Early Filial Cannibalism in Fish Revisited: Endocrinological Constraint, Costs of Parental Care, and Mating Possibility.

AbstractOffspring desertion by parents generally occurs at an early stage of parental care, which is thought to minimize the costs of parental care prior to desertion. This study investigated the effects of endocrinological constraints on early total filial cannibalism by male Rhabdoblennius nitidus in the field, a paternal brooding blennid fish with androgen-dependent brood cycling. In brood reduction experiments, cannibal males showed low levels of plasma 11-ketotestosterone (11-KT) relative to noncannibals and also similar levels of 11-KT to males in the parental care phase. Since 11-KT regulates male courtship intensity, males with decreased courtship activity would exhibit total filial cannibalism. However, there is a possibility that a transient increase in 11-KT levels at the early stage of parental care delays total filial cannibalism. In contrast, total filial cannibalism could occur before a decline to the lowest 11-KT levels, at which point males might still be able to exhibit courtships, possibly to reduce the costs of parental care. To understand how much and when caregiving males exhibit mating and parental care behaviors, it is important to consider not only the presence of endocrinological constraints but also its intensity and flexibility.

Unique Mode of Antiviral Action of a Marine Alkaloid against Ebola Virus and SARS-CoV-2.

Lamellarin α 20-sulfate is a cell-impenetrable marine alkaloid that can suppress infection that is mediated by the envelope glycoprotein of human immunodeficiency virus type 1. We explored the antiviral action and mechanisms of this alkaloid against emerging enveloped RNA viruses that use endocytosis for infection. The alkaloid inhibited the infection of retroviral vectors that had been pseudotyped with the envelope glycoprotein of Ebola virus and SARS-CoV-2. The antiviral effects of lamellarin were independent of the retrovirus Gag-Pol proteins. Interestingly, although heparin and dextran sulfate suppressed the cell attachment of vector particles, lamellarin did not. In silico structural analyses of the trimeric glycoprotein of the Ebola virus disclosed that the principal lamellarin-binding site is confined to a previously unappreciated cavity near the NPC1-binding site and fusion loop, whereas those for heparin and dextran sulfate were dispersed across the attachment and fusion subunits of the glycoproteins. Notably, lamellarin binding to this cavity was augmented under conditions where the pH was 5.0. These results suggest that the final action of the alkaloid against Ebola virus is specific to events following endocytosis, possibly during conformational glycoprotein changes in the acidic environment of endosomes. Our findings highlight the unique biological and physicochemical features of lamellarin α 20-sulfate and should lead to the further use of broadly reactive antivirals to explore the structural mechanisms of virus replication.

Antivirus activity, but not thiolreductase activity, is conserved in interferon-gamma-inducible GILT protein in arthropod.

We have previously reported that gamma-interferon inducible lysosomal thiolreductase (GILT) functions as a host defense factor against retroviruses by digesting disulfide bonds on viral envelope proteins. GILT is widely conserved even in plants and fungi as well as animals. The thiolreductase active site of mammalian GILT is composed of a CXXC amino acid motif, whereas the C-terminal cysteine residue is changed to serine in arthropods including shrimps, crabs, and flies. GILT from Penaeus monodon (PmGILT) also has the CXXS motif instead of the CXXC active site. We demonstrate here that a human GILT mutant (GILT C75S) with the CXXS motif and PmGILT significantly inhibit amphotropic murine leukemia virus vector infection in human cells without alterning its expression level and lysosomal localization, showing that the C-terminal cysteine residue of the active site is not required for the antiviral activity. We have reported that human GILT suppresses HIV-1 particle production by digestion of disulfide bonds on CD63. However, GILT C75S mutant and PmGILT did not digest CD63 disulfide bonds, and had no effect on HIV-1 virion production, suggesting that they do not have thiolreductase activity. Taken together, this study found that antiviral activity, but not thiolreductase activity, is conserved in arthropod GILT proteins. This finding provides a new insight that the common function of GILT is antiviral activity in many animals.

Application of heavy-ion-beam irradiation to breeding large rotifer.

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure.

The Spirocyclic Imine from a Marine Benthic Dinoflagellate, Portimine, Is a Potent Anti-Human Immunodeficiency Virus Type 1 Therapeutic Lead Compound.

In this study, we aimed to find chemicals from lower sea animals with defensive effects against human immunodeficiency virus type 1 (HIV-1). A library of marine natural products consisting of 80 compounds was screened for activity against HIV-1 infection using a luciferase-encoding HIV-1 vector. We identified five compounds that decreased luciferase activity in the vector-inoculated cells. In particular, portimine, isolated from the benthic dinoflagellate Vulcanodinium rugosum, exhibited significant anti-HIV-1 activity. Portimine inhibited viral infection with an 50% inhibitory concentration (IC50) value of 4.1 nM and had no cytotoxic effect on the host cells at concentrations less than 200 nM. Portimine also inhibited vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped HIV-1 vector infection. This result suggested that portimine mainly targeted HIV-1 Gag or Pol protein. To analyse which replication steps portimine affects, luciferase sequences were amplified by semi-quantitative PCR in total DNA. This analysis revealed that portimine inhibits HIV-1 vector infection before or at the reverse transcription step. Portimine has also been shown to have a direct effect on reverse transcriptase using an in vitro reverse transcriptase assay. Portimine efficiently inhibited HIV-1 replication and is a potent lead compound for developing novel therapeutic drugs against HIV-1-induced diseases.

A Genetic Map for the Only Self-Fertilizing Vertebrate.

The mangrove killifish Kryptolebias marmoratus, and its close relative Kryptolebias hermaphroditus, are the only vertebrate species known to reproduce by self-fertilization due to functional ovotestis development. To improve our understanding of their genomes, we constructed a genetic map. First, a single F1 fish was made by artificial fertilization between K. marmoratus and K. hermaphroditus strains. F2 progeny were then obtained by self-fertilization of the F1 fish. We used RAD-seq to query genomic DNAs from the two parental strains, the F1 individual and 49 F2 progeny. Results identified 9904 polymorphic RAD-tags (DNA markers) that mapped to 24 linkage groups, corresponding to the haploid chromosome number of these species. The total length of the map was 1248 cM, indicating that about one recombination occurred for each of the 24 homologous chromosome pairs in each meiosis. Markers were not evenly distributed along the chromosomes: in all chromosomes, many markers (> 8% of the total markers for each chromosome) mapped to chromosome tips. Centromeres suppress recombination, and this uneven distribution is probably due to the species' acrocentric chromosomes. Mapped marker sequences were compared to genomic sequences of medaka and platyfish, the next most closely related species with sequenced genomes that are anchored to genetic maps. Results showed that each mangrove killifish chromosome corresponds to a single chromosome of both platyfish and medaka, suggesting strong conservation of chromosomes over 100 million years of evolution. Our genetic map provides a framework for the K. marmoratus/K. hermaphroditus genome sequence and an important resource for understanding the biology of hermaphroditism.

Light-dependent transcriptional events during resting egg hatching of the rotifer Brachionus manjavacas.

Rotifer resting eggs often have to endure harsh environmental conditions during the diapause phase. They are stimulated by light to hatch. In order to study the hatching mechanism, we observed resting eggs and measured their transcriptional expression under different light exposure periods (total darkness, and after 30 min, and 4h light). By using differential-display reverse transcription PCR (DDRT-PCR), we isolated 80 genes that displayed different expression patterns in response to the three light treatments: 20 genes were expressed in total darkness, 40 different genes were differentially expressed under 30 min light, and 20 further genes were expressed after 4h of light. The resting eggs showed no phenotypic differences in embryonic development during the 4h illumination period. In general, the expression patterns of the analyzed genes in resting eggs were differentially modulated by light exposure time. In total darkness, resting eggs mainly expressed genes encoding cell defense and homeostasis functions. In the 30 min illumination group, we found enriched expression of genes encoding fatty acid metabolism-related components, including Acyl-CoA dehydrogenase (ACAD). Genes encoding cellular and embryonic developmental functions were highly observed in the 30 min-illuminated group but were not observed in the 4h-illuminated group. Real-time RT-PCR revealed that several transcripts such as encoding V-type H(+)-translocating pyrophosphatase (V-PPase) and Meckelin had prolonged expression levels when exposed to light for 4h. In the 4h illuminated group, the RecQ protein-like 5 (RECQL5) gene was enriched. This RECQL5 gene may be expressed to protect the developing embryo from continuous light exposure. The data presented in this study indicate that DDRT-PCR-aided gene screening can be helpful to isolate candidate genes involved in the hatching process.

Complete mitochondrial genome of the monogonont rotifer, Brachionus koreanus (Rotifera, Brachionidae).

The complete mitochondrial genome was obtained from the assembled genome data sequenced by next generation sequencing (NGS) technology from the monogonont rotifer Brachionus koreanus. The mitochondrial genome of B. koreanus was composed of two circular chromosomes designated as mtDNA-I (10,421 bp) and mtDNA-II (11,923 bp). The gene contents of B. koreanus were identical with previously reported B. plicatilis mitochondrial genomes. However, gene orders of B. koreanus showed one rearrangement between the two species. Of 12 protein-coding genes (PCGs), 3 genes (ATP6, ND1, and ND3) had an incomplete stop codon. The A + T base composition of B. koreanus mitochondrial genome was high (68.81%). They also showed anti-G bias (12.03% and 10.97%) on the second and third position of PCGs as well as slight anti-C bias (15.96% and 14.31%) on the first and third position of PCGs.

Differential transcript expression of selected gene batteries in two clonal strains of the self-fertilizing fish, Kryptolebias marmoratus.

The hermaphroditic fish, Kryptolebias marmoratus is known as a unique vertebrate that reproduces by internal self-fertilization, resulting in genetic homogeneity. In this study, we characterized two strains, PAN-RS (13 and 20 days after hatching, dah) and DAN (20 dah), that show different growth rates and morphometric parameters. In the same period (20 dah), the PAN-RS strain showed significantly faster growth rate than the DAN strain in all the parameters measured in this study. In the case of the 13 dah of PAN-RS strain, they showed similar morphometries and growth rate with the DAN strain (20 dah). To investigate why they showed different growth rates in both strains, we analyzed the transcript profile of several genes, such as growth-, hypothalamic-pituitary-gonadal axis-, vitellogenesis-, steroidogenesis-, and sex-related gene for both strains. Based on our results, the different growth rates with several reproduction parameters would be associated with transcript profiles of some gene batteries as the first trigger for the related protein expression in both strains. In addition, this study would provide a better understanding of the physiology and endocrinology in K. marmoratus, particularly, of the transcriptional regulation of growth- and reproduction-related genes with clonal difference.

Two circular chromosomes of unequal copy number make up the mitochondrial genome of the rotifer Brachionus plicatilis.

The monogonont rotifer Brachionus plicatilis is an emerging model system for a diverse array of questions in limnological ecosystem dynamics, the evolution of sexual recombination, cryptic speciation, and the phylogeny of basal metazoans. We sequenced the complete mitochondrial genome of B. plicatilis sensu strictu NH1L and found that it is composed of 2 circular chromosomes, designated mtDNA-I (11,153 bp) and mtDNA-II (12,672 bp). Hybridization to DNA isolated from mitochondria demonstrated that mtDNA-I is present at 4 times the copy number of mtDNA-II. The only nucleotide similarity between the 2 chromosomes is a 4.9-kbp region of 99.5% identity including a transfer RNA (tRNA) gene and an extensive noncoding region that contains putative D-loop and control sequence. The mtDNA-I chromosome encodes 4 proteins (ATP6, COB, NAD1, and NAD2), 13 tRNAs, and the large and small subunit ribosomal RNAs; mtDNA-II encodes 8 proteins (COX1-3, NAD3-6, and NAD4L) and 9 tRNAs. Gene order is not conserved between B. plicatilis and its closest relative with a sequenced mitochondrial genome, the acanthocephalan Leptorhynchoides thecatus, or other sequenced mitochondrial genomes. Polymerase chain reaction assays and Southern hybridization to DNA from 18 strains of Brachionus suggest that the 2-chromosome structure has been stable for millions of years. The novel organization of the B. plicatilis mitochondrial genome into 2 nearly equal chromosomes of 4-fold different copy number may provide insight into the evolution of metazoan mitochondria and the phylogenetics of rotifers and other basal animal phyla.

Analysis of expressed sequence tags of the cyclically parthenogenetic rotifer Brachionus plicatilis.

BACKGROUND: Rotifers are among the most common non-arthropod animals and are the most experimentally tractable members of the basal assemblage of metazoan phyla known as Gnathifera. The monogonont rotifer Brachionus plicatilis is a developing model system for ecotoxicology, aquatic ecology, cryptic speciation, and the evolution of sex, and is an important food source for finfish aquaculture. However, basic knowledge of the genome and transcriptome of any rotifer species has been lacking. METHODOLOGY/PRINCIPAL FINDINGS: We generated and partially sequenced a cDNA library from B. plicatilis and constructed a database of over 2300 expressed sequence tags corresponding to more than 450 transcripts. About 20% of the transcripts had no significant similarity to database sequences by BLAST; most of these contained open reading frames of significant length but few had recognized Pfam motifs. Sixteen transcripts accounted for 25% of the ESTs; four of these had no significant similarity to BLAST or Pfam databases. Putative up- and downstream untranslated regions are relatively short and AT rich. In contrast to bdelloid rotifers, there was no evidence of a conserved trans-spliced leader sequence among the transcripts and most genes were single-copy. CONCLUSIONS/SIGNIFICANCE: Despite the small size of this EST project it revealed several important features of the rotifer transcriptome and of individual monogonont genes. Because there is little genomic data for Gnathifera, the transcripts we found with no known function may represent genes that are species-, class-, phylum- or even superphylum-specific; the fact that some are among the most highly expressed indicates their importance. The absence of trans-spliced leader exons in this monogonont species contrasts with their abundance in bdelloid rotifers and indicates that the presence of this phenomenon can vary at the subphylum level. Our EST database provides a relatively large quantity of transcript-level data for B. plicatilis, and more generally of rotifers and other gnathiferan phyla, and can be browsed and searched at gmod.mbl.edu.

Purification and characterization of two isoforms of glucose 6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27.

Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.

Two low-temperature-inducible Chlorella genes for delta12 and omega-3 fatty acid desaturase (FAD): isolation of delta12 and omega-3 fad cDNA clones, expression of delta12 fad in Saccharomyces cerevisiae, and expression of omega-3 fad in Nicotiana tabacum.

In an attempt to clarify the involvement of fatty acid desaturases (FADs) in the freezing tolerance of Chlorella vulgaris IAM C-27, developed by hardening, we have isolated cDNA clones for two types of FADs from the Chlorella strain, based on the sequence information of genes for delta12 and omega-3 FADs, respectively desaturating oleic acid (18:1) to linoleic acid (18:2) and linoleic acid (18:2) to linolenic acid (18:3). The deduced amino acid sequence of the first clone, designated CvFad2, showed about 66% similarity to the microsomal delta12 FADs from several higher plants and this gene had delta12 FAD activity when expressed in Saccharomyces cerevisiae. The predicted protein encoded by a second gene, designated CvFad3, showed about 60% similarity to the microsomal and plastidial omega-3 FADs from several higher plants. The features of the amino acid sequences of the C- and N-terminal regions of CvFAD3 and fatty acid analysis of polar lipids in transgenic tobacco plant expressing the CvFad3 gene suggested that this gene encodes the microsomal omega-3 FAD. Southern blot analysis showed that both genes were single-copy genes in the genome of the Chlorella strain. Different transcriptional patterns were observed with the two genes during hardening in Northern blot analysis.