Evolutionary formation of melatonin and vitamin D in early life forms: insects take centre stage

Abstract

Melatonin, a product of tryptophan metabolism via serotonin, is a molecule with an indole backbone that is widely produced by bacteria, unicellular eukaryotic organisms, plants, fungi and all animal taxa. Aside from its role in the regulation of circadian rhythms, it has diverse biological actions including regulation of cytoprotective responses and other functions crucial for survival across different species. The latter properties are also shared by its metabolites including kynuric products generated by reactive oxygen species or phototransfomation induced by ultraviolet radiation. Vitamins D and related photoproducts originate from phototransformation of Δ5,7 sterols, of which 7-dehydrocholesterol and ergosterol are examples. Their Δ5,7 bonds in the B ring absorb solar ultraviolet radiation [290–315 nm, ultraviolet B (UVB) radiation] resulting in B ring opening to produce previtamin D, also referred to as a secosteroid. Once formed, previtamin D can either undergo thermal-induced isomerization to vitamin D or absorb UVB radiation to be transformed into photoproducts including lumisterol and tachysterol. Vitamin D, as well as the previtamin D photoproducts lumisterol and tachysterol, are hydroxylated by cyochrome P450 (CYP) enzymes to produce biologically active hydroxyderivatives. The best known of these is 1,25-dihydroxyvitamin D (1,25(OH)₂D) for which the major function in vertebrates is regulation of calcium and phosphorus metabolism. Herein we review data on melatonin production and metabolism and discuss their functions in insects. We discuss production of previtamin D and vitamin D, and their photoproducts in fungi, plants and insects, as well as mechanisms for their enzymatic activation and suggest possible biological functions for them in these groups of organisms. For the detection of these secosteroids and their precursors and photoderivatives, as well as melatonin metabolites, we focus on honey produced by bees and on body extracts of Drosophila melanogaster. Common biological functions for melatonin derivatives and secosteroids such as cytoprotective and photoprotective actions in insects are discussed. We provide hypotheses for the photoproduction of other secosteroids and of kynuric metabolites of melatonin, based on the known photobiology of Δ5,7 sterols and of the indole ring, respectively. We also offer possible mechanisms of actions for these unique secosteroids and summarize differences and similarities of melatoninergic and secosteroidogenic pathways in diverse organisms including insects.

I. INTRODUCTION

Melatonin and vitamin D are biologically important molecules with a variety of interactive regulatory properties that have long captured the attention of both scientific and non-scientific communities, and continue to be at the centre of public interest. Melatonin is a molecule with a indole backbone and a molecular mass (mw) of 232 g/mol (Lerner, Case & Takahashi, 1960; Zisapel, 2018) for which an important role is regulation of the circadian rhythm (Reiter, 1991), even in zooplankton (Tosches et al., 2014) and organisms devoid of an organized central nervous system (Nath et al., 2017). It also provides cytoprotection against environmental insults, oxidative stress and different types of ionizing radiation (Tan et al., 2014; Reiter et al., 2017; Vijayalaxmi et al., 2004; Nuszkiewicz, Woźniak & Szewczyk-Golec, 2020; Slominski et al., 2014c; Reiter et al., 2016). Melatonin, being an estimated 2.5 billion years old (Reiter et al., 2017), is widely produced in nature from prokaryotes (bacteria, cyanobacteria, archea) to unicellular eukaryotes and to complex multicellular organisms including plants, fungi and all animal taxa (Tan et al., 2014; Zhao et al., 2019; Reiter et al., 2015; Back, 2021; Back, Tan & Reiter, 2016). In the pineal gland of vertebrates, melatonin synthesis occurs during the night with a sharp reduction during the day due to the inhibitory action of visible light, especially blue wavelengths. In all other tissues where melatonin has been identified, its synthesis is usually non-circadian (Acuña-Castroviejo et al., 2014).

Vitamin D3 and vitamin D2 (collectively referred to as vitamin D) with respective masses of 384 and 396 g/mol) are products of phototransformation of 7-dehydrocholesterol (7-DHC) and ergosterol respectively. Absorption of ultraviolet B (UVB) energy (290–315 nm, peak at 295 nm) by the 5,7-diene in the B-ring results in the opening of the B ring to form the secosteroid previtamin D (Holick, 2003; Bikle, 2011; Wacker & Holick, 2013). Previtamin D2 and D3 are thermodynamically unstable and undergo an isomerization to form vitamin D2 and D3, respectively. During exposure to UVB radiation previtamin D2 and D3 can also absorb UVB energy and be converted to several photoproducts including lumisterol and tachysterol (MacLaughlin, Anderson & Holick, 1982; Holick et al., 1980; Bikle, 2011; Holick, Tian & Allen, 1995; Wacker & Holick, 2013; Dauben & Baumann, 1961; Havinga, 1973; Boomsma et al., 1977). In addition, any other 5,7 diene sterol with or without a side chain and with or without additional hydroxyl groups (Zmijewski et al., 2008, 2009, 2011; Slominski et al., 2013, 2004, 2020b) when exposed to solar UVB radiation will also be converted to previtamin D and then to vitamin D. Good examples are vitamins D4 and D5 which are formed through photochemical transformation of 22,23-dihydroergosterol and 7-dehydrositosterol, respectively (Phillips et al., 2012; Jäpelt & Jakobsen, 2013; Silvestro et al., 2013). Holick, Holick & Guillard (1982b,a) and Holick (1989, 2003) reported the presence of ergosterol, 7-DHC and several other unidentified 5,7-diene sterols in plankton net tows from the Sargasso Sea. In the brine shrimp (Artemia parthenogenetica) and krill (Euphausiacea) only 82% and 74%, respectively, of the 5,7-diene sterols present were 7-DHC. It was also observed by Holick et al. (1982b,a) and Holick (1989, 2003) that Emiliania huxlei, a phytoplankton species that has existed in the Sargasso Sea unchanged for more than half a billion years, had 1 μg of ergosterol/g wet mass. This large amount of ergosterol must have a vital role in its survival. The ergosterol could be an ideal sunscreen because of its absorption of the UVB and longer UVC wavelengths of solar radiation to protect ultraviolet radiation (UVR)-sensitive macromolecules including DNA, RNA and proteins from the UVR damage. Ergosterol absorbs UVB radiation to form previtamin D2. The UVR absorption spectrum for previtamin D is essentially identical to that absorbed by RNA and DNA with a maximum at 260 nm. This could have played an essential role in the survival of organisms dependent on sunlight when UVC was able to penetrate the Earth’s surface because of limited ozone in the atmosphere, potentially causing excessive DNA and RNA damage (Holick, 2003). Another hypothesis for how 5,7-diene sterols and vitamin D became so intimately related to calcium and bone metabolism in land vertebrates was the observation that the structurally rigid flat four-ring 7-DHC has a structure sandwiched in between the fatty acid side chains and the polar head group of phospholipids in the plasma membrane. After exposure to UVB this planar structure opens to form previtamin D3 leading to the planar cis-cis conformer of previtamin D3 undergoing structural rearrangement to form the more thermodynamically stable vitamin D3. Vitamin D3, which has a non-planar structure, is more easily released from the plasma membrane into the extracellular environment (Holick et al., 1995). It has been suggested that this process causes formation of an entrance pore which enhances the entry of calcium into the cell (Holick, 2003). Vitamin D was likely present from the beginning of cellular life (Bikle, 2011; Holick, 2003) since it is present in all plants, animals and fungi. It might be 2 billion years old, since ergosterol and other 5,7-dienal sterols are produced by phytoplankton, zooplankton, amoebas and other eukaryotic unicellular organisms (Voshall et al., 2021; Raederstorff & Rohmer, 1987; Darnet et al., 2021; Holick, 1989, 2003; Holick et al., 1982b,a). Thus, the presence of secosteroids in living organisms provides a record of exposure to UVB (Wacker & Holick, 2013; Slominski et al., 2018d).

While melatonin in vertebrates is generally considered the ‘messenger of the night’, and vitamin D the ‘messenger of the sun’ (Sniadecki, 1840; Mozolowski, 1939; McCollum et al., 1922; Holick et al., 1980; Slominski et al., 2018d; Holick & Slominski, 2024), it should be noted that absorption of UVB by the indole ring of melatonin leads to its breakage, with production of kynuric compounds including AFMK (N(1)-acetyl-N(2)-formyl-5-methoxykynuramine) with possible conversion to AMK (N(1)-acetyl-5-methoxykynuramine) (Fischer et al., 2006; Slominski et al., 2017c). In plants, AFMK appears to be more biologically active than melatonin in the induction of cytoprotective responses (Tan et al., 2007). Thus, during the exposure of non-vertebrate organisms to sunlight, melatonin can serve as a precursor for a family of biologically active compounds. While the production and predicted functions of melatonin across different group of organisms is highly diverse (Zhao et al., 2019; Tan et al., 2014; Back et al., 2016), there is a significant gap in our knowledge about vitamin D3 except for its function as a prohormone in vertebrates (Wacker & Holick, 2013; Holick, 2003; Bikle, 2011). For insects, the production and role of melatonin requires an informed overview with a proper perspective, while for vitamin D this field is open for investigation and interpretation.

II. INSECTS IN A ‘NUTSHELL’

Insects are the largest (80%) and most diverse group of animals on Earth. They evolved from crustaceans 480 million years ago at about the same time as terrestrial plants appeared (Misof et al., 2014), with flying insects originating around 370–440 million years ago (Schachat et al., 2022; Elias, 2023). Insects are present in almost every habitat on Earth, playing important roles as pollinators, decomposers, prey and predators. While cholesterol is the dominant tissue sterol for most insects, their cells lack enzymes to synthesize it de novo from its precursor 7-DHC, and therefore these sterols are derived from food, enzymatic transformation of plant or fungal sterols or are provided by symbioses with yeasts or fungi (Janson et al., 2009; Jing & Behmer, 2020; Nasir & Noda, 2003; Vaitsopoulou et al., 2022; Wen et al., 2023). However, cholesterol can be metabolized by an insect C-7 cholesterol dehydrogenase (Neverland, Nvd) to 7-DHC (Wen et al., 2023; Yoshiyama et al., 2006; Gilbert, Rybczynski & Warren, 2002; Zhu et al., 2019; Ekoka et al., 2021). It has also been found that sterols are absorbed from food in the midgut by Niemann-Pick type C1 (NPC1) receptors, which are also highly expressed in the intestine of mice. In Drosophila melanogaster, NPC1a and NPC1b receptors have been identified that exhibit 42% and 35% identity to the human NPC1 protein, respectively (Fluegel, Parker & Pallanck, 2006). NPC1a null mutants of D. melanogaster are lethal at an early larval stage. They can be partially rescued if the larvae are maintained on a high-cholesterol diet or a diet containing 20-hydroxyecdysone, an insect steroid hormone. It has been suggested that NPC1a promotes trafficking of sterols between cells in many D. melanogaster tissues, particularly to the ring gland where sterols are needed for ecdysone synthesis (Fluegel et al., 2006). The steroid hormone ecdysone plays a crucial role in regulating insect developmental stages and metamorphosis. In addition, many neurotransmitters present in mammals, including serotonin and serotonin receptors, are also present in all insects serving as a signalling system that regulates various aspects of their physiology, in a fashion that depends on the type of receptor involved (Blenau & Thamm, 2011; Lubawy et al., 2020; Farooqi, Ali & Amir, 2022). Serotonin is also a precursor for melatonin. Although melatonin was detected in the majority of insects tested, its role and mechanism of action are less defined (Farooqi et al., 2022; Fan et al., 2021; Zhao et al., 2019). Melatonin in insects is probably involved in regulating photoperiodism, which regulates diapause (an adaptive phenotype that allows persistence in variable environments; Hand et al., 2016) and other processes such as polyphenism (phenotypic plasticity in response to environmental changes; Simpson, Sword & Lo, 2011), reproduction, behaviour, and pigmentation in various insect species. Melatonin metabolism is controlled by arylalkylamine N-acetyltransferase (AANAT) and its enzymatic activity is affected by photoperiod. The activity of AANAT and the content of melatonin in the insect’s brain depend not only on photoperiod but also show circadian oscillations. Moreover, expression of the AANAT gene is circadian at least in some examined insect species. AANAT transcription can be downregulated by silencing with RNA interference (RNAi) cycle (cyc) or Clock (Clk) expression which are involved in the molecular mechanism of the circadian clock, while RNAi against period (per), one of the main clock genes, upregulates AANAT transcription. The clock neurons in the brain express AANAT and hydroxyindole-O-methyltransferase (HIOMT) in the Chinese oak silk moth (Antharaea pernyi) (Mohamed et al., 2014; Wang et al., 2015). In D. melanogaster two AANATs and oscillations of melatonin in the brain have been identified (Amherd et al., 2000; Hintermann et al., 1996).

Our analyses will focus on D. melanogaster because of its experimental utility (Ugur, Chen & Bellen, 2016; Hales et al., 2015; Yamaguchi & Yoshida, 2018; Costa-Rodrigues, Couceiro & Moreno, 2021; Mohr & Perrimon, 2019), and on the honey bee Apis mellifera because of the importance of this species in the production of honey (Kim et al., 2020, 2022; Crane, 1991, 2020; Waheed et al., 2019). Bees and flies undergo complete metamorphosis, however, there are differences in development and how ecdysone regulates this process. Bees are active during the daytime while D. melanogaster is active during dawn and dusk. Bees use UVB vision to locate flowers, distinguish their shape and colour, and find nectar and pollen. It appears that UVB is important for their survival and well-being (Dyer & Garcia, 2014). It has been reported that in D. melanogaster, UVR including UVB, has a negative effect such as decreasing their lifespan and leading to physical abnormalities and changes in behaviour (Wang et al., 2008), however, our unpublished results did not confirm that (K. Briedikova, A. Naumowicz & E. Pyza, unpublished data). Moreover, UVB has a positive effect on the survival of house crickets (Psarianos et al., 2022). Photoreceptor cells named R1–R8 of the D. melanogaster compound eye detect UV light via the R7 photoreceptor (Morante & Desplan, 2008; Johnston, 2013), and are protected against UV-induced DNA damage by the enzyme heme oxygenase (Damulewicz et al., 2018, 2017). Its expression is controlled by the circadian clock and is high at the beginning of the day when UV exposure is highest.

III. MELATONIN

(1). Overview

In diverse organisms on Earth including plants, fungi, bacteria, unicellular eukaryotes and animals, melatonin is synthesized from tryptophan with serotonin being an important intermediate (Fig. 1) (Zhao et al., 2019; Back et al., 2016; Reiter et al., 2015; Tan et al., 2014; Hardeland, 2015, 2016; Wang et al., 2023; Jiao et al., 2016). For example, in plants tryptophan, which is synthesized through the shikimic acid pathway, is decarboxylated to tryptamine by tryptophan decarboxylase (TDC) and then converted to serotonin by tryptamine 5-hydroxylase (T5H) (Fig. 1A) (Tan et al., 2014, 2016; Back et al., 2016; Hardeland, 2015; Back, 2021; Zhao et al., 2019). In addition, in plants serotonin is metabolized by either acetylserotonin-O-methyltransferase (ASMT) or caffeic acid-O-methyltransferase (COMT) to 5-methoxytryptamine (5MT) which can be methylated by serotonin N-acetyltransferase (SNAT) to give melatonin (Fig. 1B) (Tan et al., 2014, 2016; Zhao et al., 2019). The majority of species in the animal kingdom transform tryptophan to 5-hydroxytryptophan by tryptophan hydroxylase. This product then undergoes enzymatic decarboxylation to serotonin followed by acetylation and methylation to generate melatonin (Axelrod & Weissbach, 1960; Lerner et al., 1960; Tan et al., 2016; Slominski, Wortsman & Tobin, 2005c; Reiter et al., 2017; Reiter, 1991). These reactions are catalysed by tryptophan hydroxylase (TPH), aromatic amino acid decarboxylase (AADC), serotonin N-acetyltransferases [AANAT/SNAT or arylamine N-acetyltransferase (NAT)] and HIOMT/ASMT, or their homologs or paralogs depending on the taxon (Back et al., 2016; Reiter et al., 2016, 2017; Tan et al., 2016, 2014; Slominski et al., 2008). It should be noted that plant SNAT has significant differences in its amino acid sequence from that of human AANAT/SNAT (Hwang & Back, 2022; Liao et al., 2021; Arnao, Cano & Hernández-Ruiz, 2022). In mammals, serotonin can also be acetylated by other broad-spectrum NATs (Slominski et al., 2005c, 2002, 2003; Semak et al., 2004), explaining why melatonin is synthesized in AANAT-deficient mice (Roseboom et al., 1998).

Fig. 1.

Melatonin synthesis pathways in different species. (A) Biosynthetic pathway in plants. (B) Biosynthetic pathway in animals. AADC, aromatic amino acid decarboxylase; AcCoA, acetyl coenzyme A; ASMT, acetylserotonin-O-methyltransferase; BH3OH, 4α-hydroxytetrahydrobiopterin; BH4, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin; CoA, coenzyme A; COMT, caffeic acid-O-methyltransferase; HIOMT, hydroxyindole-O-methyltransferase; PLP, pyridoxal 5-phosphate; PMP, pyridoxamine phosphate; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine; SNAT, serotonin N-acetyltransferase; T5H, tryptamine 5-hydroxylase; TDC, tryptophan decarboxylase; TPH, tryptophan hydroxylase.

(2). Detection of serotonin, N-acetyl-serotonin (NAS), melatonin and its metabolites in insects and honey

In insects, synthesis of melatonin follows the pathway common to vertebrates (Farooqi et al., 2022; Subala & Shivakumar, 2018). Thus, TPH oxidizes L-tryptophan to 5-hydroxytryptophan using (6R)-L-erythro-5,6,7,8-tetrahydrobiopterine (BH4) as the cofactor (Fig. 1B). This is followed by decarboxylation by AAD using pyridoxal 5-phosphate (PLP) as the cofactor, to produce serotonin (Fig. 1B). Serotonin is acetylated to N-acetyl serotonin (NAS) by AANAT/SNAT (Smith, 1990; Han et al., 2012) and further methylated by HIOMT/ASMT as in vertebrates (Fig. 1B) (Farooqi et al., 2022; Subala & Shivakumar, 2018). The conversion of serotonin to NAS also requires the cofactor/co-substrate, acetyl coenzyme A. It is also likely that alternative pathways of serotonin acetylation by broad-spectrum NAT described in vertebrates (Slominski et al., 2003; Gaudet et al., 1993) operate in insects (Han et al., 2012; Bhandari, Bisht & Merkler, 2021; Battistini et al., 2019; Futahashi et al., 2022; Tang et al., 2023). Orthologues of AANAT are even present in bacteria (Tang et al., 2023). In insects, AANAT/NATs play multiple roles in addition to melatonin synthesis, being involved in the metabolism of aromatic neurotransmitters including the inactivation of biogenic amines, and cuticle sclerotization (Smith, 1990; Battistini et al., 2019). In insects, AANAT is also involved in melanin synthesis which follows the melanogenic pathway in this taxon unlike in vertebrates (Futahashi et al., 2022). In addition, 5-hydroxytryptophan can be generated from tryptophan non-enzymatically through direct action of UVA, or by reactive oxygen species (ROS) which can also be generated by UVA (Slominski et al., 2017c; Schallreuter et al., 2012). Therefore, the serotoninergic and melatoninergic pathways may not require TPH since during the daytime diurnal insects are exposed to solar light which may induce non-enzymatic conversion of tryptophan to 5-hydroxytryptophan, a direct precursor of serotonin.

The presence of serotonin, NAS and melatonin in a wide range of insects is well documented (Smith, 1990; Vieira et al., 2019; Fan et al., 2021; Farooqi et al., 2022; Nehela & Killiny, 2021; Zhao et al., 2019; Kim et al., 2021; Lima et al., 2023). Serotonin and melatonin are present in heads, eyes and other insects organs where they regulate a wide range of physiological processes, as well as protecting against pathologies and noxious environmental factors such as plasticizers and particulate matter in the air (Farooqi et al., 2022). They serve as neurotransmitters and hormones, especially serotonin. They also act as biological modifiers/regulators of a variety of developmental, behavioural, immune, endocrine, developmental and defensive and protective processes, and are involved in the regulation of biological cycles including the circadian rhythm (Farooqi et al., 2022; Wong et al., 2023; Muthusamy et al., 2023; Fan et al., 2021; Vieira et al., 2019; Paoli, Macri & Giurfa, 2023; Pantalia et al., 2023; Gajardo, Guerra & Campusano, 2023; Aprison, Dzitoyeva & Ruvinsky, 2023; Smith, 1990). In addition, melatonin plays a central role in protection against environmental insults including radiation and oxidative stress (Muthusamy et al., 2023; Li et al., 2022; Farooqi et al., 2022; Fan et al., 2021; Ertugrul et al., 2020; Zhao et al., 2019). Thus, the photoprotective, radioprotective and protective properties of these compounds against oxidative stress are conserved across different species (Fischer et al., 2006, 2008, 2013; Kleszczyński, Zillikens & Fischer, 2016; Slominski et al., 2005c; Slominski et al., 2014c, 2017d, 2018b; Reiter et al., 2015, 2016, 2017; Tan et al., 2014, 2016; Vijayalaxmi et al., 2004; Janjetovic et al., 2017; Skobowiat et al., 2018; Nuszkiewicz et al., 2020; Ertugrul et al., 2020; Zhao et al., 2019; Khatoon et al., 2019; Hardeland, 2016; Fan et al., 2021; Millet-Boureima et al., 2021; Ortega-Arellano, Jimenez-Del-Rio & Velez-Pardo, 2021; Arnao et al., 2022; Hwang & Back, 2022; Li et al., 2022; Wong et al., 2023; Muthusamy et al., 2023; Stefan et al., 2021). The regulatory actions of serotonin are mediated through interactions with membrane-bound G-protein coupled receptors (GPCRs) which are present in insects (Blenau & Thamm, 2011; Lubawy et al., 2020; Farooqi et al., 2022). For example, D. melanogaster has five types of serotonin receptors: 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, and 5-HT7, while the honey bee has three types: Am5-HT1A, Am5-HT2α, and Am5-HT7. There is a shortage of information relating to the presence of receptors in insects that are equivalent to the membrane-bound melatonin receptors, MT1 and MT2, present in vertebrates (Dubocovich et al., 2010; Slominski et al., 2012c). However, MT-like receptors were detected in bees (Li et al., 2018; Kong et al., 2021) and an MT2-like receptor in the American cockroach Periplaneta americana (Kamruzzaman et al., 2021). It is also unclear whether MT1 and MT2 homologues are present in D. melanogaster (Millet-Boureima et al., 2021). It is possible that in insects melatonin may exert its actions in a fashion independent of membrane-bound receptors as described in vertebrates (Slominski et al., 2012c; Reiter et al., 2016; Slominski et al., 2023a). Even less defined is the role of NAS, aside from being a precursor of melatonin or playing a role in sclerotization, as described above. We predict that it will be involved in protective responses against solar radiation and oxidative stress (Millet-Boureima et al., 2021; Janjetovic et al., 2017, 2014; Slominski et al., 2017d; Skobowiat et al., 2018) or regulation of other functions (Slominski et al., 2017d, 2020c) as described in humans.

Importantly, serotonin, NAS and melatonin are present in the honey produced by bees. Using analytical methods of high-performance liquid chromatography (HPLC) separation and mass spectrometry (MS), we detected the presence of tryptophan, serotonin, NAS and melatonin (Kim et al., 2021, 2022). These findings were confirmed independently by other investigators (Borges et al., 2022; Lima et al., 2023). The significance of these findings in relation to bees and their colony well-being, as well as to human nutrition and health, has been discussed in detail (Borges et al., 2022; Lima et al., 2023; Kim et al., 2021, 2022).

Interestingly, we also detected products of melatonin metabolism in honey (Kim et al., 2022, 2021). These included 2-hydroxymelatonin, AFMK and AMK (Kim et al., 2022, 2021). Melatonin can be metabolized enzymatically though the indolic pathway, and both enzymatically and non-enzymatically through kynureic pathways across different species, including plants (Slominski et al., 2017c; Slominski et al., 2008; Zhao et al., 2019; Tan et al., 2007; Back, 2021; Fischer et al., 2006). 2-hydroxymelatonin, AFMK and AMK are also produced in plants (Back, 2021; Fischer et al., 2006; Hardeland, 2015, 2016; Tan et al., 2007; Zhao et al., 2019) so its nutritional delivery to bees represents an additional explanation for its presence in honey. In addition, 2-hydroxymelatonin, AFMK and AMK can be produced non-enzymatically either through ROS-dependent mechanisms or by the direct action of UVR on melatonin (Slominski et al., 2017c; Fischer et al., 2006; Hardeland, Tan & Reiter, 2009; Slominski et al., 2008). Therefore, it is likely that when insects are exposed to solar light, melatonin will be transformed to its derivatives, some of which may even be more potent anti-oxidative molecules (Tan et al., 2007). Therefore, we speculate that UVB-induced breaking of the indole ring in some species may lead to generation of more potent protective molecules as would be expected from the logic of UVR-driven evolution (Slominski et al., 2018d).

While production of serotonin and melatonin from tryptophan in D. melanogaster is well documented (see above), there is a shortage of information on the kynuric pathway of melatonin metabolism. Therefore, we used the quadrupole time of flight liquid chromatography/mass spectrometry (qTOF-LC/MS) methodology to detect AFMK and AMK in body extracts from D. melanogaster as described in Kim et al. (2022, 2021) (see online Supporting Information, Appendix S1 and Fig. S1). Similarly to honey, AFMK and AMK were detected in D. melanogaster body extracts, confirming the ability of these insects to produce, metabolize or accumulate serotonin and melatonin derivatives.

In summary, serotoninergic/melatoninergic systems play important roles in the physiology of insects. While the serotoninergic system in insects is well defined, the melatoninergic system provides fertile ground for further investigations. We confirmed that the serotoninergic and melatoninergic systems both operate in D. melanogaster. In addition, honey contains precursor, intermediate and final products of these metabolic pathways including tryptophan, hydroxytryptophan, serotonin, NAS, melatonin, 2-hydroxymelatonin, AFMK and AMK, as well as other biogenic amines that may affect its medicinal and perhaps attractive properties.

IV. VITAMIN D PRODUCTION AND METABOLIC ACTIVATION

(1). Production and metabolism: an overview

Vitamin D is not classified as a true vitamin but as a pro-hormone based on the fact that it is generated in animals as a result of their exposure to UVB radiation (Wacker & Holick, 2013; Bikle, 2011; Deluca, 2014; McCollum, 1967; Holick et al., 1980; Carlberg, 2022). The physiological functions of vitamin D are well recognized. Vitamin D is essential for most vertebrates to absorb dietary calcium and phosphate efficiently to support circulating concentrations of these minerals in the normal range. Maintaining normal blood calcium and phosphate concentrations is critically important for most metabolic functions including the activity of neurons and muscles. In addition, maintaining a normal serum calcium-phosphorus product concentration is required for the deposition of calcium hydroxyapatite into the collagen matrix of the skeleton. This mineralization is suggested to represent a key step in the evolution of a structurally sound skeleton for vertebrates to survive on land (Wacker & Holick, 2013; Keegan et al., 2013; Holick, 2003; Holick et al., 1995). To date, most attention has focused on vitamins D3 and D2, and their role in vertebrate physiology and pathology (Hewison et al., 2024). Deficiency in vitamin D in humans has multiple health consequences (Holick, 2007), including rickets in children and osteopenia/osteoporosis and osteomalacia in adults (Holick, 2003; Bouillon et al., 2019; Bikle, 2020; Bikle & Christakos, 2020; Holick, 2007; Hewison et al., 2024).

The absorption of UVB energy by the B ring of Δ5,7 sterols generates thermodynamically unstable previtamin D which can either undergo thermal isomerization to vitamin D or can absorb UVB radiation to form photoproducts including lumisterol and tachysterol. Δ5,7 sterols are important intermediates in sterol synthesis in different species including plants, fungi, some bacteria, algae, unicellular organisms including amoebas, invertebrates and vertebrates (Phillips et al., 2012; Jäpelt & Jakobsen, 2013; Silvestro et al., 2013; Bikle, 2011; Voshall et al., 2021; Raederstorff & Rohmer, 1987; Darnet et al., 2021; Urbain, Valverde & Jakobsen, 2016; Holick et al., 1995; Keegan et al., 2013; Deluca, 2014; Guo et al., 2018; Slominski et al., 2015c; Hewison et al., 2024; Göring, 2018; Jiang et al., 2020; Nes, 2011; Lee, Wei & Welander, 2023; Pearson, Budin & Brocks, 2003; Brocks et al., 2023), and different secosteroidal configurations can be generated (Fig. 2). Some bacteria, including Carnobacterium maltaromaticum, can produce 7-DHC (Li et al., 2023), which is consistent with a report of cholesterol synthesis by some bacteria (Lee et al., 2023). There is significant variability in the content of Δ5,7 sterols and vitamin D in plants. Rye grass (Lolium perenne) exposed to sunlight generates both vitamin D2 and vitamin D3. Some plants in the Solanum family, such as Solanum alacoxylon, produce glycoside derivatives of 1,25-dihydroxyvitamin D3 (1,25(OH)₂D3). This observation explains why cattle ingesting this plant at the end of the summer become hypercalcemic and can die as a result of soft tissue calcification of their cardiovascular system. Some plants also esterify provitamin Ds. These various modifications often make detection difficult (Jäpelt & Jakobsen, 2013; Black et al., 2017; Jäpelt et al., 2011, 2013; Khan et al., 2022; Göring, 2018).

Fig. 2.

Products of ultraviolet B (UVB)-induced phototransformation of Δ5,7 sterols present in animals, plants, fungi and unicellular organisms including bacteria.

The vitamin D3 and vitamin D2 activation and inactivation pathways are similar in many vertebrates, including dogs and cats, farm animals and primates (Hewison et al., 2024). It is now recognized that vitamin D is inactive in the regulation of calcium and phosphate metabolism and requires sequential hydroxylations to become active. Vitamin D is first metabolized in the liver by one of the vitamin D-25 hydroxylases (CYP2R or CYP27A1), with the most prominent and active one being CYP2R1. As a result of this metabolism, vitamin D3 and vitamin D2 are converted to their respective 25-hydroxylated derivatives, 25(OH)D3 and 25(OH)D2, respectively (Hewison et al., 2024; Holick & Clark, 1978; Holick, Smith & Pincus, 1987; Bikle, 2020; Bikle & Christakos, 2020; Bouillon et al., 2019; Tuckey, Cheng & Slominski, 2019a; Jenkinson, 2019; Deluca, 2014; Holick & DeLuca, 1974; Zhu et al., 2013; Holick, 2007). These are the clinically important major circulating forms of vitamin D and are measured to determine a patient’s vitamin D status. 25(OH)D at physiological concentrations is also biologically inactive on calcium and phosphate metabolism and requires an essential hydroxylation in the kidneys by the 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) to place a hydroxyl group on carbon 1 in the alpha position to form the biologically active form of vitamin D, 1,25(OH)₂D (Hewison et al., 2024; Holick & Clark, 1978; Holick et al., 1987; Bikle, 2020; Bikle & Christakos, 2020; Bouillon et al., 2019; Tuckey et al., 2019a; Jenkinson, 2019; Deluca, 2014; Holick & DeLuca, 1974; Zhu et al., 2013; Holick, 2007). 1,25(OH)₂D is the hormonal form of vitamin D that is responsible for controlling intestinal calcium and phosphate absorption, renal handling of calcium and phosphate and bone resorption of calcium and phosphate. It does this by interacting with the vitamin D receptor (VDR) and unlocking genetic information to control these functions (Hewison et al., 2024; Carlberg, 2018, 2022; Zmijewski & Carlberg, 2020). At the same time it also initiates its own destruction by markedly up-regulating the expression of 1,25-dihydroxyvitamin D-24R-hydroxylase (CYP24A1). This enzyme sequentially oxidizes the side chain of both 25(OH)D3 and 1,25(OH)₂D3 to their respective 23-carboxy metabolites which are biologically inactive and excreted into the bile (Jones, Prosser & Kaufmann, 2012, 2014; Tuckey et al., 2019a; Jenkinson, 2019; Bikle, 2014, 2020; Bikle & Christakos, 2020; Tieu, Tang & Tuckey, 2014). Similarly CYP24A1 oxidizes the side chains of 25(OH)D2 and 1,25(OH)₂D2 with subsequent cleavage of the side chain between C24 and C25 (Li & Tuckey, 2023). Vitamin D is also inactivated through the 3-epimerization of 25(OH)D3 and 1,25(OH)₂D3 where the hydroxyl group in the 3β position is isomerized to the 3α position, producing 3-epi-25(OH)D3 and 3-epi-1,25(OH)₂D3, respectively (Kamao et al., 2004, 2005; Tuckey et al., 2019b). Vitamin D hydroxyderivatives can also undergo phase II conjugation reactions including sulfation and glucuronidation (Higashi et al., 2014; Gomes, Shaw & Hewavitharana, 2016; Axelson & Christensen, 1988; Huynh et al., 2021; Higashi et al., 1999; Jenkinson et al., 2022; Hewison et al., 2024).

(2). Pathways of vitamin D metabolism in unicellular organisms and plants.

For non-vertebrate organisms, the pathways of vitamin D metabolism remain to be established. However, there is evidence that insects, plants, fungi and simple eukaryotes such as algae or symbiotic organisms including lichen not only have the capacity to convert their pro-vitamin Ds to vitamin D (Holick & Stoddard, 2022; Holick, 1989; Oonincx et al., 2018) but also appear to express enzymes with similar activities to the vertebrate vitamin D-25-hydroxylases and 25-hydroxyvitamin D 1α-hydroxylase. Whether these organisms can also produce vitamin D metabolites remains to be determined but it is known is that plants in the Solanum family can produce glycoside derivatives of 1,25(OH)₂D3 (Jäpelt & Jakobsen, 2013; Göring, 2018; Black et al., 2017; Jäpelt et al., 2013; Oonincx et al., 2018; Mello, 2003). However, it must be noted that some bacteria can hydroxylate vitamin D3 to 25(OH)D3, 1,25(OH)₂D3 and other hydroxyderivatives (Ban et al., 2014; Sawada et al., 2004; Szaleniec et al., 2018; Sugimoto et al., 2008; Abdulmughni et al., 2017; Ehrhardt et al., 2016; Jozwik et al., 2023; Putkaradze et al., 2020). The bacterial CYP109E1 can also hydroxylate vitamin D2 (Putkaradze et al., 2020). In addition, it was recently reported that the gut microbiota in mice can hydroxylate vitamin D3 to active hydroxyderivatives in a cooperative manner (Li et al., 2023), however, the claim that bacteria can transform 7-DHC to vitamin D3 still awaits confirmation.

(3). Alternative pathways of vitamin D activation.

Alternative pathways of vitamin D activation have recently been discovered in mammalian systems (Slominski et al., 2023b, 2024) that include CYP11A1-catalysed hydroxylations of the side chains of vitamin D3 (Guryev et al., 2003; Slominski et al., 2005b; Tuckey et al., 2008) and vitamin D2 (Slominski et al., 2006; Nguyen et al., 2009; Slominski et al., 2011), and their precursors 7-DHC (Slominski et al., 2004; Guryev et al., 2003) and ergosterol (Slominski et al., 2005a; Tuckey et al., 2012). Hydroxylated products from the precursors may themselves undergo UVB-induced phototransformation, producing the corresponding secosteroids, as proposed previously (Slominski et al., 2004; Slominski et al., 2020a,b,d, 2018a). These pathways operate in vivo (Slominski et al., 2009, 2012b,a, 2014a, 2015b,a) and their products can be modified by other CYPs including CYP27B1 (Slominski et al., 2015c; Tuckey et al., 2019a; Slominski et al., 2021b; Hewison et al., 2024). It is unclear whether these vitamin D hydroxyderivatives can be produced by other organisms, since CYP11A1 is evolutionarily restricted to vertebrates (Slominski et al., 2021b; Slominski et al., 2015c; Miller & Auchus, 2011; Tuckey et al., 2019a). However, they may be produced by orthologs or paralogs of these enzymes since their products, including 20(OH)D3, are detectable in honey (Kim et al., 2020). In addition, it was recently documented that the previtamin D photoproducts lumisterol and tachysterol can be metabolised by CYP11A1 and CYP27A1 into biologically active products (Tuckey et al., 2014, 2018; Slominski et al., 2017a, 2021a, 2022). The current challenge is to determine whether their production is conserved across different species as shown for the classical vitamin D3 and vitamin D2 hydroxy metabolites. An additional unknown is whether other forms of vitamin D such as D4 and D5, as well as additional secosteroids such as those shown in Fig. 2, undergo similar metabolism through hydroxylation and have biological activity.

(4). Detection of vitamin D, their precursors and derivatives in honey and insects

7-DHC is the essential precursor needed to produce the critically important moulting hormone ecdysone. It is generally accepted that insects cannot synthesise sterols, including cholesterol and Δ5,7 sterols like 7-DHC and ergosterol. They obtain these essential sterols and cholesterol by ingesting them from fungi, yeast, plants and animals (Janson et al., 2009; Jing & Behmer, 2020; Nasir & Noda, 2003; Vaitsopoulou et al., 2022; Wen et al., 2023). Unlike vertebrates, insects are able to convert cholesterol by Neverland (Nvd), a C-7 cholesterol dehydrogenase, into 7-DHC (Wen et al., 2023; Yoshiyama et al., 2006; Gilbert et al., 2002; Zhu et al., 2019; Ekoka et al., 2021). Insects like D. melanogaster are able to convert ergosterol into 7-DHC. Therefore, it is possible that other Δ5,7 sterols could also be produced in insects from the corresponding plant, fungi or bacterial Δ5 sterols (Göring, 2018; Black et al., 2017; Jäpelt & Jakobsen, 2013; Gallo et al., 2020; Jiang et al., 2020; Voshall et al., 2021; Lee et al., 2023). With UVB exposure, these could be transformed into the corresponding secosteroids (see Fig. 2). In addition, ecdysteroids are synthesized by plants as a tool for interaction or warfare with insects (Savchenko et al., 2022).

The number of publications concerning vitamin D in insects is low. A recent study revealed that mosquitoes (Aedes aegypti) exposed to sunlight in Florida contained significant amounts of 7-DHC and vitamin D3 (Holick & Stoddard, 2022). There was no evidence for other vitamin D compounds. When several types of insects were fed with 7-DHC or ergosterol followed by exposure to UVB radiation, vitamin D3 and vitamin D2 were detected, respectively (Oonincx et al., 2018). These results suggest that insects exposed to sunlight may serve as good dietary sources of vitamin D for vertebrates, especially those like bats that lack sun exposure.

Bats represent the second largest order of mammals after rodents, comprising about 20% of all classified mammalian species worldwide (>1,400 species). 48.5% live in caves and underground habitats whereas the other 51.5% live in environments exposed to sunlight (Tanalgo, Oliveira & Hughes, 2022). Some cave and underground bats have found novel sources of vitamin D. The vampire bat Desmodus rotundus is a strictly sanguivorous species that ingests up to 50% of its body mass in blood from medium- and large-bodied terrestrial mammals and birds every night. These bats are able to utilize the 25(OH)D in their blood diet and maintain extremely high circulating concentrations of 25(OH)D of up to 400 ng/ml (Southworth et al., 2009). Similarly fish-eating bats including the piscivorous Noctilio leporinus obtain vitamin D from their diet and maintain circulating concentrations of 25(OH)D in the range of 236–247 ng/ml. Both nocturnal and tree-dwelling bats have the capacity to produce vitamin D3 when exposed to natural sunlight (Southworth et al., 2013). However, the same study demonstrated that at the times when cave- and ground-dwelling bats are exposed to sunlight (in the early morning and at dusk) there is minimal UVB radiation which is therefore unlikely to result in vitamin D3 production in the skin. An evaluation of the vitamin D status of three plant-visiting species revealed that they had circulating concentrations of 25(OH)D in the range of 5–32 ng/ml. These bats are thought to supplement their plant-based diet with insects. Therefore, observations that insects can produce vitamin D (Oonincx et al., 2018; Holick & Stoddard, 2022) when exposed to sunlight suggests that they may be a source of vitamin D for nocturnal insectivorous bats and tree-dwelling fruit bats (Southworth et al., 2013, 2009).

To determine whether insects are able to metabolize vitamin D3, a recent unpublished study was conducted on laboratory-reared mosquitoes (Aedes aegypti). After hatching from their pupal stage, the flying mosquitoes were fed human blood that contained tritiated vitamin D3 and then killed 24 h later. A lipid extract was obtained and analysed using HPLC to determine if classical vitamin D metabolites were present. None were detected using methodology described in Holick & Stoddard (2022) (M.F. Holick, unpublished results), although the presence of non-classical D3-hydroxyderivatives such as those produced by the action of CYP11A1 was not assessed. In addition to mosquitoes, vitamins D3 and D2 have been detected in other flying insects, with higher levels when the animals were exposed to UVB (Oonincx et al., 2018; Holick & Stoddard, 2022; Melo-Ruiz et al., 2013). Vitamin D3, lumisterol3 (L3) and 7-DHC were also detected in honey of various origins, as well as 20(OH)7-DHC, 20(OH)D3, 1,20(OH)₂D3, 25(OH)D3 and 1,25(OH)₂D3 (Kim et al., 2020) (Table 1). The detection of these hydroxyderivatives suggested that vitamin D3 has undergone sequential hydroxylation at C25 and C1α as in vertebrates or plants (see above), while hydroxylation at C20 opens up the question of which enzyme is involved in this process (Kim et al., 2020). Since CYP11A1 only appears to be expressed in vertebrates (Slominski et al., 2015c), it is possible that the enzyme involved in C20-hydroxylation of ecdysone may be responsible for this reaction (Savchenko et al., 2022; Gilbert et al., 2002; Wen et al., 2023).

Table 1.

Molecular masses (m/z) detected in honey and in Drosophila melanogaster by liquid chromatography mass spectrometry (LC-MS) corresponding to vitamins D3 and D2, lumisterol3 (L3), lumisterol2 (L2), tachysterol3 (T3) and their derivatives and precursors.

Compound	Honey	D. melanogaster
D3	1407.329 [M+Na]+	2367.337 [M+H-H2O]+
20(OH)D3	1423.324 [M+Na]+	3383.331 [M+H-H2O]+
25(OH)D3	1383.331 [M+H-H2O]+	2401.342 [M+H]+
1,20(OH)2D3*	1439.319 [M+Na]+	2399.326 [M+H-H2O]+
1,25(OH)2D3	1399.326 [M+H-H2O]+	2399.326 [M+H-H2O]+
20,23(OH)2D3	2439.319 [M+Na]+	3439.319 [M+Na]+
D2	2397.347 [M+H]+	3397.347 [M+H]+
20(OH)D2	2395.331 [M+H-H2O]+	2413.342 [M+H]+
25(OH)D2	2413.342 [M+H]+	3395.331 [M+H-H2O]+
1,20(OH)2D2	2411.326 [M+H-H2O]+	2411.326 [M+H-H2O]+
1,25(OH)2D2	2451.319 [M+Na]+	2411.326 [M+H-H2O]+
17,20(OH)2D2	4411.326 [M+H-H2O]+	2411.326 [M+H-H2O]+
L3**	1385.347 [M+H]+	2385.347 [M+H]+
20(OH)L3	2423.324 [M+Na]+	2401.342 [M+H]+
25(OH)L3	2383.331 [M+H-H2O]+	(−)
(25R)-27(OH)L3	2401.342 [M+H]+	2401.342 [M+H]+
(25S)-27(OH)L3	2383.331 [M+H-H2O]+ (?)	(−)
L2	3397.347 [M+H]+	3379.337 [M+H-H2O]+
24(OH)L2	2395.331 [M+H-H2O]+	(−)
28(OH)L2	3413.342 [M+H]+	3413.342 [M+H]+
T3	2367.337 [M+H-H2O]+	2367.337 [M+H-H2O]+
20(OH)T3	2401.342 [M+H]+	2383.331 [M+H-H2O]+
25(OH)T3	2383.331 [M+H-H2O]+	2401.342 [M+H]+
7-DHC	1407.329 [M+Na]+	3367.337 [M+H-H2O]+
20(OH)-7DHC	1383.331 [M+H-H2O]+	2383.331 [M+H-H2O]+
7-DHP	2315.232 [M+H]+	2315.232 [M+H]+
Ergosterol	2379.337 [M+H-H2O]+	2379.337 [M+H-H2O]+

7-DHC, 7-dehydrocholesterol; 7-DHP, 7-dehydropregnenolone; (−), not detected.

^*m/z of 399.326 [M+H-H₂O]⁺ also detected for honey.

^**385.347 [M+H]⁺ also detected for honey.

¹Detection procedure as reported in Kim et al. (2020).

^2–4QTof LC-MS detection of m/z with retention times corresponding to listed standards as described previously (Kim et al., 2022, 2020; Slominski et al., 2022, 2017a), or with modifications as described below.

²An Eclipse Plus C18 column (1.8 μm, 2.1 × 100 mm; Agilent Technology, Santa Clara, CA, USA) was used with methanol/water solvent containing 0.1% formic acid: 0–2 min, 85% methanol; 2–8 min, 85–97% methanol; 8–11 min, 97% methanol; 11–12 min, 85% methanol; 12–16 min, 85% methanol, all at a flow rate of 0.3 ml/min.

³An ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm; Waters, Milford, MA, USA) was used with methanol/water solvent containing 0.1% formic acid: 0–1 min, 40% methanol; 1–1.1 min, 40–85% methanol; 1.1–3 min, 85–97% methanol; 3–5 min, 97% methanol; 5–5.5 min, 97–40% methanol; 5.5–7 min 40% methanol, all at a flow rate of 0.3 ml/min.

⁴An Atlantis T3 C18 column (5μm, 4.6 × 100 mm; Waters, Milford, MA, USA) was used with methanol/water solvent containing 0.1% formic acid: 0–20 min, 857–100% methanol; 20–30 min, 100% methanol; 30–30.5 min, 100–85% methanol; 30.5–35 min, 85% methanol, all at a flow rate of 0.5 ml/min.

To obtain a fuller picture of the secosteroids produced by insects we analysed the same samples of honey previously used for detection of 7-DHC and vitamin D3 derivatives in a new analysis using qTOF-LC/MS following the methodology described in Kim et al. (2020). A wide range of compounds were detected (Table 1), and the LC/MS spectra are provided in Figs S2–S8. In addition to compounds listed previously, we detected 20,23(OH)₂D3, 20(OH)L3, vitamin D2 and its hydroxyderivatives 20(OH)D2, 17,20(OH)₂D2 and 1,20(OH)₂D2 as well as the precursor molecule, ergosterol. Tachysterol3 (T3), 20(OH)T3, 25(OH)T3 and 7-dehydropregnenolone (7-DHP) as well as lumisterol2 (L2) and its hydroxyderivatives including 24(OH)L2 and 28(OH)L2 (Table 1) were also detected. In vertebrates, the latter are the products of the action of CYP27A1 on L2 (Wu et al., 2023). The detection of T3, 20(OH)T3, 20(OH)L3 and 25(OH)T3 complements our previous analyses of photoderivatives of 7-DHC, and is consistent with the photochemistry of pre-D3 transformation (Fig. 2) and the ability of insects to hydroxylate at C20, as discussed previously (Kim et al., 2020, 2022). The detection of vitamin D2 in honey is consistent with its detection in other insects (Oonincx et al., 2018). Ergosterol must be of nutritional origin, while 20(OH)D2 could result from the action of a similar C20 hydroxylase to that producing 20(OH)D3 and 20(OH)-7DHC. Likewise, the presence of 1,20(OH)₂D3 may be a result of the action of the C1α-hydroxylase that activates 25(OH)D3 as discussed previously (Kim et al., 2020, 2022), and it is known that plants can hydroxylate 25(OH)D3 at C1α (Göring, 2018; Jäpelt & Jakobsen, 2013). The presence of L2 is likely due to the photochemical transformation of Δ5,7 sterols, or alternatively may be of nutritional origin. However, our detection of 24(OH)L2 and 28(OH)L2 requires explanation. Recent evidence shows that in humans these hydroxyderivatives are generated by the action of CYP27A1 on L2, whereas L2 does not serve as a substrate for CYP11A1 (Wu et al., 2023). By contrast, L3 can serve as a substrate for both CYP11A1 and CYP27A1 (Tuckey et al., 2014, 2018; Slominski et al., 2017a). However, there is no information available regarding enzymes that could hydroxylate lumisterol compounds in insects and plants. These could be non-homologous isofunctional enzymes which also hydroxylate at the C25 position. Good candidates are the enzymes involved in production of ecdysteroids such as the sterol 25-hydroxylase (CYP306A1). The presence of vitamin D, lumisterol and tachysterol derivatives in honey may contribute to its well-recognized health benefits (Crane, 1991, 2020; Waheed et al., 2019; Zhang et al., 2022; Almasaudi, 2021; Oduwole et al., 2018; Saikaly & Khachemoune, 2017; Ahmed & Othman, 2013; Khalil et al., 2012; El-sound, 2012; Mandal & Mandal, 2011; Al et al., 2009), and may implicate these molecules in physiology and protective mechanisms in social insects, for example in development and in colony functions as discussed previously (Kim et al., 2022, 2020).

We also detected secosteroid derivatives in D. melanogaster (Table 1; Figs S2–S8). The samples were extracted following standard protocols to extract secosteroids from different sources (Kim et al., 2022, 2020; Slominski et al., 2022, 2017a). The extracts were analysed by qTOF-LC/MS using conditions described previously (Kim et al., 2022, 2020; Slominski et al., 2022, 2017a), with some modifications as described in Table 1. We detected vitamin D3 and D2, L3, L2, T3 and their hydroxyderivatives (Table 1, Figs S2–S8). We also detected secosteroid precursor Δ7 sterols including 7-DHC, 20(OH)-7DHC and ergosterol. Thus, similarly to honey (Kim et al., 2022, 2020) and several other insects, vitamins D3 and D2 (Oonincx et al., 2018; Holick & Stoddard, 2022; Melo-Ruiz et al., 2013) are detectable in the fruit fly. In addition, we detected vitamin D hydroxyderivatives such as 25(OH)D and 1,25(OH)₂D and Δ5,7 sterols, similar to reports for plants (Silvestro et al., 2013; Jäpelt et al., 2013; Jäpelt & Jakobsen, 2013; Göring, 2018; Black et al., 2017) and honey (Kim et al., 2020). Detection of other hydroxyderivatives of vitamin D, tachysterol, lumisterol and ergosterol, including C20 hydroxyderivatives, are comparable to previous reports of their presence in honey (Kim et al., 2020). Thus, these compounds clearly can be produced by these insects or are obtained from a nutritional origin. The detection of 7-DHP is highly novel and implies that enzymatic cleavage of the side chain of 7-DHC occurs in insects, as in vertebrates (Slominski et al., 2004; Slominski et al., 2009, 2012a), however, the corresponding enzyme in insects remains to be identified. Alternatively, an isofunctional CYP to CYP11A1 has been found in plants (foxglove, Digitalis lanata) which can cleave the side chain of cholesterol and campesterol to produce pregnenolone (Carroll et al., 2022). It is feasible that this enzyme could also cleave the side chain of 7-DHC, as predicted from the action of CYP11A1 (Guryev et al., 2003; Slominski et al., 2012a, 2015c; Slominski et al., 2004), and thereby provide a nutritional source of 7-DHP to insects.

V. PERSPECTIVE ON PHYSIOLOGICAL ROLES SHARED BY VITAMIN D, MELATONIN AND THEIR DERIVATIVES IN INVERTEBRATES

A challenge that remains is to investigate the physiological roles of vitamin D, melatonin, and their derivates in insects. We focus our attention below on vitamin D derivatives, since the activation of lumisterol and tachysterol by enzymatic modifications has only been discovered recently (Tuckey et al., 2014, 2018; Slominski et al., 2017a, 2022). From studies in mammalian systems we know that these secosteroids have photoprotective, antioxidative and pro-differentiation properties via their action on nuclear receptors (Slominski et al., 2022, 2021a, 2020b, 2017a; Chaiprasongsuk et al., 2020a, 2019). In addition, they can have antiviral activities through mechanisms independent of those induced by interactions with nuclear receptors (Qayyum et al., 2021, 2022; Song et al., 2021). Similar mechanisms could operate in insects. For vitamin D, it has long been assumed that its beneficial actions in vertebrates are secondary to its activation to 1,25(OH)₂D3, which exerts phenotypic effects through interactions with the VDR (Bouillon et al., 2019; Bikle & Christakos, 2020; Hewison et al., 2024; Zmijewski & Carlberg, 2020; Carlberg, 2018, 2022; Christakos et al., 2016; Rochel, 2022), including via its non-genomic binding site (Mizwicki et al., 2004; Mizwicki & Norman, 2009). Unfortunately, neither plants nor insects have a VDR equivalent to that of vertebrates and their calcium and phosphorus metabolism is regulated by other mechanisms including (in insects) via the ecdysone receptor (EcR) (Kamiyama & Niwa, 2021; Saez et al., 2000; Yao et al., 1992). In addition, it has been shown that vitamin D3 can inhibit mutagenic and carcinogenic effects of doxorubicin in D. melanogaster, apparently through action on the EcR (Vasconcelos et al., 2020). Furthermore, insects do have orthologs or analogs of vertebrate liver X receptor (LXR), retinoic acid orphan receptor (ROR) and the aryl hydrocarbon receptor (AhR) (King-Jones & Thummel, 2005; Pan et al., 2019; Brown et al., 2005; Peng et al., 2017; Hahn, 1998; Kulkarni et al., 2022; Kim, Jan & Jan, 2006). These receptors could serve as potential targets for the action of hydroxyderivatives of vitamin D3, lumisterol or tachysterol, since they have recently been identified as alternative receptors to the VDR for these molecules in mammals (Slominski et al., 2021a, 2017a,b, 2022, 2018c, 2014b; Brzeminski et al., 2022; Song et al., 2022). Interestingly, the AhR, an alternative receptor for vitamin D3 and tachysterol metabolites, was recently defined as a nuclear receptor for melatonin and its metabolites and photoderivatives (Slominski et al., 2023a). Finally, receptor-independent mechanisms of action of lumisterol or vitamin D hydroxyderivatives have recently been described (Qayyum et al., 2022, 2021; Song et al., 2021), which are similar to the cytoprotective effects of melatonin and its metabolites that require high concentrations of the ligands and appear to be receptor independent (Janjetovic et al., 2017; Slominski et al., 2017d; Bocheva et al., 2022; Holtkamp et al., 2023; Linowiecka et al., 2023; Vijayalaxmi et al., 2004; Reiter et al., 2013, 2016; Tan et al., 2013; Reiter & Tan, 2019). Carlberg (2022) proposed that the primordial role of vitamin D was to protect DNA which is in agreement with literature showing DNA-protective and anti-oxidative actions of vitamin D derivatives including protection against UVR and various pollutants (Slominski et al., 2020d,b, 2015c; Chaiprasongsuk et al., 2020a,b, 2019; Rybchyn et al., 2018; Gordon-Thomson et al., 2014; Tongkao-On et al., 2015; Song et al., 2013; De Silva et al., 2021, 2023; Nuszkiewicz et al., 2020; Bocheva, Slominski & Slominski, 2023, 2021). Similar functions have been assigned to melatonin and its derivatives (Reiter & Tan, 2019; Zhao et al., 2019; Reiter et al., 2017; Tan et al., 2014, 2013, 2007; Vijayalaxmi et al., 2004; Slominski et al., 2014c, 2017d; Janjetovic et al., 2017; Skobowiat et al., 2018). Other actions could include anti-microbial and anti-viral activities of vitamin D derivatives (Hewison et al., 2024; Qayyum et al., 2022; Slominski et al., 2020d; Grant, 2021; Bishop et al., 2021). Again, similar functions are assigned to melatonin and its derivatives (Hernández-Ruiz et al., 2023; Anderson & Reiter, 2020; Reiter et al., 2022; He et al., 2021). Thus, similar mechanisms of action and similar functions of vitamin D and melatonin and their derivatives could operate in insects to those observed in vertebrates, opening a ‘Pandora’s Box’ of possibilities for experimental testing.

VI. CONCLUSIONS

1. We have discussed the wide distribution of melatonin in nature and the possibly of a similar distribution of vitamins D and their precursors and derivatives. While melatonin production has been shown in almost all living organisms on Earth from unicellular to complex multicellular ones (Fig. 1), a similarly wide and diverse distribution of secosteroids is likely, given the production of Δ5,7 sterols by most living organisms and their predictable phototransformation to the corresponding secosteroids when exposed to UVB (Fig. 2). In this context, detection of additional secosteroidal compounds is expected, aside from the classical products resulting from isomerization of previtamin D3 and D2 (Fig. 2).

2. In insects, melatonin metabolites are detectable across different species and are present in bee honey, an important nutritional product for animals and humans. The detection of melatonin derivatives in insects is likely in the near future, as recent descriptions of secosteroids should stimulate research in this area given the shortage of reports on the presence of vitamin D and its derivatives in this taxon. Such compounds could result from phototransformation of endogenously generated Δ5,7 sterols or be ingested from dietary sources. Current knowledge suggests that insects do not have the ability to produce sterols endogenously, but can modify them for different purposes. There remains a need for a wider search for different secosteroids in a range of organisms and natural products, including plants, simple eukaryotes and bacteria.

3. Determining the nature of common bioregulatory proteins (receptors) activated both by melatonin/derivatives and secosteroids represents a challenge. Identification and characterization of such receptors could shed light on the evolution of the functions of these molecules and their downstream signalling pathways in different species. While these molecules can act via different receptors, they may also share common receptors, as studies on the AhR have recently indicated.

4. It has been proposed that the original primary function of melatonin was protection against oxidative stress. Similarly, it was proposed that the ancient function of vitamin D was to protect DNA against UVR damage. UVR is a major environmental stressor with UVA predominantly inducing oxidative damage, while UVB directly damages biomolecules through absorption of its electromagnetic energy, including DNA, RNA and proteins. It has been proposed that UVR was a significant driver of biological evolution. Therefore, we speculate that one of the original functions of melatonin and secosteroids (vitamin D derivatives) would have been to protect against solar-light-induced damage and free radicals, with this function extended to include different cytoprotective mechanisms that are conserved across species living on the Earth’s surface.

5. Interestingly, there is analogy between UVB-induced secosteroidogenesis and non-enzymatic transformation of melatonin to generate AFMK and AMK (compounds with a broken indolic ring) after absorption of both UVB and UVA (Slominski et al., 2017c). In plants, transformation of melatonin to AFMK can enhance their resistance against environmental stress. Thus, both melatonin and vitamin D derivatives share similar protective functions against UVR-induced oxidative stress and DNA damage.