Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6–11, 8–11 and 6–9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40–50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception—increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10–12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.     

Disparities in activity levels and learning ability between Djungarian hamster (Phodopus sungorus) and Roborovskii hamster (Phodopus roborovskii)

The Djungarian hamster and the Roborovskii hamster belong to the same genus of Phodopus. However, the Djungarian hamster is tame and shows sedative behavior, while Roborovskii hamster is not tame and shows high levels of locomotor activity. Hyperactivity occurs in animals with tameless behavior. Tameness or tamelessness behavior is very important because tameness helps for breeding and controlling as well as it enables a strong human‐animal bond. In the present study, we examined the relationships between activity levels and cognitive function in Djungarian and Roborovskii hamsters. Three types of behavioral tests were performed to analyze their activity levels, memory and leaning ability. The levels of L‐ and D‐amino acids and monoamines in the brain were then determined. Roborovskii hamsters showed significantly higher locomotor activity than Djungarian hamsters. Memory ability was not significantly different between the two hamsters, but Roborovskii hamsters showed lower learning ability. Brain levels of D‐serine which is related to enhancement in memory and learning ability, were significantly higher in Djungarian hamsters, but the reverse was true for brain dopamine and serotonin levels. These results suggest that these differences in brain metabolism may be related to the behavioral differences between the two hamsters.     

Characterization of the cathelicidin cluster in the Japanese quail (Coturnix japonica)

The Japanese quail has several advantages as a low‐fat meat bird with high immunity against diseases. Cathelicidins (CATHs) are antimicrobial peptides that play an important role in innate immunity. The aim of this study was to characterize the CATH cluster in the Japanese quail (Coturnix japonica). The Japanese quail CATH (CjCATH) cluster, contains four CATH genes, as in the chicken. The coding sequences of CjCATHs exhibited >85.3% identity to chicken CATHs. The predicted amino acid sequences of the four CjCATH genes contained the cathelin‐like domain characteristic of CATH proteins. Polymorphisms were detected in the open reading frames (ORFs) of all CjCATH sequences. Two amino acid substitutions were observed in the antimicrobial region of the mature peptide of CjCATH2, and predicted to influence peptide function. CjCATH1 is expressed in lung, heart, bone marrow and bursa of Fabricius (BF). CjCATH2 is expressed in bone marrow. CjCATH3 is expressed in lung, heart, bone marrow, BF, tongue and duodenum. CjCATHB1 is expressed in bone marrow and BF. This study is the first to characterize CATH genes in the Japanese quail, and identifies novel antimicrobial peptide sequences belonging to the cathelicidin family, which may play a role in immunity in this species.     

Development and introduction of detection dogs in surveying for scats of small Indian mongoose as invasive alien species

The small Indian mongoose was introduced to the island of Okinawa in 1910 to control rats and snakes, however, it poses a threat to many endemic species in the Yambaru forest region in Okinawa Island. An important concern in eradication is that, as much as possible, capture should be carried out without affecting native species. To protect endemic species in the Yambaru region of Okinawa Island, we trained and used detection dogs to search for mongoose scats that indicated that mongoose were present in the area. We confirmed the ability of the dogs to detect scats in positive and negative control tests in the Yambaru forest region after basic, obedience, and mongoose-scat-detection specific training. In 30 trials, the dogs found 60 of 65 scats samples. For the trained alert, the dogs carried out 59 trained alerts for the 60 scats found. In the positive and negative control tests, the dogs showed no reaction to 129 of 130 negative controls. We then surveyed the forest in the Yambaru region for mongoose scats using the detection dogs. In this study, the detection dogs found 79 scats in a survey of a 69.0-km area. We divided the survey area into 43 grids and calculated the rate of detection in each grid. The rate of detection of scats was higher in the southern part of the Yambaru region. These result showed that our mongoose scat detection dogs can be useful tools to assist in determining the presence of mongoose in the Yambaru region of Okinawa Island.     

Somatic embryogenesis and Agrobacterium-mediated transformation of Japanese apricot (Prunus mume) using immature cotyledons

This study describes a successful method of somatic embryogenesis and genetic transformation using immature cotyledons of Prunus mume. Immature cotyledons from four different developmental stages of eight different P. mume cultivars were used for the experiments to optimize somatic embryogenesis and genetic transformation protocols. Somatic embryogenesis was induced when the explants were cultured on somatic embryo inducing medium consisting of MS basic medium supplemented with 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM 6-benzyladenine (BA). They were cultured for 30 days and then transferred to somatic embryo propagation medium containing 0.1 μM α-naphthaleneacetic acid (NAA) and 5 μM BA. It appeared that the developmental stage of the immature cotyledons used as explants was the most important factor for somatic embryogenesis; higher frequencies of somatic embryogenesis were observed when the immature cotyledons were less than 5 mm in length regardless of cultivars. For genetic transformation, the immature cotyledons were inoculated with Agrobacterium tumefaciens EHA101 harbouring a binary plasmid vector with neomycin phosphotransferase II and an intron-interrupted β-glucuronidase gene under the control of cauliflower mosaic virus 35S promoter, and three transgenic plant lines were obtained from inoculated “Sirakaga” immature cotyledons. Transgenic somatic embryos and shoots were selected using 25 mg l⁻¹ kanamycin. Integration of transgenes in the genome of GUS-positive putative transgenic shoots was confirmed by PCR and Southern blot analyses.     

Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification (LAMP) with species-specific primers

A loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA ITS sequence of P. aphanidermatum was developed. Results of a specificity test using 57 strains of Pythium spp. indicated that the LAMP assay gave no cross reactions in other 39 Pythium species, 11 strains of Phytophthora spp. and eight other soil borne pathogens. The detection limit was 10 fg of genomic DNA, which was ten times the sensitivity of the polymerase chain reaction. The LAMP assay was applied to hydroponic solution samples from tomato fields, and the results were compared to those of the conventional plating method. LAMP was observed to be effective for the specific detection of P. aphanidermatum. Furthermore, P. aphanidermatum was detected directly in tomato roots infected with P. aphanidermatum without DNA extraction. The LAMP method established in this study is a simple, sensitive and rapid tool for the detection of P. aphanidermatum.     

Duration of thermal support for preventing hypothermia induced by anesthesia with medetomidine-midazolam-butorphanol in mice

Hypothermia during anesthetic events is a common adverse effect of anesthesia in laboratory animals. In particular, small rodents such as mice is susceptible to hypothermia during anesthetic events. Therefore, the animals will need additional thermal support by external heating devices during and after anesthesia. In general, the time of recovery from anesthesia is typically longer in case of injectable anesthesia rather than inhalant anesthesia. However, the durations of thermal support have been almost limited to 1 hr from administration of anesthesia in general. Our study objectives are two-fold: 1) to compare the levels of hypothermia induced by injectable anesthesia with medetomidine-midazolam-butorphanol (MMB) and inhalant anesthesia with isoflurane (ISO); 2) to find the adequate durations of thermal support for preventing hypothermia induced by their anesthesia in mice. Adult male ICR mice were anesthetized during 40 min without and with the thermal support for 1 (both anesthetic groups), 2, 3, and 5 hr (in MMB group). Without thermal support, the decrease of body temperature in MMB group were more severe than that in ISO group. The durations of thermal support completely prevented hypothermia at 5 hr-support in MMB group and that at 1 hr-support in ISO group. However, the other short durations did not prevent hypothermia at 1, 2 and 3 hr-support in MMB group. These results suggest that the mice should be received thermal support over 5 hr after injection of MMB anesthesia to prevent hypothermia.     

Microbial mineralization of organic nitrogen into nitrate to allow the use of organic fertilizer in hydroponics

Hydroponics is an excellent technique for the cultivation of vegetable crops and other plants, but organic fertilizers cannot be used in conventional hydroponic systems, which generally use only inorganic fertilizers, because organic compounds in the hydroponic solutions generally have phytotoxic effects that lead to poor plant growth. Few microorganisms are present in hydroponic solutions to mineralize the organic compounds into inorganic nutrients. In this article a novel and practical hydroponic culture method that uses microorganisms to degrade organic fertilizer in the hydroponic solution has been developed. Soil microorganisms were cultured by regulating the amounts of organic fertilizer and inoculum, with moderate aeration. The microorganisms mineralized organic nitrogen via ammonification and nitrification into nitrate at an efficiency of 97.6%. The culture solution containing the microorganisms was usable as a hydroponic solution, and organic fertilizer could be directly added to it during vegetable cultivation. Vegetables grew well in the organic hydroponic system. Organic hydroponics based on this method is therefore a practical tool for the utilization of organic sources of fertilizer.     

Internal Mechanisms of Plant Adaptation to Aluminum Toxicity and Phosphorus Starvation in Three Tropical Forages

Many tropical forage grasses and legumes grow well in acid soils, adapting to excess aluminum (Al) and phosphorus (P) starvation stresses by using mechanisms that are still unclear. To determine these mechanisms, responses to Al toxicity and P starvation in three tropical forages were studied: two grasses, Brachiaria hybrid cv. ‘Mulato’ (B. ruziziensis clone 44-06 × B. brizantha cv. ‘Marandú’) and Andropogon gayanus, and one legume, Arachis pintoi. The tropical grasses tolerated high levels of Al toxicity and P starvation, with the Brachiaria hybrid maintaining very low levels of Al concentration in shoots. ²⁷Al Nuclear Magnetic Resonance spectroscopy (NMR) analysis revealed that, in the Brachiaria hybrid, Al makes complexes with some ligands such as organic-acid anions in the root symplast. The forages probably adapted to P starvation through high P-use efficiency. These experiments provide the first direct evidence we know of that organic acid anions within root tissue help detoxify Al in non-accumulator species such as the Brachiaria hybrid.     

Stimulatory effect of aluminum on the growth of tea pollen tubes

It is well known that when inorganic elements such as boron (B), calcium, cobalt, and manganese are added to media in combination or alone, they stimulate the germination and/or tube growth of various kinds of pollen (Schmücker 1933; Loo and Hwang 1944; Yamada 1958; Brewbaker and Kwack 1963). Among these elements, the stimulatory effect ofB is the most effective and generally recognized. Aluminum (Al) belongs along with B to Group IIIa in the periodic table, and Fähnrich and Ultlich (1964) reported that Al inhibited the germination and growth of the pollen tube of Petunia hybrida and Antirrhinum majus. However, stimulatory effects of Al on pollen germination or pollen tube growth had not been previously reported.