Metabarcoding of feces and intestinal contents to determine carnivorous diets in red-crowned cranes in eastern Hokkaido,Japan

The red-crowned crane Grus japonensis in Hokkaido, Japan forms a closed population as a residence that is independent of the mainland population. Based on observations of a limited number of individuals as well as cranes in captivity, red-crowned cranes are omnivores and eat fish, worms, insects and plants in their own territories except in winter, when they are fed with dent corn that is supplied in eastern Hokkaido. DNA metabarcoding based on high throughput sequencing was carried out using universal primer sets for cytochrome oxidase subunit I gene. Feces from 27 chicks collected in June and July in the period from 2016 to 2018 and intestinal contents from 33 adult and subadult cranes that were found dead almost throughout year in 2006–2013 in the field in eastern Hokkaido were used. Although compositions varied considerably in the cranes, both insects and fish were found in adults and subadults to the same extents, while insects were predominant in chicks. Both insects and fish were detected in all seasons for adults and subadults. Horse flies, scarab beetles and weevils accounted for the most of the insects regardless of the life stage. Dace, stickleback, flatfish and sculpin were the major fish species in adults, while chicks ate almost only stickleback. The results provide the first comprehensive data on carnivorous diets in wild red-crowned cranes in eastern Hokkaido as basis for conservation of red-crowned cranes, for which the life style and area continue to change.     

Changes of plasma free amino acid concentrations and myofibrillar proteolysis index by starvation in non-pregnant dry cows

The effects of 5 day starvation on urinary metabolite excretion, plasma metabolites and free amino acid concentrations in four non‐pregnant dry cows were investigated. Starvation significantly reduced (P < 0.05) the bodyweight and feces weight. The plasma glucose concentration decreased (P < 0.05) during days 2–5 of starvation. Starvation significantly increased (P < 0.05) the plasma nonesterified fatty acid concentration. The plasma urea nitrogen concentration and urinary urea nitrogen excretion increased transiently (P < 0.05) at days 1 and 2 of starvation. The plasma 3‐methlhistidine concentration was significantly higher (P < 0.05) at days 2, 3 and 5 of starvation. However, the urinary 3‐methylhistidine excretion was significantly higher (P < 0.05) at days 4 and 5 of starvation, not corresponding to the change in the plasma. Starvation did not change the plasma proline, methionine, lysine and total amino acid concentrations. On the other hand, starvation reduced (P < 0.05) the plasma aspartic acid, serine, asparagine, glutamic acid, glutamine, alanine, tyrosine, tryptophan and histidine concentrations. Plasma glycine increased during starvation. Plasma threonine, isoleucine, leucine and branched‐chain amino acid increased (P < 0.05) by prolonged starvation. The results of this study indicate that prolonged starvation accelerated myofibril protein degradation in non‐pregnant dry cows and apparently consumes most glucogenic amino acids in preference to other amino acids.     

Spawning behaviour of amemasu charr,Salvelinus leucomaenis leucomaenis,with a discussion of the macroevolutionary patterns of postspawning behaviour in the Salmoninae

Abstract – Video cameras inside underwater housings were used to record the spawning behaviour of a subspecies of the white‐spotted charr, the amemasu charr (Salvelinus leucomaenis leucomaenis) in the Tokimae and Onbetsu Rivers, Hokkaido, Japan. Unlike other Salvelinus species, in which females use lateral swings of their bodies to ventilate and distribute eggs over gravel crevices (undulating) immediately after spawning, female amemasu charr covered their eggs with beats of their tails (cover digging). Cover digging right after spawning has previously been documented in Salmo, Oncorhynchus and Parahucho. Phylogenetic analysis of postspawning behaviours in the Salmoninae indicated that (i) resting then covering the eggs is plesiomorphic for the group, (ii) undulating replaced resting in the ancestor of Salvelinus, (iii) cover digging was advanced in the behavioural sequence to occur immediately after egg deposition in the ancestor of Parahucho + Salmo + Oncorhynchus and in the amemasu.     

Quantitative PCR assay for the detection of the parasitic ciliate <Emphasis Type="Italic">Cryptocaryon irritans</Emphasis>

We developed a quantitative PCR assay for detecting the parasitic ciliate Cryptocaryon irritans, which causes “white spot disease” in marine fishes, from the natural environment. A specific primer set for C. irritans was designed and its high specificity was confirmed in silico: almost all of the sequences deposited in the GenBank nucleotide database were covered, 22/23 for the forward primer and 7/7 for the reverse primer. We estimated that there were 3,415.9 rRNA gene copies per genome of C. irritans. In artificial mixture experiments to validate whether the qPCR assay is applicable to natural samples, the estimated copy numbers showed significantly positive correlations with the number of theronts added (p < 0.001). When we applied this qPCR assay to natural samples collected bimonthly from surface and bottom seawaters at an aquaculture site (water depth, 10 m) from May 2009 to March 2010, we only detected C. irritans (112.0 ± 6.3 cells/l) in the surface seawater sample in November. This qPCR assay is a useful tool for detecting C. irritans rapidly and quantitatively in natural environments; it could also help advance our understanding of the ecology of C. irritans, as well as facilitate the diagnosis of the disease.     

Expression profiles of interferon gamma genes in response to immunostimulants and alloantigen in ginbuna crucian carp Carassius auratus langsdorfii

Interferon gamma (IFNγ), a Th1-derived cytokine is one of the key molecules inducing cell-mediated immune responses in mammals. The expression of 2 distinct IFNγ (ifng1 and ifng2) and IFNγrel (ifngrel) genes was examined in kidney leukocytes from clonal ginbuna crucian carp Carassius auratus langsdorfii. The expression of IFNγ and IFNγrel genes was induced in kidney leukocytes by stimulation with lipopolysaccharide or phytohemagglutinin in a dose-dependent manner. The expression levels of IFNγ and IFNγrel genes in kidney leukocytes from allo-sensitized fish cocultured with allogeneic cells were higher than those from fish cocultured with isogeneic cells. The highest expression of ifng1 was observed at day 1, whereas that of ifng2 and ifngrel was detected at day 2 after cocultivation with allogeneic cells. However, the expression pattern of ifng2 and ifngrel in kidney leukocytes from allo-sensitized fish by scale-grafting was similar to those from non-sensitized fish. These results indicate that ifng1 is a major isoform of IFNγ related to antigen-specific cell-mediated immunity (CMI), and will be a useful marker to evaluate induction of CMI in ginbuna crucian carp. In addition, IFNγ would be a crucial type-II interferon compared to IFNγrel, relevant to cell-mediated immunity in fish as in mammals.     

Mineral composition of leaves and bark in aluminum accumulators in a tropical rain forest in Indonesia

Mineral composition including AI, Ca, Mg, P, S, and Si and relationships between Al and other elements such as Ca, Mg, P, S, and Si in leaves and bark of trees in a tropical rain forest in West Sumatra were studied. Sixty five tree species and 12 unidentified trees were referred to as AI accumulators based on Chenery\s's definition (more than 1 g kg^-1 Al in leaves). For most of the Al accumulators, Al concentration in leaves was higher than in bark. However, some members of Euphorbiaceae, Melastomataceae, and Ulmaceae families showed a reverse trend. Most of the non-accumulators also showed a higher Al concentration in bark than in leaves. These results indicated that there was a difference in the mechanism of Al accumulation in tree bodies. Some of the Al accumulators showed an extremely high Al concentration (more than 10 g kg^-1) not only in the mature leaves, but also in the new leaves. Analysis of the relationships between the concentration of Al and the other 5 elements in leaves, revealed that Al accumulators could be separated into two groups at the Al concentration of 3 g kg^-1. This finding suggested that new criteria based on Al concentration (23 g kg^-1) or Al/Ca ratio in leaves could be proposed in order to define Al accumulators, apart from Chenery's criterion. Aluminium accumulators with an Al concentration in leaves lower than 3 g kg^-1 (AI accumulators <3 g kg^-1) showed the same trend as the non-accumulators in terms of these elemental relationships, while Al accumulators with an Al concentration in leaves higher than 3 g kg^-1 (AI accumulators 23 g kg^-1) showed a different trend from the non-accumulators. The Al accumulators 23 g kg^-1 and the other trees (AI accumulators < 3 g kg^-1 and non-accumulators) showed separately positive correlations between the concentrations of AI and Ca (or Mg) in the leaves. This observation seems to be opposite to general findings in plant nutrition, i.e. inhibition of Ca or Mg uptake by AI. A positive correlation between Al and S was also observed for all the trees. The Al accumulators ≥3 g kg^-1 showed positive correlations between the concentrations of Al and P (or Si) in the leaves, unlike the other trees. These findings suggested that Al stimulated P, S, or Si accumulation in leaves or Al was transported with P, S, or Si for the Al accumulators ≥3 g kg^-1. No negative relationships between Al and the other 5 elements in the leaves were observed for the Al accumulators ≥3 g kg^-1.     

Alpha-fetoprotein in serum and tumor tissues in dogs with hepatocellular carcinoma.

Serum alpha-fetoprotein (AFP) concentrations were measured before and after surgical removal of tumor masses in four dogs with hepatocellular carcinoma (HCC). Localization of AFP was also examined immunohistochemically in tumor tissues. In three cases, the serum AFP concentration was 10 to 20 times higher than that of normal dogs. One to two months after surgery, the serum AFP concentration had decreased to normal range. AFP was localized in the tumor tissues in these three cases. One case, which had a low serum AFP, did not show AFP localization in tumor tissue.     

Molecular detection and characterization of Haemobartonella felis in domestic cats in Japan employing sequence-specific polymerase chain reaction (SS-PCR)

A novel PCR assay was developed in order to examine the prevalence of Haemobartonella felis (H. felis) in Japanese domestic cats and which was able to differentiate of the Ohio strain and the California strain of H. felis. Blood samples from a total of 21 cats suspected of having haemobartonellosis were examined employing a novel PCR assay and demonstrated positive results in 18 cats which was confirmed by cytological examination of blood smears. Four out of 18 positive cats (22%) were infected with the California strain, whilst the other 12 cats (67%) were infected with the Ohio strain and two animals (11%) were infected with both strains. As most of the cats with moderate to severe anemia were infected with the Ohio strain, it is suggested that the most prevalent strain of H. felis in Japanese domestic cats might be the Ohio strain. In the present study, it was thought that molecular detection and characterization of H. felis may provide valuable information regarding the severity and prognosis of this illness.     

Dysferlin and animal models for dysferlinopathy

Dysferlin (DYSF) is involved in the membrane-repair process, in the intracellular vesicle system and in T-tubule development in skeletal muscle. It interacts with mitsugumin 53, annexins, caveolin-3, AHNAK, affixin, S100A10, calpain-3, tubulin and dihydropyridine receptor. Limb-girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy (MM) are muscular dystrophies associated with recessively inherited mutations in the DYSF gene. The diseases are characterized by weakness and muscle atrophy that progress slowly and symmetrically in the proximal muscles of the limb girdles. LGMD2B and MM, which are collectively termed "dysferlinopathy", both lead to abnormalities in vesicle traffic and membrane repair at the plasma membrane in muscle fibers. SJL/J (SJL) and A/J mice are naturally occurring animal models for dysferlinopathy. Since there has been no an approach to therapy for dysferlinopathy, the immediate development of a therapeutic method for this genetic disorder is desirable. The murine models are useful in verification experiments for new therapies and they are valuable tools for identifying factors that accelerate dystrophic changes in skeletal muscle. It could be possible that the genetic or immunological background in SJL or A/J mice could modify muscle damage in experiments involving these models, because SJL and A/J mice show differences in the progress and prevalent sites of skeletal muscle lesions as well as in the gene-expression profiles of their skeletal muscle. In this review, we provide up-to-date information on the function of dysferlin, the development of possible therapies for muscle dystrophies (including dysferlinopathy) and the detection of new therapeutic targets for dysferlinopathy by means of experiments using animal models for dysferlinopathy.