Establishment of intensive culture technology for a marine pelagic copepod Acartia steueri

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。創価大

Mass cultivation of marine planktonic copepods

To meet the increasing global demand for seafood, efficient and stable aquaculture production is essential. This requires mass production of fish fry and development of suitable live diets for their growth. In aquaculture and the ornamental industry, marine planktonic co- pepods are recognized as preferred live feeds for marine fish larvae over the commonly used organisms Artemia and rotifers. Marine fish larvae fed with the copepods show better survival, pigmentation and growth. Based on this, wild copepods collected from the natural ecosystem have been used as the live diet for fish larvae culturing. Mass culture of copepods under a con trolled environment is desirable due to the unstable collection amount, difficulties in obtaining consistent quality, and risk of parasite/pathogen contamination when collecting copepods from natural ecosystems. Despite being a preferred live feed, copepod use is still limited due to low productivity and cost efficiency when intensively cultured.This review focuses on the status and challenges in the mass cultivation technology of marine planktonic copepods. Section 2 summarizes the importance of live diets for marine fish produc- tion and the challenges of a conventional live diet using rotifer and Artemia from the viewpoint of prey size and nutrient demand of the fish larvae. Section 3 focuses on the characteristics of marine planktonic copepods as aquaculture live diets, and briefly introduces the research history of culturing copepods from the 1970s. Section 4 reviews which copepods have been cultured as target species to date. Section 5 presents the types of diets that have been used to cultivate cope- pods, and what factors should be considered in selecting their diets. In addition, non-microalgal diets such as yeast, bacteria and protists, which are considered to be cheaper to produce than microalgal diets, will also be introduced. Section 6 discusses copepod stocking density, which determines productivity and production cost in the mass culture, and presents how stress caused by high-density conditions affects their survival, egg production, and hatching success. Section 7 deals with cannibalism, one of the most serious problems limiting productivity in mass copepod culturing, and discuss how many eggs and larvae are lost through cannibalism based on quanti- tative data. Section 8 summarizes the various methods developed for collecting and separating eggs and nauplii from individual adults to minimize cannibalism risk. Section 9 covers the meth- ods for storing copepod eggs and nauplii, which can enable copepod products to be transported between producers and consumers, and assist producers in maintaining backup cultures.departmental bulletin pape

抱卵型カイアシ類Oithona oculata のバイオリアクターを用いた試験的培養

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

浮遊性カイアシ類Acartia steueriの幼生・幼体の培養における微細藻類餌料の検討

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

Distinguishing intrahepatic cholangiocarcinoma from poorly differentiated hepatocellular carcinoma using precontrast and gadoxetic acid-enhanced MRI

PURPOSEWe aimed to gain further insight in magnetic resonance imaging characteristics of mass-forming intrahepatic cholangiocarcinoma (mICC), its enhancement pattern with gadoxetic acid contrast agent, and distinction from poorly differentiated hepatocellular carcinoma (pHCC).METHODSFourteen mICC and 22 pHCC nodules were included in this study. Two observers recorded the tumor shape, intratumoral hemorrhage, fat on chemical shift imaging, signal intensity at the center of the tumor on T2-weighted image, fibrous capsule, enhancement pattern on arterial phase of dynamic study, late enhancement three minutes after contrast injection (dynamic late phase), contrast uptake on hepatobiliary phase, apparent diffusion coefficient, vascular invasion, and intrahepatic metastasis.RESULTSLate enhancement was more common in mICC (n=10, 71%) than in pHCC (n=3, 14%) (P < 0.001). A fat component was observed in 11 pHCC cases (50%) versus none of mICC cases (P = 0.002). Fibrous capsule was observed in 13 pHCC cases (59%) versus none of mICC cases (P < 0.001). On T2-weighted images a hypointense area was seen at the center of the tumor in 43% of mICC (6/14) and 9% of pHCC (2/22) cases (P = 0.018). Other parameters were not significantly different between the two types of nodules.CONCLUSIONThe absence of fat and fibrous capsule, and presence of enhancement at three minutes appear to be most characteristic for mICC and may help its differentiation from pHCC

カイアシ類1 個体からのDNA 抽出方法の改良とホルマリン固定期間がミトコンドリア遺伝子のPCR増幅に与える影響

　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

Practical Science and Environmental Education Workshop in Manaus, Brazil

It is an unequivocal fact that Amazonian tropical forest is the largest remaining primary forest in the world. The ecosystem in the region is e tremely comple with high biodiversity (Peres et al. 2010). Conservation and protection of the dynamic forest and river regions is e tremely important not only for the natural environments, but also for the economy and social dependence of benefits from such abundant natural environments. Important natural parameters that affect status of the natural environments include light (natural sunlight), soil, and water, which abundantly e ist in the Amazon region. Solar energy is the primary energy source for the majority of living organisms in both terrestrial and aquatic ecosystems, and drives the diurnal and seasonal cycles of biogeochemical processes (Monteith & Unsworth 2013). In particular, in situ light data remains one of the most underappreciated data measurements although having a significant impact on the physical, chemical and biological processes in the ecosystem (Johnsen 2012). Soil provides the fundamental basis for all terrestrial living organisms including the Amazonian forests as well as life-sustaining infrastructure for human society. Water is the most essential single entity to constitute all organisms from a single cell to the earth. Understanding of importance and roles of each factor and interaction of such comple dynamics in the natural environments can serve as fundamental platform for natural scientists, particularly for young scientists such as university students. The objective of this workshop was to provide hand- on scientific and environmental education for university students in Manaus, Amazonas, Brazil through practical field measurements using the three most important parameters in the natural ecosystem composed of natural sunlight, soil, and water. The workshop was divided into a series of lectures, in situ field sampling, and data processing, analysis and interpretation with the ultimate goal of empowering the undergraduate students with research-centered environmental education and e perience of developing international collaboration.departmental bulletin pape

Prognostic impact of clinical factors for immune checkpoint inhibitor with or without chemotherapy in older patients with non-small cell lung cancer and PD-L1 TPS ≥ 50%

IntroductionThe proportion of older patients diagnosed with advanced-stage non-small cell lung cancer (NSCLC) has been increasing. Immune checkpoint inhibitor (ICI) monotherapy (MONO) and combination therapy of ICI and chemotherapy (COMBO) are standard treatments for patients with NSCLC and programmed cell death ligand-1 (PD-L1) tumor proportion scores (TPS) ≥ 50%. However, evidence from the clinical trials specifically for older patients is limited. Thus, it is unclear which older patients benefit more from COMBO than MONO.MethodsWe retrospectively analyzed 199 older NSCLC patients of Eastern Cooperative Oncology Group performance status (ECOG PS) 0-1 and PD-L1 TPS ≥ 50% who were treated with MONO or COMBO. We analyzed the association between treatment outcomes and baseline patient characteristics in each group, using propensity score matching.ResultsOf the 199 patients, 131 received MONO, and 68 received COMBO. The median overall survival (OS; MONO: 25.2 vs. COMBO: 42.2 months, P = 0.116) and median progression-free survival (PFS; 10.9 vs. 11.8 months, P = 0.231) did not significantly differ between MONO and COMBO group. In the MONO group, OS was significantly shorter in patients without smoking history compared to those with smoking history [HR for smoking history against non-smoking history: 0.36 (95% CI: 0.16-0.78), P = 0.010]. In the COMBO group, OS was significantly shorter in patients with PS 1 than those with PS 0 [HR for PS 0 against PS 1: 3.84 (95% CI: 1.44-10.20), P = 0.007] and for patients with squamous cell carcinoma (SQ) compared to non-squamous cell carcinoma (non-SQ) [HR for SQ against non-SQ: 0.17 (95% CI: 0.06-0.44), P &lt; 0.001]. For patients with ECOG PS 0 (OS: 26.1 months vs. not reached, P = 0.0031, PFS: 6.5 vs. 21.7 months, P = 0.0436) or non-SQ (OS: 23.8 months vs. not reached, P = 0.0038, PFS: 10.9 vs. 17.3 months, P = 0.0383), PFS and OS were significantly longer in the COMBO group.ConclusionsECOG PS and histological type should be considered when choosing MONO or COMBO treatment in older patients with NSCLC and PD-L1 TPS ≥ 50%

Identity of the elusive IgM Fc receptor (FcμR) in humans

Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcμR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcμR in human B-lineage cDNA libraries. FcμR is defined as a transmembrane sialoglycoprotein of ∼60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcα/μR) but exhibits an exclusive Fcμ-binding specificity. The cytoplasmic tail of FcμR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcμR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcμR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcμR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcμR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis