In situ hybridization analysis of olfactory receptor expression in the sea turtle olfactory organ.

The olfactory organ of turtles consists of an upper chamber epithelium (UCE) with associated glands, and a lower chamber epithelium (LCE) devoid of glands. The UCE and LCE are referred to as the air-nose and the water-nose, respectively, because the UCE is thought to detect airborne odorants, while the LCE detects waterborne odorants. However, it is not clear how the two are used in the olfactory organ. Odorant receptors (ORs) are the major olfactory receptors in turtles; they are classified as class I and II ORs, distinguished by their primary structure. Class I ORs are suggested to be receptive to water-soluble ligands and class II ORs to volatile ligands. This study analyzed the expression of class I and II ORs in hatchlings of the green sea turtle, Chelonia mydas, through in situ hybridization, to determine the localization of OR-expressing cells in the olfactory organ. Class I OR-expressing cells were distributed mainly in the LCE, implying that the LCE is receptive to waterborne odorants. Class II OR-expressing cells were distributed in both the UCE and LCE, implying that the entire olfactory organ is receptive to airborne odorants. The widespread expression of class II ORs may increase opportunities for sea turtles to sense airborne odorants.

Helicobacter delphinicola infection and the risk of gastric disease in common bottlenose dolphin.

Gastritis and gastric ulcers are well-recognized conditions in cetaceans; bacteria of the genus Helicobacter are considered the primary cause of these diseases. Dolphins have been shown to be susceptible to infection by at least 2 gastric species of Helicobacter, H. cetorum and H. delphinicola, both of which are closely related to the human pathogen H. pylori. In the present study, we evaluated the carriage rate and relationship to gastric disease of H. cetorum and H. delphinicola, based on a study population of 82 dolphins maintained at 21 facilities in Japan. Of these 82 dolphins, 79 (96.3%) and 45 (54.9%) were positive for H. cetorum and H. delphinicola, respectively; H. delphinicola infection was significantly associated with chronic gastric diseases (odds rate: 5.9; 95% CI: 2.1-16.9), but no such association was detected for H. cetorum. Of the 21 facilities, 20 (95%) and 11 (55%) housed H. cetorum- and H. delphinicola-positive dolphins, respectively, and our study suggested that the transmission between dolphins occurs quickly within pools. These findings indicate that methods will need to be established to prevent the transmission of Helicobacter infections within facilities housing dolphins.

Genetic diversity and structure of captive gentoo penguin populations in Japan.

Until the last decade, gentoo penguins were usually split into two subspecies, northern gentoo penguins (Pygoscelis papua papua) breeding in the Falkland Islands, South Georgia, and other subantarctic islands and southern gentoo penguins (P. papua ellsworthi) breeding in the South Sandwich, South Orkney and South Shetland islands, and Antarctic Peninsula. Recent genetics research, however, suggests that the population at South Georgia is much more closely related to those further south and should be included in P. papua ellsworthi. In Japanese zoos and aquariums, captive breeding of gentoo penguins is conducted separately in three populations: "Captive-South Georgia," originating from South Georgia, "Captive-South Shetlands," originating from South Shetlands, and "Captive-Unknown," originating from at least one founder of unknown subspecies. The aims of the present study were to investigate the genetic diversity and differentiation of these captive populations using microsatellite analysis. Genetic diversity in each captive population was similar to that found in the wild, although they had much lower contemporary effective population sizes. Pairwise genetic differentiation indexes (FST ) among the three captive populations were as follows: 0.0309 ("Captive-South Georgia" and "Captive-Unknown"), 0.1094 ("Captive-South Georgia" and "Captive-South Shetlands"), and 0.1214 ("Captive-South Shetlands" and "Captive-Unknown"). Using Bayesian clustering, there was relatively high genetic differentiation between the "Captive-South Shetlands" group, which formed a distinct cluster, and individuals of the "Captive-Unknown" group, which were assigned to clusters in common with "Captive-South Georgia." The results from the present study are useful for future management of captive gentoo penguin populations in Japan.

Midazolam and butorphanol combination for sedating beluga whales (Delphinapterus leucas).

Safe sedation doses for performing minor procedures such as bronchoscopy, endoscopy, and tooth extraction for beluga whales (Delphinapterus leucas) require elucidation. This study aimed to provide suggestions for determining appropriate midazolam and butorphanol doses to adequately sedate beluga whales to complete procedures and minimize the risk of side effects. We administered midazolam and butorphanol to six captive beluga whales (9-44 years old). Topical lidocaine anesthesia was administered during bronchoscopy. The sedation doses for the beluga whales varied from 0.020 to 0.122 mg/kg for midazolam and from 0.020 to 0.061 mg/kg for butorphanol. In beluga whales, optimal midazolam and butorphanol doses were lowest in old whales. These findings contribute to knowledge regarding appropriate sedation and prevention of overdose accidents during minor procedures in beluga whales.

Bronchial Tree System Analysis of Live Beluga Whale (Delphinapterus leucas) Using Bronchoscopy.

Cetaceans, including beluga whales (Delphinapterus leucas), have high morbidity and mortality rates due to bacterial or fungal lower respiratory infections. Bronchoalveolar lavage fluid (BALF) collection by bronchoscopy is beneficial for detecting pathogenic microorganisms in the lower respiratory tract. Efficient and safe bronchoscopy requires characterizing the bronchial tree systems of beluga whales, as no reports exist on bronchial length and bifurcation. In this study, bronchoscopy was performed on five captive beluga whales (9-44 years old) to detect bronchial length and bifurcation. The lengths from the blowhole to the scope impassable points due to the minimized bronchi diameters of the left principal bronchus (LPB), right principal bronchus (RPB), and tracheal bronchus (TB) were 110-155, 110-150, and 80-110 cm, respectively, and were correlated with the body length. Bronchoscopy identified more than 10, 10, and 6 bifurcated bronchi from the LPB, RPB, and TB, respectively. This is the first report to clarify the differences in bronchial tree systems between beluga whales and other cetaceans, as well as the differences for each individual beluga whale. These results could be useful for obtaining BALF via bronchoscopy to detect pathogenic microorganisms causing infections in the lower respiratory tract of beluga whales.

In situ hybridization analysis of odorant receptor expression in the olfactory organ of the pig-nosed turtle Carettochelys insculpta.

The turtle olfactory organ consists of upper (UCE) and lower (LCE) chamber epithelium, which send axons to the ventral and dorsal portions of the olfactory bulbs, respectively. Generally, the UCE is associated with glands and contains ciliated olfactory receptor neurons (ORNs), while the LCE is devoid of glands and contains microvillous ORNs. However, the olfactory organ of the pig-nosed turtle Carettochelys insculpta appears to be a single olfactory system morphologically: there are no associated glands; ciliated ORNs are distributed throughout the olfactory organ; and the olfactory bulb is not divided into ventral and dorsal portions. In this study, we analyzed the expression of odorant receptors (ORs), the major olfactory receptors in turtles, in the pig-nosed turtle olfactory organ, via in situ hybridization. Of 690 ORs, 375 were classified as class I and 315 as class II. Some class II ORs were expressed predominantly in the posterior dorsomedial walls of the nasal cavity, while other class II ORs and all class I ORs examined were expressed in the remaining region. These results suggest that the pig-nosed turtle olfactory organ can be divided into two regions according to the expression of ORs.

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus).

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5'UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

Expression of human mutant cyclin dependent kinase 4, Cyclin D and telomerase extends the life span but does not immortalize fibroblasts derived from loggerhead sea turtle (Caretta caretta).

Conservation of the genetic resources of endangered animals is crucial for future generations. The loggerhead sea turtle (Caretta caretta) is a critically endangered species, because of human hunting, hybridisation with other sea turtle species, and infectious diseases. In the present study, we established primary fibroblast cell lines from the loggerhead sea turtle, and showed its species specific chromosome number is 2n = 56, which is identical to that of the hawksbill and olive ridley sea turtles. We first showed that intensive hybridization among multiple sea turtle species caused due to the identical chromosome number, which allows existence of stable hybridization among the multiple sea turtle species. Expressions of human-derived mutant Cyclin-dependent kinase 4 (CDK4) and Cyclin D dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. The recombinant fibroblast cell lines maintained the normal chromosome condition and morphology, indicating that, at the G1/S phase, the machinery to control the cellular proliferation is evolutionally conserved among various vertebrates. To our knowledge, this study is the first to demonstrate the functional conservation to overcome the negative feedback system to limit the turn over of the cell cycle between mammalian and reptiles. Our cell culture method will enable the sharing of cells from critically endangered animals as research materials.

Purification and Characterization of Sperm Creatine Kinase and Guanylate Cyclase of the Sea Urchin Hemicentrotus pulcherrimus.

Creatine kinase and guanylate cyclase were purified from Hemicentrotus pulcherrimus spermatozoa. The molecular weight of the purified sperm tail creatine kinase was estimated to be 137,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sperm tail guanylate cyclase was purified by chromatography on a WGA-Sepharose column connected to a Concanavalin A-Sepharose column, and a Superose 12 HR column. The molecular weight of the tail guanylate cyclase was estimated to be 128,000 by SDS-PAGE. The specific activity of the purified enzyme was 8.25 µmol of cGMP formed/min/mg protein. Sperm-activating peptide I (SAP-I) causes an electrophoretic mobility shift of H. pulcherrimus sperm guanylate cyclase from 131 kDa to 128 kDa. The 131 kDa form of guanylate cyclase was co-purified with a 76 kDa protein, whose molecular mass is similar to that of a SAP-I receptor. The purified 131 kDa form of guanylate cyclase had higher activity than the 128 kDa form. The 131 kDa and 128 kDa forms of guanylate cyclase contained 23.83 ± 0.65 and 4.16 ± 0.45 moles of phosphate per mol protein (mean ± S.D.; n = 3), respectively. The activities of guanylate cyclase and creatine kinase increased during the testis development. During spermatogenesis, sperm tail creatine kinase was detected immunohistochemically only in mature spermatozoa.

Induction of the Acrosome Reaction of Hemicentrotus pulcherrimus Spermatozoa by the Egg Jelly Molecules, Fucose-Rich Glycoconjugate and Spem-Activating Peptide I: (glycoconjugate/sperm-activating peptide/acrosome reaction/sea urchin/fertilization).

A fucose-rich glycoconjugate (FRG) was isolated from egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel filtration. FRG induced the acrosome reaction in H. pulcherrimus spermatozoa in a concentration-dependent manner, although it showed about half the activity of the original unfractionated jelly. Synthetic sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) increased the rate of the acrosome reaction induced by FRG; the maximal rate of the acrosome reaction with FRG and SAP-I being that of the unfractionated jelly. The half-maximal increase in induction of the acrosome reaction by SAP-I with FRG occurred at 4 × 10-10 M SAP-I, which was almost the same concentration inducing half-maximal stimulation of sperm respiration. Pronase digestion of FRG resulted in an 50% decrease in induction of the acrosome reaction and also in the elevation of cAMP in sperm. Some reagents (monensin and 3-isobutyl-1-methylxanthine) which increase intracellular pH, Ca2+ and cyclic nucleotides also increased the rates of the acrosome reaction induced by FRG or pronase-digested FRG. However, the rates did not reach those with FRG or pronase-digested FRG with SAP-I. These results indicate that SAP-I promotes induction of the acrosome reaction by acting as a specific co-factor of FRG.

The Participation of Speract in the Acrosome Reaction of Hemicentrotus pulcherrimus: (speract/sea urchin/sperm/acrosome reaction/cyclic nucleotides).

A speract-free macromolecular fraction was prepared from the egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel filtration and tested for ability to induce the acrosome reaction in H. pulcherrimus spermatozoa with or without exogenously added synthetic speract. The macromolecular fraction without speract showed only about half the activity of the original unfractionated jelly for induction of the acrosome reaction. The rates of the acrosome reaction induced by the fraction with speract were comparable with those induced by the unfractionated jelly at all pHs tested. Speract itself, however, did not induce the acrosome reaction in the absence of the macromolecular fraction of jelly. The acrosome reaction was associated with incerase of the cyclic AMP concentration in sperm cells, the extent of incerase depending on the concentration of the macromolecular fraction. Addition of speract to the fraction enhanced both induction of the acrosome reaction and increase in the cyclic AMP concentration induced by the fraction. These results suggest that a major factor(s) responsible for the acrosome reaction is a macromolecular component(s) of the jelly and that speract promotes the reaction as a co-factor.

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti).

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.