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Walking is a locomotion mode in which animals move over the ground using their appendages. Walking is observed in both terrestrial and aquatic animals, but the morphology and diversity of appendages in the latter group have been less extensively studied. The present paper reports on the "adhesive areas," which may represent morphological and physiological adaptations for stable aquatic walking, in the paintpot cuttlefish, Ascarosepion tullbergi. This animal employs arm IV as a forelimb and an ambulatory flap as a hindlimb for walking, resulting in a gait‐like manner of movement. The structure of the adhesive area is exclusively located on the ventral skin surface of arm IV and the ambulatory flap, which are in contact with the ground during walking. The "adhesive areas" are characterized by a dense population of adhesive mucus‐secreting cells and the development of numerous wrinkles on the surface. These features may enhance the gripping and sticking capacity of the ground‐contact area, thus improving walking stability. The use of adhesive areas for walking is a unique feature of A. tullbergi, as other cuttlefish with adhesive areas primarily use them for attaching to substrata in strong currents. Our results contribute to the understanding of the locomotion strategy of cuttlefish. [ABSTRACT FROM AUTHOR]

The coconut crab, Birgus latro, has black protrusions on the tops of its walking legs and claw fingers. In addition, there are regularly aligned small black protrusions on parts of the exoskeleton surface of the claws and leg. In this study, the elemental composition, crystal structure, tissue structure, and mechanical properties of these protrusions were studied using a materials science approach, and the results were compared with those of mineralized cuticle. These leg tips were found to be a non-calcified fibrous tissue of α-chitin connected to the mineralized cuticle. The tip of the second walking leg was elongated and had a pointed shape with an oval cavity at its center that was more than 1000 times larger than the pore tubes (100–350 nm) of the mineralized cuticle. It was very soft, with a hardness of 0.4 GPa, corresponding to 11–12% of the hardness of the hard exocuticle and 55–57% of the hardness of the soft endocuticle. The elastic modulus of 8.0 GPa obtained by means of nanoindentation testing was consistent with that of α-chitin fibers of shrimp shells obtained by means of tensile testing. These soft protrusions provide a secure grip on the surfaces of trees or rocks and protect the claw fingertips. It was concluded that the black protrusions are related to a unique ecological (engaging in vertical movements, entering and exiting limestone caves, and escape behavior) aspect of the coconut crab, the largest terrestrial crustacean. [ABSTRACT FROM AUTHOR]

The mud crab, Scylla serrata, has huge claws in comparison with its body size. The color of the claw top's finger surface changes from white to deep-mottled blue, and this discoloration was strongly associated with the change in hardness inside the finger cross section. With special attention to the discoloration points, the tissue structure of the exoskeleton was investigated via scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction (XRD), and the mechanical properties were examined using Vickers hardness and nanoindentation tests. The exocuticle in the deep blue surface exoskeleton was as thin as that in other crustaceans, and the exoskeleton was occupied by the endocuticle with a twisted plywood structure. On the other hand, in the white surface exoskeleton, the thickness of the hard and dense exocuticle accounted for 52–59% of the exoskeleton thickness. This percentage increased at the claw tip. The hardness of the exocuticle was 2.5 times that of the endocuticle, and the microstructures and mechanical properties gradually varied at the boundary between the exo- and endocuticle. The mechanical properties were almost constant in the exocuticle, but calcium (Ca) concentrations decreased from the outer surface toward the boundary in that region and magnesium (Mg) concentrations increased. The change in the unit cell volume obtained via XRD suggested that some of the Ca atoms in the calcite crystal structure in that region were replaced with Mg atoms. Changes in crustacean coloration may help us to understand the tissue structure and mechanical properties within the exoskeleton. [ABSTRACT FROM AUTHOR]

The Ryukyu-ayu population on Okinawa-jima Island went extinct suddenly in the late 1970s, but the cause remains unclear. Reports say that genetic diversity was not responsible, so we investigated food shortage as a probable driver. We examined pre-extinction specimens to look for territorial individuals, which have unique morphological characteristics and occur only in food-affluent conditions. We found that 42.1% (n = 45) of individuals possessed longer dorsal fins than non-territorial fish. Combining this with another key characteristic (blackened membrane at posterior dorsal fin ray), we found that territorial fish probably constituted 35.5% which fell in the range of the expected proportion for territorial fish in the nominotypical subspecies and higher than that observed in the extant population on Amami-oshima Island. These findings imply that the food resources in the riverine environments of Okinawa-jima Island before the extinction event might be sufficient for Ryukyu-ayu to establish feeding territories. Therefore, food shortage did not cause this population extinction. [ABSTRACT FROM AUTHOR]

The 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. [ABSTRACT FROM AUTHOR]

The present study tested the hypothesis that whether limited genetic diversity was the main factor that led the Ryukyu-ayu population on Okinawa-jima Island to extinction in the late 1970s. The usability of fluctuating asymmetry (FA) in reflecting genetic diversity was tested using two extant populations of Ryukyu-ayu on Amami-oshima Island, then used to estimate genetic diversity for the extinct population which only formalin-preserved specimens are available. The results showed that higher FA indices were associated with lower genetic diversity in the two extant populations, and the extinct population on Okinawa-jima Island had lower FA indices than the two extant populations on Amami-oshima Island. This finding suggests that the genetic diversity of the extinct population was not low enough as to cause its extinction. [ABSTRACT FROM AUTHOR]

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 (β) 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 β-tACS on iTBS using motor evoked potentials (MEPs). Eighteen and thirteen healthy participants were recruited for α and β 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 β-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, β-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 β 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 β-tACS is applied. [ABSTRACT FROM AUTHOR]

Hydrophis stokesii was collected in the coastal water of Okinawajima Island on 30 March 2021. This is the first record of the species from Japanese waters, extending its known range of occurrence toward east-northeast by more than 900 km. The snake was an adult female, 1,545 mm in snout-vent length, 203 mm in tail length, and 3,360 g in body mass. It had 11 well-developed yolked follicles in ovaries and a half-digested spiny puffer fish (Diodon sp.) in its stomach. This individual may possibly represent a stable population of H. stokesii in the Okinawan waters. [ABSTRACT FROM AUTHOR]

Gobioid larvae collected from the coast of Okinawa Island, southern Japan, were identified on the basis of a combination of morphological characters and a sequence of a hypervariable region of mitochondrial 12S ribosomal RNA (12S rRNA) gene (138–145 base pairs). A short-term formalin fixation technique enabled identification using both morphological and genetic methods. Thirteen of the 21 types of gobioid larvae assessed were identified to the species level. Additionally, we described the morphology of the postflexion larvae of wormfish Paragunnellichthys sp. and shrimp-associated goby Ctenogobiops feroculus, identified for the first time in their respective genera. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Cucumber mosaic virus (CMV) 2b protein has multiple activities as a viral suppressor of RNA silencing (VSR). Here, we characterized the 2b protein (IA2b) of a CMV-IA isolate. This IA2b protein has two nuclear localization signals, and when it was fused with GFP, the protein was found to be preferentially localized in the nucleus. A transgenic tobacco line, 2b8, expressing the IA2b gene showed an enhanced expression of an agroinfiltrated sense transgene, which indicated that IA2b has VSR activity. When 2b8 plants were crossed with plants showing sense-transgene-induced post-transcriptional gene silencing (S-PTGS), the IA2b protein caused the disappearance of small interfering RNAs (siRNAs) and the S-PTGS phenotype was converted to the overexpression phenotype. When 2b8 plants were crossed with plants showing inverted repeat-induced post-transcriptional gene silencing (IR-PTGS), the IA2b protein did not suppress the accumulation of siRNAs processed from hairpin RNAs and the silencing phenotype was maintained. When a tobacco plant showing an overexpressed phenotype of a sense transgene was crossed with IR-PTGS plants targeting the same transgene, the primary siRNAs that were produced by the IR-PTGS construct triggered the production of the secondary siRNAs through the RNA-dependent RNA polymerase6 (RDR6)-dependent pathway. The IA2b protein suppressed the secondary siRNA formation but not the primary siRNA production from hairpin RNAs, resulting in the silencing phenotype from IR-PTGS being maintained. These results indicate that IA2b suppresses RDR6-dependent siRNA production and does not interfere with the silencing step by siRNA-guided RNA degradation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Larvae and juveniles of Alectis indica reared in captivity are described based on 47 specimens (3.2-32.0 mm in body length: BL). Development was typical for the tribe Carangini except for the presence of elongated fin filaments. Elongated dorsal-fin filaments were present at preflexion (3.2 mm BL). During flexion, the anal- and pelvic-fin rays elongated and the body deepened. The full complement of fin spines and rays was present by 7.1 mm BL. The larvae of A. indica could be differentiated from those of Alectis ciliaris, which also inhabits in the Indo-Pacific waters, by the presence of a ventral series of melanophores on the tail, elongated pelvic fins, and the timing of anal-fin spine migration. The rounded body and elongated fin rays of A. indica cause it to resemble venomous Cubomedusae. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Development of eggs and larvae of the big roughy Gephyroberyx japonicus are described on the basis of specimens reared in captivity. Spherical eggs (diameter 1.26-1.35 mm) with a single oil globule were pelagic. Newly hatched larvae (2.8-3.1 mm in body length, BL) had strong linear pigmentation on the head and trunk. The mouth opened at ca. 3.5 mm BL; thereafter the yolk was absorbed. Notochord flexion started at ca. 4.5 mm BL when body depth increased rapidly, and melanophores spread to all of the body. Notochord flexion was completed at ca. 5.0 mm BL. Head spination and pelvic fins began to develop during the flexion stage. [ABSTRACT FROM AUTHOR]

Some decapod crustaceans have tooth-like white denticles that exist only on the pinching side of claws. We revealed the denticle microstructure in the coconut crab, Birgus latro, using optical and scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and a focused ion beam (FIB)-SEM. Three-dimensional analysis and fracture surface observation were performed in order to clarify the microstructural differences in two mineralized layers—the exocuticle and the endocuticle. The denticles consist of a columnar structure normal to the surface and are covered with a very thin epicuticle and an exocuticle with a twisted plywood pattern structure. Due to abrasion, the exocuticle layer was lost in the wide area above the large denticles; conversely, these layers remained on the surface of the relatively small denticles and on the base of the denticle. The results showed that the mineralized exoskeleton of the crab's claw is classified into three structures: a twisted plywood pattern structure stacked parallel to the surface for the exocuticle, a porous structure with many regularly arranged pores vertical to the surface for the endocuticle, and a columnar structure vertical to the surface for the denticle. [ABSTRACT FROM AUTHOR]

The amplification mechanism of short interfering RNAs (siRNAs) along the transgene sequence exists in RNA interference (RNAi). The RNA-dependent RNA polymerase synthesizes complementary RNAs by using the transgene mRNA as a template, and the secondary siRNAs are generated from the outside of primary RNAi target. Four independent RNAi vectors which produced primary siRNAs against distinct regions of the tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene ( NtFAD3) were transiently expressed in leaves of the NtF4D3-overexpressed transgenic plants. Regardless of the RNAi vector used, the secondary NtFAD3 siRNAs were generated preferentially from the 3’ downstream region of the transgene. Secondary siRNAs from the 5’ upstream region adjacent to the annealing site of primary siRNAs accumulated under the detection level. Our results suggest that different regulatory mechanisms are involved in the spreading of RNA silencing into 5’ upstream and 3’ downstream regions of the target sequence, respectively. [ABSTRACT FROM AUTHOR]

The spotted flagtail, Kuhlia marginata (Kuhliidae) is commonly found in streams throughout the Ryukyu Archipelago, including Okinawa Island, Japan. Although it has been suggested that they spawn at sea, little is known about their migratory history. The aim of the study was to clarify their migration history based on otolith microchemistry analysis, size composition and the gonadal development of fish collected from the Genka River on Okinawa Island. All strontium/calcium (Sr/Ca) ratios of otoliths remained high around their cores, and then dropped rapidly at some distance from the core. The estimated standard length corresponding to decreases correlated with the size of new recruits collected in the river (ca. 20 mm in standard length: SL). Multiple increases, the sign for migrations into the high saline area, were recognized intermittently around the otolith margin. As the estimated SL at these increases in the Sr/Ca ratio occurred above the minimum maturation size (female: 95.5 mm SL; male: 83.5 mm SL), such multiple increases in Sr/Ca ratios were caused by spawning migration. Therefore, we conclude that the catadromous pattern of K. marginata is as follows; this species grows in the sea during the early life stage until ca. 20 mm SL and then grow in the freshwater area until maturation, before they migrate again to the sea for spawning. Sr/Ca ratio profiles also suggested that a large number of males did not return to the freshwater area after spawning, whereas females might spawn several times during their lives around the inshore area. [ABSTRACT FROM AUTHOR]

Simple Summary: Understanding the diverse mechanisms by which organisms achieve exceptionally high mechanical properties may enable the development of unique, biologically inspired materials. We assessed the microstructure, composition, and mechanical resistance of the pinching side with denticles and of the outer side without denticles in robust claws of coconut crabs with body weight (BW) of 300 g to 1650 g. Surprisingly, they were independent of BW except for low hardness near the surface of the denticles of a small crab of 300 g. Although the microstructure of the denticles was clearly different from that of the exocuticle, their mechanical resistance indicated the same maximum value. The denticle can be regarded as a bulge of the cuticle without phosphorus. The design principles found in the exoskeleton provided promising opportunities for the research and development of novel structural materials. 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 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To estimate the spawning site of the spotted flagtail, Kuhlia marginata, sperm motility was examined under conditions of different salinities. The spermatozoa were immotile in 0 and 5 ppt test solutions and in seminal fluid but lived longer in 20–30 ppt. Sperm were most active at 25–35 ppt. Therefore, we considered that spotted flagtail spawn in 20–35 ppt salinity. Because the mean salinity of the Genka estuary is below the optimum required for spawning, the spawning site in Okinawa Island would be seawater. This evidence suggested that the spotted flagtail is a catadromous species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]