A Parasite Survey of Passerine Birds and Northern Bobwhite Quail (Colinus virginianus) in the Rolling Plains Ecoregion

by

Jessica Louise Herzog, B.S.

A Thesis

In

Environmental Toxicology

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCES

Approved

Ronald J. Kendall, Ph.D. Chair of Committee

Seshadri Ramkumar, Ph.D.

Mark Sheridan Dean of the Graduate School

May, 2020

© 2020, Jessica L. Herzog

Texas Tech University, Jessica L. Herzog, May 2020

ACKNOWLEDGMENTS

Thank you, Dr. Ronald J. Kendall, for giving me the opportunity to obtain my Master’s Degree. I appreciate the resources and support you provided me throughout my time as a student in your lab. Thank you, Dr. Seshadri Ramkumar, for being a part of my thesis committee and supporting me along the way. Thank you, Dr. Steven Presley, for your continued support and guidance during my time at TIEHH.

I would also like to thank the Texas Tech Graduate School, the Justin W. Robbe Memorial Scholarship Fund, Texas Tech University Student Housing, The AFO-WOS Travel Award Committee, and The AFO-WOS Student Travel Grant Committee for generously providing funding I needed to complete my research and degree. Thank you to Ribelin and the personnel at Matador Wildlife Management Area, Dalby Ranch, and Morrison Ranch for providing field sites and helping me with field work. I am deeply grateful to Dr. James Surles for advice concerning my statistics and Heath Garner, Joe Luksovsky, and Dr. Mike Kinsella for their assistance in identifying helminths.

I thank the members of the Wildlife Toxicology Laboratory, both past and present, for their help and support, especially with field and molecular work. I thank Tammy Henricks for all that she has done to keep supplies at hand and my project running smoothly. Thank you to the students, faculty, and staff of the Institute of Environmental and Human Health for brightening my days and regularly checking up on and encouraging me. Your support was invaluable.

Finally, thank you to my family, friends, and rabbits for being there, even on a daily basis. This list includes but is by no means limited to Joe, Maureen, Cindy, Jim, Taylor, Mary Anne, Joe, Laurie, Charlie, and Hailey Herzog, Kyle and Luke Harding, Judy Brogan, Mike Johnston, Kathy Hunko, Jennifer Olsen, Kendall Blanchard, Katy Shaw, Olivia Wright, Alyssa Hay, Regan Balko, Kalin Skinner, Maya Bastille, The Gammons Family, and Emery Hyden. To those of you listed and everyone else who truly believed in me: your love and encouragement are why I continued my pursuit of this degree and wrote this thesis. I am forever thankful for you all.

ii Texas Tech University, Jessica L. Herzog, May 2020

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ii

ABSTRACT ...... v

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

CHAPTER I: BACKGROUND AND SIGNIFICANCE ...... 1

Introduction ...... 1 The Rolling Plains Ecoregion ...... 3 General Ecology of Quail Studied ...... 4 General Ecology of Passerines Studied...... 6 Parasite Ecology ...... 7 Helminths ...... 8 Ectoparasites ...... 13 Haemoparasites ...... 14 Research Goals and Objectives ...... 16 Thesis Outline and Chapter Introductions...... 17 References ...... 19 CHAPTER II: HELMINTHS COMMON BETWEEN NORTHERN BOBWHITE QUAIL (COLINUS VIRGINIANUS) AND SELECT PASSERINES IN THE ROLLING PLAINS ECOREGION, TX ...... 29

Abstract ...... 29 Introduction ...... 30 Materials and Methods ...... 33 Ethics Statement ...... 33 Study Area and Sample Collection ...... 34 Helminth Extraction ...... 35 Helminth Processing and Identification ...... 35 Statistical Analysis ...... 36 Results ...... 36 Helminth Prevalence ...... 36 Eyeworm and Caecal Worm Infection ...... 40 Infection with Other Helminths ...... 42 Discussion ...... 45 iii Texas Tech University, Jessica L. Herzog, May 2020

References ...... 52 CHAPTER III: ECTOPARASITES AND HAEMOPARASITES OF NORTHERN BOBWHITE QUAIL (COLINUS VIRGINIANUS) AND SELECT PASSERINES IN THE ROLLING PLAINS ECOREGION, TX ...... 58

Abstract ...... 58 Introduction ...... 59 Materials and Methods ...... 63 Ethics Statement ...... 63 Sample Collection and Processing ...... 63 Ectoparasite Extraction ...... 64 Ectoparasite Identification ...... 64 Blood Smear Preparation ...... 65 Smear Quality and Examination ...... 65 Statistical Analysis ...... 66 Results ...... 67 Ectoparasite Prevalence ...... 67 Ectoparasite Counts ...... 70 Haemoparasite Prevalence ...... 70 Discussion ...... 71 References ...... 76 CHAPTER IV: CONCLUSIONS AND FUTURE DIRECTIONS ...... 82

Conclusions ...... 82 Future Directions ...... 84 References ...... 96 APPENDIX A: HELMINTH PHOTOS ...... 101

APPENDIX B: ECTOPARASITE PHOTOS ...... 107

APPENDIX C: HAEMOPARASITE PHOTOS ...... 110

iv Texas Tech University, Jessica L. Herzog, May 2020

ABSTRACT Eyeworms (Oxyspirura petrowi) and caecal worms (Aulonocephalus pennula) are heteroxenous nematodes being investigated as contributors to the decline of the Northern Bobwhite (Colinus virginianus; hereafter Bobwhite), an iconic game bird. Researchers have documented declines of over 4% annually across the range and declines in historic strongholds including the Rolling Plains of Texas. While climatic factors and habitat change were considered to be leading contributors, parasitic infection is gaining attention as a potentially important factor. Researchers have documented infection rates of eyeworms in Bobwhite as high as 90% and caecal worms as high as 100%. Furthermore, eyeworms have been documented in a Curve- billed Thrasher (Toxostoma curvirostre), Northern Mockingbirds (Mimus polyglottos), and Scaled Quail (Callipepla squamata) from the Rolling Plains. This is likely due to their diets, which include insect intermediate hosts of eyeworms. However, due to extremely few comprehensive parasite studies concerning these species and previous passerine helminth reports being compiled from incidental bycatch, the extent of shared parasitism between quail and passerines in the Rolling Plains is unknown. Thus, the potential for passerines specifically to serve as reservoir hosts or means of dispersal to introduce parasites to naïve populations may currently be overlooked. To investigate this possibility and further document shared parasitism between passerines and quail, full parasite surveys on Bobwhite, Scaled Quail, Northern Mockingbirds, Curve-billed Thrashers, and Northern Cardinals (Cardinalis cardinalis) were conducted. Birds were trapped with baited double funnel traps at three study sites in the Rolling Plains from March to October 2019. After which, specimens were fully necropsied and both endo and ectoparasite prevalence were assessed. These assessments have demonstrated instances of coinfection, with one of the most prominent cohabitations occurring between eyeworms and lice across all species. In addition to this, data elicited from this survey contributes to broadening the current knowledge of parasitic infection concerning the study species. The findings of this study are a valuable baseline for future studies given the passerines’ potential to serve as reservoir hosts and disperse parasites.

v Texas Tech University, Jessica L. Herzog, May 2020

LIST OF TABLES

2.1.1 Prevalence of collected helminth species. Percentages correspond to the total number of specific hosts necropsied. Adapted from Ferrer et al. (2004) ...... 37

2.1.2 Data pertaining to helminths aside from O. petrowi and A. pennula collected from study birds during the 2019 field season...... 43

3.1.1 Prevalence of ectoparasites across species. Percentages correspond to the total number of specific hosts examined. Adapted from Ferrer et al., (2004)...... 68

3.1.2 Prevalence of observed haemoparasite categories. Percentages correspond to the total number of specific hosts necropsied. Adapted from Ferrer et al., (2004)...... 70

vi Texas Tech University, Jessica L. Herzog, May 2020

LIST OF FIGURES

1.1 Population trends (mean annual BBS counts) for Bobwhite (blue) and Scaled Quail (green) from 1978 to 2019. The long-term means are represented by dotted lines (Texas Parks and Wildlife, 2020d)...... 2

1.2 Ecoregions of Texas. The Rolling Plains is colored red. (Texas Parks and Wildlife, 2020c)...... 4

2.1 County Locations in the Rolling Plains Ecoregion of Texas used to obtain avian hosts. Birds were trapped at counties A (Cottle County), B (Garza County), and C (Mitchell County). Bobwhite were donated from D (Stonewall County)...... 33

2.2 The count (in parenthesis) and percent distribution of eyeworms, caecal worms, and other helminths (excluding Raillietina spp. and Mesocestoides spp. tetrathyridium) out of 5,429 helminths collected...... 39

2.3 A bar graph representing the average eyeworm infection intensity at each site for each species of bird sampled...... 40

2.4 A bar graph representing the average caecal worm infection intensity at each site for each species of quail collected...... 41

2.5 A bar graph representing the percentage of Bobwhite sampled from each age class infected with each total number of helminth species. Adapted from Bruno (2014)...... 44

3.1 Examples of smears made and their quality. Smear A is too thick, B is a typical smear, C is nearly too small, and D is too small...... 65

4.1 Partial PCR amplification of COX1 for Sample 83 at 50°C and 53°C using universal nematode primers. Lane M: 100 bp DNA ladder; lanes 1 and 3-5: unamplified nematode samples; lanes 2 and 6: approximately 750 base pair amplification of Sample 83. Adapted from (Kalyanasundaram et al., 2017)...... 90

4.2 The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model. The tree with the highest log likelihood (-13542.29) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 27 nucleotide sequences. There were a total of 1654 positions in the final dataset. Evolutionary analyses were conducted in MEGA X...... 91 vii Texas Tech University, Jessica L. Herzog, May 2020

CHAPTER I

BACKGROUND AND SIGNIFICANCE

Introduction

Northern Bobwhite Quail (Colinus virginianus; hereafter Bobwhite) and Scaled Quail (Callipepla squamata) have been declining throughout their native range since the 1960s (Sauer et al., 2013; see Figure 1.1). Annual declines of over 4% gave Bobwhite the number one spot in the National Audubon Society’s 2007 “The Top 10 Common Birds in Decline” report, while Scaled Quail experienced declines of over 3% (Sauer et al., 2013). Both quail also declined in the Rolling Plains Ecoregion of West Texas, despite this being a historical stronghold for Bobwhite (Dunham and Kendall, 2017). Because Bobwhite and Scaled Quail are two of the most important and economically valuable game birds for rural communities throughout the Rolling Plains (Johnson et al., 2012), their decline garnered interest among hunters, landowners, and researchers. This interest led to investigations into possible factors of the decline, beginning with Operation Idiopathic Decline (OID) in 2010. This multi- year study spanned over 35 counties and examined approximately 2,600 quail (Rolling Plains Quail Research Foundation, 2020). Ultimately, researchers determined that infection with eyeworms (Oxyspirura petrowi) and caecal worms (Aulonocephalus pennula) were prominent contributing factors to quail decline (Rolling Plains Quail Research Foundation, 2020). Studies have since reported eyeworm prevalence of over 90% in Bobwhite and over 80% in Scaled Quail (Dunham et al., 2014, Dunham and Kendall, 2017). Caecal worm prevalence has been reported at 100% for Bobwhite and 80% for Scaled Quail (Rollins, 2007, Dunham et al., 2017b) and the detrimental effects these helminths are thought to have on individuals have been investigated (e.g. Dunham et al., 2014, Bruno et al., 2015). Thus, further investigations into the role parasites may play in Bobwhite decline are needed.

1 Texas Tech University, Jessica L. Herzog, May 2020

Figure 1.1. Population trends (mean annual BBS counts) for Bobwhite (green) and Scaled Quail (blue) from 1978 to 2019. The long-term means are represented by dotted lines (Texas Parks and Wildlife, 2020d).

Since eyeworms were first documented in Rolling Plains Bobwhite in the early 1960s (Jackson and Green, 1965) and Texas Scaled Quail in 1957 (Wallmo, 1957), they have been reported in 29 counties throughout the ecoregion (Dunham et al., 2017a). In addition to quail hosts, eyeworms can infect over 28 grassland avian species (Pence, 1972) including Northern Mockingbirds (Mimus polyglottos) and Curve-billed Thrashers (Toxistoma curvirostre) of the Rolling Plains (Dunham et al., 2014) and Northern Cardinal (Cardinalis cardinalis) in Louisiana (Pence 1972). Because eyeworms have been documented in both quail and passerines and reportedly cause lacrimal duct inflammation and corneal scarring in bobwhite (Dunham et al., 2014, Bruno et al., 2015), they may negatively impact passerines.

Shared infection of eyeworms between songbirds and quail may stem from the birds’ diets, which incorporate eyeworm insect intermediate hosts such as cockroaches, crickets, and grasshoppers (Almas et al., 2018). Additional parasites reported in Bobwhite, Scaled Quail, and Northern Cardinal like Tetrameres americana require an insect intermediate host for infection as well (Cram, 1933, Durant and

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Knight, 1941, Howard, 1981). Via utilization of insect intermediate hosts by a variety of helminths and consumption of shared insect intermediate hosts by many avian species, a range of shared helminth infection between quail and passerines likely exists.

Although the same species of nematode have been documented separately in quail and passerines, knowledge of the extent of shared parasitism between these birds is lacking. This is due in part because there have been few recent, targeted surveys. For example, few Scaled Quail helminth surveys and only one survey for Curve-billed Thrashers and Northern Mockingbirds have been conducted in the Rolling Plains to use for comparison with previous Bobwhite surveys (Dunham and Kendall, 2014). Furthermore, passerine helminth reports tend to be compiled from incidental bycatch, which may lead to underestimating the potential for passerine helminth infection. Finally, due to similar issues concerning passerine helminth surveys, even greater uncertainty surrounds ecto and haemoparasites of Rolling Plains birds. Hence, full parasite surveys are necessary to further elucidate individual and shared infection.

Understanding shared parasitism is necessary given the lack of previous parasite surveys and the idea that dispersal of infected birds may lead to introduction of parasites to naïve populations (Dunham and Kendall, 2014, Viana et al., 2016). In order to investigate this possibility and further document the extent of parasitism in passerines and quail, I conducted full parasite surveys on Bobwhite, Scaled Quail, Northern Mockingbirds, Curve-billed Thrashers, and Northern Cardinals.

The Rolling Plains Ecoregion

The Rolling Plains Ecoregion of West Texas is roughly 9.7 million hectares of mesquite-shortgrass savannah habitat spanning over 18 Texas counties (Hatch et al., 1990, Texas Parks and Wildlife 2020a, 2020b; see Figure 1.2). Elevation ranges from 244 to 914 meters above sea level (Rollins, 2007). At least one third of the ecoregions’ total area is dominated by land use practices of growing crops such as wheat, as well as grazing cattle (Hatch et al., 1990, Rollins, 2007). In the absence of wildfire, native

3 Texas Tech University, Jessica L. Herzog, May 2020 grasses have been encroached upon by honey mesquite (Prosopis glandulosa) and redberry juniper (Juniperus pinchotii Sudworth) (Parajulee et al., 1997, Texas Parks and Wildlife, 2020a). Summers are long and hot, while winters are mild (Texas Parks and Wildlife 2020b). Average annual temperatures can span -1.1°C in January to 35.6°C in July (Dunham et al., 2017a, Texas Parks and Wildlife, 2020b). Average annual rainfall is between 50.8cm to 71cm per year, reaching its maximum amount in May and September (Texas Parks and Wildlife, 2020b).

Figure 1.2. Ecoregions of Texas. The Rolling Plains is colored red. (Texas Parks and Wildlife, 2020c).

General Ecology of Quail Studied

Bobwhite are medium-sized quail ranging in size from 140-170g, with males being larger than females (Brennan et al., 2014). Although distributed from the East Coast of the United States to the Great Plains, Bobwhite tend to live year-round in habitats of successional vegetation (Stoddard, 1931, Brennan et al., 2014). Bobwhite are social and spend most of their lives in covey groups (Hernandez and Peterson, 2007). Adult forage consists primarily of seeds, but also includes vegetation and

4 Texas Tech University, Jessica L. Herzog, May 2020 insects. Insects are particularly important for breeding females, who need the associated increase in protein for egg laying. Insects comprise up to 20% of breeding Bobwhite females’ diets (Brennan et al., 2014). Insects are also vital to growing chicks, whose diet is up to 80% insects during the first two weeks after hatching (Eubanks and Dimmick, 1974).

In Texas, breeding season commences in mid-April and continues until October if heat and rainfall are favorable. During this time, Bobwhite may engage in multi-clutch polygamy, where the female’s first clutch is incubated by a male and an additional clutch which may be fertilized by a separate male is laid and incubated by her (Hernandez and Peterson, 2007). Despite the ability to maintain multiple nests, overall nest success is low due to depredation, nest abandonment, weather, and human disturbances (Roseberry, 1975, Lehmann, 1984).

Bobwhite have a life span averaging six months, with mortality up to 80% (Brennan et al., 2014). Additionally, Texas Bobwhite populations fluctuating on a boom-and-bust cycle (Lusk et al., 2007) have been continually declining across the state for years (Hernandez and Peterson, 2007). Habitat loss was thought to be a major contributor to declines (Hernandez and Peterson, 2007), while disease and parasitism were easily dismissed as they have been historically in many wildlife studies which considered the co-evolution of parasites with their host to negate harm inflicted upon the host by the parasite (Friend, 2014, Gehman et al., 2019). However, given reports of high prevalence of parasites in Bobwhite, their potential role in the decline deserves further investigation.

Scaled Quail tend to be larger than Bobwhite, with mass averaging 177g for females and 195g for males (Nelson and Martin, 1953). In Texas, their range partially overlaps that of Bobwhite (Rollins, 2000), then extends into Mexico. They are residents throughout their range, preferring desert grassland and shrubland habitats (Dabbert et al., 2009). Like Bobwhite, Scaled Quail form covey groups. Adult diet includes grains and seeds, and arthropod proportions of the diet can be dissimilar to those of Bobwhite (Campbell-Kissock et al., 1985, Dabbert et al., 2009). Nesting 5 Texas Tech University, Jessica L. Herzog, May 2020 begins any time from May to August (Dabbert et al., 2009). Only one brood is raised (Wallmo, 1956), with nesting success reported at 64% (Pleasant et al., 2006). Adult survival is variable and subject to boom-and-bust cycles every 2-3 years (Lusk et al., 2007). Like Bobwhite, Scaled Quail have declined throughout the Rolling Plains (Rollins, 2000, Cantu et al., 2005). Factors influencing their decline are similar to those influencing Bobwhite decline, including disease (Rollins, 2000).

General Ecology of Passerines Studied

The Northern Cardinal is a relatively long-lived passerine (Laskey, 1944) that can be found throughout much of the United States from the East Coast to the Midwest and south into Central America (Halkin and Linville, 1999). It is a common resident throughout Texas, preferring successional habitats and those with shrubs and woody vegetation. Infection with a variety of parasites representing nematode, protozoa, and lice groups have been reported (Halkin and Linville, 1999). Adult diet is mostly vegetable and animal matter, while nestlings are fed nearly exclusively insects (McAtee, 1908, Laskey, 1944). Fledging juveniles and adults switching territories between years account for the species’ migratory movements (Merritt, 1975, Halkin and Linville, 1999). Northern Cardinal populations are stable, and the species is experiencing northward range expansion (Halkin and Linville, 1999).

Northern Mockingbirds are another long-lived passerine found throughout most of the continental United States and Mexico. This species prefers open habitats, either natural areas like chaparral, or urban areas such as parks. Reports of blowfly larvae, protozoa, and helminth infection are not numerous, but available (Farnsworth et al., 2011). Adult diet consists of about 50% fruit and 50% arthropods, which increases to roughly 85% of the diet during breeding season (Beal et al., 1916). Only northern populations tend to be migratory, but individuals in resident populations have been known to disperse from their natal territories to breed elsewhere (Cooke, 1946, Farnsworth et al., 2011) or move from breeding to wintering territories (Laskey, 1935). In contrast to Northern Cardinals, Northern Mockingbird populations as a whole have been declining across their range (Sauer et al., 2013). 6 Texas Tech University, Jessica L. Herzog, May 2020

Curve-billed Thrashers are found throughout New Mexico, Arizona, Texas, and Mexico. They prefer open habitats such as the South Texas brushland and Sonoran Desert. Very little is known about parasites infecting this species, but ticks, nasal mites, and eyeworms have been documented (Eads and Borom, 1975, Spicer, 1987, Dunham and Kendall, 2014). Adult and nestling diet is almost exclusively arthropods and fruits (Fischer, 1981, 1983). Northern populations have been described as partially migratory (Phillips, 1986). Additionally, juveniles have dispersed “considerable distances” to breed in other populations (Russell and Monson, 1998), which has resulted in difficulty distinguishing juvenile dispersal from migration. The species is reported to be declining across its range (Sauer et al. 2013).

Parasite Ecology

Parasitism is an association of two different species where the parasite uses the host as a habitat to obtain resources, especially food (Price, 1977, Anderson and May, 1978, Friend et al., 1999). Although parasites are usually small and inconspicuous (Price, 1977), they are well adapted to a variety of environmental conditions and habitat types, and they likely account for over 50% of the earth’s organisms (Price, 1977). They are grouped into microparasites (e.g. haemoparasites) and macroparasites (e.g. helminths) (Bruno, 2014).

Parasites utilize either direct or indirect lifecycle strategies to infect their definitive host(s) (e.g. birds or mammals) (Friend et al., 1999). Indirect lifecycles are characterized by the presence of an intermediate host in which the parasite will develop to a level of sufficient maturity to be able to infect and survive in the definitive host. Multiple intermediate hosts may be used including arthropods, amphibians, and other mammals (Friend et al., 1999). In contrast, direct lifecycles do not require any sort of intermediate host; parasite eggs are incidentally consumed during foraging (Friend et al., 1999). Finally, parasites may also utilize paratenic hosts. Paratenic hosts feed on intermediate hosts, but the parasite does not develop inside them. Instead, they are a carrier used so that the parasite can still infect the

7 Texas Tech University, Jessica L. Herzog, May 2020 definitive host without the definitive host having to directly consume the intermediate host (Friend et al., 1999).

While many avian parasites do not contribute to disease in their host, they can cause harm in certain cases. Parasites depend on their host for survival yet increase their probability of infecting other hosts by producing rapidly in large numbers (Tompkins et al., 2012, Bruno, 2014). This process requires many resources, which when ultimately taken from the host can serve to decrease its fitness (Tompkins et al., 2012). Furthermore, depending on the host’s nutritional, disease, and current parasite infection status, infections that may otherwise be benign or mild can become pathogenic or lead to death (Friend et al., 1999). Thus, parasites can ultimately have the ability to influence the fluctuations of host population levels (Anderson and May, 1978, Galosi et al., 2019, Gehman et al., 2019) and should continue to be studied in avian species. The following paragraphs are general descriptions of helminths (including specific species reported in birds), ectoparasites, and haemoparasites and how these groups relate to avian hosts.

Helminths

Helminths include acanthocephalans, cestodes, and nematodes (Sepulveda and Kinsella, 2013). Instead of reproducing inside their host, helminths produce eggs that are shed into the external environment via the host’s feces (Anderson and May, 1978). Helminths utilize both direct and indirect lifecycles and various types of intermediate hosts (Friend et al., 1999, Khan et al., 2019). Because the best practice of necropsying recently euthanized hosts to ensure survival of extracted helminths is not always possible in field settings (Sepulveda and Kinsella, 2013), and hosts that have died from infection are often consumed by other wildlife before they can be examined (Friend, 2014), conducting helminth studies on wild host populations can be difficult. However, continued study of avian helminth infections is important. For example, despite helminths being documented in birds since the late 1800s and hundreds of species being found (Rausch, 1983, Bush, 1990, Friend et al., 1999), helminth data for many avian hosts is nonexistent (Rausch, 1983). Continued study will increase 8 Texas Tech University, Jessica L. Herzog, May 2020 understanding of helminths themselves (Rausch, 1983, Bush, 1990) and the role they play in reducing host fitness or causing death from helminthiasis (Anderson and May, 1978, Hudson et al., 2002).

Acanthocephalans, known as thorny headed worms, are usually identified by the presence of a proboscis covered in hooks (Richardson, 2001). These hooks are typically counted in spiral rows (Amin and Dailey, 1998, M. Kinsella, personal communication, 21 Dec 2019). Attachment in the small intestines allows the parasite to obtain nutrients directly from the host, as they do not possess their own digestive system (Friend et al., 1999, Richardson, 2001). Acanthocephalans rely on at least one intermediate host, including insects, to facilitate infection (Friend et al., 1999). Though they are the least common avian parasite (Bush, 1990), infection has been reported in birds worldwide, including passerines (Friend et al., 1999). A specific example of infection is that of Mediorhynchus papillosus. Of the eight species in the Mediorhynchus genus in North America, M. papillosus is the most common species infecting birds (Amin and Dailey, 1998) and has been reported in Bobwhite (Moore and Simberloff, 1990, Bruno et al., 2019). Generally, acanthocephalans are not pathogenic, but infection has been thought to cause emaciation and death in Common Eiders (Somateria mollissima) (Friend et al., 1999).

Cestodes, or tapeworms, are flat, segmented worms with an obvious scolex (Friend et al., 1999). The scolex is the primary feature used in species identification (M. Kinsella, personal communication, 21 Dec 2019). Segments are called proglottids and contain larval embryos that are shed with the feces and consequently consumed by beetle intermediate hosts (Reid et al., 1938). Cestode larvae (e.g. Mesocestoides spp. tetrathyridium) require a second vertebrate intermediate host, such as a mammal, to develop to the point of infecting a bird. For example, various Mesocestoides representatives have been found in ocelots, coyotes, and badgers in Texas (Pence and Dowler, 1979, Pence and Windberg, 1984). Like acanthocephalans, cestodes do not possess their own digestive tract and thus embed themselves in the host’s intestinal epithelium, using their suckers to obtain nutrients (Friend et al., 1999). Cestodes have

9 Texas Tech University, Jessica L. Herzog, May 2020 been reported in songbirds (Lincicome, 1939, Bush, 1990) and Raillietina spp. specifically has been reported in Bobwhite (Davidson et al., 1980, Bruno et al., 2019). The Raillietina genus is characterized by species typically having two rows of hooks on their rostellum and an alternating pattern of genital pores (Khalil et al., 1994). However, cestodes can easily break, especially if the host is frozen before retrieval, making identification difficult (Sepulveda and Kinsella, 2003). While cestodes do not typically kill their hosts, they can decrease the host’s fitness under high infection intensities by creating near intestinal blockages (Friend et al., 1999).

Eyeworm (Oxyspirura petrowi) (Skrjabin, 1929), are nematodes in the family Thelaziidae identifiable by their buccal capsule and males’ tail curling into a spiral (Anderson et al., 2009). They can withstand many climates (Al-Moussawi and Mohammad, 2013) and enter diapause to survive poor conditions both in and outside the host (Sommerville and Davey, 2002). Infection requires an insect intermediate host—potentially crickets, grasshoppers, and beetles (Kistler et al., 2016, Almas et al., 2018). Eyeworms may infect their hosts in a similar mechanism to that of their tropical counterpart, O. mansoni (Schwabe, 1951). After the ingested insect intermediate host reaches the crop, O. mansoni larvae migrate from the insect’s tissues up the esophagus, entering the orbital cavity via the naso-lacrimal ducts in as little as five minutes from the moment they were ingested (Schwabe, 1951).

At least 28 avian species across orders have been documented as hosts of O. petrowi (Pence, 1972). In the Rolling Plains of Texas specifically, infection has been documented in Scaled Quail (Dunham and Kendall, 2017), a Curve-billed Thrasher, and Northern Mockingbirds (Dunham and Kendall, 2014). Eyeworms have also been reported in Bobwhite of the ecoregion at a prevalence of over 90% (Dunham et al., 2014). Nematodes were found beneath the nictitating membrane and in the lacrimal duct and other tissues surrounding the eyes, as they have been in other studies (Jackson, 1969, Dunham et al., 2014, 2017a). Studies have reported corneal scarring and inflammation and lesions on ocular tissues (Bruno et al., 2015, Dunham et al, 2017a). Moreover, there have been reports of infected Bobwhite flying unpredictably

10 Texas Tech University, Jessica L. Herzog, May 2020 and crashing into fences and buildings (Jackson and Galley, 1963, Jackson, 1969). Finally, eyeworms are reportedly approximately 96% genetically similar to the human eyeworm, Loa loa (Kalyanasundaram et al., 2018), which has been known to reduce vision in humans (Jaksche et al., 2004). Given these findings, it is reasonable to suspect that eyeworm infection can negatively impact birds’ vision.

Caecal worms (Aulonocephalus pennula) (Chandler, 1935) are Subulurid nematodes identifiable by the presence of 6 trough-like grooves coming from the mouth and males having two proportionate spicules (Chandler, 1935). Like eyeworms, caecal worms also enter diapause to withstand unfavorable conditions (Sommerville and Davey, 2002) and utilize insect intermediate hosts (Henry et al., 2018). While some caecal worms have been found in a host’s intestines (Dunham et al., 2017b), the caecum is the preferred infection site (Chandler, 1935). Most birds have caeca, but those in passerines, for example, are small or vestigial and ultimately unsuitable for infection in comparison to the large, long caeca of gallinaceous birds (Clench and Mathias, 1995). Thus, gallinaceous birds like Bobwhite, are the typical hosts.

While little is known about the lifecycle of caecal worms, (Dunham et al., 2017b), their pathogenicity has been considered. Caecal worm infection may decrease absorption of vitamin A, a nutrient important during reproduction (Lehmann, 1953). The host’s digesta may be consumed by caecal worms and they may also disturb the digesta (Dunham et al., 2017b), thereby potentially impeding the digestive capabilities of the host’s (Lehmann, 1984). Infection levels of more than 150 worms per host can cause near blockage and pathological changes of the caecum (Rollins, 1980). Furthermore, prevalence of caecal worms in Bobwhite of up to 80% (Dunham et al., 2014) and an average of more than 200 worms in single individuals, along with poor body condition and minimal digesta in the caecum have been reported (Lehmann, 1984, Dunham et al., 2017b). Heavily infected individuals may die out of the population (Dunham et al., 2017b, Henry et al., 2017). In consideration with other caecal worms, A. pennula is reportedly up to 92% related to Ascarididae and Anisakidae nematodes whose species representatives can deprive the host of nutrients,

11 Texas Tech University, Jessica L. Herzog, May 2020 thereby causing weight loss (Kalyanasundaram et al., 2017). Also, the caecal worm Trichostrongylus tenuis has been implicated in regulating populations of red grouse (Lagopus lagopus scoticus) (Hudson et al., 1998) and decreased breeding success may be correlated with increasing infection levels (Potts et al., 1984). The aforementioned findings suggests harm to hosts of A. pennula is plausible.

Tetrameres pattersoni (Cram, 1933) are spirurid nematodes of the family Tetrameridae (Mollhagen, 1976). Obvious sexual dimorphism between filiform males and round females distinguishes Tetrameres spp. in general from other helminths, while the presence of two rows of somatic spines on the anterior end of males and the absence of a right spicule are characteristics specific to T. pattersoni (Mollhagen, 1976). Intermediate hosts include grasshoppers and beetles but these have only been determined through experimental studies. Infection begins after larvae inside the intermediate host encyst in the muscle of the definitive host, mature into third stage larvae, and then encyst in the proventriculus (Mollhagen, 1976). While Tetrameres spp. have been reported in Northern Cardinals (Quentin and Barre, 1976), gallinaceous birds are the specific host of T. pattersoni (Mollhagen, 1976). In Bobwhite, prevalence has been documented at over 30% (Davidson et al., 1980, Bruno et al., 2019). With the potential to reach a high prevalence of infection and the females feeding on hosts’ blood (Mollhagen, 1976), T. pattersoni may harm hosts. However, the realistic potential for the parasite to cause harm is unknown.

Subulura brumpti (Lopez-Neyra, 1922) is a small, heteroxenous species of nematode in the family Heterakidae (Alicata, 1939, Cuckler and Alicata, 1944). Identifying characteristics include 6 lips encircling the mouth and dorsal curvature on the anterior end (Cuckler and Alicata, 1944). Infection in the caeca of gallinaceous primary hosts occurs after individuals consume third stage larvae encysted in grasshoppers or beetles (Cuckler and Alicata, 1944, Nagarajan et al., 2012). S. brumpti have also been found free floating in the crop (Bruno, 2014). Though generally nonpathogenic (Ruff, 1984), Nagarajan et al. (2012) reported ruffled feathers, bloody

12 Texas Tech University, Jessica L. Herzog, May 2020 feces, and reduced egg production during an outbreak in Japanese Quail (Coturnix japonica).

Ectoparasites

Ectoparasites are arthropod parasites that maintain a close association with their vertebrate hosts for the entirety of their lifecycle (Nelson et al., 1995). Negative consequences of infection range in severity from reductions in fitness and lifespan to death (Brown et al., 1995, Friend et al., 1999). Both nestling and adult birds can be impacted, with anemia, paralysis, and myiasis, among other conditions, being reported (Friend et al., 1999). Groups reported to parasitize birds include flies, ticks, mites, and lice (Friend et al., 1999), which will be discussed further.

Chewing lice are insects of the order Phthiraptera (Price et al., 2003). They are generally dorsoventrally flattened and small, sometimes undetectable by the naked eye (Price et al., 2003, Pistone, 2016). Of the Phthiraptera, sucking lice parasitize mammals and chewing lice ubiquitously parasitize birds (Price et al., 2003). Chewing lice have been identified in at least 157 avian families (Rozsa,1997, Price et al., 2003), with an average of 2 species of chewing lice found on each avian taxon. Each of the approximately 4,500 species that has been described (Price et al., 2003) complete the entirety of their lifecycle on their host (Rozsa,1997, Catanach and Johnson, 2015). Lice are mainly transmitted by direct contact of one host with another (Bartlow et al., 2016).

Species identification is accomplished by microscopically examining chemically cleared and slide-mounted specimens, taking note of characteristics such as head to body size ratio, location of spiracles and spines, and complexity of genitalia (Price et al., 2003). Identification can be hindered by having to examine adults of both sexes and a lack of identified specimens in museums to use for reference (Price et al., 2003). However, Goniodes ortygis, Lipeurus caponis, and Oxylipeurus clavatus, among others have been documented in Bobwhite (Price et al., 2003). Furthermore, species from the Menacanthus and Brueelia genera have been documented in

13 Texas Tech University, Jessica L. Herzog, May 2020 passerine species examined for this thesis project. Species include M. eurysternus and B. brunneinucha in Northern Mockingbirds (Price et al., 2003) and M. eurysternus in Northern Cardinals (Pistone, 2016). B. dorsale and B. pallidula have been reported in Curve-billed Thrashers (Price et al., 2003). Concerning pathogenicity, lice can consume and damage the skin and feathers of their hosts (Friend et al., 1999). They can also consume the host’s blood (Waterhouse, 1953). However, overall pathogenicity of lice is low (Clayton and Tompkins, 1995).

Haemoparasites

Haemoparasites including haemosporidia of the Plasmodium and Haemoproteus genera commonly infect birds (Friend et al., 1999, Valkiunas, 2005). As a group, haemosporidians have been reported in over 10,000 avian species (Daszak et al., 2000) with an even distribution across the United States tied to that of their Dipteran intermediate hosts (Friend et al., 1999, Astudillo et al., 2013). Infection starts with sporozites in the saliva of the insects being transferred to the avian host through the location of the bite, usually exposed flesh under the feathers or on the legs and face. The sporozites develop quickly through their generational stages into merozoites (Friend et al., 1999). Merozoites enter the erythrocytes to become infectious gametocytes that proliferate in their hosts (Friend et al., 1999, Hudson et al., 2002). Infection continues via other blood-sucking insect intermediate hosts ingesting the gametocytes when they bite the bird (Friend et al., 1999). If appropriate insects intermediate hosts for the species of haemosporidia are not available, infection will not spread between infected and uninfected primary hosts (Friend et al., 1999). However, coinfection of Plasmodium spp. and Haemoproteus spp. can occur in infected individuals (Clark et al., 2009). Haemoparasites are best detected from stained blood smears examined under a microscope with oil immersion to ensure adequate detection and species identification (Clark et al., 2009).

Plasmodium spp., also known as malaria, encompass hundreds of species that infect the greatest number of avian species (Marzal et al., 2015). They are generally characterized by elongated “U”, or “V” shaped gametocytes with an amphophilic 14 Texas Tech University, Jessica L. Herzog, May 2020 nucleus and the presence of brownish to black granules in the cytoplasm. Round schizonts are present in the cytoplasm and do not usually interfere with the normal positioning of the nucleus (Clark et al., 2009). Specific examples of infection are P. elongatum in Bobwhite (Wetmore, 1941) and P. relictum in many passerines, including Northern Mockingbird (Soares et al., 2017). P. relictum in particular is one of the most pathogenic malaria, having been linked to die-offs, population declines and extinctions (Valkiunas, 2005, Marzal et al., 2015).

Haemoproteus spp. have been reported in approximately 67% of avian hosts examined, making them the most common haemosporidia (Atkinson and Van Ripper, 1991, Friend et al., 1999). In contrast to Plasmodium spp., only Haemoproteus gametocytes can be observed in avian erythrocytes (Clark et al., 2009). Gametocytes are characterized by their shape (Bennett and Pierce, 1988), which is classically an elongated halter that curves around and sometimes distorts the nucleus (Clark et al., 2009). Distinguishing Haemoproteus spp. from Plasmodium spp. can be difficult due to similarities in appearance (Friend et al., 1999, M. Borst, personal communication, 29 Oct 2019). Northern Mockingbirds and Northern Cardinals, among other passerines, are documented hosts of Haemoproteus spp. (Astudillo et al., 2013). A specific example of infection in Bobwhite is H. lophortyx (Cardona et al., 2002). Haemoproteus spp. have been linked to increased nestling deaths and decreased fledging success for passerines (Merino et al., 2000), while H. lophortyx has been reported to cause lethargy, prostration, anemia, and death in quail (O’Roke, 1930), especially in juvenile birds (Cardona et al., 2002).

Finally, microfilaria are filarial nematodes in the family Onchocercidae (Anderson, 2000). The subfamilies Lemdaninae and Splendidofilariinae infect birds, with the latter being reported in passerines (Anderson, 2000). Microfilaria species vary in length but are generally serpentine with a basophilic color (Clark et al., 2009). Like haemosporidia, they utilize hematophagous insect intermediate hosts. Eggs develop inside the insect after being obtained from eggs released into the host’s blood by the adult nematode (Anderson, 2000). Microfilaria are easier to spot than Plasmodium

15 Texas Tech University, Jessica L. Herzog, May 2020 spp. and Haemoproteus spp. because they are much larger than erythrocytes (Clark et al., 2009). However, examination of the entire smear is important due to their low abundance in blood (Clark et al., 2009). Infection with microfilaria may decrease host immunity, thus making them susceptible to coinfection with other species and further reducing host fitness (Astudillo et al., 2013, Clark et al., 2016).

Research Goals and Objectives

Despite high prevalence of eyeworms and caecal worms being documented in rapidly declining populations of Rolling Plains Bobwhite, the influence of parasites on Bobwhite decline is still largely unclear (Commons et al., 2019). Furthermore, certain passerines are suitable hosts for eyeworms, but passerine helminth surveys for the ecoregion are nearly nonexistent. These factors and a general lack of an understanding of the impact of parasitic infection on wild avian populations (Bush, 1990, Lutz et al., 2017) highlight the need to conduct more parasite surveys. Parasite retrieval and processing methods have been established (e.g. Clayton and Drown, 2001, Sepulveda and Kinsella, 2013), while investigating wildlife disease sparks increasing interest for researchers (Friend, 2014). Thus, advancing the science is a matter of actively working to capture and sample specific hosts. Conducting parasite research in the form of targeted and complete surveys will help increase understanding of the role parasites play in individual fitness and population regulation (Friesen et al., 2017, Gehman et al., 2019, Khan et al., 2019). Consequently, the research goals of this study were to 1) contribute additional data to the few existing reports of parasitism in specific Rolling Plains passerines via a targeted, full parasite survey and 2) document shared parasitic infection between specific quail and passerine species in the Rolling Plains Ecoregion. These goals are outlined below:

16 Texas Tech University, Jessica L. Herzog, May 2020

Chapter 2: Helminths Common Between Northern Bobwhite Quail (Colinus virginianus) and Select Passerines in the Rolling Plains Ecoregion, TX

1) Add to existing survey data concerning documented helminth infection in study species including Bobwhite, Scaled Quail, Northern Cardinals, Northern Mockingbirds, and Curve-billed Thrashers

2) Document shared infection between the aforementioned study species

Chapter 3: Ectoparasites and Haemoparasites of Northern Bobwhite Quail (Colinus virginianus) and select Passerines in the Rolling Plains Ecoregion, TX

1) Add to existing survey data concerning documented ectoparasite and haemoparasite infection in study species including Bobwhite, Scaled Quail, Northern Cardinals, Northern Mockingbirds, and Curve-billed Thrashers

2) Document shared infection between the aforementioned study species

Conducting full parasite surveys on Bobwhite, Scaled Quail, Northern Cardinals, Northern Mockingbirds, and Curve-billed Thrashers will contribute data for future researchers hoping to study parasites in these species in the Rolling Plains Ecoregion. On a broader scale, researchers can use such studies to begin to better understand the implications of parasitic infection for topics including host fitness, introduction of parasites to naïve populations, and population decline.

Thesis Outline and Chapter Introductions

This thesis consists of 4 chapters. Chapter 1 outlines potential implications of parasitic infection in Rolling Plains Bobwhite in relation to their decline and how eyeworm infection in quail and passerines may foreshadow shared infection with other parasites. Chapter 2 discusses helminth infection in select quail and passerine species of the Rolling Plains, including shared infection. Chapter 3 completes the full parasite survey of the study species by discussing ectoparasites and haemoparasites found in

17 Texas Tech University, Jessica L. Herzog, May 2020 individual specimens. Chapter 4 summarizes and concludes each chapter, as well as presents potential directions for future researchers.

18 Texas Tech University, Jessica L. Herzog, May 2020

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79. M. Kinsella, personal communication, 21 Dec 2019.

80. Marzal, A., García-Longoria, L., Callirgos, J.M.C., Sehgal, R.N., 2015. Invasive avian malaria as an emerging parasitic disease in native birds of Peru. Biol. Invasions. 17, 39-45.

81. McAtee, W.L., 1908. Food habits of the grosbeaks. U.S. Dept. Agric., Bur. Biol. Surv., Bull. 32.

82. Merino, S., Moreno, J., José Sanz, J., Arriero, E., 2000. Are avian blood parasites pathogenic in the wild? A medication experiment in blue tits (Parus caeruleus). Proc. R. Soc. Lond. B: Biol. Sci., 267, 2507-2510.

83. Merritt, R.E., 1975. The spatial relations within a selected population of the cardinal (Cardinalis cardinalis). Doctoral Dissertation, Univ. of Tennessee, Knoxville, USA.

84. Mollhagen, T.R., 1976. A study of the systematics and hosts of the parasitic nematode genus Tetrameres (Habronematoidea: Tetrameridae). Doctoral dissertation, Texas Tech University, USA.

85. Moore, J., Simberloff, D., 1990. Gastrointestinal helminth communities of bobwhite quail. Ecology. 71, 344-359.

86. Nagarajan, K., Thyagarajan, D., Raman, M., 2012. Subulura brumpti infection-an outbreak in Japanese quails (Coturnix coturnix japonica). Vet. Res. Forum. 3, 67.

87. Nelson, A.L., Martin, A.C., 1953. Game bird weights. J. Wildl. Manag. 17, 36-41.

88. Nelson, W.A., Keirans, J.E., Bell, J.F., Clifford, C.M., 1975. Host-ectoparasite relationships. J. Med. Entomol. 12, 143-166.

89. O'Roke, E.C., 1930. The morphology, transmission and life-history of Haemoproteus lophortyx O'Roke, a blood parasite of the California valley quail. Univ. Calif. Publ. Zool. 36, 1-50.

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90. Owen, J.C., 2011. Collecting, processing, and storing avian blood: a review. J. Field Ornithol. 82, 339-354.

91. Parajulee, M.N., Slosser, J.E., Montandon, R., Dowhower, S.L., Pinchak, W.E., 1997. Rangeland grasshoppers (Orthoptera: Acrididae) associated with mesquite and juniper habitats in the Texas Rolling Plains. Environ. Entomol. 26, 528-536.

92. Pence, D.B., Dowler, R.C., 1979. Helminth parasitism in the badger, Taxidea taxus (Schreber, 1778), from the western Great Plains. J. Helminthol. Soc. Wash. 46, 245- 253.

93. Pence, D.B., Windberg L.A., 1984. Population dynamics across selected habitat variables of the helminth community in coyotes, Canis latrans, from South Texas. J Parasitol. 70, 735-746.

94. Pence, D.B., 1972. The genus Oxyspirura (Nematoda: Thelaziidae) from birds in Louisiana. Proceedings of the Helminthology Society of Washington 39, 23- 28.

95. Phillips, A.R., 1986. The known birds of North and Middle America, Part I. Hirundinidae to Mimidae; Certhiidae. Denver Museum of Natural History, Denver, CO, USA.

96. Pleasant, G.D., Dabbert, C.B., Mitchell, R.B., 2006. Nesting ecology and survival of scaled quail in the Southern High Plains of Texas. J. Wildl. Manag. 70, 632- 640.

97. Potts, G.R., Tapper, S.C., Hudson, P.J., 1984. Population fluctuations in red grouse: analysis of bag records and a simulation model. J. Anim. Ecol. 53, 21- 36.

98. Price, P.W., 1977. General concepts on the evolutionary biology of parasites. Evolution. 1, 405-420.

99. Quentin, J.C., Barre, N., 1976. Description et cycle biologique de Tetrameres (Tetrameres) cardinalis. Ann. Parasitol. Hum. Comp. 51, 65-81.

100. Rozsa, L., 1997. Patterns in the abundance of avian lice (Phthiraptera: Amblycera, Ischnocera). J. Avian Biol. 28, 249-254.

101. Rausch, R.L., 1983. The biology of avian parasites: helminths. J. Avian Biol. 7, 367-442.

102. Reid, W.M., Ackert, J.E., Case, A.A., 1938. Studies on the life history and biology of the fowl tapeworm Raillietina cesticillus (Molin). Trans. Am. Microsc. Soc., 57, 65-76. 26 Texas Tech University, Jessica L. Herzog, May 2020

103. Richardson, D.J., 2001. Acanthocephala. e LS.

104. Rolling Plains Quail Research Foundation, 2020. Operation Idiopathic Decline. https://www.quailresearch.org/research-projects/. Accessed January 2020.

105. Rollins, D., 1980. Comparative ecology of bobwhite and scaled quail in mesquite grassland habitats. M.S. Thesis. Oklahoma State University.

106. Rollins, D., 2000. Status, ecology, and management of scaled quail in West Texas. Proceedings of the National Quail Symposium 4, 165-172.

107. Rollins, D., 2007. Quails on the rolling plains. In: Brennan L. (Ed.), Texas quails: ecology and management. Texas A&M University Press, College Station, USA, pp. 117-141.

108. Ruff, M.D., 1984. Nematodes and acanthocephalans. In diseases of poultry (Hofstad, S., Barnes, H.J., Calnek, B.W., Reid, W.M., Yoder, H.W. Eds.) 8th Ed., Iowa State University Press, Iowa, USA. pp. 614-648.

109. Russell, S.M., Monson, G., 1998. The birds of Sonora. University of Arizona Press, Tucson, AZ, USA.

110. Sauer, J.R., Hines, J.E., Fallon, J.E., Pardieck, K.L., Ziolkowski, Jr., D.J., Link, W.A., 2013. The North American breeding bird survey, results, and analysis 1966-2013. USGS Patuxent Wildlife Research Center, Laurel, MD, USA.

111. Sepulveda, M.S., Kinsella, J.M., 2013. Helminth collection and identification from wildlife. J. Vis. Exp. 82, e51000. doi:10.3791/51000.

112. Skirnisson, K., Jouet, D., Ferté, H., Nielsen, Ó.K., 2016. Occurrence of Mesocestoides canislagopodis (Rudolphi, 1810) (Krabbe, 1865) in mammals and birds in Iceland and its molecular discrimination within the Mesocestoides species complex. Parasitol. Res. 115, 2597-2607.

113. Soares, L., Marra, P., Gray, L., Ricklefs, R.E., 2017. The malaria parasite Plasmodium relictum in the endemic avifauna of eastern Cuba. Conserv. Biol. 31, 1477-1482.

114. Spicer, G.S., 1987. Prevalence and host-parasite list of some nasal mites from birds (Acarina: Rhinonyssidae, Speleognathidae). J. Parasitol. 73, 259-264.

115. Texas Parks and Wildlife, 2020a. Rolling Plains Ecological Region. https://tpwd.texas.gov/landwater/land/habitats/cross_timbers/ecoregions/rollin g_plains.phtml. Accessed January 2020.

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116. Texas Parks and Wildlife, 2020b Texas Ecoregions-Rolling Plains. https://tpwd.texas.gov/education/hunter-education/online-course/wildlife- conservation/texas-ecoregions. Accessed January 2020.

117. Texas Parks and Wildlife, 2020c. Texas Ecoregions. https://tpwd.texas.gov/education/hunter-education/online-course/wildlife- conservation/texas-ecoregions. Accessed January 2020.

118. Texas Parks and Wildlife, 2020d. TPWD Quail Roadside Survey Historical Data 1978 to 2019 https://tpwd.texas.gov/huntwild/hunt/planning/quail_forecast/forecast/. Accessed February 2020.

119. Tompkins, D.M., Dobson, A.P., Arneberg, P., De Leo, G.A., Krecek, R.C., Manfredi, M. T., Lanfranchi, P., Zaffaroni, E. 2002. Parasites and host population dynamics. Ecol. Wildl. Dis. pp. 45-62.

120. Valkiunas, G., 2005. Avian malaria parasites and other haemosporidia. CRC Press, Boca Raton, FL, USA.

121. Viana, D.S., Santamaría, L., Figuerola, J., 2016. Migratory birds as global dispersal vectors. Trends Ecol. Evol. 31, 763-775.

122. Wallmo, O.C., 1957. Ecology of scaled quail in West Texas. Doctoral Dissertation, Texas A&M University, USA.

123. Wallmo, O.C., 1956. Ecology of scaled quail in west Texas. Tex. Game and Fish Comm. 134 pp.

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CHAPTER II

HELMINTHS COMMON BETWEEN NORTHERN BOBWHITE QUAIL (COLINUS VIRGINIANUS) AND SELECT PASSERINES IN THE ROLLING PLAINS ECOREGION, TX Abstract

Steady declines of Northern Bobwhite Quail (Colinus virginianus) over the last few decades in the Rolling Plains Ecoregion of Texas are potentially influenced by prevalent eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) infection. Eyeworm infection has also been documented in Scaled Quail (Callipepla squamata), Northern Mockingbirds (Mimus polyglottos) and Curve-billed Thrashers (Toxostoma curvirostre) in the ecoregion, suggesting that more parasites may be shared between quail and passerines. However, due to a lack of helminth surveys, the extent of shared parasitism and the potential consequences for hosts other than quail have not been investigated. Thus, helminth surveys were conducted on Bobwhite, Scaled Quail, Northern Mockingbirds, Curve-billed Thrashers, and Northern Cardinals (Cardinalis cardinalis) to contribute data to existing parasitological gaps for birds in the Rolling Plains Ecoregion. Birds were trapped at three study sites across the Rolling Plains from March to October 2019. Complete necropsies were conducted on 54 individuals (36 quail and 18 passerines) and extracted helminths were microscopically identified. Nematode, cestode, and acanthocephalan helminths representing 8 genera and 6 species were found. Specifically, A. pennula and O. petrowi had the highest prevalence and O. petrowi was shared across each of the study species. Comparisons of avian orders revealed that O. petrowi and A. pennula infections were not significantly different between each sex and age. Coinfection was common and female birds harbored a significantly higher average infection of helminths aside from eyeworm, caecal worms, and cestodes than males. Future studies could incorporate investigations into temporal parameters of helminth infection, techniques to non- invasively assess infection, and molecular identification of recovered helminths.

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Introduction

The Northern Bobwhite Quail (Colinus virginianus; hereafter Bobwhite) has been experiencing an approximate 4% decline across its range every year for decades (Sauer et al., 2013). In the Rolling Plains Ecoregion of Texas, boom-and-bust cycles and stable habitat conditions would normally help maintain Bobwhite populations (Lusk et al., 2007, Rollins 2000; 2007). However, the frequency of booms has dropped over the years and Bobwhite are declining across the ecoregion overall (Dunham et al., 2017b). Scaled Quail (Callipepla squamata) in the ecoregion have been declining as well (Rollins, 2000), at an annual rate of about 3% (Sauer et al., 2013). Due to the significance and economic importance of quail hunting, especially for rural communities (Johnson et al., 2012), and the fact that the Rolling Plains was historically considered a stronghold of Bobwhite populations (Rollins, 2007; Dunham and Kendall, 2017), the declines have concerned hunters, landowners, and researchers alike. Hence, investigations into quail decline began.

Operation Idiopathic Decline (OID) was one of the first studies to investigate potential causes of quail decline in the Rolling Plains. Beginning in 2010, this collaborative three-year study examined the potential influences of habitat conditions, environmental contaminants, and disease (Rolling Plains Quail Research Foundation, 2020). Approximately 2,600 Bobwhite and Scaled Quail collected across the Rolling Plains of Texas and Oklahoma were examined (Rolling Plains Quail Research Foundation, 2020). Findings of 90% caecal worm prevalence and 50% eyeworm prevalence rendered these helminths deserving of further study (Rolling Plains Quail Research Foundation, 2020). Since OID, a number of other studies focusing on caecal worm and eyeworm infection in Bobwhite have been conducted in the Rolling Plains (e.g. Dunham et al., 2014, Bruno et al., 2015, Dunham and Kendall, 2017, Henry et al., 2017). Of these investigations, caecal worm prevalence in Bobwhite was reported at 98% (Dunham et al., 2017b), while eyeworm prevalence was documented at 95% and 73% in Bobwhite and Scaled Quail, respectively (Dunham et al., 2017a).

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Caecal worm nematodes are usually found free floating amidst digesta in the caeca of avian hosts (Chandler, 1935). Because gallinaceous birds have larger caeca than other birds (Clench and Mathias, 1995), they are the preferred host group. Caecal worms are thought to utilize grasshoppers and beetles as intermediate hosts to facilitate infection (Henry et al., 2018). Once inside the host, caecal worms may consume their host’s digesta (Dunham et al., 2017b) and disrupt their ability to digest (Lehmann, 1984). The most severe of these disruptions are partial caecum blockages at infection levels surpassing 150 individuals in a single host (Rollins, 1980). Given A. pennula’s close relationship to Ascarididae and Anisakidae nematodes, which have reportedly reduced the nutrient absorption capabilities and body masses of their hosts (Kalyanasundaram et al., 2017), interference with host digesta and various aspects of digestion itself are plausible. Caecal worms may also have the ability to influence host population dynamics given severe infection levels (Henry et al., 2017), as the caecal worm Trichostrongylus tenuis has done in populations of red grouse (Lagopus lagopus scoticus) (Hudson et al., 1998).

Eyeworms, like caecal worms, are heteroxenous nematodes that use grasshoppers and beetles (Kistler et al., 2016, Almas et al. 2018) to facilitate infection in their avian hosts, which are also their definitive hosts (Pence, 1972). They can be found underneath the nictitating membrane and in the lacrimal ducts (Jackson, 1969, Dunham et al, 2014), thereby being positioned to potentially cause corneal scarring and inflammation and lesions of ocular tissues (Bruno et al. 2015, Dunham et al., 2017a). Infection was first documented in Rolling Plains Scaled Quail in 1957 (Wallmo, 1957) and Bobwhite in the 1960s (Jackson and Green, 1965). Later, passerine host representatives for the ecoregion were documented when Dunham and Kendall (2014) found infection in Northern Mockingbirds (Mimus polyglottos), and a Curve-billed Thrasher (Toxostoma curvirostre). Shared infection is thought to be a result of these birds’ diets incorporating eyeworm intermediate hosts (Beal et al., 1916, Fischer, 1981).

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Considering shared infection of eyeworms and that all helminth groups documented across avian taxa utilize insect intermediate hosts (Rausch, 1983, Bush, 1990, Anderson, 2000), more parasites may be common between quail and passerines in the Rolling Plains. Species found could represent any of the cestode, nematode, acanthocephalan, or trematode groups (Sepulveda and Kinsella, 2013). The least prevalent of these in birds are the acanthocephalans (thorny headed worms) (Bush, 1990), though they have been found in numerous passerine species (Amin and Dailey, 1998) and Bobwhite (e.g. Mediorhynchus papillosus: Bruno et al., 2019). In contrast, nematodes are the most prevalent group, (Anderson et al., 2000) with Tetrameres spp., being reported in Northern Cardinal (Quentin and Barre, 1976) and Bobwhite (e.g. T. pattersoni: Bruno et al., 2019). Like the ocular inflammation potentially caused by eyeworm, these helminths may harm their hosts. Acanthocephalan infection could emaciate and kill Common Eiders (Somateria mollissima) (Friend et al., 1999), while female Tetrameres spp. have been known to feed on the blood of the host (Mollhagen, 1976).

Despite documentation of individual parasites in specific quail and passerine species, the extent of parasitism between these birds in the Rolling Plains has not been addressed. This may be in part due to helminths, especially nematodes being taxonomically challenging to identify (De Ley et al., 2005). Moreover, a lack of surveys may stem from parasites not historically being considered to negatively impact their hosts and consequently being overlooked (Gehman et al., 2019). In the Rolling Plains, only one helminth survey for passerines has been conducted (Dunham and Kendall, 2014), while little is known about parasitism in Scaled Quail there (Bedford, 2015). Such factors complicate comparing the extent of helminth infection in passerines and Scaled Quail with that reported in recent Bobwhite surveys (e.g. Bruno et al., 2019). Furthermore, many surveys utilize helminth data from hosts obtained as incidental bycatch, thereby underestimating the range of potential hosts available.

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Recent literature is ever shifting toward the idea that parasites may impact their hosts and populations in ways and to extents that were not previously considered. Some impacts are still obscure such as the idea of infracommunities inside hosts, potentially exacerbating the effects each parasite species would have on the host individually (Bush, 1990). Other impacts are pronounced, as the caecal worms Trichostrongylus tenuis decreasing breeding success of Red Grouse (Lagopus lagopus scoticus) (Potts et al., 1984), thereby regulating its populations (Hudson et al., 1998). If the lack of targeted, complete helminth surveys for passerines in the Rolling Plains persists, researchers will not be able to better understand host-helminth interactions and their associated consequences like the ones presented above.

In order to address knowledge gaps concerning shared parasitism in quail and passerines of the Rolling Plains, complete helminth surveys were conducted on Bobwhite, Scaled Quail, Northern Cardinals, Northern Mockingbirds, and Curve- billed Thrashers. Birds were trapped at three study sites representing the upper, central, and lower Rolling Plains from March to October 2019. Donated specimens were also received. Individuals were completely necropsied and recovered helminths were microscopically identified. The objectives of this study were to 1) add to existing survey data concerning documented helminth infection in study species and 2) document shared infection between study species.

Materials and Methods

Ethics Statement

This experiment was approved by Texas Tech University Animal Care and Use Committee under protocols 16071-08 and 19015-02. All birds were trapped and handled according to Texas Parks and Wildlife permit SRP-0715-095, United States Fish and Wildlife Permits MB88764B-0 and MB88764B-1, and Federal Bird Banding Permit 22590.

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Study Area and Sample Collection

Quail and passerines were captured from March to October 2019 at private ranches representing the Rolling Plains Ecoregion. This mesquite-shortgrass savannah is dominated by honey mesquite (Prosopis glandulosa) and juniper (Juniperus pinchotti) (Rollins 2007). Ranches are spread across the Rolling Plains as follows: northern (Cottle County: Matador Wildlife Management Area), central (Garza County: Cross H Ranch), and southern (Mitchell County: Renderbrook Spade Ranch and Howard Morrison Ranch) Rolling Plains (see Figure 2.1). Birds were trapped using baited wire walk in double funnel traps and methods described in Dunham et al., (2017a). Donated birds were also received (see Figure 2.1).

Figure 2.1. County Locations in the Rolling Plains Ecoregion of Texas used to obtain avian hosts. Birds were trapped at counties A (Cottle County), B (Garza County), and C (Mitchell County). Bobwhite were donated from D (Stonewall County).

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Helminth Extraction

Immediately after euthanasia, eyes were examined for eyeworms by completely removing the eyes from the host and teasing apart the associated tissues in a petri dish (Dunham et al., 2017a). Caeca were examined for caecal worms following a modified method of Dunham et al. (2017a) where each caecum was slit open longitudinally and the contents were washed with deionized water through a 100-mesh sieve. If other organs including the remainder of the intestines and the right pectoralis major and minor muscles could not be immediately examined, they were stored at room temperature in a 15 mL or 50 mL tube filled with 70% ethanol (England et al., 2017). All stored organs were examined for helminths within two weeks of the initial storage date. Donated birds were thawed once and examined immediately thereafter to lessen the damage to helminths (Bruno, 2014). Each organ (fresh, stored, or thawed) was examined following methods similar to that of Bruno (2014), where they were opened if needed and teased apart in a petri dish filled with water. Digesta was flushed through a sieve. The contents of the petri dish and sieve were then examined under a magnifying lamp for helminths to ensure as complete a collection as possible.

Helminth Processing and Identification

Both live and dead helminths were stored submerged in 70% ethanol in 15 mL tubes until processing (Sepulveda and Kinsella 2013). Processing involved first chemically treating acanthocephalans and cestodes as described in Sepulveda and Kinsella (2013). Then, representatives of all helminths were either temporarily wet mounted (nematodes and cestodes) in 20% lacto-phenol (M. Kinsella, personal communication, 21 Dec 2019) or stained with Semichon’s Aceto-Acid Carmine, dehydrated in a series of concentrations of ethanol, and permanently mounted in Damar Balsam mountant on a microscope slide (cestodes and acanthocephalans) (Sepulveda and Kinsella, 2013, Bruno, 2014). Subsequently, helminths were microscopically identified using an Olympus BX 50 microscope, published keys (Khalil et al., 1994, Anderson et al., 2009), descriptions in the literature (Pence, 1972, Mollhagen, 1976, Butboonchoo et al., 2016), and reference slides from the Museum of 35 Texas Tech University, Jessica L. Herzog, May 2020

Texas Tech University. Finally, helminths that could not be identified or whose identification was to be confirmed were sent to Texas Medical Veterinary Diagnostics Laboratory (College Station, TX) and Dr. Mike Kinsella (Ph.D., Missoula, MT).

Statistical Analysis

Birds were examined in this study with the intent of determining parasite prevalence. Prevalence is defined as the total count of hosts infected with a specific species of parasite divided by the number of hosts examined for the parasite species (Bush et al., 1997).

The statistical analyses were limited in this study because of the host themselves and the characteristics of the collected data. Considering hosts, not all species were captured at each study site and the number of male, female, adult, and juvenile representatives of each species were not equal. In some cases, certain representatives were not present at all which made checking for constant variance across species impossible. Additionally, the dataset was small and contained many zeroes due to not all individuals being infected with each species of helminth, which restricted the use of many statistical models. Statistical analyses could only be conducted to compare avian orders, all birds, or quail. The size of the dataset, zeroes, and the characteristics of the hosts led to 2-way ANOVAs without interaction, Student’s Two Sample T Tests, and a Poisson Regression being used to represent the data. These tests were conducted in R (R Core Team 2017).

Results

Helminth Prevalence

A total of 54 birds were sampled, five of which were donated Bobwhite. Birds were comprised of 34 Bobwhite, two Scaled Quail, and 18 passerines. The passerines included seven Northern Mockingbirds and Northern Cardinals, and four Curve-billed Thrashers. Eyeworms were the most prevalent of the helminths, at a prevalence of 79.6% across all birds sampled (see Table 2.1.1). This percentage was supported by 36 Texas Tech University, Jessica L. Herzog, May 2020 each of the Curve-billed Thrashers, 6 Northern Mockingbirds, 29 Bobwhite, and three Northern Cardinals being infected. Cecal worms were the next most prevalent helminth at 36% across quail only. All sampled quail were infected with helminths (see Table 2.1.1).

Other helminths found included Raillietina spp. and Physaloptera spp. in Bobwhite (see Table 2.1.1). Bobwhite were also infected with Subulura brumpti and Tetrameres pattersoni. Infection of Tetrameres spp. was found in one Northern Cardinal. Different Mesocestodides canislagopodis, Mesocestoides spp. tetrathyridium, and Onicola canis were collected from Bobwhite and one Curve-billed Thrasher. Finally, one unidentified nematode was collected from a Curve-billed Thrasher and an unidentified acanthocephalan was taken from a Northern Mockingbird (see Table 2.1.1). The identifiable features necessary for identification of the nematode were damaged during processing and the acanthocephalan was overstained during slide preparation, rendering these specimens unidentifiable (Joe Luksovsky and Mike Kinsella, personal comm.).

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Table 2.1.1. Prevalence of collected helminth species. Percentages correspond to the total number of specific hosts necropsied. Adapted from Ferrer et al. (2004).

Nematodes Cestodes Acanthocephalans Mesocestoides Aulonocephalus Oxyspirura Physaloptera Subulura Tetrameres Tetrameres Unidentified Raillietina Mesocestoides Onicola Unidentified spp. Host pennula Petrowi spp. brumpti pattersoni spp. nematodes spp. canislagopodis tetrathyridium canis Acanthocephalans

Scaled 2/2 ½ 0 0 0 0 0 0 0 0 0 0 Quail (100%) (50%)

Northern 0 3/7 0 0 0 1/7 0 0 0 0 0 0 Cardinal (42.9%) (14.3%)

Bobwhite 34/34 29/34 4/34 1/34 2/34 0 0 4/34 2/34 1/34 1/34 0 (100%) (85.3%) (11.8%) (2.9%) (5.9%) (11.8%) (5.9%) (2.9%) (2.9%)

Northern 0 6/7 0 0 0 0 0 0 0 0 0 1/7 Mockingbird (85.7%) (14.3%)

Curve-billed 0 4/4 0 0 0 0 1/4 0 0 0 1/4 0 Thrasher (100%) (25%) (25%)

Total 36/54 43/54 4/54 1/54 2/54 1/54 1/54 4/54 2/54 1/54 2/54 1/54 (36%) (79.6%) (7.4%) (1.9%) (3.7%) (1.9%) (1.9%) (7.4%) (3.7%) (1.9%) (3.7%) (1.9%)

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Helminths representing the nematode, cestode, and acanthocephalan groups were collected from captured birds. Raillietina spp. and Mesocestoides spp. tetrathyridium were not included in any of the following figures or statistical analysis. Counting them was foregone due to the goal of the study being presence, not intensity of infection and some hosts having cestodes that were deteriorated to the point of not having a scolex. However, a total of 5,429 helminths were counted (see Figure 2.2). Caecal worms represented the largest proportion (87%) of these specimens, with 4,719 individuals being found (see Figure 2.2). Eyeworms represented the second largest proportion, at 12% and 677 individual nematodes. Finally, other helminths (an unidentified acanthocephalan, O. canis, other nematodes, and M. canislagopodis) (<1%) and 33 individuals, completed the counted proportion of helminths.

The most common infection site for quail was the caeca, while collection sites across all birds were the ocular tissues (nictitating membrane, lacrimal ducts, and Harderian glands), small intestines, proventriculus, pectoralis major, pancreas, and inside the body cavity. Eyeworms were found in the ocular tissues and caecal worms were collected from the caeca and small intestine. Raillietina spp. and some O. canis individuals were collected from the small intestines as well, while the acanthocephalan was found attached to the epithelium. Nematodes found inside the proventriculus included Subulura brumpti, T. pattersoni, and the unidentified nematode. The Tetrameres spp. were found inside nodes on the proventriculus (see Appendix A), while the Mesocestodides spp. tetrathyridium were collected from the pancreas. The body cavity served as the infection site for the remaining O. canis specimens. M. canislagopodis and Physaloptera spp. were found embedded in the pectoralis major.

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Figure 2.2. The count (in parenthesis) and percent distribution of eyeworms, caecal worms, and other helminths (excluding Raillietina spp. and Mesocestoides spp. tetrathyridium) out of 5,429 helminths collected.

Eyeworm and Caecal Worm Infection

Eyeworm infection was present in 43 out of the 54 birds sampled. For infected Bobwhite, infection ranged from 1-40 nematodes. The maximum infection was lower for Scaled Quail, at seven eyeworms. Of the 18 passerines sampled, 13 were infected, with a range of 1-8 nematodes. The maximum infection was found in a Northern Cardinal. Curve-billed Thrashers and Northern Mockingbirds followed, with maximum infections of five and six eyeworms, respectively. A 2-way ANOVA was used to look at eyeworm infection for each sex and the output showed that the effect of order was significant, F(1,53)=24.84, P<0.0001, but not the effect of sex (P=0.65). Another 2-way ANOVA was used to look at eyeworm infection for adults and juveniles. This output also showed a significant effect of order, F(1,53)=18.55, P<0.0001, but not an effect of age (P=0.437).

Average eyeworm infection differed for each species across study sites (see Figure 2.3). For Bobwhite, the highest average infection of 22 nematodes was at

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Cottle County, the northern site. The lowest average infection of 14 nematodes was at Garza county, the central site. Scaled Quail at Mitchell County, the southern site, had an average infection of 3.5 nematodes. Average infection for passerines was also highest at Mitchell County.

Figure 2.3. A bar graph representing the average eyeworm infection intensity at each site for each species of bird sampled.

Caecal worm infection was present in all 36 quail sampled, but not in passerines due to their not having caecum. For Bobwhite, infection ranged from 1-336 nematodes, while infection for Scaled Quail ranged from 60-162 nematodes. Average caecal worm infection was relatively consistent across sites for Bobwhite, with averages ranging from 105.6 to 153.3 nematodes at each site (see Figure 2.4). For Scaled Quail, the average infection was 111 nematodes at Mitchell County.

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Figure 2.4. A bar graph representing the average caecal worm infection intensity at each site for each species of quail collected.

A Student’s Two Sample T Test determined that caecal worm infection was not significantly different between male and female quail (P=0.373), nor was it significantly different between adults and juveniles (P=0.67). The same test determined that infection in all Bobwhite vs. all Scaled Quail was not significantly different (P=0.77).

Infection with Other Helminths

The maximum infections of other helminths in Bobwhite were three O. canis and two T. pattersoni nematodes. Scaled Quail had no other helminths. For passerines, maximum infections with other helminths included four O. canis in a Curve-billed Thrasher and three Tetrameres spp. in a Northern Cardinal. Out of all birds sampled, only three adult Northern Cardinals were not infected with any helminths.

Infection with other helminths aside from eyeworms or caecal worms occurred in 16 birds sampled (see Table 2.1.2). Of these individuals, 10 were coinfected with eyeworms. For quail species, infection with other helminths was concurrent with cecal 42

Texas Tech University, Jessica L. Herzog, May 2020 worm infection. Only one of the 16 individuals, a Northern Mockingbird, was not also infected with eyeworm or caecal worm. The average count per host of other helminths was 1.06 helminths for all females and 0.5 helminths for all males. These averages were determined to be significantly different from each other (P<0.05) via use of a Poisson Regression.

Patterns of infection may be tied with month, age, and sex, but not statistical analysis were conducted on these potential relationships. For example, the acanthocephalans were collected in July and August. Aside from the O. canis found in a Curve-billed Thrasher juvenile female, the rest of the acanthocephalans were collected from juvenile male birds. Also, the Tetrameres spp. and T. pattersoni were collected in April from adult males.

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Table 2.1.2. Data pertaining to helminths aside from O. petrowi and A. pennula collected from study birds during the 2019 field season.

Host Host Host Helminth Month Eyeworm Caecal Worm Host IDa Sex Age Species Collected Present Presentb Northern 1688 Male Adult Tetrameres spp. April Yes -

Cardinal

Physaloptera Bobwhite 1695 Male Adult spp. July Yes Yes

1706 Male Adult September Yes Yes

502 Male Adult Subulura Unknown Yes Yes

brumpti

1683 Male Adult Tetrameres April No Yes

1676 Male Adult pattersoni April Yes Yes

1790 Male Juvenile Raillietina spp. September No Yes

1699 Male Adult April Yes Yes

989 Male Adult Unknown Yes Yes

1794 Male Juvenile Mesocestoides July Yes Yes

1793 Male Juvenile canislagopodis August No Yes

Mesocestoides 1676 Male Adult spp. April Yes Yes tetrathyridium

1794 Male Juvenile Onicola canis July Yes Yes

Northern 1792 Male Juvenile Unidentified August No - acanthocephalan Mockingbird

Curve-billed 1677 Male Adult Unidentified April Yes - Nematode Thrasher

1795 Female Juvenile Oncicola canis July Yes -

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Finally, all Bobwhite sampled were infected with helminths, with the highest percentage of adult and juvenile Bobwhite being infected with two different species (see Figure 2.5). The lowest number of helminth species infecting approximately 6% of adults was one. The maximum level of coinfection for approximately 3% of adults was four helminth species. Approximately 9% of juveniles were infected with the typical coinfection of two helminth species. Approximately 3% each were infected with three and four species.

Figure 2.5. A bar graph representing the percentage of Bobwhite sampled from each age class infected with each total number of helminth species. Adapted from Bruno (2014).

Discussion

Researchers found infection in the same organs as were infected in the current study, but also documented helminths in the neck muscle (Villarreal, 2012, Olsen, 2014), crop (Bruno, 2014), beneath the pericardial sac, and in the coelomic cavity (Kubečka et al., 2018). Composition of the helminth community was supported by researchers also finding a dominant proportion of caecal worms, followed by eyeworms, and other helminths (Villarreal, 2012, Bruno, 2014). Most Bobwhite had coinfection of two species, a documentation which was supported by Bruno et al. 45

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(2019). However, Olsen (2014) noted infection of only one species in most birds. Additionally, the maximum coinfection of 4 species found in this study was less than that of Villarreal (2012) and Bruno et al. (2019), who found five and six species, respectively.

All quail sampled were infected with caecal worms and infection was not significantly different between species. The research by Dunham et al. (2017b) supports this finding. For Scaled Quail specifically, despite Dancak et al. (1982) documenting a 74% prevalence of caecal worms, the finding of 100% prevalence was similar to other studies, which reported a 91% prevalence or higher (Lehmann, 1953, Olsen, 2014, Bedford, 2015). Infection ranged from 60-162 nematodes. The range of 1-263 nematodes found by Dancak et al. (1982) was wider. Caecal worm prevalence in Bobwhite was supported by previous studies reporting values ranging from 82% in 142 birds in Fisher County, TX (Villarreal, 2012) to 97% in 128 Bobwhite from the Rolling Plains (Bruno et al., 2019). Infection ranged from 1-336 nematodes, which was supported on the lower end by Villarreal (2012) and Olsen (2014), but not on the higher end with these researchers each finding over 500 worms in single individuals. More recently, Henry et al. (2017) documented a range of 51-572 nematodes. Finally, caecal worm infection was not significantly different between age or sex. These findings were supported by Lehmann (1953), Dancak et al. (1982), and Bruno et al. (2019). However, while Villarreal (2012) and Olsen (2014) found no significant difference between sexes, infection was significantly different between adult and juvenile quail in both of these studies. Mean caecal worm infection in Scaled Quail also varied between adults and juveniles in Bedford’s (2015) study, but no statistical analyses were conducted due to the small sample size.

Most quail were infected with eyeworms as well. Prevalence of 50% in Scaled Quail came from infection of seven nematodes in a single individual. While Landgrebe et al. (2007) found a similar (56%) prevalence of eyeworms, prevalence in Scaled Quail generally appears low. For example, Dancak et al. (1982) documented a 2% prevalence in 104 birds, (Olsen, 2014) found 13% prevalence for 23 birds, and 46

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Bedford (2015) found 4% prevalence for 204 birds. Infection prevalence was higher in Bobwhite, at 85.3% across 34 birds. Dunham et al. (2017) documented significantly higher eyeworm infection in Bobwhite as compared to Scaled Quail, but the high prevalence of eyeworm in Bobwhite alone receives mixed support when reviewing the literature. For example, low prevalence values such as 2% in 142 Bobwhite (Villarreal, 2012) and 9% across 209 birds (Olsen, 2014) have been reported, contrasting with the high prevalence values of 66% in 128 birds (Bruno et al., 2019) and a maximum of 100% prevalence during certain months (Henry et al., 2017). Infection ranged from 1-40 nematodes, which is similar to the 1-36 nematode range reported by Olsen (2014), but not as well reflected by the 1-23 nematode range reported by Villarreal (2012) or the 0-58 nematode range reported by Henry et al. (2017).

Passerines had eyeworm infection as well. A prevalence of 42.9% and a maximum of eight nematodes in a single individual was documented in Northern Cardinals. The only published documentation supporting these findings comes from Pence (1972). He found 2 nematodes in a single Northern Cardinal out of the 20 total birds examined. Higher prevalence values were documented form Northern Mockingbirds (85.7%) and Curve-billed Thrashers (100%). Again, the only comparable findings came from Dunham and Kendall (2014) documenting infection in these species. Regardless, the statistical analysis of eyeworm infection across orders showed that infection was not significantly different between age or sex but was for order. For Bobwhite, eyeworm infection has been reported to differ between ages, but not for sexes (Villarreal, 2012, Dunham et al., 2014, Bruno et al. 2019).

Disregarding prevalence values, other nematode findings for Bobwhite of this study are similar to reports in the literature, but the acanthocephalan findings were not necessarily supported. First, Tetrameres pattersoni and Physaloptera spp. were found in Bobwhite in the current study and by Kellog and Doster (1972), Olsen (2014), and Bruno et al. (2019). Villarreal (2012) documented T. pattersoni, but not Physaloptera spp. Also, Subulura spp. was found by Davidson et al. (1980) and S. brumpti was 47

Texas Tech University, Jessica L. Herzog, May 2020 found by Bruno (2014). However, the finding of Physaloptera spp. and the new documentations of Gongylonema spp., and Procyrnea pileate in Scaled Quail by Landgrebe et al. (2007) could not be replicated. The finding of Tetrameres spp. in the Northern Cardinal was supported by Quentin and Barre, (1976) and is one of the few documentations of such a helminth in this host. For Acanthocephalans, Cram (1931), Bruno (2014) and Olsen (2014) also documented Onicola canis in Bobwhite, but no documentation of this helminth in Curve-billed Thrashers was available at the time of this study. Olsen, (2014) also found O. canis in Scaled Quail, which the current study could not replicate. The unidentified acanthocephalan found in the Northern Mockingbird could not be compared to the findings of previous studies since no other documentation of acanthocephalans in Northern Mockingbirds was available at the time of this study.

Cestodes have also been documented in previous studies, but identification tended to be more precise. For example, while Villarreal (2012) collected Raillietina spp. as was collected in this study, Olsen (2014) found R. cesticillus, specifically. Davidson et al (1980) also found R. cesticillus, as well as R. colina. Furthermore, Mesocestoides cestodes were found, with M. canislagopodis in two Bobwhite and Mesocestodides spp. tetrathyridium in one individual. M. canislagopodis has been documented in Rock Ptarmigan, another gallinaceous bird (Skirnisson et al., 2016). During the 2016-2017 hunting season, Kubečka et al. (2018) were the first to document Mesocestoides spp. in North American birds when they found a potential new species of Mesocestoides spp. tetrathyridium in a Bobwhite and Scaled Quail from two sites in Texas, one of which was near a study site used for this project. Additionally, Mesocestoides have been documented in coyote and badger intermediate hosts in Texas (Pence and Dowler, 1979, Pence and Windberg, 1984), both of which were seen at study sites. Hence, this finding represents the second documentation of avian Mesocestoides in North America and one of the few for Bobwhite, especially because Mesocestoides are thought to be an incidental infection (Kubečka et al., 2018). Finally, unlike Davidson et al. (1980), Dancak et al. (1982), Villarreal (2012),

48

Texas Tech University, Jessica L. Herzog, May 2020 and Bruno et al. (2019), Rhabdometra odiosa was not documented in any quail from this study and the finding of Choanotaenia infundibulum in Scaled Quail (Landgrebe et al., 2007) could not be reproduced.

The results of this study may have been influenced by the nature of parasite development within and external to the hosts. Low-level infections of juvenile nematodes may become intense infections of adults (Davidson et al., 1980), which depends more so on conditions within the host, rather than host age. The internal environment of the host may regulate the rate of parasite development or initiate diapause conditions (Sommerville and Davey, 2002), leading to similar infection levels across age classes. Furthermore, infection may also depend on the climate, temperature, or precipitation of the study area, especially for eyeworms and caecal worms (Dunham et al., 2017b, Henry et al., 2017, Blanchard et al., 2019). For example, caecal worm infection has reportedly been exacerbated by drought conditions (Lehmann et al., 1984). In contrast, precipitation can influence the survival of parasite eggs and increase vegetation growth, which in turn facilitates reproduction and population growth of the intermediate hosts themselves (Dunham et al., 2014, 2016). Insect intermediate host availability in the habitat of the potential definitive host is a critical factor influencing infection (Pence, 1972). Furthermore, patterns of infection may be tied to monthly or seasonal collection of hosts (Villarreal, 2012, Dunham et al., 2014, Bedford, 2015, Henry et al., 2017) or the climatic and habitat conditions of study site (Olsen, 2014).

Shortcomings of this study may have also impacted its findings. First, Bobwhite may have been aged incorrectly in the field. They can sometimes be difficult to age based on examining the color of their wing feathers (Haugen, 1957), which was done in this study. Incorrectly ageing Bobwhite subsequently skewed infection levels and likely led to the findings of a coinfection of four species in a single juvenile and eyeworm infections being similar between age classes. Additionally, any prevalence values reported that differed from those previously published were likely the result of a small sample size. Finally, damaging cestodes 49

Texas Tech University, Jessica L. Herzog, May 2020 during bird or organ storage or upon cestode retrieval (Dancak et al., 1982, Villarreal, 2012, Olsen, 2014) and ruining the acanthocephalan in the Northern Mockingbird during staining clearly impacted the results concerning these helminths.

Finally, results may have been influenced by characteristics of the hosts themselves. For example, the varied proportions of intermediate hosts consumed by each bird may have balanced infection levels across infected definitive hosts, thus lessening the difference between infection levels across ages or sexes (Olsen, 2014). Diet may have also led to the documentation of low infection of eyeworms in Scaled Quail compared with Bobwhite, as these birds consume different proportions of insects (Campbell-Kissock et al., 1985, Dabbert et al., 2009), or contributed to not finding certain helminth species in Scaled Quail or Bobwhite. Diet may have contributed to the average of other helminths being higher than that of males, as females tend to consume more insects during breeding season. Finally, eyeworm infection may have differed by order based on host size. Physically, the orbital cavity of songbirds may not be large enough to support similar infection levels to those of quail. In terms of mass, Bedford (2015) noted a significant difference in eyeworm infection depending on mass of Scaled Quail. Because passerines captured weighed roughly 40g and quail could weigh over 150g, such a phenomenon could be possible. Additionally, the low infection of eyeworms in Scaled Quail compared to Bobwhite.

The current study contributes data to the few previous helminth surveys conducted in Texas, especially for Scaled Quail and passerines. The findings of eyeworms in passerines, the various Mesocestoides in Bobwhite, the acanthocephalan in the Northern Mockingbird, and the Tetrameres spp. in the Northern Cardinal are some of the few or only documentations of such host-parasite associations. While drawing conclusions about host-parasite relationships from small sample sizes and low numbers of retrieved helminths are not entirely reliable determinants of realistic infections (Pence, 1972), the data presented herein serve as a valuable and needed baseline for future studies. Conducting more parasite surveys will help future researchers determine if the prevalence values documented in this study are typical or 50

Texas Tech University, Jessica L. Herzog, May 2020 not, for the host or study area. Future researchers may investigate temporal parameters of infection such as season of host collection and the impacts of temperature or precipitation on parasites. Researchers may also pursue molecular techniques to assess infections within a host without euthanasia and full necropsy or to aid in helminth identification. Doing so may alleviate some of the difficulties encountered in this study.

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CHAPTER III

ECTOPARASITES AND HAEMOPARASITES OF NORTHERN BOBWHITE QUAIL (COLINUS VIRGINIANUS) AND SELECT PASSERINES IN THE ROLLING PLAINS ECOREGION, TX

Abstract

Northern Bobwhite quail (Colinus virginianus), an iconic upland gamebird, have been steadily declining across their native range since the 1960s. The Rolling Plains Ecoregion of TX was once a population stronghold, but Bobwhite are declining there as well, perhaps due to prevalent infection of eyeworm (Oxyspirura petrowi). Scaled Quail (Callipepla squamata), Northern Mockingbirds (Mimus polyglottos), and Curve-billed Thrashers (Toxistoma curvirostre) are also documented hosts in the ecoregion. Additional examples of shared parasitism between passerines and quail may exist, including those from ectoparasite and haemoparasite infection. However, understanding is limited by a lack of complete parasite surveys. To increase understanding of shared infection for birds in the Rolling Plains Ecoregion, complete parasite surveys were conducted on Bobwhite, Scaled Quail, Northern Mockingbirds, Curve-billed Thrashers, and Northern Cardinals (Cardinalis cardinalis). Birds were trapped at three study sites across the Rolling Plains from March to October 2019. Ectoparasite and blood samples were taken, and parasites were microscopically identified. A total of 50 individuals (34 quail and 17 passerines) were sampled for ectoparasites, with a 72% maximum overall prevalence across species. Lice representing five genera were found, while mites were represented by Eutrombicula spp. Total count was not significant between avian orders (P=0.206) or counties (P=0.648). A total of 48 birds (30 quail and 18 passerines) were sampled for haemoparasites, with a maximum overall prevalence of 20.8% across species. Haemoparasites were comprised of Plasmodium spp., Haemoproteus spp., and microfilaria. Future studies could investigate the idea of molecular identification of ecto and haemoparasites or the consequences of such infection on host fitness and immunity. 58

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Introduction

The Northern Bobwhite Quail (Colinus virginianus, hereafter: Bobwhite) is a well-recognized gamebird of high economic importance (Johnson et al., 2012) that has been experiencing range-wide decline for decades (Sauer et al., 2013). Bobwhite are also declining in their historic stronghold, the Rolling Plains Ecoregion, despite favorable habitat conditions (Rollins 2000, 2007, Dunham and Kendall, 2017). Perhaps prevalent eyeworm (Oxyspirura petrowi) infection (90%) is contributing to the declines more than scientists have previously anticipated (Dunham et al, 2014, Rolling Plains Quail Research Foundation, 2020). Other avian hosts documented in the ecoregion include Scaled Quail (Callipepla squamata), Northern Mockingbirds (Mimus polyglottos) and a Curve-billed Thrasher (Toxostoma curvirostre) (Dunham and Kendall, 2014, 2017).

Quail and passerines of the Rolling Plains may have additional parasites not limited to helminths in common. However, a lack of regional ecto and haemoparasite surveys on targeted hosts leaves shared parasitism between quail and passerines largely unaddressed. For example, no known Bobwhite lice surveys were conducted after the 1950s in any region of Texas and none were ever conducted in the Rolling Plains. Four lice surveys were conducted for Scaled Quail of Texas, but besides Howard (1981), they were conducted in the 1950s and did not include prevalence data (Emerson, 1950, Wallmo, 1956, Wiseman, 1959). The most recent survey for lice of Texas passerines at the time of this study was conducted by Pistone (2016). All other lice surveys investigating host-louse interactions in Texas were conducted on doves (Pistone, 2016), which highlights the notion that Texas is an understudied area for lice-host relationships (Pistone, 2016). Haemosporidia survey literature is sparse in Texas as well, with Xiang et al. (2017) being the only and most current survey for Bobwhite of the Rolling Plains. Currently, there are no surveys for haemoparasites of Scaled Quail in Texas (Peterson, 2007), nor are there any passerine haemosporidian surveys conducted in the ecoregion.

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Parasitism and disease have been overlooked for years as causing harm to their hosts (Friend, 2014, Gehman et al., 2019). Hence, the attitude that parasitological surveys were not necessary may have developed and consequently, few surveys were conducted. A lack of surveys may also stem from the sheer and increasing size of the ectoparasite and haemoparasite groups, which leaves researchers nescient of where to begin additional surveys. For example, Price et al. (2003) doubled the reported number of lice taxa published in the preceding checklist only 50 years prior, listing 4464 lice species parasitizing 324 species of birds (Smith, 2004). Certain genera alone have been reported to include over 300 species representatives (Bush et al., 2016). The list of haemosporidian parasites is growing beyond its recent count of 250 species as well (Valkiunas, 2005). Infection of these lice is spread across 10,000 species of birds (Daszak et al., 2000). Hundreds of surveys were required to obtain the wealth of information scientist have for ecto and haemoparasites. Yet, challenges and shortcomings associated with lice and haemoparasite identification, a cornerstone of full parasite surveys, may be keeping current data gaps open.

Concerning avian lice, although articles addressing specific species-host relationships have been published, there is not a comprehensive guide to all species (Galloway, 2019). Current guides such as Price et al. (2003) are only suitable for identifying collected specimens to genus (Galloway, 2019). Also, Price et al. (2003) uses obscure terminology for descriptions of genera (Smith, 2004), despite lice morphology being highly variable (Bush et al., 2016) and a limited number of researchers being well versed in lice taxonomy (Smith, 2004). Furthermore, utilizing reference collections is not necessarily easier than using written keys. Specimens in museum collections remain largely unidentified and documented for access by researchers (Smith, 2004, Bush et al., 2016, Galloway, 2019). Considering the difficulties presented with lice identification, it is no wonder certain avian species have not been surveyed (Galloway, 2019), leaving many lice species understudied and undescribed (Bush et al., 2016).

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Haemoparasites are a better studied in comparison to lice but their study presents its own challenges. First, examination of blood smears is time consuming, often taking between 20 and 35 minutes per smear for an experienced observer (Godfrey et al., 1987, Ishtiaq, et al., 2017). Also, species identification of haemosporidia in blood smears is inherently difficult (Valkiūnas et al., 2014) such that it should ideally be tasked only to an experienced lab technician (Rush et al., 2016). Haemosporidian morphology in general is highly variable, with few researchers understanding how to navigate the range of identifying characteristics across genera (Martinsen et al., 2006, Valkiūnas et al., 2014). In contrast, Haemoproteus spp. closely resemble Plasmodium spp. (Friend et al., 1999, Clark et al., 2009, M. Borst, personal communication, 29 Oct 2019), so much so that they are difficult or impossible to distinguish, especially during early developmental stages (Garnham, 1966, Valkiūnas, 2005). Lastly, mild haemosporidiasis in wildlife is common, leading to few parasites present in blood smears and consequently, lower detectability and fewer examples to compare with published keys (Valkiūnas, 2005, Valkiūnas et al., 2014). Identification problems persist from a stark lack of access to identification training (Valkiūnas et al., 2014).

Second, the field of molecular haemosporidian identification is underdeveloped. A majority of the hundreds of sequences in repositories such as GenBank are identified to genus or higher and some are misidentified (Valkiūnas et al. 2008a, Valkiūnas et al., 2014). This lack of proper and adequate identification has rendered much of the current available sequences nearly unusable (Valkiūnas et al., 2014). Additionally, molecular markers have been developed for a mere 20% of avian haemosporidia identified (Valkiūnas et al., 2014). Amelioration of these current shortcomings is complicated, however. Coinfections of morphologically related haemosporidia in wildlife hosts are common and hinder the detection of specific species (Valkiūnas et al., 2003, Valkiūnas et al., 2014), while the genetic diversity of haemosporidia is broad such that species specific primers have yet to be developed for a majority of known species (Valkiūnas et al., 2014).

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More ecto and haemoparasite surveys are needed to reduce the complications associated with identifying species of each group. Furthermore, current literature is shifting to the acceptance of parasites as having the potential to negatively impact their hosts and host populations (Friend, 2014). While lice are not typically pathogenic (Clayton and Tompkins, 1995), coinfection may influence host immunity and susceptibility to other parasites or diseases (Møller and Rozsa, 2005). Amblyceran (e.g. Menacanthus spp.) and Ischnoceran lice (e.g. Brueelia spp., Goniodes spp., Lipeurus spp., and Oxylipeurus spp.) infections have been thought to activate the host’s immune system (Fairn et al., 2012). Hence, researchers should prioritize further studies of chewing lice (Galloway, 2019), especially those for passerines whose host- lice associations are largely nonexistent (Galloway et al., 2014). Similar interest should be given to haemosporidia surveys. Further studies will aid in the understanding of host-parasite interactions, which have not been adequately documented (Valkiūnas et al., 2014). Such research is important because haemosporidia like Haemoproteus spp. has been linked to causing anemia and death in quail (O’Roke, 1930), while P. relictum has been perpetrated in passerine die-offs (Valkiunas, 2005). Additional surveys will help researchers more clearly understand host-parasite interactions and the associated consequences they may have on individuals and populations (Gehman et al., 2019, Khan et al., 2019).

In order to address knowledge gaps concerning shared parasitism in quail and passerines of the Rolling Plains, a complete ectoparasite and haemoparasite survey was conducted on Bobwhite, Scaled Quail, Northern Cardinals, Northern Mockingbirds, and Curve-billed Thrashers. Birds were trapped at three study sites representing the upper, central, and lower Rolling Plains from March to October 2019. Donated Bobwhite were also received. Ectoparasites and blood were collected from individuals and the associated blood smears and ectoparasite specimens examined. The objectives of this study were to 1) add to existing survey data concerning documented ectoparasite and haemoparasite infection in study species and 2) document shared infection between the aforementioned study species.

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Materials and Methods

Ethics Statement

This experiment was approved by Texas Tech University Animal Care and Use Committee under protocols 16071-08 and 19015-02. All birds were trapped and handled according to Texas Parks and Wildlife permit SRP-0715-095, United States Fish and Wildlife Permits MB88764B-0 and MB88764B-1, and Federal Bird Banding Permit 22590.

Sample Collection and Processing

This study was conducted at private ranches across the Rolling Plains Ecoregion from March to October 2019. The mesquite-shortgrass savannah habitat there has many honey mesquites (Prosopis glandulosa) and junipers (Juniperus pinchotii) throughout (Hatch et al., 1990, Parajulee et al., 1997). Temperatures as low as -1.1°C in January and as high as 35.6°C in July (Dunham et al., 2017) have been documented. Precipitation is less variable, with a yearly range of 50.8-71cm (Texas Parks and Wildlife, 2020). Ranches were located in Cottle, Garza, and Mitchell counties, representing the northern, central, and southern Rolling Plains, respectively. Quail and passerines were captured using the methods described in Dunham et al. (2017a) and brought back live to The Institute of Environmental and Human Health Aviary. Donated, frozen birds were occasionally received during the study. A blood sample was taken from each wild-caught individual via jugular venipuncture as soon as possible upon arrival and placed into an EDTA tube. If this method was not successful, a heart stick was performed with a capillary tube after the bird had been euthanized for a separate study. Ectoparasites were also collected from wild-caught and donated birds. They were stored at room temperature in 70% ethanol in 15 mL tubes for later processing.

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Ectoparasite Extraction

Ectoparasites were collected after wild-caught hosts had been euthanized for a separate study or donated hosts were thawed. Collection followed a modified version of the methods used by Clayton et al. (1992). Each wild-caught host was placed in its own Ziplock bag with cotton balls soaked in acetone for approximately three minutes to ensure that all ectoparasites were killed. This step was omitted for donated birds, as freezing killed any ectoparasites present on them. After, the host’s body was held over a white letter size sheet of paper and all feather tracts were vigorously ruffled for approximately one minute. Ruffling was repeated one additional time. Then, the paper was folded in half lengthwise to serve as a funnel to transfer the ectoparasites and feather debris to a 15 mL tube filled with ethanol. A separate tube was used for each host. Ectoparasites were stored at room temperature until further processing.

Ectoparasite Identification

Ectoparasites were transferred from their tubes into separate petri dishes for counting and preliminary sorting. Spraying 70% ethanol into the tipped tube ensured that all ectoparasites ended up in the petri dish. Then, ectoparasites were counted and similar ones were noted. Ectoparasites were again stored in 70% ethanol in their tubes until identification. For efficiency, identification was conducted on a random proportion representing at least 20% and up to 100% of the total count. Although representative slides are traditionally prepared with Hoyer’s Medium mountant, perfecting this method was not possible. Each slide was time consuming to create, had excessive air bubbles, and the medium crystalized upon contact with the sealant (see Appendix B), rendering the mounted parasites difficult to examine. Thus, temporary 70% ethanol wet mounts and photos were used for identification. Each slide was viewed under an Olympia BX 50 microscope at 10X and 40X. Photos of each ectoparasite were taken to identify using Price et al. (2003) and representative photos from Smith and Rycroft (2020). Once identification was finished, ectoparasites were returned to their tubes and stored at room temperature.

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Blood Smear Preparation

Blood smears were made in the laboratory immediately after blood was collected from wild-caught hosts. An average of two blood smears per individual were made using the methods described in Owen (2011). A capillary tube was used to take blood from the EDTA tube (or heart in the case of heart sticks) and place a drop on a clean slide. Then, another slide was placed ahead of the drop at an approximately 45- degree angle, drawn back until it reached the blood, and pulled forward down the slide. Once dry, smears were stored in a slide box at room temperature until they could be stained (Owen, 2011).

Staining facilitated the detection of haemoparasites. First, smears were fixed in 95% ethanol for five minutes (Weckstein et al., 2017). Then they were air dried and stained with Giemsa stain for 45 minutes (Woronzoff-Dashkoff, 2002). Stained slides were gently rinsed in deionized water, air dried, and stored in a slide box until examination (Owen, 2011). Whole blood used to make smears was stored in EDTA tubes in the − 20 °C freezer for potential future use (Owen, 2011).

Smear Quality and Examination

Quality was not consistent across smears, which can be a major pitfall for future examinations (Woronzoff-Dashkoff, 2002). While making at least two smears per wild-caught individual helped alleviate this issue, some smears were still too thick or small, lacking an adequate monolayer and feathered edge, respectively (see Figure 3.1). Some smears had mild cell lysis or sloughing, which may have been due to inadequate time in the fixative (Woronzoff-Dashkoff, 2002). Others had stain precipitate or water damage (see Appendix C), which may have been the result of staining smears too late after fixation or the stain not changed regularly enough (Woronzoff-Dashkoff, 2002). This damage could be confused with Plasmodium spp. Imperfections from improper technique, when present, decreased the quality of the

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Texas Tech University, Jessica L. Herzog, May 2020 smear and impacted interpretation of the host’s blood (Eberhard and Lammie, 1991, Clark et al., 2009).

Figure 3.1. Examples of smears made and their quality. Smear A is too thick, B is a typical smear, C is nearly too small, and D is too small.

Blood smears were examined using an Olympia BX 50 microscope. First, the entirety of each blood smear was examined at 40X to detect microfilaria. Then, one smear was examined from the beginning of the smear to the feathered edge under oil immersion for the detection of haemosporidia (Weatherhead and Bennett, 1991). Examining only one slide out of the pair ensured that one was left unoiled for future use. Photos of potential blood parasites were taken. Blood parasites were identified using published descriptions and photos (Christensen et al., 1983, Clark et al., 2009, Kelly et al., 2018, Valkiūnas and Iezhova, 2018). After examination, oil was blotted off the slides and they were returned to their slide box for storage.

Statistical Analysis

Birds were examined to determine parasite prevalence, which is the total number of hosts infected with a specific parasite divided by the total number of hosts

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Texas Tech University, Jessica L. Herzog, May 2020 examined to detect that parasite (Bush et al., 1997). Furthermore, a count of haemoparasites was not recorded due to the purpose of smear examination being to report presence or absence of haemoparasites. Finally, the statistical analysis of this study were limited in part by the avian hosts themselves and the characteristics of the collected data.

Not all species of hosts were captured at each study site, which limited the possibilities for statistical analysis. Within species, there were not equal numbers of male, female, adult, and juvenile representatives. Some hosts did not have certain representatives at all. The statistical analyses were also limited by the variation in the proportion of the total count of ectoparasites being identified (ranging from 20% to 100%. Thus, statistics could not be conducted on ectoparasite genera and had to be used only to compare avian orders. Ultimately, a 2-way ANOVA without interaction was conducted in R (R Core Team, 2017) to compare total count of ectoparasites across orders and counties.

Concerning haemoparasites, statistical analysis could not be conducted due to the aforementioned limitations from the hosts themselves and the low prevalence of haemoparasites found in all birds sampled.

Results

Ectoparasite Prevalence

A total of 50 birds, five of which were donated Bobwhite, were sampled for ectoparasites. Of these 34 quail and 17 passerines, 28 of the 32 Bobwhite were infected, one of two Scaled Quail, five of seven Northern Mockingbirds, one of six Northern Cardinals, and one of three Curve-billed Thrashers. Overall ectoparasite prevalence across hosts was 72%, which was made up of lice and mites. Of the lice, Goniodes spp. were the most prevalent, at 52% across all birds sampled (see Table 3.1.1). Lipeurus spp. were the next most prevalent species, at 32%. Finally,

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Menacanthus spp. were the third most prevalent, at 28%. Mites were represented by Eutrombicula spp., with a prevalence of 6% across hosts.

Bobwhite were infected with the greatest number of ectoparasite species, with Goniodes spp. being the most prevalent (75%) and Eutrombicula spp. being the least prevalent (3.1%). In contrast, Northern Cardinals and Curve-billed Thrashers were only infected with Eutrombicula spp., with a prevalence of 16.7% and 33.3%, respectively. Northern Mockingbirds were the only hosts infected with Brueelia spp., with a prevalence of 42.9%, while Bobwhite were the only hosts infected with Lipeurus spp., with a prevalence of 50%. Scaled Quail were the only hosts harboring Oxylipeurus spp., with a prevalence of 50%. Goniodes spp., Menacanthus spp., and Eutrombicula spp. represent instances of shared infection.

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Table 3.1.1. Prevalence of ectoparasites across species. Percentages correspond to the total number of specific hosts examined. Adapted from Ferrer et al., (2004).

Lice Mites Infected vs. Host Goniodes spp. Brueelia spp. Lipeurus spp. Menacanthus spp. Oxylipeurus spp. Eutrombicula spp. Sampled Ind. Scaled ½ 1/2 0/2 0/2 1/2 1/2 0/2

Quail (50%) (50%) (50%) (50%)

Northern 1/6 0/6 0/6 0/6 0/6 0/6 1/6 Cardinal (16.7%) (16.7%)

Bobwhite 28/32 24/32 0/32 16/32 9/32 0/32 1/32 (87.5%) (75%) (50%) (28.1%) (3.1%)

Northern 5/7 1/7 3/7 0/7 4/7 0/7 0/7 Mockingbird (71.4%) (14.3%) (42.9%) (57.1%)

Curve-billed 1/3 0/3 0/3 0/3 0/3 0/3 1/3 Thrasher (33.3%) (33.3%)

Total 36/50 26/50 3/50 16/50 14/50 1/50 3/50 (72%) (52%) (6%) (32%) (28%) (2%) (6%)

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Ectoparasite Counts

A total of 469 ectoparasites (181 lice and 9 Eutrombicula spp. mites) infected 36 out of the 50 hosts sampled. The mode infection was zero ectoparasites, while the average infection was 9.38 ectoparasites per host. Coinfection of up to three species occurred in 19 hosts. The maximum infection of 95 lice was found in a single Bobwhite. No infection was found in four of the Bobwhite sampled, while five Bobwhite were each infected with one louse. One of the two Scaled Quail examined was infected with five lice and the other was not infected. The maximum infection in a passerine was 20 lice in a Northern Mockingbird, while nine of the passerines were not infected. Mite infection (Eutrombicula spp.) was found in a single Curve-billed Thrasher (six mites) and a single Northern Cardinal (one mite). The total count of ectoparasites was not significantly different between Passeriformes and Galliformes (P=0.446), nor was it significantly different between counties (P=0.648).

Haemoparasite Prevalence

A total of 48 wild-caught birds were sampled for haemoparasites. Prevalence was 20.8% across all sampled hosts. Of these 30 quail and 18 passerines, three of the 28 Bobwhite were infected, one of two Scaled Quail, and six of seven Northern Mockingbirds. The Northern Cardinals and Curve-billed Thrashers were not infected. Plasmodium spp. were the most prevalent of the haemoparasites, at 13% across all birds sampled (see Table 3.1.2). Bobwhite were infected at 10.7% prevalence and Northern Mockingbirds at 42.9% prevalence. Microfilaria were the least prevalent of all haemoparasites, at 2% across all birds sampled. This 2% came from infection documented in a single Northern Mockingbird, which also represented 14.3% prevalence of microfilaria for this host. Haemoproteus spp. had a prevalence of 6%, infecting only Northern Mockingbird (28.6%) and Scaled Quail (50%). This 50% prevalence represented the maximum prevalence for any haemoparasite category. Northern Mockingbirds were the only host infected with all categories of haemoparasites. Cases of shared infection came from Plasmodium spp. and Haemoproteus spp. 70

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Table 3.1.2. Prevalence of observed haemoparasite categories. Percentages correspond to the total number of specific hosts necropsied. Adapted from Ferrer et al. (2004).

Infected vs. Haemoproteus Plasmodium spp. Microfilaria Host Sampled Ind. spp. Scaled 1/2 0/2 1/2 0/2 Quail (50%) (50%)

Northern 0/7 0/7 0/7 0/7 Cardinal

Bobwhite 3/28 3/28 0/28 0/28 (10.7%) (10.7%)

Northern 6/7 3/7 2/7 1/7 Mockingbird (85.7%) (42.9%) (28.6%) (14.3%)

Curve-billed 0/4 0/4 0/4 0/4 Thrasher

Total 10/48 6/48 3/48 1/48 (20.8%) (13%) (6%) (2%)

Discussion

Interpreting the results of the ectoparasite portion of the current study was limited by the shortage of published, relevant prevalence values in certain hosts and for certain species of lice. Thus, comparing prevalence values with those from other studies was difficult and findings were instead reported in terms of genera documented in a host, along with species representatives of that genera from hosts captured in other study areas. Prevalence values found for quail were compared to those of other studies because this is the only group for which relevant documentation of a prevalence metric was available. Overall, there was a 72% prevalence across hosts, which is higher than that of 15.5% reported by Pistone (2016), but lower than the maximum 93% prevalence reported by Lindell et al. (2002). The finding that ectoparasite count was

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Texas Tech University, Jessica L. Herzog, May 2020 not different between orders or counties (study sites) is supported by those of Bush and Clayton (2006) and Pistone (2016), respectively.

The results for each host species differ slightly from those of other researchers. First, considering passerines, a single Eutrombicula spp. mite was found in a Northern Cardinal, which species representative E. cinnabaris was reported in the Barrier Islands (Wilson and Durden, 2003). However, lice were not found as they were in the study by Pistone (2016). He documented M. eurysternus, Myrsidea spp., and B. pallidula. Findings in Curve-billed Thrashers were similar, as six Eutrombicula spp. were found in a single host but lice such as B. dorsale (Price et al., 2003, Pistone, 2016) or B. pallidula (Price et al., 2003) were not found. Finally, Goniodes spp., Brueelia spp., and Menacanthus spp. were documented in Northern Mockingbirds. While no additional documentation of Goniodes spp. in this host was available at the time of this study, Price et al. (2003) noted reports of B. brunneinucha and M. eurysternus.

Second, the results for quail differ slightly from those of other researchers, but more relevant prevalence values in terms of the study areas and method of lice collection were available to use for comparison. There was a maximum prevalence of 50% infection in Scaled Quail, while Howard (1981) found a higher maximum prevalence of 85%. Infection of Goniodes spp, and Menacanthus spp. was documented, which was supported by Wallmo (1956) observing G. squamatus and Menacanthus spp. in Scaled Quail. Oxylipeurus spp. was also found, which was supported by a finding of O. callipeplus by Wiseman (1959). However, there was no documentation of Colincola spp., as Emerson (1950) and Wallmo (1956) had when they found Colincola pallidua and C. numidianus, respectively. For Bobwhite, a Goniodes spp. prevalence of 75% was documented, followed by Lipeurus spp. infection (50%), and Menacanthus spp. (28.1%). Price et al. (2003) documented G. ortygis and L. caponis infection in Bobwhite. These findings are somewhat similar to Bergstrand and Klimstra (1964), who examined 339 Bobwhite in Illinois and found a high (91%) prevalence of G. ortygis as well, followed by a 43% prevalence of

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Menacanthus spp. Also, Doster et al. (1980) surveyed approximately 40 Bobwhite and found a prevalence of 47% for G. ortygis, and 13% for M. pricei. In contrast, there was no Oxylipeurus spp. infection in Bobwhite. However, Price et al. (2003) found such infection, as well as Bergstrand and Klimstra (1964) with their finding of 93% prevalence of O. clavatus. Doster et al. (1980) found this same species at a prevalence of 63%. A 3.1% prevalence of Eutrombicula spp. was documented for mites, whose species representative E. aifreddugesi alfreddugesi was documented by Doster et al. (1980). The genus was not documented by Bergstrand and Klimstra (1964) or Price et al. (2003). Instead, the latter pair found an 81% prevalence of Megninia spp. mites. Doster et al. (1980) also found infection of ticks (Ixodida), which this study did not replicate.

The findings of this study may differ from those of other researchers due to the constraints of the study and available data. For example, the overall prevalence finding may have been influenced by the dissimilar climate of the study area to those of the other studies or may have been impacted by the host species utilized versus those examined by the other researchers. Lice infection is known to be shaped by aspects of the local geography climate (Moyer et al., 2002), while specific host associations are typical (Whitaker, 1988). Also, feather ruffling was used rather than the dusting method used by some researchers. Collection method is known to impact how many and what type of lice are obtained (Whitaker, 1988). Additionally, the arrant lack of ectoparasite surveys for the Rolling Plains Ecoregion means that there are few data to compare the findings of this study with. This fact and other researchers reporting up to 31 previously undocumented host-louse associations (Wilson and Durden, 2003, Sari et al., 2013, Pistone, 2016) mean that it is not out of the realm of possibility for the finding of Goniodes spp. in a Northern Mockingbird, for example, to be a new documentation rather than a misidentification. Simply not enough data exists to be able to tell.

Like those of the ectoparasite portion of this study, the haemoparasite results show mixed support for those of other researchers. For example, one Scaled Quail was

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Texas Tech University, Jessica L. Herzog, May 2020 found to be infected only with Haemoproteus spp. In contrast, Greiner et al. (1975) found Haemoproteus spp. and Plasmodium spp. in 31 out of 134 Scaled Quail and Hungerford (1955) found infection in 32 out of 111 birds. An overall haemoparasite prevalence of 10.7% was found in Bobwhite, which is similar to the findings of Xiang et al. (2017) who documented 0% prevalence upon examining 400 blood smears and Crook et al. (2009) who also found 0% prevalence after examining 294 blood smears and conducting polymerase chain reaction (PCR) on an additional 30 blood samples. Greiner et al., (1975) reported haemoparasites in only 17 out of 947. In contrast, the findings of 0% prevalence in Northern Cardinals do not reflect the findings of microfilaria (Love et al. 1953) or a 43% prevalence across 342 Northern Cardinals examined (Greiner et al., 1975), but mirror those of Gentry (2013), who found 0% prevalence of plasmodium after examining 63 smears. The findings of 0% haemosporidian prevalence in Curve-billed Thrashers contradict those of Fokidis et al. (2008) who reported a prevalence of 39% in 23 individuals. Finally, an 85.7% haemoparasite prevalence was documented in Northern Mockingbirds, which included microfilaria. While Love et al. (1953) also documented microfilaria, they found a lower prevalence (25%) after examining blood smears from 36 birds. Despite the inconsistencies in the other findings of this study, coinfection was documented as it has been previously (Valkiūnas, 2005). Coinfection of Plasmodium spp. between Bobwhite and Northern Mockingbird was observed, as well as that of Haemoproteus spp. between Scaled Quail and Northern Mockingbird.

Contradictory findings for each species and that of a low haemoparasite prevalence of 20.8% across sampled birds may have been the result of poor technique. For example, inconsistent and inadequately sized smears were examined, when the ideal blood smear should be thin and extend up to or slightly past half the length of the slide (Woronzoff-Dashkoff, 2002). Small smears present in the sample may have physically reduced the available blood that could contain haemoparasites, thus decreasing the likelihood of detection. Furthermore, examination of at least 50,000 red blood cells is required to determine parasite prevalence (Valkiūnas et al., 2008b), but

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Texas Tech University, Jessica L. Herzog, May 2020 this amount may not have been present on particularly small smears or addressed via examining a proportion of each smear used for oil immersion.

The results of this study may be also explained by the small sample sizes coincidentally only encompassed the uninfected proportion of the population for each species. More likely, sample size skewed the host species’ actual infection levels. Furthermore, the nature of infection must be considered. For instance, the quail, Curve-billed Thrashers, and Northern Cardinals examined may have harbored too low of infection to detect microscopically, kept reduced by their immune responses to infection (Valkiūnas, 2005, Fokidis et al., 2008, Valkiūnas et al., 2014). In contrast, because all of the Northern Mockingbirds sampled were juveniles, they may not have had time to develop their immune systems to the point of reducing the infection to the chronic, less detectable levels that has been reported in adults (Valkiūnas, 2005, Bosholn et al., 2010), thereby not reflecting the low prevalence reported by Love et al. (1953). Finally, a lack of haemoparasite studies in these host species (Tweit, 1996, Peterson 2007, Gentry, 2013, Walstrom and Outlaw, 2017) complicates determining if the results presented within are typical or an anomaly. Hence, the findings may not be unreasonable for the host or region, especially for Northern Cardinals (Gentry, 2013) and Bobwhite (Xiang et al., 2017).

This study contributes data of ecto and haemoparasitism to that collected during the few existing surveys for select quail and passerine species, particularly in the Rolling Plains Ecoregion. More ecto and haemoparasite surveys such as this will help future researchers alleviate some of the issues encountered in this study, including addressing the discrepancies in this study’s findings. Future studies may incorporate molecular identification of ecto and haemoparasites. Studies that expand upon the preliminary data collected for this study will help researchers better understand the idea of lice as intermediate hosts (Pistone et al., 2018), the influences of lice on body condition (Stenkewitz et al., 2017), and the impact haemoparasites may have on host’s immunity (Møller and Rozsa, 2005, Valkiūnas, 2005).

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32. Kelly, E.J., Baldwin, T.J., Frame, D.D., Childress, A.L., Wellehan, J.F., 2018. Haemoproteus (Parahaemoproteus) spp. in captive-bred bobwhite quail (Colinus virginianus) in southern Utah, USA. J. Wildl. Dis. 54, 726-733.

33. Khan, J.S., Provencher, J.F., Forbes, M.R., Mallory, M.L., Lebarbenchon, C., McCoy, K.D., 2019. Parasites of seabirds: a survey of effects and ecological implications. Adv. Mar. Biol. 82, 1-50.

34. Lindell, C.A., Gavin, T.A., Price, R.D., Sanders, A.L., 2002. Chewing louse distributions on two Neotropical thrush species. Comp. Parasitol. 69, 212-217.

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36. M. Borst, personal communication, 29 Oct 2019.

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39. Moyer, B.R., Drown, D.M., Clayton, D.H., 2002. Low humidity reduces ectoparasite pressure: implications for host life history evolution. Oikos. 97, 223-228.

40. O'Roke, E.C., 1930. The morphology, transmission and life-history of Haemoproteus lophortyx O'Roke, a blood parasite of the California valley quail. Univ. Calif. Publ. Zool. 36, 1-50.

41. Owen, J.C., 2011. Collecting, processing, and storing avian blood: a review. J. Field Ornithol. 82, 339-354.

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43. Peterson, M.J., 2007. Diseases and Parasites of Texas Quails. In: Texas Quails: Ecology and Management. Brennan L, editor. Texas A&M University Press, College Station, USA, pp. 89-114.

44. Pistone, J.P., 2016. Evaluation of blood gas analytes and ectoparasites from South Texas birds. Doctoral dissertation, Texas A&M University, Texas, USA.

45. Pistone, D., Lindgren, M., Holmstad, P., Ellingsen, N.K., Kongshaug, H., Nilsen, F., Skorping, A., 2018. The role of chewing lice (Phthiraptera: Philopteridae) as intermediate hosts in the transmission of Hymenolepis microps (Cestoda: Cyclophyllidea) from the willow ptarmigan Lagopus lagopus (Aves: Tetraonidae). J. Helminthol. 92, 49-55.

46. Price, R.D., Hellenthal, R.A., Palma, R.L., Johnson, K.P., Clayton, D.H., 2003. The chewing lice: world checklist and biological overview. Illinois Natural History Survey Special Publication. 501 pp.

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49. Rollins, D., 2007. Quails on the rolling plains. In: Brennan L. (Ed.), Texas quails: ecology and management. Texas A&M University Press, College Station, USA, pp. 117-141.

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50. Rush, E.M., Wernick, M., Beaufrère, H., Ammersbach, M., Vergneau-Grosset, C., Stacy, N., Pendl, H., Wellehan Jr, J.F., Warren, K., Le Souef, A., Cooey, C., 2016. Advances in clinical pathology and diagnostic medicine. In: current therapy in avian medicine and surgery. WB Saunders Press, Philadelphia, USA. pp. 461-530.

51. Sauer, J.R., Hines, J.E., Fallon, J.E., Pardieck, K.L., Ziolkowski, Jr., D.J., Link, W.A., 2013. The North American breeding bird survey, results, and analysis 1966–2013. USGS Patuxent Wildlife Research Center, Laurel, MD, USA.

52. Smith, S.V., 2004. Lousy Lists. Syst. Biol. 53, 666-668. https://doi.org/10.1080/10635150490468521

53. Smith, V.S., Rycroft, S. Phthiraptera.info. Checklist dataset https://doi.org/10.15468/amm7zq. Accessed January 2020.

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57. Valkiunas, G., 2005. Avian malaria parasites and other haemosporidia. CRC Press, Boca Raton, FL, USA.

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62. Valkiūnas, G., Palinauskas, V., Ilgūnas, M., Bukauskaitė, D., Dimitrov, D., Bernotienė, R., Zehtindjiev, P., Ilieva, M. and Iezhova, T.A., 2014. Molecular characterization of five widespread avian haemosporidian parasites (Haemosporida), with perspectives on the PCR-based detection of haemosporidians in wildlife. Parasitol. Res. 113, 2251-2263.

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65. Walstrom, V.W., Outlaw, D.C., 2017. Distribution and prevalence of haemosporidian parasites in the northern cardinal (Cardinalis cardinalis). J. Parasitol. 103, 63-68.

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CHAPTER IV

CONCLUSIONS AND FUTURE DIRECTIONS

Conclusions

Prolonged, steady declines of Bobwhite (Sauer et al., 2013) have concerned and perplexed hunters, landowners, and researchers alike. Disease and parasitism were overshadowed by poor habitat conditions as potential reasons for the declines, and consequently overlooked as they have been historically (Friend, 2014, Gehman et al., 2019). However, stable habitat and a growing acceptance over the past decade of the need to study the impact of parasitism on individual Bobwhite and their populations has led to more parasite surveys and studies (e.g. Dunham et al., 2014, Dunham and Kendall, 2017, Bruno et al., 2019). Such research has led to documentations of heavy eyeworm and caecal worm infection in Bobwhite (Rollins, 2007, Dunham et al., 2014), as well as potential pathological consequences of infection (Dunham et al., 2014, Bruno et al., 2019). Furthermore, eyeworm infection in Scaled Quail and certain passerines living in the same study areas as captured Bobwhite has also been documented (Dunham and Kendall, 2014, 2017), suggesting that additional parasites may be shared between avian species. Although shared parasitism of eyeworms has been documented between quail and passerines, a stark lack of parasitological surveys for passerines and Scaled Quail in the Rolling Plains Ecoregion leaves the true extent of shared parasitism open to further examination. More complete, targeted parasite surveys are needed to ease knowledge gaps of helminth, ectoparasite, and haemoparasites infection (Galloway, 2019, Gehman et al., 2019), particularly in Rolling Plains birds. Conducting such surveys requires researchers to address difficulties inherent to parasite preparation (De Ley et al., 2005) and identification (Smith, 2004, De Ley et al., 2005, Valkiūnas et al., 2014) and capture as many species of hosts as possible. Progressing the research will require future studies to pose novel questions about parasites themselves (e.g. Pistone et al., 2018, Kalyanasundaram et al., 2019), as well as studies that investigate and

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Texas Tech University, Jessica L. Herzog, May 2020 perfect molecular techniques (De Ley et al., 2005, Wang, et al., 2018). Doing so will help address the many shortcomings of past and current studies and provide ample opportunities for collaboration and publication by future researchers with a variety of research backgrounds. As such, this thesis was written to begin to address the current scope of parasitological surveys for quail and passerines in the Rolling Plains Ecoregion and provide suggestions for improvement and potential directions for future researchers. Chapter 2 addresses previously documented helminth infections in quail and passerines and outlines a complete helminth survey conducted on targeted hosts with the intention of closing historical data gaps concerning helminth infection in the study birds. Shared infection of eyeworms was documented between quail and passerines and females of all study species tended to have a higher average infection of helminths aside from eyeworms and caecal worms in comparison to males. Rare findings included Mesocestoides spp. tetrathyridium in Bobwhite and an acanthocephalan in a Northern Mockingbird. Increasing the sample size of hosts captured would lead to more helminths being obtained, thus increasing the range of possible statistical analysis, and improving the comparability of studies and accuracy of future researchers’ results and conclusions. Chapter 3 outlines an investigation of ecto and haemoparasite infection in hosts, thereby completing the parasite survey for each host and contributing to the pronounced lack of such surveys in the Ecoregion. The findings of a 72% prevalence of ectoparasites across hosts and a 20.8% prevalence of haemoparasites along with documentation of specific genera for each parasite group help to close knowledge gaps concerning ecto and haemoparasite infection in quail and select passerines. Collecting more hosts and improving parasite preparation and identification techniques would improve the quality of the data such that more rigorous statistical analyses could be conducted. Finally, Chapter 4 is a general conclusion of the previous chapters, as well as a note to help future researchers. Future researchers would benefit from collecting more hosts and working with experienced parasitologists to improve their parasite processing and identification skills before working with specimens. Furthermore, 83

Texas Tech University, Jessica L. Herzog, May 2020 suggestions to take future research are presented, including bolstering reference collections and digital databases and using and refining molecular techniques for nonlethal assessment of infection and identification of specimens. Although parasitological studies can be challenging, the field is prime for additional studies which will inherently foster collaboration and lead to a multitude of pioneering publications. In conclusion, this thesis presents the challenges and results from conducting full parasite surveys on Bobwhite, Scaled Quail, Curve-billed Thrashers, Northern Cardinals, and Northern Mockingbirds in the Rolling Plains Ecoregion. Although recent helminth surveys for Bobwhite have been conducted, few full parasite surveys exist for them and the other study species in the Ecoregion. However, documenting shared infection is important to begin to understand the implications of infection for a range of host species. Conducting full parasite surveys will help researchers investigate the influences of single species infection and coinfection on individual hosts and their populations. The continued study of parasitism is not only important for elucidating potential causes of Bobwhite decline, but also to advance this challenging field whose prevalent knowledge gaps impact wildlife in countless ways.

Future Directions

Parasites in wildlife have been studied for generations, but the field is riddled with shortcomings and data gaps. Thus, more research in the form of complete, targeted parasite surveys is paramount. Addressing the data gaps and shortcomings offers many opportunities for collaboration and groundbreaking papers. Future researchers must improve their personal parasite processing and identification skill sets and techniques with the intention of further processing and documenting the wet- stored specimens obtained herein. Additional studies would help increase researchers’ understanding of parasites themselves, such as their lifecycles and seasonal prevalence, as well as what impacts infection of individual species and communities of parasites could have on hosts. Finally, advancing molecular techniques would potentially alleviate issues associated with identification encountered in this study and 84

Texas Tech University, Jessica L. Herzog, May 2020 facilitate infection assessment without euthanasia, provided the shortcomings of molecular identification techniques are addressed.

The results of this study were negatively impacted by a lack of expertise in slide creation and parasite identification. Utilizing different techniques in the future may lead to more complete results. First, collecting more hosts would likely increase the number of parasites obtained, which would in turn provide researchers more specimens to work with and an increased margin of error during processing. Also, future researchers should only obtain helminths from recently euthanized hosts. Doing so greatly reduces parasite damage (Sepulveda and Kinsella, 2013) and improves the effectiveness of further processing steps, ultimately leading to better-quality specimens and slides (Berland, 1984, Sepulveda and Kinsella, 2013). For haemoparasites, collecting both venous and heart blood may impact the type and detection of haemoparasites found. This is evidenced by Love et al. (1953) who noted a 4-fold increase in microfilaria detection when using heart blood over venous to make smears. Finally, regardless of the parasite group being studied, professionals should be consulted and shadowed to perfect preparation techniques on practice specimens before the study begins. The professionals’ experience-based advice to prevent over- staining or under-clearing of helminths, crystallization of ectoparasite mounting media, or cell sloughing on smears is best learned in person and is not readily available in the literature or in educational videos (De Ley et al., 2005). Conversations and interactions with professional parasitologists are imperative for correct and permanent preservation of usually delicate specimens and for achieving quality slides that are invaluable for both current researchers and those who come decades later (Huber, 1998).

After learning the proper skills to collect and prepare parasites, future researchers could take a subset of the remaining helminths and ectoparasites from this study that are stored in 70% ethanol and properly mount them. Doing so would result in producing quality slides that could be submitted to a museum and thereby help

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Texas Tech University, Jessica L. Herzog, May 2020 other researchers circumvent limited or no access to reference slides of certain parasites. Future researchers could also take photos of each parasite and label them with proper measurements. These photos could then be digitized and shared publicly with the intention of creating a digital reference database such as phthiraptera.info or bolstering the Texas Tech Natural Science Research Laboratory (NSRL) or Smithsonian Museum of Natural History (NMNH) Department of Invertebrate Zoology Collection Databases. Many specimens in the NSRL are not digitally catalogued and those that are tend not to have reference photos associated with them (H. Garner, personal communication, 21 Nov 2019). The proportion of records without photos is even greater for the approximately 50 million catalogued records of invertebrates in the NMNH database (NMNH, 2020). Having access to quality photos would increasing the utility of both the NSRL and NMNH collections and lessen NMNH’s need to ship rare slides up to 100 years old to researchers across the globe, thereby reducing the risk of damage to original specimens and slides.

In addition to better documentation of helminths, future researchers can broaden their understanding of helminths themselves. For example, future studies could continue to look into the lifecycles of nematodes including eyeworms and caecal worms, which are largely unknown (Kistler et al., 2016a, Dunham et al., 2017.) and can be achieved via experimental microscopy and molecular studies (Stenkewitz et al., 2017). Researchers may examine the development of helminths in their hosts, as Kalyanasundaram et al. (2019) did during the first study of its kind for eyeworms. Researchers may also choose to investigate how seasonal helminth prevalence changes, as Blanchard et al. (2019) did for eyeworms and caecal worms or choose to fill gaps in host and parasite collection like Bruno et al. (2019). Studies such as these are important for increasing researchers’ understanding of host-parasite relationships on a temporal scale.

Future researchers can continue to study ecto and haemoparasites as well. For example, mallophagan lice infection intensity has been linked to the size of the

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Texas Tech University, Jessica L. Herzog, May 2020 uropygial gland of Rock Ptarmigan (Lagopus muta) from which the bird draws oil from during preening to keep its feathers in good condition (Gonzalez, 2014). Furthermore, Amyrsidea lagopi infection intensity specifically has been correlated with the number of holes present in Rock Ptarmigans’ tail feathers (Stenkewitz et al., 2017). While the extent of potentially negative physiological impacts of lice infections must still be elucidated by future researchers (Stenkewitz et al., 2017), lice infections from a variety of species may decrease the body condition and flight capability of affected hosts. Additionally, researchers may investigate the idea of lice as intermediate hosts such as the study conducted by Pistone et al. (2018) suggesting that certain lice are intermediate hosts for infection of the cestode Hymenolepis microps parasitizing Willow Ptarmigan (Lagopus lagopus). Finally, future researchers may investigate how haemoparasite infection influences host immunity and susceptibility to other diseases or infections (Valkiūnas, 2005).

Continuing to advance and refine techniques in molecular parasitology will be another valuable addition to future parasitological studies. Molecular techniques would further improve upon traditional parasite identification techniques and reduce error, particularly for ecto and haemoparasite samples. Researchers must devote ample time to collected specimens when utilizing historical identification techniques. Fixing and staining blood smears takes at least an hour per set, while a researcher may spend days to clear, stain, dehydrate, and mount lice and helminths (Whitaker, 1988, Sepulveda and Kinsella, 2013). Additional time must be spent for identification, which can be anywhere from minutes to hours depending on the parasite group and skill level of the investigator (e.g. Wang, et al., 2018). In contrast, molecular techniques may facilitate faster identifications because researchers could identify ecto and haemoparasites from whole blood (Walstrom and Outlaw, 2017) or homogenized ectoparasite samples (Nielsen, et al., 2019), completely eliminating the need and associated time to make blood smears or mount individual specimens. Furthermore, using traditional methods does not always result in accurate identifications, but molecular techniques can be more reliable, especially for inexperienced researchers

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Texas Tech University, Jessica L. Herzog, May 2020 and those whose time is limited (Valkiūnas et al., 2014, Wang, et al., 2018). Finally, conducting polymerase chain reaction (PCR) on whole blood may lead to a higher detection and more accurate identification of haemoparasites, even for samples containing haemosporidia in early developmental stages (Valkiūnas, 2005, Valkiūnas et al., 2008, Walstrom and Outlaw, 2017). Molecular techniques can also be standardized, whereas traditional ecto and haemoparasite studies utilize a broad range of parasite collection and detection methods (Whitaker, 1988, Ishtiaq et al., 2017) to obtain their results, potentially rendering findings and conclusions incomparable to those of other supposedly similar studies.

Molecular techniques may also facilitate nonlethal assessment of helminth infections. For example, fecal floats are traditionally used to detect parasites by identifying eggs found in hosts’ feces, but the challenges of identification may lead to misidentification (Zajac and Conboy, 2012). Also, the technique is difficult to employ in field conditions (Kistler et al., 2016b). However, molecular techniques such as quantitative PCR (qPCR) can be used to accurately identify parasite eggs, including those of eyeworms and caecal worms in feces or on cloacal swabs obtained in the field (Kistler et al., 2016b, Blanchard et al., 2018), thereby helping researchers nonlethally assess parasite infection in Bobwhite. Additional research has shown that increasing detectability of parasite eggs via qPCR is correlated to increased eyeworm and caecal worm infection levels in Bobwhite (Kalyanasundaram et al., 2018). Thus, molecular techniques can enhance the utility of traditional methods and enable quantification of parasitic infections, both in laboratory and field settings, even on a regional scale (Blanchard et al., 2018).

Although molecular identification of nematodes via PCR is possible, the study is inherently challenging (De Ley et al., 2005, Prosser et al., 2013). Researchers experienced complications firsthand when using trial and error methods to identify using molecular techniques, a subset of helminths collected from Bobwhite examined for the helminth survey portion of the current study. DNA was extracted from each of

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Texas Tech University, Jessica L. Herzog, May 2020 five nematodes using the DNeasy Blood and Tissue Kit (Qiagen, Germany). A 10 μL PCR reaction was performed with 5 μL Red Dye Master Mix (Bioline, England), 0.5 μL of forward and reverse universal nematode primers (Nem 1, Nem 2, Nem 3) (Prosser et al., 2013), and 1 μL of templates of each of the nematode’s extracted DNA and 3 μL of nuclease free water. Amplified PCR products were checked through 1.5% agarose gels stained with ethidium bromide (Invitrogen, USA) and visualized by BioRad GelDoc XR system (BioRad, USA).

First, a series of gradient PCRs were performed with the goal of amplifying a portion of the cytochrome oxidase I gene (COX1). Gradients ranging from 45-65°C were used to determine an appropriate annealing temperature. Subsequent PCR reactions resulted in a 750 base pair amplification of Sample 83, a nematode from the proventriculus of a Bobwhite (see Figure 4.1). Amplified PCR product was purified with the GenElute PCR Clean-Up Kit (Sigma, USA) and sequenced at Genscript USA Inc. BLAST results revealed that the PCR products for Sample 83 have a high similarity (83%) to nematodes from order spirurida at the nucleotide level.

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Figure 4.1. Partial PCR amplification of COX1 for Sample 83 using universal nematode primers. Lane M: 100 bp DNA ladder; lanes 1 and 2: approximately 750 base pair amplification of Sample 83. Lames 3-5: unamplified nematode samples; lanes Adapted from (Kalyanasundaram et al., 2017).

Finally, the evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model (Tamura and Nei, 1993). The tree with the highest log likelihood (-13542.29) is shown (see Figure 4.2). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 27 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. There were a total of 1654 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

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Figure 4.2. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model. The tree with the highest log likelihood (-13542.29) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 27 nucleotide sequences. There were a total of 1654 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.

While the results were obtained from Sample 83, Sample 77, a nematode found in the proventriculus of a Curve-billed Thrasher, amplified during test gradients but a good quality amplicon was not obtained after gel purification with the QIAquick PCR

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Purification Kit (Qiagen, Germany). Thus, the original amplification of Sample 77 may have been a pseudo amplification. Because results were obtained for only one out of five helminth samples, more preliminary testing and troubleshooting is needed to eventually obtain the sequences needed to identify extracted helminth DNA to genus or species. However, the series of trial and error done by the researchers caused them to deplete the extracted DNA down to 3μL and completely exhaust the whole helminths not treated for microscopy so future research will need to be conducted on new helminth samples.

The success rate of molecularly identifying nematodes to species via PCR is approximately 50% (De Ley et al., 2005) so it is not surprising that researchers encountered instances of non and pseudo amplifications. The low success rate and results of this pilot study may have been associated with the choice of primers, a crucial element for achieving DNA amplification. The correct primers will bind to a certain sequence on the DNA; incorrect primers will bind to an undesired region, if they bind at all (Lorenz, 2012, Qu and Zhang, 2015). However, choosing an appropriate nematode primer is inherently difficult because no COX1 primers span the entire nematode phylum and many do not enable identification of nematodes to species (De Ley et al., 2005). Given the diversity of nematode mitochondrial genomes, COX1 may not be the most appropriate gene to use for nematode phylogenetic studies (De Ley et al., 2005) and researchers should consider designing a primer for the internal transcribed spacer 1(ITS1) region (e.g. Henry et al., 2020). Nonetheless, the broadest choice of primers researchers have for nematode studies are the “cocktail” nematode primers developed by Prosser et al. (2013) to target the COX1 gene of three orders and 8 families of nematodes.

Researchers interested in working with nematodes from families not addressed by the cocktail primers may look to molecular studies conducted on the same microscopically identified nematodes as they wish to extract DNA from. Such a strategy will presumably allow researchers to circumvent trial and error and choose a

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Texas Tech University, Jessica L. Herzog, May 2020 more specific primer with a better chance of amplification. However, a notable lack of available data, including in published sequence databases (De Ley et al., 2005, Nadler et al., 2007, Guo et al., 2019) renders such attempts futile for the Tetramerid and Subulurid nematodes used in this pilot study. For example, the only known primer for Tetrameres comes from a single documentation of Tetrameres fissispina in a Common Eider (Nadler et al., 2007). Using this primer instead of the Nem primers may have resolved the two cases of non-amplification from Tetramerid nematodes. Yet, Sample 83 amplified with the Nem primers, suggesting that a specific Tetrameres primer is not necessary. Also, to date, Aulonocephalus pennula is the only nematode out of 60 known species in the Subuluroidea superfamily (Baruš et al., 2013) that has been molecularly characterized using the Nem primers (Kalyanasundaram et al., 2017, Guo et al., 2019). Thus, these primers should have amplified the DNA from the Subulurid nematode, but they did not and there are no other appropriate primers to address this case. Choosing the best primer for the nematode DNA at hand is clearly challenging and must be addressed by future researchers to avoid additional instances of non and pseudo amplification and inconsistent results.

Future studies may use different techniques or parameters to potentially achieve better results. First, DNA should be quantitated with a NanoDrop or Qubit system before conducting further studies (Desjardins and Conklin, 2010). Using DNA with impurities such as proteins and organic compounds present or using DNA that contains nucleotides of insufficient mass, can have substantial negative impacts on the results of subsequent PCR reactions (Lorenz, 2012). Also, the presence of magnesium chloride (MgCl2) plays an important role in allowing DNA polymerases to function (Lorenz, 2012). Its concentration in the PCR reaction can easily be altered, yet adjustment can have the most pronounced impact of all reagents on the outcome of PCR reactions. Unsatisfactory concentrations of magnesium can prevent the reaction from occurring entirely or it can result in binding of the primers to undesirable sites on the DNA (Lorenz, 2012). Finally, future researchers should conduct a variety of test PCR reactions to determine the appropriate parameters for thermocycling, including

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Texas Tech University, Jessica L. Herzog, May 2020 annealing temperature (Rychlik et al., 1990, Lorenz, 2012). Temperatures and durations chosen for each step in the thermocycling program vary for each molecular study of nematodes (e.g. Prosser et al. 2013, Kalyanasundaram et al., 2017) and will influence the subsequent PCR reaction. High temperatures or excessive durations at certain steps can inactivate reagents and cause the PCR reaction to fail (Lorenz, 2012). Furthermore, although the researchers used temperature gradients, they may still have used an inappropriate annealing temperature during their PCR reactions. A range of annealing temperatures is available (Lorenz, 2012), but even when the best primers are chosen, too low of an annealing temperature may cause the primer to bind to nonspecific DNA sequences and cause pseudo amplification, while an excessively high temperature may not allow the primer to bind at all, leading to a non- amplification (Rychlik et al., 1990). Ultimately, future researchers would benefit from obtaining a greater volume of helminth DNA from each species of nematode before conducting another pilot study to address the aforementioned shortcomings and complications inherent to current helminth molecular identification protocols.

Although properly addressing the shortcomings aforementioned are outside the scope of the current study and molecular pilot study, doing so provides future researchers with ample opportunities. Traditional parasite preparation and slide creation techniques are the foundation of parasitology, but these techniques are misunderstood or unknown to many researchers. Improving parasite preparation and slide creation techniques will bolster museum collections with sides that last for decades, benefitting countless amateur and professional scientists and researchers (Huber, 1998), while creating a photo database of parasite specimens could have a world-wide reach. By sharing their knowledge of learning and improving traditional techniques, researchers will foster collaborations and community. Furthermore, molecular parasitologists are breaking new ground each day to address their own research questions and build the foundations of this rapidly growing field. Molecular parasitology is gaining interest from researchers whose backgrounds include host and human health (Ricklefs and Fallon, 2002, Cannell et al., 2013), vectors (Pistone et al.,

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2017), and parasite detection (Valkiūnas et al., 2014), lifecycles (Kalyanasundaram et al., 2019), and evolution (Ricklefs et al., 2004). Collaboration between a variety of researchers will help increase the current parasitological knowledge base and unite the fields of traditional and molecular parasitology (Valkiūnas et al., 2014), amplifying researchers’ understanding of both fields and creating infinite opportunities for new questions, projects, and published work.

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References

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23. Kalyanasundaram, A., Blanchard, K.R., Henry, C., Brym, M.Z., Kendall, R.J., 2018. Development of a multiplex quantitative PCR assay for eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) detection in Northern Bobwhite quail (Colinus virginianus) of the Rolling Plains Eco- Region, Texas. Vet. Parasitol. 253, 65-70.

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APPENDIX A: HELMINTH PHOTOS

Figure 4. Reddish nodes on the proventriculus of a Northern Cardinal. Three of the nodes each contained 1 Tetrameres spp. nematode.

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Oxyspirura petrowi

Figure 5. The anterior end of an eyeworm from a Bobwhite. Photo courtesy of Aravindan Kalyanasundaram.

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Raillietina spp.

Figure 6. Stained proglottids of a Raillietina spp. cestode from a Bobwhite.

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Figure 7. The scolex of a Raillietina spp. cestode from a Bobwhite. Note the hooks used to identify species of the genus. Photo courtesy of Mike Kinsella.

Figure 8. A genital pore of a Raillietina spp. cestode from a Bobwhite. Photo courtesy of Joe Luksovsky.

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Mesocestoides spp. Tetrathyridium

Figure 9. A scolex of a Mesocestoides spp. tetrathyridium from a Bobwhite. Photo courtesy of Joe Luksovsky. Tetrameres pattersoni

Figure 10. The posterior end of a Tetrameres pattersoni nematode from a Bobwhite. Photo courtesy of Mike Kinsella. 105

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Figure 11. The anterior end of a Tetrameres pattersoni nematode from a Bobwhite. Photo courtesy of Mike Kinsella.

Figure 12. Spines on a Tetrameres pattersoni nematode from a Bobwhite. Photo courtesy of Mike Kinsella.

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APPENDIX B: ECTOPARASITE PHOTOS

Figure 13. Lipeurus spp. from a Bobwhite. The slide also has crystallization from a reaction of the Hoyer’s Medium with the sealant.

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Figure 14. Goniodes spp. from a Bobwhite.

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Figure 15. Menacanthus spp. from a Bobwhite.

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APPENDIX C: HAEMOPARASITE PHOTOS

Figure 16. Erythrocytes with water damage incurred during preparation. The damage resembles Plasmodium spp.

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Figure 17. Haemoproteus spp. in erythrocytes from a Scaled Quail.

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Figure 18. Plasmodium spp. in erythrocytes from a Bobwhite.

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Figure 19. Microfilaria amidst erythrocytes from a Northern Mockingbird.

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