Wickerhamiella bidentis sp. nov., a novel yeast species isolated from flowers and insects in Japan.

Two strains were isolated from flowers and insects in Japan, namely NBRC 115686T and NBRC 115687, respectively. Based on sequence analysis of the D1/D2 domain of the 26S large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region and physiological characteristics, these strains were found to represent a novel yeast species of the genus Wickerhamiella. Considering pairwise sequence similarity, NBRC 115686T and NBRC 115687 differ from the type strain of the most closely related species, Wickerhamiella galacta NRRL Y-17645T, by 65-66 nucleotide substitutions with 12 gaps (11.65-11.83 %) in the D1/D2 domain of the LSU rRNA gene. The novel species differ from the closely related Wickerhamiella species in some physiological characteristics. For example, compared with Wickerhamiella galacta JCM 8257T, NBRC 115686T and NBRC 115687 assimilated d-galactose, and could grow at 35 and 37 °C. Hence, the name Wickerhamiella bidentis sp. nov. is proposed to accommodate this species in the genus Wickerhamiella. The holotype is NBRC 115686T (ex-type strain JCM 35540=CBS 18008).

Transcriptome Analysis in a Scleractinian Coral, Acropora tenuis, during the Spawning Season With Reference to the Gonadal Condition.

Synchronous spawning is a striking feature of coral. Although it is important for reproductive success, corals reallocate energy for reproduction to growth when they are damaged by external stimuli. To assess the transcriptome before and after spawning in the scleractinian coral Acropora tenuis, we tagged three colonies (one bleached and two unbleached) in the field around Sesoko Island (Okinawa, Japan) in November 2016, sampled them monthly from May to July 2017, and performed RNA sequencing (RNA-Seq) analysis. Histological analysis revealed that the previously bleached colony possessed gametes in June, by which time the other two colonies had already spawned. In RNA-Seq analyses, multi-dimensional scaling based on gene expression similarity among the samples reflected the differences between colonies and between months except for the sample of a non-spawned colony in May, which was similar to the samples in June. The similarity of the non-spawned colony sample in May to the samples in June was also shown in hierarchical clustering based on the expression patterns of the genes that were differentially expressed between months in the spawned colonies. These results suggest that non-spawning was already decided in May, and that the physiological condition in a non-spawned colony in May was advanced to June. RNA-Seq analysis also showed that genes related to gametogenesis and those related to apoptosis were upregulated before and after spawning, respectively.

Ovarian Development and Larval Release of the Hermit Crab Clibanarius eurysternus (Hilgendorf, 1879) (Decapoda: Anomura: Diogenidae) in Okinawa, Japan.

This study investigated ovarian development and larval release in the hermit crab Clibanarius eurysternus, which inhabits the upper intertidal zone on Okinawa, Japan. Adult females were collected monthly and had a high gonadosomatic index (GSI) from May to October. Oocytes at vitellogenic stages were observed from May to October. Some females had post-ovulatory follicles in the ovary and embryos on the abdomen (ovigerous females) in June and August, suggesting active ovarian development and larval release during these months (breeding season). When adult females were collected weekly according to the lunar cycle, a high GSI with oocytes at the tertiary yolk globule stage in the ovaries was maintained in all lunar phases. Since post-ovulated follicles were seen in ovaries collected around the last quarter and new moons, darkness at night is needed for spawning. Ovigerous females exposed to stepwise changes in hydrostatic pressure exhibited significantly higher synchrony between larval release and high pressure at night from the last quarter moon to the new moon, suggesting the importance of the tidal amplitude cycle in larval release under non-moonlight conditions. We concluded that the continuity of synchronous spawning and larval release of C. eurysternus are repeated under darkness and high tide at night during the breeding season.

Does estrogen regulate vitellogenin synthesis in corals?

Most broadcast spawner corals have a vitellogenic phase that lasts at least 6 months. It is established that estrogen regulates vitellogenin synthesis in vertebrates. Although some research have been conducted on the physiological role of sex steroids in corals, little is known about their involvement in oocyte development. This study aimed to detect steroid hormones - progesterone, testosterone, and estradiol-17ß (E2) - in Acropora tenuis and study the relationships between vitellogenesis/vitellogenin synthesis and these steroids. This study also investigated the effect of E2 on vitellogenin synthesis in corals and identified steroidogenic enzymes in A. tenuis genome. Branches from tagged coral colonies were collected monthly from March to November. Histological observations showed that oocytes were vitellogenic from March to May (Stage IV and V), but not in June, and that gonads were occupied by immature oocytes in September (Stage I). Real-time qPCR revealed that vitellogenin (vg1 and vg2) transcript levels in coral branches were high in April and May, implying that corals actively underwent vitellogenesis during these months, and spawned before June. Liquid chromatography-mass spectrometry revealed that E2 could be detected in coral branches in March, April, and May, but not in June, whereas testosterone and progesterone did not fluctuate much in the same months. Immersing branches in E2-containing seawater failed to increase vitellogenin transcript levels. The results indicate that E2 is involved in oogenesis but does not positively regulate vitellogenin synthesis. Steroidogenic enzymes (except CYP19A) were identified in A. tenuis, suggesting that corals may endogenously synthesize progestogens and androgens from cholesterol.

Verification of differentially expressed genes in relation to hydrostatic pressure in the brain of two wrasse species with high-tide preference in spawning.

Fish that inhabit shallow water are exposed to periodic changes in tidal cues, including hydrostatic pressure (HP). The present study aimed at verifying differentially expressed genes (DEGs) in the brain of the threespot wrasse Halichoeres trimaculatus (tropical species) and the honbera wrasse Halichoeres tenuispinis (temperate species), both of which were exposed to HP at 30 kPa (possible high-tide stimuli in the field) or 1 kPa (low tide) for 3 or 6 h. A de novo assembly yielded 174,710 contigs (63,530 contigs were annotated) from the brain of threespot wrasse. Following RNA sequencing, quantitative PCR confirmed DEGs that were upregulated [AT atypical cadherin 2 (FAT2)] and downregulated [neuronal leucine-rich repeat protein 3 (LRRN3), dual specificity tyrosine phosphorylation-regulated kinase 1 (DYRK), mitogen-activated protein kinase kinase 1 (MAP2K1) and phosphoinositide 3 kinase (PI3K)]. The effect of HP on the transcription of these DEGs (except for MAP2K1) disappeared within 6 h, suggesting that HP is a transitory stimulus occurring at the beginning of the tidal cycle. Similar DEG transcription was observed in the brain of honbera wrasse maintained under HP for 6 h. In situ hybridization of the brain of the threespot wrasse revealed that strong signals of MPA2K1 were seen in the telencephalon, diencephalon and pituitary, whereas those of PI3K were seen in the telencephalon, diencephalon and medulla oblongata. This result suggests that these kinases are involved in sensory function (telencephalon), somatic and visceral function (medullar oblongata) and the neuroendocrine system (diencephalon and pituitary), all of which were related to changes in HP stimuli. Following HP exposure, the transcription of c-fos increased in the pituitary of honbera wrasse, suggesting that external stimuli directly or indirectly activate hormone synthesis at the hypothalamic-pituitary-gonadal axis. It is concluded that HP alters gene expression in relation to neural development and function in the central nervous system and plays a role in exerting tidal-related reproduction and feeding in wrasses.

Effects of pars intercerebralis removal on circatidal rhythm in the mangrove cricket, Apteronemobius asahinai.

The circatidal rhythm is an endogenous rhythm corresponding to the tidal cycles, and its neural mechanism remains unknown. The mangrove cricket, Apteronemobius asahinai, possesses both circatidal and circadian clocks, and simultaneously exhibits circatidal and circadian rhythms in its locomotor activity. In a previous study, we showed that surgical removal of the optic lobes, the principal circadian clock locus in crickets, disrupted their circadian rhythm, but not their circatidal rhythm. In this study, we focused on the pars intercerebralis (PI) because surgical removal of the PI disrupts the circadian rhythm and causes arrhythmic activity in some cricket species. After surgical removal of the PI, the proportion of crickets displaying circatidal rhythm decreased, and more than half of the crickets exhibited arrhythmic activity. Surgical removal of the regions around the PI also caused a similar effect on locomotor activity. Our results indicate that the PI and/or its surrounding regions are important not only for circadian but also for circatidal rhythm. This suggests the presence of a neural or hormonal pathway in the PI and/or its surrounding regions that is common to the circatidal and circadian rhythms.

Changes in mRNA abundance of insulin-like growth factors in the brain and liver of a tropical damselfish, Chrysiptera cyanea, in relation to seasonal and food-manipulated reproduction.

Food availability can become a factor driving the reproductive activity of tropical fish, particularly when primary production within their habitats fluctuates with tropical monsoons. The present study examined the involvement of insulin-like growth factors (IGF) in controlling the reproduction of the sapphire devil Chrysiptera cyanea, a reef-associated damselfish that is capable of manipulating its reproductive activity based on food availability. We cloned and characterized the cDNAs of igf1 and igf2 and determined their transcript levels in relation to seasonal and food-manipulated reproduction. The partial cDNAs of sapphire devil igf1 and igf2 had open reading frames (ORFs) composed of 600 bp (155 amino acid residue) and 636 bp (211 aa), respectively. Phylogenetic analyses revealed that IGF1 and IGF2 of the sapphire devil were clustered into those of teleosts. The gonadosomatic index increased from March to June. Vitellogenic oocytes and ovulatory follicles were observed in ovaries from May to June, which suggests that the spawning season lasts for at least 2 months. The hepatosomatic index, but not the condition factor, increased in March and June. The transcript levels of igf1 in the brain, but not in the liver, increased in April, June (vitellogenesis) and July (post vitellogenesis). Ovarian activity during the spawning season was maintained by high food supply (HH) for 30 days, although it was suppressed in the food-restriction treatment (LL) and restored in the re-feeding treatment (LH). The transcript levels of igfs in the brain, but not in the liver, in LH were lower than those in HH and LL. Moreover, immersing fish in seawater containing estradiol-17ß suppressed transcript levels of igfs in the liver, but not in the brain. We conclude that reproductive activity during the spawning season is influenced by nutritive conditions and that crosstalk exists between the reproductive and growth network in the neural and peripheral tissues, thus controlling the reproductive activity of this species.

RNAi of the circadian clock gene period disrupts the circadian rhythm but not the circatidal rhythm in the mangrove cricket.

The clock mechanism for circatidal rhythm has long been controversial, and its molecular basis is completely unknown. The mangrove cricket, Apteronemobius asahinai, shows two rhythms simultaneously in its locomotor activity: a circatidal rhythm producing active and inactive phases as well as a circadian rhythm modifying the activity intensity of circatidal active phases. The role of the clock gene period (per), one of the key components of the circadian clock in insects, was investigated in the circadian and circatidal rhythms of A. asahinai using RNAi. After injection of double-stranded RNA of per, most crickets did not show the circadian modulation of activity but the circatidal rhythm persisted without a significant difference in the period from controls. Thus, per is functionally involved in the circadian rhythm but plays no role, or a less important role, in the circatidal rhythm. We conclude that the circatidal rhythm in A. asahinai is controlled by a circatidal clock whose molecular mechanism is different from that of the circadian clock.

Distinct decision-making properties underlying the species specificity of group formation of flies.

Many animal species form groups. Group characteristics differ between species, suggesting that the decision-making of individuals for grouping varies across species. However, the actual decision-making properties that lead to interspecific differences in group characteristics remain unclear. Here, we compared the group formation processes of two Drosophilinae fly species, Colocasiomyia alocasiae and Drosophila melanogaster, which form dense and sparse groups, respectively. A high-throughput tracking system revealed that C. alocasiae flies formed groups faster than D. melanogaster flies, and the probability of C. alocasiae remaining in groups was far higher than that of D. melanogaster. C. alocasiae flies joined groups even when the group size was small, whereas D. melanogaster flies joined groups only when the group size was sufficiently large. C. alocasiae flies attenuated their walking speed when the inter-individual distance between flies became small, whereas such behavioural properties were not clearly observed in D. melanogaster. Furthermore, depriving C. alocasiae flies of visual input affected grouping behaviours, resulting in a severe reduction in group formation. These findings show that C. alocasiae decision-making regarding grouping, which greatly depends on vision, is significantly different from D. melanogaster, leading to species-specific group formation properties.

Investigation on the validity of 3D micro-CT imaging in the fish brain.

BACKGROUND: Micro-computed tomography (CT) is a non-invasive technique that is used to obtain three-dimensional (3D) images of tissue structure in small animals. Compared with extensive mammal studies, few 3D imaging studies of fish have been conducted using micro-CT. An optimized method for imaging fish tissue structure is necessary, because they have adapted to diverse environments via functional and structural specialization. NEW METHOD: Brains of three species with different sizes and habitats were fixed in 4% paraformaldehyde and immersed in non-ionic iodinated contrast agent (Iopamiron). We examined the relationship between Iopamiron concentration and immersion time to determine universally optimal conditions for use in fish. RESULTS: We reconstructed 3D images of whole fish brains from cross-sections of brains from the Malabar grouper (Epinephelus malabaricus), bastard halibut (Paralichthys olivaceus), and threespot wrasse (Halichoeres trimaculatus). Developmental changes in brain structure were observed in the bastard halibut. Most brain regions of the threespot wrasse were distinguishable, although inner regions of the brain were less visible. COMPARISON WITH EXISTING METHODS: Histological techniques are typically used to observe fish brain structure, despite its drawbacks in terms of tissue sample preparation (shrinkage and distortion) and image capture (3D image constriction). The technique examined in the present study solves these problems and allows for the simultaneous handling of multiple specimens. CONCLUSION: Micro-CT imaging is suitable for observing the surfaces and inner structures of fish of various species.

An Overview of Monthly Rhythms and Clocks.

Organisms have evolved to cope with geophysical cycles of different period lengths. In this review, we focus on the adaptations of animals to the lunar cycle, specifically, on the occurrence of biological rhythms with monthly (circalunar) or semi-monthly (circasemilunar) period lengths. Systematic experimental investigation, starting in the early twentieth century, has allowed scientists to distinguish between mythological belief and scientific facts concerning the influence of the lunar cycle on animals. These studies revealed that marine animals of various taxa exhibit circalunar or circasemilunar reproductive rhythms. Some of these rely on endogenous oscillators (circalunar or circasemilunar clocks), whereas others are directly driven by external cues, such as the changes in nocturnal illuminance. We review current insight in the molecular and cellular mechanisms involved in circalunar rhythms, focusing on recent work in corals, annelid worms, midges, and fishes. In several of these model systems, the transcript levels of some core circadian clock genes are affected by both light and endogenous circalunar oscillations. How these and other molecular changes relate to the changes in physiology or behavior over the lunar cycle remains to be determined. We further review the possible relevance of circalunar rhythms for terrestrial species, with a particular focus on mammalian reproduction. Studies on circalunar rhythms of conception or birth rates extend to humans, where the lunar cycle was suggested to also affect sleep and mental health. While these reports remain controversial, factors like the increase in "light pollution" by artificial light might contribute to discrepancies between studies. We finally discuss the existence of circalunar oscillations in mammalian physiology. We speculate that these oscillations could be the remnant of ancient circalunar oscillators that were secondarily uncoupled from a natural entrainment mechanism, but still maintained relevance for structuring the timing of reproduction or physiology. The analysis and comparison of circalunar rhythms and clocks are currently challenging due to the heterogeneity of samples concerning species diversity, environmental conditions, and chronobiological conditions. We suggest that future research will benefit from the development of standardized experimental paradigms, and common principles for recording and reporting environmental conditions, especially light spectra and intensities.

Circatidal rhythm and the veiled clockwork.

Many intertidal organisms exhibit endogenous rhythms corresponding to the tidal cycle, i.e., circatidal rhythm. However, circatidal rhythm has been poorly investigated in insects. Several hypotheses have been proposed for the clock generating circatidal rhythm. Nevertheless, the physiological mechanisms of the clocks remain unknown, with one of the long-standing questions being whether the circatidal rhythm is generated by circadian clocks, their variations, or clocks distinct from the circadian clock. In the mangrove cricket Apteronemobius asahinai, the circatidal clock operating locomotor activity rhythm differs from the circadian clock at both the molecular and neural level. Dissections of the clock generating the circatidal rhythm are expected in the near future.

The circatidal rhythm persists without the optic lobe in the mangrove cricket Apteronemobius asahinai.

Whether the circatidal rhythm is generated by a machinery common to the circadian clock is one of the important and interesting questions in chronobiology. The mangrove cricket Apteronemobius asahinai shows a circatidal rhythm generating active and inactive phases and a circadian rhythm modifying the circatidal rhythm by inhibiting activity during the subjective day simultaneously. In the previous study, RNA interference of the circadian clock gene period disrupted the circadian rhythm but not the circatidal rhythm, suggesting a difference in molecular mechanisms between the circatidal and circadian rhythms. In the present study, to compare the neural mechanisms of these 2 rhythms, we observed locomotor activity in the mangrove cricket after surgical removal of the optic lobe, which has been shown to be the locus of the circadian clock in other crickets. We also noted the pigment-dispersing factor immunoreactive neurons (PDF-IRNs) in the optic lobe, because PDF is a key output molecule in the circadian clock system in some insects. The results showed that the circadian modulation was disrupted after the removal of the optic lobes but that the circatidal rhythm was maintained with no remarkable changes in its free-running period. Even in crickets in which some PDF-immunoreactive somata remained after removal of the optic lobe, the circadian rhythm was completely disrupted. The remnants of PDF-IRNs were not correlated to the occurrence and free-running period of the circatidal rhythm. These results indicate that the principal circatidal clock is located in a region(s) different from the optic lobe, whereas the circadian clock is located in the optic lobe, as in other crickets, and PDF-IRNs are not important for circatidal rhythm. Therefore, it is suggested that the circatidal rhythm of A. asahinai is driven by a neural basis different from that driving the circadian rhythm.

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket.

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.