Genomic and transcriptomic analyses of the subterranean termite Reticulitermes speratus: Gene duplication facilitates social evolution.

Termites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing a transitional taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome, and methylome of the Japanese subterranean termite Reticulitermes speratus Our analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression was prevalent in the R. speratus genome. The duplicated genes comprised diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense), and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but their expression patterns were highly variable, exhibiting caste biases. Some of the assayed duplicated genes were expressed in caste-specific organs, such as the accessory glands of the queen ovary and the frontal glands of soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification, leading to caste-biased expression and subfunctionalization and/or neofunctionalization conferring caste-specialized functions.

Hormone-related genes heterochronically and modularly regulate neotenic differentiation in termites.

Caste development in social insects requires the coordination of molting and metamorphosis during postembryonic development. In termites, i.e., hemimetabolous eusocial insects, caste fate is determined during postembryonic development. However, it is not fully understood how the mechanisms of molting/metamorphosis are regulated in the course of differentiation between reproductive and sterile castes. In termites, only reproductives derived from alates are imagos and other sterile castes (including developmentally-terminal soldier caste) are basically juveniles or nymphs. Furthermore, supplementary reproductives that appear when the original queens and kings die or become senescent, exhibit larval features such as winglessness, and are called neotenics. Therefore, the question of whether neotenics are larvae or imagos is still under debate. In this study, by inducing female neotenic differentiation in a damp-wood termite Hodotermopsis sjostedti, morphological investigations together with juvenile hormone (JH) quantification and expression/functional analyses of genes responsible for molting and/or metamorphosis were carried out. JH titer and expression of one of the downstream genes (Kr-h1) were shown to be temporarily lowered, but increased just prior to the molt into neotenics, while consistently lowered in imaginal molt (i.e., alate differentiation). In contrast, ecdysone-related genes (EcR and E93) were upregulated at both neotenic and alate differentiation, suggesting that the heterochronic actions of ecdysone and JH lead the neotenic differentiation. Moreover, expression analyses, supported by reverse genetic experiments, showed that EcR and E93 were specifically upregulated in genital sternites (EcR and E93) and ovaries (E93) and required for the development of imaginal characters. These results suggest that the resultant mosaic phenotype of female neotenics is due to modular responses of different body parts to hormonal actions.

Appearance of a transparent protrusion containing two pairs of legs on the apodous ring preceding the anamorphic molt in a millipede, Niponia nodulosa.

BACKGROUND: Arthropods gradually change their forms through repeated molting events during postembryonic development. Anamorphosis, i.e., segment addition during postembryonic development, is seen in some arthropod lineages. In all millipede species (Myriapoda, Diplopoda), for example, postembryonic processes go through anamorphosis. Jean-Henri Fabre proposed 168 years ago the "law of anamorphosis", that is, "new rings appear between the penultimate ring and the telson" and "all apodous rings in a given stadium become podous rings in the next stadium", but the developmental process at the anamorphic molt remains largely unknown. In this study, therefore, by observing the morphological and histological changes at the time of molting, the detailed processes of leg- and ring-addition during anamorphosis were characterized in a millipede, Niponia nodulosa (Polydesmida, Cryptodesmidae). RESULTS: In the preparatory period, a few days before molting, scanning electron microscopy, confocal laser scanning microscopy, and histological observations revealed that two pairs of wrinkled leg primordia were present under the cuticle of each apodous ring. In the rigidation period, just prior to molt, observations of external morphology showed that a transparent protrusion was observed on the median line of the ventral surface on each apodous ring. Confocal laser scanning microscopy and histological observations revealed that the transparent protrusion covered by an arthrodial membrane contained a leg bundle consisting of two pairs of legs. On the other hand, ring primordia were observed anterior to the telson just before molts. CONCLUSIONS: Preceding the anamorphic molt in which two pairs of legs are added on an apodous ring, a transparent protrusion containing the leg pairs (a leg bundle) appears on each apodous ring. The morphogenetic process of the rapid protrusion of leg bundles, that is enabled by thin and elastic cuticle, suggested that millipedes have acquired a resting period and unique morphogenesis to efficiently add new legs and rings.

Termite soldier mandibles are elongated by dachshund under hormonal and Hox gene controls.

In social insects, interactions among colony members trigger caste differentiation with morphological modifications. In termite soldier differentiation, the mandible size considerably increases through two moltings (via the presoldier stage) under the control of juvenile hormone (JH). Regulatory genes are predicted to provide patterning information that induces the mandible-specific cell proliferation. To identify factors responsible for the mandibular enlargement, expression analyses of 18 candidate genes were carried out in the termite Hodotermopsis sjostedti Among those, dachshund (dac), which identifies the intermediate domain along the proximodistal appendage axis, showed mandible-specific upregulation prior to the molt into presoldiers, which can explain the pattern of cell proliferation for the mandibular elongation. Knockdown of dac by RNAi reduced the mandibular length and distorted its morphology. Furthermore, the epistatic relationships among Methoprene tolerant, Insulin receptor, Deformed (Dfd) and dac were revealed by combined RNAi and qRT-PCR analyses, suggesting that dac is regulated by Dfd, downstream of the JH and insulin signaling pathways. Thus, caste-specific morphogenesis is controlled by interactions between the factors that provide spatial information and physiological status.

Parasitism-Induced Intersexuality in a Sexually Dimorphic Varunid Crab, Ptychognathus ishii (Decapoda: Varunidae).

Although many animals that perform sexual reproduction exhibit sexual dimorphism, individuals with intersex traits between the traits of males and females appear in some species, depending on environmental factors. Ptychognathus ishii, a varunid crab, exhibits distinctive sexual dimorphism in the morphology of its abdomen, chelipeds and setal tufts on the chelipeds. In this study, however, we report for the first time that intersex individuals with intermediate characters between those of males and females were occasionally found in wild populations. Morphological features of intersex individuals are described. Their taxonomic positions are identified based on DNA sequences of part of the mitochondrial cytochrome c oxidase I (COI) gene. It was shown that the intersexuality was induced by entoniscid parasites, because all intersex individuals were parasitized by entoniscid isopods, identified as Entionella sp. The apparent correlation between parasitism and morphological anomalies suggests that the parasitic isopods affect physiological conditions, leading to the feminization of male hosts.

Developmental Process of a Heterozooid: Avicularium Formation in a Bryozoan, Bugulina californica.

In bryozoans (phylum Bryozoa), representative colonial animals mostly found in marine environments, some species possess different types of individuals (heterozooids) specialized in different functions such as defense or structural support for their colonies. Among them, the best-known heterozooids are the avicularia, known to function as defenders. The differentiation processes of heterozooids, including avicularia, should be important keys to understand the evolutionary significance of bryozoans. However, the developmental process of avicularium formation remains to be fully understood. In this study, therefore, in order to understand the detailed developmental process and timing of avicularium formation, extensive observations were carried out in a bryozoan species, Bugulina californica (Cheilostomata, Bugulidae), that possesses adventitious avicularia, by performing stereomicroscopy on live materials, in addition to scanning electron microscopy and histological observations. The whole process can be divided into seven stages based on developmental events. Especially notably, at the earlier stages, there are three major budding events that produce proliferating cell masses corresponding to primordial tissues: (1) budding of the peduncle cushion at the outer margin of the distal part of a young autozooid, (2) budding of the head-part primordium from the peduncle cushion, and (3) budding of the polypide inside the head part. Experimental control of temperature showed that 20°C would be the best to maintain B. californica colonies.

Pattern of sucker development in cuttlefishes.

BACKGROUND: Morphological novelties have been acquired through evolutionary processes and related to the adaptation of new life-history strategies with new functions of the bodyparts. Cephalopod molluscs such as octopuses, squids and cuttlefishes possess unique morphological characteristics. Among those novel morphologies, in particular, suckers arranged along the oral side of each arm possess multiple functions, such as capturing prey and locomotion, so that the sucker morphology is diversified among species, depending on their ecological niche. However, the detailed developmental process of sucker formation has remained unclear, although it is known that new suckers are formed or added during both embryonic and postembryonic development. In the present study, therefore, focusing on two cuttlefish species, Sepia esculenta and S. lycidas, in which the sucker morphology is relatively simple, morphological and histological observations were carried out during embryonic and postembryonic development to elucidate the developmental process of sucker formation and to compare them among other cephalopod species. RESULTS: The observations in both species clearly showed that the newly formed suckers were added on the oral side of the most distal tip of each arm during embryonic and postembryonic development. On the oral side of the arm tip, the epithelial tissue became swollen to form a ridge along the proximal-distal axis (sucker field ridge). Next to the sucker field ridge, there were small dome-shaped bulges that are presumed to be the sucker buds. Toward the proximal direction, the buds became functional suckers, in which the inner tissues differentiated to form the complex sucker structures. During postembryonic development, on both sides of the sucker field ridge, epithelial tissues extended to form a sheath, covering the ridge for protection of undifferentiated suckers. CONCLUSIONS: The developmental process of sucker formation, in which sucker buds are generated from a ridge structure (sucker field ridge) on the oral side at the distal-most arm tip, was shared in both cuttlefish species, although some minor heterochronic shifts of the developmental events were detected between the two species.(325 words).

Unique Morphogenesis in the Damp-Wood Termite: Abscission of the Stylus during Female Reproductive Caste Differentiation.

Eusocial insects organize their colonies based on division of labor and caste differentiation, in which caste-specific morphologies are sculpted during postembryonic development. In the differentiation between reproductive and sterile castes, characteristics related to mating and reproduction are developed in reproductives, and degenerated in neuters, although little is known about the developmental regulations during the differentiation. In some species of termites, a sensory protrusion at the posterior end of the abdomen, the stylus, is known to disappear in female reproductives. In the present study, we performed anatomical and histological analyses in the damp-wood termite Hodotermopsis sjostedti to elucidate the developmental process underlying the disappearance of the stylus during neotenic and alate differentiation. Although it was first hypothesized that styli were hidden beneath the enlarged seventh sternite, our observation results found out that the styli were completely lost in reproductive females. Further histological observations revealed that the stylus disappearance was not accomplished by degeneration process; rather, styli were separated from the abdomen and discarded with the exoskeleton (exuviae) during the molt into the reproductive caste. This phenomenon in which live tissues are discarded through developmental processes is suggested to be a case of abscission, as seen in plant leaves.

Life Cycle of the Japanese Green Syllid, Megasyllis nipponica (Annelida: Syllidae): Field Collection and Establishment of Rearing System.

Some polychaete species in the family Syllidae exhibit distinctive life cycles, in which a posterior part of the body of an individual detaches as a reproductive individual called a "stolon". This type of reproductive mode is known as stolonization or schizogamy. Although a number of observations have been reported, and techniques using molecular markers have recently been applied to characterize this phenomenon, little is known about the developmental and physiological mechanisms underlying stolonization. In the present study, Megasyllis nipponica, a common syllid species distributed throughout Japan, is proposed as a model to reveal the developmental and physiological mechanism of stolonization, and the rearing system to maintain it in laboratory conditions is described. This species was repeatedly sampled around Hokkaido, where more dense populations were found from August to October. The animals were maintained in the laboratory under stable long-day condition (20°C, 16L:8D), and fed mainly with spinach powder. Stolonization processes, spawning, embryonic and postembryonic development were observed and documented, and the required period of time for each developmental stage was recorded. The complete generation time was around two months under the rearing condition. The information provided is valuable to maintain this and other syllid species in the laboratory, and hence contributes to the establishment of new evolutionary and developmental research lines in this group of annelids.

Body part-specific development in termite caste differentiation: crosstalk between hormonal actions and developmental toolkit genes.

In social insects, interactions among colony members trigger caste differentiation with morphological modifications. During caste differentiation in termites, body parts and caste-specific morphologies are modified during postembryonic development under endocrine controls such as juvenile hormone (JH) and ecdysone. In addition to endocrine factors, developmental toolkit genes such as Hox- and appendage-patterning genes also contribute to the caste-specific body part modifications. These toolkits are thought to provide spatial information for specific morphogenesis. During social evolution, the complex crosstalks between physiological and developmental mechanisms should be established, leading to the sophisticated caste systems. This article reviews recent studies on these mechanisms underlying the termite caste differentiation and addresses implications for the evolution of caste systems in termites.

Intracellularity, extracellularity, and squeezing in the symbiotic organ underpin nurturing and functioning of bacterial symbiont in leaf beetles.

Cassidine leaf beetles are associated with genome-reduced symbiotic bacteria Stammera involved in pectin digestion. Stammera cells appear to be harbored in paired symbiotic organs located at the foregut-midgut junction either intracellularly or extracellularly, whereas the symbiont is extracellular in the ovary-accessory glands of adult females and during caplet transmission in eggs. However, using fluorescence and electron microscopy, an intracellular symbiotic configuration of Stammera was observed in Notosacantha species. Detailed inspection of other cassidine species revealed fragmented cell membrane and cytoplasm of the symbiotic organs, wherein Stammera cells are in an intermediate status between intracellularity and extracellularity. We also identified a mitochondria-rich region adjacent to the symbiont-filled region and well-developed muscle fibers surrounding the whole symbiotic organ. Based on these observations, we discuss why the Stammera genome has been reduced so drastically and how symbiont-derived pectinases are produced and supplied to the host's alimentary tract for plant cell wall digestion.

Paleocene origin of a streamlined digestive symbiosis in leaf beetles.

Timing the acquisition of a beneficial microbe relative to the evolutionary history of its host can shed light on the adaptive impact of a partnership. Here, we investigated the onset and molecular evolution of an obligate symbiosis between Cassidinae leaf beetles and Candidatus Stammera capleta, a γ-proteobacterium. Residing extracellularly within foregut symbiotic organs, Stammera upgrades the digestive physiology of its host by supplementing plant cell wall-degrading enzymes. We observe that Stammera is a shared symbiont across tortoise and hispine beetles that collectively comprise the Cassidinae subfamily, despite differences in their folivorous habits. In contrast to its transcriptional profile during vertical transmission, Stammera elevates the expression of genes encoding digestive enzymes while in the foregut symbiotic organs, matching the nutritional requirements of its host. Despite the widespread distribution of Stammera across Cassidinae beetles, symbiont acquisition during the Paleocene (∼62 mya) did not coincide with the origin of the subfamily. Early diverging lineages lack the symbiont and the specialized organs that house it. Reconstructing the ancestral state of host-beneficial factors revealed that Stammera encoded three digestive enzymes at the onset of symbiosis, including polygalacturonase-a pectinase that is universally shared. Although non-symbiotic cassidines encode polygalacturonase endogenously, their repertoire of plant cell wall-degrading enzymes is more limited compared with symbiotic beetles supplemented with digestive enzymes from Stammera. Highlighting the potential impact of a symbiotic condition and an upgraded metabolic potential, Stammera-harboring beetles exploit a greater variety of plants and are more speciose compared with non-symbiotic members of the Cassidinae.

Upregulation of Hox genes leading to caste-specific morphogenesis in a termite.

BACKGROUND: In social insects, interactions among colony members trigger caste differentiation with morphological modifications. In termite caste differentiation, caste-specific morphologies (such as mandibles in soldiers, genital organs in reproductives or wings in alates) are well developed during post-embryonic development under endocrine controls (e.g., juvenile hormone and ecdysone). Since body part-specific morphogenesis in caste differentiation is hormonally regulated by global factors circulated throughout the body, positional information should be required for the caste-specific and also body part-specific morphogenesis. To identify factors providing the positional information, expression and functional analyses of eight Hox genes were carried out during the three types of caste differentiation (i.e., soldier, neotenic and alate differentiation) in a termite, Hodotermopsis sjostedti. RESULTS: Spatio-temporal patterns of Hox gene expression during caste differentiation were elucidated by real-time qPCR, showing the caste-specific upregulations of Hox genes during the differentiation processes. Among eight Hox genes, Deformed (Dfd) was upregulated specifically in mandibles in soldier differentiation, abdominal-A (abd-A) and Abdominal-B (Abd-B) were upregulated in the abdomen in neotenic differentiation, while Sex-comb reduced (Scr) and Antennapedia (Antp) were upregulated during alate differentiation. Furthermore, RNAi knockdown of Dfd in soldier differentiation and of abd-A and Abd-B in neotenic differentiation distorted the modifications of caste-specific morphologies. CONCLUSIONS: Gene expression and functional analyses in this study revealed that, in the caste differentiation in termites, upregulation of Hox genes provide positional identities of body segments, resulting in the caste-specific morphogenesis. The acquisition of such developmental modifications would have enabled the evolution of sophisticated caste systems in termites.

Morphological, histological and gene-expression analyses on stolonization in the Japanese Green Syllid, Megasyllis nipponica (Annelida, Syllidae).

Benthic annelids belonging to the family Syllidae (Annelida, Errantia, Phyllodocida) exhibit a unique reproduction mode called "schizogamy" or "stolonization", in which the posterior body part filled with gametes detaches from the original body, as a reproductive unit (stolon) that autonomously swims and spawns. In this study, morphological and histological observations on the developmental processes during stolonization were carried out in Megasyllis nipponica. Results suggest that the stolon formation started with maturation of gonads, followed by the formation of a head ganglion in the anteriormost segment of the developing stolon. Then, the detailed stolon-specific structures such as stolon eyes and notochaetae were formed. Furthermore, expression profiles of genes involved in the anterior-posterior identity (Hox genes), head determination, germ-line, and hormone regulation were compared between anterior and posterior body parts during the stolonization process. The results reveal that, in the posterior body part, genes for gonadal development were up-regulated, followed by hormone-related genes and head-determination genes. Unexpectedly, Hox genes known to identify body parts along the anterior-posterior axis showed no significant temporal expression changes. These findings suggest that during stolonization, gonad development induces the head formation of a stolon, without up-regulation of anterior Hox genes.

Sucker formation in a bigfin reef squid: Comparison between arms and tentacles.

Cephalopods have acquired numerous novelties and expanded their habitats to various marine environments as highly agile predators. Among cephalopod novelties, multiple arms are used for complex behaviors, including prey capture. Suckers on arms are innovative features for realizing these arm functions. In addition, tentacles in Decapodiformes (squids and cuttlefishes) are arms specialized in prey capture and tentacular suckers show unique morphologies. However, little is known about the developmental process of sucker formation that should differ between tentacles and other arms. In this study, therefore, sucker formation processes on second arms and tentacles were observed and compared in a bigfin reef squid, Sepioteuthis lessoniana, to reveal the developmental processes forming the unique sucker morphologies, especially in tentacles. Morphological and histological observations of suckers during embryogenesis showed that, in second arms, the sucker-producing area appeared at the most distal part. At the most proximal side of the sucker-producing area, new sucker buds were isolated by invagination of the epithelial tissue. At the proximal arm parts, suckers with functional structures were observed. In tentacles, although the basic sucker formation pattern was similar to that in second arms, sucker formation started at earlier embryonic stages and the number of suckers was drastically increased compared to that in second arms. In addition, although four sucker rows were observed at the tentacular club, that is, the thickest part of a tentacle, our observations suggested that two sets of two sucker rows are compressed to form the four rows. Therefore, the sucker-formation processes are temporally and spatially different between arms and tentacles. In addition, S. lessoniana shows conserved and unique patterns of sucker formation in comparison with previously described species, suggesting that sucker formation patterns were diversified among Decapodiformes lineages.

Hydrocoel morphogenesis forming the pentaradial body plan in a sea cucumber, Apostichopus japonicus.

Echinoderms constitute an animal phylum characterized by the pentaradial body plan. During the development from bilateral larvae to pentaradial adults, the formation of the multiple of five hydrocoel lobes, i.e., the buddings from the mesodermal coelom, is the firstly emerging pentameral character. The developmental mechanism underlying the hydrocoel-lobe formation should be revealed to understand the evolutionary process of this unique and highly derived body plan of echinoderms, although the morphogenetic mechanisms of hydrocoel lobes are largely uninvestigated. In this study, using the sea cucumber Apostichopus japonicus, in which hydrocoel is easily observable, the developmental process of hydrocoel lobes was described in detail, focusing on cell proliferation and rearrangement. Cell proliferation was not specifically distributed in the growing tips of the hydrocoel lobes, and inhibition of cell proliferation did not affect lobe formation. During lobe formation, the epithelium of the hydrocoel lobes was firstly thickened and then transformed into a simple epithelium, suggesting that tissue expansion via tissue remodeling contributes to the hydrocoel-lobe formation.

Spiroplasma as facultative bacterial symbionts of stinkbugs.

Many insects are associated with facultative symbiotic bacteria, and their infection prevalence provides an important clue to understand the biological impact of such microbial associates. Here we surveyed diverse stinkbugs representing 13 families, 69 genera, 97 species and 468 individuals for Spiroplasma infection. Diagnostic PCR detection revealed that 4 families (30.8%), 7 genera (10.1%), 11 species (11.3%) and 21 individuals (4.5%) were Spiroplasma positive. All the 21 stinkbug samples with Spiroplasma infection were subjected to PCR amplification and sequencing of Spiroplasma's 16S rRNA gene. Molecular phylogenetic analysis uncovered that the stinkbug-associated Spiroplasma symbionts were placed in three distinct clades in the Spiroplasmataceae, highlighting multiple evolutionary origins of the stinkbug-Spiroplasma associations. The Spiroplasma phylogeny did not reflect the host stinkbug phylogeny, indicating the absence of host-symbiont co-speciation. On the other hand, the Spiroplasma symbionts associated with the same stinkbug family tended to be related to each other, suggesting the possibility of certain levels of host-symbiont specificity and/or ecological symbiont sharing. Amplicon sequencing analysis targeting bacterial 16S rRNA gene, FISH visualization of the symbiotic bacteria, and rearing experiments of the host stinkbugs uncovered that the Spiroplasma symbionts are generally much less abundant in comparison with the primary gut symbiotic bacteria, localized to various tissues and organs at relatively low densities, and vertically transmitted to the offspring. On the basis of these results, we conclude that the Spiroplasma symbionts are, in general, facultative bacterial associates of low infection prevalence that are not essential but rather commensalistic for the host stinkbugs, like the Spiroplasma symbionts of fruit flies and aphids, although their impact on the host phenotypes should be evaluated in future studies.

Construction of a massive genetic resource by transcriptome sequencing and genetic characterization of Megasyllis nipponica (Annelida: Syllidae).

Understanding the processes and consequences of the morphological diversity of organisms is one of the major goals of evolutionary biology. Studies on the evolution of developmental mechanisms of morphologies, or evo-devo, have been extensively conducted in many taxa and have revealed many interesting phenomena at the molecular level. However, many other taxa exhibiting intriguing morphological diversity remain unexplored in the field of evo-devo. Although the annelid family Syllidae shows spectacular diversity in morphological development associated with reproduction, its evo-devo study, especially on molecular development, has progressed slowly. In this study, we focused on Megasyllis nipponica as a new model species for evo-devo in syllids and performed transcriptome sequencing to develop a massive genetic resource, which will be useful for future molecular studies. From the transcriptome data, we identified candidate genes that are likely involved in morphogenesis, including genes involved in hormone regulation, sex determination and appendage development. Furthermore, a computational analysis of the transcriptome sequence data indicated the occurrence of DNA methylation in coding regions of the M. nipponica genome. In addition, flow cytometry analysis showed that the genome size of M. nipponica was approximately 524 megabases. These results facilitate the study of morphogenesis in molecular terms and contribute to our understanding of the morphological diversity in syllids.

Four new species of Ctenodrilus, Raphidrilus, and Raricirrus (Cirratuliformia, Annelida) in Japanese waters, with notes on their phylogenetic position.

Four new species of annelids, Ctenodrilus japonicus sp. nov., Raphidrilus misakiensis sp. nov., Raphidrilus okinawaensis sp. nov., and Raricirrus anubis sp. nov., are described based on specimens collected from Japanese waters. Ctenodrilus japonicus sp. nov. inhabits the interstitial environment and can be distinguished from the other congeners by the following features: (i) total of 16 chaetigers, (ii) chaetigers 1-3 with stout hooks, (iii) minute body (approximately 1 mm in length), (iv) all parapodia with the same number of chaetae (two notochaetae; two neurochaetae), and (v) presence of dorsal and ventral papillae. Raphidrilus misakiensis sp. nov. lives under intertidal stones and can be distinguished from other congeners by having pectinate neurochaetae. Raphidrilus okinawaensis sp. nov. inhabits the interstitial environment and can be distinguished from other congeners by: (i) absence of annulation on the peristomium and achaetous segment and (ii) presence of a heart body in chaetigers 4-5. Raricirrus anubis sp. nov. inhabits whale bones and can be distinguished from other congeners by the following features: (i) presence of a heart body in chaetigers 9-14, (ii) presence of capillary neurochaetae on chaetiger 1, and (iii) presence of simple curved spines. A phylogenetic tree based on partial sequences of cytochrome c oxidase subunit I and 16S rRNA from the new species and other cirratulid worms showed that Raphidrilus is included in Cirratuliformia. This is the first record of Raphidrilus and Raricirrus from Japanese waters.

Endosymbiotic bacteria of the boar louse Haematopinus apri (Insecta: Phthiraptera: Anoplura).

Insects exclusively feeding on vertebrate blood are usually dependent on symbiotic bacteria for provisioning of B vitamins. Among them, sucking lice are prominent in that their symbiotic bacteria as well as their symbiotic organs exhibit striking diversity. Here we investigated the bacterial diversity associated with the boar louse Haematopinus apri in comparison with the hog louse Haematopinus suis. Amplicon sequencing analysis identified the primary endosymbiont predominantly detected from all populations of H. apri with some minor secondary bacterial associates. Sequencing and phylogenetic analysis of bacterial 16S rRNA gene confirmed that the endosymbionts of the boar louse H. apri, the hog louse H. suis and the cattle louse Haematopinus eurysternus form a distinct clade in the Gammaproteobacteria. The endosymbiont clade of Haematopinus spp. was phylogenetically distinct from the primary endosymbionts of other louse lineages. Fluorescence in situ hybridization visualized the endosymbiont localization within midgut epithelium, ovarial ampulla and posterior oocyte of H. apri, which were substantially the same as the endosymbiont localization previously described in H. suis and H. eurysternus. Mitochondrial haplotype analysis revealed that, although the domestic pig was derived from the wild boar over the past 8,000 years of human history, the populations of H. apri constituted a distinct sister clade to the populations of H. suis. Based on these results, we discussed possible evolutionary trajectories of the boar louse, the hog louse and their endosymbionts in the context of swine domestication. We proposed 'Candidatus Haematopinicola symbiotica' for the distinct clade of the endosymbionts of Haematopinus spp.