Search

While copepods are important prey for many wild marine fish larvae and are preferred as a live feed for culturing larval fish, their low productivity and the costs associated with culturing them typically limit their use as a live food source in all but experimental situations. In a series of experiments with four readily and commercially available microalgae (Thalassiosira weissflogii Grunow, Rhodomonas salina Wislouch, Tetraselmis suecica Kylin Butch, and Isochrysis galbana Parke), we determine which of them, when exclusively fed to a commonly occurring copepod, Oithona oculata, over a 15-day period, results in the greatest copepod survival and egg production rates. We posit that species of Oithona might be an ideal candidate for copepod taxa for commercial-scale copepod culture and that the microalga Rhodomonas salina is an ideal food source to culture them.

Considerable skill is required to identify copepods at the species level based on their morphological characteristics. However, DNA analysis does not require advanced microscopy techniques and provides objective data on the phylogenetic relationships between samples. Therefore, DNA analysis is useful as an alternative method for taxonomic studies of copepods. The lysis buffer method by Lee & Frost (2002) is a simple protocol for extracting DNA from single copepod samples. In this method, a fixative, such as formalin, is first replaced with ethanol and a buffer solution. Then, the copepod sample is lysed in the lysis buffer containing a proteolytic enzyme. Thus far, we have conducted DNA extraction of single copepod samples using this method and performed gene amplification by PCR. However, due to the low success rate of PCR amplification, genetic data could not be obtained for approximately 50% of the formalin-fixed samples. In this study, we improved the lysis buffer method with the aim of enhancing the success rate of DNA extraction and PCR amplification from single copepod samples. In addition, the effect of formalin fixation time on PCR amplification was also examined.Zooplankton samples were collected from Manazuru Port, Sagami Bay on September 14, 2017, using a plankton net with a mesh size of 180 μm and fixed with 5% neutralized formalin-seawater. Adult females of the calanoid copepod Acartia japonica were selected from these samples and stored individually in ethanol. DNA was extracted from these copepods via a modified ethanol removal method, with adjustments made to the dilution of the lysis buffer, and incubation time. The mitochondrial cytochrome b gene was amplified from these DNA samples by PCR. When the concentration of the PCR product was 20 ng μL-1 or more, PCR amplification was considered to be successful. Based on the conditions optimized by the above studies, the effect of the formalin fixation time on the PCR amplification of copepods was also investigated. A higher success rate was obtained when natural drying or vacuum drying was performed to remove ethanol during DNA extraction rather than removal by pipetting. Since there was no significant difference between the results of natural drying and vacuum drying, natural drying, which is easier to perform, was selected as the optimum method. We also confirmed that a high success rate was maintained without diluting the lysis buffer after the inactivation of proteolytic enzyme. Regarding the incubation time for lysis, changing from the conventional 60 minutes to 30 minutes did not result in a significant decrease in the success rate of PCR amplification. Thus, the success rate of PCR increased to approximately 90%. Additionally, compared with the conventional lysis buffer method, the number of steps was reduced by half, and the required time was shortened from 1.5 hours to approximately 50 minutes. Furthermore, we confirmed that this improved lysis buffer method can be applied to single cells of small protozoa such as flagellates and ciliates. The effect of formalin fixation time on PCR amplification of the mitochondrial cytochrome b gene after DNA extraction via this improved lysis buffer method was investigated. As expected, the success rate of PCR amplification decreased with the formalin fixation time. However, when the fixation period was within 1 month, PCR products with a concentration of more than 20 ng μL-1 were obtained in 95% of the individual copepod samples. Furthermore, even after 3 months, similar concentrations of PCR product were obtained in 80% of individuals.Genetic analysis of small zooplankton is increasingly important not only in taxonomy but also for biodiversity and phylogeographic studies. The data presented in this study will be very important and useful in such studies.

In aquaculture and ornamental industries, copepods are recognized as preferred live feeds for marine fish larvae over commonly used organisms such as Artemia and rotifers. Marine fish larvae fed with copepods show better survival and growth. Despite obvious advantages of copepods as the live feed, their use is still limited owing to low productivity and cost-efficiency when mass cultivated. Copepods from the genus Acartia are good candidates for a live feed because their body size, swimming behavior, and biochemical composition are suitable for many marine fish larvae which have small mouth gapes. In addition, Acartia species produce dormant eggs which can be stored and hatched to feed fish larvae. Acartia steueri Smirnov is widely distributed in the coastal waters of the western Pacific Ocean, and is an essential food source for the larvae of commercially important fish in their natural habitats. Different dietary microalgae affect the egg production rate, hatching success, survival rate, growth rate, and the population growth of copepods. One of the underlying bottlenecks in the intensive cultivation of copepods is fatally low survival rate during their larval stages. Calanoid copepods including genus Acartia feed on live microalgae. In the present study, in order to clarify the favorable dietary microalgae for larvae of Acartia steueri, the nauplii individuals were fed with four mono-microalgal diets and one mixed-microalgal diet to measure their survival rate. The present study conducted two experiments. In the first experiment, the nauplii hatched within 24 hours were individually reared in 6-well plates under three diet conditions (mono-diet of Tetraselmis suecica, Rhodomonas salina and Isochrysis galbana) in April 2019. Survival rate and development stages of the copepods were measured every two days. In the second experiment, the nauplii were reared in 600 mL beakers under three diet conditions (mono-diet of T. suecica, Chaetoceros gracilis, and a mixed diet of T. suecica + C. gracilis at 1:1 carbon ratio) in April 2020. Survival rate and development stages of the copepods were measured at day 10 and day 20 during the incubation duration.In the first experiment, the survival rate at day 20 was 26.8 ± 7.2％ when fed with T. suecica, which was the highest value among the mono-microalgal diet conditions. However, only 0.6％ of individuals fed with T. suecica were developed to the adult stage (copepodid VI). In addition, the malformation at first antennas was observed from the copepodid individuals fed with T. suecica. T. suecica is well known to be rich in amino acids but with poor fatty acid content. These results might suggest that T. suecica is the favorable diet for early developmental stages (i.e. nauplii) of the copepod A. steueri, but has a nutritional problem for the later development stages of life cycle. In the second experiment, the ratio of individuals developed until adult stages was maximized under the mixed diet condition of T. suecica and C. gracilis, and this mixed diet can be considered a favorable diet for A. steueri larvae in the present study.

In aquaculture and ornamental industries, copepods are recognized as preferred live feeds for marine fish larvae over commonly used organisms such as Artemia and rotifers. Marine fish larvae fed with copepods show better survival and growth. Despite obvious advantages of copepods as live feed, their use is still limited owing to low productivity and cost-efficiency when mass cultured. Copepods can be divided into free-spawner and sac-spawner (egg-carrying) according to their spawning style. In cultivation of egg-carrying copepod, a simple nauplii collection/harvesting method with low labor has not been established, because females carry the egg-sac until hatching. Manual collection of nauplii using a siphon hose and mesh-filters is a common method in copepod cultivation, but automation is necessary to reduce labor costs if copepods are to be viably cultured at a commercial scale. Recently, we devised a zooplankton bioreactor for rapid separation of free-spawner copepod eggs from adults in a tank. The automation collects eggs on a mesh filter in a water flow system which can also function as water exchange device. In the present study, we apply this bioreactor to egg-carrying copepod, and report the results from cultivation at laboratory-scale for 45 days. Species of Oithona are good candidates for live feed because their body size and biochemical composition are suitable for many marine fish larvae which have small gapes. Oithona oculata is widely distributed and a typical dominant species in coastal waters, and was chosen as target species of egg-carrying copepod in present study. 150 adult individuals of O. oculata were placed into a culture chamber which were immersed in a reproduction tank containing 3 L seawater. The culture chamber to retain the copepods has a 100-μm nylon mesh placed 5 mm above the bottom, while allowing passage of nauplii. The copepods were fed daily with a sufficient amount of Thalassiosira weissflogii and Isochrysis galbana and incubated at 28°C for 45 days. Produced nauplii in the reproduction tank were collected daily using water flow of 1.5 L min-1. The species abundance in each development, ovigerous rate and collected number of nauplii were measured daily. In the semi-continuous culture, the collected nauplii were cultured in maturation vessels and returned to the production tank after their maturation, and 45 days cultivation (four generations) was achieved. Total abundance was gradually increased from day 4 to day 20 and reached at 500 inds. L-1. Ovigerous rate varied from 0 to 88% during the incubation period. The maximum number of collected nauplii was 380 nauplii L-1 at day 5. The results obtained in present study suggest that the bioreactor can also be applied to egg-carrying copepod by optimization of the mesh size and the water flow velocity.

Purpose We analyzed interfractional robustness of scanning carbon ion radiotherapy (CIRT) for prostate cancer based on the dose distribution using daily in‐room computed tomography (CT) images. Materials and Methods We analyzed 11 consecutive patients treated with scanning CIRT for localized prostate cancer in our hospital between December 2015 and January 2016. In‐room CT images were taken under treatment conditions in every treatment session. The dose distribution on each in‐room CT image was recalculated, while retaining the pencil beam arrangement of the initial treatment plan. Then, the dose–volume histogram (DVH) parameters including the percentage of the clinical target volume (CTV) with 95% and 90% of the prescribed dose area (V95% of CTV, V90% of CTV) and V80% of rectum were calculated. The acceptance criteria for the CTV and rectum were set at V95% of CTV ≥95%, V90% of CTV ≥98%, and V80% of rectum < 10 ml. Results V95% of CTV, V90% of CTV, and V80% of rectum for the reproduced plans were 98.8 ± 3.49%, 99.5 ± 2.15%, and 4.39 ± 3.96 ml, respectively. Acceptance of V95% of CTV, V90% of CTV, and V80% of rectum was obtained in 123 (94%), 125 (95%) and 117 sessions (89%), respectively. Acceptance of the mean dose of V95% of CTV, V90% of CTV, and V80% of rectum for each patient was obtained in 10 (91%), 10 (91%), and 11 patients (100%), respectively. Conclusions We demonstrated acceptable interfractional robustness based on the dose distribution in scanning CIRT for prostate cancer.

Background/aim The present study aimed to compare the radiation dose distribution of carbon-ion radiotherapy (CIRT) for stage I esophageal cancer with three-dimensional conformal radiotherapy (3DCRT) and volumetric modulated arc therapy (VMAT). Patients and methods Fifteen patients with cT1bN0M0 esophageal cancer who received 3DCRT at Kanagawa Cancer Center between January 2014 and April 2019 were enrolled. The dose-volume histogram parameters of the target volume and normal organs planned with CIRT, 3DCRT, and VMAT were evaluated. Results The homogeneity index for the target volume of CIRT was significantly lower than that of 3DCRT and VMAT. In addition, the radiation dose of CIRT to the heart, lungs, spinal cord, and skin was significantly lower than that of 3DCRT and VMAT. Conclusion Favorable dose distributions with CIRT were demonstrated compared with 3DCRT and VMAT for esophageal cancer.

We compared predictive performance between dose volume histogram (DVH) parameter addition and deformable image registration (DIR) addition for gastrointestinal (GI) toxicity in cervical cancer patients. A total of 59 patients receiving brachytherapy and external beam radiotherapy were analyzed retrospectively. The accumulative dose was calculated by three methods: conventional DVH parameter addition, full DIR addition and partial DIR addition. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${D}_{2{cm}^3}$\end{document}, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${D}_{1{cm}^3}$\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${D}_{0.1{cm}^3}$\end{document} (minimum doses to the most exposed 2 cm3, 1cm3 and 0.1 cm3 of tissue, respectively) of the rectum and sigmoid were calculated by each method. V50, V60 and V70 Gy (volume irradiated over 50, 60 and 70 Gy, respectively) were calculated in full DIR addition. The DVH parameters were compared between toxicity (≥grade1) and non-toxicity groups. The area under the curve (AUC) of the receiver operating characteristic (ROC) curves were compared to evaluate the predictive performance of each method. The differences between toxicity and non-toxicity groups in \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${D}_{2{cm}^3}$\end{document} were 0.2, 5.7 and 3.1 Gy for the DVH parameter addition, full DIR addition and partial DIR addition, respectively. The AUCs of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${D}_{2{cm}^3}$\end{document} were 0.51, 0.67 and 0.57 for DVH parameter addition, full DIR addition and partial DIR addition, respectively. In full DIR addition, the difference in dose between toxicity and non-toxicity was the largest and AUC was the highest. AUCs of V50, V60 and V70 Gy were 0.51, 0.63 and 0.62, respectively, and V60 and V70 were high values close to the value of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${D}_{2{cm}^3}$\end{document} of the full DIR addition. Our results suggested that the full DIR addition may have the potential to predict toxicity more accurately than the conventional DVH parameter addition, and that it could be more effective to accumulate to all pelvic irradiation by DIR.

The purpose of the study was to compare a 3D convolutional neural network (CNN) with the conventional machine learning method for predicting intensity-modulated radiation therapy (IMRT) dose distribution using only contours in prostate cancer. In this study, which included 95 IMRT-treated prostate cancer patients with available dose distributions and contours for planning target volume (PTVs) and organs at risk (OARs), a supervised-learning approach was used for training, where the dose for a voxel set in the dataset was defined as the label. The adaptive moment estimation algorithm was employed for optimizing a 3D U-net similar network. Eighty cases were used for the training and validation set in 5-fold cross-validation, and the remaining 15 cases were used as the test set. The predicted dose distributions were compared with the clinical dose distributions, and the model performance was evaluated by comparison with RapidPlan™. Dose–volume histogram (DVH) parameters were calculated for each contour as evaluation indexes. The mean absolute errors (MAE) with one standard deviation (1SD) between the clinical and CNN-predicted doses were 1.10% ± 0.64%, 2.50% ± 1.17%, 2.04% ± 1.40%, and 2.08% ± 1.99% for D2, D98 in PTV-1 and V65 in rectum and V65 in bladder, respectively, whereas the MAEs with 1SD between the clinical and the RapidPlan™-generated doses were 1.01% ± 0.66%, 2.15% ± 1.25%, 5.34% ± 2.13% and 3.04% ± 1.79%, respectively. Our CNN model could predict dose distributions that were superior or comparable with that generated by RapidPlan™, suggesting the potential of CNN in dose distribution prediction.

Background/aim The purpose of this study was to compare the dose distribution between scanning carbon-ion radiotherapy (sCIRT) and volumetric-modulated arc therapy with stereotactic body radiation therapy (VMAT-SBRT) for stage I non-small cell lung cancer (NSCLC). Patients and methods Fifteen patients with early-stage NSCLC who underwent sCIRT at Kanagawa Cancer Center between 2018-2020 were enrolled. Dose-volume histogram parameters of the planned target volume and normal organs for sCIRT and VMAT-SBRT were evaluated. Results The homogeneity index of the target volume of sCIRT was significantly lower than that of VMAT-SBRT. The dose of sCIRT was significantly lower than that of VMAT-SBRT at low volumes in the lung, heart, spinal cord, and esophagus. Conclusion The dose distribution of sCIRT for early-stage NSCLC was better than that of VMAT-SBRT.

To clarify temporal variation of in situ egg production of the copepods, Acartia japonica Mori, 1940, in Sagami Bay, central Japan, the species’ abundance, egg morphology and hatching patterns were studied during 2015. Adults of A. japonica were observed in the water column from June to October. Results showed that adults reared in the laboratory produced three types of eggs: subitaneous, delayed-hatching and diapause. All egg types had a surface covered with short spines; diapause eggs had a thicker chorion than subitaneous eggs. During the early period of the species occurrence in the bay, most females produced only subitaneous eggs, which hatched within 72 h. From the end of September, a large proportion of females began to produce diapause eggs. Linear regression analysis showed that the ratio of diapause eggs was negatively correlated with day length and water temperature (P

This study aimed to evaluate the performance of the hybrid deformable image registration (DIR) method in comparison with intensity-based DIR for pelvic cone-beam computed tomography (CBCT) images, using intensity and anatomical information. Ten prostate cancer patients treated with intensity-modulated radiation therapy (IMRT) were studied. Nine or ten CBCT scans were performed for each patient. First, rigid registration was performed between the planning CT and all CBCT images using gold fiducial markers, and then DIR was performed. The Dice similarity coefficient (DSC) and center of mass (COM) displacement were used to evaluate the quantitative DIR accuracy. The average DSCs for intensity-based DIR for the prostate, rectum, bladder, and seminal vesicles were 0.84 ± 0.05, 0.75 ± 0.05, 0.69 ± 0.07 and 0.65 ± 0.11, respectively, whereas those values for hybrid DIR were 0.98 ± 0.00, 0.97 ± 0.01, 0.98 ± 0.00 and 0.94 ± 0.03, respectively (P

世界の水産物生産量は、1990 年代に漁獲生産が頭打ちとなり、養殖生産によってその後の右肩上がりの需要を賄っている。天然域からの種苗採集に依存しない人工種苗生産においては、仔稚魚が要求する栄養に富む小型動物プランクトンを生産できないことが大きな障壁となっている。海産浮遊性カイアシ類はワムシやアルテミアといった既存の生物餌料よりも高い栄養価を示し、仔稚魚の成長、生残、市場価値を向上させることから、理想的な生物餌料と認識され、水産分野への利用が期待されている。しかし、人工環境下でのカイアシ類の生存率と卵生産は低く、加えて不安定なためにカイアシ類の大量培養は未だ困難とされている。水産餌料として利用する場合、カイアシ類は高個体密度環境で培養されるため、成体による卵の捕食 (共食い) が発生し、槽内で生産された卵の実に30%が共食いによって損失される。そのためカイアシ類の水産分野での利用には共食い防止技術の確立が喫緊の課題である。相模湾真鶴港に優占する浮遊性カイアシ類Acartia steueri とA. japonica を培養候補種とし現場調査を行ったところ、A. steueri の出現個体密度は高く、水温範囲は12-28oC と広く、本邦の魚類種苗生産の水温帯に合致した。そこで本博士研究の目的を、浮遊性カイアシ類A. steueri の集約的な培養技術の確立とし、(1)A. steueri 発達段階前期 (幼生) から後期 (幼体)、成体の好適餌料の検討、(2)培養個体密度がA. steueri の卵生産と代謝速度に与える影響の解明、(3)実験室規模での回分式カイアシ類培養槽の運転と生産性の評価を行った。本論文は5 章から構成されている。第1 章は総合序論として、海産浮遊性カイアシ類の初期生物餌料としての有用性と、その集約的培養において生産性を損なう要因である“成体による卵・幼生の捕食”（共食い）を問題提起し、その解決方法として成体培養槽と幼生・幼体培養槽からなる2 槽式培養法を提案した。第2 章ではA. steueri を複数の餌料藻類条件で培養し、幼生から成体までの生存率と、成体の卵生産速度を測定し、それぞれの好適餌料を検討した。幼生から成体における生存率は、緑藻Tetraselmis suecica と珪藻Chaetoceros gracilis からなる混合餌料を与えた条件で最大化されたため好適餌料として選択した。成体の卵生産は、緑藻T. suecica と珪藻Thalassiosira weissflogii からなる混合餌料を与えた条件で最大化されたため、好適餌料として選択した。これらの餌料検討の結果、幼生から成体までの生存率が20%と高効率な継代培養に成功した。A. steueri を高個体密度で培養した際の生理状態を理解し、適切な培養個体密度を検討するため第3 章では、本種成体を異なる個体密度条件で培養し、生存率、卵生産速度、代謝速度を測定した。生存率と卵生産速度のいずれにおいても、各個体密度条件間で有意な差は認められなかったが、呼吸速度は高個体密度条件で有意に減少した。これらの結果より、本種の生存率と卵生産速度は個体密度によって変化せず、高個体密度条件では代謝速度を減少させ卵生産速度を維持したため、高密度培養に適した生物学的特徴を有していると考えられた。第4 章では、共食い防止機構を備えた成体培養槽を作成後、第2、3 章で決定した好適餌料と培養個体密度を用いた20 日間の運転を実施し、その生産性を評価した。その結果、既往研究の1.6 倍の生産性を達成したため、大型化に向けての方途を見出した。第5 章では総合考察として、本研究手法による大型生産システムを想定した試算を行うとともに、本手法の他カイアシ類種への応用と実利用に向けた今後の課題を論じた。