©2019 Maria Denise Gemmellaro ALL RIGHTS RESERVED

Maria Denise Gemmellaro

DISTRIBUTION AND ECOLOGY OF FORENSICALLY IMPORTANT BLOW FLIES

(DIPTERA: CALLIPHORIDAE) IN SICILY AND ECUADOR WITH A FOCUS ON

THE GEOMETRIC MORPHOMETRIC VARIATIONS AND NUTRITIONAL

ECOLOGY OF THE BLUE BOTTLE FLY CALLIPHORA VICINA.

MARIA DENISE GEMMELLARO

A dissertation submitted to the

School of Graduate Studies

Rutgers, The State University of New Jersey

In partial fulfillment of the requirements

For the degree of

Doctor of Philosophy

Graduate Program in Entomology

Written under the direction of

Dr. George C. Hamilton

And approved by

______

New Brunswick, New Jersey

MAY 2019

ABSTRACT OF THE DISSERTATION

DISTRIBUTION AND ECOLOGY OF FORENSICALLY IMPORTANT BLOW FLIES

(DIPTERA: CALLIPHORIDAE) IN SICILY AND ECUADOR WITH A FOCUS ON

THE GEOMORPHOMETRICS AND NUTRITIONAL ECOLOGY OF THE BLUE

BOTTLE FLY CALLIPHORA VICINA.

by MARIA DENISE GEMMELLARO

Dissertation Director:

Dr. George C. Hamilton

For my dissertation, I conducted a survey of blow flies (Diptera: Calliphoridae) in Sicily, Italy and Ecuador across four different altitudinal levels (Sicily: 20m, 700m, 1153m, and 1552m;

Ecuador: 561m, 1312m, 1948m, and 3336m). I determined the species richness, abundance and diversity of the blow fly communities in these two areas and across four elevations using four RESCUE!® POP! Fly Trap per baited with 100g of beef liver per trap. Twelve blow fly species were collected in Sicily while 17 species were collected in Ecuador. The main species collected in Sicily was Lucilia sericata (Meigen) (68.50%) while Consomyiops verena (Walker) was the main species collected (51.67% of total capture) in Ecuador. The total number of flies captured was highest at high elevations in Ecuador, while in Sicily it was highest at intermediate elevations. Blow fly activity was also assessed in lava fields and volcanic caves in

Sicily during the winter months and showed that colonization occurred even in the caves in the absence of light and the presence of low temperatures (between 4°C and 6°C).

Furthermore, I investigated the food preference of Calliphora vicina (Robineau-Desvoidy)

(Diptera: Calliphoridae) adults among different food sources, noticing significant differences

between males and females for certain trophic substrates. Finally, I conducted a geometric morphometric analysis of the right wing of C. vicina adults collected from different altitudinal levels and found significant differences across elevations, as well as between females and males.

iii

Acknowledgements

I would like to thank everyone that has helped me during my time at Rutgers as a graduate student. First of all, my gratitude goes to Dr. George Hamilton, who welcomed me into his lab and who has encouraged me and supported every project I have undertaken in any way he could. I would have not made it without his help and I truly consider myself lucky for having had him as my advisor. Many thanks also to Kate Hamilton for always being there for advice, support and affection. I also thank the rest of my committee, Dr. Gail Anderson, Dr.

Jeff Tomberlin, Dr. Changlu Wang, Dr. Jessica Ware and Dr. Lauren Weidner, for all their help during my research. In particular, Dr. Weidner has been a valid colleague and a precious friend since I came to Rutgers; she has helped me through every aspect of graduate school and has been there for me when I needed her the most. I would not be where I am today without her. I would also like to thank all the students that have helped me during my research. In particular I would like to thank Alessandra Pittalà and Mariela Dominguez for helping me collect insects in Sicily and Ecuador, and Melvin Delvillar, Marina Perez, Michael

Monzon, Brittany Naugle, Kayleigh Torcivia and Jessica Villata and Laura Rademaker for their help in the lab. I would also like to thank Elena Forzisi for all she has done for me in the last months; her help has played a major role in my progress and her friendship has been a priceless gift for me. Thank you also to the other graduate students in the program, especially Caryn Michel, who is one of the most resourceful individuals I know and who has helped me unconditionally in every aspect of my life, and Nakorn Pradit, a true friend with a huge heart that has enriched my life here at Rutgers. I also want to thank Dr. Richard Merrit; he has believed in me, helped and supported me since even before I started graduate school.

I want to express all my gratitude to him for helping me realize my value and being there for me every time I needed him.

I would like to thank all the students that have taken my classes, especially those who have come from abroad to attend them. Working with them has given me the opportunity to grow as a person and as an entomologist and I can’t thank them enough for that. I would also like to thank everyone in the Department of Entomology, from the administration to the rest of the faculty, for helping me with every practical aspect of graduate school.

I would like to thank my friends, the historical ones and the ones I have made in the last few years. Maria Antonella, Caterina, Lucrezia, Andrea, Cristina, Francesca, Katia, Mattia,

Simone, Nicolas and Angela: they may live across the ocean, but they are always close to me.

Finally, I would like to thank my family, my mom and my dad, my brothers Salvo, Alfio and

Antonio, my grandmother, my aunts and uncles, my cousins, Leo and Lala and everyone else at home that has been with me throughout this journey. I am deeply grateful for every one of them and could never express with words what they mean to me. Also, I want to thank

Baby Alice for her precious smiles; she has really shown me something I didn’t even know I liked this much.

Lastly, infinite thanks to Roberta, Regina and Anica, for all they have done during my time in graduate school. Thank you for making me laugh, for helping me take life less seriously and for staying with me throughout everything.

Dedication

I would like to dedicate my dissertation to my relatives who are no longer with me. In particular, I would like to dedicate it to my aunt Nunzia, who I am sure would have been very happy to see me reach this goal. I would then like to dedicate this to my grandparents

Papà Alfio and Nonna; I know they are proud of me and will always be with me.

I would also like to dedicate my dissertation to my grandfather Nonno Turi, to my aunts

Zina, Maria, Rosetta and uncles Zino, Salvatore, Biagio and Nicola.

And finally, I would like to dedicate my dissertation to Tina, my dog, who blessed me and my family with her presence for many years, and to Tobo and Micia, two other furry friends that have left only great memories.

Some of the data presented in Chapter 3 “An Analysis of Blow Fly (Diptera: Calliphoridae) Species Richness and Distribution Along Elevation Gradients in Sicily (Italy) and Ecuador” have been presented in the PhD dissertation of collaborator Mariela Alejandra Domínguez Trujillo entitled “Influencia de la altitud en la composición de comunidades de moscas necrófagas de importancia forense (Diptera: Calyptratae) en la provincia del Napo.”. Pontificia Universidad Católica del Ecuador.

The following chapter was previously published in a peer-reviewed journal: Chapter 2: Gemmellaro, M.D., C. Bucolo, E. Musumeci, G.C. Hamilton and L.M. Weidner. 2018. First Observations of Initial Blow Fly (Diptera: Calliphoridae) Activity on Lava Fields and in Subterranean Environments in Sicily in Cool Temperatures. Journal of medical entomology, 55(6): 1622-1626. (tjy099)

Table of Contents

Abstract…………………………………………………………………………..……...ii-iii

Acknowledgements……………………………………………………………………....iv-v

Dedication……………………………………………………………………………...….vi

List of Tables…………………………………………………………………………..viii-ix

List of Figures………………………………………………………………………...…x-xi

Chapter 1: Introduction ………………………………………………………………...1-18

Chapter 2: First Observations of Initial Blow Fly (Diptera: Calliphoridae) Activity on Lava

Fields and in Subterranean Environments in Sicily in Cool Temperatures……………...19-32

Chapter 3: Calliphoridae Species Richness and Distribution Along an Altitudinal Gradient in

Sicily (Italy) and Ecuador …………………………………………………………..…33-80

Chapter 4: Food preferences of the adult male and female blue bottle fly, Calliphora vicina

(Robineau-Desvoidy) (Diptera: Calliphoridae)………………………………………...81-100

Chapter 5: A Geometric Morphometric Analysis of Wing Variations in Shape and Size of the

Blue Bottle Fly Calliphora vicina (Diptera: Calliphoridae)……………………………...101-127

Chapter 6: Conclusions…………………………………………………………...…128-129

vii

List of Tables

Table 2.1: The amount of time required for blow fly colonization and development. Amount of additional days to reach the stage are noted in parentheses……………………………..31

Table 3.1: Location, level designation, elevation and geographical coordinates of collection sites in Sicily and Ecuador……………………………………………………...………….61

Table 3.2: Average temperature (±SE), average relative humidity (±SE) and total precipitation during the duration of the survey…………….……………………………....62

Table 3.3: Relative abundance (%) of blow fly species collected in Sicily. Relative abundances are shown by species per elevational gradient as well as overall relative abundance (Rel. Ab.) during the duration of the survey.…………………………………………………………63

Table 3.4: The male to female (m/f) ratio of adult blow flies found in Sicily. Ratios are depicted by species for each elevational gradient as well as an overall m/f ratio.…………..64

Table 3.5: Differences in species abundance across elevational gradients using Kruskal-Wallis one -way ANOVA and Dunn test with Benjamini-Hochberg adjustment..…………….….65

Table 3.6: Indicator species for elevational gradients in Sicily. A level designation, elevation, indicator value and p value are provided for each species. ……………………..…….……66

Table 3.7: Significant differences in blow fly communities in Sicily across elevational gradients using Multiple Response Permutation Procedure. ……………………..….…….67

Table 3.8: Relative abundance (%) of blow fly species collected in Ecuador. Relative abundances are shown by species per elevation gradient as well as overall relative abundance

(Rel. Ab.) during the duration of the survey. ………………..….…………………….....…68

viii

Table 3.9: The male to female (m/f) ratio of adult blow flies found in Sicily. Ratios are depicted by species for each elevational gradient as well as an overall m/f ratio. ……..…...69

Table 3.10: Differences in species abundance across elevational gradients using Kruskal-

Wallis one -way ANOVA and Dunn test with Benjamini-Hochberg adjustment……...…...70

Table 3.11: Indicator species for elevational gradients in Sicily. A level designation, elevation, indicator value and p value are provided for each species…………………………………71

Table 3.12: Significant differences in blow fly communities in Ecuador across elevational gradients using Multiple Response Permutation Procedure………………………………..72

Table 4.1: Food resources provided to C. vicina adults and their protein and carbohydrate content (prior to adding water)……………………………………………………………99

Table 4.2 Food preference ranking for overall (males and females), males only and females only of C. vicina………………………………………………………………………….100

Table 5.1: Principal Component Analysis eigenvalues that account for 95% of variation....118

Table 5.2: Dunn test with the Benjamini-Hochberg adjustment for pair-wise comparison of the Centroid Size (CS) of wings across altitudes………………………………………….119

Table 5.3: Canonical Variate Analysis assessing differences between wings belonging to

Calliphora vicina adults collected at different altitudes...……………………………..……..120

Table 5.4: Procrustes ANOVA analysis for Size and Shape – Altitude and Sex..…………121

List of Figures

Figure 2.1: Map of research site locations. The yellow pin represents the location of the cave, and the red pin represents the location of the terrestrial site………………………….....…32

Figure 3.1: Elevational gradient sampling sites in Sicily. Orange=20m (level 1), blue=700m

(level 2), yellow=1153m (level 3) and red=1552m (level 4) ……………………………...... 73

Figure 3.2: Elevational gradient sampling sites in Ecuador Blue=561m (level 1), yellow=1312m (level 2), red=1948m (level 3) and green= 3376m (level 4) …………….….74

Figure 3.3: Blow fly species diversity across elevational gradient in Sicily using the Shannon

(H) diversity index. ……………………………………………………………………….75

Figure 3.4: Sorenson's (CC) Similarity Index between elevational gradients in Sicily. a1=level

1 (20m), a2=level 2 (700m), a3=level 3 (1153m), and a4=level 4 (1552m) ………………...76

Figure 3.5: Nonmetric multidimensional scaling ordinations of blow fly communities across elevational gradients in Sicily. This ordination explains 91% of this ordination (minimum stress= 0.083). MRPP analysis showed significant differences between 700m and 1153m

(A=0.3035 P=0.035). Circles represent altitude 20m, triangles altitude 700m, squares altitude

1153m, crosses altitude 1552m. …………………………………………………………...77

Figure 3.6: Blow fly species diversity across elevational gradients in Ecuador using the

Shannon (H) diversity index. ………………………………………………………...……78

Figure 3.7: Sorenson's (CC) Similarity Index between elevational gradients in Ecuador. a1=level 1 (561m), a2=level 2 (1312m), a3=level 3 (1948m), and a4=level 4 (3336m)……..79

Figure 3.8: Nonmetric multidimensional scaling ordinations of blow fly communities across elevational gradients in Ecuador. This ordination explains 82% of this ordination (minimum stress= 0.14). MRPP analysis showed significant difference between all levels (561m and

1948m (A=0.2893 p=0.027); 561m and 3336m (A=0.2747 p=0.04); 1312m and 1948m

(A=0.31261 p=0.025); 1312m and 3336m (A=0.2977 p=0.028)), except between 1948m and

3336m (A=0.1031 p=0.083). Circles represent altitude 20m, triangles altitude 700m, squares altitude 1153m, crosses altitude 1552m. Circles represent altitude 561m, triangles altitude

1312m, squares altitude 1948m, crosses altitude 3336m…………………………………...80

Figure 5.1: Wing of Calliphora vicina showing the 19 landmarks used in the geometric morphometric analysis. ………………………………………………………………….122

Figure 5.2: Percentage of variance which each principle component accounts for..………123

Figure 5.3: Visual depiction of shape variation in the right wing of C. vicina (displaying PC1).

A) Lollipop graph, B) Outline graph, C) Wireframe graph……………………………….124

Figure 5.4: Projections of Procrustes-aligned landmark configurations on the first two principal components (=relative warps) of the shape covariance matrix. Males are displayed in blue, females are displayed in red..…………………………………………………….125

Figure 5.5: Canonical Variate Analysis on Altitudes of the first two Canonical variate scores. Altitudes: 20m (Red), 7oom (Green), 1153m (Blue), 1552m (Purple).……………126

Figure 5.6: Frequency plot of canonical variate 1 for the right wing of C. vicina showing separation between males and females (females=red, males=blue)...…………………..…127

CHAPTER 1

Introduction

Forensic Entomology

Forensic entomology is defined as the use of insects and other arthropods in legal investigations. The three main branches that can be distinguished in this field are urban entomology, stored product entomology and medico-legal entomology (Haskell and

Williams 2008). Urban entomology is the management of insect infestations in man-made structures, such as termites (Isoptera) or bedbugs (Hemiptera: Cimicidae). Stored product entomology refers to insect infestations of food products that have been harvested, processed and packaged, such as beetles in flour or grains. Medico-legal entomology refers to the use of insects in criminal investigations (i.e. in cases of neglect/abuse and homicide).

Insects can arrive to a body minutes to hours after death if the body is exposed (Anderson and VanLaerhoven 1996); moreover, they arrive to a carcass in a predictable sequence throughout the process of decomposition known as faunal succession (Goff 2000). This faunal succession on the body along with the developmental time of certain groups are commonly used for the estimation of the minimum postmortem interval (PMI), defined later in this chapter.

Blow flies

Blow flies (Diptera: Calliphoridae) are a cosmopolitan group of sarcosaprophagous flies, found in most terrestrial habitats, where they play various ecological roles. Blow flies can play an important part as pollinators and some species are commercially reared for the pollination of several crops (Sajjad et al. 2008; Howlett 2012). Certain blow fly species feed

2 on feces to obtain proteins; and for this reason, can vector enteric pathogens and be of great importance in medical entomology (Greenberg 1971, 1973). Colonization by calliphorids can also occur in living hosts (humans or other vertebrates), known as myiasis, and this phenomenon can have large negative impacts on the livestock industry (Wall et al. 1992).

Blow flies are well known for their being the primary colonizers of carrion. Calliphorid flies are typically the first flies to arrive on a decomposing corpse (Greenberg 1991) making them useful in a forensic context. Carrion represents an ephemeral yet rich resource to exploit for different purposes. Gravid females use it as an oviposition substrate (Norris 1965; Byrd and

Castner 2010) or to obtain a protein meal necessary for the development of their oocytes and as s substrate for egg-laying (Lee et al. 1992). A decomposing carcass also represents a source of nutrients for blow fly larvae and is a location for them to continually feed and develop if the size allows (Ireland and Turner 2006).

Blow flies are the main insect group used in forensic investigations because they have been observed to arrive and colonize a dead body shortly after death (sometimes within minutes) if the body is accessible, and can lay eggs shortly after arrival (Greenberg & Kunich 2002;

Carter et al. 2007; Johansen et al. 2014). Calliphorids are holometabolous insects and their developmental cycle depends on temperature, humidity, precipitations and other variables

(Byrd and Castner 2010). Eggs usually hatch within 24 hours into first instar larvae, if temperatures are suitable. Once hatched, blow fly larvae continuously feed on carrion and go through two molting events (three instars) prior to pupation. In the head region, larvae have a structure, called a cephaloskeleton, which terminates in two mouth hooks. These hooks allow the larvae to dig into the carrion and feed on it by shredding small pieces that are lysed through proteolytic enzymes and quickly ingested (Queiroz et al. 1997). After third instar

3 larvae have completed feeding, they migrate away from their food source in search of a protected, dark, dry place to pupate. The pupal stage is the longest stage of a blow fly life cycle. When fully developed, the adult fly emerges from its puparium using a special organ, the ptilinum, which inflates like a balloon, causing the tip of the puparium to pop open.

After eclosion, adult flies are whitish with non-functional, folded wings. This changes within

24 hours as the fly inflates its wings and the exoskeleton hardens and takes on a coloration

(Whiting 1914).

Knowing this information about their development is vital as their main contribution in forensic entomology is in the estimation of time of colonization (TOC), defined as the time elapsed from when an insect lays eggs on the body and its discovery (Catts and Goff 1992;

Amendt et al. 2004; Tomberlin et al. 2011). The use of blow flies in any type of forensic investigation is only possible if there are data available on their presence and distribution in a given area and if developmental data on the species recovered at a crime scene are available.

Understanding this type of basic information about blow flies is vital for any kind of analysis that involves their use in a forensic context.

Nutritional ecology of the adult blow fly

Calliphorid species have been studied extensively in terms of their development (Byrd and

Allen 2001; Grassberger and Reiter 2002; Owings et al. 2014) morphology (Sukontason et al.

2006, Whitworth 2010; Szpila and Villet 2011), molecular identification (Wells and Sperling

2001; Shayya et al. 2018; Sontigun et al. 2018), behavior (Podhorna et al. 2018, Hans et al.

2018; Yan et al. 2018) and feeding habits (Chaudhury et al. 2015; Bernhardt et al. 2017;

Salazar-Sousa et al. 2018). Data obtained from these studies have been useful for gaining an

4 understanding of blow fly biology and in forensic investigations. Nevertheless, most of our knowledge about calliphorids comes from what we have observed in the laboratory. In the wild, larval activity has been studied for several species; however, little is known about adult behavior. While we do see adults visiting decomposing matter when looking for an oviposition site (Byrd and Castner 2010) or protein meal (William et al. 1979), and occasionally on flowers for carbohydrates (Kevan 1973), their nutritional ecology is understudied. Exploring this area could be a way to shed more light on their behavior related to both their ecological and forensic roles.

Research has been done on the effect that different trophic substrates can have on the development and size of the larvae of several blow fly species (Kaneshrajah and Turner

2004; Clark et al. 2006; Ireland and Turner 2006; El-Moaty et al. 2013). Niederegger et al.

(2013) analyzed the development of C. vicina and Calliphora vomitoria (Lynnaeus) on beef, pork and turkey in different processed substrates (steak, liver and minced meat). They reported significant differences in the development of C. vomitoria, whose growth was more successful on processed substrates. Less has been done on the food preference of adults. Durdle et al.

(2016) observed that when offered different human and non-human substrates, there are significant difference between one day old Lucilia cuprina (Wiedemann) compared to three- day old flies.

In a crime scene involving a dead body, blow flies are commonly attracted to the body itself.

However, other potential food sources, such as leftover food, garbage, pet food or even biological traces like sperm and blood, could be present on the scene. Flies could therefore travel across different sources, feed on several of them, and create artifacts through

5 defecation and regurgitation (Durdle et al. 2013) that may mimic blood spatter patterns

(Benecke and Barksdale 2003). Genetic material can be transferred as well, making the scene difficult to interpret. Therefore, understanding the nutritional needs of blow flies can help assess their food preference; and this in turn, could provide additional information for their practical application in forensic contexts.

Species Richness and Distribution: Altitudinal Patterns

Studies have analyzed variations in the geographical patterns of species richness (Brown and

Lomolino 1998; Stevens 1992; Alahuhta 2015; Tang et al. 2017), and they have been correlated with climatic changes (Klanderud and Birks 2003), different habitats (Hortal et al.

2013), physiology and evolution (Dillon et al. 2006). Among the most researched drivers of species richness and distribution are latitudinal and altitudinal patterns (Wallace 1878; Pianka

1966; Brown and Lomolino 1998; MacArthur 1984, Stevens 1992; Gutierrez 1997; Rahbek

1997; Fleishman et al. 1998). Species richness has been found to decrease as latitude increases, a pattern that has been well documented (Wallace 1878; Pianka 1966; Brown and

Lomolino 1998) and it is commonly explained as a monotonic relationship with climatic factors or other energy related factors (Richerson and Lum 1980; Turner et al. 1987; Currie

1991; Rohde 1992; Wright et al. 1993; Austin et al. 1996; Grytnes et al. 1999). Another commonly found pattern is a humped-shaped pattern where species richness reaches its peak at intermediate elevations (Whittaker 1960; Janzen 1973; Whittaker and Niering 1975;

Shmida and Wilson 1985; McCoy 1990; Lieberman et al. 1996; Gutierrez 1997; Rahbek 1995;

Rahbek 1997; Fleishman et al. 1998). These two trends have been observed for several taxa and in different ecoregions (Terborgh 1977, Stevens 1992; Brown 1995; Rahbek 1995;

Rosenzweig 1995; Brown and Lomolino 1998); however, some studies have stated that the hump-shaped pattern is the more common with mid elevations having the highest species richness (Lees et al. 1999; Colwell and Lees 2000).

Geographic area (Schoener 1976), climate (Sanders et al. 2003; Bhattarai et al. 2004), geo- physical limits imposed by natural geographic boundaries (Colwell and Lees 2002) and anthropogenic impact (Nogués-Bravo et al. 2008) are the common drivers of species patterns along an elevational gradient. Several studies have looked at how insect species richness varies across elevational gradients (Olson 1994; Sparrow et al. 1994; Sanchez-

Rodriguez and Baz 1995; Fleishman et al. 1998; Szewczyk and McCain 2016). Studies on ants have shown that richness was high at low elevations, low at higher elevations, and no reported species above certain elevations (Collins 1980; Atkin and Proctor 1988). However, different results were observed in other studies on the relationship between ant species richness and elevation, where a peak in mid-elevation was actually reported (Fisher 1998;

Samson et al. 1997; Sanders 2002).

The geographical distribution of blow flies has been documented in many areas of the world showing variation across location and seasons (Ives 1991; Davies 1990; Richards et al. 2009,

Weidner et al. 2015). However, their distribution across an altitudinal gradient has received less attention (Tumrasvin et al. 1978; Baumgartner and Greenberg 1985; Baz et al. 2007;

Moophayak et al. 2014). The study and management of biodiversity requires baseline data on diversity, distributions and the drivers of variation. Blow flies play many ecological roles, and therefore exploring them may help understand more about the ecological dynamics of the ecosystem and their role in forensic investigations.

Geometric Morphometrics

Morphometrics can be defined as the quantitative analysis of biological shape, shape variation, and covariation of shape, in correlation with biotic or abiotic factors (Webster and

Sheets 2010). As opposed to traditional morphometrics, which rely only on a specific set of measures, such as linear distances, ratios or angles (Rohlf 1990), geometric morphometrics allows one to conduct analyses while preserving the relative spatial arrangement (Zelditch et al. 2004). This makes it possible to observe and assess variations in shape among different taxa, and within and between populations (Walker 2000).

Geometric morphometric analysis is based on multivariate statistics performed on anatomical landmarks, corresponding to biologically homologous points on a given anatomical structure or on the entire body (Bookstein 1991; Rohlf and Marcus 1993; Adams et al. 2004). Wings are of major importance to pterygote insects by providing flight, locomotion, defense, and sexual displays (Wootton 1992; Berwaerts et al. 2002). Insect wings have been analyzed extensively using geometric morphometrics to understand the effect of stress factors (such as pesticides) on the morphology of insects (Hoffmann 2002), to differentiate different geographic lineages (Hall et al. 2014), to quantify sexual dimorphisms

(Gidaszewski et al. 2009), and for species identification (Lyra et al. 2010).

Research Objectives

The research objectives of this dissertation are the following:

1. To analyze patterns of colonization and insect activities within volcanic caves and on

the surface during the cold season in Sicily.

Rationale: Few observations have been published on the activity of necrophilous insects in hypogenic environments. Common assumptions are that there is no activity in the absence of light and at low temperatures. Assessing their activity in cold, dark environments could help expand our knowledge on their behavior and lead to further research to explore their activity in different conditions.

H0: There is no difference in insect activity and colonization between a terrestrial environment and a hypogenic environments.

2. To analyze blow fly species richness, abundance and diversity across four altitudinal

levels in Sicily and Ecuador.

Rationale: No extensive research on blow flies has been conducted in these two areas. Even less has been done on the analysis of species richness and abundance of blow flies across different elevations. The results of this investigation could assess elevational patterns of richness in these areas for this group and provide a baseline checklist to be used as a reference in forensic investigations and other studies.

H0: There is no significant difference between blow fly richness, abundance and distribution in different environments and across altitudinal levels.

3. To assess the food preference of Calliphora vicina (Robineau-Desvoidy) (Diptera:

Calliphoridae) adults when offered different types of non-human food sources.

Rationale: As opposed to blow fly larval nutrition, data on the nutritional preferences of blow fly adults are lacking. Gaining an understanding of their nutritional needs and preference can help understand their biology. Moreover, it can help the interpretation of crime scenes where alternative blow fly food sources are present (left over food, pet food or biological fluids) and can improve rearing protocols.

H0: There is no significant difference in the food preference of C. vicina males and females when exposed to different food substrates.

4. To analyze variation in size and shape in the right wing of C. vicina adults collected

from different altitudinal levels.

Rationale: Insect wings’ morphology and size have been proven to be informative of the insect’s biology and to correlate with the environment where the insect lives. For example, for some taxa, body size has been observed to increase with altitude and latitude to conserving heat in cold climates (Kuclu et al. 2011). The analysis of the geometric morphometric changes observed in the wing of this blow fly can help distinguish among the elevations as well as between females and males.

H0: There is no significant difference in the shape and size of the wing of C. vicina adults among individuals collected from different altitudes and between females and males.

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CHAPTER 2

First observations of initial blow fly (Diptera: Calliphoridae) activity on lava fields

and in subterranean environments in Sicily in cool temperatures

Abstract

In criminal cases involving the recovery of human remains, as well as in cases of myiasis and pest management, the expertise of a forensic entomologist has been requested more and more frequently in Sicily. Recently, research on the insect species of forensic interest in Sicily has been investigated. The aim of this research was to raise awareness of this discipline and to build a Sicilian entomofauna checklist during cool temperatures. The predominant species observed in this study was Calliphora vicina, the first to colonize the carcasses; Lucilia spp. were also present. We wanted to explore the potential insect activity on decaying matter both on lava fields, exposed to sunlight, and in dark subterraneous environments. No activity was observed in our dark environment for 20 days; then, C. vicina was observed on one of the carcasses, laying eggs in complete darkness at a temperature of 6.4°C. Larval development under these conditions was delayed and the mortality rate was high. This preliminary trial allowed us to improve our experimental design and helped map new sites where we can expand our research to collect new data on insect distribution and their activity in caves.

Key Words: forensic entomology, Calliphora, decomposition, colonization, cold temperature, dark environments, volcanic caves

Introduction

Forensic entomology is the application of insects to any kind of legal issue, from insect infestations of food products, to the presence of insects in urban environments or in a medico-legal aspect, such as aiding in a minimum postmortem interval (m-PMI) determination (Smith 1986; Hall 1990; Greenberg 1991; Schoenly 1992; Byrd & Castner

2009). Insects in the orders Diptera and Coleoptera are usually the main insects of forensic importance. Blow flies (Diptera: Calliphoridae) are commonly the first insects to arrive at a carcass, and their arrival has been observed minutes after death (Greenberg & Kunich 2002).

For them, a carcass represents a trophic resource that is both important and ephemeral, and therefore it is important for them to access and colonize remains in the shortest possible time. When a body is recovered, if colonized by larvae, it could potentially help estimate a time of colonization (TOC) and aid in the determination of a time since death (Altamura and

Introna 1981; Byrd & Castner 2009; Reibe and Madea 2010; Reibe et al. 2010; Tomberlin et al. 2011). Since insects are poikilotherms, their development depends on the temperature of their environment (Borror and DeLong 1971; Daly et al. 1978; Byrd & Castner 2009).

Temperature is one of the most important parameters influencing the development of insects (Ratte 1984) and, specifically for blow flies, temperature seems to have an effect even more significant than photoperiod (Sharma et al. 2015). Blow fly development is one of the main methods used to estimate the m-PMI (Ames and Turner 2003). Temperature is used to calculate accumulated degree days (ADD) and accumulated degree hours (ADH), and these represent the energy units needed for a particular species to reach a particular developmental stage at a particular temperature. These are calculated by subtracting the lower threshold temperature of a species from the average daily or hourly temperature and then adding together the results for the entire period spent at that temperature (Wagner et al. 1984; Ames

21 and Turner 2003). It is assumed that no development can occur below the lower threshold temperature (Laudien 1973); however, the determination of the lower threshold temperature for a species is challenging, because even though a species cannot develop under a certain temperature, it can still remain alive. The development of Calliphora vicina and Calliphora vomitoria has been researched by Kamal (1958) and by Highely and Haskell (2001) and the lower threshold temperature for them has been determined to be 6°C; this value, however, could also be greatly dependent on the geographic origin of the species. Davies and Ratcliff

(1994), who worked on C. vicina and C. vomitoria collected from NE England, established a lower threshold temperature of 3.5°C for them. However, these results are not supported by the results of Ames and Turner (2003), who worked on the same species but collected their specimens from SE England; for these, the lower threshold temperature was calculated to be

5°C.

An aspect that needs to be considered when evaluating the temperature at which the species are developing is the variation between the recorded ambient temperature and the actual temperature the insects are experiencing. As noted by Turner (1991), the temperature that is commonly used to calculate development time and consequently m-PMI is the current ambient temperature of the scene (whether an experimental site or crime scene) or when looking for the historical temperature of a location, the temperature recorded by the closest weather station. There are, however, significant differences between the actual temperature at which the insects are developing and the recorded temperature; these differences are often neglected and could cause errors in the estimation of the m-PMI (Greenberg 1991; Turner

1991). The discrepancies in these values can be due to the so-called mesoclimatic effects,

22 which are caused by the physical characteristics of the specific geographic areas where decomposition and colonization is occurring (forest, grasslands etc.) and microclimatic effects, which are variances of the decomposing body itself and are often caused by the body’s conditions (i.e. wrapped in plastic or naked) and by larval activities (feeding, movement). In regard to the mesoclimatic effects, Reed (1958) noted their importance by showing the difference in decomposition rate between dog carcasses decomposing in pasturelands and others decomposing in woodlands, also factoring in seasonality.

The activity of fly larvae inside a carcass is known to raise the temperature of the carcass significantly compared to the temperature of the surrounding environment (Deonier 1940).

Turner and Howard (1992) observed temperatures of the larval masses in decomposing rabbit carcasses that were 5-20°C higher than the ambient temperature, reaching and maintaining peaks of more than 40°C, which is higher or close to the lethal temperature for

Calliphora spp. and Phormia regina which are 39°C and 45°C respectively (Wigglesworth

1967). One consideration that could be made is that when collected and taken to the lab, the number of larvae is lower than the number of larvae at the scene and this prevents them from reaching temperatures that are as high as the ones recorded in the larval masses at the scene; moreover, it is very likely that the temperature at which development will be recorded to occur is not the higher, larval mass temperature, but the lower, ambient temperature. Both these factors may cause the overestimation of the m-PMI (Turner and Howard 1992).

In regards to the presence and activity of specific insect groups, data for several geographic areas and for many specific eco-environments are lacking, making the application of forensic entomology problematic or impossible in these areas. The island of Sicily is an area where very little information regarding its entomofauna in general, and specifically the entomofauna of forensic interest is known. Our goal was to identify the initial colonizers of remains in lava fields and subterranean environments in Sicily during cool temperatures and in the absence of light.

Methods

Site locations

Sicily is the largest of the 20 regions of Italy; geographically, it is the biggest island of the

Mediterranean basin and its territory is characterized by different bio-geographic areas, geological assets and climatic regions (La Mela Veca et al. 2016). Sicily’s landscape is mainly dependent on agricultural activities, and along the active and abandoned farms, grasslands, pastures and shrublands can be observed (Barbera and Cullotta 2012).

The main mountain ranges are located in the northern and northeastern areas of the island; these are represented by Mt Etna (the tallest active volcano of Europe), Mts. Madonie,

Nebrodi and Sicani) (La Mela Veca 2016). Forests of non-native species (eucalypt and conifer) are widespread within typical Mediterranean assets that are extremely variable due to both anthropogenic and natural effects (Mazzoleni et al. 2004; Ruhl et al. 2005).

Next to agriculture, tourism is the other major economic resource for Sicily (Schneider and

Schneider 1976) and it is significantly influenced by seasonality (Cuccia and Rizzo 2011).

Tourists decide to visit Sicily for social or religious/cultural reasons, like special holidays or festivities or for natural reason, such as an eruption or the warm weather. Sicily is a typical

Mediterranean Island and its peak touristic season is considered to be the summer, which allows tourists to spend time by the sea, while the rest of the year, even though the weather remains quite pleasant (Cuccia and Rizzo 2011).

The landscape around Mt. Etna has been shaped in a unique way by the volcanic activity. In past 50 years, there have been several major eruptions (1971, 1983, 1991–1993, 2001 and

2002–2003) that have had an important impact on the human activities (Branca et al. 2017) and that have changed the silhouette of the volcano as well as the surrounding areas. Mt

Etna and the area around are protected by the Mount Etna Regional Nature Park, which was established in 1987 by the Sicilian Regional Authority; since 2013, Mt. Etna also became part of the Unesco World Heritage and according to some unpublished results produced by Mr.

Alfredo Pasqualino, since then, the flow of tourists in the park and to the volcano and its caves has increased by 49.55% (Sicilianetwork 2017). The Sicilian hypogenic landscape is as rich and various as its terrestrial one. The caves found in the area around Syracuse, in the central area of the island were created by water and are adorned by the typical stalactites and stalagmites structure. The caves of the northeastern part of Sicily, instead, are mainly of volcanic origin and were indeed created by subterranean rivers of lava flowing out of the volcano Mt. Etna.

The research site was located in eastern Sicily, outside of Bronte, on the western side of Mt.

Etna, the tallest active volcano of Europe. This area has no trees or major vegetation due to past lava eruptions (1651-1653), but does contain small plants such as red valerian

(Centranthus ruber, (L.) DC.), Mount Etna broom (Genista aetnensis, (Raf. ex Biv.) DC.), lichens

(Stereocaulon vesuvianum, Persoon, 1810) and stonecrop (Sedum spp.). Research sites were located on the surface of the lava field and inside a cave. The entrance to the cave was a circular opening in the ground with a diameter of 1.45 m. The main tunnel is approximately

150 m long; upon entering, the height of the cave is 2.3 m but it decreases to 80 cm after the first 30 m. Proceeding along the tunnel, with a width of approximately 3 m, the height of the tunnel became lower until the end of the cave, at which point there are very small galleries that can only be accessible by crawling.

Survey

During the winter (Dec - Feb) four carcasses were exposed (two chickens (Gallus gallus domesticus) and two piglets (Sus scrofa domesticus)), 10 m apart, on the surface of the lava field.

Each carcass was covered with a metal cage (30 x 30 x 60 cm3) to reduce predation, and five pitfall traps were placed approximately 50 cm around each carcass to collect crawling insects.

Carcasses were inspected twice a day, at 0800 and 1730 hours. Insects were collected with forceps both from the carcass and the soil beneath the carcass. Sweeps were not conducted during this experiment, as temperatures were too cold for flight (0 ± 1.6°C). Adult flies collected were placed into 30 mL plastic vials and frozen at -18°C. When larvae were collected, a sub-sample was placed in 75% ethanol, and the remaining larvae were placed on beef liver in a 0.25 L container with breathable lids to be reared to adulthood. For the

26 surface site, precipitation, humidity and temperature were recorded using the weather station located in the town of Bronte (37.8000° N, 14.8333° E).

Our subterranean site was located approximately 1.2 km from the first site (Figure 2.1). At this site we used a trap baited with beef liver, and a piglet carcass. The trap consisted of a plastic container (20 cm3) containing approximately 80 g beef liver with a small opening on top. A piglet carcass was placed 30 m away and was set up as previously mentioned. This site was visited daily, between 1600 and 1800 hours. For the subterranean site, temperature was recorded using a digital thermometer with a metal probe (Koch Electronic

Thermometer, Model 13211).

Results

In the lava fields, we put out the carcasses on 19 Dec 2012 at 1700 hours. During this trial, temperatures ranged from 1 – 7 °C with a mean temperature (± SE) of 4 ± 1°C.

Colonization occurred within the first 15 hours of exposure, and the first blow fly species to arrive was Calliphora vicina (Robineau-Desvoidy). Adults began to eclose from these carcasses by day 31 (Table 1). This species arrived at all four carcasses and was the only species to colonize the carcasses. Two adult Lucilia caesar (L.) were captured, one at a piglet carcass and one at a chicken carcass, but no colonization occurred from this species.

In the subterranean environment, we placed the carcass into the cave on February 10 2013 at

1600. The mean temperature (± SE) during this trial was 6 ± 2 °C, with temperatures

27 ranging from -8 to 10°C. No activity was observed on or around the carcass or baited trap for 20 days. On day 21, C. vicina adults were observed laying eggs on the carcass. In the days when oviposition was observed, the carcass was in complete darkness, with an ambient air temperature of 4°C. On day 22, two adults were collected in the baited trap while ovipositing. One individual was identified as C. vicina and the other as Calliphora vomitoria (L.).

The initial eggs laid on the carcass took 7 days to hatch, and by day 55 pupae were observed.

Due to the lava rock formation on the bottom of the cave not all the pupae were collected as they fell through the cracks and remained there until they eclosed, totaling 96 days (Table

2.1). Eclosed adults from within the cave were collected. These adults did not unfold their wings and had no pigmentation. They did not survive longer than 3 days and never unfolded their wings or acquired any coloration. Hatching and eclosion times are indicated in Table

2.1.

Discussion

Our study represents a preliminary observation on initial blow fly activity and colonization in dark environments and under low temperatures in Sicily. On the lava field, colonization occurred within the first 24 hours, and eclosion began by day 31. Lucilia caesar adults were observed and collected on and around the carcasses but no oviposition was recorded and no eggs or larvae were ever collected throughout the trial. A few studies have explored the activity and development of this species at different temperatures, however, none have been done using temperatures lower than 15 °C (Greenberg and Reiter 2001; Vanin et al. 2008;

Ring 1967). Therefore, at the present time, there is a lack of information on L. caesar at low temperatures.

In the cave, colonization began on day 21, and eclosion began by day 96. Both C. vicina and

C. vomitoria were found to be active and ovipositing, although only C. vicina colonized the carcass. In the cave, oviposition on the piglet carcass was observed when the temperature was recorded at 4°C. Calliphora vicina is a widely studied species of forensic interest whose development and temperature range have been analyzed in several studies (Hückesfeld et al.

2011). Moreover, C. vicina has also been the subject of research pertaining to nocturnal activity and development at low temperatures (Baqué et al. 2014). According to (Hückesfeld et al. 2011), eggs of C. vicina took seven days to hatch when reared at 5°C; in addition, these larvae did not grow in size for 5 days when kept at this temperature.

A similar delay in the hatching of the eggs was observed during a case investigation conducted in Switzerland (Faucherre et al. 1999). A body was discovered in a cave 18 days after being reported missing and egg masses of C. vicina were collected from it. In order to understand the pattern of colonization in a cave, at a depth of 10 m, with no light and a temperature of 5 °C, a bait consisting of beef liver was placed in the same spot where the body had been found and colonization by C. vicina was observed 12 days from when the bait had been placed out. The bait remained in the cave for six additional days to reconstruct the circumstances of the recovery of the body (Faucherre et al. 1999). The larvae hatched from the eggs collected from the body and those collected from the baits had the same pattern of development. This case shows that C. vicina is able to be oviposit and develop under extreme conditions, such as the complete absence of light and at a temperature that goes below what the literature has often reported as a minimum threshold. (Greenberg 1990) observed

29 nocturnal oviposition from C. vicina, but the temperatures of his study were significantly higher (18-34.5 °C). Overall, this study shows that blow fly activity is present in areas of reduced light and during periods of low temperatures, and work focusing with these variables should be further investigated.

References

Amendt J, Zehner R, Reckel, F. 2008. The nocturnal oviposition behavior of blowflies (Diptera: Calliphoridae) in Central Europe and its forensic implications. Forensic Science International 175(1): 61-64.

Baqué M, Filmann N, Verhoff MA, Amendt J. 2015. Establishment of developmental charts for the larvae of the blow fly Calliphora vicina using quantile regression. Forensic Science International 248: 1-9.

Byrd J H, Castner J L (Eds.). 2009. Forensic entomology: the utility of arthropods in legal investigations. CRC press. 1-2.

Faucherre J, Cherix D, Wyss C. 1999. Behavior of Calliphora vicina (Diptera, Calliphoridae) under extreme conditions. Journal of Insect Behavior 12(5): 687-690.

Grassberger M, Reiter C. 2001. Effect of temperature on Lucilia sericata (Diptera: Calliphoridae) development with special reference to the isomegalen-and isomorphen- diagram. Forensic Science International 120(1): 32-36.

Greenberg B. 1990. Nocturnal oviposition behavior of blow flies (Diptera: Calliphoridae). Journal of Medical Entomology 27(5): 807-810.

Greenberg, B., & Kunich, J. C. (2002). Entomology and the law: flies as forensic indicators. Cambridge University Press.

Hückesfeld S, Niederegger S, Schlegel P, Heinzel HG, Spieß R. 2011. Feel the heat: The effect of temperature on development, behavior and central pattern generation in 3rd instar Calliphora vicina larvae. Journal of Insect Physiology 57(1): 136-146.

Ring RA. 1967. Maternal induction of diapause in the larva of Lucilia caesar L.(Diptera: Calliphoridae). Journal of Experimental Biology 46(1): 123-136.

Vanin S, Tasinato P, Ducolin G, Terranova C, Zancaner S, Montisci M, Turchetto M. 2008. Use of Lucilia species for forensic investigations in Southern Europe. Forensic Science International 177(1): 37-41.

Table 2.1. The amount of time required for blow fly colonization and development. Amount of additional days to reach the stage are noted in parentheses.

Location Egg Laid (d) Eggs Hatched (d) Pupation (d) Eclosion (d)

Surface (4 carcasses) 0.5 (+1.5) 2 (+13) 15 (+16) 31

Underground I (Carcass) 21 (+7) 28 (+27) 55 (+41) 96

Underground II (baited trap) 22 (+8) 30 (+27) 57 (+43) 100

Figure 2.1. Map of research site locations. The yellow pin represents the location of the cave, and the red pin represents the location of the terrestrial site.

CHAPTER 3

An Analysis of Blow Fly (Diptera: Calliphoridae) Species Richness and Distribution

Along Elevation Gradients in Sicily (Italy) and Ecuador

Abstract

A survey of blow flies (Diptera: Calliphoridae) was conducted in Sicily, Italy and Ecuador using traps baited with beef liver across four different altitudinal levels. The elevations ranged from 20m to 1552m in Sicily, and 561m to 3336m in Ecuador. Species richness, relative abundance and diversity were calculated, as well as the ratio of females to males and community assemblage. Twelve species were collected in Sicily and 17 species were collected in Ecuador. In Sicily, the most abundant blow fly species was Lucilia sericata (Meigen) accounting for 68.50% of the total capture. While in Ecuador, Consomyiops verena (Walker) accounted for 51.67% of all species captured. In Sicily, significant differences were only observed in the relative abundance of L. sericata across elevation gradients. In Ecuador, significant differences were observed in the relative abundance of Calliphora nigribasis

(Macquart), Chrysomya albiceps (Weidemann), C. verena, Hemilucilia semidiaphana (Rondani),

Lucilia ibis (Shannon), Lucilia purpurescens (Walker), and Paralucilia sp. across elevation gradients. These data can help build a checklist of blow fly species in these two regions and can be instrumental in forensic investigations.

Key words: forensic entomology, species richness, Calliphoridae, altitudinal gradient, Sicily,

Ecuador

Introduction

Blow flies (Diptera: Calliphoridae) are the main initial arthropod decomposers of organic matter (Grassberger and Frank 2004). A study of arthropod succession on carrion in

Venezuela reported that seven of the collected 14 species of primary forensic importance were calliphorid flies (Vélasquez 2008). A similar study in Vienna, Austria, traced insect presence on a decomposing carcass for 60 days and observed that blow flies were the most abundant in the initial 15 days (Grassberger and Frank 2004).

Adult blow flies are able to detect a carcass shortly after death occurs (DeJong 1994; Gruner et al. 2007). In Colorado, DeJong (1993) observed that a dead greyhound carcass was visited by adults of Phormia regina (Meigen) 40 seconds after exposure, and oviposition occurred 13 minutes after exposure. Blow flies can travel several kilometers to reach carrion (Vogt and

Morton 1991; Spradbery et al. 1995). Blow fly species, like Lucilia cuprina (Wiedemann) require a protein meal for the maturation of their ovaries (Williams et al. 1979). Others travel to a corpse for oviposition opportunities (Norris 1965; Byrd and Castner 2010). Braak (1984) observed adults of Chrysomya albiceps (Wiedemann) and Chrysomya marginalis (Wiedemann) travelling several kilometers to find a suitable sized carcass to lay their eggs.

Functional roles of blow flies in ecosystems are diverse. Besides recycling carrion, several species use feces as a protein source and therefore can be important vectors of enteric pathogens (Greenberg 1971, 1973). They can also colonize living hosts (humans or other vertebrates); this phenomenon is known as myiasis and can have a strong impact in the livestock industry (Wall et al. 1992). Blow flies are important pollinators and they are reared commercially for pollination of different crops including carrots (Howlett 2012) and Brussel

35 sprouts (Faulkner 1977), both wild-growing and in cages (Pérez-Bañón et al. 2007; Clement et al. 2007). Since they are the first individuals to arrive and colonize a body shortly after death (Carter et al. 2007; Johansen et al. 2014), blow flies are also among the main necrophagous insects to be used in forensic investigations, and can be used to estimate the minimum post mortem interval (mPMI) defined as the time elapsed from the colonization of the body and its discovery (Catts and Goff 1992; Amendt et al. 2004; Tomberlin et al. 2011).

In order to use blow flies appropriately in forensic entomology, it is essential to have information on their species diversity, abundance and distribution for each geographic area where investigations are conducted. Studies have analyzed information about blow fly species composition and community structure in different areas around the world; including

North America (Kneidel 1984; Ives 1991; Weidner et al. 2015), Europe (Kuusela & Hanski

1982; Kuusela 1983; Hanski 1987; Davies 1990; Feddern et al. 2018) and in Asia (Bunchu et al. 2012). Although some of this work analyzed the spatial and temporal distribution of blow flies (Hanski 1987, Weidner et al 2015, Feddern et al. 2018), very little work has been done focusing on the elevational distribution of such flies (Tumrasvin et al. 1978; Baumgartner and Greenberg 1985; Baz et al. 2007; Moophayak et al. 2014).

Species distribution across different elevations have been examined for several taxa and have shown that there are two commonly observed relationship patterns between altitude and species richness. The first pattern is known as a monotonic decrease, where species richness decreases as elevation increases (MacArthur 1984, Stevens 1992). The second most common pattern is a hump-shaped pattern, where species richness peaks at intermediate elevations

(Whittaker 1960; Janzen 1973; Whittaker and Niering 1975; Shmida and Wilson 1985;

McCoy 1990; Lieberman et al. 1996; Rahbek 1995; Rahbek 1997; Fleishman et al. 1998).

These patterns have been shown to exist across various forms of taxa including fungi

(Raviraja et al. 1998), vascular plants (Bruun et al. 2006) birds (Terborgh 1977), mammals

(Owen 1990), and insects (Lawton et al. 1987, Wolda 1987; Janzen 1973; Fernandes and

Price 1988; McCoy 1990; Kearns 1992; Stevens 1992; Olson 1994, Sparrow et al. 1994,

Sanchez-Rodriguez and Baz 1995).

The distribution of species across elevational gradients can be impacted by several biotic (e.g. human and non-human animal activity) and abiotic factors (e.g. temperature, oxygen). For example, deforestation is commonly more intense at low and high altitudes, but broadly the human impact decreases as elevation increases (Nogués-Bravo et al. 2008). The disappearance of a taxon’s habitat, due to deforestation or urbanization could be a strong factor resulting in species disappearance from that area. Numerous abiotic factors play a role in the relationship between species distribution and elevation, among them are physical geometric constraints (Sanders 2002; Colwell and Lees 2002) and climate (Sanders et al.

2003; Bhattarai et al. 2004). If species are constrained by hard boundaries (large bodies of water, the bottom of a valley (lower boundaries) or eco-physiological features such as altitude increase (upper boundaries) (Grytnes and Vetaas 2002), they may face an actual barrier to dispersal (Colwell and Lees 2002).

On a global level, anthropogenic climate change is expected to produce an increase in average air temperature of 1.4 – 5.8 °C by 2100 relative to 1990 (IPCC, 2001). Along with warmer temperatures, seasonal changes in precipitation, radiation, potential evapotranspiration and other climatic regimes are predicted (Hulme and Jenkins 1998).

Studies performed across different taxa have shown that the duration of life cycle stages was affected in response to climatic changes throughout the year (Menzel et al. 2006; Root et al.

2005; Rosenzweig et al. 2008) and shifted their distribution towards higher latitudes or higher elevations (Parmesan et al. 1999; Parmesan and Yohe 2003; Hickling et al. 2006;

Rosenzweig et al. 2007). These potential climatic changes can dramatically impact species distribution, as species are expected to respond to these changes at an individual species level

(Huntley 1999), and it has already been shown that butterfly and moth diversity reacts negatively to temperature changes (Turner et al. 1987).

Gaining information about the distribution of blow flies across elevational gradients in areas rich in biodiversity could provide an understanding of the biology and behavior of this group at a species level. This information, in turn, could also become a tool in ecological investigations pertaining to habitats and climatic changes, as well as in forensic investigations. Therefore, the purpose of this study was to document the distribution of forensically important Calliphoridae across elevational gradients in two different areas, Sicily and Ecuador, for which little information is available.

Materials and Methods

Study areas

The survey was conducted in the eastern part of Sicily, on the slopes of Mt. Etna (Figure 1) and in the inner part of Ecuador (Figure 2). These areas were chosen because both locations were accessible, had substantial changes in elevation and lacked information on the biodiversity and distribution of forensically important blow flies.

Sicily (Table 3.1, Figure 3.1)

Site 1 (37.5427° N, 15.0843° E, Elevation 20m): this site was located on a green area populated by moderate Mediterranean vegetation (macchie or garrigue, Cistus sp. (Cistaceae) and Senecio sp. (Senecioneae) near the city of Catania, which has some of the highest seismic activity (Monaco et al. 2000). It is located on the south-eastern slope of Mt. Etna and is one of the more populated cities in Sicily with 314,000 people in 2017 (ISTAT, National

Institute of Statistics).

Site 2 (37.8071° N, 14.6994° E, Elevation 700m): this site was located in the area of San

Teodoro, a small village in the Province of Messina, on the western side of Mt Etna. The area is covered by Mediterranean scrubs, almond trees (Prunus dulcis, Rosaceae) and willows

(Salix sp. Salicaceae).

Site 3 (37.8521° N, 14.6919° E, Elevation 1153m): this area is known as Condrada Casale

Nuovo, though it is still part of the San Teodoro municipality. This rural area is covered in

Turkey oaks (Quercus laevis, Fagaceae) and elders (Sambucus nigra, Adoxaceae).

Site 4 (37.9258° N, 14.6702° E, Elevation 1552 m): this rural site is on Monte Soro, the highest peak of the Nebrodi mountains, located north-west of Mt. Etna. The most abundant trees in this area are beech trees (Fagus sp.).

Ecuador (Table 3.1, Figure 3.2)

Site 1 (0.5956°S, 77.5011° O, Elevation 561 m): this site is part of a megathermal rainforest, and was located in the area around Tena, in the valley of the Misahuallí river, in the Amazon

39 region. Tree crowns, in this area, are not commonly stratified because they belong to a large number of species, whose maturation period and reaction to light is different.

Site 2 (0.4143°S, 77.4837°O, Elevation 1312 m): this site was located in Sarayacu (1312m), situated in a tropical rainforest in the amazon region. In this area, trees are typically quite diverse, and gregarious dominants are usually not common. Tropical rain forest is often defined as multilayered, since the tree crowns form distinct strata.

Site 3 (0.282.31°S, 77.533365°O, Elevation 1948 m): this site is located in the area around

Baeza in the central northern part of the Ecuadorian Amazon forest. The climate is equatorial, semi-humid, and mesothermic. This area is incredibly diverse, and in a one- hectare plot of land it is possible to find 40 to 100 species of trees (Morales and Vinicius

2003).

Site 4 (0.222324°S, 78.82235°O, Elevation 3336 m): the highest of the Ecuador sites, this location was located in Papallacta, a mountainous area of the eastern Cordillera whose ecosystems ranges from tropical forests to alpine/glaciers as the altitude increases. It is characterized by an equatorial, high-mountain climate and one of the most well-known species of this area is the paper tree (Polylepis sp.), whose height can reach up to 10 meters.

Sampling procedure

Sampling was performed in all sites using four RESCUE!® POP! Fly Trap (Sterling

International, Inc., Spokane, WA), traps per site. Traps were baited with 100 g beef liver purchased from a local supermarket. The traps were deployed in different locations at each

40 sampling site, at a distance of 200-300 m from each other, depending on the area’s geographical conditions.

When deployed, the traps were baited on site, hung from trees at an average height of 1.8m

± 0.12m (SE) and left at the site for one week. During the sample periods, any traps that were lost or stolen were not replaced. Two one-week sampling sessions were conducted in the summer of 2017 in Sicily and in 2016 in Ecuador. Each sampling site was equipped with a HOBO Temperature/Light Data Logger (model UA-002-64K Onset Computer Corp.

Cape Cod, Massachusetts) which recorded temperature (C°) and light intensity (lux) every 30 minutes.

At the end of each sampling period, the traps were collected and emptied. Adult Diptera were cleaned with water, left to dry for 2 hours at ambient temperature, placed in a jar without preservatives, and frozen until they were sorted by family and pinned. Non-dipteran arthropods were discarded. All pinned calliphorid specimens were labeled with two different labels: a color label to indicate the trap, and a unique numerical label to indicate the specimen number. Pinned specimens were stored in Cornell University Drawers (Bioquip

Products, Rancho Dominguez, CA) housed at Rutgers University.

Identifications were performed using morphological characteristics with the keys of Szpila

(2017), Carvalho and Mello Patiu (2008), Amat et al. (2008) and Whitworth (2010). Voucher specimens were confirmed morphologically by Dr. Terry Whitworth and Dr. Krzysztof

Szpila. Voucher specimens were then deposited in the Rutgers Department of Entomology

Museum collection.

Data Analysis

Calliphorid species richness (number of species), relative abundance, and community structure (occurrence and abundance) were analyzed for each region and elevation. All data were subjected to a Shapiro-Wilkes test to determine if the raw data were normally distributed prior to analysis. This test revealed that relative abundance in both Sicily and

Ecuador were not normally distributed; richness was not normally distributed in Ecuador but had a normal distribution in Sicily. The relationship between species richness/abundance and elevation was analyzed using a Kruskal-Wallis test. This non-parametric test is an alternative to a one-way (between groups) ANOVA and is commonly used to compare three or more sample ranks instead of means. The null hypothesis tested was that samples used for comparisons were drawn from the same distribution or from distributions having the same median (Kruskal and Wallis 1952). To analyze species richness in Sicily, which was normally distributed, an ANOVA was performed. When significant results were obtained a post hoc test was performed; significant results after the Kruskal-Wallis analysis were further analyzed with a Dunn test (Dunn 1961) with the Benjamini-Hochberg adjustment for pair-wise comparison (Benjamini and Hochberg 1995) while significant results obtained after the

ANOVA were further analyzed through a Tukey’s HSD test. For each altitudinal level within each ecoregion, the Shannon (H) (Shannon 1948) diversity indexes and the Sorensen’s

Similarity Index (CC) (Sorensen 1948) were calculated. Sorenson’s Similarity Index CC is an index that compares the species composition of two sites taking into account the species present in each of the two sites and the species each have in common and ranges between 0 to 1, with 0 being completely dissimilar and 1 being completely similar.

Using the methods developed by Dufrene and Legendre (1997) and used by Weidner et al.

(2015), a nonmetric multidimensional scaling (NMDS) analysis followed by a multi-response permutation procedure (MRPP) for multiple pairwise comparison (McCune and Grace 2002) and an indicator species analysis (ISA) were performed. An indicator value is used to indicate which taxon or taxa are considered the best predictor of that altitude; a value of 0 shows that they are a poor predictor and a value of 100 shows a perfect predictor. In order to sort species and examine their degree of association, a cluster analysis based on abundance was performed. Distances between clusters are determined by the greatest distance between any two items in different clusters. Analyses were conducted using R Studio 1.1.463 (R Studio

Team 2018) and Microsoft® Excel 2018 version 16.21.1.

Results

Average temperatures (±SE), average humidity (±SE) and total precipitation while traps were deployed in each location was recorded (Table 3.2).

Sicily

A total of 9923 adult Diptera were collected; of these, a total of 4926 belonged to the family

Calliphoridae, encompassing 12 species within four genera (Table 3.3). The most abundant calliphorid species overall was Lucilia sericata (Meigen) (68.50%). The least abundant species was Cynomya mortuorum (Lynnaeus) (0.02%).

The altitude where the highest number of blow flies were collected was 700m (3108 individuals), followed by 1153m (1150 individuals), 1552m (397 individuals) and 20m (206 individuals). The predominant species at 20m were L. sericata (42.23%) and Calliphora vicina

(Robineau-Desvoidy) (56.79%). At 700m the dominant species was L. sericata (89.80%). At

1153m, C. vicina accounted for 50.60%; followed by L. sericata (43.22%). At the highest elevation (1552m), Calliphora vomitoria (Lynnaeus) (55.16%) and C. vicina (40.80%) were the most collected species (Table 3.3). The sex-ratio of calliphorids collected showed an overall bias towards females, with an overall value of m/f ratio is 0.15. The m/f ratio for the two most common species was 0.05 and 0.54 for L. sericata and C. vicina, respectively (Table 3.4).

There were significant differences in relative abundance across altitude for only one species,

L. sericata (H=13.224, df=3, p=0.004). Specifically, significant differences in L. sericata relative abundance were found when comparing 20m with 700m (p=0.0139) and 700m with

1550m (p=0.007) (Table 3.5). The Shannon diversity index (H) was calculated for all four altitude levels (20m H=0.746; 700m H=0.553; 1153m H=0.951; 1552m H=0.833) (Figure 3).

Sorenson Similarity Index (CC) for each pair of elevations is represented in Figure 3.4. Since species richness was normally distributed across altitudes (Shapiro-Wilkes test p=0.313) an

ANOVA analysis was performed. The test indicated significant differences between elevations (F=27.07, df=3, p=0.004). Subsequently, significant differences between 20m and

700m (p=0.004), 20m and 1153m (p=0.03), and 700m and 1552m (p=0.007) were observed.

Lucilia sericata and L. silvarum were indicator species for 700m (level 2), while C. vicina was an indicator species for 1153m (level 3); no indicator species were found for 20m (level 1) or

1552m (level 4). (Table 3.6). Blow fly communities were shown to be significantly different between altitudes with significant differences between 700m and 1153m (MRPP: A=0.304 p=0.035) (Table 3.7, Figure 3.5).

Ecuador

A total of 2890 adult Diptera were collected; of these, there were 1492 calliphorids belonging to nine genera and 17 species. The most abundant calliphorid species collected during the survey was Consomyiops verena (Walker) (51.67%). The least abundant species collected were

Cochliomyia hominivorax, Chrysomya sp. and Lucilia ochicornis each constituting 0.06% (1 individual captured per species). The largest number of flies were collected at 3336m (550 flies) and 1948m (550 flies). In the remaining sites, the flies collected were 215 at 561m and

130 at 1312m (Table 3.8).

Predominant species at 561m were Paralucilia sp. (31.63%), L. eximia (25.12%) and C. albiceps

(20.46%). At 1312m, predominant species were C. albiceps (43.85%), L. eximia (27.69%) and

Paralucilia sp. (15.38%). At 1948m, predominant species were C. verena (65.27%), followed by

Lucilia purpurescens (Walker) (13.82%). At the highest elevation (3336m), the two main species were C. verena (74.90%) and C. nigribasis (24.18%) (Table 3.8). The sex-ratio (m/f) of the blow flies collected has an overall value of 0.25. The m/f ratio of the four most abundant species was 0.27 for C. verena, 0.66 for C. nigribasis, 0.12 for L. eximia and 0.24 for C. albiceps

(Table 3.9).

Significant differences were observed across elevations for C. nigribasis (H=10.253, df=3, p=0. 017), C. albiceps (H=11.061, df=3, p=0.011), C. verena (H=10.929, df=3, p=0.012), H. semidiaphana (H=8.7542, df=3, p=0.033), L. ibis (H=8.1373, df=3, p=0.043), L. purpurescens

(H=12.913, df=3, p=0.005), Paralucilia sp. (H=11.331, df=3, p=0.010). Calliphora nigribasis numbers differed significantly between level 1 (561m) and level 4 (3336m; p=0.018;) and level 2 (1312m) and level 4 (3336m; p=0.027). The distribution of C. albiceps was significantly

45 different between level 3 and all other levels; 1948m (level 3) and 561m (level 1; p=0.038),

1948m (level 3) and 1312m (level 2; p=0.067), 1948m (level 3) and 3336m (level 4; p=0.047), as well as 3336m (level 4) and 1312m (level 2; p=0.057). Consomyiops verena was significantly different between 3336m (level 4) and 561m (level 1; p=0.027). The distribution of L. ibis was significantly different between 561m (level 1) and 1948m (level 3) (p=0.044). Lucilia purpurescens was significantly different between 561m (level 1) and 1948m (level 3; p=0.006),

1312m (level 2) and 1948m (level 3; p=0.023), 1948m (level 3) and 3336m (level 4; p=0.0129). For Paralucilia sp. significant differences were observed between 561m (level 1) and 1948m (level 3; p=0.018) and 561m (level 1) and 3336m (level 4; p=0.036) (Table 3.10).

The Shannon diversity index (H) was calculated for all four altitude levels (561m H=0.610;

1312m H=1.205; 1948m H=1.515; 3336m H=1.697) (Figure 3.6). Sorenson Similarity Index

(CC) for each pair of elevations is represented in Figure 7. Species richness was not normally distributed across altitudes (Shapiro test p=0.034) and therefore a Kruskal-Wallis analysis was performed which showed no significant difference across all altitudes (p=0.21). The

Indicator Species Analysis (ISA) showed that Paralucilia sp. was found to be a strong indicator of the first elevation level (561m), L. purporescens and L. ibis were strong indicators for the third elevation level (1948m) and C. nigribasis was a strong indicator for the highest elevation (3336m) (Table 3.11). Blow fly communities were shown to be significantly different between elevational gradients with significant differences between all levels except between levels 1 (561m) and 2 (1312m), and levels 3 (1948m) and 4 (3336m). (Table 3.12,

Figure 3.8).

Discussion

With few exceptions, the altitudinal distribution of blow flies in the Mediterranean ecoregion has not been extensively researched. Baz et al. (2007) analyzed the altitudinal distribution patterns of blow flies in Spain at nine different elevations and found eight different species with significant differences between elevation and abundance for most of the species collected. In Sicily, Gemmellaro et al. (2018) looked at blow fly colonization inside volcanic caves and surrounding areas during periods of cold temperatures and noticed a delay in hypogenic colonization by C. vicina and C. vomitoria. This study shows that blow fly species distribution can be correlated with changes in altitude. In fact, the data collected during our survey show that the relative abundance of L. sericata, the most abundant species collected in

Sicily (68.5% of total capture) changed significantly throughout the elevation gradient, going from being 42.23% of capture at 20m to be absent at the highest altitude (1552m). These differences could be due to the fact the along with altitude, average temperature and habitats changed across sites, going from an urbanized location with mean temperature of 25oC to a more rural area with abundant vegetation and a mean temperature of 16oC. This is in agreement with previous studies that have found L. sericata mainly in urban sites (Reiter

1984). However, this survey documented this species also at intermediate elevations in rural sites; this corresponds with other studies, which have reported L. sericata in less urbanized rural areas (Anderson 1995).

Calliphora vicina was the second most abundant species collected in Sicily (21.0% of total capture) and its relative abundance, although not significantly different across altitudes still showed variation in the survey, with higher values observed at low and high elevations

(56.8% at 20m, 50.6% at 1153m and 40.8% and 1552m). This pattern is consistent with data

47 found in the literature, which classify C. vicina as an urban species (Reiter 1984), although it has been observed in non-urban scenarios as well (Haskell et al. 1997). Moreover, the higher presence of C. vicina at an elevation where temperature was lower agrees with the definition of C. vicina as a thermophobic species (Martinez- Sanchez et al. 2000) and with studies on the geographical distribution of blow flies which have shown that this species is more abundant in Northern, colder, regions (Weidner et al. 2015).

At the highest altitudinal level (1552m) C. vomitoria was the main colonizer, representing

55.16% of total number of flies captured; at lower elevations, however, this species had low representation. Calliphora vomitoria is also considered a species well adapted to cold climates

(Davies and Lawrence 1992), and its higher abundance in the highest elevation, when compared to C. vicina, may be related to the fact that C. vomitoria prefers non-anthropic habitats while C. vicina prefers urban environments (Nuorteva 1963). The highest site of this survey, was characterized by low urbanization and colder temperatures, providing a suitable habitat for this species.

The comparison of males/female ratios of the two most abundant species showed large differences. For L. sericata 5% of the total capture were males. For C. vicina, however, 35.5% of the total capture were males. In another survey conducted in Manhattan, NY with baited traps, high numbers of C. vicina males were also recorded (Chu et al. 2017). This observation might be explained by the nutritional needs of C. vicina males, which, when offered multiple food sources, have shown to prefer protein-rich substrates versus carbohydrates (see

Chapter 4).

In Sicily, the most diverse sampling site was located at elevation level 3 - 1153m (H=0.95); this could be related to the habitats provided by the geographical features and vegetation of the site. The lowest diversity was observed at elevation level 2 - 700m (H=0.55); however, this was also the site where the highest number of blow flies was collected. The similarity CC index for the Sicily sites showed that species composition in relation to elevation is most dissimilar between 20m and 1552m (CC=0.025); different urbanization and average temperatures characterize these areas, creating habitats that can be conducive to different blow fly species. The most similar elevations in relation to diversity were 1153m and 1552m

(CC=0.85), where similar landscapes and vegetation are present, and the anthropogenic impact is low.

Species richness was significantly different between the first elevation level (20m) and the second (700m) and between the second (700m) and the third (1153m) levels. This may be explained by the location of the first level being in close proximity to a large city, while none of the others were. Richness was also the same at the lowest and highest elevations

(richness=4), even though there was only one species in common between the two levels (C. vicina). The number of flies captured during the survey was also lower at the two extreme elevations (206 at 20m and 397 at 1552m) than it was at the two intermediate ones (3197 at

700m and 1153 at 1153m). This is in agreement with numerous authors who have found the correlation between species richness and elevation to have a hump shaped pattern, with a peak in richness at intermediate elevations (Lees et al. 1999; Colwell and Lees 2002).

In Ecuador, the number of blow flies collected during the survey was lower than what was collected in Sicily. The same was also true when looking at all Diptera captured during the

49 study (9923 for Sicily; 2890 for Ecuador). This could be related to the high relative humidity and precipitation in the sampling sites in Ecuador. High humidity can be inversely related to fly abundance (Ngoen-Klan et al. 2011). Research in Malaysia showed that the abundance of

Muscidae diminished as humidity increased (Bong and Zairi 2009). However, the number of blow fly species was slightly higher in Ecuador (17 versus 12 species in Sicily). This could also be due to the fact that the former is an island while the latter is located on the mainland.

Similar results have been observed in other studies comparing insect diversity between islands and the mainland, where significant differences were found in both the number of species and in the number of individuals that were collected (Janzen 1973). Moreover, when compared to Sicily, Ecuador is closer to the Equator, and studies have noticed a higher number of species at lower latitudes and this has been correlated to warmer climates and to other factors (such as vegetation, human footprint and physical barriers) (Hawkins and

Felizola Diniz-Filho 2004). The sites sampled in Ecuador had a more diverse vegetation and lower levels of urbanization when compared to the Sicilian sites. This could explain the difference in the number of species sampled in the two location.

In Ecuador, just over half of the total capture was represented by C. verena (51.7%). No extensive data are present in the literature about the biology and development of this blow fly. It is known to be attracted to garbage and decomposing matter, and occasionally feces

(Baumgartner and Greenberg 1985). In a survey of the blow fly community of Peru,

Greenberg and Szyska (1984) collected this fly at an altitude of 1,430m, in an asynanthropic area and recorded an on-site egg to adult development of 17 days. In this survey, C. verena was also the most abundant blow fly collected in the highest site in our survey (Papallacta,

3336m), which is also the site where the highest number of flies was collected. This is

50 surprising considering that Papallacta was the most elevated site of this survey but could be explained by the low anthropogenic impact that characterizes this site and by the different habitats that the great diversity of this area provides.

Significant differences in abundance across all four altitudinal levels were observed for C. nigribasis with few collections at the highest altitude (3336m). Calliphora nigribasis has been observed to be a good flier and can travel up to 3.5km a day (Tsuda et al. 2009). The fact that in our survey it was only captured at the highest altitude could be interpreted as a preference for this habitat. It could also be that geographical constraints, such as mountains, are a physical impediment for the dispersal of this species. The distribution of C. albiceps and

Paralucilia sp., was also significantly different and seemed to prefer lower altitudes (561m and

1948m) altitudes given that they were not collected in the higher sites.

The Shannon diversity index indicated that the most diverse site was the highest site,

Papallacta, (3336m) (H=1.7). Overall, the habitats in this area were quite diverse, going from tropical forest to glaciers and therefore may provide living conditions for numerous species.

Sorenson’s Similarity Index showed that the species composition at the highest altitude is quite dissimilar to all the other levels and that the two lower elevations and the two intermediate elevations were very similar to each other. However, species richness, was not significantly different across the four altitudes, indicating that even though species may differ across altitudes in relation to habitats and temperature, their number does not change significantly.

One of the limitations of this survey was the use of a bait as opposed to whole animal carcasses. Bait type and size may have impacted the number and composition of the flies collected. This possibility has been suggested by other studies that compared the use of a bait as opposed to a carcass (Weidner et al. 2017; Gemmellaro et al. 2018). Additionally, the rate of decay of the liver bait used for this survey may have been impacted by the different environmental parameters at each elevation. This difference in decomposition could have affected the chemical cues released, and therefore effected the ‘attractiveness’ of the bait. A study conducted by Vogt and Woodburn (1994) reported significant differences in the number of flies collected by seven-day old baits compared to fresh baits. Future work should be considered analyzing multiple seasons across multiple years along elevational gradients to provide useful information about the seasonality of the species collected.

The results of these surveys provide baseline data about the calliphorid communities in Sicily and Ecuador, across different altitudes and can help in the interpretation of general ecological patterns of calliphorid species distribution across elevational gradients. Moreover, this information could be used as a reference in future studies as well as assist with increased implementation of the use of insects in forensic investigations.

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Table 3.1. Location, level designation, elevation and geographical coordinates of collection sites in Sicily and Ecuador.

Location Level Elevation (m) Coordinates Sicily Catania 1 20 37.5427° N, 15.0843° E

San Teodoro 2 700 37.8071° N, 14.6994° E

Contrada Casale Nuovo 3 1153 37.8521° N, 14.6919° E

Monte Soro 4 1552 37.9258° N, 14.6702° E

Ecuador Tena 1 561 0.5938° S, 77.4818° W

Sarayacu 2 1312 0.4208° S, 77.4844° W

Baeza 3 1948 0.2759° S, 77.5321° W

Papallacta 4 3336 0.2225° S, 78.0825° W

Table 3.2. Average temperature (±SE), average relative humidity (±SE) and total

precipitation during the duration of the survey.

Location Elevation Elevation( Temperature (°C) Humidity Annual Level m) (±SE) (%) (±SE) Precipitation

Sicily Catania 1 20 25.23 ± 0.64 64.13 ±1.24 0.3 mm

San Teodoro 2 700 22.46 ± 0.83 62.06 ± 1.11 7 mm Contrada 3 1153 19.72 ± 0.42 61.14 ± 0.87 8 mm Casale Nuovo Monte Soro 4 1552 16.32 ± 0.13 62.2 ±1.54 8.2 mm

Ecuador Tena 1 561 23.28 ± 1.2 99.52 ±0.44 4330 mm

Sarayacu 2 1312 19.47 ± 0.66 94.57± 2.43 1013 mm

Baeza 3 1948 17.07 ± 0.87 89.37± 1.56 2220 mm

Papallacta 4 3336 11.29 ± 0.11 93.41 ±1.32 1281 mm

Table 3.3. Relative abundance (%) of blow fly species collected in Sicily. Relative abundances are shown by species per elevation gradient as well as overall relative abundance (Rel. Ab.) during the duration of the survey.

SICILY Altitude Altitude Altitude Altitude Species Total Rel. Ab. 20m 700m 1153m 1552m Lucilia sericata 42.23% 89.80% 43.22% - 3375 68.50% Calliphora vicina 56.79% 5.44% 50.60% 40.80% 1030 21.00% Calliphora vomitoria - <1% 2.60% 55.16% 251 5.09% Lucilia ampullacea - 2.83% 2.78% <1% 121 2.40% Lucilia silvarum <1% 2.09% <1% - 67 1.36% Chrysomya albiceps - <1% <1% - 27 0.55% Calliphora subalpina - <1% - 3.78% 19 0.38% Lucilia caesar - <1% <1% - 14 0.28% Lucilia illustris - <1% - - 12 0.24% Lucilia cuprina <1% <1% <1% - 7 0.14% Calliphora loewi - <1% <1% - 2 0.04% Cynomia mortuorum - <1% - - 1 0.02%

Number of Species 4 12 9 4

Number of Specimens 206 3108 1150 397 4244 100.00%

Table 3.4. The male to female (m/f) ratio of adult blow flies found in Sicily. Ratios are depicted by species for each elevational gradient as well as an overall m/f ratio.

M/F Altitude Altitude Altitude Altitude Total Tot Species ratio 20m 700m 1153m 1552m Males Females

Lucilia sericata 42.23% 147M/2644F 16M/481F 176 3199 0.05 Calliphora vicina 44M/73F 72M/97F 195M/387F 49M/113F 360 670 0.53 Calliphora vomitoria 2F 13M/17F 55M/164F 68 183 0.37 Lucilia ampullacea 38M/50F 7M/25F 1F 45 75 0.6 Lucilia silvarum 1F 14M/51F 1F 14 53 0.26 Chrysomya albiceps 26F 1F 0 27 Calliphora subalpina 4F 6M/9F 6 13 0.46 Lucilia caesar 3M/7F 4M 7 7 1 Lucilia illustris 4M/8F 4 8 0.5 Lucilia cuprina 1M 4F 2F 1 6 0.16 Calliphora loewi 1F 1F 0 2 Cynomia mortuorum 1F 0 1

Table 3.5. Differences in species abundance across elevational gradients in Sicily using Kruskal-Wallis one -way ANOVA and Dunn test with Benjamini-Hochberg adjustment. Species Kruskal- Wallis Dunn

1 v 2 1 v 3 1 v 4 2 v 3 2 v 4 3 v 4

L. silvarum 6.771 (0.080) ------

L. sericata 13.224 (0.004) 0.014 0.211 0.670 0.210 0.007 0.114

L. ampullacea 7.313 (0.063) ------

L. cuprina 1.187 (0.756) ------

L. illustris 6.400 (0.094) ------

L. caesar 3.845 (0.279) ------

C. vicina 7.318 (0.062) ------

C. vomitoria 2.522 (0.471) ------

C. subalpina 6.450 (0.090) ------

C. loewi 2.143 (0.543) ------

C. albiceps 4.393 (0.222) ------

C. mortuorum 4.393 (0.222) ------

Numbers 1-4 relate to elevation level (1=20m, 2=700m, 3=1153 m, 4=1552m. Significant p values are bolded for each test.

Table 3.6. Indicator species for elevational gradients in Sicily. A level designation, elevation, indicator value and p value are provided for each species.

Species Level Elevation Value P value

Lucilia sericata 2 700 m 82.70 0.004 Lucilia silvarum 2 700 m 72.76 0.033 Calliphora vicina 3 1153 m 56.50 0.026

Table 3.7. Significant differences in blow fly communities in Sicily across elevational gradients using Multiple Response Permutation

Procedure.

Altitude 20m Altitude 700m Altitude 1153m

Altitude 700m A:0.1982; Obs. Delta: 0.5097; Exp. Delta: 0.6357 p=0.13 Altitude 1153m A:0.0042; Obs. A:0.3035; Obs. Delta: 0.5053; Delta: 0.3681; Exp. Delta: 0.5075 Exp. Delta: 0.5285 p=0.6 p=0.035 Altitude 1552m A:-0.002; Obs. A:0.3732; Obs. A:0.1968; Obs. Delta: 0.7864; Delta: 0.2714; Delta: 0.3648; Exp. Delta: 0.7848 Exp. Delta: 0. 5925 Exp. Delta: 0. 4541 p=0.66 p=0.2 p=0.2

Table 3.8. Relative abundance (%) of blow fly species collected in Ecuador. Relative abundances are shown by species per elevation gradient as well as overall relative abundance (Rel. Ab.) during the duration of the survey.

Altitude Altitude Altitude Altitude Total Rel. Ab. Species 561m 1312m 1948m 3336m Consomyiops verena - - 65.27% 74.90% 771 51.67% Calliphora nigribasis - - - 24.18% 138 9.25% Lucilia eximia 25.12% 27.69% 2.72% - 112 7.50% Chrysomia albiceps 20.46% 43.85% <1% <1% 108 7.24% Paralucilia sp. 31.63% 15.38% - - 88 5.90% Lucilia purpurescens - 2.30% 13.82% - 79 5.30% Hemilucilia semidiaphana 12.09% 3.08% 5.09% - 58 3.90% Chrysomia megacephala 3.72% - 7.81% - 51 3.42% Hemilucilia segmentaria 2.79% <1% <1% - 43 2.90% Lucilia ibis - 1.54% 4.00% - 24 1.60% Chloroprocta idiodea - 4.61% - - 6 0.40% Lucilia sp. 1.86% - <1% - 5 0.33% Cochliomyia macellaria 1.4% <1% - - 4 0.27% Lucilia albofusca <1% <1% - - 2 0.14% Cochliomyia hominivorax <1% - - - 1 0.06% Chrysomia sp. - - <1% - 1 0.06% Lucilia ochicornis - - <1% - 1 0.06%

Number of Species 10 10 11 3

Number of Specimens 215 130 550 550 1492 100.00%

Table 3.9. The male to female (m/f) ratio of adult blow flies found in Sicily. Ratios are depicted by species for each elevational gradient as well as an overall m/f ratio.

Altitude Altitude Altitude Altitude Tot Tot M/F Species 561m 1312m 1948m 3336m Male Female ratio Consomyiops verena 72M/287F 91M/321F 163 608 0.27 Calliphora nigribasis 53M/80F 53 80 0.66 Lucilia eximia 8M/46F 3M/32F 15F 19 93 0.12 Chrysomia albiceps 8M/36F 13M/44F 2F 5F 21 87 0.24 Paralucilia sp. 13M/55F 20F 13 75 0.17 Lucilia purpurescens 3F 7M/69F 7 72 0.09 Hemilucilia semidiaphana 4M/22F 4F 5M/23F 9 49 0.15 Chrysomia megacephala 1M/7F 6M/37F 7 44 0.16 Hemilucilia segmentaria 1M/5F 1F 2F 9 34 0.12 Lucilia ibis 2F 3M/19F 3 21 0.14

Chloroprocta idiodea 6F 0 6 Lucilia sp. 4F 1F 0 5 Cochliomyia macellaria 3F 1F 0 4 Lucilia albofusca 1F 1F 0 2

Cochliomyia hominivorax 1F 0 1 Chrysomia sp. 1F 0 1 Lucilia ochicornis 1F 0 1

Table 3.10. Differences in species abundance across elevational gradients using Kruskal-Wallis one -way ANOVA and Dunn test with

Benjamini-Hochberg adjustment.

Species Kruskal- Wallis Dunn 1 v 2 1 v 3 1 v 4 2 v 3 2 v 4 3 v 4 C. nigribasis 10.253(0.020) 1.0 1.0 0.017 1.0 0.026 0.054 C. megacephala 5.025 (0.170) ------C. idiodea 3.00 (0.392) ------C. albiceps 11.061 (0.011) 0.848 0.038 0.047 0.067 0.057 1.0 Chrysomya sp. 3.00 (0.392) ------C. hominivorax 3.00 (0.392) ------C. macellaria 4.393 (0.222) ------C. verena 10.923 (0.010) 1.0 0.074 0.026 0.099 0.053 0.615 H. segmentaria 2.987 (0.394) ------H. semidiaphana 8.754 (0.030) 0.124 0.937 0.115 0.111 0.760 0.071 L. albofusca 2.143 (0.543) ------L. eximia 5.67 (0.129) ------L. ibis 8.137 (0.040) 0.288 0.044 1.0 0.411 0.360 0.088 L. ochicornis 3.00 (0.392) ------L. purporsecens 12.913 (< 0.001) 0.706 0.006 1.0 0.023 0.882 0.013 Lucilia sp. 7.702 (0.053) ------Paralucilia sp. 11.331 (0.010) 0.441 0.018 0.036 0.098 0.130 1.0 Numbers 1-4=elevation level (1=561m, 2=1312m, 3=1948m, 4=3336m). Significant p values are bolded for each test.

Table 3.11. Indicator species for elevational gradients in Sicily. A level designation, elevation, indicator value and p value are provided for each species.

Species Level Elevation Value P value Paralucilia sp. 1 561m 77.27 0.021 Lucilia purpurescens 3 1948m 96.20 0.003 Lucilia ibis 3 1948m 68.75 0.028 Calliphora nigribasis 4 3336m 75.00 0.033

Table 3.12. Significant differences in blow fly communities in Ecuador across elevational gradients using Multiple Response Permutation Procedure.

Altitude 561m Altitude 1312m Altitude 1948m

Altitude 1312m A:0.0108; Obs. Delta:0.5555; Exp. Delta:0.5616 p=0.39 Altitude 1948m A:0.2893; A:0.3161; Obs. Delta:0.5465; Obs. Delta:0.5185; Exp. Delta:0.7689 Exp. Delta:0.7581 p=0.027 p=0.025 Altitude 3336m A:-0.2747; A:0.2977; A:0.1031; Obs. Delta:0.586; Obs. Delta:0.558; Obs. Delta:0.5489; Exp. Delta:0.808 Exp. Delta:0. 7945 Exp. Delta:0.612 p=0.04 p=0.028 p=0.083

Figure 3.1. Elevational gradient sampling sites in Sicily. Orange=20m (level 1), blue=700m

(level 2), yellow=1153m (level 3) and red=1552m (level 4).

Figure 3.2. Elevational gradient sampling sites in Ecuador Blue=561m (level 1), yellow=1312m (level 2), red=1948m (level 3) and green=3336m (level 4).

1 0.9 0.8 0.7 0.6 0.5 diversity index diversity

H 0.4 0.3 0.2 Shannon Shannon 0.1 0 0 200 400 600 800 1000 1200 1400 1600 1800 Altitude

Figure 3.3. Blow fly species diversity across elevational gradient in Sicily using the Shannon

(H) diversity index.

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 Sorenson's CCindex similarity 0.1 0 a1_a2 a1_a3 a1_a4 a2_a3 a2_a4 a3_a4 Altitude pairwise comparison

Figure 3.4. Sorenson's (CC) Similarity Index between elevational gradients in Sicily. a1=level 1 (20m), a2=level 2 (700m), a3=level 3 (1153m), and a4=level 4 (1552m).

Figure 3.5. Nonmetric multidimensional scaling ordinations of blow fly communities across elevational gradients in Sicily. This ordination explains 91% of this ordination (minimum stress=0.083). MRPP analysis showed significant differences between 700m and 1153m

(A=0.3035 p=0.035). Circles represent altitude 20m, triangles altitude 700m, squares altitude

1153m, crosses altitude 1552m.

1.8 1.6 1.4 1.2 1

diversity index diversity 0.8 H 0.6 0.4 Shannon Shannon 0.2 0 0 500 1000 1500 2000 2500 3000 3500 4000 Altitude

Figure 3.6. Blow fly species diversity across elevational gradients in Ecuador using the

Shannon (H) diversity index.

0.8

0.7

0.6

0.5

0.4

0.3

0.2

Sorenson's CCIndex similarity 0.1

0 a1_a2 a1_a3 a1_a4 a2_a3 a2_a4 a3_a4 Altitude pairwise comparison

Figure 3.7. Sorenson's (CC) Similarity Index between elevational gradients in Ecuador. a1=level 1 (561m), a2=level 2 (1312m), a3=level 3 (1948m), and a4=level 4 (3336m).

Figure 3.8. Nonmetric multidimensional scaling ordinations of blow fly communities across elevational gradients in Ecuador. This ordination explains 82% of this ordination (minimum stress=0.14). MRPP analysis showed significant difference between all levels (561m and

1948m (A=0.2893 p=0.027); 561m and 3336m (A=0.2747 p=0.04); 1312m and 1948m

(A=0.31261 p=0.025); 1312m and 3336m (A=0.2977 p=0.028)), except between 1948m and

3336m (A=0.1031 p=0.083). Circles represent altitude 20m, triangles altitude 700m, squares altitude 1153m, crosses altitude 1552m. Circles represent altitude 561m, triangles altitude

1312m, squares altitude 1948m, crosses altitude 3336m.

CHAPTER 4

Food preferences of adult male and female Calliphora vicina (Robineau-Desvoidy)

(Diptera: Calliphoridae)

Abstract

Male and female adults of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) were exposed to different food sources and their food preferences were assessed. Each fly was individually color-marked on their thorax; 20 flies (10 males, 10 females) were released at the same time in an arena where they were exposed to four different food sources (sucrose, honey, beef blood and pet food). The flies were filmed for six hours and their preference was assessed based on the number of visits to a source and the duration of each visit examining number of visits to a food source x the duration of each visit (NxL values).

Significant differences were observed between males and females in their preferences of proteins versus carbohydrates; females preferred carbohydrate-rich foods, while males preferred protein-rich foods. These data could be useful for a better understanding of the nutritional ecology of blow fly and to better interpret entomological evidence at crime scenes when multiple potential food sources are present.

Key words: forensic entomology, food preference, Calliphoridae, nutritional ecology,

Calliphora vicina

Introduction

The nutritional needs of insects strictly depend upon the physiological and morphological changes that occur throughout their development together with the specific nutritional and developmental requirements of a particular life stage. For instance, adult female mosquitoes

(Diptera: Culicidae) obtain energy from floral nectar (Smith and Gadawski 1994; Gary and

Foster 2006), but need a blood meal for maturation of their ovaries (Qiu et al. 2011), and standing water to lay eggs (Osgood 1971; Navarro-Silva et al. 2009). In contrast, adult blow flies (Diptera: Calliphoridae) have different nutritional needs and the resources they exploit depends on their developmental stage.

Blow flies are known to be the primary arthropod colonizers of decomposing vertebrate remains; however, the nutritional requirements of adults are more complex. Selection of nutritional resources is based on their age and reproductive status (Strangways-Dixon 1959;

Strangways-Dixon 1961). Adult blow flies have been shown to rely on nectar for carbohydrates (Grinfel’d 1955; Norris 1965), while protein is usually obtained from feces

(Hanski 1987).

Carrion is typically used by adults as an oviposition site (Norris 1965; Byrd and Castner

2010) but may provide a protein meal for adults (Lee et al. 1992). Carrion also provides shelter and nutrient resources for larvae (Ireland and Turner 2006). These observations, however, do not take into consideration the possible differences that exist among species, or between males and females of the same species, or those between gravid and non-gravid females (investigated by Brodie et al. 2014).

A common assumption about blow fly feeding behavior is that, as adults, they only feed on liquid food. This belief is reflected in the common protocols used to maintain calliphorid colonies (Byrd and Castner 2010). It has been observed, however, that the proboscis of an adult blow fly is actually designed to allow them to consume semisolid or solid food due to the ability to secrete digestive enzymes onto food prior to sponging (Graham-Smith 1930).

Other studies have shown that blow flies prefer dry food over wet food (Browne 1993).

The nutritional choices of blow flies are mediated by different apnuemones and kairomones released by different potential food sources (Brodie et al. 2016) or by other blow flies. For instance, several authors have shown that the presence of gravid blow flies on a carcass enhances its attractiveness to other blow flies, due to the release of semiochemical signals by the gravid insects (Denno & Cothran 1975; Kneidel 1984; Wertheim et al. 2005). However, additional studies are needed to investigate the nature of such semiochemicals and their effect on the fly’s behavior.

Calliphora vicina (Robineau-Desvoidy) is commonly known as the blue bottle fly due to the blueish metallic color of its abdomen and thorax. Calliphora vicina is found throughout

Europe and the New World, but it is also present in South Africa, Australia and New

Zealand (Dear 1985; Erzinçlioglu 1984; Wallman 2001). Calliphora vicina is one of the major colonizers of vertebrate remains and it is normally active during cold seasons or during the colder periods of the day (Howlett et al. 2016; Mohr and Tomberlin 2014; Coleman 2014,

Gemmellaro et al. 2018).

A survey of blow flies in Ecuador and Sicily, Italy, at different altitude levels, was conducted as a means to evaluate calliphorid species composition, richness and abundance (see Chapter

3). For this survey, we used traps baited with beef liver, a commonly employed method to sample insects of forensic importance (Vogt and Havenstein 1974; Spradbery 1979; Chen et al. 2004) with the intent of collecting flies attracted to the volatile compounds released from decomposing organic matter. When examining the data, male/female ratio for C. vicina adults was higher than what was found for other species (see Chapter 3). The male/female ratio of blow flies collected with baited traps has been shown to vary across species and based on the bait used. A study analyzed the differences in male/female ratios of necrophagous flies collected using carrion baited traps and the number of calliphorid females collected was significantly higher than males (Martín-Vega and Baz 2013). Differences in the male/female ratio of flies collected by baited traps has also been observed within the same genus. An analysis of the sex ratio was conducted on Chrysomya sp. collected with liver-baited traps in

New Guinea; 108 Chrysomya bezziana (Villeneuve) were collected and only one of them was a male. In contrast, for other Chrysomya sp. collected during the survey 17-26% were males

(Spradbery 1979). The age of the bait can also influence the male/female ratio of the captured adult calliphorids. Vogt and Woodburn (1994) reported that old baits, which had been exposed in traps for seven days before the traps were deployed collected significantly higher numbers of flies when compared to fresh baits that had been exposed immediately before deployment. (Vogt and Woodburn 1994). In fact, the number of C. vicina males collected in the above-mentioned survey was higher than males belonging to other species, suggesting that males are actively seeking out resources that emanate Volatile Organic

Compounds (VOCs) typical of decomposition. These volatiles are commonly associated with a carcass and can indicate an ideal oviposition site for females. This may explain why

85 more females are normally collected on a carcass or liver-baited traps. However, based on these observations, C. vicina males are also highly attracted to this resource.

Understanding food preferences of specific blow fly species could be of forensic importance, as these flies may visit and consume different resources available at a potential crime scene.

Moreover, it may shed some light on our understanding of the nutritional ecology of this group. Comparisons between the food preferences of males and females for one food over another could clarify the different physiological needs of each sex and improve our understanding of their behavior even in relation to the estimation of time of colonization

(oviposition). It has been shown that blow flies consume blood, semen, and saliva if this is the only source of food available to them (Durdle et al. 2009; Durdle et al. 2011; Durdle et al. 2013; Durdle et al. 2015), but it is unknown what their preference would be when several options are available to them. The objective of this study was to investigate the nutritional preferences of males and females of adult C. vicina when exposed to different trophic substrates.

Materials and Methods

For this study, 20 C. vicina adults (10 males and 10 females) were used to create a colony that provided the adult flies used in the comparison trial. Adults were kept in a bug dorm (30 cm3

BugDorm-1 collapsible insect rearing cage - Mega View Science Co., Ltd., Taichung,

Taiwan) at 25oC and a photoperiod of 10:14 (L:D) cycle. Larvae were kept in clear plastic

946 mL Extreme Freeze® Reditainer® Freezable deli containers, with modified breathable lids and were fed using beef liver purchased from a local super market until the post-feeding stage was reached. Resulting pupae were kept in aired individual 29 mL translucent plastic

SOLO® cups, kept at 25oC and a photoperiod of 10:14 (L:D) cycle. Once emerged, adults were put in the previously described bug dorm with water and sugar (Domino® Sugar

Premium PureCane Granulated Domino Foods, Inc. Yonkers NY) ad libitum for three days; after such time the trial was begun. Adult flies were placed in a freezer at -19oC for 10 min to anesthetize them so they could be individually marked with acrylic paint (Craft Smart®

Acrylic Paint, 2 oz./59 ml) (10 different colors for the females and 10 different colors for the males) on their thorax, in a way to not impede locomotion. The flies were left out for approximately 2 hours to acclimate and to let the paint dry; after which they were released, all at once, in the arena with all the food sources. The arena was an 80 x 80 x 80 cm clear acrylic box vented with 8 “windows” covered with fine (1mm x 1mm) acrylic mesh. Inside the arena, four different food sources were placed. Food sources were chosen based on current literature and reviewed case reports to look for other potential attractants for blow flies. The sources chosen were: two sugar sources (Shoprite® honey, and Domino sugar) and two non-human protein-based sources (H-Mart beef blood, and Hill's® Science Diet® Adult

Chicken & Barley Entrée pet food). A list of food sources utilized for the trial and their protein and carbohydrate content is provided in Table 1. Sugar, honey, pet food and beef liver were purchased at a local supermarket (Shoprite®, East Brunswick, NJ). A petri dish with water was also placed in the arena. All food sources where homogenized and mixed with enough water to render them sufficiently moist for the duration of the experiment without altering their nutritional content. They were then placed in clear petri dishes until the dishes were completely filled and were covered with white gauze (TopCare® All Purpose

Sterile Pad); to ensure that color would not be a factor in the choice made by the flies and that the flies would not drown while feeding on liquid substances. The petri dishes with the food sources were then placed in a circle inside the arena and water was placed at the center

87 of the arena; the position of each source inside the arena was chosen randomly and was changed during each session. Fly activity was then videotaped using a Digital HD Video

Camera – Model HDR-XR550V, (Sony®, Japan) for 6 consecutive hours per session for a total of three recording sessions (18 hours in total). During each session, the number of visits of each individual fly to each individual food source was documented as well as the duration of each visit in seconds.

In this experiment, two variables were used to indicate preference: the mean number of visits to each food resource and the mean length of visits. Using the protocol proposed by

Durdle et al. (2016), the two variables were combined by multiplying number of visits to a food resource and the length of each visit together to create a “NxL” value for each food resource for all flies together and for each individual sex. The median NxL values for each food resource were then calculated to evaluate the overall preference among the food resources. Using these values, the following comparisons were made: the preference of males and females combined for the four food resources, the preference of males or females alone for the four food resources, the preference of males and females alone and together for carbohydrates only and the preference of males and females alone and together for protein only. A Mann-Whitney test was run to assess the preference among food sources. Analyses were conducted using R Studio 1.1.463 (R Studio Team 2018) and Microsoft® Excel 2018 version 16.21.1.

Results

To examine the food preference of males and females of C. vicina adults, their preference was ranked using the NxL values that were calculated (Table 2). When male and female

88 preference were evaluated together and when both proteins and carbohydrates were available, sugar was the preferred food choice. Honey, another carbohydrate resource, ranked second, followed by beef blood and pet food. When males and females were analyzed separately the food preference of each sex differed. Females preferred sugar-based foods (honey followed by sugar) while males preferred beef blood (protein) followed by honey (carbohydrate) (Table 2). Another goal was to evaluate the attractiveness of the two sugar sources (Honey and Sucrose). Our results did not show a significant difference

(p<0.05; U=154; Z-score=0.25; p=0.80) between the two.

In the comparison between beef blood and pet food a non-significant difference was noticed

(p<0.05; U=86; Z-score=0.44; p=0.66) despite their different carbohydrate content (Table

1).

Discussion

The order Diptera includes about 150 families for a total of more than 160,000 species

(Evenhuis et al. 2008); it is one of the three largest animal groups in the world (Skevngton and Dang 2002), among which there are numerous flower-visiting families (at least 71 families) and at least 555 flower-visiting species (Larson et al. 2001). Diptera have a major role as pollinators; it is possible that they were essential in the angiosperm radiation

(Labandeira 1998; Endress 2001) and we know that Diptera are pollinators of more than 100 cultivated plants, including strawberries, onions, cacao and tea (Heath 1982; Ssymank et al.

2008). Calliphorids are raised commercially to be used as pollinators of several crops, such as lettuce, peppers and tomatoes (www.forkdtreeranch.com/index.html). Specifically, C. vicina has been involved in the pollination of wild-growing carrots (Pérez-Bañón et al. 2007) and,

89 in caged conditions, in the pollination of onion (Allium cepa) (Clement et al. 2007; Sajjad et al.

2008), Brussel sprouts (Brassica oleracea) (Faulkner 1977) and carrots (Howlett 2012).

Calliphora vicina is a good candidate for pollination since it is found all over the world (Dear

1985) and does not represent a threat to livestock (Dear 1985) like other calliphorids, and it is easy to rear en mass (Turner 2005; Davies 2006; van Veen 2008).

The main reason why they visit flowers is food: nectar and pollen provide essential nutrients for them, being a source for carbohydrate and proteins, respectively (Kevan 2002). In addition, flowers can also provide a place for them to find mates, offer them shelter and warmth, or trap flies by emanating odors and visual cues which mimic those of rotting flesh

(Ssymank et al. 2008). For example, a plant known as dead-horse arum (Helicodiceros muscivurus; Araceae, Aroideae) emits a smell similar to that of carrion. Stensmyr et al. (2002) confirmed that the volatile organic chemicals released by this plant is similar to that released by an animal carcass during decomposition. This study was designed with the goal of exploring the nutritional needs of adult C. vicina by analyzing their food preference when given the choice of multiple sources, with particular focus on the differences between males and females. From the data, no significant differences were observed between sugar and honey or between beef blood and pet food.

Knowing the food preferences of flies could be helpful in forensic investigations and crime scene analysis. A better understanding of what substrates flies are attracted to initially can help the interpretation of potential artifacts left by adult flies during regurgitation and defecation, which can be common at scenes (Durdle et al. 2009), and in the search for DNA traces at a crime scene (Durdle et al. 2013). The non-significant result we observed when

90 comparing the beef blood and pet food could indicate that both these foods satisfy the nutritional needs that three-day old flies have in terms of proteins as their content is about the same, but further tests are needed to confirm this. These two sources, however, have a significantly different carbohydrate content (Table 1). The fact that the flies’ preference of these foods seems to be equivalent, despite their difference in terms of carbohydrates, may be due to the fact that other carbohydrate sources were available simultaneously. Further studies where nothing but these two food sources are offered are necessary to evaluate whether or not the difference between these two food sources remains non-significant.

When given access to both honey and sucrose, along with other sources, adults of C. vicina were shown to have a preference for sugar, even though they visited both sources. Both sources seem to be commonly used in rearing protocols (Orr 1963; Stoffolano et al. 1995;

Mackley and Snow 1992; Thomas 1993; Blystone and Hansen 2014). Females preferred sugar-based foods (honey followed by sugar). The two sugars are normally employed in laboratory rearing protocols as the main food sources for adults, while the blood is commonly used as a protein meal to help the development of the oocytes and therefore encourage oviposition. while males preferred food choice was beef blood (protein) followed by honey (carbohydrate) and pet food, another protein resource (Table 2).

These findings contradict what has been found by others where females were believed to be attracted and always arrive first to carrion to have a protein meal thus favoring oviposition

(Glaser 1923; Mackerass 1933; Evans 1935; Dorman et al. 1938; Hobson 1938; Rasso and

Fraenkel 1954; Orr 1964). This analysis also shows that males are more attracted to protein- rich resources than non-gravid females. This agrees with observations during work

91 conducted in Sicily and Ecuador using protein baited traps, during which C. vicina males were more abundant than males from other species (see Chapter 3).

We know that female blow flies require protein for the development of their oocytes

(Rachman 1980) and therefore for oviposition; however, it is also often stated that several species of flies can survive for up to two months feeding exclusively on water and carbohydrates (sugar) (Fraenkel 1940), and it has been hypothesized that protein meals are only useful to females for the development of eggs and are otherwise detrimental to the health of the flies (Fraenkel 1940).

The quest for food and mates in blow flies is mediated by chemical and visual cues that are captured by the flies based on their age, sex and development status. We know that

Calliphoridae rely on visual cues when looking for food (Wall and Fisher 2001), when they look for a suitable place to lay their eggs (Brodie et al. 2014) and when they are in search of a mate (Boeddeker et al. 2003). Blow flies display a marked sexual dimorphism in the size of their eyes. Males have significantly larger eyes than females (Cronin et al. 2014) and this suggests that males rely more on visual cues than females. For this trial, we tried to minimize the effect of colors and visual stimuli, by covering the Petri dishes with white gauze.

The reason food was homogenized and water was added to the solid sources was in order to remain compliant with standard blow fly feeding protocols, which usually include diets consisting of liquid foods; calliphorids mouthparts are best suited for the consumption of liquids (Durdle et al. 2016). Nevertheless, it is possible for blow flies to actually feed on solid or semiliquid food. Experiments carried out on the black blow fly, Phormia regina (Meigen)

92 have shown that the chemoreceptors present on the tarsal segments of the flies are triggered by several sugars once they reach an acceptance threshold (Hassett et al. 1950). The successful stimulation of these receptors leads the insect to respond by extending their proboscis and if the substrate is accepted, by drinking (Hassett et al. 1950).

When in presence of solid or semisolid foods whose contents trigger the acceptance threshold, the fly extends its proboscis to make contact with the food source. If unable to sponge solid food, digestive salivary enzymes are secreted which, together with structures called prestomal teeth, reduce the food into small particles suitable for consumption

(Graham-Smith 1914). This process requires extra time and energy for the flies to consume solid food.

Despite the extra time and energy expended there could still be benefits in consuming non- liquid food compared to liquid food. The nutrients in solid and semisolid foods may actually be more concentrated. After the first sponging of liquid food, flies have been observed suspending drops of liquid food from their proboscis for some time before re-consuming it; they repeat this several times with the same meal before they fully consume it (Hendrichs et al. 1993). The reason behind this behavior may lay in the fact that flies try to let most of the water present in the food evaporate so that they can consume a less volume but a more nutritious meal (Hendrichs et al. 1993). This study expands on the work already conducted on other blow fly species (to assess their food preferences (Durdle et al. 2016). It could encourage new research on the food preferences of different species or on the same species using adults of different age or different food sources.

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Table 4.1. Food resources provided to C. vicina adults and their protein and carbohydrate content (prior to adding water).

Protein Carbohydrates Food Resource per 100 g per 100 g Sucrose (Domino sugar) 0 100 Honey (ShopRite® Honey) 0 80.9 Beef Blood (HMart®) 90 Pet Food (Hill's® Science Diet® Adult Chicken & 7 13 Barley Entrée)

100

Table 4.2. Food preference ranking for overall (males and females), males only and females only of C. vicina.

Overall (N x L) Males (N x L) Females (N x L)

1st preference Sugar (5278543) Blood (35250) Honey (6448) 2nd preference Honey (2250987) Honey (10698) Sugar (1715.5) 3rd preference Blood (1112654) Pet Food (2024) Blood (1112) 4th preference Pet Food (738456) Sugar (455) Pet Food (738)

101

CHAPTER 5

A Geometric Morphometric Analysis of Wing Variations in Shape and Size of the

Blue Bottle Fly Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae)

Abstract

A geometric morphometric analysis was performed on the right wing of adult Calliphora vicina

(Robineau-Desvoidy) (Diptera: Calliphoridae) collected across four altitudinal levels in Sicily

(20m, 700m, 1153m, 1552m). The objective of this study was to assess differences in shape and centroid size between females and males and across elevations. Significant differences were observed in wing shape between males and females but not between all altitudinal levels. Female wings were found to be significantly larger than males (p <0.01). Wings of flies collected at the highest altitudinal level resulted in significantly larger wings than those collected at lower altitudes (p <0.001), with centroid size values ranging from 12.1- 14.1.

Key words: geometric morphometrics, Calliphoridae, Calliphora vicina, wing, shape, size, altitudinal gradient

102

Introduction

Blow flies (Diptera: Calliphoridae) are a group of necrophagous insects of major importance in decomposition ecology (Byrd and Castner 2010). They are the most consistent part of the first wave of invertebrate colonization of corpses (Grassberger and Frank 2004) and can provide information useful for the calculation of the colonization interval (Amendt et al.

2004; Tomberlin et al. 2011). Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae:) known as the blue bottle fly because of its blueish metallic color (Szpila 2009), is a cosmopolitan species present in Europe (Szpila 2009), North America (Weidner et al. 2015),

South Africa (Williams and Villet 2006), Australia (Wallman 2001) and New Zealand

(Erzinçlioglu 1984; Dear 1985). Calliphora vicina is commonly observed during cold seasons or during the colder hours of the day (Howlett et al. 2016; Mohr and Tomberlin 2014;

Coleman et al. 2014; Gemmellaro et al. 2018).

Variations in body size and biological shape are generally correlated with the biology of several species (Kuclu et al. 2011). For instance, in mosquitos in the genus Aedes, a larger size is associated with more frequent blood meals, higher longevity and additional factors that could potentially impact their vectorial ability (Vargas et al. 2010). These changes in size and shape can also play an important role in ecological investigations analyzing potential correlations between size and morphology of an organism and the environment (Van’T

Land et al. 1999). Kuclu et al. (2011) examined the geometric morphometric variation of size and shape in populations of Aedes vexans (Meigen) and noticed that the grouping patterns were consistent with the geographical regions where the specimens were collected. Two size trends have been observed in relation to altitude and latitude (Chown and Klok 2003). The first pattern is known as the Bergmann’s rule (Atkinson 1994) and it states that as latitude

103 and altitude increase, so does body size. Bergmann (1847) hypothesized that in larger individuals the surface/volume ratio is more conductive to conserving heat in cold climates.

The second pattern, opposite to the Bergmann’s rule, describes a negative correlation between body size and latitude/altitude. This pattern has also been observed in ectothermic organisms (Masaki 1967). For instance, the average body size of Homoptera, Hemiptera,

Formicidae and other Hymenoptera has been observed to be larger in tropical areas than in more temperate regions (Schoener and Janzen 1968). This pattern might have evolved because of the length of seasonal patterns (Mousseau 1997), although there is no consistent pattern across taxa (Silver and Renshaw 1999). Shape is the most noticeable phenotypic component of an organism (Ricklefs and Miles 1994). Variations in shape have been observed to correlate with changes in temperature or in geographical areas (Kucklu et al.

2011). In Drosophila populations, changes in temperature results in changes in wing shape

(Cavicchi et al. 1985), and Van’T Land et al. (1999) observed significant differences in wing shape from Drosophila populations coming from different geographical areas, observing clusters or latitudinal clines.

Morphometrics is defined as the quantitative analysis of biological shape, shape variation and covariation of shape correlated with biotic or abiotic factors (Webster and Sheets 2010).

Traditional morphometric approaches rely on a specific set of measures such as linear distances, ratios or angles (Rohlf 1990). Geometric morphometrics makes it possible to conserve the relative spatial arrangement throughout the analysis (Zeldich et al. 2004), thus allowing evaluation of variations in shape between and within populations (Walker 2000).

Multivariate statistical analyses of anatomical landmarks based on biologically homologous points should be utilized to more easily determine if unique relationships among landmarks

104 exist and to draw and visualize diagrams of morphological changes. (Bookstein 1991; Rohlf and Marcus 1993; Adams et al. 2004; Webster and Sheets 2010).

The morphology of an insect’s wing has received ample attention because it is crucial for several aspects of an adult insect’s life including flight aerodynamics, foraging, thermal regulation, defense mechanisms, and sexual displays (Betts & Wootton 1988; Wootton 1992;

Berwaerts et al. 2002, Berwaerts et al. 2006). Insect wing morphology has been the object of numerous ecological and evolutionary studies (Moraes et al. 2004; Carreira et al. 2005; Soto et al. 2008; Hernández et al. 2010). Hall et al. (2014) performed geometric morphometric analysis of the wing of the Old World Screwworm Chrysomya bezziana (Villeneuve) (Diptera:

Calliphoridae) to look at potential differences between different geographic lineages. They also looked at geometric morphometric differences in wings that had been flattened and mounted on slides and wings that were still attached to the specimen. Their findings showed significant differences in both comparisons.

Wing morphology has also been used as an indicator of stressful or changing environmental conditions (Hoffmann et al. 2005). For example, studies to evaluate the effects of a pyrethroid pesticide and low temperatures on specimens of Helicoverpa punctigera (Wallengren)

(Lepidoptera: Noctuidae) showed significant stress-specific differences in wing shape

(Hoffmann et al. 2002). Changes in the morphology of an insect can be triggered by climatic factors (decreasing temperature) and environmental factors (atmospheric pressure, oxygen availability, and increased solar radiation related to high-altitude environments) (Hodkinson

2005; Dillon & Frazier 2006). The effect of temperatures on wing morphology has been studied; however, the results were not consistent among taxa (Silver and Renshaw 1999). It

105 has also been observed that lower temperatures/higher altitudes correlate with larger bodies and wings and reduced wing loading (body mass/wing area) (Norry et al. 2001; Altshuler and

Dudley 2002; Gilchrist & Huey 2004).

Recent studies have analyzed sexual shape dimorphism in blow fly species (Hall et al. 2014).

Nuñez-Rodríguez and Liria (2017) examined sexual dimorphism in three blow flies species and observed significant differences between males and females with females being larger.

Other studies have focused on the differences among species for the purpose of identification (Vasquez,́ M. and J. Liria. 2012; Sontigun et al. 2017). A geometric morphometric analysis on blow fly larvae belonging to three different species showned significant differences in the shape of their cephaloskeleton. Data produced by this research can create new tools for the identification of calliphorid species that could be used with traditional dichotomous keys and molecular barcoding. Moreover, analyses conducted within the same species across different regions/altitudes can provide valuable information for understanding the species biology and how it correlates with climatic and environmental factors. The purpose of this study was to investigate the variations in size and shapes in the wings of C. vicina across an altitudinal gradient in Sicily, Italy.

Materials and Methods

A survey of necrophagous Diptera was conducted in the Eastern part of Sicily across four different altitude levels (20m, 700m, 1153m, 1552m) (Chapter 3), using RESCUE!® POP! Fly

Traps (Sterling International, Inc., Spokane, WA), baited with 100g beef liver. Collected specimens were cleaned, sorted by sex, and morphologically identified. Morphological identifications were made using the keys of Spzilia (2017). For this study only adults of C.

106 vicina with intact or minimally damaged wings were used. The number of individuals analyzed per elevational gradient was as follows: 20m=53 individuals, 700m=112 individuals,

1153m=158 individuals, and 1552m=39 individuals, totaling 362 individuals (204 females and 158 males).

The right wing of each fly was removed with forceps and mounted on a microscope slide using a drop of Hoyer's Microscope Slide Mounting Medium (Hempstead Halide) and covered with a cover slip (CS-24/60 Rectangular Cover Glass, No. 2 Thickness, 24 x 60mm,

Harvard Apparatus). The wing was then photographed using a Zeiss SteREO Discovery.V8, equipped with a ZEISS Axiocan ERc 5s (5M pixels) digital camera. JPEG Images were converted into tps files using TpsUtil V. 1.64. Nineteen landmarks were chosen and digitally marked each wing using TpsDig2 V.2.20 software (Figure 5.1). The tps files were then imported into MorphoJ software for analyses (Klingenberg 2011). To exclude variations caused by differences in position, orientation and scale, the raw landmark coordinates were aligned and superimposed using the software’s Procrustes Fit function. A Procrustes superimposition is a method that translates of all objects to be analyzed to a common centroid. They are then scaled to the same centroid size, rotated and centered to minimize summed squared distances between the corresponding landmarks. Centroid size was found by calculating the square root of the sum of the squared distances between each landmark and the center of the landmark configuration. The new Procrustes coordinates were then used for the statistical analyses.

Size for each wing was estimated using its centroid size (Alibert et al. 2001; Beniteź et al.

2011). Variations in size, and shape between males and females, and across altitudes,

107 centroid size was analyzed using a Procrustes Analysis of Variance (ANOVA). Results from this analysis are indicated as sums of squares (SS) and mean squares (MS), which by definitions are dimensionless (Klingenberg and McIntry 1998; Klingenberg et al. 2002).

Following this, shape variations among all specimens were analyzed using a Principal

Component Analysis (PCA). This analysis is based on the covariance matrix of the variation in the shape of the wing. Centroid size data were not normally distributed (Shapiro-Wilk normality test). To examine the difference in centroid size between females and males a

Wilcoxon test. A Shapiro-Wilk normality test on the centroid size distribution across altitudes showed that the distribution was not normal (p=0.007) and therefore a Kruskal-

Wallis (Kruskal and Wallis 1952) test was performed to analyze CS across altitude followed by a Dunn test (Dunn 1961) with a Benjamini-Hotchberg adjustment for pairwise comparisons (Benjanmini and Hotchberg 1995). To analyze CS between sexes a Wilcoxon

Test was performed. Following the methods of Sanzana et al. (2013), differences between locations (pair-wise) were measured using Procrustes distances, which are the product of a

Canonical Variate Analysis (CVA). The results and the respective p values of each pair-wise analysis were calculated after permutation tests based on 10,000 runs.

To describe how size can affect shape, and to calculate the allometric effect, a regression analysis between sexes and across attitude was done using Procrustes distance (dependent variable), and the centroid size (independent variable). All statistical and morphometric analyses were performed using MorphoJ (Klingenberg 2011), R studio 1.1.463 (R Studio

Team 2018) and Microsoft® Excel 2018 Version 16.21.1

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Results

The Principal Components Analysis showed that the first three principle components (PC) accounted for 53.9% (PC1: 28.8%, PC2: 15.8%, PC3: 9.3%) of variance in wing shape (Table

5.1; Figure 5.2). The remaining PC values were lower than 7%. Variations in shape are represented in a lollipop graph, outline graph and a wireframe graph with the axis corresponding to PC1 (Figure 5.3a-5.3c). Males and females were successfully distinguished using PC1 and PC2 (Figure 5.4).

The average (±SE) Centroid Size (CS) for flies across elevational gradients was as follows:

20m: 13.1 (± 0.1); 700m: 12.1 (±0.1); 1153m: 13 (±0.1); 1552m: 14.1 (±0.2). Average CS for females was 13.3 (±0.1) while for males averaged 12.3 (±0.1). Differences in centroid size across elevational gradients were found to be significant (H=54.0298, df=3, p<0.001). Use of a Dunn Test with a Benjamini-Hochberg adjustment showed significant differences between the wing centroid size of flies collected at 20m and 700m (p<0.001), 20m and

1552m (p=0.0109), 700m and 1153m (p<0.001), 700m and 1552m (p<0.001), and 1153m and 1552m (p<0.001) (Table 5.2). No significant difference was observed between 20m and altitude 1153m (p=0.349). The Wilcoxon Test also showed significant differences in centroid size between sexes (w=21536, p<0.001).

The results of the CVA analysis of altitude (Figure 5.5) and sex (Figure 5.6) showed significant differences between all elevations (20m and 700m, p<0.002; 20m and 1153m, p<0.001; 20m and 1552m, p<0.001; 700m and 1153m, p<0.001; 700m and 1552m, p=0.002, and 1153m and 1552m p<0.001) (Table 5.3). Both sexes were also separated along the CV1 axis (Procrustes distance=0.0215, p<0.001) (Figure 5.6).

109

The Procrustes ANOVA showed significant differences in shape and size for both elevation

(CS: p<.001 and Geometric Shape HS: p<.001) and sex (CS: p<0.001 and Geometric Shape

HS: p<0.001) (Table 5.4 and Table 5.5). The pooled-within group regression analysis showed significant differences for females and males (p<0.001, 10,000 permutations), and allometry explained 4.149% of variations found in shape. The regression analysis on Procrustes coordinates and centroid size across elevations showed significant results (p<0.001, 10,000 permutations), and allometry explained 1.82% of variation.

Discussion

Geometric morphometric analyses showed significant variation in the shape and size of the right wing of C. vicina. The differences found were significant among all populations collected at different altitudes. Bergmann’s law states that as latitude increases so does the size of an organism (Bergmann 1847; Mayr 1956) and this trend has been also observed in altitude increases. The results of this study are in agreement with Bergmann’s Law, showing wing size increases with increasing altitudes.

Similar studies have related the variation observed in wing shape with environmental conditions (Adams & Funk 1997; Hoffmann et al. 2002; Hoffmann et al. 2005; Takahashi et al. 2011). Variations in wing shape can impact thermoregulation and therefore effect oxygen content, temperature, atmospheric pressure and solar radiations can have on an insect’s body and activity levels (Hodkinson 2005). In addition, one study involving Drosophila flavopilosa

(Frey) (Diptera: Drosophilidae) in Chile reported that both wing length and breadth increased with altitude (Budnik et al. 1988) while Dillon et al. (2006) analyzed the

110 locomotion of Drosophila melanogaster (Meigen) at high altitudes and noticed reduced walking speeds and flight performance as altitude increased.

At high elevations and lower temperatures, larger wings could mean less energy when flying to increase body temperature (Brodsky 1994); individuals with larger wings could have the ability to fly in high winds, have more strength and better aerodynamic abilities (Berwaerts et al. 2002; Frazier et al. 2008). Nylin & Svard̈ (1991) showed that the size of European butterflies depends on seasonality and the duration of periods favorable for their growth. In this study, significant differences in size and shape of the right wing of C. vicina adults collected at different altitudes were observed, with size increasing with elevations. This could provide flies living at high elevations with the ability to fly more efficiently when searching for mates, food and oviposition substrates. This study also showed that males and females of

C. vicina display significant differences in the shape and size of their right wing. This result was expected as males and females of other blow fly species have been successfully distinguished by the geometric morphometric analysis of their wing (Hall et al. 2014). The differences between sexes could be related to the different need for dispersal to find food and mates that exist between male and female blow flies. For example, larger size in females can be interpreted as a fecundity advantage (Nuñez and Liria 2017). Females also can travel up to several kilometers to find a suitable carcass to lay eggs (Braak 1986) and would require better flight performance than males. This study confirms the value of geometric morphometrics on the analysis of intraspecific variations; it additionally provides information on the differences in size and shape between males and females and among populations collected in different geographical locations. These analyses could be further extended to other populations of C. vicina, located in different geographic areas and

111 elevations. They could also be applied to other species or used to analyze potential interspecific differences among other species of forensically important Diptera.

112

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Table 5.1. Principal Component Analysis eigenvalues that account for 95% of variation.

PC Eigenvalues Variance % Cumulative %

1 0.00015585 28.767 28.767

2 0.00008562 15.804 44.57

3 0.00005018 9.262 53.833

4 0.00003792 7 60.833

5 0.00003035 5.602 66.434

6 0.00002718 5.017 71.451

7 0.0000183 3.377 74.828

8 0.00001678 3.098 77.926

9 0.00001327 2.45 80.376

10 0.00001246 2.3 82.676

11 0.00001103 2.036 84.713

12 0.00000919 1.697 86.41

13 0.00000787 1.452 87.861

14 0.00000711 1.313 89.174

15 0.00000651 1.202 90.376

16 0.00000538 0.994 91.37

17 0.00000473 0.873 92.243

18 0.00000433 0.798 93.041

19 0.00000403 0.744 93.786

20 0.00000382 0.705 94.491

21 0.00000367 0.678 95.168

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Table 5.2. Dunn test with the Benjamini-Hochberg adjustment for pair-wise comparison of the Centroid Size (CS) of wings across altitudes.

Altitude 20m Altitude 700m Altitude 1153m

Altitude 700m 4.29 (p<0.001)

Altitude 1153m 0.93 (p=0.349) -4.59 (p<0.001)

Altitude 1552m -2.60 (p=0.109) -6.8 (p<0.001) -3.90 (p<0.001)

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Table 5.3. Canonical Variate Analysis assessing differences between wings belonging to

Calliphora vicina adults collected at different altitudes.

Altitude 20m Altitude 700m Altitude 1153m

Altitude 700m p=0.001 - -

Altitude 1153m p<0.001 p<0.001 -

Altitude 1552m p<0.001 p=0.002 p<0.001

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Table 5.4. Procrustes ANOVA analysis for Size and Shape – Altitude and Sex.

Elevational Gradient

Effect SS MS df F P

Centroid Individual 129.655749 43.218583 3 20.20 <0.001

Size

Shape Individual 0.00935129 0.0000916793 102 5.99 <0.001

Sex

Effect SS MS df F P

Centroid Individual 77.539442 77.539442 1 34.12 <.001

Size

Shape Individual 0.04131596 0.0012151753 34 96.42 <.001

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Figure 5.1. Wing of Calliphora vicina showing the 19 landmarks used in the geometric morphometric analysis.

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Figure 5.2. Percentage of variance which each principle component accounts for.

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Figure 5.3. Visual depiction of shape variation in the right wing of C. vicina (displaying PC1).

A) Lollipop graph, B) Outline graph, C) Wireframe graph.

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Figure 5.4. Projections of Procrustes-aligned landmark configurations on the first two principal components (=relative warps) of the shape covariance matrix. Males are displayed in blue, females are displayed in red.

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Figure 5.5. Canonical Variate Analysis on Altitudes of the first two Canonical variate scores. Altitudes: 20m (Red), 7oom (Green), 1153m (Blue), 1552m (Purple).

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Figure 5.6. Frequency plot of canonical variate 1 for the right wing of C. vicina showing separation between males and females (females=red, males=blue).

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CHAPTER 6

Conclusions

The survey conducted in the volcanic caves of Sicily showed that in dark, cold environments blow fly activity and colonization can still occur. A delay in colonization was also observed, which is in agreement with what was previously observed in Switzerland when a body was found in a cave. These data refute the assumption that blow fly activity ceases in the absence of light and below temperatures which have been previously suggested. These results show the need for more research on blow fly activity in cold, dark environments.

During the altitudinal study, 12 blow fly species were captured in Sicily while 17 species were captured in Ecuador. In this study it was shown that certain species prefer low altitudes while others are were more likely to be found at high elevations. Significant differences were found for seven species across four altitude levels in Ecuador (C. nigribasis, C. albiceps, C. verena, H. semidiaphana, L. ibis, L. purpurescens, Paralucilia sp.), while only one species, L. sericata, was significantly different in Sicily. This is in line with the biodiversity of Ecuador, both in terms of entomofauna and the variety of habitats present as compared to the uniform landscape in Sicily. These data can be used to assess the ecology of calliphorid flies in these data-deficient areas and represent the first checklist of blow flies in these areas, which in turn, could be instrumental in forensic investigations.

The nutritional preferences of C. vicina adults among different non-human food sources have been assessed and significant difference were observed between females and males.

Compared to females, males showed a higher preference for protein meals. It has been

129 documented that females need proteins for the development of their oocytes, but little is known about their impact on male physiology. Moreover, understanding the overall food preferences of C. vicina can help with the interpretation of crime scenes where multiple potential food sources (i.e. leftovers, bodily fluids) are present. These results suggest the need for further research in this area.

The variations in shape and size of the right wing of C. vicina were analyzed and significant differences were observed. A PCA and a Kruskal-Wallis analysis showed significant differences in wing shape and size of males and females. The shape and size of wings of individuals collected at different altitudes was significantly different, with size increasing with altitude. These data represent the first geometric morphometric analysis conducted on C. vicina across an elevational gradient in Sicily.