A Comparative Study on the Microstructures, Mineral Content, and Mechanical Properties of Non-Avian Reptilian Eggshells.

We analyze 214 freshly laid eggs belonging to 16 species across three orders of Class Reptilia. Using mechanical compression tests, we measure each egg's absolute stiffness (K, unit: N m-1) and relative stiffness (C number). The effective Young's modulus, E, was obtained by combining experimental and numerical methods. The mineral (CaCO3) content was measured by acid-base titration, the microstructures by scanning electron microscopy (SEM), and the crystallography by electron backscatter diffraction (EBSD). We find that the C number of reptilian eggs is, on average, higher than that of bird eggs, indicating that reptilian eggs are stiffer with respect to the egg mass than birds. However, Young's moduli of the reptilian eggshells (32.85 ± 3.48 GPa) are similar to those of avian eggshells (32.07 ± 5.95 GPa), even though those eggshells have different crystal forms, microstructures, and crystallography. Titration measurement shows that the reptilian eggshells are highly mineralized (>89% for nine Testudines species and 96% for Caiman crocodilus). Comparing the species with aragonite and calcite crystals, we find that calcite shells, including those of the Kwangsi gecko (inner part) and spectacled caiman (outer part), generally have larger grains than the aragonite ones. However, the grain size is not correlated to the effective Young's modulus. Also, as measured by the C number, the aragonite shells are, on average, stiffer than the calcite ones (except for the Kwangsi gecko), primarily due to their thicker shells.

Responses of medaka (Oryzias latipes) ammonia production and excretion to overcome acidified environments.

Anthropogenic acidification of water is an on-going environmental disaster for freshwater fishes. Fishes rely on ammonia excretion to eliminate the excess acid and mitigate the harmful effects; however, it remains largely unknown how ammoniagenesis occurs and is coordinated with ammonia excretion upon acidic stress. Medaka (Oryzias latipes) was used to examine the effects of acidic stress on ammonia production and excretion. We reveal an undiscovered ammonia-producing cell type that is rich in glutaminase (GLS) and located adjacent to the ammonia-excreting ionocytes, Na+/H+ exchanger (NHE) cells, in the gills. The gills, comparing with other ammoniagenetic organs, is the quickest to respond to the acidic stress by triggering GLS-dependent ammonia production. The unique division of labor between GLS and NHE cells in the gills allows medaka to simultaneously upregulate GLS activity and ammonia excretion shortly after exposure to acidic environments. Pharmacological experiment with a GLS inhibitor abolished the activated ammonia excretion, further suggesting the essential role of the unique feature in the responses to acidic stress. Our study shades light on a novel physiological mechanism to timely and efficiently mitigate adverse effects of acidification, providing a new way to assess the impact of on-going environmental acidification on fish.

External light-dark cycle shapes gut microbiota through intrinsically photosensitive retinal ganglion cells.

Gut microbiota are involved in many physiological functions such as metabolism, brain development, and neurodegenerative diseases. Many microbes in the digestive tract do not maintain a constant level of their relative abundance but show daily oscillations under normal conditions. Recent evidence indicates that chronic jetlag, constant darkness, or deletion of the circadian core gene can alter the composition of gut microbiota and dampen the daily oscillation of gut microbes. However, the neuronal circuit responsible for modulating gut microbiota remained unclear. Using genetic mouse models and 16s rRNA metagenomic analysis, we find that light-dark cycle information transmitted by the intrinsically photosensitive retinal ganglion cells (ipRGCs) is essential for daily oscillations of gut microbes under temporal restricted high-fat diet conditions. Furthermore, aberrant light exposure such as dim light at night (dLAN) can alter the composition, relative abundance, and daily oscillations of gut microbiota. Together, our results indicate that external light-dark cycle information can modulate gut microbiota in the direction from the brain to the gut via the sensory system.

Egg Incubation Mechanics of Giant Birds.

Finite element analysis (FEA) was used to conduct mechanical analyses on eggshells of giant birds, and relate this to the evolution and reproductive behavior of avian species. We aim to (1) investigate mechanical characteristics of eggshell structures of various ratite species, enabling comparisons between species with or without reversed sexual size dimorphism (RSSD); (2) quantify the safety margin provided by RSSD; (3) determine whether the Williams' egg can have been incubated by an extinct giant bird Genyornis newtoni; (4) determine the theoretical maximum body mass for contact incubation. We use a dimensionless number C to quantify relative shell stiffness with respect to the egg size, allowing for comparison across wide body masses. We find that RSSD in moas significantly increases the safety margin of contact incubation by the lighter males. However, their safety margins are still smaller than those of the moa species without RSSD. Two different strategies were adopted by giant birds-one is RSSD and thinner shells, represented by some moa species; the other is no RSSD and regular shells, represented by the giant elephant bird. Finally, we predicted that the upper limit of body mass for contact incubation was 2000 kg.

Body-size Scaling is Related to Gut Microbial Diversity, Metabolism and Dietary Niche of Arboreal Folivorous Flying Squirrels.

Thermal homeostasis of mammals is constrained by body-size scaling. Consequently, small mammals require considerable energy to maintain a high mass-specific metabolic rate (MSMR) and sustain target body temperature. In association with gut microbiota, mammalian hosts acquire absorbable molecules and fulfill their metabolic requirements. Our objective was to characterize gut microbes in wild mammals and relate those findings to host body-size scaling. Two large (Petaurista philippensis grandis and P. alborufus lena), one medium (Trogopterus xanthipes) and one small (Pteromys volans orii) species of flying squirrels (FS) were studied. Using 16S rRNA genes, 1,104 OTUs were detected from four FS, with 1.99% of OTUs shared among all FS. Although all FS gut microbiota were dominated by Firmicutes, they were constituted by different bacterial families. Moreover, Bacteroidetes accounted for up to 19% of gut microbiota in small FS, but was absent in large FS. Finally, based on metagenome predictions, carbohydrate and amino acid metabolism genes were enriched in small body-size FS. In conclusion, gut microbiota compositions and predictive metabolic functions were characteristic of body-size in FS, consistent with their adaptations to folivorous dietary niches.

The gene structure and hypervariability of the complete Penaeus monodon Dscam gene.

Using two advanced sequencing approaches, Illumina and PacBio, we derive the entire Dscam gene from an M2 assembly of the complete Penaeus monodon genome. The P. monodon Dscam (PmDscam) gene is ~266 kbp, with a total of 44 exons, 5 of which are subject to alternative splicing. PmDscam has a conserved architectural structure consisting of an extracellular region with hypervariable Ig domains, a transmembrane domain, and a cytoplasmic tail. We show that, contrary to a previous report, there are in fact 26, 81 and 26 alternative exons in N-terminal Ig2, N-terminal Ig3 and the entirety of Ig7, respectively. We also identified two alternatively spliced exons in the cytoplasmic tail, with transmembrane domains in exon variants 32.1 and 32.2, and stop codons in exon variants 44.1 and 44.2. This means that alternative splicing is involved in the selection of the stop codon. There are also 7 non-constitutive cytoplasmic tail exons that can either be included or skipped. Alternative splicing and the non-constitutive exons together produce more than 21 million isoform combinations from one PmDscam locus in the P. monodon gene. A public-facing database that allows BLAST searches of all 175 exons in the PmDscam gene has been established at http://pmdscam.dbbs.ncku.edu.tw/ .

Variations in Gut Microbiota of Siberian Flying Squirrels Correspond to Seasonal Phenological Changes in Their Hokkaido Subarctic Forest Ecosystem.

Gut microbial communities of animals are influenced by diet and seasonal weather changes. Since foraging strategies of wild animals are affected by phenological changes, gut microbial communities would differ among seasons. However, interactions of plant-animal-microbiota with seasonal changes have not been well characterized. Here, we surveyed gut microbial diversity of Siberian flying squirrels (Pteromys volans orii) from a natural forest in Hokkaido during spring and summer of 2013 and 2014. Additionally, we compared microbial diversity to temperature changes and normalized difference vegetation index (NDVI). Changes in both seasonal temperature and phenology were significantly associated with alterations in gut microbiota. There were two clusters of OTUs, below and above 20 °C that were significantly correlated with low and high temperatures, respectively. Low-temperature cluster OTUs belonged to various phyla, whereas the high-temperature cluster was only constituted by Firmicutes. In conclusion, gut microbiota of Siberian flying squirrels varied with environmental changes on an ecological scale.

Identification of TMAO-producer phenotype and host-diet-gut dysbiosis by carnitine challenge test in human and germ-free mice.

OBJECTIVE: The gut microbiota-derived metabolite, trimethylamine N-oxide (TMAO) plays an important role in cardiovascular disease (CVD). The fasting plasma TMAO was shown as a prognostic indicator of CVD incident in patients and raised the interest of intervention targeting gut microbiota. Here we develop a clinically applicable method called oral carnitine challenge test (OCCT) for TMAO-related therapeutic drug efforts assessment and personalising dietary guidance. DESIGN: A pharmacokinetic study was performed to verify the design of OCCT protocol. The OCCT was conducted in 23 vegetarians and 34 omnivores to validate gut microbiota TMAO production capacity. The OCCT survey was integrated with gut microbiome, host genotypes, dietary records and serum biochemistry. A humanised gnotobiotic mice study was performed for translational validation. RESULTS: The OCCT showed better efficacy than fasting plasma TMAO to identify TMAO producer phenotype. The omnivores exhibited a 10-fold higher OR to be high TMAO producer than vegetarians. The TMAO-associated taxa found by OCCT in this study were consistent with previous animal studies. The TMAO producer phenotypes were also reproduced in humanised gnotobiotic mice model. Besides, we found the faecal CntA gene was not associated with TMAO production; therefore, other key relevant microbial genes might be involved. Finally, we demonstrated the urine TMAO exhibited a strong positive correlation with plasma TMAO (r=0.92, p<0.0001) and improved the feasibility of OCCT. CONCLUSION: The OCCT can be used to identify TMAO-producer phenotype of gut microbiota and may serve as a personal guidance in CVD prevention and treatment. TRIAL REGISTRATION NUMBER: NCT02838732; Results.

Functional Characteristics of the Flying Squirrel's Cecal Microbiota under a Leaf-Based Diet, Based on Multiple Meta-Omic Profiling.

Mammalian herbivores rely on microbial activities in an expanded gut chamber to convert plant biomass into absorbable nutrients. Distinct from ruminants, small herbivores typically have a simple stomach but an enlarged cecum to harbor symbiotic microbes; however, knowledge of this specialized gut structure and characteristics of its microbial contents is limited. Here, we used leaf-eating flying squirrels as a model to explore functional characteristics of the cecal microbiota adapted to a high-fiber, toxin-rich diet. Specifically, environmental conditions across gut regions were evaluated by measuring mass, pH, feed particle size, and metabolomes. Then, parallel metagenomes and metatranscriptomes were used to detect microbial functions corresponding to the cecal environment. Based on metabolomic profiles, >600 phytochemical compounds were detected, although many were present only in the foregut and probably degraded or transformed by gut microbes in the hindgut. Based on metagenomic (DNA) and metatranscriptomic (RNA) profiles, taxonomic compositions of the cecal microbiota were dominated by bacteria of the Firmicutes taxa; they contained major gene functions related to degradation and fermentation of leaf-derived compounds. Based on functional compositions, genes related to multidrug exporters were rich in microbial genomes, whereas genes involved in nutrient importers were rich in microbial transcriptomes. In addition, genes encoding chemotaxis-associated components and glycoside hydrolases specific for plant beta-glycosidic linkages were abundant in both DNA and RNA. This exploratory study provides findings which may help to form molecular-based hypotheses regarding functional contributions of symbiotic gut microbiota in small herbivores with folivorous dietary habits.

Genetic variants of Cao Bang hantavirus in the Chinese mole shrew (Anourosorex squamipes) and Taiwanese mole shrew (Anourosorex yamashinai).

To determine the genetic diversity and geographic distribution of Cao Bang virus (CBNV) and to ascertain the existence of CBNV-related hantaviruses, natural history collections of archival tissues from Chinese mole shrews (Anourosorex squamipes) and Taiwanese mole shrews (Anourosorex yamashinai), captured in Guizho Province, People's Republic of China, and in Nantou County, Taiwan, in 2006 and 1989, respectively, were analyzed for hantavirus RNA by RT-PCR. Pair-wise alignment and comparison of the S-, M- and L-segment sequences indicated CBNV in two of five Chinese mole shrews and a previously unrecognized hantavirus, named Xinyi virus (XYIV), in seven of 15 Taiwanese mole shrews. XYIV was closely related to CBNV in Vietnam and China, as well as to Lianghe virus (LHEV), recently reported as a distinct hantavirus species in Chinese mole shrews from Yunnan Province in China. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, showed that XYIV shared a common ancestry with CBNV and LHEV, in keeping with the evolutionary relationship between Anourosorex mole shrews. Until such time that tissue culture isolates of CBNV, LHEV and XYIV can be fully analyzed, XYIV and LHEV should be regarded as genetic variants, or genotypes, of CBNV.

Gene Structure and Sequence Polymorphism of the Coat Color Gene, Mc1r, in the Black-Bellied Vole (Eothenomys melanogaster).

Yung-Chih Lai, Shiao-Wei Huang, and Hon-Tsen Yu (2016) Color polymorphism is a long-standing issue in ecological and evolutionary biology. The black-bellied vole (Eothenomys melanogaster) with complete melanic and brown forms provides an outstanding opportunity to study the genetic polymorphism underpinning color variation. Mutations in the coding region of melanocortin 1 receptor (Mc1r) have been shown to cause color variation in a wide range of species. However, the contribution to color variation produced by the Mc1r regulatory regions have rarely been studied in wild animals. To this end, the Mc1r promoter sequence in black-bellied voles was cloned and characterized in this study. At least 11 distinct transcription initiation sites were identified using 5'-RACE. Furthermore, a candidate core promoter region in the upstream GC-rich sequence was identified based on key transcription factor binding motifs. The black-bellied vole Mc1r coding region was conserved with that found in the house mouse and demonstrated characteristics that are consistent with the structure of a G-protein coupled receptor, e.g. seven transmembrane domains. We found a negative association between coat color variations and polymorphisms of either regulatory or coding regions. This implies that Mc1r might reflect geographic cline rather than adaptive evolution. Although we found a negative association, the extra information we obtained in the Mc1r promoter of the black-bellied vole can be beneficial to other studies in exploring the association between regulatory mutations and adaptive phenotypes in wild animals.

Metabolic characteristics of dominant microbes and key rare species from an acidic hot spring in Taiwan revealed by metagenomics.

BACKGROUND: Microbial diversity and community structures in acidic hot springs have been characterized by 16S rRNA gene-based diversity surveys. However, our understanding regarding the interactions among microbes, or between microbes and environmental factors, remains limited. RESULTS: In the present study, a metagenomic approach, followed by bioinformatics analyses, were used to predict interactions within the microbial ecosystem in Shi-Huang-Ping (SHP), an acidic hot spring in northern Taiwan. Characterizing environmental parameters and potential metabolic pathways highlighted the importance of carbon assimilatory pathways. Four distinct carbon assimilatory pathways were identified in five dominant genera of bacteria. Of those dominant carbon fixers, Hydrogenobaculum bacteria outcompeted other carbon assimilators and dominated the SHP, presumably due to their ability to metabolize hydrogen and to withstand an anaerobic environment with fluctuating temperatures. Furthermore, most dominant microbes were capable of metabolizing inorganic sulfur-related compounds (abundant in SHP). However, Acidithiobacillus ferrooxidans was the only species among key rare microbes with the capability to fix nitrogen, suggesting a key role in nitrogen cycling. In addition to potential metabolic interactions, based on the 16S rRNAs gene sequence of Nanoarchaeum-related and its potential host Ignicoccus-related archaea, as well as sequences of viruses and CRISPR arrays, we inferred that there were complex microbe-microbe interactions. CONCLUSIONS: Our study provided evidence that there were numerous microbe-microbe and microbe-environment interactions within the microbial community in an acidic hot spring. We proposed that Hydrogenobaculum bacteria were the dominant microbial genus, as they were able to metabolize hydrogen, assimilate carbon and live in an anaerobic environment with fluctuating temperatures.

Spatial heterogeneity of gut microbiota reveals multiple bacterial communities with distinct characteristics.

We analyzed bacterial communities of six distinct gut sites (the food bolus and mucus layer of the proximal small intestine, cecum and distal large intestine), using wild folivorous flying squirrels. We found significant spatial heterogeneity in composition, diversity, and species abundance distributions (SADs) of gut microbiota, corresponding to physicochemical conditions. High diversity was detected in the mucus layer of small intestine and the food bolus of cecum, followed by the food bolus of large intestine and the mucus layer of cecum, and relatively low diversity in the food bolus of small intestine and the mucus layer of large intestine, likely due to disturbance and resource partitioning. The SADs showed succession-like patterns in the food bolus communities from the proximal to distal gut. Notably, each mucus layer community had a unique pattern different from the food bolus community of the same compartment, with distinct relative abundances of dominant species. In combination with data from other mammalian fecal samples, we concluded that gut microbiota were apparently dynamic in community structure, from low species richness with unequal abundances to high species richness with equal abundances; these findings were interpreted as strong habitat effects on bacterial communities.

Phylogeography of Chinese house mice (Mus musculus musculus/castaneus): distribution, routes of colonization and geographic regions of hybridization.

House mice (Mus musculus) are human commensals and have served as a primary model in biomedical, ecological and evolutionary research. Although there is detailed knowledge of the biogeography of house mice in Europe, little is known of the history of house mice in China, despite the fact that China encompasses an enormous portion of their range. In the present study, 535 house mice caught from 29 localities in China were studied by sequencing the mitochondrial D-loop and genotyping 10 nuclear microsatellite markers distributed on 10 chromosomes. Phylogenetic analyses revealed two evolutionary lineages corresponding to Mus musculus castaneus and Mus musculus musculus in the south and north, respectively, with the Yangtze River approximately representing the boundary. More detailed analyses combining published sequence data from mice sampled in neighbouring countries revealed the migration routes of the two subspecies into China: M. m. castaneus appeared to have migrated through a southern route (Yunnan and Guangxi), whereas M. m. musculus entered China from Kazakhstan through the north-west border (Xinjiang). Bayesian analysis of mitochondrial sequences indicated rapid population expansions in both subspecies, approximately 4650-9300 and 7150-14 300 years ago for M. m. castaneus and M. m. musculus, respectively. Interestingly, the migration routes of Chinese house mice coincide with the colonization routes of modern humans into China, and the expansion times of house mice are consistent with the development of agriculture in southern and northern China, respectively. Finally, our study confirmed the existence of a hybrid zone between M. m. castaneus and M. m. musculus in China. Further study of this hybrid zone will provide a useful counterpart to the well-studied hybrid zone between M. m. musculus and Mus musculus domesticus in central Europe.

An invertebrate Warburg effect: a shrimp virus achieves successful replication by altering the host metabolome via the PI3K-Akt-mTOR pathway.

In this study, we used a systems biology approach to investigate changes in the proteome and metabolome of shrimp hemocytes infected by the invertebrate virus WSSV (white spot syndrome virus) at the viral genome replication stage (12 hpi) and the late stage (24 hpi). At 12 hpi, but not at 24 hpi, there was significant up-regulation of the markers of several metabolic pathways associated with the vertebrate Warburg effect (or aerobic glycolysis), including glycolysis, the pentose phosphate pathway, nucleotide biosynthesis, glutaminolysis and amino acid biosynthesis. We show that the PI3K-Akt-mTOR pathway was of central importance in triggering this WSSV-induced Warburg effect. Although dsRNA silencing of the mTORC1 activator Rheb had only a relatively minor impact on WSSV replication, in vivo chemical inhibition of Akt, mTORC1 and mTORC2 suppressed the WSSV-induced Warburg effect and reduced both WSSV gene expression and viral genome replication. When the Warburg effect was suppressed by pretreatment with the mTOR inhibitor Torin 1, even the subsequent up-regulation of the TCA cycle was insufficient to satisfy the virus's requirements for energy and macromolecular precursors. The WSSV-induced Warburg effect therefore appears to be essential for successful viral replication.

Metagenomic analysis reveals a functional signature for biomass degradation by cecal microbiota in the leaf-eating flying squirrel (Petaurista alborufus lena).

BACKGROUND: Animals co-evolve with their gut microbiota; the latter can perform complex metabolic reactions that cannot be done independently by the host. Although the importance of gut microbiota has been well demonstrated, there is a paucity of research regarding its role in foliage-foraging mammals with a specialized digestive system. RESULTS: In this study, a 16S rRNA gene survey and metagenomic sequencing were used to characterize genetic diversity and functional capability of cecal microbiota of the folivorous flying squirrel (Petaurista alborufus lena). Phylogenetic compositions of the cecal microbiota derived from 3 flying squirrels were dominated by Firmicutes. Based on end-sequences of fosmid clones from 1 flying squirrel, we inferred that microbial metabolism greatly contributed to intestinal functions, including degradation of carbohydrates, metabolism of proteins, and synthesis of vitamins. Moreover, 33 polysaccharide-degrading enzymes and 2 large genomic fragments containing a series of carbohydrate-associated genes were identified. CONCLUSIONS: Cecal microbiota of the leaf-eating flying squirrel have great metabolic potential for converting diverse plant materials into absorbable nutrients. The present study should serve as the basis for future investigations, using metagenomic approaches to elucidate the intricate mechanisms and interactions between host and gut microbiota of the flying squirrel digestive system, as well as other mammals with similar adaptations.

dysregulation of CD1d-restricted type ii natural killer T cells leads to spontaneous development of colitis in mice.

BACKGROUND & AIMS: CD1d-restricted natural killer (NK) T cells are a subset of immunoregulatory T cells that comprise type I (express the semi-invariant T-cell receptor [TCR] and can be detected using the α-galactosylceramide/CD1d tetramer) and type II (express diverse TCRs and cannot be directly identified). Studies in mouse models of inflammatory bowel disease revealed a complex role for type I NKT cells in the development of colitis. Type II NKT cells have been associated with intestinal inflammation in patients with ulcerative colitis. METHODS: To investigate whether dysregulation of type II NKT cells, caused by increased expression of CD1d, can contribute to colitis, we generated transgenic mice that express high levels of CD1d and a TCR from an autoreactive, type II NKT cell (CD1dTg/24αßTg mice). RESULTS: CD1dTg/24αßTg mice had reduced numbers of 24αß T cells compared with 24αßTg mice, indicating that negative selection increases among type II NKT cells engaged by abundant self-antigen. The residual 24αß T cells in CD1dTg/24αßTg mice had an altered surface phenotype and acquired a cytokine profile distinct from that of equivalent cells in 24αßTg mice. Interestingly, CD1dTg/24αßTg mice spontaneously developed colitis; adoptive transfer experiments confirmed that type II NKT cells that develop in the context of increased CD1d expression are pathogenic. CONCLUSIONS: Aberrant type II NKT cell responses directly contribute to intestinal inflammation in mice, indicating the importance of CD1d expression levels in the development and regulation of type II NKT cells.

Use of a cytogenetic whole-genome comparison to resolve phylogenetic relationships among three species: implications for mammalian systematics and conservation biology.

The objective was to apply a novel modification of a genome-wide, comparative cytogenetic technique (comparative genomic hybridization, comparative genomic hybridization (CGH)), to study species belonging to the myrmecophagous (ant/termite eating) mammalian orders/superorders (Pholidota, Tubulidentata, Carnivora, and Xenarthra), as a model for other applications in mammalian systematics and conservation biology. In this study, CGH was applied to high-quality metaphase spreads of pangolin (Pholidota), using probes of sloth and canine (Xenarthra and Carnivora, respectively) genomic DNA labeled with different fluorophores, thereby facilitating analysis of the visible color spectrum on pangolin karyotypes. Our results posited that pholidotes are closer to carnivores than to xenarthrans, which confirmed the current consensus that myrmecophagy in these mammalian lineages was more likely because of homoplasy (convergent evolution) than being an ancestral character. Since the modified CGH technique used is genome-wide, has chromosome-level resolution, and does not need full genome sequencing, it has considerable potential in systematics and other fields.

Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon.

BACKGROUND: The black tiger shrimp (Penaeus monodon) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the P. monodon genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximately 11.1 Mb of fosmid end sequences (FESs) from 20,926 non-redundant reads representing 0.45% of the P. monodon genome were obtained for repetitive and protein-coding sequence analyses. RESULTS: We found that microsatellite sequences were highly abundant in the P. monodon genome, comprising 8.3% of the total length. The density and the average length of microsatellites were evidently higher in comparison to those of other taxa. AT-rich microsatellite motifs, especially poly (AT) and poly (AAT), were the most abundant. High abundance of microsatellite sequences were also found in the transcribed regions. Furthermore, via self-BlastN analysis we identified 103 novel repetitive element families which were categorized into four groups, i.e., 33 WSSV-like repeats, 14 retrotransposons, 5 gene-like repeats, and 51 unannotated repeats. Overall, various types of repeats comprise 51.18% of the P. monodon genome in length. Approximately 7.4% of the FESs contained protein-coding sequences, and the Inhibitor of Apoptosis Protein (IAP) gene and the Innexin 3 gene homologues appear to be present in high abundance in the P. monodon genome. CONCLUSIONS: The redundancy of various repeat types in the P. monodon genome illustrates its highly repetitive nature. In particular, long and dense microsatellite sequences as well as abundant WSSV-like sequences highlight the uniqueness of genome organization of penaeid shrimp from those of other taxa. These results provide substantial improvement to our current knowledge not only for shrimp but also for marine crustaceans of large genome size.

Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals.

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.