Sex determination by ultrasonography prior to artificial breeding of the pufferfish Arothron manilensis (Tetraodontiformes, Tetraodontidae).

Artificial breeding was induced in the pufferfish Arothron manilensis following ultrasonographic sex determination. Hormonal treatment of mature male and female specimens followed the collection (and measurement) of fully developed eggs by cannulation. Fertilized eggs (0.85 ± 0.02 mm diameter) were spherical, demersal and individually adhesive. Hatching occurred 5 days after fertilization, larvae being 2.23 ± 0.15 mm in total length and 2.08 ± 0.14 mm in notochord length. The larvae had all died within 14 days of hatching. To improve artificial breeding techniques for A. manilensis, it is necessary to determine more appropriate timing for hormone injection, as well as feeding nutrient-enhanced SS type Brachionus sp. to newly hatched larvae.

Arothron: An R package for geometric morphometric methods and virtual anthropology applications.

OBJECTIVES: The statistical analysis of fossil remains is essential to understand the evolution of the genus Homo. Unfortunately, the human fossil record is straight away scarce and plagued with severe loss of information caused by taphonomic processes. The recently developed field of Virtual Anthropology helps to ameliorate this situation by using digital techniques to restore damaged and incomplete fossils. MATERIALS AND METHODS: We present the package Arothron, an R software suite meant to process and analyze digital models of skeletal elements. Arothron includes tools to digitally extract virtual cavities such as cranial endocasts, to statistically align disarticulated or broken bony elements, and to visualize local variations between surface meshes and landmark configurations. RESULTS: We describe the main functionalities of Arothron and illustrate their usage through reproducible case studies. We describe a tool for segmentation of skeletal cavities by showing its application on a malleus bone, a Neanderthal tooth, and a modern human cranium, reproducing their shape and calculating their volume. We illustrate how to digitally align a disarticulated model of a modern human cranium, and how to combine piecemeal shape information on individual specimens into one. In addition, we present useful visualization tools by comparing the morphological differences between the right hemisphere of the Neanderthal and the modern human brain. CONCLUSIONS: The Arothron R package is designed to study digital models of fossil specimens. By using Arothron, scientists can handle digital models with ease, investigate the inner morphology of 3D skeletal models, gain a full representation of the original shapes of damaged specimens, and compare shapes across specimens.

Endomaker, a new algorithm for fully automatic extraction of cranial endocasts and the calculation of their volumes.

OBJECTIVES: Reproducing cranial endocasts is a major goal of researchers interested in vertebrate brain evolution. We present a new R software, named endomaker, which allows the automatic extraction of endocasts from skull meshes along with the calculation of its volume. MATERIALS AND METHODS: We applied endomaker on non-primate and primate skulls including the Australopithecus africanus specimen Sts-5. RESULTS: We proved endomaker is faster, more feature-rich and possibly more accurate than competing software. DISCUSSION: Endomaker is the only available program endowed with the possibility to process an entire mesh directory straight away, promising to expand the scope and phylogenetic breadth of comparative studies of brain evolution.

Complete mitochondrial genome and the phylogenetic position of the stellate puffer (Arothron stellatus).

The complete mitochondrial genome of the stellate puffer (Arothron stellatus) was first determined in this study. It was a 16,475 bp circular molecule and consisted of 37 genes with typical gene order in vertebrate mitogenome. Its nucleotide content was 29.0% A, 32.0% C, 15.9% G and 23.1% T. This mitogenome had 30 bp short intergenic spaces located in 12 gene junctions and 28 bp overlaps located in 7 gene junctions. In the protein-coding genes, two start codons (GTG and ATG) and two stop codons (TAG and TAA/TA/T) were found. The 22 tRNA genes ranged from 64 bp (tRNA-Cys) to 74 bp (tRNA-Leu1 and tRNA-Lys). The phylogenetic result showed that Takifugu xanthopterus was most closely related to the reef-related A. stellatus among all Tetraodontiformes species.

Pufferfish Saxitoxin and Tetrodotoxin Binding Protein (PSTBP) Analogues in the Blood Plasma of the Pufferfish Arothron nigropunctatus, A. hispidus, A. manilensis, and Chelonodon patoca.

Pufferfish saxitoxin and tetrodotoxin (TTX) binding protein (PSTBP) is a glycoprotein that we previously isolated from the blood plasma of the pufferfish Takifugu pardalis; this protein was also detected in seven species of the genus Takifugu. We proposed that PSTBP is a carrier protein for TTX in pufferfish; however, PSTBP had not yet been found in genera other than Takifugu. In this study, we investigated the presence of PSTBP-like proteins in the toxic pufferfish Arothron nigropunctatus, A. hispidus, A. manilensis, and Chelonodon patoca. On the basis of ultrafiltration experiments, TTX was found to be present and partially bound to proteins in the plasma of these pufferfish, and Western blot analyses with anti-PSTBP antibody revealed one or two bands per species. The observed decreases in molecular mass following deglycosylation with glycopeptidase F suggest that these positive proteins are glycoproteins. The molecular masses of the deglycosylated proteins detected in the three Arothron species were larger than that of PSTBP in the genus Takifugu, whereas the two bands detected in C. patoca had molecular masses similar to that of tributyltin-binding protein-2 (TBT-bp2). The N-terminal amino acid sequences of 23⁻29 residues of these detected proteins were all homologous with those of PSTBP and TBT-bp2.

Revision of the Megaperidae Manter, 1934 n. comb. (Syn. Apocreadiidae Skrjabin, 1942) including a reorganization of the Schistorchiinae Yamaguti, 1942.

Modified and/or new keys to the four subfamilies now recognized within the Megaperidae Manter, 1934 n. comb. (Syn. Apocreadiidae Skrjabin, 1942) as well as the genera within each subfamily are presented. Two new genera, Paraschistorchis n. gen. and Plesioschistorchis n. gen., both within the Schistorchiinae Yamaguti, 1942, are erected and keys are provided to the species considered in both new genera-distinguished by possessing caeca that end either in separate ani or blindly. Plesioschistorchis callyodontis (Yamaguti, 1942) n. comb. and Plesioschistorchis haridis (Nagaty, 1957) n. comb. are re-described from new material collected from the common parrotfish, Scarus psittacus Forsskål (Perciformes: Scaridae), inhabiting the Red Sea off Egypt; S. psittacus represents a new host record for both species. The taxonomic status of Schistorchis sensu stricto Lühe, 1906 is examined and revised, a key to the four species we consider in this genus offered, and the monotypic genus Megacreadium Nagaty, 1956 declared a junior synonym of Schistorchis. Members of Schistorchis sensu stricto possess a unique "complex" (i.e. highly cellular/glandular) instead of "simple" (i.e. entirely muscular) type of oral sucker that is quite large in relation to body size; an elongate, somewhat sub-rectangular-shaped body; 5+ testes arranged in at least two rows; caeca that open via separate ani; a long post-testicular region; a median genital pore either at the anterior margin of or just anterior to the ventral sucker; and species of Schistorchis sensu stricto parasitize the intestine of marine fish within the Order Tetraodontiformes Berg. With the revision of this genus, we re-describe Schistorchis carneus Lühe, 1906 from the lower and mid-intestine of the white-spotted puffer, Arothron hispidus (Linnaeus) (Tetraodontiformes: Tetraodontidae), collected in the Red Sea off Egypt. Finally, a plea is made for further study of the Megaperidae n. comb. focusing, in particular, on the following: (1) obtaining new type/voucher materials of Plesioschistorchis manteri (Gupta & Tandon, 1984) n. comb. and Schistorchis paruchini Kurochkin, 1974; (2) elucidating the life histories (i.e. intermediate hosts) of members of the Postporinae Yamaguti, 1958 and Schistorchiinae; and (3) generating DNA sequence data for more species of megaperids to help future workers produce increasingly accurate taxonomic classifications that better reflect phylogenetic relationships within this ecologically diverse group of digeneans.

Design and characterization of 3D hybrid collagen matrixes as a dermal substitute in skin tissue engineering.

The highly interconnected porous dressing material was fabricated with the utilization of novel collagen (COL-SPG) for the efficient healing of the wound. Herein, we report the fabrication of 3D collagen impregnated with bioactive extract (COL-SPG-CPE) to get rid of infection at the wound site. The resultant 3D collagen matrix was characterized physiochemically using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and mechanical property. The dressing substrate possesses the high swelling ability, increase in the porosity, in vitro enzymatic degradability and antibacterial property. The in vitro biocompatibility and fluorescence activity of the collagen scaffold against both NIH 3T3 fibroblast and Human keratinocyte (HaCaT) cell lines assisted in excellent cell adhesion and proliferation over the collagen matrix. Furthermore, the in vivo evaluation of the COL-SPG-CPE 3D sponge exhibited with enhanced collagen synthesis and aids in faster reepithelialization. However, the rate of wound healing was influenced by the expression of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and transforming growth factor (TGF-ß) growth factors promotes the collagen synthesis, thereby increases the healing efficiency. Based on the results, COL-SPG-CPE has a potential ability in the remodeling of the wound with the 3D collagen as wound dressing material.

Isolation and Identification of Cytotoxic and Biological Active Toxin from the Puffer Fish Arothron stellatus.

This study is to investigate the biological, biochemical and cytotoxic effects of puffer fish (Arothron stellatus) toxin extracts under in-vitro condition. Extracted toxins from various organs of puffer fish were purified by using active charcoal column, and Bio-gel-P2 column chromatography. The lethality of toxin was tested in crabs, which consists of neurotoxic compounds. The degree of the brine shrimp lethality assay was found directly proportional to the concentration of the toxin extracts, which was well supported by hemolytic assay. The experimental results suggested that the gonad was found higher toxins than the liver and muscles. The mortality rate of brine shrimp nauplii was increased with the raise of concentrations of toxin level. Among the different doses and time dependent cytotoxic effect of human cervical carcinoma (HeLa) cells were showed 4.0 µg/mL of toxin, which was effectively inhibited cancer cell proliferation. HPLC and TLC analysis was revealed that the A. stellatus toxin contains tetrodotoxin (TTX), related compounds 4-epi TTX and anhydro-TTX. The present results suggested that the A. stellatus contain TTX as a major and anh-TTX as a minor toxin. It could be the potential candidate in the field of anticancer drug discovery against human cervical cancer cells. The present data is confirming that the puffer fish toxin as an interesting source of novel bioactive natural compounds with potent applications in pharmacology.

Tetrodotoxin and Its Analogues in the Pufferfish Arothron hispidus and A. nigropunctatus from the Solomon Islands: A Comparison of Their Toxin Profiles with the Same Species from Okinawa, Japan.

Pufferfish poisoning has not been well documented in the South Pacific, although fish and other seafood are sources of protein in these island nations. In this study, tetrodotoxin (TTX) and its analogues in each organ of the pufferfish Arothron hispidus and A. nigropunctatus collected in the Solomon Islands were investigated using high resolution LC-MS. The toxin profiles of the same two species of pufferfish from Okinawa, Japan were also examined for comparison. TTXs concentrations were higher in the skin of both species from both regions, and relatively lower in the liver, ovary, testis, stomach, intestine, and flesh. Due to higher TTX concentrations (51.0 and 28.7 µg/g at highest) detected in the skin of the two species from the Solomon Islands (saxitoxin was <0.02 µg/g), these species should be banned from consumption. Similar results were obtained from fish collected in Okinawa, Japan: TTX in the skin of A. hispidus and A. nigropunctatus were 12.7 and 255 µg/g, respectively, at highest, and saxitoxin was also detected in the skin (2.80 µg/g at highest) and ovary of A. hispidus. TTX, 5,6,11-trideoxyTTX (with its 4-epi form), and its anhydro forms were the most abundant, and 11-oxoTTX was commonly detected in the skin.

Synthesis of highly interconnected 3D scaffold from Arothron stellatus skin collagen for tissue engineering application.

The substrate which is avidly used for tissue engineering applications should have good mechanical and biocompatible properties, and all these parameters are often considered as essential for dermal reformation. Highly interconnected three dimensional (3D) wound dressing material with enhanced structural integrity was synthesized from Arothron stellatus fish skin (AsFS) collagen for tissue engineering applications. The synthesized 3D collagen sponge (COL-SPG) was further characterized by different physicochemical methods. The scanning electron microscopy analysis of the material demonstrated that well interconnected pores with homogeneous microstructure on the surface aids higher swelling index and that the material also possessed good mechanical properties with a Young's modulus of 0.89±0.2 MPa. Biocompatibility of the 3D COL-SPG showed 92% growth for both NIH 3T3 fibroblasts and keratinocytes. Overall, the study revealed that synthesized 3D COL-SPG from fish skin will act as a promising wound dressing in skin tissue engineering.

Scorpion envenomation in pygmies from Democratic Republic of Congo, the example of Pelenge Center, Lomela, DRC

In a survey among the pygmies of central Democratic Republic of Congo, the incidence of scorpion stings seemed very high with a severity greater than expected. Species responsible were not identified. Specific studies are needed to clarify the risk emerging in the equatorial African forest.

Toxic marine puffer fish in Thailand seas and tetrodotoxin they contained.

A total of 155 puffers caught from two of Thailand's seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000-2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992-1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication.

Biomedical and pharmacological potential of tetrodotoxin-producing bacteria isolated from marine pufferfish Arothron hispidus (Muller, 1841)

Specimens of the pufferfish Arothron hispidus collected at Parangipettai, on the southeast coast of India, were subjected to bacterial isolation and identification. Three species were identified, namely Bacillus sp., Kytococcus sedentarius and Cellulomonas fimi. Partially-purified microbial filtrates exhibited hemolytic activity on chicken and human erythrocytes of O, B and AB blood groups, with maximum activity of 32 HU. The microbial filtrates also presented ATPase, Mg2+-ATPase, Na+K+-ATPase and AchE enzymatic activities of positive neuromodulation in Kytococcus sedentarius with 1300, 300.1, 1549.98 and 140.55%, in Cellulomonas fimi with 620, 300, 10 and 128.42%, and in Bacillus species with 40, 200, 849.98 and 158.69%, respectively. Toxicity symptoms were observed when the bacterial filtrate was intraperitoneally injected into mice. The bacterial filtrate caused adverse effects on viability of the mouse muscle cell line (L929) and leukemia cell line (P388). Maximum level of inhibition was observed on the growth of L929 cell line. Bacillus lentimorbus inhibited the cell line from 84.03 to 94.43% whereas Bacillus species inhibited the growth in a range between 77.25 and 86.16% at the lowest dilution.Specimens of the pufferfish Arothron hispidus collected at Parangipettai, on the southeast coast of India, were subjected to bacterial isolation and identification. Three species were identified, namely Bacillus sp., Kytococcus sedentarius and Cellulomonas fimi. Partially-purified microbial filtrates exhibited hemolytic activity on chicken and human erythrocytes of O, B and AB blood groups, with maximum activity of 32 HU. The microbial filtrates also presented ATPase, Mg2+-ATPase, Na+K+-ATPase and AchE enzymatic activities of positive neuromodulation in Kytococcus sedentarius with 1300, 300.1, 1549.98 and 140.55 percent, in Cellulomonas fimi with 620, 300, 10 and 128.42 percent, and in Bacillus species with 40, 200, 849.98 and 158.69 percent, respectively. Toxicity symptoms were observed when the bacterial filtrate was intraperitoneally injected into mice. The bacterial filtrate caused adverse effects on viability of the mouse muscle cell line (L929) and leukemia cell line (P388). Maximum level of inhibition was observed on the growth of L929 cell line. Bacillus lentimorbus inhibited the cell line from 84.03 to 94.43 percent whereas Bacillus species inhibited the growth in a range between 77.25 and 86.16 percent at the lowest dilution.(AU)

Ecología trófica del pez Arothron meleagris (Tetraodontiformes: Tetraodontidae) en el arrecife de Los Frailes, Baja California Sur, México

Trophic ecology of the fish Arothron meleagris (Tetraodontiformes: Tetraodontidae) from Los Frailes reef, Southern Baja California, Mexico. Monthly samples were taken from November 2004 to October 2005 in the Los Frailes reef, BCS, Mexico. The objective was to determine the feeding habits of the pufferfish, Arothron meleagris, as well as possible variations in feeding patterns due to size and/or sex. We captured 101 specimens, with all stomachs contained food. The index of relative importance (IRI) was used to determine the main items; which were non-identified organic matter (NIOM) (30.44%), Echinometra vanbrunti (26.25 %), Porifera (12.63 %), Pocillopora spp. (11.84%), Bryozoa (5.37 %) and Porites spp. (4.83 %). Levin’s index indicated a low trophic niche breadth (Bi= 0.12) and allowed us to determine that A. meleagris is a specialist predator with strong preferences towards certain food types. The Morisita-Horn index showed a high trophic overlap between sexes ( or = 0.78), seasons (hot and cold or = 0.95) and among sizes (small and medium or = 0.94; small and large or = 0.74; medium and large or = 0.83). Rev. Biol. Trop. 57 (1-2): 113-123. Epub 2009 June 30.

Con la finalidad de determinar los hábitos alimenticios del tamboril de oro Arothron meleagris, así como sus posibles variaciones de alimentación por talla y sexo, se realizaron muestreos mensuales de noviembre 2004 a octubre 2005, en el arrecife de Los Frailes, Baja California Sur, México. Se capturaron un total de 101 ejemplares, en los cuales todos los estómagos contenían alimento. Se aplicó el índice de importancia relativa (IIR) para determinar los principales categorías alimenticias (ítems), los cuales fueron: Echinometra vanbrunti (26.25 %), Porifera (12.63 %), Pocillopora spp. (11.84%), Bryozoa (5.37 %) y Porites spp. (4.83 %). El índice de Levin indica baja amplitud del nicho trófico (Bi= 0.12) por lo que se le puede considerar como un depredador especialista con marcadas preferencias hacia algunos tipos alimentarios. El índice de Morisita-Horn indicó que existe un traslape alto entre las dietas por sexo (hembras y machos o = 0.78), temporadas (cálida y fría o = 0.95) y entre tallas (chica y mediana o = 0.94, chica y grande o = 0.74, mediana y grande o = 0.83).