Corazonin signaling integrates energy homeostasis and lunar phase to regulate aspects of growth and sexual maturation in Platynereis

Significance Gonadotropin Releasing Hormone (GnRH) acts as a key regulator of sexual maturation in vertebrates, and is required for the integration of environmental stimuli to orchestrate breeding cycles. Whether this integrative function is conserved across phyla remains unclear. We characterized GnRH-type signaling systems in the marine worm Platynereis dumerilii, in which both metabolic state and lunar cycle regulate reproduction. We find gnrh-like (gnrhl) genes upregulated in sexually mature animals, after feeding, and in specific lunar phases. Animals in which the corazonin1/gnrhl1 gene has been disabled exhibit delays in growth, regeneration, and maturation. Molecular analyses reveal glycoprotein turnover/energy homeostasis as targets of CRZ1/GnRHL1. These findings point at an ancestral role of GnRH superfamily signaling in coordinating energy demands dictated by environmental and developmental cues.


Adult Platynereis heads express orthologs of the vertebrate glycoprotein hormone system
In vertebrates, GnRH controls not only the pulsatile secretion, but also the expression of pituitary gonadotropins (6)(7)(8)(9)(10). Interestingly, a GnRH-like peptide from Octopus vulgaris that acts through a Corazonin-type receptor (11) stimulates LH-release from cultured quail pituitary cells (12). The vertebrate gonadotropins (LH and FSH), as well as the metabolicrelated factors thyroid-stimulating hormone (TSH) and thyrostimulin (TS), are glycoprotein hormone (GPH) heterodimers (13,14). The α and β subunits of GPHs form distinct groups that are found across all bilaterians, suggesting that a GPH-like system already existed in early animal ancestors (15,16). Given that also GnRH-like systems are part of this ancestral repertoire, we investigated if a functional relationship between GnRH-like peptides and GPHs existed in the bristleworm.
In Platynereis dumerilii, a GPHβ-like subunit is referred to as GPB (1). Our independent searches yielded not only this subunit, but also a partial sequence encoding a putative glycoprotein α-like subunit that we named GPA (Table S3). In line with previous phylogenetic analyses (15)(16)(17), GPA and GPB cluster with GPHα and GPHβ subunits from other invertebrates, respectively (Fig. S5A). Moreover, our analysis suggests that the common bilaterian ancestor possessed single GPA and GPB subunits, and that these diversified further in the evolutionary lineage leading to vertebrates, giving rise to (i) vertebrate thyrostimulin (GPHα2 and GPHβ5) and (ii) vertebrate gonadotropin/thyroid hormone (GPHα1 and FSHβ/LHβ/TSHβ) systems, a diversification that is likely linked to the whole genome duplications that occurred in vertebrate evolution. We therefore refer to the invertebrate glycoprotein hormone subunits simply as GPA and GPB, respectively. To investigate whether the GnRH-like system in Platynereis could also be involved in the regulation of vertebrate glycoprotein hormone orthologs, we analyzed the expression levels of both gpa and gpb in sexually mature and fed premature worms, two conditions that we knew to exhibit higher crz1/gnrhl1 levels (see Fig. 2B and H). When comparing crz1-/samples with their respective wild-type counterparts, transcript levels for both gpa and gpb did not significantly differ in both of these settings (Fig. S5B-E). While these results do not support a direct hierarchical regulation between gonadotropin-like signals and glycoprotein hormone orthologs on transcript level, they also do not exclude that such a regulation could exist at the level of peptide release.

Consequences of lysosomal α-mannosidase impairments in other species
One of the molecular consequences of knocking out the Platynereis crz1/gnrhl1 gene that we reveal in this study is the strong reduction of lysosomal α-mannosidase. In humans, αmannosidase deficiency leads to α-mannosidosis, a lysosomal storage disorder which causes the accumulation of mannose-rich polysaccharides, with consequent impairment of glycoprotein turnover and cellular functions (18). No defects in growth rate have been associated with patients affected by α-mannosidosis. Yet stunted growth characterizes guinea pigs with a deficiency in lysosomal α-mannosidase activity (19). Moreover, honeybee larvae show a developmental delay in pupal metamorphosis when fed with swainsonine (20), an alkaloid causing similar effects to α-mannosidosis (21). These findings are in line with the developmental delay we observe in crz1-/-worms, which may be caused by the accumulation of unprocessed mannose-rich glycans and related impairment in glycoprotein turnover (see ref. 18). Glycans processed by lysosomes can be used as additional energy sources, and in particular mannose can be converted into fructose-6-phosphate, which fuels both gluconeogenesis and glycolysis. Moreover, mannose-6-phosphate is also crucial as targeting signal, especially for proteins directed to lysosomes.

Corazonin-and GnRH signaling systems are involved in orchestrating life-history transitions
GnRH-like peptides have been already suggested to play a role in the regulation of developmental and life-history transitions in various animal systems. As mentioned in the introduction of our study, changes in GnRH signaling activity regulate puberty onset in vertebrates, an event which, on the one hand, requires the attainment of specific metabolic conditions, and, on the other hand, involves major physiological and morphological changes.
Moreover, in the tunicate Ciona intestinalis, GnRH-like hormones promote tail absorption in larvae, one of the two major events in ascidian metamorphosis (22). Finally, CRZ regulates density-dependent polyphenism in the locusts, Schistocerca gregaria and Locusta migratoria (23), whereas in the ant, Harpegnatos saltator, a CRZ peptide controls caste transition from worker to reproductive gamergate (a queen-like state) (24). It is interesting to note that CRZ promotes the release of preecdysis-and ecdysis-triggering hormones from the Inka cells of Manduca sexta (25). In line with this, a corazonin receptor was found to be expressed in the Y-organ (the gland which represents the source of steroid hormones and regulates moulting in decapod crustaceans) in the green shore crab, Carcinus maenas (26). Our observations in the crz1/gnrhl1 mutant worms suggest that also outside arthropods, Corazonin signaling may engage in the regulation of key and particularly energy-demanding developmental transitions, potentially triggered also as a response to a physiologically-controlled metabolic stress (see (27)(28)(29)(30)).
In vertebrates, GnRH has been proposed as a key endocrine regulator of both seasonal and semi-lunar/lunar reproduction (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Yet, these notions are largely correlational, as it is hard to decouple the direct role that GnRH has in reproduction from its potential involvement as a downstream effector of seasonal and lunar timers. Indeed, outcomes of these infradian rhythms are often reproductive events requiring per se GnRH signaling (see ref. 39). The temporal separation between metamorphosis onset and spawning make the bristleworm an interesting model to distinguish between these processes. In fact, our expression data support the notion that GnRH-like signaling is more directly linked to lunar reproductive rhythmicity. In a broader perspective, our results thereby help to shed light on the still elusive endocrine basis of lunar-regulated life-histories.

Worm culture and phenotypic analysis
For maturation timing experiments, data were retrieved from the analysis of culture boxes in which worms were growing at similar densities. To assess growth rate, we first measured the length of 2-months old worms kept in comparable density conditions and fed ad libitum (algae suspension). Then, 2-months old crz1-/-and +/+ worms were isolated in boxes with the same final density, and fed normally with the same amount of food (spinach and tetramin). After 3 months, worms were anesthetized and photographed. The length was measured using ImageJ software, and segments counted. For regeneration experiments, the 15 terminal segments of the tail were amputated from age-matched ~50 segments-long premature worms, and the number of re-grown segments counted every week under the microscope (after brief anesthesia) for 4 weeks. After the amputation, animals were placed in filtered sea water with the addition of ampicillin and streptinomycin (final concentration 62.5µg/mL and 250µg/mL, respectively) for two days. For the first (acute) feeding assay, we starved 12 age-matched premature worms of each genotype for 12 days, and then individually isolated them. We fed each worm with a single circular spinach disc, and we placed some leaves in control wells with no worms. At 1 and 3 days post leave administration, we used bright field microscopy, combined with image analysis, to quantify the area of residual leaves, and thereby estimated the consumed amount of spinach (see

Proteome data analysis
For positive protein identification, as a minimum two peptides, at least one of them being unique, had to be detected. Trypsin/P was specified in the digestion mode. Peptide mass tolerance was set to 50 and 25 ppm for the first and the main search, respectively. The false discovery rate (FDR) was set to 0.01 both on peptide and protein level. Peptides were mapped against a reference proteome set established before (43). Carbamidomethylation was set as fixed modification, methionine oxidation and N-terminal acetylation as variable modifications. Each peptide was allowed to have a maximum of two missed cleavages and two modifications. "Match between runs" was enabled and the alignment and match time window set to 10 and 1 min, respectively. The analysis of the quantitative protein abundance has been performed using the Perseus software platform (44) Fig. S1. Sequence alignment of GnRH/AKH/CRZ prepropeptides from different bilaterian taxa. The magenta box highlights the region corresponding to the GnRHassociated peptide (GAP), illustrating that there is no detectable conservation of GAP across the diverse groups.      Maximum Likelihood phylogeny for metabolic enzymes investigated in this study; black arrowheads point at the respective Platynereis genes whose expression was analysed in this work (see Fig. 4). (A) Lysosomal alpha-mannosidases; (B) tobi-like/alpha-glucosidases;

Supplementary Figures
(C) phosphoenolpyruvate carboxykinases, and (D) glycogen synthases. Sequences were retrieved from the NCBI repository; color-codes were used to highlight specific subsets.