Seasonal Fluctuations in Fly Density and Pathogen Carriage in Urban Villages of Pudong New Area

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Many urban villages in Shanghai's Pudong New Area exhibit conditions conducive to fly proliferation and pathogen dissemination. Methods : Our study was conducted from June to October 2024 in Taiping Village, a representative urban village in Pudong. Fly density and species composition were monitored across six environments (residential indoor/outdoor, green belts, wet market exteriors, restaurant exteriors, public toilet exteriors) using standardized cage traps and sticky ribbons following protocols. Captured flies were taxonomically identified, and 149 pooled samples were screened via RT-PCR and microfluidic chip technology for 31 enteric pathogens (viruses, bacteria, parasites). Statistical analyses employed Excel 2019 and R 4.4.3. Results : Sarcophagidae dominated the fly community (60.75%), followed by A. illocata (17.29%) and L. sericata (13.55%). Peak fly density occurred in June-August (1.48 flies/trap). Public toilet exteriors exhibited the highest density (1.33 flies/trap). Pathogen carriage was detected in 61.75% of samples, with Cryptosporidium (51.68%) and diarrheagenic E. coli (DEC, 22.82%) most prevalent. Multi-pathogen co-infection occurred in 44.12% of DEC-positive samples. Residential indoor environments showed the highest pathogen detection rate (71.43%), significantly exceeding public toilet exteriors (38.89%). Conclusion : This study identifies Sarcophagidae as the primary fly vector in Pudong’s urban villages, carrying diverse enteric pathogens—notably Cryptosporidium and DEC—with peak transmission risk during summer. The high overall pathogen carriage rate, particularly in crowded residential interiors, underscores the critical need for enhanced sanitation infrastructure, targeted vector control and community hygiene education to mitigate outbreak risks of enteric infections in these high-density settlements. Urban village Fly population Enteric pathogens Diarrhea Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Flies are important mechanically vectors of a variety of zoonotic diseases, and their body surfaces can carry pathogens including Salmonella, Shigella, norovirus and parasites [ 1 – 3 ] . There are some microorganisms that can survive up to 35 days on the surface or body of flies [ 4 ] . In urban environments, flies habitually shuttle between human/animal excreta and human food sources [ 5 ] . Their characteristic feeding behavior—simultaneous regurgitation and defecation during feeding—significantly amplifies the risk of pathogen transmission through contamination of food, water, and household items [ 6 , 7 ] . Pathogens can be transmitted by flies from contaminated areas to healthy human habitats, causing them to become infected with diseases [ 8 ] . This transmission mechanism is particularly pronounced in areas with poor sanitation and has become an important driver of diarrhoeal disease outbreaks worldwide [ 9 ] . Pudong New Area has a northern subtropical oceanic monsoon climate, with relatively high temperatures and abundant precipitation, which provides favorable breeding conditions for vectors such as flies and other insects [ 10 ] . In addition, Pudong New Area is an important international gateway to China, with major transportation hubs such as Pudong International Airport and Yangshan Deep-water Port. Frequent international passenger flows and trade in goods create potential avenues for cross-border pathogen importation. Studies have shown that outbreaks can spread through international travel, where infected flies are transported to other countries by plane or ship, causing international spread of pathogens [ 11 ] . Therefore, it is very important to study the pathogen spectrum of flies in Pudong New Area and control the density of flies. An Indian study showed that 72% of flies caught with fly ribbons in residential environments tested positive for intestinal bacteria, viruses or parasites. Poor sanitary conditions (e.g., untreated waste near living areas) and inadequate hygiene practices are significant risk factors for high fly density. As fly density increases, the probability of residents contracting infectious diarrhea rises correspondingly [ 12 ] . Despite accelerated urbanization in recent years, sanitary conditions remain substandard in certain areas of Pudong New Area, such as urban villages, where the population is mostly low-income and low-education, with high population density, poor hygiene awareness, and a large amount of untreated garbage in the living environment, which makes urban villages very suitable for the breeding and reproduction of flies, and are prone to the epidemic of intestinal infectious diseases. In recent years, infectious diarrhea in Pudong New Area has demonstrated distinct seasonal patterns, and its reported incidence rate has remained consistently high among notifiable infectious diseases. This imposes a substantial disease burden. Consequently, research on flies as vectors for infectious diarrhea transmission is imperative. In this study, we conducted an on-site investigation on the changes in fly density, species and pathogen carriage in urban villages in Pudong New Area, and analyzed the density, species and pathogen carriage of flies, so as to provide a scientific reference for formulating fly control and early warning and prediction of infectious diarrhea diseases in urban villages. MATERIALS AND METHODS Study sites. This study selected Taiping Village in Kangqiao Town, Pudong New Area as the research site to investigate the density, species composition, and pathogen-carrying status of flies in urban villages, as illustrated in Fig. 1 . Taiping Village was chosen due to its prominent sanitation issues, complex environment, and high population density. Located in the urban-rural fringe of Kangqiao Town, Pudong New Area, the village covers a total area of 1.24 square kilometers and comprises five residential teams.The village has an estimated population of 7,000 residents, the vast majority of whom are migrants. It contains approximately 400 residential buildings, with an average of over 15 occupants per building, primarily rental households. Sanitation and ventilation conditions are generally poor. The village has insufficient toilets, with several households typically sharing one facility. Kitchens are also commonly located in shared public areas. Some residents live in temporary sheds constructed adjacent to the houses, with a per capita living area of less than 5 square meters. To ensure representative sampling of the study area, six distinct environments were selected for investigation following an initial field survey of Taiping Village: exterior environments of restaurants, exterior environments of wet markets, exterior environments of public toilets, green belts, indoor residential environments, and outdoor residential environments. Furthermore, the residential environments were subdivided into five geographical sectors (eastern, southern, western, northern, and central) for grouped analysis. Location Codes for Research Sites as Table 1 shown. Table 1 Location Codes for Research Sites Location Code A1 A2 A3 B1 B2 B3 Study Site East Green Belt East Residential Outdoor East Residential Indoor West Green Belt West Residential Outdoor West Residential Indoor Location Code C1 C2 C3 D1 D2 D3 Study Site South Green Belt South Residential Outdoor South Residential Indoor North Green Belt North Residential Outdoor North Residential Indoor Location Code E1 E2 E3 F G H Study Site Central Green Belt Central Residential Outdoor Central Residential Indoor Wet Market Outdoor Restaurant Outdoor Public Toilet Outdoor Fly Sample Collection. 1. Outdoor Environment Sampling Fly samples in outdoor environments were collected using the cage trap method. All flies captured within 24 hours were transported to the laboratory for taxonomic identification. Sampling Equipment: Canopy-style (suspended type) fly traps compliant with “GB/T 23796 − 2009 Surveillance Methods for Vector Density—Flies”were deployed. The main body measured 40 cm in height and 25 cm in diameter, with an inner conical core of 35 cm height and a top aperture diameter of 2 cm. Attractant: Decayed fish (100g per trap, prepared one day in advance and stored at room temperature) was used as bait, purchased from fangyuan wet markets. Sampling Period: Sampling was conducted at 7-day intervals from June to September 2024. Fly traps were deployed at 09:00 on the sampling day and retrieved at 09:00 the following day, constituting a 24-hour collection cycle. In the event of severe winds or heavy rainfall, sampling was postponed until the day immediately following cessation of adverse weather, with subsequent sampling resuming the 7-day cycle. Outdoor Fly Density Calculation: $$\:D=\frac{{N}_{f}}{{N}_{t}}$$ D: Fly density (flies/trap). N f : Total number of flies captured (unit: individual). N t : Number of traps deployed (unit: trap). 2. Indoor Environment Sampling Fly samples in indoor environments were collected using the sticky ribbon method, in accordance with“GB/T 23796 − 2009 Surveillance Methods for Vector Density—Flies”. Sampling Tool: Sticky fly ribbons (length: 400 mm, width: 35 mm) were used. Procedure: Ribbons were suspended 2.5 m above the floor during sampling. A minimum distance of 3 meters was maintained between adjacent ribbons, with one ribbon placed per standard room. Sampling Period: Sampling was conducted every 7 days from June to September 2024. Sticky fly ribbons were deployed at 09:00 on the sampling day and retrieved at 09:00 the following day, constituting a 24-hour collection cycle. In the event of severe winds or heavy rainfall, sampling was postponed until the day immediately following cessation of adverse weather, with subsequent sampling resuming the 7-day cycle. Indoor Fly Density Calculation: $$\:D=\frac{{N}_{f}}{{N}_{s}\times\:T}$$ D: Fly density[flies/(ribbon·hour)]. N f : Total number of flies captured (unit: individual). N s : Number of traps deployed (unit: ribbon). T: Sampling duration (hours) Fly sample collection, transportation, and species identification Transportation requirements: After each sampling, use disinfected and sterilized vector organism breeding cages to transport the samples to the Pudong New Area CDC(Center of disease control) Vector Laboratory. During transportation, provide life support for water source and temperature to ensure the fly samples arrive in a living state. If fly samples are found dead prior to transportation, place them into biohazard sample bags and transport using a -20℃ freezer. Upon arrival at the laboratory, immediately transfer the samples to a -80℃ freezer for freezing and storage for future testing. Sampling and transportation containers are provided by the Pudong CDC Vector Laboratory. The sample delivery personnel shall fill in the record form in detail, and the CDC sample receiver shall sign for it. Fly species identification: After receiving the cages, use the freezing method to stun or kill the flies. According to the Common Vector Organisms: Classification and Identification Manual edited by Zhou Minghao and Chu Hongliang [ 14 ] , use a stereo microscope (Nikon SMZ800N) to classify and count the flies on an ice plate based on their morphological characteristics. Sample Preprocessing Detection of pathogens carried on the surface and inside flies 1. Extraction of surface-carried pathogens: Separate fly samples by species. Place 10-20 flies of each species into a single tube. Transfer them to a glass conical flask containing 15mL sterile physiological saline. Oscillate repeatedly to fully wash off pathogens carried on the fly surface. Reserve the washing solution for later use. 2.Extraction of internally-carried pathogens: Place surface-washed flies in a culture dish. Add 75% ethanol to fully disinfect the surface for 5 minutes to eliminate surface pathogens. Rinse three times with sterile physiological saline to remove ethanol. Cut open the fly abdomen with sterile ophthalmic scissors and place it in a 2mL sampling tube. Add 1mL Hanks solution and 15μL proteinase K. Vigorously oscillate for 5 minutes using a Kayaode G100 high-throughput tissue grinding homogenizer until the fly viscera are thoroughly macerated. Reserve the homogenate for later use. PCR Detection Viral RNA was extracted using a fully automated nucleic acid extraction instrument (Tianlong-GeneRotex96). Detection was performed by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR). Experimental results were interpreted according to the determination criteria specified in the manufacturer's (Thermo Fisher SCIENTIFIC) manual for intestinal pathogen screening microfluidic chips. Sealing was performed using a chip sealer, and quantification was conducted with a QuantStudio7 fluorescent quantitative PCR instrument. Determination was based on the morphology of amplification curves and corresponding Ct values. Qualitative detection covered 31 intestinal pathogens, including: 7 Viruses: Adenovirus, Norovirus (GI + GII), Sapovirus (I,II,IV&V), Astrovirus, Para-enteric orphan virus, Enterovirus (excluding D68), Rotavirus A + B + C 18 Bacteria: Enteropathogenic Escherichia coli, Enterotoxigenic E. coli, Enteroadherent E. coli, Intestinal invasive Escherichia coli or Shigella, Enterohemorrhagic E. coli, E. coli O157, Vibrio (V. parahaemolyticus, V. vulnificus, V. cholerae), Yersinia enterocolitica, Aeromonas hydrophila, Campylobacter (C. jejuni, C. coli, C. upsaliensis), Plesiomonas shigelloides, Clostridium difficile (Toxin A/B), Highly virulent C. difficile O27, Salmonella, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Shiga toxin-producing E. coli (STEC)(stx1/stx2) 6 Parasites: Blastocystis hominis, Dientamoeba fragilis, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Cryptosporidium Results were analyzed using Design and Analysis software. Data Analysis After collecting information on fly populations, density, and pathogen detection results, data were organized using Excel 2019. Statistical analysis was performed using R 4.4.3, with P<0.05 considered statistically significant. Data preprocessing and statistical analysis figures were generated in Excel 2019. Redundancy analysis result plots were created by R 4.4.3. RESULT Species Composition of Captured Flies From June to October 2024, we deployed a total of 234 fly traps in outdoor environments across Taiping Village, capturing 202 flies. The fly density was 0.863 flies/trap. Indoors in residential households, 90 sticky fly strips were placed, capturing 12 flies, with a density of 0.133 flies/(ribbon·hour). The most frequently captured species was Sarcophagidae , accounting for 60.748% of the total. This was followed by A.illocata and L.sericata , representing 17.290% and 13.551%, respectively. Additionally, a small number of M.domestica were captured, constituting 5.607% of the total. The least captured species were C.megacephala and F.prisca , each accounting for 1.402%.The distribution of captured fly species across different months is illustrated in Fig. 2 . Captured Fly Density Dynamics: The overall density trend across monitored fly species manifested an initial decline followed by a modest recovery over the observational months. In the outdoor environments across Taiping Village, the highest total fly density occurred in June (1.48 flies/trap), dominated by Sarcophagidae (1.02 flies/trap). Density declined sharply in July (0.52 flies/trap) but gradually recovered in August (0.77 flies/trap) and September (0.83 flies/trap). By October, density decreased to 0.62 flies/trap. Indoor fly density was significantly lower than outdoors. Activity was confined to June (0.028 flies/strip·h) and July (0.002 flies/strip·h), with no captures from August to October. L.sericata were captured in both June and July, with the highest density observed in June (0.0175 flies/(strip·h)). Concurrently, minor quantities of Sarcophagidae and A.illocata were also captured in June. As illustrated in Fig. 3 , temporal trends in fly density across distinct environments from June to October revealed an overall decline in greenbelts, residential outdoor areas, and residential interiors, while agricultural market exteriors exhibited a significant surge in October. Spatial stratification identified public toilet exteriors as the highest-density locus (1.33 flies/trap), followed sequentially by market exteriors (1.06 flies/trap), greenbelts (0.92 flies/trap), and residential exteriors (0.73 flies/trap), with restaurant exteriors registering the lowest density (0.55 flies/trap). Pathogen Detection Results Following weekly species identification of the captured flies, we tested them for pathogen carriage. Flies within each trap were grouped by species. From June to October, a total of 149 sample groups were tested, with a pathogen positivity rate of 61.745%. Based on our analysis of these samples, we detected a total of 13 pathogens, including: 9 bacterial species (EAEC, EHEC, EIEC, EPEC, STEC, O157, Aeromonas hydrophila , Campylobacter ), 3 kind of viruses (Sapovirus, Norovirus, Astrovirus), and 2 kind of parasites ( Cryptosporidium , Blastocystis hominis ). The status of intestinal pathogens in various fly species is shown in Fig. 4 . Cryptosporidium was the most widely distributed pathogen in the environment, with a detection rate of 51.68%. The overall detection rate of diarrheagenic Escherichia coli (DEC) was 22.82%, among which EPEC (10.07%), EHEC (6.71%), and STEC (5.37%) were the predominant subtypes. Blastocystis hominis was detected in 9.40% of samples, while lower detection rates were observed for Aeromonas hydrophila (2.68%), sapovirus (0.67%), Campylobacter (0.67%), Astrovirus (0.67%), and Norovirus (0.67%). Notably, 44.12% of DEC-positive samples showed co-infection with other pathogens (e.g., Cryptosporidium or Blastocystis hominis ), indicating a prevalent multi-pathogen co-infection phenomenon in fly samples from urban villages. The distribution of CT values for different pathogens is shown in Fig. 5 , where Cryptosporidium exhibits the highest detection rate and the lowest average CT value. The temporal variation in pathogen detection rates in flies, as shown in Fig. 6 reveals peak periods of pathogen detection during Weeks 4–7 and 10–14. The positive rates of various pathogens in fly samples across different environments and the results of Fisher's exact tests are as shown as Table 2 . The highest overall pathogen detection rate occurred in residential indoor environments (71.43%), while the lowest was observed in public toilet outdoor environments (38.89%). The Fisher's exact test for DEC showed p < 0.05, indicating a statistically significant difference in DEC detection rates among environments; no such significance was found for other pathogens. Table 2 Number of pathogen-positive samples across environments and Fisher's exact test results Pathogen Number of pathogen-positive samples P-value (P<0.05) Green Belt(n = 55) Residential Outdoor(n = 47) Residential Indoor(n = 7) Market Outdoor(n = 13) Restaurant Outdoor(n = 9) Public Toilet Outdoor(n = 18) Cryptosporidium 28 27 4 5 6 7 0.62 Blastocystis hominis 4 5 1 3 1 0 0.26 Sapovirus 9 9 0 3 1 2 0.86 Aeromonas hydrophila 2 1 1 0 0 0 0.52 Campylobacter 0 1 0 0 0 0 0.63 Astrovirus 0 0 0 1 0 0 0.19 Norovirus 0 0 0 0 0 1 0.32 DEC 13 17 1 2 1 0 0.03 EPEC 4 5 0 1 0 0 0.79 EHEC 3 5 1 0 0 0 0.44 STEC 2 3 0 0 0 0 0.90 EAEC 1 2 0 0 1 0 0.55 EIEC 1 0 0 1 0 0 0.36 O157 2 2 0 0 0 0 1.00 Overall detection rate 63.64% 65.96% 71.43% 61.54% 66.67% 38.89% - DISCUSSION This study revealed that the fly community in Taiping Village is dominated by Sarcophagidae , followed by A.illocata and L.sericata . This distribution pattern is likely closely related to the unique microenvironment of the urban village: Sarcophagidae exhibit a strong attraction to decaying meat and animal excrement, enabling them to proliferate rapidly in areas where kitchen waste accumulates, public toilets lack adequate cleaning, and stray animal feces pile up [ 14 – 16 ] . In Taiping Village, uncleared kitchen waste is present in outdoor areas of residential zones and around markets. Resident toilets are mostly communal and not regularly cleaned by the occupants. Concurrently, the area has a large population of stray animals such as cats and dogs, resulting in significant amounts of animal feces in the outdoor environment. All these factors provide ideal breeding conditions for Sarcophagidae . Notably, L.sericata exhibit higher densities in green belts and around public toilets. This may be associated with the damp vegetation in green belts and the concentrated fecal contamination surrounding public toilets. This finding suggests the need for targeted sanitation management enhancement in these specific areas [ 17 ] . From the perspective of seasonal fluctuation, the peak density of flies occurred from June to August, which aligns with the high-temperature and high-humidity summer climate in the Shanghai area [ 18 ] . Elevated temperatures accelerate the decomposition of organic matter, while precipitation may cause delays in waste removal, further promoting fly reproduction [ 16 ] . However, a significant decline in fly density was observed in October, likely associated with the drop in temperature and the onset of the fly overwintering period. This pattern is consistent with the conclusion drawn from previous studies that "fly density exhibits a positive correlation with temperature" [ 19 , 20 ] . It also highlights the need to prioritize fly control efforts during the summer months. Among the 13 pathogens detected in this study, Cryptosporidium and Campylobacter exhibited higher CT values, indicating higher pathogen loads carried by flies. This suggests their potential role as significant vectors for intestinal infectious diseases in the urban village. Cryptosporidium infection is a common cause of gastroenteritis, typically resulting in diarrhea lasting approximately one to two weeks post-infection. It poses a greater risk to immunocompromised individuals and is primarily transmitted via the fecal-oral route, often through contaminated water or food [ 21 , 22 ] . Campylobacter , on the other hand, can disrupt the intestinal permeability barrier, triggering acute enteritis [ 23 – 25 ] . The high detection rates of these pathogens in outdoor residential areas and green belts are directly linked to the flies' behavioral patterns of frequent contact with both human excreta and food sources [ 4 , 5 ] . Additionally, norovirus was detected in only one positive sample from the exterior environment of public toilets. This low detection rate may be associated with the relatively small sample size or the limited viability duration of the virus on the surface of flies. Further validation with an expanded sample size may be required. Despite this, the overall pathogen detection rate reached 71.43% within the interior environments of residential areas, significantly higher than the 38.89% observed in public toilet exteriors. This disparity suggests that indoor sanitary conditions—such as poor ventilation and shared toilet facilities—may exacerbate the retention of pathogens within the human living environment. Consequently, specific improvements to residents' living facilities are necessary [ 26 ] . The main limitation of this study lies in the lack of direct support from epidemiological investigation data. Although we detected various human intestinal pathogens on the body surfaces of flies and analyzed their spatial distribution patterns, the absence of synchronized stool pathogen testing among community residents—particularly those with diarrhea symptoms—prevents us from quantifying the statistical association between fly breeding density, pathogen carriage rate, and human infection rates. It is recommended that future research incorporate monitoring of diarrheal symptoms in the population simultaneously with fly sampling, along with collecting stool samples from volunteers for comparative pathogen analysis. This would provide the most direct evidence supporting the role of flies in the transmission of intestinal infectious diseases. Based on the findings of this study, the following control measures are recommended: Firstly, environmental remediation in key areas: Enhance the frequency of waste removal in environments surrounding markets and restaurant kitchens, and promote the use of sealed waste containers to reduce potential fly breeding sites [ 27 , 28 ] . Secondly, seasonal fly control interventions: During the peak fly density period from June to August, implement residual spraying and toxic bait placement in areas such as green belts and around public toilets to suppress fly populations [ 29 ] . Thirdly, resident hygiene education: Conduct targeted health campaigns for the floating population in urban villages, advocating for the timely disposal of kitchen waste and consistent hand-washing after toilet use to disrupt the fecal-oral transmission route. CONCLUSION This study revealed that the fly population in the urban villages of Pudong New Area is predominantly composed of Sarcophagidae . These flies were found to carry various enteric pathogens, including Cryptosporidium , Blastocystis hominis , EPEC, EHEC and etc. Summer was identified as the period of peak fly density and consequently, heightened transmission risk. Although the distribution of pathogens across different environments did not reach statistical significance, the overall high detection rate underscores the necessity of enhancing environmental sanitation management and fly vector control within urban villages to mitigate the risk of enteric infectious disease outbreaks. Declarations Ethics approval and consent to participate (Not applicable) Consent for publication Prior to the deployment of fly traps, informed consent was obtained from residents. They were explicitly informed that the collected fly samples would be transported to the laboratory for pathogen detection, and that the final analytical results would be published in a scientific paper. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests All authors read and approved the final manuscript. Funding This work was supported by Health System Subject Leader Training Program of Pudong New Area in Shanghai, Grant No. PWRd2023-12. Authors' contributions J.L. , Y.T. and C.L. wrote the main manuscript text, R.G.and H.L. prepared figures 1-2, Y.G.and J.L. prepared figures 3-4, Q.L. and G.G. prepared figures 5-6. H.L. and L.H. provided the profesional guidance and revisions. All authors reviewed the manuscript. Acknowledgements (Not applicable) References World Health Organization. Vector-borne Diseases.2017. Available from: https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases. Retrieved on 31-10-2017. Onwugamba, F. C., Fitzgerald, J. R., Rochon, K., Guardabassi, L., Alabi, A., Kühne, S., Grobusch, M. P., & Schaumburg, F. (2018). 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15:29:29","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108757,"visible":true,"origin":"","legend":"","description":"","filename":"dae9b4cecc5f458e81db7b9b0ae968211structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7586460/v1/9745926e5ce78a91a7e274ad.xml"},{"id":94469369,"identity":"e0618fde-7b29-4608-a786-a9f83af14512","added_by":"auto","created_at":"2025-10-27 15:28:29","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117469,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7586460/v1/c04268ce8a00ab0e02374fc7.html"},{"id":94469489,"identity":"249e0248-47fa-4f1f-979a-57ce81f064b6","added_by":"auto","created_at":"2025-10-27 15:29:42","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54186,"visible":true,"origin":"","legend":"\u003cp\u003eThe Location of Taiping Village\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7586460/v1/d0f6d2f846338179216baf87.jpeg"},{"id":94469432,"identity":"c4d7564c-7f61-4fa3-923e-4cc7b0c7f7e4","added_by":"auto","created_at":"2025-10-27 15:29:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40197,"visible":true,"origin":"","legend":"\u003cp\u003eSpecies Composition of Captured Flies Monthly from June to October\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7586460/v1/8d5bc06bdc557ad5e96a5cdd.png"},{"id":94469500,"identity":"581a20aa-1d3b-4b9e-908c-300ea66a88ee","added_by":"auto","created_at":"2025-10-27 15:29:53","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":65262,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal Trends in Fly Density Across Environmental Types and Sampling Sites, June-October\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7586460/v1/cce49e76f0f045ebbe14d04a.jpeg"},{"id":94469402,"identity":"5a394324-49e9-4395-aebe-e834f5835de7","added_by":"auto","created_at":"2025-10-27 15:28:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":25669,"visible":true,"origin":"","legend":"\u003cp\u003eIntestinal Pathogens in Different Fly Species\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7586460/v1/33775fde9bd6f0b93ad931bd.png"},{"id":94491158,"identity":"13b089b7-4017-4f27-b64f-5b7d51db27a7","added_by":"auto","created_at":"2025-10-27 17:23:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":875806,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7586460/v1/8594b8db-0b62-49f7-b450-08aa2adfaaf0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seasonal Fluctuations in Fly Density and Pathogen Carriage in Urban Villages of Pudong New Area","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eFlies are important mechanically vectors of a variety of zoonotic diseases, and their body surfaces can carry pathogens including Salmonella, Shigella, norovirus and parasites\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. There are some microorganisms that can survive up to 35 days on the surface or body of flies\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In urban environments, flies habitually shuttle between human/animal excreta and human food sources\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Their characteristic feeding behavior\u0026mdash;simultaneous regurgitation and defecation during feeding\u0026mdash;significantly amplifies the risk of pathogen transmission through contamination of food, water, and household items \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Pathogens can be transmitted by flies from contaminated areas to healthy human habitats, causing them to become infected with diseases\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. This transmission mechanism is particularly pronounced in areas with poor sanitation and has become an important driver of diarrhoeal disease outbreaks worldwide\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePudong New Area has a northern subtropical oceanic monsoon climate, with relatively high temperatures and abundant precipitation, which provides favorable breeding conditions for vectors such as flies and other insects\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In addition, Pudong New Area is an important international gateway to China, with major transportation hubs such as Pudong International Airport and Yangshan Deep-water Port. Frequent international passenger flows and trade in goods create potential avenues for cross-border pathogen importation. Studies have shown that outbreaks can spread through international travel, where infected flies are transported to other countries by plane or ship, causing international spread of pathogens\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is very important to study the pathogen spectrum of flies in Pudong New Area and control the density of flies.\u003c/p\u003e\u003cp\u003eAn Indian study showed that 72% of flies caught with fly ribbons in residential environments tested positive for intestinal bacteria, viruses or parasites. Poor sanitary conditions (e.g., untreated waste near living areas) and inadequate hygiene practices are significant risk factors for high fly density. As fly density increases, the probability of residents contracting infectious diarrhea rises correspondingly\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Despite accelerated urbanization in recent years, sanitary conditions remain substandard in certain areas of Pudong New Area, such as urban villages, where the population is mostly low-income and low-education, with high population density, poor hygiene awareness, and a large amount of untreated garbage in the living environment, which makes urban villages very suitable for the breeding and reproduction of flies, and are prone to the epidemic of intestinal infectious diseases. In recent years, infectious diarrhea in Pudong New Area has demonstrated distinct seasonal patterns, and its reported incidence rate has remained consistently high among notifiable infectious diseases. This imposes a substantial disease burden. Consequently, research on flies as vectors for infectious diarrhea transmission is imperative.\u003c/p\u003e\u003cp\u003eIn this study, we conducted an on-site investigation on the changes in fly density, species and pathogen carriage in urban villages in Pudong New Area, and analyzed the density, species and pathogen carriage of flies, so as to provide a scientific reference for formulating fly control and early warning and prediction of infectious diarrhea diseases in urban villages.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eStudy sites.\u003c/p\u003e\n\u003cp\u003eThis study selected Taiping Village in Kangqiao Town, Pudong New Area as the research site to investigate the density, species composition, and pathogen-carrying status of flies in urban villages, as illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Taiping Village was chosen due to its prominent sanitation issues, complex environment, and high population density. Located in the urban-rural fringe of Kangqiao Town, Pudong New Area, the village covers a total area of 1.24 square kilometers and comprises five residential teams.The village has an estimated population of 7,000 residents, the vast majority of whom are migrants. It contains approximately 400 residential buildings, with an average of over 15 occupants per building, primarily rental households. Sanitation and ventilation conditions are generally poor. The village has insufficient toilets, with several households typically sharing one facility. Kitchens are also commonly located in shared public areas. Some residents live in temporary sheds constructed adjacent to the houses, with a per capita living area of less than 5 square meters.\u003c/p\u003e\n\u003cp\u003eTo ensure representative sampling of the study area, six distinct environments were selected for investigation following an initial field survey of Taiping Village: exterior environments of restaurants, exterior environments of wet markets, exterior environments of public toilets, green belts, indoor residential environments, and outdoor residential environments. Furthermore, the residential environments were subdivided into five geographical sectors (eastern, southern, western, northern, and central) for grouped analysis. Location Codes for Research Sites as Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shown.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eLocation Codes for Research Sites\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLocation Code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB3\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStudy Site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEast Green Belt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEast Residential Outdoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEast Residential Indoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWest Green Belt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWest Residential Outdoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWest Residential Indoor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLocation Code\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStudy Site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSouth Green Belt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSouth Residential Outdoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSouth Residential Indoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNorth Green Belt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNorth Residential Outdoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNorth Residential Indoor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLocation Code\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStudy Site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCentral Green Belt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCentral Residential Outdoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCentral Residential Indoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWet Market Outdoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRestaurant Outdoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePublic Toilet Outdoor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eFly Sample Collection.\u003c/p\u003e\n\u003cp\u003e1. Outdoor Environment Sampling\u003c/p\u003e\n\u003cp\u003eFly samples in outdoor environments were collected using the cage trap method. All flies captured within 24 hours were transported to the laboratory for taxonomic identification.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eSampling Equipment: Canopy-style (suspended type) fly traps compliant with \u0026ldquo;GB/T 23796\u0026thinsp;\u0026minus;\u0026thinsp;2009 Surveillance Methods for Vector Density\u0026mdash;Flies\u0026rdquo;were deployed. The main body measured 40 cm in height and 25 cm in diameter, with an inner conical core of 35 cm height and a top aperture diameter of 2 cm.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eAttractant: Decayed fish (100g per trap, prepared one day in advance and stored at room temperature) was used as bait, purchased from fangyuan wet markets.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eSampling Period: Sampling was conducted at 7-day intervals from June to September 2024. Fly traps were deployed at 09:00 on the sampling day and retrieved at 09:00 the following day, constituting a 24-hour collection cycle. In the event of severe winds or heavy rainfall, sampling was postponed until the day immediately following cessation of adverse weather, with subsequent sampling resuming the 7-day cycle.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eOutdoor Fly Density Calculation:\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:D=\\frac{{N}_{f}}{{N}_{t}}$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eD: Fly density (flies/trap).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eN\u003csub\u003ef\u003c/sub\u003e: Total number of flies captured (unit: individual).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eN\u003csub\u003et\u003c/sub\u003e: Number of traps deployed (unit: trap).\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e2. Indoor Environment Sampling\u003c/p\u003e\n\u003cp\u003eFly samples in indoor environments were collected using the sticky ribbon method, in accordance with\u0026ldquo;GB/T 23796\u0026thinsp;\u0026minus;\u0026thinsp;2009 Surveillance Methods for Vector Density\u0026mdash;Flies\u0026rdquo;.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eSampling Tool: Sticky fly ribbons (length: 400 mm, width: 35 mm) were used.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eProcedure: Ribbons were suspended 2.5 m above the floor during sampling. A minimum distance of 3 meters was maintained between adjacent ribbons, with one ribbon placed per standard room.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eSampling Period: Sampling was conducted every 7 days from June to September 2024. Sticky fly ribbons were deployed at 09:00 on the sampling day and retrieved at 09:00 the following day, constituting a 24-hour collection cycle. In the event of severe winds or heavy rainfall, sampling was postponed until the day immediately following cessation of adverse weather, with subsequent sampling resuming the 7-day cycle.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eIndoor Fly Density Calculation:\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e$$\\:D=\\frac{{N}_{f}}{{N}_{s}\\times\\:T}$$\u003c/div\u003e\u003c/div\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eD: Fly density[flies/(ribbon\u0026middot;hour)].\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eN\u003csub\u003ef\u003c/sub\u003e: Total number of flies captured (unit: individual).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eN\u003csub\u003es\u003c/sub\u003e: Number of traps deployed (unit: ribbon).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eT: Sampling duration (hours)\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003cp\u003eFly sample collection, transportation, and species identification\u003c/p\u003e\u003cp\u003eTransportation requirements: After each sampling, use disinfected and sterilized vector organism breeding cages to transport the samples to the Pudong New Area CDC(Center of disease control) Vector Laboratory. During transportation, provide life support for water source and temperature to ensure the fly samples arrive in a living state. If fly samples are found dead prior to transportation, place them into biohazard sample bags and transport using a -20℃ freezer. Upon arrival at the laboratory, immediately transfer the samples to a -80℃ freezer for freezing and storage for future testing. Sampling and transportation containers are provided by the Pudong CDC Vector Laboratory. The sample delivery personnel shall fill in the record form in detail, and the CDC sample receiver shall sign for it.\u003c/p\u003e\u003cp\u003eFly species identification: After receiving the cages, use the freezing method to stun or kill the flies. According to the Common Vector Organisms: Classification and Identification Manual edited by Zhou Minghao and Chu Hongliang\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, use a stereo microscope (Nikon SMZ800N) to classify and count the flies on an ice plate based on their morphological characteristics.\u003c/p\u003e\u003cp\u003eSample Preprocessing\u003c/p\u003e\u003cp\u003eDetection of pathogens carried on the surface and inside flies\u003c/p\u003e\u003cp\u003e1. Extraction of surface-carried pathogens: Separate fly samples by species. Place 10-20 flies of each species into a single tube. Transfer them to a glass conical flask containing 15mL sterile physiological saline. Oscillate repeatedly to fully wash off pathogens carried on the fly surface. Reserve the washing solution for later use.\u003c/p\u003e\u003cp\u003e2.Extraction of internally-carried pathogens: Place surface-washed flies in a culture dish. Add 75% ethanol to fully disinfect the surface for 5 minutes to eliminate surface pathogens. Rinse three times with sterile physiological saline to remove ethanol. Cut open the fly abdomen with sterile ophthalmic scissors and place it in a 2mL sampling tube. Add 1mL Hanks solution and 15\u0026mu;L proteinase K. Vigorously oscillate for 5 minutes using a Kayaode G100 high-throughput tissue grinding homogenizer until the fly viscera are thoroughly macerated. Reserve the homogenate for later use.\u003c/p\u003e\u003cp\u003ePCR Detection\u003c/p\u003e\u003cp\u003eViral RNA was extracted using a fully automated nucleic acid extraction instrument (Tianlong-GeneRotex96). Detection was performed by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR). Experimental results were interpreted according to the determination criteria specified in the manufacturer\u0026apos;s (Thermo Fisher SCIENTIFIC) manual for intestinal pathogen screening microfluidic chips. Sealing was performed using a chip sealer, and quantification was conducted with a QuantStudio7 fluorescent quantitative PCR instrument. Determination was based on the morphology of amplification curves and corresponding Ct values.\u003c/p\u003e\u003cp\u003eQualitative detection covered 31 intestinal pathogens, including:\u003c/p\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e7 Viruses: Adenovirus, Norovirus (GI\u0026thinsp;+\u0026thinsp;GII), Sapovirus (I,II,IV\u0026amp;V), Astrovirus, Para-enteric orphan virus, Enterovirus (excluding D68), Rotavirus A\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e18 Bacteria: Enteropathogenic Escherichia coli, Enterotoxigenic E. coli, Enteroadherent E. coli, Intestinal invasive Escherichia coli or Shigella, Enterohemorrhagic E. coli, E. coli O157, Vibrio (V. parahaemolyticus, V. vulnificus, V. cholerae), Yersinia enterocolitica, Aeromonas hydrophila, Campylobacter (C. jejuni, C. coli, C. upsaliensis), Plesiomonas shigelloides, Clostridium difficile (Toxin A/B), Highly virulent C. difficile O27, Salmonella, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Shiga toxin-producing E. coli (STEC)(stx1/stx2)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e6 Parasites: Blastocystis hominis, Dientamoeba fragilis, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Cryptosporidium\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003cp\u003eResults were analyzed using Design and Analysis software.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eAfter collecting information on fly populations, density, and pathogen detection results, data were organized using Excel 2019. Statistical analysis was performed using R 4.4.3, with P\u0026lt;0.05 considered statistically significant. Data preprocessing and statistical analysis figures were generated in Excel 2019. Redundancy analysis result plots were created by R 4.4.3.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULT","content":"\u003cp\u003eSpecies Composition of Captured Flies\u003c/p\u003e\n\u003cp\u003eFrom June to October 2024, we deployed a total of 234 fly traps in outdoor environments across Taiping Village, capturing 202 flies. The fly density was 0.863 flies/trap. Indoors in residential households, 90 sticky fly strips were placed, capturing 12 flies, with a density of 0.133 flies/(ribbon\u0026middot;hour). The most frequently captured species was \u003cem\u003eSarcophagidae\u003c/em\u003e, accounting for 60.748% of the total. This was followed by \u003cem\u003eA.illocata\u003c/em\u003e and \u003cem\u003eL.sericata\u003c/em\u003e, representing 17.290% and 13.551%, respectively. Additionally, a small number of \u003cem\u003eM.domestica\u003c/em\u003e were captured, constituting 5.607% of the total. The least captured species were \u003cem\u003eC.megacephala\u003c/em\u003e and \u003cem\u003eF.prisca\u003c/em\u003e, each accounting for 1.402%.The distribution of captured fly species across different months is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eCaptured Fly Density Dynamics:\u003c/p\u003e\n\u003cp\u003eThe overall density trend across monitored fly species manifested an initial decline followed by a modest recovery over the observational months. In the outdoor environments across Taiping Village, the highest total fly density occurred in June (1.48 flies/trap), dominated by \u003cem\u003eSarcophagidae\u003c/em\u003e (1.02 flies/trap). Density declined sharply in July (0.52 flies/trap) but gradually recovered in August (0.77 flies/trap) and September (0.83 flies/trap). By October, density decreased to 0.62 flies/trap. Indoor fly density was significantly lower than outdoors. Activity was confined to June (0.028 flies/strip\u0026middot;h) and July (0.002 flies/strip\u0026middot;h), with no captures from August to October. \u003cem\u003eL.sericata\u003c/em\u003e were captured in both June and July, with the highest density observed in June (0.0175 flies/(strip\u0026middot;h)). Concurrently, minor quantities of \u003cem\u003eSarcophagidae\u003c/em\u003e and \u003cem\u003eA.illocata\u003c/em\u003e were also captured in June.\u003c/p\u003e\n\u003cp\u003eAs illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, temporal trends in fly density across distinct environments from June to October revealed an overall decline in greenbelts, residential outdoor areas, and residential interiors, while agricultural market exteriors exhibited a significant surge in October. Spatial stratification identified public toilet exteriors as the highest-density locus (1.33 flies/trap), followed sequentially by market exteriors (1.06 flies/trap), greenbelts (0.92 flies/trap), and residential exteriors (0.73 flies/trap), with restaurant exteriors registering the lowest density (0.55 flies/trap).\u003c/p\u003e\n\u003cp\u003ePathogen Detection Results\u003c/p\u003e\n\u003cp\u003eFollowing weekly species identification of the captured flies, we tested them for pathogen carriage. Flies within each trap were grouped by species. From June to October, a total of 149 sample groups were tested, with a pathogen positivity rate of 61.745%. Based on our analysis of these samples, we detected a total of 13 pathogens, including: 9 bacterial species (EAEC, EHEC, EIEC, EPEC, STEC, O157, \u003cem\u003eAeromonas hydrophila\u003c/em\u003e, \u003cem\u003eCampylobacter\u003c/em\u003e), 3 kind of viruses (Sapovirus, Norovirus, Astrovirus), and 2 kind of parasites (\u003cem\u003eCryptosporidium\u003c/em\u003e, \u003cem\u003eBlastocystis hominis\u003c/em\u003e). The status of intestinal pathogens in various fly species is shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCryptosporidium\u003c/em\u003e was the most widely distributed pathogen in the environment, with a detection rate of 51.68%. The overall detection rate of diarrheagenic Escherichia coli (DEC) was 22.82%, among which EPEC (10.07%), EHEC (6.71%), and STEC (5.37%) were the predominant subtypes. \u003cem\u003eBlastocystis hominis\u003c/em\u003e was detected in 9.40% of samples, while lower detection rates were observed for \u003cem\u003eAeromonas hydrophila\u003c/em\u003e (2.68%), sapovirus (0.67%), \u003cem\u003eCampylobacter\u003c/em\u003e (0.67%), Astrovirus (0.67%), and Norovirus (0.67%). Notably, 44.12% of DEC-positive samples showed co-infection with other pathogens (e.g., \u003cem\u003eCryptosporidium\u003c/em\u003e or \u003cem\u003eBlastocystis hominis\u003c/em\u003e), indicating a prevalent multi-pathogen co-infection phenomenon in fly samples from urban villages.\u003c/p\u003e\n\u003cp\u003eThe distribution of CT values for different pathogens is shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, where \u003cem\u003eCryptosporidium\u003c/em\u003e exhibits the highest detection rate and the lowest average CT value.\u003c/p\u003e\n\u003cp\u003eThe temporal variation in pathogen detection rates in flies, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e reveals peak periods of pathogen detection during Weeks 4\u0026ndash;7 and 10\u0026ndash;14.\u003c/p\u003e\n\u003cp\u003eThe positive rates of various pathogens in fly samples across different environments and the results of Fisher\u0026apos;s exact tests are as shown as Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The highest overall pathogen detection rate occurred in residential indoor environments (71.43%), while the lowest was observed in public toilet outdoor environments (38.89%). The Fisher\u0026apos;s exact test for DEC showed p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, indicating a statistically significant difference in DEC detection rates among environments; no such significance was found for other pathogens.\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ctable id=\"Tab2\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber of pathogen-positive samples across environments and Fisher\u0026apos;s exact test results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePathogen\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eNumber of pathogen-positive samples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003cp\u003e(P\u0026lt;0.05)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGreen Belt(n\u0026thinsp;=\u0026thinsp;55)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResidential Outdoor(n\u0026thinsp;=\u0026thinsp;47)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResidential Indoor(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMarket Outdoor(n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRestaurant Outdoor(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePublic Toilet Outdoor(n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCryptosporidium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBlastocystis hominis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSapovirus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAeromonas hydrophila\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCampylobacter\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAstrovirus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNorovirus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEPEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEHEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSTEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEAEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEIEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eO157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOverall detection rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.64%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.96%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.43%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.54%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.89%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study revealed that the fly community in Taiping Village is dominated by \u003cem\u003eSarcophagidae\u003c/em\u003e, followed by \u003cem\u003eA.illocata\u003c/em\u003e and \u003cem\u003eL.sericata\u003c/em\u003e. This distribution pattern is likely closely related to the unique microenvironment of the urban village: \u003cem\u003eSarcophagidae\u003c/em\u003e exhibit a strong attraction to decaying meat and animal excrement, enabling them to proliferate rapidly in areas where kitchen waste accumulates, public toilets lack adequate cleaning, and stray animal feces pile up \u003csup\u003e[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In Taiping Village, uncleared kitchen waste is present in outdoor areas of residential zones and around markets. Resident toilets are mostly communal and not regularly cleaned by the occupants. Concurrently, the area has a large population of stray animals such as cats and dogs, resulting in significant amounts of animal feces in the outdoor environment. All these factors provide ideal breeding conditions for \u003cem\u003eSarcophagidae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eNotably, \u003cem\u003eL.sericata\u003c/em\u003e exhibit higher densities in green belts and around public toilets. This may be associated with the damp vegetation in green belts and the concentrated fecal contamination surrounding public toilets. This finding suggests the need for targeted sanitation management enhancement in these specific areas \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFrom the perspective of seasonal fluctuation, the peak density of flies occurred from June to August, which aligns with the high-temperature and high-humidity summer climate in the Shanghai area\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Elevated temperatures accelerate the decomposition of organic matter, while precipitation may cause delays in waste removal, further promoting fly reproduction \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. However, a significant decline in fly density was observed in October, likely associated with the drop in temperature and the onset of the fly overwintering period. This pattern is consistent with the conclusion drawn from previous studies that \"fly density exhibits a positive correlation with temperature\" \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. It also highlights the need to prioritize fly control efforts during the summer months.\u003c/p\u003e\u003cp\u003eAmong the 13 pathogens detected in this study, \u003cem\u003eCryptosporidium\u003c/em\u003e and \u003cem\u003eCampylobacter\u003c/em\u003e exhibited higher CT values, indicating higher pathogen loads carried by flies. This suggests their potential role as significant vectors for intestinal infectious diseases in the urban village. \u003cem\u003eCryptosporidium\u003c/em\u003e infection is a common cause of gastroenteritis, typically resulting in diarrhea lasting approximately one to two weeks post-infection. It poses a greater risk to immunocompromised individuals and is primarily transmitted via the fecal-oral route, often through contaminated water or food \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eCampylobacter\u003c/em\u003e, on the other hand, can disrupt the intestinal permeability barrier, triggering acute enteritis \u003csup\u003e[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The high detection rates of these pathogens in outdoor residential areas and green belts are directly linked to the flies' behavioral patterns of frequent contact with both human excreta and food sources \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAdditionally, norovirus was detected in only one positive sample from the exterior environment of public toilets. This low detection rate may be associated with the relatively small sample size or the limited viability duration of the virus on the surface of flies. Further validation with an expanded sample size may be required.\u003c/p\u003e\u003cp\u003eDespite this, the overall pathogen detection rate reached 71.43% within the interior environments of residential areas, significantly higher than the 38.89% observed in public toilet exteriors. This disparity suggests that indoor sanitary conditions\u0026mdash;such as poor ventilation and shared toilet facilities\u0026mdash;may exacerbate the retention of pathogens within the human living environment. Consequently, specific improvements to residents' living facilities are necessary \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe main limitation of this study lies in the lack of direct support from epidemiological investigation data. Although we detected various human intestinal pathogens on the body surfaces of flies and analyzed their spatial distribution patterns, the absence of synchronized stool pathogen testing among community residents\u0026mdash;particularly those with diarrhea symptoms\u0026mdash;prevents us from quantifying the statistical association between fly breeding density, pathogen carriage rate, and human infection rates. It is recommended that future research incorporate monitoring of diarrheal symptoms in the population simultaneously with fly sampling, along with collecting stool samples from volunteers for comparative pathogen analysis. This would provide the most direct evidence supporting the role of flies in the transmission of intestinal infectious diseases.\u003c/p\u003e\u003cp\u003eBased on the findings of this study, the following control measures are recommended: Firstly, environmental remediation in key areas: Enhance the frequency of waste removal in environments surrounding markets and restaurant kitchens, and promote the use of sealed waste containers to reduce potential fly breeding sites \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Secondly, seasonal fly control interventions: During the peak fly density period from June to August, implement residual spraying and toxic bait placement in areas such as green belts and around public toilets to suppress fly populations\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Thirdly, resident hygiene education: Conduct targeted health campaigns for the floating population in urban villages, advocating for the timely disposal of kitchen waste and consistent hand-washing after toilet use to disrupt the fecal-oral transmission route.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study revealed that the fly population in the urban villages of Pudong New Area is predominantly composed of \u003cem\u003eSarcophagidae\u003c/em\u003e. These flies were found to carry various enteric pathogens, including \u003cem\u003eCryptosporidium\u003c/em\u003e, \u003cem\u003eBlastocystis hominis\u003c/em\u003e, EPEC, EHEC and etc. Summer was identified as the period of peak fly density and consequently, heightened transmission risk. Although the distribution of pathogens across different environments did not reach statistical significance, the overall high detection rate underscores the necessity of enhancing environmental sanitation management and fly vector control within urban villages to mitigate the risk of enteric infectious disease outbreaks.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate (Not applicable)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to the deployment of fly traps, informed consent was obtained from residents. They were explicitly informed that the collected fly samples would be transported to the laboratory for pathogen detection, and that the final analytical results would be published in a scientific paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Health System Subject Leader Training Program of Pudong New Area in Shanghai, Grant No. PWRd2023-12.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.L. , Y.T. and C.L. wrote the main manuscript text, R.G.and H.L. prepared figures 1-2, Y.G.and J.L. prepared figures 3-4, Q.L. and G.G. prepared figures 5-6. H.L. and L.H. provided the profesional guidance and revisions. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements (Not applicable)\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization. Vector-borne Diseases.2017. Available from: https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases. Retrieved on 31-10-2017.\u003c/li\u003e\n\u003cli\u003eOnwugamba, F. C., Fitzgerald, J. R., Rochon, K., Guardabassi, L., Alabi, A., K\u0026uuml;hne, S., Grobusch, M. P., \u0026amp; Schaumburg, F. (2018). The role of \u0026apos;filth flies\u0026apos; in the spread of antimicrobial resistance. Travel medicine and infectious disease, 22, 8\u0026ndash;17. https://doi.org/10.1016/j.tmaid.2018.02.007\u003c/li\u003e\n\u003cli\u003eAbdAllah, O. R., Gabre, R. M., Mohammed, S. A., Korayem, A. M., Hussein, H. E., \u0026amp; Ahmad, A. A. (2025). 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The Cochrane database of systematic reviews, 2007(1), CD004932. https://doi.org/10.1002/14651858.CD004932.pub2\u003c/li\u003e\n\u003cli\u003eDavydova, A., Fastl, C., Mughini-Gras, L., Bai, L., Kubota, K., Hoffmann, S., Rachmawati, T., \u0026amp; Pires, S. M. (2025). Source attribution studies of foodborne pathogens, 2010-2023: a review and collection of estimates. Food microbiology, 131, 104812. https://doi.org/10.1016/j.fm.2025.104812\u003c/li\u003e\n\u003cli\u003eTalukdar, P. K., Dines, M. C., Shelden, E. A., Toy, B. A., Kale, A. S., Driskell, R. R., Gloss, L. M., \u0026amp; Konkel, M. E. (2025). Campylobacter jejuni regulates cell cycle progression to potentiate host cell invasion. 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One health outlook, 7(1), 36. https://doi.org/10.1186/s42522-025-00153-9\u003c/li\u003e\n\u003cli\u003eLin, L. F. (2020). Epidemic status of emerging and re-emerging vector-borne infectious diseases and control strategies for vector organisms. Chinese Journal of Vector Biology and Control, 26(3), 193\u0026ndash;196. https://doi.org/10.19821/j.1671-2781.2020.03.001\u003c/li\u003e\n\u003cli\u003eYu, G. H., Wu, X., Zhou, B., et al. (2023). Fly density and seasonal fluctuation in Shenyang from 2019 to 2023. Chinese Journal of Vector Biology and Control, 29(6), 510\u0026ndash;513.\u003c/li\u003e\n\u003cli\u003eSong B, Zhang Y, Min Y, et al. Ecological monitoring of flies and insecticide resistance in Musca domestica in Nanjing, 2022[J]. Chinese Journal of Vector Biology and Control, 2023, 34(5): 637-641.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Urban village, Fly population, Enteric pathogens, Diarrhea","lastPublishedDoi":"10.21203/rs.3.rs-7586460/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7586460/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Flies are significant mechanical vectors of zoonotic pathogens, posing substantial risks for diarrheal disease transmission in urban environments with substandard sanitation. Many urban villages in Shanghai's Pudong New Area exhibit conditions conducive to fly proliferation and pathogen dissemination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Our study was conducted from June to October 2024 in Taiping Village, a representative urban village in Pudong. Fly density and species composition were monitored across six environments (residential indoor/outdoor, green belts, wet market exteriors, restaurant exteriors, public toilet exteriors) using standardized cage traps and sticky ribbons \u0026nbsp;following protocols. Captured flies were taxonomically identified, and 149 pooled samples were screened via RT-PCR and microfluidic chip technology for 31 enteric pathogens (viruses, bacteria, parasites). Statistical analyses employed Excel 2019 and R 4.4.3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: \u003cem\u003eSarcophagidae\u003c/em\u003edominated the fly community (60.75%), followed by \u003cem\u003eA. illocata\u003c/em\u003e (17.29%) and \u003cem\u003eL. sericata\u003c/em\u003e (13.55%). Peak fly density occurred in June-August (1.48 flies/trap). Public toilet exteriors exhibited the highest density (1.33 flies/trap). Pathogen carriage was detected in 61.75% of samples, with \u003cem\u003eCryptosporidium\u003c/em\u003e (51.68%) and diarrheagenic E. coli (DEC, 22.82%) most prevalent. Multi-pathogen co-infection occurred in 44.12% of DEC-positive samples. Residential indoor environments showed the highest pathogen detection rate (71.43%), significantly exceeding public toilet exteriors (38.89%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: This study identifies \u003cem\u003eSarcophagidae\u003c/em\u003e as the primary fly vector in Pudong’s urban villages, carrying diverse enteric pathogens—notably \u003cem\u003eCryptosporidium\u003c/em\u003e and DEC—with peak transmission risk during summer. The high overall pathogen carriage rate, particularly in crowded residential interiors, underscores the critical need for enhanced sanitation infrastructure, targeted vector control and community hygiene education to mitigate outbreak risks of enteric infections in these high-density settlements.\u003c/p\u003e","manuscriptTitle":"Seasonal Fluctuations in Fly Density and Pathogen Carriage in Urban Villages of Pudong New Area","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 13:52:02","doi":"10.21203/rs.3.rs-7586460/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-13T12:03:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-27T19:18:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218993851351655359538235398733092864716","date":"2025-11-12T12:23:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T22:00:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158391490296552080242973268015519942978","date":"2025-10-15T20:01:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307899861874417985474108002258753830864","date":"2025-10-15T18:28:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71895383529176779576917974683791244642","date":"2025-10-15T12:55:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-13T08:40:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-15T11:56:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-12T07:04:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-12T07:02:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2025-09-11T00:34:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"22ff61d6-d65e-4056-adcc-3e7c35e7ed54","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T13:10:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 13:52:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7586460","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7586460","identity":"rs-7586460","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}