects of reproduction and development such as male sex determination in branchiopod crustaceans. Phenotypic endpoints regulated by the receptor have been well-documented, but molecular interactions involved in receptor activation remain elusive. We hypothesized that the MfR subunits, methoprene-tolerant transcription factor (Met) and steroid receptor coactivator (SRC), would be expressed coincident with the timing of sex programming of developing oocytes by methyl farnesoate in daphnids. We also hypothesized that methyl farnesoate activates MfR assembly. Met mRNA was expressed rhythmically during the reproductive cycle, with peak mRNA accumulation just prior period of oocytes programming of sex. Further, we revealed evidence that Met proteins self-associate in the absence of methyl farnesoate, and that the presence of methyl farnesoate stimulates dissociation of Met multimers with subsequent association with SRC. Results demonstrated that the Met subunit is highly dynamic in controlling the action of methyl farnesoate through temporal variation in its expression and availability for receptor assembly.

Triphenyl phosphate is an organophosphate flame retardant and plasticizer that has been detected in a variety of environmental media and shown to cause weight gain in rats. We hypothesized that triphenyl phosphate would modify the activity of the PPAR: RXR signalling network in a manner that would favor lipid accumulation. Gal4-driven luciferasebased transcription reporter gene assays were used to evaluate the responses of human PPARα: RXRα, PPARγ: RXRα, and the individual receptor subunits, to triphenyl phosphate. Triphenyl phosphate was a potent inhibitor of PPARα: RXRα signalling. The flame retardant interacted with both the PPARα and RXRα subunits to inhibit their respective activities at concentrations that were not overtly toxic to the cells. Bioluminescence resonance energy transfer experiments revealed that triphenyl phosphate actually inhibited the dimerization of PPARα and RXRα. In contrast, triphenyl phosphate modestly activated the PPARγ: RXRα receptor complex. This net activation of the complex was due to strong activation of the PPARγ receptor subunit and modest inhibition of the RXRα subunit. Further experiments revealed that triphenyl phosphate stimulated pre-adipocyte differentiation to lipid-laden adipocytes at a concentration that disrupted the PPAR signalling network. This dual activity of triphenyl phosphate, as an inhibitor of PPARα: RXRα signalling and an activator of PPARγ: RXRα signalling provides a regulatory scenario that could lead to weight gain and other symptoms of metabolic syndrome.

Branchiopod crustaceans (e.g., Daphnia sp.) and some other taxa utilize both asexual and sexual reproduction to maximize population sustainability. The decision to switch from asexual to sexual reproduction is triggered by environmental cues that foretell a potentially detrimental change in environmental conditions. This review describes the cascade of events beginning with environmental cues and ending with changes in gene expression that dictate male sex determination in daphnids, the initial event in the switch to sexual reproduction. Several environmental cues have been identified which, either in isolation or in combination, stimulate male sex determination. These cues are typically associated with change of season, exhaustion of resources or loss of habitat. Maternal daphnids receive and respond to these cues, we propose, through the secretion of neuropeptides, which suppress (hyperglycemic hormone‐like neuropeptides, allatostatin) or stimulate (allatotropin) the male sex differentiation program. In response, maternal daphnids produce the male sex‐determining hormone, methyl farnesoate. Methyl farnesoate binds to a protein MET that dimerizes with the protein SRC forming an active transcription factor. This complex then regulates the expression of genes, primarily doublesex (dsx), involved in programming the single‐celled embryo to develop into a male. In the absence of methyl farnesoate programming, the embryo develops into a female. Epigenetic modifications of the genome as a possible mode of methyl farnesoate action and the utility of this model to decipher the role of epigenetics in sex differentiation in other species are discussed.

Environmental signals to maternal organisms can result in developmental alterations in progeny. One such example is environmental sex determination in Branchiopod crustaceans. We previously demonstrated that the hormone methyl farnesoate could orchestrate environmental sex determination in the early embryo to the male phenotype. Presently, we identify a transcription factor that is activated by methyl farnesoate and explore the extent and significance of this transgenerational signaling pathway. Several candidate transcription factors were cloned from the water flea Daphnia pulex and evaluated for activation by methyl farnesoate. One of the factors evaluated, the complex of two bHLH-PAS proteins, dappuMet and SRC, activated a reporter gene in response to methyl farnesoate. Several juvenoid compounds were definitively evaluated for their ability to activate this receptor complex (methyl farnesoate receptor, MfR) in vitro and stimulate male sex determination in vivo. Potency to activate the MfR correlated to potency to stimulate male sex determination of offspring (pyriproxyfen>methyl farnesoate>methoprene, kinoprene). Daphnids were exposed to concentrations of pyriproxyfen and physiologic responses determined over multiple generations. Survivial, growth, and sex of maternal organisms were not affected by pyriproxyfen exposure. Sex ratio among offspring (generation 2) were increasingly skewed in favor of males with increasing pyriproxyfen concentration; while, the number of offspring per brood was progressively reduced. Female generation 2 daphnids were reared to reproductive maturity in the absence of pyriproxyfen. Sex ratios of offspring (generation 3) were not affected in this pyriproxyfen lineage, however, the number of offspring per brood, again, was significantly reduced. Results reveal likely components to a hormone/receptor signaling pathway in a crustacean that orchestrates transgenerational modifications to important population metrics (sex ratios, fecundity of females). A model is provided that describes how these signaling processes can facilitate population sustainability under normal conditions or threaten sustainability when perturbed by environmental chemicals.