Bloodthirsty Bites: A Study of Sandfly Feeding Patterns in the Aegean Region of Türkiye

Bloodthirsty Bites: A Study of Sandfly Feeding Patterns in the Aegean Region of Türkiye | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bloodthirsty Bites: A Study of Sandfly Feeding Patterns in the Aegean Region of Türkiye Metin Pekagirbas, Fatma Bursali, Serkan Bakirci This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6202222/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jul, 2025 Read the published version in BMC Veterinary Research → Version 1 posted 16 You are reading this latest preprint version Abstract Background This study determined the blood-feeding patterns of wild-caught sandflies collected from provinces (Aydin and Mugla) in the Aegean region in Türkiye. Adult sandflies were collected using three CDC light traps per village in August and October 2024. A total of 280 sandfly specimens (194 females and 86 males) were collected during the study. Of the 194 female specimens, 38 (23 from Aydin and 15 from Mugla) were found to be blood-fed, 137 were non-blood-fed, and 19 were gravid. In total, DNA was extracted from 38 blood-fed sandflies using Invitrogen PureLink genomic DNA isolation kit. To determine the blood-feeding patterns of these insects, the mitochondrial cytochrome b ( cytb ) gene region was amplified via multiplex PCR using specific primers. Results The PCR results showed that 23 sand fly females sampled from Aydin locality fed mostly from cows (60.5%) followed by dogs (21%) and chickens (18.4%). All the samples collected from Aydin blood fed from cows whereas for females sampled from Mugla locality, 53% blood-fed from dogs and 46% from birds. Host preference of the samples was also subjected to ELISA reaction to identify samples whose host preference could not be determined by PCR and to see if there were multiple host preferences. Our bird primers used in PCR methods, could not identify the blood meal from chickens, probably due to low amounts of the blood ingested having been digested or DNA was denatured. However, the ELISA studies clearly elucidated the host of these samples. Despite the limitations and disadvantages inherent to both methods, which are the most frequently employed in blood meal analysis, all samples were successfully analyzed in the present study, and the results obtained by both methods were highly consistent with each other. Conclusion In conclusion, to the best of our knowledge, this is the first study in the region to analyse ELISA and PCR methodologies in a comparative manner in relation to host feeding patterns of sand flies. The detection of blood meal in field-caught sand flies has the potential to facilitate a more comprehensive understanding of the eco-epidemiology of vector-borne diseases, thereby contributing to the planning of strategic control methods. Blood-meal ELISA PCR Sand fly Türkiye Figures Figure 1 Figure 2 Background Phlebotomine sandfly species are considered to be significant vectors for various pathogens, including protozoa (e.g. Leishmania spp.), bacteria (e.g. Bartonella sp.), and viruses (e.g. Toscana virus, Leishmania RNA virus) (Depaquit et al. 2010 ; Munstermann 2019 ; Nalcaci et al. 2019). Among the over 1,000 identified worldwide, only a small fraction (about 100 species) in the genera Phlebotomus , Sergentomyia , and Lutzomyia can act as vectors for diseases, transmitting pathogens present in their saliva or ingested blood to their host during their blood-feeding activities. Not only because of the diseases they transmit but also because of their painful bites, their presence can be a nuisance (Tsirigotakis et al. 2018 ; Cecílio et al. 2022 ; Bursali and Touray 2024 ). Leishmaniasis, a protozoan disease that occurs in various forms (cutaneous, visceral, mucocutaneous, diffuse cutaneous, canine, feline) and has a high global burden, particularly in certain regions such as Brazil, Bangladesh, Ethiopia, Sudan, Afghanistan, Ethiopia, Iran, Syria, and Türkiye (Ready 2013 ; Alten et al. 2016 ; Özbel et al. 2022 ). Sandflies also transmit phleboviruses, which cause fever, nausea, vomiting, and neurological complications (Alkan et al. 2016 ). The blood-feeding patterns of hematophagous insects are of particularly interesting in public health, as they determine the frequency with which they feed on vertebrate hosts. These insects typically feed regularly, at intervals of 3–6 days, to obtain the nutrients, they require for survival. The blood meals of such insects, such as sandflies and mosquitoes, can provide valuable insights into their host-feeding preferences and patterns (Moraes et al. 2018 ; Costa et al. 2021 ). This information is crucial for understanding the transmission dynamics of vector-borne diseases (Bursali and Simsek 2022; McGregor and Lewis 2023 ; Bursali et al. 2024 ). A comprehensive understanding of the blood-feeding patterns exhibited by these insects is of paramount ecological and epidemiological importance. As demonstrated by several studies (Chaves and Añez 2004 ; Yared et al. 2019 ; Azmi et al. 2020 ; Costa et al. 2021 ; Yetismis et al. 2022 ; Jibreel et al. 2023 ; Karagul and Kasap 2024 ) that have analyzed the blood meals of captured sandflies using molecular and biochemical techniques, it is evident that sandflies exhibit a remarkable diversity in their feeding preferences. Host preferences vary widely, from highly specific to opportunistic, both between different geographical regions and even within the same species. Old World sandflies belonging to the genera Phlebotomus and Sergentomyia have been observed to exhibit zoophilic and anthropophilic feeding tendencies, depending on the specific species (Tiwananthagorn et al. 2012 ; Macedo-Silva et al. 2014 ; Sales et al. 2015 ; Cera-Vallejo et al. 2024 ). Furthermore, sandflies have been observed to consume a mixture of blood from different hosts (González et al. 2015 ; Yared et al. 2019 ; Azmi et al. 2020 ). While sandflies exhibit a certain degree of phenotypic plasticity in their feeding behavior, they are also influenced by environmental factors and the availability of resources (Bursali & Touray, 2024 ). Furthermore, the type of habitat (e.g. forest, savannah) influences the availability of potential blood sources and thus, feeding patterns (Bursali and Touray 2024 ). To date, different methodologies have been utilized to analyze blood samples from sand flies, which have been observed feeding on a variety of hosts (Nery et al 2004 ). In recent years, several molecular techniques such as quantitative polymerase chain reaction (qPCR), multiplex PCR, including PCR-RFLP targeting cytochrome b and cytochrome oxidase c genes have been frequently used. These techniques are complemented by expensive and time-consuming methods such as DNA sequence analysis (Kent et al. 2009; Karakus et al. 2017; Yetismis et al. 2022 ). In addition, the MALDI-TOF method for protein profiling is characterized by its high sensitivity. However, it is important to note that this method is associated with significant financial costs and requires expertise for its successful implementation (Hlavackova et al.2019). In contrast, serology-based techniques, including precipitation and ELISA, continue to be utilized with success in contemporary clinical practice due to their cost-effectiveness when compared to alternative methods (Gebre-Michael et al. 2010 ). This study determined the blood-feeding patterns of wild sandflies collected from provinces (Aydın and Mugla) in the Aegean region in Türkiye, which has 28 sandfly species (Erisoz-Kasap et al. 2019), including 24 Phlebotomus and 4 Sergentomyia species. Material & Method Sandf fly collection and morphological identification The study areas, which include Gokceovacik village (36° 80’15’’N; 28°97’68’’E) in the Dalaman district of Mugla province and Asagikayacik village (37° 88’46’’N; 27°94’05’’E) in the inner parts of the province of Aydin, were selected based on previous cases of visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), and canine leishmaniasis (CanL) in humans. Adult sandflies were collected using three CDC light traps (John W. Hock, Gainesville, FL, USA) per village in August and October 2024. The trap locations were determined based on possible breeding and resting sites of sand flies, such as animal barns, chicken nests, etc. Data on the number of households, their geographic coordinates, the presence or absence of domestic animals, and the number of occupants in sampled rooms were recorded for each village. The traps were set at 18:00 h and collected at 06:00 am. The sand flies were collected, counted and stored in eppendorf tubes at − 80°C in liquid nitrogen for subsequent morphological identification. Blood-fed female sand fly specimens were separated and dissected under a stereo microscope. These were then placed on microscope slides for morphological identification according to identification keys and drawings that had previously been published (Lewis 1982 ; Artemiev and Neronov1984; Killick-Kendrick et al 1991 ). The thorax and abdomen of female sandflies were stored in eppendorf for blood-meal analyzis until DNA isolation. Blood feeding pattern determination In total, DNA was extracted from 38 blood-fed sandflies using Invitrogen PureLink genomic DNA isolation kit. To determine the blood-feeding patterns of these insects, the mitochondrial cytochrome b ( cytb ) gene region was amplified via multiplex PCR using specific primers (Kent and Norris, 2005 ; Pitzer et al., 2014; Lee et al., 2002 ). PCR reactions were conducted under standard conditions, with a final reaction volume of 2 µL containing 50 ng DNA, 2X 12.5 µl Master mix, 0.25 µl each of primers and 10.25 µl ddH 2 0. The PCR reaction was carried out with an initial denaturation at 95°C for 5 minutes, 40 cycles of denaturation at 95°C for 30 seconds, annealing at 57°C for 1 minute, extension at 72°C for 1 minute, then final extension: 72°C for 5 minutes. Negative controls were included to monitor contamination. Amplified PCR products were visualized on 2% agarose gel to confirm successful amplification. The band sizes for potential hosts were as follows: humans (334 bp), goats (132 bp), dogs (680 bp), cows (561 bp), horses (500 bp), and birds (383 bp) (Bursali and Simsek 2022). Also, direct enzyme-linked immunosorbent assay (ELISA) technique was used to confirm blood feeding patterns and to determine the host preference of the samples that were negative during PCR reaction (Beier et al., 1988). The method relies on the reaction between antibodies specific to potential host blood (human, cow, dog, horse, chicken) and the ingested blood meal within the sandfly. Individual insects were homogenized in phosphate-buffered saline (PBS). The resulting homogenates were subjected to an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies against various host species according to (Bursali et al. 2024 ). Wells of a microplate (Corning 96-well Clear Round Bottom Polystyrene Not Treated Sterile Microplate) were coated with antibodies (anti-human IgG, anti-horse IgG, anti-bovine IgG, anti-dog IgG, and anti-chicken IgG) specific to various host species. These five antibodies were used to identify potential blood meal sources. Peroxidase-conjugated anti host IgG antibody was added to the wells and incubated. After washing, ABTS substrate was added, resulting in a colorimetric reaction at 405 nm using a microplate reader (BioTek ELx808 Absorbance Plate Reader). Resulting color changes were indicative of the presence of specific host blood. Negative and positive controls were included to ensure assay accuracy. The choice of antibodies was based on the prevalence of these host species in the study region. Results Blood feeding pattern determination A total of 280 sandfly specimens (194 females and 86 males) were collected from two different localities during the study. Of the 194 female specimens, 38 (23 from Aydin and 15 from Mugla) were found to be blood-fed, 137 were non-blood-fed, and 19 were gravid. Following the dissection and identification of blood-fed sandflies, four and three Phlebotomus species were found in Mugla and Aydin, respectively. In total, DNA from 38 sand flies was utilized for the amplification of the cyt b gene region to ascertain their feeding patterns, and the results of the PCR showed that sand flies females in the sampling locations predominantly fed on cows (60.5%), followed by dogs (21%) and chickens (18.4%), respectively (Fig. 1 ). Of the 23 females sampled from Aydin (20 Phlebotomus papatasi , three P. tobbi ), all were fed on cows. In contrast, of the 15 female sand flies (14 P. major s.l., one P. alexandri ) sampled from Mugla, eight were fed on dog blood and seven on chicken blood (Fig. 2 ). A total of 38 blood meals were tested by ELISA for host identification successfully. In all localities, cows were the most common hosts, followed by dog and chicken hosts. The majority of blood meals were derived from a single vertebrate host among the five tested species (human or animal) (Table 1 ). Table 1 Natural blood-feeding preferences of sand flies collected from Aydin and Mugla provinces in Türkiye Locality Number of blood fed sandflies Method used Hosts Cow Dog Chicken Human Horse Aydin 23 PCR 23/23(100%) - - - - ELISA 23/23 (100%) - - - - Mugla 15 PCR - 8/15 (53%) - - - ELISA - 8/15 (53%) 7/15 (46%) - Discussion This study determined the natural feeding patterns of sandfly populations from the Aegean region of Türkiye using the PCR and ELISA methods. A total of 38 blood-fed sandflies were collected from various locations between August and October 2024. Sandfly specimens were analyzed to identify the blood source of these wild-type populations, and the results showed that cow blood meals were found as the most common source for all tested sandfly species. In addition, dog and chicken blood meals were identified in all species, albeit at much lower frequencies compared to the preference ratio for cow blood. Mixed blood meals from two different hosts were not observed. It is important to acknowledge the limitations of this study, which was conducted in a single location in both study areas. This precludes the possibility of reflecting the real fauna of these locations as reported in previous studies conducted in the study areas (Pekagirbas et al. 2021; Arserim et al. 2022 ). Sandflies have been demonstrated to display a broad spectrum of host preferences, encompassing humans, animals, and even ectothermic vertebrates such as amphibians (de Ávila et al. 2018 ; Elaagip et al. 2020 ; Jibreel et al. 2023 ; Bursali and Touray 2024 ). The blood-feeding patterns of these insects can be influenced by the availability of potential hosts as well as local environmental conditions, host body size and attractiveness (Bursali and Touray 2024 ). Numerous species are opportunistic feeders, acquiring blood meals from accessible hosts irrespective of species. For example, that P. perniciosus from Spain, Italy and Portugal displayed opportunistic feeding habits, with no clear host preference (De Colmenares et al. 1995 ; Bongiorno et al. 2003 ; Maia et al. 2013 ). Azmi et al ( 2020 ) found that P. papatasi and P. sergenti in Palestine fed on humans, hyraxes, rats, livestock, and birds, with some individuals even taking mixed blood meals. Jaouadi et al ( 2018 ) reported that sandflies in Tunisia primarily fed on humans, rodents, and livestock. Salah et al ( 2020 ) found that P. sergenti in Palestine fed on humans, livestock, birds, and dogs. González et al ( 2015 ) observed that P. perniciosus in Spain primarily fed on wild animals, but also took blood meals from humans, sheep, and goats. Palit et al ( 2005 ) and Srinivasan et al ( 2015 ) discovered that sandflies in India fed on multiple hosts, including humans, cattle, and other animals with P. argentipes having a significant preference for humans. A small percentage of sandflies had mixed blood meals from both avian and mammalian sources. At the species level, chicken blood was the most frequently detected avian source. Human, cow, and dog blood were also identified among the mammalian meals. In contrast to the findings of other studies on the P. papatasi , which were previously described as opportunistic (Svobodoba et al. 2003) and generally reported to be highly anthropophilic (Burniston et al. 2010 ), human blood was not identified in this study. The literature on the feeding patterns of sand fly species in Türkiye have been limited. Karakuş et al ( 2017 ) found that the most common food preference of sand flies was dog blood, followed by humans, mice, cats, and cows in terms of frequency in their study in Aydin, the same study revealed that the P. neglectus species showed a clear preference for human blood. This finding was later shown by Ozbel et al (2015) to be the most common blood-feeding preference (80%) among the P. tobbi species. In contrast to the findings of the aforementioned studies, no human samples were detected in the blood-feeding female sandflies captured in the wild in the present study. Yetismis et al ( 2022 ) reported that, in accordance with the results of the present study, the majority of blood sources of blood-fed sand flies belonged to the species Bos tauru s. Karagul and Kasap ( 2024 ) identified bovine animals, particularly cows, as the most common blood source followed by chickens and goats. Humans were identified as a minor blood source. Among the studied species, Paraphlebotomus sand flies were unique in their broader host range, with a particular preference for avian hosts. These results suggest that the host feeding preferences of different sand fly species in Mugla and Aydin provinces are mainly cows, dogs, and chickens, which may be related to the fact that local people keep their animal barns, chicken nests, and their dogs close to their homes. In line with the hypothesis of Dinesh et al ( 2001 ), that sand flies tend to feed on cows, cows were the most common source of blood as a food source in this study. This may be also related to the higher proportion of cows compared to other hosts in terms of body size, carbon dioxide output level, host-derived volatile organic compounds (Bezerra-Santos et al. 2024 ) and other odour secretions. On the other hand, as stated by Yetismis et al ( 2022 ), although the feeding rate from cows is higher than from other hosts, it may not be correct to define this situation as a real host preference because the settlements in the current study are not environments where all hosts coexist. The efficacy of molecular blood-meal identification is contingent upon the quantity of blood ingested and the duration of blood digestion in the midgut of the insect (Kent and Norris, 2005 ). It is important to note that the quantity of blood ingested by sand flies (1 µl or less) (Daba et al. 2004 ) is less than that of mosquitoes (2–6 µl), which is considered to be a biological difference that complicates the generalisation of results (Clements, 1992 ). The process of blood digestion can cause DNA denaturation, which can make DNA detection difficult (Kent et al. 2005; Sant’Anna et al 2008 ), therefore it is essential to use protocols that will detect the minimum amount of DNA. Another factor that must be considered when attempting to identify blood sources using molecular methods is the inhibitory effect of substances found in the tissues of insects, particularly those found in their exoskeleton, head and thorax (Paiva et al., 2007 ). Substances such as haem in blood (Kent, 2009 ) can also reduce the efficiency of the PCR reaction. In contrast to molecular methods, conventional serological methodologies for blood meal identification necessitate the production of species-specific antibodies against all potential hosts. These techniques are also constrained by factors such as the unavailability of products for exotic animals, diminished sensitivity, and the possibility of cross-reactivity between species (Valinsky et al. 2014 ). Despite the limitations and disadvantages inherent to both methods, which are the most frequently employed in blood meal analysis, all samples were successfully analyzed in the present study, and the results obtained by both methods were highly consistent with each other. The bird primers utilized in the PCR methods failed to identify the blood meal from chickens, likely due to the low quantities of the blood ingested having been digested or the DNA denatured. However, the ELISA studies unequivocally elucidated the host of these samples. Determining the feeding patterns of sand flies will facilitate the identification of species that are susceptible to zoophagic feeding in designated study areas (Gebre-Michael et al. 2010 ). This will enable the determination of the suitability of mammals for zooprophylaxis against diseases caused by sand flies (Morrison et al. 1993 ). Moreover, a range of control measures, including the use of insecticide-impregnated collars on vertebrate hosts, can be applied for a variety of diseases for which sand flies are vectors. In conclusion, To the best of our knowledge, this is the first study in the region to analyze ELISA and PCR methodologies in a comparative manner in relation to host feeding patterns of sand flies. Regardless of the methodology used, the detection of blood meal in field-caught sand flies has the capacity to substantiate the hypothesis that there exists a robust relationship between sand flies in rural areas and reservoir hosts of the diseases they carry. This may facilitate comprehension of the role of endemic domestic animals and further contribute to the planning of strategic control methods by providing a more comprehensive understanding of the eco-epidemiology of vector-borne diseases. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no conflicts of interest. Funding This study was supported by Aydın Adnan Menderes University BAP (project number: BAP-FEF 21005). Fatma Bursali is funded by TÜBİTAK (2219 Postdoctoral Fellowship Programme) Author credit information FB designed the original project. MP made sample collection. Experimental studies were carried out by FB, MP. FB, MT, MP and SB revised the manuscript. All authors contributed to the final version of the text. Acknowledgements The authors have nothing to report. References Alkan C, Erisoz Kasap O, Alten B, et al (2016) Sandfly-Borne Phlebovirus Isolations from Türkiye: New Insight into the Sandfly fever Sicilian and Sandfly fever Naples Species. 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J Med Entomol 51:237–244. https://doi.org/10.1603/me12131 Maia C, Dionísio L, Afonso MO, et al (2013) Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal. Memórias do Instituto Oswaldo Cruz 108:481. https://doi.org/10.1590/0074-0276108042013014 McGregor BL, Lewis A (2023) Host Associations of Culicoides Biting Midges in Northeastern Kansas, USA. Animals (Basel) 13:2504. https://doi.org/10.3390/ani13152504 Moraes CS, Aguiar-Martins K, Costa SG, et al (2018) Second Blood Meal by Female Lutzomyia longipalpis: Enhancement by Oviposition and Its Effects on Digestion, Longevity, and Leishmania Infection. Biomed Res Int 2018:2472508. https://doi.org/10.1155/2018/2472508 Morrison AC, Ferro C, Tesh RB: Host preferences of the sandfly Lutzomyia longipaplpis at an endemic focus of American visceral leishmaniasis in Colombia. Am J Trop Med Hyg 1993, 49:68-75. Munstermann LE (2019) Chapter 12 - Phlebotomine Sand Flies and Moth Flies (Psychodidae). In: Mullen GR, Durden LA (eds) Medical and Veterinary Entomology (Third Edition). Academic Press, pp 191–211 Nalçacı M, Karakuş M, Yılmaz B, Demir S, Özbilgin A, Özbel Y, et al.Detection of Leishmania RNA virus 2 in Leishmania species from Turkey.Trans R Soc Trop Med Hyg 2019; 113: 410-7 Nery LC, Lorosa NE, Franco AM (2004) Feeding preference of the sand flies Lutzomyia umbratilis and L. spathotrichia (dip- tera: Psychodidae, Phlebotominae) in an urban forest patch in the city of Manaus, Amazonas, Brazil. Mem Inst Oswaldo Cruz 99(6):571–574. https://doi.org/10.1590/s0074-027620040006000 06 Özbel, Y., Karakus, M., Arserim, S.K., Kalkan, S.O. & Töz, S. (2015) Molecular detection and identification of Leishmania spp. in naturally infected Phlebotomus tobbi and Sergentomyia dentata in a focus of human and canine leishmaniasis in western Turkey. Acta Tropica , 155 , 89–94. Özbel Y, Töz S, Muñoz C, et al (2022) The current epidemiology of leishmaniasis in Türkiye, Azerbaijan and Georgia and implications for disease emergence in European countries. Zoonoses Public Health 69:395–407. https://doi.org/10.1111/zph.12977 Paiva, B. R. D., Secundino, N. F. C., Pimenta, P. F. P., Galati, E. A. B., Andrade Junior, H. F., & Malafronte, R. D. S. (2007). Padronização de condições para detecção de DNA de Leishmania spp. em flebotomíneos (Diptera, Psychodidae) pela reação em cadeia da polimerase. Cadernos de saúde pública , 23 (1), 87-94. Palit A, Bhattacharya SK, Kundu SN (2005) Host preference of Phlebotomus argentipes and Phlebotomus papatasi in different biotopes of West Bengal, India. International Journal of Environmental Health Research 15:449–454. https://doi.org/10.1080/09603120500392525 Pekağırbaş, M., Karakuş, M., Kasap, O. E., Demir, S., Nalçacı, M., Töz, S., ... & Özbel, Y. (2021). Investigation of Phlebotominae (Diptera: Psychodidae) fauna, seasonal dynamics, and natural Leishmania spp. infection in Muğla, Southwest of Turkey. Acta tropica , 216 , 105827. Pitzer JB, Kaufman PE, Tenbroeck SH, Maruniak JE (2011) Host blood meal identification by multiplex polymerase chain reaction for dispersal evidence of stable flies (Diptera:Muscidae) between livestock facilities. J Med Entomol 48:53–60. https://doi.org/10.1603/me10123 Ready PD (2013) Biology of phlebotomine sand flies as vectors of disease agents. Annual review of entomology 58:227–250 Sant’Anna MR, Jones NG, Hindley JA, Mendes-Sousa AF, Dillon RJ, Cavalcante RR, et al. Blood meal identification and parasite detection in laboratory-fed and field captured Lutzomyia longipalpis by PCR using FTA databasing paper. Acta Trop. 2008;107:230–7. Salah I, Abbasi I, Warburg A, et al (2020) Ecology of Leishmaniasis in an urbanized landscape: Relationship of sand fly densities, and Leishmania tropica infection rates with reservoir host colonies. Acta Trop 204:105332. https://doi.org/10.1016/j.actatropica.2020.105332 Sales KG da S, Costa PL, de Morais RCS, et al (2015) Identification of phlebotomine sand fly blood meals by real-time PCR. Parasit Vectors 8:230. https://doi.org/10.1186/s13071-015-0840-3 Srinivasan R, Jambulingam P, Kumar NP, et al (2015) Temporal distribution and behaviour of sand flies (Diptera: Psychodidae) in a cutaneous leishmaniasis focus of the Kani Tribe settlements in the Western Ghats, India. Acta Tropica 148:147–155. https://doi.org/10.1016/j.actatropica.2015.04.015 Svobodová, M., Sádlová, J., Chang, K. P., & Volf, P. (2003). Distribution and feeding preference of the sand flies Phlebotomus sergenti and P. papatasi in a cutaneous leishmaniasis focus in Sanliurfa, Turkey. The American journal of tropical medicine and hygiene , 68 (1), 6-9. Tiwananthagorn S, Bhutto AM, Baloch JH, et al (2012) Zoophilic feeding behaviour of phlebotomine sand flies in the endemic areas of cutaneous leishmaniasis of Sindh Province, Pakistan. Parasitol Res 111:125–133. https://doi.org/10.1007/s00436-011-2808-3 Tsirigotakis N, Pavlou C, Christodoulou V, et al (2018) Phlebotomine sand flies (Diptera: Psychodidae) in the Greek Aegean Islands: ecological approaches. Parasites & Vectors 11:97. https://doi.org/10.1186/s13071-018-2680-4 Valinsky, L., Ettinger, G., Bar-Gal, G. K., & Orshan, L. (2014). Molecular identification of bloodmeals from sand flies and mosquitoes collected in Israel. Journal of medical entomology , 51 (3), 678–685. https://doi.org/10.1603/me13125 Yared S, Gebresilassie A, Abbasi I, et al (2019) A molecular analysis of sand fly blood meals in a visceral leishmaniasis endemic region of northwestern Ethiopia reveals a complex host-vector system. Heliyon 5:e02132. https://doi.org/10.1016/j.heliyon.2019.e02132 Yetismis K, Mert U, Caner A, et al (2022) Blood mealanalysis and molecular detection of Leishmania DNA in wild-caught sand flies inleishmaniasis endemic areas of Turkey and Northern Cyprus. Acta Parasitol. 67,932–942. https://doi.org/10.1007/S11686-022-00542- Additional Declarations No competing interests reported. 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Munstermann \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nalcaci et al. 2019). Among the over 1,000 identified worldwide, only a small fraction (about 100 species) in the genera \u003cem\u003ePhlebotomus\u003c/em\u003e, \u003cem\u003eSergentomyia\u003c/em\u003e, and \u003cem\u003eLutzomyia\u003c/em\u003e can act as vectors for diseases, transmitting pathogens present in their saliva or ingested blood to their host during their blood-feeding activities. Not only because of the diseases they transmit but also because of their painful bites, their presence can be a nuisance (Tsirigotakis et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cec\u0026iacute;lio et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bursali and Touray \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Leishmaniasis, a protozoan disease that occurs in various forms (cutaneous, visceral, mucocutaneous, diffuse cutaneous, canine, feline) and has a high global burden, particularly in certain regions such as Brazil, Bangladesh, Ethiopia, Sudan, Afghanistan, Ethiopia, Iran, Syria, and T\u0026uuml;rkiye (Ready \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Alten et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; \u0026Ouml;zbel et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Sandflies also transmit phleboviruses, which cause fever, nausea, vomiting, and neurological complications (Alkan et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The blood-feeding patterns of hematophagous insects are of particularly interesting in public health, as they determine the frequency with which they feed on vertebrate hosts. These insects typically feed regularly, at intervals of 3\u0026ndash;6 days, to obtain the nutrients, they require for survival. The blood meals of such insects, such as sandflies and mosquitoes, can provide valuable insights into their host-feeding preferences and patterns (Moraes et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This information is crucial for understanding the transmission dynamics of vector-borne diseases (Bursali and Simsek 2022; McGregor and Lewis \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Bursali et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A comprehensive understanding of the blood-feeding patterns exhibited by these insects is of paramount ecological and epidemiological importance.\u003c/p\u003e \u003cp\u003eAs demonstrated by several studies (Chaves and A\u0026ntilde;ez \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Yared et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Azmi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yetismis et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jibreel et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Karagul and Kasap \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) that have analyzed the blood meals of captured sandflies using molecular and biochemical techniques, it is evident that sandflies exhibit a remarkable diversity in their feeding preferences. Host preferences vary widely, from highly specific to opportunistic, both between different geographical regions and even within the same species. Old World sandflies belonging to the genera \u003cem\u003ePhlebotomus\u003c/em\u003e and \u003cem\u003eSergentomyia\u003c/em\u003e have been observed to exhibit zoophilic and anthropophilic feeding tendencies, depending on the specific species (Tiwananthagorn et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Macedo-Silva et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sales et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cera-Vallejo et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, sandflies have been observed to consume a mixture of blood from different hosts (Gonz\u0026aacute;lez et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yared et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Azmi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While sandflies exhibit a certain degree of phenotypic plasticity in their feeding behavior, they are also influenced by environmental factors and the availability of resources (Bursali \u0026amp; Touray, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, the type of habitat (e.g. forest, savannah) influences the availability of potential blood sources and thus, feeding patterns (Bursali and Touray \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo date, different methodologies have been utilized to analyze blood samples from sand flies, which have been observed feeding on a variety of hosts (Nery et al \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In recent years, several molecular techniques such as quantitative polymerase chain reaction (qPCR), multiplex PCR, including PCR-RFLP targeting cytochrome b and cytochrome oxidase c genes have been frequently used. These techniques are complemented by expensive and time-consuming methods such as DNA sequence analysis (Kent et al. 2009; Karakus et al. 2017; Yetismis et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, the MALDI-TOF method for protein profiling is characterized by its high sensitivity. However, it is important to note that this method is associated with significant financial costs and requires expertise for its successful implementation (Hlavackova et al.2019). In contrast, serology-based techniques, including precipitation and ELISA, continue to be utilized with success in contemporary clinical practice due to their cost-effectiveness when compared to alternative methods (Gebre-Michael et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study determined the blood-feeding patterns of wild sandflies collected from provinces (Aydın and Mugla) in the Aegean region in T\u0026uuml;rkiye, which has 28 sandfly species (Erisoz-Kasap et al. 2019), including 24 \u003cem\u003ePhlebotomus\u003c/em\u003e and 4 \u003cem\u003eSergentomyia\u003c/em\u003e species.\u003c/p\u003e"},{"header":"Material \u0026 Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSandf fly collection and morphological identification\u003c/h2\u003e \u003cp\u003eThe study areas, which include Gokceovacik village (36\u0026deg; 80\u0026rsquo;15\u0026rsquo;\u0026rsquo;N; 28\u0026deg;97\u0026rsquo;68\u0026rsquo;\u0026rsquo;E) in the Dalaman district of Mugla province and Asagikayacik village (37\u0026deg; 88\u0026rsquo;46\u0026rsquo;\u0026rsquo;N; 27\u0026deg;94\u0026rsquo;05\u0026rsquo;\u0026rsquo;E) in the inner parts of the province of Aydin, were selected based on previous cases of visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), and canine leishmaniasis (CanL) in humans. Adult sandflies were collected using three CDC light traps (John W. Hock, Gainesville, FL, USA) per village in August and October 2024. The trap locations were determined based on possible breeding and resting sites of sand flies, such as animal barns, chicken nests, etc. Data on the number of households, their geographic coordinates, the presence or absence of domestic animals, and the number of occupants in sampled rooms were recorded for each village. The traps were set at 18:00 h and collected at 06:00 am. The sand flies were collected, counted and stored in eppendorf tubes at \u0026minus;\u0026thinsp;80\u0026deg;C in liquid nitrogen for subsequent morphological identification. Blood-fed female sand fly specimens were separated and dissected under a stereo microscope. These were then placed on microscope slides for morphological identification according to identification keys and drawings that had previously been published (Lewis \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Artemiev and Neronov1984; Killick-Kendrick et al \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). The thorax and abdomen of female sandflies were stored in eppendorf for blood-meal analyzis until DNA isolation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBlood feeding pattern determination\u003c/h3\u003e\n\u003cp\u003eIn total, DNA was extracted from 38 blood-fed sandflies using Invitrogen PureLink genomic DNA isolation kit. To determine the blood-feeding patterns of these insects, the mitochondrial cytochrome b (\u003cem\u003ecytb\u003c/em\u003e) gene region was amplified via multiplex PCR using specific primers (Kent and Norris, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Pitzer et al., 2014; Lee et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). PCR reactions were conducted under standard conditions, with a final reaction volume of 2 \u0026micro;L containing 50 ng DNA, 2X 12.5 \u0026micro;l Master mix, 0.25 \u0026micro;l each of primers and 10.25 \u0026micro;l ddH\u003csub\u003e2\u003c/sub\u003e0. The PCR reaction was carried out with an initial denaturation at 95\u0026deg;C for 5 minutes, 40 cycles of denaturation at 95\u0026deg;C for 30 seconds, annealing at 57\u0026deg;C for 1 minute, extension at 72\u0026deg;C for 1 minute, then final extension: 72\u0026deg;C for 5 minutes. Negative controls were included to monitor contamination. Amplified PCR products were visualized on 2% agarose gel to confirm successful amplification. The band sizes for potential hosts were as follows: humans (334 bp), goats (132 bp), dogs (680 bp), cows (561 bp), horses (500 bp), and birds (383 bp) (Bursali and Simsek 2022).\u003c/p\u003e \u003cp\u003eAlso, direct enzyme-linked immunosorbent assay (ELISA) technique was used to confirm blood feeding patterns and to determine the host preference of the samples that were negative during PCR reaction (Beier et al., 1988). The method relies on the reaction between antibodies specific to potential host blood (human, cow, dog, horse, chicken) and the ingested blood meal within the sandfly. Individual insects were homogenized in phosphate-buffered saline (PBS). The resulting homogenates were subjected to an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies against various host species according to (Bursali et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Wells of a microplate (Corning 96-well Clear Round Bottom Polystyrene Not Treated Sterile Microplate) were coated with antibodies (anti-human IgG, anti-horse IgG, anti-bovine IgG, anti-dog IgG, and anti-chicken IgG) specific to various host species. These five antibodies were used to identify potential blood meal sources. Peroxidase-conjugated anti host IgG antibody was added to the wells and incubated. After washing, ABTS substrate was added, resulting in a colorimetric reaction at 405 nm using a microplate reader (BioTek ELx808 Absorbance Plate Reader). Resulting color changes were indicative of the presence of specific host blood. Negative and positive controls were included to ensure assay accuracy. The choice of antibodies was based on the prevalence of these host species in the study region.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBlood feeding pattern determination\u003c/h2\u003e \u003cp\u003eA total of 280 sandfly specimens (194 females and 86 males) were collected from two different localities during the study. Of the 194 female specimens, 38 (23 from Aydin and 15 from Mugla) were found to be blood-fed, 137 were non-blood-fed, and 19 were gravid. Following the dissection and identification of blood-fed sandflies, four and three \u003cem\u003ePhlebotomus\u003c/em\u003e species were found in Mugla and Aydin, respectively. In total, DNA from 38 sand flies was utilized for the amplification of the \u003cem\u003ecyt b\u003c/em\u003e gene region to ascertain their feeding patterns, and the results of the PCR showed that sand flies females in the sampling locations predominantly fed on cows (60.5%), followed by dogs (21%) and chickens (18.4%), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of the 23 females sampled from Aydin (20 \u003cem\u003ePhlebotomus papatasi\u003c/em\u003e, three \u003cem\u003eP. tobbi\u003c/em\u003e), all were fed on cows. In contrast, of the 15 female sand flies (14 \u003cem\u003eP. major\u003c/em\u003e s.l., one \u003cem\u003eP. alexandri\u003c/em\u003e) sampled from Mugla, eight were fed on dog blood and seven on chicken blood (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA total of 38 blood meals were tested by ELISA for host identification successfully. In all localities, cows were the most common hosts, followed by dog and chicken hosts. The majority of blood meals were derived from a single vertebrate host among the five tested species (human or animal) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNatural blood-feeding preferences of sand flies collected from Aydin and Mugla provinces in T\u0026uuml;rkiye\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLocality\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNumber of blood fed sandflies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMethod used\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c8\" namest=\"c4\"\u003e \u003cp\u003eHosts\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChicken\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHuman\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHorse\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAydin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23/23(100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23/23 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMugla\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8/15 (53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8/15 (53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7/15 (46%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study determined the natural feeding patterns of sandfly populations from the Aegean region of T\u0026uuml;rkiye using the PCR and ELISA methods. A total of 38 blood-fed sandflies were collected from various locations between August and October 2024. Sandfly specimens were analyzed to identify the blood source of these wild-type populations, and the results showed that cow blood meals were found as the most common source for all tested sandfly species. In addition, dog and chicken blood meals were identified in all species, albeit at much lower frequencies compared to the preference ratio for cow blood. Mixed blood meals from two different hosts were not observed. It is important to acknowledge the limitations of this study, which was conducted in a single location in both study areas. This precludes the possibility of reflecting the real fauna of these locations as reported in previous studies conducted in the study areas (Pekagirbas et al. 2021; Arserim et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSandflies have been demonstrated to display a broad spectrum of host preferences, encompassing humans, animals, and even ectothermic vertebrates such as amphibians (de \u0026Aacute;vila et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Elaagip et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jibreel et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Bursali and Touray \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The blood-feeding patterns of these insects can be influenced by the availability of potential hosts as well as local environmental conditions, host body size and attractiveness (Bursali and Touray \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Numerous species are opportunistic feeders, acquiring blood meals from accessible hosts irrespective of species. For example, that \u003cem\u003eP. perniciosus\u003c/em\u003e from Spain, Italy and Portugal displayed opportunistic feeding habits, with no clear host preference (De Colmenares et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Bongiorno et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Maia et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Azmi et al (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that \u003cem\u003eP. papatasi\u003c/em\u003e and \u003cem\u003eP. sergenti\u003c/em\u003e in Palestine fed on humans, hyraxes, rats, livestock, and birds, with some individuals even taking mixed blood meals. Jaouadi et al (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that sandflies in Tunisia primarily fed on humans, rodents, and livestock. Salah et al (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that \u003cem\u003eP. sergenti\u003c/em\u003e in Palestine fed on humans, livestock, birds, and dogs. Gonz\u0026aacute;lez et al (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) observed that \u003cem\u003eP. perniciosus\u003c/em\u003e in Spain primarily fed on wild animals, but also took blood meals from humans, sheep, and goats. Palit et al (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and Srinivasan et al (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) discovered that sandflies in India fed on multiple hosts, including humans, cattle, and other animals with \u003cem\u003eP. argentipes\u003c/em\u003e having a significant preference for humans. A small percentage of sandflies had mixed blood meals from both avian and mammalian sources. At the species level, chicken blood was the most frequently detected avian source. Human, cow, and dog blood were also identified among the mammalian meals. In contrast to the findings of other studies on the \u003cem\u003eP. papatasi\u003c/em\u003e, which were previously described as opportunistic (Svobodoba et al. 2003) and generally reported to be highly anthropophilic (Burniston et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), human blood was not identified in this study.\u003c/p\u003e \u003cp\u003eThe literature on the feeding patterns of sand fly species in T\u0026uuml;rkiye have been limited. Karakuş et al (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) found that the most common food preference of sand flies was dog blood, followed by humans, mice, cats, and cows in terms of frequency in their study in Aydin, the same study revealed that the \u003cem\u003eP. neglectus\u003c/em\u003e species showed a clear preference for human blood. This finding was later shown by Ozbel et al (2015) to be the most common blood-feeding preference (80%) among the \u003cem\u003eP. tobbi\u003c/em\u003e species. In contrast to the findings of the aforementioned studies, no human samples were detected in the blood-feeding female sandflies captured in the wild in the present study. Yetismis et al (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that, in accordance with the results of the present study, the majority of blood sources of blood-fed sand flies belonged to the species \u003cem\u003eBos tauru\u003c/em\u003es. Karagul and Kasap (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) identified bovine animals, particularly cows, as the most common blood source followed by chickens and goats. Humans were identified as a minor blood source. Among the studied species, \u003cem\u003eParaphlebotomus\u003c/em\u003e sand flies were unique in their broader host range, with a particular preference for avian hosts. These results suggest that the host feeding preferences of different sand fly species in Mugla and Aydin provinces are mainly cows, dogs, and chickens, which may be related to the fact that local people keep their animal barns, chicken nests, and their dogs close to their homes. In line with the hypothesis of Dinesh et al (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), that sand flies tend to feed on cows, cows were the most common source of blood as a food source in this study. This may be also related to the higher proportion of cows compared to other hosts in terms of body size, carbon dioxide output level, host-derived volatile organic compounds (Bezerra-Santos et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and other odour secretions. On the other hand, as stated by Yetismis et al (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), although the feeding rate from cows is higher than from other hosts, it may not be correct to define this situation as a real host preference because the settlements in the current study are not environments where all hosts coexist.\u003c/p\u003e \u003cp\u003eThe efficacy of molecular blood-meal identification is contingent upon the quantity of blood ingested and the duration of blood digestion in the midgut of the insect (Kent and Norris, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). It is important to note that the quantity of blood ingested by sand flies (1 \u0026micro;l or less) (Daba et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) is less than that of mosquitoes (2\u0026ndash;6 \u0026micro;l), which is considered to be a biological difference that complicates the generalisation of results (Clements, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The process of blood digestion can cause DNA denaturation, which can make DNA detection difficult (Kent et al. 2005; Sant\u0026rsquo;Anna et al \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), therefore it is essential to use protocols that will detect the minimum amount of DNA. Another factor that must be considered when attempting to identify blood sources using molecular methods is the inhibitory effect of substances found in the tissues of insects, particularly those found in their exoskeleton, head and thorax (Paiva et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Substances such as haem in blood (Kent, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) can also reduce the efficiency of the PCR reaction. In contrast to molecular methods, conventional serological methodologies for blood meal identification necessitate the production of species-specific antibodies against all potential hosts. These techniques are also constrained by factors such as the unavailability of products for exotic animals, diminished sensitivity, and the possibility of cross-reactivity between species (Valinsky et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Despite the limitations and disadvantages inherent to both methods, which are the most frequently employed in blood meal analysis, all samples were successfully analyzed in the present study, and the results obtained by both methods were highly consistent with each other. The bird primers utilized in the PCR methods failed to identify the blood meal from chickens, likely due to the low quantities of the blood ingested having been digested or the DNA denatured. However, the ELISA studies unequivocally elucidated the host of these samples.\u003c/p\u003e \u003cp\u003eDetermining the feeding patterns of sand flies will facilitate the identification of species that are susceptible to zoophagic feeding in designated study areas (Gebre-Michael et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This will enable the determination of the suitability of mammals for zooprophylaxis against diseases caused by sand flies (Morrison et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Moreover, a range of control measures, including the use of insecticide-impregnated collars on vertebrate hosts, can be applied for a variety of diseases for which sand flies are vectors.\u003c/p\u003e \u003cp\u003eIn conclusion, To the best of our knowledge, this is the first study in the region to analyze ELISA and PCR methodologies in a comparative manner in relation to host feeding patterns of sand flies. Regardless of the methodology used, the detection of blood meal in field-caught sand flies has the capacity to substantiate the hypothesis that there exists a robust relationship between sand flies in rural areas and reservoir hosts of the diseases they carry. This may facilitate comprehension of the role of endemic domestic animals and further contribute to the planning of strategic control methods by providing a more comprehensive understanding of the eco-epidemiology of vector-borne diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed 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\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Aydın Adnan Menderes University BAP (project number: BAP-FEF 21005). Fatma Bursali is funded by T\u0026Uuml;BİTAK (2219 Postdoctoral Fellowship Programme)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor credit information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFB designed the original project. MP made sample collection. Experimental studies were carried out by FB, MP. FB, MT, MP and SB revised the manuscript. All authors contributed to the final version of the text.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have nothing to report.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlkan C, Erisoz Kasap O, Alten B, et al (2016) Sandfly-Borne Phlebovirus Isolations from T\u0026uuml;rkiye: New Insight into the Sandfly fever Sicilian and Sandfly fever Naples Species. 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Acta Parasitol. 67,932\u0026ndash;942. https://doi.org/10.1007/S11686-022-00542-\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-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Blood-meal, ELISA, PCR, Sand fly, Türkiye","lastPublishedDoi":"10.21203/rs.3.rs-6202222/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6202222/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThis study determined the blood-feeding patterns of wild-caught sandflies collected from provinces (Aydin and Mugla) in the Aegean region in T\u0026uuml;rkiye. Adult sandflies were collected using three CDC light traps per village in August and October 2024. A total of 280 sandfly specimens (194 females and 86 males) were collected during the study. Of the 194 female specimens, 38 (23 from Aydin and 15 from Mugla) were found to be blood-fed, 137 were non-blood-fed, and 19 were gravid. In total, DNA was extracted from 38 blood-fed sandflies using Invitrogen PureLink genomic DNA isolation kit. To determine the blood-feeding patterns of these insects, the mitochondrial cytochrome b (\u003cem\u003ecytb\u003c/em\u003e) gene region was amplified via multiplex PCR using specific primers.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe PCR results showed that 23 sand fly females sampled from Aydin locality fed mostly from cows (60.5%) followed by dogs (21%) and chickens (18.4%). All the samples collected from Aydin blood fed from cows whereas for females sampled from Mugla locality, 53% blood-fed from dogs and 46% from birds. Host preference of the samples was also subjected to ELISA reaction to identify samples whose host preference could not be determined by PCR and to see if there were multiple host preferences. Our bird primers used in PCR methods, could not identify the blood meal from chickens, probably due to low amounts of the blood ingested having been digested or DNA was denatured. However, the ELISA studies clearly elucidated the host of these samples. Despite the limitations and disadvantages inherent to both methods, which are the most frequently employed in blood meal analysis, all samples were successfully analyzed in the present study, and the results obtained by both methods were highly consistent with each other.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn conclusion, to the best of our knowledge, this is the first study in the region to analyse ELISA and PCR methodologies in a comparative manner in relation to host feeding patterns of sand flies. 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