A New Species of Branchellion (Hirudinea: Piscicolidae) Parasitizing the Gills of Short-Tail Stingrays (Batoidea: Dasyatidae) from the West Pacific.

A new fish leech, Branchellion brevicaudatae sp. n., is described based on specimens parasitizing the gills of the short-tail stingray, Bathytoshia brevicaudata (Hutton, 1875), collected from Japanese waters. The new species can be distinguished from other congeners by having: i) pulsating vesicles emerging from posterior base of branchiae, one pair per somite; ii) dorsal white spots, not arranged in longitudinal row; and iii) blackish body. A phylogenetic tree based on partial sequences of the mitochondrial cytochrome c oxidase subunit I gene from the new species and other piscicolid worms showed that the new species is sister to Branchellion torpedinis Savigny, 1822. This is the first record of Branchellion Savigny, 1822 from Japanese waters.

Ontogenetic Expressions of Sexually Dimorphic Traits in the Skeleton Shrimp Caprella scaura (Crustacea: Amphipoda).

In sexual dimorphism, males often exhibit exaggerated characters as weapons or ornaments. Among the numerous amphipod species (Amphipoda, Crustacea) showing sexual dimorphism, caprellids (Caprellidae) are characterized by considerably larger males that possess weapons, although the developmental processes underlying these sex-related differences are largely unknown. Therefore, to clarify the process of sexual differentiation during postembryonic development in caprellids, morphometric analyses of Caprella scaura were conducted. Principal component analysis using 31 morphometric traits showed drastic allometric changes occurring at two ontogenetic body length (BL) points (i.e., 3.8 and 8.8 mm). In individuals larger than 3 mm, head spines appeared in both sexes, and penises did only in males, allowing the discrimination of juveniles from larvae. Moreover, in larger males (BL > 8.8 mm), traits used in reproductive behavior, i.e., the first antenna, second gnathopod, and first to fifth pereonites, were extremely exaggerated. Observations of pre-copulatory behavior along with morphological assays revealed that sexually mature males could be identified by the size ratio between the triangular projection and palmar spine on the propodus of the second gnathopod. In contrast, female maturation could be determined by the marginal setae of oostegites forming a brood pouch. The body size distribution of sexually mature females was concentrated within a narrow range of BLs (6-9 mm), whereas that of sexually mature males showed a broader range (BL 9-18 mm), suggesting that, in C. scaura, males continue to molt and grow even after sexual maturation via indeterminate growth, to increase their lifetime reproductive success.

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.

Reduction of a nymphal instar in a dampwood termite: heterochronic shift in the caste differentiation pathways.

BACKGROUND: Generally in termites, alates differentiate through multiple nymphal instars which gradually develop wing buds. However, in a dampwood termite, Hodotermopsis sjostedti, alates molt directly from a single nymphal instar with short wing buds. In this study, to examine the mechanism underlying the wing formation during the alate differentiation in H. sjostedti, histological and morphological observations were carried out on the developmental process of wing formation during the nymphal instar, in comparison with those in Zootermopsis nevadensis, which has two nymphal instars. Furthermore, the expression patterns of genes that are thought to be responsible for wing formation, i.e., wing-patterning genes and genes encoding hormone-related factors, were quantified during alate differentiation and compared between the two species. RESULTS: The results showed that, in H. sjostedti, wings were formed in a complicatedly folded shape, not only inside the wing buds as seen in Z. nevadensis, but also under the dorsal thoracic cuticle, where the wing tips shifted toward the median thoracic part. Accordingly, the wing expansion pattern also differed from that in Z. nevadensis. Furthermore, the results of real-time qRT-PCR on overall expression profiles of wing-patterning genes and hormone-related genes suggest that the single nymphal instar in H. sjostedti well resembles to the second nymphal instar in Z. nevadensis. In particular, significant upregulation of vestigial (vg) and downregulation of Krüppel homolog 1 (Kr-h1) that were observed at the second nymphal instar in Z. nevadensis apparently occurred during the single nymphal instar in H. sjostedti. CONCLUSION: The developmental events for wing formation are compacted into a single nymphal instar in H. sjostedti, and as a result, the unique wing formation is seen to compensate for the spatial restriction inside small wing buds, leading to the completion of functional wings.

Development of Cerata in the Cladobranchian Sea Slug Pteraeolidia semperi (Mollusca: Gastropoda: Nudibranchia).

Cladobranchian sea slugs are characterized by a number of dorsal projections, called "cerata," which are presumably involved in such biological functions as kleptocnidal defense, gas exchange, and symbiotic photosynthesis. Here, we investigated the developmental pattern of ceras formation in a cladobranchian, Pteraeolidia semperi, using field-collected individuals at various postembryonic developmental stages. As the body length increased, the total number of cerata increased in a logistic manner, up to 280 per individual. On the dorsal side of the body, the cerata exhibited a conspicuous formation of repeated, laterally-paired clusters, or rows, along the antero-posterior axis of the animals. As the body length increased, the number of ceras rows increased in a logistic manner, reaching a plateau at around 15 rows per individual. Two types of ceras clusters were observed: well-developed ceras clusters forming a glove-like structure with a basal bulge, which tended to be found in larger animals and at the anterior body region, and less-developed ceras clusters without the bulge, which tended to be found in smaller animals and at the posterior body region. Statistical and simulation analyses suggested that bulge formation underlies increased ceras number, even after the plateaued formation of new ceras rows. These results indicate that, in the postembryonic development of P. semperi, the increase of dorsal cerata entails the following processes: (i) increase of the number of ceras rows, (ii) formation of the basal bulge in each ceras cluster, and (iii) increase of the number of cerata per ceras cluster.

The activin signaling transcription factor Smox is an essential regulator of appendage size during regeneration after autotomy in the crayfish.

Members of the phylum Arthropoda, comprising over 80% of total animal species, have evolved regenerative abilities, but little is known about the molecular mechanisms mediating this process. Transforming growth factor ß (TGF-ß) signaling mediates a diverse set of essential processes in animals and is a good candidate pathway for regulation of regeneration in arthropods. In this study we investigated the role of activin signaling, a TGF-ß superfamily pathway, in limb regeneration in the crayfish. We identified and cloned a downstream transcription factor in the activin pathway, Smox, and characterized its function with regard to other elements of the activin signaling pathway. Gene knockdown of Smox by RNAi induced regeneration of complete but smaller pereopods after autotomy. This indicates that activin signaling via Smox functions in regulation of pereopod growth and size. The expression levels of both Smox and the activin receptor babo were closely correlated with molting. The expression level of Smox increased when babo was knocked down by RNAi, indicating that Smox and babo transcription are linked. Our study suggests that the Babo-Smox system in activin signaling is conserved in decapods, and supports an evolutionary conservation of this aspect of molecular signaling during regeneration between protostomes and deuterostomes.

Dynamics of vitellogenin and vitellogenesis-inhibiting hormone levels in adult and subadult whiteleg shrimp, Litopenaeus vannamei: relation to molting and eyestalk ablation.

Levels of vitellogenin (VG) and vitellogenesis-inhibiting hormone (VIH) in the whiteleg shrimp, Litopenaeus vannamei, were measured by time-resolved fluoroimmunoassay in relation to the molting cycle and ovarian maturation induced by eyestalk ablation. During the molt cycle, VG mRNA expression levels and VG concentrations showed similar patterns of fluctuation. VG levels increased significantly at early intermolt (stage C0) in adults, but not in subadults. Unilateral and bilateral eyestalk ablation increased VG levels in adults, whereas only bilateral eyestalk ablation affected subadults. VIH levels showed contrasting patterns between adults and subadults. In adults, levels were high in late postmolt adults (stage B) and then low thereafter, whereas they increased from postmolt (stage A) to intermolt (stage C0) in subadults and remained high. Unilateral eyestalk ablation increased VIH levels 10 days following ablation in adults, after which levels decreased at 20 days. VIH levels decreased from 10 to 20 days after bilateral ablation. Both unilateral and bilateral ablation led to increased VIH levels in subadults. Eyestalk ablation induced ovarian maturation, but did not reduce VIH concentrations in the hemolymph. This phenomenon was perhaps due to other crustacean hyperglycemic hormone peptides having cross-reactivity with VIH antibodies. This is the first report to quantify concentrations of VG and VIH together in L. vannamei hemolymph, and to examine their relative dynamics.

Molecular cloning of prophenoloxidase and the effects of dietary ß-glucan and rutin on immune response in hemocytes of the fleshy shrimp, Fenneropenaeus chinensis.

To investigate the effects of dietary ß-glucan (0.5 or 1 g kg⁻¹ diet: 0.5-BG, 1-BG) and rutin (0.5 or 1 g kg⁻¹ diet: 0.5-RT, 1-RT) after 10 days in the absence of pathogen challenge on the immune response of Fenneropenaeus chinensis, we determined total hemocyte count (THC) and the expression of four immune-related genes in hemocytes: those for prophenoloxidase (proPO), peroxinectin (PX), lipopolysaccharide and/or ß-glucan binding protein (LGBP), and c-type lectin (CL). As a prerequisite for subsequent experiments, cDNA encoding proPO of the fleshy shrimp, Fenneropenaeus chinensis (f-proPO) was obtained from hemocytes; it had a full length of 3023 bp, with an open reading frame (ORF) of 2061bp, a 105-bp 5'-untranslated region, and a 906-bp 3'-untranslated region containing the poly A signal. The THCs of shrimp fed ß-glucan of 1 g kg⁻¹ diet, and rutin of 1 g kg⁻¹ diet were significantly higher than that of the control (P < 0.05). The expression of proPO mRNA was slightly downregulated and that of LGBP mRNA was upregulated (except in 1-RT). PX and CL mRNA remained constitutively expressed in all groups. Our results reveal that ß-glucan and rutin dietary supplements have minimal effect on immune response in the absence of pathogen challenge.

Metabolism of amino acids during hyposmotic adaptation in the whiteleg shrimp, Litopenaeus vannamei.

The penaeid prawn, Litopenaeus vannamei, was employed to investigate intracellular isosmotic regulation in situations where invertebrates encounter hyposmosis. Hemolymph osmolality was first analyzed to confirm osmoregulatory conditions in the experimental animals, followed by analysis of amino acids in muscle and hemolymph using high-performance liquid chromatography. Total muscle amino acid levels decreased when hemolymph osmolality was extremely low, whereas glycine and L-serine levels increased in the hemolymph. These results suggest that tissue amino acids were released into the hemolymph to lower the osmolality of the tissues for purposes of low-salinity adaptation. Next, oxygen consumption and ammonia excretion rates were examined, and the O/N ratio was determined. Oxygen consumption levels and ammonia excretion rates increased, and the O/N ratio decreased when the animals were exposed to low salinity. These results suggest that amino acids were abundantly consumed as an energy source when animals were exposed to low salinity. To confirm the consumption of particular amino acids, the specific activity of L-serine ammonia lyase was also examined. Specific activity was highest when L-serine levels in the hemolymph were highest. Thus, it appears that L-serine levels increased under hyposmotic conditions due to the consumption of L-serine as an energy source. It was concluded that particular amino acids as osmolytes are likely metabolized as energy sources and consumed for purposes of hyposmotic adaptation.