Vitamin D 1 versus ecdysteroids : Growth effects on cell regeneration and malignant growth in insects are similar to those in humans

Polyhydroxylated derivatives of 6-keto,7-dehydrocholesterol (ecdysteroids) are common constituents of various plants. In 1965, they were accidentally discovered in the search for the insect moulting hormone. These biologically important natural compounds are neither insect hormones nor inducers of insect ecdysis. Due to their strong anabolic, vitamin D-like effects in insects, domestic animals and humans, I propose the use of the arbitrary term vitamin D1. The present paper describes the effects of vitamin D1 on the growth and regeneration of excised epidermal cells of the tobacco hornworm, Manduca sexta (Sphingidae). The periods of programmed cell death and cell proliferation (histolysis and histogenesis, respectively) exactly coincide in insects with endogenous peaks of increased concentration of vitamin D1. Epidermal cells communicate with each other, creating a mutually integrated tissue, connected by mechanical, chemical, electrical, ionic or other so far incompletely known factors. After natural cell death, or after the artifi cial removal of some epidermal cells, the neighbouring cells that lose communication integrity, begin to divide mitotically to replace the disconnected part. Cell divisions are arrested as soon as the integrity of the living tissue is established. During insect ontogeny, the application of juvenile hormone causes regenerating epidermal cells to repeat the previous morphogenetic programme (i.e., development of patches of larval tissue on the body of a pupa, or metathetely). Conversely, the application of vitamin D1 (20-hydroxyecdysone) caused the regenerating cells to prematurely execute a future morphogenetic programme (i.e., development of patches of pupal tissue on the body of a larva, or prothetely). Among the key features of insect regeneration, is the arrest of cell divisions when tissues resume living cell-to-cell integrity. This prevents the formation of aberrant groups of cells, or tumours. It is well established that the main physiological systems of insects (e.g., circulatory, respiratory, neuro-endocrine) are structurally and functionally similar to corresponding systems in humans. Thus the basic principles of cell regeneration and the role of vitamin D1 in insects may also be valid for humans. The common vitamins D2 (ergocalciferol) or D3 (cholecalciferol), are exclusively lipid soluble secosterols, which require activation by UV irradiation and hydroxylation in the liver. By contrast, the neglected vitamin D1 is a natural derivative of polyhydroxylated 7-dehydrocholesterol of predominantly plant origin, which is both partly a water and partly a lipid soluble vitamin. It neither requires UV irradiation, nor hydroxylation due to 6 or 7 already built-in hydroxylic groups. Like other vitamins, it enters insect or human bodies in plant food or is produced by intestinal symbionts. Vitamin D1 causes strong anabolic, vitamin D-like effects in domestic animals and in humans. I am convinced that avitaminosis associated with a defi ciency of vitamin D1 in human blood may be responsible for certain hitherto incurable human diseases, especially those related to impaired nerve functions and somatic growth, aberrant cell regeneration or formation of malignant tumours.


INTRODUCTION
Recent progress in biology and medicine has been largely infl uenced by technical advances and innovations.During the twentieth century, human health care became dependent on the massive use of pharmaceutical drugs.Yet, some old medical problems remained unresolved.One example of this probably occurred approximately a century ago, in the search for the growth promoting, antirachitic (to cure or prevent rickets) action of vitamin D. In 1930, organic chemists found provitamin D in skin, which was thought to be tentatively converted into vitamin D 3 (calciferol) by UV irradiation.ity and Hess et al. (1925) propose that "it would seem quite possible that cholesterol in the skin is normally activated by UV-irradiation and rendered anti-rachitic".It was assumed that the cholesterol obtained from brains of rats, contained a small amount of an impurity that could be a precursor of the vitamin.Thus, to shed more light on this problem, A.F. Hess asked the famous German steroid chemist, A. Windaus, to collaborate on the elucidation of the chemical structure of the antirachitic agent.In 1937, Windaus & Bock (1937) isolated and identifi ed a compound related to 7-dehydrocholesterol (see Fig. 1A), from pig, rat and human skin, as well as from animal sources, such as whole milk or liver.The active compound was named vitamin D 3 (cholecalciferol, see Fig. 1B and see Wolf, 2004 for a review).
Investigations on vitamin D were directed to purely lipid soluble animal sterols, because 7-dehydrocholesterol was supposed to be exclusively an animal sterol (zoosterol).The research concentrated on animal fats, such as milk, butter or codfi sh oils.Cholesterol and its derivatives were considered purely lipid-soluble compounds (the existence of partly water soluble, polyhydroxylated sterols was unknown at that time).The polar, water soluble fractions were thus disposed of without being tested for the antirachitic properties of vitamin D.Moreover, the most serious confusion in the research on vitamin D was the fact that, unlike other vitamins, vitamin D was not expected to occur in plants, because 7-dehydrocholesterol was erroneously not considered to be present in plants.Since that time, the biochemical status of vitamin D was defi ned as a purely lipid soluble animal sterol.The preparations of commercial vitamin D 3 (calciferol) are available under the name Vigantol, the more active derivative originating by metabolic hydroxylation of vitamin D 3 in liver and kidneys is known as vitamin D 3 -triol (commercial name, Rocaltrol).Exact structure of vitamin D 1 was not completely assigned at that time, which could explain the absence of D 1 -based pharmacological drugs.
According to Karlson (1981), the status of vitamin D 1 was not clear.It was a crystalline substance isolated by Windaus et al. (1931) from ultraviolet irradiated ergosterol.Previous investigators predicted that vitamin D might be somewhat related to 7-dehydrocholesterol (Hess et al., 1925).When Karlson (1966) isolated ecdysone, which is a derivative of 7-dehydrocholesterol, he was so impressed by Williams's (1952) idea of an "Arthropod moulting in the human body can be compared with its effects on the growth of insect exoskeleton, which is manifested by the formation of a new, enlarged integumental cover.As a matter of fact, both in insects and mammals, essential growth factors with a similar function to vitamin D are structurally related to 7-dehydrocholesterol (Sláma, 1993(Sláma, , 2014(Sláma, , 2016;;Sláma & Zhylitskaya, 2016).
Previous studies on the effects of vitamins and hormones in insects were made on immature larvae and pupae of the greater wax moth, Galleria mellonella L. (Sláma, 1975) and the carpet beetle, Dermestes vulpinus De Geer (Sláma et al., 1974;Sláma 1999Sláma , 2015)).Similar investigations were done using the tobacco hornworm, Manduca sexta L., which is substantially larger.These experiments mainly involved isoprenoid analogues of insect juvenile hormone (JH) (Sláma et al., 1974;Sláma, 1999) and the polyhydroxylated derivatives of 6-keto,7-dehydrocholesterol (ecdysone, ecdysteroids; Sláma et al., 1974Sláma et al., , 1993;;Sláma, 2015), which were initially thought to be arthropod moulting hormones.During the last three decades, extensive data on the effects of ecdysteroids have been published on insects (Sláma, 2014(Sláma, , 2015a;;Sláma et al., 1993) and also on anabolic, vitamin D-like effects of these compounds in mammals (Sláma & Lafont 1995;Dinan & Lafont, 2006).The existence of polyhydroxylated sterols was unknown to the pioneers of vitamin D research as these were discovered three decades later by Karlson (1996).Ecdysteroids, long believed by organic chemists to be the arthropod hormones stimulating insect ecdysis (Dinan et al., 2009;Kumpun et al., 2011;Lafont et al., 2011) are according to my view the erroneously assigned, true vitamin D. Since these polyhydroxylated derivatives of 6-keto,7-dehydrocholesterol are neither hormones produced by the prothoracic gland of insects nor inducers of insect ecdysis (see below) and to avoid further confusion of earlier work, I proposed earlier to use the term vitamin D 6 , due to its strong anabolic, vitamin D-like effects both in insects and mammals (index 6 for the six hydroxylic groups in the molecule and the Latin "Hexapoda" for six-legged creatures, the class Insecta) (Sláma, 1993(Sláma, , 2015a(Sláma, , b, 2016;;Sláma & Zhylitskaya, 2014).

A brief history of vitamin D research
A brief survey of the history of vitamin D indicates its close relations to rickets (rachitis), which is a bone disease caused by vitamin D defi ciency.In 1921, it was found that the occurrence of rickets was related to seasonal variations in sunlight (Hess & Unger, 1921).Sunlight would cure rickets just as well as cod-liver oil.Irradiation of food items (e.g., milk butterfat, dietary oil) could also impart antirachitic potency.Due to this, the history of vitamin D research was closely related to UV-irradiated compounds (Windaus et al., 1931;Karlson, 1981).The story of D-vitamin research was perhaps best reviewed by Wolf (2004).Herein, I quote only a few essential points from his 2004 review.
After 1924, it was found that the antirachitic properties of certain animal fats were increased by UV irradiation.Irradiated cholesterol also shows increased biological activ- hormone secreted by prothoracic gland", that he did not consider the possibility that the biological activities of ecdysone and vitamin D are similar.
In the bioassays used by Karlson, ecdysone stimulated formation of new epidermis in headless maggots of blowfl y.According to the hormonal theory of Williams (1952), the effects were ascribed to a hormone secreted by insect prothoracic glands (Karlson, 1966).However, almost immediately after Karlson disclosed the crystallographic structure of ecdysone, various phytochemists reported that this compound is present in extracts of various, taxonomically unrelated species of plants (Nakanishi et al., 1966;Jizba et al., 1967;Sláma et al., 1974 for review).The most abundant derivative found in plants as well as in insects and crustaceans was 20-hydroxyecdysone, currently known as ecdysterone (Fig. 1C).Some species of plants contained enormous amounts of this compound, assumed by organic chemists to be the "Arthropod moulting hormone".For example, just one gram of the rhizomes of the fern, Polypodium vulgare, contain the same amount of ecdysterone es 500 kg of silkworm pupae (Jizba et al., 1967).Additional screening of plants done mainly by Japanese phytochemists revealed that these partly water soluble, polyhydroxylated derivatives of 7-dehydrocholesterol are widely distributed in the botanical world (Hikino et al., 1968).Despite the common occurrence of vitamin-like ecdysteroids in plants (Sláma et al., 1974;Sláma, 1979), organic chemists still distinguish between zooecdysteroids and phytoecdysteroids (Kumpun et al., 2011).The fi rst controversial defi nition of 20-hydroxyecdysone (vitamin D 1 ) proposed that: "Ecdysteroids were the reserve materials for the growth of tissues in plants" (Sláma, 1979).
Extensive physiological investigations have revealed that ecdysteroids are not insect hormones (Sláma, 1983(Sláma, , 1993(Sláma, , 2014)).Prothoracic glands do not stimulate insect ecdysis (Sláma, 1983(Sláma, , 1998) ) and the peaks in endogenous concentrations of vitamin D 1 in insects occur in the absence of prothoracic glands (Delbecque & Sláma, 1980).The hypothesis regarding the role of prothoracic glands proposed by Williams (1952) was later refuted by Williams (1987).Unfortunately, most biochemists did not comprehend the signifi cance of this refutation by Williams and continued to consider the disintegrating prothoracic glands of Bombyx to be the source of the moulting hormone (Iga et al., 2014;Nakaoka et al., 2017).In addition, the alternative biological defi nition proposed that: "Ecdysteroids were the homeostatic tissue factors synchronising the growth of tissue and cells with the essential developmental features of the moulting cycle" (Sláma, 1980), was ignored.Later it was experimentally confi rmed (Sláma, 1980) that ecdysteroids do not stimulate, but actually inhibit insect ecdysis in a dose-dependent way.There is strong presumptive evidence (Sláma, 1993) that ecdysteroids (here vitamin D 1 ) are biologically far more important natural compounds of medical importance, than just being a hormone produced by the tiny prothoracic glands (Sláma, 1993;Sláma & Lafont, 1995).
Herein, I describe the effects of vitamin D 1 (20-hydroxyecdysone) and juvenile hormone (JH) on the regeneration of epidermal tissue in the tobacco hornworm, Manduca sexta L. The replacement of dead cells is the most important aspect of regeneration in all animals and humans.According to H.E. Hinton (quoted in Sláma et al., 1974), "the terms of science should correspond with the facts in nature".As ecdysteroids are neither arthropod moulting hormones (Sláma, 1983) nor stimulate ecdysis (Sláma, 1980) and have strong anabolic, vitamin D-like effects, it is more appropriate to use the generic term vitamin D 1 , although it was originally proposed for a structurally unidentifi ed UVirradiation product of ergosterol by Windaus et al. (1931).

MATERIAL AND METHODS
Larvae and pupae of M. sexta were obtained from stock cultures reared on artifi cial diets (Sláma, 1960).Procedures used for epidermal excisions or producing local lesions in epidermal cells using thermocautery are described in Sláma (1975).Cauterization was used only on fully grown larvae of the last larval instar, other experiments were based on epidermal excisions.The exact size and location of epidermal injuries are depicted in Fig. 2.
Prior to excisions, selected larvae and pupae were immobilised by submersion in water for 15 to 35 min (Sláma, 1980(Sláma, , 2016)).Pupae in diapause, which have relatively low gaseous exchange rates, were submerged in water for two hours.The areas from which larval epidermis was excised were covered by rapidly polymerizing cyanoacryllic glue (Fig. 2A).Epidermal excisions in the rigid cuticle of pupae were covered by a strip of a broken cover slip.The margins were sealed using bee's wax melted using the platinum wire used for thermocautery (Fig. 2B).A 10% ethanolic solution of vitamin D 1 (20-hydroxyecdysone; natural compound isolated from Leuzea carthamoides (Willd.)(Buděšínský et al., 2009), was injected into larvae and pupae (Sláma, 1980).Local application of vitamin D 1 was made by covering the wound with a small piece of solidifi ed, 12% gelatine containing 2 mg/ ml of vitamin D 1 .The strip of gelatine was covered externally by a polymerized polyester layer produced by a small drop of cyanoacryllic glue.For topical application of a juvenile hormone analogue, we used methyl, 10,11-epoxy,7-ethyl-3,11-dimethyl-2,6-tridecadienoate (JH-I) (Sláma et al., 1974;Sláma, 1985Sláma, , 1999;;Paroulek & Sláma, 2014).Determination of the content of vitamin D 1 in insect bodies, expressed in 20-hydroxyecdysone equivalents, is described by Sláma (1980) and Delbecque & Sláma (1980) and in the blood of Japanese quails by (Koudela et al., 1995;Sláma et al., 1996).
The excisions or lesions in epidermal cells by thermocautery were initially done on groups of ten specimens.The results were individually evaluated, documented and stored in a personal computer database.In the case of equivocal results or increased mortality, experiments were repeated using a new group of ten specimens.When necessary, the total number of experimental specimens is reported in the text or in fi gure captions.The anatomical and morphological structure of the regenerated epidermal patches was evaluated after the following moult, using light (Sláma, 1975(Sláma, , 1980) ) or scanning electron microscopy (Sláma & Weyda, 1997).In exceptional circumstances, epidermal fragments were fi xed in 70% ethanol for histological observations or prepared for scanning electron microscopy (Sláma & Weyda, 1997.

Endogenous peaks in the content of vitamin D 1 during insect development
Most insects obtain vitamin D 1 from food or symbiotic bacteria.It is used in the construction of hydro/lipophilic cell membranes by newly proliferating or regenerating cells.When the dietary supply of vitamin D 1 is superfl uous to requirements, the danger of hypervitaminosis (hyperecdysonism of Williams, 1970), which is manifested in the precocious, pathophysiological secretion of new cuticle, is prevented by the excretion of vitamin D 1 .During the non-feeding stages, vitamin D 1 is retrieved from disintegrating tissues and reutilized for the construction of cell membranes in proliferating pupal or adult tissues (Sláma, 1982).The schematic drawing in Fig. 3 shows the relationships between endogenous concentrations of vitamin D 1 and total body metabolism (O 2 consumption), which is valid for most endopterygote insects.It is important to note that the peaks in endogenous content of vitamin D 1 exactly coincide with minimum metabolic activity.This inverse relationship results from the low metabolic effi ciency of old, disintegrating larval tissue and organs (histolysis), which is associated with the simultaneous proliferation of newly  developed pupal or adult tissues (histogenesis).The homeostatic turnover of vitamin D 1 is usually less pronounced during the initial larval-pupal transformation (the sterol reutilisation theory of Sláma, 1998).The pupal peaks are larger, due to the extensive transformations involved in the development of adult digestive and muscular systems (Fig. 3).

Cell-to-cell communication among living cells
The postembryonic development of insects involves distinctive moulting and reproductive cycles.The cycles are triggered by hormones of the central neuroendocrine system, which track external environmental signals (e.g., availability of food, suitable temperature and photoperiod) and transforms them into developmental instructions coded in the genome.During larval somatic growth, the body increases in size by the multiplication of cells in peripheral tissues, or increase in cell size by polyploid endomitosis, as in some Diptera.Hormones from the central neuroendocrine system instruct genes on chromosomes in the peripheral cells when to execute their inherited programmes.There are two principal types of developmental cycles regulated by centrally produced hormones: (1) The epigenetic or "status quo" developmental cycles under the infl uence of JH (stationary larval-larval or pupalpupal cycles), that take place without structural changes in the DNA.
(2) The morphogenetic developmental cycles that occur in the complete absence of JH (embryonic cycles, larvalpupal, pupal-adult, larval-adult cycles).These moulting cycles are associated with the reprogramming of genetical information.Some insects, which do not require environmental signals, exhibit autonomic (hormone independent) development (Sláma, 2015a).As reported in Fig. 3, the morphogenetic cycles are intimately associated with endogenous peaks in the concentration of vitamin D 1 .
During normal development, epidermal cells excised at the beginning of an instar (before the endogenous peak of vitamin D 1 ) regenerated into the homochronic, one and the same population of epidermal cells, matching the structure of the integument of the host.An example of homochronic regeneration is depicted in Fig. 4, which shows that the excised patch of epidermal cells that regenerated during metamorphosis is indistinguishable from the surrounding epidermal cells.The microscope observations revealed that the excised epidermis was replaced by cells proliferating from the disconnected margins of the wound.In other words, epidermal cells removed from penultimate larvae regenerated during metamorphosis into epidermis of the last larval instar (Fig. 4A, B).Cells removed from the last larval instar regenerated into pupal epidermal structures (Fig. 4C, D).Moreover, cells removed from freshly developed pupae regenerated into adult structures with hairs and scales, indistinguishable from those of the host (Fig. 4E,  F).The excisions were made at the beginning of an instar, however, the remodelling of fi nal structures occurred later, during the endogenous peak in vitamin D 1 , as can be seen in Fig. 3.The larval architecture of epidermal cells, with folded cuticle and sharply delimited, distinctly different, fl at pupal cells of the heterochronic epidermal patches in Manduca were monitored earlier by scanning electron microscopy (Sláma & Weyda, 1997).
The activity of JH can be artifi cially prolonged by applying JH analogues, which is usually followed by the development of larval-pupal intermediates or supernumerary larval instars.This type of suspended appearance of structural characters of previous, older ontogenetic stages in the next stage has been described many times as a heterochronic developmental deviation, which is called metathetely by Sir V.B. Wigglesworth (1970).Conversely, the premature appearance of structural characters of a future ontogenetic stage is called prothetely (Sláma, 1975(Sláma, , 1980) ) (usually manifested by precocious pupal wing lobes in larvae) and are associated with exogenous applications of vitamin D 1 (Williams, 1970;Sláma, 1975Sláma, , 1985Sláma, , 1999)).
The above results have several physiological implications: ( 1  the ontogenetic status of the neighbouring cells, e.g.larval, pupal or adult status of the host.( 5) During the endogenous peak of vitamin D 1 , the regenerating epidermal cells metamorphose synchronously with the epidermal cells of the host.This shows that living epidermal cells communicate and establish a unit of physiologically integrated tissue.When living cell integrity is disturbed by cell death or epidermal injury, order is re-established by multiplication of disconnected neighbouring cells.
The principles of regeneration in insects may be common to all multicellular organisms, including plants (e.g., formation of a callus), invertebrate or vertebrate animals, as well as humans (wound healing).It is very diffi cult to determine the exact nature of the factors responsible for the maintenance of the integrity of living cells, which arrest further cell divisions when regeneration is complete.It may be mechanical, chemical, electrical, osmotic, or ionic, among other possibilities like an undisturbed extracellular stromal sheath, which protects the external cells of each organ.A failure in resuming cell integrity can lead to aberrant cell divisions and formation of tumours.
Fig. 5 shows a schematic outline of cytological changes during regeneration of an epidermal excision in Manduca sexta.The excised epidermal section (Fig. 5B) is invaded by mitotically dividing cells from the margin of the wound (Fig. 5C), which replace the missing epithelium (Fig. 5D).During the endogenous peak of vitamin D 1 , the newly formed cells secrete a new cuticle simultaneously with the host cells (Fig. 5E).When there is a physical obstacle to living cell continuity, such as the insertion of a cover slip into a pupa in diapause (Fig. 5F), the disconnected cells began dividing and formed a layer of cells around the object until cell-to-cell continuity was achieved.

Regeneration of epidermal cells under the infl uence of JH
It is pointed out in Fig. 4, that under normal developmental conditions, epidermal excisions always regenerate into homochronic structures, indistinguishable from the epidermis of the host.However, this is not the case when regenerating epidermal cells are exposed to juvenile hormone (JH) activity at the time when the host cells became JH-insensitive (see Fig. 6).The integument of fully grown last instar larvae is fully expanded and very fl exible.Due to this, the patch of epidermal cells shown in Fig. 6A was not excised but cut using thermocautery.The regenerating epidermal areas developed into heterochronic, metathetelic (or previous stage), larval epidermal patches on pupae (Fig. 6B).Epidermal excisions and JH treatment of pupae resulted into the metathetelic pupal patches on the adult body (Fig. 6D).The excisions were done on 3-day-old larvae or pupae (Fig. 6A and C), because epidermal cells of uninjured specimens become insensitive to exogenous JH at this time.In contrast to normal cells, regenerating epidermal cells still retained their sensitivity to JH.They developed epidermis of the preceding ontogenetic stage (Fig. 6B), e. g. patches of larval cuticle on pupae and of pupal cuticle on adults.

Developmental fates of metathetelic epidermal patches
According to the long standing advocated hormonal concept of Piepho (1951) and Schneiderman & Gilbert (1964), the sequence of larval, pupal, and adult structures in insects is determined by respectively high, intermediate or low concentrations of JH.A heterochronic specimen should thus develop into the homochronic structures of either larval, pupal or adult structures, depending on the concentration of JH.
In this study, I investigated the developmental fate of 37 heterochronic patches during metamorphosis in Manduca.Fig. 7 shows the structural changes that occurred in the heterochronic patches during the pupal-adult transformation.The results can be briefl y described as follows: (1) Larval metathetelic patches produced by the regeneration of larval epidermal cells metamorphosed into heterochronic patches of pupal cuticle on the adults (Fig. 7A and B); (2) After treatment with a juvenile hormone (JH) analogue, pupae bearing these metathetelic larval patches, developed into heterochronic patches of secondary larval cuticle on secondary pupal instars.In other words, this result manifests the "status quo" effect of JH (Fig. 7C and D).The most important aspect of this physiological feature is that the heterochronic character of the developing tissues was fully preserved.This effect is an example of the epigenetic action of JH and its 4000 bio analogues.The extant mixture of different morphogenetic structures can be infl uenced repeatedly many times by JH and reproduced without the morphogenetic instructions on the genome being changed.
The results in Fig. 7 indicate that there is no homochronisation of the heterochronic tissues during insect metamorphosis, contrary to what is predicted by current hormonal theories (Piepho, 1951;Gilbert, 2012).In the absence of JH, larval cells metamorphosed into pupal cells, while pupal cells simultaneously metamorphosed into adult cells.This shows that the selective physiological condition for induction of a given ontogenetic stage cannot depend simply on the concentration of juvenile hormone.This depends on the previously attained morphogenetic stage, which is associated with irreversible changes and reprogramming of genes on chromosomes in peripheral target tissues (Sláma, 1985).The epigenetic role of JH is based on informing the genes in peripheral cells when to execute their inherited genetical programmes.Fig. 7C and D demonstrate that the "reversal of metamorphosis" under the infl uence of high concentrations of JH (the dosages of JH analogue were 500-fold greater) is not possible.The heterochronic tissues in Fig. 7 were analyzed earlier using scanning electron microscopy (Sláma & Weyda, 1997).The cuticle of larvae is wrinkled and folded while the pupal epidermal cells are covered by a smooth, rigid cuticle.The boundaries of larval patches of epidermal cells on pupae are sharply delimited.

The effect of vitamin D 1 on regeneration of epidermal cells
Due to its partial water solubility, vitamin D 1 does not penetrate the lipid coating of insect integument.To be active, vitamin D 1 needs to be injected into the body cavity or ingested in food.Phytophagous insect larvae commonly feed on the leaves of plants containing about 0.01% of vitamin D 1 (Sláma et al., 1974;Zelený et al., 1997).Excess vitamin D 1 in their diets is eliminated by excretion (Sláma, 1985(Sláma, , 1999;;Zelený et al., 1997).Injections of copious amounts of vitamin D 1 (20 μl of 0.5 mg/ml solution of 20-hydroxyecdysone in 10% ethanol) cause pathophysiological (hyperecdysonic) syndromes of hypervitaminosis, associated with adverse growth effects, like ceasing to feed, immobilisation due to muscular paralysis or formation of larval-pupal intermediates, which die due to failure to moult (Sláma et al., 1974;Sláma, 1975Sláma, , 1980)).In our experiments, the larvae of Manduca tolerated injections of enormous amounts (up to 1000 μg) of vitamin D 1 (20-hydroxyecdysone).In contrast, as little as 2 μg injected into a non-feeding pupa produced lethal hyperecdysonic syndromes (Sláma, 1999).
The lack of integumental penetration rules out the possibility of localized epidermal effects of vitamin D 1 (except for the dipping in methanol).To achieve the local prothetelic effects of vitamin D 1 in Manduca, it was necessary to apply the vitamin selectively to regenerating epidermal cells, without increasing its content in the whole body.Finally, as shown in Fig. 8, I achieved this by supplying vitamin D 1 to the excised area of epidermis by covering it by a piece of solidifi ed, 12% gelatine, containing 2 mg of 20-hydroxyecdysone per ml.The gelatine cover was overlain by a polyester coating consisting of a drop of quickly polymerizing cyanoacryllic glue (Fig. 8A).
The advantage of this technique was that the regenerating epidermal cells received the vitamin D 1 by diffusion from the gelatine.Unfortunately, for most (88%, n = 37) of the smaller (L 4 ) larvae, some physiologically active amounts of vitamin D 1 passed through the excision into the body cavity.These larvae succumbed to hypervitaminosis, such as not feeding, immobilisation and precocious secretion of new larval cuticle.Fortunately, despite these obstacles, 18 larvae survived and successfully moulted into the last larval instar.Eight of these larvae exhibited beautiful regenerated patches of sclerotized, prothetelic (precociously developed), brown pupal cuticle (Fig. 8B).
The great scientifi c value of prothetelic patches, shown in Fig. 8B, depends on the fact that the regenerating larval epidermal cells can be reprogrammed by vitamin D 1 prematurely to form the future, pupal epidermis.Despite the relatively high content of endogenous JH in the penultimate larval instar, the regenerated pupal cells were fully integrated with the surrounding larval cells of the previous morphogenetic stage.These experiments were more successful when done using the larger larvae of the last instar (Fig. 8C and D) and resulted in the development of prothetelic adult cuticular patches on the pupae.
The cells of the prothetelic adult epidermal patches, which were produced by the action of vitamin D 1 directly from the larval stage, actually bypassed the externally exposed pupal stage.According to my view, the regenerating larval cells were at fi rst reprogrammed by diffusion of vitamin D 1 from the gelatine into the covert pupal stage.Then, during the subsequent transfer through the prepupal peak of vitamin D 1 in the absence of JH, the regenerated cells of the pupal ontogenetic stage developed further and realized the initial part of the pupal-adult transformation.As already seen in Fig. 3, this period is characterized by the prepupal peak in the endogenous concentration of vitamin D 1 .Thus, when the larval cells of the host underwent the larval-pupal transformation, the regenerating prematurely transformed pupal cells simultaneously entered the initial stages of the pupa-adult morphogenetic programme.Therefore, pupae that successfully developed from the larvae that were operated on regenerated patches that consisted of thin, transparent adult cuticle without scales (Fig. 8D).Cytological analysis (Sláma & Weyda, 1997) revealed that the morphological structure of the prothetelic, delicate, white epidermal regeneration patches in Fig. 8D are indeed imaginal, and unlike the thick, elastic metathetelic larval patches in Fig. 7.The absence of scales and setae in the adult cuticular patches can be explained by the different time intervals needed for the larval-pupal transformation (3-5 days) and that required for the pupal-adult transformation in Manduca, which is much longer (10 days).Apparently the short 3-day period of the larval-pupal transformation is insufficient for the differentiation of adult setae and scales on the heterochronic prothetelic patch.

Developmental fate of the prothetelic epidermal patches
General validity of the above is corroborated by developmental fates of the prothetelic, pupal and adult epidermal patches.The data in Fig. 9 show that the vitamin D 1 induced regeneration of prothetelic pupal patches on larvae (Fig. 9A) developed into prothetelic adult patches on pupae.As already mentioned, the adult cuticle was incompletely differentiated, without adult hairs and bristles (Fig. 9B).During the next period of the pupal-adult transformation, the prothetelic patches of incompletely differentiated adult cuticle developed into patches of thin and transparent adult cuticle, without setae and scales (Fig. 9C).This indicates that during the short (3-day) period associated with the larval-pupal moult, pupal prothetelic epidermal cells only completed the initial phase of the 10-day long pharate adult period.Obviously, there was not enough time for the complete differentiation of adult setae and scales.It is more diffi cult to completely develop the primitive, secondary adult cuticle.Occasionally, some heterochronic specimens exhibited adult cuticles with partly formed adult setae, but the primitive adult prothetelic epidermis was unable to develop into secondary adult cuticle with differentiated setae.I conclude that the inherited morphogenetic programme of lepidopteran insects does not contain information for repeating the development of specifi c adult structures.
Experience based on working with more than 4,000 JH bio analogues ( Sláma et al., 1974;Sláma, 1999), revealed multiple repetitions of epidermal cells in all immature stages, except the adult stage, when treated with JH.In-terestingly, it was possible to induce the local regeneration patches by using a combination of JH and vitamin D 1 , and produce substantially different cuticular patterns of all immature stages on one insect body (Fig. 10).
In conclusion, heterochronic tissues, either JH-induced metathetelic or vitamin D 1 induced prothetelic, persist during insect metamorphosis and do not become homochronic with the surrounding cells of the host.The physiological reasons of this phenomenon are quite prosaic.Decisive factors are the required periods of time needed at a given tem-  perature for the execution of cell divisions and subsequent differentiation of the structural elements.An important condition is the absence of "reversal of metamorphosis".Recognition of these rules in insect morphogenesis and understanding the actions of JH and vitamin D 1 , have enabled us to obtain unprecedented combinations of heterochromic stages such as, for instance, the local cuticular patches of all immature ontogenetic stages exhibited in Fig. 10.
Unlike to vertebrate animals, avitaminosis associated with the defi ciency of vitamin D 1 and malignant tumours is very diffi cult to be found.There are insects, like termites, feeding on pure cellulose, obtaining vitamin D 1 exclusively from intestinal symbiotic fl ora.In Manduca, the lack of vitamin D 1 occurs between the two endogenous peaks, befor the time of pupal ecdysis (see Fig. 3).At this time, specimens with excised epidermal window succumbed and died due to ecdysial failures (62.5%, n = 32).Surviving specimens showed malformed, tumour-like epidermal regenerates, such as the one marked "e" in Fig. 10.

Brief recapitulation of the role of D 1 in insects
The data presented in Figs 3-9 reveal several biologically key features: (1) The periods of extensive cell death and cell proliferation (histolysis-histogenesis) are always linked with an increase in endogenous concentrations of vitamin D 1 (Fig. 3).( 2) The living cells communicate and create a mutually integrated unit of tissue.(3) Naturally dead cells or artifi cially removed cells disrupt the living cell integrity resulting in cell divisions associated with the reconstruction of the extant morphological structure (homochronic regeneration) (Fig. 4).( 4) The disconnected cells associated with wounds divide mitotically and restore living cell integrity (Fig. 5; Sláma, 1975;Sláma & Weyda, 1997).( 5) The mitotic divisions stop as soon as the cellular integrity of epidermal tissue is re-established, which is clearly manifested by the shape and size of the regenerated heterochronic epidermal patches (Figs 5-9).( 6) The application of exogenous JH to regenerating larval epidermal cells produces a local "status quo" effect, manifested by local patches of previous larval cuticle on pupae (metathetely) (Fig. 6).( 7) The established metathetelic character of the cuticular patterns (larval patches on pupal body) is preserved during metamorphosis, i.e. local patches of larval cuticle on pupae develop into pupal patches on adults (Fig. 7).( 8) In contrast to the metathetelic action of JH, the application of vitamin D 1 to regenerating epidermal cells caused the reciprocal, premature development of structural characters of a future ontogenetic stage (prothetely), manifested by local patches of regenerated pupal cuticle on larvae (Fig. 8).
Evidently, the most important feature of regeneration is the induction of mitotic divisions in those living cells that are deprived of mutual contact within the tissue.The second most important feature is that cell division ceases immediately after tissue integrity is achieved.In insects and probably other organisms (e.g., plants, vertebrates, including humans), the fi nal signal of cell and tissue integrity is closely related to vitamin D 1 .Failure to develop functional cellular membranes due to a lack of vitamin D 1 prevents the release of the end-point signal for tissue integrity, which may result in uncontrolled cell divisions and the development of tumours.In insects, disintegrating tissues change into a polynucleated syncytia (chromatic droplets of Wigglesworth, 1970), with distorted cell membranes.These tumour-like malformations are eliminated by an invasion of phagocytotic haemocytes.In higher animals and humans this protective mechanism does not occur and tumours with polynuclear cells continue to be produced from faulty, vitamin D 1 defi cient stem cells.The most crucial factor leading to the development of regenerating epidermal cells in insects is the previously attained morphogenetic stage (embryonic, larval, pupal or adult; Novák, 1966Novák, , 1975)), not the concentration of JH (Sláma et al., 1974;Sláma, 1975Sláma, , 1980Sláma, , 1983Sláma, , 1985)).

Sequestration of vitamin D 1 from prothoracic glands and other organs
The results are consistent with the earlier conclusions of Novák (1975) and Sláma (1975Sláma ( , 1980Sláma ( , 1985) ) that once attained morphogenetic structures can be repeated by the action of JH, but they cannot develop in the opposite direction, towards a previous ontogenetic stage.Unfortunately, the controversial concepts of "reversal of metamorphosis", proposed by Schneiderman & Gilbert (1959) and Sehnal (1984) are still cited (Riddiford, 2008;Gilbert, 2012).The metathetelic regeneration patches recorded in this study on Manduca (Figs 6 and 7) clearly support the epigenetic role of insect JH, manifested by the repeated formation of existing morphogenetic stages, like multiple larval or pupal stages, without structural changes in the DNA.This function of JH was corroborated over the past three decades by results of experiments using 4000 synthetic bioanalogues of insect JH (Sláma et al., 1974;Sláma, 1999Sláma, , 2015a, b), b).
The coincidence between endogenous peaks of vitamin D 1 and cell proliferation (Fig. 3) have been known for a long time (Sláma, 1975(Sláma, , 1982(Sláma, , 1988(Sláma, , 1998(Sláma, , 1999;;Gilbert, 2012;Smagghe, 2009, for review).The old hormonal theories (Schneiderman & Gilbert, 1959, 1964) propose that the endogenous peaks of vitamin D 1 (ecdysteroids) are the result of the PG responding to prothoracicotropic hormone (PTTH) secreted by the brain (Gilbert & Warren, 2005).Williams who proposed the brain-PG theory (Williams, 1952) and later refuted it (Williams, 1987) after fi nding that disintegrating gut is a major source of ecdysteroid.According to Sláma (1999), the whole concept concerning the role of PTTH is erroneous.The more recent studies by Sláma (1982Sláma ( , 1983Sláma ( , 1988Sláma ( , 1998) ) demonstrate that the PG are under the control of JH, not PTTH.Their physiological function is the metabolic production of water from dietary lipids (Sláma & Lukáš, 2016).The release of vitamin D 1 from PG occurs, as from many other peripheral organs, only during the short period when old larval organs disintegrate during the prepupal period (Sláma, 1998).In addition, PG in species that feed on dry food in the adult stage remain functional until the end of their life.
It is diffi cult to challenge the biochemical, PTTH-PG theory, mainly because of the heavily branched anatomical structure of the PG.Nevertheless, I managed to remove the PG from several hundred living Galleria larvae (Sláma 1980(Sláma , 1983(Sláma , 1998)).Removal of the glands had no effect on the regulation of moulting and development in this insect.In the carpet beetle, Dermestes vulpinus, the PG are located within the head capsule.Removal of PG by decapitation had no effect on its moulting cycles and did not affect the endogenous peaks of vitamin D 1 in its body (Delbecque & Sláma, 1980;Sláma, 2015a).These results were discredited in 1988 by Sehnal and his colleagues (Sehnal et al., 1988) who expressed doubts about whether the PG were successfully removed from the living larvae of Galleria.They assume that the disintegrating prepupal PG is the sole source of vitamin D 1 in this species.
A new, alternative theory proposes that the peaks in endogenous concentration of vitamin D 1 are due to the reutilisation of the built-in vitamin D 1 during the non-feeding stages in metamorphosis.The theory that vitamin D 1 is re-utilised (Sláma, 1998) proposes that structurally bound vitamin D 1 , found in the cell membranes of disintegrating larval tissues, could be enzymatically hydrolysed and the free vitamin D 1 , as the polar metabolite, could be reutilized for growth of the newly proliferating tissues (Sláma, 1998(Sláma, , 2015a, b;, b;Sláma & Zhylitskaya, 2014).Physiological reasons for the re-utilisation of vitamin D 1 during insect metamorphosis are related to the fact that insects do not synthesize the sterol nucleus de novo (Svoboda & Thomson, 1985).They receive dietary sterols from plants or microbial symbionts and convert them into cholesterol by dealkylation (Ikekawa et al., 1993).The reports of the release of vitamin D 1 from the PG of Galleria (Bollenbacher et al., 1978;Sehnal et al., 1988) or Bombyx (Iga et al., 2014;Nakaoka et al., 2017) are only for the vitamin D 1 that is released when the prepupal PG disintegrates (Galleria and Bombyx do not feed in the adult stage; their PG disintegrate and release vitamin D 1 shortly before pupation).

Earlier investigations on the regeneration of epidermal cells, hypervitaminosis
The regeneration of insect epidermal cells was carefully investigated half a century ago by Sir Vincent B. Wigglesworth (1970) in an hemipteran species, Rhodnius prolixus Stål (Reduviidae).He describes the regeneration of epidermal cells after local lesions of the integument, including the regeneration of specifi c bristles and dermal plaques.Unfortunately, vitamin D 1 and JH analogues were not available at that time.The results of the current study (Fig. 4) on Manduca are fully consistent with Wigglesworth's results on epidermal regeneration in Rhodnius.However, these results are in direct confl ict with the early conclusions of Piepho (1951), who studied the regeneration of pieces of epidermis implanted into the body cavity of Galleria.Essentially, Piepho proposed a hypothesis, which was used by Schneiderman & Gilbert (1959, 1964) as a corner stone of the hormonal theory, which has dominated insect endocrinology for more than 60 years (Smagghe, 2009;Gilbert, 2012;Sláma, 2015a, b).Piepho (1951) assumes that insect epidermal cells are just like a ball of hormones.He assumes that epidermal implants develop into larval, pupal, or adult structures simply in response, respectively, to high, intermediate or low concentrations of JH.According to Piepho, when the concentration of JH is high, pupal epidermal cells can dedifferentiate and then differentiate into previous, outlived morphogenetic stages.For example, pupal cells could differentiate back into larval cells under high concentrations of JH.However, this has never been independently experimentally confi rmed.The concept of Piepho (1951) and the related hormonal theories (Gilbert & Warren, 2005;Riddiford, 2008) were refuted in 1997 based on the results of scanning electron-microscopy study of regenerating epidermal cells (Sláma & Weyda, 1997).The effects of JH on epidermal cells followed the all-ornone rule in terms of previous and future ontogenetic structures.Studies using JH and its 4000 bio analogues (Sláma, 1999) indicate that developmental instructions are coded in the genome.A minimum physiologically active dose of JH and a 500-million-fold higher concentration have the same effect.In contrast to this more or less qualitative action of JH, injections of vitamin D 1 cause a quantitative, dose-dependent acceleration of the moulting cycle (Sláma, 1980).Overdosage usually results in precociously formed, incompletely differentiated intermediate forms (Sláma et al., 1974(Sláma et al., , 1993;;Sláma & Lafont, 1995;Sláma, 1999;Sláma & Zhylitskaya, 2014).In this respect, the difference in the effect of different physiological concentrations of vitamin D 1 (Fig. 3) and of artifi cially increasing the concentrations by injection is very important.Because of this, the prothetelic regeneration of patches induced by vitamin D 1 shown in Figs 7 and 8 are rather unique features observed for the fi rst time only in Galleria (Sláma, 1975).
During animal evolution, vitamin D 1 acquired a special function in the regulation of insect growth, which involves periodical changes in the structure of the exoskeleton.Therefore, the main growth function of vitamin D 1 in insects does not depend on growth of bones and muscles, as in vertebrates, but on the repeated exchange and enlargement of the integument (Sláma & Zhylitskaya, 2014).During moulting in insects, epidermal cells fi rst detach from the old cuticle (apolysis).During this pharate stage, the cells proliferate and differentiate, before forming a new epidermis and secreting a new cuticle.Moulting is controlled by endogenous peaks in the concentration of vitamin D 1 (Fig. 3).The peaks have a homeostatic function in synchronising the different stages in the moulting cycle (Sláma, 1980).Injections of vitamin D 1 made before the peak accelerate, while those made after the peak inhibit the onset of insect ecdysis.The secretion of new cuticle can only occur at a particular stage in the moulting cycle.The importance of the above was revealed by biochemists and molecular biologists investigating the effects of vitamin D 1 at the levels of receptors and genes (Gilbert & Warren, 2005;Smagghe, 2009).
The above indicate that injections of vitamin D 1 made before the endogenous peak can result in a serious developmental abnormality, manifested by pathophysiological syndromes of D 1 hypervitaminosis.The syndromes were fi rst described soon after the discovery of ecdysone by C.M. Williams (1970) in pupae of the Cecropia silkworm, Hyalophora cecropia (see Sláma et al., 1974).The pathogenicity of vitamin D 1 does not depend only on the injections of high concentrations, but also on the non-physiological, instantaneous delivery of an abundant amount.The endogenous peaks show a physiological course represented by the successive rise and decline in the concentration of vitamin D 1 .Recent attempts to moderate the effects of hypervitaminosis are based on the preparation of synthetic, vitamin D 1 complexes with porphyrins (Sláma & Zhylitskaya, 2014).These complexes are enzymatically hydrolysed, slowly releasing the free vitamin D 1 , thus, imitating the natural endogenous peaks.
The secretion of the new and reabsorption of the old cuticle is the most vulnerable period in the moulting cycle.These stages are usually immobile.Feeding larvae have the lowest titres of vitamin D 1 because they have high rates of metabolism and excretion.It has been assumed that high amounts of these compounds in certain plants protect them against herbivores (see below).Experimental injection of vitamin D 1 into feeding larvae produce rather dramatic results.They cause an immediate cessation of feeding, which is associated with reduced locomotion and neuromuscular paralysis ("preecdysial sleep").In the present experiments with Manduca, these syndromes of D 1 hypervitaminosis were frequently encountered in association with the prothetelic effects described in Fig. 8.The affected larvae did not feed and move, quickly desiccated and died.Ignoring the hypervitaminosis features recorded in vivo can lead to misleading results related to in vitro investigations of its effects on vitamin D 1 receptors (Gilbert & Warren, 2005;Smagghe, 2009).As in insects, large hypervitaminic amounts of vitamin D 1 result in embryo toxicity in vertebrates (Košár et al., 1977) and aberrant tissue growth (Lagova & Valueva, 1981).
During the discovery of JH-active materials in plants, Sláma & Williams (1965) speculated that "by containing insect hormones and other effective compounds, plants could possibly evolve an incredibly sophisticated self-defence against insect predation".Despite criticisms (Sláma, 1979) the insects living on the vitamin D 1 rich Siberian plant, Leuzea carthamoides, were studied, which revealed that the caterpillars of several insect species, especially polyphagous Noctuid moths, are virtually resistant to the relatively large amounts of vitamin D 1 in their food (Zelený et al., 1997).
It is also possible that, as in the case of structurally related brassinosterols, vitamin D 1 could also act as a phytohormone.This was experimentally investigated using special bioassays, in which the effect of vitamin D 1 (20-hydroxyecdysone) was compared with the effects of certain commonly known phytohormones (Macháčková et al., 1995).The assays used were the wheat coleoptile bioassay, gibberellin bioassays using dwarf maize or rice, cytokinin bioassays using tobacco callus, brassinolide-related ethylene formation in dwarf maize and alfalfa, fl owering assays using Chenopodium and special assays based on somatic embryogenesis in cell cultures of alfalfa.The results provide unambiguous evidence that the effects of vitamin D 1 are not similar to that of any of the above phytohormones (Macháčková et al., 1995).
I used insects as a sensitive biological assay (0.1 μg detection limits of 20-hycdroxyecdysone equivalents) (Sláma et al., 1993;Sláma & Zhylitskaya, 2014), to study the translocation of vitamin D 1 in the plant Leuzea carthamoides, during an entire vegetative season.The dry winter roots contain 0.5 to 2.1% of vitamin D 1 complex (95% of 20-hydroxyecdysone + 5% mixture of related ecdysteroids) and dry seeds 2.1 to 3.1%.The highest content was found in the early unfurling leaves in spring.This shows that vitamin D 1 and the related derivatives of 7-dehydrocholesterol are biosynthesized within green leaves during photosynthesis (average content of dry leaves is 0.01 to 0.05%).At the end of a season, vitamin D 1 is translocated from the shoots to the roots and seeds.Curiously enough, the seeds of Leuzea contain approximately 500,000-fold more vitamin D 1 (Stránský et al., 1998) than an average insect pupa (Karlson, 1966).Moreover, the content of this derivative of 7-dehydrocholesterol (zoosterol?) in the plant is 700 fold greater than that of phytosterols, ergosterol and β-sitosterol.This disproportion is due to the fact that cholesterol derivatives are preferentially hydroxylated in plants and thus disappear from the pool of free lipid soluble plant sterols (Stránský et al., 1998).
Plants of Leuzea carthamoides (Fig. 11) are a rich and convenient source of vitamin D 1 for pharmacological use.
Pure vitamin D 1 is a white crystalline substance with a sweet taste due to its six or seven hydroxylic groups.Paradoxically, it is a partly water soluble, physiologically important "cholesterol sugar" (Sláma et al., 1996).The essential structural feature of vitamin D 1 is the conjugated, ɑ, β-unsaturated, 6-keto,7-dehydro grouping in the B ring of cholesterol, which stabilizes the molecule and provides a strong electron accepting property (Sláma et al., 1974).

Similarities between insect and human physiological systems
It has been already pointed out in this paper that regenerating epidermal cells of Manduca require vitamin D 1 for the maintenance of tissue integrity and prevention of uncontrolled mitotic divisions.Similar roles in regeneration can be envisaged for vitamin D 1 in the human body.Such comparisons were generally ignored because protostomes, such as insects, and deuterostomes, like humans, have evolved separately over approximately 550 million years.However, despite such a large phylogenetic divergence, 37% of the genes in insect genomes (Drosophila) are homologous and used for similar morphogenetic and physiological functions both in insects and humans (Devillers, 2013).For instance, the primordial formation of insect and human hearts is orchestrated by similar sets of genes (tinman) (Bodmer, 1995;Sláma, 2012).In both instances the hearts perform identical, involuntary, purely myogenic contractions based on the discharge of electrical potentials from depolarized myocardial cells.In insects, as in the human heart, rhythmical beating is regulated by similar sets of special pacemaker nodi (e.g., sinoatrial, atrioventricular and Hiss bundle in the human heart; posterior nodi in the insect heart) (Sláma, 2012).
Analogous insect-human similarities were recently reported for structures and functions of their central neuroendocrine systems.For example, the neurosecretory cells in the insect brain are similar structurally and functionally to neurosecretory cells located in the human hypothalamus (Sláma, 2015a, b).Additional evidence for insect-human similarities are reported in the fi eld of respiratory physiology.Curiously enough, insects actually breath like humans.They mechanically inspire and expire air through particular spiracles and ventilation is regulated by an autonomic, cholinergic neuroendocrine system, known as the coelopulse (Sláma & Santiago-Blay, 2017).This system is structurally and functionally like the human parasympathetic nervous system.These similarities indicate that the role and physiological functions of vitamin D 1 in regeneration and cell divisions described here for Manduca (Figs 4-9) could also be true for human tissues.This statement is further supported by the similar physiological role of vitamin E (γ-and δ-tocopherols) in the regulation of reproduction both in insects and humans (Jedlička et al., 2009).
The above indicated similarities between the effects of vitamin D 1 in insects and humans can be extended to other vertebrates, like Japanese quail (Koudela et al., 1995).Studies have revealed strong anabolic, vitamin D-like growth effects of vitamin D 1 (20-hydroxyecdysone), which surpass the effects of the commercial dietary additives for chickens.The quails stored the dietary vitamin D 1 in their blood depending on the amounts of the vitamin in their food (0.5, 6.0 and 8.0 ng per 100 μl of blood serum) (Sláma et al., 1996).The strong anabolic effects of vitamin D 1 in birds (Koudela et al., 1995) confi rm previous reports of Ukrainian, Belorussian and Kazakhstan authors, working with pigs, cattle, and other domestic animals (Syrov, 1984;Dzukharova et al., 1984;Sláma & Lafont, 1995;Kholodova, 2001;Smagghe, 2009).Stopka et al. (1999) investigated the pharmacological effects of pure vitamin D 1 in mice, using daily peritoneal injections of from 2.8 mg/g to 10 mg/g, during their juvenile and reproductive periods.The injections of vitamin D 1 signifi cantly enhanced the growth rate in juvenile females, but not in juvenile males.In the adult stage, the injections caused substantial increase in growth of both the male and female mice.These results are consistent with previous observations on the effects of vitamin D 1 in mice, rats and other animals, and in humans (Sláma et al., 1974;Sláma & Lafont, 1995;Kholodova, 2001).Historically, the fi rst reports of vitamin D 1 in humans were for patients infested with parasitic worms.They excreted vitamin D 1 in their urine (Koolman et al., 1986;Baswaid et al., 1991).Later, however, it was discovered, that vitamin D 1 is also present in urine and blood samples of healthy people (Gharib et al., 1991).

Th e neglected role of vitamin D 1 in human health care
Clinical investigations on vitamin D 1 were hindered for a long time by the persistent belief it is an insect hormone and by the limited availability of the pure substance.In the former Soviet Union, the research was stimulated by the accidental fi nding that dietary additions of a drug prepared from the Siberian plant, Leuzea (syn.Rhaponticum) carthamoides increased the production of milk and meat in cattle (see Sláma & Lafont, 1995 for a review).They used a partly purifi ed extract of vitamin D 1 from Leuzea for development of the pharmacological preparation called ECDIS-TEN, in 1980.Prescriptions recommend 5 mg capsules, 3-times per day.The advertisements promise a plethora of benefi cial pharmacological effects like anabolic improvement of physical condition, removal of astheno-depressive states, tonic effects, elimination of weakness, impairment of chronic intoxication, neurasthemia, neurosis, hypotension, fatigue and recovery from infectious disease (Sláma & Lafont, 1995, for review).According to Syrov (1984), the preparation was used as an anabolic drug by Soviet Olympic athletes (Syrov, pers. commun. to KS).The medication advertised a monthly increase of half a pound of muscular tissue.Due to its partial water solubility and natural occurrence, vitamin D 1 has never been classifi ed as a prohibited anabolic steroid.
Evidently, the general role of the antirachitic vitamin D in adult stages depends mainly on the enhanced growth and regeneration of certain tissues and organs.Based on what happens in Manduca, vitamin D 1 should also provide functional cell membranes for preventing uncontrolled endomitotic cell divisions in human organs.So far, I have not found any reports of tests on the role of vitamin D 1 on the ratchet bone disease, using randomized controlled trials, the gold standard in medical testing.The original reports on vitamin D, completed almost 100-years ago, noted an increased antirachitic property of samples collected during summer (Hess & Unger, 1921;Hess et al., 1925).The effects were attributed to increased UV-radiation, increasing the conversion of cholesterol into the antirachitic 7-dehydrocholesterol.I feel that seasonal effects on antirachitic properties can be explained in a more prosaic way.Namely, sunshine increases the photosynthesis of vitamin D 1 in the green leaves of plants.The pioneers of vitamin D research did not know this, because they did not know that cholesterol derivatives such as vitamin D 1 are present in plants.
The credibility of the theory of UV-based activation of secosterol vitamins D 2 and D 3 in the skin (Windaus et al., 1931;Windaus & Bock, 1937), however, needs to be reexamined.Who knows where vegetarians get their essential vitamin D from, if it is an animal sterol?In addition, where do Nordic people get their vitamin D, if their skin is permanently covered and not exposed to UV radiation?Polyhydroxylated, 6-keto, 7-dehydrocholesterol (ecdysone, vitamin D 1 ) was discovered in 1965 (Karlson, 1966).This is almost three decades after the end of the basic antirachitic investigations.In 1965, nobody suspected that an "insect moulting hormone" could be the true vitamin D. The early suggestion that ecdysteroids could be the neglected vitamin D (Sláma, 1993), was ignored.The respected authority on the chemistry of vitamins, Prof. P. Karlson (Karlson, 1981) and the associated group of chemists, generally known as "ecdysonists", did not comprehend the possibility of ecdysone being a vitamin and adhered strictly to the old concept of it being an insect hormone.The results reported here of the study on Manduca (Figs 4-10) confi rm the key role of vitamin D 1 during tissue regeneration (Sláma, 1993(Sláma, , 2014(Sláma, , 2015a, b), b).These fi ndings are in good agreement with the role of endogenous peaks of vitamin D 1 in the control of cell divisions in insects (Fig. 3) and it is highly likely that vitamin D 1 has a similar role in cell division and regeneration in humans.Confi rmation of this will depend on the availability of a suffi cient quantity of vitamin D 1 for pharmacological investigations.
As already mentioned, vitamin D 1 is present in human blood and urine (Koolman & Moeller, 1986;Gharib et al., 1991).Its concentration in human blood should refl ect the concentration of vitamin D 1 in the diet.The storage of vitamin D 1 in the blood of Japanese quail (Sláma et al., 1996) indicate that humans could also store vitamin D 1 in their blood and excrete the excess in their urine.Experimental proof of the above can be easily obtained using radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), which are well documented and widely used in studies on insects (Delbecque & Sláma, 1980;Sláma et al., 1996;Smagghe, 2009;Gilbert, 2012).

The D 1 avitaminosis theory of malignant growth
According to the data presented in previous sections, vitamin D 1 should be included in the sterolic vitamin D group with anabolic, growth stimulating and antirachitic properties.It is widely produced by microorganisms, fungi and plants, and due to its amphoteric solubility, can be placed in both the partly water soluble and lipid soluble categories of vitamins.In juvenile vertebrates, vitamin D 1 stimulates anabolic growth of bones and musculature (antirachitic activity); in the adult stage it is essential for growth and regeneration of individual tissues and cells.The structurally well-formed cellular membranes, produced in the presence of vitamin D 1 result in the resumption of living cell integrity in regenerating tissues.The resumed integrity between living cells stops further divisions of regenerating cells not only in invertebrate, but also in vertebrates.By contrast, the defi ciency of vitamin D 1 during somatic growth or regeneration may lead to the formation of aberrant cell divisions (Syrov, 1984;Sláma & Lafont, 1955;Kholodova, 2001).This may fi nally result in the formation of polynucleated syncytia of pernicious malignant cells.In insects, the polynucleated syncytia appear in disintegrating tissues and are swallowed and destroyed by phagocytotic haemocytes.This protective, anti-tumour mechanism does not operate in humans, where the defective polynucleated cells persist and create malignant tumours.
The chemical structure of Vitamin D 1 is derived from 7-dehydrocholesterol and includes many hydroxylic groups on the secondary or tertiary carbon atoms.Due to this, its biological activity does not depend on hydroxylation in the liver or kidneys.In addition, its biological activity equally does not depend on the conversion of cholesterol into 7-dehydrocholesterol by UV-radiation.Moreover, the most pronounced pharmacological effects of vitamin D 1 in humans are manifested straight by strong anabolic, vitamin D-like effects on the growth of the body (Sláma & Lafont, 1995).As already mentioned, there is a plethora of reports of the benefi cial health effects of vitamin D 1 , such as rejuvenation, increased growth, enhanced muscle performance, tonic, neurasthenic, neurogenic, anti-depressive, anti-fatigue, immuno-stimulating and similar vitamin D-like effects (review by Sláma & Lafont, 1995).
Similar to the effects in insects depicted in Figs 4-10, a defi ciency of vitamin D 1 in human blood might result in defective regeneration of naturally dead cells or artifi cially wounded tissue.As a consequence of uncontrolled mitotic or endomitotic divisions (malignant tumour growth) can occur in vitamin D 1 defi cient tissue (Sláma, 2018).
Chemotherapy, which abruptly stops all mitotic divisions, can temporarily abolish malformed, vitamin D 1 defi cient regeneration.It is likely that success in restraining malignant growth will be related to the accidental presence or absence of vitamin D 1 during and after chemotherapy.When vitamin D 1 defi ciency is prolonged, the oncological problems can come back again in the form of metastases.Naturally, the most vulnerable and most susceptible tissues to vitamin D 1 defi ciency are the relatively large, acinose cells, whose cell membranes are adapted to secrete proteinaceous products (such as mammary glands, salivary glands, prostate cells, pancreatic cells, intestinal epithelium).External factors increasing the incidence of malignant growth (hereditary, malnutrition, adverse chemicals, senescence, obesity, repeated injury, viral infections, alcoholism, smoking, etc.), have a common denominator, a defi ciency of vitamin D 1 in the blood.As far as I know, nobody has tested whether the addition of the newly defi ned vitamin D 1 could stop or shrink already developed malignant tumours.However, there are many lucrative research proposals for destroying malignant growth by destroying already existing, polynuclear tumour cells.According to my view, these cells are the consequences, not the cause, of the defective growth of stem cells.Solution of the problem with malignant tumours would be to eliminate the cause, not just the consequence of the malignant growth.In other words, the goal should be to prevent vitamin D 1 defi cient stem cells from producing malformed tumour cells.
In addition to these oncological problems, there are other incurable diseases such as spinal muscular atrophy or neural dysfunctions (Alzheimer disease, Parkinson's disease in non-dividing nerve cells).Based on what is here presented on the role of vitamin D 1 , I conclude that the hydro-lipophilic properties of vitamin D 1 may be essential for the faultless electrophysiological functioning of nerve cells.
I hope sincerely that this 50-year long story of the hunt for the fi ctious insect hormone and 100-year neglect of vitamin D 1 , may stimulate investigations by professionals working in the fi eld of human health.Medical research was too expensive for me as it is far greater than that required for studying insects.I trust, however, that a simple analysis of the vitamin D 1 content and defi ciency in blood of healthy and oncological patients might help resolve the previously neglected and now re-defi ned role of vitamin D 1 in the formation of pernicious malignant tumours.

Fig. 2 .
Fig. 2. A -size and location of the epidermal area excised from the last instar larva; B -window excised from the integument of a freshly ecdysed pupa of Manduca sexta.

Fig. 3 .
Fig. 3. Relationships between endogenous concentration of vitamin D 1 (full line), total body metabolism (dotted line) and transformation of larval-pupal and pupal-adult tissues (dotted area) during the metamorphosis of the wax moth, Galleria mellonella L.
) Disturbed cell integrity, caused by the natural death or artifi cial removal of a living cell, causes neighbouring cells to divide.(2) The dividing epidermal cells stop dividing as soon as the living cell-to-cell integrity is re-established.(3) This mechanism prevents the formation of tumours.(4) The regenerating epidermal cells remember

Fig. 5 .
Fig. 5. Schematic outline of the regeneration of epidermis (A) excised from Manduca sexta.The interrupted integrity of epidermal cells (B) was repaired by mitotic divisions of the marginal cells (C) that divided until the new cells (D) reunifi ed the living epithelium.The reconstituted epithelium remodelled extracellular stromal elements and a new cuticle externally (E).Epidermal cells separated by a cover slip divide and grow all the way around the inorganic object until they achieve living cell integrity (F).

Fig. 6 .
Fig. 6.Heterochronic regeneration of epidermal excisions under the effects of juvenile hormone (JH).The excisions were made on 3-day old feeding last instar larvae (A) or 3-day old developing pupae (C) of Manduca sexta.At this time, epidermal cells of the larvae and pupae are insensitive to JH.The dividing and regenerating cells, however, are sensitive to JH, which results in the development of metathetelic (or previous stage) larval cuticle on pupae (B) or pupal cuticle on adults (D).
The results in Fig.6perhaps represent the best examples of the local metathetelic actions of insect JH.Physiological implications of these results are: (1) Regenerating epidermal cells independently execute their own, JH conditioned, epigenetic or "status quo" morphogenetic programmes, although the surrounding host cells simultaneously follow substantially different, larval-pupal or pupal-adult morphogenesis; (2) Regenerating epidermal cells that develop into larval structures (larval metathetely) are fully compatible with the morphological more advanced surrounding pupal cells; (3) Regenerating epidermal cells of heterochronic tissues are able to cease dividing when tissue integrity is achieved.

Fig. 7 .
Fig. 7.A and B -metamorphosis of the metathetelic, larval epidermal patches during the pupal-adult transformation of Manduca sexta.C and D -heterochronic pupae treated with a JH analogue (100 μg of JH-1 per specimen) develop into secondary pupae with secondary pupal cuticle and a metathetelic larval cuticular patch in the middle (a -primary pupal cuticle, partially removed; b -secondary pupal cuticle; c -local patch of heterochronic, secondary larval cuticle).

Fig. 8 .
Fig. 8.A -a fourth instar larva (L 4 ) of Manduca sexta with the area of excised epidermis covered by solidifi ed gelatine containing vitamin D 1 .B -regenerated patch of prothetelic pupal cuticle on the last instar larva (L 5 ).C -area of epidermis excised from a last instar larva covered by gelatine containing vitamin D 1 .D -local prothetelic patch of incompletely developed, scale less adult cuticle after the next moult.

Fig. 9 .
Fig. 9. Developmental fates of the prothetelic patches induced by vitamin D 1 on Manduca sexta.A -prothelic regeneration of pupal cuticle on a last (L 5 ) instar larva.B -prothetelic regeneration of adult cuticle (white patch) after the moult into pupa.Note the absence of setae ("hairs") that are abundant on adults.C -prothetelic patch of undifferentiated adult cuticle after the moult to adulthood.Note the absence of setae ("hairs").

Fig. 10 .
Fig. 10.Histological specimen of a multiple heterochronic patch on an adult Manduca sexta subjected to regeneration combined with an application of juvenile hormone (JH) and vitamin D 1 during the immature period.a -adult epidermis with setae; b -incompletely differentiated adult cuticle with minimal setation; c -primary pupal cuticle; d -secondary pupal cuticle; e -larval cuticle.