Larval Development of Non-Insect Arthropods: Metamorphosis and Sexual Differentiation

*Kenji Toyota, Yuta Sakae and Taisen Iguchi*

## **Abstract**

In insects, metamorphosis is one of the most important research topics. Their drastic morphological and physiological changes from larvae to pupae, and then to adults, have fascinated many people. These changing life history patterns are tightly regulated by two endocrine systems, the ecdysteroids (molting hormones) and the juvenile hormones. Metamorphosis is also the most universal phenomenon in noninsect arthropods (especially crustaceans). Additionally, as dwarf males (e.g., barnacle crustaceans) show distinct sexual dimorphism during the larval developmental stage, larval development and sexual differentiation are also intimately associated. Our knowledge of endocrinology and gene cascades underlying metamorphosis and sexual differentiation in non-insect arthropods is rudimentary at best and relies heavily on well-studied insect models. Advances in newly developed applications, omics technologies and gene-targeting, are expected to lead to explorative molecular studies that reveal components and pathways unique to non-insect arthropods. This chapter reconciles known components of metamorphosis and sexual differentiation in noninsect arthropods and reflects on our findings in insects to outline future research.

**Keywords:** metamorphosis, ecdysteroid, juvenile hormone, sex determination, *doublesex*

## **1. Introduction**

Arthropods are among the best-known animals on the earth and have fascinated many people, including researchers. Extant arthropods can be classified into four subfamilies: Chelicerata, Myriapoda, Crustacea, and Hexapoda. Although it is now widely accepted that Crustacea and Hexapoda are integrated as Pancrustacea, Crustacea and Hexapoda (insects) are used to focus on non-insect arthropods in this chapter. Their phylogenetic relationship has been debated for many years. However, recent progress in next-generation sequencing has provided their exact position (**Figure 1**) [1–3]. Genetics and developmental biology using model insects such as fruit fly *Drosophila melanogaster*, red flour beetles *Tribolium castaneum*, and silkworm *Bombyx mori*, have long been a driving force in not only basic biology but also a wide range of sciences including medical, agricultural, and so on. On the other hand, for non-insect arthropods, there is a long history of physiological knowledge of some crustaceans (especially

#### **Figure 1.**

*Phylogenetic tree of extant arthropods. The branching pattern is constructed based on previous studies [1–4] with the exclusion of some clades for clarity. Crustacea are not monophyletic and include Ostracoda, malacostraca, decapods, and Branchiopoda. Detection of synthesizing enzyme genes in the genomes is indicated by the presence of putative juvenile hormone acid O-methyltransferase (JHAMT) or farnesoic acid O-methyltransferase (FAMeT) for JH, and of putative Halloween genes for ecdysteroids, respectively. Likewise, in terms of these receptor genes in the genome, it is indicated by the presence of methoprene tolerant (met) orthologs for JH and of ecdysone receptor (EcR) for ecdysteroids, respectively.*

decapods) that are important for fisheries and aquacultures and some mites that are problematic as agricultural pests, but molecular insights are still limited due to lack of genome information and reverse genetic approaches. In the last decade, the research environment surrounding biology has drastically improved with the advances of sequencing, imaging, handling of large-scale data (bioinformatics), and new-generation genome modification technologies such as genome editing. Based on these developments, many findings on embryogenesis, larval metamorphosis, sex determination, and sexual differentiation have been reported in many non-insect arthropods. In this chapter, we will provide an overview of the knowledge of canonical metamorphosis and sex determination of insects, and the attempt to compare them with non-insect arthropods, particularly branchiopod and decapod crustaceans and spider chelicerates.

## **2. Larval metamorphosis**

Tadpoles developing into frogs, and insect larvae or pupae into adults, are classical examples of metamorphosis, and biologists have long been fascinated with these dramatic and obviously spontaneous transformations [5–7]. Previous studies showed that such morphological alterations were accompanied by major changes in the chemical composition and biochemical function of almost all larval tissues. In the last century, the discovery that metamorphosis is centrally regulated by endocrine systems has allowed us to understand how these post-embryonic developmental and physiological processes are brought about and controlled [7, 8]. This section outlines the endocrine and molecular mechanisms of canonical complete metamorphosis (holometaboly) in insects and then overviews current research findings on larval metamorphosis in the decapod crustaceans and arachnid chelicerates.

*Larval Development of Non-Insect Arthropods: Metamorphosis and Sexual Differentiation DOI: http://dx.doi.org/10.5772/intechopen.105395*

#### **2.1 Insects with complete metamorphosis**

Holometabolous insects go through a series of discrete stages (larva, pupa, and adult) that hardly resemble one another, but are finely adapted to specific roles in their life cycle. Juvenile hormone (JH) and ecdysteroids (the representative active form is 20-hydroxyecdysone: 20E) are the two key endocrine factors that together coordinate molting and metamorphosis (**Figure 2**) [7, 9–11].

JHs are a family of acyclic sesquiterpenoids that are involved in a range of physiological processes in insects such as not only metamorphosis, but also ovarian development, reproductive behavior, and various types of phenotypic plasticity including caste differentiation in social insects and weapon traits development in beetles [12, 13]. In terms of metamorphosis, JH acts as the best-known anti-metamorphic hormone which prevents larvae/nymphs from undergoing precocious metamorphosis and is often referred to as the *status quo* hormone [14]. Its representative *status quo* action is associated with a surge of ecdysteroids that triggers molting [15]. At the beginning of the ecdysteroids surge, the presence or absence of JH decides whether the molt will be a *status quo* molt that repeats the same stage (larva-larva) or a molt with metamorphosis (larva-pupa or pupa-adult) [16].

More than half a century of arthropod endocrinology research has revealed that molting is precisely regulated by complex multiple hormone systems, and ecdysteroids are a key hormone mediating a variety of physiological and behavioral changes that are essential for molting and metamorphosis [7, 10, 17]. Their primary function is to induce molting and they serve this function throughout the arthropods [10, 18]. In insects, circulating ecdysteroids typically come from the prothoracic glands. The prothoracic glands secrete ecdysone which is then further converted to 20E in peripheral tissues [19]. The functions of ecdysteroids in the control of insect metamorphosis have striking parallels with those of the thyroid hormones in directing the metamorphosis of amphibians [5, 6].

## **2.2 Larval metamorphosis in decapod crustaceans**

Recent molecular phylogeny has supported the theory that the Crustacea clade is not monophyletic, but is divided into at least three extant clades (Ostracoda, Malacostraca, and Branchiopoda) (**Figure 1**) [4]. As aforementioned, both Crustacea and Hexapoda form a new clade known as Pancrustacea [1–3]. This theory spurs the notion that a comparative analysis between crustaceans and insects is essential to

## **Figure 2.**

*Chemical structures of JHIII, methyl farnesoate (MF), and 20-hydroxyecdysone (20E).*

understanding the evolutionary origins of various traits considered unique to insects. Indeed, crustaceans and insects share various basic traits, such as endocrine-driven developmental and reproductive processes, which are regulated primarily by JHs and ecdysteroids. In non-insect Arthropods, methyl farnesoate (MF) is thought to be the equivalent of JH in insects. In insects, MF is finally converted to JH III (active JH form in insects) by CYP15A1, except for the Lepidoptera [20]. However, CYP15A1 orthologs have never been found in non-insect Arthropoda [21, 22], indicating that the CYP15A1 gene acquisition might have been an important event enabling JH biosynthesis in insects [20]. In fact, detection of insect-type JH (e.g., JH III) has not been reported in crustaceans; instead, MF has been widely regarded as the functional crustacean JH since its discovery in various crustaceans [8, 23, 24]. Research on crustacean larval metamorphosis and endocrine pathways such as MF and ecdysteroids has been vigorously pursued in the fisheries-important decapod order (crabs and shrimps). Production of MF and ecdysteroids in decapod crustaceans is thought to take place in the mandibular organ and Y-organ, the glands unique to Malacostraca crustaceans which are considered to be functionally analogous to the insect corpus allatum and prothorathic glands [21]. Unlike insects, the synthesis of MF and ecdysteroids in decapod crustaceans is inhibitory regulated by mandibular organ-inhibiting hormones (MOIHs) and molt-inhibiting hormones (MIHs), cryptic members of the crustacean hyperglycemic hormone (CHH)-like neuropeptides secreted from the X-organ/sinus gland complex in the eyestalk [25].

In the marine decapod species, each larva differs from conspecific juveniles and adults based on morphological, ecological, behavioral, physiological, and/or other biological traits. Moreover, the juveniles and adults are benthic feeders or predators, whereas larvae grow in the pelagic environment, preying on phyto- and/or zooplankters [26]. In addition, those larval forms are so different from the conspecific adults that some of them have been described as distinct species, and many larval names were originally genus names, for example, nauplius, cypris, zoea, megalopa, mysis, nisto, puerurus, and phyllosoma in decapod species [26]. This disorganization of larval names causes confusion in advancing comparative developmental and physiological biology in decapods; however, no unified nomenclature has yet been defined. To resolve this problem, clarification of the endocrine regulation of larval metamorphosis and the associated gene regulatory network among decapod species would facilitate interspecific comparative analysis and advance our understanding of this phenomenon, as is the case with insects. Although several studies have challenged the current understanding of the effects of MF and 20E on larval metamorphosis, no consistent results have been obtained, as follows.

Our group demonstrated that treatment of either MF or 20E induced high mortality caused by disruption of molting-associated metamorphosis in the kuruma prawn *Marsupenaeus japonicus* (larval stages: nauplius, zoea, mysis, post larva, and juvenile; **Figure 3**) [27]. Likewise, treatment of MF to the larvae of the freshwater prawn *Macrobrachium rosenbergii* resulted in an inhibitory effect on metamorphosis [28]. On the other hand, the administration of MF has been shown to accelerate metamorphosis in late-stage shrimp and barnacle larvae [23, 29–31]. These data suggest that the role of MF in regulating decapod metamorphosis is still unclear. As a distinct approach, eyestalk ablation of intermediate larvae led to failure or impedance of metamorphosis to the megalopa stage in several crabs *Rhithropanopeus harrisii*, *Callinectes sapidus* and *Portunus trituberculatus*, and American lobster *Homarus americanus* [32–34]. Later, the impact of eyestalk ablation on metamorphosis was demonstrated to be accompanied by a rise in circulating MF concentrations,

*Larval Development of Non-Insect Arthropods: Metamorphosis and Sexual Differentiation DOI: http://dx.doi.org/10.5772/intechopen.105395*

**Figure 3.**

*Larval developmental staging (nauplius, zoea, and mysis) and adult (dorsal and frontal views) of kuruma prawn. Each scale bar indicates 100 μm.*

confirming that these observations were potentially the result of inhibition by MF [35]. Additionally, as for the new mystery, our previous work demonstrated that nauplius larvae of kuruma prawn have higher tolerance against the lethal effect of MF and 20E treatment than other later stages (e.g., zoea and mysis), suggesting that there are different endocrine cassettes regulating transition from nauplius to zoea and later metamorphosis [27]. Similarly, larval transcriptomics during metamorphosis in spiny lobster *Sagmariasus verreauxi* has suggested that the puerulus (megalopa)-juvenile metamorphosis is regulated by the MF signaling pathway, whereas the phyllosoma (zoea)-puerulus (megalopa) metamorphosis is preceded by sustained the expression level of FAMeT, which encodes the enzyme synthesizing MF [36]. These data could suggest that MF is not involved in early-stage (nauplius in the kuruma prawn and phyllosoma in spiny lobster) metamorphosis through the conventional inhibitory mechanism.

#### **2.3 Larval metamorphosis in chelicerates**

Chelicerates are classified into a large and ancestral sister group of arthropods (**Figure 1**). Chelicerates have generally been considered to be ametabolous because the larvae display a very similar morphology to adults (**Figure 4**). However, detailed morphological observations in several chelicerates cast doubt that the chelicerates are "ametabolous."

Observations with a scanning electron microscope show that scorpion larvae have incomplete mechanoreceptors and chemoreceptors [37]. Larvae differ in the morphology of the tip of tarsi and aculeus compared to that of nymphs and adults [37, 38]. The scorpion larvae ride on the mother's back after the delivery, at which point the

**Figure 4.**

*The appearance of larvae and adult in Parasteatoda tepidariorum. Scale bars indicate 1.0 mm.*

structure at the tip of the larva's tarsi works, the tip of the tarsi functions like a sucker, before developing into a claw after molting [37, 38]. In addition, the exoskeleton of adult scorpions fluoresces when exposed to UV light, whereas the exoskeleton of firstinstar larvae does not fluoresce [39, 40]. In ticks, the genital primordium opens to the exoskeleton through multiple molting [41]. The fourth leg, not observed in first-instar mite larvae, appears in molted nymphs [42]. In spiders (*Parasteatoda tepidariorum*), the development of the pedipalp, which is the male copulatory organ, progresses at the sub-adult stage similarly to the metamorphic manner of insects and is completed through molting to the adult [43]. Thus, morphological observations of larvae and sub-adults in scorpions, ticks, and spiders suggest that chelicerates exhibit hemimetaboly rather than ametaboly.

In insects and crustaceans, it is demonstrated that ecdysteroids and MF regulate metamorphosis, but the molecular mechanism of metamorphosis in chelicerates is unknown [8, 12, 44–46]. In chelicerates, 20E has been detected in ticks, scorpions, horseshoe crabs, and spiders [47–52]. The ecdysone receptor (EcR) and retinoid X receptor (RXR), which act as 20E receptors, have also been identified in ticks, scorpions, and spiders [53–59]. In fact, the administration of 20E demonstrates to induce molting of ticks, horseshoe crabs, and spiders [60–63]. Although there have been no reports of identification of sesquiterpenoid hormones in Chelicerata, genomic and transcriptomic data suggest that ticks and spiders may have precursors of JH, MF [64, 65]. Therefore, it is possible that ecdysteroids and sesquiterpenoid hormones cause molting and associated metamorphosis in chelicerates, despite this not being direct evidence.

### **3. Sex determination and differentiation**

Sex determination is the most fundamental developmental process that establishes sexually dimorphic traits. In many animals, males and females have distinct sex-related characteristics such as body size, ornamentation, and color [66]. These extreme phenotypic differences make sexual dimorphism one of the most interesting aspects of animal morphology, physiology, and behavior. This outstanding diversity of sexually dimorphic traits is reflected in the underlying molecular mechanisms by a series of systems ranging from sex-specific gonadal steroid hormones sealing sexual fate in mammals and other vertebrates [67], to cell-autonomous sex-specific splicing loops that maintain the sexual state in holometabolous insects [68, 69].

#### *Larval Development of Non-Insect Arthropods: Metamorphosis and Sexual Differentiation DOI: http://dx.doi.org/10.5772/intechopen.105395*

The DMRT gene, which is conserved in metazoans, is an important transcriptional factor that has a key role in sex determination [70–72]. Four DMRT genes (*doublesex, dmrt11E, dmrt93B, dmrt99B*) have been found in insects, and *doublesex* (*dsx*) is known as a conserved master switch regulating the development of sexual dimorphic traits [72, 73]. *Dsx* contributes to insect sex determination via sex-specific splicing cascades [68, 72]. All insect species have cell-autonomous sex determination mechanisms, in which the sex-determining cascade operates on a cell-to-cell basis [74].

Focusing on the mechanisms inducing the sex-specific traits by DSX function, there are major differences between insects and non-insect arthropods. As described above, in insects, sex-specific splicing of *dsx* makes sexual dimorphic traits, although the upstream signals are extremely variable in the insect order [75]. On the other hand, previous studies using the water flea, which is a well-studied species in the branchiopod crustaceans, have revealed that its *dsx* has no sex-specific splicing isoforms and apparently shows the male-biased expression pattern resulting in the promotion of masculinization [76–79]. As described below, there is no sex-specific splicing of *dsx* in non-insect arthropods, and the mechanism of sexual differentiation by male-biased expression is conserved.

#### **3.1 Decapod crustaceans as a treasury box of diverse sex differentiation pathways**

In general, sex determination and sexual differentiation in arthropods are cellautonomous in manner, whereas in most mammals and other vertebrates, sexual identity is unified throughout the body by sex steroids (estrogens and androgens) secreted by the gonads. Interestingly, only Malacostraca crustaceans including decapods exceptionally have a cell-nonautonomous sexual differentiation manner, and unlike gonad-dependent endocrine regulation in vertebrates, have male-specific endocrine glands known as the androgenic glands (AG), which are located on the terminal of the vas deferens [80]. Physiological roles of the AGs have been historically revealed to play a key function in male sexual differentiation, by AG ablation and implantation in the amphipod *Orchestia gammarella* [80]. Other studies have been conducted in the woodlouse *Armadillidium vulgare* using AG implantation [81], AG ablation [82], and injections of AG extracts [83]. Then, the androgenic gland hormone has been purified and identified [84, 85]. After that, physiological roles of AGH have further been demonstrated in decapod species using, for instance, AG implantation in the red claw crayfish *Cherax quadricarinatus*, and the marbled crayfish *Procambarus fallax f. virginalis* [86]. Moreover, our group has recently succeeded in chemically synthesizing *P. fallax f. virginalis* insulin-like androgenic gland factor (IAG) [87]. Although injection of the synthetic IAG to female crayfish did not induce masculinization on the external morphology, this treatment apparently suppressed oocyte maturation *in vivo* [87], suggesting that its IAG has a pivotal role in the suppression of female secondary sex characteristics. Several attempts to understand the regulatory mechanism of IAG expression have shown that male eyestalk ablation causes AG hypertrophy and hyperplasia [88, 89]. There is a unique developmental axis defined as the X-organ/sinus gland/neuroendocrine complex (XO-SG)-AG-testis axis. Although the fine details are controversial, it has been demonstrated that IAG interacts with its binding protein and receptor to activate downstream pathways [90–92].

Several studies have demonstrated that the *dsx* regulates the IAG expression. For example, the *dsx* is predominantly expressed in the testis, and its expression levels gradually increase with larval development in the Chinese shrimp *Fenneropenaeus* 

*chinensis*. Its knockdown resulted in suppression of *IAG* expression, suggesting that *dsx* promotes male sexual differentiation via IAG signaling [93]. While, the *dsx* mainly expresses in the ovary, and its knockdown increased *IAG* expression in the red claw crayfish *C. quadricarinatus*, implying that *Cqdsx* is involved in female sexual differentiation [94]. Both *dsx* genes have no sex-specific splicing forms, therefore, there is male- or female-biased expression to promote sexual differentiation pathways.

In addition to IAG, the crustacean female sex hormone (CFSH) was discovered as a novel eyestalk-derived neuropeptide that induces the development of secondary female characteristics in two crab species *C. sapidus* and *Carcinus maenas* [95]. CFSH is synthesized and secreted in the X-organ/sinus gland complex in the eyestalks.

Currently, its homolog has been successfully identified in several other decapod species such as the swimming crab *P. trituberculatus* [96], the Chinese mitten crab *Eriocheir sinensis* [97], the green shore crab *C. maenas* [98] and the mud crab *Scylla paramamosain* [99–101], the kuruma prawn *M. japonicus* [102], the Pacific white shrimp *Litopenaeus vannamei* [97], the banana shrimp *Fenneropenaeus merguiensis* [103], the Antarctic shrimp *Chorismus antarcticus* [104], the Eastern rock lobster *S. verreauxi* [105], the giant freshwater prawn *M. rosenbergii* [97, 106, 107], the peppermint shrimp *Lysmata vittata* [108, 109], the red swamp crayfish *Procambarus clarkii* [96], and the Australian crayfish *C. quadricarinatus* [110]. RNA interference of *CFSH* caused the anomalous development of female reproductive characteristics including ovigerous setae, gonopores, and extended parental brood care in *Callinectes sapidus* [95], and the formation of gonopores in juvenile stages in the mud crab [100]. These reports indicate that the CFSH plays a pivotal role in the development of femalespecific reproductive characteristics. On the other hand, it has been discovered that CFSH expression can be detected in the eyestalks of both females and males in the kuruma prawn [111] and several crab species [95, 99, 100], and moreover, two distinct CFSH subtypes have been identified as eyestalk- and ovary-types in the kuruma prawn [111]. Based on immunohistochemistry and *in situ* hybridization analyses of both CFSH subtypes, the ovary-type is predominantly expressed in oogonia and previtellogenic oocytes during vitellogenesis. These data suggest that ovary-type CFSH may take part in reproductive processes, although the differences in physiological function between both subtypes are still unclear. Besides, in the Australian crayfish, CFSH expression has been detected in the central nervous system, antennal gland, and gut [110]. Taken together, accumulating CFSH studies indicate that it might regulate female secondary reproductive phenotypes in some crab species, and is not a female-specific hormone in other decapod species. Although a new potential function of CFSH has been reported to be its involvement in growth [108], more comparative analysis will be required for comprehensively understanding its physiological roles.

Some recent studies have demonstrated the crosstalk between CFSH and IAG to facilitate sexual differentiating processes. In the mud crab *S. paramamosain*, it has been demonstrated that CFSH plays a pivotal role in the development of female reproductive traits and suppresses the *IAG* expression in AG *in vitro* [99]. Furthermore, the transcriptional relationship of *CFSH* to *IAG* expression has also been demonstrated with respect to the involvement of signal transducers and activators of the transcription-binding site [100]. Additionally, it has recently demonstrated that feedback regulation of both IAG and CFSH in peppermint shrimp *L. vittata*, a species that possesses a protandric simultaneous hermaphroditism reproductive system [108, 109]. To date, the CFSH receptor has not been identified. Further studies on the CFSH receptor and its downstream signaling pathways are necessary to understand the mechanisms underlying endocrine crosstalk among CFSH, IAG and *dsx* in sexual differentiation of decapods.

*Larval Development of Non-Insect Arthropods: Metamorphosis and Sexual Differentiation DOI: http://dx.doi.org/10.5772/intechopen.105395*

#### **3.2 Sex determination and differentiation in chelicerates**

Since chelicerates is an ancestral sister group among arthropods, it is an important group for considering the evolutionary diversity of sex determination and sexual differentiation mechanisms including arthropods and vertebrates. Among chelicerates, spiders display a particularly clear morphological sexual dimorphism, females are 3–14 times larger than males and, in some species, females are 75.2 times heavier than males [112, 113]. In addition, several species of males, such as the banksia peacock spider, show a brilliant appearance like a peacock male and perform the mating dances [114, 115]. While studies on morphological and behavioral sexual dimorphism have been proceeding, studies on sex determination and sexual differentiation are completely unclear. Not only in spiders but also in other chelicerates, has research on sex determination and sexual differentiation remained almost untouched. However, recent studies have begun to find clues to the mechanism of sex determination and/or sexual differentiation in Chelicerata, referring to the sex determination cascade of insects.

In tick (*Metaseiulus occidentalis*), a comparative analysis of genomic sequences of insects and water fleas identified *dsx* (*MoccDsx1* and *MoccDsx2*), *dmrt11E* (*MoccDmrt11E*), and *dmrt99B* (*MoccDmrt99B*) [116, 117]. Quantitative RT-PCR in adult ticks indicates that *MoccDsx1* and *MoccDsx2* are highly expressed in males [116, 117]. Furthermore, the transcripts of *MoccDsx1* and *MoccDsx2* in adult males and females exhibit the same size, suggesting that they are not subject to sex-specific splicing [116, 117]. However, it is still unclear whether they actually exhibit transcriptional patterns and levels similar to adults at the sex determination point.

Spiders and scorpions experienced whole-genome duplication (WGD) after diverging from other chelicerates [118]. Seven *dsx*-like genes are found in the genome sequence of the spider (*P. tepidariorum*) [119]. Probably, it is expected that one of eight *dsx*-like genes in the ancestor disappeared after WGD and so the total became seven. The *dsx*-like genes of *P. tepidariorum* are classified by comparative analysis of gene and amino acid sequences of *dsx* in fly (*Drosophila melanogaster*) and water flea (*Daphnia magna*): *dsx* (*PtDsx1*), *dmrt11E* (*PtDsx2*), *dmrt93B* (*PtDsx3*), *dmrt99B* (*PtDsx1A*, *PtDsxA2* and *PtDsxA2-like*), other (*PtLOC107443841* and *PtLOC110283461*) [119]. A high transcript level of *dsx* (*PtDsx1*) and *dmrt99B* (*PtDsxA2-like*) in adult males and female bias expression of *dmrt93B* (*PtDsx3*) is detected [119]. While the sequence analysis of the cloned *dsx*-like genes cDNA detects splicing variants in *dsx* (*PtDsx1*) and *dmrt11E* (*PtDsx2*), there is no sexual difference in their expression. In addition, whole-mount *in situ* hybridization using embryos cannot provide information about the sex difference of each gene due to the lack of genetic sex markers in *P. tepidariorum*. Therefore, the results of expression analysis in sub-adults and adults of ticks and spiders suggest that sex determination in chelicerates might be caused by high expression of *dsx* in males, the same as in crustaceans.

### **4. Conclusions and future directions**

This chapter focuses on larval metamorphosis, sex determination, and sexual differentiation in non-insect arthropods, especially in decapod crustaceans and spider chelicerates. Insects have long been the frontrunners in the study of these phenomena in arthropods, however, new emerging methods such as next-generation sequencing, 3D and high-resolution imaging techniques [120, 121], and genome

editing methods [122–125] are opening the door for every non-model species. It is of great benefit to the wealth of available knowledge of insects. Indeed, although this chapter referred primarily to holometabolous insects, the latest work has revealed that some hemimetabolous insects such as termites do not have the sex-specific splicing isoforms of *dsx* observed in decapods and chelicerates [126–128]. Thus, new discoveries that overturn established theories are being found in insects, and it is expected that the developmental and physiological biology of non-insect arthropods will advance dramatically in the near future.

Although not described extensively in this chapter, the research environment is paving the way for Myriapoda species using the centipede *Strigamia maritima* as a model [129]. In 2014, the genome of this species was also released, revealing that the biosynthesis and receptor systems for JH and ecdysteroids are conserved (**Figure 1**) [129, 130]. The repertoire of DM domain-containing genes, including *dsx*, has also been reported, and the regulatory mechanism of *dsx* expression is expected to be elucidated in the near future.

To date, chelicerates have generally been considered ametabolous because their offspring display similar morphology to adults. However, detailed observations of several species revealed the morphological changes associated with molting. These results suggest that we need to change our perception to chelicerates as being hemimetabolous organisms.

Chelicerates tend to only be seen as pest organisms because they are a source of allergies and have venom. However, in recent years, among the chelicerates, spiders, and scorpions have begun to be emphasized as material sources and models that can play an active role in various industries. Spiders spin up to seven types of thread called "spider silk" [131–133]. It had been used in sutures and fishing lines in ancient times due to its lightweight, strong and extensible properties [131, 133, 134]. In addition to these, spider silk and its constituent spidroins are currently being considered for application in adhesives, cosmetics, humidity sensors, and the aerospace industry [132, 135, 136]. It is also attracting attention as a biomaterial for biomedical applications (artificial blood vessels, matrigels, porous sponges, and microcapsules) due to its high cell compatibility, low immunogenicity, and slow *in vivo* degradability [132, 137, 138]. In fact, spider silk is used as a guiding material and scaffolding for transplanted cells as demonstrated in a preclinical study for regenerative treatment of bone, skin, myocardium, and nerve [139, 140]. Spider silk, which is useful in various industries, has recently been found to have sex differences in composition [114]. If either male or female spider silk may be more suitable for the desired application, the development of a technique that can control the sex of the spider in order to increase the production of spider silk of either sex may be expected. Other benefits of developing techniques to control the sex of spiders and other chelicerates are the application of spider and scorpion venoms to pharmacology, medical science, and the development of agricultural pesticides. Spider and scorpion venom is a complex cocktail containing a lot of mineral salts, small organic molecules, and small polypeptides [141–143]. This cocktail contains unknown molecules that have a biomolecular activity and is of great interest. In fact, some poisons contribute to the development of pharmacologic tools (e.g., PcTx1; tool for elucidating the roles of acid-sensing ion channels and related pathologies), clinical trials (e.g., GsMTx4; for the treatment of Duchenne muscular dystrophy, and Chlorotoxin; an anticancer drug for neuroectodermal tumors), and pesticides (e.g., GS-ω/κ-HXTX-Hv1a; a bioinsecticide for aphids, spider mites, spotted-winged drosophila, thrips, and whiteflies recognized as major greenhouse pests: Spear® T (Vestaron Corporation, Durham, NC, USA)) [143, 144]. In recent

*Larval Development of Non-Insect Arthropods: Metamorphosis and Sexual Differentiation DOI: http://dx.doi.org/10.5772/intechopen.105395*

years, it has been reported that spider and scorpion venom components also exhibit sex differences [145–149]. This suggests that the development of control techniques for chelicerates sex may be useful for efficient venom constituent identification and increased production. However, most sex-related research, including sex determination and sexual differentiation, remains unclear in chelicerates. This is due to a lack of analytical tools for late-stage embryos and larvae, including the markers for the typing of genetic sex. We hope that we will overcome these barriers in the future and deepen our understanding of sex in chelicerates, which is important from a biological, evolutionary, and industrial perspective.

## **Acknowledgements**

The authors would like to thank Dr. Mike Roberts, Independent Consultants, UK for his critical readings of this manuscript.

## **Conflict of interest**

The authors declare no conflicts of interest associated with this manuscript.

## **Author details**

Kenji Toyota1,2,3\*, Yuta Sakae4,5 and Taisen Iguchi6

1 Niigata University, Marine Biological Station, Sado Center for Ecological Sustainability, Sado, Niigata, Japan

2 Faculty of Science, Department of Biological Sciences, Kanagawa University, Hiratsuka, Kanagawa, Japan

3 Faculty of Industrial Science and Technology, Department of Biological Science and Technology, Tokyo University of Science, Katsushika-ku, Tokyo, Japan

4 Department of Integrated Biosciences, The University of Tokyo, Graduate School of Frontier Sciences, Kashiwa, Chiba, Japan

5 Division of Translational Genomics, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, Chiba, Japan

6 Yokohama City University, Graduate School of Nanobioscience, Yokohama, Kanagawa, Japan

\*Address all correspondence to: megabass0719@yahoo.co.jp

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*Larval Development of Non-Insect Arthropods: Metamorphosis and Sexual Differentiation DOI: http://dx.doi.org/10.5772/intechopen.105395*

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## **Chapter 2**
