Immune cells

Marsupial milk contains numerous immune cell types that provide passive immunity to the developing pouch young^{(Reference Smith and Keyte28)} (Table 1). Macrophages, neutrophils and lymphocytes have been identified in the colostrum and throughout lactation in the tammar wallaby, quokka and koala^{(Reference Young, Basden and Cooper29–Reference Cockson and McNeice31)}. Neutrophils are the most abundant immune cells in koala milk, and their numbers increase until the end of lactation, highlighting their importance to phagocytic protection of the joey even at this late stage^{(Reference Young, Basden and Cooper29,Reference Young and Deane30)} . Preference for a high level of neutrophils may be due to their superior capacity to deal with pathogenic bacteria and fungi^{(Reference Young, Basden and Cooper29)}. A range of other immune cells are likely to be present in milk, given the identification of immune receptors and cell markers in milk transcriptomes and proteomes^{(Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20)} . These include dendritic cells, helper (CD4) and cytotoxic (CD8) T lymphocytes, and other granulocytes such as eosinophils and basophils^{(Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20)} .

Table 1. Bioactive components present in mammary gland, milk and pouch environment

IgA, immunoglobulin A; IgE, immunoglobulin E; IgG, immunoglobulin G; IgM, immunoglobulin M; MM1, marsupial milk 1; VELP, very early lactation protein; WAP, whey acidic protein; WFDC2, WAP four-disulphide domain protein-2.

Immunoglobulins

Immunoglobulins (Igs) play an important role in adaptive immunity. There are four types of Igs in marsupials (IgA, IgE, IgM and IgG) that have different functions^{(Reference Morris, Prentis and O’Meally32)} (Table 1). IgA is involved in mucosal immunity, IgE is involved in defence against parasites and hypersensitivity, IgG is the most abundant immunoglobulin in the extracellular fluid and blood, and IgM is the first immunoglobulin produced after exposure to an antigen^{(Reference Morris, Prentis and O’Meally32)}. All four Ig types are present in marsupial milk and mammary glands. Once transferred through the milk, they can provide passive immunity to the altricial young^{(Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20,Reference Hurley and Theil33)} . IgA and IgG are the predominant Igs in marsupial milk, which is also the case in eutherians^{(Reference Deane and Cooper34–Reference Adamski and Demmer36)}.

Immunoglobulins are generally transferred via the milk during two periods in marsupials: in the colostrum immediately after birth at the start of phase 2A, and when the young first emerges from the pouch at the onset of phase 3^{(Reference Adamski and Demmer36,Reference Daly, Digby and Lefèvre37)} . At these times, high levels of Igs provide passive protection as young are exposed to new environments and microorganisms^{(Reference Cheng and Belov11)}. However, the expression of Ig types at these two timepoints can differ between species. IgG is absorbed across the pouch young gut epithelium, facilitated by the neonatal Fc receptor, and enters peripheral circulation, thereby protecting against infection^{(Reference Yadav38)}. IgG was detected in wallaby, kangaroo and possum colostrum, although the concentration was low compared with eutherian colostrum^{(Reference Deane and Cooper34,Reference Deane, Cooper and Renfreec35)} . In possums, IgG was significantly elevated during late lactation compared with early lactation^{(Reference Adamski and Demmer36)}, whereas in koalas, IgG levels remained constant through early and late lactation, although the neonatal Fc receptor was expressed only during early lactation^{(Reference Hewavisenti, Morris and O’Meally18,Reference Adamski and Demmer36,Reference Daly, Digby and Lefévre39)} .

IgA and IgM are not absorbed across the gut epithelium but are involved in immune defence at these sites^{(Reference Woof and Kerr40)}. IgA was identified in the colostrum of the tammar wallaby and brushtail possum^{(Reference Deane, Cooper and Renfreec35,Reference Adamski and Demmer41)} . Higher IgA levels were observed during late lactation compared with early lactation in the koala and brushtail possum^{(Reference Morris, O’Meally and Zaw20,Reference Adamski and Demmer41)} . In the koala milk proteome, IgA accounted for 2% of peptides in late lactation, compared with only 0·08% in early lactation. Conversely, IgM was identified only during early lactation in the koala and was not found in a late lactation milk proteome^{(Reference Morris, O’Meally and Zaw20)}. Tasmanian devil milk has been investigated only during mid-lactation, and IgA levels were the highest of the four Ig types present^{(Reference Hewavisenti, Morris and O’Meally18)}.

The S100 family of calcium-binding proteins

The S100 protein family consists of S100A1–S100A16, S100A19, S100B, S100G, S100P and S100Z proteins^{(Reference Kwek, Wynne and Lefèvre42)}. These proteins have well-known antimicrobial and immunomodulatory functions in eutherians^{(Reference Ryckman, Vandal and Rouleau43,Reference Büchau, Hassan and Kukova44)} . In humans, S100A8 and S100A9 are highly expressed in colostrum and at lower levels in mature milk^{(Reference Pirr, Richter and Fehlhaber45)}. Gene knockout experiments in mice have shown that S100A8 and S100A9 are absorbed across the neonatal gut, enter peripheral circulation and may protect against neonatal sepsis via direct antimicrobial activity^{(Reference Pirr, Richter and Fehlhaber45,Reference Wang, Song and Wang46)} . In marsupials, S100A8 and S100A9 are expressed in the milk and mammary gland^{(Reference Peel, Cheng and Djordjevic17)}, whereas S100A9 and S100A15 are expressed in the pouch skin^{(Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20,Reference Kwek, Wynne and Lefèvre42)} (Table 1). Interestingly, S100A8 and S100A9 were not present in the koala early lactation milk transcriptome or proteome and have not been investigated in the colostrum of any marsupial species. Instead, these proteins were highly abundant in mid- and late lactation in devils and koalas^{(Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20)} . S100A9 was the seventh most highly expressed immune transcript in the Tasmanian devil mid-lactation milk transcriptome (0·07%)^{(Reference Hewavisenti, Morris and O’Meally18)}. Similarly, S100A8 and S100A9 were highly abundant in the koala late lactation milk proteome, being the eighth (1·67%) and twenty-fifth (0·59%) most abundant protein, respectively^{(Reference Hewavisenti, Morris and O’Meally18)}. The high abundance of S100A8 and S100A9 indicates that these proteins may have important functions in marsupial milk, likely having anti-inflammatory and antimicrobial properties similar to human S100A8 and S100A9. The marsupial S100A8–S100A9 complex may also enter peripheral circulation in the young and protect against systemic infection, as in humans^{(Reference Pirr, Richter and Fehlhaber45)}.

Marsupials also have a unique S100 protein not found in eutherians, S100A19. This protein is expressed in the tammar wallaby mammary gland during pregnancy and involution but not lactation^{(Reference Kwek, Wynne and Lefèvre42)}. S100A19 is also expressed in the foregut of joeys during early pouch life when their diet consists only of milk^{(Reference Kwek, Wynne and Lefèvre42)}. Marsupial S100A19 is closely related to eutherian S100A15 and A7^{(Reference Kwek, Wynne and Lefèvre42)}, both of which are antimicrobial^{(Reference Büchau, Hassan and Kukova44,Reference Gläser, Harder and Lange47)} . As such, S100A19 likely provides antimicrobial protection to the mammary gland and pouch young gut during development.

Lysozymes

The lysozyme family comprises lysozyme C and calcium-binding lysozyme. Among these, lysozyme C is the most prevalent across mammals, while calcium-binding lysozyme is less common^{(Reference Ragland and Criss48,Reference Irwin, Biegel and Stewart49)} . Lysozyme is widely expressed in milk and is renowned for its antibacterial activity and immunomodulatory roles within the innate immune system^{(Reference Ragland and Criss48)}. Variation exists between mammalian species in the number of lysozyme C genes, with one gene identified in humans, four in the grey short-tailed opossum (Monodelphis domestica) and two complete and six incomplete genes in the tammar wallaby^{(Reference Irwin, Biegel and Stewart49)}. Gene duplications in the lysozyme family have not been well characterised across marsupials despite the recent release of numerous high-quality reference genomes across the marsupial tree^{(Reference Peel, Silver and Brandies50–Reference Stammnitz, Gori and Kwon52)}. In marsupial milk, lysozyme has been identified in the early milk proteome of the tammar wallaby^{(Reference Joss, Molloy and Hinds53)}, mid-lactation milk transcriptome of the Tasmanian devil^{(Reference Hewavisenti, Morris and O’Meally18)}, mid-lactation milk of the ringtail possum^{(Reference Nicholas, Loughnan and Messer54)} and late lactation milk proteome of the koala^{(Reference Morris, O’Meally and Zaw20)}, and in the whey proteins and mammary gland RNA of all lactation stages of the brushtail possum^{(Reference Piotte, Marshall and Hubbard55)} (Table 1). Up-regulation of lysozyme gene expression in the mammary gland and greater lysozyme C abundance in the milk as the lactation cycle progresses^{(Reference Demmer, Ross and Ginger6,Reference Vander Jagt, Whitley and Cocks56,Reference Kuy, Kelly and Smit57)} suggests an important role of lysozyme C as the joey detaches from the teat and starts to move out of the pouch. Lysozyme has also been identified by proteomics in post-reproductive tammar wallaby pouch secretions^{(Reference Ambatipudi, Joss and Deane58)}, and lysozyme C was the most highly abundant transcript in the woylie pouch skin^{(Reference Peel, Silver and Brandies50)}. Although lysozyme has well-characterised antibacterial activity in eutherians^{(Reference Irwin, Biegel and Stewart49)}, possible antimicrobial and immunomodulatory roles of lysozyme in marsupials remain to be confirmed.

Transferrin and lactoferrin

The glycoproteins transferrin and lactoferrin are both integral components of the transferrin family and major components of milk. Lactoferrin exists in three isoforms: lactoferrin-α, lactoferrin-β and lactoferrin-γ. Lactoferrin-α is the only isoform capable of binding iron. The iron-binding and sequestration capabilities of transferrin and lactoferrin-α are crucial in inhibiting bacterial growth^{(Reference Kell, Heyden and Pretorius59)}. Lactoferrin has demonstrated antibacterial, antiviral, antifungal, anti-inflammatory and anticarcinogenic activity^{(Reference Kell, Heyden and Pretorius59)} (Table 1). Furthermore, enzymatic cleavage of the N-lobe of lactoferrin yields crucial peptides lactoferricin and lactoferrampin, which exhibit antimicrobial activity and are under positive selection in primates^{(Reference Barber, Kronenberg and Yandell60)}. Marsupial N lobe sequences from the opossum and Tasmanian devil cluster separately to eutherians^{(Reference Hughes and Friedman61)}, but neither antimicrobial function nor diversity within marsupials has been explored.

Lactoferrin was the most abundant protein in the koala late lactation milk proteome (Fig. 1), accounting for almost half of all transcripts expressed^{(Reference Morris, O’Meally and Zaw20)}. Possible antimicrobial or other functions should be investigated to better understand the role of lactoferrin in marsupials.

Figure 1. Differential expression of lactoferrin between early and late proteomes. Lactoferricin accounted for 49·4% of all transcripts expressed in koala milk during late lactation, but just 0·19% of transcripts produced during early lactation.

Transferrin has been detected around the time of birth in the brushtail possum^{(Reference Adamski and Demmer36)} and tammar wallaby^{(Reference Ambatipudi, Joss and Raftery21)} (Table 1). Elevated levels of transferrin have also been observed at mid-lactation, when the joey begins exiting the pouch, in both the ringtail^{(Reference Nicholas, Loughnan and Messer54)} and brushtail possums^{(Reference Adamski and Demmer36,Reference Adamski and Demmer41,Reference Grigor, Bennett and Carne62)} . Transferrin was also identified in the early lactation milk proteome^{(Reference Joss, Molloy and Hinds53)} and mid-lactation mammary gland transcriptome^{(Reference Lefèvre, Digby and Whitley19)} of the tammar wallaby. Evidence for increased expression of transferrin coinciding with birth and first pouch exit suggests that it may play an important role in the immunological protection of marsupial young.

Whey proteins

The whey acidic protein (WAP) is a type of whey protein characterised by cysteine-rich domains, known as disulfide core (4-DSC) domains^{(Reference Wilkinson, Roghanian and Simpson63)}. The WAP is classified as antimicrobial and involved in inhibiting neutrophil serine proteases^{(Reference Wiesner and Vilcinskas64)}. WAP inhibits the growth of Staphylococcus aureus ^{(Reference Iwamori, Nukumi and Itoh65)} and also has non-immune functions, such as stimulating the proliferation of mammary epithelial cells^{(Reference Topcic, Auguste and De Leo66)}. WAP has been identified as a major whey protein in the milk of numerous eutherian and marsupial species, as well as monotremes^{(Reference Sharp, Lefèvre and Nicholas67)} (Table 1). Marsupial WAP has an additional 4-DSC domain to the two 4-DSCs in eutherian WAP^{(Reference Sharp, Lefèvre and Nicholas67)}. WAP was one of the top 200 most highly expressed transcripts in the Tasmanian devil mid-lactation milk transcriptome^{(Reference Hewavisenti, Morris and O’Meally18)} and has also been detected at mid-lactation in the red kangaroo^{(Reference Nicholas, Fisher and Muths68)} and brushtail possum^{(Reference Demmer, Stasiuk and Adamski69)}. The tammar wallaby mammary gland WAP four-disulphide domain protein-2 (WFDC2) has demonstrated bactericidal activity against Salmonella enterica, Pseudomonas aeruginosa and S. aureus but no activity against commensal bacteria^{(Reference Watt, Sharp and Lefevre70)}. In koalas, WFDC2 was abundant in the early lactation milk proteome and present in the late lactation proteome^{(Reference Morris, O’Meally and Zaw20)}. The abundance of WFDC2 in early lactation may coincide with the need for increased immune protection around birth, while the expression of WAP in mid-lactation suggests a possible immunological role in protecting the joey as it begins to exit the pouch.

Whey proteins with specific primary roles can also have secondary bioactive functions as pre-proteins, aside from contributing to the macronutrient profile of milk. Prominent preproteins in marsupial milk include β-lactoglobulin, which is a lipocalin protein that transports iron and other hydrophobic molecules^{(Reference Roth-Walter, Pacios and Gomez-Casado71)}, and α-lactalbumin, which is intimately involved in lactose metabolism^{(Reference Brew72)} and iron absorption^{(Reference Wang and Zhang73)}. Evidence from eutherians suggests possible bioactivity of these two whey proteins. Proteolytic digestion of bovine β-lactoglobulin and α-lactalbumin resulted in numerous sequences with activity against Gram-positive bacteria^{(Reference Pellegrini, Thomas and Bramaz74,Reference Pellegrini, Dettling and Thomas75)} (Table 1). β-Lactoglobulin and α-lactalbumin have been widely identified in marsupial milk: both were among the top 200 most highly expressed transcripts in the Tasmanian devil milk transcriptome^{(Reference Hewavisenti, Morris and O’Meally18)} and were the predominant proteins in the mature reproductively active tammar wallaby pouch^{(Reference Ambatipudi, Joss and Deane58)}. α-Lactalbumin was expressed in the mammary gland of the brushtail possum at all phases of lactation and increased in phase 3^{(Reference Demmer, Ross and Ginger6,Reference Piotte, Marshall and Hubbard55)} and was present in all samples of ringtail possum milk tested^{(Reference Nicholas, Loughnan and Messer54)}. β-Lactoglobulin was the most abundant transcript in the koala mammary gland transcriptome^{(Reference Morris, O’Meally and Zaw20)}; was secreted at constant levels in red kangaroo mid-lactation^{(Reference Nicholas, Fisher and Muths68)}; was present in the early milk protein and expressed in the mammary gland at constant levels throughout lactation in the brushtail possum^{(Reference Demmer, Ross and Ginger6,Reference Kuy, Kelly and Smit57)} ; and was present in the tammar wallaby mid- and late lactation mammary gland transcriptomes^{(Reference Lefèvre, Digby and Whitley19)}. The main chain of β-lactoglobulin was also identified in the milk proteins of the tammar wallaby throughout lactation, with a second isoform present in phase 2A and a third isoform at a high concentration in phase 2B^{(Reference Joss, Molloy and Hinds7)}. In pouch secretions of the tammar wallaby, β-lactoglobulin was present in high concentrations. Although there was no evidence of direct antimicrobial activity of β-lactoglobulin against Escherichia coli or S. aureus, the antimicrobial potential of cleaved peptides has been hypothesised^{(Reference Ambatipudi, Joss and Raftery21)}. Both α-lactalbumin and β-lactoglobulin are present in the milk, mammary glands and pouches of numerous marsupials. It is possible they provide immune protection when cleaved, but given that they have other functions, their expression likely has other effects, too, and further investigation is required to confirm their bioactivity.

Additional immune proteins

Marsupial milk is also a rich source of other immune proteins, accounting for 9% and 6·6% of all proteins in koala and Tasmanian devil milk, respectively^{(Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20)} (Table 1). These include immune receptors such as the major histocompatibility complex proteins (MHC) classes I and II, toll-like receptors (TLRs) and natural killer (NK) cell receptors^{(Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20,Reference Lefèvre, Digby and Whitley76)} . The presence of these receptors indicates the diversity of immune cells present in the milk and mammary gland, and potential protective functions involving antigen recognition and binding.

Immune signalling molecules, such as cytokines and chemokines, are also present in marsupial milk and mammary glands. The chemokine CCL25 was one of the most highly expressed immune signalling molecules in both koala and Tasmanian devil milk^{(Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20)} . CCL25 has only recently been identified in human milk and is highly expressed in colostrum^{(Reference Abe, Onoda and Furushima77)}. In humans, CCL25 is essential for thymocyte development^{(Reference Uehara, Hayes and Li78)} and may play a similar role in pouch young. Other chemokines, such as CCL28, were also identified in the devil mammary gland transcriptome^{(Reference Morris, O’Meally and Zaw20)}. In humans, CCL28 and CCL25 facilitate the transfer of IgA into the milk by attracting IgA-antibody-producing immune cells to the mammary gland epithelium^{(Reference Wilson and Butcher79,Reference Bowman, Kuklin and Youngman80)} . CCL28 is also antibacterial against Gram-negative and Gram-positive bacteria, and the fungus Candida albicans ^{(Reference Hieshima, Ohtani and Shibano81)}. As such, these chemokines may be involved in both direct and indirect protection of the pouch young gastrointestinal tract.

Complement factors are present in both marsupial milk and the mammary gland. Proteins involved in the classical complement pathway (C2, C3 and C4A) were identified in the koala milk proteome and mammary gland transcriptome^{(Reference Morris, O’Meally and Zaw20)}. These factors were highly abundant during early lactation, accounting for 2·4% of peptides. Complement proteins bind to pathogens, thereby enabling phagocytosis by immune cells, and some are also bactericidal^{(Reference Janeway, Travers and Walport82)}.

Antimicrobial peptides

Cathelicidins and defensins

Cathelicidins and defensins are two major families of antimicrobial peptides in mammals. They form part of the innate immune system through antimicrobial and immunomodulatory activity and may also be involved in immune defence against cancer^{(Reference Kościuczuk, Lisowski and Jarczak83–Reference Petrohilos, Patchett and Hogg85)}. Cathelicidins and defensins are small, cationic, amphipathic peptides that are expressed in immune and epithelial cells^{(Reference Kościuczuk, Lisowski and Jarczak83,Reference Bals and Wilson86)} . Both cathelicidins and defensins are encoded as pre-propeptides that undergo enzymatic cleavage to release the C-terminal active mature peptide^{(Reference Kościuczuk, Lisowski and Jarczak83,Reference Ganz87)} . Defensin mature peptides contain six highly conserved cysteine residues that form three disulphide bonds that define the α-, β- and θ-subfamilies^{(Reference Yang, Biragyn and Hoover84,Reference Hazlett and Wu88)} . Theta defensins are found only in Old World primates and so are not discussed further in this review. Both cathelicidins and defensins from eutherian mammals have antimicrobial activity against fungi, enveloped viruses, Gram-positive and Gram-negative bacteria and parasites^{(Reference Yang, Biragyn and Hoover84,Reference van-Harten, van-Woudenbergh and van-Dijk89)} . They are also immunomodulatory; they enhance phagocytosis, are chemotactic, induce degranulation of neutrophils and mast cells, and induce migration of epithelial cells^{(Reference Yang, Biragyn and Hoover84,Reference van-Harten, van-Woudenbergh and van-Dijk89)} .

Marsupials have a large repertoire of diverse cathelicidins, likely driven by the need for additional immune protection of altricial young during development in the pouch (Table 1). This differs from eutherians, such as humans and mice, that have only a single cathelicidin peptide^{(Reference van-Harten, van-Woudenbergh and van-Dijk89)}. Cathelicidins have only been characterised in the grey short-tailed opossum, koala, Tasmanian devil and tammar wallaby, with between six and nineteen cathelicidin genes identified in a single species^{(Reference Peel, Cheng and Djordjevic16,Reference Peel, Cheng and Djordjevic17,Reference Daly, Digby and Lefévre39,Reference Peel, Cheng and Djordjevic90,Reference Belov, Sanderson and Deakin91)} . Similar to human cathelicidins, marsupial peptides have broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant S. aureus, and fungi (Fig. 2)^{(Reference Peel, Cheng and Djordjevic16,Reference Peel, Cheng and Djordjevic17,Reference Peel, Cheng and Djordjevic90)} . Koala cathelicidin PhciCath5 was active against Chlamydia pecorum, one of the aetiological agents that cause chlamydiosis that has severely impacted koala populations^{(Reference Peel, Cheng and Djordjevic17)}. Recent work has shown that Tasmanian devil cathelicidins have anticancer activity against devil facial tumour disease (DFTD) cells and may also have immunomodulatory functions (Fig. 2)^{(Reference Petrohilos, Patchett and Hogg85)}.

Figure 2. Bioactive roles of cathelicidin.

Marsupial cathelicidins are expressed in the milk and mammary gland throughout lactation^{(Reference Peel, Cheng and Djordjevic16,Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20,Reference Carman, Simonian and Old92)} . This is unlike other immune proteins that are generally expressed in only two periods as discussed in previous sections^{(Reference Edwards, Hinds and Deane12)}. In the tammar wallaby and koala, individual cathelicidins were differentially expressed during early and late lactation^{(Reference Joss, Molloy and Hinds7,Reference Morris, O’Meally and Zaw20,Reference Wanyonyi, Sharp and Khalil93)} . Some of these peptides had antimicrobial activity, whereas others were not active against the strains tested and may have immunomodulatory functions^{(Reference Peel, Cheng and Djordjevic17,Reference Wanyonyi, Sharp and Khalil93)} . In addition, cathelicidins may also be involved in mammary gland proliferation and remodelling during involution in the tammar wallaby (Fig. 2)^{(Reference Wanyonyi, Sharp and Khalil93)}. Cathelicidins are also expressed in the pouch skin^{(Reference Peel, Cheng and Djordjevic16,Reference Peel, Silver and Brandies50)} and may provide direct protection. Tasmanian devil cathelicidins expressed in the pouch were active against bacteria identified in the pouch microbiome. Cathelicidins may also indirectly protect pouch young by contributing to modulation of bacterial communities in the pouch, although this hypothesis has not been proven^{(Reference Peel, Cheng and Djordjevic16)}.

Defensins are the second major family of antimicrobial peptides in mammals. Defensins have been characterised in the greater bilby (Macrotis lagotis), brown antechinus (Antechinus stuartii), common wombat (Vombatus ursinus), fat-tailed dunnart (Sminthopsis crassicaudata), eastern barred bandicoot (Perameles gunnii), red kangaroo, western ringtail possum, numbat (Myrmecobius fasciatus), woylie, grey short-tailed opossum, koala, tammar wallaby and Tasmanian devil^{(Reference Belov, Sanderson and Deakin91,Reference Jones, Yuanyuan and O’Meally94,Reference Peel, Hogg and Belov95)} (Table 1). Marsupials encode multiple α- and β-defensin genes within their genomes, similar to eutherians. However, some α- and β-defensin lineages are specific to marsupials and, hence, may have novel functions^{(Reference Jones, Yuanyuan and O’Meally94)}. α-Defensins have not been detected in any marsupial milk, mammary gland or pouch skin transcriptome or proteome to date, so their bioactive role at these sites is unknown^{(Reference Hewavisenti, Morris and O’Meally18,Reference Morris, O’Meally and Zaw20,Reference Jones, Yuanyuan and O’Meally94)} . A small number of β-defensins were expressed during early lactation in the koala^{(Reference Morris, O’Meally and Zaw20,Reference Jones, Yuanyuan and O’Meally94)} and mid-lactation in the Tasmanian devil^{(Reference Hewavisenti, Morris and O’Meally18)}. β-Defensins were also found in the transcriptomes of the koala’s lactating mammary gland, the bilby’s non-lactating mammary gland and the pouch skin of both bilby and the woylie^{(Reference Peel, Hogg and Belov95)}. Human defensins are expressed in the mammary gland and provide antibacterial defence against infections such as mastitis as well as neonatal gastrointestinal infections^{(Reference Baricelli, Rocafull and Vázquez96,Reference Tunzi, Harper and Bar-Oz97)} . Given the function of marsupial defensins is unknown, future studies should focus on understanding the antimicrobial and immunomodulatory functions of these peptides, and their potential role in protecting marsupial pouch young.

Casein

Compared with bovine milk, where protein is approximately 80% casein and 20% whey proteins^{(Reference Jäkälä and Vapaatalo104)}, marsupial milk has relatively less casein with a ratio of approximately 50:50. This is generally constant throughout lactation, although concentration tends to increase^{(Reference Nicholas105)}. The most abundant caseins are β-casein, followed by α-casein, then κ-casein^{(Reference Morris, O’Meally and Zaw20,Reference Vercruysse, Van Camp and Dewettinck106)} . κ-Casein was once thought to be absent in marsupials until the genes encoding κ-casein were discovered in the brushtail possum^{(Reference Stasiuk, Summers and Demmer107)}. The amount of casein increases throughout lactation, predominantly owing to β-casein while α-casein remains relatively stable. In wallabies, caseins form micelles similar in structure to the well-studied bovine milk, despite the notable evolutionary distance^{(Reference Horne, Anema and Zhu108)}. Although caseins and whey proteins are well-known sources of nourishment, the peptides generated from these proteins can also play key roles in supporting an array of biological processes. Bioactive peptides generated from eutherian milk caseins have demonstrated an array of immunomodulatory and antimicrobial functionalities. Casocidin-1 generated from α_s2-casein in cow has potent in vitro antimicrobial activity against E. coli and Staphylococcus carnosus. Isracidin is another casein (α_s1-casein)-derived peptide that has displayed antibacterial activity in vivo against S. aureus and Candida albicans in cow and sheep^{(Reference Silva and Malcata109)}. Apart from these direct antimicrobial roles, β- and κ-casein-derived peptides in cow milk may promote lymphocyte proliferation, whereas β-casein identified from human milk induces chemokine production^{(Reference Nielsen, Liang and Rathish110)}. Peptides derived from casein have been found in peripheral circulation in neonates but not in adults, indicating that they can cross the gastrointestinal barrier and enter systemic circulation in infants. This finding highlights the possible age-specific functions that these peptides may have in promoting juvenile health and development^{(Reference Fox, Uniacke-Lowe and McSweeney111)}. However, a focus on milk peptides derived from caseins in marsupials has been notably insufficient or absent. Given that they likely represent a large proportion of the milk proteome in marsupials, a deeper understanding of casein-derived peptides will be essential for the development of tailored marsupial immune milk supplements to ensure orphaned joeys’ survival.

Vitamins and minerals

Milk provides essential dietary vitamins and minerals that are required for a variety of bioactive functions, including bone mineralisation, enzyme cofactors and hormonal/nutritive signalling. Marsupial milk mineral content has been reviewed recently^{(Reference Stannard, Miller and Old4)}, although the marsupial-specific literature on milk vitamins is limited and varies across species.

Kangaroo milk contains a similar diversity and concentration of vitamins to cow milk^{(Reference Poole, Sharman and Scott112)}, including lipophilic (vitamin A) and hydrophilic (B1, B2, B3, B5, B6, B7 and B12) vitamins^{(Reference Gaucheron113)}. Similar to other milk nutrients, the concentration of vitamins differs between lactation phases 2 and 3^{(Reference Poole, Sharman and Scott112)}. Folic acid (vitamin B9) is the most stable form of folate, which is an essential bioactive vitamin for neural tube closure and guides spinal cord development in mammals. In eutherians, this occurs early in gestation as folic acid is delivered via maternal blood. Suboptimal uptake of folic acid during pregnancy in eutherians leads to perinatal morbidity and mortality, signifying its importance^{(Reference Denny, Jeanes and Fathe114)}. In marsupials, neural development occurs ex utero within the pouch; hence, folic acid is delivered via the milk. This finding could explain why red and grey kangaroos’ (Macropus giganteus) early lactation milk contains elevated levels of folic acid, which then decreases throughout the lactation period^{(Reference Poole, Sharman and Scott112)}. The fluctuation of these vitamins based on their lactation stages has been descriptively studied in Poole’s 1982 study^{(Reference Poole, Sharman and Scott112)}.