RESUMEN
Background & Objectives: American foulbrood (AFB), caused by the highly virulent, spore-forming bacterium Paenibacillus larvae, poses a significant threat to honey bee brood. The widespread use of antibiotics not only fails to effectively combat the disease but also raises concerns regarding honey safety. The current computational study was attempted to identify a novel therapeutic drug target against P. larvae, a causative agent of American foulbrood disease in honey bee. Methods: We investigated effective novel drug targets through a comprehensive in silico pan-proteome and hierarchal subtractive sequence analysis. In total, 14 strains of P. larvae genomes were used to identify core genes. Subsequently, the core proteome was systematically narrowed down to a single protein predicted as the potential drug target. Alphafold software was then employed to predict the 3D structure of the potential drug target. Structural docking was carried out between a library of phytochemicals derived from traditional Chinese flora (n > 36,000) and the potential receptor using Autodock tool 1.5.6. Finally, molecular dynamics (MD) simulation study was conducted using GROMACS to assess the stability of the best-docked ligand. Results: Proteome mining led to the identification of Ketoacyl-ACP synthase III as a highly promising therapeutic target, making it a prime candidate for inhibitor screening. The subsequent virtual screening and MD simulation analyses further affirmed the selection of ZINC95910054 as a potent inhibitor, with the lowest binding energy. This finding presents significant promise in the battle against P. larvae. Conclusions: Computer aided drug design provides a novel approach for managing American foulbrood in honey bee populations, potentially mitigating its detrimental effects on both bee colonies and the honey industry.
Asunto(s)
Paenibacillus larvae , Proteoma , Animales , Abejas/microbiología , Paenibacillus larvae/efectos de los fármacos , Paenibacillus larvae/genética , Paenibacillus larvae/metabolismo , Proteoma/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
Phytochemical screening of aqueous extract from six medicinal wild plants grown in South-eastern of Tunisia: Atriplex halimus, Teucrium polium, Moricandia arvensis, Deverra tortuoa, Haplophyllum tuberculatum and Polygonum equisetiforme were evaluated. Both decoction and ultrasound assisted extraction were used. Antioxidant, antibacterial proprieties, and phenolic profiling, using LC-ESI-MS method, were assessed. Total polyphenols, flavonoids, and condensed tannins contents ranged from 7.47±0.19 to 22.25±0.49â mg GAE/g Dw, 5.47±0.06 to 7.55±0.07â mg RE/g Dw, and 0.33±0.02 to 19.43±0.64â mg TAE/g Dw, respectively. Moreover, the reducing power and DPPH tests showed that P. equisetiforme (EC50 : 12.50±0.50â µg/ml; DPPHâ + : 213.49±4.24â mg TEAC/g DW), T. polium (EC50 : 25.00±1.00â µg/ml; DPPHâ + : 181.39±9.47â mg TEAC/g DW) as well as H. tuberculatum (EC50 : 56.25±0.25â µg/ml; DPPHâ + : 177.83±5.85â mg TEAC/g DW) extracts were the most effective natural antioxidants. For anti-bacterial activity, the ultrasonic extract of H. tuberculatum showed the highest activity against both P. aeruginosa (13.50±0.71â mm) and S. aureus (13.00±0.00â mm) at 10â mg/ml. Furthermore 24 phenolic compounds were identified, with predominance of quinic acid, gallic acid, protocatechuic acid, syringic acid, p-coumaric acid, trans-ferulic acid, catechin (+), trans-cinnamic and silymarin. These results were further consolidated by to heatmap clustering with P. equisetiforme, H. tuberculatum, T. polium as the main antioxidant and antibacterial sources which supports their domestication and industrial use.
Asunto(s)
Antioxidantes , Plantas Medicinales , Antioxidantes/farmacología , Antioxidantes/química , Túnez , Staphylococcus aureus , Extractos Vegetales/farmacología , Extractos Vegetales/química , Fenoles/farmacología , Fenoles/análisis , Flavonoides , Antibacterianos/farmacologíaRESUMEN
Epidemiologic studies keep up the proposition that Allium vegetables can lower the risk of cancers. Acute myeloid leukemia (AML) cells exhibit high proliferative potency and have a reduced capacity of undergoing apoptosis and maturation. The beneficial effects of Allium seem related to the organosulfur products generated upon processing of these species. For this purpose, the aim of this study was to test Allium roseum fresh (FAE), crude (CAE) and dried (DAE) aqueous extracts for activity against the human acute leukemia cell line (U937). As assessed by flow cytometry, inhibited cell proliferation was in a dose-dependent manner. Firstly, study showed that cell growth was inhibited with 20 mg/mL using FAE and CAE (60% and 73% respectively). Secondly, our experiments clearly indicate that all A. roseum extracts do not induce cell apoptosis. This was confirmed by the soft binding of Annexin V to phosphatidylserine. Finally, the high expression of macrophage's marker CD11 associated with adequate morphological changes proves clearly the differentiation aspect produced by A. roseum extract. Taken together, these data suggest that A. roseum could be a promising candidate for the alternative medicine in the field of cancer therapy.
RESUMEN
Rosemary (Rosmarinus officinalis L.) is a popular herb in cooking, traditional healing, and aromatherapy. This study was conducted to evaluate the effects of meteorological conditions plant growth stage and genetic factors on the yield, quantitative and qualitative composition, on the antioxidant and antimicrobial activities of rosemary essential oil from two Tunisian locations (El Fahs and Matmata) during two successive years. The composition of the essential oils obtained by hydrodistilation from rosemary plants were carried out annually using GC and GC/MS. Results showed the the main constituents were camphor (18.2-28.1%), 1,8-cineole (6.4-18.0%), α-pinene (9.7-13.5%), borneol (4.4-9.5%), and camphene (5.1-8.7%). The principal component and heatmapper analyses showed group segregation of the two studied varities based on major essential oil compounds. Additionally, in vitro antimicrobial and antioxidant activities showed that rosemary essential oils had an important ability in scavenging DPPH, as well as a higher bactericidal effect. The seasonal variation, growth stage and genetic pools seemed to be a factors of significant variation of the composition, antimicrobial and the antioxidant activities of the rosemary essential oils. These finding would be taken to use the chemotaxonomy tools to develop a program for Rosmary protection conservation and identification based on essential oil composition.
Asunto(s)
Antiinfecciosos , Aceites Volátiles , Rosmarinus , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Aceites Volátiles/química , Aceites Volátiles/farmacología , Rosmarinus/química , Estaciones del AñoRESUMEN
BACKGROUND: Hyperactivation of mechanistic target of rapamycin (mTOR) signaling pathway is involved in the regulation of cellular growth, proliferation, and more in general, is a common phenomenon in most types of cancers. Thus, natural substances targeting this pathway can be of great therapeutic potential in supporting the treatment of tumor patients. Rhus tripartita (Ucria) Grande is a plant growing in desertic areas which is traditionally used for the treatment of several diseases in Tunisia. In the present work, the biochemical profile of the main compounds present in the plant leaf extract was determined and the anti-leukemic potential of the plant extracts against acute monocytic leukaemia (AML) THP-1 cells was investigated. METHODS: After HPLC identification of some phenolic compounds present in the plant extract and the quantification of saponin content, the cytotoxic effect of Rhus tripartita extracts on THP-1 cell culture was evaluated using the colorimetric MTT assay for cell viability. THP-1 cells were incubated with medium containing the relative IC50 concentrations of total plant extract, saponin extract and some standard compounds (rutin (R); kaempferol (K); mixture of catechin, epicatechin, and epicatechin-gallate (CEEG); ellagic acid (EA). Finally, qRT-PCR and western blotting analysis were used to evaluate the effect of some flavonoids present in a crude extract of polyphenols and the total extract of saponins on cell survival and apoptosis. RESULTS: Analysis of expression level of some gene (PIK3CA, PTEN, AKT1, mTOR, EIF4E, RPS6KB1, and TSC1) involved in the mTOR pathway and the phosphorylation of S6 and AKT proteins allowed to observe that a total Rhus tripartita extract and some of the compounds found in the extract controls THP-1 cell proliferation and apoptosis via regulation of the PI3K-Akt-mTOR signaling pathway. CONCLUSION: Rhus tripartita-induced inhibition of cell cycle and induction of apoptosis may involve the mTOR pathway. Therefore, Rhus tripartita extract may be a useful candidate as a natural anti-cancer drug to support the treatment of AML.
Asunto(s)
Apoptosis/efectos de los fármacos , Polifenoles/farmacología , Rhus/química , Saponinas/farmacología , Transducción de Señal/efectos de los fármacos , Antineoplásicos/farmacología , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Extractos Vegetales/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células THP-1 , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The aim of this study was to investigate the polyphenolic profile and biological properties of leaves acetonic extracts from six Tunisian spontaneous plants of Marrubium vulgare L., Rhus tripartita (Ucria) D.C., Hernaria fontanesii J. Gay subsp. fontanesii, Ziziphus lotus L., Plantago ovata Forsk., Thymelaea hirsuta (L.) Endl. Bioassay-guided and HPLC-PDA-ESI-MS procedures demonstrated that R. tripartita contained the highest amount of phenolic compounds (1475.1 µg/g), followed by Z. lotus (1087.8 µg/g) and P. ovata (1027.6 µg/g). Interestingly, in R. tripartita myricetin-3-O-galactoside turned out to be the most abundant one. The plant extracts showed antimicrobial efficacy against Listeria monocytogenes, Staphylococcus aureus and S. epidermidis including methicillin resistant strains; no activity was detected against Gram-negative bacteria. R. tripartita revealed the best MIC and MBC values and caused significant decrease of S. aureus biofilm. Both R. tripartita and Z. lotus did not display any toxicity against Artemia salina Leach (LC50 > 1000 µg/mL).
Asunto(s)
Antibacterianos/farmacología , Artemia/efectos de los fármacos , Extractos Vegetales/toxicidad , Hojas de la Planta/química , Polifenoles/análisis , Pruebas de Toxicidad , Animales , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Bioensayo , Cromatografía Líquida de Alta Presión , Dosificación Letal Mediana , Pruebas de Sensibilidad Microbiana , Polifenoles/química , Staphylococcus aureus/efectos de los fármacos , Staphylococcus epidermidis/efectos de los fármacos , ZiziphusRESUMEN
Allium roseum is an important medicinal and aromatic plant, specific to the North African flora and a rich source of important nutrients and bioactive molecules including flavonoids and organosulfur compounds whose biological activities and pharmacological properties are well known. In the present study, the inhibition of amyloid beta protein toxicity by the ethanolic extract of this plant is investigated for the first time. Preliminary biochemical analyses identified kæmpferol and luteolin-7-o-glucoside as the more abundant phenolic compounds. The effects of A. roseum extract (ARE) on aggregation and aggregate cytotoxicity of amyloid beta-42 (Aß42), whose brain aggregates are a hallmark of Alzheimer's disease, were investigated by biophysical (ThT assay, Dynamic light scattering and transmission electron microscopy) and cellular assays (cytotoxicity, aggregate immunolocalization, ROS measurement and intracellular Ca2+ imaging). The biophysical data suggest that ARE affects the structure of the Aß42 peptide, inhibits its polymerization, and interferes with the path of fibrillogenesis. The data with cultured cells shows that ARE reduces Aß42 aggregate toxicity by inhibiting aggregate binding to the cell membrane and by decreasing both oxidative stress and intracellular Ca2+. Accordingly, ARE could act as a neuroprotective factor against Aß aggregate toxicity in Alzheimer's disease.
Asunto(s)
Allium/química , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/metabolismo , Extractos Vegetales/farmacología , Agregación Patológica de Proteínas/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Calcio/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Citosol/metabolismo , Evaluación Preclínica de Medicamentos , Dispersión Dinámica de Luz , Etanol/química , Humanos , Microscopía Electrónica de Transmisión , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/aislamiento & purificación , Fármacos Neuroprotectores/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Agregación Patológica de Proteínas/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The present study was conducted to compare the antibacterial activity of oven-dried and freeze-dried Allium sativum along with its spray-dried microencapsulated essential oil in the preservation of minced beef meat. Allium sativum extracts were tested against mesophilic aerobic microorganisms, coagulase-positive staphylococci, Escherichia coli, Salmonella sp., and the sulfite-reducing anaerobes. A difference between the chemical compositions of powders obtained by the conventional oven-drying and freeze-drying has been verified by HPLC-MS2, freeze-dried fresh garlic powder contains 74% of allicin, and 12% cysteine sulfoxides comparing to the oven-drying garlic powder in which is detected two thiosulfinate isomers: allicin (67%) and allyl-1-propenyl thiosulfinate (21%). CIELAB color analysis was performed to assess the effect of drying temperature on powders. The microflora-inhibiting effect of freeze-dried fresh garlic and the spray-dried microencapsulated essential oil at a concentration of 20% represents a promising way to be used in food systems such as meat and meat products preservation, at 4-8°C.
RESUMEN
Jujube (Ziziphus lotus L.) fruit has multiple functional properties and represents an interesting source of bioactive compounds. The purpose of this study was to improve the functionality and the sensory properties of sponge cake enriched with Z. lotus fruit. The polyphenols and flavonoids levels in the sponge cake and its antioxidant potential increased with the addition of 0-10 g of jujube powder/100 g of wheat flour. The crumb color parameters, L* and b*, decreased with the addition of jujube powder, whereas the a* value increased. In the texture analysis, addition of jujube powder resulted in an increase of the hardness and chewiness, but the springiness was reduced. The sensory evaluation showed that supplementation of jujube powder did not manifest any undesirable organoleptic response and showed satisfactory consumer acceptability. Overall, the addition at 5% jujube powder showed the finest sensory properties of the sponge cake.
Asunto(s)
Sensación/fisiología , Bocadillos , Ziziphus/química , Antioxidantes/análisis , Color , Flavonoides/análisis , Frutas/química , Humanos , Extractos Vegetales/química , Polifenoles/análisis , PolvosRESUMEN
Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has emerged as a promising strategy to overcome tumor drug resistance. Here, we investigated the anticancer activity of Allium roseum L. extracts, a wild edible species in North Africa, on human Chronic Myeloid Leukemia (CML) K562 cells. The dehydrated aqueous extract (DAE) disturbed the cell cycle progression and induced the apoptosis of K562 cells. Chemical analysis of DAE showed a diversity of organosulfur compounds S-alk(en)yl-cysteine sulfoxides (RCSO) and high amount of allicin, suggesting that such molecule may be behind its antitumor effect. DAE was efficient in inhibiting K562 cell viability. DAE inhibitory effect was associated with the dephosphorylation of the BCR-ABL kinase and interfered with ERK1/2, Akt, and STAT5 pathways. Furthermore, we found that DAE-induced inactivation of Akt kinase led to the activation of its target FOXO3 transcription factor, enhancing the expression of FOXO3-regulated proapoptotic effectors, Bim and Bax, and cell cycle inhibitor p27. Finally, we found that DAE reduced the secretion of vascular endothelial growth factor. Overall, our data suggest that A. roseum extract has great potential as a nontoxic cheap and effective alternative to conventional chemotherapy.
Asunto(s)
Allium/química , Antineoplásicos Fitogénicos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Extractos Vegetales/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteína Forkhead Box O3/metabolismo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT5/metabolismoRESUMEN
Context The present study deals with new biological properties of the wild edible Diplotaxis simplex (Viv.) Spreng (Brassicaceae). Objectives The current study evaluates the antioxidant, the anti-inflammatory and the anti-cancer properties of ethyl acetate and ethanol extracts from D. simplex flowers. Materials and methods The anti-proliferative activity of the extracts (10-70 µg/mL) was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) against human colon cancer cell line Caco-2. The anti-inflammatory potential was evaluated by the inhibitory effect of the extracts (1.5-7.5 mg/mL) on phospholipase A2 activity as well as on carrageenan-induced paw oedema in mice. Extracts (200 mg/kg) or indomethacin (50 mg/kg) as positive control were injected intraperitoneally for albino mice prior to the induction of the oedema by carrageenan. Antioxidant activities were investigated using various complementary methods. Results Flower extracts contained a high level of polyphenolics (17.10-52.70 mg GAE/g) and flavonoids (74.20-100.60 mg QE/g), which correlate with its appreciable antioxidant potential in ß-carotene peroxidation (IC50 value: 12.50-27.10 µg/mL), DPPH(â¢) radical-scavenging (IC50 value: 0.20-0.40 mg/mL), Fe(3+ )reducing (EC50 value: 0.10-0.14 mg/mL) and Fe(2+ )chelating (IC50 value: 0.20-0.60 mg/mL) assays. These extracts were effective in inhibiting cancer cell growth (IC50 value: 62.0-63.25 µg/mL). Besides, the ethyl acetate extract inhibited phospholipase A2 activity (IC50 value: 2.97 mg/mL) and reduced the paw oedema in mice (from 0.38 ± 0.01 to 0.24 ± 0.01 cm), 4 h post-carrageenan challenge. Conclusion These data suggest that D. simplex may be useful as a candidate in the treatment of inflammation and the colon cancer.
Asunto(s)
Antiinfecciosos/farmacología , Antineoplásicos Fitogénicos/farmacología , Brassicaceae , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Edema/prevención & control , Extractos Vegetales/farmacología , Acetatos/química , Animales , Antiinfecciosos/aislamiento & purificación , Antineoplásicos Fitogénicos/aislamiento & purificación , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Compuestos de Bifenilo/química , Brassicaceae/química , Células CACO-2 , Carragenina , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Etanol/química , Flores , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Inhibidores de Fosfolipasa A2/aislamiento & purificación , Inhibidores de Fosfolipasa A2/farmacología , Fosfolipasas A2/metabolismo , Fitoterapia , Picratos/química , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales , Solventes/químicaRESUMEN
The present study deals with the valorization of an edible spontaneous plant of the Tunisian arid areas: Allium roseum. This plant is traditionally used for therapeutic and culinary uses. Thin-layer drying behavior of Allium roseum leaves was investigated at 40, 50 and 60 °C drying air temperatures and 1 and l.5 m/s air velocity, in a convective dryer. The increase in air temperature significantly affected the moisture loss and reduced the drying time while air velocity was an insignificant factor during drying of Allium roseum leaves. Five models selected from the literature were found to satisfactorily describe drying kinetics of Allium roseum leaves for all tested drying conditions. Drying data were analyzed to obtain moisture diffusivity values. During the falling rate-drying period, moisture transfer from Allium roseum leaves was described by applying the Fick's diffusion model. Moisture diffusivity varied from 2.55 × 10(-12) to 8.83 × 10(-12) m(2)/s and increased with air temperature. Activation energy during convective drying was calculated using an exponential expression based on Arrhenius equation and ranged between 46.80 and 52.68 kJ/mol. All sulfur compounds detected in the fresh leaves were detected in the dried leaves. Convective air drying preserved the sulfur compounds potential formation.
RESUMEN
Melanin performs a crucial role in protecting the skin against harmful ultraviolet light. However, hyperpigmentation may lead to aesthetic problems and disorders such as solar lentigines (SL), melasma, postinflammatory hyperpigmentation and even melanoma. Arthrophytum scoparium grows in the desert in the North African region, and given this type of environment, A. scoparium exhibits adaptations for storing water and produces useful bioactive factors. In this study, the effect of A. scoparium ethanol extract (ASEE) on melanogenesis regulation in B16 murine melanoma cells was investigated. Cells treated with 0.017% (w/v) ASEE showed a significant inhibition of melanin biosynthesis in a time-dependent manner without cytotoxicity. To clarify the mechanism behind the ASEE-treated melanogenesis regulation, the expressions of tyrosinase enzyme and melanogenesis-related genes were determined. Results showed that the expression of tyrosinase enzyme was significantly decreased and Tyr, Trp-1, Mitf and Mc1R mRNA expressions were significantly down-regulated. LC-ESI-TOF-MS analysis of the extract identified the presence of six phenolic compounds: coumaric acid, cinnamic acid, chrysoeriol, cyanidin, catechol and caffeoylquinic acid. The melanogenesis inhibitory effect of ASEE may therefore be attributed to its catechol and tetrahydroisoquinoline derivative content. We report here that ASEE can inhibit melanogenesis in a time-dependent manner by decreasing the tyrosinase protein and Tyr, Trp-1, Mitf and Mc1R mRNA expressions. This is the first report on the antimelanogenesis effect of A. scoparium and on its potential as a whitening agent.
Asunto(s)
Caryophyllaceae/química , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Melanocitos/citología , Monofenol Monooxigenasa/metabolismo , Extractos Vegetales/farmacología , Actinas/metabolismo , Animales , Catecoles/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Interferón Tipo I/metabolismo , Melaninas/metabolismo , Melanocitos/efectos de los fármacos , Melanoma Experimental , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismo , Fenoles/farmacología , Proteínas Gestacionales/metabolismo , ARN Mensajero/metabolismo , Receptor de Melanocortina Tipo 1/metabolismo , Tetrahidroisoquinolinas/farmacologíaRESUMEN
Extracts of aerial parts and roots of wild Astragalus gombiformis Pomel were tested for their antibacterial, antioxidant, and insecticidal activities and contents of phenolic compounds. Antibacterial activity was tested by the paper disk agar diffusion method and determination of the minimal inhibitor concentration. Among the tested extracts, three extracts (methanol, chloroform, and ethyl acetate) from aerial parts and two extracts (water, methanol) from roots exhibited diameters of inhibition zone equal or above 12 mm (at 150 microg/ disk) and minimal inhibitor concentrations ranging between 233 and 1250 microg/ml. Spectrophotometric and HPLC analyses showed that contents of both total polyphenols and flavonoids, as well as antioxidant activity were higher in the methanolic extract of aerial parts as compared to roots. No insecticidal activity of the extracts of the aerial parts was found against Culex pipiens.