Predation by zooplankton on <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis>: biological control of the deadly amphibian chytrid fungus?

Batrachochytrium dendrobatidis (hereafter Batrachochytrium), a fungal pathogen of amphibians, causes the disease chytridiomycosis which is responsible for unprecedented population declines and extinctions globally. Host defenses against chytridiomycosis include cutaneous symbiotic bacteria and anti-microbial peptides, and proposed treatment measures include use of fungicides and bioaugmentation. Efforts to eradicate the fungus from localized areas of disease outbreak have not been successful. Instead, control measures to mitigate the impacts of the disease on host populations, many of which are already persisting with Batrachochytrium in an endemic state, may be more realistic. The infective stage of the fungus is an aquatic zoospore, 3–5 μm in diameter. Here we show that zoospores of Batrachochytrium are consumed by the zooplankter Daphnia magna. This species inhabits amphibian breeding sites where Batrachochytrium transmission occurs, and consumption of Batrachochytrium zoospores may lead to effective biological control of Batrachochytrium.     

Combining ethidium monoazide treatment with real-time PCR selectively quantifies viable Batrachochytrium dendrobatidis cells

Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10^® Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.     

Seasonal Ecology and Behavior of an Endangered Rainforest Frog (Litoria rheocola) Threatened by Disease

One of the most devastating wildlife diseases ever recorded is chytridiomycosis, a recently emerged amphibian disease that is caused by the chytrid fungus Batrachochytrium dendrobatidis. Understanding, predicting, and managing the impacts of chytridiomycosis on any amphibian species will require detailed information on its ecology and behavior because this pathogen is transmitted by contact with water or other individuals, and pathogen growth rates are thermally sensitive. The common mistfrog (Litoria rheocola) is an endangered tropical rainforest frog that has declined due to chytridiomycosis. We tracked L. rheocola during the winter (cool/dry) and summer (warm/wet) seasons at a low- and high-elevation site. We found that seasonal differences in environmental temperatures and frog behavior should render this species most vulnerable to B. dendrobatidis during cooler months and at higher elevations, which matches observed patterns of infection prevalence in this species. During winter, frogs moved shorter distances than during summer, and they spent less time in vegetation and more time in the stream, which should increase exposure to aquatic B. dendrobatidis zoospores. At a low-elevation site (40 m ASL), estimated body temperatures were within the optimal range for B. dendrobatidis growth (15-25°C) most of the time during winter, but they reached temperatures above this threshold frequently in summer. At a higher elevation (750 m ASL), estimated body temperatures were within the range most favorable for B. dendrobatidis year-round, and did not exceed 25°C, even during summer. Our study provides the first detailed information on the ecology and behavior of L. rheocola and suggests ecological mechanisms for infection dynamics that have been observed in this endangered species.     

Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay.In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined.     

Motile Zoospores of Batrachochytrium dendrobatidis Move Away from Antifungal Metabolites Produced by Amphibian Skin Bacteria

Chytridiomycosis is an amphibian skin disease that threatens amphibian biodiversity worldwide. The fungal agent of chytridiomycosis is Batrachochytrium dendrobatidis. There is considerable variation in disease outcomes such that some individuals and populations co-exist with the fungus and others quickly succumb to disease. Amphibians in populations that co-exist with the B. dendrobatidis have sublethal infections on their skins. Symbiotic skin bacteria have been shown in experiments and surveys to play a role in protecting amphibians from chytridiomycosis. Little is known about the mechanisms that antifungal skin bacteria use to ameliorate the effects of B. dendrobatidis. In this study, we identified that B. dendrobatidis isolate JEL 310 zoospores display chemotaxis, in the presence of two bacterially-produced metabolites (2,4-diacetylphloroglucinol and indole-3-carboxaldehyde). In the presence of either metabolite, B. dendrobatidis zoospores move more frequently away from the metabolite. Using parameters estimated from this study, a simple stochastic model of a random walk on a lattice was evaluated. The model shows that these individual behaviors over short time-scales directly lead to population behaviors over long time–scales, such that most zoospores will escape, or not infect a tryptone substrate containing the bacterially-produced metabolite, whereas many zoospores will infect the tryptone substrate containing no metabolite. These results suggest that amphibians that have skin bacteria produce antifungal metabolites that might be able to keep B. dendrobatidis infections below the lethal threshold and thus are able to co-exist with the fungus.     

Crossing the threshold: an amphibian assemblage highly infected with Batrachochytrium dendrobatidis in the southern Brazilian Atlantic forest

The fungal infection caused by Batrachochytrium dendrobatidis (Bd) in amphibians is known to be lethal when infection intensity values exceed loads of 10,000 zoospores per individual. We investigated Bd infection intensity in 100 anurans of southern Brazil. Almost half of the individuals were infected and the intensity ranged from four to about 156,000 zoospore genomic equivalents. We found no clinical signs of chytridiomycosis and no evidence of mortality. However, we observed a reduction in the number of infected individuals with loads above 10,000 zoospores. This fact could be considered indirect evidence that individuals with high loads are removed from the population.     

The effect of salinity on the survival,growth, sporulation and infection of Phytophthora ramorum

Phytophthora ramorum has been found in waterways outside infested nurseries, but little is known about its behavior in water. This study examined the effect of salinity on survival, growth, sporulation, and infection. P. ramorum survival and growth was negatively correlated with salt concentration (range of 0–45 g l⁻¹), but showed a level of tolerance even at 45 g l⁻¹. No sporangia were observed in cultures with higher than 20 g l⁻¹ of salt and zoospores were not released from sporangia above 14 g l⁻¹. Water sources with different salinity were used to understand the environment where P. ramorum can survive and infect host material. Water from natural bodies and water amended with different salt concentrations were added to P. ramorum-infested sand and baited with rhododendron leaf disks. Infection decreased with increasing salt concentration and increased with higher initial concentration of P. ramorum. This research helps to better understand the effects of water quality on survival and infectivity of P. ramorum, expanding the potential survey range.     

Screening capacity of UV-absorbing compounds in spores of Arctic Laminariales

The functional significance of phlorotannins as ultraviolet radiation screens in brown algae is presented. Spectral analysis of zoospore suspensions of the three Arctic Laminariales Saccorhiza dermatodea, Alaria esculenta and Laminaria digitata showed strong absorption in the UV waveband, characteristic of phlorotannins. An induction in the synthesis of the UV-absorbing compound in zoospore suspensions of S. dermatodea and A. esculenta was observed as an increase in absorbance in the UV region after 8 h exposure to the whole light spectrum. Transmission of UVR was also negatively correlated with zoospore density in both these species but not in L. digitata. ‘Biofilters’ constructed from UV-transparent acrylic sheet, containing zoospore suspensions or solutions of phloroglucinol showed varying capacity to protect zoospore cultures from the lethal effects of ultraviolet radiation. Phloroglucinol protects the zoospores from damage by screening out the much harmful shorter UV-B spectra (280-290 nm). Cultured spores of A. esculenta and L. digitata after exposure to the whole light spectrum covered by filters containing phloroglucinol showed high rates of germination, unlike controls covered by seawater-only filters that showed 100% mortality. Biofilters containing zoospore suspensions act as buffers and showed variable UV-protection properties on the germination of its conspecies. At highest zoospore density (∼ 4 × 10⁶ spores ml^− 1), zoospores were observed to screen UV radiation maintaining viability among shielded spores in all species investigated. The protective function of zoospore film is, however, density-dependent in L. digitata. At lower spore density, UV-screening function in S. dermatodea and A. esculenta is attributed to their capacity to accumulate and release UV-absorbing compounds into the medium. Ultraviolet radiation transmission by zoospore suspensions of Saccorhiza and Alaria decreased during exposure to the whole light spectrum which is consistent with the earlier observation of enlarged phenolic vesicles following UVR exposure. The increase in vesicle size and the corresponding increase in UV-absorbing capacity may contribute to greater tolerance of UVR exposure in both species.     

The relationship between the emergence of Batrachochytrium dendrobatidis,the international trade in amphibians and introduced amphibian species

Chytridiomycosis is an emerging infectious disease of amphibians caused by the chytrid Batrachochytrium dendrobatidis. The disease has been associated with global amphibian declines and species extinctions, however the principle drivers that underly the emergence of chytridiomycosis remain unclear. Current evidence suggests that the world trade in amphibians is implicated in the emergence of chytridiomycosis. Here, we review the evidence that the amphibian trade is driving the emergence of chytridiomycosis by (1) spreading infected animals worldwide, (2) introducing non-native infected animals into naïve populations and (3) amplifying infection of amphibians by co-housing, followed by untreated discharge of infectious zoospores into water supplies. We conclude that the evidence that the amphibian trade is contributing to the spread of Batrachochytrium dendrobatidis is strong, and that specific actions are necessary to prevent the introduction of the pathogen into thus-far uninfected areas. Specifically, we recommend the development of national risk-abatement plans, focused on firstly preventing introduction of Bd into disease free areas, and secondly, decreasing the impact of the disease on populations that are currently infected.     

Nikkomycin Z is an effective inhibitor of the chytrid fungus linked to global amphibian declines

Fungal infections in humans, wildlife, and plants are a growing concern because of their devastating effects on human and ecosystem health. In recent years, populations of many amphibian species have declined, and some have become extinct due to chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis. For some endangered amphibian species, captive colonies are the best intermediate solution towards eventual reintroduction, and effective antifungal treatments are needed to cure chytridiomycosis and limit the spread of this pathogen in such survival assurance colonies. Currently, the best accepted treatment for infected amphibians is itraconazole, but its toxic side effects reduce its usefulness for many species. Safer antifungal treatments are needed for disease control. Here, we show that nikkomycin Z, a chitin synthase inhibitor, dramatically alters the cell wall stability of B. dendrobatidis cells and completely inhibits growth of B. dendrobatidis at 250 μM. Low doses of nikkomycin Z enhanced the effectiveness of natural antimicrobial skin peptide mixtures tested in vitro. These studies suggest that nikkomycin Z would be an effective treatment to significantly reduce the fungal burden in frogs infected by B. dendrobatidis.     

Electrotaxis of zoospores of Phytophthora palmivora at physiologically relevant field strengths

Plant roots generate electrical fields in the rhizosphere as a consequence of their ion transport activities. We show here that zoospores of the plant pathogen Phytophthora palmivora exhibit anodal electrotaxis in electrical fields ≥0.5 V m⁻¹ comparable in size to the physiological fields around roots. An experimental protocol for applying weak electrical fields and quantifying electrotaxis is described. In this system, zoospore suspensions are isolated from the electrodes and their products using agarose bridges. Therefore, electrotaxis was not due to movement or trapping of zoospores in chemical, oxygen, pH or inhibitor gradients established by electrolysis. The electrophoretic and electroosmotic mobilities of encysted zoospores were measured. These forces did not influence the distribution of zoospores in electrotactic experiments at physiological field strengths. The electrotactic response saturated at fields above 10 V m⁻¹ was inhibited in media of osmotic strength below 400 Osmol m⁻³, was maximal at pH 7.5 and increased at high zoospore densities. These data suggest that electrotaxis may be a useful adjunct to chemotaxis in root targeting by zoospores.     

Differential interactions between the nematocyst-bearing mixotrophic dinoflagellate Paragymnodinium shiwhaense and common heterotrophic protists and copepods: Killer or prey

To investigate interactions between the nematocyst-bearing mixotrophic dinoflagellate Paragymnodinium shiwhaense and different heterotrophic protist and copepod species, feeding by common heterotrophic dinoflagellates (Oxyrrhis marina and Gyrodinium dominans), naked ciliates (Strobilidium sp. approximately 35 μm in cell length and Strombidinopsis sp. approximately 100 μm in cell length), and calanoid copepods Acartia spp. (A. hongi and A. omorii) on P. shiwhaense was explored. In addition, the feeding activities of P. shiwhaense on these heterotrophic protists were investigated. Furthermore, the growth and ingestion rates of O. marina, G. dominans, Strobilidium sp., Strombidinopsis sp., and Acartia spp. as a function of P. shiwhaense concentration were measured. O. marina, G. dominans, and Strombidinopsis sp. were able to feed on P. shiwhaense, but Strobilidium sp. was not. However, the growth rates of O. marina, G. dominans, Strobilidium sp., and Strombidinopsis sp. feeding on P. shiwhaense were very low or negative at almost all concentrations of P. shiwhaense. P. shiwhaense frequently fed on O. marina and Strobilidium sp., but did not feed on Strombidinopsis sp. and G. dominans. G. dominans cells swelled and became dead when incubated with filtrate from the experimental bottles (G. dominans + P. shiwhaense) that had been incubated for one day. The ingestion rates of O. marina, G. dominans, and Strobilidium sp. on P. shiwhaense were almost zero at all P. shiwhaense concentrations, while those of Strombidinopsis sp. increased with prey concentration. The maximum ingestion rate of Strombidinopsis sp. on P. shiwhaense was 5.3 ng C predator⁻¹d⁻¹ (41 cells predator⁻¹d⁻¹), which was much lower than ingestion rates reported in the literature for other mixotrophic dinoflagellate prey species. With increasing prey concentrations, the ingestion rates of Acartia spp. on P. shiwhaense increased up to 930 ng C ml⁻¹ (7180 cells ml⁻¹) at the highest prey concentration. The highest ingestion rate of Acartia spp. on P. shiwhaense was 4240 ng C predator⁻¹d⁻¹ (32,610 cells predator⁻¹d⁻¹), which is comparable to ingestion rates from previous studies on other dinoflagellate prey species calculated at similar prey concentrations. Thus, P. shiwhaense might play diverse ecological roles in marine planktonic communities by having an advantage over competing phytoplankton in anti-predation against potential protistan grazers.     

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica,a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea—a suitable algal prey item—heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d⁻¹. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13 ng C predator⁻¹d⁻¹ (0.06 cells predator⁻¹d⁻¹). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019 ng C predator⁻¹d⁻¹ (266 bacteria predator⁻¹d⁻¹), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 10⁷ cells ml⁻¹ was 0.01 ng C predator⁻¹d⁻¹ (48 Synechococcus predator⁻¹d⁻¹). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.     

Mixotrophy in the newly described dinoflagellate Yihiella yeosuensis: A small,fast dinoflagellate predator that grows mixotrophically,but not autotrophically

To investigate tropical roles of the newly described Yihiella yeosuensis (ca. 8 μm in cell size), one of the smallest phototrophic dinoflagellates in marine ecosystems, its trophic mode and the types of prey species that Y. yeosuensis can feed upon were explored. Growth and ingestion rates of Y. yeosuensis on its optimal prey, Pyramimonas sp. (Prasinophyceae), as a function of prey concentration were measured. Additionally, growth and ingestion rates of Y. yeosuensis on the other edible prey, Teleaulax sp. (Cryptophyceae), were also determined for a single prey concentration at which both these rates of Y. yeosuensis on Pyramimonas sp. were saturated. Among bacteria and diverse algal prey tested, Y. yeosuensis fed only on small Pyramimonas sp. and Teleaulax sp. (both cell sizes = 5.6 μm). With increasing mean prey concentrations, both specific growth and ingestion rates of Y. yeosuensis increased rapidly before saturating at a mean Pyramimonas concentration of 109 ng C mL⁻¹ (2725 cells mL⁻¹). The maximum growth rate (mixotrophic growth) of Y. yeosuensis fed with Pyramimonas sp. at 20 °C under a 14:10-h light-dark cycle of 20 μE m⁻² s⁻¹ was 1.32 d⁻¹, whereas the growth rate of Y. yeosuensis without added prey was 0.026 d⁻¹. The maximum ingestion rate of Y. yeosuensis fed with Pyramimonas sp. was 0.37 ng C predator⁻¹ d⁻¹ (9.3 cells predator⁻¹ d⁻¹). At a Teleaulax concentration of 1130 ng C mL⁻¹ (66,240 cells mL⁻¹), growth and ingestion rates of Y. yeosuensis fed with Teleaulax sp. were 1.285 d⁻¹ and 0.38 ng C predator⁻¹ d⁻¹ (22.4 cells predator⁻¹ d⁻¹), respectively. Thus, Y. yeosuensis rarely grows without mixotrophy, and mixotrophy supports high growth rates in Y. yeosuensis. Y. yeosuensis has the highest maximum mixotrophic growth rate with the exception of Ansanella graniferaamong engulfment feeding mixotrophic dinoflagellates. However, the high swimming speed of Y. yeosuensis (1572 μm s⁻¹), almost the highest among phototrophic dinoflagellates, may prevent autotrophic growth. This evidence suggests that Y. yeosuensis may be an effective mixotrophic dinoflagellate predator on Pyramimonas and Teleaulax, and occurs abundantly during or after blooms of these two prey species.     

Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli

Enantiopure l-tert-leucine (l-Tle) was synthesized through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant Escherichia coli coexpressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH). The leudh gene from Lysinibacillus sphaericus CGMCC 1.1677 encoding LeuDH was cloned and coexpressed with NAD⁺-dependent FDH from Candida boidinii for NADH regeneration. The batch reaction conditions for the synthesis of l-Tle were systematically optimized. Two substrate feeding modes (intermittent and continuous) were addressed to alleviate substrate inhibition and thus improve the space-time yield. The continuous feeding process was conveniently performed in water at an overall substrate concentration up to 1.5 M, with both conversion and ee of >99% and space-time yield of 786 g L⁻¹ d⁻¹, respectively. Furthermore, the preparation was successfully scaled up to a 1 L scale, demonstrating the developed procedure showed a great industrial potential for the production of enantiopure l-Tle.     

Swabbing Often Fails to Detect Amphibian Chytridiomycosis under Conditions of Low Infection Load

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naïve amphibian populations. Intervention strategies to rapidly respond to Bd incursions require sensitive and accurate diagnostic methods. Chytridiomycosis usually is assessed by quantitative polymerase chain reaction (qPCR) amplification of amphibian skin swabs. Results based on this method, however, sometimes yield inconsistent results on infection status and inaccurate scores of infection intensity. In Asia and other regions where amphibians typically bear low Bd loads, swab results are least reliable. We developed a Bd-sampling method that collects zoospores released by infected subjects into an aquatic medium. Bd DNA is extracted by filters and amplified by nested PCR. Using laboratory colonies and field populations of Bombina orientalis, we compare results with those obtained on the same subjects by qPCR of DNA extracted from swabs. Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected. Infection loads determined from filtered water were at least 1000 times higher than those estimated from swabs. Subjects significantly varied in infection load, as they intermittently released zoospores, over a 5-day period. Thus, the method might be used to compare the infectivity of individuals and study the periodicity of zoospore release. Sampling methods based on water filtration can dramatically increase the capacity to accurately diagnose chytridiomycosis and contribute to a better understanding of the interactions between Bd and its hosts.     

Short-term feeding of Temora longicornis Müller in the laboratory and the field

Short-term feeding (min to h) of the copepod Temora longicomis (Müller) was investigated in the laboratory and in the field by monitoring the increase in gut fluorescence of adult females. Laboratory feeding studies using the diatoms Thalassiosira pseudonana (Hust.), T. weissflogii (Hust.) and Ditylumbrightwellii (West) showed that ingestion rates increased with increased food concentration and cell size. The maximum ingestion rate was 9.5 ng chl. equiv. female ⁻¹. h ⁻¹ on a diet D. brightwellii. Clearance rates decreased with increased food concentration and decreased cell size. Maximum clearance rate was 1.9 ml. female⁻¹·h⁻¹ also on a diet of D. brightwellii. On two of three occasions, ingestion and clearance rates in the field were twice as high at night as during the day. Night rates were similar to laboratory rates in the chlorophyll range found in the field. Diel differences in feeding activity in the field appeared to have been behaviorally induced rather than being related to vertical migration of individuals into food rich layers.     

Distribution and Pathogenicity of Batrachochytrium dendrobatidis in Boreal Toads from the Grand Teton Area of Western Wyoming

The pathogen Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis, has been linked to amphibian population declines and extinctions worldwide. Bd has been implicated in recent declines of boreal toads, Bufo boreas boreas, in Colorado but populations of boreal toads in western Wyoming have high prevalence of Bd without suffering catastrophic mortality. In a field and laboratory study, we investigated the prevalence of Bd in boreal toads from the Grand Teton ecosystem (GRTE) in Wyoming and tested the pathogenicity of Bd to these toads in several environments. The pathogen was present in breeding adults at all 10 sites sampled, with a mean prevalence of 67%. In an experiment with juvenile toadlets housed individually in wet environments, 10⁶ zoospores of Bd isolated from GRTE caused lethal disease in all Wyoming and Colorado animals within 35 days. Survival time was longer in toadlets from Wyoming than Colorado and in toadlets spending more time in dry sites. In a second trial involving Colorado toadlets exposed to 35% fewer Bd zoospores, infection peaked and subsided over 68 days with no lethal chytridiomycosis in any treatment. However, compared with drier aquaria with dry refuges, Bd infection intensity was 41% higher in more humid aquaria and 81% higher without dry refuges available. Our findings suggest that although widely infected in nature, Wyoming toads may escape chytridiomycosis due to a slight advantage in innate resistance or because their native habitat hinders Bd growth or provides more opportunities to reduce pathogen loads behaviorally than in Colorado.     

Timing and abundance of sporangia production and zoospore release influences the recovery of different Phytophthora species by baiting

Analysis of soil samples using High Throughput Sequencing (HTS) frequently detects more Phytophthora species compared with traditional soil baiting methods. This study investigated whether differences between species in the timing and abundance of sporangial production and zoospore release could be a reason for the lower number of species isolated by baiting. Stems of Eucalyptus marginata were inoculated with ten Phytophthora species (P. nicotianae, P. multivora, P. pseudocryptogea, P. cinnamomi, P. thermophila, P. arenaria, P. heveae, P. constricta, P. gondwanensis and P. versiformis), and lesioned sections for each species were baited separately in water. There were significant differences between species in timing of sporangia production and zoospore release. P. nicotianae, P. pseudocryptogea, P. multivora and P. thermophila released zoospores within 8–12 h and could be isolated from lesioned baits within 1–2 days. In contrast, P. constricta did not produce zoospores for over 48 h and was only isolated 5–7 days after baiting. P. heveae and P. versiformis did not produce zoospores and were not recovered from the baits. When species were paired in the same baiting tub, those that produced zoospores in the shortest time were isolated most frequently, while species slow to produce zoospores, or which produced them in lower numbers, were isolated from few baits or not at all. Thus, species differences in the timing of sporangia production and zoospore release may contribute to the ease of isolation of some Phytophthora species when they are present together with other Phytophthora species in an environmental sample.     

High cell density fed-batch fermentation for the production of a microbial lipase

Extracellular lipase of the yeast Candida rugosa was produced via high cell density fed-batch fermentations using palm oil as the sole source of carbon and energy. Feeding strategies consisted of a pH-stat operation, foaming-dependent control and specific growth rate control in different experiments. Compared to foaming-dependent feeding and the pH-stat operation, the specific growth rate control of feeding proved to be the most successful. At the specific growth rate control set at 0.05 h⁻¹, the final lipase activity in the culture broth was the highest at ∼700 U L⁻¹. This was 2.6-fold higher than the final enzyme activity obtained at a specific growth rate control set at 0.15 h⁻¹. The peak enzyme concentration achieved using the best foaming-dependent control of feeding was around 28% of the peak activity attained using the specific growth rate control of feeding at 0.05 h⁻¹. Similarly, the peak enzyme concentration attained using the pH-stat feeding operation was a mere 9% of the peak activity attained by specific growth rate control of feeding at a set-point of 0.05 h⁻¹. Fed-batch fermentations were performed in a 2 L stirred-tank bioreactor (30 °C, pH 7) with the dissolved oxygen level controlled at 30% of air saturation.