Introduction

Spiders are one of the most important groups of arthropods that play a crucial role in ecosystems because they belong to the most abundant group of terrestrial predators (Turnbull 1973). Analysis of available data indicates that spiders as a bioindicators of environments (e.g., Bednarek et al. 2016), their venom and silk (e.g., Konwarh et al. 2016; Wang and Wang 2016) are top researching aspects of spider life. In contrast, knowledge about the neuroendocrine system of this group of arthropods, which includes the presence of the main hormones (juvenile hormones, ecdysteroids) and neuropeptides, and their role in ontogenesis, is still not well understood and explored (compare with Table 1). On the other hand, the neuroendocrine system of insects is probably the best understood of all of the arthropods (for reviews, see Davey 2000; Roe et al. 2014; Weaver and Audsley 2009).

Table 1 Current knowledge about role of the neuroactive compounds in molting and reproduction in spiders

Every scientist who has tried to investigate the physiology of spiders knows what the reason is for this great divergence in knowledge between groups of arthropods. The fact that spiders do not tolerate any invasive manipulation is the first and most important reason. It should be emphasized that spiders can be divided into two infraorders—Mygalomorphae and Araneomorphae that differ in several aspects. Mygalomorphae spiders are larger in size that can be helpful in micromanipulations. Moreover, it should be noted that the smaller size of spider body is correlated with a decrease in the tolerance for these kinds of procedures. Obviously, injecting and administering active compounds is possible. However, the high mortality rate and the weaker effect of used substances should be expected (personal communications). The efficient detoxification system in spiders may be the reason for this phenomenon (Pourié and Trabalon 2003). Therefore, the use of a higher concentrations of chemical substances than those shown in studies on insects is required (personal communications). However, the using of Mygalomorphae spiders is restricted, because the majority of these spiders is protected by the Washington Convention. Moreover, long-term experiments require spiders with a short period of ontogenesis (to obtain many generations in a brief time) and easy to laboratory breeding. Mygalomorphae spiders have a long development cycle that prolongate the time of the experiment to achieve appropriate stages of ontogenesis. Thus, it seems that using of Araneomorphae spiders with a short time of development is more appropriate. However, selecting a suitable species within this infraorder of spider will determine the possibility of carrying out the experiments (doing the appropriate number of replications resulting from the spider size) and their success (the high mortality should be borne in mind). It should be noticed that there are only a few Araneomorphae spiders that can be successfully grown via laboratory breeding (e.g., Parasteatoda tepidariorum, Cupiennius salei; Mcgregor et al. 2008).

Despite the fact of quite accurate description of the anatomy and morphology of the nervous and neuroendocrine systems of spiders (Gabe 1955; Legendre 1953, 1959), the knowledge about their role in the synthesis of neuroactive compounds is still insufficient and unclear. Therefore, until now, the potential role of the molting organ (MO) in the ecdysteroids synthesis is not proven (Bonaric 1980; Bonaric and De Reggi 1977; Bonaric and Juberthie 1983). Moreover, the analogue organs to insect corpora cardiaca (CC) and corpora allata (CA) are still not discovered, although the data indicate that Schneider organ 1 and Neurohemal organ 1 can refer to the insect CC, and Schneider organ 2—CA (Bonaric 1980; Bonaric and Juberthie 1983; Bonaric et al. 1984). It should be emphasized that the CA plays an essential role in JH synthesis in insects (Tobe and Stay 1985), whereas an allatotectomy (CA removal) is a method that is widely used to understand the functions of the hormones (compare with Table 2). Thus, studies about the presence and roles of JH in spiders by observing changes in the morphology of the analogs of the CA and allatotectomy are not possible because data about this organ in spiders are not known. Similarly, the removal of any other organ that may be responsible for JH synthesis is not possible because only rudimentary data about the morphology of the central nervous system of spiders are available. In addition, the localization and the protection mechanism of the nervous system prevents the removal of its elements in vivo.

Table 2 Examples of possible direction of research about hormones and neuropeptides

Another very important issue is connected with the differences in the physiology of spiders, compared with insects. For instance, spiders do not have a true phenoloxidase (a component of the immune system), which is in contrast to other arthropods (Decker and Rimke 1998), and their RNA isolation yield is lower than in insects (personal communication). Thus, optimization of all of the available protocols, such as determining the enzyme activity, detecting various proteins, isolating RNA and determining gene expression, is necessary (personal communications; Babczyńska et al. 2011; Bednarek et al. 2016; Posnien et al. 2014). In addition, some methods cannot be used in spider studies (Western blot, ELISA, IHC) because commercially available antibodies that are dedicated to spiders are not present. The antibodies that are dedicated for other animals do not always give a positive response to the material from spiders (personal communication).

Finally, studies about the physiology of spiders at the gene level (detection, expression, knockout) are also limited for several reasons. The sequence of the genome is only available for a few species of spiders (thanks to the Project i5K). Comprehensive genomic or transcriptomic resources are not available. Therefore, previous studies about spiders have relied on cloning the genes that are involved in the process being analyzed in a model organism, such as Drosophila melanogaster (Posnien et al. 2014).

To sum up, it seems that the analysis of the role of the neuropeptides, JHs and ecdysteroids in the well-known physiological processes in spiders with short period of development (Araneomorphae) is an appropriately chosen, safe and easy method with which to explore these compounds in this group of arthropods. Furthermore, our experience with spiders indicates that the processes of reproduction and molting are a good starting point to begin the exploration of the neuroendocrine system. Hence, in this paper, we emphasize the influence of selected hormones and neuropeptides in the control of these processes.

This paper is the first article that summarizes the current knowledge about the role of juvenile hormones, ecdysteroids and neuropeptides in the reproduction, molting induction and development in spiders from Araneomorphae infraorder. In addition, the authors suggest possible ways to detect these substances and identify their function in the ontogenesis of spiders what is unique and cannot be found in the arachnological literature.

Juvenile hormones

A juvenile hormone (JH) is a sesquiterpenoid that is synthesized from a farnesoic acid derivative in the classical mevalonate pathway (Riddiford 1994).

Insect JHs are biosynthesized by the CA (Tobe and Stay 1985). This process is regulated by the antagonistic action of neuropeptides—allatostatins (Stay and Tobe 2007) and allatotropins (Elekonich and Horodyski 2003). Six various forms of the JHs have been described in insects, and their occurrence is dependent on the insect order (Riddiford 1994). However, JH III is widely distributed in this group of arthropods (Crone et al. 2007). JHs are secreted into the hemolymph and transported (Hidayat and Goodman 1994) to the target cells by juvenile hormone binding proteins (JHBP). Then, the hormone enters the cell and interacts with the dimer of the nuclear receptor. The two main candidates for the JH receptor were selected—ultraspiracle (USP) (Riddiford et al. 2000) and Methoprene-tolerant (Met) proteins (Konopova et al. 2011; Konopova and Jindra 2007; Wilson and Fabian 1986). It is generally believed that the Met protein is actually the JH receptor (Miura et al. 2005). The lack of JHs in the cell induces the formation of Met–Met homodimers. However, the appearance of the hormone induces the disintegration of this complex and Met binds with a co-activator (e.g., Taiman, FITC, SRC). The complex of Met-JH-co-activator acts by activating or repressing specific target genes in the regulatory sequences (Riddiford 2012; Zhang et al. 2011). JHs together with ecdysteroids play a crucial role in the regulation of metamorphosis and development. This sesquiterpenoid hormone is responsible, among others, for the “status quo” of larvae (Riddiford 1994). Moreover, JHs are involved in reproduction (for example, vitellogenesis, the development of gonads, egg production) (Davey 2000; Handler and Postlethwait 1977; Kethidi et al. 2005; Venugopal and Kumar 2000) and the regulation of diapause and lifespan (Liu et al. 2008).

It should be emphasized that while knowledge about JHs in insects is quite extensive, data about this sesquiterpenoid hormone in Arachnida are rudimentary and equivocal.

The presence of juvenile hormone or its analogue is still not confirmed in Acari (mites, hard and soft tick) (Neese et al. 2000). However, Zhu et al. (2016) suggested the methyl farnesoate (MF) may play a role of JHs in this group of arthropod. MF is the immediate precursors of JH, which plays a similar role in crustaceans as insects JHs (Laufer et al. 1986; Tobe et al. 1989). Moreover, some evidence about the gonadotrophin from the synganglion–lateral organ complex in the tick (Marzouk et al. 1985), which acts in a similar manner as insect JHs, was found (Aeschlimann 1968). Studies about exogenous JHs (Obenchain and Mango 1980) and the anti-juvenoid (Leahy and Booth 1980) on tick reproduction indicate the occurrence of specific JH receptors and JHBP (Kulcsar et al. 1989) in this group of arthropods. However, the only data about the identification of the JH receptor in Acari resulted from in silico analysis, which indicate the presence of the Met protein only in tick Ixodes scapularis (Qu et al. 2015). The role of JH-like substances (e.g., MF, gonadotrophin) in ticks in reproduction (e.g., egg development, the induction of vitellogenesis and oviposition) and in suppressing molting is unclear (see reviews Belozerov 2006; Rees 2004). For instance, vitellogenesis is stimulated by administration of a JH analog, JH I or JH III in virgin female Ornithodoros moubata (Connat et al. 1983), while the lack of this effect was observed in other similar experiment on the same species (Chinzei et al. 1991).

Until now the occurrence of juvenile hormones or other similar substances, such a MF is not confirmed in spiders. Despite the fact that Bonaric (1988) confirmed the presence of substances similar to the insect hormones in spider Pisaura mirabilis (Pisauridae), until now their chemical nature is still undefined. The site of JHs and/or their analog synthesis is constantly undetermined. Nonetheless, it is emphasized that such hypothetical organ should occur (Bonaric 1995). It is suggested that Schneider organ 2 (part of stomatogastric ganglion; Legendre 1953, 1959) can be a possible homolog of the insects CA, and hence, it can be a structure responsible for juvenile hormone or analogues synthesis (Bonaric 1980; Bonaric et al. 1984; Juberthie 1983; Bonaric and Juberthie 1983). Molecular mechanism of action of this group of hormones is still unknown. Furthermore, no protein receptors are identified. Few studies on the role of JHs and/or their analogues in the physiological processes of spiders indicate that this group of hormones probably plays a similar role in the development and reproduction in spiders. For instance, it was described that application of the insect JH prolonged the intermolting times in P. mirabilis (Bonaric 1979, 1986) as like as in insects, (e.g., Konopova and Jindra 2007). Moreover, the presence of JH analogues during the vitellogenesis in female spider P. mirabilis was noticed (Bonaric 1988). Thus, it seems that this group of hormones may play a role in the regulation of this process in spiders.

Ecdysteroids

Zooecdysteroids are a group of animal steroid hormones that include ecdysone (α-ecdysone) and 20-hydroxyecdysone (20E, initially named β-ecdysone). In insects and ticks, 20E is the major active hormone (Dees et al. 1984; Lafont 1997; Lafont et al. 2005; Riddiford 1993).

In insects, ecdysone is produced by a prothoracic gland until the metamorphosis, at which time the gland degenerated (Sakurai and Williams 1989). In adults, ecdysteroids are synthesized by the ovaries (Carney and Bender 2000; Hagedorn et al. 1975) and male accessory glands (Hentze et al. 2013). The response of tissues to ecdysteroids is mediated by a heterodimer of the nuclear receptors: the ecdysone receptor (EcR; Koelle et al. 1991) and ultraspiracle protein (USP; Yao et al. 1992, 1993)—EcR–USP. Low titer of 20E in cell induces the EcR–USP binding with co-repressors. The EcR–USP-co-repressor complex has the ability to bind to DNA and to repress the transcription of target genes. However, in the case of the elevated 20E titer, the 20E-EcR–USP complex binds to the co-activator and then, by attaching to the specific DNA sequence (ecdysone response elements), induces transcription of the gene (King-Jones and Thummel 2005; Riddiford et al. 2000; Thummel 1996). Ecdysteroids coordinate the critical developmental events, including embryonic morphogenesis (Kozlova and Thummel 2003), larval molting and metamorphosis (Riddiford 1993). Additionally, in adults, ecdysone signaling is essential for particular aspects of oogenesis such as vitellogenesis and follicle development (Carney and Bender 2000; Hodin and Riddiford 1998), as well as for courtship (Ganter et al. 2012).

Compared to knowledge about juvenile hormones, data about ecdysteroids in arachnids are more extensively explored. The site of ecdysteroids synthesis is prothoracic glands in larvae and in the nymphal integument in adult ticks (Lomas et al. 1997; Zhu et al. 1991). Similar to insects, these steroid hormones act by EcR and USP receptors (Palmer et al. 1999). The results of various experiments indicate that the function of ecdysteroids in ticks is quite similar to that in insects (the induction of molting—Solomon et al. 1982, sperm maturation—Oliver and Dotson 1993). Data about the role of these steroid hormones in the regulation of oogenesis and oviposition are ambiguous (stimulatory effect—Sankhon et al. 1999; inhibition effect—Diehl et al. 1986).

Similar to Acari, data about ecdysteroids in spiders are also described in detail than about juvenile hormones and/or their analogues (see Table 1.). Until now, the presence of ecdysteroids has been documented in the females of eight arachnid species: P. mirabilis (Pisauridae) (Bonaric and De Reggi 1977), Tegenaria domestica (Agelenidae), Coelotes terrestris (Agelenidae) (Trabalon et al. 1992), T. atrica (Agelenidae) (Trabalon et al. 1998), Brachypelma albopilosum (Theraphosidae) (Trabalon and Blais 2012), Schizocosa avida, S. rovneri, S. uetzi (Lycosidae) (Stubbendieck et al. 2013). Data about the occurrence of ecdysteroids in spider males exclusively come from research Trabalon and Blais (2012), and Stubbendieck et al. (2013) about B. albopilosum and S. uetzi. The site of ecdysteroid synthesis in this group of arthropods is still not confirmed. However, the presence of the molting organ (MO) was shown. The MO can be responsible for ecdysteroids synthesis, because the correlation between ultrastructural changes in these cells of the organ and the following molting was observed (Bonaric and Juberthie 1983). Moreover, the relationships between the release of the secret from the MO and the highest titer of ecdysone in P. mirabilis were noticed (Bonaric 1980; Bonaric and De Reggi 1977). The structure of this gland was described for the first time in spider P. mirabilis (Bonaric 1980; Bonaric and Juberthie 1979; Gabe 1966; Kühne 1959; Legendre 1958, 1959). The MO consists of groups of cells, mainly located in the lower part of the prosoma, and it is an endocrine tissue in the meaning of Millot (1930). The localization and volume of this organ are dependent on sex, stage of ontogenesis and age of the animal. The hypothetical model of regulation of the molting process was put forward by Bonaric (1995) based on changes in ecdysteroids titer fluctuation and ultrastructural alternation in the MO cells in P. mirabilis. The cyclic MO activity is controlled by the neuroendocrine system via the retrocerebral system. Neurosecretory cells from the brain, mainly from protocerebrum, produce the neurosecret under stimuli (e.g., proprioceptors or climatic factors). It was transported to the Neurohemal organ 1 by Schneider organ 1, where is the release to hemolymph. Neurosecret contains molting hormone activation, which is responsible for the MO activation. Moreover, the neurosecretory components can stimulate the hypothetical organ to synthesize juvenile hormones. However, it should be emphasized that the role of MO in the synthesis of ecdysteroids has not yet been confirmed. Mechanism of molecular action on the gene transcription level is still not known. Nevertheless, research about the EcR and USP receptors are restricted to only short communications about the spider Agelena silvatica (Agelenidae) (Honda et al. 2009). It seems that ecdysteroids play a similar role in the regulation of the molting process, as like as in insects. It was observed the correlation between the ecdysteroid level and the molt appearance in P. mirabilis (Eckert 1967; Bonaric and De Reggi 1977). Moreover, the application of ecdysone and 20E reduced a shortening of intermolting time and induced a molting in P. mirabilis, Larinioides cornutus (Araneidae), Aphonopelma hentzi (Theraphosidae) (Bonaric 1976, 1977; Krishnakumaran and Schneiderman 1970). Ecdysteroids can be responsible for the spider ovaries development (Trabalon et al. 1992), pheromone production, sexual cannibalism (Stubbendieck et al. 2013; Trabalon et al. 1998, 2005) and regulation of metabolism (Trabalon and Blais 2012). It seems that ecdysteroids are involved in the control of vitellogenesis. However, contradictory data are present. On the one hand, the correlation between the ecdysteroids titer and the start of vitellogenesis and the probable induction of this process are observed in C. terrestris, T. domestica and T. atrica (Pourié and Trabalon 2003; Trabalon et al. 1992, 1998). On the other hand, observation of the ecdysteroids titers in different stages of ontogenesis in spiders from genus Schizocosa indicates the lack of role of these groups of hormone in the induction of the vitellogenesis (Stubbendieck et al. 2013).

Neuropeptides

The neuroendocrine system of insects regulates most critical metabolic, behavioral, homeostatic, developmental and reproductive processes. The corpora cardiaca (CC) glands, which store and release the neurohormones that are synthesized in neuroendocrine cells of the brain, are one of the most important structures of the neuroendocrine system. CC glands can also produce their own peptides due to the presence of specific secretory cells (De Loof and Hoffmann 2001; Marciniak et al. 2011).

One group of proteins that are secreted by the neuroendocrine system in insects is neuropeptides. They belong to neurosecret together with steroids and sesquiterpenoids. Neuropeptides represent the largest single class of signal compounds. They are diversified chemical messengers that can act as neuromodulators, neurotransmitters and neurohormones. They are produced in the central nervous system of insects by the neurosecretory cells and interneurons (Duve et al. 1999; Bendena 2010; Harshini et al. 2002; Marciniak and Rosinski 2007; Sarkar et al. 2003).

Insect neuropeptides are grouped into families that are based on the structural similarity of the amino acid sequence of each substance and their main physiological functions. After the first successful attempt of sequencing the genome of the insect D. melanogaster, a classification system that was based on the peptide precursor genes that encode them was added. The main assumption of the categorization is the fact that the neuropeptides that belong to the same family are encoded by a single precursor gene. It is supposed that peptides encoded by ortholog genes in other species of insects are members of the same family of neuropeptides (Marciniak and Rosiński 2007; Marciniak et al. 2011; Nassel 2002).

Among the previously identified neuropeptides are the peptides that regulate the majority of physiological and behavioral processes in insects, the most well known of which is their impact on growth and reproduction and the control of JH synthesis by the CA.

Allatoregulating neuropeptides

Allatoregulating neuropeptides are multifunctional peptides. One of their main functions is to regulate the biosynthesis of JHs. Allatostatins have an inhibitory effect, whereas allatotropins stimulate biosynthesis and are released by the CA (Bendena 2010; Gade and Hoffmann 2005; Harshini et al. 2002; Marciniak and Rosiński 2007; Sarkar et al. 2003).

Allatostatin

Allatostatins (ASTs) are a superfamily of invertebrate neuropeptides that were originally defined by their action as inhibitors of JH biosynthesis in vivo. ASTs act on the G-protein-coupled receptors (GPCRs) in the cells of the CA to inhibit JH biosynthesis in insects. To date, more than 60 allatostatins have been isolated and characterized from a variety of insect species. Structurally similar neuropeptides have also been identified in crustaceans and ticks (Stay and Tobe 2007; Zhu and Oliver 2001). These peptides can be classified into three groups: the FGL—allatostatin superfamily (A type), the W(X)6W allatostatins (B type) and the lepidopteran (M. sexta) allatostatin (C type) (Bendena 2010).

FGL-allatostatin superfamily (A type)

Allatostatins of the FGLamide (phenylalanine–glycine–leucine) type were originally identified and isolated from brain extracts of the cockroach Diploptera punctata. Subsequently, the members of this peptide family were isolated from other insect orders, for example from the cricket Gryllus bimaculatus, the locust Schistocerca gregaria and the blowfly Calliphora vomitoria. The A-type allatostatins are 5–18 amino acids long, which is the minimum required for biological activity, and are characterized by a common C-terminal pentapeptide (F/YXFGLamide) (Marciniak et al. 2011; Tobe et al. 2000). The rapid and reversible inhibition of JH synthesis after the application of the synthetic or isolated allatostatin A has only been observed in cockroaches, crickets and, most recently, in termites (Belles et al. 1994; Lorenz et al. 1995; Pratt et al. 1991; Woodhead et al. 1989). To date, the A-type allatostatin has not been reported to have any significant effect of JH synthesis by the CA in any lepidopteran (Audsley et al. 2008).

The W(X)6W-allatostatins (B type)

The second family of allatostatins was isolated from the brains of the cricket G. bimaculatus and has the general sequence W(X)6Wamide. These C-terminal nonapeptides contain the amino acid tryptophan at positions 2 and 9 and have a high sequence similarity to the myoinhibiting peptides that were previously isolated from Locusta migratoria (Locmi-MIP) and Manduca sexta. Neuropeptides with a similar structure were also isolated from the stick insect Carausius morosus and the silkworm Bombyx mori (Audsley et al. 2008; Wang 2004). They are active in JH biosynthesis in crickets but are less potent than the A-type neuropeptides. They have also not been investigated as precisely as the FGLamide type (Stay et al. 2002).

Lepidopteran (M. sexta) allatostatin (C type)

The first peptide of this group Manse-AST (pEVRFRQCYFNPISCF) was purified from the brains of the moth M. sexta. The C-type allatostatin is a non-amidated, N-terminally blocked, 15-residue peptide that contains a disulfide bridge between the C residues at positions 7 and 14. It strongly inhibited JH biosynthesis in vitro by the CA of fifth-instar larvae and adult females of the moth. It also had an inhibitory effect on the activity of the CA from adult females of other lepidopterans but was ineffective in other orders of insects (Abdel-Latief and Hoffmann 2014; Stay et al. 2002; Tobe et al. 2000).

Among arachnids, allatostatins have also been identified in ticks. A-type allatostatins were detected by immunocytochemistry methods in the synganglion of Dermacentor variabilis by Zhu and Oliver (2001) and I. scapularis (Simo and Park 2014). The immunoreactive cells were detected in the different regions of the synganglion: protocerebral, cheliceral, palpal, stomodeal, postesophageal and opisthosomal. The stronger positive reaction in the preesophageal part of the synganglion was observed. Therefore, authors suggest that this may be a part of neuroendocrine system where allatostatins are released. Another type of allatostatin (Manse-AST) in the central nervous system of I. scapularis was also described (Neupert et al. 2009). Despite the presence of these peptides in ticks, their role is still unknown. As mentioned, Zhu et al. (2016) suggested that the methyl farnesoate may act as JHs in ticks and allatoregulatory neuropeptides may stimulate or inhibit the mevalonate–farnesol pathway in I. scapularis.

Allatotropin

The allatotropin (AT) peptide was first isolated from M. sexta (Manse-AT). Its sequence (GFKNVEMMTARGF-NH2) was confirmed based on cDNA. Allatotropin exhibits stimulating activity for biosynthesis in vivo and release of juvenile hormone by corpora allata (Dyker et al. 2001). This effect was only confirmed in adult members of M. sexta, whereas the stimulation in larval stages and in pupae was not observed (Kataoka et al. 1989). Allatotropin compared to other allatoregulatory neuropeptides are characterized by high species specificity. Therefore, Bogus and Scheller (1996) reported that allatotropin caused increased secretion of juvenile hormone II from corpora allata in larvae in stage 6 (L6) in the Galleria mellonella. Allatotropins have a number of other physiological functions, although their exact role is still not fully understood (Dyker et al. 2001).

Allatoregulating neuropeptides in spiders

The current knowledge about this family of neuropeptides comes from studies that were carried out on insects. There are only a few publications (Table 1) that are based on the immunohistochemical distribution and localization of allatostatins in the model spider species C. salei. These studies have shown the presence of allatostatins in the arcuate body, which is a principal part of the central nervous system of spiders (Loesel et al. 2011). Recent studies have also indicated the presence of allatoregulating neuropeptides in spiders. Christie (2015a) identified peptides that belong to allatostatin A, allatostatin B, allatostatin C and allatotropin in Latrodectus hesperus based on an in silico characterization of the neuropeptidome. Subsequently, he performed a similarity analysis using L. hesperus as the reference. He detected the same allatoregulating neuropeptides in Latrodectus tredecimguttatus, Stegodyphus mimosarum, Stegodyphus lineatus, Stegodyphus tentoriicola and Acanthoscurria geniculata (Christie and Chi 2015). The available results of the study of allatoregulating neuropeptides in spiders are presented in Table 1. Until now, there are no data about potential functions of allatostatins and allatotropin and/or their analogues on the development, reproduction and molting induction in spiders.

Future prospects

We believe that future research should be directed toward the confirmation of the presence of JHs and/or their analogues in spiders. The knowledge about ecdysteroids and neuropeptides requires revision and needs more details. The evolutionary relationship between spiders and the other arthropods described (e.g., ticks, mites) suggests the possibility of the presence of JHs and/or their analogues in spiders and indicates that neuroactive compounds may play a similar role in all arthropods. Analysis of the studies about JHs and their analogues, ecdysteroids and neuropeptides in other arthropods (see Table 2) indicates several possible directions for the research of these compounds.

Determining the concentration of hormones and neuropeptides in various tissues during the different stages of ontogenesis seems to be the first appropriate method to study these compounds (Abdel-Latief et al. 2004; Dong et al. 2009; Edwards et al. 1995; Elliott et al. 2010; Maestro and Bellés 2006; Neese et al. 2000; Pascual et al. 1992; Wang 2004; Williamson et al. 2001b; Zhu et al. 1991). Identifying the relationship between the concentration of these substances and the age of the animal is a good starting point for analyzing the functions of these compounds in ontogenesis of spiders. However, it should be noted that the studies on the concentration of JHs and its presence in various tissues are difficult due to the fact that the molecules of these hormones adhere to various surfaces, including glass, in a non-selective manner. Thus, not only is the determination of the level of JHs difficult to implement, but also the identification of its receptors (Konopova and Jindra 2007).

In recent years, a number of researches about the neuroactive compounds that are based on an analysis of the presence of various genes and their expression level have been undertaken. Detecting the genes that encode the enzymes of the biosynthesis pathway of these substances is the most accurate way of determining the tissues and organs that are responsible for the synthesis of hormones and neuropeptides de novo. Analysis of the available data about the determination of the presence of genes: spook (and its paralogs), phantom, disembodied, shadow (for pathway of ecdysteroids; Ono et al. 2006; Mitchell and Smith 1986; Rewitz et al. 2006; Warren et al. 2002, 2004), jhamt (JH acid o-methyltransferase gene) (for JH synthesis; Marchal et al. 2011; Shinoda and Itoyama 2003), and the gene that encodes the AST and At preprohormone (for neuropeptides pathway, Maestro and Bellés 2006) indicates that this approach is very appropriate for this purpose. Determining the tissues whose physiology is regulated by hormones and neuropeptides can be carried out by detecting the genes that encode the receptors (or potential candidates for JH and/or their analogues) or enzymes from the last step of the biosynthesis pathway (in the case of ecdysteroids, 20E is synthesized by ecdysone conversion by 20-hydroxylase, Petryk et al. 2003). Moreover, this approach is widely used in the literature. Studies about the genes that encode the ecdysteroid receptors EcR and (Bortolin et al. 2011; Koelle et al. 1991; Nakagawa et al. 2007; Palmer et al. 1999; Yao et al. 1993), enzyme 20-hydrolase, potential JH and its analogues receptor candidates Met, USP, Gce (Baumann et al. 2010; Gong et al. 2016) and the neuropeptide receptors for allatostatin A (AlstR/DAR-1 and DAR-2 receptor genes) (Larsen et al. 2001; Lungchukiet et al. 2008a), allatostatin C (AeAS-CrA and AeAS-CrB) (Mayoral et al. 2010) and the allatotropin receptor (AeATr) (Nouzova et al. 2012) have been observed (compared with Table 2). It should be emphasized that studies at the gene level allow the correlation between the expression level of the genes in the various tissues and the stages of ontogenesis be identified. This enables the identification of the critical organ of the synthesis and action of neuroactive compounds. Hence, determining the crucial physiological processes that are controlled by JHs and/or their analogues, ecdysteroids or neuropeptides is possible (Buszczak et al. 1999; Hassanien et al. 2014; Larsen et al. 2001; Schwedes et al. 2011; Williamson et al. 2001b, compared with Table 2). It seems that the silencing of the genes that encode the receptors or the enzymes of the biosynthesis pathway of JHs and/or their analogues, ecdysteroids and neuropeptides and then identifying the occurrence and course of various physiological processes (e.g., vitellogenin levels or the time between moltings) is the best way to study the role of these compounds (Griebler et al. 2008; Hassanien et al. 2014; Jia et al. 2013; Konopova et al. 2011; Konopova and Jindra 2007; Maestro and Bellés 2006; Mello et al. 2014; Wan et al. 2014a, b).

However, the problems that are associated with the studies of the genomes of spiders (see Sect. 1) have not been solved. Therefore, another possible method should be used to test neuroactive compounds. The synthetic hormones and neuropeptides and (in the case of JHs and/or their analogues) chemical analogs and anti-juvenoids (which are commercially available) can be provided to an organism. This would allow the differentiated roles of these compounds to be detected in several physiological processes. The duration of their action and the point at which they are deactivated in various tissues, depending on sex and age, can be determined by applying various concentrations of synthetic neuroactive compounds for different periods of incubation (for the treatment of spiders with the appropriate concentration of substances—see Sect. 1).

We would like to emphasize once again that choosing the appropriate physiological processes to investigate is the key stage of any study about JHs and/or their analogues, ecdysteroids and neuropeptides. It seems that the site of the synthesis of these compounds de novo, as well as the site and mechanisms of their action in the context of the reproduction and molting induction and regulation of Araneomorphae spiders, is the best approach in the initial stages of the discovery of these hormones and neuropeptides. We believe that these preliminary studies are necessary as starting points for further studies, which could lead to the rapid development of research about the neuroendocrine system in spiders.