Assignment 2: Paper Review
This assignment offers a critical analysis of "Microanatomy of Passerine Hard-Cornified Tissues: Beak and Claw Structure of the Black-capped Chickadee (Poecile atricapillus)" by Caroline Van Hemert,1,2*
Colleen M. Handel,1 John E. Blake,3 Rhonda M. Swor,3
and Todd M. O’Hara2, published in 2012 by the Journal of Morphology.
Disclaimer: Figures 2, 3, & 4 were directly obtained from the study conducted by Van Hemert et al., (2012) to assist in an accurate description and critique of the study, belonging fully to the authors listed above.
INTRODUCTION
This study by Van Hemert et al. (2012) reviews the
microanatomy of the healthy beak of the Black-capped Chickadee (Poecile atricapillus), a passerine
bird species. While the paper focuses predominantly on the histology of the chickadee beak, claw histology was also investigated to a degree. The major reason these individuals conducted this research was
due to the lack of literature currently available about the microanatomy of keratinized structures in passerine birds, irrespective of their abundance and diversity
across the globe. |
| Figure 1: A Black-capped Chickadee in Homer, Alaska with a contorted beak, a symptom of avian keratin disorder (Photograph by Martin Renner, 2016). |
Keratinized structures such as the bird beak and claw are
highly susceptible to disease, caused by nutritional, viral, parasitic, and
toxic agents (Pass & Perry, 1984). In recent years, an epizootic disease resulting
in beak deformation has emerged in wild passerines, mainly in Alaska and
the Pacific Northwest region of North America. Similarly, the Black-capped
chickadee is a species particularly susceptible to the avian keratin disorder,
which disturbs the layers of keratinized epidermal tissue making up the beak.
As a result, it is of vital importance to clarify the anatomy of a healthy
passerine, which can be used as a comparison point for pathological conditions.
Because specializations of the avian beak allow the Class Aves to occupy an
immense diversity of ecological niches, understanding the configuration of
these tissues is critical both biologically and ecologically.
METHODOLOGY
 |
| Figure 2: High-magnification radiograph showing underlying bone structure of the beak in relation to external margins of the hard-cornified epidermis of the rhamphotheca (rhinotheca, gnatotheca) in Black-capped Chickadee (Van Hemert et al., (2012). |
This study was began in the
spring and fall of 2008, where 18 adult Black-capped Chickadee individuals were
collected from areas of south central and interior Alaska. 13 other samples
from previously collected birds were also used, following collection in the
fall season yielding a sample size of 31. To trap these birds, funnel traps and
mist nets were used. The birds were euthanized using isoflurane, and kept cool
prior to necropsy. Following necropsy,
the bone structure of the chickadees was analyzed using high magnification radiography.
This is a useful method that uses radiation to provide a highly magnified and
resolved image of the underlying tissues, organs, bones, and vessels of the specimen (“What is Radiography?”, 2018).
A major aspect of this paper was
outlining the histological methodology used to accurately prepare histological
sections for observation of the tissues. To process the 31 samples, the beaks and
claws were both treated with formalin-based formic acid for decalcification.
The tissues were trimmed, placed in a 70% isopropanol solution for 60 full days
prior to processing, and then dehydrated in increasing concentrations of
isopropanol. The samples were cleared with toluene, infiltrated using paraffin,
then embedded in paraffin blocks. A microtome was used for sectioning the
tissues and then staining was conducted using the classic combination stain;
hematoxylin and eosin (H&E). Special stains such as Masson’s trichrome were
also used for proper staining during this study. Masson’s stain was used
because it binds to glycogen and mucin, allowing visualization of the
respiratory epithelia and basement membranes when observing the histological
slides of the tissues.
FINDINGS
Following analysis by microscopic
techniques, it was confirmed that the pre-maxillary and mandibular bones extend
through the majority of the rhamphotheca, and are covered in both dermal and
epidermal tissue. The pre-maxillary and mandibular bones showed extension just
prior to the beak tip, where wide layers of dermal tissue replace the bony
tissues, and the epidermis increases in thickness. It was observed that the
beak of the Black-capped chickadee has many specialized structures for optimal
respiration and olfactory techniques. In
the upper half of the beak, the nasal passages were lined with hyaline
cartilage bearing large basophilic cells.
The cartilage layer was surrounded by connective tissue with collagen
fibers, which are hypothesized to add extra support to the nasal passage of the
chickadee. The nasal chamber also showed pseudo-stratified ciliated columnar
epithelia, with many mucus glands for assistance in the lubrication and
protection of this passage.
 |
| Figure 3: Examples of specialized structures in upper beak of Black-capped Chickadee: (A) PAS-positive basement membrane, salivary glands, and cartilage of rostral nasal passages; (B) hyaline cartilage, pseudo-stratified, ciliated respiratory epithelium, and
mucous glands of middle nasal concha; (C) salivary glands and cuboidal olfactory epithelium in lower beak; (D) hyaline cartilageand stratified squamous epithelium of rostral nasal passage. PAS (A), hematoxylin and eosin (B–D) (Van Hemert et al., 2012).
Van Hemert et al. (2012) also
determined that the dermis within the beak of the Black-capped Chickadee is
highly vascularized, containing connective tissue, blood vessels, and nerves
such as the trigeminal and facial nerves, veins, and arteries. Dermal papillae
were also discovered under the bird’s soft skin, but ending near the transition
into keratinized epidermis. Because the
dermal papillae lie at the base of feather follicles, is expected that these
structures assist in the proliferation of the epidermal cells and growth of the
feathers that surround the beak. Histological analyses also showed Herbst
corpuscles within the dermis, often near the premaxillary and mandibular bones
of the beak. While these mechanoreceptors were surely identified, they were
only localized in small clusters within the dermis. Because the Black-capped
chickadee beaks are characteristically black, it was revealed that the dark
coloration of the beak is attributed to the presence of melanocytes spanning
the dermal layer.
When observing the epidermal
components of the chickadee beak, Van Hemert et al. (2012) determined that the
length of the stratum germinativum (which can be divided in a stratum basale, a
stratum intermedium and a stratum transitivum) is consistent among the upper
and lower sections of the beak. The nuclei of the stratum intermedium showed
less definition and appeared granular, while the cells of the stratum
transitivum demonstrated a distinct polyhedral configuration. The cells of the
stratum corneum (localized above the stratum germinativum) appeared squamous
and de-nucleated, permitting the onset of keratinization. This layer had many
longitudinally arranged corneocytes, lining the surface of the rhamphotheca.
These cells did not appear to be highly uniform, and many areas showed signs of
abrasion. Because this aspect of the bird beak is fully exposed to the external
environment, a moderate amount of wear and tear from the activities of daily
bird life is expected. Melanocytes contributing to the coloration of the
Black-capped chickadee were also observed in the epidermal layers, but
dramatically less abundant relative to the dermis. In the epidermal layers, the
authors were able to determine that pigmentation of the beak was also due to
melanin granules that concentrated around the nuclei of the cells migrating to
the outer layer of the beak, most specifically within the superficial/tomial
layers.
While Van Hemert et al. (2012)
were able to successfully determine many general characteristics about the
beaks and claws of the Black-capped Chickadee, they were most importantly
effective in discovering unique characteristics about this particular species.
Because bird beaks are so diverse, understanding special characteristics about
this species of chickadee may explain it’s specialized ecological niche of
woodlots and orchards, where the bird digs holes in the soft, rotting wood and
feeds on a mixture of seeds, insects, and spiders (Canadian Wildlife
Federation, 2018). Research determined that the rhamphotheca of the
Black-capped Chickadee is much thicker relative to chickens (Gallus gallus), which is often used as a
type species for comparison studies with other birds. Because the goal of this
study was to provide a healthy point of comparison for diseased passerine
species, determining an alteration in the passerine condition relative to
chickens was an exciting discovery. The hard-cornified layer of the
Black-capped Chickadee was determined to be similar to other free-ranging
species, including the Great-spotted Woodpecker (Dendrocopus major) the Bohemian Waxwing (Bombycilla garrula) and several species of psittacines. Because the
Black-capped Chickadee and these other species nest in cavities, probe under
bark for food, and must open partially frozen seeds during the winter season,
and the beaks used to break into the tree bark must be strong and capable of
handling a large degree of mechanical stress.
 |
As previously described, small
clusters of Herbst corpuscles were observed within the dermis of the chickadee beak.
Species of birds localized near aquatic regions such as shorebirds, geese, and
ducks possess densely packed Herbst corpuscles that contribute to a bull tip
organ. Black-capped chickadees and other species of passerine birds do not
possess this food-locating structure, and are considered visual foragers.
Because this species does not rely on beak mechanoreceptors for feeding, the small
clusters of these corpuscles within the beak are consistent with that of other
passerine species.
CRITIQUE
In a personal critique of this
publication, I was very impressed by the study conducted by Van Hemert et al.
(2012) about the microanatomy of the Black-capped Chickadee keratinized
structures. To begin, the authors ensured the euthanasia of the birds involved
in the study was done ethically and in correspondence with the legal
requirements of the Alaska Science Center Institutional Animal Use & Care
committee. When conducting studies about animals or when using animal models,
it is crucial to act ethically and with respect for the individuals involved,
which was surely completed by this team of researchers. The methodology was well done and the depth of findings were both very detailed, and the published study included several
figures of the histological slides prepared and analyzed by the researchers.
This allows the readers to follow along visually with the findings outlined in
the text, which provides clarity to the claims offered by the researchers and
improves the overall understanding of the study.
I thought that a unique and
productive outcome of this publication was outlining several useful resources
for continued studies in beak gross and microanatomy, and detailing the methodology
used for the histological staining of hard-cornified tissues. The researchers provided
an outline for other researchers to use when attempting to investigate
hard-cornified structures such as the avian beak, which may be extrapolated to
the use of horse hooves, deer antlers, and many more tissues of veterinary
significance. Van Hemert et al. (2012) also made a point to acknowledge areas
of their histological procedures that could have been improved. It was
explained that the usage of a paraffin as an embedding medium and sectioning
using a regular microtome was generally successful during this study, but some
troubles arose when attempting to re-section blocks that were previously cut
and stored for approximately one year. Van Hemert et al. (2012) hypothesized
that these issues occurred due to excessive dehydration of the tissues, an
error that could be improved by re-treating the blocks before re-sectioning and
choosing a paraffin with a melting point closer to that of the avian beak.
 |
| (Walgreens, 2018). |
At last, the authors ensured that
prior to completing the study, several test runs about how to properly analyze
hard-cornified tissues in a histological context were conducted to understand
the perfect timeline for the decalcification procedure. The researchers
understood the damage acids may cause to tissues, but carefully decided that
formic acid was slow acting acid that demonstrated no negative impact on the appearance
and viability of the tissues. In the study, Van Hemert et al. (2012) used Nair
to break the disulfide bonds in keratin-rich tissues, due to the presence of
thioglycolate salts in this formula. The use of this product was only ever
informally acknowledged amongst histopathologists for the analysis of hooves in
the past. This paper was the first to conduct a trial and error experiment for
use of Nair as a softening agent, which uniquely converting a product normally
used for hairy legs into a product with great potential for increasing the
breadth of knowledge about the microanatomy of hard-cornified tissues!
In the future, I think it would be
beneficial to use a greater sample size in a repeat study, to investigate any
forms of interspecific or intraspecific differences amongst Black-capped
Chickadees and other bird species. Van Hemert et al. (2012) outlined small inconsistencies in results
compared to studies by Lucas & Stettenheim (1972) when analyzing the
discontinuity of the laminae between the inner and outer surfaces of the
chickadee beak. While Lucas & Stettenheim (1972) discovered lateral and
medial columns of transitional cells within the beak and noted the smooth
configuration of the stratum corneum layer lining the tomial edges of the
beak, Van Hemert et al. (2012) discovered a reduction in the adhesion of
individual corneocytes along the tip and tomial edges. It was noted by the
authors that while differences in the histological preparation mechanisms could
have been slightly different between the two studies, interspecific variation amongst Black-capped
Chickadee individuals also could have been the cause of this inconsistency. In
future studies, it would be interesting to investigate these findings more
extensively, amongst Black-capped Chickadee populations and in comparison to other species of
passerine birds. For now, I look forward to future studies relating the
findings of Van Hemert et al. (2012) to birds experiencing pathological
conditions, such as the avian keratin disease or other epizootic beak deformities.
I hope that the research efforts provided by this team simultaneously offers
assistance in the fields of veterinary medicine and ornithology.
Literature Cited
Canadian Wildlife Federation. (2018). Black-capped Chickadee.
Retrieved from
http://cwf-fcf.org/en/resources/encyclopedias/fauna/birds/black-capped-chickadee.html
Cornell Lab of Ornithology. (2018). Black-capped Chickadee Identification. Retrieved from https://www.allaboutbirds.org/guide/Black-capped_Chickadee/id
ETSU Homepage. (2018). What is Radiology? Retrieved from
https://www.etsu.edu/crhs/alliedhealth/radiologic/whatis.php
Lucas AM,Stettenheim PR. 1972. Avian anatomy: Integument. In: Agriculture Handbook 362. Washington, DC: US Department of Agriculture.
O’Loughlin, M. (n.d.). Retrieved from http://www.longfellownokomismessenger.com/feeding-birds-in-winter-seeds-of-wisdom-from-local-experts
O’Loughlin, M. (n.d.). Retrieved from http://www.longfellownokomismessenger.com/feeding-birds-in-winter-seeds-of-wisdom-from-local-experts
Pass, D. A., & Perry, R. A. (1984). The pathology of
psittacine beak and feather disease. Australian
Veterinary Journal, 61(3), 69-74. doi:10.1111/j.1751-0813.1984.tb15520.x
Soniak, M. (2016). What's Got Bird Beaks in a Twist?
Retrieved from https://www.audubon.org/news/whats-got-bird-beaks-twist
Van Hemert, C., Handel, C. M., Blake, J. E., Swor, R. M.,
& Ohara, T. M. (2012). Microanatomy of passerine hard-cornified tissues:
Beak and claw structure of the black-capped chickadee (Poecile atricapillus). Journal of Morphology, 273(2), 226-240.
doi:10.1002/jmor.11023
Walgreens. (2018). Nair Hair Remover Lotion For Body &
Legs Baby Oil. Retrieved from
https://www.walgreens.com/store/c/nair-hair-remover-lotion-for-body-&-legs-baby-oil/ID=prod2033112-product
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