A suspected parasite spill-back of two novel Myxidium spp. (Myxosporea) causing disease in Australian endemic frogs found in the invasive Cane toad

Abstract

Infectious diseases are contributing to the decline of endangered amphibians. We identified myxosporean parasites, Myxidium spp. (Myxosporea: Myxozoa), in the brain and liver of declining native frogs, the Green and Golden Bell frog (Litoria aurea) and the Southern Bell frog (Litoria raniformis). We unequivocally identified two Myxidium spp. (both generalist) affecting Australian native frogs and the invasive Cane toad (Bufo marinus, syn. Rhinella marina) and demonstrated their association with disease. Our study tested the identity of Myxidium spp. within native frogs and the invasive Cane toad (brought to Australia in 1935, via Hawaii) to resolve the question whether the Cane toad introduced them to Australia. We showed that the Australian brain and liver Myxidium spp. differed 9%, 7%, 34% and 37% at the small subunit rDNA, large subunit rDNA, internal transcribed spacers 1 and 2, but were distinct from Myxidium cf. immersum from Cane toads in Brazil. Plotting minimum within-group distance against maximum intra-group distance confirmed their independent evolutionary trajectory. Transmission electron microscopy revealed that the brain stages localize inside axons. Myxospores were morphologically indistinguishable, therefore genetic characterisation was necessary to recognise these cryptic species. It is unlikely that the Cane toad brought the myxosporean parasites to Australia, because the parasites were not found in 261 Hawaiian Cane toads. Instead, these data support the enemy-release hypothesis predicting that not all parasites are translocated with their hosts and suggest that the Cane toad may have played an important spill-back role in their emergence and facilitated their dissemination. This work emphasizes the importance of accurate species identification of pathogens relevant to wildlife management and disease control. In our case it is paving the road for the spill-back role of the Cane toad and the parasite emergence.

Figure 1. Myxosporea in the Green and Golden Bell frog (Litoria aurea) and the Southern Bell frog (Litoria raniformis).

Affected tadpoles of Green and Golden Bell frog suffered from chronic hepatitic lesions characterised by biliary hyperplasia, hepatitis with lymphoplasmacytic infiltration and fibrosis (A), parasite development was observed inside the bile ducts (arrows). Liver tissue of tadpoles from a semicaptive population of Green and Golden Bell frogs contained early plasmodia found within bile ducts (B, C). Round myxosporean stages (arrow), were identified in the brain and spinal cord and meninges of tadpoles of the Southern Bell frog (D, E). Brain stages were accompanied by a multifocal nonsuppurative meningoencephalitis (D). Each consisted of several secondary cells enclosed in a primary cell wall (E). Scale bars: A - 50 µm; B, C, D, E - 10 µm. A, D, E - H&E, B, C - Giemsa.

Figure 2. Transmission electron microscopy (TEM) of the developmental stages in the Green and Golden Bell frog tadpoles (Litoroa aurea).

Brain multinucleated (A) and single nucleated (B) extrasporogonic developmental stages found exclusively within axons (a), enclosed by myelin sheets (black arrows). Developmental stages possessed characteristic cell in cell development consistent with myxosporean extrasporogonic development. The histozoic primary (blue) cells, inside myelinated axon (green), had distinct intracellular cleavages defining the secondary (red) cells (an enlarged area in the white quadrangle; inset A). The parasitised myelinated axon is several times larger in size than the normal myelinated fiber (white arrows). Liver bile duct plasmodia (C) with cell in cell development, primary cell (p) and secondary cell (s). Based on TEM these stages appeared to be unattached to the biliary epithelium (c). The coelozoic plasmodia in bile ducts had numerous mitochondria (m) in the cytoplasm with pinocytic channels (gray arrows) and dispersed lipid inclusions in the cytoplasm (D). Scale bars: A - 1 µm; B - 1 µm; C, D - 2 µm.

Figure 3. Relationship of genotypes of Myxidium spp. in Australian frogs.

(A) Phylogenetic tree based on SSU rDNA sequences including the North American M. melleni and rooted using a shrew parasite Soricimyxum fegati. Parasite hosts and their status in Australia are indicated for the brain and liver genotype. (B) Phylogenetic trees based on SSU rDNA, LSU rDNA, ITS rDNA and ITS2 rDNA with genotype names and mean distances are indicated on the right. (C) The intraspecific and interspecific distance of the brain genotype (blue •), liver (red ▵), Brazil-1 (green ▵), Brazil-2 (brown ▵) and pooled liver+Brazil-1+Brazil-2 genotypes (black outlined ▵) were placed on the graph to evaluate whether they represent candidate species. The graphs are divided into four quadrants that represent different categories of “species” : top left - species concordant with current taxonomy; top right - probable composite species, i.e. candidates for taxonomic split; bottom left - species that have undergone recent divergence, hybridization, or synonymy; bottom right - probable specimen misidentification. Notice that if the liver+Brazil-1+Brazil-2 genotypes (black outlined ▵) are treated as a single species they are resolved in the top right quadrangle suggestive of cryptic species; however when split into the three individual genotypes they became resolved in the top left quadrangle supporting their species status. Trees and distances were inferred using the Minimum Evolution: Maximum Composite Likelihood method in MEGA4 with bootstrap test (1,000 replicates, >50% are shown).

Figure 4. Myxospores of Myxidium species - liver genotype.

Scanning electron microscopy of myxospores recovered from the gall bladder of the Striped Marsh frog (Limnodynastes peronii). Myxospores are ellipsoidal, shell valves have ridges and suture line cross-sectioning the spore. Scale bar, 5 µm.

Figure 5. Collection sites of Cane toads (Bufo marinus, syn. Rhinella marina) surveyed for the presence of Myxidium species.

Names of the localities are shown together with the number of animals surveyed in February 2010. The Cane toad was introduced (149 individuals) to the Hawaiian island of Oahu from Puerto Rico in April 1932 and introduced to the Manoa Arboretum at the upper end of Manoa Valley and a taro patch adjoining the HSPA Waipio substation and immediately spread throughout the island . In 1935, juvenile and adult toads were collected and in total 101 live individuals arrived in Australia . Two localities recorded as source populations for translocation to Australia are indicated by ★.

Figure 6. Phylogenetic tree of Myxozoa based on SSU rDNA gene sequence.

The Bayesian tree was reconstructed using MrBayes 3.1.2 with a GTR+G+I nucleotide model from the stringent Alignment 3, for details about alignment and phylogenetic reconstruction see Materials and Methods and Figure S1. For details on biological traits and GenBank accession numbers see Table S4. Bayesian posterior probabilities are shown at the nodes. The myxosporean sequences fall into marine and fresh clades; the exceptions are Sphaeromyxa spp. and Zschokkella icterica sequences from marine fish that cluster in the fresh water clade . Myxosporean species with documented actinospore development are underlined.