The opportunistic fish pathogen, Enterococcus faecalis has been reported to cause mass mortality in several fish species in different countries. The objectives of this study were to (i) identify E. faecalis from the diseased fishes through molecular techniques; (ii) assess the antibiotic susceptibility profile of E. faecalis isolates; and (iii) control disease in tilapia fish by treatment with medicinal plant extracts. A total of 48 isolates were phenotypically identified as Enterococcus species from tilapia, stinging catfish and walking catfish cultivated in several fish farms in Gazipur. Ten randomly selected isolates were identified as E. faecalis by 16S rRNA gene sequencing. Artificial infection revealed that most of the isolates caused moderate to high mortality in fishes with characteristic disease symptoms. These isolates exhibited resistance to multiple antibiotics in vitro. Bioassay revealed that organic extracts of Tamarindus indica and Emblica officinalis leaves, Allium sativum bulb, and Syzygium aromaticum bud inhibited the growth of E. faecalis. Methanol extracts of A. sativum and methanol and acetone extracts of S. aromaticum significantly reduced the mortality of fish artificially infected with E. faecalis as both preventive and therapeutic agents. This is the first report on molecular identification, and herbal control of fish pathogenic E. faecalis in Bangladesh.

The DUT gene encodes Deoxyuridine triphosphatase (dUTPase), which is involved in nucleotide metabolism. dUTPase prevents uracil misincorporation in DNA by balancing the intracellular ratio between dUTP and dTTP. This study aimed to investigate the role of Dr-dut gene in the planarian Dugesia ryukyuensis by assessing the consequences of Dr-dut silencing on known phenomena, including regeneration following amputation and radiation damage. We functionally disrupted planarian Dr-dut mRNA by feeding RNAi-containing food to animals. Dr-dut RNAi resulted in the death of planarians in 28 days, and elevated double-stranded DNA breakage. Expression of the DNA damage response gene Dr-atm and the DNA repair genes Dr-rad51 and Dr-rad51c temporarily increased, and then decreased following the onset of feeding. When RNAi-treated planarians were amputated, both head and tail parts failed to regenerate, and the animals died in 25 and 29 days, respectively. Administration of 5-fluorouracil (5-FU) also resulted in death and DNA damage, and synergistically caused higher genotoxicity in planarian fed Dr-dut RNAi-containing food.

ObjectivePlanarians including Dugesia ryukyuensis (Dr) have strong regenerative abilities that require enhanced DNA replication. Knockdown of the DUT gene in Dr, which encodes deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase), promotes DNA fragmentation, inhibits regeneration, and eventually leads to death. dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate. dUTPase is known to prevent uracil misincorporation in DNA by balancing the intracellular ratio between dUTP and dTTP, and contributes to genome stability. Nevertheless, the catalytic performance of Dr-dUTPase has not been reported.ResultsTo confirm the catalytic activity of Dr-dUTPase, we cloned and expressed Dr-DUT in E. coli. Then, we purified Dr-dUTPase using His-tag and removed the tag with thrombin. The resulting Dr-dUTPase had the leading peptide Gly–Ser–His– originating from the vector at the amino terminus, and a mutation, Arg66Lys, to remove the internal thrombin site. We observed the hydrolysis of dUTP by Dr-dUTPase using Cresol Red as a proton sensor. The Km for dUTP was determined to be 4.0 µM, which is similar to that for human dUTPase. Dr-dUTPase exhibited a preference for dUTP over the other nucleotides. We conclude the Dr-dUTPase has catalytic activity.Electronic supplementary materialThe online version of this article (10.1186/s13104-019-4191-6) contains supplementary material, which is available to authorized users.

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca . Yet, the contribution of signaling molecules downstream of cAMP and Ca in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.