J Arthropod-Borne Dis, December 2018, 12(4): 426–431 M Fatemi et al.: Designing and Introducing … 426 http://jad.tums.ac.ir Published Online: December 25, 2018 Short Communication Designing and Introducing a New Artificial Feeding Apparatus for Sand Fly Rearing Mahboubeh Fatemi 1, Zahra Saeidi 1, Parviz Noruzian 2, *Amir Ahmad Akhavan 1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Tajhiz Gostar Omid Iranian Co, Tehran, Iran (Received 26 June 2018; accepted 13 July 2018) Abstract Background: Due to strict ethical rules, the risk of accidental disease transmission and the most importantly, inconven- ience regarding using of live animals, artificial feeding apparatus has been developed for colonization of haematophagous insects. Rearing of sandfly is more difficult than other haematophagous insects. Methods: In the current study, a new apparatus for membrane feeding of Phlebotomus papatasi was designed, made and compared with available apparatus in Sand Fly Insectary, Tehran University of Medical Sciences, Tehran, Iran, in 2014. Results: In comparison to other apparatus designed for artificial feeding of other arthropods, our designed apparatus had the highest performance which after up to 1h, the majority of sand flies landed and took blood and among tested membranes, chicken skin was proved the most efficient membrane. Conclusion: Sand fly artificial feeding apparatus can be used at least for rearing of Ph. papatasi. Keywords: Phlebotomus papatasi, Artificial feeding apparatus, Blood-feeding insects, Sandfly Introduction Insect colonization has been intensified by increasing of interest in understanding of their life cycle, physiology, anatomy, genetics, and study on arthropod-borne diseases, insecticide and repellency tests to control the diseases (1). Blood-feeding is one of the most critical and difficult steps in rearing process of haematoph- agous insects. Animal maintenance for blood feeding of the colony, not only is costly and time-consuming but also requires ethical con- siderations. Due to these restrictions, risk of ac- cidental disease transmission and inconven- ience of using live animals, artificial feeding apparatus has been developed. Artificial feed- ing is a useful technique in nutritional and be- havioral studies on insects such as feeding stim- ulants and interaction between parasite and in- vertebrate host (2). Moreover, one of the most advantages of this technique in the parasite and vector interaction studies is capability of adding a certain concentration of parasite to feeding mixture (3). Precise control over the test con- ditions is considered as another advantage of this method compared to feeding on live hosts. Based on advantages mentioned above, mem- brane feeding is preferred to feeding on animals. The earliest attempt at feeding tsetse flies through rat skin was made by Rodhain et al. (4). Subsequently, this technique has been used suc- cessfully to feed various haematophagous in- sects including vectors and their transmittable pathogens to study developmental stages and transmission cycle of micro-organism inside their body (5-15). Establishment and mainte- nance of sandflies are far more difficult than other haematophagous insects (16-17). The aim of the current study was to design and introduce a new apparatus for artificial feed- ing of Phlebotomus papatasi in the insectary condition. *Corresponding author: Dr Amir Ahmad Akhavan, E-mail: aaakhavan@tums.ac.ir J Arthropod-Borne Dis, December 2018, 12(4): 426–431 M Fatemi et al.: Designing and Introducing … 427 http://jad.tums.ac.ir Published Online: December 25, 2018 Materials and Methods In order to membrane feeding of Ph. pa- patasi four artificial feeding apparatus includ- ing: Tick artificial feeding apparatus, Mosquito artificial feeding apparatus based on the idea of Cosgrove et al. (18), modified Cosgrove et al. apparatus and a new artificial feeding apparatus which was designed and made in this study in Sand Fly Insectary, Tehran University of Medical Sciences, Tehran, Iran, in 2014, (The patent number: 86836, Islamic Republic of Iran) were compared. Tick artificial feeding apparatus has a cham- ber to hold the blood, 5.5cm in diameter and 12.5cm deep. The minimum required volume of blood is 5ml. It was connected to a water bath that circulates warm water inside the ap- paratus. A glass tube was oriented vertically in the chamber that is 4.5×7cm. Membranes were stretched over one end of the glass tube which is in contact with blood mixture. Fine mesh gauze is fitted on another end of the apparatus to transfer sand flies through it (Fig. 1). The second apparatus consisted of an 8×7 cm aluminum chamber with a volume of 2.8 ml heated by electrical elements. A thermostat has been connected to adjust the desired tem- perature (Fig. 2). Chamber in modified Cosgrove et al. ap- paratus has a volume of 9ml and the tempera- ture is displayed on a digital screen that is ad- justable with buttons (Fig. 3). The idea of the design of sand flies artifi- cial feeding apparatus was inspired by the two previously available equipment (19, 20). The apparatus contains a stainless steel container in a volume of 6L and a steel plate embedded on the top of the container. The steel plate is used as a container for a water bath if needed. To as- sure a constant temperature in the blood feeders, a pair of water outlet and inlet pipes was de- signed for each blood feeder of the apparatus. In order to offer blood supply for the insects, 7.5×2.5cm glass blood-feeders were designed (Fig. 4). A cavity at the top was designed to hold the blood mixture (up to 2ml) and a pair of water outlet and inlet pipes for circulating warm water inside the blood-feeder. In each ex- periment, up to four blood-feeders can be con- nected to apparatus at the same or different an- gles, simultaneously. All the pipes were con- sidered 120cm in length in order to make the same condition. A temperature sensor was placed inside the outlet, and a built-in display installed to moni- tor experiment process. The temperature and time were adjustable with buttons on the screen. The Alarm sound could be heard after finishing the time given to apparatus (Fig. 5). To mimic natural condition of blood feeding, the mem- brane temperature was adjusted to 35±1 °C. The bottom of feeding containers (5.5cm height, 4cm diameter) was moistened with dis- tilled water before the introduction of the sand flies and placed in close contact with the mem- brane by the clamp. Therefore, the membrane is accessible to the sand flies for blood-feeding. In four mentioned artificial feeding appa- ratus, Parafilm, Nescofilm and skin of three- week-old chicken as membrane were tested and compared. Membranes were stretched over the blood mixtures in one end of each feeder and fastened with elastic band. In each case, a new membrane and fresh blood were used and Ph. papatasi were starved for 2h before the blood meal. Moreover, three sources of defibrinated or heparinized blood of human, BALB/c mice and Rhombomys opimus were used to achieve the highest blood feed- ing. All experiments were carried out under the same condition at 26±2 °C temperature and 80 % relative humidity. Animal protocol was approved by the Eth- ics Committee of Tehran University of Medi- cal Sciences, Tehran, Iran. Results In tick and Cosgrove's artificial feeding ap- paratus, no sand fly landed to blood chamber. J Arthropod-Borne Dis, December 2018, 12(4): 426–431 M Fatemi et al.: Designing and Introducing … 428 http://jad.tums.ac.ir Published Online: December 25, 2018 In modified Cosgrove's artificial feeding apparatus, the sand flies landed on the mem- branes but no Ph. papatasi took blood. In three above-mentioned apparatuses, because of pres- ence of only one blood feeder, each blood sam- ple was tested separately. In sand fly artificial feeding apparatus made in the current study, after up to 1h the majority of sand flies landed and took blood. Moreover, three blood sources were checked simultaneously. Among the tested membranes, the chicken skin was the best and the heparinized blood had the most efficient because it was not clotted even after 4h. No preference among the tested blood was observed. Fig. 1. Tick artificial feeding apparatus Fig. 2. Mosquito artificial feeding apparatus of based on the idea of Cosgrove et al. Fig. 3. Modified Cosgrove’s apparatus Fig. 4. Glass blood feeder Fig. 5. Phlebotomus papatasi artificial feeding apparatus J Arthropod-Borne Dis, December 2018, 12(4): 426–431 M Fatemi et al.: Designing and Introducing … 429 http://jad.tums.ac.ir Published Online: December 25, 2018 Discussion Feeding through membrane has been utilized for various hematophagous arthropods to pur- pose of mass rearing of insects or to artificial- ly infect vectors with parasites (3, 5, 18, 20-22). There were disadvantages in the available ar- tificial feeding equipment such as the temper- ature which could only be measured in the wa- ter tank with no temperature sensor in the vi- cinity of membrane, and also the time that was not adjustable. The current new designed feed- ing apparatus does not have the above-men- tioned weakness points. Perhaps the lack of cir- culation of warm water and the moisture, that is critical for sand fly feeding, were responsi- ble for unattractiveness of blood chamber for Ph. papatasi in modified and Cosgrove's arti- ficial feeding apparatus. Each blood-sucking arthropod has a pref- erence host to take blood, determining the most appropriate blood lead to the highest blood feed- ing that has direct effect on egg production. Be- cause of capability of supporting several blood feeder by this apparatus various blood sources can be tested simultaneously to achieve the best result. Results of artificial-feeding of Ph. papa- tasi showed no significant differences in blood- feeding proportion of sand flies between hu- man, BALB/c mice, and Rh. opimus. In agree- ment with our results, there was no preference between human blood and blood from differ- ent types of host (cow, dog, guineapig, ham- ster, horse, pig, rabbit) in Ph. papatasi blood feeding (23). Angle of blood- feeder can affect on per- cent of Lutzomyia shannoni blood feeding (20). Therefore, ability to change the angle on this device has been provided. Besides, different kinds of membrane includ- ing natural or synthetic such as guinea pig mes- entery and large bowel, batwing, human perito- neum, human kidney capsule, Ox liver capsule, Baudruche, chick skin, Parafilm, Chitosan, di- alyzing bag and so on have been tested to stim- ulate insects to taking a blood-meal through membrane (8, 19, 24-26). However, different studies, same to our study, have shown that chicken skin is more effective than other mem- branes (25, 27, 22, 28). Conclusion New designed artificial feeding apparatus could be used for rearing of Ph. papatasi in- stead of live animals. Acknowledgements This study was supported financially by Re- search deputy of Tehran University of Medical Sciences (TUMS) and the Center for Research and Training in Skin Diseases and Leprosy, Teh- ran, Iran, (project no. 93-03-27-26140). 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