Background & objectives: The treatment of wounds with live green bottle fly larvae is receiving considerableattention in many countries. Laboratory rearing of Lucilia sericata is crucially important for the treatment ofwounds.Study design: The study was carried out for mass rearing of green bottle flies from April to November 2010.Hand catch and net trap baited with beef and cattle liver were used to collect adult flies from the field. Thecollected samples were placed in appropriately labeled tubes and sent to the laboratory.  Adult stage flies rearedin the insectary were used for species identification using specific keys.Results: A total of 89 flies (55 females and 34 males) were collected from Hashtgerd area. In the first generation,299 flies were produced in the laboratory including 105 (35.12%) males, and 194 (64.88%) females. The female/male sex ratio was 1.61 for parents, whereas it was 1.84, 1.30 for F1 and F2 generations respectively. In total,432 flies were reared in F3 generation including 173 (40.04%) males, and 259 (59.96%) females, and the sexratio was 1.49.Conclusion: Setting up the mass rearing of sheep blowfly at the School of Public Health, Tehran University ofMedical Sciences is an important step in producing candidate flies for the treatment of myiasis by maggottherapy in future

F. Firoozfar , S.H. Mosa Kazemi, K. Shemshad , M. Baniardalani  , M. Abolhasani  , A. Biglarian  , A. Enayati   & J. Rafinejad

Journal of Vector Borne Diseases

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J Vector Borne Dis 49, March 2012, pp. 23–26Laboratory colonization of Lucilia sericata Meigen (Diptera: Caliphoridae)strain from Hashtgerd, IranF. Firoozfar1,2, S.H. Mosa Kazemi1, K. Shemshad3, M. Baniardalani1, M. Abolhasani1,A. Biglarian4, A. Enayati5 & J. Rafinejad1,61Department of Medical Entomology and Vector Control, School of Public Health;  2Students Scientific Research Center, Tehran Universityof Medical Sciences, Tehran, Iran; 3Department of Entomology, Science and Research Branch, Islamic Azad University, Tehran; 4Departmentof Biostatistics, University of Social Welfare and Rehabilitation (USWR), Tehran; 5Department of Medical Entomology, School of PublicHealth and Health Sciences Research Center, Mazandaran University of Medical Sciences, Sari; 6Evaluation Management and DevelopmentCenter, Deputy of Research Ministry of Health and Medical Education, Tehran, IranABSTRACTBackground & objectives: The treatment of wounds with live green bottle fly larvae is receiving considerableattention in many countries. Laboratory rearing of Lucilia sericata is crucially important for the treatment ofwounds.Study design: The study was carried out for mass rearing of green bottle flies from April to November 2010.Hand catch and net trap baited with beef and cattle liver were used to collect adult flies from the field. Thecollected samples were placed in appropriately labeled tubes and sent to the laboratory.  Adult stage flies rearedin the insectary were used for species identification using specific keys.Results: A total of 89 flies (55 females and 34 males) were collected from Hashtgerd area. In the first generation,299 flies were produced in the laboratory including 105 (35.12%) males, and 194 (64.88%) females. The female/male sex ratio was 1.61 for parents, whereas it was 1.84, 1.30 for F1 and F2 generations respectively. In total,432 flies were reared in F3 generation including 173 (40.04%) males, and 259 (59.96%) females, and the sexratio was 1.49.Conclusion: Setting up the mass rearing of sheep blowfly at the School of Public Health, Tehran University ofMedical Sciences is an important step in producing candidate flies for the treatment of myiasis by maggottherapy in future.Key words   Insectary; Iran; Lucilia sericata; maggot therapyINTRODUCTIONThe sheep blowfly Lucilia sericata has a long historyin medicine and its maggots have been used in wound heal-ing. Some unsuccessful surgical and antibiotic treatmentsof infections such as temporal mastoiditis and perinealgangrene were treated using maggot therapy. Wounds re-sulting from cuts, wounds or even trauma resulting fromdiseases like diabetes, bed sores, gangrene, and burns arethe problems of human societies1. In many of these cases,surgery is the only treatment, sometimes leading to ampu-tation. Leg amputation due to diabetes is frequently re-ported from around the world. According to World HealthOrganization, this happens every 30 seconds. Diabetic footulcer is responsible in half of diabetic patients’ visits tothe hospitals. In many of these patients, it may lead toamputation of one or both feet2. Treatment by flies’ lar-vae as a suitable alternative method in the treatment of thewounds has introduced3. Up to now, maggot therapy wasperformed for treating many different kinds of wounds.Centrally important to maggot therapy is that the lar-vae used in the treatment of wounds should reduce theinfections and improve the nutrition of tissues for rapidimprovement of wounds. There are 1100 known speciesof sheep flies, with 228 species in the Neotropics, Africaand southern Europe. Lucilia species has been reported inthe countries, namely India, Japan, central America, andsouthern United States of America4 .Considering the significance of maggot therapy, es-tablishing an insectary for mass rearing of larvae to pro-vide hospitals and medical centers with Lucilia larvae isimportant. The aim of this study was mass rearing andmaintenance of L. sericata (Phaenicia sericata) maggotunder laboratory conditions at the School of Public Health,Tehran University of Medical Sciences.MATERIAL & METHODSStudy areaThe experimental study was performed from April to J Vector Borne Dis 49, March 201224November 2010. Sampling methods used were hand catchby net and bait traps. Sampling was carried out in placesincluding gardens, around livestock, slaughterhouse inHashtgerd county, Alborz province, northern Iran.Adult fly collectionThe samples were caught from different places ofAlborz province and were sent to the insectary of MedicalEntomology at School of Public Health, Tehran Univer-sity of Medical Sciences in appropriately labeled tubes5.Beef and Hamburger were used as bait for collecting adultstage of the fly in open area. Information such as placeand date of collection, sampling method and weather con-dition was recorded5.Species identificationAdult flies were identified using standard keys6–8. Thediagnosis of larvae is performed based on the respiratorypores and posterior of cephalo-pharyngeal skeleton of lar-vae, therefore, definitive diagnosis was carried out on deadlarvae. In order to identify the live specimens, adults wereanesthetized by CO2, ether or cold shock for a short time.After identification, the adults were transferred to newcages for oviposition.MaintenanceThe flies were maintained in the insectary under con-trolled conditions of mean temperature of 27±1°C, rela-tive humidity of 80±5%, and daily light /dark period of16:8 h. The average temperature for larval developmentwas also 27±1°C. The electrical fly killer was hung in theinsectary area to kill the free flying flies in the insectary9,10.Layers of red meat with tissue papers in between wereplaced in the petri dish and, then a cup container with cutedge was placed on the meat11. In order to rear the larvae,sheep blood agar was used because of its simplicity foodsource12,13. Supervision of rearing cages was essential andlarvae were isolated from rearing cages. Upon emerging,the adults were placed in new cages and provided withessential food.Statistical analysisThe hatching dishes were filled with larval mediumand were visited daily according to method ofFleischmann13. All values are expressed as the mean ±standard error of the means (SEM).RESULTSIn all, 89 adult flies were collected from slaughter-houses of Hashtgerd, Alborz province and were transferredto the Tehran School of Public Health. The life cycle ofgreen flies is shown (Figs. 1–4) and the duration of lifecycle of male and female L. sericata is given (Table 1).The average number of female parents emerged per eggsbatch was 6.1. The time required for the eggs to hatchwas 52±2 h. The mean time for larval and pupal develop-ment in F1, F2, and F3 generations was 96±2, and176±2.33 h respectively. The time for emerging from pu-pae to laying eggs was 200±2.66 h. In F1 generation, 507maggots age I were reared to 299 adults, hence, a mortal-ity rate during the F1 generation of 41.03% was reported.Mortality rate of Lucilia sericata was 53.15% for F2 gen-eration, whereas in F3 generation, it was 27.28 %.  Num-ber of eggs, larvae and pupae stages I, II, III and adultsyielded in F1, F2 and F3 generations are shown (Table 2).Totally, 299 flies were reared including 105 (35.12%)males, and 194 (64.88%) females in the first-generation.They were transferred to spawn cages and nine eggsbatches were obtained. In F1 generation, the average num-ber of female flies emerged per eggs batch was 21.55%.Fig. 1: The batch of eggs of Lucillia sericata at the insec-tary, School of Public Health, Tehran University ofMedical Sciences.Fig. 2: The Lucillia sericata maggots at the insectary,School of Public Health. 25Firoozfar et al: L. sericata laboratory from Hashtgerd, IranDISCUSSIONThe different time periods required for the completionof three generations of Lucilia sericata in our study showedthat the life stags are affected by temperature and humid-ity of the Insectary. The first generation of wild flies tooklonger than their parents to lay eggs. This delay may bejustified by the time required for the adaptation to the cap-tivity conditions. Also the first generation took longer tomate. Lysek in 1991 reported the optimal temperature of37± 3°C for rearing this fly. The duration of larval stagewas reported to be 4 to 13 days14. In our study, the dura-tion of larval stage was 96 ± 2 h with an average tempera-ture of 27 ± 1°C. Sherman and Wyle11; and Wolff andHansson1 colonized this species in insectary and reporteda cycle from egg to adult of about 1–2 weeks a periodwhich was calculated 8.33 days in our study. In parallel,the egg to egg period was reported as 2–3 weeks15. In ourstudy, the period from egg to adult was noticeably longerin F2 generation, which might be justified by different diet,nutrition and new environment. Comparison of the resultsof studies by Sherman and Wyle11; and Wolff andHansson1  indicated that in the second generation in ourstudy less eggs were developed to larvae and adults suc-cessfully (46.85%). This decrease in the second genera-tion was more than that in the first generation, this factmight be explained by the specific conditions includinglight, temperature, humidity, food and larvae nutrition. Theeffect of different kinds of food on larval development ofFig. 3: The pupa of Lucilia sericata at the insectary,School of Public Health.Fig. 4: The adult of Lucilia sericata at the insectary,School of Public Health.Table 1. Duration of life stage of Lucilia sericata in the insectary 2010Generation Adult Egg period Larval stage    Pupa stage       Emergence to egg laying (h)Male Female Time (h)   Mean ± SEM Time (h) Mean ± SEM Time (h)   Mean ± SEM Time(h) Mean ± SEMParents 34 55 – – – – – – – –F1 105 194 60 ± 3 52±2 72 ± 1 96 ± 2 192 ± 3 176 ± 2.33 312 ± 3 200 ± 2.66F2 84 110 48 ± 1 96 ± 3 168 ± 2 168 ± 3F3 173 259 48 ± 2 120 ± 2 168 ± 2 120 ± 2Table 2. Number of eggs, batch, and larvae stages I, II, III, pupae and adults of Lucilia sericata duringF1, F2 and, F3 generations in insectary 2010Generation Egg batch Larval stage Pupae Adults Totalration batch (No.)I  II III Male FemaleF1 9 507 (100) 431 (85) 375 (87) 330 (87) 105 (35.1) 194 (64.9) 299F2 11 414 (100) 348 (84.05) 293 (84.19) 214 (73.03) 84 (43.3) 110 (56.7) 194F3 4 594 (100) 553 (93.09) 509 (92.04) 464 (91.15) 173 (40.1) 259 (59.9) 432Figures in parentheses indicate percentages. J Vector Borne Dis 49, March 201226Lucilia sericata is considerable. The size of larvae fed onhamburger was smaller than those fed on beef and liver.Similar to our results, the growth rate of Lucilia larvaefed on beef liver16 was significantly lower than those grownon the chicken flesh and beef. Protein intake is necessaryin the first generation of in vitro rearing. The average pe-riod between emerging of adults, mating, spawning, andlaying eggs in our study was 8.33 days, whereas Shermanand Wyle11; Wallman and Day17 stated two weeks fromemergence to laying of eggs. Mortality rate during the lifecycle of Lucilia sericata was 41.03, 53.15 and 27.28% inF1, F2, and F3 generations respectively. The mortalityrate of gravid female was more than non-gravid and malein F2 generation. The susceptibility of the flies to tem-perature and humidity was noted in our study. The stagesI and II larvae are quite sensitive to dryness and food short-age, whereas the III instar larvae are sensitive to humid-ity. Establishing a colony of this fly in insectary in Iran isof high importance for undertaking maggot therapy to treatthe disease.ACKNOWLEDGEMENTSThe authors are grateful to Dr Ladonni and Mr.Akbarzadeh, Department of Medical Entomology andVector Control, for their valuable suggestions. The au-thors are also grateful to Mr Hossaini for his kind coop-eration throughout the study in flies’ insectary. Thisproject was financially supported by a grant from TehranUniversity of Medical Sciences Project No. 89-03-27-11453. The authors declare that they have no conflictsof interest.REFERENCES1. Wolff  H, Hansson J. Rearing larvae of Lucilia sericata forchronic ulcer treatment – an improved method. Sweden: Depart-ment of Dermatology. Acta Derm Venereol 2005; 85: 126–31.2. Cetinkaya M, Özkan H, Köksal N, COkun SZ. Neonatal myia-sis: a case report. Turkish J Pediatrics 2008; 50: 581–4.3. Bani-Ardalani M. Weekly Medical Report 2005; No. 578, 15years.4. UniProt.“Calliphoridae”(HTML). Available from: http://beta.uniprot.org/taxonomy/7371 (accessed on May 31, 2008).5. Hall MJ. Trapping the flies that cause myiasis: their responsesto host stimuli. Ann Trop Med Parasitol 1995; 89(4): 333–57.6. James MT. The flies that cause myiasis in man. United StatesDepartment of Agriculture Miscellaneous Publication 1974; 631:1–175.7. Zumpt F. Calliphorinae. In: Lindner E, editor. Die fliegen derpalaearktischen Region XI, 64i. Schweizerbart, Stuttgart 1959;8: 1–140.8. House-flies, blow-flies and their allies (Calyptrate: Diptera).In:  Lane RP, Crosskey RW, editors. Medical Insects Arach-nids.   Chapman and Hall 1993; p. 403–28.9. Greenberg B. Flies through history. Flies and Disease, I edn.,Princeton, New York: Princeton University Press 1973;p. 1–447.10. Sherman RA, My-Tien Tran JM. A simple sterile food sourcefor rearing the larvae of Lucilia sericata (Diptera: Caliphoridae).Med Vet Entomol 1995; 9(4): 393–8.11. Sherman R, Wyle F. Low-cost, low-maintenance rearing of mag-got in hospitals, clinics and schools. Am J Trop Med Hyg 1996;54: 38–41.12. Spiller D. Insect colonization and mass production. In: SmithCN, editor. Houseflies: Chapter 14. New York and London: Aca-demic Press  1996; 203–25.13. Fleischmann W, Grassberger M, Sherman R. Maggot therapy: ahandbook of maggot-assisted wound healing complementarymedicine. Stuttgart 93 pp. George Thieme Verlag 14, 70469,Germany 2004; p. 93. Available from: www.thieme.de14. Lysyk TJ. Effects of temperature, food, and sucrose feeding onlongevity of the housefly (Diptera: Muscidae). Environ Entomol1991; 20: 1176–80.15. Nsary J, Ladani M, Mirab-Zadeh A, Mashayekhi M, SharifiSH. The final report of the project “Using of larvae LuciliaSericata to treatment of infectious wounds.  Scientific and In-dustrial Research Organization of Iran 2006; p. 1–51.16. Salimi M, Goodarzi D, Karimfar MH, Edalat H. Human uro-genital myiasis caused by Lucilia sericata (Diptera,Caliphoridae) and Wohlfahrtia magnifica (Diptera:Sarcophagidae) in Markazi Province of Iran. Iranian J Arthro-pod Borne Dis 2010; 4(1): 72–6.17. Wallman JF, Day DM. Influence of food substrate tissue typeon larval growth in Calliphora augur and Lucilia cuprina(Diptera: Caliphoridae). J Forensic Sci 2006; 51: 57–63.Correspondence to: Dr Javad Rafinejad, Department of Medical Entomology and Vector Control, School of Public Health, Tehran Univer-sity of Medical Sciences and Evaluation Management and Development Center, Deputy of Research Ministry of Healthand Medical Education, P.O.Box 14155-6446, Tehran, Iran.Received: 2 September 2011 Accepted in revised form: 18 February 2012

Laboratory colonization of Lucilia sericata Meigen (Diptera: Caliphoridae) strain from Hashtgerd, Iran

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Laboratory colonization of Lucilia sericata Meigen (Diptera: Caliphoridae) strain from Hashtgerd, Iran

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