The Ecology of Carrion Decomposition: Necrophagous Invertebrate

THE ECOLOGY OF CARRION DECOMPOSITION: NECROPHAGOUS INVERTEBRATE

ASSEMBLY AND MICROBIAL COMMUNITY METABOLIC ACTIVITY

DURING DECOMPOSITION OF SUS SCROFA CARCASSES

IN A TEMPERATE MID-WEST FOREST

Thesis

Submitted to

The College of Arts and Sciences of the

UNIVERSITY OF DAYTON

In Partial Fulfillment of the Requirements for

The Degree

Master of Science in Biology

Andrew Joseph Lewis

UNIVERSITY OF DAYTON

Dayton, OH

December, 2011

THE ECOLOGY OF CARRION DECOMPOSITION: NECROPHAGOUS INVERTEBRATE

ASSEMBLY AND MICROBIAL COMMUNITY METABOLIC ACTIVITY DURING

DECOMPOSITION OF SUS SCROFA CARCASSES IN A TEMPERATE MID-WEST

FOREST

Name: Lewis, Andrew Joseph

APPROVED BY:

______M. Eric Benbow, Ph.D. Faculty Advisor

______Carl Friese, Ph.D. Committee Member

______Ryan McEwan, Ph.D. Committee Member

______Jayne Robinson, Ph.D. Department Chairperson

ii ABSTRACT

THE ECOLOGY OF CARRION DECOMPOSITION: NECROPHAGOUS INVERTEBRATE

ASSEMBLY AND MICROBIAL COMMUNITY METABOLIC ACTIVITY

DURING DECOMPOSITION OF SUS SCROFA CARCASSES

IN A TEMPERATE MID-WEST FOREST

Name: Lewis, Andrew Joseph University of Dayton

Advisor: Dr. M. Eric Benbow

Decomposition is a fundamental process to ecosystem function and energy flow where nutrients are recycled and reintroduced into food webs. Vertebrate carrion decomposition can provide significant resource pulses for habitats and can range from large whale carcasses to small rodents. Necrophagous invertebrates have been documented to be a predominant driver of vertebrate carrion decomposition. However, microorganisms also participate in the utilization of this common food fall, microorganisms. Little is known about the structure and composition of the microbial communities associated with carrion, or if they follow a pattern of succession as decomposition progresses, although this process is important for nutrient and energy cycling in ecosystems. The objective of this study was to evaluate both the microbial and invertebrate community succession during

iii carrion decomposition. It was hypothesized that microbial and invertebrate communities on decomposing carcasses would vary both over decomposition time and between seasons, as well demonstrates inter-carcass variation among the replicates. To test these hypotheses, Sus scrofa carcasses (N=3-6) were placed in a forested habitat near Xenia, OH during spring (15 March – 8 June 2009) summer (23

July – 31 August 2009), autumn (11 November 2009 – 1 May 2010) and winter (2

February – 1 May 2010). For the microbial sample collections skin biopsies and swabs of the anus and buccal of each carcass along with cores of soil underneath and 1m away were collected to compare with microbial community metabolic succession during decomposition. Biolog EcoPlates, phenotypic microarray 96 well plates were used to monitor the differential use of 31 different carbon sources to provide a community level physiological profile (CLPPs) as a measure of microbial community metabolic activity. In addition, standardized insect samples involving aerial sweep nets, pitfall traps and hand collections were used to evaluate the arthropod communities through the entire process of carrion decomposition.

One and two-way ANOVA with Bonferroni Post-tests, non-metric multidimensional scaling (NMDS), multi-response permutation procedure (MRPP) and indicator species analysis (ISA) were employed to evaluate the microbial community metabolic activity and invertebrate community change over decomposition, between replicates, and between seasons. For the microbial communities, there were significant differences (p<0.001) between seasons for both carrion and soil samples. Carrion samples were significantly different from soil samples (p<0.001), but the control and soil under body were not (p=0.271). For the invertebrates,

iv while most taxa remained constant among seasons, five taxa demonstrated significant differences (One-way ANOVA; p<0.05) in presence across seasons.

Necrophagous insect communities had significant differences across the different stages of decomposition (MRPP; p<0.001) for each season in multivariate analyses.

According to pairwise comparisons while there were significantly different necrophagous insect communities between summer and fall (p=0.001) and summer and winter (p< 0.001), the communities were similar between fall and winter trials

(p=0. 073). Another observation was a undocumented phenomenon concerning en masse larval dispersal of the blow fly Phormia regina.. We highly recommend that future studies related to carrion decomposition increase replication of carcasses and make comparisons across seasons and year.

v DEDICATION

To my mother and father for showing me the way.

To my brothers and sister for pushing me in the right direction.

To my grandparents for providing guidance.

To my wife for keeping me going when times get rough.

To my friends who help make sure I stay sane.

And lastly to my advisor, who gave me the chance to prove myself.

vi ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Eric Benbow for providing me with the chance to go to graduate school and receive my Master’s. Thank you to my committee members, Dr. Carl Friese and Dr. Ryan McEwan for providing guidance and figuring out the best course I should take to reach my goals with school. Thanks to all of the undergraduates who helped out with the research; Tiffany Blair,

Maureen Berg, Allison Gansel, Elizabeth Gazdick, An Lai, Jessica Teater, Carolyn

Teter, Sandra Tilton, and Jon White. Thanks as well to the past and present graduate students that aided me as well; Meg Shoda, Kathy Gorbach, Ryan Kimirakaus, Jen

Lang, Mollie MacIntosh, Jennifer Pechal, and Jennifer Rosati. I greatly appreciate all the other professors and graduate students for teaching me and lending a helping hand or sound advice when needed. Thanks to my parents Alice and Kevin Lewis and my wife Caitlin Powell for going out into the field with me when I needed help and for always being willing to do some proof reading. I am also grateful to the

Greene County Parks District for allowing me to use the Morris Bean Reserve for my research. Lastly, thanks to the University of Dayton for providing me with the education and ability to perform this research and obtain my degree.

vii TABLE OF CONTENTS

ABSTRACT………………………………………………………………………………………………………….iii

LIST OF ILLUSTRATIONS……………………………………………………………………………………..ix

LIST OF TABLES…………………………………………………………………………………………………xii

CHAPTER 1: Microbial Metabolic Community Change During Vertebrate Carrion Decomposition……………………………………………………………………………………………………..1

ABSTRACT……………………………………………………………………………………...... 1 INTRODUCTION…………………………………………………………………………………...2 METHODS……………………………………………………………………………………………8 RESULTS……………………………………………………………………………………………15 DISCUSSION……………………………………………………………………………………….18

CHAPTER 2: Necrophagous Insect Community Assembly During Vertebrate Carrion Decomposition: Seasonal and Inter-Carcass Variation..………………...... 34

ABSTRACT………………………………………………………………………………………...34 INTRODUCTION…………………………………………………………………………………36 METHODS………………………………………………………………………………………….39 RESULTS……………………………………………………………………………………………44 DISCUSSION……………………………………………………………………………………….47

CHAPTER 3: When Entomological Evidence Crawls Away: Phormia regina En Masse Larval Dispersal..………………………………………………………………………………………………..62

ABSTRACT…………………………………………………………………………………………62 INTRODUCTION…………………………………………………………………………………63 METHODS………………………………………………………………………………………….66 RESULTS……………………………………………………………………………………………68 DISCUSSION……………………………………………………………………………………….71

BIBLIOGRAPHY………………………………………………………………………………………………….84

viii LIST OF ILLUSTRATIONS

Chapter 1

Figure 1: Mean (SD) non-normalized (A) and normalized (B) carrion microbial metabolic community activity during each season compared to non-normalized (C) and normalized (D) activity for soil underneath (Under) and 1 m away from the carcass (Control). Each figure shows the overall microbial community metabolic activity for each date, standardized for ADH, through the process of decomposition in each season. In Figure 1A, the number beside each point represents the number of days since death for each sampling date in each season. ………………………………….22

Figure 2: Non-metric multidimensional scaling ordinations of microbial community level physiological profiles for carrion communities (A) during decomposition time is represented by ADH ranges and among seasons (B), and soil communities during decomposition (C) represented by ADH ranges and among seasons (D). The MRPP statistics indicate significant differences where p<0.05, and the amount of variation explained by each axis is also provided in the axes labels…………………………………….23

Figure 3: Non-metric multidimensional scaling ordinations of microbial community level physiological profiles for carrion communities over decomposition time represented as ADH during each season: A) spring, B) summer, C) autumn, and D) winter. The MRPP statistics indicate significant differences where p<0.05, and the amount of variation explained by each axis is also provided in the axes labels……...24

Figure 4: Non-metric multidimensional scaling ordinations of microbial community level physiological profiles for soil communities over decomposition time represented as ADH during each season; A) spring, B) summer, C) autumn, and D) winter. The MRPP statistics indicate significant differences where p<0.05, and the amount of variation explained by each axis is also provided in the axes labels……...25

ix Figure 5: Mean (SD) normalized microbial metabolic activity of each carbon source for carrion communities during A) spring, B) summer, C) autumn, and D) winter over decomposition time represented as ranges in ADH.…………………………………...... 26

Figure 6: Mean (SD) normalized microbial metabolic activity of each carbon source for soil communities under each carcass during A) spring, B) summer, C) autumn, and D) winter over decomposition time represented as ranges in ADH.………………..27

Figure 7: Mean (SD) normalized microbial metabolic activity of each carbon source for soil communities 1 m away from each carcass during A) spring, B) summer, C) autumn, and D) winter over decomposition time represented as ranges in ADH.…..28

Chapter 2

Figure 1: Mean (SD) necrophagous community taxa richness across decomposition expressed as ADH for each season. The duration of each seasonal trial, expressed as number of days, is given adjacent to the highest ADH datum…………………………...…...52

Figure 2: Shannon Diversity Index for each sampling date during decomposition expressed as ADH……………………………………………………………………………………………....53

Figure 3: Adult insect mean percent composition of the five dominant taxa for every sampling date (expressed as ADH) in each season; (A) spring, (B) summer, (C) autumn, and (D) winter………………………………………………………………………………………54

Figure 4: Non-metric multidimensional scaling ordination of the necrophagous invertebrate communities across seasons, with significant differences in communities among stages of decomposition (p < 0.05). The amount of variation explained by each axis is also provided for each axis. …………………………………………55

Figure 5: Non-metric multidimensional scaling ordination of the insect communities across the different stages of decomposition for the summer trial with significant differences among stages of decomposition (p<0.05). The amount of variation explained by each axis is also provided for each axis.…………………………………………. 56

Figure 6: Non-metric multidimensional scaling ordination of the insect communities across the different stages of decomposition for the autumn trial with significant differences among stages of decomposition (p<0.05). The amount of variation explained by each axis is also provided for each axis. ………………………………………....57

Figure 7: Non-metric multidimensional scaling ordination of the insect communities across the different stages of decomposition for the winter trial with significant differences among stages of decomposition (p<0.05). The amount of variation explained by each axis is also provided for each axis……………………………………………………………………………………………………………………58

Chapter 3:

Figure 1: A swine carcass during decomposition but before the formation of a larval mass, showing the ground cover conditions and the scavenger exclosure. The carcass length is about 0.9 m………………………………………………………………………………76

Figure 2: (A) Phormia regina larval mass in the shape of a swine carcass (about 0.4 m at the widest point) before dispersal and (B) the same mass (about 2.9 m long and 0.36 m wide) after 45 min en masse dispersal……………………………………………………...77

Video S1: A video capturing a large larval mass during the initial stages of en masse post-feeding dispersal through the understory of the wooded study site. (http://academic.udayton.edu/BenbowLab/Benbow_Lab/Videos.html) ……………..79

Video S2: A video capturing the most likely route of an en masse post-feeding dispersal of larvae through the understory of the wooded study site before they began to individually spread about 14 m from the point of origin. The larvae appear rice-like on the ground and among leaf litter. (http://academic.udayton.edu/BenbowLab/Benbow_Lab/Videos.html)…...... 80

xi LIST OF TABLES

Chapter 1

Table 1: Indicator carbon sources for the carrion samples. The values represent the indicator value (IV), the higher the value, the stronger the indicator that carbon source was for that particular ADH and season. Only carbon sources that had a p<0.05 were used within this table…………………………………………………………………..…29

Table 2: Indicator carbon sources for the soil samples. The values represent the indicator value (IV), the higher the value, the stronger the indicator that carbon source was for that particular ADH and season. Only carbon sources that had a p<0.05 were used within this table……………………………………………………………………..30

Table 3: Two-Way ANOVA statistics demonstrate the effects of sampling date and carrion microbial community metabolic use of specific carbon sources during decomposition for each season…………………………………………………………………………...31

Table 4: Two-Way ANOVA statistics demonstrate the effects of sampling date and soil microbial community metabolic use of specific carbon sources underneath the carcass during decomposition for each season…………………………………………………….32

Table 5: Two-Way ANOVA statistics demonstrate the effects of sampling date and soil microbial community metabolic use of specific carbon sources 1 m away from the carcass during decomposition for each season………………………………………….…...33

Chapter 2

Table 1: Mean necrophagous community taxa composition and variability (SD and coefficient of variation [CV]) variation within and among seasons. If no values are given the taxon was not present. An * denotes that there are significant differences (p<0.05) for those taxa between seasons (One-Way ANOVA with Bonferroni's Multiple Comparison Test) and are elaborated in Table 2…………………………………….59

Table 2: One-Way ANOVA and Bonferroni's Multiple Comparison Test results for taxa that were significantly different between seasons. (Sp = spring, Su = summer,

xii Au = Autumn, Wi = winter, and ns = no significant difference between those seasons)………………………………………………………………………………………………………...... 60

Table 3: Accumulated degree-hour (ADH) ranges for each stage of decomposition during each season (- indicates that the carcasses for those seasons never reached that stage of decomposition)………………………………………………………………………………61

Chapter 3:

Table 1: Characteristics of post-feeding larval dispersal (Phormia regina) from replicate swine carcasses from May-June 2009. Variables that could potentially influence larval dispersal are provided which include date of initial dispersal, the number of dispersal events per carcass, estimated distance traveled, percent slope at carcass, accumulated degree hours (ADH) and median ambient temperature (and range) from time of death to first observed dispersal event, relative humidity, rainfall, and first and second Coleoptera taxa to colonize each carcass………………....81

Table 2: Characteristics of aggregate en masse larval (Phormia regina) dispersal from replicate swine carcasses from July-August 2009. Factors evaluated for association with dispersal distances are given for each carcass in addition to the first and second Coleoptera taxa to colonize each carcass, accumulated degree hours (ADH) and median ambient temperature (and range) from time of death to first observed dispersal event, humidity and rainfall on the date of dispersal. Larval mass temperatures are provided to evaluate possible larval mass thermogenisis influence on insect development and dispersal…………………………………………………………………..82

Table 3: Soil characteristics near all carcass plots for both the spring and summer trials, including percent moisture, pH, soil color, and soil type. With soil color, the first number is the hue, the second is the value, and the third is the chroma; values are from (Munsell Color Company Inc., Baltimore, MD)………………………………………..83

xiii

CHAPTER 1: Microbial Metabolic Community Change During Vertebrate Carrion

Decomposition

ABSTRACT

Necrophagous invertebrates are noted for aiding in the decomposition process. However, there are other organisms that participate in the utilization of this common food fall, microorganisms. Little is known about the structure and composition of the microbial communities associated with carrion, or if they follow a pattern of succession as decomposition progresses, although this process is important for nutrient and energy cycling in ecosystems. The objective of this study was to evaluate microbial succession during carrion decomposition. It was hypothesized that microbial communities on decomposing carcasses compared to the soil underneath them and 1m away would be different during decomposition, and that they would vary both over decomposition time and between seasons. To test the effect of season on the microbial metabolic activity of this process, Sus scrofa carcasses (N=3-6) were placed in a forested habitat near Xenia, OH during spring

(15 March – 8 June 2009) summer (23 July – 31 August 2009), autumn (11

November 2009 – 1 May 2010) and winter (2 February – 1 May 2010). Skin biopsies and swabs of the anus and buccal of each carcass and replicate (N = 3)

1 cores of soil underneath and 1m away were collected to evaluate microbial community metabolic succession during decomposition. Biolog EcoPlates, phenotypic microarray 96 well plates monitor the differential use of 31 different carbon sources, were used to provide a community level physiological profile

(CLPPs) as a measure of variation in overall microbial community metabolic activity.

Two-way ANOVA with Bonferroni Post-tests, non-metric multidimensional scaling

(NMDS), multi-response permutation procedure (MRPP) and indicator species analysis (ISA) were employed to evaluate the microbial community metabolic activity over decomposition, between replicates, and between seasons. There was significant differences in the microbial metabolic function (p<0.001) between seasons for both carrion and soil samples. Carrion samples were significantly different from soil samples (p<0.001), but the control and soil under body were not found to be significantly different (p=0.271). Hypotheses about microbial community succession, variation amongst replicates and variation across seasons were all supported by the data.

INTRODUCTION

Decomposition plays a large role in recycling nutrients and organic matter in ecosystems (Campbell et al. 2008), and often forms the base of food webs (Putman

1978). Having knowledge of the decomposition process and organisms involved can provide mechanistic understanding of these ecosystem processes (Janetski et al.

2009). Necrophagous insects are the predominant eukaryotes that contribute to

2 vertebrate carrion decomposition (Payne 1965, Benecke 2001, Archer 2003,

Matuszewski et al. 2010). Data on the consumption rates and life cycle dynamics of these necrophagous insects provide important understanding for ecological succession (VanLaerhoven 2010), with direct application to forensic entomology

(Payne et al. 1968, Tomberlin et al. 2011a, Tomberlin et al. 2011b).

Until recently, other organisms also involved in decomposition have been overlooked – those that are predominantly microscopic. Microbes (e.g., bacteria and fungi) also play a dominant role in decomposition ecology; they are part of the living flora of vertebrates (Janaway 1996), initially exist in the environment where carrion fall (Wilson 2005), and respond to biochemical changes of a carcass immediately after death (Janaway 1996). Microbes have been documented to be one of the largest components in decomposition and nutrient cycling of plant decaying matter

(Jenkinson 1977), which accounts for approximately 99% of the organic matter on

Earth (Swift et al. 1979). Also, microbes can be found in every ecosystem and biome, including temperate forests (Gallo et al. 2004), lakes (Christian and Lind

2006), oceans (Verschuere et al. 1997) and more extreme environments such as the

Antarctic (Ellis-Evan et al. 1998), salt marshes (Brock 1978) and hot springs

(Skirnisdottir et al. 2000). Current research indicates that microbes are formidable competitors with other consumers (e.g., crustaceans and insects) for decomposing resources (Burkepile et al. 2006, Trienens et al. 2010), but this has not been studied in detail.

3 Stages of Carrion Decomposition:

There are several stages of carrion decomposition: fresh, bloating, active decay, advanced decay, and dry (Payne et al. 1968). There are few notable changes to the body in early decomposition that are recognizable by humans, but necrophagous insects, such as blow flies (Diptera: Calliphoridae), quickly detect an organism that has entered the fresh stage (Goff 2009). Immediately after death cells begin to undergo autolysis, or cell death, that begins when circulation and oxygen exchange cease. As cells break down they release enzymes, which initiates a cascade sequence of additional enzymes signaling continuous cell apoptosis in the decomposing remains (Gill-King 1997). During this stage microbes found on and in the body begin utilizing the nutrients being produced by this cascade of enzymatic activity, changes in pH and cell death; this process is known as putrefaction (Vass et al. 2002). Bloating forms from gas accumulation caused by microbial metabolism soon after death, resulting in carrion ‘inflation’ as the internal organs begin to expand (Vass et al. 2002, Matuszewski et al. 2010). Bloating can cause gas and fluids to exit the body through the natural orifices. However, post-mortem gas build up can be so significant that it causes the organs and skin to rupture, creating new postmortem lesions that can be mistaken for wounds in forensic cases (Vass 2001). The active decay stage ensues once necrophagous insects have colonized the carrion and begin to utilize it for food acquisition or as an oviposition/breeding site (Payne

1965). The advanced decay stage primarily consists of skin, cartilage, and bone, as the insects have consumed most of the soft tissue (Goff 2009). When the remains

4 consist primarily of dried bones this is considered to be the dry stage of decomposition (Matuszewski et al. 2010).

Microbial Community Ecology During Decomposition:

There are several factors known to affect microbial community assembly during vertebrate carrion decomposition; they include temperature (Ward et al.

1998, Carter and Tibbett 2006), moisture and humidity (Schimel et al. 1999), tissue type (Dickson et al. 2010), surrounding vegetation (Ibekwe et al. 2002, Kuske et al.

2002) and soil pH (Haslam and Tibbett 2009). However, there are a variety of challenges for researchers studying microbial assemblages of these systems.

Literature exists but does not explicitly examine the amount of variation in the number and transition of microbial species present, relative abundance of specific species (Burkepile et al. 2006), functional (or metabolic) changes or other factors such as antibiotics (Rozen et al. 2008) that can affect microbial community assembly during decomposition. Whereas there are studies of microbial communities in the soil underneath and adjacent to carrion (Carter and Tibbett 2006, Carter et al. 2007,

Benninger et al. 2008, Stokes et al. 2009), we are aware of only a few studies that have examined microbial community assembly (i.e., assessed genomically or functionally) of decomposing vertebrate carcasses throughout decomposition; those by Vass (2001), Vass et al. (2002), Burkepile (2006) and Dickson et al. (2010).

There are several areas of research emerging to understand microbial community ecology of carrion decomposition. These are 1) how microbial communities assemble during decomposition, 2) the role of temperature in driving this assembly, 3) how the microbial communities interact with necrophagous

5 invertebrate colonization and succession, and 4) intra- and inter-seasonal variation in these processes and interactions (Tomberlin et al. 2011a, Tomberlin et al. 2011b).

Characterizing Environmental Microbial Communities:

To address the above questions, there are several techniques used to determine microbial community structure, function and their role in the environment. These include, but are not limited to, using fatty acid methyl esters

(FAMEs) to produce fatty acid profiles (Ibekwe and Kennedy 1999); phospholipid fatty acids (PLFAs) that are structural components specific to microbes and can aide in taxa identification (Dunfield 2008); terminal restriction fragment length polymorphism (T-RFLP) uses PCR and fluorescent tagging of terminal ends of rRNA fragments (Kirk et al. 2004); and automated ribosomal intergenic spacer analysis

(ARISA) which amplifies the intergenic region of rRNA which can be variable in size

(Danovaro et al. 2006). These processes utilize genomic and cell membrane composition to identify specific taxa, or groups of taxa, to give a structural assessment of microbial communities. Another practical and affordable approach to understanding the functional role of microbes in ecological processes is the use of physiological, or metabolic, profiling. The study of metabolomics uses the systematic assessment of metabolic by-products to understand biological processes

(Daviss 2005), including those of entire microbial communities using differential carbon resource, such as Biolog EcoPlates (Fiehn 2002, Weber and Legge 2010).

In this study, we used metabolic profiling to characterize microbial community assembly associated with decomposing carcasses using Biolog EcoPlates

(Insam and Goberna 2004a, Stefanowicz 2006, Weber and Legge 2010). These 96

6 well plate phenotypic microarrays provide community level physiological profiles

(CLPPs) determined by the differential use of carbon sources from environmental samples (Insam and Goberna 2004a, Bucher and Lanyon 2005). Plates similar to

Biolog EcoPlates were originally developed for use in the medical field; however,

EcoPlates have found successful application in ecological studies (Insam and

Goberna 2004a, Bucher and Lanyon 2005, Calbrix et al. 2005, Stefanowicz 2006,

Thottathil et al. 2008). This technique has been used to understand the culturable component of the microbial community, including those of both terrestrial (Miller et al. 2007, Papatheodorou et al. 2008, Ros et al. 2008) and aquatic habitats (Sala et al.

2006, Richardson et al. 2008, Thottathil et al. 2008). They are used to evaluate the functional aspect of microbial assembly, not individual species or taxa within the community; thus, they provide a community-level functional signature that can be compared between sample types or over time to monitor community metabolic change (Richardson et al. 2008). (See the Materials and Methods for additional details on this technique.)

The overall aim of this study was to use metabolic/physiological profiling to understand microbial community change during carrion decomposition. This research contributes to the broader field of decomposition ecology while also having potential use in the forensic sciences for better estimating a minimum postmortem interval (mPMI) under varying environmental conditions. Three hypotheses were tested: 1) there would be notable changes in the microbial CLPPs during decomposition, suggesting a pattern of community assembly; 2) the soil microbial CLPPs immediately underneath carrion would follow similar trends in

7 carrion community change during decomposition but with different profiles, while control soil profiles would not change during that time; and, 3) there would be seasonal variation in these assembly patterns related to local ambient environmental (e.g. temperature) conditions.

METHODS

Study Site & Design:

This study was conducted within Morris Bean Reserve of Greene County, OH, a Midwest temperate forest of 12.2 hectares surrounded by agricultural fields with a small tributary stream running adjacent to the reserve that empties into the Little

Beavercreek River. The predominant trees are honey locust (Gleditsia triacanthos) and a variety of maples (Acer spp), while the most common sub-canopy cover is

Amur honeysuckle (Lonicera maackii). Five 1m2 plots, along each of six, 50 m transects were established running north-south in the reserve for a total of 30 plots, within 10-80 m from each other (Lewis and Benbow 2011).

Sus scrofa (swine) carcasses weighing 14-18 kg were used as models of vertebrate (Schoenly et al. 2007) carrion decomposition during four seasonal trials: spring (March-June 2009), summer (July-August 2009), autumn (November 2009-

March 2010), and winter (February-March 2010). Six replicate carcasses were used in the spring and summer trials whereas three were used for the autumn and winter trials; the latter trials only included three replicates because of cost constraints and longer decomposition time expected during the colder months. Previous research

8 has shown that swine carcasses are suitable models for understanding the process of human decomposition, making this research relevant to the forensic sciences

(Catts and Goff 1992, Carvalho et al. 2000, Schoenly et al. 2006, Schoenly et al.

2007), especially because human remains are difficult and expensive to acquire.

As described in a companion study (Lewis and Benbow 2011) carcasses were purchased from a local farm immediately after (i.e., within minutes) being euthanized. Each carcass was double bagged in black heavy duty garbage bags that were tightly taped in order to prevent access by any invertebrates. The carcasses remained bagged for 2-3 h during transport to the study site and were placed at randomly chosen plots along each transect within 20 min of each other. Each plot was new, without any previous carcass decomposition from a preceding trial.

Carcasses were exposed to the environment approximately 2 hrs before sunset

(NOAA) on 15 April 2009 (1730-1745 hrs), 22 July 2009 (1800-1815 hrs), 11

November 2009 (1545-1600 hrs) and 8 February 2010 (1630-1645), for the spring, summer, autumn and winter trials, respectively. Each carcass was placed under wire mesh exclosure cages (0.6 x 0.9 x 0.6 m) to prevent access and disturbance by large scavengers (e.g., coyotes, vultures).

Temperature was recorded using NexSens DS1921G micro-T data loggers

(Fondriest Environmental Inc., Beavercreek, OH) every 15 min attached to the cages over each carcass through the duration of the study. Rainfall data was recorded from a local weather station (Station code: KSGH, Springfield, OH) approximately 8 km away from the study site. The ground slope was measured for each carcass location and videos and pictures were taken using an Olympus Stylus 1050 SW

9 (Olympus, Center Valley, PA) camera. Soil characterization for this site was determined using the Munsell Soil Color Chart (Munsell Color Company Inc.,

Baltimore, MD) and Green County, Ohio Soil Survey (1978) from samples taken approximately one meter away and underneath each carcass location (Lewis and

Benbow 2011).

Microbial Community Assessment:

Microbial communities were sampled from the carcasses and soil every three days, weather and field conditions permitting, until the dry stage of decomposition.

After all samples were collected for a seasonal trial, three dates representing three different phases of decomposition were chosen for analysis. The spring and summer trials followed the same sampling regime, with the first sampling date corresponding with the initial oviposition of Calliphoridae (Order: Diptera) signifying early decomposition (e.g. fresh and bloat stages) . The second sampling date took place when the carcasses reached middle decomposition (e.g. active decay stage), while the last sampling date represented late decomposition (e.g. advanced decay and dry stages). For the autumn trial the first sampling date corresponded with initial Calliphoridae oviposition; however, the second sampling date occurred prior the first snow of the season. It was decided at the beginning of this study that the carcasses would not be disturbed once they were covered with snow, in order to determine the effect of snow on the microbial community functional change during decomposition. The third sampling date took place in January of 2011, which corresponded with increased temperatures and snowmelt. For the winter trial, there were four sampling dates instead of three due lack of invertebrate activity

10 during the beginning of the trial. The first sampling date took place immediately after carcass exposure since a delay in initial oviposition was expected during winter temperature conditions. The second date coincided with the next snowmelt.

Both a third and fourth sampling date were chosen to represent different decomposition time points, even though there appeared to be negligible change to the carcasses and little adult or larval blow fly activity was noted on the carcasses during the entire trial.

For each carcass, sterilized swabs were taken from three regions: the buccal cavity, skin on the front shoulder and the interior anal cavity. Skin biopsies were also taken from the shoulder adjacent to the location of a preceding swab. To characterize the soil communities a Humboldt cork borer was used (Humboldt

Manufacturing, Chesterfield, MO) (1 cm diameter) to sample a 2.5 cm deep core of soil representing the O and A horizons. Three random core sub-samples (each 2cm3) were taken under the carcass and then composited for one sample, while another three random control samples were taken one meter away from the carcass and composited as well. Each core location was noted to avoid re-sampling.

Functional diversity changes of heterotrophic microbial communities were defined by CLPPs using culture-based Biolog EcoPlate techniques with details described in recent literature (Insam and Goberna 2004a, Stefanowicz 2006, Miller et al. 2007, Papatheodorou et al. 2008, Ros et al. 2008). We used this technique to trace the bacterial carbon source utilization profile during the course of carrion decomposition. The plates are 96 well microtiter plates containing 31 different carbon sources replicated in three wells on each plate and include carbohydrates,

11 amino acids, carboxylic acids, amines, alcohols, polymers, surfactants, and an ester

(Cederlund et al. 2008). Each well also includes tetrazolium violet dye which turns to violet formazan under reduced conditions that occurs with the metabolic use of the substrate (Stefanowicz 2006); the degree of carbon source metabolism represented by the degree of violet formazan formation is measured using a spectrophotometer at 590 nm (Kelly and Tate 1998). When environmental samples from the same substrate are taken over time, this technique describes the CLPP change of the microbial community of the substrate (Ros et al. 2008).

The samples taken for microbial assessments were stored at -20C for several months, slowly thawed and then processed using a modified protocol described by Insam and Goberna (2004b). For each sample, 40 ml of 25% strength

Ringer solution was added to sterile 50 ml centrifuge tubes and then agitated for 10 min with 15 sterilized 3 mm glass beads using a Burrell Wrist-Action Shaker

(Burrell Scientific, Pittsburgh, PA) at 385 Osc/min at an amplitude setting of 2.3 cm.

Swabs and skin biopsies were added directly to the Ringer solution within the centrifuge tube. The composite soil samples were homogenized using a sterilized spatula, and a 1 g subsample was added to the Ringer solution. All samples were centrifuged for 2 min at 2000 rpm using a Damon/IEC Division, IEC HN-SII (GMI,

St. Paul, MI) swing-bucket centrifuge.

Using an 8-channel Finnpipette II micropipette (Fisher Brand, Pittsburgh,

PA), the plates were inoculated with 100 l supernatant aliquots per well. Soil supernatant was diluted by 10-1 before inoculation. All sample processing occurred under a Purifier Class II Safety Cabinet (Labconco Corporation, Kansas City, MO).

12 The plates were then incubated at 22C, based on preliminary experiments for these types of samples (unpublished data), and read using a Wallac 1420 Victor2

Multilabel Counter (Perkin/Elmer, Waltham, MA) for optical density (OD) at 590 nm every 12 h until 0.7 OD was reached, or 10 readings (120 h), whichever occurred first. This was performed as a first step to understand the average plate microbial physiological activity that was used in standardized comparisons, accounting for cell density differences (Weber and Legge 2010).

Data Analysis:

Analyses were performed according to Stefanowicz (2006) and Weber and

Legge (2010). Briefly, to evaluate CLPPs, data were standardized by using the average metabolic activity that approached or met 0.7 OD, or after 10 readings. This was done to account for possible density differences among samples and provided a means to assess overall average microbial activity (Weber and Legge 2010). First, the average well color development (AWCD) was determined by taking the average carbon use of each plate. These data were then normalized using methods modified from Thottathil et al (2008) and Calbrix (2005) by subtracting the average water well (background) activity for each plate, then subtracting that value from each carbon source usage and then dividing by the average of all normalized carbon resource of the plate, we using the following equation to determine the average normalized plate activity (AWCD) (Weber et al. 2010):

13 Ai represents the carbon use for each well, A0 is the average of the water wells, and

Ak represents the normalized carbon source use and A k is the mean normalized plate microbial activity (Weber and Legge 2010).

̅ ∑

Initially, we used the mean plate activity at 0.7 OD, or the 10th reading, from both the non-normalized (raw activity values) and normalized carbon use to determine if there was a change in the overall metabolic activity of microbial communities during decomposition (Figure 1). For the remaining analyses, the mean normalized metabolic activity for each carbon source was used for statistical analyses. We used 2-way-ANOVA with Bonferroni post-test for pair wise comparisons in GraphPad Prism 5.0 (GraphPad Inc, La Jolla, CA) to test differences in specific carbon source utilization and variation over decomposition. Non-metric multidimensional scaling (NMDS) followed by multi-response permutation procedures (MRPP), and indicator species (or in this case, carbon source) (McCune and Grace 2002) analysis (ISA) were performed using PCOrd (MJM Software Design,

Gleneden Beach, OR) to analyze differences in the overall microbial community

CLPPs among sample dates, replicate carcasses, and sample location (i.e. anal, buccal, skin), between carrion and soil, and overtime and decomposition stage using date and after adjusting time to accumulated degree hours (ADH). For each of the ordinations, the axes that explained the most variation and the strongest orthogonality (lowest stress) were used for representing the data in multidimensional space (McCune and Grace 2002)

14 RESULTS

Overall Metabolic Activity:

For all seasons, except for autumn, mean carrion microbial metabolic activity

(non-normalized) decreased during decomposition (Figure 1A), and varied substantially for communities of soil under the carcass and 1 m away (Figure 1C), and this was true among seasons. Some differences were noted between control soil that showed a decrease in metabolic activity and the soil underneath the body, which remained stable (Figure 1C). However, when metabolic activity was normalized, the trend for microbial metabolic activity of both carrion and soil communities was consistent over decomposition for all seasons (Figures 1B and

1D).

Microbial Community Metabolic/Physiological Profiles:

Using multivariate analyses we found no significant differences in normalized metabolic profiles among the sample locations (i.e., buccal, skin, anus) of each carcass (MRPP, T=0.116, p=0.490); therefore, this potential effect was ignored

(i.e., data pooled) for the remainder of analyses. There were significant differences between microbial metabolic activity for all seasons (Figure 2B). In order to evaluate different, more discrete stages of decomposition, the CLPPs for similar decomposition stages (e.g. early decay, active decay) were grouped together allowing all seasons to be compared based upon similar ADH ranges. Significant differences (p<0.05) were documented between all of the different ADH ranges

15 among seasons according to the pairwise multiple comparison post-test with regards to the carrion samples (e.g. buccal swab, skin biopsy) (Figure 2A).

We found significant differences in normalized microbial metabolic activity between communities of carrion and both soil types (i.e., underneath and 1 m away from carcass) (MRPP pairwise, p<0.001 for both), but there was no significant difference between the two soil communities (MRPP pairwise, p=0.271).

Since the soil microbial community metabolic profiles did not significantly differ under the carcass or 1 m away, for the remaining analyses we ignored this effect. When grouped, however, there were significant differences in the microbial metabolic profiles of the soil communities among the decomposition stages represented by ranges in ADH (Figure 2C) and among seasons (Figure 2D). The ordinations were robust with the two-axis solution representing approximately

90% variation among the communities. Because we found significant differences in microbial metabolic profiles both over decomposition and between seasons, we analyzed each season separately.

There were significant differences in the CLPPs over decomposition within each season for both the carrion (p<0.001) and soil samples (p<0.001) (Figure 3A and 3C). Indicator species analysis determined several carbons sources that followed patterns of utilization across decomposition among the seasons.

Indicators for the carrion samples included α -D-Lactose, D-Cellobiose, D-Galactonic

Acid γ –Lactone, and Putrescine during early decomposition, D-Malic Acid, Glycyl-L-

Gglutamic Acid and L-Phenylalanine for the middle decomposition, and α-

Cyclodextrin, γ-Hydroxybutyric Acid, α -Ketobutyric Acid, 2-Hydroxy Benzoic Acid,

16 and Phenylethylamine for the late decomposition (Table 1). There were no significant carbons sources that showed repetitive use across seasons for the different stages of decomposition (Table 2). For soil communities, there were only significant metabolic profile signature differences over decomposition in spring and summer (Figure 4).

Two-way ANOVA was used to determine significant differences between carbon source utilization, stage of decomposition, and interaction effects for each substrate. With the carrion samples, significant differences existed between every

ADH, except for autumn (Figure 5, Table 3). For the all soil communities there were no significant differences among the different decomposition stages for all seasons

(Figures 6 and 7, Tables 4 and 5). Carrion microbial metabolic activity had significant differences in 20 of the of the carbon sources for spring trial (Figure 5A) and 22 carbon sources for the summer (Figure 5B) among the different phases of decomposition. Autumn and winter had only 3 and 2 significant differences in carbon source utilization respectively (Figure 5C and 5D). Soil metabolic activity for samples under the body had few significant differences among the different phases of decomposition with spring having 2 (Figure 6A), summer 2 (Figure 6B), autumn 2

(Figure 6C) and winter 5 (Figure 6D). Control soil had low variation in the metabolic activity across phases of decomposition as well with spring having 3

(Figure 7A), summer 2 (Figure 7B), autumn 1 (Figure 7C), and winter 3(Figure 7D).

17 DISCUSSION

This is the first study to employ microbial metabolic profiling using Biolog

EcoPlates™ to evaluate microbial community activity during carrion decomposition.

These results demonstrate that there is a great potential to use this technique for carrion decomposition research and forensic applications. Current research in this field has been aimed at determining what microorganisms compose communities involved in carrion decomposition (Burkepile et al. 2006, Dickson et al. 2010). Our results demonstrate that the microbial metabolic profiles describe significant functional changes in the community during decomposition both within and among seasons, similar to other studies in aquatic habitats (Burkepile et al. 2006, Dickson et al. 2010). This demonstrates community-level functional changes occurring over decomposition; however, more research involving which species are changing over time is needed using deep sequencing approaches such as 454 pyrosequencing (Kirk et al. 2004, Mardis 2008).

Seasonal variance in abiotic conditions was hypothesized to significantly impact microbial community assembly. Differences in microbial metabolic activity existed between the seasons using the whole plate metabolic activity. NMDS ordinations derived from normalized microbial metabolic activity for each carbon source per sample (e.g. buccal swab, soil under body) revealed significant differences between the groupings for both carrion (Figure2A) and soil (Figure 2C) samples, but not between soil underneath the body and the control soil samples.

18 Temperature can have great influence on microbial communities (Ward et al.

1998, Carter and Tibbett 2006). We found significant differences between different stages of decomposition within each season except for the autumn carrion and soil, as well as the winter soil samples. However, why did microbial metabolic activity increase in the autumn trial as temperatures outside decreased. Carter and Tibbett

(2006) demonstrated when chicken carcasses (Ovis aries) were subjected to different temperatures, the efficiency of the microbial metabolic activity during decomposition varied as measured by the amount of carbon dioxide production during respiration. They reported that when carcasses decomposed under 2°, 12°, and 22° C, the process was most efficient at 2°. This may explain the increased metabolic activity between the second and last sampling dates for the autumn trial.

This could also aide in explaining why the microbial metabolic activity decreased during warmer temperature seasons. For the spring and summer trials, large

Calliphoridae larval masses colonized and consumed the carcass throughout the decomposition process. Carter and Tibbett (2006) speculated that higher ambient temperatures might have negative impacts on microbial community catabolic reactions. These larval masses have the potential to generate increased temperatures from 30°C to >50°C higher than the surrounding ambient temperature through collective thermogenesis (Slone and Gruner 2007). The depletion of the carrion resource by an invertebrate competitor, but also increased aggregate temperatures resulting from larval masses of that competitor may be affecting microbial community assembly during carrion decomposition. Additional studies are warranted to understand this process.

19 The CLPPs of the replicate carcasses within and among seasons were predicted to be significantly different due to individual differences in the flora existing within and on the carrion at time of death and in the environmental flora found at each plot. There were multiple carbon sources that were significant indicators for each stage of decomposition (Table 1 and 2). Some of these carbon sources followed patterns across seasons, which only occurred for the carrion samples and not the soil. Among the carrion samples, spring and summer had the most similarities in the carbon source utilization across decomposition due most likely to the similar abiotic variables experienced (e.g. temperature and humidity).

While other substrates have been marked as potential indicators of different stages of decomposition (Vass et al. 2002), our results suspect that the Biolog EcoPlate™ carbon sources could be potentially good indicators of the decomposition process and thus, useful in microbial PMI (mPMI) estimates

Preston-Mafham (2002) discussed how the density of microbial communities can greatly impact the rate and profile of Biolog Ecoplates™, making it important to either know the density before plate inoculation, or use normalization techniques to remove, or reduce, the effect of density on the profiles (Weber and Legge 2010). We believe it is necessary to compare the normalized carbon usage back to the raw usge to provide insight into understanding this effect. For example, in analyzing the soil samples from the autumn and winter, the raw physiological activity of the control soil was decreasing compared to soil underneath the carcass. This could be explained by the fact that during the colder temperatures, the carcass provided shelter from the elements for the microbial communities underneath the body,

20 allowing the microbial communities to maintain or potentially grow while the control soil microbial communities could not. Another potential factor involved in decreasing the microbial community density is the saturation of the soil with metabolic byproducts resulting from decomposition. The spring trial soil communities showed a dramatic decrease in metabolic activity, which could be attributed to the longer decomposition time, which allowed the soil to become more saturated with metabolic byproducts.

Further, Biolog Ecoplates™ were a successful means of analyzing the vertebrate microbial community metabolic succession during the decomposition of terrestrial carrion. We found that Biolog EcoPlates™ were a cost-effective and practical technique to determine microbial community assembly functional differences among seasons and different substrates (i.e., carrion vs soil), and over the decomposition process. To gain more insight into the microbial community structure and assembly process of species in future studies Biolog Ecoplates™ should be used in tandem with genomic or molecular techniques (Ibekwe and

Kennedy 1999, Kirk et al. 2004, Danovaro et al. 2006, Dunfield 2008). This way, the

Biolog plates™ can provide the CLPP for a community, and due to their relative ease of use and low cost, narrow down samples of interest where genomic techniques can identify the individuals that make up the microbial community assembly.

Figure 2: Non-metric multidimensional scaling ordinations of microbial community level physiological profiles for carrion communities (A) during decomposition time is represented by ADH ranges and among seasons (B), and soil communities during decomposition (C) represented by ADH ranges and among seasons (D). The MRPP statistics indicate significant differences where p < 0.05, and the amount of variation explained by each axis is also provided in the axes labels.

Figure 3: Non-metric multidimensional scaling ordinations of microbial community level physiological profiles for carrion communities over decomposition time represented as ADH during each season: A) spring, B) summer, C) autumn, and D) winter. The MRPP statistics indicate significant differences where p < 0.05, and the amount of variation explained by each axis is also provided in the axes labels.

Figure 4: Non-metric multidimensional scaling ordinations of microbial community level physiological profiles for soil communities over decomposition time represented as ADH during each season; A) spring, B) summer, C) autumn, and D) winter. The MRPP statistics indicate significant differences where p < 0.05, and the amount of variation explained by each axis is also provided in the axes labels.

Figure 5: Mean (SD) normalized microbial metabolic activity of each carbon source for carrion communities during A) spring, B) summer, C) autumn, and D) winter over decomposition time represented as ranges in ADH.

28 Table 1: Indicator carbon sources for the carrion samples. The values represent the indicator value (IV), the higher the value, the stronger the indicator that carbon source was for that particular ADH and season. Only carbon sources that had a p<0.05 were used within this table.

29 Table 2: Indicator carbon sources for the soil samples. The values represent the indicator value (IV), the higher the value, the stronger the indicator that carbon source was for that particular ADH and season. Only carbon sources that had a p<0.05 were used within this table.

30 Table 3: Two-Way ANOVA statistics demonstrate the effects of sampling date and carrion microbial community metabolic use of specific carbon sources during decomposition for each season.

31 Table 4: Two-Way ANOVA statistics demonstrate the effects of sampling date and soil microbial community metabolic use of specific carbon sources underneath the carcass during decomposition for each season.

33 CHAPTER 2: Necrophagous Insect Community Assembly During Vertebrate Carrion

Decomposition: Seasonal and Inter-Carcass Variation

ABSTRACT

Decomposition is a fundamental process to ecosystem function and energy flow where nutrients are recycled and reintroduced into food webs. Vertebrate carrion decomposition can provide significant resource pulses for habitats and can range from large whale carcasses to small rodents. Necrophagous invertebrates have been documented to be a predominant driver of vertebrate carrion decomposition. The objective of this study was to evaluate the arthropod species composition, richness and diversity change over decomposition, and to determine if these were consistent between replicate carrion and among seasons. It was hypothesized that necrophagous arthropod (primarily insects) taxa composition as overall community structure, richness, and diversity would vary between replicates and across decomposition as well as different seasons. To test this hypothesis, Sus scrofa carcasses (N=3-6) were placed in a forested habitat near Xenia, OH during spring (15 March – 8 June, 2009) and summer (23 July – 31 August, 2009), autumn

34 (11 November, 2009 – 1 May, 2010) and winter (2 February - 1May, 2010).

Standardized insect samples involving aerial sweep nets, pitfall traps, and hand collections were used to evaluate the arthropod communities through carrion decomposition. One-way ANOVA, non-metric multidimensional scaling (NMDS), multi-response permutation procedure (MRPP), and indicator species analysis (ISA) were employed to evaluate the insect communities over decomposition, between replicates, and among seasons. Taxa richness and diversity were low during early and late decomposition and higher during active decay for all seasons. While most invertebrate communities were the same among seasons, five taxa demonstrated significant differences in presence (One-way ANOVA; p<0.05) in relative abundance across seasons. For each stage of decomposition, there were significantly different necrophagous insect communities (MRPP; p<0.001) within each season. There were significantly different necrophagous insect communities between summer and autumn (p=0.001) and summer and winter (p<0.001), the communities were similar between autumn and winter trials (p=0. 073For each season there were significant indication for each stage of decomposition, but those taxa varied across seasons for each stage. This study demonstrated substantial variability in necrophagous communities and assembly among carrion over decomposition and among seasons. We highly recommend that future studies related to carrion decomposition increase replication of carcasses and season.

35 INTRODUCTION

The decomposition process is critical to nutrient cycling and energy flow of most ecosystems (Carter et al. 2007). Thus, understanding this process has broad application to ecological and environmental science. Necrophagous invertebrates are responsible for the majority of vertebrate carcass decomposition (Payne 1965,

Carvalho and Linhares 2001, Goff 2009). The species composition of this invertebrate group on carrion can vary among habitats, regions, days and seasons

(Matuszewski et al. 2010). Many species predominantly consume the carrion organic material directly (Diptera: Calliphoridae) (Campobasso et al. 2001), while others use the resource as habitat or as a location to find other prey insects attracted to the carrion as food sources (Coleoptera: Silphidae) (Gibbs and Stanton

2001). The Diptera and Coleoptera comprise the majority of arthropod taxa that contribute most to terrestrial carrion decomposition. During decomposition there is a qualitative, recognizable, pattern of necrophagous insect succession that describes the invertebrate communities associated with the different stages of decomposition

(Arnaldos et al. 2005, Kreitlow 2010); however, most studies of this process have been qualitative and without replication. Little is known how invertebrate carrion community assembly varies among replicate carcasses.

There are several stages of decomposition: fresh, bloat, active decay, advanced decay, and dry/skeletal stages (Payne et al. 1968). The fresh stage begins at death and continues until the carcass begins to bloat, the beginning of the bloating stage (Payne 1965, Tomberlin et al. 2011b). Bloating occurs due to

36 microbial metabolic activity that produce gaseous byproducts that cause the carrion to inflate which also attracts/repels certain necrophagous insect taxa to the carcass

(LeBlanc 2008, Matuszewski et al. 2010). Active decay follows bloating and is obvious when the body begins to rapidly decompose due to insect activity (Centeno et al. 2002). Advanced decay is signaled by a decrease in entomological activity as the resource is consumed (Goff 2009). When all that remains is bones, dry skin, and hair the carcass is considered to be in the dry stage (Payne 1965).

While this arthropod successional pattern provides a model for ecological research (VanLaerhoven 2010), it also has some applied, civic uses. Forensic entomologists are able to use this pattern of arthropod community assembly to make entomologically-based estimates of how long a person has been deceased, otherwise known as the post-mortem interval (PMI) (Merritt and Benbow 2009,

Wells and Lamotte 2010). Researchers try to determine the period of insect activity

(PIA), often closely related to the PMI, that can be useful in criminal investigations

(Tomberlin et al. 2011b). This can be an effective tool in legal cases, as it can enable the investigators to accuse or acquit a suspect in a crime (Schoenly et al. 1992). This tool requires that researchers are objective and quantitative in describing the pattern of succession to make a claim that could sentence a person to prison.

However, there are exceptions where the PIA is not synonymous with the PMI, potentially providing error in making entomologically-based PMI estimates.

Recently it was brought to the attention of the forensic entomological field through the United States National Research Council that there is a need to determine the error rate and predictability of the use of entomological evidence in legal case work

37 (Natl. Res. Counc. (U.S.) et al. 2009, Tomberlin et al. 2011a, Tomberlin et al. 2011b).

Therefore, all factors related to the pattern of arthropod carrion succession should be studied in detail, allowing for better statistical predictions and generalization of study results beyond local habitats or certain seasons (e.g., summer).

In forensic cases, there have been several sources of error in entomologically-based PMI estimates that originate from modifications to the accessibility of arthropods to the carrion source, or through mediations of larval growth and development. For instance, the effect of clothing on delaying fly colonization (Kelly et al. 2008), whether nocturnal oviposition by blow flies can occur (Amendt et al. 2007a), the distance and migration patterns of larval blow flies

(Gomes et al. 2006, Lewis and Benbow 2011), and the effects that drugs and other chemicals can have on larval growth and development (Goff et al. 1989) are all known to influence the rate and dynamics of arthropod colonization of carrion/corpses. Further, additional research has shown that variation can occur in the necrophagous communities depending on the surrounding environment (e.g. forest, prairie, urban) (Matuszewski et al. 2010) and time (e.g. seasons, years)

(Archer 2003, Arnaldos et al. 2004). It has been demonstrated that Sus scrofa

(domesticated swine) are suitable models of human decomposition due to similarity in size, a similar omnivore digestive tract (Centeno et al. 2002), and attraction by similar necrophagous insects species (Schoenly et al. 2007); however, researchers do not have a strong understanding of how arthropod colonization and assembly vary among other carrion species compared to swine. All of these issues are further exacerbated by a history of using few, if any, replicates in carrion-arthropod

38 succession studies, and fewer using replicate human models for inferences in forensics (Matuszewski et al. 2008).

It was hypothesized that necrophagous arthropod (primarily insects) taxa composition as overall community structure, richness, and diversity would vary between replicates and across decomposition as well as different seasons. Another goal was to document, for first time, the forensically important necrophagous invertebrates for Southwest Ohio using Sus scrofa carcasses.

METHODS

Study Site & Design:

This study was conducted within Morris Bean Reserve of Greene County, OH, a Midwest temperate forest of 12.2 hectares surrounded by agricultural fields.

There is a small tributary stream running adjacent to the reserve that empties into the Little Beavercreek River. The predominant trees are honey locust (Gleditsia triacanthos) and a variety of maples (Acer spp), while the most common sub-canopy cover is Amur honeysuckle (Lonicera maackii). Five 1m2 plots, along each of six, 50 m transects were established north to south in the reserve for a total of 30 plots that were from 10-80 m from each other.

Sus scrofa (swine) carcasses weighing from 14-18 kg were used as models of vertebrate carrion decomposition (Schoenly et al. 2007) during four seasonal trials: spring (March-June 2009), summer (July-August 2009), autumn (November 2009-

March 2010), and winter (February-March 2010). Six replicate carcasses were used

39 in the spring and summer trials whereas three were used during the autumn and winter trials; the latter trials only included three replicates because of cost constraints and longer expected decomposition time during the colder months.

Research has shown that swine carcasses are suitable models for understanding the process of human remains decomposition making this research relevant to the forensic sciences (Catts and Goff 1992, Carvalho et al. 2000, Schoenly et al. 2006,

Schoenly et al. 2007), especially since human remains are difficult and usually expensive to acquire.

The carcasses were purchased from a local farm immediately after (i.e., within minutes) being euthanized. Each carcass was immediately double bagged in

1mm thick garbage bags that were tightly sealed with tape in order to prevent access by any invertebrates. The carcasses remained in the bags for two to three hours during transport to the study site where one carcass was placed at a randomly chosen plot along each of three or six transects (depending on season).

Each plot was new, without any previous carcass decomposition from a preceding trial. Carcasses were exposed at 2 hrs before sunset defined by the National Oceanic and Atmosphere Administration (NOAA) on 15 April 2009, 22 July 2009, 11

November 2009 and 8 February 2010, for the spring, summer, autumn and winter trials, respectively. Each carcass was placed under a wood exclosure cage (0.6 x 0.95 x 0.6 m) with 2.5 cm mesh chicken wire to prevent disturbance by vertebrate scavengers (e.g. raccoons, coyotes, vultures).

Temperature was recorded every 15 min through the duration of the study using NexSens DS1921G micro-T data loggers (Fondriest Environmental Inc.,

40 Beavercreek, OH) that were attached to each exclosure cage. These were used to calculate accumulated degree hours (ADH) that are units for accumulated heat over time that is related to invertebrate growth rates (Byrd et al. 1996; Amendt et al.

2007a), and can be used to thermally standardize the progression of decomposition.

Rainfall was calculated using data accumulated at a local weather station (Station code: KSGH, Springfield, OH) approximately 8 km away from the study location. The ground slope was measured for each carcass location and videos and pictures were taken using an Olympus Stylus 1050 SW (Olympus, Center Valley, PA) camera. Soil conditions were relatively homogenous throughout the study site and among carcass replicates as described by Lewis and Benbow (2011).

Invertebrate Community Assessment:

Sampling for carrion invertebrate communities was as follows: daily from fresh through the active decay, every second day through post decay, and every fourth day through the dry stage and until activity of necrophagous insects was no longer prevalent. Aerial sweeps using a 36cm wide net were used to collect adult flies (primarily Calliphoridae) over the carcass. This method was standardized with a maximum of three passes over each carcass; with flies only kept from two passes to represent the adult fly community per replicate carcass. We used hand collections with forceps to sample ground and carcass dwelling invertebrates from three different areas of each plot: the first area (A1) included the ground and vegetation surrounding the carcass for a 5 m radius; the second area (A2) included collections of invertebrates that were taken from the carcass not in contact with the soil; and the third area (A3) was underneath the carcass and was done by lifting it up while it

41 was still intact which in most cases took place until the dry stage. We used standardized hand collections to collect representative taxa in each of these areas and to minimally disturb the carcass invertebrate community during decomposition, while also maintaining consistent sampling effects among replicates and seasons.

To standardize sampling for non-blow flies in the A1 and A2 areas, specimens were collected for five minutes or for a total of 10 specimens, which ever occurred first. For A3, sampling occurred for 30 sec or until 10 specimens were collected. This standardization protocol as well as the pit-fall traps were established after the completion of the spring trial. Blow fly larvae were collected in a tiered approach to minimize the effects of sampling on larval interactions and their activity during decomposition: when total larval abundance was estimated to be low (100-

500 larvae) only about 10% of the population was collected (usually from 10-50 larvae), while for larger masses (>1000 larvae) a total of 100 larvae were collected from all larval masses on or off of the carcass. Blow fly larvae were placed into vials and transported to the laboratory for rearing to adulthood or preserved by boiling for 30 seconds and then preservation in ethanol (Amendt et al. 2007b). The rearing process involved placing the larvae or eggs onto a 3-5 oz piece of beef liver in a 1 qt glass jar or insect breeder (Bioquip, Rancho Dominguez, CA) filled with 2-5 cm of wood chip substrate. Additional pieces of beef liver were added when the previous piece was consumed; when the larvae reached the post-feeding, dispersal stage they were transferred to a new container filled with 10-15 cm of fresh substrate (Byrd et al. 2010). After the larvae pupated and emerged, the adult blow flies were then dispatched using ethyl acetate and preserved in ethanol. Lastly, except for the

42 spring trial, pitfall traps were used to collect mobile invertebrates that were drawn to the carcass when sampling was not conducted (e.g., at night). For this we used three pitfall traps per carcass (32 oz plastic cups with cup lip flush to ground) filled approximately halfway with water and randomly placed with regard to direction 1 m away from the carcass. Insects were collected every sample date and redeployed on the same day. All insects collected (or reared) from the sweep nets, hand collections (except blow fly larvae) and pitfall traps were preserved in 70% ethanol and stored for later identification. Diptera identifications were made using Hall

(1977), Cutter and Dahlem (2004) and Whitworth (2006). Arnett (2000, 2001,

2002) was used for all of Coleoptera and incidental taxa identifications.

Data Analysis:

For all analyses, organisms counted and identified from each of the sampling methods (i.e. aerial sweep nets, pitfall traps, hand collection) were pooled together to represent the community for each carcass on each sampling date. We formulated a taxa list as well as calculated taxa composition and relative abundance, taxa richness and diversity for each season (Table 1). To ease data interpretation and visualization, any taxa in the order Coleoptera were grouped to family level.

One-way ANOVA was performed for each taxon to determine significant differences in relative abundance among seasons (Table 3).

Overall invertebrate community structure was described using non-metric multidimensional scaling (NMDS), followed by multi-response permutation procedures (MRPP), and indicator species (or in this case, taxa) analysis (ISA) using

PCOrd (MJM Software Design, Gleneden Beach, OR) and arcsine square root

43 transformed data following recommendations by McCune et al (2002). These analyses only involved the summer, autumn and winter seasons because slightly different sampling techniques were used (e.g., pitfall traps and limited hand collections) in the spring trial. An overall ordination was performed using all of the seasons (except spring) pooled together, and then separate ordinations (using only the taxa >1.5% of the total population to reduce outliers) for each individual season after significant community differences among seasons were statistically evaluated with MRPP. For each of the ordinations, the axis that explained the most variation and the strongest orthogonality (lowest stress) were used for representing the data in multidimensional space (McCune and Grace 2002).

Further, we classified each stage of decomposition by ranges of ADH to examine community structure differences during each stage of decomposition in relation to accumulated heat. To avoid confusion, hereafter we use the stage terminology set forth by Payne (1968), standardized and described with the field- measured ADH ranges that are given in Table 3.

RESULTS

Taxa Richness and Diversity:

A total of 6,383 insects were collected and identified that predominantly represented Diptera and Coleoptera. Taxa richness for spring and summer had similar patterns through decomposition (Figure1). During the fresh stage, richness was low (1-3 taxa) and uniform among replicates (≈ ±1 SD), while the bloat and

44 active decay stages were characterized by increased richness (3-12 taxa) and higher variation (≈ ±4 SD), and the advanced decay and dry stage communities had both low richness and variation (≈ ±2.5 SD) (Figure 1). Autumn and winter trials followed similar trends to spring and summer, except that they never entered advanced decay (Figure 1). The winter and autumn trials had high taxa richness, (Figure 1), while the summer was characterized by the highest diversity (Figure 2).

Taxa Composition:

We collected a total of 16 taxa that represented seven taxa of Diptera and nine Coleoptera families. When performing a rare taxa analysis (i.e. any taxa composing >1.5% of the total population), three taxa were omitted from further analyses such as (e.g. NMDS, MRPP, and ISA) due to having outlier effects; these taxa included Lucilia illustris, L. sericata, and Pollenia pediculata. Several genera of

Silphidae family showed seasonal preference during this study. Further field observations indicated that Oiceptoma inaequalis and O. novaboracense were present during the spring trial in high abundance, sometimes estimated to have hundreds of adults and larvae on and around a carcass at one time. However during the summer trial, O. inaequalis was not present while O. novaboracense had less than

10 individuals collected during the entire trial. Necrodes, and Nicrophorus along with Necrophila americana were present during the summer trial, but were never collected during the spring trial. For the majority of taxa, relative abundance was consistent across seasons (Table 1). The relative abundance of several taxa was significantly different among seasons: Phormia regina (p<0.001), L. coeruleiviridis

(p=0.002), Muscidae (p=0.045), Staphylinidae (p<0.001), and Silphidae (p<0.001),

45 but the differences depended on certain seasons (Table 2). Staphylinidae only accounted for < 22% of the populations for spring, autumn and winter, while in summer this beetle family accounted for > 61% across replicates. The five most dominant taxa for each season were used to evaluate taxa composition changes during decomposition for each season (Figure 3). The taxa of the winter trial were very different compared to the other seasons. Blow fly species were not among the most the dominant taxa as the with seasons, as well as it followed a different pattern of succession where members of Coleoptera were the first primary colonizers and remained the dominant order throughout decomposition.

Community Structure:

There were significantly different necrophagous insect communities between summer and autumn (p=0.001) and summer and winter (p<0.001), while the communities were similar between autumn and winter trials (p=0.073) (Figure 4).

Further, there were significantly different necrophagous communities that characterized each stage of decomposition within each season (MRPP; p<0.001), with indicator taxa (ISA) different for each stage and between seasons (Figures 5-7).

The indicator taxa for summer were the following: L. coeruleiviridis (indicator value

[IV] = 50.7, p=0.001) for fresh stage, P. regina (IV=47.8, p<0.001) for bloat stage,

Muscidae (IV=30.8, p = 0.025) for active decay, Staphylinidae (IV=33.3, p<0.001) and Histeridae (IV=25.9, p=0.043) for advanced decay and Cleridae (IV=25.0, p=0.049) for the dry stage (Figure 5). The indicator taxa for autumn were L. coeruleiviridis (IV=69.3, p=0.001) for the fresh stage, Staphylinidae (IV=55.3, p=0.007) for bloat, and Silphidae (IV=82.4, p<0.001), Muscidae (IV=41.2, p = 0.034),

46 and Nitidulidae (IV=41.2, p=0.034) for the active decay stage. The indicator taxa for winter were Staphylinidae (IV=64.9, p=0.004) for fresh stage, Muscidae (IV=59.9, p=0.038) for bloat stage and Silphidae (IV=62.0, p<0.001) and Carabidae (IV=55.6, p=0.039) for the active decay stage.

DISCUSSION

Eleven families of arthropods were responsible for carrion decomposition in this study. The successional pattern of the necrophagous communities followed patterns similar to those reported in previous research (Payne 1965, Archer 2003,

Tabor et al. 2004, Watson and Carlton 2005), but with some notable differences.

The spring and summer trials all began with little Coleopteran activity and greater adult and larval Calliphoridae activity. The Calliphoridae larvae were responsible for the majority of organic tissue decomposition (Carter et al. 2007), with the

Coleoptera either aiding in the consumption of the carcass or preying on blow fly larvae that were also there. During the advanced decay and dry stages, the only invertebrates present at the body were a few remaining Calliphoridae larvae and

Coleoptera consuming the dry remains of the carcass similar to previous studies

(Arnaldos et al. 2004). The spring and summer communities followed similar trends as those described in other research (Watson and Carlton 2003, Tabor et al.

2004, Matuszewski et al. 2008). The autumn and winter carcasses underwent a different pattern of decomposition. While the carcasses in the autumn trial did have blow fly oviposition, the eggs did not hatch. Winter carcasses never experienced

47 any oviposition, which drastically reduced the rate of decomposition (Gomes et al.

2006). The carcasses in the autumn trial appeared to only achieve the end of active decay, due to mummification and progression of the microbial community decomposition, while the winter carcasses appeared to proceed through bloat just into the active decay which has been noted by Watson et al. (2005). While fly activity was minimal for both autumn and winter, the beetle communities followed a similar pattern to the spring and summer trials. It is hypothesized that the cold weather that occurred at the onset of the autumn and winter trials along with a large amount of beetle activity, could have delayed the flies from ovipositing on the carcasses. While the seasonal differences noted here share similarities with other necrophagous invertebrate successional research, there are discrepancies. Most successional decomposition research has been conducted in either spring and summer during the warmer weather months (Tabor et al. 2004, Schoenly et al.

2007) or different geographical regions, with milder winter temperature (Richards and Goff 1997, Carvalho and Linhares 2001, Archer 2003, Bharti and Singh 2003,

Watson and Carlton 2005, Martinez et al. 2007).

The carcasses in the spring and summer trials both underwent the expected patterns of community assembly. There was low taxa richness at the beginning of decomposition; then as the carrion entered the bloat and active decay stages the taxa richness and variation greatly increased. This indicates the importance of using replicate carcasses in decomposition research and highlights areas of research interest related to community assembly rules in nature. With the bloating stage, the volatile byproducts produced by the microbial communities could act as signaling

48 agents to attract or repel certain insects during the decomposition process

(Tomberlin et al. 2011b). As the carcasses progressed to the advanced decay and dry stages, the taxa richness was reduced. With the autumn and winter trials the carcasses experienced the same pattern of decomposition as the spring and summer trials through the bloat stage, but progressed much more slowly, probably due to little Diptera activity when compared to the other seasons. This left enough of the carcass around to continue to attract new taxa several months into the decomposition process for both the autumn and winter trials.

Certain taxa demonstrated relative abundance changes among seasons or were more prevalent in specific seasons. P. regina and L. coeruleiviridis and the families Muscidae, Staphylinidae, and Silphidae did have significant differences across seasons (Table 2). There was also notable differences in the appearance of specific taxa in different seasons, such as Cocholiomayia macellaria only collected in spring (Table 1) which was similar to that reported by Centeno et al. (2002), Tabor et al. (2004), Arnaldos et al. (2004), Gill (2005), and Sharanowski et al. (2008).

Within each season through decomposition, Staphylinidae and Silphidae were the only consistent taxa throughout the seasons (Figure 3)and Silphidae were dominant for every season except summer where Staphylinidae was composed of > 60% of the population (Table 2). Further, Silphidae predominantly colonized the carcasses that were farthest from the tributary that flowed through the reserve and the carcasses that were located near the floodplains had higher colonization of Staphylinidae. For overall successional patterns, except for the winter trial, there were similar patterns

49 of succession. The initial dipteran activity followed by coleopterans, but winter had little dipteran activity and was dominated by Coleoptera.

This is one of the first studies to use multivariate analysis to describe invertebrate communities over decomposition, as well as among seasons for carrion decomposition. Using this approach, it was determined that significant seasonal differences among seasons (Figure 4). These data were further supported by the taxa composition (Table 2 and Figure 3) as well as other research (Centeno et al.

2002, Arnaldos et al. 2004, Tabor et al. 2004, Gill 2005, Sharanowski et al. 2008).

Further, there were significant differences (p = < 0.05) among the stages of decomposition; which has been described subjectively (Payne 1965, Schoenly et al.

2007) but, never quantitatively or statistically. Fresh stage was indicated by Diptera colonization, except for the winter trial that had minimal dipteran activity. The bloat and active decay stages for all seasons were best represented by taxa such as

Silphidae, Staphylinidae, Nitidulidae, and Carabidae, which can be attracted to the carrion, as well as the invertebrates colonizing the carcasses (Watson and Carlton

2003, Arnaldos et al. 2004, Tabor et al. 2004). In the summer the advanced stage was represented by Histeridae and the dry stage by Cleridae which is consistent with previous literature (Byrd and Castner 2010).

The relationship between ADH and initial oviposition by flies was also examined in these experiments. For the spring, summer, and autumn trials ADHs were 671, 345, and 121respectively, while the actual post mortem interval in days was 3, 1, and 2 respectively. ADH was not a reliable means of predicting when initial oviposition occurred. Instead initial oviposition was more often explained by the

50 amount of time since death for the carcasses. The summer ADH was half that of spring, but was 66%. Another impact factor was the weather. The reason the spring trial initial oviposition might have occurred later than the other two seasons was due to rain for the first two days at the beginning of the trial, which can inhibit adult dipteran activity (Mahat et al. 2009). The lack of oviposition within the first several days of death for the carcasses in the winter trial can be attributed to the weather since adult dipterans are not able to function at colder temperatures (e.g. 0

- 10°C) and will enter diapause (Ames and Turner 2003).

In conclusion, this study contributes to a call to enhance the science of forensics (Natl. Res. Counc. (U.S.) et al. 2009, Tomberlin et al. 2011a, Tomberlin et al.

2011b). The use of multiple replicate carcasses allowed for strong statistical analyses and ability to evaluate variation within and among seasons. The use of multiple seasons in this research demonstrated that taxa composition and community assembly can vary across seasons (Archer 2003, Tabor et al. 2004,

Matuszewski et al. 2010), so more research is required that spans not only all seasons in a geographic area, but those areas over multiple years. Further, there has been limited studies that have taken place in the autumn or winter colder weather regions and for those that have been done, very few take into consideration the winter months (Gill 2005, Sharanowski et al. 2008, Matuszewski et al. 2010). Little is known about carcass decomposition after freezing temperatures and thawing and how this might affect necrophagous invertebrate assembly.

Figure 2: Shannon Diversity Index for each sampling date during decomposition expressed as ADH.

Figure 3: Adult insect mean percent composition of the five dominant taxa for every sampling date (expressed as ADH) in each season; (A) spring, (B) summer, (C) autumn, and (D) winter.

58 Table 1: Mean necrophagous community taxa composition and variability (SD and coefficient of variation [CV]) variation within and among seasons. If no values are given the taxon was not present. An * denotes that there are significant differences

(p<0.05) for those taxa between seasons (One-Way ANOVA with Bonferroni's

Multiple Comparison Test) and are elaborated in Table 2.

59 Table 2: One-Way ANOVA and Bonferroni's Multiple Comparison Test results for taxa that were significantly different between seasons. (Sp = spring, Su = summer,

Au = Autumn, Wi = winter, and ns = no significant difference between those seasons).

60 Table 3: Accumulated degree-hour (ADH) ranges for each stage of decomposition during each season (- indicates that the carcasses for those seasons never reached that stage of decomposition).

61 CHAPTER 3: When Entomological Evidence Crawls Away: Phormia regina En Masse

Larval Dispersal

ABSTRACT

In criminal and civil legal investigations the forensic entomologist usually assists in providing an estimate of the postmortem interval (PMI), which closely couples with the time or period of insect activity (PIA). A minimum PIA is often interpreted and estimated by dipteran larval developmental age of the oldest specimens collected at a crime scene and/or autopsy. In entomological evidence protocols investigators are recommended to search a 2-10 m perimeter area for the oldest larvae that may have begun to disperse away from the body for burial and pupation. In this study, we described a case of a large aggregate (>90% larvae) en masse post-feeding dispersal of blow fly larvae away from replicate swine carcasses serving as models of human decomposition. Larval dispersal was evaluated for a spring and a summer trial, with en masse characteristics only occurring during the latter. This en masse dispersal occurred in five out of six replicate carcasses and masses moved from 2-26 m away. These data and observations suggest the

62 importance of performing searches greater than 10 m from human remains for entomological evidence at crime scenes. By missing the oldest larvae at a crime scene, interpretation of entomological evidence can be compromised and erroneous.

Based on these data and observations we recommend the crime scene investigators and researchers consider increasing the search radius around crime scene remains to increase the likelihood that the oldest larvae have been collected for analysis.

INTRODUCTION

Recent recommendations have been made to encourage the use of basic science to inform forensic entomology as a response to a US National Research

Council report (2009) (Tomberlin et al. 2011a, Tomberlin et al. 2011b). In this paper we offer novel information related to forensic entomology that is commensurate with this response. Even with enhanced technological advances in the forensic sciences, insects are often a valuable form of crime scene evidence that is easily and quickly collected (Arnaldos et al. 2005, Byrd et al. 2010). The dominant taxa most often used in forensic entomology include species from the order Diptera

(primarily the family Calliphoridae) and Coleoptera (Schoenly et al. 1992, Goff

2009). Blow flies (Diptera: Calliphoridae) are often the initial colonizers during invertebrate succession on decomposing animal remains, including humans (Hanski

1987, Byrd and Castner 2010). Blow flies go through holometabolous development with egg, larval, pupae, and adult stages (Castner 2010); the larvae are responsible for much of human body decomposition and are often used to provide an estimate of

63 the postmortem interval (PMI) by quantifying the period of insect activity (PIA)

(Amendt et al. 2007; Goff 2009; Byrd et al. 2010b). The PMI and PIA are often, but not always, closely coupled in duration (Tomberlin et al. 2011a, Tomberlin et al.

2011b). Deviations between the two can occur when a body has been concealed or treated in a way that impedes or delays insect colonization (Campobasso et al. 2001;

Amendt et al. 2007; Anderson 2010). In the latter part of the third instar, larvae stop feeding on the carcass and begin dispersal. These are referred to as post- feeding third instars (Arnott and Turner 2008), and typically undergo dispersal from the carcass as individuals or in small groups, but not as a collective mass that makes up >90% of the larval population associated with the carcass, as described below. We differentiate between individual and en masse dispersal based upon the number of larvae concentrated in a defined area on and away from the carcass.

Quantifying the PIA requires collecting and identifying the oldest (often, but not always, the largest (Goff et al. 1989)) larvae at a crime scene either on the body, in the surrounding environment near human remains or during the post-mortem examination. The developmental stages of these specimens are then compared to local environmental temperatures as accumulated degree hours (ADH) or accumulated degree days (ADD). The ADH and ADD are accumulated heat units that are associated with, and influence, insect growth and development and can be used to predict insect development. Each species requires a threshold range of accumulated heat units to develop from an egg to adulthood (Hall 2010). By determining the stage and age of the oldest larvae and the associated ADH/ADD required for the species to reach that stage, a forensic entomologist can then

64 estimate the time of initial blow fly colonization as eggs (or larvae for some species

[i.e. Sarcophagidae]), which often occurs within hours of death (Haskell et al. 1990;

Anderson 2000; Ames et al. 2003; Amendt et al. 2007); this is considered the PIA

(Amendt et al. 2007; Tomberlin et al. 2011a; Tomberlin et al. 2011b). Thus, the PMI is often very similar to the PIA if the oldest larvae are collected from bodies that have been placed in the natural environment without any barriers/inhibitors, such as clothing or submersion in water, to invertebrate access (Turner 2009). Finding and collecting the oldest larvae can be challenging for inexperienced investigators because third instars go through a post-feeding dispersal stage where in some blow fly species larvae move away from the body in search of a suitable location for pupation (Gomes et al. 2006). Once the larvae find an appropriate location, they burrow into the soil, under leaf litter, and undergo pupation, after which adults emerge (Merritt and Benbow 2009, Castner 2010).

Post-feeding dispersing larvae are most often reported as radiating out individually in a uniform, 360 pattern from the decomposing remains (Tessmer and Meek 1996). These dispersal events are most often documented in waves of individuals and not in large masses or aggregate groups (Smith et al. 1981, Smith

1986, Byrd et al. 2010). For the purposes of this paper we define en masse post- feeding dispersal when >90% of all larvae on the carcass move at the same time and in the same direction as one or two collective aggregates. To the best of our knowledge this phenomenon has not been formally described in the peer-reviewed literature, whereas there have been reports of smaller groups of larvae moving collectively from remains (Smith et al. 1981, Smith 1986, Byrd et al. 2010), but not

65 nearly the entire population at the same time and in the same direction for > 5 m as described here. For most forensic entomological evidence collection protocols, a 2-

10 m search perimeter around the remains is recommended to account for larval dispersal away from the body (Green 1951; Nuorteva 1977; Greenberg 1990;

Haskell et al. 1990; Amendt et al. 2007). In the search area, investigators are recommended to visually search for moving larvae while also taking random soil samples to uncover buried pupae (Lundt 1964, Vogt and Woodburn 1982).

The objectives of this paper were to 1) document and describe a unidirectional large aggregate en masse dispersal of larvae from replicated decomposing carcasses; 2) compare this en masse dispersal to the more commonly reported individual-based radial larval dispersal, where the majority of larvae disperse in a 360 pattern; and 3) discuss the importance of including a larger search area around human remains to detect the oldest larvae or pupae at a crime scene, having direct bearing on entomologically-based estimates of the PMI.

METHODS

In a companion study evaluating insect and microbial succession of carrion decomposition ecology in a forested habitat, six replicate Sus scrofa L. carcasses were used as models of human decomposition in each of two seasons (May-June and

July-August 2009) (Catts and Goff 1992, Carvalho et al. 2000). The study site was located within Morris Bean Reserve of Greene County, OH, and is a Midwest temperate forest lot surrounded by agricultural fields. There is a small tributary

66 stream that runs through the reserve, which empties into the Little Beavercreek

River that is adjacent to the property. The predominant trees are honey locust

(Gleditsia triacanthos [Leguminosae]) and a variety of maples (Acer spp.), while the most common sub-canopy cover is Amur honeysuckle (Lonicera maackii Rupr.). The ground cover is uniform throughout the study site.

Five plots along each of six, 50 m transects were established running north to south in the reserve. Each plot was a minimum of 10 m from others with a maximum of 80 m apart. For both trials, six 14-18 kg swine carcasses were purchased from a local farm. Immediately after death (i.e., within minutes) each pig was double bagged in black heavy duty garbage bags that were tightly duct taped in order to prevent access by any invertebrates. The pigs remained bagged for 2-3 h during transport to the study site and were placed at randomly chosen plots along each transect from 1800-1815 h. For the spring and summer trial, carcasses were exposed on 15 May and 22 July, respectively. Each carcass was placed under wire mesh exclosure cages (0.6 x 0.9 x 0.6 m) (Fig. 1) to prevent access and disturbance by large scavengers (e.g., coyotes, vultures). Blow fly larvae were collected in a tiered approach to minimize the effects of sampling on larval interactions and their activity during decomposition: when total larval abundance was low (100-500 larvae) only an estimated 10% of the population was collected (usually from 10-50 larvae), while for larger masses (>1000 larvae) a total of 100 larvae were collected from all larval masses on or off of each carcass. Temperature was recorded using

NexSens DS1921G micro-T data loggers (Fondriest Environmental Inc., Beavercreek,

OH) every 15 min through the duration of the study. Rainfall was calculated using

67 data accumulated at a local weather station (Station code: KSGH, Springfield, OH) that is approximately 8 km away from the study location. The ground slope was measured for each carcass location and videos and pictures were taken using an

Olympus Stylus 1050 SW (Olympus, Center Valley, PA) camera. Soil characterization was determined using the Munsell Soil Color Chart (Munsell Color

Company Inc., Baltimore, MD) and Green County, Ohio Soil Survey (1978) from samples taken about one meter and uphill from each carcass location. Larval identifications were made using Hall (1977), Cutter and Dahlem (2004) and

Whitworth (2006).

RESULTS

Spring Dispersal:

During late May 2009, we observed post-feeding larval dispersal patterns that conform to what has been reported in the literature (Smith et al. 1981, Smith

1986, Byrd et al. 2010) of larval activity commensurate with established protocols for entomological evidence collection (Amendt et al. 2007b). Individual post- feeding third instar larvae traveled in every direction (nearly 360), seemingly at random with respect to direction and to each other, and then began to burrow for pupation within about a 2-6 m radius of the carcasses. For four of the carcasses there was successive oviposition resulting in multiple waves of larvae demonstrating individual-based radial dispersal at different time intervals but in the same pattern (Table 1); these dispersal events are sometimes called oscillations

68 (Smith et al. 1981). Initial oviposition for these carcasses took place on 17 March

2009 within 48 hours of death. The adults of two species were noted ovipositing on the carcasses during this spring trial: Cochliomyia macellaria Fabricus (Diptera:

Calliphoridae) and Phormia regina Meigen (Diptera: Calliphoridae) (Hall 1977).

Approximately 100 larvae from various stages of development were collected from each carcass, reared to adults and identified only as P. regina (Hall 1977). The environmental characteristics (e.g., temperature and rainfall) of each dispersal event were relatively similar among carcasses, and Staphylinidae and Silphidae were the beetle families found during the spring (Table 1). Families of Coleoptera were given to evaluate the potential for predation pressure differences among carcasses that may have been important to aggregate en masse dispersal documented in the summer trial.

Summer En Masse Dispersal:

In late July and early August 2009 we documented en masse post-feeding larval dispersal of P. regina from the replicate carcasses. During this time post- feeding third instar larvae from five of six carcasses moved as en masse aggregates away from the carcasses (Video S1, Fig. 2, Table 2); a gregarious dispersal activity very different from the previous spring and from previous reports on smaller masses of larvae moving < 10 m from remains (Green 1951; Nuorteva 1977;

Greenberg 1990; Haskell et al. 1990; Amendt et al. 2007). The larvae were traveling in a single direction and in one or two large masses from an individual carcass

(Video S1). As an example, the larval mass in Fig. 2B was 0.36 m at its widest point and 2.9 m long giving an estimated area of 1.0 m2, and moved 1.3 m in 45 min. The

69 masses were variable in depth but estimated to be from 1-6 cm deep, and were observed traveling in the same direction as an aggregate (usually north to northeast) from 2-26 m and then would begin to disperse in a more radial, 360 pattern, similar to the spring dispersal at the carcasses that were individual-based and most often reported in the published literature (Video S2) (Green 1951;

Nuorteva 1977; Greenberg 1990; Haskell et al. 1990; Amendt et al. 2007).

For the summer trial, initial oviposition on all carcasses took place on 23 July

2009 between 0800-1100 except for Carcass 4 that experienced oviposition during the evening of 22 July between 1900-2000 (Table 2). The environmental characteristics (e.g., temperature, rainfall and slope) of each en masse dispersal event were relatively similar among carcasses including the arrival date of the first two Coleoptera families (Staphylinidae and Silphidae) (Table 2). However, there was rain documented the two days prior (or day of) to the longest distance of en masse dispersal events on 31 July and 1 August (Table 2), suggesting rainfall and perhaps soil moisture may have been associated with long distance dispersal.

Photographic and video documentation (Videos S1 and S2) was taken of this dispersal activity. As shown in Fig. 2, the larvae were beginning to disperse en masse uphill at two time points about 45 min apart. By comparing the photographs to distances measured in the field, the mass moved an estimated 1.3 m in just 45 min giving a rate of 1.7 m/h. One of the larval masses traveled a distance of 26 m away from the body before dispersing in an individual-based radiating pattern, having traveled the last 9 m in 5 h (Table 2, Carcass 2). For four out of the five carcasses the larvae traveled north to northeast, upslope and along deer paths,

70 perhaps as the routes of least resistance. Carcass 1 was an exception, where the larval mass dispersed 2 m to the southeast of the carcass (Table 2).

Soil characteristics for each of the carcass locations in both the spring and summer trial are detailed in Table 3. Comparing the soil percent moisture in the summer trial, there was a higher level of water retention in the two plots that had the longest dispersal of the larval masses (Carcasses 2 and 3) and the lowest was soil near the plots where no en masse dispersal was documented (Carcass 6). Other than soil drainage, no other potential environmental differences were documented

(Table 2).

DISCUSSION

The results and observations described here are very different from larval dispersal patterns that have been reported in previous literature (Smith et al. 1981,

Smith 1986, Byrd et al. 2010). For instance, a study by Greenberg (1990) detailed larval dispersal behavior of several species of Diptera and documented that 98% of

P. regina remained at a food site (i.e., various species of carcasses) and the other 2% did not travel more than 3.3 m away. In another study by Green (1951) it was reported that larvae could travel up to 10 m away from the source. In a recent paper by Amendt et al. (2007b), the search radius for post-feeding larvae was recommended to be in a 2-10 m 360 radius surrounding the body in forensic entomological collections. The maximum distances reported here are about twice as far than what has been heretofore reported and recommended in the literature.

71 This en masse behavior has been noted before but only as much smaller clusters of larvae leaving live sheep (Smith et al. 1981). The aggregate en masse exodus from carrion defined and reported here may have been stimulated from other larvae that had begun to move, similar to individual dispersal behavior reported in previous studies on larval blow flies (Smith et al. 1981, Smith 1986, Bass

1997). We suggest three hypotheses that may explain the behavior reported in this study. First, due to accumulated rainfall (Table 2), the soil downhill could have become too saturated and would have been poor conditions for larval burrowing and pupation (Bornemissza 1957); this could have been associated with the larvae moving to higher ground (usually to the northeast) where they would be able to disperse and find suitable soil. A second hypothesis may be that the larvae were part of the rapid decomposition of the carcasses during this summer (about 7 d), with the soil from underneath and immediately around the body being quickly saturated with decompositional fluids, ammonium, and other microbial metabolic byproducts that could have influenced P. regina to alter normal dispersal behavior

(Greenberg 1990) and leave the body en masse to search for unsaturated soil

(Bornemissza 1957) or potentially for another food source (Gomes et al. 2006). A third hypothesis may be associated with predator presence and/or abundance, such as Coleoptera (Staphylinidae) that were first present on all carcasses that experienced en masse dispersal during the summer trial. Additionally, but not studied here, would be the influence of other insect and bird or rodent predators during the summer period that may have influenced this aggregate larval dispersal.

It is beyond the scope of this paper to evaluate all of the ecological factors that may

72 have contributed to this phenomenon. We recommend future studies that include both replicate study organisms and increased sampling regimes to better understand the frequency of this larval activity. Gomes et al. (2006) provide an excellent review of blow fly post-feeding larval dispersal that discusses additional ecological factors that might influence larval dispersal. It is obvious that more research is needed to explain this forensically important blow fly (P. regina) activity.

IMPLICATIONS FOR FORENSICS

The recommended search radius around human remains for forensic entomological evidence in the form of dispersing larvae is 2-10 m in a 360 search area (Amendt et al. 2007b). This search radius covers the dispersal patterns and distances for some species of blow flies, such as those that undergo pupation immediately adjacent to or underneath the carcass (Lundt 1964, Haskell and Catts

1990, Gomes et al. 2006). Further, Greenberg (1990) reported on species-specific dispersal in artificial channels, indicating that 98% of P. regina larvae did not move from the food source itself, contrary to the field measurements of this study. This study demonstrated that at least two of the replicate carcasses in the same habitat demonstrated aggregate larval en masse dispersal that was much greater than the currently recommended search radius, one traveling 14 m and the other traveling

26 m. Thus, in a forensic case with insect evidence there would be a 33% chance that about 90% of the dispersing larvae would not be potentially located and collected.

Because of these findings, we propose that when entomological evidence is part of a crime scene during forensic investigations that investigators and

73 researchers consider that the 360 search radius be extended to 20-25 m from the remains. Another aspect to consider is that these larvae traveled as aggregates en masse in one direction for extensive distances. The importance of a predominate northeasterly direction of dispersal is unclear but could be important in forensic investigations when searching for post-feeding larvae. If one direction predominates during en masse dispersal of certain seasons, this could be included in recommendations for entomological evidence collection that would extend beyond the currently recommended 2-10 m 360 search area (Tessmer and Meek 1996).

Lastly, we found that this behavior happens quite rapidly, resulting in observations of an en masse dispersal event being potentially missed during crime scene investigations. For example, one larval mass traveled 26 m over an estimated

12-14 h, a very short amount of time that could easily be missed during an investigation. If the carcasses would have been visited a few hours before or after that dispersal event, very different larval evidence would have been collected and there would have been no observation of this en masse event. In order to determine if this phenomenon occurs more often then what has been observed in most carcass decomposition studies (especially those with no replication), we recommend that researchers make more frequent collections or observations (e.g., every 2 hours) during these studies in order to document and study this event. This paper provides novel information about a blow fly activity that is part of the ecology of carcass decomposition, demonstrating the potential aspects of this process that scientist do not understand. As such, reporting such events to the broader forensic audience is a response to the National Research Council (US) report (2009) calling for better

74 basic science as the foundation for forensics as recently advocated by Tomberlin et al. (2011a, 2011b) .

Figure 1: A swine carcass during decomposition but before the formation of a larval mass, showing the ground cover conditions and the scavenger exclosure. The carcass length is about 0.9 m.

77 Figure 2: (A) Phormia regina larval mass in the shape of a swine carcass (about 0.4 m at the widest point) before dispersal and (B) the same mass (about 2.9 m long and

0.36 m wide) after 45 min en masse dispersal.

Video S1: A video capturing a large larval mass during the initial stages of en masse post-feeding dispersal through the understory of the wooded study site.

(http://academic.udayton.edu/BenbowLab/Benbow_Lab/Videos.html)

80 Table 1: Characteristics of post-feeding larval dispersal (Phormia regina) from replicate swine carcasses from May-June 2009. Variables that could potentially influence larval dispersal are provided which include date of initial dispersal, the number of dispersal events per carcass, estimated distance traveled, percent slope at carcass, accumulated degree hours (ADH) and median ambient temperature (and range) from time of death to first observed dispersal event, relative humidity, rainfall, and first and second Coleoptera taxa to colonize each carcass.

a Distances were estimated.

81 Table 2: Characteristics of aggregate en masse larval (Phormia regina) dispersal from replicate swine carcasses from July-August 2009. Factors evaluated for association with dispersal distances are given for each carcass in addition to the first and second Coleoptera taxa to colonize each carcass, accumulated degree hours

(ADH) and median ambient temperature (and range) from time of death to first observed dispersal event, humidity and rainfall on the date of dispersal. Larval mass temperatures are provided to evaluate possible larval mass thermogenisis influence on insect development and dispersal.

a Larvae dispersed away from the body, but not en masse as the other larval masses.

Individuals dispersed in all directions but most went to the southeast. b These temperatures were taken from the larval mass the day before migration since the en masse groups were not aggregates near the end of the dispersal event.

83 BIBLIOGRAPHY

Amendt, J., R. Zehner, and F. Reckel. 2007a. The nocturnal oviposition behavior of blowflies (Diptera: Calliphoridae) in Central Europe and its forensic implications. Forensic Science International in press.

Amendt, J., C. P. Campobasso, E. Gaudry, C. Reiter, H. N. LeBlanc, and M. J. R. Hall. 2007b. Best practice in forensic entomology — standards and guidelines. Int J. Legal Med 121: 90-104.

Ames, C., and B. Turner. 2003. Low temperature episodes in development of blowflies: implications for postmortem interval estimation. Medical and Veterinary Entomology 17: 178-186.

Archer, M. S. 2003. Annual variation in arrival and departure times of carrion insects at carcasses: implications for succession studies in forensic entomology. Australian Journal of Zoology 51: 569-576.

Arnaldos, M. I., E. Romera, J. J. Presa, A. Luna, and M. D. Garcia. 2004. Studies on seasonal arthropod succession on carrion in the southeastern Iberian Peninsula. Int J. Legal Med 118: 197-205.

Arnaldos, M. I., M. D. Garcia, E. Romera, J. J. Presa, and A. Luna. 2005. Estimation of postmortem interval in real cases based on experimentally obtained entomological evidence. Forensic Science International 149: 57-65.

Arnett, R. H., Jr. 2000. American Insects: A Handbook of the Insects of America North of Mexico. CRC Press, Boca Raton, FL.

Arnett, R. H., Jr., and M. C. Thomas. 2001. American Beetles: Archostemata, Myxophaga, Adephaga, Polyphaga: Staphyliniformia. CRC Press, Boca Raton, FL.

Arnett, R. H., Jr., M. C. Thomas, P. E. Skelley, and J. H. Frank. 2002. American Beetles: Polyphaga: Scarabaeoidea through Curculionoidea. CRC Press, Boca Raton, FL

84 Arnott, S., and B. Turner. 2008. Post-feeding larval behaviour in the blowfly, Calliphora vicina: Effects on post-mortem interval estimates. Forensic Science International 177: 162-167.

Bass, B. 1997. The body farm. Popular Science: 76-82.

Benecke, M. 2001. A brief history of forensic entomology. Forensic Science International 120: 2-14.

Benninger, L. A., D. O. Carter, and S. L. Forbes. 2008. The biochemical alteration of soil beneath a decomposing carcass. Forensic Science International 180: 70-75.

Bharti, M., and D. Singh. 2003. Insect faunal succession on decaying rabbit carcasses in Punjab, India. J Forensic Sci 48: 1-11.

Bornemissza, G. F. 1957. An analysis of arthropod succession in carrion and effect of this decomposition on the soil fauna. Aust. J. Zool 5: 1-12.

Brock, T. D. 1978. Thermophilic microorganisms and life at high temperatures. New York: Springer.

Bucher, A. E., and L. E. Lanyon. 2005. Evaluating soil management with microbial community-level physiological profiles. Applied Soil Ecology 29: 59-71.

Burkepile, D. E., J. D. Parker, C. B. Woodson, H. J. Mills, J. Kubanek, P. A. Sobecky, and M. E. Hay. 2006. Chemically mediated competition between microbes and animals: microbes as consumers in food webs. Ecology 87: 2821-2831.

Byrd, J. H., and J. L. Castner. 2010. Insects of Forensic Importance, pp. 39-126. In J. H. Byrd and J. L. Castner [eds.], Forensic Entomology; the utility of arthropods in legal investigations, Second ed. CRC Press, Boca Raton, FL.

Byrd, J. H., W. D. Lord, J. R. Wallace, and J. K. Tomberlin. 2010. Collection of Entomological Evidence during Legal Investigations, pp. 127-176. In J. H. Byrd and J. L. Castner [eds.], Forensic Entomology; the utility of arthropods in legal investigations, Second ed. CRC Press, Boca Raton, FL.

Calbrix, R., K. Laval, and S. Barray. 2005. Analysis of the potential functional diversity of the bacterial community in soil: a reproducible procedure using sole-carbon-source utilization profiles. European Journal of Soil Biology 41: 11-20.

85 Campbell, N. A., J. B. Reece, L. A. Urry, M. L. Cain, S. A. Wasserman, P. V. Minorsky, and R. B. Jackson. 2008. Biology. Pearson Benjamin Cummings, San Francisco, CA.

Campobasso, C. P., G. Di Vella, and F. Introna. 2001. Factors affecting decomposition and Diptera colonization. Forensic Science International 120: 18-27.

Carter, D. O., and M. Tibbett. 2006. Microbial decomposition of skeletal muscle tissue (Ovis aries) in a sandy loam soil at different temperatures. Soil Biology and Biochemistry 38: 1139-1145.

Carter, D. O., D. Yellowlees, and M. Tibbett. 2007. Cadaver decomposition in terrestrial ecosystems. Naturwissenschaften 94: 12-24.

Carvalho, L. M. L., and A. X. Linhares. 2001. Seasonality of Insect Succession and Pig Carcass Decomposition in a Natural Forest Area in Southeastern Brazil. Journal of Forensic Sciences 46: 604-608.

Carvalho, L. M. L., P. J. Thyssen, A. X. Linhares, and F. A. B. Palhares. 2000. A checklist of arthropods associated with pig carrion and human corpses in Southeastern Brazil. Mem. Inst. Oswaldo Cruz, Rio de Janeiro 95: 135-138.

Castner, J. L. 2010. General Entomology and Insect Biology, pp. 17-38. In J. H. Byrd and J. L. Castner [eds.], Forensic Entomology; the utility of arthropods in legal investigations, Second ed. CRC Press, Boca Raton, FL.

Catts, E. P., and M. L. Goff. 1992. Forensic Entomology in Criminal Investigations. Annu. Rev. Entomol 37: 253-272.

Cederlund, H., T. Thierfelder, and J. Stenstrom. 2008. Functional microbial diversity of the railway track bed. Science of the Total Environment 397: 205-214.

Centeno, N., M. Maldonado, and A. Oliva. 2002. Seasonal patterns of arthropods occurring on sheltered and unsheltered pig carcasses in Buenos Aires Province (Argentina). Forensic Science International 126: 63-70.

Christian, B. W., and O. T. Lind. 2006. Key Issues Concerning Biolog Use for Aerobic and Freshwater Bacerial Community-Level Physiological Profiling. International Review of Hydrobiology 91: 257-268.

Cutter, R. M., and G. Dahlem. 2004. Identification Key to the Common Forensically Important Adult Flies (Diptera) of Northern Kentucky Department of Biological Sciences, Northern Kentucky University, Highland Heights, KY.

86 Danovaro, R., G. M. Luna, A. Dell'Anno, and B. Pietrangeli. 2006. Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments. Applied Environmental Microbiology 27: 5982-5989.

Daviss, B. 2005. Growing pains for metabolomics. The Scientist 19: 25-28.

Dickson, G. C., R. T. M. Poulter, E. W. Maas, P. K. Probert, and J. A. Kieser. 2010. Marine bacterial succession as a potential indicator of postmortem submersion interval. Forensic Science International.

Dunfield, K. E. 2008. Lipid based community analysis, pp. 557-566. In M. R. Carter and E. G. Gregorich [eds.], Soil sampling and methods of analysis, 2nd ed. CRC Press, Boca Raton.

Ellis-Evan, J. C., J. Layboum, P. R. Bayliss, and S. J. Perriss. 1998. Physical, chemical and microbial community characteristics of lakes of the Larsemann Hills, Continental Antarctica. Archiv fur Hydrobiologie 141: 209-230.

Fiehn, O. 2002. Metabolomics - the link between genotypes and phenotypes. Plant Molecular Biology 48: 155-171.

Gallo, M., R. Amonette, C. Lauber, R. L. Sinsabaugh, and D. R. Zak. 2004. Microbial Community Structure and Oxidative Enzyme Activity in Nitrogen- Amended North Temperate Forest Soils. Microbial Ecology 48: 218-229.

Gibbs, J. P., and E. J. Stanton. 2001. Habitat Fragmentation and arthropod community change: carrion beetles, phoretic mites, and flies. Ecological Applications 11: 79-85.

Gill, G. J. 2005. Decomposition and arthropod succession on above ground pig carrion in rural Manitoba. University of Manitoba, Winnipeg, Manitoba.

Gill-King, H. 1997. Chemical and Ultrastructural Aspects of Decomposition pp. 93- 108. In W. D. Haglund and M. H. Sorg [eds.], Forensic Taphonomy: The postmortem fate of human remains. CRC Press, London.

Goff, M. L. 2009. Early post-mortem changes and stages of decomposition in exposed cadavers. Experimental and Applied Acarology 49: 21-36.

Goff, M. L., A. I. Omori, and J. R. Goodbrod. 1989. Effect of cocaine in tissues on the development rate of Boettcherisca peregrina (Diptera: Sarcophagidae). J. Med. Entomol 26: 91-93.

87 Gomes, L., W. Augusto, C. Godoy, and C. V. Zuben. 2006. A review of postfeeding larval dispersal in blowflies: implications for forensic entomology. Naturwissenschaften 93: 207-215.

Green, A. A. 1951. The control of blowflies infesting slaughterhouses. Ann. Appl. Biol 38: 475.

Greenberg, B. 1990. Behavior of postfeeding larvae of some Calliphoridae and a Muscid (Diptera). Ann. Entomol. Soc. Am. 83: 1210-1214.

Hall, R. D. 1977. The Blowflies of Virginia (Diptera: Calliphoridae). The Insects of Virginia 11.

Hall, R. D. 2010. The Forensic Entomologist as Expert Witness, pp. 453-476. In J. H. Byrd and J. L. Castner [eds.], Forensic Entomology; the utility of arthropods in legal investigations, Second ed. CRC Press, Boca Raton, FL.

Hanski, I. 1987. Carrion fly community dynamics: patchiness, seasonality and coexistence. Ecol. Entomol. 12: 257-266.

Haskell, N. H., and E. P. Catts. 1990. Entomology and Death - A Procedural Guide. Forensic Entomology Partners.

Haslam, T. C. F., and M. Tibbett. 2009. Soils of contrasting pH affect the decomposition of buried mammalian (Ovis aries) skeletal muscle tissue. Journal of Forensic Sciences 54: 900-904.

Ibekwe, A. M., and A. C. Kennedy. 1999. Fatty acid methyl ester (FAME) profiles as a tool to investigate community structure of two agricultural soils. Plant and Soil 206: 151-161.

Ibekwe, A. M., A. C. Kennedy, P. S. Frohne, S. K. Papiernik, C. H. Yang, and D. E. Crowley. 2002. Microbial diversity along a transect of agronomic zones. FEMS Microbiology Ecology 39: 183-191.

Insam, H., and M. Goberna. 2004a. Use of Biolog for the Community Level Physiological Profiling (CLPP) of Environmental Samples. Mol. Micro. Ecol. Man. 5.3.2: 1-8.

Insam, H., and M. Goberna. 2004b. Use of Biolog for the community level physiological profiling (CLPP) of environmental samples, pp. 853-860. In G. A. Kowalchuk, F. J. de Bruijn, I. M. Head, A. D. L. Akkermans and J. D. van Elsas [eds.], Molecular Microbial Ecology Manual. Kluwer Academic Publishers, Netherlands.

88 Janaway, R. C. 1996. The decay of human buried remains and their associated materials, pp. 58-85. In J. Hunter, C. Roberts and A. Martin [eds.], Studies in crime: An introduction to forensic archaeology. B.T. Batsford Ltd., London.

Janetski, D. J., D. T. Chaloner, S. D. Tiegs, and G. A. Lamberti. 2009. Pacific Salmon effects on stream ecosystems: a quantitative synthesis. Oecologia 159: 583-595.

Jenkinson, D. S. 1977. The soil biomass. NZ Soil News 25: 213-218.

Kelly, J. A., T. C. Van Der Linde, and G. S. Anderson. 2008. The Influence of Clothing and Wrapping on Carcass Decomposition and Arthropod Succession: A Winter Study in Central South Africa. Can. Soc. Forensic Sci. J. 41: 135-147.

Kelly, J. J., and R. L. Tate. 1998. Use of Biolog for the analysis of microbial communities from zinc-contaminated soils. Journal of Environmental Quality 27: 600-608.

Kirk, J. L., L. A. Beaudette, M. Hart, P. Moutoglis, J. N. Klironomos, H. Lee, and J. T. Trevors. 2004. Methods of studying soil microbial diversity. Journal of Microbiological Methods 58: 169-188.

Kreitlow, K. L. T. 2010. Insect Succession in a Natural Environment, pp. 251-270. In J. H. Byrd and J. L. Castner [eds.], Forensic Entomology: The Utility of Arthropods in Legal Investigations, 2nd ed. CRC Press, Boca Raton, FL.

Kuske, C. R., L. O. Ticknor, M. E. Miller, J. M. Dunbar, J. A. Davis, S. M. Barns, and J. Belnap. 2002. Comparison of soil bacterial communities in rhizospheres of three plant species and the interspaces in an arid grassland. Applied Environmental Microbiology 68: 1854-1863.

LeBlanc, H. N. 2008. Olfactory stimuli associated with the different stages of vertebrate decomposition and their role in the attraction of the blowfly Calliphora vomitoria (Diptera: Calliphoridae) to carcasses. The University of Derby.

Lewis, A. J., and M. E. Benbow. 2011. When Entomological Evidence Crawls Away: Phormia regina En Masse Larval Dispersal. Journal of Medical Entomology In Press.

Lundt, H. 1964. Ecological observations about the invasion of insects into carcasses buried in soil. Pedobiologia 4: 158-180.

Mahat, N. A., Z. Zafarina, and P. T. Jayaprakash. 2009. Influence of rain and malathion on the oviposition and development of blowflies (Diptera:

89 Calliphoridae) infesting rabbit carcasses in Kelantan, Malaysia. Forensic Science International 192: 19-28.

Mardis, E. R. 2008. Next-Generation DNA Sequencing Methods. Annual Review of Genomics and Human Genetics 9: 387-402.

Martinez, E., P. Duque, and M. Wolff. 2007. Succession pattern of carrion-feeding insects in Paramo, Colombia. Forensic Science International 166: 182-189.

Matuszewski, S., D. Bajerlein, S. Konwerski, and S. Krzysztof. 2008. An initial study of insect succession and carrion decomposition in various forest habitats of Central Europe. Forensic Science International 180: 61-69.

Matuszewski, S., D. Bajerlein, S. Konwerski, and K. Szpila. 2010. Insect succession and carrion decomposition in selected forests of Central Europe. Part 1: Pattern and rate of decomposition. Forensic Science International 194: 85-93.

McCune, B., and J. Grace. 2002. Analysis of Ecological Communities. MjM, Gleneden Beach, OR.

Merritt, R. W., and M. E. Benbow. 2009. Entomology, pp. 1-12. In A. Jamieson and A. Moenssens [eds.], Wiley Encyclopedia of Forensic Science. John Wiley & Sons, Ltd., New Jersey.

Miller, C. D., R. Child, J. E. Hughes, M. Benscai, J. P. Der, R. C. Sims, and A. J. Anderson. 2007. Diversity of soil mycobacterium isolates from three sites that degrade polycyclic aromatic hydrocarbons. J. App. Micro. 102: 1612- 1624.

Natl. Res. Counc. (U.S.), Comm. Indentifying Needs Forensic Sci. Community, Comm. Sci. Law Policy Glob. Aff., and Comm. Appl. Theor. Stat. Div. Eng. Phys. Sci. 2009. Strenghting Forensic Science in the United States: A Path Forward. Natl. Acad. Press: 352.

Papatheodorou, E. M., E. Efthimiadou, and G. P. Stamou. 2008. Functional diversity of soil bacteria as affected by management practices and phenological stage of Phaseolus vulgaris. Euro. J. Soil Biol. 44: 429-436.

Payne, J. A. 1965. A summer carrion study of a baby pig Sus Scrofa Linnaeus. Ecology 46: 592-602.

Payne, J. A., E. W. King, and G. Beinhart. 1968. Arthropod succession and decomposition of buried pigs. Nature 219: 1180-1181.

90 Preston-Mafham, J., L. Boddy, and P. F. Randerson. 2002. Analysis of microbial community functional diversity using sole-carbon-source utilisation profiles - a critique. FEMS Microbiology Ecology 42: 1-14.

Putman, R. J. 1978. Flow of energy and organic matter from a carcass during decomposition. Decomposition of small mammal carrion in temperate systems 2. Oikos 31: 58-68.

Richards, E. N., and M. L. Goff. 1997. Arthropod Succession on Exposed Carrion in Three Contrasting Tropical Habitats on Hawaii Island, Hawaii. Journal of Medical Entomology 34: 328-339.

Richardson, N. F., J. L. Ruesink, S. Naeem, S. D. Hacker, H. M. Tallis, B. R. Dumbauld, and L. M. Wisehart. 2008. Bacterial abundance and aerobic microbial activity across natural and oyster aquaculture habitats during summer conditions in a northeastern Pacific estuary. Hydrobiol. 596: 269- 278.

Ros, M., M. Gobema, J. A. Pascual, S. Larnmer, and H. Insain. 2008. 16S rDNA analysis reveals low microbial diversity in community level physiological profile assays. J. Microbiol. Methods 72: 221-226.

Rozen, D. E., D. J. P. Engelmoer, and P. T. Smiseth. 2008. Antimicrobial strategies in burying beetles breeding on carrion. Proceedings of the National Academy of Sciences 105: 17890-17895.

Sala, M. M., J. Pinhassi, and J. M. Gasol. 2006. Estimation of bacterial use of dissolved organic nitrogen compounds in aquatic ecosystems using Biolog plates. Aqu. Micro. Ecol. 42: 1-5.

Schimel, J. P., J. M. Gulledge, J. S. Clein-Curley, J. E. Lindstrom, and J. F. Braddock. 1999. Moisture effects on microbial activity and community structure in decomposing birch litter in the Alaskan taiga. Soil Biology and Biochemistry 31: 831-838.

Schoenly, K., M. L. Goff, and E. M. 1992. A Basic Algorithm for Calculating the Postmortem Interval with Arthropod Successional Data. Journal of Forensic Sciences 37: 808-823.

Schoenly, K. G., N. H. Haskell, R. D. Hall, and J. R. Gbur. 2007. Comparative Performance and Complementarity of Four Sampling Methods and Arthropod Preference Tests from Human and Porcine Remains at the Forensic Anthropology Center in Knoxville, Tennessee. Journal of Medical Entomology 44: 881-894.

91 Schoenly, K. G., N. H. Haskell, D. K. Mills, C. Bieme-Ndi, K. Larsen, and Y. Lee. 2006. Using Pig Carcasses as model corpses to teach concepts of forensic entomology and ecological succession. The American Biology Teacher 68: 402-410.

Sharanowski, B. J., E. R. 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.

Skirnisdottir, S., G. O. Hreggvidsson, S. Hjorleifsdottir, V. T. Marteinsson, S. K. Petursdottir, O. Holst, and J. K. Kristjansson. 2000. Influence of Sulfide and Temperature on Species Composition and Community Structure of Hot Spring Microbial Mats. Applied Environmental Microbiology 66: 2835-2841.

Slone, D. H., and S. V. Gruner. 2007. Thermoregulation in larval aggregations of carrion-feeding blow flies (Diptera : Calliphoridae). Journal of Medical Entomology 44: 516-523.

Smith, K. G. V. 1986. A manual of forensic entomology. Cornell Univ. Press, Ithaca.

Smith, P. H., R. Dallwitz, K. G. Wardhaugh, W. G. Vogt, and T. L. Woodburn. 1981. Timing of larval exodus from sheep and carrion in the sheep blowfly, Lucilia cuprina. Ent. exp. & appl. 30: 157-162.

Stefanowicz, A. 2006. The biolog plates technique as a tool in ecological studies of microbial communities. Polish J. Environ. Stu. 15: 669-676.

Stokes, K. L., S. L. Forbes, L. A. Benninger, D. O. Carter, and M. Tibbett. 2009. Decomposition Studies Using Animal Models in Contrasting Environments: Evidence from Temporal Changes in Soil Chemistry and Microbial Activity. In K. Ritz, L. Dawson and D. Miller [eds.], Criminal and Environmental Soil Forensics. Springer Science, London.

Swift, M. J., O. W. Heal, and J. M. Anderson. 1979. Decomposition in terrestrial ecosystems. Blackwell Scientific Publications, Berkely and Los Angeles.

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.

Tessmer, J. W., and C. L. Meek. 1996. Dispersal and distribution of Calliphoridae (Diptera) immatures from animal carcasses in Southern Louisiana. J. Med. Entomol 33: 665-669.

Thottathil, S. D., K. K. Balachandran, K. V. Jayalakshmy, G. V. M. Gupta, and S. Nair. 2008. Tidal switch on metabolic activity: Salinity induced responses on

92 bacterioplankton metabolic capabilities in a tropical estuary. Est. Coastal Shelf Sci. 78: 665-673.

Tomberlin, J. K., M. E. Benbow, A. M. Tarone, and R. Mohr. 2011a. Basic research in evolution and ecology enhances forensics. Trends in Ecology and Evolution 26: 53-55.

Tomberlin, J. K., R. Mohr, M. E. Benbow, A. M. Tarone, and S. VanLaerhoven. 2011b. A roadmap for bridging basic and applied research in forensic entomology. Annual Review of Entomology 56: 401-421.

Trienens, M., N. P. Keller, and M. Rohlfs. 2010. Fruit, flies and filamentous fungi - experimental analysis of animal - microbe competition using Drosophila melanogaster and Aspergillus mould as a model system. Oikos 119: 1765- 1775.

Turner, B. 2009. Experimental validation of forensic evidence: a study of the decomposition of buried pigs in a heavy clay soil. Forensic Science International 101: 113-122.

VanLaerhoven, S. 2010. Ecological Theory and Its Application in Forensic Entomology, pp. 493-518. In J. H. Byrd and J. L. Castner [eds.], Forensic Entomology; the utility of arthropods in legal investigations, Second ed. CRC Press, Boca Raton, FL.

Vass, A. A. 2001. Beyond the grave: understanding human decomposition. Microbiology Today 28.

Vass, A. A., S. A. Barshick, G. Sega, J. Caton, J. T. Skeen, J. C. Love, and J. A. Synstelien. 2002. Decomposition chemistry of human remains: a new methodology for determining the postmortem interval. J. For. Sci. 47: 542- 553.

Verschuere, L., V. Fievez, L. Van Vooren, and W. Verstraete. 1997. The contribution of individual populations to the Biolog pattern of model microbial communities. FEMS Microbiology Ecology 24: 353-362. Vogt, W. G., and T. L. Woodburn. 1982. Dispersal of post-feeding larvae of Lucilia cuprina J. Aust. Entomol. Soc. 21: 289-291.

Ward, D. M., M. J. Ferris, S. C. Nold, and M. M. Bateson. 1998. A natural view of microbial biodiversity within hot spring cyanobacterial mat communities. Micrbiology and Molecular Biology Review 62: 1353-1370.

Watson, E. J., and C. E. Carlton. 2003. Spring succession of necrophilous insects on wildlife carcasses in Louisiana. Journal of Medical Entomology 40: 338-347.

93 Watson, E. J., and C. E. Carlton. 2005. Insect succession and decomposition of wildlife carcasses during fall and winter in Louisiana. Journal of Medical Entomology 42: 193-203.

Weber, K. P., and R. L. Legge. 2010. Community-Level Physiological Profiling. Bioremediation, Methods in Molecular Biology 599: 263-281.

Wells, J. D., and L. R. Lamotte. 2010. Estimating the Postmortem Interval, pp. 367- 388. In J. H. Byrd and J. L. Castner [eds.], Forensic Entomology: The Utility of Arthropods in Legal Investigations, 2nd ed. CRC Press, Boca Raton, FL.

Whitworth, T. 2006. Keys to the Genera and Species of Blow Flies (Diptera: Calliphoridae) of America North of Mexico. Proc. Entomol. Soc. Wash. 108: 689-725.

Wilson, M. 2005. Microbial inhabitants of humans: their ecology and role and health and disease. Cambridge University Press, New York.