Cladophialophora bantiana metabolites are efficient in the larvicidal and ovicidal control of Aedes aegypti, and Culex quinquefasciatus and have low toxicity in zebrafish embryo.

Mosquitoes' current insecticide resistance status in available public health insecticides is a serious threat to mosquito control initiatives. Microbe-based control agents provide an alternative to conventional pesticides and insecticides, as they can be more targeted than synthetic insecticides. The present study was focused on identifying and investigating the mosquitocidal potential of Cladophialophora bantiana, an endophytic fungus isolated from Opuntia ficus-indica. The Cladophialophora species was identified through phylogenetic analysis of the rDNA sequence. The isolated fungus was first evaluated for its potential to produce metabolites against Aedes aegpti and Culex quinquefasciatus larvae in the 1-4th instar. The secondary metabolites of mycelium extract were assessed at various test doses (100, 200, 300, 400, and 500 µg/mL) in independent bioassays for each instar of selected mosquito larvae. After 48 h of exposure, A. aegypti expressed LC50 values of 13.069, 18.085, 9.554, and 11.717 µg/mL and LC90 = 25.702, 30.860, 17.275, and 19.601 µg/mL; followed by C. quinquefasciatus LC50 = 14.467, 11.766, 5.934, and 7.589 µg/mL, and LC90 = 29.529, 20.767, 11.192, and 13.296 µg/mL. The mean % of ovicidal bioassay was recorded 120 h after exposure. The hatchability (%) was proportional to mycelia metabolite concentration. The enzymatic level of acetylcholinesterase in fungal mycelial metabolite treated 4th instar larvae indicated a dose-dependent pattern. The GC-MS profile of C. bantiana extracts identified five of the most abundant compounds, namely cyclobutane, trans-3-undecene-1,5-diyne, 1-bromo-2-chloro, propane, 1,2,3-trichloro-2-methyl-, 5,5,10,10-tetrachlorotricyclo, and phenol, which had the killing effect in mosquitoes. Furthermore, the C. bantiana fungus ethyl acetate extracts had a strong larvicidal action on A. aegypti and C. quinquefasciatus. Finally, the toxicity test on zebrafish embryos revealed the induction of malformations only at concentrations above 1 mg/mL. Therefore, our study pioneered evidence that C. bantiana fungal metabolites effectively control A. aegypti and C. qunquefasciastus and show less lethality in zebrafish embryos at concentrations up to 500 µg/mL.

Biological effects of Avicennia marina (Forssk.) vierh. extracts on physiological, biochemical, and antimicrobial activities against three challenging mosquito vectors and microbial pathogens.

Mosquitoes are principal vector of several vector-borne diseases affecting human beings leading to thousands of deaths per year and responsible for transmitting diseases like malaria, dengue, chikungunya, yellow fever, Zika virus, Japanese encephalitis, and lymphatic filariasis. In the present study, we evaluated the different solvent extracts of mangrove Avicennia marina for their toxicity against larvae of three major mosquito vectors, as well as selected microbial pathogens. The larvicidal mortality of third instars was observed after 24 h. Highest larval mortality was found for the acetone extract of A. marina against Culex quinquefasciatus (LC50 = 0.197 mg/ml; LC90 = 1.5011 mg/ml), Anopheles stephensi (LC50 = 0.176 mg/ml; LC90 = 3.6290 mg/ml), and Aedes aegypti (LC50 = 0.164 mg/ml; LC90 = 4.3554 mg/ml). GC-MS analysis of acetone extract revealed 5 peaks, i.e., 1-hexyl-2-nitrocyclohexane (3.229%), eicosanoic acid (40.582%), cis-9-hexadecenal (70.54%), oleic acid (4.646%), and di-N-decylsulfone (5.136%). Parallel to larvicidal assay, sub-lethal dosage acetone extracts severely affected the enzyme regulations (α,ß-carboxylesterase, GST and CYP450) of third instars. Larval and pupal durations increased in all treatment sub-lethal dosage (0.127, 0.151, 0.177, and 0.197 mg/ml), whereas egg hatchability and means of fecundity decreased compared to control. The survival rate was reduced statistically in Cx. quinquefasciatus (χ2 = 23.77, df = 1, P = 0.001) in all the treatment dosages as compared to the control. Antimicrobial activity assays showed significant growth inhibition post treatment with acetone and methanol extracts against Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus pneumoniae, Escherichia coli, and Shigella flexneri. Overall, these results indicated the potential employment of A. marina extracts as a source of natural mosquitocidal and antimicrobial compounds of green-based environment.

Larvicidal, Histopathological, Antibacterial Activity of Indigenous Fungus Penicillium sp. Against Aedes aegypti L and Culex quinquefasciatus (Say) (Diptera: Culicidae) and Its Acetylcholinesterase Inhibition and Toxicity Assessment of Zebrafish (Danio rerio).

Fungal metabolites are considered to be most efficient tools to overcome the issues related to insecticide resistance and environmental pollution. The present study focus on the evaluation of the mosquito larvicidal efficacy of metabolites of seven indigenous fungal isolates (Penicillium sp. Aspergillus niger, A. flavus, A. parasiticus, Rhizopus sp. Mucor sp. and Aspergillus sp.) on the larvae of Aedes aegypti and Culex quinquefasciatus under the laboratory condition. The preliminary screening of the isolate, Penicillium sp. showed better larvicidal effect when compared to other fungi. The fungus was grown on Potato Dextrose Broth (PDB) in the laboratory (at 25°C) and maintained in the relative humidity (at 76 ± 4% for 15 days). Larvicidal potency of mycelial ethyl acetate extract (MEAE) of Penicillium sp. was performed against 1st to 4th instars larvae of Ae. aegypti and Cx. quinquefasciatus using four different concentrations (100, 200, 300, and 500 µg/ml) that showed better larval mortality values (µg/ml) of LC50 = 6.554, 5.487, 6.874, 6.892, and the LC90 = 11.486, 10.366, 12.879, 13.865 for Ae. aegypti and LC50 = 7.000, 13.943, 18.129, 25.212 and the LC90 = 12.541, 23.761, 30.923, 41.696 for Cx. quinquefasciatus. Exposure of metabolite to larvae resulted in behavior changes i.e., excitation, up and down with aggressive movement, anal papillae biting behavior. Further, the larvae treated with Penicillium sp. metabolite exhibited significant reduction in the levels of acetylcholinesterase. The 4th instar mosquito larvae treated with the 500 µg/ml mycelia extract showed severe histological damages. During the antibacterial analysis of Penicillium sp.- mycelium the maximum growth inhibition zone was recorded in Shigella dysenteriae (31.2 mm) and Klebsiella pneumoniae (31.1 mm) followed by others. In addition, to check the toxicity of Penicillium sp. MEAE against embryos of Zebrafish, a model system, using different concentrations of metabolites (1.0, 0.5, 0.125 mg/ml, 30, 3.0, and 0.5 µg/ml) and life-stage parameters were observed at 124 hpf. Furthermore, the Fourier Transformed Infrared and GCMS spectrum analysis of mycelium reflected several chemical compounds. The outcome of the study clearly shows that Penicillium sp. metabolites could serve as an ideal eco-friendly, single-step and inexpensive source for the control of Ae. aegypti and Cx. quinquefasciatus larvae.

Structural characterization, teratogenicity and in vitro avian antimicrobial activity of posterior salivary gland (PSG) toxin from cuttlefish, Sepia prashadi.

A low molecular weight posterior salivary gland (PSG) toxin was isolated and purified from the cuttlefish Sepia prashadi by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The protein and neutral sugar content of the PSG toxin was determined to be 1.033 mg/g and 282 µg/g. Fourier Transform Infrared (FT-IR) spectroscopy revealed the presence of υ-OH, υ-CO and δ-NH functional groups. Circular Dichroism (CD) spectroscopy and K2D2 analysis quantified the presence of 38.39% α-helix and 9.25% ß-sheet and 52.36% of ß-turn. Matrix Assisted Laser Desorption/Ionization-Time-of Flight/Mass Spectrometry (MALDI-TOF/MS) and MASCOT analysis revealed the amino acid sequence of MEMQSKQQNSKAPANRKIFPWMKTSAVATASKRVEMASLLNLQERQIKIWFQNRMKQKSQQPQTR (1.92 kDa) homologous to homeobox protein H4 of pufferfish, T. rubripes. The PSG toxin showed differential stability with pH and induced premature hatching in Zebrafish eggs and dose dependant developmental malformations in embryos with a Maximum tolerated dose of 1.85 µM. The PSG toxin exhibited significant antibacterial activity with pronounced zone of inhibition against S. typhimurium (12.94 mm) and inhibited avian RBC binding of Newcastle Disease virus (NDV) at a titre value of 1/4. The present study strongly advocates the biomedical potential of the PSG toxin from S. prashadi and illustrates its promise as a potential avian antimicrobial agent of the future.

Low Molecular Weight Sulfated Chitosan: Neuroprotective Effect on Rotenone-Induced In Vitro Parkinson's Disease.

The present investigation was an attempt to study the effect of low molecular weight sulfated chitosan (LMWSC) on in vitro rotenone model of Parkinson's disease (PD) by evaluating cell viability, oxidative stress, mitochondrial membrane potential, DNA fragmentation, and apoptosis. Incubation of SH-SY5Y cells with 100 nm rotenone resulted in neuronal cell death, redox imbalanced mitochondrial dysfunction, DNA fragmentation, condensation, and apoptotic cellular morphology. Rotenone exposure enhanced the expression of preapoptotic (cytochrome C (cyto c), caspase-3, -8, -9, and Bax) and down-regulated the expression of anti-apoptotic (Bcl-2) markers. Reduction of the intracellular reactive oxygen species (ROS) levels ensued due to pretreatment of LMWSC along with consequent normalization of antioxidant enzymes, mitigation of rotenone induced mitochondrial dysfunction and apoptosis. Our current findings suggested that LMWSC exhibit the pronounced neuroprotective effects, which could be due to its antioxidant, mitochondrial protection, and anti-apoptotic properties. We thus conclude that LMWSC could be developed as a novel therapeutic molecule for the benefit of reducing the consequences of PD. However, further extensive preclinical and clinical studies are warranted.

Toxicity, teratogenicity and antibacterial activity of posterior salivary gland (PSG) toxin from the cuttlefish Sepia pharaonis (Ehrenberg, 1831).

Toxins from the posterior salivary gland (PSG) of cuttlefishes are known toxins with pronounced toxicity. In the present study, ionic peptide rich PSG toxin from the cuttlefish S. pharaonis was isolated by ion exchange chromatography and purified by Reversed Phase High Performance Liquid Chromatography (RP-HPLC), with active fraction at a retention time of 26min. The net protein content of the PSG toxin was estimated to be 46.6mg at a proximate molecular weight of∼50kDa. Fourier Transform Infrared Spectroscopy (FT-IR) of PSG toxin revealed the presence of alcoholic OH, primary NH, alkyl CH and conjugated CONH functional groups. Circular Dichroism (CD) spectroscopy and K2D analysis of the PSG toxin confirmed the presence of secondary structure with 36.77% α-helix,12.31% ß sheet and 50.92% random coil. Scanning Electron Microscopy (SEM) of the PSG toxin eluted amberlite IRA 900 Cl- resin showed surface abrasion and corrosive blebbing. Energy Dispersive X-ray Spectrometry (EDX) analysis of PSG toxin treated resin revealed increase in nitrogen and sulphur content corresponding to amino acid composition. Teratogenicity of PSG toxin against Zebrafish embryo demonstrated developmental malformations and premature hatching at a maximum tolerated dose of 1.25µM. The PSG toxin (50µM) exhibited commendable inhibitory activity with pronounced zone of inhibition against gram E. coli (10mm) and K. pneumonia (10mm). The results strongly demonstrate the toxicity of the ionic peptide rich PSG toxin from S. pharaonis and its exploitation for its promise as a potential antibacterial agent of the future.

Data supporting the anticancer activity of posterior salivary gland (PSG) toxin from the cuttlefish Sepia pharaonis Ehrenberg (1831).

The data presented illustrated the in vitro anti-proliferative effect of the PSG toxin from the cuttlefish, Sepia pharaonis. The cytostatic potentials of the PSG toxin were determined by the lymphocyte migration inhibition assay. The PSG toxin (50 µg/ml) exhibited commendable inhibition of the migration of lymphocytes across the agarose gel matrix under the presence of lipopolysaccharide mitogen, with a mean migration index of 0.625. The cytotoxicity of the PSG toxin against selected cancer cell lines was determined using the MTT assay. The PSG toxin exhibited dose-dependent cytotoxicity against the MCF-7 breast cancer cells followed by KB (oral), HeLa (cervical) and A549 (lung) cancer cell lines. The PSG toxin also exhibited proportional release of LDH leakage by mitochondrial damage with an IC50 of 13.85 µM against MCF-7 breast cancer cells. The in vitro anticancer activity of the PSG toxin against the selected cell lines was evaluated by Karthik et al. (2017) [1].

In vitro and in vivo anticancer activity of posterior salivary gland toxin from the cuttlefish Sepia pharaonis, Ehrenberg (1831).

Posterior salivary gland (PSG) toxins are high molecular weight toxins secreted by cephalopods and gastropods which possess immense potentials in biomedical applications. In the present study, the biomedical potentials of the PSG toxin from the cuttlefish, S. pharaonis was determined in vitro and in vivo. The cytostatic potentials of the PSG toxin was determined by the lymphocyte migration inhibition assay. The PSG toxin (50 µg/ml) effectively inhibited the migration of lymphocytes across the agarose gel matrix under the presence of lipopolysaccharide mitogen. The cytotoxicity of the PSG toxin against cancer cell lines was determined using the MTT assay. The PSG toxin exhibited highest cytotoxicity against the MCF-7 breast cancer cells (IC50-10.64 µM) followed by KB, HeLa and A549 cells. The PSG toxin also exhibited proportional release of LDH leakage by mitochondrial damage with an IC50-13.85 µM against MCF-7 breast cancer cells. Flow cytometry analysis revealed that the PSG toxin induced apoptosis in MCF-7 cells by cell cycle arrest at G0/G1 phase. The PSG toxin (80 mg/kg b.w.) exhibited pronounced reduction (29%) in tumor growth in experimentally induced breast carcinoma in female Balb/C mice, in vivo. Hematological analysis illustrated the restoration of blood and biochemical parameters by the PSG toxin in mice induced with tumor. Histopathology studies also revealed the restitution of morphological features in the mammary tumor and vital organs in mice treated with the PSG toxin without any observed toxicity and adverse effects. The PSG toxin further exhibited commendable potentials in the prevention of tumor metastasis into immediate organs viz lungs, thus functioning as an anti-metastatic agent. The results of the present study showed that the PSG toxin exhibited immense promise as a potential peptide based anticancer agent, in future.

Structural characterization and in vitro biomedical activities of sulfated chitosan from Sepia pharaonis.

A low molecular weight sulfated chitosan (SP-LMWSC) was isolated from the cuttlebone of Sepia pharaonis. Elemental analysis established the presence of C, H and N. The sulfation of SP-LMWSC was confirmed by the presence of characteristic peaks in FT-IR and FT-Raman spectra. The thermal properties of SP-LMWSC were studied by thermogravimetric analysis and differential scanning calorimetry. Electrolytic conductivity of SP-LMWSC was measured by cyclic voltammetry and the molecular weight was determined by MALDI-TOF/MS. The molecular structure and sulfation sites of SP-LMWSC were unambiguously confirmed using (1)H, (13)C, 2D COSY and 2D HSQC NMR spectroscopy. SP-LMWSC exhibited increased anticoagulant activity in avian blood by delaying coagulation parameters and displayed cytostatic activity by inhibiting the migration of avian leucocytes. SP-LMWSC demonstrated avian antiviral activity by binding to Newcastle disease virus receptors at a low titer value of 1/64. These findings suggested that SP-LMWSC isolated from an industrial discard holds immense potentials as carbohydrate based pharmaceuticals in future.