Collection of environmental DNA from stemflow for monitoring arboreal biodiversity: Preliminary validation using lichens.

The forest canopy harbors a diverse array of organisms. However, monitoring their biodiversity poses challenges due to limited accessibility and the vast taxonomic diversity. To address these challenges, we present a novel method for capturing arboreal biodiversity by harnessing stemflow as a source of DNA from organisms inhabiting trees. Our method involves encircling the tree trunk with gauze, directing the stemflow along the gauze into a funnel, and collecting it in a plastic bag. We employed dual collection systems to retrieve environmental DNA (eDNA) from the stemflow: the gauze trap, designed to capture macroscopic biological fragments, and the plastic bag trap, which collected the stemflow itself. The trapped fragments and stemflow were separately filtered, and eDNA was subsequently extracted from the filter membranes. To validate our method, we focused on foliose lichens, which are easily observable on tree surfaces. We performed eDNA metabarcoding and successfully detected a majority of the observed foliose lichen species, including those not identified through visual observation alone.•We have developed a non-invasive and straightforward method for monitoring arboreal biodiversity by collecting eDNA from stemflow, which has been validated using lichens for its efficacy.•This cost-effective approach minimizes disruptions to tree ecosystems and is expected to provide an efficient means of sampling and monitoring arboreal organisms.

Quadrupedal Walking with the Skin: The Ambulatory Flaps in "Walking" Cuttlefish (Paintpot Cuttlefish, Metasepia tullbergi).

AbstractThe locomotion strategy of cephalopods is an important factor that influences their ability to exploit various oceanic environments. Particularly, Metasepia cuttlefish have a unique locomotion strategy; they prefer slow walking (ambling) on the seafloor over swimming. For this locomotion, they use their ventral arms as forelimbs and ambulatory flaps as hindlimbs. This locomotion is similar to the gait of quadruped vertebrates, where the forelimbs and hindlimbs on the left and right move alternately. The original description and some textbooks have considered these flaps to be muscular; however, this has not been proven. Here, we report the histological morphology of the ambulatory flaps of Metasepia tullbergi and their ambling locomotion. Histological observations indicated that the ambulatory flaps had a papillae structure comprising papillae musculature (dermal erector or retractor muscles) and connective tissue in the skin. Behavioral observations indicated that the ambulatory flaps changed their shape during ambling, which could explain the existence of the skin papillae. Our results suggest that ambulatory flaps are skin papillae, which can change shape by using their papillae musculature and connective tissue. This is a unique feature of Metasepia species that use the skin papillae for locomotion.

Gravity filtration of environmental DNA: A simple, fast, and power-free method.

Filtration is required during the collection of trace amounts of environmental DNA (eDNA) from water samples to achieve a concentration sufficient for downstream molecular experiments. To date, collected water samples have been filtered by humans or electric power using various instruments. We developed a simple gravity filtration system that does not need for an external force. The system comprises a plastic bag filled with a water sample (1 L), a filter cartridge, and a long plastic tube (e.g., 2 m). When hung at a height equal to the tube length, this filtration unit can enable power-free collection and concentration of eDNA at any remote location within a reasonable time (10-60 min).•A simple, rapid, power-free, practical filtration system for environmental DNA analysis is reported.•If there is a place to hang the filtration system, filtration can be performed anywhere.•The filtration speed increased when the system was hung higher.

Detailed clinical features and genotype-phenotype correlation in an OTOF-related hearing loss cohort in Japan.

Mutations in the OTOF gene are a common cause of hereditary hearing loss and the main cause of auditory neuropathy spectrum disorder (ANSD). Although it is reported that most of the patients with OTOF mutations have stable, congenital or prelingual onset severe-to-profound hearing loss, some patients show atypical clinical phenotypes, and the genotype-phenotype correlation in patients with OTOF mutations is not yet fully understood. In this study, we aimed to reveal detailed clinical characteristics of OTOF-related hearing loss patients and the genotype-phenotype correlation. Detailed clinical information was available for 64 patients in our database who were diagnosed with OTOF-related hearing loss. As reported previously, most of the patients (90.6%) showed a "typical" phenotype; prelingual and severe-to-profound hearing loss. Forty-seven patients (73.4%) underwent cochlear implantation surgery and showed successful outcomes; approximately 85-90% of the patients showed a hearing level of 20-39 dB with cochlear implant and a Categories of Auditory Performance (CAP) scale level 6 or better. Although truncating mutations and p.Arg1939Gln were clearly related to severe phenotype, almost half of the patients with one or more non-truncating mutations showed mild-to-moderate hearing loss. Notably, patients with p.His513Arg, p.Ile1573Thr and p.Glu1910Lys showed "true" auditory neuropathy-like clinical characteristics. In this study, we have clarified genotype-phenotype correlation and efficacy of cochlear implantation for OTOF-related hearing loss patients in the biggest cohort studied to date. We believe that the clinical characteristics and genotype-phenotype correlation found in this study will support preoperative counseling and appropriate intervention for OTOF-related hearing loss patients.

After-Effects of Intermittent Theta-Burst Stimulation Are Differentially and Phase-Dependently Suppressed by α- and ß-Frequency Transcranial Alternating Current Stimulation.

Intermittent theta-burst stimulation (iTBS) using transcranial magnetic stimulation (TMS) is known to produce excitatory after-effects over the primary motor cortex (M1). Recently, transcranial alternating current stimulation (tACS) at 10 Hz (α) and 20 Hz (ß) have been shown to modulate M1 excitability in a phase-dependent manner. Therefore, we hypothesized that tACS would modulate the after-effects of iTBS depending on the stimulation frequency and phase. To test our hypothesis, we examined the effects of α- and ß-tACS on iTBS using motor evoked potentials (MEPs). Eighteen and thirteen healthy participants were recruited for α and ß tACS conditions, respectively. tACS electrodes were attached over the left M1 and Pz. iTBS over left M1 was performed concurrently with tACS. The first pulse of the triple-pulse burst of iTBS was controlled to match the peak (90°) or trough (270°) phase of the tACS. A sham tACS condition was used as a control in which iTBS was administered without tACS. Thus, each participant was tested in three conditions: the peak and trough of the tACS phases and sham tACS. As a result, MEPs were enhanced after iTBS without tACS (sham condition), as observed in previous studies. α-tACS suppressed iTBS effects at the peak phase but not at the trough phase, while ß-tACS suppressed the effects at both phases. Thus, although both types of tACS inhibited the facilitatory effects of iTBS, only α-tACS did so in a phase-dependent manner. Phase-dependent inhibition by α-tACS is analogous to our previous finding in which α-tACS inhibited MEPs online at the peak condition. Conversely, ß-tACS reduced the effects of iTBS irrespective of its phase. The coupling of brain oscillations and tACS rhythms is considered important in the generation of spike-timing-dependent plasticity. Additionally, the coupling of θ and γ oscillations is assumed to be important for iTBS induction through long-term potentiation (LTP). Therefore, excessive coupling between ß oscillations induced by tACS and γ or θ oscillations induced by iTBS might disturb the coupling of θ and γ oscillations during iTBS. To conclude, the action of iTBS is differentially modulated by neuronal oscillations depending on whether α- or ß-tACS is applied.

Structural Changes and Mechanical Resistance of Claws and Denticles in Coconut Crabs of Different Sizes.

The exoskeleton of the pinching side of claws with denticles and of the outer side without them on the coconut crab, Birgus latro, which is a rare organism, were studied using a materials science approach. The mechanical resistance of three claws of different sizes was investigated along the exoskeleton thickness from the outer surface to the inner surface, and the results were compared, including the contribution of the microstructure and chemical compositions. Mechanical properties, hardness (H) and stiffness (Er), were probed through nanoindentation tests. The results showed the H, Er, microstructures, and chemical components of the exocuticle and endocuticle layers were almost the same, in a BW range of 300 g to 1650 g. At the same time, the H and Er near the surface of the denticles of a small coconut crab of 300 g were lower than those of other large coconut crabs. The microstructure of the denticles was clearly different from that of the exocuticle, but the maximum mechanical properties near their surface indicated almost the same values, Hmax = 4 GPa and Er(max) = 70 GPa, regardless of being on the pinching side or the outer side. A denticle can be regarded as a bulge of the cuticle without phosphorus and with high magnesium. The results provided novel information that expanded our knowledge about the claw microstructure of coconut crabs with different body sizes, and may be used in further studies.

First observation of larval oarfish, Regalecus russelii, from fertilized eggs through hatching, following artificial insemination in captivity.

BACKGROUND: Little is known about the life history of oarfish of the genus Regalecus, although it is a famous deep-sea fish and an apparent origin of sea serpent legends. We successfully performed artificial insemination using a recently dead pair of sexually mature individuals. We report for the first time development from fertilized eggs to early larvae in the Lampridiformes. RESULTS: Eggs required 18 days of development from fertilization to hatching under 20.5-22.5 °C conditions. Oarfish larvae had similar morphological features as other lampridiform larvae hatched in the ocean. Larvae typically faced downward and swam using pectoral fins; they frequently opened their mouths. This mouth-opening behavior and swimming ability were both consistent with osteological development. The larvae did not eat and died four days after hatching. CONCLUSIONS: This is the first successful instance of artificial insemination and hatching in the oarfish, as well as the first reliable morphological and behavioral description of lampridiform larvae.

Clinical Characteristics and In Vitro Analysis of MYO6 Variants Causing Late-Onset Progressive Hearing Loss.

MYO6 is known as a genetic cause of autosomal dominant and autosomal recessive inherited hearing loss. In this study, to clarify the frequency and clinical characteristics of hearing loss caused by MYO6 gene mutations, a large-scale genetic analysis of Japanese patients with hearing loss was performed. By means of massively parallel DNA sequencing (MPS) using next-generation sequencing for 8074 Japanese families, we found 27 MYO6 variants in 33 families, 22 of which are novel. In total, 2.40% of autosomal dominant sensorineural hearing loss (ADSNHL) in families in this study (32 out of 1336) was found to be caused by MYO6 mutations. The present study clarified that most cases showed juvenile-onset progressive hearing loss and their hearing deteriorated markedly after 40 years of age. The estimated hearing deterioration was found to be 0.57 dB per year; when restricted to change after 40 years of age, the deterioration speed was accelerated to 1.07 dB per year. To obtain supportive evidence for pathogenicity, variants identified in the patients were introduced to MYO6 cDNA by site-directed mutagenesis and overexpressed in epithelial cells. They were then assessed for their effects on espin1-induced microvilli formation. Cells with wildtype myosin 6 and espin1 co-expressed created long microvilli, while co-expression with mutant constructs resulted in severely shortened microvilli. In conclusion, the present data clearly showed that MYO6 is one of the genes to keep in mind with regard to ADSNHL, and the molecular characteristics of the identified gene variants suggest that a possible pathology seems to result from malformed stereocilia of the cochlear hair cells.

Sound production in the coconut crab, the largest terrestrial crustacean.

Sound production in terrestrial crustaceans, including the coconut crab, Birgus latro, is not fully understood. Here, we present the first description of the acoustic features and sound production mechanisms of coconut crabs. The sound production system was determined based on X-ray videography and anatomical observations. The results indicated that the crabs produced a tapping sound by beating the scaphognathite, which is also used for ventilation, in the efferent branchial channel. The frequencies of the produced sounds were diverse, and the sound interval also varied within the same individual. From observations under captivity, differences in the sounds were confirmed at each mating phase. Although the relationship between the sounds and actions was not clarified in this study, it is probable that the crabs deliberately produce various types of sounds for different occasions. The coconut crab is known to use visual and chemical communication mechanisms, but these results suggest that a diverse set of sounds is an additional communication pathway during agonistic and mating interactions.

Mutational Spectrum and Clinical Features of Patients with LOXHD1 Variants Identified in an 8074 Hearing Loss Patient Cohort.

Variants of the LOXHD1 gene, which are expressed in hair cells of the cochlea and vestibule, have been reported to cause a progressive form of autosomal recessive non-syndromic hereditary hearing loss, DFNB77. In this study, genetic screening was conducted on 8074 Japanese hearing loss patients utilizing massively parallel DNA sequencing to identify individuals with LOXHD1 variants and to assess their phenotypes. A total of 28 affected individuals and 21 LOXHD1 variants were identified, among which 13 were novel variants. A recurrent variant c.4212 + 1G > A, only reported in Japanese patients, was detected in 18 individuals. Haplotype analysis implied that this variation occurred in a mutational hot spot, and that multiple ancestors of Japanese population had this variation. Patients with LOXHD1 variations mostly showed early onset hearing loss and presented different progression rates. We speculated that the varying severities and progression rates of hearing loss are the result of environmental and/or other genetic factors. No accompanying symptoms, including vestibular dysfunction, with hearing loss were detected in this study. Few studies have reported the clinical features of LOXHD1-gene associated hearing loss, and this study is by far the largest study focused on the evaluation of this gene.

Evaluation of detection probabilities at the water-filtering and initial PCR steps in environmental DNA metabarcoding using a multispecies site occupancy model.

Environmental DNA (eDNA) metabarcoding is a recently developed method to assess biodiversity based on a high-throughput parallel DNA sequencing applied to DNA present in the ecosystem. Although eDNA metabarcoding enables a rapid assessment of biodiversity, it is prone to species detection errors that may occur at sequential steps in field sampling, laboratory experiments, and bioinformatics. In this study, we illustrate how the error rates in the eDNA metabarcoding-based species detection can be accounted for by applying the multispecies occupancy modelling framework. We report a case study with the eDNA sample from an aquarium tank in which the detection probabilities of species in the two major steps of eDNA metabarcoding, filtration and PCR, across a range of PCR annealing temperatures, were examined. We also show that the results can be used to examine the efficiency of species detection under a given experimental design and setting, in terms of the efficiency of species detection, highlighting the usefulness of the multispecies site occupancy modelling framework to study the optimum conditions for molecular experiments.

Development of the Lunate-Shaped Caudal Fin in White Shark Embryos.

The lunate-shaped caudal fin in lamnid sharks is a morphological specialization for their thunniform mode of locomotion, but its developmental process during gestation has been poorly investigated. Observations of 21 embryonic specimens of the white shark (Carcharodon carcharias) revealed that their caudal fin morphology drastically changes from strongly heterocercal to lunate-shaped through ontogeny. This morphological change involves (1) rapid elongation of the ventral lobe, (2) increased upward curvature of the vertebra within the caudal fin, and (3) formation of keels at both lateral sides of the caudal fin base. These morphological changes are probably shared among the members of the family Lamnidae and are in contrast with the developmental process of the heterocercal tail in the lamniform Carcharias taurus, in which the caudal fin morphology is almost unchanged through the late gestation period. Anat Rec, 301:1068-1073, 2018. © 2018 Wiley Periodicals, Inc.

Dental ontogeny of a white shark embryo.

Unlike most viviparous vertebrates, lamniform sharks develop functional teeth during early gestation. This feature is considered to be related to their unique reproductive mode where the embryo grows to a large size via feeding on nutritive eggs in utero. However, the developmental process of embryonic teeth is largely uninvestigated. We conducted X-ray microcomputed tomography to observe the dentitions of early-, mid-, and full-term embryos of the white shark Carcharodon carcharias (Lamniformes, Lamnidae). These data reveal the ontogenetic change of embryonic dentition of the species for the first time. Dentition of the early-term embryos (∼45 cm precaudal length, PCL) is distinguished from adult dentition by 1) the presence of microscopic teeth in the distalmost region of the paratoquadrate, 2) a fang-like crown morphology, and 3) a lack of basal concavity of the tooth root. The "intermediate tooth" of early-term embryos is almost the same size as the adjacent teeth, suggesting that lamnoid-type heterodonty (lamnoid tooth pattern) has not yet been established. We also discovered that mid-term embryos (∼80 cm PCL) lack functional dentition. Previous studies have shown that the maternal supply of nutritive eggs in lamnoid sharks ceases during mid- to late-gestation. Thus, discontinuation of functional tooth development is likely associated with the completion of the oophagous (egg-eating) phase. Replacement teeth in mid-term embryos include both embryonic and adult-type teeth, suggesting that the embryo to adult transition in dental morphology occurs during this period. J. Morphol. 278:215-227, 2017. © 2016 Wiley Periodicals,Inc.

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes ≤4 L or >4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

A Mighty Claw: Pinching Force of the Coconut Crab, the Largest Terrestrial Crustacean.

Crustaceans can exert a greater force using their claws than many animals can with other appendages. Furthermore, in decapods, the chela is a notable organ with multifunctional roles. The coconut crab, Birgus latro, is the largest terrestrial crustacean and has a remarkable ability to lift weights up to approximately 30 kg. However, the pinching force of this crab's chelae has not been previously investigated. In the present study, we measured the pinching force of the chelae in 29 wild coconut crabs (33-2,120 g in body weight). The maximum force ranged from 29.4 to 1,765.2 N, and showed a strong positive correlation with body mass. Based on the correlation between pinching force and body weight, the force potentially exerted by the largest crab (4 kg weight) reported in a previous study would be 3300 N, which greatly exceeds the pinching force of other crustaceans as well as the bite force of most terrestrial predators. The mighty claw is a terrestrial adaptation that is not only a weapon, which can be used to prevent predator attack and inhibit competitors, but is also a tool to hunt other terrestrial organisms with rigid exteriors, aiding in these organisms to be omnivores.

Eye retraction in the giant guitarfish, Rhynchobatus djiddensis (Elasmobranchii: Batoidea): a novel mechanism for eye protection in batoid fishes.

Eye retraction behavior has evolved independently in some vertebrate linages such as mudskippers (fish), frogs and salamanders (amphibians), and cetaceans (mammals). In this paper, we report the eye retraction behavior of the giant guitarfish (Rhynchobatus djiddensis) for the first time, and discuss its mechanism and function. The eye retraction distance was nearly the same as the diameter of the eyeball itself, indicating that eye retraction in the giant guitarfish is probably one of the largest among vertebrates. Eye retraction is achieved by unique arrangement of the eye muscle: one of the anterior eye muscles (the obliquus inferior) is directed ventrally from the eyeball and attaches to the ventral surface of the neurocranium. Due to such muscle arrangement, the obliquus inferior can pull the eyeball ventrally. This mechanism was also confirmed by electrical stimulation of the obliquus inferior. The eye retraction ability of the giant guitarfish likely represents a novel eye protection behavior of elasmobranch fishes.

Northernmost record of a poorly known tuskfish, Choerodon margaritiferus (Perciformes: Labridae), from southern Japan, and first description of a female.

We describe 5 specimens of Choerodon margaritiferus Fowler and Bean, 1928 (94.5-107.0 mm standard length, 1 male and 4 females) collected near Okinawa Island, southern Japan. This is the northernmost record of this species and the first record of it in Japanese waters; until now, C. margaritiferus was recorded only from the western central Pacific. This species was originally described on the basis of 1 male specimen, and all additional specimens described in the literature were also male. Morphological characters of both sexes are described, and new diagnostic characters of the female are presented.

Growth of the Coconut Crab, Birgus latro, at Its Northernmost Range Estimated from Mark-Recapture Using Individual Identification Based on Carapace Grooving Patterns.

Although populations of the coconut crab, Birgus latro, have declined in the tropical Indo-Pacific region, insufficient knowledge exists for the management of this species. We investigated the growth of the northernmost coconut crab population, located at Ocean Expo Park, Okinawa, southern Japan, using a mark-recapture method based on the identification of individual carapace grooving patterns. Of the 485 crabs photographed (264 males, 221 females; 14.3-68.8 mm thoracic length [ThL]), 64 males and 62 females were recaptured (recapture rate 26%). The liberty period ranged from two to 2384 days. The annual data indicated that most crabs molted during winter, except for juveniles and crabs near the maximum size. Using the GROTAG program, the asymptotic ThL (L∞) was estimated as 80.72 and 49.89 mm for males and females, respectively. The Brody growth coefficient (K) was 0.063 for both sexes. The growth curves from these parameters showed that males grew larger than females because of a difference in growth speed. Longevity was estimated at approximately 50 years for both sexes. The growth data obtained in the present study, which are the most precise gathered for the coconut crab to date, can be compared with the results of studies performed in other regions.

Sense transgene-induced post-transcriptional gene silencing in tobacco compromises the splicing of endogenous counterpart genes.

Sense transgene-induced post-transcriptional gene silencing (S-PTGS) is thought to be a type of RNA silencing in which ARGONAUTE1 directs the small interfering RNA (siRNA)-mediated cleavage of a target mRNA in the cytoplasm. Here, we report that the altered splicing of endogenous counterpart genes is a main cause for the reduction of their mature mRNA levels. After the S-PTGS of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) gene, 3'-truncated, polyadenylated endo-NtFAD3 transcripts and 5'-truncated, intron-containing endo-NtFAD3 transcripts were detected in the total RNA fraction. Although transcription proceeded until the last exon of the endogenous NtFAD3 gene, intron-containing NtFAD3 transcripts accumulated in the nucleus of the S-PTGS plants. Several intron-containing NtFAD3 transcripts harboring most of the exon sequences were generated when an endogenous silencing suppressor gene, rgs-CaM, was overexpressed in the S-PTGS plants. These intron-containing NtFAD3 splice variants were generated in the presence of NtFAD3 siRNAs that are homologous to the nucleotide sequences of these splice variants. The results of this study indicate that the inhibition of endo-NtFAD3 gene expression is primarily directed via the alteration of splicing and not by cytoplasmic slicer activity. Our results suggest that the transgene and intron-containing endogenous counterpart genes are differentially suppressed in S-PTGS plants.