Zoological Letters最新文献

{"title":"Developmental fates of shark head cavities reveal mesodermal contributions to tendon progenitor cells in extraocular muscles.","authors":"Shunya Kuroda,&nbsp;Noritaka Adachi,&nbsp;Rie Kusakabe,&nbsp;Shigeru Kuratani","doi":"10.1186/s40851-021-00170-2","DOIUrl":"https://doi.org/10.1186/s40851-021-00170-2","url":null,"abstract":"<p><p>Vertebrate extraocular muscles (EOMs) function in eye movements. The EOMs of modern jawed vertebrates consist primarily of four recti and two oblique muscles innervated by three cranial nerves. The developmental mechanisms underlying the establishment of this complex and the evolutionarily conserved pattern of EOMs are unknown. Chondrichthyan early embryos develop three pairs of overt epithelial coeloms called head cavities (HCs) in the head mesoderm, and each HC is believed to differentiate into a discrete subset of EOMs. However, no direct evidence of these cell fates has been provided due to the technical difficulty of lineage tracing experiments in chondrichthyans. Here, we set up an in ovo manipulation system for embryos of the cloudy catshark Scyliorhinus torazame and labeled the epithelial cells of each HC with lipophilic fluorescent dyes. This experimental system allowed us to trace the cell lineage of EOMs with the highest degree of detail and reproducibility to date. We confirmed that the HCs are indeed primordia of EOMs but showed that the morphological pattern of shark EOMs is not solely dependent on the early pattern of the head mesoderm, which transiently appears as tripartite HCs along the simple anteroposterior axis. Moreover, we found that one of the HCs gives rise to tendon progenitor cells of the EOMs, which is an exceptional condition in our previous understanding of head muscles; the tendons associated with head muscles have generally been supposed to be derived from cranial neural crest (CNC) cells, another source of vertebrate head mesenchyme. Based on interspecies comparisons, the developmental environment is suggested to be significantly different between the two ends of the rectus muscles, and this difference is suggested to be evolutionarily conserved in jawed vertebrates. We propose that the mesenchymal interface (head mesoderm vs CNC) in the environment of developing EOM is required to determine the processes of the proximodistal axis of rectus components of EOMs.</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-021-00170-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25375152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Functional identification of an opsin kinase underlying inactivation of the pineal bistable opsin parapinopsin in zebrafish.","authors":"Baoguo Shen,&nbsp;Seiji Wada,&nbsp;Haruka Nishioka,&nbsp;Takashi Nagata,&nbsp;Emi Kawano-Yamashita,&nbsp;Mitsumasa Koyanagi,&nbsp;Akihisa Terakita","doi":"10.1186/s40851-021-00171-1","DOIUrl":"https://doi.org/10.1186/s40851-021-00171-1","url":null,"abstract":"<p><p>In the pineal organ of zebrafish larvae, the bistable opsin parapinopsin alone generates color opponency between UV and visible light. Our previous study suggested that dark inactivation of the parapinopsin photoproduct, which activates G-proteins, is important for the regulation of the amount of the photoproduct. In turn, the photoproduct is responsible for visible light sensitivity in color opponency. Here, we found that an opsin kinase or a G-protein-coupled receptor kinase (GRK) is involved in inactivation of the active photoproduct of parapinopsin in the pineal photoreceptor cells of zebrafish larvae. We investigated inactivation of the photoproduct in the parapinopsin cells of various knockdown larvae by measuring the light responses of the cells using calcium imaging. We found that GRK7a knockdown slowed recovery of the response of parapinopsin photoreceptor cells, whereas GRK1b knockdown or GRK7b knockdown did not have a remarkable effect, suggesting that GRK7a, a cone-type GRK, is mainly responsible for inactivation of the parapinopsin photoproduct in zebrafish larvae. We also observed a similar knockdown effect on the response of the parapinopsin photoreceptor cells of mutant larvae expressing the opsin SWS1, a UV-sensitive cone opsin, instead of parapinopsin, suggesting that the parapinopsin photoproduct was inactivated in a way similar to that described for cone opsins. We confirmed the immunohistochemical distribution of GRK7a in parapinopsin photoreceptor cells by comparing the immunoreactivity to GRK7 in GRK7a-knockdown and control larvae. These findings suggest that in pineal photoreceptor cells, the cone opsin kinase GRK7a contributes greatly to the inactivation of parapinopsin, which underlies pineal color opponency.</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2021-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-021-00171-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25363806","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Visualization of antennal lobe glomeruli activated by nonappetitive D-limonene and appetitive 1-octen-3-ol odors via two types of olfactory organs in the blowfly Phormia regina.","authors":"Toru Maeda, Tomoyosi Nisimura, Shunnya Habe, Tatsuya Uebi, Mamiko Ozaki","doi":"10.1186/s40851-020-00167-3","DOIUrl":"10.1186/s40851-020-00167-3","url":null,"abstract":"<p><p>Appetite or feeding motivation relies significantly on food odors. In the blowfly Phormia regina, feeding motivation for sucrose is decreased by the odor of D-limonene but increased by the odor of 1-octen-3-ol odor. These flies have antennal lobes (ALs) consisting of several tens of glomerular pairs as a primary olfactory center in the brain. Odor information from different olfactory organs-specifically, the antennae and maxillary palps-goes to the corresponding glomeruli. To investigate how odors differently affect feeding motivation, we identified the olfactory organs and glomeruli that are activated by nonappetitive and appetitive odors. We first constructed a glomerular map of the antennal lobe in P. regina. Anterograde fluorescence labeling of antennal and maxillary afferent nerves, both of which project into the contralateral and ipsilateral ALs, revealed differential staining in glomerular regions. Some of the axonal fiber bundles from the antennae and maxillary palps projected to the subesophageal ganglion (SOG). We visualized the activation of the glomeruli in response to odor stimuli by immunostaining phosphorylated extracellular signal-regulated kinase (pERK). We observed different glomerulus activation under different odor stimulations. Referring to our glomerular map, we determined that antennal exposure to D-limonene odor activated the DA13 glomeruli, while exposure of the maxillary palps to 1-octen-3-ol activated the MxB1 glomeruli. Our results indicated that a nonappetitive odor input from the antennae and an appetitive odor input from the maxillary palps activate different glomeruli in the different regions of ALs in the blowfly P. regina. Collectively, our findings suggest that compartmentalization of glomeruli in AL is essential for proper transmission of odor information.</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-020-00167-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38689544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Management of flying insects on expressways through an academic-industrial collaboration: evaluation of the effect of light wavelengths and meteorological factors on insect attraction.","authors":"Masahiro Komatsu,&nbsp;Keigo Kurihara,&nbsp;Susumu Saito,&nbsp;Mana Domae,&nbsp;Naoki Masuya,&nbsp;Yuta Shimura,&nbsp;Shunichiro Kajiyama,&nbsp;Yuna Kanda,&nbsp;Kouki Sugizaki,&nbsp;Kouji Ebina,&nbsp;Osamu Ikeda,&nbsp;Yudai Moriwaki,&nbsp;Naohiro Atsumi,&nbsp;Katsuyoshi Abe,&nbsp;Tadashi Maruyama,&nbsp;Satoshi Watanabe,&nbsp;Hiroshi Nishino","doi":"10.1186/s40851-020-00163-7","DOIUrl":"https://doi.org/10.1186/s40851-020-00163-7","url":null,"abstract":"<p><p>Insect outbreaks often occur in the absence of natural enemies and in the presence of excess suitable host materials. Outbreaks of gypsy moths are especially problematic in remote areas located in high-latitude regions in Japan because the majority of adults emerge during the short summer season and initiate synchronous mass flight toward artificial lights. The aggregation of moths in public facilities not only is an annoyance to visitors but also permits the establishment of new populations the following year. The aim of this study was to establish a method to reduce the numbers of large moths that are attracted to lights in the rest areas of expressways in Hokkaido based on the results of research on their behavioral ecology and physiology. First, we conducted extensive insect surveys using light traps that emit light at different wavelengths; the traps were set along the expressways in the summers of 2014-2018. The insects attracted to the light were roughly classified into those showing a preference for broadband light wavelengths (from UV-A to green) and short light wavelengths (from UV-A to blue). The former included aquatic insects and winged ants, and the latter included moths and beetles. Next, we analyzed correlations between moth emergence and daily meteorological data. When gypsy moths were abundant during an outbreak, the daily catch of gypsy moths was positively correlated with the highest ambient temperature on the catch day but not with the visibility range, wind speed, or moon phase. In contrast, the daily catch of oak silkmoths did not correlate with any of these parameters. Our results provide guidance for the management of forest insects inhabiting cool-temperate to subarctic regions based on light wavelengths with reference to weather variables.</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-020-00163-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38351101","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"New insights into the morphology and evolution of the ventral pharynx and jaws in Histriobdellidae (Eunicida, Annelida).","authors":"Alexander Tzetlin,&nbsp;Nataliya Budaeva,&nbsp;Elena Vortsepneva,&nbsp;Conrad Helm","doi":"10.1186/s40851-020-00168-2","DOIUrl":"https://doi.org/10.1186/s40851-020-00168-2","url":null,"abstract":"<p><p>The jaw apparatus in several annelid families represents a powerful tool for systematic approaches and evolutionary investigations. Nevertheless, for several taxa, this character complex has scarcely been investigated, and complete comparative analyses of all annelid jaws are lacking. In our comprehensive study, we described the fine structure of the jaw apparatus and the ventral pharyngeal organ (VPO) in Histriobdella homari - a minute ectocommensal of lobsters putatively belonging to the Eunicida - using different comparative morphological approaches, including SEM, TEM, CLSM and subsequent 3D reconstruction. The H. homari jaw apparatus is composed of ventral paired mandibles and dorsal symmetrical maxillae consisting of numerous dental plates, ventral carriers and an unpaired dorsal rod, and the general assemblage and arrangement of the different parts are highly comparable to those of other eunicid families. The jaw ultrastructure of histriobdellids resembles that of the families Dorvilleidae and (juvenile) Onuphidae. Furthermore, our data reveal that in the process of development of the jaw apparatus, the mandibles, maxillae II and unpaired dorsal rod are formed first, and the remaining maxillae and ventral carriers appear later. Notably, the muscular apparatus differs from that in Dorvilleidae and Onuphidae in terms of the number and arrangement of muscle fibers encompassing the jaws - not only because of the very small size of Histriobdella but also because histriobdellid maxillary protraction occurs due to straightening of the dorsal rod and thus requires a different muscular scaffold. Based on our investigations, the arrangement of the muscular apparatus of the jaws, the presence of paired ventral carriers and the dorsal rod, and the morphology of the ventral pharyngeal organ represent a histriobdellid autapomorphy. Our datasets form a basis for further comparative analyses to elucidate the evolution of Eunicida and jaw-bearing Annelida.</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-020-00168-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38351087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Coloration principles of the Great purple emperor butterfly (Sasakia charonda).","authors":"Doekele G Stavenga,&nbsp;Hein L Leertouwer,&nbsp;Kentaro Arikawa","doi":"10.1186/s40851-020-00164-6","DOIUrl":"https://doi.org/10.1186/s40851-020-00164-6","url":null,"abstract":"<p><p>The dorsal wings of male Sasakia charonda butterflies display a striking blue iridescent coloration, which is accentuated by white, orange-yellow and red spots, as well as by brown margins. The ventral wings also have a variegated, but more subdued, pattern. We investigated the optical basis of the various colors of intact wings as well as isolated wing scales by applying light and electron microscopy, imaging scatterometry and (micro)spectrophotometry. The prominent blue iridescence is due to scales with tightly packed, multilayered ridges that contain melanin pigment. The scales in the brown wing margins also contain melanin. Pigments extracted from the orange-yellow and red spots indicate the presence of 3-OH-kynurenine and ommochrome pigment. The scales in the white spots also have multilayered ridges but lack pigment. The lower lamina of the scales plays a so-far undervalued but often crucial role. Its thin-film properties color the majority of the ventral wing scales, which are unpigmented and have large windows. The lower lamina acting as a thin-film reflector generally contributes to the reflectance of the various scale types.</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-020-00164-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38351097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus.","authors":"Yasuaki Tomiyama,&nbsp;Tsugumichi Shinohara,&nbsp;Mirai Matsuka,&nbsp;Tetsuya Bando,&nbsp;Taro Mito,&nbsp;Kenji Tomioka","doi":"10.1186/s40851-020-00166-4","DOIUrl":"https://doi.org/10.1186/s40851-020-00166-4","url":null,"abstract":"<p><p>The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwo^RNAi crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-020-00166-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38689057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Morphology of Stephanella hina (Bryozoa, Phylactolaemata): common phylactolaemate and unexpected, unique characters.","authors":"Thomas F Schwaha,&nbsp;Masato Hirose","doi":"10.1186/s40851-020-00165-5","DOIUrl":"https://doi.org/10.1186/s40851-020-00165-5","url":null,"abstract":"<p><p>Stephanella hina is a little studied freshwater bryozoan belonging to Phylactolaemata. It is currently the only representative of the family Stephanellidae, which in most reconstructions is early branching, sometimes even sister group to the remaining phylactolaemate families. The morphological and histological details of this species are entirely unknown. Consequently, the main aim of this study was to conduct a detailed morphological analysis of S. hina using histological serial sections, 3D reconstruction, immunocytochemical staining and confocal laser scanning microscopy techniques. The general morphology is reminiscent of other phylactolaemates; however, there are several, probably apomorphic, details characteristic of S. hina. The most evident difference lies in the lophophoral base, where the ganglionic horns/extensions do not follow the traverse of the lophophoral arms but bend medially inwards towards the mouth opening. Likewise, the paired forked canal does not fuse medially in the lophophoral concavity as found in all other phylactolaemates. Additional smaller differences are also found in the neuro-muscular system: the rooting of the tentacle muscle is less complex than in other phylactolaemates, the funiculus lacks longitudinal muscles, the caecum has smooth muscle fibres, latero-abfrontal tentacle nerves are not detected and the medio-frontal nerves mostly emerge directly from the circum-oral nerve ring. In the apertural area, several neurite bundles extend into the vestibular wall and probably innervate neurosecretory cells surrounding the orifice. These morphological characteristics support the distinct placement of this species in a separate family. Whether these characteristics are apomorphic or possibly shared with other phylactolaemates will require the study of the early branching Lophopodidae, which remains one of the least studied taxa to date.</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-020-00165-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38350383","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction to: Natural selection, selective breeding, and the evolution of resistance of honeybees (<i>Apis mellifera)</i> against <i>Varroa</i>.","authors":"Jacques J M van Alphen,&nbsp;Bart Jan Fernhout","doi":"10.1186/s40851-020-00162-8","DOIUrl":"https://doi.org/10.1186/s40851-020-00162-8","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.1186/s40851-020-00158-4.].</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-020-00162-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38053974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sea as a color palette: the ecology and evolution of fluorescence.","authors":"Marie-Lyne Macel,&nbsp;Filomena Ristoratore,&nbsp;Annamaria Locascio,&nbsp;Antonietta Spagnuolo,&nbsp;Paolo Sordino,&nbsp;Salvatore D'Aniello","doi":"10.1186/s40851-020-00161-9","DOIUrl":"https://doi.org/10.1186/s40851-020-00161-9","url":null,"abstract":"<p><p>Fluorescence and luminescence are widespread optical phenomena exhibited by organisms living in terrestrial and aquatic environments. While many underlying mechanistic features have been identified and characterized at the molecular and cellular levels, much less is known about the ecology and evolution of these forms of bioluminescence. In this review, we summarize recent findings in the evolutionary history and ecological functions of fluorescent proteins (FP) and pigments. Evidence for green fluorescent protein (GFP) orthologs in cephalochordates and non-GFP fluorescent proteins in vertebrates suggests unexplored evolutionary scenarios that favor multiple independent origins of fluorescence across metazoan lineages. Several context-dependent behavioral and physiological roles have been attributed to fluorescent proteins, ranging from communication and predation to UV protection. However, rigorous functional and mechanistic studies are needed to shed light on the ecological functions and control mechanisms of fluorescence.</p>","PeriodicalId":54280,"journal":{"name":"Zoological Letters","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2020-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40851-020-00161-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38043321","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}