ABUNDANCE AND MOLECULAR IDENTIFICATION OF HOUSE FLY MUSCA DOMESTICA (DIPTERA: MUSCIDAE) WITH STUDY TO ASSOCIATED PATHOGENIC BACTERIA

OQ784762-OQ784764. The highest bacterial pathogen presence were Gram-positive Staphylococcus spp. and Bacillus spp. (77.0 %). Gram-negative Escherichia coli , Pseudomonas aeruginosa , and Klebsiella pneumonias had the lowest presence (total of 23.0 %). On the other hand, the results of antibiotic sensitivity revealed that Bacillus spp., Pseudomonas aeruginosa , and Klebsiella pneumonia are more resistant to several antibiotics than Staphylococcus spp. and Escherichia coli .


INTRODUCTION
Most tropical and subtropical developing nations face a major threat from vectorborne illnesses, which account for millions of yearly fatalities in these areas.2004) demonstrated that indirect and direct microbiota-induced phenotypes can affect an insect vector's capacity to spread human illnesses which can impact the host-vector competence.Musca domestica L. (Diptera: Muscidae), known as the house fly, is the most widespread species of fly, found in livestock facilities and cattle feedlots all over the globe (Khamesipour et al., 2018;Neupane et al., 2019).Musca domestica is an earnest a public health pest and pest to livestock; it acts as a mechanical vector of many pathogens to humans and animals through contaminated water and unsanitary food handlers (Douglass and Jesse, 2002; Mian et al., 2002;Issa, 2019).Many pathogenic microbes, including viruses, bacteria, parasites, and fungi are known to be carried by M. domestica (Greenberg, 1973).Adult M. domestica remains in areas rich in microbes, such as those polluted with animal excrement, to feed on decaying matter, including manure and animal fluids, as well as spoiled food and drink (Neupane et al., 2019).On the other hand, investigating of various aspects of host physiology can be influenced by the microbiota of M. domestica, providing resistance to the colonization of pathogenic organisms (Bahrndorff et al., 2017).The microbiota of insects can support the host's physiology in a number of ways, including by giving extra nutrients, supplying colonization resistance against harmful organisms, enhancing tolerance to environmental changes, and promoting the growth and maturation of the host immune system (Bahrndorff et al., 2016; Cerf-Bensussan et al., 2010).Thus, it has been proposed that changes in variations in the human microbiome can account for variations in the phenotypic, like vector competence.Few research, however, has looked at differences in the microbiota through space and time between individuals in natural settings.Also, studying the microbiota of M. domestica helps to understand the relationship between the house fly and pathogenic agents, as well as its ability to transmit disease (EL-Ghwas et al., 2021).More than 100 pathogens can be transmitted to humans and animals by M. domestica including diarrhoea, cholera, bacillary dysentery, typhoid and ophthalmia, and various bacterial strains such as Escherichia coli, Salmonella, and Shigella spp.(Issa, 2019).Evidence showed that adult house flies might contaminate food and spread illness by ingesting pathogens and then transmitting them through their spongy mouthparts, vomit, body hairs, sticky feet, and digestive tracts (De Jesús et al., 2004).Although few studies have addressed the activity pattern of the house fly M. domestica and variation in its associated microbes under field conditions, the present study aimed at investigating the abundance of M. domestica adults in an urban region of Cairo Governorate, Egypt, throughout one year as well as bacterial diversity on its body surface.

Collection of adult flies
Musca domestica adults were collected monthly by hunting from El-Muqattam region (30 °02 ' 20 '' N, 31 °16 ' 28 '' E), Cairo Governorate, Egypt (Figure 1) through the period from May 2022 to April 2023 using commercial pheromone baited fly trap (produced by AL RAWDA Company).Collected adults were sacrificed using ethanol 70% and immediately transferred to sterile Falcon tubes, kept cool on ice for transportation to the laboratory (Neupane et al., 2019), where they were identified morphologically under a binocular microscope according to the description of Al-Ghamdi et al., (2015).Only bacterial isolation specimens were put in sterile saline immediately after collection.LCD digital thermometer and hygrometer were used to determine temperature and relative humidity (HTC-1).
The common house fly, Musca domestica L., is a mechanical vector of numerous diseases that may be spread from one living thing to another by sponging their mouthparts on their body and leg hairs after they have vomited.House flies (Musca domestica) have been known as a mechanical vector in spreading infectious diseases such as cholera, shigellosis, salmonellosis and skin infections.The present study aims to investigate the abundance of M. domestica adults in an urban region of Cairo Governorate, Egypt, throughout one year and bacterial diversity on its body surface.Adults of M. domestica were collected monthly by hunting.Collected adults were subjected to molecular identification.Bacteria were isolated from the surface of flies' body pieces and identified by automated identification systems (VITEK).Obtained data revealed that the highest abundance (201.67±15.57)was recorded in May 2022, while the lowest abundance (17.33±0.57)was recorded in April 2023.Also, M. domestica abundance positively correlated significantly (P≤0.001) with temperature (r= 0.895) and relative humidity (r= 0.827).Musca domestica species were molecularly identified, and the sequences were then banked on NCBI under the accession numbers OQ784762-OQ784764.The highest bacterial pathogen presence were Gram-positive Staphylococcus spp.and Bacillus spp.(77.0 %).Gram-negative Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumonias had the lowest presence (total of 23.0 %).On the other hand, the results of antibiotic sensitivity revealed that Bacillus spp., Pseudomonas aeruginosa, and Klebsiella pneumonia are more resistant to several antibiotics than Staphylococcus spp.and Escherichia coli.

Extraction of DNA
Tissue samples of Musca domestica were put into 1.5 mL Eppendorf tubes.An Invitrogen PureLink® Genomic DNA Kit (Waltham, Massachusetts, USA) was used to extract DNA.To sum up, between 180 and 250 µL of lysis tissue buffer was added to each sample, followed by the addition of proteinase K (10 µL for every 180 µL of tissue lysis buffer) was added and incubated at 56°C for 4 hours.Following the protocol provided by the manufacturer (Invitrogen, Waltham, Massachusetts, USA), the supernatant was put into a new tube.Before vertexing, the lysate was treated with 200 µL of lysis/binding buffer and 200µL of ethanol.The sample was centrifuged for one minute at 10,000 x g.An elution buffer (50 L) was used to elute the DNA after being washed twice with wash buffers and preserved at -20°C.

Polymerase chain reaction (PCR)
The reverse primer LCO1490-R:5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′ and the forward primer LCO1490:5′-GGTCAACAAATCATAAAGATATTGG-3′ were used to amplify the mitochondrial DNA of the COI (cytochrome oxidase subunit I) (Folmer et al., 1994).The final reaction volume for the PCR amplification was 2X (50 L), and it contained 14.5 L of nuclease-free water, 0.2 M (2 L) of each primer, 25 L of 2X master mix solution (i-Taq, iNtRON, Seongnam, Korea), 4 L of template DNA, and 0.2 mg/ml of BSA.After that, it went through 40 times each for denaturation for 1 minute at 95 o C, annealing for 1 minute at 46 o C, and extension for one minute at 72 o C. The denaturation phase lasted for ten minutes at 95°C.At 72 degrees, a final 10-minute extension was carried out.A 1% agarose gel stained with ethidium bromide was used in conjunction with a transilluminator run the PCR amplicon (U.V. transilluminator, Spectroline, Westbury, USA) in order to examine the purity and quantity of the PCR result.Purification of PCR products was achieved using a reagent (Seoul, Korea).Alignment M. domestica COI nucleotide sequences followed sequencing of a single strand of DNA.

Bioinformatics
Chromas Pro 1.5 beta was used to compile the obtained sequences (Technelysium Pty., Tewantin, QLD, Australia).For molecular identification, the BLAST (Basic Local Alignment Search Tool) was employed to evaluate the newly gained M. domestica COI sequences (accession numbers: OQ784762-OQ784764) to those already present in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi).To align the sequences, muscle alignment was performed in MEGA 11.0.Sequence divergences are determined using the Tamura-3 parameter (Tamura, 1992).NJ trees use the Tamura 3-parameter approach to depict the patterns of species divergence (Tamura, 1992).The bootstrapping method with 1000 replicates was carried out in MEGA 11.0 (Kumar et al., 2004).ITOl software for better visuals (Letunic and Bork, 2021).PopArt v.3.0 was utilized to assess the minimal spanning network for haplotype divergence.The phylogenetic tree was inferred using evolutionary distances, and its branches are drawn to size to reflect these distances.The 3-parameter Tamura approach was used in order to calculate evolutionary distances, which appear as the mean number of base substitutions per location (Tamura, 1992).Thirty nucleotide sequences were analyzed in this study.First, second, third, and non-coding positions were all accounted for.The final dataset included 434 positions.MEGA11 was used for evolutionary analysis.

Isolation and identification of associated pathogenic bacteria
Bacteria were isolated from the surface of five flies' body pieces by using sterile saline and centrifugation.The solution spread into nutrient, blood agar, and

Statistical analysis
Data from our results were input and coded using SPSS V.22.Data were tested for satisfying assumptions of parametric tests, and continuous variables were subjected to Shapiro-Wilk and Kolmogorov-Smirnov test for normality.Data were presented as mean and standard deviation.ANOVA analyses were done regarding M. domestica abundance, temperature, and relative humidity; post-hoc analysis was evaluated using Tukey pairwise comparison; P-values were considered significant at <0.05 using MiniTab V 14.The correlation coefficient was estimated between species abundance and observed physical parameters using SigmaPlot V12.0.Data were visualized when possible, using R studio V 2022.02.4

Abundance of Musca domestica adults
Data in Table (1) revealed that the highest abundance of adult Musca domestica (201.67±15.57)was recorded in August 2022, while the lowest abundance (17.33 ± 0.57) was recorded in February 2023.A substantial variation was also detected (P<0.05) in the abundance of M. domestica over the studied periods (Figure 2).

Genetic identification of domestica
According to Saitou and Nei, 1987 the neighbor-joining method employed to deduce the evolutionary history of the species.The best tree choice is shown (Figure 5a).The proportion of bootstrapped value (1000 repetitions) that groups together linked taxa is shown as a colour grade for the branches.Using the minimal haplotype spanning network, the studied nucleotide sequences were also mapped in terms of haplotype diversity.A p-value of 0.117941 indicates nucleotide diversity; 46 segregating sites and 38 parsimony-informative sites were found.The value of Tajima's D was -1.97762, and the probability that D is greater than -1.97762 was found to be 0.98698% (Figure 5b).

Bacterial diversity on Musca domestica body surface
The results of bacterial isolation showed that sixty-four (64) bacteria isolates were obtained for this study; forty-nine (49) were Gram-positive with 77% and fifteen (15) were Gram-negative with 23 % from the houseflies' external surfaces.As illustrated in Figure ( 6).On the other hand, the automated identification systems (VITEK) illustrated that there were six (6) different bacterial genera which are Escherichia coli (12) with (8.0%), Pseudomonas aeruginosa (3) with (2.0%), and Klebsiella pneumonia (9) with (5.0%), Staphylococcus epidermidis ( 14   On the other hand, the results presented in Table (3) for antibiotics sensitivity revealed that bacillus subtilis.,Pseudomonas aeruginosa, and Klebsiella pneumonia are more resistant to several antibiotics than Staphylococcus spp.and Escherichia coli.On the other hand, streptomycin, followed by cefuroxime, has recorded potency against almost all tested pathogens.

DISCUSSION
The findings demonstrated a monthly preponderance of the Musca domestica, a common house fly in the Cairo Governorate of Egypt.Differences in the abundance that is significant (P<0.05) of M. domestica over the studied months were recorded; these differences can be attributed to climatic changes, especially temperature.These results agree with the previous results recorded by Bacillus spp.suggests a potential risk of the infections being transmitted from houseflies to people, which results in illnesses.Thus, raising awareness of this issue and working toward controlling this pest is vital.They must be managed and various vector control strategies must be used to lower their population's density.Thus, the present study's results considered a step in drawing an updated map for insect vectors in Egypt that helps in national control programs Attention has recently been drawn to the function of insect vectors' microbiota in the spread of human infections (Weiss and Aksoy, 2011; Cirimotich et al., 2011; Azambuja et al., 2005).The microbiota of numerous insects known to transmit human infections, particularly those that feed on blood, has been documented (Jones et al., 2010; Wei et al., 2014; Osei-Poku et al., 2012; Gupta et al., 2104; Aksoy et al., 2014).Also, Dillon and Dillon, (

Figure 1
Figure 1 The Study area El-Muqattam region in Cairo, Egypt.
McKanky (Merck, Germany), incubated for 24 hours at 37 ˚C.Colonies  and differentiation were carried out using Gram stain.The pure bacterial isolates were identified by Automated Identification Systems (VITEK) (Kassiri et al., 2012; Kababian et al., 2020; El-Ghwas et al., 2021; Nazari et al., 2017).Sensitivity Bacteria strains to antibiotics The disc diffusion technique was used to examine the susceptibility of bacterial strains isolated from fly parts according to the recommendations of the Laboratory Standards Institute (CLSI) and Clinical (Wayne, 2013; Humphries et al., 2018; El-Waseif et al., 2022).A suspension was adjusted turbidity at 0.5 McFarland for each bacterial strain.Then, antibiotics disks were added to the surface of the medium and incubated for 24 hours at 37°C.The antibiotics covered the four categories: tobramycin, cefotaxime, novobiocin, ampiclox, imipenem, linezolid, azithromycin, tetracycline, rifamycin, piperacillin/tazobactam, kanamycin, and streptomycin.

Figure 2
Figure 2 Stream graph represents the total number of adult Musca domestica collected from El-Muqattam region, Cairo Governorate, Egypt, throughout the study period.In addition, temperature and relative humidity (RH) varied significantly (P<0.05) from one month to another, with the temperature peaking during Aug. 2022 (36.4 ± 0.52a ˚C) and the temperature getting very low during Feb. 2023 (17.26 ± 0.32g ˚C); and the temperature getting very low during Apr.2023 (31.46 ± 0.32˚C); during the same month relative humidity gets as low as 41.26±0.54%(Table2and Figure3).

Figure 3
Figure 3 Line chart of recorded temperature and relative humidity in El-Muqattam region, Cairo Governorate, Egypt, throughout the study period.On the other hand, as shown in Figure (4), M. domestica abundance positively correlated significantly (P ≤ 0.001) with temperature (r= 0.895) and relative humidity (r= 0.827).

Figure 4
Figure 4 Correlation matrix of Musca domestica abundance with temperature relative humidity

Figure 6
Figure 6 Bacterial isolates diversity and distribution on Musca domestics' body surface.

Figure 7
Figure 7 % of Bcterial isoltes from the houseflies external surface.

Table 1
Relative Abundance of adult Musca domestica collected from El-Muqattam region, Cairo Governorate, Egypt, from May 2022 to April 2023.
efThis means in a column that does not share a letter is significantly different.

Table 2
Relative Abundance of adult Musca domestica collected from El-Muqattam region, Cairo Governorate, Egypt, from May 2022 to April 2023.

Table 3
The antibiotics sensitivity of bacterial isolates from Musca domestics' body surface.

Abdel Latif et al., (2004), who
found that the highest abundant of M. domestica in different localities of Alexandria Governorate, Egypt, was recorded in West Districts and El-Amreya where mean grid counts were 75.0±40.8and 72.9±46.3,while the lowest abundance was recorded in East Districts and Borg El-Arab where a mean grid counts were 37.7±19.7 and 39.5±28.1,respectively.Also, Abd El-Halim et al., (2005) who recorded a high abundance of M. domestica in fourteen Egyptian governorates throughout the from 1999 to 2001.Additionally, according to Kenawy

et al., (2014); Desoky et al., (2020),
M. domestica was more plentiful in the summer, autumn, and spring in Cairo and Qalyoubiya Governorates, Egypt, than it was in the winter.Also, they noted seasonal variations in M. domestica abundance in Egypt's Sohag Governorate as a result of climate changes.Moreover, the identification of M. domestica has been a challenge for entomologists due to its morphological similarity to other fly species.However, the use of molecular techniques has provided an effective solution to this problem.The cytochrome oxidase subunit I (COI) gene has been widely used for species identification due to its high variability and conserved regions.Several studies have successfully identified M. domestica using the COI gene, including a study by Archana et al., (2016) that identified M. domestica from different regions of India using COI barcoding.Similarly, a study by Mashaly et al., (2017) used COI gene sequencing to identify M. domestica from different locations in Saudi Arabia.The success of these studies highlights the effectiveness of the COI gene in identifying M. domestica and its potential for use in other insect species.On the other hand, the results of bacterial isolation showed that sixty-four (

Babak et al., 2008; Mawak and Olukose, 2006).
According to research by several scientists, the housefly is a potent carrier of infections such Shigella spp.and Campylobacter spp.and a carrier of bacteria, like Salmonella spp.(

Bahrndorff et al., (2017). Furthermore
, houseflies in Spanish broiler farms were shown to carry Escherichia coli strains that produce β-lactamase.Also, houseflies may be involved in the spread of a disease, the spread of resistance genes, and ecological niches according to Solar

-Gines et al., (2015). Additionally, El-Ghwas et al., (2021) previously
(Nazari et al., 2017)l variety discovered in Musca domestica samples from several places in the Makkah region of Saudia Arabia.Also, there were 18 different bacterial isolates in all; 14 were from Bacillus species, two from Pseudomonas species, one from Micrococcus species, and one from Staphylococcus species.Moreover, most isolates belonged to Bacillus spp., Staphylococcus spp., and Bacillus spp. was shown to be the most common species of bacteria found in different environments, accounting for 31.1% and 22.9% of all detected strains, respectively, in prior research(Nazari et al., 2017).Salmonella and Shigella were the most common types of bacteria detected in hospitals, whereas Enterococcus spp.were the least common.However, Kababian et al., (2020) found that 160 M. domestica house flies collected from animal husbandry in the Iranian province of Qom harbored 23 different bacterial species.P. aeruginosa (100%) and E. coli (73.8% of the total) were the most often isolated bacteria from the skin and the digestive system, respectively.CONCLUSIONThe results demonstrated a monthly preponderance of the Musca domestica, a common house fly in the Cairo Governorate of Egypt, where the highest abundance of adult Musca domestica (201.67±15.57)was recorded in August 2022, while the lowest abundance (17.33±0.57)was recorded in February 2023.Additionally, the presence of various pathogens, such as Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumonia, Staphylococcus aureus, and