Funding for fieldwork during which our methodologies were developed was provided by Environment Canada&#39;s Canadian Wildlife Service, Environment Canada&#39;s Strategic Technologies Application of Genomics in the Environment Research Fund, the Endangered Species Recovery Fund, the Interdepartmental Recovery Fund, the Natural Sciences and Engineering Research Council and the Ontario Ministry of Training, Colleges and Universities (scholarships to A.A.C.), Queen&#39;s University (Duncan and Urlla Carmichael Fellowship to A.A.C.) and Wildlife Preservation Canada. We wish to thank the Editor and four anonymous reviewers for their comments, which greatly improved this manuscript. Thanks to Wildlife Preservation Canada staff and the members of the North American Loggerhead Shrike Working Group for discussions that assisted in development of this methodology. We extend our thanks to all the Citizen Scientists and staff at African Lion Safari, Cambridge, Ontario, for their assistance in completing the survey. We especially thank Erin Sills, Marketing and PR Coordinator, African Lion Safari, for her assistance in producing the on-line survey and summarizing results.

The research protocol presented herein complies with African Lion Safari&#39;s Animal Care Committee guidelines.
1. Sexing Loggerhead Shrikes by Color and Patern of the Sixth Primary Wing Feather
NOTE: Shrikes can be sexed in hand based on the coloration and pattern in the sixth primary wing feather (P6). In brief, the technique requires the observer to visually extrapoloate a line along the lower edge of the primary wing coverts, and then to assess how far the brown extends in the rachis (shaft) through the white portion of the feather visible below the primary coverts.
<ol>
	<li>Hold the bird firmly in the banders grip and carefully extend one wing to allow the sixth primary wing feather to be viewed (Figure 1). Do not over-extend as this can cause harm to the bird&#39;s musculature.</li>
	<li>Locate the P6 feather: Loggerhead Shrikes have 10 primary feathers, with the last (10th) being the most distal (outermost) feather and reduced in size compartive to the other 9 primary feathers. Unlike the secondary feathers, and with the exception of the reduced 10th primary feather, all primaries have a degree of white coloration. It may be easier to count backwards from the 10th primary than to locate the first primary and count forward to locate the P6.</li>
	<li>Assess the brown in the rachis (hereafter shaft): Does it extend at least half way through the white, and touch, or nearly touch the distal end (furthest from the body and point of emergence) of the primary covert feathers as they lay naturally over the primary feathers? If &#34;yes&#34;, the bird is female (Figure 2). If &#34;no&#34;, then the bird is male (Figure 2).</li>
	<li>The pattern and extent of coloration in the primary feathers varies among individuals. If unsure of whether the brown in the shaft extends at least half way through the white patch to the distal tips of the primary coverts, or if the brown is somewhat indistinct, use a secondary technique. Specifically, assess the symmetry of the brown to white transition in the feather vane on each side of the shaft (Figure 2). If the brown at the point where the coloration changes to white in the vane meets at the same spot on either side of the shaft, the bird is male. If there the line meets asymetrically at the shaft, creating a step or notch, the pattern indicates that the bird is a female (Figure 2).</li>
	<li>If there is still uncertainty as to the individual&#39;s sex, examine the angle of the transition between the brown and the white coloration. If the brown in the feather vane has a steep angle where it meets the white in the vane at the shaft, forming an upside down &#34;V&#34;, the pattern indicates that the bird is a female (Figure 2). If the pattern is more that of an &#34;M&#34;, it indicates a male bird (Figure 2).</li>
</ol>

The authors have nothing to disclose.

Herein, we describe a simple and efficient method whereby loggerhead shrike can be sexed based only on visual cues, and provide an assessment of the method&#39;s accuracy. Our simple method is easily and quickly undertaken, with results indicating a high accuracy rate that increases with a small amount of training. Our results support those of previous work13 that indicated the method originally developed for use in northern shrike12 had utility for sexing adult loggerhead ...

The loggerhead shrike (Lanius ludovicianus) is a North American passerine with a broad geographical range encompassing most of North America and a variety of habitats that can generally be described as grassland1. It is one of only two species of shrikes (Order Passeriformes) that occur in North America. Shrikes are best known for the unique raptor-like bill, which allows them to take vertebrate prey, and their unique behavior of impaling food items on thorns or other sharp objects. The loggerhead shrike is the only species of &#39;true shrike&#39; (Family Laniidae) endemic to the continent. Shrikes breeding above 40&#176;N are generally obligate migrants1,2,3, with wintering grounds almost entirely encompassed within that of non-migratory conspecifics1,4.
North American Breeding Bird Survey data5 for loggerhead shrike indicate a significant (3.18% yr-1) range-wide population decline. The loggerhead shrike is one of Partners in Flight&#39;s 24 &#34;Common Birds in Steep Decline&#34;-i.e., those that have lost more than 50% of their populations over the past 40 years, but which lack other elevated vulnerability factors that would warrant higher &#34;Watch List&#34; status6. Habitat loss due to succession and human development likely contributed to the initial declines4,7, but continued population declines are outpacing habitat loss in the breeding season, suggesting other limiting factors, in particular in areas where the species is an obligate migrant4,8. Results of a population viability analysis conducted for the critically endangered population of loggerhead shrike in Ontario suggests that over-wintering success of birds in their first year of life is a driver of the population trends9,10. Results further indicate that the conservation breeding effort, which is augmenting the wild population, has kept the species from extirpation in this area9,10.
Understanding sex differences is an important component of both ecological and evolutionary hypotheses. The plumage of loggerhead shrikes (Laniusludovicianus) is sexually monochromatic and therefore individuals cannot reliably be sexed in the hand. However, based on a method applicable to the northern shrike (Lanius excubitor)11,12, it has been shown to be possible to sex at least some populations of adult loggerhead shrikes using the coloration pattern in the sixth primary wing feather13. We have revised this methodology13 to include consideration of a second variable, specifically the extent of pigmentation in the rachis of the sixth primary, which allows reliable identification of sex in the majority of individuals in eastern populations, and tested its application (previously only applied to adult birds) to fledged young of the year. The method requires no specialized equipment or costly lab assays, and no measurements are required that would be subject to observer bias. Based on our results, the method is easily learned and, once mastered, highly accurate. Here, we present detailed instructions on how to sex shrike in the hand using our method and discuss the wider implications of including sex assessment in future research and conservation efforts for this unique and enigmatic species of conservation concern.

visually sexing loggerhead shrike (lanius ludovicianus) using plumage coloration and pattern

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, the species has undergone a drastic decline since the mid-1960s. The cause of decline is unknown, and research is actively underway to address this knowledge gap. These efforts are hindered by an inability to sex the species in hand, which to date was only possible using molecular markers. Here, we present a protocol to sex loggerhead shrikes by visually analyzing the coloration and pattern in the sixth primary feather. The application of the method will facilitate our ability to identify threats on a finer scale than has been possible to date and to address various ecological and evolutionary hypotheses. The methodology is simple and results reliable&#8211;we encourage including this method for research of both in situ and ex situ populations.

Male and female plumage is, overall, monomorphic in the loggerhead shrike. However, it has been established that sex can be discerned based on the pattern of coloration in the 6th primary in the population that occurs on mainland California13 and northern shrike12. We tested Sustaita et al.'s (2014) protocol13 to determine if it was applicable to northeastern populations of loggerhead shrike and to younger ...

Watch this Scientific Journal Video about Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern at JoVE.com

Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern

We present a protocol to characterize the sex of loggerhead shrike visually based on the coloration and pattern of the sixth primary wing feather.

Visually Sexing Loggerhead Shrike (Lanius Ludovicianus) Using Plumage Coloration and Pattern

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, ...

visually-sexing-loggerhead-shrike-lanius-ludovicianus-using-plumage

Research

JoVE Journal

Biology

6.1K Views.  African Lion Safari. Loggerhead shrike heeft verloren meer dan 50% van de bevolking in Noord-Amerika in de afgelopen 40 jaar. De mogelijkheid om geslacht te bepalen in loggerhead heiligdom heeft ons een ander instrument om te helpen begrijpen wat de oorzaak is van de daling en hoe het behoud fokken kan het beste helpen compenseren de bedreigingen. Het verenkleed van loggerhead heiligdom is seksueel monomorf, zodat vogels niet betrouwbaar kunnen worden geslacht in de hand, met behulp van fysieke kenmerken zoals in...

Video: Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the milli-scale structure of barbs and barbules largely determines the directional pattern of reflected light; and through the macro-scale spatial structure of overlapping, curved feathers, these directional effects create the visual texture. Milli-scale and macro-scale effects determine where on the organism's body, and from what viewpoints and under what illumination, the iridescent colors are seen. Thus, the highly directional flash of brilliant color from the iridescent throat of a hummingbird is inadequately explained by its nano-scale structure alone and questions remain. From a given observation point, which milli-scale elements of the feather are oriented to reflect strongly? Do some species produce broader "windows" for observation of iridescence than others? These and similar questions may be asked about any organisms that have evolved a particular surface appearance for signaling, camouflage, or other reasons.
In order to study the directional patterns of light scattering from feathers, and their relationship to the bird's milli-scale morphology, we developed a protocol for measuring light scattered from biological materials using many high-resolution photographs taken with varying illumination and viewing directions. Since we measure scattered light as a function of direction, we can observe the characteristic features in the directional distribution of light scattered from that particular feather, and because barbs and barbules are resolved in our images, we can clearly attribute the directional features to these different milli-scale structures. Keeping the specimen intact preserves the gross-scale scattering behavior seen in nature. The method described here presents a generalized protocol for analyzing spatially- and directionally-varying light scattering from complex biological materials at multiple structural scales.

We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the ...

Engineering

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

Genetics

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Environment

Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate hosts into raising their young and how hosts detect parasitism provide important insights in the field of coevolutionary biology. Brood parasites, such as cuckoos and cowbirds, gain an evolutionary advantage because they do not have to pay the costs of rearing their own young. However, these costs select for host defenses against all developmental stages of parasites, including eggs, their young, and adults. Egg rejection experiments are the most common method used to study host defenses. During these experiments, a researcher places an experimental egg in a host nest and monitors how hosts respond. Color is often manipulated, and the expectation is that the likelihood of egg discrimination and the degree of dissimilarity between the host and experimental egg are positively related. This paper serves as a guide for conducting egg rejection experiments from describing methods for creating consistent egg colors to analyzing the findings of such experiments. Special attention is given to a new method involving uniquely colored eggs along color gradients that has the potential to explore color biases in host recognition. Without standardization, it is not possible to compare findings between studies in a meaningful way; a standard protocol within this field will allow for increasingly accurate and comparable results for further experiments.

This protocol provides guidelines for running egg rejection experiments: outlining techniques for painting experimental egg models to emulate the colors of natural bird eggs, conducting fieldwork, and analyzing the collected data. This protocol provides a uniform method for conducting comparable egg rejection experiments.

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate ...

Behavior

Manipulation of Color Patterns in Jumping Spiders for Use in Behavioral Experiments

In the field of behavioral ecology, many experiments are designed to investigate the evolutionary purposes of colorful traits in the context of sexual selection and predation. Methods are various but mostly consist of modifying the color patterns of individuals with diverse colorants. Such techniques have been used across many vertebrate taxa, particularly in birds, but have remained underdeveloped for invertebrates because of the difficulty of effectively manipulating color in small organisms. Instead, to manipulate the appearance of invertebrates, scientists have usually modified the lighting environment to filter out certain wavelengths. However, such a method affects not only the phenotypic trait of interest but the entire appearance of the individual and its surrounding. Here, scaling down the techniques previously used on colorful birds, we present ways of manipulating the colors of small arthropods, using equally emblematic but understudied species: the colorful jumping spiders.

The goal of this protocol is to manipulate the color patterns of jumping spiders and other very small arthropods with paint in order to study questions related to sexual selection, sexual cannibalism, predation, aposematism, or any other field of animal coloration.

In the field of behavioral ecology, many experiments are designed to investigate the evolutionary purposes of colorful traits in the context of sexual ...

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

Neuroscience

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.

This protocol provides a convenient set of methods, which enables extremely fast, easy, non-invasive, reliable and low-cost, molecular sex determination of birds and their non-invasive, quick, safe and easily recognizable marking shortly after hatching. Only limited handling of chicks is required. This convenient toolbox of methods complies entirely with the RRR-guidelines.

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of thermoregulation in birds across a variety of ecological circumstances. However, few studies characterize the contribution of the feathers alone to thermoregulation. Several previous studies have established methods for measuring the insulation value of animal pelts, but they require destructive sampling methods that are problematic for birds, whose feathers are not distributed evenly across the skin. More information is needed about 1) how the contribution of feathers to thermoregulation varies both across and within species and 2) how feather coats may change over space and time. Reported here is a method for rapidly and directly measuring the thermal performance of feather coats and the skin using dried whole skin specimens, without the need to destroy the skin specimen. This method isolates and measures the thermal gradient across a feather coat in a way that measurements of heat loss and metabolic cost in live birds, which use behavioral and physiological strategies to thermoregulate, cannot. The method employs a thermal camera, which allows the rapid collection of quantitative thermal data to measure heat loss from a stable source through the skin. This protocol can easily be applied to various research questions, is applicable to any avian taxa, and does not require destruction of the skin specimen. Finally, it will further the understanding of the importance of passive thermoregulation in birds by simplifying and accelerating the collection of quantitative data.

This protocol describes the quantification of heat transmission through a flat skinned avian specimen using a thermal camera and hot water bath. The method allows obtaining of quantitative, comparative data about the thermal performance of feather coats across species using dried flat skin specimens.

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of ...

Visually Sexing Loggerhead Shrike (Lanius Ludovicianus) Using Plumage Coloration and Pattern

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