Using pentosidine and hydroxyproline to predict age and sex in an avian species.

All living organisms are subject to senescence accompanied by progressive and irreversible physiological changes. The error damage and cross-linking theories suggest that cells and tissues are damaged by an accumulation of cross-linked proteins, slowing down bodily processes and resulting in aging. A major category of these cross-linked proteins are compounds called advanced glycation end products (AGEs). We investigated the relationship between accumulation of the AGE, pentosidine (Ps), and hydroxyproline (HYP) a post-translationally modified amino acid, with age, sex, and breeding status (breeder/nonbreeder) from skin samples of known age (i.e., banded as fledglings), free-ranging Double-crested Cormorants (Phalacrocorax auritus, Lesson 1831). We developed multivariate models and evaluated the predictive capability of our models for determining age and breeding versus nonbreeding birds. We found significant relationships with Ps and HYP concentration and age, and Ps concentration and sex. Based on our two-class model using Ps and HYP as explanatory variables, we were able to accurately determine whether a cormorant was a breeder or nonbreeder in 83.5% of modeled classifications. Our data indicate that Ps and HYP concentrations can be used to determine breeding status of cormorants and potentially age of cormorants although sex-specific models may be necessary. Although the accumulation of Ps explained the greatest amount of variance in breeding status and age, importantly, Ps covaried with HYP and combined improved prediction of these demographics in cormorants. Our data support the error damage and cross-linking theories of aging. Both Ps and HYP increase predictably in cormorants and are predictive of age and breeding status. Given the ubiquity of these biomarkers across taxa, their use in estimating demographic characteristics of animals could provide a powerful tool in animal ecology, conservation, and management.

An evaluation of the use of pentosidine as a biomarker for ageing turtles.

Concentrations of the biomarker pentosidine have been shown to be useful measures of age for a number of avian and mammalian species. However, no study has examined its usefulness as an age marker in a long-lived ectotherm despite the fact that such a marker could prove useful in understanding age distributions of populations subject to conservation programmes. Therefore, we evaluated pentosidine concentrations in the interdigital webbing of 117 female yellow mud turtles (Kinosternon flavescens) at a 35 year study site in western Nebraska where nearly all turtles are of known age. Pentosidine concentrations were extraordinarily low and positively correlated with age in this turtle, but concentrations were too variable to permit precise estimates of age for turtles of unknown age. These results may reflect the remarkable physiological adaptations of this turtle to low temperatures and oxygen deprivation in a highly seasonal environment requiring prolonged hibernation. Whether pentosidine concentrations in other ectotherms occupying less seasonal environments would be more highly correlated with age remains to be determined. However, our results suggest that patterns of accumulation of pentosidine in ectotherms may be fundamentally different from those in endotherms.

Feeding of grey squirrels (Sciurus carolinensis) with the contraceptive agent DiazaCon™: effect on cholesterol, hematology, and blood chemistry.

Grey squirrels (Sciurus carolinensis) are an invasive species in Britain and Italy. They have replaced native red squirrels (Sciurus vulgaris) throughout most of Britain, and cause damage to trees. Currently, lethal control is used to manage grey squirrel populations in Britain, but nonlethal methods might be more acceptable to the public. One such method is contraception with 20,25-diazacholesterol dihydrochloride (DiazaCon™). DiazaCon™ inhibits the conversion of desmosterol to cholesterol, resulting in increasing desmosterol concentrations and decreasing cholesterol concentrations. Because cholesterol is needed for the synthesis of steroid reproductive hormones, such as progesterone and testosterone, inhibition of cholesterol synthesis indirectly inhibits reproduction. Desmosterol is used as a marker of efficacy in laboratory studies with species that do not reproduce readily in captivity. Grey squirrels were gavaged with a DiazaCon™ solution for 2 days, and then fed DiazaCon™-coated peanuts for an additional 8 days at target doses of 50 and 100 mg DiazaCon™ per kg body weight. There was a significant difference in cholesterol concentrations in the treatment groups compared to the control group. Cholesterol was reduced by ≥ 40% for 2 months in both treatment groups. There were no differences among groups with respect to blood chemistry and hematology parameters, and mean values are reported. The mean overall dose of DiazaCon™ received was 29.0 ± 1.6 and 55.3 ± 4.3 mg/kg in the low (50 mg/kg) and high dose (100 mg/kg) groups, respectively. DiazaCon™ might provide an effective, acceptable alternative to lethal control.

Characterizing 3-chloro-p-toluidine hydrochloride on rough-hulled rice and ethyl-cellulose-coated rice baits using high-performance liquid chromatography.

Methods are developed to extract and quantitate the avicide 3-chloro-p-toluidine hydrochloride (CPT HCl) from rough-hulled rice and ethyl-cellulose-coated rice baits using high-performance liquid chromatography. The mobile phase used in the ethyl-cellulose-coated rice matrix method is an acetonitrile(ACN)-phosphate buffer (60:40) at pH 8, and the rough-hulled rice matrix method uses an CAN-phosphate (70:30) buffer at pH 2. Increased retention time is observed for CPT HCl at the higher pH. The two methods have been useful in characterizing different bait formulations in an ongoing pesticide formulation improvement program.

Development of a high performance liquid chromatography/mass spectroscopy method for the determination of strychnine concentrations in insects used to assess potential risks to insectivores.

A high performance liquid chromatography/mass spectrometry (HPLC/MS) method was developed in support of a study to assess potential tertiary risks posed to insectivores by strychnine baited pocket gophers (Thomomys sp.). Necropholous insects are primary consumers of pocket gopher carcasses. A field study was conducted to collect insects from strychnine-baited and control pocket gopher carcasses. The majority of the insects collected were from the orders Diptera (flies, assayed separately as adults and larvae), Coleoptera (beetles), and Hymenoptera (ants and wasps, assayed separately). Samples (0.5 g) were extracted in acetic acid (2%) and analyzed with the mass spectrometer configured for tandem mass spectrometry. For most of the samples the strychnine concentrations were less than the method limit of detection. However, strychnine concentrations as high as 0.338, 0.341, 0.698, and 0.034 microg/g were detected in ants, fly adults, fly larvae, and beetles, respectively. This information collected with the HPLC/MS method is critical for assessing potential non-target hazards for insectivores.

Desmosterol: a biomarker for the efficient development of 20,25-diazacholesterol as a contraceptive for pest wildlife.

20,25-Diazacholesterol is being evaluated as a contraceptive for the nonlethal control of avian and mammalian wildlife pests. The identification of an analyte in blood which was highly correlated with absorbed dose and efficacy is valuable for determining effective formulations and dosing variables. Such an analyte or biomarker is also valuable for determining the percentage of pest populations that consume an effective dose of the active ingredient in the field. HPLC analyses of blood collected from dosed animals failed to detect 20,25-diazacholesterol but indicated that levels of free cholesterol and related compounds were affected by 20,25-diazacholesterol absorption. The greatest percent change in chromatographic peak area associated with 20,25-diazacholesterol administration was observed for desmosterol, a cholesterol precursor. 20,25-Diazacholesterol appeared to block the conversion of desmosterol to cholesterol, resulting in an elevated concentration of the precursor. The elevation of blood desmosterol levels is being used as an indicator of 20,25-diazacholesterol absorption and to facilitate the development of a 20,25-diazacholesterol-based contraceptive for pest wildlife.