Larvicidal potential, antimicrobial properties and molecular docking analysis of Egyptian Mint (Mentha rotundifolia) against Culex pipiens L. (Diptera: Culicidae) and Midgut-borne Staphylococcus aureus.

Mosquitoes prefer stagnant areas near hospitals to live and easily spread pathogenic bacteria. Our current study aims to isolate multidrug-resistant (MDR) Staphylococcus aureus isolates from midguts of Mosquito Culex pipiens and study the potential of mint as a biocontrol strategy against C. pipiens larvae and their midgut-borne S. aureus. Samples of the third and fourth larval instars of C. pipiens were collected from water ponds around three Cairo hospitals. Ciprofloxacin, gentamycin and tetracycline, as well as various concentrations of mint leaf extract (MLE) were tested for antibiotic susceptibility. Sixty-five isolates were obtained and showed antibiotic resistance to tetracycline, gentamycin, ciprofloxacin, and undiluted MLE with resistant percentages (%) of 27.69, 30.76, 17.46, and 23.08%, respectively. Undiluted MLE inhibited 61.53% of the multidrug S. aureus isolates, whereas it couldn't inhibit any of these isolates at dilutions less than 50 µg/mL. The MIC of MLE was ≤ 700 µg/mL, while the MIC of the antibiotics ranged from 0.25 to 5.0 µg/mL for the three antibiotics. The most inhibited S. aureus isolate was identified by 16SrRNA sequencing approach and registered in GenBank as S. aureus MICBURN with gene accession number OQ766965. MLE killed all larval stages after 72 h of exposure, with mortality (%) reaching 93.33 and 100% causing external hair loss, breakage of the outer cuticle epithelial layer of the abdomen, and larvae shrinkage. Histopathology of treated larvae showed destruction of all midgut cells and organelles. Gas chromatography (GC) of MLE revealed that menthol extract (35.92%) was the largest active ingredient, followed by menthone (19.85%), D-Carvone (15.46%), Pulegone (5.0579%). Docking analysis confirmed that alpha guanine and cadinol had the highest binding affinity to both predicted active sites of Culex pipiens acetylcholinesterase. As a result, alpha-guanine and cadinol might have a role as acetylcholinesterase inhibitors.

Larvicidal potential, toxicological assessment, and molecular docking studies of four Egyptian bacterial strains against Culex pipiens L. (Diptera: Culicidae).

Mosquito control in Egypt depends on applying chemical synthetic pesticides that impact negatively on human health and the environment as well as the development of antibiotic and chemical resistance. This study aims to control the 3rd and 4th instars of Culex pipiens larvae using four bacterial strains. According to Phenotypic and molecular identification, the four isolates were identified as Bacillus subtilis MICUL D2023, Serratia marcescens MICUL A2023, Streptomyces albus LARVICID, and Pseudomonas fluorescens MICUL B2023. All strains were deposited in GenBank under accession numbers OQ764791, OQ729954, OQ726575, and OQ891356, respectively. Larvicidal activity of all microbial strain metabolites against a field strain of C. pipiens explored low LC50 results and reached its lowest values on the 3rd day with values of 6.40%, 38.4%, and 46.33% for P. fluorescens, S. albus, and S. marcescens, respectively. In addition, metabolites of P. fluorescence were more toxic than those of S. albus, followed by S. marcescens. B. subtilis shows no larvicidal effect on both field and lab mosquito strains. Microscopic alterations of 3rd and 4th instars showed toxic effects on different body parts (thorax, midgut, and anal gills), including losing external hairs, abdominal breakage, and larvae shrinkage, as well as different histological malformations in the digestive tract, midgut, and cortex. GC-MS analysis detected 51, 30, and 32 different active compounds from S. albus, S. marcescens, and P. fluorescens, respectively. GC detected 1, 2-BENZEA2:A52NEDICARBOXYLIC ACID, 2-Cyclohexene-1-carboxylic-acid-5-2-butenyl-methyl ester, and 3 octadecahydro2R3S4Z9Z-11R-12S from S. albus, S. marcesens, and P. fluorescens, respectively. Total protein, Total carbohydrate, and Acetylcholine esterase activity indicated significantly low levels on the 3rd day. All strain metabolites were safe against HSF cell lines. The docking results confirmed the role of the produced metabolites as larvicidal agents and Acetylcholine esterase inhibition. Such a problem need more studies on applying more and more natural pesticides.

Midguts of Culex pipiens L. (Diptera: Culicidae) as a potential source of raw milk contamination with pathogens.

Despite their importance, only few studies focused on the mosquitoes borne microbial diseases, especially bacterial and fungal diseases, their vectorial capacity toward microorganisms, and their important role in raw milk contamination with pathogens in some unsanitary dairy farms. In Egypt, where Culex pipiens is the historical main vector of lymphatic filariasis, only few studies discussed the isolation of pathogens from the midguts of different stages of C. pipiens. This study aims to isolate and identify the pathogenic symbiotic microorganisms inside the midgut of adult female C. pipiens as well as investigate its ability to transmit their midgut pathogens to raw milk. A total of 750 field strain C. pipiens larvae of the second and third larval instars were collected from ponds water around the livestock farms in Mariotteya, Giza, Egypt, for microbial pathogen isolation and identification. All collected larval instars were transported to the laboratory at the Research and Training Center on Vectors of Diseases (RTC), Ain Shams University, where they were maintained for further studies. Six groups of C. pipiens were tested for the incidence of various pathogenic microorganisms in their midguts and their possibility to contaminate commercial sterilized milk. Traditional PCR assays and sequencing method detected and identified 16srRNA genes of the predominant hemolytic isolates from milk and midguts of female C. pipiens. The phylogenetic analyses of the obtained isolates were performed based on NCBI data. Three strains of Bacillus anthracis strain CPMESA 2021, Staphylococcus warneri strain CPSAME 2021, and Bacillus cereus strain CPSEMA 2021, which represent most food pathogens, were found in the midguts of C. pipiens and were submitted to the GenBank database with the accession numbers OK585071, OK576651, and OK585052, respectively. The isolation of these strains from mosquitoes raises contemporary issues concerning milk safety, such as bacterial isolates, the degree of the vectorial capacity of mosquitoes, milk production and processing conditions, and human pathogenicity. Such serious issues need further investigation.

Impact of anti-sandfly saliva antibodies on biological aspects of Phlebotomus papatasi (Diptera: Psychodidae), vector of cutaneous leishmaniasis.

Sandflies are the main vectors of Leishmania parasites in tropical and subtropical areas. The immunization of vertebrate hosts with vector components through repeated bites may offer an alternative method for sandfly control. Aliquots of female Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) were weekly blood fed on 12 individual hamsters throughout 18 successive weeks. Significant biological and biochemical changes resulting from antibodies developed by immunized host sera against repeated biting were observed in sandfly females. Blood feeding and fertility rates of females significantly gradually declined to the end of the study period. No appreciable difference was observed in mortality rates among flies repeatedly fed on individual hamsters throughout weeks 9 and 18, compared to flies fed on naïve hamsters. Total salivary gland proteins of female sandflies were compared to proteins in sera of sensitized hamsters. SDS-page revealed bands common to both flies and hosts, indicating the development of anti-saliva antibodies in hamster sera. The importance of anti-sandfly saliva antibodies as a potential tool for vector control leading to the interruption of leishmaniasis is discussed.

Analysis of phoretic relation between chewing lice and hippoboscid flies of Columba livia.

Phoresy is a biologically mechanical phenomena where an immobile organism hitches on a other mobile organism to translocate. This behaviour is not studied very well on the field level especially between two important parasites infesting the same host. Parasite/parasite interaction is rarely studied through most biological host-parasite systems. Here, we evaluated the phoretic relation between parasitic chewing lice and hippoboscid flies (Pseudolynchia canariensis) on rock pigeons. A total of 69 captivated rock pigeons, Columba livia, were examined for the parasitic chewing lice and hippoboscid flies in Giza Zoo and two local farms near Cairo, Egypt. Results indicated that there is a positive correlation between infestation of hippoboscid flies and chewing lice. Also, the analysis of louse/louse interaction using ANOVA indicated a significant difference between the three chewing louse species which were recorded on the rock pigeons with relatively high abundance of two species, Columbicola columbae and Campanulotes compar. The analysis of hippoboscid flies' abundance and its relation with chewing lice infestation indicate a significant increase of lice intensity in case of high infestation with the fly. The level of hygiene of nest may be inversely related to level of parasite infestation. This work forms a step in the process of understanding parasite/parasite and host/parasite interactions using two parasitic species with a characteristic phoretic behaviour in nature.

Exploring Simplified Methods for Insect Chitin Extraction and Application as a Potential Alternative Bioethanol Resource.

Chitin, the second most plentiful biopolymer in nature, is a major component of insect cuticle. In searching for alternative resources for fossil fuels, some fungal strains of Mucor circinelloides from an insect-source were found to produce bioethanol directly using insect chitin as a substrate. Herein, simplified methods for insect chitin extraction and application as a substrate in submerged fermentation for bioethanol production were explored. Chitin of the American cockroach (Periplaneta americana (L.)) was isolated by refluxing the cockroaches dried exoskeletons with 4% NaOH. The purity of the extracted chitin was assessed to be high when the physicochemical properties of the extracted chitin matched these of commercially available crab and shrimp samples. The extracted chitin was employed as a substrate in submerged fermentation using two strains of M. circinelloides. One of these, strains M. circinelloides 6017 showed immense potential for bioethanol production directly. It could to bio-transform 15 g/L of colloidal chitin directly to 11.22 ± 0.312 g/L of bioethanol (74% of the initial chitin mass) after 6 days of incubation. These results confirm the possibility of using insect biomass as a potential alternative resource for bioethanol production in a simple manner thus contributing to the creation of an alternate energy source.

New records of chewing lice (Phthiraptera: Amblycera, Ischnocera) from Egyptian pigeons and doves (Columbiformes), with description of one new species.

Little information is available about the chewing lice of wild birds of Egypt, including common groups such as pigeons and doves (Columbiformes). Through this work, parasitic chewing lice of common columbiformes of Egypt were revised including new data. Three species of pigeons and doves (Streptopelia decaocto Frivaldszky 1838, Spilopelia senegalensis Linnaeus 1766 and Columba livia Gmelin 1789) were examined for chewing lice at three different localities. A total of 124 specimens of lice were collected. Nine species were identified from these samples; one species (Columbicola joudiae n. sp.) was considered a new species to science, six species were recorded from Egypt for the first time, and two species have been identified in Egypt before. Taxonomic and ecological remarks for all identified chewing lice samples are provided along with known and local hosts, measurements and material examined. Description and images of the new species are also provided.