Developmental variation of the blow fly Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) by different substrate tissue types

    Developmental variation of the blow fly Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) by different substrate tissue types Zeinab A. El-Moaty, Abd Elmoneim M. Kheirallah PII: DOI: Reference:

S1226-8615(13)00044-7 doi: 10.1016/j.aspen.2013.03.008 ASPEN 416

To appear in:

Journal of Asia-Pacific Entomology

Received date: Revised date: Accepted date:

22 October 2011 23 February 2013 21 March 2013

Please cite this article as: El-Moaty, Zeinab A., Kheirallah, Abd Elmoneim M., Developmental variation of the blow fly Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) by different substrate tissue types, Journal of Asia-Pacific Entomology (2013), doi: 10.1016/j.aspen.2013.03.008

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Developmental variation of the blow fly Lucilia sericata (Meigen,

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1826) (Diptera: Calliphoridae) by different substrate tissue types.

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Zeinab A. El-Moaty*, Abd Elmoneim M. Kheirallah

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*Zoology Department, Faculty of Science, Alexandria University, Alexandria, Egypt. Corresponding author: [email protected]

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Key words: Calliphorid larvae, development, Forensic Entomology, postmortem interval,

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tissue types.

Abstract

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The green bottle blow fly Lucilia sericata is recognized as being among the first wave of the faunal succession on human cadavers. Thus, it is used to estimate the postmortem interval

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(PMI). The nutritional intake of larvae is likely to vary subject to the part of a corpse on which they are feeding. A study was therefore conducted to investigate the effect for the type

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of food substrate on larval growth in this species. Larvae were reared on cow liver, brain, heart, lung, kidney, intestine or minced meat. Results showed significant differences in the duration of the feeding, postfeeding larval, and pupal stages from different substratum (F=

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42.43; 74.71 and 655.71; P<0.0001, respectively). Larval growth measured(Ed. note: Unclear. Please confirm) as length varied significantly between different tissues (F=3.56; P<0.05). Larvae that were reared on heart were smaller than those reared on other tissues. Also, there was a significant effect for the type of tissue on the adult size of males (F= 2.41, P<0.05) and females (F= 2.85, P<0.05). These results may have important implications for forensic entomologists, since initial infestations commonly occur in wounds or in the cranial area. Therefore, the position at which larvae have been feeding on a body will be a crucial observation at a crime scene and is very important in PMI estimation.

Introduction Blow flies (Diptera: Calliphoridae) are recognized as being among the first wave of the faunal succession on human cadavers (Smith, 1986; Nuorteva, 1977). Therefore, their life

ACCEPTED MANUSCRIPT 2 cycles and development are used in criminal investigations to estimate the postmortem interval (PMI), the time between death, and discovery of a corpse (Nuorteva, 1977;

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Anderson, 2000).

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The green bottle blow fly Lucilia sericata is widespread throughout the major

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zoogeographical regions (Greenberg and Povolny, 1971; Smith, 1986; Spradbery, 1991). This species is an economically important ectoparasite of domestic sheep in many temperate parts

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of the world (Hall and Wall, 1995). L. sericata is considered to be a poor interspecific competitor relative to the other abundant species (Cragg, 1955; Macleod and Donnelly, 1962;

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Smith and Wall, 1997a) breed successfully in carrion and minimize the effect of interspecific competition(Ed. note: Unclear. Please review and confirm). L. sericata must act as a pioneer

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species (Denno and Cothran, 1975; Easton and Feir, 1991; Fisher et al., 1998). Therefore, this

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species is the primary and most accurate forensic indicator of time of death (Nuorteva, 1977).

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One approach for PMI estimation involves killing the larvae collected from a crime scene and comparing the measured length with reference data derived from rearing larvae in the

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laboratory where they are most commonly fed on the liver (Williams and Richardson, 1984; Anderson, 2000; Grassberger and Reiter, 2001; Grassberger et al., 2003) or ground beef (Wells and LaMotte, 1995; Greenberg and Tantawi, 1993) of various mammalian species. However, on a corpse, a range of tissues are available as food sources for colonizing blow fly larvae. Maggot invasion of a corpse often occurs through the eyes and into the skull cavity (Greenberg and Kunich, 2002). Hence, for a large part of their development, such larvae are feeding on brain. Therefore, it is possible that the organ, on which the larvae feed on a corpse, might significantly alter the rate of growth resulting in an underestimation of the PMI. A study by Kaneshrajah and Turner (2004) on Calliphora vicina found that an error of up to 2

ACCEPTED MANUSCRIPT 3 days might be expected in a postmortem interval estimate using larvae grown on pig’s liver compared with brain, heart, kidney, and lung. Moreover, the development of larvae of

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Calliphora augur and Lucilia cuprina fed sheep’s liver was adversely affected compared with

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larvae fed meat and brain. They moulted later, reached maximum length more slowly and

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sometimes produced significantly smaller pupae (Day and Wallman, 2006). While larvae of Lucilia sericata grew significantly faster, wandered from the food source 31 h earlier when reared on lung compared to liver and gave rise to larger adults when reared on lung and heart

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compared to liver(Unclear. Is something missing? Please confirm) (Clark et al., 2006).

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In a recent study, the developmental rate of Chrysomya albiceps reared on bovine liver and chicken heart did not change. However, larvae reared on liver and raw muscles presented

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a lower adult emergence rate. While(Do you mean, ‘rate, while’? Please confirm) the stomach

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showed similarities with the control group (meat) in the efficiency parameters; rate and

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emergence interval (Estrada et al., 2009). Also, the larval growth and weight of Cochliomyia macellaria did not differ between larvae reared on equine versus porcine muscle (Boatright and Tomberlin, 2010). This work was done to determine growth rates and developmental

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characteristics for L. sericata reared on different tissues.

2. Materials and Methods Laboratory colonies of L. sericata used in this study were originated from third feeding instars collected from exposed rabbit carcasses at the botanical garden of Faculty of Science, Moharrem Bey District, Alexandria, Egypt. Adult flies were maintained in rearing cages (50cm3) at room temperature (25-29ºC). Each cage of insects was given water, sugar and powdered milk. Three days after emergence, adult flies were provided with beef liver daily as a protein source to promote the ovarian development of females. It should be mentioned that

ACCEPTED MANUSCRIPT 4 the experiments were initiated from F2-F4 individuals to reduce selection and inbreeding effects.

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When eggs were required, a dish containing approximately 100g of fresh beef liver was

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placed in the cage and removed once sufficient eggs had been laid. Newly deposited eggs

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were placed on water-moistened filter paper in Petri dishes and inspected every hour until hatching was initiated.

Within one hour, twenty newly hatched first instar larvae were transferred into 100 ml jars

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each containing 50g of cow liver, brain, heart, lung, kidney, intestine or minced meat. Each

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jar was placed in a larger high sided beaker (500 ml). These beakers contained a 2 cm deep layer of dry sawdust on the bottom and were covered with a fine, nylon mesh, held in place with elastic bands. Experimental jars were kept at a constant temperature of 25±2°C, relative

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humidity 60±10% and photoperiod 12h:12h. Treatments were replicated ten times; at least three replicates for each tissue were left undisturbed until pupation to obtain valid mortality

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data.

The larvae were sampled throughout their development at different time-intervals.

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Samples of larvae were removed from the medium, rinsed in water and then killed by immersion in boiling water for 30 s (Adams and Hall, 2003). The length of larvae from each medium was measured to the nearest 0.1 mm using an eye piece micrometer on a light microscope. Approximately 100 gm of different types of fresh substrate tissue and ten active feeding third instar larvae were selected and frozen till determination of total protein and total lipid contents were performed. As larvae finished feeding and reached the wandering phase, they left the food and migrated out of the jar to the sawdust in the beaker from which they were unable to escape and where they remained until they pupariated. The time taken for larvae to wander was recorded.

ACCEPTED MANUSCRIPT 5 Once all larvae pupated, the jar containing any remaining food was removed from the larger beaker. The first adult eclosion was noted for each tissue and after a minimum of seven

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days had elapsed, all flies were killed by freezing. All adults from the undisturbed

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experimental jar for each tissue were counted and sorted by sex. The size of adults was then

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determined by measuring the length of the posterior cross vein (dm-cu) on the left wing. Indication of the adult size by the measurement of the posterior cross vein is well-documented by Saunders and Bee (1995), Smith and Wall (1997) and Clark et al. (2006). The left wing of

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each adult was removed and affixed to a white paper using clear nail polish. The paper was

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then oven dried at 40-50oC for a minimum of 24 h (Ireland & Turner, 2006). The wings were scanned as digital images using the Microtek Stylus Colour 5800 scanner, adopting the settings of 300dpi resolution and 400% magnification suggested by Hwang (2004). The

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length of the cross vein was measured using the computer software "ImageJ v.1.36b", obtained via the internet (Rasband, 2002). As suggested by Hwang (2004), the program was

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calibrated to 47 pixels=1mm.

Statistical analysis

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The effects of various tissues on the growth and development of L. sericata were subjected to a one-way analysis of variance (ANOVA) (Sokal and Rohlf, 1981). The LSD test at a level of significance of P = 0.05 was used to determine the separation and significance of means. The data for percent emergence were normalized by arcsine square root transformation before analysis. All statistical analyses were performed with the software SPSS (Norušis, 2005).

3. Results 3.1. Growth rate.

ACCEPTED MANUSCRIPT 6 The larval lengths of L. sericata reared on different tissues are presented in Fig. 1. Larvae were significantly larger, being 1.5 mm larger on average when reared on brain and

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lung than when reared on other tissues (F=42.31 ; P<0.000 ). Larvae were smaller by 3 mm

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when reared on intestine compared to brain or lung (Fig. 1). The post hoc test for the

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difference between pairs of substrates showed that there is no significant difference between the maximum length for larvae reared on brain or lung. Also, the maximum length did not differ significantly between larvae reared on kidney, heart or meat.

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The lipid and protein contents (mg /g) in various tissues and in the third feeding larvae of

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L. sericata are presented in Table 1. Analysis of tissue substrates and the third feeding instar larvae demonstrated that the highest lipid and protein contents in tissues were found in brain tissue, whereas meat and intestine contain the lowest lipid and protein content, respectively.

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On the other hand, the third feeding instar larvae reared on liver and brain showed the highest lipid and protein content respectively while those reared on lung and intestine contain the

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lowest content of lipid and protein, respectively.

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3.2. Development rate

The time taken by each stage to complete its development at different types of tissue is presented in Fig. 2. The time taken by the feeding larvae to reach the wandering stage is significantly differed among different substratum (F= 582.86; P<0.0001). The fastest larval development was recorded for larvae reared on brain and lung (70 h) whereas the longest duration recorded for larvae was reared on intestine (106 h). The post hoc test showed that there is no significant difference between the larval development on brain or lung as well as between larvae reared on kidney, heart or meat. Significant differences in the duration of the postfeeding larval and pupal stages from different tissues (F=101.43 and 65.00; P<0.0001, respectively) were recorded. The post hoc

ACCEPTED MANUSCRIPT 7 test showed that there is no significant difference between the duration of pupariation for larvae reared on brain or lung, and between pupariation on liver or meat as well as between

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kidney, heart or intestine. Furthermore, adult eclosion for larvae reared on brain occurred 10 h

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between different substrates (F=1460.86; P<0.0001).

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earlier than intestine (130 and 140 h, respectively). The total development varied significantly

3.3. Survivorship and adult size

Survivorship of L. sericata adults, which was determined as the percentage number of

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individuals reaching adulthood, was higher when larvae were reared on brain (85%) compared

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with those reared on minced meat (60%) (Fig.3). Statistical analysis (ANOVA) shows a significant difference in the adult survival percentages among different tissues (F= 267.86; P< 0.0001).

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Fig. 4 showed the variations in the adult size of both males and females, when larvae were reared on different types of tissues. There was a significant effect for the type of tissue on the

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adult size of males (F= 8.24; P<0.001) and females (F= 23.14; P< 0.000). Adult flies of both

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sexes were smaller when reared on intestine compared to lung or brain.

4. Discussion

The analysis of the insect colonies of corpses can be a valuable forensic tool in the determination of PMI (Keh, 1985). Many variables that influence the rate of development for insects are not usually incorporated into the commonly-applied models for estimating PMI, largely because of the paucity of relevant data (Villet et al., 2010). However, the current models which are used to age blow fly larvae are produced by extrapolation from analysis of the development rates of larvae, usually reared on a single medium; liver (Levot et al., 1979; Byrd and Butler, 1996) or ground beef ( Anderson, 2000; Grassberg et al., 2003).

ACCEPTED MANUSCRIPT 8 In this study, larvae grew on brain faster and became larger compared with liver and meat. These results are consistent with those obtained by Day and Wallman (2006) who found

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significantly smaller pupae from larvae grown on liver twice with L. cuprina but only once

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with C. augur. However, in a study conducted by Kaneshrajah and Turner (2004), it was

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observed that larvae of C. vicina grown on brain and heart showed a marked wet weight loss during the postfeeding stage leading to pupae of reduced weight and size. Investigations of Lucilia larvae feeding on meat have suggested that the main factors

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involved in the breakdown of the meat are mechanical maceration, an alkaline reaction

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resulting from bacterial action (Hobson 1932a, b, c) and proteolytic enzymes present in larval excreta (Mackerras and Freney, 1933). Therefore, differences in growth rate are contributing to the amount of soluble protein in tissue and the activity of proteolyic enzymes of larvae.

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Larvae of Calliphora are able to build fat from proteins and accumulate it in the fat bodies to be freely spent during metamorphosis (Uvarov, 1928). Generally, fatty substances are used

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and stored for energy. It follows that larvae consuming more fat will be expending less energy on metabolism than those feeding on substances which are purely proteinaceous. These larvae

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can thus direct more energy into growth (Uvarov, 1928). In this study, a comparison of nutrients in the tissues indicated that liver has less total lipid content than brain. Thus, larvae reared on brain consumed more lipids and metabolize it to increase in size. Liver acts as a filter and accumulates(Ed. note: Unclear. Is something missing? Please review and confirm), metabolizes toxic and other foreign substances. It also serves as a store for vitamin A. Liver differs structurally from the other tissues in that it is denser and contains less fat and more connective tissue. It is likely that a larger output of energy is necessary to liberate the nutrients in liver for feeding larvae and that upon consumption of nutrients some toxins may also be ingested. These reflect on the inability of larvae to consume more tissue and convert it into biomass.

ACCEPTED MANUSCRIPT 9 In the present study, the larval development is faster in brain and lung as larvae wandered 24 h earlier than those reared on minced meat. Studies by Kaneshraja and Turner (2004) on C.

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vicina , Clark et al. (2006) on L. sericata and Estrada et al. (2009) on Ch. albiceps suggested

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that larval development varied considerably with different tissues and that development was

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poorest on liver compared to other organs.

Analysis of data shows that the effect of food type is significant over the whole development time where adult eclosion was earlier in the brain than those of other tissues.

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This might be attributed to the softer consistency and slightly higher water content of these

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tissues that appear to promote rapid consumption and fast development compared to other studied tissues.

Differences in the adult survivorship reared on alternate food resources significantly

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reflected the effect regarding the nature of food on the ability of larvae to complete their development. Ireland and Turner (2006) found significant differences in the adult

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survivorship between brain and liver or muscles. In conclusion, larvae of L. sericata grown on liver were often smaller than those grown on

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brain and lung. They also molted later reaching their maximum length more slowly. The present results suggest that there may be limitations in the forensic application of data that derive from a type of animal tissue other than that on which larvae at a death scene have been feeding; the initial infestations are commonly in wounds or in the cranial area. Therefore, considering the tissue from which the larvae were taken from the corpse is very important in PMI estimation.

References Adams, Z. J. O., Hall, M. J. R., 2003. Methods used for the killing and preservation of blowfly larvae, and their effect on post-mortem larval length. Forensic Sci. Int. 13, 50-61.

ACCEPTED MANUSCRIPT 10 Anderson, G. S., 2000.

Minimum

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forensically important Calliphoridae (Diptera). J. Forensic Sci. 45, 824-832.

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Boatright, S. A., Tomberlin, J. K., 2010. Effects of temperature and tissue type on the

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development of Cochliomyia macellaria (Diptera: Calliphoridae). J. Med. Entomol. 47, 917-

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Byrd, J. H., Butler, J. F., 1996. Effects of temperature on Cochliomyia macellaria (Diptera:

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Calliphoridae) development. J. Med. Entomol. 33, 901-905.

Clark, K., Evans, L., Wall, R., 2006. Growth rates of the blowfly, Lucilia sericata, on

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different body tissues. Forensic Sci. Int. 156, 145-149.

Cragg, J. B., 1955. The natural history of sheep blow flies in Britain. Ann. App. Biol. 42, 197-

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Day, D. M., Wallman, J. F., 2006. Influence of substrate tissue type on larval growth in

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Denno, R. F., Cothran, W. R., 1975. Niche relationships of a guild of necrophagous flies.

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Ann. Entomol. Soc. Am. 68, 741-754. Easton, C., Feir, D., 1991. Factors affecting the oviposition of Phaenicia sericata (Meigen) (Diptera: Calliphoridae). J. Kansas Entomol. Soc. 64, 287-294. Estrada, D. A., Grella, M. D., Thyssen, P. J., Linhares, A. X., 2009. Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae) developmental rate on artificial diet with animal tissues for forensic purpose. Neotrop. Entomol. 38, 203-207. Fisher, P., Wall, R., Ashworth, J. R., 1998. Attraction of the sheep blow fly, Lucilia sericata (Diptera: Calliphoridae) to carrion bait in the field. Bull. Entomol. Res. 88, 611-616.

ACCEPTED MANUSCRIPT 11 Grassberger, M., Reiter, C., 2001. Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen-and isomorphen-

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diagram. Forensic Sci. Int. 120, 32-36.

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Grassberger, M., Friedrich, E., Reiter, C., 2003. The blow fly Chrysomya albiceps

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(Wiedemann) (Diptera: Calliphoridae) as a new indicator in Central Europe. Int. J. Legal Med. 117, 75-81.

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Greenberg, B., Povolny, D., 1971. Bionomics of flies, In Greenberg, Flies and disease, vol. 1. Princeton University Press, Princeton, NJ. 57-83pp.

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Hall, M., Wall, R., 1995. Myiasis of humans and domestic animals. Adv. Para. 35, 258-334. Hobson, R. P.,1932a. Studies on the nutrition of blow-fly larvae. II. Role of the intestinal flora

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in digestion. J. Exp. Biol. 9, 128–38. Hobson, R. P., 1932b. Studies on the nutrition of blow-fly larvae. III. The liquification of muscle. J. Exp. Biol. 9, 359–65. Hobson, R. P., 1932c. Studies on the nutrition of blow-fly larvae. IV. The normal role of microorganisms in larval growth. J. Exp. Biol. 9, 366–77. Hwang, C. C., 2004. Urban ecology of necrophagous flies in Greater London, Ph.D. Thesis, King’s College London. Ireland, S., Turner, B., 2006. The effects of larval crowding and food type on the size and development of the blow fly, Calliphora vomitoria. Forensic Sci. Int. 159, 175-181.

ACCEPTED MANUSCRIPT 12 Kaneshraja, G., Turner, B., 2004. Larvae grow at different rates on different body tissues. Int. J. Legal Med. 118, 242-244.

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Keh, B., 1985. Scope and applications of forensic entomology. Ann. Rev. Entomol. 30, 137–

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Levot, G. W., Brown, K. R., Shipp, E., 1979. Larval growth of some calliphorid and sarcophagid Diptera. Bull. Entomol. Res. 69, 469-475.

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Mackerras, M. J., Freney, M. R., 1933. Observations on the nutrition of maggots of Australian blow-flies. J. Exp. Biol. 10, 237–46.

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MacLeod, J., Donnelly, J., 1962. Microgeographic aggregations in blow fly populations. J. Anim. Ecol. 31, 525-543.

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Norušis, M. J., 2005. SPSS 13.0 Guide to data analysis. Prentice Hall, Inc., New Jersey, USA. Nuorteva, P., 1977. Sarcosaprophagous insects as forensic indicators, pp. 1072-1095, vol. 2.

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In: C.G. Tedeschi, W.G. Eckert and L.G. Tedeschi (eds.), Forensic medicine: A study in trauma and environmental hazards. Saunders, Philadelphia.

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Rasband, W., 2002. ImageJ v.1.26t, http://rsb.info.nih.gov/ij. Saunders, D. S., Bee, A., 1995. Effects of larval crowding on size and fecundity of the blow fly Calliphora vicina (Diptera: Calliphoridae). Eur. J. Entomol. 92, 615-622. Smith, K. G. V., 1986. A manual of forensic entomology. British Museum (Natural History), London, and Cornell University Press, Ithaca, New York. Spradbry, J. P., 1991. A manual for the Diagnosis of Screw-Worm Fly. Goanna, Australia. Smith, K. E., Wall, R., 1997a. The use of carrion as breeding sites by the blow fly Lucilia sericata and other Calliphoridae. Med. Vet. Entomol. 11, 38-44.

ACCEPTED MANUSCRIPT 13 Smith, K. E., Wall, R., 1997b. A symmetric competition between larvae of the blow flies Calliphora vicina and Lucilia sericata in carrion. Ecol. Entomol. 22, 468-474.

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biological research, 2nd ed. W.H. Freeman, New York.

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Sokal, R. R. , Rohlf, F. J., 1981. Biometry: the principles and practice of statistics in

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Uvarov, B. P., 1928. Insect nutrition and metabolism. A summary of the literature. Trans. Entomol. Soc. London. 2, 255–343.

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Villet, M., Richards, C., Midgley, J., 2010. Contemporary precision, bias and accuracy of

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minimum post-mortem intervals estimated using development of carrion-feeding insects. In: Amendt, J., Campobasso, C. P., Goff, M. L., Grassberger, M., eds. Current concepts in

LaMotte, L. R., 1995. Estimating maggot age from weight using inverse

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Wells, J. D.,

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forensic entomology. Heidelberg: Springer, 109-137.

prediction. J. Forensic Sci. 40, 585–90.

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Williams, H., Richardson, A. M. M., 1984. Growth energetics in relation to temperature for

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larvae of four species of necrophagous flies (Diptera: Calliphoridae). Aust. J. Ecol. 9, 141–52.

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Fig.1. The mean larval length (mm) of L. sericata from hatching to pupation at seven different tissues.

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Brain Lung Liver Kidney Heart Meat Intestine

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Fig.2. The mean minimal time duration of the feeding, postfeeding larvae,pupae and total development of L. seicata reared at seven different tisssues. Error bars are ± S.D.

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Fig.4. The mean adult wing length (mm ) of L. sericata reared on seven different tissues. Error bars are ± S.D.

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Table 1. Lipid and protein contents (mg /g) in various tissues and in the third feeding larvae of L. sericata. Type of Tissue Larvae tissue Total Total lipid Total Protein Ratio lipid Total Protein Brain 33.8 85 0.173 14.7 83 Lung 20.4 75 0.063 4.6 73 Liver 30.9 74 0.096 19.5 63 Kidney 22 71.2 0.14 10 61.2 Heart 33 54 0.087 4.7 56 Meat 14 51 0.18 9.2 50.9 Intestine 18.8 50.6 0.385 7.1 50.6

Ratio 0.407 0.279 0.611 0.432 0.524 0.228 0.336

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Fig.1. The mean larval length (mm) of L. sericata from hatching to pupation at seven different tissues.

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Fig.2. The mean minimal time duration of the feeding, postfeeding larvae,pupae and total development of L. seicata reared at seven different tisssues. Error bars are ± S.D.

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Highlight abstract This study investigates effect of seven tissues on larval growth of Lucilia sericata > There were significant differences in the duration and the adult size between different tissues > The

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smallest larvae were obtained from those reared on heart > the position of feeding larvae on a

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coropse will be very important in PMI estimation.