Experimental Exposures of Boreal Toads (Bufo boreas) to a Pathogenic Chytrid Fungus (Batrachochytrium dendrobatidis)

One of the major causes of worldwide amphibian declines is a skin infection caused by a pathogenic chytrid fungus (Batrachochytrium dendrobatidis). This study documents the interactions between this pathogen and a susceptible amphibian host, the boreal toad (Bufo boreas). The amount of time following exposure until death is influenced by the dosage of infectious zoospores, duration of exposure, and body size of the toad. The significant relation between dosage and the number of days survived (dose-response curve) supports the hypothesis that the degree of infection must reach a particular threshold of about 10⁷–10⁸ zoosporangia before death results. Variation in air temperature between 12°C and 23°C had no significant effect on survival time. The infection can be transmitted from infected to healthy animals by contact with water containing zoospores; no physical contact between animals is required. These results are correlated with observations on the population biology of boreal toads in which mortalities associated with B. dendrobatidis have been identified.     

Selection of entomopathogenic fungi for use in combination with sub-lethal doses of imidacloprid: perspectives for the control of the leaf-cutting ant <Emphasis Type="Italic">Atta sexdens</Emphasis><Emphasis Type="Italic">rubropilosa</Emphasis> Forel (Hymenoptera: Formicidae)

In the first part of this study, four isolates of the fungus Beauveria bassiana (Bals.) Vuillemin (LPP1, LPP2, CG05 and CG24) and one isolate of Metarhizium anisopliae (Metsch.) Sorokin (CG46) were tested against adult foragers of Atta sexdens rubropilosa. Ants were allowed to walk on filter paper discs, inside Petri dishes, previously impregnated with 1 ml of a conidia suspension (2 × 10⁷ conidia ml⁻¹), maintained at 80% RH and 26°C for 24 h and subsequently, transferred to sterile Petri dishes, maintained at 23°C, 80% RH, 24 h dark. The mean values of LT₅₀ for LPP2, LPP1, CG46, CG24 and CG05 were 3.5, 3.7, 3.8, 4.2 and 4.4 days, respectively. Control insects for all tests in this study showed less than 10% mortality. Experiments were carried out to test the toxicity of imidacloprid (IMI) to A. sexdens rubropilosa. Mortality was evaluated 10 days following a 24 h exposure to the insecticide. Percent mortality caused by 500, 200, 100 and 10 ppm IMI was 77.8, 56.7, 45.5 and 5.5 respectively. Insects treated with 10 ppm IMI were observed to have reduced locomotor activity 24 h after exposure to the insecticide. The LC₅₀ of IMI was 154.3 ppm. Subsequent tests were carried out to evaluate the combination of a sub-lethal dose of IMI (10 ppm) and infection by CG24 (1 × 10⁷ conidia ml⁻¹). Mortality due to fungal infection alone was 43.3%. Mortality of insects treated with IMI followed by exposure to the fungus was 64.3%. These results indicate that IMI significantly increases the susceptibility of ants to infection by B. bassiana CG24.     

Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms

Pythium porphyrae is a fungal pathogen responsible for red rot disease of the seaweed Porphyra (Rhodophyta). Infection forecasts of Porphyra by P. porphyrae were estimated from the epidemiological observations of Porphyra thalli and numbers of zoospore of P. porphyrae in laboratory and cultivation areas. Four features of forecasting infections were determined by relating zoospore concentrations to the incidence of thallus infection; infection (in more than 1000 zoospores L⁻¹), microscopic infection [less than 2 mm in diameter of lesion (in from 2000 to 3000 zoospores L⁻¹)], macroscopic infection [more than 2 mm in diameter of lesion (in from 3000 to 4000 zoospores L⁻¹), and thallus disintegration (in more than 4000 zoospores L⁻¹). High zoospore concentrations led to more infection. The tendency that zoospore concentration of P. porphyrae increased with the rate of infection of Porphyra thalli was generally observed in forecasting infections in both the laboratory and in cultivation areas. Based on the Porphyra cultivation areas, the accuracy and consistency of forecasting infections suggest that this method could be employed to manage and control red rot disease.     

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.     

Temperature, hydric environment, and prior pathogen exposure alter the experimental severity of chytridiomycosis in boreal toads

Prevalence of the pathogen Batrachochytrium dendrobatidis (Bd), implicated in amphibian population declines worldwide, is associated with habitat moisture and temperature, but few studies have varied these factors and measured the response to infection in amphibian hosts. We evaluated how varying humidity, contact with water, and temperature affected the manifestation of chytridiomycosis in boreal toads Anaxyrus (Bufo) boreas boreas and how prior exposure to Bd affects the likelihood of survival after re-exposure, such as may occur seasonally in long-lived species. Humidity did not affect survival or the degree of Bd infection, but a longer time in contact with water increased the likelihood of mortality. After exposure to approximately 10(6) Bd zoospores, all toads in continuous contact with water died within 30 d. Moreover, Bd-exposed toads that were disease-free after 64 d under dry conditions, developed lethal chytridiomycosis within 70 d of transfer to wet conditions. Toads in unheated aquaria (mean = 15 degrees C) survived less than 48 d, while those in moderately heated aquaria (mean = 18 degrees C) survived 115 d post-exposure and exhibited behavioral fever, selecting warmer sites across a temperature gradient. We also found benefits of prior Bd infection: previously exposed toads survived 3 times longer than Bd-na?ve toads after re-exposure to 106 zoospores (89 vs. 30 d), but only when dry microenvironments were available. This study illustrates how the outcome of Bd infection in boreal toads is environmentally dependent: when continuously wet, high reinfection rates may overwhelm defenses, but periodic drying, moderate warming, and previous infection may allow infected toads to extend their survival.     

Exposure of Fischerella [Mastigocladus] to high and low temperature extremes: strain evaluation for a thermal mitigation process

In conjunction with a proposed algal cultivation scheme utilizing thermal effluent, twelve Fischerella strains were tested for tolerance to temperatures above and below their growth range. Exposure to 65 °C or 70 °C for 30 min caused bleaching and death of most or all cells. Effects of 60 °C exposure for periods of up to 2 h ranged from undetectable to severe for the various strains. Chlorophyll a content typically decreased 21–22% immediately following 60 °C or 65 °C (1 h) exposure. However, the 60 °C-shocked cultures regained normal Chl a content after 24 h at 45 °C, whereas Chl a in 65 °C-shocked cultures immediately lost visible autofluorescence and was later degraded. Exposure to 15 °C virtually stopped growth of all strains during a 48 h exposure period. Most strains grew as rapidly as 45 °C controls when restored to 45 °C, while a few strains recovered more slowly. Comparison with dark-incubated controls indicated that photooxidative damage did not occur during cold shock. Certain strains exhibited relatively rapid recovery from both heat and cold exposure, thus meeting the temperature tolerance criteria for the proposed algal cultivation process.     

The effect of temperature on growth characteristics and competitions of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Oscillatoria mougeotii</Emphasis> in a shallow,eutrophic lake simulator system

Blue-green algal blooms formed by Microcystis and Oscillatoria often occur in shallow eutrophic lakes, such as Lake Taihu (China) and Lake Kasumigaura (Japan). Growth characteristics and competitions between Microcystis aeruginosa and Oscillatoria mougeotii were investigated using lake simulator systems (microcosms) at various temperatures. Oscillatoria was the superior competitor, which suppressed Microcystis, when temperature was <20°C, whereas the opposite phenomenon occurred at 30°C. Oscillatoria had a long exponential phase (20 day) and a low growth rate of 0.22 day⁻¹ and 0.20 day⁻¹ at 15°C and 20°C, respectively, whereas Microcystis had a shorter exponential phase (2–3 days) at 30°C and a higher growth rate (0.86 day⁻¹). Interactions between the algae were stronger and more complex in the lake simulator system than flask systems. Algal growth in the lake simulator system was susceptible to light attenuation and pH change, and algae biomasses were lower than those in flasks. The outcome of competition between Microcystis and Oscillatoria at different temperatures agrees with field observations of algal communities in Lake Taihu, indicating that temperature is a significant factor affecting competition between Microcystis and Oscillatoria in shallow, eutrophic lakes.     

Bioassay for in vitro differentiation of pineapple cultivar resistance levels to heart rot disease

Summary An in vitro bioassay to differentiate pineapple plant resistance levels to Phytophthora nicotianae var. parasitica (heart rot disease) is deseribed here. Conditions to cause death of in vitro-cultured plants were defined using a cultivar previously found to be susceptible to this fungus in our Field-Grown Pineapple Germplasm Bank (ev. Smooth Cayenne Serrana). The effects of zoospore concentration, inoculation technique, and disease progress during the course of time after infection were evaluated. The highest rates of plant death were observed with the use of 10⁸ zoospores ml⁻¹, and the inoculation technique of needle-mediated leaf base wound. One hundred percent plant death was observed at 144h after infection. Different susceptible varieties along with a resistant pineapple relative were additionally compared. In vitro results confirmed previous observations obtained under field conditions. The protocol described here may be used for early selection (in vitro) of new pineapple genotypes showing resistance to this fungus. At present, this protocol is extensively used in the Biotechnology-assisted Cuban Program for Pineapple Breeding.     

Ty1-copia group retrotransposon sequences in amphibia and reptilia

We have isolated sequences belonging to Tyl-copia group retrotransposons from the genomes of an amphibian (Pyxicephalus adspersa) and three reptiles (Conolophus subscristatus, Amblyrynchus cristatus and Pytas mucosus). Two different seqences were found in the amphibian (Tpa1 and Tpa2). Each is present in several copies per genome and absent from the genomes of two other amphibian species. The C. subcristatus sequence Tcs1 is present in multiple copies in both its host genome (Galapagos land iguana) and the genome of the related Galapagos marine iguana (A. cristatus). There is little or no polymorphism in Tcs1 insertions between different individual animals, suggesting that this sequence is not transposing rapidly in either iguana genome. The P. mucosus sequence Tpm1 shows a discontinuous distribution in snake species, suggesting that it has either been lost from many lineages during vertical germline transmission or has been transferred horizontally in some snake species. Phylogenetic comparisons of all these sequences with each other and with other members of this retrotransposon group from other animals and plants show that sequences within a particular vertebrate species are most closely related to each other, consistent with a vertical transmission model for their evolution.     

Effects of ambient temperature and of temperature acclimation on nitrogen excretion and differential catabolism of protein and nonprotein resources in the intertidal snails,Littorina saxatilis (Olivi) and L. obtusata (L.)

Nitrogenous excretion in two snails, Littorina saxatilis (high intertidal) and L. obtusata (low intertidal) was studied in relation to temperature acclimation (at 4° and 21°C), including total N excretion rates, the fraction of urea in N excretion, corresponding O:N ratios and the partitioning of deaminated protein between catabolic and anabolic processes at 4°, 11° and 21°C. Aggregate N excretion rates in both species showed no significant compensatory adjustments following acclimation. Total weight specific N excretion rates at 21°C were higher in standard 3 mg L. saxatilis (739 ng N mg⁻¹ h⁻¹) than standard 5 mg L. obtusata (257 ng N mg⁻¹ h⁻¹) for snails acclimated to 21°C. Comparisons of Q₁₀ values of total weight specific N excretion to Q₁₀ values for weight specific oxygen consumption ({xxV}O₂) between 4° to 11 °C and 11° to 21°C indicated that, while total rates of catabolic metabolism ({xxV}O₂) and protein deamination in L. obtusata were essentially parallel, the relationship between N excretion and {xxV}O₂ in L. saxatilis revealed the partitioning of a larger share of deaminated protein carbon into anabolism at 4° and 21°C than at 11°C. Urea N accounted for a larger share of aggregate N excreted in L. saxatilis than in L. obtusata, but in both species urea N is a greater proportion of total N excreted when acclimated at 4°C (urea N: ammonia N ratio range: 1 to 2.15) than in snails acclimated to 21°C (urea N: ammonia N ratio range: 0.46 to 1.39). Molar O:N ratios indicate that the proportion of metabolism supported by protein catabolism is greater in L. saxatilis (O:N range: 2.5–8.4) than in L. obtusata (O:N range: 7.3–13.0). In both species, regardless of acclimation temperature, the O:N ratios are generally lowest (high protein catabolism) at 4°C and highest at 21°C.     

Effect of crab shell size on bio-demineralization with lactic acid-producing bacterium, <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">tolerans</Emphasis> KCTC-3074

Demineralization (DM) from crab shell (CS) waste was carried out using a lactic acid-producing bacterium, Lactobacillus paracasei subsp. tolerans KCTC-3074 for 7 days at 25, 30, and 35°C. DM rates were 89∼92% and slightly affected by temperature. DM was also performed for four particle-sized shell samples (0.84∼3.35, 3.35∼10, 10∼20, and 20∼35 mm) with 10% inoculum, 5% shell, and 10% glucose at 30°C and 180 rpm for 7 days. It was found out that the shell size had a slight effect on the rate of DM. Negative relationships were found between DM and residual dry weight (r² = 0.960), and between DM and pH (r² = 0.906). Conversely, positive relationships were found between DM and medium protein (r² = 0.696), and between DM and total titratable acidity (r² = 0.630).     

The culture ofEntomophthora muscae (C) Fres. in carrot flies (Psila rosae F.) and the effect of temperature on the pathology of the fungus

A method for maintaining anin vivo culture ofEntomophthora muscae (C) Fres. on its original host, adult carrot flies (Psila rosae F.), is described. The lethal time for adult carrot flies was greatly influenced by temperature, both for infected and for uninfected flies. In the range 8.2°C–20.2°C the LT₅₀ for infected flies was about 5.4 times shorter than the estimated average life-span for uninfected flies. The discharge of primary spores was also strongly dependent on temperature. The total number of primary spores discharged per fly at 100% RH and in darkness ranged between 1.2×10⁴ and 9.6×10⁴ with a mean of 5.1×10⁴.      

Mycorrhizal responses of barley cultivars differing in P efficiency

The purpose of this study was to investigate how barley cultivars which are different in dry matter yield at low phosphorus (P) supply (i.e. they differ in agronomic P efficiency) respond to mycorrhizal infection. In a preliminary experiment, six mycorrhizal fungi were tested for their ability to colonize barley (Hordeum vulgare L.) roots at a soil temperature of 15°C.Glomus etunicatum was the most effective species and was used in the main experiment. The main experiment was conducted under glasshouse conditions in which soil temperature was maintained at 15°C. Treatments consisted of a factorial arrangement of 8 barley cultivars, 2 mycorrhiza (inoculated and non-inoculated), and 3 rates of P (0, 10 and 20 mg kg^-1). P utilization efficiency (dry matter yield per unit of P taken up) and agronomic P efficiency among the barley cultivars was significantly negatively correlated with mycorrhizal responses. However, the response to mycorrhizal infection was positively correlated with response to P application. Poor correlation was observed between P concentration when neither mycorrhiza nor P were supplied and the percentage of root length infected. The extent of mycorrhizal infection among the barley cultivars in soil without P amendment varied from 8.6 to 28.6%. Significant interactions between cultivar and P addition, and between mycorrhiza and P addition were observed for shoot dry weight but not root dry weight.     

Temperature management strategy for efficient gene expression in a thermally inducible <Emphasis Type="Italic">Escherichia coli</Emphasis>/bacteriophage system

In a two-phase operation, E. coli containing λSNNU1 (Q ⁻ S ⁻) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least 40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast, when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min, and a production phase at 37°C, the highest level of cloned gene expression was achieved.     

Effects of food limitation and temperature on cladocerans from a tropical Brazilian lake

Food limitation was tested in the laboratory by individual growth and reproduction of two cladoceran species, Ceriodaphnia richardi and Daphnia gessneri, from the shallow tropical Brazilian Lake Monte Alegre. The cladocerans were fed cultivated green alga Scenedesmus spinosus in concentrations of 0.20, 0.10, 0.05, and 0.025 mg C l⁻¹. Higher biomass and growth rates occurred in the two highest-food concentrations; the two lowest ones negatively affected clutch size and first reproduction. The threshold food concentration is lower than 0.025 mg C l⁻¹ and the incipient limiting level is a value between 0.10 and 0.20 mg C l⁻¹. The largest species, D. gessneri, was more sensitive to low food concentrations. The effects of low and high temperatures (19 and 27°C) were evaluated by life table experiments with three cladocerans from the lake—Daphnia ambigua, D. gessneri, and Moina micrura—with no food limitation (1 mg C l⁻¹ of S. spinosus). Higher population growth rates for the three species were found at 27°C; better performance in most life table parameters was observed for the former two species at the highest temperature, D. gessneri being the most sensitive to the lowest temperature. There are indications that temperature is an important abiotic factor that constrains populations of cladocerans for a short period in winter in the lake, when temperature decreases to 18–19°C. However, its influence cannot be separated from a biotic factor such as food, whose effect is stronger in the cool season, when concentrations are lower and contribution of inedible algae is relatively higher.     

Life history and coldhardiness ofAthrycia cinerea (Dipt.: Tachinidae) in western Canada

Athrycia cinerea Coq. is a univoltine parasitoid of the bertha armyworm,Mamestra configurata Walker (Lepidoptera: Noctuidae), in western Canada. This parasitoid overwinters as a pupa in the soil. These pupae are less sensitive than their host to increased cold stress caused by lower temperatures or danger durations of exposure. Exposure to −7.5°C for 140 days did not significantly reduce survival. Survival decreased with exposure to temperatures from −10 to −20°C, but survival was 48% even after 40 days exposure to −20°C. The frequency distribution ofA. cinerea puparia per host is highly contagious. Other aspects of the life history are described. Contribution No. 1403 Winnipeg Research Station.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> pathogenicity to the cherry slugworm, <Emphasis Type="Italic">Caliroa cerasi</Emphasis> (Hymenoptera: Tenthredinidae) larvae

The cherry slugworm Caliroa cerasi is a significant destructive pest of sour cherries (Prunus cerasus) in Turkey. The potential of entomopathogenic fungi for controlling C. cerasi was evaluated. The effects of exposure methods and conidial concentrations (1 × 10⁶, 1.5 × 10⁶, 1 × 10⁷ and 1.5 × 10⁷ conidia/ml) on mature larvae of C. cerasi infected by Beauveria bassiana were investigated under laboratory conditions. Larvae sprayed directly with B. bassiana conidial suspensions and larvae exposed to B. bassiana-treated leaves resulted in 100% mortality within 2.90 and 2.77 days, respectively. The median lethal time (LT₅₀) and days to mortality were highest in the 1.0 × 10⁷ concentrations of conidia for both direct spray and leaf exposure. The present study suggests that B. bassiana has good potential for control of the cherry slugworm, C. cerasi.     

Oxygen consumption by ammocoetes of the lampreyGeotria australis in air

 When covered by moistened lint-free gauze, the larvae (ammocoetes) of the lamprey Geotria australis survived, without apparent discomfort, for 4 days in water-saturated air at 10, 15 and 20 °C. In air, the mean standard rates of O₂ consumption of medium to large ammocoetes of G. australis (xˉ=0.52 g) at 10, 15 and 20 °C were 14.5, 35.7 and 52.1 μl⋅g^-1⋅h^-1, respectively. At 15 °C, the slope of the relationship between log O₂ consumption (μl O₂⋅h^-1) and log body weight for ammocoetes over a wide range in body weight was 0.987. The Q ₁₀s for rate of O₂ consumption between 10 and 15 °C, 15 and 20 °C and 10 and 20 °C were 4.9, 2.9 and 3.6, respectively. Our results and observations of the ammocoetes suggest that, when out of water, larval G. australis derives most of its O₂ requirements from cutaneous respiration, particularly at lower temperatures. This would be facilitated by the small size and elongate shape (and thus a relatively high surface-to-volume ratio), low metabolic rate, thin dermis, extensive subdermal capillary network and high haemoglobin concentration of larval G. australis. Accepted: 28 March 1996     

Laboratory studies on the biology ofSpalangia nigra [Hym.: Pteromalidae]

At 21 °C,Spalangia nigra Latreille (Hymenoptera: Pteromalidae) averaged 29.3 days between exposure and emergence of 1^st progeny from host house flies,Musca domestica L. (Diptera: Muscidae). At 27 °C, the average developmental time to 1^st emergence was reduced to 26.6 days, and a majority of adult wasps emerged from host house fly puparia between 29 and 40 days postoviposition. The sex ratio of progeny ranged from 1.4 to 1.8 female-to-male, but all progeny of virgin females were male. Male wasps lived from 6.8–15 and females 11–17.8 days at 27 °C; honey as a food source increased longevity. No significant differences in parasitism byS. nigra were associated with host house fly pupal densities ranging from 1 to 200 pupae per female-male pair of wasps, but average percent parasitism decreased at host densities greater than 50. House fly pupae exposed to parasitism at ages ranging from 4 to 96 h did not differ in subsequent production of adult flies.S. nigra did not demonstrate preference for house flies or stable flies,Stomoxys calcitrans (L.) (Diptera: Muscidae) as hosts. The results of these studies indicate thatS. nigra may contribute significantly to previously unexplained mortality of house flies and stable flies.