Date of Award

2026

Keywords

marsupials, milk, pouch secretions, conservation, proteomics

Document Type

Thesis - ECU Access Only

Publisher

Edith Cowan University

Degree Name

Doctor of Philosophy

School

School of Science

First Supervisor

Michelle L. Colgrave

Second Supervisor

Angéla Juhász

Third Supervisor

Mitchell G. Nye-Wood

Fourth Supervisor

Katherine A. Farquharson

Abstract

Marsupials, a distinct infraclass of mammals, are predominantly native to the Australian continent and include iconic species like the koala (Phascolarctos cinereus) and the Tasmanian devil (Sarcophilus harrisii). Unfortunately, their populations have been severely impacted by diseases, domestic animal attacks, roadkill, habitat losses, low genetic diversity, and wildfires, intensifying the need for effective conservation strategies. Improving the survival of orphaned joeys represents an important avenue to support these efforts. However, current hand-rearing practices often achieved limited success, largely due to incomplete compositional understanding of marsupial milk and pouch secretions.

Unlike eutherian mammals, marsupials give birth to altricial young that are immunologically naïve and lack mature immune tissues and cells. As immune tissues develop over the first four months of life, joeys rely heavily on passive immunity provided by the pouch environment and milk. The limited ability to reproduce the immune protection normally provided by maternal milk and the pouch environment contribute significantly to the low survival of hand-reared orphaned marsupial joeys.

The research presented herein optimised sample preparation and data analysis workflows for precious marsupial samples and investigated the role of marsupial milk and pouch secretions in supporting healthy joey growth, using koala milk and pouch secretions from breeding Tasmanian devils. This study applied liquid-chromatography tandem mass spectrometry (LC-MS/MS) to characterise protein composition of marsupial milk and pouch secretions. To begin, milk fractionation, protein extraction, sample preparation, data acquisition, and data analysis workflows were optimised using eutherian cow and Arabian camel milk. This optimisation established a systematic protocol that improved disulfide bond reduction, alkylation efficiency, enzymatic digestion, and downstream LC-MS/MS data analysis methodologies, which were later adapted for the analysis of marsupial milk and pouch secretions. On average, the present study on koala milk managed to quantitatively profile 874, 1,220, and 498 protein groups from early-, mid- and late-lactation milk, respectively. Moreover, the study identified a core set of highly abundant proteins conserved across all major lactation stages, including β lactoglobulin, caseins, clusterin, trichosurin, albumin and lactotransferrin underscoring their key nutritional, immunological, and detoxification functions. Furthermore, the study managed to identify core sets of interacting protein clusters involved in translation, protein transport, energy metabolism, and immune function, processes essential for both healthy joey development and lactation in mothers. These protein clusters varied in abundance in correspondence with the demands of the growing pouch young at each stage. An emphasis on immune proteins revealed stage-specific regulation of immune pathway, with early- and mid-lactation milk supporting foundational immune development and neuroimmune protection, respectively, and late-lactation associated with a more immunocompetent joey.

Proteomic profiling of Tasmanian devil pouch secretions revealed 403 proteins native to the pouch environment. Of these, 153 proteins were consistently detected across all samples, while 200 were found in at least three, reflecting a largely overlapping protein expression profile. This core protein set included a diverse array of proteins with known direct and indirect roles in immune function, such as immunoglobulins, lysozymes, complement cascade proteins, S100 family proteins and other antimicrobial proteins and peptides. Moreover, the immune pathways involving these proteins were significantly overrepresented, and localised to cellular immune complexes, supporting the long standing hypothesis that the pouch secretions play an essential role in providing immunological protection to the developing joey.

These findings provide valuable insights into marsupial-specific developmental strategies, which may inform improved hand-rearing protocols for both the focal species and other members of the marsupial infraclass.

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Link to publisher version (DOI)

10.25958/0wd6-2296