Survey of Forensically Important Present at Different Heights in Toco, Trinidad, West Indies Jessica Uriostigui Texas A&M University Abstract Many species of flies are important in a forensic investigation because they allow the time of colonization to be approximated. This research focuses on three types of flies that are widely considered to be forensically important. These are cuprina (Diptera:) (Wiedemann), Lucilia sericata (Diptera:Calliphoridae) (Meigen), and Sarcophagidae (Diptera:Sarcophagidae) (Macquart). traps with a cadaverine attractant were set up at different heights above ground at four different locations. Many other than the three species mentioned were collected, but these were the ones that had more data and were identified to the lowest taxonomic level possible. There was a higher number of overall collected specimens at lower positions. The preference of each species was unique, and in most cases the preference was very slight due to the few specimens collected of those species. Key words: Calliphoridae, Sarcophagidae, cadaverine, Trinidad Introduction involves the application of knowledge about insects and their role in decomposition to the investigation of a crime. Flies are particularly important in a forensic investigation, as a forensic entomologist may be able to approximate the time of colonization of a specific fly, which may give insight into the approximate time of death of a corpse. Flies may lay eggs on decomposing bodies, which is why it is possible to find an approximate time of death by determining the stage of development that a fly larva is in, taking into consideration the duration in each development stage of the specific fly and the temperature of the surroundings (Greenberg 1991). Lucilia coeruleiviridis, , Chrysomya rufifaces, , , Calliphora livida, and were identified as forensically important flies in Florida (Gruner 2007). The forensic importance of certain species will vary by location based on their presence. Cochliomyia macellaria, Chrysomya megacephala, Chrysomya rufifaces, Musca domestica, Sarcophagidae, and Piophilidae were found to be forensically important species in Jamaica, which is in the Caribbean and should therefore be a list of species that are more likely to be encountered in Trinidad (Cranston 2008). The presence of blow flies at different elevations was researched in Spain by Arturo Baz in 2007. This research used pitfall traps, had varying terrestrial elevations, and the traps were baited with squid. There is more diversity of species and a greater number of blow flies present at lower elevations, but some species such as Lucilia sericata, Chrysomya albiceps, and Lucilia caesar have little presence anywhere higher than 1500 feet (Baz 2007). The objective of this research is to determine whether the diversity and number of forensically important flies vary based on the height above ground. Greater diversity near the ground is expected, as there are more resources available near the ground such as food and protection from the sun, wind, and rain. The focus of this study includes , Lucilia sericata, and Sarcophagidae, as more data was collected from these three groups. Plenty of data on was collected, and it would have been included had they been identified down to species. Materials and Methods While keeping in mind accessibility and the likelihood that the fly traps would remain relatively undisturbed, the exact location of each fly trap was determined on day one. After determining the overall locations, the fly traps were individually labeled based on location and relative height. The fly traps require water to allow for the attractant to work and to prevent the collected insects from leaving after a while. The location for each set of three remained consistent by placing the traps vertically along the same horizontal point or within a 10 ft horizontal radius. There were four separate locations and three different heights at each location. Twine was used to suspend the fly traps at each position. Higher positions were reached by flinging a rope over tree branches to suspend the fly bags higher above ground. The labeling system consisted of an alphanumeric designation. The particular location was designated by a letter and the relative height of the three bags per location designated numerically. The locations were represented as A through D and the relative heights were labeled 1 through 3. The number 1 representing the fly traps closer to the ground and the number 3 representing the farthest from the ground. Using a measuring tape, the height of each bag above ground was measured from the entrance of the trap to the ground directly below it in centimeters. In order to measure the height above ground of traps that were higher up, a long stick was used and then measured. The fly traps were checked daily to ensure that they were not being disturbed. On day five the fly traps were removed and closed to prevent other specimens from entering at unintended heights in between removal of traps and collection of specimens. The collected specimens were removed from the fly traps on days five, six, and eight. Large straight serrated forceps along with small forceps were used to remove the specimens from the fly traps. The specimens were placed in a total of 12 labeled glass vials following the label system mentioned, and placed in the freezer to prevent decay. 95% alcohol was later used to preserve the specimens long enough to identify them. Days nine to twelve consisted of identifying the specimens collected by looking at them under a USB microscope and the use of two identification keys made by Claudio Jose Barros de Carvalho and Terry Whitworth. Two small forceps were used to move the limbs out of the way in order to see certain structures that were key to the identification of the insects collected. Results There was a total of 324 specimens collected from the cadaverine fly traps. A total of 14 Lucilia cuprina, 8 Lucilia sericata, 37 Sarcophagidae, 209 Muscidae, 14 Termitoidae, 14 Vespoidea, 7 Apoidea, 1 Nematocera, 13 Formicidae, 1 Araneae, 3 Coleoptera, 1 Blattidae, 1 Caelifera, and 1 Ensifera were collected and identified. The presence and quantity of specimens at each height above ground varied by location. Trap 1C had the most specimens collected compared to the other traps. Location C, which was near a runoff water source and much vegetation, had the highest number of collected specimens overall. Location A had the least number of specimens collected, and it was a dry area with much shade from trees and buildings. Location B, which resulted in 72 collected species, had the least vegetation, had more exposed soil, and was near pavement. Location D, which was more exposed to the sun, had a total of 35 specimens. (Table 1).

Table 1: Total insects collected 1A: 2A: 3A: 1B: 2B: 3B: 1C: 2C: 3C: 1D: 2D: 3D: Total 71 184 227 cm 75 cm 169 cm 201 cm 39 cm 183 cm 207 cm 33 cm 206 cm 290 cm cm cm Lucilia 2 3 1 1 3 0 2 0 0 1 0 1 14 cuprina Lucilia 1 0 0 0 3 1 2 0 0 0 0 1 8 sericata Sarcophagidae 0 0 0 0 4 1 22 0 1 5 0 4 37

Muscidae 0 4 1 5 34 2 93 18 40 9 2 1 209

Termitoidae 4 2 0 0 7 0 0 1 0 0 0 0 14 Vespoidea 2 3 2 0 3 2 0 2 0 0 0 0 14

Apoidea 0 1 2 0 1 2 0 1 0 0 0 0 7 Nematocera 1 0 0 0 0 0 0 0 0 0 0 0 1 Formicidae 0 0 0 1 0 0 5 0 0 7 0 0 13 Araneae 0 0 0 1 0 0 0 0 0 0 0 0 1 Coleoptera 0 0 0 0 1 0 0 0 0 2 0 0 3

Blattidae 0 0 0 0 0 0 1 0 0 0 0 0 1 Caelifera 0 0 0 0 0 0 0 0 0 0 0 1 1 Ensifera 0 0 0 0 0 0 0 0 0 0 0 1 1 Total 10 13 6 8 56 8 125 22 41 24 2 9 324 In locations A and B, Lucilia cuprina had a slight preference for the intermediate trap, a slight preference for the lower trap at location C, and an equal preference for the higher and lower traps at location D. Lucilia sericata showed a slight preference for lower traps at location A and C, a slight preference for the intermediate trap at location B, and a slight preference for the higher trap at location D. Sarcophagidae had no presence at location A, a slight preference for the intermediate trap at location B, a strong preference for the lower trap at location C, and a slight preference for the lowest trap at location D. (Figure 1). Figure 1: Presence of Specimens at Every Point

Lucilia cuprina, Lucilia sericata and Sarcophagidae Present at Each Point

25

20

15 22 Sarcophagidae

10 Lucilia sericata 4 Lucilia cuprina

Number of specimens 5 3 5 4 1 2 3 3 1 1 0 2 1 1 1 2 1 1 1 1A 2A 3A 1B 2B 3B 1C 2C 3C 1D 2D 3D Fly Traps

If the data is compiled and separated by height regardless of location, the lowest traps demonstrate a higher presence of Sarcophagidae and nearly the same presence of Lucilia sericata and Lucilia cuprina in both intermediate and low traps. Overall, there were more total specimens collected at the lower traps (Figure 2). Figure 2: Presence of Specimens Based Only on Height Presence of Specimens at Varying Positions

High 2 1 5

Intermediate 6 4 5

Low 6 3 27 PLACEMENT OF FLY TRAPS

0 5 10 15 20 25 30 35 40 NUMBER OF SPECIMENS

Lucilia Cuprina Lucilia Sericata Sarcophagidae

Fig. 2. Low placement represents traps 1D, 1C, 1A and 1B which range in height from 33 cm to 75 cm. The intermediate traps are 2B, 2C, 2A and 3B which range in height from 169 cm to 201 cm. High represents traps 2D, 3C, 3A and 3D which range in height from 206 cm to 290 cm.

Discussion When comparing the data from each location individually, there is not a consistent preference. Which is why it is possible that other factors such as shade, proximity to other resources, or height above ground near the area could have affected the variation in results. For instance, trap 2D was under a palm leave, which may be why only two Muscidae and zero of the specimens of interest to this particular research were collected. The ground on the property where the research was conducted was rugged and had rapid variation in elevation within a few feet. Therefore, it is possible for a fly to be flying near the ground at one point and at a higher position later if it makes a turn without changing its vertical position. The results suggest a slight preference regarding Lucilia cuprina and Lucilia sericata towards low and intermediate traps, however very few were collected to demonstrate a strong preference. 72.97 % of Sarcophagidae were collected in the highest traps, while 42.86% of Lucilia cuprina were collected equally in intermediate and high traps, and 50% of Lucilia sericata were collected in the intermediate traps. The difference between the number of flies present on the preferred height traps and the second most preferred traps is a total of 17 for Sarcophagidae, 1 for Lucilia sericata, and 4 for Lucilia cuprina. These are not representative of a strong correlation, but they do demonstrate a slight trend that can be studied further. Therefore, the results from this research do not necessarily suggest that there is an inverse relationship between height above ground and the number of Lucilia cuprina, Lucilia sericata, and Sarcophagidae present, as this was not specifically noticed at most of the locations and there is not much data for at least two of these. There was a variety of other species of insects that were also collected from the traps. It is possible that some were attracted to the cadaverine, but it is also likely that the traps happened to be in the way of certain insects and they just fell in as a coincidence, not necessarily due to the cadaverine attractant. Perhaps flying too high may be more dangerous due to the presence of birds, exposure to the sun, or lack of protection from wind. Near the ground there are different places to land quickly and protection by buildings or leaves. Flight exhaustion may be a reason for flies to stay near the ground. Conclusion From the collected data, there appears to be an overall preference closer to the ground. In the future, it would be interesting to replicate this experiment with a greater difference in height, as greater variance in height might provide more definitive results. Other factors could have played a big part in this research. The terrain, shade, and direct sunlight placement not considered at first. Identifying would have been easier if the traps had been collected earlier and placed in alcohol right away. More research is needed as relatively few flies were found to reach a definitive conclusion. Identification down to species of the other insects that were trapped such as Muscidae, Termitoidae, Vespoidea, Formicidae, and Apoidea might provide more insight into the role that these insects can play in forensics. These five families showed up quite a bit, and some even had an equal presence as Lucilia cuprina and most had a greater presence than Lucilia sericata. Although there was enough data to include Muscidae, it was not included as it would have been more beneficial to identify them down to species in order to obtain clearer conclusions. As there were plenty of Muscidae, taking them into consideration once fully identified might provide some interesting data. Acknowledgements I would like to express my gratitude to Dr. Adrienne Brundage and Dr. Kevin Conway for their help throughout this research. Special thanks for teaching me how to identify, and for dealing with the strong smell from the fly traps. I would like to thank all the people in Trinidad and Tobago that were kind enough to feed us, lend us a place to stay, and were nice in general. I am grateful for the 12 other undergraduate students and two graduate students that I spent time with, as they helped when it was needed and were always kind. References Baz A, Cifrián B, Díaz-äranda L, Martín-Vega D (2007) The distribution of adult blow-flies (Diptera: Calliphoridae) along an altitudinal gradient in Central Spain. Annales de la Société entomologique de France (N.S.), 43(3): 289-296.

Carvalho CJB, Mello-Patiu CA (2008) Key to the adults of the most common forensic species of Diptera in South America. Revista Brasileira de Entomologia. 52(3), 390-406.

Cranston, AW (2008) Survey of forensically-important Calliphoridae in Kingston and St. Andrew, Jamaica, West Indies. LSU Master's Theses. 3464. https://digitalcommons.lsu.edu/gradschool_theses/3464

Greenberg B (1991) Flies as Forensic Indicators. Journal of Medical Entomology, 28(5): 565– 577.

Gruner S, Slone D, Capinera J (2007) Forensically Important Calliphoridae (Diptera) Associated with Pig Carrion in Rural North-Central Florida, Journal of Medical Entomology, 44(3): 509–515.

Whitworth, T. (2006). Keys to the genera and species of blow flies (Diptera: Calliphoridae) of America North. Proceedings of the Entomological Society of Washington,108: 689-725.