Can Newts Cope with the Heat? Disparate Thermoregulatory Strategies of Two Sympatric Species in Water

Many ectotherms effectively reduce their exposure to low or high environmental temperatures using behavioral thermoregulation. In terrestrial ectotherms, thermoregulatory strategies range from accurate thermoregulation to thermoconformity according to the costs and limits of thermoregulation, while in aquatic taxa the quantification of behavioral thermoregulation have received limited attention. We examined thermoregulation in two sympatric newt species, Ichthyosaura alpestris and Lissotriton vulgaris, exposed to elevated water temperatures under semi-natural conditions. According to a recent theory, we predicted that species for which elevated water temperatures pose a lower thermal quality habitat, would thermoregulate more effectively than species in thermally benign conditions. In the laboratory thermal gradient, L. vulgaris maintained higher body temperatures than I. alpestris. Semi-natural thermal conditions provided better thermal quality of habitat for L. vulgaris than for I. alpestris. Thermoregulatory indices indicated that I. alpestris actively thermoregulated its body temperature, whereas L. vulgaris remained passive to the thermal heterogeneity of aquatic environment. In the face of elevated water temperatures, sympatric newt species employed disparate thermoregulatory strategies according to the species-specific quality of the thermal habitat. Both strategies reduced newt exposure to suboptimal water temperatures with the same accuracy but with or without the costs of thermoregulation. The quantification of behavioral thermoregulation proves to be an important conceptual and methodological tool for thermal ecology studies not only in terrestrial but also in aquatic ectotherms.     

Ail Proteins of Yersinia pestis and Y. pseudotuberculosis Have Different Cell Binding and Invasion Activities

The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ∼50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30–50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.     

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.     

Proportion of beta-D-glucuronidase-negative Escherichia coli in human fecal samples.

Convenient assays and reports that almost all clinical isolates of Escherichia coli produce beta-D-glucuronidase (GUR) have led to great interest in the use of the enzyme for the rapid detection of the bacterium in water, food, and environmental samples. In these materials, E. coli serves as an indicator of possible fecal contamination. Therefore, it was crucial to examine the proportion of GUR-negative E. coli in human fecal samples. The bacterium was isolated from 35 samples, and a mean of 34% and a median of 15% were found to be GUR negative in lauryl sulfate tryptose broth with 4-methylumbelliferyl-beta-D-glucuronide. E. coli from three samples were temperature dependent for GUR production: very weakly positive at 37 degrees C but strongly positive at 44.5 degrees C. These results remind us of differences between fecal and clinical E. coli populations, of diversity in GUR regulation and expression in natural populations of E. coli, and of the need for caution in using GUR for the detection of fecal E. coli.     

The induction and suppression of in vitro IgG anti-tetanus toxoid antibody synthesis by human lymphocytes stimulated with tetanus toxoid in the absence of in vivo booster immunizations

This report presents a new approach that by-passes booster immunizations with tetanus toxoid (TT) before in vitro studies of antibody (Ab) production. The methodology for optimal TT-induced synthesis of specific IgG anti-tetanus toxoid Ab (IgG anti-TT) by peripheral blood mononuclear cells (PBMC) from randomly selected TT immune individuals without recent booster immunizations is described. PBMC from most normal immune subjects could be repeatedly induced to produce in vitro IgG anti-TT; PBMC from subjects with high TT titers are not required for this new approach. This approach uses high cell concentrations in multiple replicate microcultures and TT washout to obtain optimal IgG anti-TT synthesis. Washed cultures produced more Ab than nonwashed cultures (p less than or equal to 0.005). The readdition of TT (2.5 to 250 ng/ml) to the culture media after washout of TT on day 4 suppressed specific Ab formation, whereas diphtheria toxoid added at comparable doses did not inhibit specific Ab formation. Suppression of antibody synthesis mediated by T cells could be induced by TT per se, and was not due to binding of synthesized Ab to TT in the latter 8 days of culture. In addition, suppression could not be induced in the first 4 days of culture by IgG anti-TT, IgG, or IgM. This approach permits the analysis of antigen-specific regulatory circuits in the steady and activated immune states, and the evaluation of in vivo and in vitro effects of biologic response modifiers on specific Ab production.     

Pharmacological studies on the potentiation of phenytoin teratogenicity by acetaminophen

An in vivo murine model was developed to measure maternal phenytoin biotransformation along with the covalent binding of phenytoin to fetal tissues in the same fetuses which were assessed for fetal anomalies. Acetaminophen was administered to pregnant CD-1 mice 1 hour prior to phenytoin, both given i.p. at varying doses and gestational times between days 11 and 13. Dams were killed between days 12 and 19. Metabolites reflecting the enzymatic bioactivation of phenytoin were quantified in maternal plasma and urine with high-performance liquid chromatography (HPLC). Acetaminophen pretreatment caused a threefold increase in phenytoin-induced fetal cleft palates without increasing resorptions. The covalent binding of radiolabeled phenytoin to fetal and placental tissues measured on day 13 was increased twofold and threefold, respectively, by acetaminophen pretreatment. Phenytoin covalent binding measured on day 16 was significantly increased in the livers of fetuses with cleft palates, but not in the livers of dams with fetuses having cleft palates. Binding to fetal brain on day 16 was over fourfold higher than that in maternal brain. Acetaminophen pretreatment differentiated dams into poor and extensive metabolisers of phenytoin, with only the latter group carrying fetuses with cleft palates. The incidence of fetal cleft palates correlated positively with maternal urinary levels of phenytoin (r = +.81, P less than .01) and its dihydrodiol metabolite (r = +.61, 0.05 less than P less than .1), and negatively with levels of para-hydroxylated phenytoin (r = -.85, P less than .01). These findings related both to the mechanism of phenytoin teratogenicity and its potentiation by acetaminophen.     

Assay for characterization of human follicular oocyte maturation inhibitor using Xenopus oocytes

Human follicular fluid from healthy mature Graafian follicles and from pathologic ovarian cyst fluid was found to be inhibitory to progesterone-induced meiotic maturation of oocytes from the South African clawed toad, Xenopus laevis. Human follicular fluid but not human serum, collected from the same individuals, demonstrated a linear dose-response inhibition on the maturation of oocytes in the Xenopus assay system. These findings indicate that the human follicular and cyst fluids contain oocyte maturation inhibitor (OMI). This human OMI was inactivated when subjected to a boiling water bath for 2 min. The OMI action was shown to be reversible in its inhibitory action. The fact that OMI can act directly on the oocyte was demonstrated by its inhibitory action on maturation in defolliculated oocytes. The findings demonstrate that the inhibitory action of human OMI is not species-specific. Xenopus oocytes provide a simple, readily available, year-round bioassay material for testing follicular oocyte maturation inhibitor.     

Toxigenic fungi associated with stored corn

A total of 246 fungal isolates were obtained from 25 moldy corn samples collected in central Iowa. Either water or ether extracts of all corn samples except one exhibited some degree of toxicity in mice and/or ducklings. At least one toxigenic isolate was obtained from each corn sample. Extracts of cultures of 99 of the fungal isolates, involving 13 genera, produced death in one or more of the assay animals. The majority of the toxigenic isolates belonged to the generaAspergillus andPenicillium. Three isolates ofTrichothecium roseum were highly toxic to ducklings and mice. The ducklings exhibited a flaccid paralysis shortly after receiving an oral dose of extracts of these three isolates. Death frequently occurred following a second oral dose given 24 hr later. Mice given intraperitoneal injections of extracts ofT. roseum first exhibited lethargy and intermittent tremors, then hyperemotivity, roughening of the hair coat, abdominal respirations, incoordination, dyspnea and clonic convulsions with death occurring usually within 10 to 20 min.

---

Zusammenfassung Insgesamt sind 246 Pilzisolierungen von 25 verschimmelten Getreideproben, die in Zentral-Iowa gesammelt worden sind, erhalten worden. Sowohol Wasser- wie Aether-Extrakte von allen Getreideproben zeigten eine gewisse Toxizität in Mäusen als auch in Entchen außer einer einzigen Probe. Kulturextrakte von 99 der Pilzisolierungen, die 13 Gattungen umfaßten, verursachten den Tod in einem oder in mehreren Versuchstieren. Die Mehrheit der toxischen Isolierungen gehörte den GattungenAspergillus undPenicillium an. Drei Isolierungen vonTrichothecium roseum waren hoch toxisch für Entchen und Mäuse. Die Entchen zeigten eine schlaffe Paralyse kurz nachdem sie eine orale Dosis der Auszüge dieser drei Isolierungen erhalten hatten. Tod erfolgte häufig nach der zweiten oralen Dosis innerhalb 24 Stunden. Mäuse, die eine intraperitoneale Injektion der Auszüge vonT. roseum erhielten, zeigten erst eine Lethargie und einen intermittierenden Tremor, dann eine Hyperemotivität, aufgerauhte Haarbedeckung, abdominale Atmung, Inkoordination, Dyspnoe, klonische Zuckungen und Tod gewöhnlich innerhalb 10 bis 20 Minuten.

     

Biological effects of toxic products from Trichothecium roseum link

Extracts of rice cultures of 8 of 16 isolates ofTrichothecium roseum killed one or more ducklings or mice or both. One of the isolates (MC-156) obtained from shelled corn was the most toxigenic; extracts of this isolate killed all treated ducklings and mice.Doses of purified toxic fraction TR-1 of 166 mg/kg body weight given intraperitoneally killed all test mice but none of the mice given 100 mg/kg doses died. However, a partially purified fraction (Fraction VII), from which toxic fraction TR-1 was derived, killed two of three mice given 78 mg/kg doses.Crude ether extracts of rice cultures ofT. roseum (MC-156) produced death when injected intraperitoneally in rabbits and a 19-day-old pig and also produced dermal necrosis when applied to the skin of rabbits. The latter phenomenon was not observed when 5 mg of toxic fraction TR-1 was applied to the skin of rabbits.The greatest production of toxin occurred in rice cultures when incubated at 28° C and with 20 % (ml/g) water added to the rice. Incubation of rice cultures ofT. roseum (MC-156) under CO₂ tension suppressed toxin production.

---

Zusammenfassung Auszüge von Reiskulturen von acht der 16 Stämme vonTrichothecium roseum haben mehrere Entchen und/oder Mäuse getötet. Einer der Auszüge (MC-156) von abgeschälten Mais war das toxischste; Auszüge dieses Isolates hat alle Entchen und Mäuse getötet. Dosen der gereinigten toxischen Fraktion TR-1, 166 mg/Kg Körpergewicht, intraperitonial verabreicht, haben alle Mäuse getötet, aber keine der Mäuse starb mit der Dose von 100 mg/Kg. Jedoch hat eine teilweise gereinigte Fraktion (Fraktion VII), von welcher die toxische Fraktion TR-1 gewonnen war, zwei von drei Mäusen mit 78 mg/Kg Dose getötet. Ungereinigte Ätherauszüge vonT. roseum (MC-156) waren tödlich, wenn sie in Kaninchen, in ein 19 Tage altes Schweinchen intraperitonel injiziert worden sind. Sie haben auch eine Hautnekrose hervorgerufen, wenn sie auf die Haut von Kaninchen gebracht worden sind. Das letztere Phänomen war nicht beobachtet, wenn 5 mg der toxischen Fraktion TR-1 an Kaninchenhaut gebracht worden ist. Die größte Produktion des Toxins fand in Reiskulturen statt, wenn sie bei 28° C und mit 20 Perzent (ml/g) Wasser bebrütet worden sind. Inkubation von Reiskulturen vonT. roseum (MC-156) unter CO₂-Druck hat die Toxinproduktion unterdrückt.