الفهرس 
INTRODUCTION
Fungi associated with mosquitos’ immature stagesOnly 14 pages are availabe for public view
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Abstract
Mosquitoes are important and dangerous vectors of parasitic diseases such as malaria and filariasis, and arboviral diseases such as yellow fever, Chikungunya, West Nile, dengue fever, and Zika. Mosquitoes are considered the main reason for the loss of more than seven million human lives annually all over the world.
In Assiut Governorate, El-Nikhila village, El-Matiaa village and Sahel Seleem district are known filarial endemic areas included in National Filaria Elimination Program (NFEP).
Chemical control of vector insects cussed health, environmentaland climatic hazard and insecticide resistance and unsustainable interventions represent challenges in reaching sustainable development goals (SDGs). Developing an alternative strategy for larval mosquito control necessitates exploring eco-friendly and biological control methods.
So, the present work has been initiated with the aim to determine the species composition of mosquitoes and their associated insects and fungi inhabiting agricultural drain water in different areas of Assiut Governorate (31° 11’ 21.4188’’ E: 27° 10’ 48.4824’’ N), Egypt.The work was carried out during72 inspection visits and 164 examinations of drain water agriculture pools in the seven areas from 2021 to 2022 years. The seven areas include the villages of El-Nikhila and El-Matiaa, in addition to the districts of Dairout, Manfalout, Abnoub, Sahel Seleem, and El-Badary.
Also, todetermine the effect of prevailing entomopathogenic fungi on Culexpipiens mosquito and the extracellular enzymes.
The following results were achievedcan be summarized as follow:
1. Species composition and incidence of mosquitoes and associated insects inhabiting agricultural drain water
1.1 Species composition and incidence of mosquitoes
1.1.1. Identification of mosquitoes by key
Six mosquito species were identified namely,Anopheles muliticolor, Aedes caspius, Culex antennatus, Cx perexigaus, Cx pipiens, and Uranotaenia unguiculata.
1.1.2. Molecular identification of mosquito species
UsingDNA sequences of the COI gene confirmed the identity of five species in agriculture drain water as Anopheles triannulatus, Culex pipiens,Culex quinquefasciatus, Ochlerotatus caspius and Uranotaenia unguiculata. Sequenceswere deposited in the GenBank under accession numbers OR145812.1, OR145813.1, OR145814.1, OR145815.1 and OR145816.1, respectively.
Resultsof Incidence indicated thatthe highest Incidence Area Weight value IAW inEl-Badary area 21. However, Culexpipiens was the most frequent mosquito species with an Incidence Species Weight ISWvalue of 14.
1.1.3. Species composition of associated insects
The taxonomic list of associated insects with mosquitoes indicatedthe presence of five insect orders, 7families, 9genus and 9identified species most of them are considered predaceous insects associated with mosquitoes, of which Pantala flavescens (Odonata:Libellulidae) was the most frequent in all areas. Incidence Area Weight IAW for each identified species indicated that P. flavescens had the maximum value of IAW 14. Also, the maximum value of incidence species weight was ISW in El-Badary area (22).
2.Surveyand incidence of fungiinhabiting different agriculture drain water pools in Assiut area
2.1. Surveyand incidence of fungi
Fungal species isolated from the drain water were classified under 18 genera namely Fusarium (12species), Aspergillus (10), Penicillium (4), Mucor, Clonostachys,and Sarocladium(3species for each).Also,Talaromyces (2),Pseudoallesheria, Trichoderma, Albifembria,CladosporiumMicroascus, Geotrichum,Cunninghamella,Purpureocillium, Alternaria,Actinomucor, andRhizopus(1species for each). from the fungal species recorded, (45) species were isolated from agricultural drain water,(39) isolatedfrom mosquito larvae,(5) from pupae,and (9) from associated insects.
The incidence patterns of the isolated fungi were high infall and summer (25), compared withwinter and spring season(23).
Concerning isolated entomopathogenic fungi inhabiting different agricultural drain water pools. Nien specieswere isolated from agricultural drain water, (4) species from the associated insects, (5) species isolated mosquito larvae. The incidence patterns of the isolated entomopathogenic fungi during different seasons revealed that the incidence value was(6) in all seasons except thefall season was(3).
2.2.Molecular identification of prevailing entomopathogenic fungusAlbifimbria associated with mosquitoes
Sample isolated in the present study (Albifimbria verrucaria AUMMC16014, arrowed) aligned with closely related strains accessed from the GenBank. This strain showed 98.94% - 100% identity and 97%-99% coverage with several strains of the same species. Beauveriabassiana represents an outgroup strain. A.= Albifimbria, M.= Myrothecium, B.= Beauveria. Note: Myrothecium verrucaria is the homotypic synonym of Albifimbria verrucaria.
Sequencing results ofthe internal transcribed spacer ITS region of rDNA of Albifimbria verrucaria AUMC16014 confirmed its morphological identification. The fungal sequence was submitted to NCBI given a GenBank accession numberOR039064.
3. Effects of entomopathogenic fungusA. verrucaria on the mosquito Culex pipiens (Diptera: Culicidae)
Lethal time fifty LT50 revealed that the three concentrationsvalues1x106,1x107, and 1x108ofthe applied funguswere 74.741, 65,034 and 56.175h, respectively. The corresponding values for LT90were 142.78 ,133.614, and 122.239, respectively. According to LT50 and LT90 values, the efficiency of fungus increased as the concentration increases. The slop value of LTP lines was 0.19 for the three concentrations. The low value of slop indicates heterogeneity response of mosquito larvae to the fungus effect.
4.Determination the extracellular enzymes of isolated and identified entomopathogenicfungus A. verrucaria
A. verrucaria produce chitinolytic and lipolytic enzymes within a wide range of pH values. The highest chitinase concentration (4.053IU/ml) was detected when the culture medium was adjusted at pH6, whereas the lowest (1.27IU/ml) was measured at pH3. The highest activity of lipase (10.63IU/ml) was measured at pH6. Specific activity of lipase was also high at pH5 and pH6 (153.061 and 123.564IU/mg protein, respectively). Also, protease was produced by A. verrucaria with activity of (458.16U/ml) and specific activity equal to (2.76IU/mgprotein).