Cultured fibroblasts of the Okinawa rail present delayed innate immune response compared to that of chicken.

The Okinawa rail is endemic to Okinawa Island and is categorized as an endangered animal. In this study, we focused on innate immunity because it is the first line of host defense. In particular, signals recognizing foreign RNA (e.g., viruses) are important for host defense because they activate the host immune system. The retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) families (RIG-I, MDA5, and LGP2) are sensors that activate innate immunity. Therefore, we analyzed these functions in the Okinawa rail using genomic and cellular analyses of fibroblasts. Fibroblasts can be obtained from dead individuals, allowing these cells to be obtained from dead individuals, which is particularly useful for endangered species. The MDA5 gene of Okinawa rail was sequenced using the Sanger method following PCR amplification and extraction of the amplified sequence from agarose gel. Additionally, mRNA expression analysis of cultured fibroblasts exposed to poly I:C was done. The MDA5 gene was found to be a mutated nonfunctional gene in the Okinawa rail. The mRNA expression rates of inflammatory cytokine genes type I IFN, and Mx1 were slower in Okinawa rail than in chicken cultured fibroblasts. Similar to the mRNA expression results, cell number and live cell ratio also slowly decreased in the Okinawa rail compared with chicken cultured fibroblasts, indicating that the innate immune reaction differs between chicken and the Okinawa rail. To the best of our knowledge, this is the first experimental evaluation of the loss of function of the Okinawa rail innate immune genes. In conclusion, our results provide a basis for conservation strategies for the endangered Okinawa rail.

Induced pluripotent stem cells of endangered avian species.

The number of endangered avian-related species increase in Japan recently. The application of new technologies, such as induced pluripotent stem cells (iPSCs), may contribute to the recovery of the decreasing numbers of endangered animals and conservation of genetic resources. We established novel iPSCs from three endangered avian species (Okinawa rail, Japanese ptarmigan, and Blakiston's fish owl) with seven reprogramming factors (M3O, Sox2, Klf4, c-Myc, Nanog, Lin28, and Klf2). The iPSCs are pluripotency markers and express pluripotency-related genes and differentiated into three germ layers in vivo and in vitro. These three endangered avian iPSCs displayed different cellular characteristics even though the same reprogramming factors use. Japanese ptarmigan-derived iPSCs have different biological characteristics from those observed in other avian-derived iPSCs. Japanese ptarmigan iPSCs contributed to chimeras part in chicken embryos. To the best of our knowledge, our findings provide the first evidence of the potential value of iPSCs as a resource for endangered avian species conservation.

Antifungal drugs-low-susceptibility in Aspergillus flavus isolated from a captively reared Okinawa rail (Hypotaenidia okinawae).

A four-month old female Okinawa rail (Hypotaenidia okinawae) presented with respiratory distress. Despite antifungal treatment with voriconazole (VRZ), micafungin (MCF), and itraconazole (ITZ), respiratory distress did not improve and the bird died 167 days after initiating treatment. Necropsy revealed multifocal pyogranulomatous necrotic nodular lesions with numerous whitish-green fungal hyphae in the left air sac. Aspergillus flavus was isolated from the left air sac lesion. Antifungal susceptibility tests indicated that the isolate showed low susceptibility to amphotericin B (AMB), fluconazole (FLZ), VRZ and MCF.

Correction: Establishment of immortalized primary cell from the critically endangered Bonin flying fox (Pteropus pselaphon).

[This corrects the article DOI: 10.1371/journal.pone.0221364.].

Establishment of immortalized primary cell from the critically endangered Bonin flying fox (Pteropus pselaphon).

The Bonin flying fox (Pteropus pselaphon) is one of the most critically endangered species of animals. The number of this species is estimated to be around 150; being classified at the top rank in the list by International Union of Animal Conservation. Our group previously showed that expression of CDK4, CYCLIN D1, and telomerase reverse transcriptase (TERT) efficiently induce immortalization of human, bovine, swine, monkey, and buffalo-derived cells. In this manuscript, we successfully established the primary cells from Bonin flying fox. We introduced CDK4, CYCLIN D1, and TERT into the primary cells. The established cells showed efficient expression of introduced genes at the protein level. Furthermore, the established cells were free from senescence, indicating it reached to immortalization. Moreover, we showed that interspecies somatic cell nuclear transfer of Bonin flying fox derived cell into bovine embryo allowed the development of the embryo to 8 cell stages. Our established cell has the potential to contribute to species conservation.

Reproductive traits of the Ryukyu long-furred rat (Diplothrix legata) on Okinawa-jima Island.

The Ryukyu long-furred rat, Diplothrix legata, is a large rodent distributed only on Amami-ohshima Island, Tokuno-shima Island and Okinawa-jima Island, Japan. This animal is endangered as a result of deforestation, predation by introduced carnivores and mortality caused by vehicles. We performed theriogenological examinations of 32 male and 25 female Ryukyu long-furred rats carcasses collected from wild populations on northern Okinawa-jima Island from December 2005 to September 2013. Adult males had remarkably large preputial glands. Seminiferous diameter of adult was significantly small (136 ± 28 µm, n=8) from April to August. Numerous spermatozoa were observed from September through February, and seminiferous diameter was significantly large (216 ± 27 µm, n=12) during this time in adults; testes length changed in a similar pattern. These findings indicate that the mating season may occur from September through February. Size (body length) at sexual maturity was estimated to be >560 mm in both sexes. From observation of corpora lutea and placental scars, litter size was estimated to range from 2 to 12 (average=6, n=4). These results provide fundamental knowledge that will be beneficial for in situ and ex situ conservation of this rare species.

Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in the Ryukyu Islands tree rat (Diplothrix legata).

Angiostrongylus cantonensis is a zoonotic nematode with rodents serving as natural definitive hosts. We report A. cantonensis in the Ryukyu Islands tree rat (Diplothrix legata, Thomas, 1906), a native endangered species in Japan. Adult and larvae of A. cantonensis were macroscopically, histologically, and genetically detected in three tree rats collected between August 2011 and January 2012 in the Yambaru area of Okinawa Prefecture, Japan. Pathologic observations of the lungs of rats showed that infection may be lethal. We also conducted a retrospective genetic survey of helminths parasitic in lung in cryopreserved lung samples of Ryukyu Islands tree rats collected between 2007 and 2011 in the Yambaru area and found A. cantonensis DNA in one of 29 samples, which was collected in December 2010.

Reproductive cycle observation of the Okinawa rail (Gallirallus okinawae) in the wild.

The captive breeding program of the Okinawa rail started in 2008. For successful captive breeding, information related to reproduction, such as age at sexual maturity, testicular cycles and ovulatory cycles, is essential to predict when reproduction is possible and when certain reproductive behaviors are most likely to occur. We made gross and histological observations of the reproductive organs of Okinawa rails to gain understanding of sexual maturity, the testicular cycle and the ovulatory cycle. We found that the weight of the testis was smallest in December and largest in March. Changes in the diameter of the seminiferous tubule showed the same pattern. Mature sperm were observed from March to June. The heaviest ovary was observed in April. A single peak of reproduction, from March to April, was observed in males and females. Our observations suggested that the Okinawa rail is a seasonal breeder. Establishing suitable breeding pairs will be critical to ensure success of the Okinawa rail captive breeding program. Our results suggested that pairing must be started before March. If supportive breeding is used, semen should be collected from March to June and artificial insemination conducted in April.

T-cell lymphoma in a wild Okinawa rail (Gallirallus okinawae).

The Okinawa rail (Gallirallus okinawae) is an endangered species that inhabits the northern part of Okinawa Main Island in southern Japan. A wild Okinawa rail was rescued from a road in Kunigami Village in Okinawa in October 2009. The bird subsequently died and underwent necropsy. Tumors were found in the liver, spleen and part of the small intestine. Microscopically, lymphoid neoplasm was confirmed in these tissues. The tumor cells were mainly positive for CD3 and CD8α by immunohistochemistry. No Marek's disease virus genes were detected by PCR of a liver tumor. This is the first report of T-cell lymphoma in the Okinawa rail.

Receptor-activating peptides for PAR-1 and PAR-2 relax rat gastric artery via multiple mechanisms.

Receptor-activating peptides for protease-activated receptors (PARs) 1 or 2 enhance gastric mucosal blood flow (GMBF) and protect against gastric mucosal injury in rats. We thus examined and characterized the effects of PAR-1 and PAR-2 agonists on the isometric tension in isolated rat gastric artery. The agonists for PAR-2 or PAR-1 produced vasodilation in the endothelium-intact arterial rings, which was abolished by removal of the endothelium. The mechanisms underlying the PAR-2- and PAR-1-mediated relaxation involved NO, endothelium-derived hyperpolarizing factor (EDHF) and prostanoids, to distinct extent, as evaluated by use of inhibitors of NO synthase, cyclo-oxygenase and Ca2+-activated K+ channels. The EDHF-dependent relaxation responses were significantly attenuated by gap junction inhibitors. These findings demonstrate that endothelial PAR-1 and PAR-2, upon activation, dilate the gastric artery via NO and prostanoid formation and also EDHF mechanisms including gap junctions, which would enhance GMBF.

Distinct roles for protease-activated receptors 1 and 2 in vasomotor modulation in rat superior mesenteric artery.

OBJECTIVE: Protease-activated receptors (PARs) 1 and 2 are expressed in various blood vessels including rat aorta, modulating vascular tone. We investigated the roles of PAR-1 and PAR-2 in vasomotor modulation in rat superior mesenteric artery. METHODS AND RESULTS: Effects of the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-amide) and the PAR-1-activating peptide Thr-Phe-Leu-Leu-Arg-amide (TFLLR-amide) on isometric tension were examined in isolated rat superior mesenteric artery or aorta. Both SLIGRL-amide and TFLLR-amide caused relaxation in the precontracted rat aortic rings. The latter peptide, but not the former, produced contraction in the resting rings. NG-nitro-L-arginine methyl ester (L-NAME), but not apamin/charybdotoxin known to block the endothelium-derived hyperpolarizing factor (EDHF) pathway, abolished the relaxation and facilitated the contraction. In the precontracted rat superior mesenteric artery, SLIGRL-amide, but not TFLLR-amide, elicited endothelium-dependent relaxation, which was only partially inhibited by L-NAME with and without indomethacin. The residual relaxation was abolished by apamin/charybdotoxin. Carbenoxolone, a gap junction inhibitor, significantly attenuated the SLIGRL-amide-evoked, EDHF-dependent relaxation, although neither 17-octadecynoic acid, a P450 epoxygenase inhibitor, nor catalase, a hydrogen peroxide scavenger, revealed inhibitory effects. The residual response resistant to carbenoxolone was unaffected by ouabain/BaCl2. In the resting artery, TFLLR-amide, but not SLIGRL-amide, produced only slight contraction, which was dramatically facilitated by combination of L-NAME and apamin/charybdotoxin or by removal of the endothelium. CONCLUSIONS: Our data suggest that, in rat superior mesenteric artery, endothelial PAR-2, upon activation, causes relaxation via both NO and EDHF pathways, and that activation of muscular PAR-1 exhibits potential contractile activity that is largely masked by NO and EDHFs pathways triggered by endothelial PAR-1. Gap junctions might be involved in the EDHF mechanisms in this artery.

A protective role of protease-activated receptor 1 in rat gastric mucosa.

BACKGROUND AND AIMS: On activation, protease-activated receptor (PAR)-2 modulates multiple gastric functions and exerts mucosal protection via activation of sensory neurons. The role of PAR-1, a thrombin receptor, in the stomach remains unknown. We thus examined if the PAR-1 agonist could protect against gastric mucosal injury in rats. METHODS: Gastric mucosal injury was created by oral administration of ethanol/HCl or absolute ethanol in conscious rats. Gastric mucosal blood flow and acid secretion were determined in anesthetized rats. Immunohistochemical analyses of PAR-1 and cyclooxygenase (COX)-1 were also performed in rat and human stomach. RESULTS: The PAR-1 agonist TFLLR-NH(2), administered intravenously in combination with amastatin, protected against the gastric mucosal injury induced by ethanol/HCl or absolute ethanol. The protective effect of TFLLR-NH(2) was abolished by indomethacin or a COX-1 inhibitor but not by ablation of sensory neurons with capsaicin. TFLLR-NH(2) produced an NO-independent increase in gastric mucosal blood flow that was partially inhibited by blockade of the endothelium-derived hyperpolarizing factor pathway. This inhibitory effect was promoted by indomethacin. TFLLR-NH(2) suppressed carbachol-evoked acid secretion in an indomethacin-reversible manner. Immunoreactive PAR-1 and COX-1 were expressed abundantly in rat gastric muscularis mucosae and smooth muscle, and the former protein was also detectable in blood vessels. Similar staining was observed in human gastric muscularis mucosae. CONCLUSIONS: The PAR-1 agonist, given systemically, protects against gastric mucosal injury via COX-1-dependent formation of prostanoids, modulating multiple gastric functions. Our data identify a novel protective role for PAR-1 in gastric mucosa, and the underlying mechanism is entirely different from that for PAR-2.

Involvement of EDHF in the hypotension and increased gastric mucosal blood flow caused by PAR-2 activation in rats.

1. Agonists for protease-activated receptor-2 (PAR-2) cause hypotension and an increase in gastric mucosal blood flow (GMBF) in vivo. We thus studied the mechanisms underlying the circulatory modulation by PAR-2 activation in vivo, especially with respect to involvement of endothelium-derived hyperpolarizing factor (EDHF). 2. Arterial blood pressure and GMBF were measured in anesthetized rats in vivo. Vascular relaxation was assessed in the precontracted rat gastric arterial rings in vitro. 3. The PAR-2-activating peptide SLIGRL-NH2 and/or trypsin, administered i.v., produced largely NO-independent hypotension and increase in GMBF accompanied by decreased gastric mucosal vascular resistance (GMVR) in rats. 4. Combined administration of apamin and charybdotoxin, but not each of them, specifically abolished the hypotension, increased GMBF and decreased GMVR caused by the PAR-2 agonists. 5. In the isolated rat gastric artery, SLIGRL-NH2 elicited endothelium-dependent relaxation even in the presence of an NO synthase inhibitor and indomethacin, which was abolished by apamin plus charybdotoxin. 6. Our data suggest involvement of apamin/charybdotoxin-sensitive K+ channels in the PAR-2-triggered hypotension and increased GMBF, predicting a role of EDHF-like factors.