Coelacanth genomes reveal signatures for evolutionary transition from water to land.

Coelacanths are known as "living fossils," as they show remarkable morphological resemblance to the fossil record and belong to the most primitive lineage of living Sarcopterygii (lobe-finned fishes and tetrapods). Coelacanths may be key to elucidating the tempo and mode of evolution from fish to tetrapods. Here, we report the genome sequences of five coelacanths, including four Latimeria chalumnae individuals (three specimens from Tanzania and one from Comoros) and one L. menadoensis individual from Indonesia. These sequences cover two African breeding populations and two known extant coelacanth species. The genome is ∼2.74 Gbp and contains a high proportion (∼60%) of repetitive elements. The genetic diversity among the individuals was extremely low, suggesting a small population size and/or a slow rate of evolution. We found a substantial number of genes that encode olfactory and pheromone receptors with features characteristic of tetrapod receptors for the detection of airborne ligands. We also found that limb enhancers of bmp7 and gli3, both of which are essential for limb formation, are conserved between coelacanth and tetrapods, but not ray-finned fishes. We expect that some tetrapod-like genes may have existed early in the evolution of primitive Sarcopterygii and were later co-opted to adapt to terrestrial environments. These coelacanth genomes will provide a cornerstone for studies to elucidate how ancestral aquatic vertebrates evolved into terrestrial animals.

Polydimethylsiloxane-coated partitioning sample collection device for the precise quantification of polycyclic aromatic hydrocarbons in air.

A novel partitioning collection device comprising a glass cartridge packed with poly(dimethylsiloxane)-coated macroporous silica particles was developed for the precise quantification of polycyclic aromatic hydrocarbons in air. The analyte collection and elution performances achieved using different amounts of poly(dimethylsiloxane) coating were quantitatively evaluated. The sample retention power increased with increasing the coating, and more than 250 L of air could be collected without analyte breakthrough at a sampling temperature of 35°C. During the air collection, the moisture in the air was not retained on the particles due to the hydrophobic surface of the sorbent. A complete and rapid elution of the collected analytes from the device was accomplished by the passage of only 10 mL of acetone with ultrasonication for 1 min. The proposed method was successfully applied for the determination of airborne polycyclic aromatic hydrocarbons in tunnel air.

Partitioning sample collection/solvent extraction cartridge packed with octadecyl-derivatized macroporous silica particles for the analysis of sesquiterpenes in air samples.

A novel sampling device based on the partition of target analytes to the extraction medium was developed for the determination of sesquiterpenes in air samples. The extraction medium was prepared by the chemical derivatization of a specially prepared macroporous silica, with a specific surface area of 2.0 m2 /g. Taking advantage of the sample extraction, which was mainly based on the partition of sesquiterpenes between air and a C18 -bonded phase, the extracted analytes were rapidly and quantitatively desorbed just by passing a small amount of desorption solvent for subsequent analysis by gas chromatography with mass spectrometry. Several experimental conditions, such as the sampling volume and temperature, were systematically evaluated. Under optimized conditions, desorption of the extracted analytes was completed within 1 min with a desorption efficiency of more than 99.9%, achieved using 5 mL of acetone for all the evaluated sesquiterpenes. The applicability of the developed device was confirmed by the determination of several sesquiterpenes from coniferous trees. Although further improvements of the device are required for collecting large volumes or high-temperature air samples, the potential of the developed sampling device was confirmed by determining traces of semivolatile organic compounds in air samples.

Development of the lateral plate mesoderm in medaka Oryzias latipes and Nile tilapia Oreochromis niloticus: insight into the diversification of pelvic fin position.

The position of the pelvic fins among teleost fishes has tended to shift rostrally during evolution. This positional shift seems to have led to the diversification of feeding behavior and allowed adaptation to new environments. To understand the developmental basis of this shift in pelvic fin position among teleosts, we investigated the embryonic development of the lateral plate mesoderm, which gives rise to the pelvic fins, at histological levels in the medaka Oryzias latipes (abdominal pelvic fins) and Nile tilapia Oreochromis niloticus (thoracic pelvic fins). Our histological analyses revealed that the lateral plate mesodermal cells expand not only ventrally but also rostrally to cover the yolk during embryogenesis of both medaka and Nile tilapia. In medaka, we also found that the lateral plate mesoderm completely covered the yolk prior to the initiation of the pelvic fin buds, whereas in Nile tilapia the pelvic fin buds appeared in the body wall from the lateral plate mesoderm at the thoracic level when the lateral plate mesodermal cells only covered one-third of the yolk. We discuss the relevance of such differences in the rate of the lateral plate mesoderm expansion on the yolk surface and the position of the pelvic fins.

Water properties and quality of the largest rice production region in Japan and their influence on the reproduction of zebrafish.

The health of freshwater aquarium fish and their breeding success depend critically on the quality of tap water. In general, tap water in Japan is potable, although the properties of tap water vary among regions in Japan. The city of Niigata is located in the largest rice production region of Japan. We have faced challenges concerning the reproduction of freshwater aquarium fish in Niigata. To determine whether water properties and quality affect the reproduction of aquarium fish in Niigata, we investigated the chemical properties of water and raised zebrafish in water from three different sources, namely tap water of Niigata in May, artificial freshwater (i.e., prepared via reverse osmosis), and natural spring water of Gosen, to document any effects on their sexual maturation and reproduction. We found that the tap water of Niigata was not stable throughout a year (median electrical conductivity = 147.1 µS/cm; SD = 25.6), with springtime lower than the first quartile. We also found that low concentrations of four pesticides in the tap water have been detected in May (max. concentration in 2020, bromobutide 2,000 ng/L, butachlor 600 ng/L, pyraclonil 200 ng/L, ipfencarbazone 20 ng/L). Moreover, rearing zebrafish in tap water negatively influenced both fish growth and reproduction: The sex ratio of adults was male biased (proportion of F0 male 70.8%); the average total length (30.5 mm) and weight (182 mg) of F0 males was decreased; the GSI of F0 females (9.7%) was decreased; the fecundity (the mating success 58.7%; the number of F1 eggs 63.1) of adults was reduced. Rearing in artificial freshwater could improve these outcomes (the sex ratio 55.7%; the total length of F0 males 31.8 mm; the weight of F0 males 211 mg; the GSI of F0 females 11.7%; the mating success 72.6%; the number of F1 eggs 99.0), whereas rearing in natural spring water from Gosen could improve the sex ratio (56.3%) and the weight of F0 males (200 mg), but not the others. Therefore, artificial freshwater made via reverse osmosis should be used for breeding freshwater aquarium fish in rice production region like Niigata. Finally, our results demonstrate that the reproduction of freshwater aquarium fish can serve as a bioindicator of low levels of organic pollutants in tap water and thus provide a basis for evaluating the safety of tap water for human consumption.

Volatile anticancer drug determination by thermal desorption technique with polydimethylsiloxane-coated macroporous silica adsorbent in gas chromatography-mass spectrometry.

An analytical method for quantifying the volatile anticancer drugs ifosfamide (IF) and cyclophosphamide (CP) in air was developed on the basis of thermal desorption (TD)-gas chromatography-mass spectrometry. Polydimethylsiloxane-coated macroporous silica was used as the adsorbent. The extraction tube was prepared by packing 0.2 g of adsorbent particles into a glass tube. The extraction and desorption efficiencies of the proposed method were quantitatively investigated in this study. The limits of detection of the proposed method for IF and CP were 3.3 ng L-1 at an air sampling volume of 3.0 L (30 min). The sensitivity of the proposed method was compared with using a Tenax TA packed tube that is widely used as the extraction medium in TD analysis. Finally, detection of IF and CP that evaporated from aqueous standard solution was investigated.

Allometric growth of the trunk leads to the rostral shift of the pelvic fin in teleost fishes.

The pelvic fin position among teleost fishes has shifted rostrally during evolution, resulting in diversification of both behavior and habitat. We explored the developmental basis for the rostral shift in pelvic fin position in teleost fishes using zebrafish (abdominal pelvic fins) and Nile tilapia (thoracic pelvic fins). Cell fate mapping experiments revealed that changes in the distribution of lateral plate mesodermal cells accompany the trunk-tail protrusion. Presumptive pelvic fin cells are originally located at the body wall adjacent to the anterior limit of hoxc10a expression in the spinal cord, and their position shifts rostrally as the trunk grows. We then showed that the differences in pelvic fin position between zebrafish and Nile tilapia were not due to changes in expression or function of gdf11. We also found that hox-independent motoneurons located above the pelvic fins innervate into the pelvic musculature. Our results suggest that there is a common mechanism among teleosts and tetrapods that controls paired appendage positioning via gdf11, but in teleost fishes the position of prospective pelvic fin cells on the yolk surface shifts as the trunk grows. In addition, teleost motoneurons, which lack lateral motor columns, innervate the pelvic fins in a manner independent of the rostral-caudal patterns of hox expression in the spinal cord.

Possible involvement of SINEs in mammalian-specific brain formation.

Retroposons, such as short interspersed elements (SINEs) and long interspersed elements (LINEs), are the major constituents of higher vertebrate genomes. Although there are many examples of retroposons' acquiring function, none has been implicated in the morphological innovations specific to a certain taxonomic group. We previously characterized a SINE family, AmnSINE1, members of which constitute a part of conserved noncoding elements (CNEs) in mammalian genomes. We proposed that this family acquired genomic functionality or was exapted after retropositioning in a mammalian ancestor. Here we identified 53 new AmnSINE1 loci and refined 124 total loci, two of which were further analyzed. Using a mouse enhancer assay, we demonstrate that one SINE locus, AS071, 178 kbp from the gene FGF8 (fibroblast growth factor 8), is an enhancer that recapitulates FGF8 expression in two regions of the developing forebrain, namely the diencephalon and the hypothalamus. Our gain-of-function analysis revealed that FGF8 expression in the diencephalon controls patterning of thalamic nuclei, which act as a relay center of the neocortex, suggesting a role for FGF8 in mammalian-specific forebrain patterning. Furthermore, we demonstrated that the locus, AS021, 392 kbp from the gene SATB2, controls gene expression in the lateral telencephalon, which is thought to be a signaling center during development. These results suggest important roles for SINEs in the development of the mammalian neuronal network, a part of which was initiated with the exaptation of AmnSINE1 in a common mammalian ancestor.

Tol2-mediated transgenesis in tilapia (Oreochromis niloticus).

The Nile tilapia (Oreochromis niloticus) is an important species in aquaculture and an excellent model system for laboratory studies. Functional genetic analysis using this species has been difficult because existing methods for producing transgenics are inefficient. Here we show that the Tol2 transposon system can be used to create transgenic tilapia with high efficiency. We constructed a line that is transgenic for GFP under control of a Xenopus elongation factor 1α (EF1α) promoter. The germline transmission rate of the Tol2 construct to the first generation was about 30%, which is much higher than conventional methods. GFP expression was strong and ubiquitous in the embryos. Application of the Tol2 system for constructing transgenics in tilapia and related species will promote research in many areas, but will be especially useful for studies of evolutionary developmental biology in the cichlid fishes of East Africa.

The involvement of Nile tilapia (Oreochromis niloticus) Neu4 sialidase in neural differentiation during early ontogenesis.

Neurogenesis is an important process for the formation of the central nervous system during ontogenesis. Mammalian sialidases are involved in neurogenesis through desialylation of sialo-glycoconjugates. However, the significance of fish sialidases, unlike that of mammals, in neurogenesis has not been investigated. The present study focuses on Nile tilapia (Oreochromis niloticus) because of its unique profiles of sialidases related to enzymatic properties, subcellular localization, and tissue-specific gene expression. First, the fish were cultured under aphotic condition, which is known to cause the delayed development of the retina and brain in various fish. Next, we investigate the effect of aphotic condition on the levels of tilapia sialidases. Our results revealed that the tilapia showed a decrease in the number of ganglion cell in the retina. The expression level of neu4 mRNA is up-regulated in the eyes from tilapia reared in Dark accompanied by the increase of retinal differentiation markers. These results indicated that tilapia Neu4 is involved in retinal development in Nile tilapia. Furthermore, we tried to clarify the function of tilapia Neu4 in the neuronal cells using two neuroblast cell lines (SH-SY5Y and Neuro2a cell lines). Tilapia Neu4 decreased sialic acid level of both nuclear glycoproteins as well as glycolipids. Moreover, tilapia Neu4 accelerated neurite formation in both two neural cell lines and, increased the acetylcholinesterase activity, but it did not affect cell proliferation. Collectively, these results suggest that Neu4 accelerates neurite differentiation during ontogenesis in tilapia.

Extraction of Volatile Anticancer Drugs in Air Using a Solid-Phase Extraction Type Device Followed by Gas Chromatography-Mass Spectrometric Analysis.

Ifosfamide (IF), cyclophosphamide (CP), and bendamustine (BD) are widely used anticancer drugs. These drugs have slight volatility; therefore, medical-staff exposure is of concern in the medical field. However, an accurate and quantitative detection method of these volatile drugs in air has not been reported. In this study, we developed the quantitative extraction and detection method of these volatile anticancer drugs in air. For the extraction of analytes, a solid-phase extraction-type collection device packed with styrene-divinylbenzene polymer particles was used. The extracted analytes were quantitatively eluted with 5 mL of ethanol, and the solution was concentrated to 100 µL with nitrogen purging. The analytes were analyzed using gas chromatography-mass spectrometry (GC-MS). The limit of detection of the proposed method for IF and CP was 0.017 and 0.033 ng L-1, respectively in air at an air sampling volume of 300 L. IF and CP showed slight volatility, whereas BD was not detected in GC-MS due to its lower volatility. The spiked recoveries of IF and CP in the proposed method were within the range of 95.5 to 101%. Finally, the proposed method was applied to determine the exposure of IF and CP during the dispensing of CP within a hospital dispensary room. The investigated volatile anticancer drugs were not detected in real air samples, indicating that the protection measures employed are sufficient.

Simultaneous Extraction and Determination of Volatile Organic Compounds and Semi-volatile Organic Compounds in Indoor Air Using Multi-bed Solid Phase Extraction Device.

A method for the simultaneous extraction and determination of indoor volatile compounds, including volatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs), was developed using a multi-bed solid phase extraction (SPE)-type collection device. The collection device was prepared by packing styrene-divinylbenzene polymer particles and activated carbon particles. The collected analytes were completely desorbed by passing 7 mL of acetone, and the solvent was then injected into a gas chromatograph-mass spectrometry without the concentration process. Because the proposed method does not require ultrasonication and a concentration process of eluted solvent, quantitative determination of a relatively volatile compound could be achieved. The total recovery including extraction and elution recoveries for all the investigated analytes were in the range from 91.6 to 109%. The limit of quantification was less than 4.0 ng L-1 for all the investigated analytes, and relative standard deviations of the peak area of the analytes in indoor air were less than 12%. The collection device could be reused for over 50 samplings.

Novel Nile tilapia Neu1 sialidases: Molecular cloning and biochemical characterization of the sialidases Neu1a and Neu1b.

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.

Heterotypy in the N-terminal region of growth/differentiation factor 5 (GDF5) mature protein during teleost evolution.

Heterotypy is now recognized as a generative force in the formation of new proteins through modification of existing proteins. We report that heterotypy in the N-terminal region of the mature growth/differentiation factor 5 (GDF5) protein occurred during evolution of teleosts. N-terminal length variation of GDF5 was found among teleost interfamilies and interorders but not within teleost families or among tetrapods. We further show that increase of proline and glutamine to the N-terminal region of mature GDF5 occurred in Eurypterygii, the higher lineage of teleosts. Because the basic amino acids, believed to control diffusion, are conserved in this region across all species examined, we suggest that the N-terminal elongation of the mature GDF5 protein during evolution has altered the protein diffusion in Eurypterygii, leading to high concentrations of the protein in the joint of the pharyngeal skeleton, the location of cartilage formation during development.

Purge-and-trap Determination of Ammonia in Water Samples Using Needle-type Extraction Coupled with Gas Chromatography-Barrier Discharge Ionization Detection.

This manuscript describes the determination of ammonia (NH3) in aqueous samples by purge-and-trap extraction using a needle-type extraction device with gas chromatography-barrier discharge ionization detector (GC-BID). NH3 was purged from the aqueous samples in basic conditions and then salted out with sodium chloride. Purged ammonia was trapped onto non-volatile carboxylic acid-coated macroporous terephthalic acid particles, which were contained within the needle-type extraction device. The analyte (NH3) was thermally desorbed by heating the extraction needle in the GC injection port, and detected by BID. After the optimization of both purge and extraction conditions, the linearity and sensitivity of the proposed method were evaluated. The limit of detection was found to be 2.0 mg L-1 at a headspace sampling volume of 100 mL. The method applicability was confirmed by the determination of spiked NH3 in tap water and river water samples.

Quantitative Determination of 2-Ethyl-1-hexanol, Texanol and TXIB in In-door Air Using a Solid-Phase Extraction-type Collection Device Followed by Gas Chromatography-Mass Spectrometry.

In this study, in-door air semi-volatile organic compounds (SVOCs) including 2-ethyl-1-hexanol, 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate (texanol), and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB), which are scheduled for adding as regulated compounds concerning indoor air reference values in Japan, were quantitatively extracted using a solid-phase extraction-type collection device, followed by sensitively determined by gas chromatography-mass spectrometry. The developed method has shown a good extraction recovery up to an air sampling volume of 900 L. The extracted analytes were quantitatively and rapidly eluted by 7 mL of acetone. The limit of quantification of the analytes were 0.7, 2.1 and 0.2 ng L-1 in air sample at a sampling volume of 300 mL without any concentration of a desorption solvent. The developed method was applied to simultaneous determinations of the investigated target analytes and phthalate esters in real indoor air samples.

Shaping of the lower jaw bone during growth of Nile tilapia Oreochromis niloticus and a Lake Victoria cichlid Haplochromis chilotes: a geometric morphometric approach.

East African cichlids have evolved feeding apparatus morphologies to adapt to diverse feeding environments. However, little is known about how the morphologies are formed during development. Here, we assessed the shape changes of the lower jaw bone during growth of the Nile tilapia Oreochromis niloticus and a Lake Victoria cichlid Haplochromis chilotes using geometric morphometric methods. 'Early Juvenile to Late Juvenile' and 'Late Juvenile to Adult' transitions of the shape change during growth of both O. niloticus and H. chilotes were detected. The 'Early Juvenile to Late Juvenile' transition of the shape change in H. chilotes occurred slightly earlier than in O. niloticus. We also compared the shape changes during growth of the two cichlids. Principal component analysis showed both commonalities and differences in the morphological changes between the cichlids. Our data suggest that most shape change may have a similar pattern during the growth of O. niloticus and H. chilotes, and that the differences in adult shapes may be due to differences arising early in development, not to the difference of shape change during growth. These data provide a basis for understanding the developmental mechanisms underlying this adaptive trait of East African cichlids.

Bone development in the jaw of Nile tilapia Oreochromis niloticus (Pisces: Cichlidae).

East African cichlids have evolved feeding apparatus morphologies adapted to their diverse feeding behaviors. The evolution of the oral jaw morphologies is accomplished by the diversity of bone formation during development. To further understand this evolutionary process, we examined the skeletal elements of the jaw and their temporal and sequential emergence, categorized by developmental stages, using the Nile tilapia Oreochromis niloticus as a model cichlid. We found that chondrogenesis started in Stage 17. The deposition of osteoid for the dermal bones commenced in Stage 18. The uptake of calcium dramatically shifted from the surface of larvae to the gills in Stage 20. The bone mineralization of the skeleton began in Stage 25. These data provide important information regarding the sequential events of craniofacial development in East African cichlids and lay the groundwork for studying the molecular mechanisms underlying adaptation of jaw structure to feeding behavior.

Quantitative Determination of Phthalate Esters from Air Samples Using a Solid-Phase Extraction-type Collection Device.

In this study, a solid-phase extraction-type collection device, with styrene-divinylbenzene polymer particles (Sunpak-H) as the adsorbent, was used for the quantitative determination of phthalate esters in air samples. The collection and elution recoveries of eight volatile phthalate esters, i.e., dimethyl phthalate, diethyl phthalate, dipropyl phthalate, diisobutyl phthalate, dibutyl phthalate, butyl-benzyl phthalate, di(2-ethylhexyl) phthalate, and dioctyl phthalate, were quantitatively evaluated. All analytes were collected using the device up to a sampling volume of 10000 L at a sampling temperature of 35°C without breakthrough. During air collection, moisture was not trapped on the adsorbent. The collected analytes were completely eluted from the device by passing 3 mL of acetone. The eluted solvent was injected into a gas chromatography-mass spectrometry system after the eluted solvent was concentrated, if necessary. After washing the adsorbent using acetone, the device could be reused more than 50 times. The limit of quantification for the analytes was less than 1 ng L-1 in air at a sampling volume of 600 L with solvent concentration. This device was successfully applied for the quantitative determination of phthalate esters in real air samples, including indoor and in-car air.

A Newly Designed Solid-Phase Extraction-type Collection Device for Precise Determination of Polycyclic Aromatic Hydrocarbons in Air.

A newly designed styrene-divinylbenzene copolymer adsorbent packed solid-phase extraction (SPE)-type collection device for the quantitative determination of airborne polycyclic aromatic hydrocarbons (PAHs) containing two to five rings is reported in this manuscript. This SPE-type collection device offers rapid, easy and quantitative elution of the analytes and easier reuse. A small collection device was initially developed for investigating the basic collection and elution performances of the adsorbent with respect to PAHs. The analytes were quantitatively collected on the adsorbent up to 3 m3 of air sampling at a sampling temperature of 35°C. The collected analytes were then completely eluted from the adsorbent by passing 3 mL of dichloromethane without carry-over of the analyte. During air collection, because no moisture was trapped on the adsorbent, the subsequent gas chromatography-mass spectrometric analysis was not influenced by moisture. Based on these successful performances, a wide-bore collection device was introduced for collecting larger air samples. After a quantitative investigation of the collection and elution performances of the wide-bore collection device, it device was successfully applied for precise determinations of PAHs in atmospheric air. Further application and employment of the device for the precise determination of semi-volatile organic compounds in environmental air samples is expected due to these excellent results.