Antibacterial and bacteriostatic potential of coelomic fluid and body paste of Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) against ampicillin resistant clinical bacterial isolates

Pheretima posthuma (Vaillant, 1868), a native earthworm of Pakistan and Southeast Asia, has wide utilization in vermicomposting and bioremediation process. In this study, P. posthuma coelomic fluid (PCF) and body paste (PBP) was evaluated as antibacterial agent against ampicillin (AMP) resistant five Gram positive and four Gram negative clinical isolates. The antibacterial effect of different doses (i.e. 25-100 µg/ml) of PCF and PBP along with AMP and azithromycin (AZM) (negative and positive controls, respectively) were observed through disc diffusion and micro-dilution methods. All nine clinical isolates were noticed as AMP resistant and AZM sensitive. Antibacterial effects of PCF and PBP were dose dependent and zone of inhibitions (ZI) against all clinical isolates were between 23.4 ± 0.92 to 0 ± 00 mm. The sensitivity profile of PCF and PBP against clinical isolates was noticed as 44.44 and 55.56%, respectively. Both PCF and PBP showed bacteriostatic (BTS) action against S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Moreover, the cumulative BTS potential of PCF and PBP against all isolates was 66.67 and 55.56%, respectively. The MICs of PCF and PBP were ranged from 50-200 µg/ml against selected isolates. The bacterial growth curves indicated that PCF and PBP inhibited the growth of all isolates at their specific MIC concentrations. However, PBP has better antibacterial potential compared to PCF against selected isolates. Therefore, it is concluded that both PCF and PBP of P. posthuma possess antibacterial and BTS potential against ampicillin resistant clinical isolates. This organism might be considered as a second choice of antibacterial agents and can further be utilized in pharmaceutical industries for novel drug manufacturing by prospecting bioactive potential agents.

Cloning, Functional Characterization and Response to Cadmium Stress of the Thioredoxin-like Protein 1 Gene from Phascolosoma esculenta

Cadmium (Cd) is a heavy metal toxicant and is widely distributed in aquatic environments. It can cause excessive production of reactive oxygen species (ROS) in the organism, which in turn leads to a series of oxidative damages. Thioredoxin (Trx), a highly conserved disulfide reductase, plays an important role in maintaining the intracellular redox homeostasis in eukaryotes and prokaryotes. Phascolosoma esculenta is an edible marine worm, an invertebrate that is extensively found on the mudflats of coastal China. To explore the molecular response of Trx in mudflat organisms under Cd stress, we identified a new Trx isoform (Trx-like protein 1 gene) from P. esculenta for the first time, designated as PeTrxl. Molecular and structural characterization, as well as multiple sequence and phylogenetic tree analysis, demonstrated that PeTrxl belongs to the Trx superfamily. PeTrxl transcripts were found to be ubiquitous in all tissues, and the highest expression level occurred in the coelomic fluid. Exposure to three sublethal concentrations of Cd resulted in the upregulation and then downregulation of PeTrxl expression levels over time in coelomic fluid of P. esculenta. The significant elevation of PeTrxl expression after 12 and 24 h of Cd exposure at 6 and 96 mg/L, respectively, might reflect its important role in the resistance to Cd stress. Recombinant PeTrxl (rPeTrxl) showed prominent dose-dependent insulin-reducing and ABTS free radical-scavenging abilities. After exposure to 96 mg/L Cd for 24 h, the ROS level increased significantly in the coelomic fluid, suggesting that Cd induced oxidative stress in P. esculenta. Furthermore, the injection of rPeTrxl during Cd exposure significantly reduced the ROS in the coelomic fluid. Our data suggest that PeTrxl has significant antioxidant capacity and can protect P. esculenta from Cd-induced oxidative stress.

Diagnosis and treatment of Candida glabrata proventriculitis in an eclectus parrot (Eclectus roratus)

CASE DESCRIPTION An 8-year-old sexually intact female eclectus parrot (Eclectus roratus) with a 4-day history of hyporexia and lethargy and a 1-day history of tenesmus was examined. CLINICAL FINDINGS Severe leukocytosis characterized by severe heterophilia and moderate monocytosis was present. Marked dilation of the proventriculus and ventriculus and ascites were identified by means of radiography, coelomic ultrasonography, and contrast-enhanced CT, with no clinically relevant motility noted on ultrasonography. Results of coelomic fluid analysis were consistent with pyogranulomatous effusion. Endoscopy of the upper gastrointestinal tract following proventricular and ventricular lavage showed a thick caseous plaque occupying 30% of the caudal proventricular mucosa. Abundant yeast organisms were evident during cytologic examination of a proventricular and ventricular wash sample, and fecal culture yielded Candida glabrata. TREATMENT AND OUTCOME The bird was treated with SC fluids, assisted feedings, nystatin, fluconazole, amoxicillin–clavulanic acid, enrofloxacin, gastroprotectants, maropitant, and analgesics and slowly improved during hospitalization. A marked decrease in proventricular dilation was evident on serial radiographs obtained over a 12-month period. One year after diagnosis, the bird was presented with a 1-week history of hyporexia and lethargy, and fecal culture grew C glabrata. Antifungal treatment was resumed for 3 months. The bird had no clinical signs of infection 16 months after this recurrence, and subsequent fecal cultures were negative for fungal growth. CLINICAL RELEVANCE Findings illustrate the importance of upper gastrointestinal endoscopy in diagnosing proventricular and ventricular dilation in birds and emphasize the need for long-term antifungal treatment and monitoring in birds with fungal infections.

Galactose-Binding C-Type Lectin Promotes Cellular Aggregation of Coelomocytes in Sea Cucumber

Echinoderms have a large coelomic cavity containing coelomocytes. When the coelomic fluid is removed from the cavity, the cells aggregate immediately. We found that a fraction or an extract of the intestine of the sea cucumber, Apostichopus japonicus, markedly accelerated cellular movement and aggregation on a glass slide, and this effect was clearly inhibited by galactose. We successfully purified the aggregation-promoting factor, a 16 kDa protein, from the intestine. TOF-MS analysis followed by de novo sequencing revealed that the protein is a C-type lectin. RNA-seq data and cDNA cloning demonstrated the factor to be a novel lectin, named AjGBCL, consisting of 158 aa residues in the mature form. Microscopic observation revealed that most of the aggregating cells moved toward aggregates and not to an intestinal fragment, suggesting that AjGBCL is not a chemoattractant but a cellular aggregation-inducing factor that may induce aggregates to release chemoattractant. We report, for the first time, an endogenous molecule that promotes coelomocyte aggregation in echinoderms.

Micropropagation of Solanum lycopersicum L. using chemical free formulated organic plant growth media

Micropropagation is currently the most commercially effective plant biotechnology that helps in the rapid generation of a large number of clonal plants of many plant species in a short period. Effective protocol was developed for in vitro micropropagation of Solanum lycopersicum (tomato) using chemical-free organic vermicompost (VC) extract medium. This experiment aims to expose the advancement in plant tissue culture technique by using synthetic Murashige and Skoog medium (MS) medium and a chemical-free organic VC (30%) extract along with 4% coelomic fluid (obtained from the earthworms Eudrilus eugeniae) as a growth medium for in vitro studies. The response of roots, leaves, shoots, and nodes in both synthetic (MS) medium and chemical-free organic VC extract medium was compared statistically using Student’s t-test. Statistically significant differences for the number of roots (P =0.011), leaves (P=0.012), and nodes (P=0.001) between the two media were reported. Both media showed the same shoot induction where no significant differences were reported with a P value of 1.000 for shoots. The protocol has led to a 100% plant survival rate on chemical-free organic vermicompost extract media. The results have also shown a significant difference in growth rate between two different media. Vermicompost and coelomic fluid used in media have antimicrobial activities which led to minimal contamination level and this consequently minimized timely subculturing. Through our studies, we found that chemical-free organic vermicompost extract media proved to be more economical and a better prospective than MS media for in vitro propagation of tomato.

A novel herpes-like virus inducing branchial lesions in a tiger shark (Galeocerdo cuvier)

A juvenile, male tiger shark ( Galeocerdo cuvier) developed illness after capture in Florida waters and was euthanized. Gross lesions included mild skin abrasions, hepatic atrophy, and coelomic fluid. Histologically, gills contained multifocal lamellar epithelial cell necrosis and thromboses. Scattered gill and esophageal epithelial cells had large, basophilic, intracytoplasmic, and intranuclear inclusions. Ultrastructurally, lamellar epithelial cells contained arrays of intracytoplasmic viral particles and scattered intranuclear nucleocapsids. Capsulated virions were 148 ± 11 nm with an 84 ± 8 nm icosahedral nucleocapsid and an electron-dense core. Next-generation sequencing, quantitative polymerase chain reaction, and in situ hybridization performed on formalin-fixed tissue confirmed a herpes-like viral infection. The viral polymerase shared 24% to 31% protein homology with other alloherpesviruses of fish, indicating a divergent virus. This report documents the pathologic findings associated with a molecularly confirmed novel herpes-like virus in an elasmobranch.

Antifungal Activity of Earthworm Coelomic Fluid Obtained from Eisenia andrei, Dendrobaena veneta and Allolobophora chlorotica on Six Species of Phytopathogenic Fungi

The functioning of soil ecosystems greatly depends on the interactions occurring between soil biota communities. It is well known that earthworms are an important soil component that substantially affects its function, including their meaningful impact on the development of different phytopathogenic soil fungi. Phytopathogenic fungi are responsible for crop disease and cause great economic damage. It has previously been established that earthworms’ coelomic fluid can suppress the growth of phytopathogenic fungi, but the exact molecular mechanism is unknown. The present study aimed at broadening the proof of this observed phenomenon by investigating the effects of the coelomic fluid extract of three different earthworm species (Eisenia andrei, Dendrobaena veneta and Allolobophora chlorotica) on the growth of six different phytopathogenic fungi species (Berkeleyomyces basicola, Fusarium culmorum, Globisporangium irregulare, Rhizoctonia solani, Macrophomina phaseolina, and Sclerotinia sclerotiorum). Coelomic fluid extract was obtained by electrostimulation or usage of extraction buffer (only in case of A. chlorotica) and prepared in three different concentrations by diluting the obtained coelomic fluid with physiological saline. The coelomic fluid extract of the three investigated earthworm species had an inhibitory effect on the growth of all six phytopathogenic fungi species. The greatest inhibitory effect was achieved with the E. andrei coelomic fluid extract reducing the growth of R. solani fungi. The findings of this research confirm the antifungal activity of coelomic fluid obtained from earthworm species belonging to different ecological categories and may be of potential use in crop protection against phytopathogenic fungi.

Presence and biodistribution of perfluorooctanoic acid (PFOA) in Paracentrotus lividus highlight its potential application for environmental biomonitoring

The first determination of presence and biodistribution of PFOA in ninety specimens of sea urchin Paracentrotus lividus from two differently contaminated sites along Palermo’s coastline (Sicily) is reported. Analyses were performed on the sea urchins’ coelomic fluids, coelomocytes, gonads or mixed organs, as well as on seawater and Posidonia oceanica leaves samples from the collection sites. PFOA concentration ranged between 1 and 13 ng/L in seawater and between 0 and 794 ng/g in P. oceanica. The analyses carried out on individuals of P. lividus from the least polluted site (A) showed PFOA median values equal to 0 in all the matrices (coelomic fluid, coelomocytes and gonads). Conversely, individuals collected from the most polluted site (B) showed median PFOA concentrations of 21 ng/g in coelomic fluid, 153 ng/g in coelomocytes, and 195 ng/g in gonads. Calculated bioconcentration factors of log10BCF > 3.7 confirmed the very bioaccumulative nature of PFOA. Significant correlations were found between the PFOA concentration of the coelomic fluid versus the total PFOA concentration of the entire sea urchin. PERMANOVA (p = 0.001) end Welch's t-test (p < 0.001) analyses showed a difference between specimens collected from the two sites highlighting the potential application of P. lividus as sentinel species for PFOA biomonitoring.

Extracellular Vesicle Signatures and Post-Translational Protein Deimination in Purple Sea Urchin (Strongylocentrotus purpuratus) Coelomic Fluid—Novel Insights into Echinodermata Biology

The purple sea urchin (Strongylocentrotus purpuratus) is a marine invertebrate of the class Echinoidea that serves as an important research model for developmental biology, cell biology, and immunology, as well as for understanding regenerative responses and ageing. Peptidylarginine deiminases (PADs) are calcium-dependent enzymes that mediate post-translational protein deimination/citrullination. These alterations affect protein function and may also play roles in protein moonlighting. Extracellular vesicles (EVs) are membrane-bound vesicles that are released from cells as a means of cellular communication. Their cargo includes a range of protein and RNA molecules. EVs can be isolated from many body fluids and are therefore used as biomarkers in physiological and pathological responses. This study assessed EVs present in the coelomic fluid of the purple sea urchin (Strongylocentrotus purpuratus), and identified both total protein cargo as well as the deiminated protein cargo. Deiminated proteins in coelomic fluid EVs were compared with the total deiminated proteins identified in coelomic fluid to assess putative differences in deiminated protein targets. Functional protein network analysis for deiminated proteins revealed pathways for immune, metabolic, and gene regulatory functions within both total coelomic fluid and EVs. Key KEGG and GO pathways for total EV protein cargo furthermore showed some overlap with deimination-enriched pathways. The findings presented in this study add to current understanding of how post-translational deimination may shape immunity across the phylogeny tree, including possibly via PAD activity from microbiota symbionts. Furthermore, this study provides a platform for research on EVs as biomarkers in sea urchin models.