This set of pharmacological experiments on quail hypothalamus explants and homogenates and the manipulations performed with immunoprecipitated human aromatase indicated that the inhibition of AA by phosphorylation is likely to be catalyzed by the activity of two Ser/Thr kinases, protein kinase A (PKA) and C (PKC), although the implication of tyrosine kinase and other Ser/Thr kinases cannot be completely ruled out (Balthazart et al., 2003; Charlier et al., 2011). The quail and human aromatase protein sequence were analyzed using bioinformatic tools to identify the potential phosphorylation sites, highly conserved amongst several avian and mammalian species. Fifteen and nineteen putative phosphorylation sites were identified for quail and human sequences respectively using the open access NETPHOS 2.0 program. These sites included 10 of the 27 serines, 5 of the 24 threonines, and 4 of the 17 tyrosines residues on the human sequence and we paralleled these findings with the results obtained using NETPHOSK, highlighting consensus sequences corresponding to PKC and PKA, protein kinases shown to affect quail and human AA during the pharmacological experiments (Balthazart et al., 2003; Charlier et al., 2011). The output pointed to 6 different residues, five serines (S118, S167, S247, S267, S497), and one threonine (T143) on the human sequence. It should be pointed out that other kinases might also be involved, as consensus binding sequence were observed on both quail and human aromatase. These were however not investigated. From this analysis, we decided to focus our attention on 6 different residues: S247, S267, and S497 that had high scores in both the predictive phosphorylation sites and PKA or PKC recognition consensus sequence; T462 and T493 that correspond to positions S455 and S486 in quail aromatase, two 2 residues that were predicted to be involved in the phosphorylation of aromatase (Balthazart et al., 2003). In addition, in vitro studies of the mouse aromatase performed by 2 different groups suggested that 2 other amino acid residues, namely serine S118 (Miller et al., 2008) or tyrosine Y361 (Catalano et al., 2009) can be phosphorylated. Indeed, the phosphorylation of Y361 triggered by the tyrosine-kinase c-Src seems involved in the E2-dependent upregulation of AA in breast cancer cell lines (Catalano et al., 2009) while phosphorylation of S188 by AGC-like kinases, including PKC and PKA, seems important for the stabilization of the protein, and therefore for the expression of AA (Miller et al., 2008). It should be noted that the effects of the phosphorylation status of these 2 residues on the rapid change of AA was not investigated and we explored the potential implication of S188 phosphorylation (not Y361) in the rapid control of AA.

Altogether, we investigated the consequence of mutating 6 S/T residues into alanine (A) using the human aromatase as template. All mutants expressed normal baseline AA and pre-incubation with ATP/Ca/Mg for 15 min markedly reduced this enzymatic activity in the 6 different mutants alone (S118A, S247A, S267A, T462A, T493A, S497A) or in combination (S267A-S247A, T493A-T462A-S267A, T493A-T462A-S267A-S247A, S497A-T493A-T462A-S267A-S247A) roughly to the same extent as in wild type enzyme, clearly showing that these single or combined mutations did not block the rapid inhibition of aromatase by phosphorylating conditions. The absence of effect, against all expectations, was thoroughly discussed in the original article (Charlier et al., 2011). In summary, it is likely that a combination of several phosphorylated residues that was not tested here is required to control AA. The control by multiple kinases reinforces the idea that several amino acids must concomitantly be phosphorylated to modify AA. It is possible that other consensus sites for phosphorylations and for other types of kinases that were also predicted on the quail and human aromatase sequences might be involved. Alternatively, it is possible that the control of AA by phosphorylations depends on the modulation of certain factors interacting with aromatase, in addition to the enzyme itself. Aromatase requires the presence of an active NADPH-cytochrome P450 reductase for electron transfer (Thompson and Siiteri, 1974; Miller, 2005; Hong et al., 2009). The phosphorylation of the reductase could be a control mechanism affecting the electron transfer and this idea should be further investigated.

## Behavior and Rapid Modulation of Aromatase Activity

It is important to note that the change in enzymatic activity resulting from the phosphorylation of aromatase is physiologically and behaviorally relevant. Indeed, rapid changes in AA were demonstrated to occur in vivo in response to social interactions in a few species investigated to date, including birds and fish. For example, expression of appetitive or consummatory sexual behavior rapidly reduces AA in specific brain regions in the preoptic-hypothalamic area of quail after 5–30 min of sexual interaction with a receptive female (Cornil et al., 2005a; de Bournonville et al., 2013) while exposure to acute restraint stress, or stress-related hormones such as AVT and corticosterone lead to an increase in AA in both male and female quail (Dickens et al., 2011, 2013). Male removal from a socially stable group of the

involved in the rapid control of aromatase activity.

Phosphorylations (PO4 ) rapidly modulate aromatase activity, inhibiting the transformation of testosterone (T) into 17β-estradiol (E2). It is likely that these modifications are induced by calcium-voltage channels, by

glutamatergic receptors and/or by dopaminergic receptors. The increase of intracellular calcium (Ca++), either from intracellular storage or from the activation of voltage-gated channel is in most cases a prerequisite for the inhibition of aromatase activity. Change in phosphorylation level could also affect the hydroxylase activity of aromatase.

sex-changing fish Lythrypnus dalli resulted in rapid increases in aggression associated with a rapid reduction in brain AA in the dominant female (Black et al., 2005, 2011). Conversely in zebra finches, a brief exposure to song (30 min) resulted in an increase of AA in NCM within the telencephalon (Remage-Healey et al., 2009). While behavior can affect AA, the reverse is also true and recent studies showed that rapid changes in AA modulate behavior and sensory processing. Indeed, an acute systemic or central administration of aromatase inhibitors such as Vorozole or Androstatrienedione significantly and rapidly reduced male sexual behavior in quail and mice (Cornil et al., 2005b; Taziaux et al., 2007; Seredynski et al., 2013), approach behavior toward females in male goldfish (Lord et al., 2009), aggressive behavior in Beach and California mice (Trainor et al., 2007, 2008) and vocalizations in midshipman fish (Remage-Healey and Bass, 2004). Fadrozole, another aromatase inhibitor, rapidly suppressed the preference for a bird's own song in male zebra finches (Remage-Healey et al., 2010) and reduced song-elicited neuronal firing rate in zebra finches and prevented the expression of hearing-driven gene expression in NCM, a brain regions involved in song discrimination in songbirds (Tremere et al., 2009). Altogether, this clearly confirms that the rapid modulation of AA, likely by phosphorylation, is of physiological importance. This also shows that the rapid inhibition of AA rapidly influences the physiology and behavior.

## Catabolism of Estrogens

Therefore, there must exist some mechanisms that are able to rapidly clear locally-produced estrogens to terminate their effects. The half-life of E2 in the brain is not known, although calculations derived from pharmacokinetic data estimated that its halflife in the blood is below 30 min (Johnson and van Tienhoven, 1981; Tsang and Grunder, 1984). The liver is known to express several enzymes involved in the clearance of circulating estrogens, notably CYP1A1, CYP1A2, CYP1B1, and CYP3A (Whirl-Carrillo et al., 2012). These enzymes catalyze the conversion of estrogen into inactive (or less active) water-soluble metabolites by oxidative metabolism and allow for further sulfation by sulfotransferases, glucuronidation by glucoronyltransferases, or methylation by catecholmethyltransferases. The elimination of plasma estrogens from the organism is ensured through excretion following their conjugation. Although this type of degradation of estrogens occurs mainly in the liver, some catabolic activity is also observed in the brain, albeit to a much lower extent. We hypothesized that the rapid elimination of estrogens in the brain involves the enzyme aromatase itself. Indeed, a few studies suggest that, in addition to its estrogen synthase activity, aromatase also catalyzes estrogen hydroxylation. A previous study in Japanese quail showed that 2-hydroxylase enzymatic activity is present in all brain regions that are known to contain a high level of AA, including the median preoptic nucleus and the ventromedial nucleus of the hypothalamus (Balthazart et al., 1994), suggesting that the same protein is involved in both the production and the conversion of estrogens. However, no sex difference in 2-hydroxylase activity was identified here, while AA is higher in males than in females in almost every brain region. The first evidence that aromatase protein also had estrogen hydroxylase activity came from Osawa et al. (1993). They demonstrated that purified aromatase obtained from placental microsomes demonstrates an estrogen-2-hydroxylase activity in certain conditions. Both aromatase and estradiol 2-hydroxylase activities were observed after purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution. Moreover, they confirmed the concomitant existence of estradiol hydroxylase activity with AA by expressing human aromatase in CHO cells. Not only did the purified aromatase exhibit the hydroxylase activity but testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and conversely, estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Similarly, equine aromatase expressed in vitro has a clear hydroxylase activity (Almadhidi et al., 1996). A specific antiserum raised against purified testicular equine P450arom and known to inhibit AA was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes. Furthermore, the estrogen hydroxylase activity was inhibited in a dose-dependent manner by different aromatase inhibitors, including fadrozole and 4-hydroxyandrostenedione (Almadhidi et al., 1996). It remains to be determined whether the phosphorylation of the same residue(s) leads to the rapid reduction of both estrogen synthase and estrogen hydroxylase activity.

## Conclusions

Altogether, a few laboratories have accumulated strong evidence showing that AA can be rapidly modulated via posttranslational modifications, most notably via phosphorylation (see **Figure 1** for general model). The rapid modulation of AA by phosphorylating conditions is a widespread mechanism present in different tissues affecting aromatase from various species, including humans. The mechanisms leading to these modifications remain poorly understood, but it is increasingly clear that the enzymatic changes must result in a local rapid modulation of estrogens availability and consequently in a modification of cellular estrogen-dependent events that are not mediated by the genomic actions of these steroids. The phosphorylation/dephosphorylation processes provide a new widespread mechanism by which estrogens concentration could be rapidly altered in the brain and other tissues, albeit likely in different directions. It would be interesting to test whether precise phosphorylation patterns affect one type of enzymatic activity while leaving the other untouched or whether phosphorylation is a general shut-down switch for all enzymatic activity displayed by the aromatase protein.

## Acknowledgments

Some of these data were obtained thanks to the Fonds Leon Frederic (ULg, Belgium) and FNRS-FRS to TDC and grant from NIH/NIMH (R01MH50388) to GF Ball and JB. TDC was a F.R.S.-FNRS Post-doctoral Researcher. CAC is a F.R.S.-FNRS Research Associate.

## References

cyp19 gene. Proc. Natl. Acad. Sci. U.S.A. 95, 6965–6970. doi: 10.1073/pnas.95. 12.6965

Roselli, C. E., and Resko, J. A. (2001). Cytochrome P450 aromatase (CYP19) in the non-human primate brain: distribution, regulation, and functional significance. J. Steroid Biochem. Mol. Biol. 79, 247–253. doi: 10.1016/S0960-0760(01)00141-8

and plasticity-associated gene expression. J. Neurosci. 29, 5949–5963. doi: 10.1523/JNEUROSCI.0774-09.2009

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Charlier, Cornil, Patte-Mensah, Meyer, Mensah-Nyagan and Balthazart. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Farnesol-like endogenous sesquiterpenoids in vertebrates: the probable but overlooked functional "inbrome" anti-aging counterpart of juvenile hormone of insects?

### **Arnold De Loof 1\*, Elisabeth Marchal 2 , Crisalejandra Rivera-Perez 3 , Fernando G. Noriega3 and Liliane Schoofs 1**

1 Functional Genomics and Proteomics Group, Department of Biology, KU Leuven, Leuven, Belgium

2 Molecular Developmental Physiology and Signal Transduction Group, Department of Biology, KU Leuven, Leuven, Belgium

3 Department of Biological Sciences, Florida International University, Miami, FL, USA

### **Edited by:**

Maximilian Bielohuby, Ludwig-Maximilians University, Germany

### **Reviewed by:**

Andreas Hoeflich, Leibniz-Institute for Farm Animal Biology, Germany Muhammad Safder, University of Karachi, Pakistan

### **\*Correspondence:**

Arnold De Loof , Functional Genomics and Proteomics Group, Department of Biology, KU Leuven–University of Leuven, Naamsestraat 59, Leuven 3000, Belgium e-mail: arnold.deloof@bio. kuleuven.be

Literature on the question whether the juvenile stage of vertebrates is hormonally regulated is scarce. It seems to be intuitively assumed that this stage of development is automated, and does not require any specific hormone(s). Such reasoning mimics the state of affairs in insects until it was shown that surgical removal of a tiny pair of glands in the head, the corpora allata, ended larval life and initiated metamorphosis. Decades later, the responsible hormone was found and named "juvenile hormone" (JH) because when present, it makes a larva molt into another larval stage. JH is a simple ester of farnesol, a sesquiterpenoid present in all eukaryotes.Whereas vertebrates do not have an anatomical counterpart of the corpora allata, their tissues do contain farnesol-like sesquiterpenoids (FLS). Some display typical JH activity when tested in appropriate insect bioassays. Some FLS are intermediates in the biosynthetic pathway of cholesterol, a compound that insects and nematodes (=Ecdysozoa) cannot synthesize by themselves. They ingest it as a vitamin. Until a recent (2014) reexamination of the basic principle underlying insect metamorphosis, it had been completely overlooked that the Ca2+-pump (SERCA) blocker thapsigargin is a sesquiterpenoid that mimics the absence of JH in inducing apoptosis. In our opinion, being in the juvenile state is primarily controlled by endogenous FLS that participate in controlling the activity of Ca2+-ATPases in the sarco(endo)plasmic reticulum (SERCAs), not only in insects but in all eukaryotes. Understanding the control mechanisms of being in the juvenile state may boost research not only in developmental biology in general, but also in diseases that develop after the juvenile stage, e.g., Alzheimer's disease. It may also help to better understand some of the causes of obesity, a syndrome that holometabolous last larval insects severely suffer from, and for which they found a very drastic but efficient solution, namely metamorphosis.

**Keywords: farnesol, insect hormones, sex steroids, development, puberty, Alzheimer, Caenorhabditis elegans, obesity**

### **INTRODUCTION**

All animals, plants and fungi pass through a juvenile stage before becoming reproductively active and next becoming aged. In holometabolous insects a high titer of juvenile hormone (JH), an ester of the endogenous sesquiterpenoid alcohol farnesol (**Figure 1**) keeps larvae in their juvenile state. JH can occur is several isoforms1 (1). It makes a larva molt into another larval stage. This is known as the "*status quo* effect" of JH. When the JH titer drops to zero at the end of larval life, metamorphosis is initiated. When metamorphosis nears completion, the JH titer rises again and the enclosing adult enters or completes (if the gonads started developing already during metamorphosis) the reproductive phase, and concurrently ages rapidly.

Hitherto, it was assumed that vertebrates do not have such a juvenilizing hormone, or, if they do have one, thyroid hormones, in particular triiodothyronine (T3) is likely candidates. Like the absence of JH has a key role in initiating metamorphosis in all holometabolous insects, the presence of T3 has a prominent role in initiating metamorphosis in some fish and amphibian species (3). Remarkably T3 has been shown to be active in some insect bioassays (4, 5). However, functional evidence supporting this view remains unconvincing (2). In particular the lack of a suitable bioassay(s) which is indispensable for monitoring the successive steps in the purification procedure represents the bottleneck for identifying the JH of vertebrates, if it exists. As long as this situation continues, one has no choice but exploring the possibilities of comparative endocrinology between vertebrates and invertebrates for finding an alternative strategy for unraveling the control mechanisms for initiating and maintaining the juvenile state in vertebrates.

1http://en.wikipedia.org/wiki/Juvenile\_hormone

The leading idea in this paper is that being in the juvenile state as an animal, follows from controlling principles that were already present in the common ancestor of plants, fungi, protostomian, and deuterostomian animals. Hence the signaling pathways involved are probably largely over a billion years old. It is therefore more likely that their key components have been well conserved in evolution, rather than that deuterostomes (to which vertebrates belong) "all of a sudden"invented a completely new strategy for generating and temporarily maintaining a juvenile state. It is well-known that insects, which are protostomes, use JH as their juvenilizing agent. Hence, the question is: could it be that in vertebrates, and by extension in all deuterostomes, the basic elements of this endogenous sesquiterpenoid system continue to be present and operate in a similar way, perhaps in a slightly modified form?

In this concept paper, we will argue that this is probably the case. We will also outline that research into aging-related diseases may benefit from the novel insights.

### **WORDING IN SHORT OF THE PARADIGM**

Both deuterostomes and protostomes must have inherited the basic principles of passing through a juvenile state before becoming sexually mature from their common ancestor. These principles are probably still operational in both. Insects, in particular holometabolous ones which in this respect are best studied, use JHs as their key hormonefor realizing and maintaining the juvenile state. The Ca2+-ATPase present in the sarco(endo)plasmic reticulum (SERCA) likely functions as the largely overlooked membrane receptor for JH and for at least some other endogenous farnesollike sesquiterpenoids (FLS). No functional hormonal counterpart of JH has yet been identified in any vertebrate. Like all eukaryotes, vertebrates do have farnesol and some other FLS, but their role is only partially understood. We think that being a juvenile vertebrate depends upon the interaction of FLS with the Ca2+ homeostasis system, like this is the case in insects. However, the key difference with insects is that in vertebrates farnesol/FLS is probably not a hormone. Instead, its precursor farnesyl-pyrophosphate (FPP) is present and probably synthesized in all cells of the body as one of the precursors in the cholesterol biosynthetic pathway. Because insects and nematodes either lost or never had the gene coding for the enzyme squalene synthase that converts squalene (that is formed from FPP) into cholesterol, they cannot make cholesterol by themselves. They solved this problem by ingesting cholesterol as a vitamin and by using a farnesol ester, namely JH as a circulating hormone to control at least one of the components of the Ca2+-homeostasis system, namely the SERCA-pump system.

### **CONSTRUCTING THE PARADIGM**

### **HOW IS THE JUVENILE STATE DEFINED?**

A juvenile is an individual organism that has not yet reached its adultform, sexual maturity or size. In particular in holometabolous insects, the juvenile state corresponds to all larval stages before metamorphosis is initiated. In amniote vertebrates, the embryo represents the larval stage, and "juvenile" applies to the time between hatching/birth and reaching maturity.

Animals in which the transition from larva/juvenile to sexually mature adult takes only days (most insects) are better experimental models for studying the principles of being a juvenile than the ones in which this transition takes months to even years.

### **Extracts of mammalian tissues do contain "JH bioactivity"**

In the pioneering decades (1960–1980) following the identification of the chemical nature of the two non-peptidergic key insect hormones, JH, and the steroid molting hormone ecdysone, various tissues from insects but also from other species, including mammals, were analyzed for the presence of compounds with JHlike activity in specific insect bioassays. Some remarkable results were obtained. In insects the most unexpected one was that not the *corpora allata*, tiny glands in the head thought to be the only site of JH synthesis, were the richest source of JH, but the adult male accessory glands of the moth *Hyalophora cecropia*, an important experimental model at that time. The huge JH concentrations present in these glands which are the physiological counterpart of the prostate gland of mammals enabled the purification of JH. It turned out to be an ester of farnesol. At that time, FPP was already known as a precursor of cholesterol in vertebrates. The fact that insects cannot synthesize cholesterol by themselves made researchers logically conclude that farnesol/FLS in insects only served as the precursor for JH. A second remarkable result was that Williams et al. (6) showed that extracts from several mammalian tissues (thymus, human placenta, and others) displayed activity when tested in typical bioassays for insect JH.

Analysis of the chemical nature of the JH-bioactive substance in extracts of mammalian tissues showed that the active factor was farnesol, the same as the active factor present in excrements of the mealworm *Tenebrio molitor* and in yeast (7, 8). Thus vertebrates do have farnesol, and this farnesol, along with other compounds, some of them being farnesol-like (9) is active in insect JH-bioassays. Yet, although this finding was intriguing, it was not further analyzed in depth because it seemed normal that a direct precursor of JH, of which the chemical identity was elucidated by Röller and Dahm (10), could also have moderate bioactivity. The data got forgotten because it was deemed irrelevant for vertebrate physiology. Moreover, one should keep in mind that in those days, the idea that the endocrine systems of vertebrates and invertebrates might have some elements in common, was far from readily accepted. To date, farnesol is best known in the perfume industry where it is used as an adjuvant. It was named after the Farnese acacia tree (*Vachellia farnesiana*) of which the flowers are a rich source of farnesol. Its exact role in plants remains to be elucidated.

### **LITTLE PROGRESS IN THE PAST 45 YEARS**

To date, we know that the basic principles of the endocrine systems of both vertebrates and insects are very similar, probably because they were already present in their common ancestor. We also

have a good idea which mutations resulted in differences in their steroids, endogenous sesquiterpenoids, and peptide hormones (11, 12). Current vertebrates no longer have the genes/enzymes needed for synthesizing ecdysteroids which are probably more "ancient" than the vertebrate-type steroids. On the other hand, insects either never had (2, 13) or lost the enzymes needed for biosynthesizing most "modern vertebrate-type steroids". Yet, some such "vertebrate-type" steroids like pregnenolone, testosterone, eestradiol, etc. are present in some insect species, but their function, if any, has not yet been found (14, 15). The biosynthetic pathway of FLS is well known (16) (**Figure 1**). However, the exact mode of action of JH is still a matter of discussion (2, 17–19).

### **NEVER TOTAL ABSENCE OF FARNESOL IN THE LIFE CYCLE OF VERTEBRATES**

In insects there is a natural condition in which JH is totally absent, namely during metamorphosis of holometabolous insects. When synthetic JH is applied before metamorphosis is initiated, larval life is usually extended. Depending upon the species, one or more extra larval instars can be formed, proving that JH is indeed a hormone that maintains the juvenile state.

In vertebrates, the situation"zerofarnesol/FLS,"a necessary tool for defining the role of farnesol is never realized during lifetime. This is due to the fact that unlike in insects where – in larvae – farnesol and JH are only synthesized in the *corpora allata*, FLS are present in all cells of the vertebrate body (**Table 1**, see later). This is probably the case throughout all developmental stages.

In vertebrate physiology, the question whether, perhaps, farnesol/FLS may have a function on its own, additional to that of FPP in prenylation and in serving as a precursor of cholesterol has not (yet) been an issue.

### **TOTAL ABSENCE OF JH IN INSECTS MIMICS SOME OF THE EFFECTS OF THE SERCA-PUMP BLOCKER THAPSIGARGIN**

What happens in insects when the JH/farnesol titer drops to zero? In holometabolous insects total absence of the sesquiterpenoid JH

**Table 1 | Occurrence of farnesol, its precursor farnesyl pyrophosphate (FPP) and its derivatives, farnesal and farnesoic acid in a variety of tissues of a male mouse (Mus musculus)**.

Metabolites were extracted from about 20 mg of tissue and 5µl of blood. FPP, farnesyl pyrophosphate; FA, farnesoic acid.

Meaning of other symbols: compared to values present the corpora allata of insects, the best studied tissue in this respect: −: close to the detection limit; +: relatively high concentrations; +: moderate concentrations.

is a regular part of the life cycle. Indeed, the *corpora allata*, the only site of farnesol and JH synthesis in juvenile insects become completely inactive at the end of larval life. Furthermore, all JH that still circulates in the hemolymph is also fully degraded by specific esterases. This results in the zero JH/FLS situation. Exactly this absence of JH rather than the release of the prothoracicotropic hormone (13) initiates metamorphosis. The most dramatic aspect of metamorphosis is the programed cell death/apoptosis of the tissues that actively secrete proteins such as the fat body, the alimentary canal, the salivary- and prothoracic glands, etc. (2).

What happens in vertebrates when an endogenous FLS can no longer exert its normal function becomes apparent from the effects of administration of the plant toxin thapsigargin, which is, like farnesol/JH also a sesquiterpenoid. Thapsigargin blocks the SER-CAs of both vertebrates and insects. This indicates that not only the overall structure of SERCAs has been very well conserved in evolution, but that the binding site of thapsigargin on the SERCAs has also been conserved. De Loof (20) argued that this binding site of sesquiterpenoids on the endoplasmic reticulum functions as a third type/family of receptors that complements the much better documented plasma membrane receptors and nuclear receptors for (some types) of hormonal ligands.

In thapsigargin-sensitive cells both the absence of JH as well as the administration of thapsigargin make the SERCAS stop pumping Ca2+ into the lumen of the smooth- and rough endoplasmic reticulum (SER/RER). As a result the Ca2+-concentration in the cytosol increases up to the level that Ca2+-induced apoptosis (21) is induced.

### **WHY DID NEMATODES, IN PARTICULAR CAENORHABDITIS ELEGANS, RETAIN THE GENES FOR SYNTHESIZING FARNESOL DURING HUNDREDS OF MILLIONS YEARS?**

Like in insects, the enzyme squalene synthase which is essential for the biosynthesis of cholesterol is absent in nematodes. Either these Ecdysozoa lost the gene or they never had it (20). This situation probably dates from before the divergence of deuterostomian and protostomian animals, or, perhaps from even before the divergence of plants, fungi, and animals, thus from at least one billion years ago. Yet, nematodes still express the genes coding for the enzymes that are instrumental to the biosynthesis of farnesol, its precursors and some metabolites, but not of cholesterol (22, 23). The fact that these genes have been conserved for so long indicates that farnesol/FLS must have a function different from that of merely serving a role in the biosynthesis of cholesterol; otherwise the genes would have become redundant. In worm research, this question triggered some interest, but for lack of performing quantification methods of FLS in small samples, a conclusive answer has not yet been given. What could that missing function be? The comparative approach with insects may provide the answer.

We performed an orienting experiment with the method of Rivera-Perez et al. (24) which will be outlined next. The results were that all FLS listed in **Table 1** are present in juvenile and adult *C. elegans*. In due time, they will be published elsewhere.

### **A NOVEL, HPLC-BASED METHOD FOR QUANTIFYING FARNESOL AND ITS PRECURSORS HAS RECENTLY BEEN DESCRIBED**

The ancient data from Williams et al. (6) and of Schmialek (7) on the presence of farnesol in a few tissues of mammals did not give any indication on its origin, either from the ingested food or from an endogenous site of synthesis. All cells of the animal body need cholesterol. A commonly held view (25) is that the few major production sites of cholesterol, namely the liver and the intestine, secrete enough cholesterol into the bloodstream to comfort all peripheral tissues. In this scenario, one should not find the typical precursors and some metabolites of farnesol in these peripheral tissues. The other possibility is that all tissues express the genes for biosynthesizing farnesol, its precursors and some derivatives, not so much because they would not receive sufficient amounts of cholesterol from the blood and thus to compensate for such theoretical insufficiency, but because they all need farnesol/FLS for another reason than for using it for cholesterol synthesis.

Sesquiterpenoids are notoriously difficult to quantify accurately and robustly because of their lipophilic and labile nature and their tendency to bind non-specifically. Mass spectrometry approaches could be accurate, but are expensive and complex to interpret (1, 24).

Fortunately, a novel, very sensitive method developed by Rivera-Perez et al. (24) allows analyzing the whole biosynthetic pathway, from mevalonate up to farnesol and some derivatives (further to JH if needed) at once. The assay is based on the derivatization of analytes with fluorescent tags, with subsequent analysis by reverse phase HPLC coupled to a fluorescent detector (HPLC-FD).

### **PRESENCE OF FRS IN MAMMALIAN TISSUES**

To verify if the old data of Williams et al. (6) are correct, we performed an orienting experiment with the Rivera-Perez et al. (24) method using extracts of tissues of a male mouse (*Mus musculus*). Although the method is able to generate both qualitative and quantitative data, we have limited ourselves to the qualitative ones. The reason is that the imposed deadline for submission of this paper did not allow generating a sufficient number of replicate experiments for an accurate quantification of all intermediates. Nevertheless, the results were convincing enough to state that all analyzed tissues of a male adult mouse seem to have different metabolites in the FLS pathway, and by extrapolation the different necessary enzymes for biosynthesizing several FLS, as shown in **Table 1**.

Contrary to what could be expected, FPP the direct precursor in the biosynthesis of squalene is only present in high concentrations in brain tissue and less in tissues known to be actively synthesizing cholesterol. FPP is also important for prenylation. The presence of FPP, farnesol, farnesal, and farnesoic acid in blood indicates that a role as a hormone may not *a priori* be ruled out, on condition that none originates from the food. In the literature there are no data suggesting a hormonal role. The high concentrations of farnesoic acid are intriguing. Farnesol and farnesal are quite toxic molecules in living cells. Rizzo and Craft (26) who studied the Sjögren–Larsson syndrome, demonstrated that conversion of farnesal to farnesol by an aldoketo reductase reduces the toxicity. The produced farnesol leaks out of the cells. The same effect was observed in insects in the JH pathway; the excess of farnesal in the *corpora allata* is converted back to farnesol that can leak out of the gland (27).

These preliminary results urge for an in depth qualitative and quantitative study of the farnesol/FLS biosynthetic pathway in all tissues of the body throughout development.

### **INBROME SIGNALING VERSUS A ROLE AS A HORMONE**

The term "inbrome" stands for "intramembrane signaling substance." It was introduced by De Loof et al. (2) to denote and categorize chemicals that act in the plane of membranes by binding to receptors that are located in membranes, in particular in intracellular membranes. A typical example is the plant toxin thapsigargin that binds to the Ca2+-ATPases located in the sarco(endo)plasmic reticulum.

In the past neitherfarnesol nor any other FLS has been advanced as a hormone of vertebrates. The fact that all tissues seem to have the biosynthetic machinery for some FLS, suggests that if these compounds do have a role in controlling some cell-physiological process, e.g.,Ca2+-homeostasis they can do so from within the cell, without the need of having to be secreted into the blood stream first. The fact that single-celled organisms, like yeast, produce sesquiterpenoids, including farnesol, via the mevalonate pathway (28) supports this view. The "inbrome model" may explain some aspects of the mode of action of lipophilic hormones, in particular of sex steroids and JH. There is a major problem with the widely accepted Karlson model of steroid hormone action (29). It says that a steroid (ecdysone in Karlson's experiments) diffuses right through the plasma membrane "unnoticed" (=without changing the permeability or electrical properties of the plasma membrane), next through the cytoplasm where it binds to a cytoplasmic receptor (=a later finding), and that finally this complex passes the nuclear pore complexes into the nucleus and binds there to the promoter regions of specific genes. Very few researchers are aware of the fact that this model was not at all readily accepted at the time it was launched. To the contrary, it was heavily disputed for good reasons. The most convincing counterargument was that the so called "specific" hormone-induced puffs could as well be induced in the absence of any hormone by changing the ionic concentration of some inorganic ions in medium in which the salivary glands were incubated (2, 30).

Karlson's model is too simple. It is much more probable that once a lipophilic signaling molecule enters a membrane, it can cause electrical changes at the level of the plasma membrane, and even more important, it will rapidly diffuse throughout the whole continuum of all interconnected intracellular membranes. Because the plasma membrane forms a continuum with the endoplasmic reticulum and the outer part of the nuclear envelope, this means that lipophilic signaling molecules are omnipresent in membranes. For figures illustrating this chain of events, see De Loof (20) and De Loof et al. (2). The action of steroid hormonereceptor complexes inside the nucleus, which has been thoroughly studied in the past decades, is only one of the aspects of steroid hormone action, not at all the full story.

### **THE JUVENILE STATE ENDS AT PUBERTY: WHY AND HOW?**

At puberty the juvenile state transits into the reproductive state. In vertebrates, increased GnRH secretion causes increased synthesis and release of gonadotropins. In endocrinology the general consensus is that the gonadotropins LH and FSH "stimulate"

the production of sex-related steroids (progesterone, estrogens, androgens, etc.). However, another interpretation is possible. De Loof et al. (13) stated that the appearance of peak concentrations of ecdysteroids in the hemolymph of metamorphosing and prediapausing insects coincides with the programed cell death of several tissues, in particular those that actively secrete proteins during larval life. Therefore, the appearance of such steroid hormone peaks indicates that, somewhere in the body, some tissue(s) is undergoing apoptosis, e.g., the fat body of insects or the follicle cell layer surrounding growing oocytes. According to this view the primordial role of the gonadotropins FSH and LH is not the "stimulate" the ovarian follicle cells to engage in producing steroid sex hormones, but to force them to undergo apoptosis. This has also been suggested before in our concept that reproduction is in fact a strategy of the cellular defense system (31). Later, De Loof et al. (13) evaluated whether a similar mechanism with a key role for apoptosis might be operational in the context of the onset of insect metamorphosis which marks the end of the juvenile state.

From"puberty"on, egg production starts. Eggs accumulate yolk proteins, lipids, glycogen, and last but not least huge amounts of Ca2+. The liver of vertebrates and its counterpart in insects, the fat body, secrete lots of yolk protein precursors, vitellogenins, into the bloodstream under the influence of female sex steroids. Any protein secretion through the RER–Golgi system is accompanied by the secretion of Ca2+ (20). A typical example is the production of milk in mammals. The Ca2+-concentration in milk amounts to about 50 mM. This should be compared to the Ca2+ concentration in the cytosol of unstimulated cells in general, which is as low as 100 nM. The onset of puberty therefore involves a drastic change in Ca2+-homeostasis, in particular in females. These changes have to be controlled one way or another. The FLS-Ca2+ homeostasis system is a primordial candidate to play a role in this process.

### **THE POSSIBILITY THAT SOME SERCA PUMP ISOFORMS ACT AS PROMISCUOUS RECEPTORS**

In endocrinology, the consensus is that a ligand should bind with high affinity and specificity to a receptor. This holds true for plasma membrane receptors of the G-protein coupled type (GPCRs), and probably for intranuclear receptors as well as. We doubt that it also applies to lipophilic steroids and other lipophilic signaling substances like JH.

The question is raised whether the end of the juvenile state is caused by increasing concentrations of steroid hormones in the intracellular membrane continuum of cells that compete with FLS for binding to the receptor site on (some isoforms of) the SER-CAs? In other words, does the endocrinological explanation for ending the juvenile state reside in the fact that the receptor site for farnesol/FLS on the SERCAs is (very?) promiscuous?

A major argument in favor of this view is that about 4,200 compounds have been synthesized or extracted from natural sources that are all active in bioassays for JH [=Juvenile Hormone Analogs or JHAs; (8)]. Some are farnesol-like; others have very different chemical structures. The most bioactive ones are synthetic peptides that are selectively active in insect species belonging to the Pyrrhocoridae. There are also anti-juvenile compounds (32), e.g., some benzodioxoles that act as non-mutagenic insect chemosterilants. It is improbable that all these compounds would bind to a different nuclear receptor but nevertheless generate similar morphological and physiological effects. The reason is that the number of nuclear receptors is estimated to be around 1,000. We think that it is more probable that many/most JHAs and sex steroids bind with a different affinity to a promiscuous receptor residing in the SER/RER. For other arguments, both in favor and against this view see De Loof (20).

Males and females do not have different sex steroids. In vertebrates both have estrogens and androgens. In insects both have 20-OH-ecdysone (=estrogen counterpart) and ecdysone (=testosterone counterpart) (20, 33). This raises the question which signaling system can handle a balance of hormones? We think that a promiscuous receptor can do so, although other possibilities cannot be excluded (20). Such a system takes into account not only the affinity of the ligand but the concentration of other competing ligands as well. A physiological effect is generated when the balance is "right."

### **SEVERE OBESITY IN INSECTS NEAR THE END OF THE JUVENILE STATE: A CAUSAL LINK WITH FLS/Ca2**+**-HOMEOSTASIS?**

The juvenile state is characterized by fast growth. This requires that tissues deposit more proteins than lipids. At the end of the end of the juvenile state, this situation is reversed in most species. Storing more lipids is often considered as beneficial "because accumulating nutrient reserves will facilitate the growth of reproductive organs and production of gametes, etc." This reasoning is incorrect because it is teleological. Individual tissues, such as adipose tissue, do not plan for the future at all. They do not deposit lipids and glycogen because that would be beneficial for the fitness of the whole organism, but because a signal inside the adipose tissue is causal to the increased lipid production and accumulation. The raises the question as to the nature of that signal. Systems in which such drastic accumulation occurs in a short time are good experimental models, e.g., during pre-metamorphosis and during pre-diapause in insects.

The literature on obesity in vertebrates, in particular in humans is truly vast. Indeed, a Pubmed search with "obesity" as query (05/10/2014) yielded 205,506 references. Yet, one cannot escape the impression that, despite the enormous research effort, there is still no consensus about the primordial cause(s).Maybe something has been overlooked. Could it be FLS/Ca2+-homeostasis?

The query "obesity and calcium" yielded 3,155 references while "obesity and farnesol" resulted in only 3 refs of which one was quite interesting. Duncan and Archer (34) administered farnesol to rats. They found that oral administration lowers serum triglyceride levels. They think that the effect is mediated by the downregulation of retinoid X receptor beta. These authors assumed that farnesol, as a dietary component, could play a role in lipogenesis and fatty acid oxidation, both impaired in obesity. The possibility that farnesol/FLS are secreted into the bloodstream from tissues that produce large amounts of farnesol/FLS and cholesterol was not considered. Our data in **Table 1** show that one has to consider the possibility that FLS present in various tissues and in blood are synthesized by the body itself.

### **METAMORPHOSIS**

We think that the FLS-lipogenesis connection deserves an in depth analysis. For this view,we focus on the situation in holometabolous insects like flies, beetles, butterflies, moths, etc. They differ from hemimetabolous ones like cockroaches, crickets, etc. in that they undergo a complete metamorphosis. The larval stage molts into a pupa and next into an adult. Prior to entering the pupal stage, the pre-metamorphic larvae undergo drastic changes. One in particular is that the penultimate and early last larval instars eat voraciously. A result is that their fat body accumulates huge amounts of lipids, glycogen, and some proteins as well with a fast gain in weight as a result. This eating comes to a complete stop during the last larval instar. Next they enter the wandering stage during which they leave the food in search for a suitable place to molt into a pupa. They empty their gut, by vomiting and/or defecation. In some species like, e.g., silk moths in which the silk glands are modified salivary glands, or *Drosophila* that uses secretions from their salivary glands as a glue to adhere to a substrate, these parts of the alimentary canal are also emptied. The larvae will become more and more immobile: they give the impression to enter in a coma-like state which lasts for some time. Meanwhile, the body is drastically reshaped. One of the changes is that the fat body is almost completely lysed. Like most other tissues that actively secreted proteins during the larval stage (2), they are replaced by adult-type tissues. Also the pupal cuticle is replaced by an adult one. At eclosion, a drastically "novel" organism makes its entrance into the world. Its larval morbid obesity is completely cured. Concurrently, the alimentary canal of the adult is drastically reshaped. Adults can still can ingest food (not the case anymore in some species that do no longer have mouthparts), but the food they can handle can be totally different from what they ate as larvae (e.g., in flies and in butterflies and moths; some beetle species do not change their diet). One could say that they underwent a severe but efficient bypass of the stomach in combination with adaptation to a novel diet.

For physiologists and endocrinologists, the interesting question is: What causes this larval obesity and how is it cured? The key endocrinological event/change that takes place at the end of larval life is that the production of JH by the CA comes to a complete standstill. Also, all JH that circulates in the hemolymph is completely metabolized by specific esterases. Therefore, it is likely that an important cause of aggravating obesity is the drop to zero of the JH titer. This is evidenced by the fact that application of synthetic exogenous JH can overrule the morbid obesity syndrome. This treatment induces, in some species at least, a larval molt into a supernumerary instar (8).

### **PREPARING FOR DIAPAUSE**

Accumulation of lipids in the fat body in the last larval instar is not just a matter of more lipids that are deposited. In the absence of JH, the whole physiology of the fat body changes. This has been shown long ago by De Loof and Lagasse (35). They studied the changes in ultrastructure of the fat body of adult Colorado potato beetles raised in two different photo-regimes. Beetles raised in long day conditions (more than 12 hr light per day) will reproduce. Their fat body cells are rich in RER (for the production of yolk protein precursors). Not much lipids or glycogen accumulate and no protein vesicles. The whole fat body volume is small. At the height of vitellogenesis, it is so small that it is difficult to imagine that it could account for all yolk protein synthesis needed for the large number of eggs (up to about 50–60 per day) the females produce.

When raised in short day conditions (less than 12 hr light per day), reproduction is inhibited and the animals prepare for entering diapause.  Not only lipids and glycogen accumulate but also large amounts of proteins, packed in vesicles. Short day conditions inactivate the *corpora allata*. Thus, it is the absence of JH that causes all these changes. Similar changes take place in the fat body of last instar beetle larvae, a stage in which JH is also absent (unpublished results).

With the insights gained in unraveling the mode of action of absence of JH in pre-metamorphosis (2) the cell biological explanation for the cited effects is probably as follows. As already cited repeatedly, SERCA pumps have a binding site for sesquiterpenoids. As long as the JH titer is high, the SERCA pumps transport Ca2+ into the lumen of the RER which causes a secretion of proteins along with Ca2+ through the RER–Golgi system. The SERCAs in the SER also pump Ca2+ into the lumen of the SER. It is a general rule that SER membranes harbor enzymes for lipid- and steroid biosynthesis (25). As long as the [Ca2+] in the SERs lumen is high, these enzymes seem to be inhibited. The result is that no extreme accumulation of lipids in the fat body will occur. When the JH titer drops to zero, Ca2+ is no longer pumped into the lumina of both RER and SER. The classical Ca2+-stores start releasing part of their stored Ca2+. The RER can no longer secrete proteins out of the cell. They remain inside the cell in the form of protein vesicles. In the SER the inhibition of enzymes active in lipogenesis and ecdysteroid synthesis is lifted, resulting in the accumulation of lipid droplets in the fat body and a rising ecdysteroid titer in the hemolymph. All this probably corresponds to early events in the apoptosis pathway.

Thus, as long as the JH titer is high, the apoptosis pathway is inhibited. When the JH titer drops, the apoptosis pathway is induced, resulting in the temporary benefit that willy-nilly reserves are stored that will get a secondary role, beneficial at the organismal level, for remodeling tissues during metamorphosis (13).

We think that the initial steps, namely the roles of a high JH titer and of high farnesol concentrations are probably very similar in inducing obesity in both insects and vertebrates. Morbid lipid deposition in obese humans may reflect some malfunctioning of the FLS/Ca2+-ATPase system. A problem is that in vertebrates a situation, in which all FLS can be made totally absent, like this is the case in pre-metamorphosing holometabolous insects, never occurs in life. Whether it can be realized by RNAi is not known. In our opinion, the link FLS-Ca2+-obesity deservesfurther exploration.

### **PERSPECTIVES FOR FUTURE RESEARCH**

The outline of a practical method for analyzing the biosynthetic pathway of FLS (24), in combination with the awareness that this pathway is highly conserved in all eukaryotes, from yeast to mammals, arthropods, and nematodes, opens quite some perspectives for innovative research. It urges for evaluating whether, perhaps this signaling pathway with a causal links to the universal Ca2+ homeostasis system might play a key role in maintaining the juvenile state in vertebrates. We hypothesize that the comparison of the pathway in various tissues during successive developmental stages may yield indications whether changes might be causal to state change. Furthermore, a renewed interest in endogenous sesquiterpenoids could, perhaps, contribute to solving some standing problems in medicine.

1. The Hutchinson–Gilford progeria premature aging syndrome has a link to FLS, but its exact mode of action is only partially understood. This type of progeria is a lamin disease, a disease of the nuclear envelope. The cause of this genetic disorder is known. The gene LMNA encodes a protein named prelamin A. Through a process known as prenylation which is not only active in this syndrome, a farnesyl group gets attached to the carboxy-terminus of prelamin A. The farnesyl group allows prelamin to temporarily attach to the nuclear rim. Once the protein is attached, the farnesyl group is removed in normal persons/cells, while in progeria patients it remains attached. The result is that the no longer-farnesylated prelamin, which is now called lamin, does not remain anchored to the nuclear rim. The nucleus displays a normal shape. Failure to remove the farnesyl group permanently affixes the abnormal protein, now called progerin to the nuclear rim. This results in an abnormal shape of the nucleus2 (36) (**Figure 1**).

It remains unclear how an abnormal shape of the nucleus can cause accelerated aging. There are numerous theories on the causes of aging; for a concise summary see De Loof et al. (19). But if the commonly held view that the nucleus is not an ionically isolated compartment is correct, the form of the nuclear envelope should not matter much. However, if the nucleus has the necessary tools to create its own nucleus-specific environment, a largely overlooked additional level of control of gene expression by inorganic ions (37) emerges. Although the published experimental data are scarce, there is now enough evidence to conclude that the nuclear envelope harbors ion pumps and channels to create a specific intranuclear environment (38–40). The discovery of Ca2+-sensitive transcription factors (41) is one of the arguments in favor of the view that the nucleus has its own specific Ca2+-homeostasis system (20).

2http://en.wikipedia.org/wiki/Progeria

a better understanding of what can go wrong after the juvenile state ends.

4. Ca2+-Homeostasis and the skeleton.

In juveniles, the skeleton of girls is on the average lighter than that of boys. Which elements of the Ca2+-homeostasis system are involved and what exactly the role of sex steroids is, remains poorly understood. Osteoporosis, a multifaceted syndrome can become problematic in later life, in particular (but not exclusively) in postmenopausal women. Apparently the system that makes that females extrude more Ca2+ from their body than males (20) is already operational from very early in development on until very late in life. Whether manipulating the mevalonate-farnesol pathway may reduce the Ca2+-loss may be worth investigating.

### **FINAL CONCLUSION**

The evolutionarily ancient and very well conserved biosynthetic pathway leading from mevalonate to farnesol and derivatives is well documented in insects, but in vertebrate endocrinology and physiology hardly any attention has been paid to it. Yet, we think that not only in insects but in vertebrates as well endogenous FLS play a key role in many aspects of development and physiology. Our ideas can be summarized in the – admittedly simplistic – oneliner: "Farnesol/FLS help to smell well, to keep [Ca2+]i low and to stay young and slim."

## **DISCUSSION**

Time has come to abandon the commonly held view that in vertebrates the juvenile state is automated, meaning that it is purely genetically controlled and that, unlike in insects no specific functional counterpart of a"JH-type signaling substance"is needed. All physiological processes and states of all organisms are controlled one way or another, not necessarily all by hormones.

The theory of evolution states that all contemporary organisms are the progeny of LUCA, the Last Universal Common Ancestor. One should not too readily assume that LUCA was a primitive organism. The environment in which LUCA lived was most probably hostile in several ways. The concentration of Ca2+ in the watery environment had probably risen far above the concentration that can hardly be tolerated in the cytosol of contemporary cells, namely about 100 nM. Above that threshold, Ca2+ starts changing the conformation and activity of some macromolecules, in particular proteins and chromatin. In other words it becomes toxic. Exactly this property makes that it can act as a secondary messenger in cells, on condition that its concentration rise does not last long. When the rise is more substantial and lasts longer, excess Ca2+ can become so toxic that the apoptosis-programed cell death pathway is induced (21). This could be worded as "the Ca2+ paradox: intracellularly toxic at low concentrations, but at the organismal level often beneficial at very high concentrations."This implies that Ca2+-homeostasis was already of outmost importance a couple of billion years ago. At least in some species, its underlying mechanisms must have been shaped to near perfection long ago; otherwise "life" would have become extinct. The fact that the structure of Ca2+-ATPases of plants, insects, mammals, etc. resembles each other so very well, indicates that the Ca2+-ATPases they inherited from LUCA were already shaped to

near perfection, and could not undergo substantial changes without becoming less efficient. Another implication is that the way in which the Ca2+-homeostasis system is regulated must be very well conserved as well.

All this may sound very logical but it raises the question: What does Ca2+-homeostasis and the way it is controlled have to do with being in the juvenile state? It took a touch of serendipity to find the link. It was first found in insects (20), in the following way. Already in Kope´c (43), a Polish biologist and pioneer in insect endocrinology, described that the brain of the moth *Lymantria dispar* is needed for normal development (43). Later, it was found that a pair of tiny glands, the *corpora allata*, which form part of the whole "brain complex," secrete the hormone "JH."When these glands are extirpated, precocious metamorphosis is initiated. As cited before, JH turned out to be a simple ester(s) of farnesol. The exact mode of action of JH remained ambiguous until very recently when De Loof et al. (2) re-analyzed the published data. They observed that it had been overlooked that in holometabolous insects the drop to zero of JH titer caused apoptosis in those tissues that had been actively secreting proteins during larval life. The calcium-induced apoptosis paradigm of Orrenius et al. (21) gave the hint that, perhaps, the presence of a high JH titer is required to keep the [Ca2+] in the cytosol low. In other words, the juvenile state of insects requires that [Ca2+]i is kept low, therefore that the Ca2+-pumps keep pumping. The lucky touch of serendipity came when De Loof et al. (2) started searching for known blockers of the Ca2+-pump. Evidently, the well-known SERCA blocker thapsigargin showed up. A search for the chemical structure of thapsigargin showed that thapsigargin is, like farnesol and JH, a sesquiterpenoid. Because administration of thapsigargin induces apoptosis in both vertebrates and invertebrates, the conclusion was reached that at least the SERCA pump has a receptor site for sesquiterpenoids. Thus this type of Ca2+- pump (to our knowledge no data on PMCAs in the literature) is subject to regulation by endogenous sesquiterpenoids. If the receptor site is promiscuous as suggested by De Loof (20), other lipophilic signaling molecules may compete, in a concentration- and affinity-dependent way in binding to this overlooked type of receptor.

The next step was to evaluate whether the basics of the system that insects use to maintain their juvenile state might also apply to vertebrates. The rationale of this idea was that the control mechanisms for being in the juvenile state have probably been very well conserved since LUCA. This is apparent from the low degree of variability in Ca2+-homeostasis systems among plants, vertebrates, invertebrates, etc. The major differences in the role of a FLS among animals are twofold. In insects and nematodes the JH/FLS reaches all cells of the body as a hormone, and, in insects at least its titer in the hemolymph can fall to zero prior to metamorphosis. In vertebrates FLS are synthesized in all cells and throughout life. This corresponds to a mode of action as an "inbrome." The final effect on Ca2+-homeostasis is probably very similar in all eukaryotes.

The concept we outlined here introduces a novel way of thinking, not only on how being a juvenile is regulated, but also on quite some other aspects of evo-devo. It emphasizes that in developmental processes researchers should leave more room for the controlling role of particular inorganic ions, especially Ca2+, as well as to the electrical aspects of cell physiology (37, 44, 45). These have come into tribulation by the enormous success of molecular biological techniques that gradually stressed ever more genes and transcription factors.

We are very well aware that our concept is still in the hypothesis and theory phase, and that a lot of additional experimental work is needed before our concept will gain "more body." The major breakthrough will probably come when the binding site of thapsigargin on the SERCA pump will be chemically defined. The next step would then be to determine the degree of promiscuity of that largely overlooked receptor site.

The advent of the novel method of Rivera-Perez et al. (24) for analyzing which endogenous FLS are synthesized in any tissue via the mevalonate pathway may open new avenues in the numerous fields in which Ca2+-homeostasis plays a role, e.g., in metabolism and reproduction (46). The data shown in **Table 1** are preliminary and do not exclude that some compounds could have been ingested with the food, but they nevertheless show that FLS are (differentially) omnipresent in the body. The fact that the compounds listed in **Table 1** are the same as the ones present in the *corpora allata* of insects, the only tissue that makes them during the larval state, and in nematodes as well (unpublished results) indicates that the mevalonate-FLS biosynthetic pathway is probably as essential in vertebrates as it is in insects and probably all other eukaryotes.

### **ACKNOWLEDGMENTS**

Elisabeth Marchal is a recipient of a postdoctoral mandate of the KU Leuven. Thanks to Marijke Christiaens for preparing the figure. This work was supported by NIH Grant No AI 45545 to Fernando G. Noriega.

### **REFERENCES**

share a common evolutionary origin. *Front Endocrinol (Lausanne)* (2011) **2**:16. doi:10.3389/fendo.2011.00016

down-regulation of fatty acid synthase in hepatocytes. *Lipids* (2008) **43**:619–27. doi:10.1007/s11745-008-3192-3

**Conflict of Interest Statement:** All authors declare that the research outlined in this paper was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 03 November 2014; paper pending published: 24 November 2014; accepted: 03 December 2014; published online: 06 January 2015.*

*Citation: De Loof A, Marchal E, Rivera-Perez C, Noriega FG and Schoofs L (2015) Farnesol-like endogenous sesquiterpenoids in vertebrates: the probable but overlooked functional "inbrome" anti-aging counterpart of juvenile hormone of insects? Front. Endocrinol. 5:222. doi: 10.3389/fendo.2014.00222*

*This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2015 De Loof, Marchal, Rivera-Perez, Noriega and Schoofs. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Reconstructing SALMFamide neuropeptide precursor evolution in the phylum Echinodermata: ophiuroid and crinoid sequence data provide new insights

### **Maurice R. Elphick 1\* † , Dean C. Semmens 1† , Liisa M. Blowes 1 , Judith Levine2 , Christopher J. Lowe2 , Maria I. Arnone3 and Melody S. Clark 4**

1 School of Biological and Chemical Sciences, Queen Mary University of London, London, UK

2 Hopkins Marine Station, Stanford University, Pacific Grove, CA, USA

3 Stazione Zoologica Anton Dohrn, Naples, Italy

4 British Antarctic Survey, Cambridge, UK

### **Edited by:**

Hubert Vaudry, University of Rouen, France

### **Reviewed by:**

Cornelis J. P. Grimmelikhuijzen, University of Copenhagen, Denmark Pascal Favrel, University of Caen Lower Normandy, France Jozef Vanden Broeck, Katholieke Universiteit Leuven, Belgium

### **\*Correspondence:**

Maurice R. Elphick, School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK e-mail<EMAIL_ADDRESS>
†Maurice R. Elphick and Dean C. Semmens have contributed equally to this work.

The SALMFamides are a family of neuropeptides that act as muscle relaxants in echinoderms. Analysis of genome/transcriptome sequence data from the sea urchin Strongylocentrotus purpuratus (Echinoidea), the sea cucumber Apostichopus japonicus (Holothuroidea), and the starfish Patiria miniata (Asteroidea) reveals that in each species there are two types of SALMFamide precursor: an L-type precursor comprising peptides with a C-terminal LxFamide-type motif and an F-type precursor solely or largely comprising peptides with a C-terminal FxFamide-type motif. Here, we have identified transcripts encoding SALMFamide precursors in the brittle star Ophionotus victoriae (Ophiuroidea) and the feather star Antedon mediterranea (Crinoidea). We have also identified SALM-Famide precursors in other species belonging to each of the five echinoderm classes. As in S. purpuratus, A. japonicus, and P. miniata, in O. victoriae there is one L-type precursor and one F-type precursor. However, in A. mediterranea only a single SALMFamide precursor was found, comprising two peptides with a LxFamide-type motif, one with a FxFamide-type motif, five with a FxLamide-type motif, and four with a LxLamide-type motif. As crinoids are basal to the Echinozoa (Holothuroidea + Echinoidea) and Asterozoa (Asteroidea + Ophiuroidea) in echinoderm phylogeny, one model of SALMFamide precursor evolution would be that ancestrally there was a single SALMFamide gene encoding a variety of SALMFamides (as in crinoids), which duplicated in a common ancestor of the Echinozoa and Asterozoa and then specialized to encode L-type SALMFamides or F-type SALMFamides. Alternatively, a second SALMFamide precursor may remain to be discovered or may have been lost in crinoids. Further insights will be obtained if SALMFamide receptors are identified, which would provide a molecular basis for experimental analysis of the functional significance of the "cocktails" of SALMFamides that exist in echinoderms.

**Keywords: neuropeptide, echinoderm, SALMFamide, evolution, ophiuroid, crinoid**

### **INTRODUCTION**

The SALMFamides are a family of neuropeptides that occur in species belonging to the phylum Echinodermata (e.g., starfish, sea cucumbers, and sea urchins) (1). The prototypes for this neuropeptide family were discovered in the starfish species *Asterias rubens* and *Asterias forbesi* and are known as SALMFamide-1 (S1) and SALMFamide-2 (S2) (2, 3). S1 was identified as a C-terminally amidated octapeptide with the amino acid sequence Gly-Phe-Asn-Ser-Ala-Leu-Met-Phe-NH2 (GFNSALMFamide) and S2 was identified as a C-terminally amidated dodecapeptide with the amino acid sequence Ser-Gly-Pro-Tyr-Ser-Phe-Asn-Ser-Gly-Leu-Thr-Phe-NH2 (SGPYSFNSGLTFamide). Both peptides have the C-terminal motif FNSxLxFamide (where *x* is variable), which suggested that S1 and S2 may have evolved as a consequence of gene duplication or intragenic DNA duplication. Immunocytochemical investigation of the expression of S1 and S2 in *A. rubens* revealed

widely distributed patterns of expression in the nervous system but in separate populations of neurons (4, 5). Furthermore, S1 and S2 are present in the innervation of neuromuscular organs and, consistent with this finding, both peptides cause relaxation of starfish cardiac stomach, tube foot, and apical muscle preparations *in vitro* (6–9).

Subsequent to the discovery of S1 and S2 in starfish, SALMFamide-type neuropeptides were identified in other echinoderms. Thus, SALMFamide-type peptides were isolated from two sea cucumber species – *Holothuria glaberrima* and *Apostichopus japonicus* – and, consistent with the actions of S1 and S2 in starfish, these peptides cause muscle relaxation in sea cucumbers (10–12). It appears, therefore, that the relaxing action of SALMFamides on echinoderm muscle may be a general property of this neuropeptide family (9). Discovery of SALMFamides in sea cucumbers also revealed structural heterogeneity in SALMFamides. Two

SALMFamides isolated from *Holothuria glaberrima* were identified as GFSKLYFamide and SGYSVLYFamide, which share with the starfish SALMFamides S1 and S2 the C-terminal motif SxLx-Famide (i.e., L-type SALMFamides) (11). However, two SALM-Famides isolated from *Apostichopus japonicus* were identified as GYSPFMFamide and FKSPFMFamide, which have the C-terminal motif SxFxFamide (i.e., F-type SALMFamides) (12).

Sequencing of the genome and transcriptome of the sea urchin *Strongylocentrotus purpuratus* (class Echinoidea) provided the first insight into the genetic basis of SALMFamide-type neuropeptide diversity in an echinoderm species (13). Thus, in *S. purpuratus* there are two genes encoding SALMFamide precursor proteins: one gene encodes a precursor comprising one L-type SALM-Famide and one L-type-like (SxIxFamide) SALMFamide (14) and the second gene encodes a precursor comprising seven F-type SALMFamides (15). However, a more complicated picture has emerged as sequence data from other echinoderms has become available. The Holothuroidea (sea cucumbers) are a sister group to the class Echinoidea and, as in *S. purpuratus*, analysis of transcriptome sequence data from *A. japonicus* revealed two SALMFamide precursor transcripts. One of the precursors comprises three Ltype or L-type-like SALMFamides and is homologous to the L-type SALMFamide precursor in *S. purpuratus* that contains two L-type or L-type-like SALMFamides (16). The second SALMFamide precursor in *A. japonicus* is largely comprised of F-type or F-type-like SALMFamides (five in total) but interestingly, unlike the precursor that gives rise to F-type SALMFamides in *S. purpuratus*, it also contains three L-type/L-type-like SALMFamides (16, 17). Thus, the "cocktail" of SALMFamides in *A. japonicus* is more complex than in *S. purpuratus*. What is not clear from these data, however, is which condition is ancestral and which is derived and to address this issue sequence data from other echinoderms is required.

Recently, genome sequence data for the starfish *Patiria miniata* has been obtained and this has revealed a SALMFamide profile similar to the sea cucumber *A. japonicus*. Thus, in *P. miniata* one SALMFamide precursor is solely comprised of Ltype SALMFamides, which include S1 and six other structurally related peptides (16). The other SALMFamide precursor in *P. miniata* is largely comprised of F-type or F-type-like SALM-Famides (eight in total) but it also contains an S2-like peptide with an L-type C-terminal motif (16). These predicted *P. miniata* SALMFamide precursor sequences now require confirmation by transcript sequencing. Nevertheless, the data currently available suggest that the occurrence of a precursor comprising several Ftype SALMFamides and one or more L-type SALMFamides may be the ancestral condition, with the absence of L-type SALM-Famides in the sea urchin F-type SALMFamide precursor being a derived condition. However, there remains the possibility that the occurrence of L-type SALMFamides in the F-type SALMFamide precursor is a feature that has arisen independently in both the holothurian and asteroid lineages. To gain further insight on this issue it will be necessary to determine the sequences of SALM-Famide precursors in species belonging to two other extant echinoderm classes: the Ophiuroidea (brittle stars) and the Crinoidea (feather stars and sea lilies). As a sister group to the Asteroidea (starfish), the Ophiuroidea could provide key insights on SALM-Famide precursor evolution. Thus, if the F-type SALMFamide precursor in brittle stars also contains L-type or L-type like SALM-Famides, as in starfish and sea cucumbers, this would add weight to the notion that this is a feature that dates back to the common ancestor of the Asterozoa (Asteroidea + Ophiuroidea) and the Echinozoa (Holothuroidea + Echinoidea). The Crinoidea are basal to the Asterozoa and the Echinozoa (18) and determination of the sequences of SALMFamide precursors in species belonging to this class of echinoderms could provide insight into the ancestral condition in the common ancestor of all extant echinoderms.

Here, we have analysed transcriptome sequence data from the starfish *P. miniata*, which in combination with genome sequence data has enabled definitive determination of the sequences of SALMFamide precursors in this species. Importantly, these data have also enabled comparison of SALMFamide gene structure in an asterozoan species (*P. miniata*) and an echinozoan species (*S. purpuratus*). Furthermore, here we report the sequences of novel SALMFamide precursors that we have discovered by analysis of transcriptome sequence data from the ophiuroid *Ophionotus victoriae*, a brittle star species that has a circumpolar distribution around Antarctica, and the crinoid*Antedon mediterranea*, a feather star species that is (as its name implies) native to the Mediterranean Sea*.* Having identified SALMFamide precursors in single species from each of the five echinoderm classes, we investigated the generality of our findings by analysis of genome/transcriptome sequence data from other echinoderm species.

### **MATERIALS AND METHODS**

### **TRANSCRIPTOME SEQUENCING OF THE STARFISH PATIRIA MINIATA**

*Patiria miniata* transcriptome sequence was produced using RNA from several developmental stages from blastula to juveniles, including bipinnaria and brachiolaria larvae. Sequencing was carried out at Huntman Genome Center University of Utah using Illumina HiSeq 101 paired end sequencing. 172,091,442 paired end reads were lightly trimmed and adapter sequences removed using Trimmomatic (19) with the following parameters: ILLUMI-NACLIP:illuminaClipping.fa:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:25. Post-quality trimming was assessed using FastQC1 . High-quality, trimmed reads were assembled *de novo* using the Trinity suite of programs, with default parameters except min\_kmer\_cov = 2 (20). The assembly yielded 203,888 transcripts, representing 101,664 transcript groups (genes). The contig N50 was 1717 bp, the median contig length was 502 bp, and the total assembled bases were 194,282,131. If only the longest isoform in each transcript group was considered, then the contig N50 dropped to 1490 bp, the median contig length was 389 bp, and the total assembled bases were 81,494,371.

### **TRANSCRIPTOME SEQUENCING OF THE BRITTLE STAR OPHIONOTUS VICTORIAE AND THE FEATHER STAR ANTEDON MEDITERRANEA**

Arms dissected from a single adult specimen of *O. victoriae* and arms dissected from a single adult specimen of *A. mediterranea* were used for RNA isolation (Total RNA Isolation System, Promega, Southampton, UK). Library preparation (TruSeqv2 kit, Illumina, Little Chesterford, Essex, UK) was performed at the

1http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

QMUL Genome Center and sequencing (Illumina HiSeq 2500 platform) was performed at the BRC Genomics Core Facility at Guy's and St Thomas' NHS Foundation Trust and King's College London.

Illumina HiSeq sequencing yielded 155931609 and 116089417 paired 101 bp long reads for *O. victoriae* and *A. mediterranea*, respectively. Raw sequence data were assembled using the de Brujn graph assembler Short Oligonucleotide Analysis Package SOAPdenovo-Trans-31mer 1.032 with the Kmer value set to 31, employing use of a high performance computing system (Apocrita3 ). The *O. victoriae* assembly yielded 669,744 contigs, with a mean length of 243 bp and N50 of 297 bp, and 17,616 contigs were >1000 bp in length. 76% of the contigs were assembled within scaffolds with a mean length of 300 bp and 30,859 scaffolds were >1000 bp in length. The *A. mediterranea* Kmer 31 assembly yielded 675,534 contigs, with a mean length of 254 bp and N50 of 310 bp, and 16,755 contigs were >1000 bp in length. 82% of the contigs were assembled within scaffolds with a mean length of 301 bp and 26,884 scaffolds were >1000 bp in length.

### **BLAST ANALYSIS OF THE ASSEMBLED TRANSCRIPTOMES OF P. MINIATA, O. VICTORIAE, AND A. MEDITERRANEA**

To enable identification of transcripts encoding SALMFamide precursors in *P. miniata*, *O. victoriae*, and *A. mediterranea*, the contig and scaffold datasets generated from assembly of Illumina HiSeq sequence data obtained for these species were set up for BLAST (Basic Local Alignment Search Tool) analysis using SequenceServer4 . BLAST searches were performed using the protein sequences of known SALMFamide precursors from *S. purpuratus*, *A. japonicus*, and *P. miniata* as queries (16). In addition, the sequence data were analysed by BLAST using short SALMFamidetype neuropeptide sequences as queries, with the *E* value set to 1000.

### **CLONING AND SEQUENCING OF SALMFAMIDE PRECURSOR CDNAS FROM O. VICTORIAE AND A. MEDITERRANEA**

Total RNA that had been generated for Illumina HiSeq sequencing (see above) was also used for cDNA synthesis (Quantitect Reverse Transcription Kit, QIAGEN, Manchester, UK). Fulllength cDNAs of SALMFamide precursors, including 50 and 30 untranslated regions (UTR), were amplified through PCR (Phusion High-Fidelity PCR Master Mix, NEB, Hitchin, Hertfordshire, UK) using the oligos: 50 -GTGACATTACTACTCCTGAT-3 0 /50 -CAACAAGACAGACTAATGAC-30 (*O. victoriae* L-type SALMFamide precursor), 50 -GAAGTGGTTGCTAATACC-30 /50 - ACTTTAGTCCTTCCGTAC-30 (*O. victoriae* F-type SALM-Famide precursor), and 50 -ATACAACGGGATAGAGAG-30 /50 - ACACTCGGAACTTGTCTA-30 (*A. mediterranea* SALMFamide precursor), designed using Primer3 software5 . The PCR products were gel-extracted and purified (QIAquick Gel Extraction Kit, QIAGEN, Manchester, UK) before being blunt-end cloned into a pBluescript SKII (+) vector (Agilent Technologies, Stockport, Cheshire, UK) cut with the *Eco*RV-HF restriction endonuclease (NEB, Hitchin, Hertfordshire, UK). The clones were then sequenced (Eurofins Genomics GmbH, Ebersberg, Germany) from the T7 and T3 sequencing primer sites.

### **IDENTIFICATION OF SALMFAMIDE PRECURSORS IN OTHER ECHINODERM SPECIES**

Our efforts to determine the sequences of SALMFamide precursors in this study or in previous studies (16) have targeted a single species for each of the extant echinoderm classes: *S. purpuratus* (Echinoidea), *A. japonicus* (Holothuroidea), *P. miniata* (Asteroidea), *O. victoriae* (Ophiuroidea), and *A. mediterranea* (Crinoidea). To assess the generality of our findings, ideally transcriptome and/or genome sequence data from multiple species for each echinoderm class would be analysed. As a step toward this level of analysis, here we have analysed genome or transcriptome sequence data from at least one additional species from each of the five echinoderm classes.

To search for SALMFamide precursors in a second echinoid species, genome sequence data from the sea urchin *Lytechinus variegatus* was analysed using a BLAST facility6 made publicly available by Andy Cameron and colleagues at CalTech (USA). To search for SALMFamide precursors in species belonging to the four other echinoderm classes, transcriptome sequence data that have recently been obtained for an investigation of echinoderm phylogenetic relationships was analysed (21). The data analysed included transcriptome sequences from a holothurian species (*Leptosynapta tenuis*), an asteroid species (*Luidia senegalensis*), a crinoid species (*Aporometra wilsoni*), and 52 ophiuroid species (21).

### **RESULTS**

### **DETERMINATION OF THE SEQUENCES OF TRANSCRIPTS ENCODING SALMFAMIDE PRECURSORS IN THE STARFISH P. MINIATA**

Previously, analysis of *P. miniata* genome sequence data enabled identification of two genes encoding SALMFamide-type neuropeptides in this species. Firstly, a gene encoding seven L-type SALMFamides (L-type gene) and secondly a gene encoding eight F-type SALMFamides and one L-type SALMFamide (F-type gene) (16). However, the predicted gene products reported by Elphick et al. (16) have yet to be confirmed by transcript sequencing. Therefore, here we analysed *P. miniata* transcriptome sequence data to identify SALMFamide precursor transcripts.

BLAST analysis of *P. miniata* transcriptome data using the predicted 174-residue *P. miniata* L-type SALMFamide precursor sequence as a query identified a 1657 bp transcript (contig 378809) encoding a 212-residue protein (**Figure 1A**; Figure S1 in Supplementary Material). Residues 63–212 of this protein were found to be identical to residues 25–174 of the predicted 174 residue protein. However,the N-terminal region of the 212-residue protein (residues 1–62) did not share sequence identity with the Nterminal region of the predicted 174-residue protein (residues 1– 24). Thus,the predicted 174-residue sequence of the L-type SALM-Famide precursor in *P. miniata*, which was based on analysis of

2http://soap.genomics.org.cn/SOAPdenovo-Trans.html

3www.hpc.qmul.ac.uk

4http://www.sequenceserver.com

5http://bioinfo.ut.ee/primer3-0.4.0/

6http://www.echinobase.org/Echinobase/Blast/LvBlast/blast.php

genomic sequence data, may be partially incorrect. Furthermore, BLAST analysis of *P. miniata* genome sequence data revealed that residues 1–62 of the 212-residue protein are encoded by an exon located on scaffold JH775329.1 (34230 bp), whereas residues 63– 212 are encoded by an exon located on scaffold JH770521.1 (or 1914; 55,595 bp). Thus, the presence of the two exons on different genomic scaffolds provided an explanation for why the first exon was not identified when analysing genomic sequence data. The putative exon encoding residues 1–24 of the predicted 174-residue L-type SALMFamide precursor was identified on the same scaffold

**FIGURE 1 | Patiria miniata SALMFamide precursors**. **(A)** L-type SALMFamide precursor. **(B)** F-type SALMFamide precursor. The predicted signal peptide of the precursor proteins is shown in blue and the putative SALMFamide neuropeptides are shown in red, with C-terminal glycine residues that are potential substrates for amidation shown in orange. Putative cleavage sites are shown in green.

(JH770521.1) as the exon encoding the C-terminal region of the protein and was a plausible candidate exon because it encodes a polypeptide that has the expected properties for the N-terminal signal peptide region of a neuropeptide precursor. Transcriptome sequencing indicates that this prediction may have been wrong. However, there remains the possibility that this gene is subject to alternative splicing and transcripts encoding the predicted 174 residue L-type SALMFamide precursor occur naturally but are less abundant than transcripts encoding the 212-residue protein.

BLAST analysis using the predicted 258-residue *P. miniata* Ftype SALMFamide precursor sequence as a query identified a 2041 base transcript (contig 387722) encoding a 258-residue protein that was identical to the query sequence (**Figure 1B**; Figure S2 in Supplementary Material). Thus, in this case analysis of transcriptome sequence data has provided confirmation of a sequence predicted from analysis of genome sequence data.

### **COMPARISON OF THE STRUCTURE OF SALMFAMIDE PRECURSOR GENES IN S. PURPURATUS AND P. MINIATA**

Identification of transcripts encoding SALMFamide precursors in the starfish *P. miniata* (see above) has enabled analysis of the structure of the genes that encode these proteins and comparison with the structure of genes encoding SALMFamide precursors in the sea urchin *S. purpuratus*. **Figure 2A** shows a diagrammatic representation of the structure of genes encoding L-type SALMFamide precursors in *S. purpuratus* and *P. miniata* and **Figure 2B** shows a diagrammatic representation of the structure of genes encoding F-type SALMFamide precursors in *S. purpuratus* and *P. miniata.*

**FIGURE 2 | Comparison of the structure of SALMFamide genes in the sea urchin S. purpuratus and the starfish P. miniata**. **(A)**. Diagrammatic representation of the structure of the L-type SALMFamide precursor gene in S. purpuratus and P. miniata. **(B)**. Diagrammatic representation of the structure of the F-type SALMFamide precursor gene in S. purpuratus and P. miniata. Exons are drawn to scale as rectangles and introns are shown as lines but not to scale (intron lengths are shown in parentheses). Non-coding exons or regions of exons are shown in white; note that the 50 and 30 of the genes are shown as a dashed line because it is possible that additional non-coding bases are transcribed in vivo but are not present in transcripts that have been sequenced. The protein-coding exons or regions of exons are shaded (gray) or colored, with the N-terminal signal peptide shown in blue

and the predicted neuropeptide products of the precursors shown in red (L-type SALMFamides) or yellow (F-type SALMFamides). The numbers above the protein-coding exons are amino-acid residue positions in the protein products of the genes. A feature that appears to distinguish L-type and F-type SALMFamide genes is the structure of the first protein-coding exons. Thus, in the two L-type SALMFamide genes the first protein-coding exon also encodes a 50 non-coding region, whilst in the two F-type SALMFamide genes the first protein-coding exon does not have a 50 non-coding region. These differences in gene structure provide important evidence that the L-type SALMFamide precursor genes in S. purpuratus and P. miniata are orthologs and the F-type SALMFamide precursor genes in S. purpuratus and P. miniata are orthologs.

The L-type SALMFamide precursors in the two species have a similar gene structure comprising two exons. The first exon encodes a 50 non-coding region and the N-terminal region of the precursor proteins, including the signal peptide. The second exon encodes the C-terminal region of the precursor proteins (including the L-type SALMFamides) and a long 30 non-coding region.

The F-type SALMFamide precursors in the two species also have a similar gene structure but, importantly, the gene structure is distinctly different to the structure of the L-type SALMFamide genes. Thus, the F-type SALMFamide genes have one (*S. purpuratus*) or two (*P. miniata*) 50 non-coding exons followed by an exon that encodes the N-terminal region of the precursor protein, with the first codon of the exon encoding the starter methionine. This contrasts with L-type SALMFamide precursor genes (see above), where the exon encoding the N-terminal region of the precursor also encodes a 50 non-coding region. In common with L-type SALMFamide precursor genes, the C-terminal region of the F-type SALMFamide precursor proteins (which includes multiple copies of SALMFamide peptides) is encoded by a large exon with a long 3 0 non-coding region.

Thus, L-type SALMFamide precursor and F-type SALMFamide precursor genes are similar in that the N-terminal and C-terminal regions of the proteins are encoded by two exons. However, the presence (L-type) or absence (F-type) of a 50 non-coding region in the first protein-encoding exons is a distinguishing feature. This provides evidence that the L-type SALMFamide precursors in *S. purpuratus* and *P. miniata* are orthologous and F-type SALM-Famide precursors in *S. purpuratus* and *P. miniata* are orthologous, which is an important finding because evidence for orthology based solely on comparison of protein sequences is not very strong, particularly for L-type SALMFamide precursors. Thus, whilst Ltype SALMFamide precursors are characterized by the presence of peptides with a C-terminal SxLxLamide (L-type) motif, the number of copies of these peptides is very different in *P. miniata* (seven) and *S. purpuratus* (two). Indeed the presence of seven SALMFamides in the *P. miniata* L-type SALMFamide precursor makes this protein, at least superficially, more similar to F-type SALMFamide precursors in *P. miniata* and *S. purpuratus*, which contain nine and seven SALMFamides, respectively. In conclusion, therefore, the evidence of orthology provided by similarities/differences in gene structure provides an important basis for classifying SALMFamide genes in echinoderms as either L-type SALMFamide precursor genes or F-type SALMFamide precursor genes.

### **IDENTIFICATION OF AN L-TYPE SALMFAMIDE PRECURSOR IN THE OPHIUROID O. VICTORIAE**

BLAST analysis of *O. victoriae* transcriptome sequence data revealed a 2422 bp contig (2059646) encoding a 169-residue protein comprising a predicted 20-residue N-terminal signal peptide and four putative SALMFamide neuropeptides bounded by monobasic or dibasic cleavage sites. A cDNA encoding this protein was cloned and sequenced, which confirmed the coding sequence predicted from assembled RNAseq data (**Figure 3A**; Figure S3 in Supplementary Material; GenBank accession number: KM979353). Analysis of the sequences of the four putative SALMFamide neuropeptides derived from this precursor

### **FIGURE 3 | Ophionotus victoriae and Antedon mediterranea**

**SALMFamide precursors**. **(A)** O. victoriae L-type SALMFamide precursor. **(B)** O. victoriae F-type SALMFamide precursor. **(C)** A. mediterranea SALMFamide precursor. The predicted signal peptide of the precursor proteins is shown in blue, putative neuropeptides derived from the precursors are shown in red with C-terminal glycine residues that likely substrates for amidation shown in orange. Putative cleavage sites are shown in green.

revealed that the first (i.e., N-terminally located) is a dodecapeptide with a canonical L-type SALMFamide motif (SxLxFamide) and the fourth (i.e., C-terminally located) is a putative 14-residue neuropeptide with an L-type-like SALMFamide motif (SxMx-Famide). The intervening second putative neuropeptide is an 11-residue peptide with the C-terminal pentapeptide sequence SGLMQamide, which is L-type-like based on the presence of the leucine residue but is otherwise very unusual in having a C-terminal glutamine residue. The third neuropeptide is also a putative 11-residue peptide but with the C-terminal pentapeptide sequence TGFMMamide, which is F-type-like based on the presence of the phenylalanine residue but it has C-terminal methionine residue. Thus, this is predominantly a precursor of L-type or L-type-like SALMFamide precursor, homologous with L-type SALMFamide precursors that have been identified in other echinoderms. However, it is atypical in containing an F-type-like SALMFamide with a C-terminal methionine.

### **IDENTIFICATION OF AN F-TYPE SALMFAMIDE PRECURSOR IN THE OPHIUROID O. VICTORIAE**

BLAST analysis of *O. victoriae* transcriptome sequence data revealed a 2343 bp scaffold (64,804) encoding a partial protein sequence comprising a predicted 23-residue N-terminal signal peptide and 11 putative SALMFamide neuropeptides bounded by dibasic cleavage sites. Because the protein-coding region of scaffold 64,804 was interrupted by a segment of unknown nucleotides it was necessary to clone and sequence a cDNA encoding this protein to determine its complete sequence. This revealed that it is a 310-residue protein comprising 12 putative SALMFamide neuropeptides, 11 of them F-type SALMFamides, and 1 of them an L-type SALMFamide (**Figure 3B**; Figure S4 in Supplementary Material; GenBank accession number: KM979352). Thus, this precursor protein is similar to the F-type SALMFamide precursor in the starfish *P. miniata*, which comprises eight F-type or F-type-like SALMFamides and one L-type SALMFamide [**Figure 1**; (16)].

### **IDENTIFICATION OF A SALMFAMIDE PRECURSOR IN THE CRINOID A. MEDITERRANEA**

BLAST analysis of *A. mediterranea* transcriptome sequence data identified a putative 1683 bp transcript encoding a SALMFamide precursor protein, which was assembled manually from three overlapping contigs (1781194, 446850, and 1694464) and then confirmed by cDNA cloning and sequencing (**Figure 3C**; Figure S5 in Supplementary Material; GenBank accession number: KM979351). The SALMFamide precursor is a 370-residue protein comprising a predicted 22-residue N-terminal signal peptide and 12 putative SALMFamide-type neuropeptides bounded by dibasic cleavage sites. Two of the putative peptides have a Cterminal LxFamide motif (i.e., L-type SALMFamide) and one of the putative peptides has a C-terminal FxFamide motif (i.e., Ftype). However, the other nine putative peptides have a variety of C-terminal motifs that include FxLamide, FxMamide, YxLamide, YxMamide, and LxLamide. The motif F/YxL/Mamide has previously been referred to as "F-type-like" and the motif LxLamide has previously been referred to as "L-type-like" (16). However, as discussed below, discovery of the *A. mediterranea* SALMFamide precursor may require reclassification of SALMFamide neuropeptides in echinoderms.

### **IDENTIFICATION OF SALMFAMIDE PRECURSORS IN OTHER ECHINODERM SPECIES**

Our findings from analysis of transcriptome or genome sequence data from other echinoderm species were consistent with those reported previously (16) or above, as described below and as illustrated in Figures S6–S10 in Supplementary Material.

An L-type SALMFamide precursor and an F-type SALMFamide precursor were identified in the sea urchin *Lytechinus variegatus* (class Echinoidea; Figure S6 in Supplementary Material), and as in *S. purpuratus*, the *L. variegatus* L-type SALMFamide precursor comprised two L-type SALMFamides (Figure S6A in Supplementary Material) and the *L. variegatus* F-type SALMFamide precursor comprised seven F-type SALMFamides (Figure S6B in Supplementary Material).

Interestingly, in the sea cucumber *Leptosynapta tenuis* (class Holothuroidea) two L-type SALMFamide precursors and one F-type SALMFamide precursor were identified (Figure S7 in Supplementary Material). Comparison of the sequences of the two L-type SALMFamide precursors in *L. tenuis* revealed high levels of sequence similarity (Figure S7C in Supplementary Material), indicating that these have arisen by gene duplication in this species or in a lineage that includes this species and other closely related holothurian species. As in the *A. japonicus* L-type SALMFamide precursor, both of the *L. tenuis* L-type SALMFamide precursors comprised three L-type or L-type-like SALMFamide neuropeptides (Figure S7A,B in Supplementary Material). The general characteristics of the F-type SALMFamide precursor in *L. tenuis* were

similar to the F-type SALMFamide precursor in *A. japonicus* in being largely comprised of F-type SALMFamides. However, the overall level of sequence identity was quite low and in the positions occupied by two L-type SALMFamides in *A. japonicus* there are F-type SALMFamides in *L. tenuis*. Analysis of a wider range of species will be required to determine which of these represents the ancestral/derived condition.

In the starfish *Luidia senegalensis* (class Asteroidea), an L-type SALMFamide precursor and an F-type SALMFamide precursor were identified (Figure S8 in Supplementary Material). The *L. senegalensis* L-type SALMFamide precursor comprises seven Ltype SALMFamides, which share high levels of sequence similarity with the seven corresponding L-type SALMFamides in the *P. miniata* L-type SALMFamide precursor (Figure S8A in Supplementary Material). The *L. senegalensis* F-type SALMFamide precursor comprises eight SALMFamides, six of which are F-type or F-type-like, and two of which are L-type. By way of comparison, the *P. miniata* F-type SALMFamide precursor comprises nine SALMFamides, eight F-type, and one L-type (Figure S8B in Supplementary Material). Thus, whilst the overall organization of the F-type SALMFamide precursors in the two species is similar and clearly indicative of orthology, lineage specific gain/loss of peptides has occurred as well as conversion of peptides from F-type to L-type or vice versa. Analysis of a wider range of species will therefore be necessary to identify the ancestral characteristics of F-type SALMFamide precursors in asteroids.

Recent molecular phylogenetic analysis of the class Ophiuroidea has identified three clades: clade A, clade B, and clade C (21). Here, an L-type SALMFamide precursor and an F-type SALMFamide precursor were identified in species belonging to each of these clades. In clade A, which includes *O. victoriae*, these were *Ophiomusium lymani* (Ophiolepididae), *Asteronyx loveni* (Asteronychidae), and *Asteroschema bidwillae* (Asterochematidae). In clade B *Clarkoma canaliculata* (Ophiocomidae) and in clade C *Ophiactis abyssicola* (Ophiactidae) and *Ophiothrix angulata* (Ophiotrichidae). The characteristics of the L-type SALM-Famide and F-type SALMFamide precursor in these species were similar to those in *O. victoriae*, and this is illustrated using sequence data from *Ophiothrix angulata* as an example in Figure S9 in Supplementary Material. Thus, the L-type SALMFamide precursors in *O. victoriae* and *O. angulata* comprise four homologous peptides. However, as reported above for other classes, there is variable conservation of the sequences of peptides that occupy equivalent positions in the precursor proteins. For example, the C-terminal region of the first SALMFamide in the precursor is very similar in both species (LNSGLxFamide), whereas the third SALMFamide exhibits sequence divergence – TGFMMamide in *O. victoriae* and SAMLLamide *O. angulata* (Figure S9A in Supplementary Material). The F-type SALMFamide precursor in *O. angulata* comprises 10 F-type SALMFamides and 1 L-type SALMFamide. This is similar to the F-type precursor in *O. victoriae*, which comprises 11 F-type SALMFamides and 1 L-type SALMFamide, with the L-type SALMFamide occupying the same position in both species (Figure S9B in Supplementary Material).

In the feather star *Apotometra wilsoni* (class Crinoidea), only a single SALMFamide precursor was identified, consistent with our findings from *A. mediterranea* (Figure S10 in Supplementary Material). Comparison of the sequences of the SALMFamide precursor in *A. mediterranea* and *A. wilsoni* revealed high levels of similarity, with both precursors comprising 14 putative SALMFamide neuropeptides.

An example where there is sequence divergence is the seventh peptide, which has the C-terminal sequence LMLamide in *A. mediterranea* and FMLamide in *A. wilsoni.* However, peptides occupying equivalent positions in the two precursors are largely similar in their sequence characteristics.

## **DISCUSSION**

Previously, analysis of genome/transcriptome sequence data has enabled identification of genes/transcripts encoding SALMFamide neuropeptide precursors in species from three of the five extant echinoderm classes: the sea urchin *S. purpuratus* (Echinoidea), the sea cucumber *A. japonicus* (Holothuroidea), and the starfish *P. miniata* (Asteroidea) (16). This has revealed that in each species there are two SALMFamide precursor genes: firstly, a gene that encodes peptides with a C-terminal LxFamide-type motif (L-type) and secondly, a gene that either only encodes peptides with a Cterminal FxFamide-type motif (F-type) (*S. purpuratus*) or encodes F-type SALMFamides plus one or more L-type SALMFamides (*A. japonicus*, *P. miniata*) (16). However, the *P. miniata* sequences were based on predictions made from genome sequence data,without supporting evidence from transcript sequences. Furthermore, a deeper understanding of SALMFamide precursor evolution in the phylum Echinodermata will require analysis of sequence data from at least one species from the two other extant echinoderm classes – the Ophiuroidea and Crinoidea.

Here, we have determined the sequences of transcripts encoding the L-type SALMFamide precursor and F-type SALMFamide precursor in the starfish *P. miniata*, which has enabled comparison of the structure of genes encoding these precursors with SALM-Famide precursor genes in the sea urchin *S. purpuratus*. This has revealed that F-type SALMFamide precursor genes have one or two introns in the 50 non-coding region of the genes, whereas L-type SALMFamide genes do not have this feature. The overall level of sequence identity when comparing L-type SALMFamide precursors or F-type SALMFamide precursors protein sequences from different echinoderm classes is low [see Figures 5 and 6 in Ref. (16)]. Consequently, the categorization of SALMFamide precursors into two types (L-type and F-type) has hitherto been based upon the relative abundance of constituent peptides that have either an LxFamide motif or FxFamide motif. Obtaining evidence of orthology based on gene structure now provides an important additional criterion for classification of SALMFamide precursors as either L-type or F-type.

The F-type SALMFamide precursors in *A. japonicus* (echinozoan clade) and *P. miniata* (asterozoan clade) are largely comprised of F-type SALMFamides but they also contain one or more Ltype SALMFamides. This suggests that the occurrence of L-type SALMFamides in F-type precursors may be a characteristic of Ftype precursors traceable to the common ancestor of the Asterozoa and Echinozoa, with the absence of L-type SALMFamides in the F-type precursors of sea urchins (Echinoidea, Echinozoa) presumably a derived feature. Here, we have obtained evidence in support of this hypothesis with the identification of a transcript encoding a F-type precursor in the brittle star *O. victoriae* (Ophiuroidea, Asterozoa). Thus, in *O. victoriae* there is an F-type precursor that largely comprises F-type SALMFamides (11) but which also contains an L-type SALMFamide. Similarly, the F-type precursor in the brittle star *O. angulata* comprises 10 F-type SALMFamides and 1 L-type SALMFamide. By way of comparison, the L-type SALMFamide precursors in *O. victoriae* and *O. angulata* comprise four putative peptides, including two L-type SALMFamides and an unusual peptide with a C-terminal LxQamide motif. This latter feature is atypical of L-type precursors that have been identified in other echinoderms and therefore it appears to be derived characteristic of ophiuroids.

Analysis of transcriptome sequence data from the crinoid *A. mediterranea* revealed only a single SALMFamide-type precursor. A homolog of this precursor was found in the feather star *A. wilsoni* and, as in *A. mediterranea*, this was the only SALMFamide precursor identified in this species. Analysis of the sequences of the putative neuropeptides derived from the *A. mediterranea* precursor reveals that it comprises five peptides with a FxLamide-type motif and four peptides with a LxLamide-type motif but only two peptides with a LxFamide-type motif and one peptide with a FxFamide-type motif. Based upon these characteristics, the *A. mediterranea* SALMFamide precursor does not appear to be an L-type-like precursor, which are largely comprised of peptides with a LxFamide motif, or an F-type-like precursor, which are largely comprised of peptides with a FxFamide motif. Thus, discovery of SALMFamide precursors in crinoid species has broadened our perspective on the structural characteristics of SALMFamides.

Hitherto SALMFamides have been characterized as either Ltype (LxFamide) or F-type (FxFamide). Peptides that deviate from these motifs have been identified in echinozoans and asterozoans; for example, one of the peptides in the *A. japonicus* Ltype SALMFamide precursor has the C-terminal motif IxLamide, which previously was categorized as L-type-like. Similarly, one of the peptides in the *A. japonicus* F-type SALMFamide precursor has the C-terminal motif FxLamide, which was categorized as Ftype-like. Our discovery that the crinoid SALMFamide precursor largely comprises peptides with a LxLamide motif or LxLamidelike motif (e.g., LxMamide) and peptides with a FxLamide motif has provided a basis for categorization of SALMFamides into four types: 1. FxFamide, 2. FxLamide or FxMamide, 3. LxFamide, and 4. LxLamide (**Figure 4**). However, we retain the use of the terms L-type (LxFamide) and F-type (FxFamide) because these are useful for categorization of SALMFamide peptides and SALM-Famide precursors in echinozoans and asterozoans. Furthermore, identification of transcripts encoding SALMFamide precursors in species representing all five extant echinoderm classes, the feather stars *A. mediterranea* and *A. wilsoni* (Crinoidea), the brittle stars *O. victoriae* and *O. angulata* (Ophiuroidea), the starfishes *P. miniata* and *L. senegalensis* (Asteroidea), the sea cucumbers *A. japonicus* and *L. tenuis* (Holothuroidea), and the sea urchins *S. purpuratus* and *L. variegatus* (Echinoidea), provides a basis for formulation of hypotheses on the evolution of these proteins.

Because crinoids are basal to the Echinozoa (Echinoidea and Holothuroidea) and the Asterozoa (Asteroidea and Ophiuroidea) in echinoderm phylogeny, one model of SALMFamide precursor

**(A) and putative SALMFamide peptides (B) in species representing each of the five extant echinoderm classes**. **(A)** SALMFamide precursors are shown in a phylogenetic diagram according to the phylogeny of Telford et al. (18) and O'Hara et al. (21), with crinoids basal to the Echinozoa (Holothuroidea and Echinoidea) and the Asterozoa (Asteriodea + Ophiuroidea). The estimated

according to O'Hara et al. (21) are: 1. 501-542 Ma, 2. 482-421 Ma, 3. 464-485 Ma, and 4. at least 479 Ma. S. pur is the sea urchin Strongylocentrotus purpuratus (Echinoidea), A. jap is the sea cucumber Apostichopus japonicus (Holothuroidea), P. min is the starfish Patiria miniata (Asteroidea),

(Continued)

### **FIGURE 4 | Continued**

O. vic is the brittle star Ophionotus victoriae (Ophiuroidea), and A. med is the feather star Antedon mediterranea (Crinoidea). Signal peptides are shown in blue and dibasic or monobasic cleavage sites are shown in green. L-type SALMFamides with a C-terminal LxFamide motif or with an L-type-like motif (e.g., IxFamide) are shown in red. F-type SALMFamides with a FxFamide motif or with an F-type-like motif (e.g., YxFamide) are shown in yellow. SALMFamides with a FxLamide-type motif are shown in orange and SALMFamides with a LxLamide motif are shown in dark red. Peptides that do not conform with any of the four color-coded categories are shown in white (e.g., GVPPYVVKVTYamide in A. japonicus and SRLPFHSGLMQamide in O. victoriae). The diagram shows how in a

evolution in the phylum Echinodermata is that ancestrally there was a single SALMFamide gene encoding a variety of SALM-Famides (as in*A. mediterranea* and*A. wilsoni*),which duplicated in a common ancestor of the Echinozoa and Asterozoa. Then one of the duplicated genes specialized to encode L-type SALMFamides and the other specialized to exclusively or predominantly encode F-type SALMFamides (as in Echinozoa and Asterozoa). In the context of this evolutionary scenario, it is of interest to compare the structural features of SALMFamide precursors in the five echinoderm classes, using the precursors identified in *S. purpuratus*, *A. japonicus*, *P. miniata*, *O. victoriae*, and *A. mediterranea* as examples (see **Figure 4A**). Starting with the single SALMFamide precursor identified in the crinoid *A. mediterranea* and with reference to the key, which shows the color coding for the different types of SALM-Famides, the predominance of peptides with a FxLamide-type or FxMamide-type motif (orange) or a LxLamide-type motif (dark red) is apparent, whilst the prototypical L-type SALMFamides (LxFamide; red) and F-type SALMFamides (FxFamide; yellow) are minor components. What cannot be deduced from this single precursor in an extant crinoid is whether or not this precursor structure is representative of an ancestral-type SALMFamide precursor that would have existed in extinct crinoid species ~550 million years ago, before the emergence of echinozoan and asterozoan echinoderms (18, 21, 22). In the lineage leading to extant feather stars (order Comatulidina; infraorder Comatulidia) such as*A. mediterranea*, intragenic duplication events may have resulted in expansion of segments of DNA encoding some SALMFamide neuropeptide types, whilst mutational changes or deletion events may have resulted in loss of other SALMFamide neuropeptide types. Further insight on this issue may be obtained if SALM-Famide precursor transcript/gene sequences are obtained in the future with broader taxonomic sampling of extant crinoids. Nevertheless, we speculate that the occurrence of multiple types of SALMFamides (FxFamide-type, FxLamide-type or FxMamidetype, LxFamide-type, and LxLamide-type) in the *A. mediterranea* SALMFamide precursor is reflective of the ancestral condition in extinct crinoids that predated the emergence of asterozoans and echinozoans, although of course the precise number of copies of each type may be variable between extant crinoid species.

If the *A. mediterranea* precursor is representative of an ancestral-type SALMFamide precursor in being comprised of a variety of SALMFamides that have C-terminal FxFamidetype, FxLamide-type or FxMamide-type, LxFamide-type, and LxLamide-type motifs, then the occurrence of SALMFamide presumed ancestral-type precursor in crinoids the majority of the putative peptides have a FxLamide-type motif or a LxLamide-type motif and there is only one L-type SALMFamide and one F-type SALMFamide. However, as a consequence of specialization following a presumed duplication of the ancestral-type gene in a common ancestor of the Echinozoa and Asterozoa, two types of SALMFamide precursor have evolved: one that is predominantly comprised of L-type SALMFamides (red) and another that is exclusively or predominantly comprised of F-type SALMFamides (yellow). **(B)** C-terminal alignments of SALMFamide neuropeptides derived from the precursor proteins shown in **(A)**. The C-terminal regions of each peptide are color-coded according to the key shown in **(A)**.

precursors in asterozoans and echinozoans that are exclusively or predominantly comprised of peptides with a LxFamide-type motif (L-type precursor) or FxFamide-type motif (F-type precursor) is interesting from both an evolutionary and functional perspective. It suggests that following the putative duplication of a gene encoding an ancestral-type SALMFamide precursor in a common ancestor of echinozoans and asterozoans, there was specialization toward on the one hand a precursor of L-type peptides (LxFamide) and on the other hand a precursor of F-type peptides (FxFamide). Conversely, peptides with a LxLamide-type motif, FxLamide-type motif, or FxMamide-type motif that predominate in the *A. mediterranea* precursor are either absent or are minor components of SALMFamide precursors in echinozoans and asterozoans (see **Figure 4B**). This specialization of SALM-Famide precursors as sources of either L-type or F-type SALM-Famide neuropeptides would presumably reflect acquisition of distinct physiological roles. Therefore, it will be of interest to compare the patterns of expression of L-type precursors and F-type precursors and the pharmacological actions of their constituent neuropeptides in echinozoan and asterozoan species.

An alternative hypothesis to that outlined above is that a second SALMFamide-type precursor remains to be identified in crinoid species and that it remains undiscovered because of sequence divergence or incomplete transcriptome data. Another scenario is that two SALMFamide-type precursors existed ancestrally in crinoids but there has been loss of one precursor in a lineage that gave rise to extant feather star species such as *A. mediterranea* and *A. wilsoni*. If either of these scenarios is correct, then the question arises as to the relationship of the crinoid SALMFamide-type precursor identified here with the L-type and F-type SALMFamide precursors in asterozoa and echinozoa. Based on the number of constituent peptides, the crinoid SALMFamide-type precursor identified here is more similar to F-type SALMFamide precursors than L-type SALMFamide precursors. Further insights on the evolutionary relationships of SALMFamide neuropeptides and their precursors will be gained if the receptors that mediate their effects are identified.

### **AUTHOR CONTRIBUTIONS**

This study was conceived and co-ordinated by Maurice R. Elphick. *P. miniata* transcriptome sequence data were obtained by Judith Levine and Christopher J. Lowe. *O. victoriae* and *A. mediterranea* transcriptome sequence data were obtained by Melody S. Clark, Maria Ina. Arnone, Dean C. Semmens, Liisa M. Blowes, and Maurice R. Elphick. Analysis of *P. miniata, O. victoriae*, and *A. mediterranea* transcriptome sequence data was performed by Maurice R. Elphick, Dean C. Semmens, and Liisa M. Blowes. Cloning and sequencing of the *O. victoriae* and *A. mediterranea* SALMFamide precursor cDNAs was performed by Dean C. Semmens. The manuscript was written by Maurice R. Elphick and Dean C. Semmens, with all other authors editing or commenting on the final draft of the manuscript.

### **ACKNOWLEDGMENTS**

This work was supported by a Ph.D. studentship awarded to Dean C. Semmens by Queen Mary University of London. Sequencing of the *O. victoriae* and *A. mediterranea* transcriptomes was funded by the Natural Environmental Research Council (NE-C300-62202-3215; British Antarctic Survey, Cambridge). This research was also supported by the National Institute for Health Research (NIHR) Biomedical Research Center based at Guy's and St Thomas' NHS Foundation Trust and King's College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We are very grateful to Dr. Timothy O'Hara and Dr. Andrew Hugall (Museum Victoria, Melbourne, VIC, Australia) for providing access to their echinoderm transcriptome sequence data.

### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at http://www.frontiersin.org/Journal/10.3389/fendo.2015.00002/ abstract

### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 17 October 2014; accepted: 10 January 2015; published online: 02 February 2015.*

*Citation: Elphick MR, Semmens DC, Blowes LM, Levine J, Lowe CJ, Arnone MI and Clark MS (2015) Reconstructing SALMFamide neuropeptide precursor evolution in the phylum Echinodermata: ophiuroid and crinoid sequence data provide new insights. Front. Endocrinol. 6:2. doi: 10.3389/fendo.2015.00002*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2015 Elphick, Semmens, Blowes, Levine, Lowe, Arnone and Clark. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The "ram effect": new insights into neural modulation of the gonadotropic axis by male odors and socio-sexual interactions

Claude Fabre-Nys 1 \*, Keith M. Kendrick 2 and Rex J. Scaramuzzi 3

 UMR 7247 Physiologie de la Reproduction et des Comportements, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Institut Français du Cheval et de L'équitation, Université de Tours, Nouzilly, France, Key Laboratory for Neuroinformation, University of Electronic Science and Technology of China, Chengdu, China, Department of Comparative Biological Sciences, Royal Veterinary College, South Mimms, UK

### Edited by:

Hubert Vaudry, University of Rouen, France

### Reviewed by:

Berta Levavi-Sivan, The Hebrew University, Israel Ruud Buijs, Universidad Autónoma de México, Mexico Graeme Bruce Martin, University of Western Australia, Australia

### \*Correspondence:

Claude Fabre-Nys, Physiologie de la Reproduction et des Comportements, Centre INRA Val de Loire, 37380, Nouzilly, France<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience

> Received: 18 December 2014 Accepted: 16 March 2015 Published: 09 April 2015

### Citation:

Fabre-Nys C, Kendrick KM and Scaramuzzi RJ (2015) The "ram effect": new insights into neural modulation of the gonadotropic axis by male odors and socio-sexual interactions. Front. Neurosci. 9:111. doi: 10.3389/fnins.2015.00111 Reproduction in mammals is controlled by the hypothalamo-pituitary-gonadal (HPG) axis under the influence of external and internal factors such as photoperiod, stress, nutrition, and social interactions. Sheep are seasonal breeders and stop mating when day length is increasing (anestrus). However, interactions with a sexually active ram during this period can override the steroid negative feedback responsible for the anoestrus state, stimulate luteinizing hormone (LH) secretion and eventually reinstate cyclicity. This is known as the "ram effect" and research into the mechanisms underlying it is shedding new light on HPG axis regulation. The first step in the ram effect is increased LH pulsatile secretion in anestrus ewes exposed to a sexually active male or only to its fleece, the latter finding indicating a "pheromone-like" effect. Estradiol secretion increases in all ewes and this eventually induces a LH surge and ovulation, just as during the breeding season. An exception is a minority of ewes that exhibit a precocious LH surge (within 4 h) with no prior increase in estradiol. The main olfactory system and the cortical nucleus of the amygdala are critical brain structures in mediating the ram effect since it is blocked by their inactivation. Sexual experience is also important since activation (increased c-fos expression) in these and other regions is greatly reduced in sexually naïve ewes. In adult ewes kisspeptin neurons in both arcuate and preoptic regions and some preoptic GnRH neurons are activated 2 h after exposure to a ram. Exposure to rams also activates noradrenergic neurons in the locus coeruleus and A1 nucleus and increased noradrenalin release occurs in the posterior preoptic area. Pharmacological modulation of this system modifies LH secretion in response to the male or his odor. Together these results show that the ram effect can be a fruitful model to promote both a better understanding of the neural and hormonal regulation of the HPG axis in general and also the specific mechanisms by which male cues can overcome negative steroid feedback and trigger LH release and ovulatory cycles.

Keywords: ram effect, odor, LH, estradiol, main olfactory system, experience, noradrenaline, kisspeptin

## Introduction

Reproduction is essential for the survival and evolution of species and in most vertebrates it is controlled by similar networks of hormonal signals. The key regulator of the network is the hypothalamic neuropeptide, gonadotrophin releasing hormone (GnRH), which controls the release of the pituitary hormones, luteinizing hormone (LH), and follicle stimulating hormone (FSH). These latter hormones then stimulate the gonads to produce functional gametes and secrete estradiol, progesterone and testosterone that sustain reproductive function and auto-regulate the activity of the gonadotrophic axis by modulating the secretion of GnRH, LH, and FSH through positive and negative feedback systems. The mechanisms involved in the regulation of this network, often referred to as the hypothalamo-pituitary gonadal axis (HPG), have been the object of abundant research for several decades (Knobil, 1981; Karsch, 1984) but a central question which remained unresolved was how sex steroids modulate the activity of GnRH neurons while the latter lack receptors for steroids involved in feedback action (Herbison, 1998). However, the discovery of kisspeptin-containing neurons as being the most potent secretagogues of GnRH (Messager et al., 2005), and the recent observation that, in mice, all kisspeptin neurons projecting on GnRH neurons have estradiol receptors (Kumar et al., 2015) make them the most probable target of steroid action (Clarkson and Herbison, 2009) and has opened up a new era in this field of research.

The HPG axis is also modulated by many internal and external factors such as nutrition, stress, immunological status, physical, and social environment (Signoret, 1980; Tomaszewska-Zaremba and Herman, 2009; Dobson et al., 2012; Follett, 2014; Roa and Tena-Sempere, 2014) but the mechanisms involved are largely unclear. The effects of the social environment are particularly intriguing because they are very diverse. In mammals they can inhibit reproduction such as in naked mole rat social groups in which reproduction is restricted to a few individuals (Goldman et al., 2006). This is also the case in marmoset family groups where the presence of the mother inhibits reproduction of her daughters (Abbott et al., 1981) or in mice where overcrowding can block reproduction (Whitten, 1959). In contrast, the presence of a sexual partner in many mammalian species can stimulate reproduction (Signoret, 1980; Vandenbergh, 2006) and may even be necessary for females to ovulate, such as in the cat, rabbit, and camel (Bakker and Baum, 2000). In sheep contact with a sexual partner has profound effects on reproductive events at all stages of reproductive life; it hastens puberty (Dyrmundsson and Lees, 1972), induces ovulation during seasonal anestrus (see review by Ungerfeld, 2007a,b) or lactational anestrus (Mauléon and Dauzier, 1965), and modifies the timing of the LH surge during the breeding season (Lindsay et al., 1975). The most spectacular and best known effect is the induction of ovulation in sexually quiescent females by exposure to a sexually active male, a phenomenon known as the "ram effect" in sheep (Martin et al., 1986; Ungerfeld, 2007a).  The "ram effect" was discovered in 1944 (Underwood et al., 1944) but studies on the mechanisms involved only started in the 80's when reliable LH assays became readily available and when the sheep, because of their size, availability and economic importance became a widely used model for the study of the HPG axis (Karsch et al., 1997). The object of this review is to summarize what we have learned in the last few decades about the mechanisms involved in the "ram effect" and to discuss how this knowledge could help us to understand the regulation of the HPG axis more generally.

## Description of the "Ram Effect"

Sheep are seasonal breeders. Ewes have regular 17 days oestrous cycles when day length is decreasing (the breeding season) and give birth in the spring when the environmental conditions are most favorable for the survival of their young. As day length increases ewes stop cycling (anestrus) but the introduction of a sexually active ram into a group of seasonally anoestrous animals will induce a pulse of LH (the short-term LH response **Figure 1A**) within minutes, whereas FSH does not undergo such a rapid change (Martin et al., 1980; Poindron et al., 1980). Exposure to a ram, or his odor, does not induce marked behavioral changes in ewes, but seems to focus their attention and induce urination (Gelez et al., 2004a). If contact with the male is maintained, the increase in pulsatile LH secretion initiates a sequence of physiological events that in some ewes will culminate in a LH surge 6–54 h later (Oldham et al., 1978; Chanvallon et al., 2011). Ovulation nearly always follows the LH surge (100% cases in Chanvallon et al., 2011; 97% in Scaramuzzi et al., 2014) but is described as "silent" because it is not associated with estrous behavior. Some females will then have a normal estrous cycle with a luteal phase lasting approximately 10 days and display estrous behavior 17– 20 days after the introduction of rams (**Figure 1B**). In others, the corpus luteum from this first "silent" ovulation does not develop normally and regresses after a few days with a resultant short cycle; the ewe starts a new cycle but without a display of estrus (Oldham and Martin, 1978). Sexual behavior in these females only appears 22–28 days after the introduction of ram at the time of the third ovulation (**Figure 1B**). If ewes are mated at the time of the male induced estrus, a synchronized pattern of lambing occurs with two peaks 164 and 172 days afterwards. This singular pattern of births (**Figure 1C**) is the origin of the discovery of this phenomenon (Underwood et al., 1944) and could be used a convenient tool to identify those breeds responding to the "ram effect."

Nearly all adult ewes have a short-term LH response after exposure to a sexually active ram during anestrus (93% in Chanvallon et al., 2011; 92% in Scaramuzzi et al., 2014). However, the intensity of this short term response varies and LH pulsatility after the "ram effect" is lower in ewes with low as opposed to high body condition (Scaramuzzi et al., 2014). An analysis of the LH response to a bolus of GnRH (75 ng) given to the same animals the day before they were exposed to the "ram effect" indicated that at least part of this variability was due to altered sensitivity of the pituitary. This was due to ewes with a low body condition having LH pulses of a lower amplitude in response to the GnRH

bolus than those with a high body condition (Scaramuzzi et al., 2014).