Life stage and proximity to roads shape the skin microbiota of eastern newts (Notophthalmus viridescens).

Host-associated microbiomes play an essential role in the health of organisms, including immune system activation, metabolism and energy uptake. It is well established that microbial communities differ depending on the life stage and natural history of the organism. However, the effects of life stage and natural history on microbial communities may also be influenced by human activities. We investigated the effects of amphibian life stage (terrestrial eft vs. aquatic adult) and proximity to roadways on newt skin bacterial communities. We found that the eft and adult life stages differed in bacterial community composition; however, the effects of roads on community composition were more evident in the terrestrial eft stage compared to the aquatic adult stage. Terrestrial efts sampled close to roads possessed richer communities than those living further away from the influence of roads. When accounting for amplicon sequence variants with predicted antifungal capabilities, in the adult life stage, we observed a decrease in anti-fungal bacteria with distance to roads. In contrast, in the eft stage, we found an increase in anti-fungal bacteria with distance to roads. Our results highlight the need to consider the effects of human activities when evaluating how host-associated microbiomes differ across life stages of wildlife.

Presence of low virulence chytrid fungi could protect European amphibians from more deadly strains.

Wildlife diseases are contributing to the current Earth's sixth mass extinction; one disease, chytridiomycosis, has caused mass amphibian die-offs. While global spread of a hypervirulent lineage of the fungus Batrachochytrium dendrobatidis (BdGPL) causes unprecedented loss of vertebrate diversity by decimating amphibian populations, its impact on amphibian communities is highly variable across regions. Here, we combine field data with in vitro and in vivo trials that demonstrate the presence of a markedly diverse variety of low virulence isolates of BdGPL in northern European amphibian communities. Pre-exposure to some of these low virulence isolates protects against disease following subsequent exposure to highly virulent BdGPL in midwife toads (Alytes obstetricans) and alters infection dynamics of its sister species B. salamandrivorans in newts (Triturus marmoratus), but not in salamanders (Salamandra salamandra). The key role of pathogen virulence in the complex host-pathogen-environment interaction supports efforts to limit pathogen pollution in a globalized world.

Transcriptomics reveal immune downregulation of newts overwhelmed by chytrid co-infection.

What happens when two emergent diseases infect the same host? In a From the Cover article in this issue of Molecular Ecology, McDonald et al. (2020) compare transcriptomic responses to co-infection by the two chytrid fungi in the skin, liver and spleen of Eastern newts (Notophthalmus viridescens). Novel molecular tools, such as high-throughput DNA sequencing for genome discovery and transcriptomics, have revolutionized our understanding of host-pathogen interactions and disease ecology (Güimil et al. 2005; Rosenblum et al. 2012). For example, epidemiologists are using genomic data to track the spread of the emergent SARS-CoV-2 in real time, both locally and globally. RNA sequencing (RNA-Seq) is routinely employed to study response to disease in humans, improving disease diagnostics, profiling and development of intervention strategies. Transcriptomic profiles may be particularly informative for emergent diseases, whose pathologies and effect on host phenotype are poorly known. Fungal pathogens increasingly threaten a variety of wild and domesticated organisms (Fisher et al. 2012), and two chytrid fungi attacking amphibians are causing one of the worst losses of vertebrate biodiversity ever recorded (Scheele et al. 2019).

The skin microbiome facilitates adaptive tetrodotoxin production in poisonous newts.

Rough-skinned newts (Taricha granulosa) use tetrodotoxin (TTX) to block voltage-gated sodium (Nav) channels as a chemical defense against predation. Interestingly, newts exhibit extreme population-level variation in toxicity attributed to a coevolutionary arms race with TTX-resistant predatory snakes, but the source of TTX in newts is unknown. Here, we investigated whether symbiotic bacteria isolated from toxic newts could produce TTX. We characterized the skin-associated microbiota from a toxic and non-toxic population of newts and established pure cultures of isolated bacterial symbionts from toxic newts. We then screened bacterial culture media for TTX using LC-MS/MS and identified TTX-producing bacterial strains from four genera, including Aeromonas, Pseudomonas, Shewanella, and Sphingopyxis. Additionally, we sequenced the Nav channel gene family in toxic newts and found that newts expressed Nav channels with modified TTX binding sites, conferring extreme physiological resistance to TTX. This study highlights the complex interactions among adaptive physiology, animal-bacterial symbiosis, and ecological context.

Rough-skinned newts produce tetrodotoxin or TTX, a deadly neurotoxin that is also present in some pufferfish, octopuses, crabs, starfish, flatworms, frogs, and toads. It remains a mystery why so many different creatures produce this toxin. One possibility is that TTX did not evolve in animals at all, but rather it is made by bacteria living on or in these creatures. In fact, scientists have already shown that TTX-producing bacteria supply pufferfish, octopus, and other animals with the toxin. However, it was not known where TTX in newts and other amphibians comes from. TTX kills animals by blocking specialized ion channels and shutting down the signaling between neurons, but rough-skinned newts appear insensitive to this blockage, making it likely that they have evolved defenses against the toxin. Some garter snakes that feed on these newts have also evolved to become immune to the effects of TTX. If bacteria are the source of TTX in the newts, the emergence of newt-eating snakes resistant to TTX must be putting evolutionary pressure on both the newts and the bacteria to boost their anti-snake defenses. Learning more about these complex relationships will help scientists better understand both evolution and the role of beneficial bacteria. Vaelli et al. have now shown that bacteria living on rough-skinned newts produce TTX. In the experiments, bacteria samples were collected from the skin of the newts and grown in the laboratory. Four different types of bacteria from the samples collected produced TTX. Next, Vaelli et al. looked at five genes that encode the channels normally affected by TTX in newts and found that all them have mutations that prevent them from being blocked by this deadly neurotoxin. This suggests that bacteria living on newts shape the evolution of genes critical to the animals' own survival. Helpful bacteria living on and in animals have important effects on animals' physiology, health, and disease. But understanding these complex interactions is challenging. Rough-skinned newts provide an excellent model system for studying the effects of helpful bacteria living on animals. Vaelli et al. show that a single chemical produced by bacteria can impact diverse aspects of animal biology including physiology, the evolution of their genes, and their interactions with other creatures in their environment.

Host density and habitat structure influence host contact rates and Batrachochytrium salamandrivorans transmission.

Batrachochytrium salamandrivorans (Bsal) is an emerging invasive pathogen that is highly pathogenic to salamander species. Modeling infection dynamics in this system can facilitate proactive efforts to mitigate this pathogen's impact on North American species. Given its widespread distribution and high abundance, the eastern newt (Notophthalmus viridescens) has the potential to significantly influence Bsal epidemiology. We designed experiments to 1) estimate contact rates given different host densities and habitat structure and 2) estimate the probability of transmission from infected to susceptible individuals. Using parameter estimates from data generated during these experiments, we modeled infection and disease outcomes for a population of newts using a system of differential equations. We found that host contact rates were density-dependent, and that adding habitat structure reduced contacts. The probability of Bsal transmission given contact between newts was very high (>90%) even at early stages of infection. Our simulations show rapid transmission of Bsal among individuals following pathogen introduction, with infection prevalence exceeding 90% within one month and >80% mortality of newts in three months. Estimates of basic reproductive rate (R0) of Bsal for eastern newts were 1.9 and 3.2 for complex and simple habitats, respectively. Although reducing host density and increasing habitat complexity might decrease transmission, these management strategies may be ineffective at stopping Bsal invasion in eastern newt populations due to this species' hyper-susceptibility.

Treatment of Chytridiomycosis in Laboratory Axolotls (Ambystoma mexicanum) and Rough-skinned Newts (Taricha granulosa).

Chytridiomycosis is an infectious disease of amphibians caused by the fungal species Batrachochytrium dendrobatidis and B. salamandrivorans and has been implicated in the population decline of amphibian species worldwide. This case report describes a successful treatment protocol for chytridiomycosis in laboratory-maintained colonies of axolotls (Ambystoma mexicanum) and rough-skinned newts (Taricha granulosa). Over 12 mo, axolotls (n = 12) in a laboratory-reared colony developed multifocal erythematous dermatitis, mainly on the distal limbs and tails. Wild-caught newts handled by the same lab personnel were housed in an adjacent room and occasionally presented with abdominal distension and lethargy. Differentials included poor water quality, pathogen infection, parasitic infestation, and trauma. Antibiotic treatment of animals according to results of bacterial culture and sensitivity, combined with bleach disinfection of aquaria, did not resolve clinical signs. Skin swabs from clinically affected axolotls submitted for a newly available commercial screen were positive for B. dendrobatidis. Additional PCR and sequencing analysis revealed chytrid-positive animals among group-housed newts in 2 clinically unaffected aquaria and suspected PCR-positives for 2 affected newt aquaria and an additional axolotl. Axolotls with skin lesions (n = 2) and newts with abdominal distension and lethargy (n = 2) underwent experimental treatment with itraconazole submersion (0.002% to 0.0025%; 5 min daily for 10 d). This pilot treatment was well tolerated and led to clinical resolution. Subsequent itraconazole treatment of the entire colony led to regrowth of extremities and restoration of normal coloration among axolotls. During treatment, the facility was decontaminated, and additional biosecurity measures were developed. PCR results after the pilot treatment and subsequent full-colony treatments (at 1 wk, 1 mo, and 6 mo after treatment) were negative for the presence of B. dendrobatidis. Because chytridiomycosis is a reportable animal disease in our state, colonies officially remained quarantined until negative PCR results were obtained at least 6 mo after treatment.

Keeping the UK's newts free from chytrid fungi.

Josh Loeb discusses recent research on the devastating Batrachochytrium salamandrivorans fungus.

Pathogen invasion and non-epizootic dynamics in Pacific newts in California over the last century.

Emerging infectious disease is a growing threat to global biodiversity. The infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to the decline and extinction of hundreds of amphibian species. Severe Bd-caused epizootics have been documented in North, Central and South America-with many of the research focused on anurans. California, where Bd-related epizootics and amphibian declines have been reported, has some of the highest diversity of salamanders. After more than a decade since the first known epizootic in California, little is known about Bd disease dynamics in salamanders. Pacific newts (Genus: Taricha) are ideal study species because of their abundance, wide geographic range, occurrence in both aquatic and terrestrial habitats, and how little is known about Bd infection dynamics for this group. We conducted a retrospective study to determine the relationship between Pacific newts and the fungal pathogen. We tested 1895 specimens collected between 1889-2009 and found no evidence of Bd-infected Pacific newts until the late 1940's. Although we estimate that Bd emerged in this genus and rapidly spread geographically throughout California, we did not find evidence for epizootic dynamics. Bd infection prevalence and intensity, two measures commonly used to estimate dynamics, remained consistently low over time; suggesting Pacific newts may not be highly susceptible. Also, we found the timing of first Bd emergence in Pacific newts predate Bd emergence in other California salamander species. In addition, we found several environmental and anthropogenic factors correlated with Bd prevalence which may help explain Bd disease dynamics in the genus Taricha. Pacific newts may be a reservoir species that signal pathogen invasion into California salamanders, though further studies are needed.

Amphibian skin microbiota exhibits temporal variation in community structure but stability of predicted Bd-inhibitory function.

Host-associated microbiomes are increasingly recognized to contribute to host disease resistance; the temporal dynamics of their community structure and function, however, are poorly understood. We investigated the cutaneous bacterial communities of three newt species, Ichthyosaura alpestris, Lissotriton vulgaris and Triturus cristatus, at approximately weekly intervals for 3 months using 16S ribosomal RNA amplicon sequencing. We hypothesized cutaneous microbiota would vary across time, and that such variation would be linked to changes in predicted fungal-inhibitory function. We observed significant temporal variation within the aquatic phase, and also between aquatic and terrestrial phase newts. By keeping T. cristatus in mesocosms, we demonstrated that structural changes occurred similarly across individuals, highlighting the non-stochastic nature of the bacterial community succession. Temporal changes were mainly associated with fluctuations in relative abundance rather than full turnover of bacterial operational taxonomic units (OTUs). Newt skin microbe fluctuations were not correlated with that of pond microbiota; however, a portion of community variation was explained by environmental temperature. Using a database of amphibian skin bacteria that inhibit the pathogen Batrachochytrium dendrobatidis (Bd), we found that the proportion of reads associated with 'potentially' Bd-inhibitory OTUs did not vary temporally for two of three newt species, suggesting that protective function may be maintained despite temporal variation in community structure.

Temporal changes in cutaneous bacterial communities of terrestrial- and aquatic-phase newts (Amphibia).

Animal-associated bacterial communities play essential roles for their host's ecology, physiology and health. Temporal dynamics of these communities are poorly understood, but might be of high relevance for amphibians with a well-expressed biphasic biology of adults where the structure of their skin changes drastically between the aquatic and terrestrial phases. Here, we investigated the temporal dynamics of cutaneous bacterial communities of Lissotriton boscai and Triturus marmoratus by monthly sampling populations from a pond and surrounding terrestrial habitats near A Coruña, Spain. These communities were characterized by 16S rRNA gene amplicons from DNA isolated from skin swabs. Newt bacterial communities displayed variation at three levels: between larvae and aquatic adults, between adult life phases (terrestrial versus aquatic), and temporally within life phases. The skin bacterial communities tended to differ to a lesser extent temporally and between larvae and adults, and more strongly between life phases. Larvae had a higher proportion of reads associated with antifungal taxa compared with adults, while no differences were found among adult life phases. Terrestrial specimens exhibited the highest community diversity. The regular transitions of adult newts between aquatic and terrestrial environments might contribute to the diversity of their skin microbiota and could increase disease resistance.

Mycolactone-producing Mycobacterium marinum infection in captive Hong Kong warty newts and pathological evidence of impaired host immune function.

A mass mortality event of captive Hong Kong warty newts Paramesotriton hongkongensis with non-granulomatous necrotic lesions occurred in Taipei Zoo, Taiwan, in 2014. Clinically, the sick newts were lethargic and often covered with water mold Saprolegnia sp. on the skin of the body trunk or extremities. Predominant pathological findings were multifocal non-granulomatous necrotic lesions in the liver, spleen, and kidneys and severe skin infection with Saprolegnia sp., with deep invasion and involvement of underlying muscles. The possibility of ranavirus infection was ruled out by negative PCR results. Unexpectedly, abundant intralesional acid-fast positive bacilli were found in the necrotic lesions of the liver, spleen, and kidney in all 14 sick newts. PCR targeting the hsp65, ITS region, and partial 16S rRNA genes was performed, and the sequence identity from amplified amplicons of hsp65 and partial 16S rRNA genes was 100% identical to that of the corresponding gene fragment of Mycobacterium marinum. Further molecular investigations demonstrated that the current M. marinum was a mycolactone-producing mycobacterium with the presence of esxA/esxB genes. Mycolactone is a plasmid-encoded, immunosuppressive, and cytotoxic toxin. The possible immunosuppression phenomenon characterized by systemic non-granulomatous necrotic lesions caused by M. marinum and the unusual deep invasive infection caused by water mold might be associated with the immunosuppressive effect of mycolactone. Therefore, it should be noted that non-granulomatous necrotic lesions in amphibians can be caused not only by ranavirus infection but also by mycobacteriosis.

Genomic innovations linked to infection strategies across emerging pathogenic chytrid fungi.

To understand the evolutionary pathways that lead to emerging infections of vertebrates, here we explore the genomic innovations that allow free-living chytrid fungi to adapt to and colonize amphibian hosts. Sequencing and comparing the genomes of two pathogenic species of Batrachochytrium to those of close saprophytic relatives reveals that pathogenicity is associated with remarkable expansions of protease and cell wall gene families, while divergent infection strategies are linked to radiations of lineage-specific gene families. By comparing the host-pathogen response to infection for both pathogens, we illuminate the traits that underpin a strikingly different immune response within a shared host species. Our results show that, despite commonalities that promote infection, specific gene-family radiations contribute to distinct infection strategies. The breadth and evolutionary novelty of candidate virulence factors that we discover underscores the urgent need to halt the advance of pathogenic chytrids and prevent incipient loss of biodiversity.

Impact of asynchronous emergence of two lethal pathogens on amphibian assemblages.

Emerging diseases have been increasingly associated with population declines, with co-infections exhibiting many types of interactions. The chytrid fungus (Batrachochytrium dendrobatidis) and ranaviruses have extraordinarily broad host ranges, however co-infection dynamics have been largely overlooked. We investigated the pattern of co-occurrence of these two pathogens in an amphibian assemblage in Serra da Estrela (Portugal). The detection of chytridiomycosis in Portugal was linked to population declines of midwife-toads (Alytes obstetricans). The asynchronous and subsequent emergence of a second pathogen - ranavirus - caused episodes of lethal ranavirosis. Chytrid effects were limited to high altitudes and a single host, while ranavirus was highly pathogenic across multiple hosts, life-stages and altitudinal range. This new strain (Portuguese newt and toad ranavirus - member of the CMTV clade) caused annual mass die-offs, similar in host range and rapidity of declines to other locations in Iberia affected by CMTV-like ranaviruses. However, ranavirus was not always associated with disease, mortality and declines, contrasting with previous reports on Iberian CMTV-like ranavirosis. We found little evidence that pre-existing chytrid emergence was associated with ranavirus and the emergence of ranavirosis. Despite the lack of cumulative or amplified effects, ranavirus drove declines of host assemblages and changed host community composition and structure, posing a grave threat to all amphibian populations.

Experimental evidence for a cost of resistance to the fungal pathogen, Batrachochytrium dendrobatidis, for the palmate newt, Lissotriton helveticus.

BACKGROUND: Batrachochytrium dendrobatidis (Bd), the causative agent of chytridiomycosis, is decimating amphibians worldwide. Unsurprisingly, the majority of studies have therefore concentrated on documenting morbidity and mortality of susceptible species and projecting population consequences as a consequence of this emerging infectious disease. Currently, there is a paucity of studies investigating the sub-lethal costs of Bd in apparently asymptomatic species, particularly in controlled experimental conditions. Here we report the consequences of a single dose of B. dendrobatidis zoospores on captive adult palmate newts (Lissotriton helveticus) for morphological and behavioural traits that associate with reproductive success. RESULTS: A single exposure to ~2000 zoospores induced a subclinical Bd infection. One week after inoculation 84% of newts tested positive for Bd, and of those, 98% had apparently lost the infection by the day 30. However, exposed newts suffered significant mass loss compared with control newts, and those experimental newts removing higher levels of Bd lost most mass. We found no evidence to suggest that three secondary sexual characteristics (areas of dorsal crest and rear foot webbing, and length of tail filament) were reduced between experimental versus control newts; in fact, rear foot webbing was 26% more expansive at the end of the experiment in exposed newts. Finally, compared with unexposed controls, exposure to Bd was associated with a 50% earlier initiation of the non-reproductive terrestrial phase. CONCLUSIONS: Our results suggest that Bd has measureable, but sub-lethal effects, on adult palmate newts, at least under the laboratory conditions presented. We conclude that the effects reported are most likely to be mediated through the initiation of costly immune responses and/or tissue repair mechanisms. Although we found no evidence of hastened secondary sexual trait regression, through reducing individual body condition and potentially, breeding season duration, we predict that Bd exposure might have negative impacts on populations of palmate newts through reducing individual reproductive success and adult recruitment.

What drives chytrid infections in newt populations? Associations with substrate, temperature, and shade.

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) is considered responsible for the population declines and extinctions of hundreds of amphibian species worldwide. The panzootic was likely triggered by human-assisted spread, but once the pathogen becomes established in a given region, its distribution is probably determined by local drivers. To assess the relative importance of potential drivers of infection in red-spotted newts (Notophthalmus viridescens), we measured Bd levels in 16 populations throughout central Pennsylvania. Infected individuals were detected in all but four populations, indicating that Bd is widespread in this region. We quantified local factors hypothesized to influence Bd, and found that infection levels were best predicted by the proportion of the pond substrate consisting of leaf litter or vegetation, along with a significant effect of water temperature. Bd infection in amphibians is temperature-dependent, and one possible explanation of the apparent substrate effect is that tree cover and vegetation provide shade, reducing the availability of shallow, warm-water patches in which newts might reduce or clear Bd infections. Alternatively, leaf litter and emergent vegetation might increase Bd infection more directly, perhaps by providing substrates for environmental growth of the fungus. We also observed a curvilinear relationship between Bd load and snout-vent length (a proxy for age), hinting that newts might develop acquired resistance to Bd infection. Though correlational, these results add to a growing body of evidence suggesting that environmental temperature is an important driver of Bd infection dynamics.

New findings from an old pathogen: intraerythrocytic bacteria (family Anaplasmatacea) in red-backed salamanders Plethodon cinereus.

During a recent study of red-backed salamanders (Plethodon cinereus), we discovered an intraerythrocytic organism typified by violet-staining, intracellular inclusions, consistent with descriptions of Cytamoeba or Aegyptianella (bacteria). Here we characterize its taxonomic status using molecular techniques and ask basic questions about its nature. Blood smears from 102 salamanders were examined from Pennsylvania, New York, and Virginia to determine prevalence, and whole blood from several infected animals was tested using a PCR which targets the 16S rRNA gene of bacteria. Phylogenetic analysis of partial 16S rRNA gene sequence (1201 bp) indicated this organism was in the order Rickettsiales and is likely a member of the family Anaplasmatacea. The organism differed from currently described taxa and was clearly differentiated from Aegyptianella pullorum of birds and "Candidatus Hemobacterium ranarum" (formally A. ranarum) of frogs. Of all salamanders, 17 (16.7%) were infected and these were significantly larger (snout-vent length) and had higher body condition scores than uninfected ones, and males were more likely to be infected than females. Erythrocytes affected by the pathogen were 5% larger than unaffected ones, but otherwise similar in morphology. Infected animals tended to have a greater number of circulating white blood cells, based on estimates from smears, indicating a nonspecific response to the pathogen by the innate immune system. Given its phylogenetic position, this pathogen is likely transmitted by an arthropod vector, and the male-biased prevalence strongly implicates trombiculid mites, which also live in leaf litter and affect male salamanders more so than females.

Detection of chytridiomycosis caused by Batrachochytrium dendrobatidis in the endangered Sardinian newt (Euproctus platycephalus) in southern Sardinia, Italy.

The chytridiomycete fungus Batrachochytrium dendrobatidis is known to be focally distributed across Europe, but has only been linked to "chytridiomycosis at a few locations in Spain. Here we report the second occurrence of chytridiomycosis in European amphibians. We found a population of endangered Sardinian newts (Euproctus platycephalus) exhibiting clinical signs of disease including loss of digits and patchy, discolored skin. Molecular examination of skin samples tested positive for B. dendrobatidis. The population of E. platycephalus has been in decline on a timescale consistent with the global emergence of chytridiomycosis, and the ecology of this salamander suggests that the disease in this species warrants concern.

Chytridiomycosis impacts predator-prey interactions in larval amphibian communities.

Despite ecologists increasingly recognizing pathogens as playing significant roles in community dynamics, few experimental studies have quantified patterns of disease impacts on natural systems. Amphibians are experiencing population declines, and a fungal pathogen ( Batrachochytrium dendrobatidis; Chytridiomycota) is a suspected causal agent in many declines. We studied the effects of a pathogenic fungus on community interactions between the gray treefrog, Hyla chrysoscelis, and eastern newts, Notophthalmus viridescens. Recent studies have characterized chytridiomycosis as an emerging infectious disease, whose suspected rapid range expansion and widespread occurrence pose a significant risk for amphibian populations worldwide. We reared larvae in outdoor polyethylene experimental tanks and tested the effects of initial larval density, predator presence, and fungal exposure on Hyla recruitment and predator-prey interactions between Hyla and Notophthalmus. Newts reduced treefrog survival, and high intraspecific density decreased metamorphic body mass independent of B. dendrobatidis. The presence of fungi reduced treefrog body mass at metamorphosis by 34%, but had no significant main effect on survival or larval period length. B. dendrobatidis differentially affected larval development in the presence of predators; Hyla developed slower when reared with the pathogen, but only when newts were present. This significant predator-by-pathogen interaction suggests that the impact of chytridiomycosis on larval amphibians may be exacerbated in complex communities. Our data suggest that B. dendrobatidis effects on host life history may be complex and indirect. Direct measurements of the community-level effects of pathogens offer an important opportunity to understand a significant threat to global biodiversity-declining amphibian populations.

Importation of salmonellae with aquarium species.

An examination of shipments of aquarium species arriving in Canada over a 2-year period revealed that one-third of the shipments of aquarium snails and aquarium frogs were contaminated with Salmonella. Salmonella were also isolated from a shipment of aquarium newts but were not recovered from goldfish. Included among these salmonellae were four of the serovars most commonly associated with human salmonellosis in Canada. Several unusual salmonellae were also isolated including a Salmonella florida that produced a delayed, weakly positive o-nitrophenyl-beta-D-galactosidase reaction, and a bacteriophage 14 lysogenized Salmonella mbandaka. The association of salmonellae with aquarium species is clearly adding to the pool of this human pathogen in Canada, and may contribute to human infections.