RESEARCH NOTE

Regurgitation of the koilin layer in chinstrap penguins (Pygoscelis antarcticus) and its association with gastric parasites

Han-Kyu Kim, Chang-Yong Choi, Min-Su Jeong, Hwa-Yeon Kang & Woo-Shin Lee

Department of Forest Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea

Abstract

The koilin membrane, formed by the secretions of the ventricular and pyloric glands, functions as a protective layer in the gizzards of most bird species. However, the ecological functions of koilin have never been studied in free-ranging penguins. During the two austral summers from 2012 to 2014, we observed the regurgitated koilins of chinstrap penguins (Pygoscelis antarcticus) at Narębski Point on King George Island, South Shetland Islands, and we detected a significant difference in the daily accumulation of regurgitated koilins between the pre-hatching and post-hatching periods in the rookery. We also found 233 gastrointestinal parasites, all Stegophorus macronectes (Nematoda, Acuariidae), from 26 out of 45 koilins freshly regurgitated by chinstrap penguins. We suggest that the regurgitation of koilins may benefit adult chinstrap penguins in the wild by reducing parasitic loads when they fast during incubation; it may also help decrease the risk of parasite transmission to chicks. Our results present the first observations of regurgitated koilins among breeding chinstrap penguins. How koilin regurgitation functions in penguins requires further study. Among the gentoo penguins (P. papua) co-occurring at the study site, we observed no regurgitated koilin layers.

Keywords
Cuticula gastris; host-parasite interaction; nematodes; parasitic load; regurgitation; Stegophorus macronectes.

The koilin membrane, also known as the cuticula gastris (Hofmann & Pregl 1907; Eglitis & Knouff 1962), is the variably developed internal lining of a carbohydrate/protein complex (probably a mucoprotein), not keratinous, secreted by the ventricular and pyloric glands in avian gizzards (Akester 1986; Baumel et al. 1993; Bels 2006; Svihus 2011). The layer protects the muscles of the gizzard from the degrading effects of gastric acids and mechanical actions of food particles (Bels 2006). The koilin layer may be relatively soft in the poorly-muscled type of gizzards (Baumel et al. 1993).

In general, the koilin layer is replaced slowly as it wears by mechanical abrasion. McAtee (1917) suggested that the shedding of koilin layers may be observed in most species of birds. The shedding occurs as the layer gradually wears away in a few pieces or is detached from the stomach wall and digested (McAtee 1906, 1917). However, a few cases of regurgitating behaviours have been reported in some bird species such as American kestrels (Falco sparverius; Vyas et al. 2009), anhingas (Anhinga anhinga; Bartlett & Bartlett 1899), common cuckoos (Cuculus canrous; Newton et al. 1896), common starlings (Sturnus vulgaris; Newton et al. 1896), hornbills (Buceros sp.; Flower 1869), little owls (Athene noctua; Newton et al. 1896), mistle thrushes (Turdus viscivorous; Newton et al. 1896) and neotropical cormorants (Phalacrocorax brasilianus; Bartlett & Bartlett 1899).

Although the presence of this layer has been mostly reported from post-mortem studies in various species of wild birds, most studies of koilin have focused on their structure (Akester 1986) or how abnormal koilins affect poultry health (Webb & Colvin 1964; Ono et al. 2004; Kaldhusdal et al. 2012). A recent report on chemical irritations causing regurgitation of koilin suggests that koilin layers may protect against chemical toxicity as well as providing mechanical protection (Vyas et al. 2009). However, there is little information on the functions of koilin in wild birds and, to the best of our knowledge, no clear description of koilin regurgitation in penguins has been published.

During our study to monitor the breeding performances of chinstrap (Pygoscelis antarcticus) and gentoo penguins (P. papua) at Narębski Point during the austral summers of 2012/13 and 2013/14, we often observed koilin regurgitation in chinstrap penguins, but not in gentoo penguins, and their regurgitated koilins in and around the penguin rookery as well as along two major passages to the nearby shoreline. On the basis of these observations, we speculated that koilin regurgitation in chinstrap penguins could be related to the chick-rearing activity, which requires active foraging and delivery of foods.

In this paper, we report our observations of the regurgitated koilins and associated parasites, and the temporal change of koilin regurgitation in chinstrap penguins. We discuss the possible benefits of koilin regurgitation for free-ranging chinstrap penguins.

Methods

Study area

Narębski Point is on the south-east coast of Barton Peninsula, King George Island, in the South Shetland Islands, Antarctica. At the Narębski Point study site (62° 14′ 14.3″ S, 58° 46′ 27.1″ W), about 3000 pairs of chinstrap and 2300 pairs gentoo penguins breed every austral summer (Republic of Korea 2014). Chinstrap penguin rookeries are located on rocky slopes close to the shore and are divided into several sub-groups (colonies) by huge pillars of rocks or cliffs.

Sample collection and analysis

Temporal change in koilin regurgitation among chinstrap penguins was assessed by daily counts in six plots for 15 days from 19 December 2013 to 6 January 2014. Each plot (2.5 m×2.5 m) was set within the same chinstrap penguin colony (1475 nests) and had 7.3±0.5 breeding pairs of chinstrap penguins. Koilin was removed from the plot before the daily counts at 18 December 2013. We searched daily for freshly regurgitated koilins in the plots, and removed all found koilins from the plots to avoid double counts. When a survey was interrupted by unfavourable weather conditions (20 and 30 December 2013), we also excluded the next day’s results for further analysis. We assessed the temporal change in the daily accumulation number of koilins through Spearman correlation analysis, and compared the numbers before (pre-hatching period: 5 days) and after (post-hatching period: 10 days) the mean hatching date of the colony of chinstrap penguins (25 December 2013; Kim, unpubl. data) using the Mann-Whitney rank-sum test. Although snowy sheathbills (Chionis albus) were occasionally observed to consume the koilin in the study area, we assumed that there was no temporal change in the effect of their scavenging activity. All statistical analyses in this study were conducted using SigmaPlot version 12.0 (SysStat 2012).

We collected the koilins on the ground in the chinstrap penguin’s rookery from 7 to 30 December 2013; only freshly regurgitated layers that were intact, cylindrically shaped, and not damaged by scavengers such as snowy sheathbills nor contaminated by faeces of nearby penguins were collected (Fig. 1). At the laboratory at King Sejong Station on Barton Peninsula, the koilins were microscopically examined for gastrointestinal parasites (see Vidal et al. 2012). The number of parasites in each koilin was recorded, and they were kept in cryovials filled with 70% ethyl alcohol for further identification. Descriptive statistics, prevalence (proportion of koilins containing parasites) and the mean intensity (average number of parasites in each koilin containing parasites) with standard deviations, range and 95% confidence intervals (CIs) were calculated using the Clopper-Pearson interval (for prevalence) and 2000 bootstrap replications (for mean intensity) with QP 3.0 software (Rozsa et al. 2000; Reiczigel et al. 2013). The collected parasites, which were all nematodes, were identified later under microscopes at the laboratory at Seoul National University in the Republic of Korea. Identifications were based on taxonomic keys and morphological descriptions, in particular regarding the structure of their cephalic collars, following Mawson (1953), Zdzitowiecki & Drózdz (1980) and Vidal et al. (2016).

Fig. 1  A koilin layer regurgitated by an adult chinstrap penguin (Pygoscelis antarcticus), 21 January 2014.

Results

The daily accumulation of regurgitated koilins, which are yellow with a green tip (Fig. 1), per plot showed the temporal decline along the breeding season (Spearman’s rank-order correlation; r_s=−0.795, P<0.001). The number was higher in the pre-hatch period (mean and SD: 3.0±0.3, median: 3.0, range: 0–8, n=30) than that in the post-hatching period (mean and SD: 0.7±0.1, median: 0.0, range: 0–3, n=60) (Mann-Whitney U=181.0, df=1, P<0.001; Fig. 2).

Fig. 2  Temporal change in the daily accumulation of regurgitated koilins at six plots in the breeding colony of chinstrap penguins. The size of filled circles represents the number of plots (ranging from one to six).

A total of 45 intact and fresh koilin layers were collected in the rookery of chinstrap penguins during the survey. We also found 233 parasites, all identified as Stegophorus macronectes (Nematoda, Acuariidae), in 26 out of 45 koilins. Anchored in the regurgitated koilins, the parasitic nematodes were alive at the time of collection (range: 0–61, prevalence: 57.8%, 95% CI for prevalence: 42.2–72.3%, mean intensity: 8.96, 95% CI for mean intensity: 5.96–16.70).

Discussion

Based on field observations in the austral summer of 2012/13, we confirmed that chinstrap penguins regurgitate yellow koilins, which were partially coloured green, possibly from regurgitated bile (Bels 2006), mostly before the hatching dates. Though the regurgitated koilins within the colony can be easily trampled by penguins or partially fed on by scavenging birds, the regurgitated koilins were still common in terms of distribution and quantity. The fresh koilins were cylindrically shaped when undamaged and untrampled, suggesting that free-ranging wild chinstrap penguins may replace the whole koilin layer at once during the early breeding season. Considering the live parasites found in the regurgitated koilins, it may be better to regurgitate the koilin than to send it to the duodenum or further along in the intestines, to prevent pathogenic effects, such as ectopic infection.

More regurgitated koilin layers were found in the pre-hatching period, compared to the post-hatching period, indicating that chinstrap penguins are more likely to regurgitate koilin layers when they are incubating eggs compared to when they are rearing chicks. Because this layer protects the stomach wall from mechanical and chemical irritation, digestive activity is paused when this layer is regurgitated and gizzards of birds in this stage must be kept empty until the koilin is renewed (McAtee 1917; Vyas et al. 2009). Breeding parents of chinstrap penguins take turns sitting on the nest in a 5–10 day period during incubation (Lishman 1985; Borboroglu & Boersma 2013), giving them time to empty their stomachs and replace koilin layers. In contrast, when chicks hatch, parental turn-taking occurs on a daily basis (Borboroglu & Boersma 2013). Therefore, we suggest that breeding chinstrap penguins in the wild have a strategy of disposing of and renewing the whole koilin before hatching, because it cannot coincide with food delivery to chicks.

The most important role of such regurgitation must be to replace the old lining in the ventriculus for renewed gizzard protection (Bels 2006). However, as McAtee (1917) suggested, shedding koilins may help birds to reduce their parasitic load, particularly nematodes, which are usually anchored between the koilin layer and the gizzard wall. As chinstrap penguins are a marine predator that consume mainly Antarctic krill (Euphausia superba; Ratcliffe & Trathan 2012), their gizzards’ koilin layers may not be mechanically abraded in the way that they are in granivorous birds. These penguins’ gastrointestinal tracts may be under greater stress from parasites, acquired from the marine food web, than stress by mechanical abrasions. Many parasitic Stegophorus macronectes were observed in the regurgitated koilins in this study. This nematode was the only gastrointestinal parasite species found in the gizzard of chinstrap penguins in prior studies (Barbosa & Palacios 2009; Vidal et al. 2012; Brandao et al. 2014).

The previously reported prevalence (67%) of S. macronectes in three adult chinstrap penguins on Deception Island, South Shetland Islands (Vidal et al. 2012), falls into the range of the prevalence estimated from the koilin analysis in this study (42.2–72.3%). Given the mean intensity of the nematode (39.5±43.9) reported from the same species in the same region (Vidal et al. 2012), getting rid of a koilin harbouring nine nematodes—the mean intensity found in our samples—may result in a roughly 23% reduction in the gastrointestinal nematode load. However, because of the small sample size in the study by Vidal et al. (2012), this quantitative comparison is not reliable and the effect of such removal on the body condition of its host is unclear. Nonetheless, these findings are suggestive of the potential role of koilin regurgitation in reducing parasites.

Parental provisioning can transfer gastric parasites to chicks (Muzaffar & Jones 2004; Vidal et al. 2012), and reducing gastric parasites in the parent’s gizzard may decrease the risk of transmission through food delivery. Although the pathogenicity and the removal effects of S. macronectes in adult chinstrap penguins are not well understood, increased growth rates were observed in experimentally de-wormed chinstrap nestlings compared to their control siblings, which shows the negative effects of gastrointestinal parasites on nestling body conditions, survival rate and fitness in this species (Palacios et al. 2012). Therefore, we suggest that the koilin regurgitation by fasting adults of wild chinstrap penguins may benefit their chicks to an unknown extent.

In contrast to chinstrap penguins, we never observed any regurgitated koilin layers among the gentoo penguins co-occurring at the study site. Whether, and in what manner, gentoo penguins replace their koilin layers is unknown.

To the best of our knowledge, this is the first study of koilin regurgitation in penguin species and the first report on the prevalence of parasites in regurgitated koilin layers. However, our knowledge is limited to this small study site and short time period. Further observations as well as experimental studies across other penguin species in different areas and seasons are required to better understand the details concerning the mechanisms and functions of koilin regurgitation and replacement.

Acknowledgements

We thank the Korea Polar Research Institute and King Sejong Station for their support on our field studies in Antarctica. We thank especially to Mr. Jin-Woo Jeong for suggesting initial steps of this study. This study was funded by the Ministry of Environment, Republic of Korea, as part of a long-term ecosystem monitoring on the Antarctic Specially Protected Areas (ASPA) No. 171 on King George Island (PN13130). This study was performed with permissions (ILAD-2692 [2013.10.18]) from the Korean Ministry of Foreign Affairs and Trade, according to the Act on Antarctic Activities and Protection of Antarctic Environment.

References

Akester A. 1986. Structure of the glandular layer and koilin membrane in the gizzard of the adult domestic fowl (Gallus gallus domesticus). Journal of Anatomy 147, 1–25. PubMed Abstract | PubMed Central Full Text

Barbosa A. & Palacios M.J. 2009. Health of Antarctic birds: a review of their parasites, pathogens and diseases. Polar Biology 32, 1095–1115. Publisher Full Text

Bartlett A.D. & Bartlett E. 1899. Wild animals in captivity, being an account of the habits, food, management and treatment of the beasts and birds at the “Zoo.” London: Chapman and Hall.

Baumel J.J., King A.S., Breazile J.E., Evans H.E. & Vanden Berge J.C. 1993. Handbook of avian anatomy: nomina anatomica avium. Cambridge, MA: Nuttall Ornithological Club.

Bels V.L. (ed.). 2006. Feeding in domestic vertebrates: from structure to behaviour. Wallingford: Cabi Publishing.

Borboroglu P.G. & Boersma P.D. 2013. Penguins: natural history and conservation. Seattle: University of Washington Press.

Brandao M.L., Moreira J. & Luque J.L. 2014. Checklist of Platyhelminthes, Acanthocephala, Nematoda and Arthropoda parasitizing penguins of the world. Check List 10, 562–573. Publisher Full Text

Eglitis I. & Knouff R. 1962. An histological and histochemical analysis of the inner lining and glandular epithelium of the chicken gizzard. American Journal of Anatomy 111, 49–65. PubMed Abstract | Publisher Full Text

Flower W.H. 1869. Note on a substance ejected from the stomach of a hornbill (Buceros corrugatus). Proceedings of the Zoological Society of London 1869, 150.

Hofmann K. & Pregl R. 1907. Uber koilin. (About koilin.). Hoppe-Seyler′s Zeitschrift für Physiologische Chemie 52, 448–471. Publisher Full Text

Kaldhusdal M., Hetland H. & Gjevre A.G. 2012. Non-soluble fibres and narasin reduce spontaneous gizzard erosion and ulceration in broiler chickens. Avian Pathology 41, 227–234. PubMed Abstract | Publisher Full Text

Lishman G.S. 1985. The comparative breeding biology of Adélie and chinstrap penguins Pygoscelis adeliae and P. antarctica at Signy Island, South Orkney Islands. Ibis 127, 84–99. Publisher Full Text

Mawson P.M. 1953. Parasitic Nematoda collected by the Australian National Antarctic Research Expedition: Heard Island and Macquarie Island, 1948–1951. Parasitology 43, 291–297. PubMed Abstract | Publisher Full Text

McAtee W.L. 1906. The shedding of the stomach lining by birds. Auk 23, 346.

McAtee W.L. 1917. The shedding of the stomach lining by birds, particularly as exemplified by the Anatidae. Auk 34, 415–421. Publisher Full Text

Muzaffar S.B. & Jones I.L. 2004. Parasites and diseases of the auks (Alcidae) of the world and their ecology—a review. Marine Ornithology 32, 121–146.

Newton A., Gadow H., Lydekker R., Roy C.S. & Shufeldt R.W. 1896. A dictionary of birds. London: Adam and Charles Black.

Ono M., Okuda Y., Shibata I., Sato S. & Okada K. 2004. Pathogenicity by parenteral injection of fowl adenovirus isolated from gizzard erosion and resistance to reinfection in adenoviral gizzard erosion in chickens. Veterinary Pathology Online 41, 483–489. Publisher Full Text

Palacios M., Valera F. & Barbosa A. 2012. Experimental assessment of the effects of gastrointestinal parasites on offspring quality in chinstrap penguins (Pygoscelis antarctica). Parasitology 139, 819–824. PubMed Abstract | Publisher Full Text

Ratcliffe N. & Trathan P. 2012. A review of the diet and at-sea distribution of penguins breeding within the CAMLR convention area. CCAMLR Science 19, 75–114.

Reiczigel J., Rozsa L., Reiczigel A. & Fabian I. 2013. Quantitative parasitology. QPweb, version 1.0. Accessed on the internet at http://www2.univet.hu/qpweb on 07 April 2014.

Republic of Korea 2014. Monitoring and management report of Narębski Point (ASPA no. 171) during the past 5 years (2009–2014). Background Paper 7, Revision 1. XXXVII Antarctic Treaty Consultative Meeting. 28 April–07 May 2014, Brasilia.

Rozsa L., Reiczigel J. & Majoros G. 2000. Quantifying parasites in samples of hosts. Journal of Parasitology 86, 228–232.

Svihus B. 2011. The gizzard: function, influence of diet structure and effects on nutrient. World’s Poultry Science Journal 67, 207–224. Publisher Full Text

SysStat. 2012. SigmaPlot for Windows, version 12.0. Chicago: SysStat Software.

Vidal V., Ortiz J., Diaz J.I., de Ybañez M.R., Amat M., Palacios M., Benzal J., Valera F., De La Cruz C. & Motas M. 2012. Gastrointestinal parasites in chinstrap penguins from Deception Island, South Shetlands, Antarctica. Parasitology Research 111, 723–727. PubMed Abstract | Publisher Full Text

Vidal V., Ortiz J., Diaz J.I., Zafrilla B., Bonete M.J., Ruiz De Ybañez M.R., Palacios M.J., Benzal J., Valera F., De La Cruz C., Motas M., Bautista V., Machordom A. & Barbosa A. 2016. Morphological, molecular and phylogenetic analyses of the spirurid nematode Stegophorus macronectes (Johnston & Mawson, 1942). Journal of Helminthology 90, 214–222. PubMed Abstract | Publisher Full Text

Vyas N.B., Spann J.W. & Hill E.F. 2009. Acute oral toxicities of wildland fire control chemicals to birds. Ecotoxicology and Environmental Safety 72, 862–865. PubMed Abstract | Publisher Full Text

Webb T. & Colvin J.R. 1964. The composition, structure, and mechanism of formation of the lining of the gizzard of the chicken. Canadian Journal of Biochemistry 42, 59–70. Publisher Full Text

Zdzitowiecki K. & Drózdz J. 1980. Redescription of Stegophorus macronectes (Johnston et Mawson, 1942) and description of Stegophorus arctowskii sp. n. (Nematoda, Spirurida) from birds of South Shetlands (the Antarctic). Acta Parasitologica Polonica 27, 205–212.