Consistent members of the echidna microbiome
Core ASVs (ASVs with ≥ 0.01% relative abundance in at least 70% of samples) were consistent between male and female echidnas, with the identification of 25 core ASVs. A total of 14 of these ASVs were recognised to be in the 20 most abundant for both males and females (Table S2), with a number of these genera, Fusobacterium, Bacteroides and Lactobacillus, also found in other native Australian animals [19]. Native Australian animals such as koalas and wallabies who also use pouches for young development have similar gut microbial composition, specifically being dominated by Firmicutes [19], the phyla which includes Lactobacillus genera. The presence of these abundant genera within our samples is consistent with gut microbiome composition of other captive short-beaked echidnas found in other zoo locations within Australia [6].
The presence of these key bacterial identities is not surprising, as a similar composition of bacterial phyla are also found in other myrmecophagous organisms, animals that feed primarily on ants and termites, such as pangolins, giant anteaters and aardvarks [20, 21].
The short-beaked echidna evolved eating a diet mostly comprised of ants, termites, and occasionally larval scarab beetles, and are one of the two only myrmecophagous mammals in Australia, the other being the numbat (Myrmecobius faciatus) [22, 23]. The exoskeleton of ants and termites is made of chitin, an amino-polysaccharide that is indigestible for the short-beak echidna. However, certain bacterial communities that were identified from the samples of this study, such as Bacteroides, are considered to be chitinolytic bacteria and have the ability to metabolise the chitin polymer into chitin oligomers that can be processed by the digestive system [21, 24]. While a functional analysis of the bacterial groups isolated in this study was not conducted, based on the presences of Bacteroides in other myrmecophagous mammals [20, 21, 25, 26], it is likely that the isolates within our short-beaked echidna samples may also assist the echidna’s digestive system with the processing of chitin. When investigating the gut microbial composition of another myrmecophagous mammal, the wild Sunda pangolin (Manis javanica), bacteria belonging to Firmicutes, Bacteroidetes, and Proteobacteria were also the most dominating groups identified [21].
However, there are two notable differences amongst the gut microbiota of the short-beaked echidnas used in this study when compared results from other myrmecophagous mammals. While the bacteria genera Fusobacterium is highly abundant within our samples, it is found in little to no abundance in other myrmecophagous mammals such as pangolins and aardvarks [20, 21, 26]. It is also generally absent or in low abundance in most mammals, and when found in humans is usually associated with colorectal cancer and regarded as an opportunist pathogen [27–29]. However, Fusobacterium have been recently identified in high abundances within healthy zoo-enclosed short-beaked echidnas, as well as in the gut microbiota of wild short-beaked echidnas [6]. Consistent with these results, samples used in this study also came from healthy short-beaked echidna candidates, hence suggesting that Fusobacterium may have a more commensal role within short-beaked echidnas. Fusobacterium have also been isolated from other healthy scavengers, such as vultures and healthy domesticated dogs [30, 31]. More efforts into understanding the functional properties of Fusobacterium within healthy short-beaked echidnas must be conducted to understand its role as a possible gut commensal microbe.
Relative to wild populations, the generalized pattern of gut microbiomes in captivity are reduced α-diversity and a significant shift in community composition [32]. Many conditions of captivity (antibiotic exposure, altered diet composition, homogenous environment, increased stress, and altered intraspecific interactions) likely lead to changes in the host-associated microbiome. α-diversity for captive echidna in our study, specifically richness, was similar to previous captive and wild echidna data [6]. Corresponding with this work, we found captive echidna lacking the genus Acinetobacter, which were dominant in wild echidnas [6]. Acinetobacter is a prevalent soil bacterium that is likely to be ingested by the short-beaked echidnas during food foraging, hence it is expected for the genera to appear within the echidna’s microbiome [6, 33]. However, feeding strategies may vary from one wildlife facility to another and limit soil intake by echidna. In this way, microbiota studies can inform conservation and management approaches – here, we might suggest that echidnas in captivity are fed in a manner to allow them to ingest soil particulates and obtain a microbiome more similar to wild individuals noting previous echidna research suggesting that the presence of dirt in the diet to be beneficial [34].
The structure of male and female microbiomes is significantly different from each another.
Ten ASVs were identified as belonging to the 20 most abundant ASVs in male samples only, the male core microbiome, indicator taxa, or a combination of those. These ten consist of genera that were present within the female samples (Bacteroides and Lactobacillus) and the genera Cetobacterium and Streptococcus, which appear to be exclusive to males only. ASV024, a taxon who presence indicated the sample originated from a male and was one of the top 20 most abundant ASVs in males, was most closely related to Lactobacillus gallinarum, which was not found within the female samples. L. gallinarum have been recognised to slow-down intestinal tumour growth when present in the gut microbiota of mice, and when given as a probiotic to broiler chickens, have the ability to prevent Salmonella infections [35, 36]. The other indicator taxa for male samples, ASV040, was mostly closely related to B. sartorii.
Cetobacterium (ASV015), a member of Fusobacteriaceae like the highly abundant Fusobacterium, are not common within the gut microbiota of terrestrial animals but have been isolated from the intestines of porpoises as well as being a key gut commensal bacterium in a range of freshwater fish species [37, 38]. This genus can assist in carbohydrate and peptide fermentation and is suggested to produce vitamin B12 to support host health [39]. Streptococcus (ASV022), on the other hand, is associated with disease in echidnas [34].
Differences in the gut microbial communities between male and female samples is not uncommon in other species, with significant differences in gut microbiota based on sex of the individual being observed in mice and human studies [40–42], explained by differences in sex hormones [40]. Sex hormones such as estrogen have the ability to regulate bacteria metabolism to assist in food digestion [43]. When the testostrone source in male mice was eliminated, resulting in male mice losing the ability to produce testostrone, their gut microbiota began to resemenble that of a female mouse microbiota [40–42]. This result was reversed once testostorne was once again provided to the male mice lacking the hormone source, with significant differences between male and female mice being restablished [41]. Therefore, the signifcant variations in gut microbial contents between sexes in this study could be explained by the influence of sex hormones, particularly because the female echidnas were in various points of gestation during the sampling points, and would hence have many flacutations in female sex hormones such as progestorne and oestrogen, when compared to males [43].
The echidna microbiome is stable across gestation stages.
During pregnancy the echidna undergoes different adaptations to provide an optimal environment for fetal growth. Such changes also involve all the microorganisms [14], which we found to be stable in composition and diversity during the five stages monitored: non-breeding, pre-gestation, early gestation, late gestation, and incubation. Human and animal findings on the gut microbiota composition across pregnancy is varied, with some studies reporting stability [18, 44] while others observe great fluctuations across pregnancy stages [45, 46]. There is also evidence of stability in the gut microbiota across the perinatal period (the period one year before and after infant birth) in healthy women, with their microbiota being particularly dominated by Bacteroides and Firmicutes [14, 17], which is also consistent with our short-beaked echidna samples. Unlike the gestion of the large placental mammals described in the aforementioned studies, the short-beaked echidna’s gestation period begins following copulation and continues for a duration of ~ 17 days until the egg is laid [47]. The gut microbiota of echidnas may not have had sufficient time to undergo changes. Diet plays a significant role in shaping the gut/faecal microbiota in mammals [48], including echidna [6]; the diet of the Currumbin echidnas remained stable across the gestation period sampling, which may have contributed to the relative stability of their gut microbiota.