The structural and functional modularity of ovarian follicle epithelium in the pill-millipede Hyleoglomeris japonica Verhoeff, 1936 (Diplopoda: Glomerida: Glomeridae).

Ovarian somatic tissues typically surround developing oocytes and play a crucial role in oogenesis across various metazoans, often displaying structural properties specific to their functions. However, there is an absence of evident structural modularity in the follicle epithelium of Myriapoda. We report here two structurally and developmentally distinct domains within the follicle epithelium of the Japanese pill millipede, Hyleoglomeris japonica. The follicle epithelium of H. japonica exhibits a thick cell mass at the apex of the follicle. These cells harbor abundant rough endoplasmic reticulum, mitochondria, Golgi complexes, and numerous microvilli, indicative of synthetic/secretory activities. Moreover, their height increases as oogenesis progresses. In contrast, another region of the epithelium lacks these features. Our findings highlight the presence of structural and functional modularity in the follicle epithelium of H. japonica. We suggest classifying the follicle epithelium of Myriapoda into three types: homogenous epithelia with enhanced synthetic activities, homogenous epithelia with diminished such activities, and heterogeneous epithelia with varying synthetic activities. These findings prompt a reevaluation of the nature of ovarian somatic tissues in Myriapoda as well as in Arthropoda.

Taxonomy of a New Parasitic Euglenid, Euglenaformis parasitica sp. nov. (Euglenales, Euglenaceae) in Ostracods and Rhabdocoels.

Parasitic euglenids have rarely been studied. We found parasitic euglenids in two species of ostracods (Cyprinotus cassidula, Dolerocypris sinensis) and two species of rhabdocoels (Mesostoma lingua, Microdalyellia armigera) in a rice field. These parasites grew and proliferated inside the host body. These parasites had pellicle strips, one emergent flagellum, and a red stigma, but no chloroplasts, and showed euglenoid movement. Inside the living host, they did not have emergent flagella and moved only by euglenoid movement, but when the host died or the parasites were isolated from the host, they extended their flagella and switched to swimming movement. We conclude that the parasites found in the four hosts that we examined are of the same species, considering the morphological characteristics and identities in the nSSU and nLSU rDNA sequences of those parasites. Molecular phylogenetic analysis showed that the parasite formed a clade with the free-living photoautotrophic species of Euglenaformis, with moderate statistical support. Therefore, the parasite is a secondary osmotroph derived from a photoautotrophic ancestor. Based on the results of morphological observation and molecular phylogenetic analysis, we propose a new species of parasitic euglenid, Euglenaformis parasitica sp. nov.

Reviewing the Effects of Skin Manipulations on Adult Newt Limb Regeneration: Implications for the Subcutaneous Origin of Axial Pattern Formation.

Newts are unique salamanders that can regenerate their limbs as postmetamorphic adults. In order to regenerate human limbs as newts do, it is necessary to determine whether the cells homologous to those contributing to the limb regeneration of adult newts also exist in humans. Previous skin manipulation studies in larval amphibians have suggested that stump skin plays a pivotal role in the axial patterning of regenerating limbs. However, in adult newts such studies are limited, though they are informative. Therefore, in this article we have conducted skin manipulation experiments such as rotating the skin 180° around the proximodistal axis of the limb and replacing half of the skin with that of another location on the limb or body. We found that, contrary to our expectations, adult newts robustly regenerated limbs with a normal axial pattern regardless of skin manipulation, and that the appearance of abnormalities was stochastic. Our results suggest that the tissue under the skin, rather than the skin itself, in the intact limb is of primary importance in ensuring the normal axial pattern formation in adult newt limb regeneration. We propose that the important tissues are located in small areas underlying the ventral anterior and ventral posterior skin.

Signs of the plastid: Enzymes involved in plastid-localized metabolic pathways in a eugregarine species.

Apicomplexa mainly comprises parasitic species and some of them, which infect and cause severe diseases to humans and livestock, have been extensively studied due to the clinical and industrial importance. Besides, apicomplexans are a popular subject of the studies focusing on the evolution initiated by a secondary loss of photosynthesis. By interpreting the position in the tree of eukaryotes and lifestyles of the phylogenetic relatives parsimoniously, the extant apicomplexans are predicted to be the descendants of a parasite bearing a non-photosynthetic (cryptic) plastid. The plastid-bearing characteristic for the ancestral apicomplexan is further strengthened by non-photosynthetic plastids found in the extant apicomplexans. The research on apicomplexan members infecting invertebrates is much less advanced than that on the pathogens to humans and livestock. Gregarines are apicomplexans that infect diverse invertebrates and recent studies based on transcriptome data revealed the presence of cryptic plastids in a subset of the species investigated. In this study, we isolated gregarine-like organisms (GLOs) from three arthropod species and conducted transcriptome analyses on the isolated cells. A transcriptome-based, multi-gene phylogenetic analysis clearly indicated that all of the three GLOs are eugregarines. Significantly, the transcriptome data from the GLO in a centipede appeared to contain the transcripts encoding enzymes involved in the non-mevalonate pathway for isopentenyl diphosphate biosynthesis and C5 pathway for heme biosynthesis. The enzymes involved in the two plastid-localized metabolic pathways circumstantially but strongly suggest that the particular GLO possesses a cryptic plastid. The evolution of cryptic plastids in eugregarines is revised by incorporating the new data obtained from the three GLOs in this study.

Morphological study of the ovary in Hanseniella caldaria (Myriapoda; Symphyla): The position of oocyte-growth and evolution of ovarian structure in Arthropoda.

In Arthropoda, the ovary is classified into Chelicerata-type and Mandibulata-type, based on the oocyte-growth position within the ovary. By contrast, oocytes of Diplopoda and Chilopoda grow within the hemocoelic space. However, as the position of oocyte-growth in Symphyla and Pauropoda has not been confirmed, whether the hemocoelic nature of oocyte-growth is common among myriapods remains ambiguous. This study described the ovarian structure of Hanseniella caldaria to reveal the oocyte-growth position in Symphyla. The oocyte is surrounded by the follicle epithelium, and the inner surface of the follicle epithelium, i.e., the space between follicle cells and oocytes, is lined with a basement membrane. The follicle epithelial layer continues to the ovarian epithelium via the follicle extension with a continuous layer of basement membrane. Data on the architecture of the follicle suggest that the follicle pouch opens to the hemocoel. Hence, the oocyte of H. caldaria grows within the hemocoelic space. Based on our findings in H. caldaria and previous studies in a millipede and in centipedes, the hemocoelic nature of oocyte-growth is considered as a common feature among myriapods and a synapomorphy of the Myriapoda for which morphological synapomorphies have been ambiguous.

Novel erythrocyte clumps revealed by an orphan gene Newtic1 in circulating blood and regenerating limbs of the adult newt.

The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known. Here we report novel behavior of erythrocytes in the newt. We identified an orphan gene Newtic1, whose transcripts significantly increased in the blastema. Newtic1 was expressed in a subset of erythrocytes that formed a novel clump (EryC). EryC formed a complex with monocytes and was circulating throughout the body. When the limb was amputated, EryCs were newly generated in the stump and accumulated into a distal portion of the growing blastema. Our data suggested that the newt erythrocytes carried multiple secretory molecules including growth factors and matrix metalloproteases, and were capable of delivering these molecules into the blastema as a form of EryCs. This study provides insight into regulations and roles of nucleated erythrocytes, that are independent of oxygen delivery.

Transgenic Expression of the piRNA-Resistant Masculinizer Gene Induces Female-Specific Lethality and Partial Female-to-Male Sex Reversal in the Silkworm, Bombyx mori.

In Bombyx mori (B. mori), Fem piRNA originates from the W chromosome and is responsible for femaleness. The Fem piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masc gene. Masc encodes a novel CCCH type zinc-finger protein and is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. In the present study, several silkworm strains carrying a transgene, which encodes a Fem piRNA-resistant Masc mRNA (Masc-R), were generated. Forced expression of the Masc-R transgene caused female-specific lethality during the larval stages. One of the Masc-R strains weakly expressed Masc-R in various tissues. Females heterozygous for the transgene expressed male-specific isoform of the Bombyx homolog of insulin-like growth factor II mRNA-binding protein (ImpM) and Bmdsx. All examined females showed a lower inducibility of vitellogenin synthesis and exhibited abnormalities in the ovaries. Testis-like tissues were observed in abnormal ovaries and, notably, the tissues contained considerable numbers of sperm bundles. Homozygous expression of the transgene resulted in formation of the male-specific abdominal segment in adult females and caused partial male differentiation in female genitalia. These results strongly suggest that Masc is an important regulatory gene of maleness in B. mori.

Phylogeographic Study of Whip Scorpions (Chelicerata: Arachnida: Thelyphonida) in Japan and Taiwan.

Whip scorpions (Thelyphonida), comprising an order in the class Arachnida, are distributed from tropical to temperate zones. Two species occur exclusively in Japan and Taiwan, but the border of their distributional ranges is ambiguous in the Central Ryukyus (Japan). We collected new specimens from the Central Ryukyus and revealed that the border of distribution of the two species lies between the Central and Southern Ryukyus, i.e., the Kerama Gap. Moreover, the estimated divergence time (15.8 Mya) of the two species, based on the mitochondrial COI gene, was older than the recently estimated time (1.55 Mya) of formation of the Kerama Gap. These results highlight the risks of a priori assumption solely on the basis of geological data for applying it as a calibration point to some terrestrial animals in this region. Typopeltis stimpsonii was genetically divided into four lineages, two of which turned out to be endemic to the Okinawa Islands. All specimens from the main island of Japan and Shikoku were in one lineage, which was also found in the Amami Islands and Hachijojima Island. This suggests that these specimens may have been dispersed by human activity. Typopeltis crucifer included five genetic lineages. Species collected from Ishigakijima and Iriomotejima Islands were genetically diversified not between the borders of these islands but within Ishigakijima Island. This study also suggests that phylogenetic diversity of the species in the Southern Ryukyus have increased through two times of invasions from Taiwan.

Phylogeography of Semiterrestrial Isopod, Tylos granuliferus, on East Asian Coasts.

We conducted phylogenetic analyses using mitochondrial COI gene sequences of Tylos granuliferus, a semiterrestrial coastal isopod in East Asia, to clarify possible phylogeographic patterns and to assess relationships between present and past marine environments and genetic population structures. Our results strongly suggest the presence of four clades of T. granuliferus, one of which consists of three subclades. The distribution pattern of clades and subclades seems to have been affected by ocean current activities. Our results also suggest that historical changes in oceanic environments and the subsequence bifurcation of current streamlines affected the first and second divergences of T. granuliferus during the late Miocene and near the beginning of the Pliocene, respectively.

Molecular phylogeny of Myriapoda provides insights into evolutionary patterns of the mode in post-embryonic development.

Myriapoda, a subphylum of Arthropoda, comprises four classes, Chilopoda, Diplopoda, Pauropoda, and Symphyla. While recent molecular evidence has shown that Myriapoda is monophyletic, the internal phylogeny, which is pivotal for understanding the evolutionary history of myriapods, remains unresolved. Here we report the results of phylogenetic analyses and estimations of divergence time and ancestral state of myriapods. Phylogenetic analyses were performed based on three nuclear protein-coding genes determined from 19 myriapods representing the four classes (17 orders) and 11 outgroup species. The results revealed that Symphyla whose phylogenetic position has long been debated is the sister lineage to all other myriapods, and that the interordinal relationships within classes were consistent with traditional classifications. Ancestral state estimation based on the tree topology suggests that myriapods evolved from an ancestral state that was characterized by a hemianamorphic mode of post-embryonic development and had a relatively low number of body segments and legs.

Embryonic development and expression analysis of Distal-less in Caprella scaura (Crustacea, Amphipoda, Caprellidea).

The Caprellidea generally possess rudimentary abdomens and degenerated third and fourth pereopods. Previous molecular phylogenetic studies support the concept that their unique body plan is derived from a gammarid-like body plan from which the abdomen or third and fourth pereopods have been lost in the Caprellidea. To understand the developmental and genetic mechanisms for the morphological evolution of the Caprellidea, we observed the embryonic development of Caprella scaura. Although in the early embryonic stage limb buds appeared in all of the pereonites, we found that elongation of the limb buds did not occur in the third and fourth pereonites; instead, only oval projections (possibly primordial gills) were observed. We next examined the gene expression of Distal-less (Dll) by in situ hybridization and found that Dll was not expressed in the third and fourth pereonites. This suggests that the suppression of Dll expression is responsible for the reduction of Caprellidea pereopods.

Complicated evolution of the caprellid (Crustacea: Malacostraca: Peracarida: Amphipoda) body plan, reacquisition or multiple losses of the thoracic limbs and pleons.

The Caprellidea (Crustacea) have undergone an interesting morphological evolution from their ancestral gammarid-like form. Although most caprellid families have markedly reduced third and fourth pereopods (the walking thoracic limbs) and pleons (the posterior body parts), one family, Caprogammaridae, has developed pleon with swimming appendages (pleopods), whereas another family, Phtisicidae, possesses well-developed functional third and fourth pereopods. The unique character status of these families implies that there has been reacquisition or multiple losses of both pereopods and the pleon within the Caprellidea lineages. Although the Caprellidea are fascinating animals for the study of morphological evolution, the phylogenetic relationships among the Caprellidea are poorly understood. One obstacle to studying the evolution of the Caprellidea is the difficulty of collecting samples of caprogammarid species. In this study, we obtained live samples of a Caprogammaridae species and confirmed that its pleon and pleopods could perform similar locomotive functions and swimming movements as observed in gammarids. From the phylogenetic analyses on 18S ribosomal RNA gene sequences, we identified three distinct clades of Caprellidea. The ancestral state reconstruction based on the obtained phylogeny suggested that once lost, the third and fourth pereopods were regained in the Phtisicidae, while the pleon was regained in the Caprogammaridae, while we could not exclude the possibility of independent losses. In either case, the caprellid lineage underwent a quite complicated morphological evolution, and possibly the Caprellidea may be an exception to Dollo's law.

Evidence of cross-fertilization in a gonochoric population of the tadpole shrimp Triops numidicus (Crustacea: Branchiopoda: Notostraca).

Cross-fertilization was evident in a gonochoric population of the notostracan Triops numidicus, in which a thick, hard eggshell had been suspected of preventing the sperm from reaching the egg. Most of the eggs from copulated females hatched into larvae, but the eggs from virgin females did not develop. In the larvae, paternal DNA fragments were detected by AFLP. In histological sections, several spermatozoa were found in the shell of newly oviposited eggs in the brood pouches of copulated females, suggesting that the shell was still soft enough to be penetrated by spermatozoa. These results showed that copulation and subsequent fertilization achieved by penetration of sperm through the newly deposited eggshell were indispensable for the normal development of the eggs.

Phylogenetic significance of the structure of adult ovary and oogenesis in a primitive chilognathan diplopod, Hyleoglomeris japonica Verhoeff (Glomerida, Diplopoda).

Some histological details of the adult ovary of Hyleoglomeris japonica are described for the first time in the glomerid diplopods. The ovary is a single, long sac-like organ extending from the 4th to the 12th body segment along the median body axis, lying between the alimentary canal and the ventral nerve cord. The ovarian wall consists of a layer of thin ovarian epithelium which surrounds a wide ovarian lumen. A pair of longitudinal "germ zones," including female germ cells, runs in the lateral ovarian wall. Each germ zone consists of two types of oogenetic areas: 1) 8-12 narrow patch-shaped areas for oogonial proliferation, arranged metamerically in a row along each of the dorsal and ventral peripheries, and 2) the remaining wide area for oocyte growth. Oogonial proliferation areas include oogonia, very early previtellogenic oocytes, and young somatic interstitial cells, among the ovarian epithelial cells. The larger early previtellogenic oocytes in the oogonial proliferation areas are located nearer to the oocyte growth area, and migrate to the oocyte growth area. They are surrounded by a layer of follicle cells and are connected with the ovarian epithelium of the oocyte growth area by a portion of their follicles. They grow into the ovarian lumen, but their follicles are still connected with the oocyte growth area. Various sizes of the previtellogenic and vitellogenic oocytes in the ovarian lumen are connected with the oocyte growth area; the smaller oocytes are connected nearer to the dorsal and ventral oogonial proliferation areas, while the larger ones are connected nearer to the longitudinal middle line of the oocyte growth area. Following the completion of vitellogenesis and egg membrane formation in the largest primary oocytes, the germinal vesicles break down. Ripe oocytes are released from their follicles directly into the ovarian lumen to be transported into the oviducts. Ovarian structure and oogenesis of H. japonica are very similar to those of other chilognathan diplopods. At the same time, however, some characteristic features of the ovary of H. japonica are helpful for understanding the structure and evolution of the diplopod ovaries. Some aspects of the phylogenetic significance in the paired germ zones of H. japonica are discussed. J. Morphol 231:277-285, 1997. © 1997 Wiley-Liss, Inc.

Phylogenetic implications of structure of adult ovary and oogenesis in the penicillate diplopod, Eudigraphis nigricians (miyosi) (diplopoda: Myriapoda).

We describe some significant structures of the adult ovary in a Japanese penicillate diplopod, Eudigraphis nigricans, with respect to phylogenetic implications. The ovary is a long, saclike organ lying between the alimentary canal and the ventral nerve cord from the fourth through the ninth body segment. The ovarian wall consists of a thin ovarian epithelium and a sparse muscle covering. There are two types of oogenetic sites: a single, mound-shaped germarium sitting on the center of the ventral ovarian epithelium, and ∼ 10 pairs of patchlike vitellarial areas metamerically arranged anterior and posterior to the germarium. The germarium consists of oogonia, early previtellogenic oocytes, and some somatic interstitial cells. In contrast, the vitellarial areas are composed of more advanced oocytes, follicle cells surrounding the oocytes, and some interstitial cells, but no oogonia. A few larger previtellogenic oocytes rise up from each vitellarial area into the ovarian lumen. Each of these oocytes is still connected with its own vitellarial area by a partial extension of its follicle. Vitellogenesis takes place in these oocytes rising in the ovarian lumen. The ripe primary oocytes leave their follicles to be transported forward into the oviducts. Some phylogenetic implications of the basic characteristics in ovarian structure and oogenesis of E. nigricans are discussed. © 1994 Wiley-Liss, Inc.