ECOLOGICAL ASPECTS of CHEWING LICE in a NORTHERN MICHIGAN POPULATION of BROWN-HEADED COWBIRDS Emily Shea Durkin Northern Michigan University

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ECOLOGICAL ASPECTS OF CHEWING LICE IN A NORTHERN MICHIGAN POPULATION OF BROWN-HEADED COWBIRDS

Emily Shea Durkin

THESIS

Submitted to Northern Michigan University In partial fulfillment of the requirements For the degree of

MASTER OF SCIENCE

Office of Graduate Education and Research

2013

ABSTRACT

ECOLOGICAL ASPECTS OF CHEWING LICE IN A NORTHERN MICHIGAN

POPULATION OF BROWN-HEADED COWBIRDS

Emily S. Durkin

Brown-headed cowbirds are brood parasites and come in direct contact with a greater number of bird species than non-brood-parasitic birds. The male and female sub populations as well as the young and older male subpopulations engage in different behaviors that could affect chewing louse communities. 401 brown-headed cowbirds collected from northern Michigan were examined for chewing lice. Chewing louse prevalence, intensity and species richness were compared between male and female cowbirds. Males had significantly greater louse prevalence and intensity compared to females (65% and 49% respectively, p<0.01; Mdn=8 and 5 respectively, p=0.08). Males and females had similar species richness. Chewing louse prevalence and intensity were also compared between young and older male brown-headed cowbirds. Young males had significantly greater louse prevalence and older males had significantly greater louse intensity when compared to each other (72% and 61% respectively, p=0.05; Mdn=5.5 and

9 respectively, p=0.03). Five different chewing louse genera were collected from brown- headed cowbirds: Brueelia , Philopterus , Machaerilaemus , Myrsidea and Menacanthus .

However, Brueelia made up the overwhelming majority of lice collected.

Emily Shea Durkin

July 1, 2013

DEDICATION

I dedicate this thesis to my family who encouraged and nurtured my desire to explore since day one.

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ACKNOWLEDGMENTS

I would like to thank the following people who made this study possible.

Dr. Jackie Bird: I cannot express my appreciation for your time, guidance, patience and understanding as my thesis advisor. You taught me so much. You surpassed your duties as an advisor and helped make this experience an enjoyable adventure!

Dr. Patrick Brown and Jill Leonard: For your time, knowledge and encouragement. I would also like to thank you for standing in for Jackie, signing papers and helping me out in areas Jackie could not after she retired.

Daniel Beckman: For your help collecting birds and your company while I collected and identified lice. I would also like to thank you and your family for everything you did to help make my life a bit easier during hectic times.

United States Fish and Wildlife Service, Daniel Elbert and Trappers: For your time, and knowledge. You made the bird collection process a pleasure!

Jim Beckman Family: For letting my volunteers and me stay at your cottage while trapping; it was spectacular!

Dr. Jason Garvon: For offering your “blasting” technique for louse collection.

William Durkin: For designing and building me two custom bird blasters.

Megan Porter: For your help with bird blasting; you made my life a lot easier.

Dr. Sarah Bush and Emily DiBlassi: For your help in louse identification.

Dr. Ricardo Palma: For your help in louse identification and confirmation.

Suzie Piziali: For your support and encouragement while I was a graduate student at Northern Michigan University.

Fellow Grad students at NMU including Steve Schaar, Kari Farkas, Saya Iwanaga, Emily and Quentin Sprengelmeyer Lexi Goodman and Kurt VanMullekom: For your insight and friendship during my time at Northern Michigan University as a graduate student.

NMU Excellence in Education Grant and NMU Biology Department Development Fund Grant: For financial support of this project.

Marquette Friends including Jesse DeCaire, Emily Schneeberger, Chris and Renee’Moore, The Walkers, Jay DeHut, Anna Eby and Sierra Cheatham: For your friendship and encouragement.

And finally to my family: For your love, support and encouragement.

This thesis follows the guidelines and format prescribed by The Journal of Paraitology.

TABLE OF CONTENTS

LIST OF TABLES ...... vii

LIST OF FIGURES ...... viii

INTRODUCTION ...... 1

OBJECTIVES ...... 10

METHODS ...... 11

RESULTS ...... 18

DISCUSSION ...... 21

SUMMARY AND CONCLUSIONS ...... 28

LITERATURE CITED ...... 29

APPENDIX A ...... 36

LIST OF TABLES

Table I . Number of male and female brown-headed cowbirds collected and examined from each collection date during 2011 in northern Michigan...... 37

Table II . The number of louse species reported on non-brood-parasitic and brood- parasitic icterid hosts and the number of studies that examined lice on each host...... 37

Table III . The number of infected and examined female, male, young male and older male brown-headed cowbirds collected from northern Michigan in 2011 and their louse prevalence ...... 38

Table V . The number of young and older male brown-headed cowbirds collected from northern Michigan in 2011 infected with lice and their median (range) louse intensity. . 38

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LIST OF FIGURES

Figure 1 . External anatomy of chewing lice...... 40

Figure 2 . Chewing louse reproductive anatomy...... 41

Figure 3 . Enclosures used by United States Fish and Wildlife Service for brown-headed cowbird trapping in northern Michigan...... 42

Figure 4 . Euthanized brown-headed cowbirds ...... 43

Figure 5 . Ventral side of wing from an older (after-second-year) male brown-headed cowbird ...... 44

Figure 6 . Ventral side of wing from a young (second-year) male brown-headed cowbird...... 45

Figure 7 . Brown-headed cowbird orientation for head and neck louse collection ...... 46

Figure 8 . Plastic chamber built by William Durkin and used for louse collection ...... 47

Figure 9 . Brown-headed cowbird being “blasted” for louse collection ...... 48

Figure 10 . Brown-headed cowbird orientation for body louse collection ...... 49

Figure 11 . Chewing louse nymphs at 40X magnification...... 50

Figure 12 . Female abdomen of Brueelia ornatissima at 100x magnification...... 51

Figure 13 . Male abdomen of Brueelia ornatissima at 100X magnification...... 52

viii

Figure 14 . Two female Brueelia ornatissima at 40X magnification ...... 53

Figure 15 . Female Machaerilaemus at 40X magnification ...... 54

Figure 16 . Male Philopterus at 40X magnification...... 55

Figure 17 . Louse species richness of brood-parasitic and non-brood-parasitic icterid hosts. Species richness values were compiled from previous studies...... 56

INTRODUCTION

Lice are true insects belonging to the order Phthiraptera. Four suborders make up

Phthiraptera: Anoplura, Rhyncophthirina, Ischnocera and Amblycera. Members of

Anoplura possess mouthparts for piercing and sucking blood and are termed “sucking lice.” The three remaining suborders are termed “chewing lice” and have mandibles for feeding on keratinized tissues. Rhyncophthirina is made up of only three species, all of which infect mammals (Price et al., 2003). Ischnocera and Amblycera infect both mammals and birds, and those infecting birds are the main focus of this study.

Chewing Lice

Chewing lice feed on their host’s keratinized tissues by shearing and scraping skin and feathers with their mandibles. The resulting feather damage can negatively affect flight (Barbosa et al., 2002), mate attraction (Clayton, 1990; Clayton and Tompkins,

1995) and temperature regulation (Booth et al., 1993). The amount of melanin present in a feather can influence its susceptibility to chewing louse damage. Barrowclough and

Sibley (1980) found the albinistic feathers of a partially albino yellow-rumped warbler were more susceptible to abrasion than its normal feathers suggesting the importance of melanin to feather “toughness.” Further, the feathers making up the white spots of barn swallows contained more holes from chewing lice than the darker feathers (Kose and

Moller, 1999).

Like all chewing lice, avian species have a hemimetabolous life cycle which includes an egg, three nymphal stages and an adult stage. Females possess special glands that produce a cement secretion used to adhere eggs to feathers (Marshall, 1981). The eggs require 4 to 10 days of incubation depending upon species. Each nymphal stage requires 3 to 12 days for completion. Adult chewing lice vary in length ranging from

0.8mm to 11mm with females being larger than males in most species. Nymphs are smaller than adults in size and lack genitalia. Adult lice live for about a month with females producing an average of one egg per day. Chewing lice have no free-living stages, a characteristic that defines as them “permanent residents” (Price et al., 2003;

Roberts and Janovy, 2009).

As permanent residents, chewing lice constantly encounter their hosts’ defenses, and the main defense against avian lice is preening (Nelson and Murray, 1971; Brown,

1972). Preening is a regularly performed grooming behavior during which birds run their feathers through their bills to restructure them, distribute preening oil on them and remove debris and ectoparasites including chewing lice. Generally, it is presumed that birds in poor body condition preen less which could increase louse intensity and species richness (Marshall, 1981). The head and neck regions of birds are not accessible for preening using the bill, an effective way to remove ectoparasites. Some chewing louse species prefer to lay their eggs on their host’s head and neck region more than other regions thereby evading their host’s main defense (Marshall, 1981; Saxena et al., 2012).

Although lice inhabiting these regions avoid preening using the bill, they are vulnerable to their host’s scratching. Clayton (1991) found that birds not capable of scratching had

2 increased numbers of lice and eggs on their heads and necks. Lice can also be dropped from their host while it is molting.

Avian chewing louse morphologies have been associated with a louse’s ability to evade host defenses. Lice with long and slender bodies can slip between the barbs of wing and tail feathers to avoid removal during preening. Species with shorter, squat bodies run away from preening sites; their speed enhanced by the increased muscle power-to-mass ratio, a consequence of decreased size (Gullan and Cranston, 2010).

Traditionally, morphology and host species have been used to classify chewing lice (Price et al., 2003). Head shape, antennae and mouth-parts are good indicators of a chewing louse’s suborder. Members of Ischnocera and Amblycera have blunt, broad heads with mouthparts on the ventral side and 1-2 claws on their tarsi. Ischnocera have fully exposed, filiform antennae while Amblycera have clubbed antennae which are partially to fully concealed in grooves. Amblycerans possess maxillary palps, but

Ischnocerans have lost them. Family can be identified by further morphological description, including abdominal segment number, antenna segment number, spiracle number and location, claw number and location, as well as the segmentation of the mesa- notum and meta-notum (Figure 1). Important species-defining characters include genital morphology (Figure 2), spiracle number and location, and seta number and location. The taxonomy of chewing lice is disorganized because in the past, many entomologists assumed host-louse specificity (Price et al., 2003). Thus, lice with the same morphology as an already defined species found on a different host species were often misidentified, under this assumption, as a new species.

Transmission of chewing lice occurs through direct contact between hosts. For birds, direct contact occurs during copulation, the brooding of young, fights, roosting and foraging. In rare cases, ischnoceran lice travel to new hosts by hitching rides on hippoboscid flies (Keirans, 1975). Unfortunately for lice, some birds recognize lousy birds and will avoid contact. An experimental study showed that lousy rock dove

(Columba livia ) males displayed less than clean males and that females chose clean males as mates significantly more often than lousy males (Clayton, 1990).

Like many parasite populations, chewing louse populations have an aggregated distribution within a host population (i.e., most hosts have few or no lice while few hosts have many lice). More than one louse species can infect a host species. In a study of neotropical birds, species richness ranged from one to nine louse species (Clayton et al.,

1992). Although louse transfer most often occurs within a single host species, not all louse species are confined to a single host species. For example, Menacanthus eurysternus has been found to infect 118 different species of birds belonging to 70 genera and 20 families (Price, 1975). A host’s species richness of lice has not been consistently correlated with any feature of the host’s biology such as age, weight, geographical location and body condition (Clayton and Walther, 2001; Price et al., 2003).

Spatial segregation has been used to infer competition in chewing lice (Clay,

1949). Under conditions of interspecific competition, long term host-parasite associations would result in body region-specific louse species. Different louse species inhabit different regions of their host’s body as well as different microhabitats of their host like the skin, feathers or, in a few cases, within the quills of primary feathers (Marshall,

1981). Spatial segregation has been described in the chewing louse species infecting

4 pigeons. Some of the species restricted their lives to the wings, including egg-laying, while another species was found to lay their eggs on the head of the bird, but nymphs and adult lice were found all over the bird (Nelson and Murray, 1971). The microhabitats on birds has not been measured but each locale likely offers a range of chewing louse habitats which could influence how a louse species is distributed. In shearwaters

(Colonectris diomedea diomedea , C. d. borealis and C. edwardsii ) one louse species was found exclusively on the host’s head while two others heavily populated the host’s body region (Gomez-Diaz et al., 2008). Louse distribution can also be influenced by their host’s defenses. When preening with their bill, birds are able to reach nearly every region of their bodies except their heads and necks. For this reason, the head and neck should be a preferred area for chewing lice to inhabit and multiple louse species have been found to prefer laying their eggs on the head and neck region (Nelson and Murray, 1971; Saxena et al. 2012).

Brown-headed cowbirds

Brown-headed cowbirds ( Molothrus ater ) belong to the blackbird family,

Icteridae. Icterids are medium- to large-sized passerines with long, straight bills, long tails, strong legs and well developed facial muscles for strong bills (Jaramillo and Burke,

1999). There are three recognized subspecies of brown-headed cowbirds: M. a. ater , M. a. artemisiae and M. a. obscurus . The birds used in this study were M. a. ater .

Currently, the brown-headed cowbird inhabits the entire United States, northern

Mexico and much of Canada. They are associated with disturbed habitats and can be found inhabiting forest edges, riparian zones, thickets, prairies, fields, cattail marshes,

5 pastures, orchards and suburban landscapes (Ortega, 1998). Brown-headed cowbirds are ground feeders and are often found near large grazing ungulates which stir up arthropods.

They mainly feed on arthropods and seeds. Females also consume mollusc shells for calcium to support the production of eggs (Ortega, 1998).

Cowbird breeding season normally begins in early April and lasts through early

August (Ortega, 1998). During the breeding season, mornings are designated for courtship and mating and afternoons are spent foraging by both sexes. Foraging areas are not defended by either sex and are shared, often in large flocks (Ortega, 1998).

Females nest-search and lay eggs in the morning as well. Brown-headed cowbirds are brood parasites, meaning they lay their eggs in the nests of host bird species. Female brown-headed cowbirds have been reported to lay their eggs in the nests of 226 different bird species. After eliminating host species that are rarely parasitized and species that reject cowbird eggs, there are 132 fostering host species which accept the egg and raise the cowbird young (Johnsgard, 1997; Ortega, 1998). Females usually silently watch their potential hosts building nests from afar and wait for the host bird’s absence to lay, but some have been observed flushing a host bird from her nest to lay in it (Norman and

Robertson, 1975). Females are reported to defend nesting areas from other female cowbirds during the breeding season (Dufty, 1982; Darley, 1983). However, Yokel

(1989a) did not report female territoriality of the cowbirds he observed. Likely, territorial behavior depends upon cowbird density and the availability of host nests (Ortega, 1998).

Average laying time for brown-headed cowbirds is 41 seconds; as a comparison, laying times for six other icterid species range from 21.5 to 53.4 minutes (Ortega, 1998). Scott and Ankey (1980) calculated that female cowbirds lay an average of 40 eggs during a

6 single breeding season. In captivity, with unlimited calcium consumption, a female laid

77 eggs during a season (Holford and Roby 1993).

Along with mating, males spend their mornings establishing and maintaining dominance hierarchies mainly through song and some display (Dufty, 1986; Ortega,

1998). The perch song sung by males used for both dominance establishment and courtship is accompanied by a display in which the male arches his wings, spreads his tail, fluffs his body feathers and bows. What makes a male dominant over others is unknown, but generally speaking, larger brown-headed cowbirds are dominant and in most cases, older males are larger (Yokel, 1989b).

Brown-headed cowbird eggs require 10-12 days of incubation. Once hatched, both male and female offspring quickly develop gray and downy juvenile plumage.

About 35 days after hatching cowbirds go through a post-juvenile molt. Nearly all juvenile feathers are replaced with adult plumage with this molt. At this point in time, the young cowbird is labeled a “hatching-year” individual and remains in this category until

January 1. As of January 1, all hatching year cowbirds are called “second-year” birds until they go through their annual post-nuptial molt after the next breeding season. After this post-nuptial molt, a cowbird is considered an “after-second-year” bird. Second-year and after-second-year males can be distinguished by the color of their secondary inner coverts. After-second-year males have black secondary inner coverts while second-year males retain some brown and gray juvenile coverts (Pyle et al., 1997; Ortega, 1998).

Females remain a brownish-gray color for the rest of their lives, and aging them is unreliable once they have gone through their post-juvenile molt.

In late summer/early fall, brown-headed cowbird flocks join together as they travel south from their breeding grounds to their wintering grounds. Brown-headed cowbirds disperse widely, and flocks from different areas often join up and winter together (Ortega, 1998). Large flocks of brown-headed cowbirds spend the winter months with various other icterids and European starlings (Sturnus vulgaris ).

Brood parasites have the opportunity to obtain chewing lice from their host bird species. Balakrishnan and Sorenson (2006) found that chewing louse communities on brood-parasitic finches (genus Vidua ) and their finch hosts were distinctly different. In contrast, Lindholm et al. (1998) found both nestling and adult diederik cuckoos

(Chrysococcyx caprius ) were infected with chewing louse species also found on their avian hosts. Both brood parasites mentioned in the previous studies have relatively small host repertoires compared to the brown-headed cowbird. Having about 132 host species raising brown-headed cowbird young gives broad exposure to a wide variety of chewing louse species compared to the young of non-brood parasitic birds. Adult brown-headed cowbirds have been found infected with the following chewing louse species: Brueelia ornatissima , Menacanthus eurysternus , Menacanthus quiscali , Menacanthus chrysophaeus , Myrsidea fuscomarginata , Machaerilaemus sp., Philopterus agelaii and

Ricinus sp. (Hahn et al., 2000; Price et al., 2003).

For ectoparasites like chewing lice, a larger host body provides greater surface area and thus, greater resources. Male cowbirds have larger bodies and larger feathers than female cowbirds (Johnsgard, 1997), and therefore, they can support a greater number of lice than can females.

Because lice are transferred through direct contact, behaviors that increase or decrease direct contact with other hosts can affect louse prevalence. During the breeding season, the behavior repertoire of males and females differ. A male’s establishment and maintenance of a dominance hierarchy through song and display involves minimal direct contact with other birds and therefore, this behavior should not affect male louse communities. In contrast, when female cowbirds lay eggs in nests belonging to other bird species they may come in contact with displaced lice from their original host. Thus, egg- laying has the potential to increase exposure of a female cowbird to lice from a different bird species, increasing female cowbird louse prevalence and changing community composition.

Although age could also affect behavior and body size, host-age preference in chewing lice has not been observed in adult birds (Potti and Merino, 1995; Darolova et al., 2001; Hamstra and Badyaev, 2009). Hahn et al.(2000) studied the louse communities in fledgling and adult brown-headed cowbirds. They found fledglings and adults to have similar louse prevalence and louse abundance. The subpopulations shared 4 louse genera but one louse genus was found only on adults and two louse genera were found only on fledglings. Because brown-headed cowbirds join up in large flocks in the winter, they can transfer lice amongst each other while foraging and roosting. Therefore, after their first winter, cowbirds may not retain the same louse community they had when they fledged.

Younger males spend more time socially foraging than older males (Rothstein et al.,

1987) which could increase the amount of direct contact they have with other birds and as a result increase louse prevalence. Also, young males are smaller than older males

(Yokel, 1989b) therefore, older males can support greater louse intensities than young males.

Objectives The objectives of this study were to 1) develop a baseline list of louse species that infect adult brown-headed cowbirds in the Great Lakes region; 2) compare louse species richness between brood-parasitic cowbirds and other non-brood-parasitic icterid species to test whether brood parasitism adds louse species to an icterid species; 3) compare louse prevalence, intensity and species richness between cowbird sexes. The effect of size and behavior differences should increase louse intensity in males and increase louse species richness in females; 4) compare louse prevalence and intensity between older and younger male cowbirds. The effect of size and behavior should increase louse prevalence in young males and increase louse intensity in older males; 5) examine the relationship between body condition and louse intensity. Cowbirds in poorer body condition should have greater louse intensity if they preen less; 6) examine the distribution of lice between the head and neck region and the body region of cowbirds. Cowbirds are not able to preen their head and neck region, and therefore lice should prefer this region;7) examine the distribution of different louse stages (nymphs and adults) and louse sexes between the head and neck region and body region of cowbirds. Greater nymph and female distribution on the head and neck region will be used to infer egg-laying preference.

METHODS

Birds

Collection- As part of a program to protect the Kirtland’s warbler (Setophaga kirtlandii ) from nest parasitization, the United States Fish and Wildlife Service (USFWS) trapped and euthanized (by thoracic compression) brown-headed cowbirds in Northern

Michigan during spring and summer of 2011. They had a total of 52 traps spread amongst four transects ranging 161-322 km long across an approximately 10,000 square km area.

The chicken-wire and wood traps were large enough for a human to enter and the ceiling of the traps had a drop-down mechanism made of wire with larger openings to allow birds to pass through (Figure 3). Cowbirds were attracted to the traps using white millet and live decoy cowbirds. Ideally, traps contained 12 decoy cowbirds, 6 male and 6 female. Each decoy cowbird was identified by colored tape on the tarsus. Cowbirds were allowed to gather until they exceeded a threshold number at which time excess birds were collected. The first birds collected from the traps were kept in a stock cage to act as future decoys if current decoys escaped or expired. On the day of collection, a cowbird collector followed each USFWS trapper. Immediately after euthanasia of each bird, a cowbird collector placed it in a labeled 3.8 L Ziploc ® bag and stored it in an ice-filled cooler. The bodies remained on ice until they were transported to Northern Michigan University where they were stored at -20°C until processed.

Birds arrived in Michigan later in the spring of 2011 than in past years. As a result, culling began almost a month later than usual on May 13 and continued through

June 30. Because so few birds arrived May 13-June 5, all birds collected during this time

11 were examined. The majority of the birds were collected June 30 and of those collected then, approximately equal numbers of males and females were randomly selected until a total of 401 birds had been examined (Table I).

Sex, Age and Body Condition- Females are a brownish color all over their bodies while males are black with brown head (Pyle, 1997; Figure 4). Males were considered

ASY if their secondary coverts were completely black (Figure 5). Males were considered

SY if they had their characteristic black adult plumage color, but with retained gray/brown secondary coverts (Pyle, 1997; Figure 6).

As a quick measure of body condition, cowbirds were assessed by following the protocol of Gregory and Robins (1998). The degree of protuberance of the keel and the development and shape of the breast muscles alongside the keel was palpated and from this examination, cowbirds were placed in one of two ranks. If the keel was prominent and breast muscle was concave relative to the keel, the bird was considered “emaciated”

(rank 0). If breast muscle was more developed with no concavity to the keel it was considered “good” (rank 1).

For a scaled measure of body condition, each bird was weighed and the length of its keel was measured. A ratio was then calculated by dividing the weight of the bird by the length of its keel; thus, the larger the ratio, the better the condition of the bird

(Johnson et al., 1985).

Lice

Collection- Frozen cowbirds were removed from their storage bag and placed in an open plastic box to thaw. Before louse collection from each bird, dislodged lice in the bag and thawing-box were collected and placed in a “container” vial. To collect lice from

12 the head and neck region, each cowbird was placed back in its bag with only the head and neck exposed (Figure 7). The head and neck were then blown with compressed air

(termed “blasting”) for 2 minutes within a chamber placed over a sheet of white paper.

The chamber was an upside-down, plastic 69.1L storage container with two holes made for the insertion of hands, a small hole for the air hose and a small hole for air to escape

(Figures 8 and 9). The bottom of the container was lined with foam weather-stripping to create a seal between the chamber and the white paper. The debris on the sheet of paper was funneled into a glass petri-dish and examined using a dissecting scope. If lice were not present, the head and neck region of the bird was considered negative. If lice were collected from the head and neck region after the initial 2 minutes in the chamber, the bird was blasted for consecutive 1 minute bouts until no lice were collected. Once lice were collected from the head and neck region, the storage bag was examined for any dislodged lice from the body. Any lice found in the storage bag were collected and placed in the “container” vial. The head and neck region of the bird was then placed back in the clean storage bag in order to expose the cowbird’s body (Figure 10) which was then blasted as described for the head and neck.

Fallen lice were counted and transferred to glass vials containing 95% ethyl alcohol (EtOH) using a fine-tipped paintbrush. Lice collected from each region of the bird (head/neck and body) received its own separate vial. Lice that fell off the bird in its storage bag and/or in its thawing box were considered “container” lice. Container lice were collected and placed in a separate vial for each bird.

Once blasting was completed for a bird, it was placed back in its bag, and returned to the freezer. The chamber was blasted with compressed air for 1 minute

13 between birds to minimize cross-contamination. Thawing boxes and collecting dishes were wiped out with 95% EtOH after each use.

Mounting - Lice were mounted on slides in 1-2 drops of lactophenol (Salmon,

1947) and covered with a cover slip. After 24 hours of clearing, slides were viewed under an inverted compound microscope (Olympus CKX41) at 100X total magnification for identification.

Identification - Nymphal lice were identified by their smaller size and their absence of genitalia (Figure 11). Nymphal lice are not reliably identified to species because they lack species identifying characters. Adult females were identified by the presence of a vulva and spermatheca (Figure 12). Adult males were identified by their large basal apodemes and parameres (Figure 13). Lice were identified to genus using the keys of Price et al. (2003). Because all lice were identified to the level of genus only in this study, genus richness was used instead of species richness. Nonetheless, genus richness is informative because birds, including brood parasites, are rarely reported to be infected by more than a single louse species from each genus (Clayton et al., 1992; Potti and Merino, 1995; Darolova et al., 2001; Price et al., 2003; Brooke, 2010). The possibility of infection by multiple louse species belonging to the same genus still exists.

Therefore, the calculated diversities may be underestimates. For each genus, a specific key was used to confirm the genus identification and to determine species (Clay, 1951;

Clay, 1969; Cicchino and Castro, 1996; Marshall, 2003; Price et al., 2002). Digital images of lice belonging to each genus and species were obtained using an Olympus

DP71 ® camera and CellSens Standard ® imaging software. After lice were identified, they were returned to their vial where they were stored in 95% EtOH.

Attempts to further clear highly pigmented lice were limited to puncturing their abdomens and soaking their whole bodies in 10% potassium hydroxide for 12 hrs. These lice were washed in 10% acetic acid and then rinsed in distilled water (Palma, 1978).

Other clearing techniques were not used to avoid damaging the specimen and thus eliminating future attempts at identification.

Analyses

All statistical analyses were performed using SPSS version 20 and Microsoft

Excel 2007.

Louse Species Richness in Icterid Species- The louse species richness in icterid species is defined as all louse species reported on an icterid host species. Icterid host species were selected for comparison if they inhabit North America and are social feeders. The number of reported louse species found on icterid host species was based on

Hahn et al. (2000) and Price et al. (2003). Host subspecies were pooled under the host species name. Because this number could be biased by the number of examined individuals for each bird host species, the number of studies reporting lice from each host species was used as an index of the number of birds examined. The relationship between louse species richness and the number of published studies was compared using

Spearman’s rank correlation. To find studies performed on the lice of each host species, a google scholar search was performed using a combination of the terms “chewing lice”,

“phthiraptera”, the host species name and the host common name. The studies referenced in Price et al. (2003) were also used in formulating the index. If no correlation existed between species richness and number of studies, then the louse species richness of brood- parasitic and non-brood-parasitic icterids could have been compared using a Mann-

Whitney U (M-W). 15

Effects of Sex- Louse prevalence of male and female birds were compared using a chi-squared test of independence. The normal approximation interval was calculated and is reported following prevalence. Louse infection intensities of male and female birds were compared using a M-W test. Louse species richness of male and female birds were compared using a M-W test.

Effects of Age- Louse prevalence of SY (younger) male and ASY (older) male birds were compared using a chi-squared test of independence. The normal approximation interval was calculated and is reported following prevalence. Louse infection intensities of younger and older male birds were compared using a M-W test.

Effects of Body Condition - The relationship between bird body condition and louse infection intensity was tested using Spearman’s rank correlation.

Distribution of Lice on Body Regions of Cowbirds- Because the surface area of the cowbird’s head and neck region to that of the body is less than 1:1, a 1:1 or greater distribution of lice would indicate that lice prefer the head and neck region. A chi- squared goodness of fit test was used to test for a 1:1 distribution. If a greater number of lice than expected were collected from the body, no conclusion about preference of the body could be made because this region has a larger surface area and could support a greater number of lice regardless of preference.

Distribution of Adults and Nymphs on Anatomical Regions of Cowbirds- To test whether nymphal lice are distributed on the head and neck region and the body region differently than an adult, a chi-squared test of independence was performed for each bird in the top 10% of louse intensity within females, young males and older males. Louse populations in the top 10% were likely large enough to meet the criteria to perform a chi-

16 squared test of independence. If no host age or host sex affect was seen, a chi squared test would have been run with pooled data.

Distribution of Louse sexes on Anatomical Regions of Cowbirds - To test whether female lice are distributed on the head and neck region differently than males, a chi- squared test of independence was performed on the component population of each infected cowbird. A separate analysis was performed for the lice of male and female cowbirds. If no sex affect was seen, a chi-squared test would have been run with pooled data.

RESULTS

A total of 762 cowbirds (588 males and 174 females) were collected and 401 (293 males and 104 females) were examined. Louse infection prevalence was 60% ±4%

(242/401) for this population of cowbirds. Median (range) louse intensity was 7 (1-242).

A total of 3,160 lice (1614 nymphs and 1563 adults) were collected. There were 959 female and 601 male Brueelia ornatissima resulting in a 1.59:1 sex ratio.

Louse Identification : Five genera of lice were found: Brueelia ornatissima (Figure

14), Myrsidea sp., Machaerilaemus sp. (Figure 15), Philopterus sp. (Figure 16) and

Menacanthus sp. Only Brueelia ornatissima was identified to species because members of this lightly pigmented genus cleared well. Unfortunately, neither clearing method revealed the characters needed for species identification in the darkly pigmented specimens.

Louse Species Richness of Icterid Species – The brood-parasitic icterid species used for comparison were Molothrus aeneus, M. ater, and M. bonariensis. The non- brood-parasitic icterid species used for comparison were Agelaius phoeniceus ,

Dolichonyx oryzivorus, Euphagus carolinus, Icterus galbula, Quiscalus major, Q. mexicanus, Q. quiscula and Xanthocephalus xanthocephalus . Louse species richness was similar between brood-parasitic (median=3) and non-brood-parasitic (median=3) icterid host species (p=0.50) (Figure 17). The louse species richness of icterid hosts was strongly correlated with the number of studies examining each host’s lice (r=0.946; p<0.001;

Table II).

Effects of Sex - Male brown-headed cowbirds’ louse prevalence of 65% ±6% was significantly higher than that of females’ (49%±10%); (χ2= 7.85; df=1; p<0.01; Table

III). Median (range) louse infection intensity was greater in males than females (8(1-243) and 5(1-62), respectively) albeit not significantly (p=0.08; Table IV). Median (range) louse species richness of males (1(1-3)) was similar to females (1(1-3); p=0.63; Table

IV).

Effects of Age - Younger male cowbird louse prevalence of 72% ±9% was significantly higher than that of older males (61% ±8%); (χ2=3.72; df=1; p=0.05); Table

III). Older males had significantly greater median (range) louse infection intensity (9 (1-

242)) than younger males (5.5(1-97); p=0.03; Table V).

Body Condition - All birds but one were ranked as “1” or “good”. The median weight-to-keel length ratio (range) was 1.43(0.43-1.85). There was no relationship between body condition and louse infection intensity (r=0.047; p=0.47).

Distribution of Lice on Anatomical Regions of Cowbirds - There were small numbers of lice collected from Philopterus , Machaerilaemus , Menacanthus and

Myrsidea . Brueelia made up the overwhelming majority of lice collected and had more members collected from the body than the head and neck region (Table VI). Myrsidea were collected from birds early on in the study before the head and neck region and body were blasted separately. Thus, the regions of the body that Myrsidea members were collected from was unknown. Only Brueelia ornatissima had a large enough sample size to test for a region preference. There were significantly more Brueelia ornatissima on the body regions of cowbirds than on the head and neck regions ( χ2=62.6 df=41; p=0.016).

Distribution of Adults and Nymphs on Anatomical Regions of Cowbirds - Only one female cowbird had an appropriate number of lice for the analysis, and adult and nymphal lice were distributed independently across body regions ( χ2=3.0; df=1; p=0.08).

Only two young males had appropriate number of lice for analysis. One young male had independently distributed adult and nymphal lice ( χ2= 0.1; df=1; p=0.07) and the other had a greater number of adult lice on the head and neck than expected ( χ2=4.2; df=1; p=0.04). There were 10 older male cowbirds that had appropriate counts for a chi-squared test. Eight of these males had independently distributed adult and nymphal lice (p=0.08-

0.84). One older male had more adult lice than expected on the head and neck ( χ2=3.9; df=1; p=0.05) and one older male had more nymphs than expected on the head and neck

(χ2=14.6; df=1; p<0.01). No further analyses were performed because the limited analyses performed indicated individual bird variability.

Distribution of Louse Sexes on Anatomical Regions of Cowbirds - Louse sexes were independently distributed on the head and neck regions of female cowbirds

(χ2=0.22; df=1; p=0.65) and male cowbirds ( χ2=1.4; df=1; p=0.24).

DISCUSSION

The ratio of male to female cowbirds collected was 3.4 (Table I). This sex ratio is within the range of those from other trapping studies (range 1.2-6.3:1; Ortega, 1998;

Yokel, 1989a; Rothstein et al., 1987; Darely, 1971) and collection method likely has minimal effect on it (Donald, 2007). A higher mortality rate in females will bias a sex ratio towards males and greater female brown-headed cowbird mortality has been reported during the breeding season (Darley, 1971). In general, the sex with greater metabolic demands has been associated with greater mortality (Donald, 2007). Female brown-headed cowbirds lay 25-77 eggs a breeding season (Ortega, 1998) a metabolically demanding activity that may contribute to this biased sex ratio.

In this northern Michigan study, 60% of the brown-headed cowbirds examined were infected with at least one louse genus. Using a similar trapping set-up in Maryland,

Hahn et al. (2000) found 20.5% of 219 adult cowbirds infected with lice. One basic difference in these two studies is louse collection method. Hahn et al. (2000) captured live cowbirds and subsequently placed their bodies in a jar filled with ethyl acetate. The breast, back, and wing feathers of each bird were then manually ruffled to dislodge dead lice. In contrast to my study, Hahn et al. (2000) did not collect lice from the head and neck region of their birds, and they did not ruffle lice from all regions of the body.

Twenty-one birds in my study (8.7%) had lice found only on the head and neck which

Hahn et al. (2000) would have designated uninfected. Sixty-nine birds in my study

(28.5%) had only 1-2 lice found on the body and if these lice were on an unruffled region

21 of the body, the methods of Hahn et al. (2000) could have missed such a low population.

Interestingly, a 37.2% increase (8.7% + 28.5%) in the Hahn et al. (2000) study would result in a 57.7% prevalence, startlingly close to 60%. Unfortunately, the relative loss of lice caused by maneuvering live birds into jars and by my method in which birds were captured, quickly euthanized and immediately placed into a bag is unknown.

We collected lice belonging to five genera (Brueelia , Myrsidea , Machaerilaemus ,

Philopterus and Menacanthus ), all previously reported on brown-headed cowbirds (Hahn et al., 2000; Price et al., 2003). This suggests that although brown-headed cowbirds are exposed to lice from different bird species, host-switching of lice is not a common phenomenon.

Louse Species Richness in Icterid Species - Louse species richness was strongly correlated (r=0.946) with the number of studies that examined host lice, a common problem in parasitology. Thus, the effect of brood-parasitism on louse species richness cannot be examined in this study.

Effects of Sex –Males had higher louse prevalence (65%) than females (49%). In other bird species, contributory factors affecting prevalence include sociality (Rozsa and

Reiczigel, 1996), age (Garamszegi et al., 2005) and stress (Raouf et al., 2006).

Sociality increases physical contact which facilitates louse transfer. Physical contact occurs during mating, nest visits, head-down displays and group foraging. Both sexes mate, so transfer should be equal between sexes. If contact during nest visits was a significant source of louse transfer, females should have greater louse prevalence, but this was not the case. The head-down display is another source of contact. Both sexes express this behavior so it is less likely a source for the resulting prevalence difference between

22 the sexes. Younger males spend more time group foraging (Rothstein et al., 1987), so as a subpopulation, males spend more time group foraging than females. Thus, the increased louse prevalence in males supports group foraging as a contributing factor.

Garamszegi et al. (2005) examined the chewing louse load of male barn swallows and found younger males to have greater louse loads than older males, but offered no basis for an age-related bias. An age-effect may contribute to the difference in louse communities observed between male and female cowbirds. Cowbirds have equal sex ratios at hatching (Weatherhead, 1989). During their first year, male and female cowbirds have similar mortality rates (Darley, 1971), but during the winter season, adult males have higher mortality, and older males make up the majority of these expired cowbirds

(Arnold and Johnson, 1983). Thus, it may be that the female subpopulation is older than the male subpopulation during the breeding season. In my study, unfortunately, I have no age data on female cowbirds. Interestingly though, younger males in my study had higher louse prevalence than older males. If the same pattern holds true for females, then the lower louse prevalence in females may be due in part to an age effect.

Stressors have been associated with increased parasitism (Poiani et al., 2000).

Using levels of corticosterone as a measure of stress in animals, Nephew et al. (2003) found that stress significantly increased in starlings after the introduction of an acute stressor and, as a result, stressed birds preened less. Carere et al. (2003) investigated corticosterone levels of male great tits (Parus major ) after the introduction of a second male (a chronic stressor). They, too, found corticosterone levels increased significantly after the introduction of another male, but unfortunately they did not quantify preening.

Male brown-headed cowbirds spend their mornings establishing and maintaining

23 dominance among other males and have been observed engaging in significantly more aggressive behavior, including bowing and chasing other birds, while foraging than females (Morris and Thompson, 1998).

The relative stress levels of brown-headed cowbird males and females during the breeding season are unknown. Female cowbirds spend their mornings nest searching and producing eggs and some females have been reported defending nesting territories

(Dufty, 1982; Darley, 1983). Territoriality has not been studied in Michigan cowbirds.

However, even if Michigan cowbirds are not territorial, they still have the stress of nest searching and egg production. Landys et al. (2010) studied corticosterone levels in female

European nuthatches (Sitta europaea ) year round and found an increase in corticosterone during the periods of nest-building and egg-laying (typically 5-7 eggs). Although brown- headed cowbirds do not build nests, they may lay 25-77 eggs during a single breeding season (Ortega, 1998). Both brown-headed cowbird sexes deal with different stressors and it is unknown whether one is more stressed than the other. More importantly it is unknown if stress in brown-headed cowbirds affects preening behavior. Without this information, the importance of stress on chewing louse communities is uncertain.

Males had greater louse intensity (8) when compared to females (5), but it was not significantly different. Regardless, the p-value was low and warrants further study as well as biological meaning. Male cowbirds typically weigh more than females (49g and 38.8g, respectively) and have larger mean wing (110.5 and 101.1mm, respectively) and tail lengths (75.2mm and 66.8mm, respectively) than females (Johnsgard, 1997). Thus, males provide more resources for chewing lice to support larger populations. In addition, if

24 males are more stressed than females, and if this results in them preening less, the intensity of chewing lice would be greater on males than on females.

Male and female cowbirds had similar louse genus richness and were infected with the same genera of lice. This suggests that female cowbirds rarely encounter straggling lice in the nests of host birds, or that straggling lice picked up at the nest rarely survive. Successful host switching by lice from a host bird to an egg-laying brood parasitic female, if it occurs, is likely a rare event.

Effects of Age - Younger (second-year) males had higher louse prevalence (72%) than older (after-second-year) males (61%). In general, brown-headed cowbird males allocate the morning hours to establishing and maintaining dominance, attracting mates and copulating while foraging is left for the afternoon. In brown-headed cowbirds, size is positively correlated with dominance and breeding success, and most large birds tend to be older (Yokel, 1989b). These dominant males have greater access to females, spending more time engaged in courtship than subordinate males (Ortega, 1998). Rothstein et al.

(1987) trapped younger cowbird males at social foraging sites earlier in the day than older males suggesting that younger males spend more time foraging than older males.

Increased social foraging time increases the chances of direct contact and louse transfer in their efforts to grow larger and possibly more dominant.

More lice were found on older infected male cowbirds than on younger infected males. A greater proportion of younger males (42%) were infected with 2 or fewer lice compared to older males (22%). Only 1-2 lice are transferred horizontally because birds are in contact for only a few seconds and lice are reluctant to leave their hosts. Small louse populations have a greater risk of extinction. Because younger males spend more

25 time socially foraging, they increase their chances of reinfection over that of older males

As a result, younger males should have higher louse prevalence and lower intensity compared to older males. My study showed a smaller proportion of older males were infected, but those that were infected had more lice which increased their louse intensity.

Effects of Body Condition - There was no correlation between body condition and louse intensity. Generally, it has been assumed that unhealthy birds may have greater louse intensity as a result of decreased grooming behaviors (Marshall, 1981). The evidence supporting this is based upon limited observations of dead birds or birds near death found with incredibly high numbers of lice (Ash, 1960; Nelson and Murray, 1971).

The trapped brown-headed cowbirds in my study had unlimited access to seed and from the simple measure I used, only one bird was labeled “0” or in poor condition, but even this bird appeared healthy despite its relatively concave breast muscles. There may be a threshold of extreme emaciation that must be met for a bird to discontinue preening, increasing louse numbers drastically.

Distribution of Adults Nymphs on Anatomical Regions of Cowbirds - Adult and nymphal lice were distributed similarly across the body regions of most of the cowbirds examined. Only three of the examined birds had dependent distributions. Two of which had more adults than expected on the head and neck and only one with more nymphs than expected on the head and neck. These data do not support a regional egg-laying preference for Brueelia ornatissima . Actual egg distribution should be determined for egg-laying preference because louse distribution is likely affected by the behaviors of an individual bird such as preening and head-down displays.

Birds have been estimated to spend about 9% of their day on self-maintenance activities and 93% of this time is spent preening (Cotgreave and Clayton, 1994). Because preening with the bill is an essential part of daily activity, it is likely more effective at removing eggs and nymphs than scratching, which is an elicited behavior. If this were true, then a louse that lays eggs all over the body would eventually produce more eggs and nymphs on the head and neck. Unfortunately, the relative effectiveness of preening and scratching against different louse stages has not been measured.

If some cowbirds are more stressed than others, and if this stress results in less preening, then a greater number of nymphs and eggs would survive on their body regions compared to their head and neck regions.

Brown-headed cowbirds perform “head-down” displays. The function of the

“head-down” display is unknown but has been hypothesized as a behavior to test the agonistic tendencies of another bird (Scott and Grumstrup-Scott, 1983). One of the responses by the display recipients is to preen the “head-down” displaying bird. “Head- down” displaying was not examined in my study. If some cowbirds in my study performed the head-down display more often than others and had their head and necks preened more often, it could affect the distributions of lice found on individual birds.

Investigations of “who” performs this display (male, female, younger or older cowbirds) and how often, and the duration a recipient bird preens the displaying cowbird, are needed to determine the effect of the “head-down” display on the distribution of lice.

SUMMARY AND CONCLUSIONS

The population of cowbirds studied had a louse prevalence of 60% and five different genera of lice were collected from the infected birds, all of which have been previously recorded on brown-headed cowbirds. Male brown-headed cowbirds had greater louse prevalence and probably greater intensity compared to females. These results could be predicted by an age-effect based on differential mortality between species and increased foraging time. Stress and its effects on preening are unknown for brown-headed cowbirds, but may also contribute to the differences in louse prevalence and intensity observed between sexes. The male and female subpopulations had similar louse species richness suggesting that although females have a greater chance of picking up straggling cowbird-host species at the nest, the event is rare.Young male cowbirds had greater louse prevalence and lower louse intensity compared to older males. Increased group foraging in younger males increases risk of reinfection by low numbers of lice and thus, more are infected with lice and those infected have fewer lice compared to older males. The brown-headed cowbirds in this study were primarily infected with Brueelia ornatissima . Brueelia ornatissima distribution on the cowbirds was independent of their age and sex and could be influenced by variability in individual bird behavior. Further investigation of chewing lice on brown-headed cowbirds could reveal information about cowbird mate selection, behavior, and overall fitness.

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APPENDIX A

GLOSSARY

Component community - Refers to all infrapopulations of parasites associated with some subset of a host species (Bush et al., 1997).

Infrapopulation - Includes all individuals of a species in an individual host at a particular time (Bush et al., 1997).

Intensity (of infection)- The number of individuals of a particular species in a single infected host (Bush et al., 1997).

Median Intensity - The median intensity of a particular species of parasite among infected members of a particular host species (Bush, 1997).

Prevalence - The number of hosts infected with one or more parasites of a particular species (or taxonomic group) divided by the number of hosts examined for the parasite species (Bush et al., 1997).

Table I. Number of male and female cowbirds collected and examined from each collection date during 2011 in northern Michigan. “M”= Male; “F”=Female

M Collected M Examined F Collected F Examined 13-May 36 36 6 6 14-May 11 11 4 4 15-May 30 30 4 4 28-May 38 38 0 0 4-Jun 52 52 0 0 5-Jun 38 38 0 0 30-Jun 383 89 160 94 Total 588 294 174 108

Table II . The number of louse species reported on non-brood-parasitic and brood- parasitic icterid hosts and the number of studies that examined lice on each host. Non-Brood-Parasitic Hosts Louse species Richness # Studies Icterus galbula 1 1

Dolichonyx oryzivorus 1 1 Xanthocephalus xanthocephalus 1 1 Quiscalus major 2 2

Euphagus carolinus 4 5 Q. quiscula 5 6 Q. mexicanus 5 4 Agelaius phoeniceus 5 7 Brood Parasitic Hosts Molothrus bonariensis 2 2 M. aeneus 3 3 M. ater 7 6

Table III. The number of infected and examined female, male, young male and older male brown-headed cowbirds collected from northern Michigan in 2011 and their louse prevalence. “SY”=Second-Year; “ASY”=After-Second-Year.

Female Male SY Male ASY Male # Infected 53 189 72 117 # Examined 108 293 100 193 Prevalence (%) 49.07 64.51 72.00 60.62

Table IV. The number of male and female brown-headed cowbirds collected from northern Michigan in 2011 infected with lice, their median (range) louse intensity and median (range) louse species richness. Female Male p-value # Infected 108 293 Median Intensity (Range) 5 (1-62) 8(1-242) 0.08 Median spp. Richness (Range) 1 (1-3) 1 (1-3) 0.63

Table V. The number of young and older male brown-headed cowbirds collected in northern Michigan in 2011 infected with lice and their median (range) louse intensity. “SY”=Second-Year; “ASY”=After-Second-Year.

SY ASY p-value # Infected 100 193 Median (Range) Intensity 5.5(1-97) 9 (1-242) 0.03

Table VI. A list of louse genera identified, the number of brown-headed cowbirds collected from northern Michigan in 2011 infected with each genus, the number of lice collected from the head and neck region and body region of cowbirds and the number of lice collected from unknown origin for each genus. # of Lice # of Birds Louse Genus Infected Head and Neck Body Unknown Menacanthus 1 0 1 0 Philopterus 7 14 2 4 Myrsidea 2 0 0 3 Machaerilaemus 8 11 3 5 Brueelia 230 799 2137 181 Total 824 2143 193

Figure 1. External anatomy of chewing lice. A. Dorsal-ventral view of female Amblycera. B. Ventral-dorsal view of female Ischnocera. [Adapted from Clayton et al. (2003)].

Figure 2 . Chewing louse reproductive anatomy. C. Male reproductive tract of Ischnoceran D. Female reproductive tract of Ischnoceran. [Adapted from Price et al. (2003)]

Figure 3 . Enclosures used by United States Fish and Wildlife Service for brown-headed cowbird trapping in northern Michigan.

Figure 4. Euthanized brown-headed cowbirds. Left. Male. Right. Female.

Figure 5. Ventral side of wing from an older (after-second-year) brown-headed cowbird male. Secondary coverts are black

Figure 6. Ventral side of wing from a young (second-year) brown-headed cowbird male. Secondary coverts are black and brown.

Figure 7. Brown-headed cowbird orientation for head and neck louse collection.

Figure 8: Plastic chamber built by William Durkin and used for louse collection

Figure 9 . Bird being “blasted” for louse collection

Figure 10 . Brown-headed cowbird orientation for body louse collection.

Figure 11 . Chewing louse nymphs at 40X magnification. Left. Philopterus nymph. Right. Brueelia nymph.

Figure 12 . Female abdomen of Brueelia at 100x magnification. A. Vulva B. Spermatheca

B A

Figure 13. Male abdomen of Brueelia at 100X magnification. A. Parameres B. Basal apodeme

Figure 14. Two female Brueelia ornatissima at 40X magnification.

Figure 15. Female Machaerilaemus at 40X magnification.

Figure 16. Male Philopterus at 40X magnification .

3.5

2.5

1.5

1 *

Number Number oficterid host species 0.5

0 1 2 3 4 5 6 7 Louse Species Richness

Figure 17 . Louse species richness of brood-parasitic (gray) and non-brood-parasitic (black) icterid hosts. Species richness values were compiled from previous studies and my study. * Indicates where my data contribute.