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In this project, red-backed salamanders, Plethodon cinereus, were collected from the heart of their range (Mountain Lake, Virginia, USA), in an effort to document the typical, or baseline, leukocyte profile of this species in its natural state. From microscopic examination of thin blood films, we determined relative proportions of all leukocyte types and determined the ratio of neutrophils to lymphocytes, which is a useful index of stress levels. In addition, individuals were examined for three naturally occurring endoparasites: two gastro-intestinal (trematodes and a ciliated protozoan in the genus Cepedietta) and one in the blood (a Rickettsial bacteria). This allowed us to conduct statistical comparisons of leukocyte data across parasite infection groups. Of 36 salamanders, 11 (31%) had trematode parasites, 7 (19%) contained Cepedietta parasites, 6 (17%) were parasitized by Rickettsia, and 12 had no parasites. The most common leukocytes were lymphocytes (87.4%), followed by neutrophils (4.2%), monocytes (3.3%), eosinophils (2.6%), and basophils (2.5%). The average neutrophil/lymphocyte ratio for these salamanders was remarkably low (0.05) for an amphibian population. Statistical comparisons of leukocyte profiles among parasite infection groups indicated parasitized salamanders had twice as many circulating leukocytes as nonparasitized individuals, which could be considered an "inflammation response". There was a slight but significant elevation in relative eosinophil abundance in parasitized salamanders. There were few differences in leukocyte subsets between parasite types, nor was there evidence of any proliferation of phagocytic cells in any parasitized individual. [ABSTRACT FROM AUTHOR]

Abstract: Measuring stress in animals is important in many ecological, zoological and veterinary research settings. A common method is to measure plasma levels of glucocorticoid hormones (cortisol, corticosterone, hereafter CORT). Over the past decade, an alternative method has become widely popular: assessing leucocyte profiles; in other words, the heterophil‐to‐lymphocyte ratio (H/L) in birds and reptiles or the neutrophil‐to‐lymphocyte ratio (N/L) in mammals, amphibians and fish. Recent studies have shown that although both indices reliably change after acute stress, they are not always correlated at baseline, and thus are not interchangeable. This lack of correlation has led to some confusion about which of these two measures is “better”; in other words, more biologically relevant and a truer readout of stress. In this review, we first document the dramatic expansion of the use of leucocyte profiles to assess stress over the past decade. We then review published evidence that may explain the discrepancies between leucocyte profiles and plasma CORT. We argue that these discrepancies stem from the nature of the stress reaction and the role of corticosterone (or cortisol) versus leucocytes during the stress response. The differing roles of the two responses translate to differences in the timing of each, which is important for researchers studying either acute or chronic stress. Although plasma glucocorticoids rise within minutes of the onset of acute stress and return to baseline within 1 or 2 hr after the stressor passes, H/L or N/L ratios remain low for at least 30–60 min or sometimes more. The temporal nature of these metrics differs also in the context of long‐term chronic stress. The glucocorticoid response wanes in repeatedly stressed animals, or those exposed to chronic environmental stress, which can often (but not always) lead to low baseline levels and more typically, an attenuated acute stress response. Meanwhile, H/L or N/L ratios appear to remain high under the same conditions. Understanding the temporal differences in the responses should help researchers decide which metric is the better method in their particular study, or whether both would be useful for elucidating different elements of the stress response. [ABSTRACT FROM AUTHOR]

The article presents a study on the impact of larval density on physiological stress of postmetamorphic individuals, particularly salamanders, using a hematological approach. It states that larval density is a factor that governs the quality of larval environments in amphibians wherein environments with high densities could lead to the reduction of larval growth rates, smaller size of metamorphosis and long-term postmetamorphic effects. It indicates that salamanders from high-density treatments had higher neutrophil to lymphocyte ratios and shows that estimated stress levels of salamanders' stress hormones were similar in any of the animals' metamorphosis case.

Measuring stress in animals is an important component of many research studies, and it has traditionally been performed via sampling levels of corticosterone in plasma. A secondary, "hematological" approach used most commonly by researchers of birds, mammals, and other taxa involves evaluating leukocyte profiles from blood smears. Such research has shown that leukocytes have a characteristic response to stress, although in amphibians this phenomenon is not as well studied. In general, stress can induce a rise in the ratio of neutrophils to lymphocytes. We evaluated the hematological response of paedomorphic Mole Salamanders (Ambystoma talpoideum) to captivity stress, specifically focusing on this parameter, but also examining other white blood cell types. Individuals captured in the wild and held in captivity for ten days before sampling had significantly more neutrophils, fewer lymphocytes, and higher ratios of neutrophils to lymphocytes than those captured from the same locations and sampled within one hour. Captive individuals also had significantly higher numbers of eosinophils. These results are consistent with hematological research in birds and other taxa and highlight the utility of this approach for measuring stress in amphibians. [ABSTRACT FROM AUTHOR]

Reproduction in amphibians is stressful and perhaps more so in females than males because of the higher energetic costs of producing eggs than sperm. The ratio of two white blood cells, neutrophils and lymphocytes, has been shown in birds, turtles, and amphibians to increase with stress. We captured breeding and nonbreeding, paedomorphic Mole Salamanders (Ambystoma talpoideum), to determine whether the stress of reproduction is reflected by neutrophil:lymphocyte ratios in this species. In blood smears of all individuals, we observed approximately 25.5 leukocytes per 1000 erythrocytes, with 13% of the leukocytes being neutrophils, 41% lymphocytes, and 46% eosinophils. Less than 1% of leukocytes were basophils and monocytes. Neutrophil:lymphocyte ratios of gravid females were significantly higher (and also more variable) than those of reproductive males and of nonreproductive individuals, indicating a higher degree of physiological stress in reproductive females. Reproductive males did not have higher ratios than nonreproductive individuals. We found no effect of body size on neutrophil:lymphocyte ratios. Our hematological stress results are consistent with studies of other amphibians where different methods were used and with other taxa. [ABSTRACT FROM AUTHOR]