ADULT BLOW FLY (DIPTERA: CALLIPHORIDAE) COMMUNITY STRUCTURE ACROSS URBAN-RURAL LANDSCAPE CHANGE IN MICHIGAN By Nicholas J. Babcock A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Entomology—Master of Science 2018 ABSTRACT ADULT BLOW FLY (DIPTERA: CALLIPHORIDAE) COMMUNITY STRUCTURE ACROSS URBAN-RURAL LANDSCAPE CHANGE IN MICHIGAN By Nicholas J. Babcock Necrophagous insects play an important role in the decomposition of vertebrate carrion in the environment. The well documented colonization, development and succession of blow flies (Diptera: Calliphoridae) on decomposing carcasses makes studying their communities relevant for use in forensic investigations to establish post-colonization intervals. during an investigation. The main objective of this research was to conduct a baseline survey of adult Calliphoridae communities among urban – rural landscape types in the Great Lakes region. It was hypothesized that Calliphoridae communities would vary in composition, diversity and distribution across multiple cities and between two landscape types (urban and rural) in Mid-Michigan. To test how adult blow fly distribution varied with changing landscape conditions in Mid-Michigan, sampling with baited jars and hanging traps were implemented over the summer months of June, July, and August in 2017. To determine how blow communities changed from an urban to rural landscape, seven cities were selected with site locations ranging from high intensity developed areas to cultivated crop fields. Over 97,000 individual flies were captured with a total of 11 Calliphoridae species identified. The adult Calliphoridae communities were primarily structured by landscape type and month of collection, with these two factors having an interactive effect as well. The two most abundant species, Phormia regina (Meigen) and Lucilia sericata (Meigen), cumulatively comprised 88.5% of the identified adult flies. These finding are important to provide a helpful taxonomic baseline of Calliphoridae species in the Great Lakes region that forensic scientists may potentially use in both future research and case work. To my amazing wife Jody, for always supporting me in my crazy adventures, and to my awesome kids Ari, Ana, and Ethan for making everyday a new adventure! iii ACKNOWLEDGEMENTS I would like to first thank the members of my committee for providing helpful insight and guidance during this most unique journey. Thank you to Dr. M. Eric Benbow, for taking a chance on a nontraditional student and allowing me to experience a whole new aspect of science and Dr. David Foran for providing helpful insight into subsampling of collection sites. Thank you to Dr. Jennifer L. Pechal for your support and guidance both in and out of the lab, and Dr. Peter White for help with support staff and writing. I would also like to thank Mr. Tom Smith for giving me the opportunity to work with Veterans at Michigan State University. Thank you to the support from the Department of Entomology and Dr. Bill Ravlin for encouraging Veterans to pursue advance degrees. All of this work could not have been possible without the help from so many others. I am very grateful for the undergraduate students who supported this research: McKinley Brewer, Ryan Himmel, Nolan Pakizer, Megan Pastrick, Makayla Scott, Katelyn Smiles, and Olivia Wood. Courtney Larson, Courtney Weatherbee from Benbow lab for their help and support. A big thank you to Joe Receveur for all of the statistical support. I would also like to thank those organizations who have provided financial support allowing me to work on this project: the Department of Entomology, The College of Animal and Natural Resources, Merritt Endowed Fellowship in Entomology, and the Ray and Bernice Hutson Memorial Entomology Endowment Travel Fund for presentations of this research. Most importantly I would like to thank my wife Jody, and three children Ari, Ana, and Ethan for encouraging me, supporting me, and loving me during this entire process. To my parents Greg and Carol Babcock who have always supported me, and encouraged me to follow after my dreams, thank you! iv TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………………….…vii LIST OF FIGURES……………………………………………………………………................ix INTRODUCTION……………..........................................................................................….........1 Decomposition…………………………………………………………………………….3 Arthropods associated with decomposition……………………………………………….4 Calliphoridae in the Midwest……………………………………………………………...6 Hypothesis…………………………………………………………………………………7 MATERIALS AND METHODS……………………………….………………………….….......9 Study location………………………………………………………………….…………10 Trap design……………………………………………………………………………….11 Necrophagous Dipteran Attraction Resources…………………………………………...11 Necrophagous Dipteran Collections...………………………………………….………...12 Necrophagous Dipteran Subsampling and Identification …………………….………….13 Statistical Analysis……………………………..………………………………………...14 RESULTS……...……………………………………………………………………………...…15 Overall Calliphoridae Community……...………………………………………………..16 Community Diversity and Structure…..………………….………………………………17 Calliphoridae Population Changes Over Time………………………….………………..18 Calliphoridae Populations Related to Landscape Differences…………………………...18 Calliphoridae Populations by City………………………………………………………..19 DISCUSSION……………………………………………………………………………………20 CONCLUSION……………………………………………………………………………..........26 APPENDICES…………………………...………………………………………………………29 APPENDIX A: TABLES…………..………………………………………………..…...30 APPENDIX B: FIGURES……………….…..….………………………...……………...48 APPENDIX C: RECORD OF DEPOSITION OF VOUCHER SPECIMENS …….....…62 v REFERENCES………………………………………………………………………………......64 vi LIST OF TABLES Table 1. Total overall number of Calliphoridae adult specimens captured during the summer of 2017 for each city location and site in urban and rural locations. NA indicates that there was not a rural location sampled for Lansing, MI. *Traps that experienced a failure during the collection period. ** Sample was lost after collection was completed.…………..………….……................31 Table 2. Calliphoridae species mean (SE) percent relative abundance in Mid-Michigan by city over the months of June, July, and August 2017. For Charlotte, Dewitt, Grand Ledge, Perry and Williamston N=24, Lansing N=12, and Mason N=23.…………………………..........................32 Table 3. The overall monthly mean (SE) relative abundance of Calliphoridae collected in Mid- Michigan over the 2017 summer. In June and July all species were represented by N = 52, while for August it was N = 51................................................................................................................33 Table 4. Two-way ANOVA statistics that tested Calliphoridae diversity by both month and landscape type during the summer of 2017………………………………………..……………...34 Table 5. Permutational multivariate analysis of variance (PERMANOVA) of Calliphoridae species from the cities of: Charlotte, DeWitt, Grand Ledge, Lansing, Mason, Perry, and Williamston in Mid-Michigan during significant effects…………………………………………………………………………………………….34 summer of 2017. Bolded the text indicates Table 6. Tukey’s Honest Significant Difference test of the four major Calliphoridae species (C. macellaria, L. sericata, L.illustris, P. regina) collected in Mid-Michigan and compared among months of the 2017 summer. Only pairwise comparisons that were found significant with alpha at or below 0.05 are given……………………………………………………………………..........35 Table 7. Tukey’s Honest Significant Difference test of the predominant Calliphoridae species collected in Mid-Michigan and compared between landscape types during the summer of 2017. Only pairwise comparisons that were found significant with alpha at or below 0.05 are given...35 Supp. Table S1. A summary of previous adult blow fly studies assed by location, length of study, bait type, and landscape type….…………………………………………………………………36 Supp. Table S2. Urban to Rural Landscape type research sites, land use, and GPS locations from the summer of 2017. …………………………………………………………………………….37 Supp. Table S3. Calliphoridae species in Mid-Michigan expressed as relative abundance by location during the summer months of June, July, and August in 2017…………………………38 Supp. Table S4. Shannon Diversity Index of adult Calliphoridae distributed amongst seven cities in Mid-Michigan during the summer of 2017……………………………………………………41 vii Supp. Table S5. Tukey’s Honest Significant Difference test for pairwise comparisons of the predominant Calliphoridae species evaluated by city collected in Mid-Michigan during the summer of 2017. Only results are displayed where p-values were considered significant with alpha at or below 0.05………………………………………………………………………..…………45 Supp. Table S6. Average daily temperatures in degree Celsius for the Mid-Michigan area during the summer of 2017. Temperature data was recorded using the Capital City Weather Station at the Capitol Regional Airport (LAN), Lansing, MI 48906……………...…………………………….47 viii LIST OF FIGURES Figure 1. Urban and Rural research site locations and surrounding land cover types in Mid- Michigan. City populations as indicated by the 2010 United States Census Bureau. Land use types defined by using Geographic Information System (GIS).……………..………………....…...…..49 Figure 2. Total land use types for urban locations in Charlotte, DeWitt, Grand Ledge, Lansing, Mason, Perry, and Williamston. Land use types defined by using Geographic Information System (GIS)………………………………………………………...…………………………………...50 Figure 3. Total land use types for rural locations in Charlotte, DeWitt, Grand Ledge, Lansing, Mason, Perry, and Williamston. Land use types defined by using Geographic Information System (GIS)……………………………………………………………………………………...……...51 Figure 4. Williamston South urban trap placement with Shepard’s hook, bait jar, and guide lines. 15JUN2017 (278 flies captured in this trapping event)………………………………..……...…..52 Figure 5. Williamston South rural trap after placement in field for 4 hours. Photo taken 15JUN2017 (1,416 flies captured in this trapping event)…………………... ……………...……………….....53 Figure 6. Percent relative abundance of Calliphoridae species for urban and rural landscape types, city and month over the 2017 in Mid-Michigan…………………………………………………..54 Figure 7. Phormia regina mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017. Tukey’s Honest Significant Different Test: (A) *Adjusted P-value <0.0020 **Adjusted P-value <0.0001 (B) *Adjusted P-value= 0.0030....55 Figure 8. Lucilia sericata mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017. Tukey’s Honest Significant Different Test: (A) *Adjusted P-value <0.0020 **Adjusted P-value <0.0001 (B) *Adjusted P-value <0.0001...….....55 Figure 9. Shannon diversity of Calliphoridae species indicated by landscape type and month in Mid-Michigan during the summer of 2017. Kruskal Wallis Rank Sum test: p-value < 0.030* p- value < 0.001**…………………………………………………………………………………..56 Figure 10. Principal Coordinates Analysis (PCoA) ordination of relative abundance distribution by month (A) and landscape type (b) during the summer of 2017. The ellipses represent 95% confidence intervals for the mean of each group. See PERMANOVA results for statistical tests of these factors. ……………………………………………………………….…………...……......57 Figure 11. Cochliomyia macellaria mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017. Tukey’s Honest Significant Different Test: (A) * Adjusted P-value <0.0020 **Adjusted P-value <0.0001 (B) *Adjusted P-value <0.0030…………………………………………………………………………………………..58 ix Figure 12. Lucilia illustris mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017…………………………………………….........58 Figure 13. Lucilia coeruleiviridis mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017……………………………….……………..........59 Figure 14. Protophormia terraenovae mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017………………………………………...…….59 Supp. Figure F1. Percent of Calliphoridae species by abundance for urban and rural trapping locations and trap placement site (Cardinal direction) distinguished by city, month and location during 2017 in Mid-Michigan……………………………………………………………………60 Supp. Figure F2. Shannon diversity of Calliphoridae species indicated by landscape change, month and city in Mid-Michigan during the summer of 2017…………………………….……………...61 x INTRODUCTION 1 Forensic entomology is where the study of arthropods intersects with the judicial system (Byrd 2002). Crimes can be committed leaving little physical evidence of what took place. The ability to determine how and where that crime was committed can take a team of investigators and forensic scientists. Forensic entomology can be relevant in both criminal and civil litigation (Joseph et al. 2011) and can be broadly broken down into three sub-areas: (i) urban, (ii) stored- product, (iii) medico-legal. Urban forensic entomology is using entomological evidence in a dwelling, commercial and public buildings, garden, or misuse of pesticides for litigation purposes. Where stored-product entomology is the use of arthropods in contamination of food sources and usually includes issues where litigation may be taking place. The most easily recognized subarea of forensic entomology is medico-legal. It involves using arthropods as evidence to locate bodies, estimate the post-mortem interval (PMI), aid in cause of death determination (Nolte et al. 1992), establish if a body was moved after death (Benecke 1998), and identify criminal suspects (Catts and Goff 1992). Medico-legal cases can also include issues of human or animal abuse, neglect, or poaching (Anderson 1999, Amendt et al. 2004). Investigating a homicide will require a wide range of tools to be used by forensic scientists who are supporting an investigation. When using information like body temperature or livor/rigor mortis to estimate a time of death, accuracy may only be possible for the first few days; however, by using necrophagous insects that time can be extended to weeks or months (Amendt et al. 2004). In recent years, there have been legal cases that used less common taxa used as evidence. Lice (order Phthiraptera) for example, were used to determine if an elderly woman had been neglected before her death (Pilli et al. 2016). While mites (class Arachnida) have been found to be forensically important in all three sub-sets of forensic entomology (OConnor 2009). Forensic entomology needs to be built on a stronger scientific foundation like 2 other physical evidence such as blood stains, hairs, fibers, and fingerprints, and be (Amendt et al. 2007, Tomberlin et al. 2010). Decomposition The decomposition of both humans and animals creates a unique and ephemeral environment for a wide range of insects and other arthropods to use (Benbow et al. 2015). There are varying ideologies amongst scientists regarding the stages of decomposition. However, it is most important to understand that the stages of decomposition are simply used to help communicate descriptions of decomposition. Goff (2009) suggested that there are five stages of decomposition which are: (i) fresh, (ii) bloated, (iii) decay, (iv) post decay, and (v) skeletal/remains. The conditions under which carrion are found can not only impact the stages of decay, but what types of arthropods can be discovered (Galloway et al. 1989). One study in Hawaii found ten orders, twenty-seven families, and forty genera of arthropods reported on domestic swine carcasses over a twenty-day span (Tullis and Goff 1987). During the fresh stage Creophilus maxillosus (Linnaeus) (Coleoptera: Staphylinidae) and Thyreocephalus albertisi (Fauvel) (Coleoptera: Staphylinidae) were found on the carrion, as well as two Calliphoridae (Diptera) species, Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart)). As decomposition progressed to the bloat stage more Staphylinidae species were observed, and while there was no increase in Diptera species it was the stage where there was the highest number of adults on the carcass. The same trend occurred during the decay stage and post-decay stage where there was no increase in Diptera species while the Staphylinidae increased. Although there was no change in Diptera species on the carrion the increase in Staphylinidae can be associated with the fact that they feed on the larval flies (Payne 1965). A 2001 study in Austria 3 also evaluated succession rates of arthropods on swine carcasses, but provided different results. Fourteen dipteran species were found on pig carrion fitted with human clothes. The clothes became soiled with blood and fluids leaking from the corps, which allowed for larger larval masses resulting in faster decomposition (Grassberger and Frank 2004). In Michigan forty-one swine carcasses were buried at a depth of 60 cm in an agricultural field and both Sarcophagidae and Muscidae species were found to colonize the buried carrion (Pastula and Merritt 2013). Arthropods associated with decomposition There are four categories of species that make up the carrion necrophagous community: (i) necrophagous species that feed on the carrion, (ii) predators and parasites of necrophagous insects, (iii) omnivorous species feeding on the carrion and necrophagous insects, and (iv) insects that use the carrion as an extension of their environment, such as spiders (Smith 1986). The two most common insect orders evaluated in forensic investigations are Diptera (flies) and Coleoptera (beetles) (Joseph et al. 2011, Payne 1965). The most common dipteran families found associated with decomposition are Calliphoridae (blow flies), Sarcophagidae (flesh flies), and Muscidae (house flies) (Campobasso et al. 2001). Entomological evidence is important and useful tool to determine when decomposition began. During the earlier stages of decomposition, the life cycles of Calliphoridae are most beneficial to help estimate a PMI. As remains undergo multiple stages of decomposition, it is more difficult to establish a PMI since the life cycles of insects come to be difficult to interpret (Goff 1993). At this point succession-based PMI becomes a better tool. This method evaluates the changes in arthropod communities that occur throughout the process of decomposition. From the consumers to predators and parasitoids there are a wide range of entomological factors that could influence PMI calculations. 4 Necrophagous insects and the parasites and predators who prey upon them are two of the most significant groups of arthropods used to establish a PMI (Goff 2009). Since some of these insects have the ability to feed on the skin and flesh of animals within minutes of death (Joseph et al. 2011) it makes them a reliable indicator for PMI within the first few weeks after death (Amendt et al. 2004). The predators who consume the necrophagous insects are also helpful because of their predictable arrival during the various life stages of their prey (Joseph et al. 2011). However, when there are high abundances of predators, parasites, and omnivorous species on carrion it could interfere with PMI calculations. Mello and Aguiar-Coelho (2009) showed in laboratory conditions how parasitoids can alter the development cycle of Calliphoridae and cause the PMI to be miscalculated by weeks. Skin beetles (Coleoptera: Dermestidae) have also been used to estimate PMI since they arrive on carrion in late stages of decay (Souza and Linhares 1997). To establish the amount of time that has passed since death a PMI can be established for a body using factors such as temperature, moisture, pH, and partial pressure of oxygen (Vass 2011). Using entomological evidence to establish a PMI can be misleading since some insect colonization can be altered based upon the circumstances surrounding a death. Goff (1992) was able to delay the oviposition of Diptera by 2.5 days by wrapping a body tightly in a blanket. Additionally, Pechal et al. (2014) revealed that delaying insect access to carrion changed the insect community structure and decomposition process. Therefore, an alternative way to define the time from insect colonization until discovery as a post-colonization interval (PCI) (Tomberlin et al. 2010, Weatherbee et al. 2017). Evaluating the distribution of blow fly species within a geographic region can be important particularly when practitioners only have outdated, difficult to access and restricted 5 taxonomic keys (Sanford 2017). Distribution databases of Calliphoridae, Muscidae and Sarcophagidae species could assist law enforcement in determining PCI for a given region. The distribution information could also help identify if a body had been moved from one location to another. For forensic entomology data to be useful within a case it is critical to have a dataset on the species present within the region (Weidner et al. 2017). There have been numerous studies of forensically important blow fly distribution around the world (Supp. Table S1) In Ankara Province China, it was determined that Calliphora vomitoria (Linnaeus) and Calliphora vicina (Robineau-Desvoidy) can be used to establish PCI when ambient temperatures are low in spring and autumn (Sabanoğlu and Sert 2010). While forty-eight species of blow flies belonging to eighteen genera were detected across Pakistan (Kurahashi and Afzal 2002). During a two-year span in Peru, 34,389 Calliphoridae were captured and examined with the major two taxa being Compsomyiops callipes (Bigot) (38%) and Phormia regina (Meigen) 23% (Baumgartner and Greenberg 1985). In South Africa Chrysomya albiceps (Wiedemann) and Chrysomya marginalis (Robineau-Desvoidy) were the widest spread species while Chrysomya chloropyga (Wiedemann) and Calliphora croceipalpis (Jaennicke) were found in high altitudes (Richards, Williams, and Villet 2009). A seasonal adult Calliphoridae distribution study in Spain covered 7,000 km2 and found ten blow fly species with C. vicina (35%) and C. vomitoria (23%) (Zabala et al. 2014). With a wide range of studies, and equally varying results of dominate species it is important to have more regional specific studies to understand local distribution (Weidner et al. 2017). Calliphoridae in the Midwest The only Great Lakes study of necrophagous Calliphoridae distribution took place in the spring of 1980 and only focused on urban locations. Nearly 1,200 flies were collected on rat 6 carrion, enhanced with chemicals such as ethyl mercaptan and ammonium carbonate, throughout Chicago, Illinois. Two main abundant species collected were Lucilia sericata and Phormia regina (Baumgartner 1988); however, this study did not account for how summer month temperatures effect the distribution of these two Calliphoridae species. Previous research has shown that P. regina has also been found to be intolerant of warmer temperatures (Byrd and Allen 2001), and therefore abundances could be expected to change during the summer. If the abundances of any one particular species should decrease within a region it could be expected that another species will increase to fill the void within the ecosystem. It has also been shown that Calliphoridae can vary based upon geographic region. For example, P. regina have varied in distribution from 29.2% (Weidner et al. 2017), 23% (Brundage et al. 2011), and 17% (Baumgartner 1988) within their respective study areas. Hypothesis This study will provide one of the first surveys of Calliphoridae distribution between an urban and rural landscape change for the Great Lakes region. It will also provide a baseline database from 2017 that forensic entomologist in the region will be able to use as a resource. While this study was designed as a descriptive study, based on previous understanding of Calliphoridae from other regions of the USA, a set of hypotheses were developed. The null hypothesis (H0) is that Calliphoridae communities will be similar and that species will be evenly distributed across an urban to rural landscape change in Mid-Michigan. The alternative hypothesis (Ha) is that Calliphoridae communities and species will vary in distribution across an urban to rural landscape. As L. sericata is a synanthrope blow fly (Mariluis et al. 2008, Marshall et al. 2011), it is predicted that this species will be most likely be predominant in more urban landscapes. Previous research showed that P. regina is attracted to both manure and carrion 7 (Marshall et al. 2011), thus it is predicted that this species will be collected at higher abundances in rural areas where these food sources would be more plentiful than in urban areas. 8 MATERIALS AND METHODS 9 Study Location This study was conducted in Mid-Michigan, a region that allowed for evaluating the distribution of carrion flies across urban to rural landscape change. The City of Lansing and the surrounding communities has mixed land use of both industrial and agricultural landscape cover that allowed for development of a sampling array that represented an urban-rural landscape change. The urban locations were predominantly low intensity developed areas, whereas the rural locations were cultivated crops and developed low intensity/open space (Figures 1- 3). Cities in the greater Lansing Capital Area selected for sampling adult Calliphoridae were: Charlotte, DeWitt, Grand Ledge, Mason, Lansing, Perry, and Williamston. The 2010 US Census showed the populations of the cities ranged from 116,020 in Lansing to 2,103 in Perry (Figure 1). To survey adult Calliphoridae communities at replicate urban-rural land cover types four traps were used at each 1 km (urban) and 10 km (rural) distance from each city center, with the four traps placed in a cardinal direction (Figure 1). The city center was determined as the pin placement within Google Earth (Earth version 7.3.2) when a city was searched. The ruler function within Google Earth was used to establish the urban and rural locations around each city center. Google Earth was used to determine the urban to rural landscape transition, and then was ground-truthed by vehicle to confirm where housing subdivisions ended and agricultural land became predominant. Permission from landowners and businesses was acquired for the placement of traps resulting in some small distance variation from both the urban and rural locations. Of the seven cities surveyed, Lansing was the only city that did not have rural landscape cover due to the distance needed to establish a rural location from Lansing. Thus, the Lansing 10 10 km sampling resulted in placement within a surrounding city suburb (e.g., primarily subdivision housing) and was not a representation of a rural location. Additionally, some cities did not allow for a true 10 km distance because the trap would have been within an urban or suburban land use setting. In this situation, a trap was placed at a location halfway between the two urban locations. The placement of the trap was in an area that was as close to a rural setting as possible. Trap Design The traps used to collect adult Calliphoridae (and other taxa) were built using an inverted cone design, similar to a butterfly trap (Figure 4, Figure 5). Each trap was 76.2 cm in length and 25.4 cm wide; traps were constructed using plywood for the frame and polyester mosquito netting (180 holes per 6.5cm2) for the walls. In the center of the base, a 15.2 cm hole was cut out of netting and the edges were suspended from the top of the trap using 58.4 cm long rope to make the inverted cone. This trap design allowed flies to enter but not exit the trap at the base. At the base and top of each trap a bead of hot glue was applied to prevent flies from exiting the trap once they had entered. A zipper running the entire height of the trap was used for specimen removal after sample collection. Each trap had a zinc-plated threaded eyehook placed at the top with a 45.7 cm rope used for hanging the trap. At each location a trap was hung from a 1.2 m garden shepherd’s hook and two 35.5 cm guide lines were placed at the base of each trap to prevent the trap for moving with the wind (Figure 4). When the trap was placed in the field the zipper was duct taped in the closed position preventing the trap from coming open. Necrophagous Dipteran Attraction Resource To attract adult necrophagous insects, bait jars were use at each trap. Bait jars were made using pig liver purchased from Michigan State University’s Meat Lab. Frozen liver was purchased in a vacuum sealed bag and allowed to thaw to room temperature before being placed 11 into a jar. Each bait jar consisted of 300 g of pig liver and 100 ml of reverse osmosis water. The jars were sealed using standard Mason jar lids and bands, and placed at an average room temperature of 25oC for 350 accumulated degree hours (ADH), calculated using the methods provided in Vass et al. (2002). Bait was allowed to decompose for a total of seven days as previous research has shown maximum insect activity associated with aged bait between seven to seventeen days (Fisher, Wall, and Ashworth 1998). Each bait was used in the field for an additional seven sampling days. During placement in the field, an individual 1.4 L Mason jar with bait was placed with the bottom of the trap suspended approximately 2-4 cm above the top of the jar; the standard Mason jar lid was removed and replaced with screen to prevent insects from falling into the bait. Necrophagous Dipteran Collections Trapping took place during the summer months of June, July and August in 2017. All traps for any given city were placed into the field and collected one day during each month. The traps were left at the field site for four hours each day: placement occurred at the first site at 1000 and all traps were sequentially collected in the order they were placed beginning at 1400. This time frame was chosen since these hours of the day were previously reported to have the highest diurnal Calliphoridae activity in the Midwest (Central Ohio) (Berg and Benbow 2011). It took approximately two hours to place all eight traps within a city. When collecting each trap, the entry hole was spun closed by hand preventing flies from exiting. At the end of each day all traps were transported to the laboratory and placed in a -20C freezer for no less than twelve hours, to ensure the flies had been euthanized prior to preservation. Flies were placed in 50 ml conical tubes with locality labels that included collection date, city, and trap location (Supp. Table S2) and placed into the -20C freezer until identification. 12 Necrophagous Dipteran Subsampling and Identification Once flies were removed from the freezer they were presorted by visual morphology into Calliphoridae and non-Calliphoridae taxa. Non-Calliphoridae were placed in a 50 ml conical tube, labeled and stored in a -20C freezer. Calliphoridae were identified to species under a stereomicroscope using the Blow flies (Diptera: Calliphoridae) of Eastern Canada with a Key to Calliphoridae Subfamilies and Genera of Eastern North America, and a Key to the Eastern Canadian species of Calliphorinae, Luciliinae and Chrysomyiinae (Marshal et. al. 2011). Traps returned a broad range of captured number of flies from 25 – 3,011 (Table 1). To facilitate sample processing and insect identification a subsampling study was performed to determine the minimum number of flies per trap that would be needed to represent the Calliphoridae community at each location. Four sites for the Williamston location were used to evaluate blow fly species in the following sample subsets: 100 specimens and 25%, 50% and 100% of total specimens. The total number of flies for each of the four sites were 1332, 1416, 170, and 175. These site totals were selected because they represented the two highest counts, and the two lowest counts. For each sample, the flies were thawed, placed in a 1.4 L Mason jar and homogenized by hand inversions for 10 seconds. For the first subsampling approach 100 flies were randomly collected from each sample. For the 25%, 50% and 100% subsamples the respective fly number for each percentage was calculated and then individual flies were randomly selected. From this pilot study, it was determined that a count of 100 flies provided an equivalent Calliphoridae species representation as that of the three percentage subsampling levels. For all of the remaining samples, the following protocol was used: 1) flies were removed from the -20C freezer and allowed to thaw; 2) after thawing all specimens were placed in a 13 Mason jar and homogenized by hand using inverted movements for 10 s; and 3) 100 flies were randomly selected and identified using the previously described methods. All data were entered into a table by city, date, and location. Statistical Analysis A Permutational Multivariate Analysis of Variance (PERMANOVA) test was used with a Bray-Curtis dissimilarity matrix to test for Calliphoridae community differences among spatial [cities, location (rural/urban)] and temporal (month) scales. This nonparametric, multivariate method has been used in other studies to test for community differences among covariates (Pechal et al. 2018). A nonparametric test is best for testing community data that does have a normal distribution or homogeneity of variance (Caruso and Migliorini 2006, McArdle and Anderson 2001). Community differences were visualized using a Principal Coordinates Analysis (PCoA). In addition, the non-parametric Kruskal-Wallis Rank Sum test was used to compare beta diversity among the urban and rural locations. Tukey’s Honest Significant Difference (Tukey) post-hoc test was performed to test for pairwise differences in individual species abundances among cities, locations, and months. To run the Tukey test the relative abundance data were transformed using the arcsine square root transformation. All p-values were considered significant with alpha at or below 0.05. All statistical analysis was done using RStudio 1.1.453 (RStudio Inc., Boston, MA, USA). 14 RESULTS 15 Overall Calliphoridae Community The Calliphoridae communities collected in urban-rural landscape types in Mid-Michigan were composed of eleven species: Calliphora vomitoria (L.), Calliphora vicina (Robineau- Desvoidy), Chrysomya rufifacies (Macquart), Cochliomyia macellaria (Fabricius), Cynomya cadaverina (Robineau-Desvoidy), Lucilia illustris (Meigen), Lucilia sericata (Meigen), Lucilia silvarum (Meigen), Lucilia coeruleiviridis (Macquart), Protophormia terraenovae (Robineau- Desvoidy), and Phormia regina (Meigen). There were over 97,000 flies captured in June, July and August of 2017 across the sites in Mid-Michigan (Table 1). The city of Grand Ledge yielded the highest number of flies at 18,237 while Mason had the lowest at 13,565. The urban Lansing sampling returned 8,029 flies, but used only half the number of traps since there were no rural locations (Table 1). At the species level, there were four predominant species: P. regina was the most abundant (10,800 flies) and accounted for 69.7% of the total Calliphoridae community; the L. sericata abundance was second highest at 18.8% (2,913 flies); and C. macellaria and L. illustris were equally abundant at 4.0% (619 flies) each. The least abundant Calliphoridae was C. vomitoria, which only represented 0.08% (13 flies) of the overall community. For each of the seven cities P. regina and L. sericata were the two most abundant species, with P. regina the most predominate blow fly species at each sampling location and L. sericata the second most abundant (Table 2, Supp. Table S3). While P. regina was the most dominate overall there was an inverse relationship with L. sericata within each city (Table 2). From June to August three species increased in overall abundance each month (C. macellaria, L. sericata, P. terraenovae) while P. regina decreased (Table 3, Figure 6-8) across all cities. Lucilia illustris relative abundances were similar over the study summer (Table 3). 16 Community Diversity and Structure Calliphoridae community diversity was highest at rural landscape locations, with rural sites accounting for 66% of the most diverse locations (Supp. Table S4). DeWitt had five of the top ten locations with the highest diversity of all samples at 1.68 (Supp. Table S4). There was one rural location in Grand Ledge sampled in June with no diversity, since P. regina was the only identified Calliphoridae species. Of the covariates tested, month (p < 2e-16, F = 51.34) was the most significant factor influencing the Calliphoridae diversity (Table 4) followed by landscape type (p = 0.001, F = 10.53), and their interaction (p = 0.017, F = 4.16). The diversity of each landscape type changed significantly by month with the greatest increase taking place from July to August for rural locations and June to July for urban locations (Figure 9, Supp. Figure F2). Calliphoridae community was further confirmed to be structured by month (p = 0.001, F = 26.8), landscape type (p = 0.001, F = 24.20), and their interaction (p = 0.001, F =4.79) (Table 5). Although city had a significant influence on community structure (p = 0.002, F = 3.8) it was the weakest (Table 5) and its interaction with landscape type (p= 0.161, F=1.4) and month (p= 0.055, F=1.5) were not significant. For this reason, the subsequent statistical analyses focused on the two factors that had the strongest and interactive effects on the Calliphoridae communities; the effect of city was evaluated and can be found in supplemental material (Supp. Table S5) but is not the focus here. Additionally, subsequent analyses focused on those species with more than 1% of the overall abundance (C. macellaria, L. coeruleiviridis, L. illustris, L. sericata, and P. regina, and P. terraenovae), and how they were affected by month and landscape type (Figure 10), as species with these abundances had enough power for meaningful statistical analyses. 17 Calliphoridae Population Changes Over Time Most of the predominant Calliphoridae species populations significantly changed over the 2017 summer at both urban and rural landscape types (Table 6, Figures 6-8, Figures 11-14). From June to August, C. macellaria, L. sericata, P. terraenovae relative abundances increased each month while P. regina decreased, and Lucilia illustris had consistent values over the summer (Table 3, Figures 6-8, Figures 11-14). There were also significant differences for P. regina, L. sericata, and C. macellaria among each of the months (Table 6), with the biggest differences between June and August (p < 0.0001) (Table 6). There was a significant increase in abundance from June to August for L. sericata and C. macellaria, but not P. regina (Table 3, Figure 6, Supp. Figure F1). While not as strong, there were significant increases in relative abundance between July and August for L. sericata (p = 0.0002), C. macellaria (p = 0.0020) and L. coeruleiviridis (p = 0.0010). There was no significant difference in L. illustris relative abundance amongst the months. Calliphoridae Populations Related to Landscape Differences There were significant differences in the mean relative abundances of P. regina, L. sericata, and C. macellaria between urban and rural landscape types (Table 7; Figures 7-8, Figure 11). Phormia regina was collected in significantly higher mean proportions (p = 0.0025) in rural (76.9% ± 5.45% SE) than urban (63.6% ±5.47% SE) areas (Figure 7), compared to L. sericata which made up 27.4% (± 5.2% SE) of the Calliphoridae communities at urban locations and only 8.6% (± 3.2% SE ) in rural (Figure 8). The populations of L. coeruleiviridis and L. illustris were not significantly different between urban and rural locations, likely because of their overall low relative abundances and variance among samples. 18 Calliphoridae Populations by City While not the primary focus of the analyses, the Calliphoridae community structure results showed the effect of city was significant but not as important as month and landscape type, there were significant associations among cities and species (Table 5). Phormia regina was the most abundant in all of the cities, but only showed a significant decrease between Williamston and Lansing (p = 0.010) and between Williamston and Charlotte (p = 0.029) with the change between Lansing and Williamston being the greatest (Table 2, Supp. Table S5). Lucilia sericata significantly decreased in relative abundance from Charlotte to DeWitt, Grand Ledge, and Williamston. The greatest decrease in relative abundance was between Charlotte and Grand Ledge (p = 0.001) (Supp. Table S5), while P. terraenovae had the highest abundance within Lansing and showed a significant change with the other six cities (all p < 0.001) (Supp. Table S5). The greatest increase of P. terraenovae was the change from Lansing to Mason, while the largest decrease in abundance was Lansing to Charlotte. Lucilia illustris was found to be the most abundant in DeWitt, which was significantly greater than Grand Ledge, Lansing, and Williamston. The most significant decrease for L. illustris was from DeWitt to Charlotte (p = 0.007) (Table 2, Supp. Table S5). Lucilia coeruleiviridis was most abundant in Grand Ledge and was significantly greater in Grand Ledge than Williamston (p = 0.015) (Supp. Table S5). Cochliomyia macellaria was captured in all of the cities (Table 2) but did not significantly vary amongst them. 19 DISCUSSION 20 The overall goal of this research was to provide an initial database of adult Calliphoridae species in the Mid-Michigan region for potential use in future forensic investigations. The study provided a survey of Calliphoridae community diversity and structure and species population differences between urban and rural landscape types over a summer in the Mid-Michigan region. In addition to Calliphoridae, other Diptera of forensic interest collected were flesh flies (Diptera: Sarcophagidae), rove beetles (Coleoptera: Staphylinidae), and carrion beetles (Coleoptera: Silphidae), but were not the focus of the present study. Similar research has been conducted on the west coast in California where Lucilia sericata (Meigen), Lucilia cuprina (Wiedemann), C. vomitoria, and P. regina were the most common species (Brundage et al. 2011). In New Jersey, L. sericata, L. coeruleiviridis and P. regina were the three most predominant species respectively (Weidner et al. 2017). In Ohio, Calliphoridae were found to colonize carrion within an hour of placement, and stopped occupying the carrion in less than an hour after sunset (Berg and Benbow 2013), and limited blow fly colonization was detected during fresh decomposition during the winter months (Benbow et al. 2013). In Illinois, L. sericata and P. regina were found to be the two predominant species in Chicago (Baumgartner 1988). In addition, research evaluating oviposition during sunrise, sunset, and nighttime in Mid-Michigan only captured seven Calliphoridae species (Cochliomyia macellaria, Pollenia rudis, L. coeruleiviridis, C. vomitoria, C. vicina, L. sericata, and P. regina) (Zurawski et al. 2009). By studying the distribution of Calliphoridae within a geographic region resulting information could provide an important resource for investigators when establishing a post-colonization interval, or the time from insect colonization until discovery, for homicide investigations (Anderson 2000). Overall, in the Mid-Michigan area blow fly communities were most affected by the month of the summer and landscape type. There was a significant but less strong impact of city 21 especially when compared to landscape type and month. These findings suggest that city is likely confounded with landscape; therefore, is may be that the flies were not responding to a city, but rather to landscape type of a particular city. The null hypothesis of the study was that there would be an even distribution of blow fly species in the Mid-Michigan region; however, the results from the study calls for rejection of the null hypothesis. This is supported by P. regina being found at higher abundance rates in rural landscapes, and C. macellaria and L. sericata in urban landscapes. The Calliphoridae communities were also affected most by the month providing additional evidence that blow fly species do vary by not only landscape, but time as well. For that reason, the alternative hypothesis of Calliphoridae species varying in distribution is accepted. The Tukey tests showed multiple blow fly species were significantly different between both landscape type and between months. The Calliphoridae community in Mid-Michigan was diverse and there were species dependent changes over time and between landscape types. In this study, L. sericata supported previous research that reported higher abundances in urban areas (Mariluis et al. 2008, Marshall et al. 2011), and being a synanthropic species (Mariluis et al. 2008, Marshall et al. 2011). Phormia regina was found to be in higher abundance in rural areas, reflecting their preferred resources that include both carrion and manure as suggested by Marshall et al. (2011). Cochliomyia macellaria was collected in higher abundances within rural habitats, and this finding could be related to being attracted to similar food sources as P. regina (Baumgartner and Greenberg 1985). A unique observation from previous research was the abundance of L. silvarium previously found in Grand Haven during a multi-state Calliphoridae survey (Schoof et al. 1956). A study in Grand Haven resulted in anywhere between 1.0 and 2.5% of the flies captured as L. silvarium (Schoof et al. 1956). The research in the present study only identified 22 sixteen specimens of L. silvarium accounting for on 0.1% of the total fly community abundance and the populations did not change over the summer. Some of the Calliphoridae populations changed in relative abundance as the summer progressed. The overall relative abundance of P. regina was 69.7% for the study and was much higher than reported in previous research in California (23%) (Brundage et al. 2011), and New Jersey (28.6%) (Weidner et al. 2017). The abundance rate of P. regina in Mid-Michigan declined throughout the summer which coincided with previous Calliphoridae research in Texas, and Florida. In Texas, the decline of P. regina was confirmed by evaluating arthropods from more than 200 forensic entomology cases (Sanford 2017), and in Florida the same trend was seen in a regional survey using swine carcasses (Gruner et al. 2007). The increase in abundance for both L. sericata and C. macellaria as overall temperatures increased was also reported in previous research (Weidner et al. 2017, Brundage et al. 2011, Goddard and Lago 1985). Previous research has shown that fluctuation between high and low environmental temperatures, and change in seasons alters adult Calliphoridae activity (Payne 1965, Watson and Carlton 2005, Tabor et al. 2004). For this reason a more in-depth study should be considered for multiple years. This would help identify if the results from this study were affected by the seasonal conditions during the summer of 2017. In Mid-Michigan the average monthly temperature (oC) was for the study was 28.3, 28.9, and 26.1 for June, July and August respectively. Compared to the historic temperatures of June (25.6) and July (27.8) both months were above average while August (26.7) was below the normal. The rainfall (cm) during the summer was higher in June (9.5), and lower in July (6.9) and August (5.1) compared to the historical amounts of 8.8 (June), 7.2 (July), and 8.2 (August) (“Climate-United States-Monthly averages” 2018, Supp. Table S6). 23 While there were some weak, but significant differences among cities, they are likely reflective in small differences more related to urban to rural landscape type transitions. Since using the term city does not define a particular population size or land cover type there can be difference among them. For instance, the city of Mason has a higher development intensity and population compared to Williamston. Of the four Williamston urban locations three sites were classified as developed open space compared to all four of the Mason urban sites being listed as developed low intensity. The changes of cover class within any region could also have an impact on the Calliphoridae community by creating microhabitats. For example, within a city if there are more developed open spaces the food resources in that area could be different than where the land cover is developed high intensity. For the blow flies within a rural environment they are most likely using trash as a food source. Using the premise that more trash would be found in high intensity areas compared to open space areas it is possible that the blow fly species preferring trash as a food source would be higher in high intensity areas. The low intensity areas for urban locations were about double the number of open spaces but collected nearly three times the number of flies. The same relationship can be seen with the rural locations. The number of cultivated crops collection sites were double the amount developed open space sites and accounted for nearly three times more of the number of flies collected. By using the term city, it helped define a given collection area; however, it is important to recognize that each defined city had its own unique landscape. This study was affected by several limiting factors. One observation was that the age of the bait may have affected collection yields, as the odor of the bait was qualitatively different between the first time it was used and subsequent sampling events. Although there were flies captured during all trapping dates, if the odor profiles change with bait age that may affect the 24 species that are attracted. Additionally, this was a one summer survey, with flies collected only once per month. To make more broadly applicable conclusions, future studies could increase fly sampling frequency to weekly events over multiple years. This would allow for finer temporal scale evaluation of how Calliphoridae communities change over time and with landscape, both aspects that would be useful for future forensic entomology research. The dataset established by this research could provide a helpful tool for forensic scientists in both future research and case work. The results show that depending on the time of the year, and landscape in which a body is found the insect evidence may inform about postmortem body movement and if insect colonization was delayed. For example, a hypothetical situation in Mid- Michigan could be where a body was found in an urban landscape type during the month of August, then the expectation would be that there would be a higher abundance of L. sericata than P. regina. If more than 70% of the Calliphoridae specimens collected were P. regina it could point to the possibility that the body was moved from a more rural environment. To some extent this was observed during the fly identification process of the research project. When blindly provided specimens from a random sample the landscape type of the sample could be routinely predicted. While highly qualitative, this observation supports the potential that this database holds promise for future research and importance to forensic investigations; however, additional studies are need that could be designed to test and validate this potential application. 25 CONCLUSION 26 In summary, this study provided the first Calliphoridae survey across an urban to rural landscape change for the state of Michigan and the Great Lakes region, including a list of blow fly species and the communities vary over summer months and to urban to rural landscape types. Further, this baseline assessment will help provide a resource as regional temperatures change either seasonally or long term. It will also provide an initial community assessment of the indigenous Calliphoridae species in the event of potential non-indigenous Calliphoridae species invasion into Michigan. Although this study has provided important baseline survey information that has potential use in future forensic investigations, several questions regarding Calliphoridae dispersion remain. The fact that P. regina is found more in rural landscape types has been suggested by previous research as being driven by food source selection, but it is possible that rural areas provide more land cover that keep ambient temperatures cooler. Additionally, it is not known how late into the fall L. sericata will continue to increase in relative abundance, or an understanding of how many of the adult Calliphoridae species respond to both acute and chronic changes in temperature. These are new and exciting areas for future research. There are also a wide range of studies that could use this new baseline adult Calliphoridae survey to develop and test new hypothesis and answer applied research questions related to forensic entomology. While insects have been used in many cases to assist in estimating a postmortem (or post-colonization) interval, one of the driving ideas behind this research was to provide a species list of Calliphoridae that could potentially be used to determine if a body had been moved after death. In this scenario, if a body that is recovered in an urban environment is heavily colonized by a species predominately found in rural landscapes, additional investigation may be warranted. However, a regional baseline understanding of 27 Calliphoridae diversity and distribution over time and related to landscape was needed. It will also be important to replicate this study not only in Michigan but other areas of the United States as well to provide a broader representation of how these species change by region and by multiple seasons. There is potential that the Great Lakes have prevented some species from entering Michigan by creating a natural barrier, and influencing the states weather patterns. Future studies should also evaluate every month of the year to provide a better understanding of when Calliphoridae species enter diapause. As research continues on the distribution and abundances of Calliphoridae communities more information will become available that can potentially be useful in the forensic sciences. 28 APPENDICES 29 APPENDIX A TABLES 30 Table 1. Total overall number of Calliphoridae adult specimens captured during the summer of 2017 for each city location and site in urban and rural locations. NA indicates that there was not a rural location sampled for Lansing, MI. *Traps that experienced a failure during the collection period. ** Sample was lost after collection was completed. June July August Total City Charlotte DeWitt Grand Ledge Lansing Mason Perry Williamston Total Site North 1,010 432 South 485 East 235 West 410 North 114 South 430 East 39* West North 417 South 1,276 623 East 722 West 161 North 416 South 414 East 221 West 153 North 322 South East 431 West 1,705 353 North 51* South East 41 42 West 400 North South 278 619 East West 431 Urban Rural Urban Rural Urban Rural 671 37* 1,483 107 753 504 829 1,756 164 674 4,886 387 NA NA NA NA 400 0** 100 85 344 225 605 443 567 476 541 332 Site 4,156 2,496 5,302 2,247 3,792 4,109 2,706 5,133 4,183 6,821 8,350 2,883 3,866 1,563 1,328 1,272 1,613 2,229 3,331 3,550 3,082 4,212 2,616 3,655 2,595 3,534 3,038 3,371 12,231 12,540 16,378 21,759 17,756 16,369 97,033 207 784 2,283 169 844 2,197 857 1,999 542 2,292 467 699 NA NA NA NA 240 204 362 483 323 1,102 1,092 1,351 755 936 1,122 449 1,018 649 391 88 618 615 159 201 1,472 957 706 615 3,011 617 782 821 268 541 612 718 141 662 321 441 438 142 296 456 60 375 197 1,199 210 354 334 796 37* 857 79 74 NA NA NA NA 387 734 1,801 233 27* 574 148 971 170 1,416 175 1,332 1,190 219 463 449 957 325 97 342 1,551 765 1,589 386 694 530 132 230 165 428 25 326 1,894 1,598 409 407 265 286 285 371 City 14,201 15,740 22,237 8,029 10,723 13,565 12,538 31 Table 2. Calliphoridae species mean (SE) percent relative abundance in Mid-Michigan by city over the months of June, July, and August 2017. For Charlotte, Dewitt, Grand Ledge, Perry and Williamston N=24, Lansing N=12, and Mason N=23. Charlotte DeWitt Grand Ledge Lansing Mason Perry Williamston Calliphora vicina 0.00 (0.00) 0.04 (0.04) 0.00 (0.00) 0.25 (0.18) 0.04 (0.04) 0.38 (0.33) 0.00 (0.00) Calliphora vomitoria 0.00 (0.00) 0.38 (0.21) 0.00 (0.00) 0.17 (0.17) 0.00 (0.00) 0.08 (0.08) 0.00 (0.00) Chrysomya rufifacies 0.35 (0.14) 0.63 (0.24) 0.5 (0.28) 0.08 (0.08) 0.47 (0.19) 0.08 (0.06) 0.33 (0.22) Cochliomyia macellaria 4.18 (1.21) 3.47 (1.06) 5.46 (1.48) 3.17 (1.3) 4.53 (1.33) 2.08 (0.63) 4.67 (1.59) Cynomya cadaverina 0.00 (0.00) 0.17 (0.10) 0.00 (0.00) 0.25 (0.25) 0.00 (0.00) 0.5 (0.38) 0.04 (0.04) Lucilia coeruleiviridis 0.96 (0.29) 2.16 (0.59) 2.46 (1.28) 1.33 (0.58) 1.00 (0.25) 2.07 (1.11) 0.21 (0.12) Lucilia illustris 2.82 (0.81) 7.14 (1.26) 2.03 (0.43) 1.25 (0.43) 3.47 (0.79) 7.62 (1.34) 2.25 (0.43) Lucilia sericata 30.68 (6.14) 12.99 (3.66) 14.1 (14.08) 28.6 (0.35) 18.91 (4.59) 18.9 (4.25) 12.3 (12.3) Lucilia silvarum 0.00 (0.00) 0.13 (0.09) 0.04 (0.04) 0.00 (0.00) 0.35 (0.19) 0.13 (0.13) 0.04 (0.040 Phormia regina 61.00 (6.63) 72.9 (4.48) 75.2 (3.68) 55.7 (7.79) 68.5 (5.72) 68.1 (5.01) 79.4 (79.4) Protophormia terraenovae 0.00 (0.00) 0.04 (0.04) 0.29 (0.11) 9.25 (2.35) 2.74 (2.74) 0.04 (0.04) 0.83 (0.750 32 Table 3. The overall monthly mean (SE) relative abundance of Calliphoridae collected in Mid-Michigan over the 2017 summer. In June and July all species were represented by N = 52, while for August it was N = 51. June July August Species Urban Rural Urban Rural Urban Rural Calliphora vicina 0.11 (0.08) 0.33 (0.33) 0.04 (0.04) 0.00 (0.00) 0.04 (0.04) 0.04 (0.04) Calliphora vomitoria 0.21 (0.13) 0.25 (0.18) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.04 (0.04) Chrysomya rufifacies 0.00 (0.00) 0.00 (0.00) 0.04 (0.04) 0.04 (0.04) 0.92 (0.20) 1.28 (0.36) Cochliomyia macellaria 0.25 (0.10) 0.17 (0.10) 2.86 (0.63) 5.08 (1.06) 4.11 (0.59) 12.64 (1.90) Cynomya cadaverina 0.11 (0.11) 0.04 (0.04) 0.08 (0.08) 0.04 (0.04) 0.04 (0.04) 0.52 (0.40) Lucilia coeruleiviridis 1.09 (0.30) 1.33 (0.70) 0.86 (0.22) 0.38 (0.22) 1.47 (0.35) 3.95 (1.61) Lucilia illustris 3.44 (0.83) 4.41 (0.94) 3.47 (0.73) 4.21 (1.03) 2.79 (0.51) 6.11 (1.58) Lucilia sericata 9.27 (1.61) 3.70 (0.84) 27.51 (4.02) 7.38 (2.42) 45.40 (4.26) 15.05 (4.02) Lucilia silvarum 0.11 (0.08) 0.00 (0.00) 0.14 (0.14) 0.00 (0.00) 0.07 (0.05) 0.30 (0.16) Phormia regina 84.37 (1.85) 89.56 (1.92) 62.43 (4.57) 82.83 (3.11) 43.89 (3.56) 57.46 (5.62) Prorophormia terraenovae 1.04 (0.50) 0.21 (0.17) 2.57 (1.12) 0.04 (0.04) 1.29 (0.95) 2.61 (2.47) 33 Table 4. Two-way ANOVA statistics that tested Calliphoridae diversity by both month and landscape type during the summer of 2017. Landscape type Month Landscape:Month Residuals Pr(>F) Degree of Freedom Sum of squares Mean sum of squares F-value 10.534 0.001 ** 51.337 < 2e-16 *** 0.017 * 4.162 0.643 6.264 0.508 9.090 0.642 3.131 0.253 0.061 1 2 2 149 Table 5. Permutational multivariate analysis of variance (PERMANOVA) of Calliphoridae species from the cities of: Charlotte, DeWitt, Grand Ledge, Lansing, Mason, Perry, and Williamston in Mid-Michigan during the summer of 2017. Bolded text indicates significant effects. R2 P - value 0.09299 0.001*** 0.08815 0.002** 0.20654 0.001*** 0.02769 0.161 0.03678 0.001*** 0.0707 0.055 0.03149 0.701 0.44566 1 Landscape City Month Landscape:City Landscape:Month City:Month Landscape:City:Month Residuals Total Degree of freedom 1 6 2 5 2 12 10 116 154 Sum of squares 1.2448 1.18 2.7648 0.3707 0.4924 0.9465 0.4215 5.9659 13.3866 Mean sum of squares 1.24479 0.19666 1.3824 0.07414 0.24618 0.07887 0.04215 0.05143 F. Model 24.2034 3.8239 26.8791 1.4416 4.7866 1.5336 0.8196 34 Table 6. Tukey’s Honest Significant Difference test of the four major Calliphoridae species (C. macellaria, L. sericata, L.illustris, P. regina) collected in Mid-Michigan and compared among months of the 2017 summer. Only pairwise comparisons that were found significant with alpha at or below 0.05 are given. Species Month Diff Lower Upper P-adj Lucilia sericata JUL-AUG 0.0002 Lucilia sericata JUN-AUG -0.3669 -0.4616 -0.2723 < 0.0001 Lucilia sericata JUN-JUL -0.1630 -0.2576 -0.0684 -0.2039 -0.2981 -0.1098 < 0.0001 Phormia regina JUL-AUG 0.2472 0.1362 0.3582 < 0.0001 Phormia regina JUN-AUG 0.4259 0.3149 0.5368 < 0.0001 Phormia regina JUN-JUL Cochliomyia macellaria JUL-AUG 0.0020 Cochliomyia macellaria JUN-AUG -0.2122 -0.2608 -0.1636 < 0.0001 Cochliomyia macellaria JUN-JUL Lucilia coeruleiviridis JUL-AUG -0.1407 -0.1891 -0.0924 < 0.0001 -0.0715 -0.1201 -0.0229 -0.0623 -0.1024 -0.0221 0.1787 0.0682 0.2891 0.0006 0.0010 Table 7. Tukey’s Honest Significant Difference test of the predominant Calliphoridae species collected in Mid-Michigan and compared between landscape types during the summer of 2017. Only pairwise comparisons that were found significant with alpha at or below 0.05 are given. Species Landscape Lower Upper Cochliomyia macellaria Urban - Rural -0.0669 -0.1001 -0.0338 Phormia regina Urban - Rural -0.1180 -0.1937 -0.0422 0.0025 Lucilia sericata Urban - Rural 0.2664 0.2018 0.3310 < 0.0001 0.0001 P-adj Diff 35 Supp. Table S1. A summary of previous adult blow fly studies assed by location, length of study, bait type, and landscape type. Citation Baumgartner 1988 Baumgartner and Greenberg 1985 Berg and Benbow 2011 Brundage et al. 2011 Grassberger and Frank 2004 Gruner et al. 2007 Pastula and Merritt 2013 Payne 1965 Sabanoğlu and Sert 2010 Schoof et al. 1956 Tullis and Goff 1987 Weidner et al. 2017 Zabala et al. 2014 Zurawski et al. 2009 Number of Months Carrion Source Landscape Type 3 36 Rats Fish, liver and pigs 5 (over 2 years) Pig carcasses Urban Rural Rural Beef liver Urban, rural, riparian Location Illinois Peru Ohio California Austria Florida Michigan 24 7 24 3 South Carolina 6 (over 2 years) China New York, Michigan, Kansas, Arizona Hawaii New Jersey Western Europe 12 5 4 12 12 Michigan 6 (over 2 years) Pig carcasses Pig carcasses Pig carcasses Pig carcasses Pig carcasses Pig carcasses Chicken and fish Pig carcasses Pig carcasses Pig liver Urban Rural Rural Rural Rural Urban Rural Urban Urban, rural Rural 36 Supp. Table S2. Urban to Rural Landscape type research sites, land use, and GPS locations from the summer of 2017. Location Cover Class North West Heading (42o) Heading (084o) 33.738 34.055 34.394 40.184 33.305 27.406 33.738 34.055 34.394 40.184 33.305 56.666 46.439 47.061 50.578 50.511 45.127 45.076 45.754 51.001 44.743 41.301 45.198 45.051 43.995 44.400 43.618 43.890 34.718 34.589 35.286 40.174 34.292 28.820 34.779 34.828 49.546 50.305 50.060 55.279 49.118 43.481 49.630 42.598 50.170 51.412 50.133 50.753 49.630 42.598 50.170 51.412 50.133 34.080 34.139 33.702 34.952 44.184 44.077 37.829 44.824 45.473 44.747 44.665 45.369 51.920 32.557 33.281 33.320 33.947 25.785 18.032 26.528 27.400 26.643 27.012 27.350 34.939 12.787 05.178 13.241 13.888 13.116 12.764 City Charlotte Charlotte Charlotte Charlotte Charlotte Charlotte Charlotte Charlotte Charlotte Charlotte Charlotte DeWitt DeWitt DeWitt DeWitt DeWitt East Urban Developed, Low Intensity East Rural Cultivated Crops North Urban Developed, Low Intensity North Rural Developed, Open Space South Urban Developed, Medium Intensity South Rural Developed, Open Space East Urban Developed, Low Intensity East Rural Cultivated Crops North Urban Developed, Low Intensity North Rural Developed, Open Space South Urban Developed, Medium Intensity North Rural Cultivated Crops South Urban Developed, Open Space South Rural Developed, Open Space West Urban Developed, Open Space West Rural Cultivated Crops Grand Ledge East Urban Developed, Open Space Grand Ledge East Rural Developed, Low Intensity Grand Ledge North Urban Developed, Low Intensity Grand Ledge North Rural Cultivated Crops Grand Ledge South Urban Developed, Open Space Grand Ledge South Rural Cultivated Crops Grand Ledge West Urban Developed, Low Intensity Grand Ledge West Rural Cultivated Crops Lansing Lansing Lansing Lansing Mason Mason Mason Mason Mason Mason Mason Mason Perry Perry Perry Perry Perry Perry East Urban Developed, High Intensity North Urban Developed, Medium Intensity South Urban Developed, High Intensity West Urban Developed, Low Intensity East Urban Developed, Low Intensity East Rural Hay/Pasture North Urban Developed, Low Intensity North Rural Developed, Low Intensity South Urban Developed, Low Intensity South Rural Developed, Low Intensity West Urban Developed, Low Intensity West Rural Deciduous Forest East Rural Cultivated Crops East Urban Developed, Low Intensity North Rural Developed, Open Space North Urban Developed, Medium Intensity South Urban Developed, Low Intensity South Rural Cultivated Crops 37 Supp. Table S3. Calliphoridae species in Mid-Michigan expressed as relative abundance by location during the summer months of June, July, and August in 2017. Location Species Phormia regina Cochliomyia macellaria Lucilia sericata Lucilia illustris Chrysomya rufifacies Lucilia coeruleiviridis Calliphora vicina Cynomya cadaverina Lucilia silvarum Prorophormia terraenovae Calliphora vomitoria Phormia regina Lucilia sericata Cochliomyia macellaria Lucilia illustris Lucilia coeruleiviridis Calliphora vomitoria Chrysomya rufifacies Prorophormia terraenovae Calliphora vicina Cynomya cadaverina Lucilia silvarum Phormia regina Lucilia sericata Lucilia illustris Cochliomyia macellaria Prorophormia terraenovae Lucilia coeruleiviridis Chrysomya rufifacies Calliphora vomitoria Lucilia silvarum Cynomya cadaverina Calliphora vicina Phormia regina Lucilia sericata Lucilia illustris North Rural (N=18) South Rural (N=17) East Rural (N=18) West Rural N=18 Mean (%) SE (%) 4.26 1.97 1.61 1.58 0.36 0.32 0.44 0.12 0.12 0.12 0.11 5.94 4.15 2.35 0.85 0.06 0.06 0.06 0.06 0.00 0.00 0.00 6.28 3.47 1.56 1.50 3.16 1.74 0.20 0.22 0.17 0.08 0.06 5.33 3.60 1.36 80.52 6.89 6.37 3.98 0.61 0.57 0.44 0.17 0.17 0.17 0.11 80.18 7.66 7.42 3.30 1.22 0.06 0.06 0.06 0.00 0.00 0.00 72.29 9.54 6.56 4.28 3.44 2.94 0.33 0.22 0.22 0.11 0.06 74.74 10.85 5.60 38 Supp. Table S3. (cont’d) West Rural N=18 North Urban N=18 South Urban N=21 East Urban N=21 Cochliomyia macellaria 4.97 1.62 Lucilia coeruleiviridis 2.65 1.46 Chrysomya rufifacies 0.69 0.33 Cynomya cadaverina 0.50 0.50 Calliphora vicina 0.00 0.00 Calliphora vomitoria 0.00 0.00 Lucilia silvarum 0.00 0.00 Prorophormia terraenovae 0.00 0.00 Phormia regina 63.59 4.91 Lucilia sericata 26.81 4.74 Lucilia illustris 3.03 0.44 Prorophormia terraenovae 2.86 1.64 Cochliomyia macellaria 2.00 0.61 Lucilia coeruleiviridis 1.33 0.34 Chrysomya rufifacies 0.24 0.12 Lucilia silvarum 0.10 0.10 Calliphora vomitoria 0.05 0.05 Calliphora vicina 0.00 0.00 Cynomya cadaverina 0.00 0.00 Phormia regina 64.98 5.48 Lucilia sericata 27.38 5.58 Lucilia illustris 3.41 1.00 Cochliomyia macellaria 2.43 0.60 Lucilia coeruleiviridis 0.71 0.30 Prorophormia terraenovae 0.62 0.36 Chrysomya rufifacies 0.24 0.14 Calliphora vicina 0.14 0.10 Calliphora vomitoria 0.10 0.10 Cynomya cadaverina 0.00 0.00 Lucilia silvarum 0.00 0.00 Phormia regina 62.60 5.09 Lucilia sericata 27.35 5.14 Lucilia illustris 3.10 0.89 Cochliomyia macellaria 2.57 0.65 Prorophormia terraenovae 2.00 1.01 Lucilia coeruleiviridis 1.25 0.35 Chrysomya rufifacies 0.49 0.24 Lucilia silvarum 0.29 0.20 39 Supp. Table S3. (cont’d) East Urban N=21 West Urban N=21 Cynomya cadaverina 0.15 0.11 Calliphora vomitoria 0.14 0.14 Calliphora vicina 0.05 0.05 Phormia regina 63.10 6.42 Lucilia sericata 28.02 5.46 Lucilia illustris 3.40 0.83 Cochliomyia macellaria 2.63 0.84 Lucilia coeruleiviridis 1.27 0.37 Prorophormia terraenovae 1.05 0.67 Chrysomya rufifacies 0.30 0.14 Cynomya cadaverina 0.14 0.14 Calliphora vicina 0.05 0.05 Lucilia silvarum 0.05 0.05 Calliphora vomitoria 0.00 0.00 40 Supp. Table S4. Shannon Diversity Index of adult Calliphoridae distributed amongst seven cities in Mid-Michigan during the summer of 2017. City Month Landscape Type Shannon Diversity DeWitt AUG DeWitt AUG Perry Mason AUG JUL DeWitt AUG Charlotte AUG DeWitt AUG Lansing AUG DeWitt AUG Grand Ledge AUG Lansing JUL Mason AUG Charlotte JUL Grand Ledge AUG Perry Lansing JUN JUL Mason AUG Mason Perry Mason JUL AUG AUG DeWitt AUG Charlotte AUG Williamston AUG Lansing AUG Williamston JUL Perry AUG Grand Ledge AUG DeWitt AUG Mason Mason Perry JUL AUG AUG Lansing AUG Lansing JUL Mason AUG Grand Ledge AUG Rural Rural Rural Urban Rural Rural Urban Urban Urban Urban Urban Rural Rural Urban Rural Urban Rural Urban Rural Urban Urban Urban Urban Urban Urban Rural Rural Rural Urban Urban Urban Urban Urban Urban Urban 1.6755 1.4415 1.4041 1.3971 1.3274 1.2671 1.2365 1.2112 1.1891 1.1759 1.1625 1.1517 1.1269 1.1243 1.1234 1.1198 1.1070 1.0994 1.0835 1.0755 1.0689 1.0496 1.0421 1.0312 1.0016 1.0011 1.0008 0.9830 0.9779 0.9705 0.9657 0.9651 0.9606 0.9428 0.9424 41 Supp. Table S4. (cont’d) Mason AUG Williamston JUL Lansing Lansing JUN JUN Charlotte AUG Williamston AUG Grand Ledge JUN Perry JUN Charlotte AUG Lansing AUG Charlotte JUL Perry AUG Williamston AUG Grand Ledge AUG Mason Perry Mason JUL JUL JUL Grand Ledge AUG Perry JUL Williamston AUG Williamston AUG Grand Ledge AUG Charlotte JUL Charlotte AUG Perry JUL Williamston AUG Williamston JUN Perry JUL Grand Ledge JUL Grand Ledge AUG Perry Perry AUG JUL Grand Ledge JUL Charlotte AUG Grand Ledge JUL Williamston JUL Lansing JUL Urban Urban Urban Urban Urban Rural Urban Rural Rural Urban Urban Rural Rural Urban Urban Urban Rural Rural Rural Urban Urban Rural Urban Urban Rural Urban Urban Urban Urban Rural Urban Rural Urban Rural Urban Urban Urban 0.9345 0.9327 0.9323 0.9193 0.9139 0.9135 0.8997 0.8985 0.8951 0.8854 0.8831 0.8761 0.8574 0.8540 0.8513 0.8426 0.8325 0.8321 0.8252 0.8195 0.8191 0.8134 0.8130 0.8130 0.8055 0.8033 0.7950 0.7906 0.7896 0.7868 0.7822 0.7734 0.7700 0.7684 0.7563 0.7543 0.7515 42 Supp. Table S4. (cont’d) DeWitt AUG Perry AUG Charlotte JUN Grand Ledge JUL Perry DeWitt Perry Perry DeWitt Charlotte DeWitt Charlotte DeWitt JUN JUN AUG JUN JUL JUN JUL JUN JUL Mason AUG Mason DeWitt DeWitt JUN JUN JUL Williamston JUL Grand Ledge JUN Charlotte Charlotte DeWitt Mason JUL JUL JUN JUN Williamston JUL Grand Ledge JUN DeWitt JUL Williamston JUL DeWitt JUN Williamston JUL Grand Ledge JUL Grand Ledge JUL Williamston AUG DeWitt JUN Williamston JUN Charlotte AUG Mason Mason JUL JUL Urban Urban Urban Urban Urban Rural Urban Urban Rural Urban Urban Rural Urban Rural Urban Urban Urban Rural Urban Rural Urban Urban Rural Urban Urban Rural Rural Rural Rural Rural Rural Rural Urban Urban Rural Rural Rural 0.7479 0.7432 0.7368 0.7144 0.7054 0.6880 0.6860 0.6491 0.6400 0.6398 0.6350 0.6307 0.6294 0.6291 0.6256 0.6244 0.6219 0.6117 0.6066 0.5925 0.5917 0.5875 0.5837 0.5654 0.5651 0.5560 0.5542 0.5506 0.5498 0.5486 0.5455 0.5430 0.5299 0.5138 0.5112 0.5067 0.5042 43 Supp. Table S4. (cont’d) Grand Ledge JUN Perry Lansing JUL JUN Charlotte AUG Charlotte Perry Charlotte DeWitt Perry Charlotte JUN JUN JUN JUL JUL JUL Grand Ledge JUL DeWitt Charlotte Mason Perry JUL JUN JUN JUN Grand Ledge JUL DeWitt Mason Charlotte Perry DeWitt Mason JUN JUN JUL JUN JUN JUN Williamston JUN Williamston JUL Grand Ledge JUN Lansing Perry Perry DeWitt Mason JUN JUN JUL JUN JUN Williamston JUN Williamston AUG Grand Ledge JUN Williamston JUN DeWitt Charlotte Mason JUL JUN JUL Rural Urban Urban Urban Rural Urban Rural Rural Urban Urban Rural Urban Urban Urban Urban Rural Urban Urban Rural Rural Rural Rural Rural Rural Urban Urban Rural Rural Rural Rural Rural Rural Rural Urban Rural Rural Rural 0.5040 0.5000 0.4941 0.4896 0.4887 0.4744 0.4717 0.4664 0.4531 0.4422 0.4280 0.4216 0.4210 0.4210 0.4196 0.4149 0.4119 0.4053 0.3862 0.3746 0.3576 0.3567 0.3508 0.3094 0.2929 0.2877 0.2877 0.2790 0.2540 0.2322 0.2270 0.2235 0.2103 0.2095 0.2095 0.1904 0.1904 44 Supp. Table S4. (cont’d) Williamston JUN Charlotte Mason JUL JUN Grand Ledge JUN Charlotte JUN Williamston JUN Mason JUN Williamston JUN Grand Ledge JUN Urban Rural Rural Rural Urban Rural Urban Rural Rural 0.1679 0.1677 0.1119 0.1119 0.0980 0.0980 0.0560 0.0560 0.0000 45 Supp. Table S5. Tukey’s Honest Significant Difference test for pairwise comparisons of the predominant Calliphoridae species evaluated by city collected in Mid-Michigan during the summer of 2017. Only results are displayed where p-values were considered significant with alpha at or below 0.05. Species Phormia regina Williamston-Lansing Phormia regina Williamston-Charlotte City Diff Lower Upper P-adj 0.2971 0.0455 0.5487 0.0100 0.2188 0.0134 0.4242 0.0289 Lucilia coeruleiviridis Williamston-Grand Ledge - - -0.0848 0.1591 0.0105 0.0146 Lucilia illustris DeWitt-Charlotte 0.1102 0.0197 0.2008 0.0069 Lucilia illustris Grand Ledge-DeWitt Lucilia illustris Lansing-DeWitt Lucilia illustris Williamston-DeWitt - - -0.1213 0.2119 0.0308 0.0019 - - -0.1376 0.2485 0.0267 0.0055 - - -0.0977 0.1883 0.0072 0.0255 Lucilia illustris Perry-Grand Ledge 0.1009 0.0104 0.1915 0.0185 Lucilia illustris Perry-Lansing 0.1172 0.0063 0.2281 0.0310 - - -0.2367 0.4119 0.0615 0.0017 - - -0.2420 0.4172 0.0668 0.0012 - - -0.2389 0.4140 0.0637 0.0015 2.7794 e-1 0.1858 0.3700 0.0001 2.7376 e-1 0.1817 0.3658 0.0001 < < 2.5116 e-1 0.1591 0.3432 -2.2581 e- - - < 0.0001 < 1 0.3186 0.1331 0.0001 - - < -2.7377 0.3186 0.1817 0.0001 - - < -2.5133 0.3434 0.1593 0.0001 Lucilia sericata DeWitt-Charlotte Lucilia sericata Grand Ledge-Charlotte Lucilia sericata Williamston-Charlotte Protophormia terraenovae Protophormia terraenovae Protophormia terraenovae Protophormia terraenovae Protophormia terraenovae Protophormia terraenovae Lansing- Charlotte Lansing-DeWitt Lansing- Grand Ledge Mason-Lansing Perry-Lansing Williamston-Lansing 46 Supp. Table S6. Average daily temperatures in degree Celsius for the Mid-Michigan area during the summer of 2017. Temperature data was recorded using the Capital City Weather Station at the Capitol Regional Airport (LAN), Lansing, MI 48906. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 June July Aug 22 25 23 17 18 19 20 19 22 22 22 20 19 23 23 23 25 21 22 22 26 21 17 14 14 17 20 17 19 20 16 24 23 23 22 23 26 26 22 22 23 26 26 26 23 22 23 22 24 26 26 24 24 24 20 19 23 23 19 19 21 22 17 18 21 24 17 17 18 19 23 24 28 29 28 27 26 24 26 23 21 19 19 24 23 20 18 17 17 19 24 25 47 APPENDIX B FIGURES 48 Figure 1. Urban and Rural research site locations and surrounding land cover types in Mid- Michigan. City populations as indicated by the 2010 United States Census Bureau. Land use types defined by using Geographic Information System (GIS). 49 Urban Landscape by Cover Class s e t i S f o r e b m u N 18 16 14 12 10 8 6 4 2 0 Developed, Low Intensity Developed, Open Space Developed, Medium Developed, High Intensity Intensity Land Use Types Figure 2. Total land use types for urban locations in Charlotte, DeWitt, Grand Ledge, Lansing, Mason, Perry, and Williamston. Land use types defined by using Geographic Information System (GIS). 50 Rural Landscape by Cover Class s e t i S f o r e b m u N 12 10 8 6 4 2 0 Cultivated Crops Developed, Low Developed, Open Hay/Pasture Deciduous Forest Woody Wetland Intensity Space Land Use Types Figure 3. Total land use types for rural locations in Charlotte, DeWitt, Grand Ledge, Lansing, Mason, Perry, and Williamston. Land use types defined by using Geographic Information System (GIS). 51 Figure 4. Williamston South urban trap placement with Shepard’s hook, bait jar, and guide lines. 15JUN2017 (278 flies captured in this trapping event). 52 Figure 5. Williamston South rural trap after placement in field for 4 hours. Photo taken 15JUN2017 (1,416 flies captured in this trapping event). 53 Figure 6. Percent relative abundance of Calliphoridae species for urban and rural landscape types, city and month over the 2017 in Mid-Michigan. 54 Figure 7. Phormia regina mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017. Tukey’s Honest Significant Different Test: (A) *Adjusted P-value <0.0020 **Adjusted P-value <0.0001 (B) *Adjusted P-value= 0.0030 Figure 8. Lucilia sericata mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017. Tukey’s Honest Significant Different Test: (A) *Adjusted P-value <0.0020 **Adjusted P-value <0.0001 (B) *Adjusted P-value <0.0001 55 Figure 9. Shannon diversity of Calliphoridae species indicated by landscape type and month in Mid-Michigan during the summer of 2017. Kruskal Wallis Rank Sum test: p-value < 0.030* p-value < 0.001** 56 Figure 10. Principal Coordinates Analysis (PCoA) ordination of relative abundance distribution by month (A) and landscape type (b) during the summer of 2017. The ellipses represent 95% confidence intervals for the mean of each group. See PERMANOVA results for statistical tests of these factors. 57 Figure 11. Cochliomyia macellaria mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017. Tukey’s Honest Significant Different Test: (A) * Adjusted P-value <0.0020 **Adjusted P-value <0.0001 (B) *Adjusted P-value <0.0030 Figure 12. Lucilia illustris mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017. 58 Figure 13. Lucilia coeruleiviridis mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017. Figure 14. Protophormia terraenovae mean (SE) relative abundance by month with trend line (A) and landscape type (B) during the summer of 2017. 59 Supp. Figure F1. Percent of Calliphoridae species by abundance for urban and rural trapping locations and trap placement site (Cardinal direction) distinguished by city, month and location during 2017 in Mid-Michigan. 60 Supp. Figure F2. Shannon diversity of Calliphoridae species indicated by landscape change, month and city in Mid-Michigan during the summer of 2017. 61 APPENDIX C RECORD OF DEPOSITION OF VOUCHER SPECIMENS 62 RECORD OF DEPOSITION OF VOUCHER SPECIMENS The specimens listed below have been deposited in the named museum as samples of those species or other taxa, which were used in this research. Voucher recognition labels bearing the voucher number have been attached or included in fluid preserved specimens. Voucher Number: 2018-03 Author and Title of thesis: Nicholas J. Babcock Adult blow fly community structure across urban to rural landscape change in Michigan. Museum(s) where deposited: Albert J. Cook Arthropod Research Collection, Michigan State University (MSU) Specimens: Family Calliphoridae Life Stage Quantity adult Genus-Species Calliphora vomitoria 1M/1F Preservation pinned Calliphoridae Calliphora vicina adult Calliphoridae Chrysomya rufifacies adult Calliphoridae Cochliomyia macellaria adult Calliphoridae Cynomya cadaverina adult Calliphoridae Lucilia illustris Calliphoridae Lucilia sericata Calliphoridae Lucilia silvarum adult adult adult Calliphoridae Lucilia coeruleiviridis adult Calliphoridae Protophormia terraenovae adult Calliphoridae Phormia regina adult 63 1M/1F pinned 1M/1F pinned 1M/1F pinned 1M/1F pinned 1M/1F pinned 1M/1F pinned 1M/1F pinned 1M/1F pinned 1M/1F pinned 1M/1F pinned REFERENCES 64 REFERENCES Alvarez Garcia, D. M., A. Pérez-Hérazo, and E. Amat. 2017. Life History of Cochliomyia macellaria (Fabricius, 1775) (Diptera, Calliphoridae), a Blowfly of Medical and Forensic Importance. Neotropical Entomology. 46: 606–612. Amendt, J., R. Krettek, and R. Zehner. 2004. Forensic entomology. Naturwissenschaften. 91: 51–65. Amendt, J., C. P. Campobasso, E. Gaudry, C. Reiter, H. N. LeBlanc, and M. J. R. Hall. 2007. Best practice in forensic entomology—standards and guidelines. Int J Legal Med. 121: 90–104. Anderson, G. S. 2000. Minimum and Maximum Development Rates of Some Forensically Important Calliphoridae (Diptera). Journal of Forensic Sciences. 45: 14778J. Anderson, G. S. 1999. Wildlife Forensic Entomology: Determining Time of Death in Two Illegally Killed Black Bear Cubs. Journal of Forensic Sciences. 44: 14567J. Anderson, G. S., and S. L. VanLaerhoven. 1996. Initial Studies on Insect Succession on Carrion in Southwestern British Columbia. Journal of Forensic Sciences. 41: 13964J. Baumgartner, D. L., and B. Greenberg. 1985. Distribution and Medical Ecology of the Blow Flies (Diptera: Calliphoridae) of Peru. Ann Entomol Soc Am. 78: 565–587. Baumgartner, D. L. 1988. Spring Season Survey of the Urban Blowflies (Diptera: Calliphoridae) of Chicago, Illinois. 4. Benbow, M. E., J. K. Tomberlin, and A. M. Tarone. 2015. Carrion Ecology, Evolution, and Their Applications. CRC Press. Benbow, M. E., A. J. Lewis, J. K. Tomberlin, and J. L. Pechal. 2013. Seasonal Necrophagous Insect Community Assembly During Vertebrate Carrion Decomposition. Journal of Medical Entomology. 50: 440–450. Benecke, M. 1998. Six Forensic Entomology Cases: Description and Commentary. Journal of Forensic Sciences. 43: 14309J. Berg, M. C., and M. E. Benbow. 2013. Environmental Factors Associated With Phormia regina (Diptera: Calliphoridae) Oviposition. J Med Entomol. 50: 451–457. Brundage, A., S. Bros, and J. Y. Honda. 2011. Seasonal and habitat abundance and distribution of some forensically important blow flies (Diptera: Calliphoridae) in Central California. Forensic Science International. 212: 115–120. 65 Byrd, J. H., and J. C. Allen. 2001. The development of the black blowy, Phormia regina (Meigen). Forensic Science International. 10. Campobasso, C. P., G. Di Vella, and F. Introna. 2001. Factors affecting decomposition and Diptera colonization. Forensic Science International, Forensic Entomology. 120: 18–27. Caruso, T., and M. Migliorini. 2006. Micro-arthropod communities under human disturbance: is taxonomic aggregation a valuable tool for detecting multivariate change? Evidence from Mediterranean soil oribatid coenoses. Acta Oecologica. 30: 46–53. Catts, E. P., and M. L. Goff. 1992. Forensic Entomology in Criminal Investigations. Annu. Rev. Entomol. 37: 253–272. Climate - United States - Monthly averages. 2018. Climate - United States - Monthly averages. https://www.usclimatedata.com/. Fisher, P., R. Wall, and J. R. Ashworth. 1998. Attraction of the sheep blowfly, Lucilia sericata (Diptera: Calliphoridae) to carrion bait in the field. Bulletin of Entomological Research. 88: 611. Galloway, A., W. H. Birkby, A. M. Jones, T. E. Henry, and B. O. Parks. 1989. Decay Rates of Human Remains in an Arid Environment. Journal of Forensic Sciences. 34: 12680J. Goddard, J., and P. K. Lago. 1985. Notes on blow fly (Diptera: Calliphoridae) succession on carrion in northern Mississippi. Journal of Entomological Science. 20: 312–317. Goff, M. L. 1993. Estimation of postmortem interval using arthropod development and successional patterns. Forensic Science Review. 5: 81–81. Goff, M L. 1992. Problems in estimation of postmortem interval resulting from wrapping of the corpse: a case study from Hawaii. Journal of Agricultural Entomology. 9(4): 237-243. Goff, M. L., A. I. Omori, and K. Gunatilake. 1988. Estimation of Postmortem Interval by Arthropod Succession: Three Case Studies from the Hawaiian Islands. The American Journal of Forensic Medicine and Pathology. 9: 220–225. Goff, M. L. 2009. Early post-mortem changes and stages of decomposition in exposed cadavers. Experimental & applied acarology. 49: 21–36. Grassberger, M., and C. Frank. 2004. Initial Study of Arthropod Succession on Pig Carrion in a Central European Urban Habitat. J Med Entomol. 41: 511–523. Gruner, S. V., D. H. Slone, and J. L. Capinera. 2007. Forensically Important Calliphoridae (Diptera) Associated with Pig Carrion in Rural North-Central Florida. J Med Entomol. 44: 509–515. Kurahashi, H., and M. Afzal. 2002. The blow flies recorded from Pakistan, with the description of one new species: Diptera: Calliphoridae. Med. Entomol. Zool. 53: 213–230. 66 Mariluis, J. C., J. A. Schnack, P. P. Mulieri, and L. D. Patitucci. 2008. Calliphoridae (Diptera) from wild, suburban, and urban sites at three Southeast Patagonian localities. Revista de la Sociedad Entomológica Argentina. 67: 107–114. Marshall, S.A., Whitworth, T. and Roscoe, L. 2011. Blow flies (Diptera; Calliphoridae) of eastern Canada with a key to Calliphoridae subfamilies and genera of eastern North America, and a key to the eastern Canadian species of Calliphorinae, Luciliinae and Chrysomyiinae. Canadian Journal of Arthropod Identification No. 11. McArdle, B. H., and M. J. Anderson. 2001. Fitting Multivariate Models to Community Data: A Comment on Distance-Based Redundancy Analysis. Ecology. 82: 290–297. Mello, R. da S., and V. M. Aguiar-Coelho. 2009. Durations of immature stage development period of Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) under laboratory conditions: implications for forensic entomology. Parasitol Res. 104: 411–418. Nolte, K. B., R. D. Pinder, and W. D. Lord. 1992. Insect Larvae Used to Detect Cocaine Poisoning in a Decomposed Body. Journal of Forensic Sciences. 37: 13304J. OConnor, B. M. 2009. Astigmatid mites (Acari: Sarcoptiformes) of forensic interest. Experimental and Applied Acarology. 49: 125–133. Pastula, E. C., and R. W. Merritt. 2013. Insect Arrival Pattern and Succession on Buried Carrion in Michigan. Journal of Medical Entomology. 50: 432–439. Payne, J. A. 1965. A Summer Carrion Study of the Baby Pig Sus Scrofa Linnaeus. Ecology. 46: 592–602. Pechal, J. L., C. J. Schmidt, H. R. Jordan, and M. E. Benbow. 2018. A large-scale survey of the postmortem human microbiome, and its potential to provide insight into the living health condition. Scientific Reports. 8: 5724. Pechal, J. L., M. E. Benbow, T. L. Crippen, A. M. Tarone, and J. K. Tomberlin. 2014. Delayed insect access alters carrion decomposition and necrophagous insect community assembly. Ecosphere. 5: art45. Pilli, E., A. Agostino, D. Vergani, E. Salata, I. Ciuna, A. Berti, D. Caramelli, and S. Lambiase. 2016. Human identification by lice: A Next Generation Sequencing challenge. Forensic Science International. 266: e71–e78. Reed, H. B. 1958. A Study of Dog Carcass Communities in Tennessee, with Special Reference to the Insects. The American Midland Naturalist. 59: 213–245. Richards, C. S., K. A. Williams, and M. H. Villet. 2009. Predicting geographic distribution of seven forensically significant blowfly species (Diptera: Calliphoridae) in South Africa. African Entomology. 17: 170–182. 67 Sabanoğlu, B., and O. Sert. 2010. Determination of Calliphoridae (Diptera) Fauna and Seasonal Distribution on Carrion in Ankara Province. Journal of Forensic Sciences. 55: 1003–1007. Sanford, M. R. 2017. Insects and associated arthropods analyzed during medicolegal death investigations in Harris County, Texas, USA: January 2013- April 2016. 23. Schoof, H. F., E. P. Savage, and H. R. Dodge. 1956. Comparative Studies of Urban Fly Populations in Arizona, Kansas, Michigan, New York, and West Virginia Island. Seasonal Abundance of Minor Species. Annals of the Entomological Society of America. 49: 59–66. Sharanowski, B. J., E. G. Walker, and G. S. Anderson. 2008. Insect succession and decomposition patterns on shaded and sunlit carrion in Saskatchewan in three different seasons. Forensic Science International. 179: 219–240. Smith, K. G. V. 1986. A manual of forensic entomology. A manual of forensic entomology. Souza, A. M. D., and A. X. Linhares. 1997. Diptera and Coleoptera of potential forensic importance in southeastern Brazil: relative abundance and seasonality. Medical and Veterinary Entomology. 11: 8–12. Tabor, K. L., C. C. Brewster, and R. D. Fell. 2004. Analysis of the Successional Patterns of Insects on Carrion in Southwest Virginia. Journal of Medical Entomology. 41: 785–795. Tomberlin, J. K., R. Mohr, M. E. Benbow, A. M. Tarone, and S. VanLaerhoven. 2010. A Roadmap for Bridging Basic and Applied Research in Forensic Entomology. Annu. Rev. Entomol. 56: 401–421. Tullis, K., and M. L. Goff. 1987. Arthropod Succession in Exposed Carrion in a Tropical Rainforest on O’ahu Island, Hawai’i. Journal of Medical Entomology. 24: 332–339. Vass, A. A. 2011. The elusive universal post-mortem interval formula. Forensic Science International. 204: 34–40 Watson, E. J., and C. E. Carlton. 2005. Insect Succession and Decomposition of Wildlife Carcasses During Fall and Winter in Louisiana. J Med Entomol. 42: 193–203. Weatherbee, C. R., J. L. Pechal, T. Stamper, and M. E. Benbow. 2017. Post-Colonization Interval Estimates Using Multi-Species Calliphoridae Larval Masses and Spatially Distinct Temperature Data Sets: A Case Study. Insects. 8: 40. Weidner, L. M., M. D. Gemmellaro, J. K. Tomberlin, and G. C. Hamilton. 2017. Evaluation of bait traps as a means to predict initial blow fly (Diptera: Calliphoridae) communities associated with decomposing swine remains in New Jersey, USA. Forensic Science International. 278: 95–100. Zabala, J., B. Díaz, and M. I. Saloña-Bordas. 2014. Seasonal Blowfly Distribution and Abundance in Fragmented Landscapes. Is It Useful in Forensic Inference about Where a Corpse Has Been Decaying? PLoS ONE. 9: e99668. 68 Zurawski, K. N., M. E. Benbow, J. R. Miller, and R. W. Merritt. 2009. Examination of Nocturnal Blow Fly (Diptera: Calliphoridae) Oviposition on Pig Carcasses in Mid- Michigan. J Med Entomol. 46: 671–679. 69