Study on the life history and protein content of Sarcophaga ruficornis (Diptera: Sarcophagidiae) a forensically important insect

Transcription

1 2016; 3(6): The Journal of Zoology Studies ISSN JOZS 2016; 3(6): JOZS 2016 Received: Accepted: Kamal Adhikari Post Graduate Student, Bulbuli Khanikor Assistant Professor, Riju Sarma Research scholar, Sudarshana Mahanta Research scholar, Jatin Kalita Professor, Corresponding Author: Bulbuli Khanikor Assistant Professor, Study on the life history and protein content of Sarcophaga ruficornis (Diptera: Sarcophagidiae) a forensically important insect Authors: Kamal Adhikari, Bulbuli Khanikor, Riju Sarma, Sudarshana Mahanta, Jatin Kalita Abstract Since past few decades insects have been serving as an important tool in forensic entomology i.e. in determining the time elapsed since death. The present investigation aims at studying one of the primary colonizer of carcass namely Sarcophaga ruficornis (Diptera: Sarcophagidiae) in Guwahati, Assam. The growth and development of S. ruficornis like other insects depends strictly on the climatic conditions prevailing in the area and level of exposure of the corpse. For laboratory culture of the flesh fly S. ruficornis, chick liver was taken as bait. The total protein content of different developmental stages of S. ruficornis and the fresh as well as rotten chick liver was determined by using the method of Lowry et al. The developmental time of S. ruficornis was found as 25±3 days during the investigation period (23±1 days during May and 28±2 days during the month of February). The protein content of the liver was found to decrease during its decomposition stage and the protein content of the developing stages of the fly was found to increase linearly. Keywords: Carcass, Sarcophaga ruficornis, Protein, Diptera: Sarcophagidiae, Insect 1. Introduction Arthropods are among the most evolved groups of animals on earth. So they are found almost everywhere on earth. Insects like other arthropods play a crucial role in different fields of modern science like forensic entomology. Knowledge of the distribution, biology and behavior of insects found at a crime scene can provide information on when, where and how the crime was committed (Kashyap and Pillai, 1989 [1] ; Anderson and Carvenka, 2001 [2] ; Hall, 2008 [3] ). Concepts of algor mortis, rigor mortis, and livor mortis play an important role during the first few hours of death and hardly can be determined up to 3 days by these methods. However, all of these parameters are affected by many other factors such as body size, age, illness, exertion period to death etc. and become less valuable as time passes (Simpson and Knight 1985 [4] ; Henssge et al, 1995 [5] ). Insects are never affected by all these parameters so they play a major role in determining the post mortem interval, among which flies are of primary significance. These insects feed on the corpse, oviposit and the larvae hatches into successive instars and finally emerges as an adult. Blow flies and flesh flies are among the first colonizer (Luna et al., 2001 [6] ; Bharti and Singh, 2003 [7] ). The study of their first mature maggots can provide the data of time elapsed since death. Attraction of the arthropod species varies according to the decomposition state of the corpse (such as fresh, bloat, decay, putrefaction, mummification, and skelotization). A particular species never stay in the corpse during the whole process of decomposition (Bornemissza, 1957 [8] ; Braack, 1981 [9] ). There is a succession of species of arthropods. Each species stay only for a limited period of time (Anderson, 2009 [10] ). Page 1

2 In the present study a forensically important species, Sarcophaga ruficornis (Diptera: Sarcophagidae) was chosen to study the life cycle by keeping them in a close culture chamber and provided chick liver as bait within the chamber. Generally S. ruficornis is found abundant in carcass in the early stage of decomposition. It is generally not found after 3-4 days. It is a medium sized to large sized fly. Front broad in the female somewhat narrower in the male. Distal portion of the arista is bare. Abdomen consists of 4 visible segments. External genitalia in male are prominent. The puparium is reddish brown and ovoid in shape. Mode of reproduction of Sarcophaga is ovoviviparous, i.e. they lay first instar maggot on the flesh.(sukontason et al.,2014 [11] ). The purpose of studying the life cycle of a single species was to get a more accurate and a firm report of the life cycle. All the life cycle of the necrophagous flies is more or less similar with the life cycle of Sarcophaga ruficornis. 2. Methodology 2.1. Obtaining the specimen Sarcophaga ruficornis was collected by exposing a bait of broiler. Flies were attracted to the bait. They oviposit in the bait and the maggots grow into successive instars and finally reached the pupal stage. Pupae at this stage were collected in an insect proof container and waited till they hatch.. After few days of adult fly emergence, few other species of the family Muscidae, Calliphoridae and Sarcophagidae that also emerged simultaneously in the cage such as were removed. Thus, a pure culture of Sarcophaga ruficornis was achieved and maintained solely by providing chick liver as bait. The species was identified as Sarcophaga ruficornis by the experts from Zoological Survey of India, Kolkata. In the present study it was found that the species of Sarcophaga were more abundant in the late winter than the species of other forensically important insects. So their collection was easier. Fig 1: Broiler exposed for decomposition showing infestation and larviposition by flies Culture of S. ruficornis: The first generation of the adults that emerged was collected by allowing a bait for larviposition as mentioned earlier. These adults were again provided with the chick liver as bait to continue their life cycle. The culture chamber was made of a glass container of which the upper open portion was covered with a mosquito net. Sufficient dry mud with sand was provided at the bottom of the chamber so that the pre pupa does not move to and fro in search of the suitable place for pupation. Fig 2: Culture chamber Page 2

3 Here it was found that the fly life cycle was shorter in wild than in culture in the laboratory. Due to its small size the broiler was found to skeletonize after 8 days of exposure period. After skeletonization, normal visitors of the corpse disappeared. Fig 3: 2 nd instar maggot of Sarcophagaruficornis growing in culture. In this way, four such generations were maintained. During the culture period no other insects were allowed to mix with the culture. During the experimental period broiler was also exposed separately in the open condition and its decomposition along with the infestation by different flies were observed. The broiler was kept inside a wire gauze of considerable perforation so as to prevent the attack of other predators in open condition. 2.3 Estimation of Protein Estimation of protein content was done following the method of Lowry et al., (1951) [12]. The total protein of 1 st instar larvae, 3 rd instar larvae, pupae and adults of the fly were taken as the sample. Protein of fresh and decomposing liver on 5 th day of decomposition were also estimated following the same method Statistical Analysis The Tukey test of the protein content of the meat sample and different instars of S. ruficornis were done with the help of SPSS (Version 16) software. 3. Results and Discussion Fig 4: Life cycle of Sarcophaga ruficornis Page 3

4 Table 1: Duration of different developmental stages of Sarcophaga ruficornis in different temperature. S. no Time Average Developmental stages Total Temperature 1 st instar 2nd 3rd Pre- Pupa Adult lengh of ( 0 C) (Days) instar instar pupal (Days) (Days) Life (Days) (Days) stage cycle(da (Days) ys) 1 February 28±1 3±1 2±1 2±2 3±2 13±1 5±1 28±2 2 March 30±1.33 2±1 2±1 2±1 3±2 12±2 5±2 26±2 3 April 31±2 2±1 2±1 2±1 2±1 11±1 5±1 24±1 4 May 32±2 2±1 2±1 2±1 1±1 11±1 5±1 23±1 As shown in the table (Table1) above the developmental period of the fly was found to strictly dependent on the temperature and level of exposure of the corpse. In the month of April and May when the temperature was higher the developmental of the fly was found to accelerate. Whereas in the month of February when the temperature was lower the flies took longer time to develop. This result was in conformity with the findings of earlier researchers (Byrd and Butler, 1997 [13] ; Wells and Kurahashi, 1994 [14] ; Boatright et al, [15] ). Flies oviposit only when the carcass is fresh (Archer, 2003 [16],[17] ; Hall et al., 1993 [18] ). This has been found true in the present study. But in the favorable season, in case of the small carcass the fly infestation was recorded maximum and therefore the oviposition rate was also found maximum. The huge number of the larvae was found to feed on the carcass voraciously and skeletonize it within a very short period of time. Moreover, the oviposition time was observed upto early bloated period. Flies were observed to visit the carcass till later part of the early bloated stage but they were not found to deposit maggot on it. The flies were rarely seen in the late decay phase and almost never seen on the dry phase. During the investigation period, the culture of the flesh fly Sarcophaga ruficornis was almost successfully completed. The adult fly lived for 3-7 days during which it deposit maggot in the bait until it was fresh. After 3-4 days the flies were resting on the wall of the culture chamber and avoided the bait. Life cycle depended on the temperature and humidity. It was observed that the life cycle which was studied in February was longer than the life cycle that was studied in May. Likewise with the advent of summer life cycle shortened. The average life cycle in culture was found to be 25±3. It was also found that the fly grown in culture had a longer life cycle than the fly growing in the wild. 3.1 Protein Estimation Proteins of different stages of S. ruficornis were determined by following the method described by Lowry et al., (1951) [11]. Table 2: Showing the protein content of developmental stages Sl No. Sample Protein(mg/ml tissue ±SE) 1 Fresh Liver 9.8± Rotten Liver 8.4± st instar 7.0± Last instar 11.84± Pupa 20.3± Adult 17.22±0.08 Page 4

5 Fig 5: Bar diagram showing the relationship between standard protein and optical density Fig 6: Bar diagram showing the protein content of different developmental stages Fig 7: Bar diagram showing the gradual decrease of protein content during decomposition and the increase of the protein content in the developing stages of Sarcophaga ruficorins Page 5

6 Table 3: Result of Tukey test for the protein content Serial No I group J group 95% confidence intervel Mean difference(i- Significance J) Lower Upper bound bound rotten liver * st instar * Fresh liver 2 Rotten liver 3 1 st instar 4 Last instar 5 Pupa 6 Adult * The mean difference is significant at the 0.05 level. Last instar * pupa * adult * fresh liver * st instar * Last instar * pupa * adult * fresh liver * rotten liver * Last instar * pupa * adult * fresh liver * rotten liver * st instar * pupa * adult * fresh liver * rotten liver * st instar * Last instar * adult * fresh liver * rotten liver * st instar * Last instar * pupa * From result of Tukey test (table 3) for the protein content of fresh and rotten liver along with the different developmental stages of S. ruficornis, the values were found significantly different from each other. The amount of protein content in these flies was found to totally dependent on the protein content of the carcasses. We know that liver contains relatively more proteins than other macromolecules (Guinez et al., 2011 [19] ) Page 6

7 The protein content of insects depends on the metamorphosis stage; adults usually have higher protein content than other instars (Ademolu et. al. 2007) [20]. But in the present study the result did not match exactly with the findings of Ademolu et al. (2007) [20] From the experiment it was found that the pupal stage contained the highest amount of protein among all the developmental stages. Sarcophaga ruficornis has an ovoviviparous mode of reproduction, in which eggs hatch into first instar maggot in the female reproductive system and the 1 st instar maggots are deposited in the carcass. Generally S. ruficornis adults feed on the sap of the flesh (as the fly is in the culture chamber). The carcass in the summer dries up very fast. So, possibly it may happen that the carcass was not in the state in which the flesh fly feed (Byrd and Castner, 2001) [21]. First instar maggot after being laid on carcass feed voraciously and attained successive instars. During this feeding period they store sufficient nutrients for the adult stage. From the experiment it was found that the total protein contents accumulate in the larvae gradually and reach its peak during the pupal stage (Fig.6 and Table.2). When a graph was plotted to show the total protein content of different developmental stages of S. ruficornis it was found that the total protein content rose linearly till the pupal stage and again fell to some extent in the adult stage. From this finding, it can be inferred that the quantity of protein that was lost in adult was utilized during the pupal to adult transformation process. An attempt was also made to correlate the protein content in the fresh liver where the first instar maggots were usually thrive and rotten liver where the last instar maggots were usually found. From the experiment it was found that protein content of the liver degraded gradually during decomposition process and the protein content of the developing stages of the flies increased gradually during development period. The total protein content of pupa was found to be 20.3±0.25, which was relatively higher in comparison to the other developmental stages (Fig.6 & Fig.7). From these findings it can be inferred that the protein content of different developmental stages of the flies was dependent on the protein profile of the carcass. 4. Conclusion Forensic entomology is evolving as an inevitable branch of forensic studies. The insects that colonize the carcass serve as clock for the estimation of post mortem interval. Here in the investigation life cycle of a forensically important insect Sarcophaga ruficornis was studied. To quantify the amount of protein present in the carcass and the growing larva, protein of various developmental stages and the larvae were estimated using the method of Lowry et al., (1951). The results of the investigation reveal that the time required for the development of a fly varies within a narrow range of 25±3 during the study period. Protein content of the developing stage increased linearly till pupa and in the adult it slightly decreased. The result signifies that heavy infestation of the fly of interest was abundant in the earlier time, i.e. when the carcass was fresh. Gradually adult disappeared and maggots began to grow. The most important tool here is the growing larvae through which the time of death can be estimated. The developmental stages of the fly did not vary much during the time period. The gradual increase in the protein content of the developmental stages till pupal stage and slight reduction on adult stage was a clear indication that the adults feed on the sap of the flesh (as the fly was in the culture chamber). The carcass in the summer dries up very fast. So, possibly it might happen that the carcass was not in the state in which the flesh fly feed. Further, it is important in poultry farm to feed the chick with a proteinaceous diet, so the last instar or the pupal stage can be recommended as a protein rich source. 5. Acknowledgement The authors are very much grateful to the UGC for their financial assistance and the head of the Department of Zoology Prof. Dr Jatin Kalita and Prof. R. K. Bhola for their guidance and help. The authors also express their heartfelt gratitude to ZSI, Kolkatta for their help in identification of the specimens. 6. References 1. Kashyap VK, Pillai VV. Efficacy of entomological method in estimation of postmortem interval: A comparative analysis. Forensic Science International. 1989; 40: Anderson GS, Cervanka VJ, Insects associated with body; their use and analysis, in advances in forensic Taphonomy method. Theory of archeological perspective, Haglund, W D and Sorg M. eds CRC press Boca Raton FL. 2002; Pp Hall. The use of forensic entomology in criminal investigations: how it can be of benefit to SIOs. The journal of homicide and major incident investigation Simpson K, Knight B. Forensic medicine, 9 th edition, Edward Arnold, London Henssge C, Knight B, Krompecher T, Madea B, Nokes L. In: Knight B (ed) The estimation Page 7

8 of the time since death in the early postmortem period. Arnold, London Luna et.al. An initial study on the succession of sarcosaprophagous Diptera (Insecta) on carrion in the southeastern Iberian Peninsula. International Journal of Legal Medicine Bharti M, Singh D. Insect faunal succession on decaying rabbit carcasses in Punjab, Journal of Forensic Sciences. 2003; 48: Bornemissza GF. An analysis of arthropod succession in carrion and the effect of its decomposition on the soil fauna. Australian Journal of Zoology. 1957; 5: Braack LEO. Visitation patterns of principal species of the insect complex at carcasses in the Kruger National Park. Koedoe. 1981; 24: Anderson GS. Insect Succession on carrion and its relationship to determining time since death. IN Forensic Entomology: The utility of arthropods in legal investigations. Castner, E. and Byrd, J. CRC TomberlinJ K., Sukontason K. Sarcophaga (Lisosarcophaga) dux (dipteral: Sarcophagidae). A flesh fly of medical importance. Journal of Biological Research Lowry OH. Rosenborough NJ, Farr AL, Rindall RJ. Protein measurement with the Folin Phenol Reagent, J Biol Chem. 1951; 193: Byrd JH, Butler JF. Effects of Temperature on Chrysomya rufifacies (Diptera: Calliphoridae) Development. Journal of medical entomology Wells JD, Kurahashi H. Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) development: rate, variation and the implications for forensic entomology. Jpn J Sanit Zool. 1994; 45(4): Boatright SA, Tomberlin JK. Effect of temperature and tissue type on the development of Cochliomyia macellaria (Diptera: Calliphoridae). J Med Entomol. 2010; 47(5): Archer MS, Elgar MA. Effects of decomposition on carcass attendance in a guild of carrion-breeding flies. Medical and Veterinary Entomology. 2003a; 17: Archer MS, MA Elgar. Yearly activity patterns in southern Victoria (Australia) of seasonally carrion insects. Forensic Science International. 2003b; 132: Hall RD, Doisy KE. Length of time after death: effect on attraction and Oviposition or larviposition of midsummer blowflies (Diptera: Calliphoridae) and Flesh Flies (Diptera: Sarcophagidae) of Medicolegal Importance in Missouri. Annals of the Entomological Society of America. 1993; 86: Guinez C, Filhoulaud G, Benhamed FR, Marmier S, Dubuquoy C, Dentin R, et. al. O- GlcNAcylation Increases ChREBP Protein Content and Transcriptional Activity in the Liver. American Diabetes Association (5): Ademolu KO, Idowu AB, Amusan AAS. Chemical analysis of tissues of Zonocerus variegatus (1) (Orthoptera: Pyrgomorphidae) duringpost embryonic development in Abeokuta, southwestern Nigeria. Nigerian J. Entomol. 2007; 24: Jason HB, James LC. Forensic entomology; The utility of arthropods in forensic investigation. 2 nd edition. CRC Press, Adhikari K, Khanikor B, Sarma R, Mahanta S, Kalita J. Study on the life history and protein content of Sarcophaga ruficornis (Diptera: Sarcophagidiae) a forensically important insect. Journal of Zoology Studies. 2016; 3(6): ************************************************************ Page 8