Genetic variation and a fitness tradeoff in the tolerance of gray treefrog (Hyla versicolor) tadpoles to the insecticide carbaryl.

One of the major unanswered questions in the study of global amphibian declines is why only some species or populations suffer declines. A possible explanation is that species and populations vary in the genetic basis of their tolerance to environmental stress such as chemical contamination. The presence of genetic variation in tolerance to chemicals and in fitness traits of amphibians is essential for persistence of species populations through survival and successful reproduction in contaminated environments. We tested for the presence of genetic variation in the tolerance of amphibian larvae to the insecticide carbaryl using gray treefrog tadpoles (Hyla versicolor). We also assessed whether tolerance of tadpoles is negatively associated with larval performance traits directly related to adult fitness, thereby providing a test of the "cost of tolerance" hypothesis. Our results demonstrate significant variation in tolerance of tadpoles to the insecticide carbaryl within a single population of the gray treefrog, Hyla versicolor. Our half-sibship design indicates that variation among sires explains a significant amount of the variation in chemical tolerance thereby suggesting a heritability genetic basis. Our results also indicate the presence of a fitness tradeoff with tolerance to the chemical carbaryl being negatively correlated, or traded off, with survival of tadpoles reared in the field in the absence of the chemical. Knowledge of genetic tradeoffs with chemical tolerance under realistic environmental conditions will be important for predicting the rate of adaptation and potential for persistence of species. Finally, the partitioning of environmental and genetic variation in tolerance to chemicals is critical to identifying which species are most susceptible, the amount of genetic variance present, the potential for adaptation to contaminants, and the presence of fitness tradeoffs. Such information is necessary to clearly understand the persistence of populations, and ultimately, the processes leading to species declines.

Call duration as an indicator of genetic quality in male gray tree frogs.

The "good genes" hypothesis predicts that mating preferences enable females to select mates of superior genetic quality. The genetic consequences of the preference shown by female gray tree frogs for long-duration calls were evaluated by comparing the performance of maternal half-siblings sired by males with different call durations. Offspring of male gray tree frogs that produced long calls showed better performance during larval and juvenile stages than did offspring of males that produced short calls. These data suggest that call duration can function as a reliable indicator of heritable genetic quality.

Autoimmune effector cells. I. Transfer of experimental encephalomyelitis with lymphoid cells cultured with antigen.

Relatively low numbers of spleen and lymph node cells from Lewis rats previously challenged with myelin basic protein efficiently transfer experimental allergic encephalomyelitis (EAE) to normal syngeneic recipients after in vitro culture with antigen. Moreover, cells obtained from rats that have recovered and are resistant to EAE can also transfer disease. Cell separation studies show that a nylon wool-adherent cell is responsible for transfer of EAE. Density gradient ultracentrifugation revealed that these effector cells are probably lymphoblasts.

Regulation of experimental allergic encephalomyelitis. II. Appearance of suppressor cells during the remission phase of the disease.

Lewis rats are susceptible to experimental autoimmune encephalomyelitis (EAE). Most rats recover from paralysis and are subsequently resistant to the disease. In an adoptive transfer system, we found that lymph node cells (LNC) from rats that had recovered from EAE protect syngeneic recipients from the disease when the latter are challenged with encephalitogenic myelin basic protein and adjuvant after receiving donor cells. Suppression is antigen-specific and requires viable LNC. In contrast to the suppressor cells we previously studied in tolerized rats, which were nonadherent T lymphocytes, the suppressor cells found in rats that have recovered from EAE adhere to glass wool. However, they are not retained on Sephadex G-10 columns to which macrophages adhere. Suppressor activity is enriched in the nylon wool-adherent LNC population (which consists of approximately 80% Ig+ cells). Our findings suggest that activation of adherent suppressor cells may be implicated in recovery from EAE. These may be adherent T cells, or B cells that produce anti-BP blocking antibodies.

Some factors affecting mutant numbers in the Salmonella/microsome assay.

An examination has been made of some of the parameters which can affect mutant numbers in the Salmonella/microsome assay. The type of minimal media plates used for the assay and the concentration of glucose-6-phosphate, one of the co-factors necessary for mono-oxygenase action, had no effect on mutant numbers. Increases in mutated bacteria resulted from the use of (1) log-phase bacteria, (2) higher NADP concentrations than those normally recommended, and (3) higher phosphate buffer concentrations. Six mutagens, i.e., 2-acetylaminofluorene (AAF), 3,3'-dichlorobenzidine (3,3'-DCB), cyclophosphamide (CY), aflatoxin B1 (AFB1), 3-methylcholanthrene (3MC) and benzo[a]pyrene (BP), all requiring mono-oxygenase activation, were studied with two Salmonella typhimurium strains, TA98 and TA100, and liver preparations from rats given different inducing agent treatments using optimum conditions. Phenobarbitone induction was generally superior to Aroclor-1254 in converting these substrates to mutagens except for the polycyclic hydrocarbon substrates. A comparison of 3-methylcholanthrene, Aroclor-1254, beta-naphthoflavone or phenobarbitone as inducing agents revealed the first three of these to be equally effective in activating BP or 3MC to mutagens, whereas phenobarbitone was less active. Dual administration of 3-methylcholanthrene and phenobarbitone to rats did not result in an additive mutagenic effect using AAF, AFB1 or 3,3'-DCB as substrates, the numbers of mutant bacteria obtained being only equal to that seen with 3-methylcholanthrene alone. These differences were not due to there being different liver protein optima for the various inducing agent treatments. The foregoing results are discussed in relation to attempts to draw up a rigid protocol for mutagenicity testing.

Biological and chemical studies on 8,9-dihydroxy-8,9-dihydro-aflatoxin B1 and some of its esters.

A number of aflatoxin B1 (AFB1) derivatives have been synthesized including 8-acyloxy- and 8-benzoyloxy-9-hydroxy-8,9-dihydro-AFB1 compounds, AFB1-8,9-diol and [3H] AFB1 labelled at the 9 position. AFB1-hydroxyesters appear to be models of AFB1-8,9-oxide in that they are bacterial mutagens, stimulate unscheduled DNA synthesis in HeLa cells and react with DNA to give trans-8,9-dihydro-8(7-guanyl)-9-hydroxy-AFB1 as the major adduct after hydrolysis. The potency of the hydroxyesters increases with ease of release of the ester grouping at position 8. Absence of the hydroxyl at position 9 gives compounds which are readily hydrolysed in water but are not biologically active. The hydroxyesters hydrolyse in water to give AFB1-diol, providing a convenient means of synthesis of this compound. Studies with AFB1-diol show that it reacts with one molecule of Tris base, probably through the ring-opened furan form, with the amino group of the Tris. Acidification results in ring closure in an analogous manner to AFB1-diol. AFB1-diol binds to DNA in vitro as well as to liver slice DNA. The compound is mutagenic towards S. typhimurium TA100 without metabolic activation. The implications of these findings are discussed in relation to the mechanisms of AFB1 carcinogenicity.