- Original Article
- Open Access
Temperature requirements for the growth of immature stages of blowflies species, Chrysomya albiceps and Calliphora vicina, (Diptera:Calliphoridae) under laboratory conditions
© The Author(s) 2018
- Received: 7 October 2017
- Accepted: 14 March 2018
- Published: 20 March 2018
In forensic entomology, a way to estimate Post Mortem Interval (PMI) is to use development data of the first colonizing insects on a dead body. Chrysomya albiceps and Calliphora vicina, two members of the family Calliphoridae, are the first waves of necrophagous insects that are the most entomologically important fly species because of its consistent time of arrival and colonization of the body following death. Regarding the bioclimatic, environmental conditions and difference in geographic population, it seems necessary to collect data about the forensically important species in each region. In this research, the growth rate of immature stages of C.albiceps and C.vicina was studied in controlled laboratory conditions.
The results of this study showed that with increasing temperature, the duration of development was reduced. The highest period of development, for both species, is related to pupae stage and involves about half of the entire development period. The amount of Accumulated Degree Days (ADD) for the egg stage to mature of the species C.vicina, at two temperatures of 15°C and 20°C, are calculated as 416 and 396 DD, respectively.
This study showed, temperature had significant effects on rate of the development on Chrysomya albiceps and Calliphora vicina and the amount of heat energy needed to development for these species is specific. Actually, the relationship between the development rate and temperature was significant and dependent to species and geographical regions.
Generally, there is a bilateral relationship between temperature and development of insects. The insect growth is a linear function of temperature variation (Defilippo et al., 2013). The rate of development and growth of insect larvae can be obtained using different methods, the most common of which is to calculate ADD/ ADH (Grassberger & Reiter, 2001; Marchenko, 1989; Anderson, 2000; Oshaghi et al., 2009). The difference in geographic populations of insects makes them tolerate a range of temperature, exclusive to each species, during different stages of growth. Therefore, ADD/ADH is different for the insect population of each geographic region (Davies & Ratcliffe, 1994; Donovan et al., 2006). The goal of this research is studying the growth parameters of pre-mature stages of forensic dominant species, i.e. C.albiceps and C.vicina, under constant laboratory temperature conditions and calculating ADD for them as well as comparing the development data of these species with populations living in other geographical areas.
Sampling location and period
This research was done under both field and laboratory conditions. The field of research was conducted in the Bisetoon protected area, west of Iran, 13 kilometers far of the city of Kermanshah, 34° 24° 37° N and 47° 14° 42° E, comprising a land area of 54,950 km2 and elevation about 1320 m above sea level. The average precipitation here is 400–450 mm per year and the average maximum and minimum temperatures are 30°-35°C and -2°C, respectively. The flora of this region is mostly meadow and it has four distinct seasons in terms of temperature conditions.
Rearing in field conditions
In this research, inbreeding rabbits, about 2. 5 kg in weight, were used. The rabbits were first killed by chloroform and then, weighed and immediately transferred to the field. The carcasses were put in cages (150 × 90 × 90) with 1.25 cm meshes with their floors being directly in contact with the ground. The sampling was done from mature flies using insect net through sweeping from the corpse in immature stages with forceps. The corpse had degeneration in four stages, i.e. decay, bloated, fresh and dry in the field and the duration for each stage depends on the season during which it happens. Eggs and larvae clusters of the stage one are collected from the corpse using forceps and put in transparent plastic containers (15× 10× 10) cm that have lace fabric lids for air conditioning. Each container is supplied with a 3 cm layer of sawdust, a piece of rabbit meat covered by an aluminum sheet and daily humidity. The goal was to detect the mature species as well as to compare the adults out of pupa with the sampled ones in terms of number. The adult samples were transferred to cages (50 × 50 × 50) cm with fabric laces containing rabbit meat substrate with humidity and a few hours later, the egg and larvae samples were put into other breeding containers and then transferred immediately to the laboratory of Kermanshah School of Public Health. In this study, larval and adult of blowflies were identified based on descriptive and pictorial keys (Szpilla, 2010a; Szpilla, 2010b).
Rearing in laboratory conditions
The identified samples were bred and evolved under constant-temperature conditions of the laboratory. In order to avoid making mistake during all breeding stages, both in the field and laboratory, rabbit meat substrate was used to feed the maggots. In the laboratory, the samples were put into incubator with constant temperature, humidity and photoperiod similar to the field conditions. Then, the growth stages of the samples were investigated regularly.
Development zero (Dz) temperature
The development of different growth stages of insects proceeds between two ranges of lower and upper development threshold temperature outside of which the growth stops. Development zero temperature or lower development threshold temperature is different based on the species, different stages of development of each species and geographic conditions. By reviewing previous studies, i.e. Marchenko, (Marchenko, 1989) Queiroz, (Queiroz, 1996) Grassberger and Reiter,(Grassberger & Reiter, 2001) Richard et al., (Richards et al., 2008) and Al-Shareef and Al-Qurashi, (Al-Shareef & Al-Qurashi, 2016) conducted in different geographic populations, some numbers were calculated for the Dz temperature of the species C.albiceps and regarding all of which an average temperature was determined for each stage as follows: egg stage to first larvae (9.72 °C); larvae stage one to second stage (11. 14°C); second stage to third stage (13°C); larvae stage three to pupal (14.48 °C) and for pupal stage to adult (14. 32°C). Overall, 10. 11°C and 15. 38°C were determined for the egg to adult and first larval to adult, respectively. Referring the studies done before, different temperatures were also suggested for the Dz temperature of the species C.vicina: Kamal (Kamal, 1958) (Dz= 1°C); Vingradova (Vinogradova & Reznik, 2015) and Marchenko (Marchenko, 1989) (Dz= 2°C); Davies and Ratcliffe (Davies & Ratcliffe, 1994) (Dz= 3. 5°C); Haskell (Haskell et al., 2000) (Dz= 6°C); Ames and Tuner (Ames & Turner, 2003) (Dz= 1°C), Donovan et al., (Donovan et al., 2006) based on linear approximation estimation method (Dz= 1°C) and Defilippo et al. (Defilippo et al., 2013) (Dz= 1. 6°C) were concidered. In this study, we considered 2°C for all the growth stages of the species C.vicina. ADD was calculated in the laboratory based on the formula suggested by Wilson and Barnett (Gennard, 2007):
ADD = Days (T constant - T basment ).
Rearing in field conditions
The results obtained from breeding eggs and larvae clusters that were collected from the rabbit body during spring and summer with average temperatures 17. 5°C and 27°C, respectively, showed that the C.albiceps was dominant compared with other Calliphoridae species. In autumn, the results obtained from breeding eggs and larvae clusters showed that the abundance of C.vicina was 70% more than other species. In the winter, the results confirmed that C.vicina was only active species.
Development of C.vicina and C.albiceps immature stages in laboratory
Development duration and thermal requirements (ADD) of Chrysomya albiceps (second generation) at constant temperatures (RH = 50%, Photoperiod = 14: 10)
Development duration (Day+/-SD)
0. 5–1+/− 0.0
10. 1–20. 3
1st larval instar (L1)
2nd larval instar (L2)
3rd larval instar (L3)
3. 5+/− 0.0
First larval instar to adult eclosion
Egg to Adult eclosion
Development duration and thermal requirements (ADD) of Calliphora vicina (second generation) at constant temperatures (RH = 70%, Photoperiod= 10: 14)
Development duration (Day+/-SD)
1st larval instar (L1)
2nd larval instar (L2)
3rd larval instar (L3)
Egg to adult eclosion
Temperature requirement in laboratory condition
The results showed that the development period was in inverse proportion to temperature. In other words, as the temperature increases to upper threshold, growth is accelerated and the length of the development period is reduced. During the growth process, the base threshold temperature for any stage was increasing in such a way that the minimum threshold of growth temperatures for the egg to first larval instar, first to second instar, second to third instar, third larval to pupal and pupal to adult were considered as 9.72 °C, 11. 14°C, 13°C, 14.48 °C and 14.317 °C, respectively. In C.albiceps, the length of the development of each stage relative to the total development period at the temperatures 25°C and 30°C were as follows: egg to first larval (9%, 6. 25%), first to second larvae (9%, 12. 5%), second to third larvae (18%, 12. 5%), third larva to pupa (27%, 25%), pupal to adult stage (45. 5%, 44%) and first larvae to adult was (90%, 94%). For C.vicina, the greatest contribution of the development period, in temperatures 15 and 20°C, is assigned to the pupal to adult period that accounted for 62 and 56 percentage of the total development period, respectively. The development time of third instar larvae to pupa stage were calculated 24 and 25 percentage of the total pre-mature time at 15°C and 20 °C respectively. Also, the development time from egg to the end of third instar larvae were observed about 14. 24 and 18.57 percentage of the total time at 15°C and 20°C respectively. Similar to C.albiceps, the greatest contribution of the development period of C.vicina is assigned to the pupal period. It was quite evident that temperature had a key role, especially in low temperatures, in making significant differences in temperature requirement of growth of ADD in C.vicina at 15°C and 20°C. For C.albiceps, the majority of required ADD was related to the immature period, and the same applied to C.vicina. As the larvae development, the amount of ADD increased too such a way that, at temperatures 15°C and 25°C, the amount of ADD for the third larvae were determined as 104°C and 90°C respectively, and for the pupal stage, it was similar and equal to 234°C (Table 2).
In this study, as other researches (Al-Shareef & Al-Qurashi, 2016; Grassberger et al., 2003; Queiroz, 1996; Grassberger & Reiter, 2001; Augul, 2009) we found that as the temperature increases, the length of the development is shortened. In C.albiceps, the length of larvae stage (first larval to Pupal instar) in 25°C and 30°C were 6 and 4 days, respectively. Different researchers determined different values for the length of this stage: Velez and Wolff (Velez & Wolff, 2008) (8–10 days at 25°C); Grassberger (Grassberger et al., 2003) (8–10 days); Al-Shareef and Al-Qurashi (Al-Shareef & Al-Qurashi, 2016) (6 days); Queiroz (Queiroz, 1996) (5 days at 27°C); Augul and Jassim (Augul, 2009) (3 days at 30°C); AL-Shareef and Al-Qurashi(Al-Shareef & Al-Qurashi, 2016) (5 days); Richard et al. (Richards et al., 2008) (5. 5 days); Shiravi et al.(Shiravi et al., 2011) (2–3 days at 28°C). In our research, the length of the pupal period for C.albiceps at 25°C and 30°C were determined as 5 and 3. 5 days, respectively. Again, other researchers determined different numbers: Grassberger (Grassberger et al., 2003). 1 and 5. 9 days at 25°C and 30°C, respectively; Al-Shareef and Al-Qurashi, (Al-Shareef & Al-Qurashi, 2016) 5. 5 and 4 days at 25°C and 30°C, respectively; Queiroz, (Queiroz, 1996) 4. 7 and 3 days at 27°C and 32°C respectively and Shiravi (Shiravi et al., 2011) (4–6 days at 28°C). In addition, in our study, the length of egg to adult eclosion, at 25°C and 30°C were determined as 12 and 8–8. 5 days, respectively. Other researchers got the followings: Grassberger (Grassberger et al., 2003) 13 and 9. 9 days at 25°C and 30°C; Velez and Wolff (Augul, 2009) (14 days at 25°C); Al-Shareef and Al-Qurashi, (Al-Shareef & Al-Qurashi, 2016) 11. 5 and 9 days at 25°C and 30°C respectively; Shiravi et al. (Shiravi et al., 2011) (8–12 days at 28°C); and Queiroz(Queiroz, 1996) (7 days at 32°C). The figures calculated for the life cycle of the speceis C.albiceps were more similar to the ones obtained in Al-shareef (Al-Shareef & Al-Qurashi, 2016) and Shiravi (Shiravi et al., 2011) studies and were different from others. The reason seems to be natural. As noted in various studies, inherent biogeographically variation between population (Marchenko, 1989; Donovan et al., 2006; Szpilla, 2010a; Greenberg & Kunich, 2002) food substrate (Greenberg & Kunich, 2002; Kounouz & Kamel, 2017) humidity, population density and other intrinsic factors are considered as the reason of such differences (Faria & Godoy, 2001; Rosa et al., 2006). It seems that histolysis and histogenesis of the adult organs in immature stage were the reason of the highest thermal requirement of ADD in both 25°C and 30°C, during the pupal period, and the length of this development stage has been mentioned in most researches (Al-Shareef & Al-Qurashi, 2016; Queiroz, 1996; Bharti, 2009; Defilippo et al., 2013; Donovan et al., 2006; Richards et al., 2008; Augul, 2009; Velez & Wolff, 2008). Our findings about the life cycle of C.vicina showed that this species will also have shorter life cycle as the temperature increases such a way that at 15°C, its life cycle, from the egg stage to adult, it took 32 days while at 20°C, it took 21 days. For this species, other researchers obtained similar results: Reibe et al. (Reibe et al., 2009) at 13°C and 20°C, 45 and 23 days, respectively; Defilippo et al. (Defilippo et al., 2013) at 15°C and 20°C, 30. 5 and 20 days, respectively; Bharti (Bharti, 2009) at 20°C and 25°C, 19 and 15. 29 days. The results obtained from our study were more near to the ones obtained from the study done by Defilippo et al. (Defilippo et al., 2013). In terms of rate of development in C.vicina and C.albiceps, the results of this study were compatible with the finding of the studies of Greenberg,(Greenberg, 1991) (Lowne 1890), Defillipo et al (Defilippo et al., 2013) Such a way that the length of post-feeding and pupal periods were determined one third and half the total immature development stage, respectively. The amount of ADD was calculated for C.albiceps at 25°C and 30°C respectively, as 179 and 159 which correspond to the results obtained from the study done by Shiravi et al. (Shiravi et al., 2011) the amount of ADD, at 28°C, was calculated between 130 and 195 DD. Moreover, the amount of ADD for the first larvae instar to adult of this species at 25°C and 30°C was calculated as 96°C and 110°C respectively. In the study done by AL-Shareef, (Al-Shareef & Al-Qurashi, 2016) the length of this period, at the same temperature, has been determined as 110 and 129. The life cycle of these species depends on some other factors, such as the geographic differences of population and genetics. Actually, the rate of development in necrophagous insects is the response of organisms to their thermal environment. Determining the rate of development of forensically important insects such as C.albiceps and C.vicina can give us valuable information concerning the circumstance of the death including the postmortem interval.
This study emphasizes the importance of determining of developmental data for these species and other first colonizers on the carcass in each geographical region. The results of this study will be usable to estimate of the PMI in the western of Iran and similar regions.
This work was supported by Tehran University of Medical Sciences, Tehran, Iran project No.34233.
This work was supported by Tehran University of Medical Sciences, Tehran, Iran project No.34233.
Availability of data and materials
They are available.
MS Sampling of insects on carcass and samples breeding. YR Managing of Project and writing of Project and Manuscript. MO Help in writing the project. OC Assistance in data analysis. ML Assistance in field and Lab. SR Assistance in Lab and editing of paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Necessary ethical approval was obtained from School of Public Health, Tehran University of Medical Sciences Ethics Committee, code No.IR.TUMS.SPH.REC.1395.1634.
Consent for publication
All authors have complete consent to print article information.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Al-Mesbah H, Al-Osaimi Z, El-Azazy OME (2011) Forensic entomology in Kuwait: the first case report. Forensic Sci Int 206:25–26View ArticleGoogle Scholar
- Al-Shareef LAH, Al-Qurashi SID (2016) Study of some biological aspects of the blowfly Chrysomya albiceps (Wiedemann 1819) (Diptera: Calliphoridae) in Jeddah, Saudi Arabia. Egyptian Journal of Forensic Sciences 6:11–16View ArticleGoogle Scholar
- Ames C, Turner BD (2003) Low temperature episodes in the development of blowflies: implications for postmortem interval estimation. Med Vet Entomol 17:178–186View ArticlePubMedGoogle Scholar
- Anderson G (2000) Minimum and maximum development rates of some forensically important Calliphoridae (Diptera). J Forensic Sci 45:824–832View ArticlePubMedGoogle Scholar
- Augul RS, Jassim SY. Study of some biological and ecological aspects of the Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae). J Al-Anbar Univ Pure Sci 2009; 3(1)Google Scholar
- Bansode SA, More VR, Zambare SP (2016) Effect of different constant temperature on the life cycle of a fly of forensic importance Lucilia cuprina. Entomol Ornithol Herpetol 5:183. https://doi.org/10.4172/2161-0983.1000183 Google Scholar
- Bharti M (2009) Studies on life cycles of forensically important flies, Calliphora vicina and Musca domestica nebulo at different temperatures. J ent Res 33(3):273–275Google Scholar
- Bharti M, Singh D (2003) Insect faunal succession on decaying rabbit carcasses in Punjab, India. J Forensic Sci 48:1133–1143View ArticlePubMedGoogle Scholar
- Chua TH (2013) Use of temperature model in estimating postmortem interval in forensic entomology. Int J Entomol Res 1:11–15Google Scholar
- Davies L, Ratcliffe GG (1994) Development rates of some pre-adult stages in blowflies with reference to low temperatures. Med Vet Entomol 8:245–254View ArticlePubMedGoogle Scholar
- Defilippo F, Rubini S, Dottori M, Bonilauri P (2013) Effect of temperature on six different developmental landmarks within the Pupal stage of the forensically important blowfly Calliphora vicina (Robineau-Desvoidy). J Forensic Sci. https://doi.org/10.1111/1556-4029.12199. Available online at: onlinelibrary.wiley.com
- Donovan SE, Hall MJR, Turner BD, Moncrieff CB (2006) Larval growth rates of the blowfly, Calliphora vicina, over a range of temperatures. Med Vet Entomol 20:106–114View ArticlePubMedGoogle Scholar
- Erzinclioglu Z (1996) Blowflies. The Richmond Publishing Co, LtdGoogle Scholar
- Faria LDB, Godoy WAC (2001) Prey choice by facultative predator larvae of Chrysomya albiceps (Diptera: Calliphoridae). Mem Inst Oswaldo Cruz, Rio de Janeiro 96(6):875–878View ArticleGoogle Scholar
- Faucherre J, Cherix D, Wyss C (1999) Behavior of Calliphora vicina(Diptera, Calliphoridae) under extreme conditions. Journal of insect behavior 12:687–690View ArticleGoogle Scholar
- Gennard DE (2007) Forensic entomology: an introduction. John Wiley & Sons Ltd, ChichesterGoogle Scholar
- Grassberger M, Friedrich E, Reiter C (2003) The blowfly Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae) as a new indicator in Central Europe. Int J Legal Med 117:75–81PubMedGoogle Scholar
- Grassberger M, Reiter C (2001) Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalenand isomorphen-diagram. Forensic Sci Int 120(1–2):32–36View ArticlePubMedGoogle Scholar
- Greenberg B (1991) Flies as forensic indicators. J Med Entomol 28(5):565–577View ArticlePubMedGoogle Scholar
- Greenberg B, Kunich JC (2002) Entomology and the law: flies as forensic indicators. Cambridge University Press, Cambridge, United KingdomGoogle Scholar
- Haskell NH, Lord WD, Byrd JF. Collection of entomological evidence during death investigations. Forensic Entomology: The Utility of Arthropods in Legal Investigations (ed. by J. H. Byrd and J. L. Castner), Florida. CRC Press; 2000. pp. 81–120Google Scholar
- Kamal AS (1958) Comparative study of thirteen species of sarcosaprophagous Calliphoridae and Sarcophagidae (Diptera) I. Bionomics. Ann Entomol Soc Am 51:261–271View ArticleGoogle Scholar
- Kounouz SH, Kamel L (2017) Development of Calliphora vicina (Robineau- Desvoid) (Diptera: Calliphoridae) under different biotic and abiotic conditions. Journal of Entomology and Zoology Studies 5(1):683–691Google Scholar
- Kumara TK, Abu Hassan A, Bhupinder S (2017) Forensic Entomology-Insect Development and Temperature. Journal of Forensic Sciences & Criminal Investigation 4(2):555636. https://doi.org/10.19080/JFSCI.2017.04.555636 Google Scholar
- Lowne B (1890) The anatomy, physiology, morphology and development of the blowfly. RH Porter, LondonGoogle Scholar
- Marchenko MI (1989) Method of retrospective determination of insect development onset in a cadaver. Sud Med Ekspert 32(1):17–20PubMedGoogle Scholar
- Oshaghi MA, Maleki-Ravasan N, Javadian E, Rassi Y, Sadraei J, Enayati AA et al (2009) Application of predictive degree day model for field development of sand fly vectors of visceral leishmaniasis in northwest of Iran. J Vector Borne Dis 46(4):247–254PubMedGoogle Scholar
- Queiroz MMC (1996) Temperature requirement of Chrysomya albiceps (Wiedemann, 1819) (Diptera: Calliphoridae) under laboratory conditions. Mem Inst Oswaldo Cruz 91(6):785–788View ArticlePubMedGoogle Scholar
- Reibe S, Schmitz J, Madea B (2009) Molecular identification of forensically important blowfly species (Diptera: Calliphoridae) from Germany. Parasitol Res 106(1):257–261View ArticlePubMedGoogle Scholar
- Richards CS, Paterson ID, Villet MH (2008) Estimating the age of immature Chrysomya albiceps (Diptera: Calliphoridae), correcting for temperature and geographical latitude. Int J Legal Med 122:271–279View ArticlePubMedGoogle Scholar
- Rosa GS, Carvalho LRD, Reis SFD, Godoy WAC (2006) The dynamics of Intraguild predation in Chrysomya albiceps Wied. (Diptera: Calliphoridae): interactions between instars and species under different abundances of food. Neotropical Entomology 35(6):775–780View ArticlePubMedGoogle Scholar
- Shiravi AH, Mostafavi R, Akbarzadeh K, Oshaghi MA (2011) Temperature requirements of some common forensically important blow and flesh flies (Diptera) under laboratory conditions. Iran J Arthropoda Borne Dis 5(1):54–62Google Scholar
- Smith KGVA (1986) Manual of forensic entomology. London. In: UK: the British museum (natural history) and Cornell University pressGoogle Scholar
- Szpila K, Matuszewski S, Bajerlein D, Konwerski S (2008) Chrysomya albiceps (WIEDEMANN, 1819), a forensically important blowfly (Diptera: Calliphoridae) new for the polish fauna. Polish Journal of Entomology 77:351–355Google Scholar
- Szpilla K. Key for identification of European and Mediterranean blowflies (Diptera, Calliphoridae) of forensic importance Adult flies 2010aGoogle Scholar
- Szpilla K. Key for identification of European and Mediterranean blowflies (Diptera, Calliphoridae) of forensic importance third instars 2010bGoogle Scholar
- Tabor KL, Brewster CC, Fell RD (2004) Analysis of the successional patterns of insects on carrion in Southwest Virginia. J Med Entomol 41:785–795View ArticlePubMedGoogle Scholar
- Velez CM, Wolff M (2008) Rearing five species of dipteral (Calliphoridae) of forensic importance in Colombia in semi controlled field conditions. Papeis Avuloso de Zoologia (Sao Paulo) 48(6):41–47Google Scholar
- Vinogradova EB, Reznik SY (2015) Influence of constant and changing temperatures on the larval development of Calliphora vicina (Diptera: Calliphoridae). Acta Soc Zool Bohem 79:149–154Google Scholar
- Williams H (1984) A model for the aging of fly larvae in forensic entomology. Forensic Sci Int 25:191–199View ArticlePubMedGoogle Scholar