Parasite Infections Influence Immunological Responses But Not Reproductive Success of Male Hellbender Salamanders (Cryptobranchus alleganiensis)

Abstract

The emergence and spread of infectious diseases is a significant contributor to global amphibian declines, requiring increased surveillance and research. We assessed host-vector-parasite dynamics using a population of eastern hellbender salamanders (Cryptobranchus alleganiensis) that harbor leeches (Placobdella appalachiensis) that transmit endoparasitic blood parasites (Trypanosoma spp) to the host, with coinfection frequently occurring. We centered our study on adult males throughout their extended 8-month paternal care period because recent research indicates that nest failure caused by lack of paternal care and filial cannibalism is contributing to hellbender population declines. Recognizing the potential for parasites to modulate host physiology and behavior, we explored how infection severity influences paternal health and reproductive success. We assessed white blood cell profiles of adult male hellbenders in response to parasites, coinfection, and seasonal temperature fluctuations, while also investigating whether parasite infection or coinfection was predictive of nest success. We found that hellbenders exhibited seasonal shifts in white blood cell indices; as temperatures increased across seasons (from 5°C to 20°C), the proportion of neutrophils and eosinophils decreased (by 14% and 46%, respectively) in circulation while the proportion of lymphocytes and basophils increased (by 8% and 101%, respectively). Moreover, the proportion of neutrophil precursors increased by 80% under colder temperatures, which signifies seasonal immune cell recruitment. We demonstrated that neutrophils and eosinophils increased while lymphocytes decreased in response to leech infection. However, as leech and trypanosome infection intensity increased together, the proportion of lymphocytes increased while neutrophils and eosinophils decreased, underscoring the complex interactions between coinfection and immune responses of hellbenders that warrant future research. Despite the influence of infection and coinfection on hellbender physiology, we detected no evidence to support the hypothesis that parasites influence the likelihood of nest failure or whole-clutch filial cannibalism. In light of amphibian declines being exacerbated by climate change and disease, our study emphasizes the need to establish hematological reference values that account for physiological adaptations to seasonal fluctuations in temperature and different life history stages and to study the physiological responses of imperiled amphibian species to parasites.

Fig. 1

Microscopic images of hellbender WBCs and trypanosomes. Images include (A) two eosinophils (pink staining) alongside a thrombocyte, (B) a basophil (arrow) next to a polychromatic (immature) red blood cell and mature red blood cells, (C) a healthy mature neutrophil (arrow) next to partial mature red blood cells, (D) a lymphocyte (arrow) adjacent to partial mature red blood cells and polychromatic red blood cells, (E and F) mature neutrophils (arrow) with toxic granulation with partial mature red blood cells, (G) band neutrophil (arrow) with visible secondary granules, (H) band neutrophil (arrow) with visible secondary granules, alongside a lymphocyte and mature red blood cells, (I–K) trypanosomes from buffy coat smears, and (L) a trypanosome from whole blood smear alongside mature and polychromatic (young) red blood cells. Images were taken on a compound light microscope at 400× magnification. The black scale bar represents 20 µm.

Fig. 2

PCA biplot illustrating the relationships among WBC types in adult male hellbenders during the 8-month paternal care period. PC1 explained 50.4% of the variance and was positively loaded with neutrophils (0.616) and eosinophils (0.374) but negatively loaded with lymphocytes (−0.687). PC2 explained 30.7% of the variance and was positively loaded with basophils (0.749) and neutrophils (0.279) but negatively loaded with eosinophils (−0.578) and lymphocytes (−0.166). Arrows represent WBC loadings, with length indicating the strength of their influence, while points represent raw data points.

Fig. 3

The relationship between temperature and the PC scores for WBC profiles in adult male hellbenders across the 8-month paternal care period. (A) The relationship between stream temperature (°C) and WBC PC1, which explained 50.4% of the variance and was positively loaded with neutrophils (0.616) and eosinophils (0.374), but was negatively loaded with lymphocytes (−0.687) (B) The relationship between stream temperature (°C) on WBC PC2, which explained 30.7% of the variance and was positively loaded with neutrophils (0.279) and basophils (0.749) but was negatively loaded with lymphocytes (−0.166) and eosinophils (−0.587). Lines represent model predictions from the top-ranking models using AICc, while points represent PC scores.

Fig. 4

The relationship between the abundance of parasites and WBC PC1 in hellbenders, demonstrating the interactive effects between leeches and trypanosomes. Trypanosomes represent the total number of trypanosomes counted within 50 random fields of view across duplicate buffy coat slides while leeches represent the number of leeches attached to the individual at the time of capture. Corresponding to our results from the top-ranked model using AICc, as the size of the circles (leech abundance) increases, PC1 values exhibit a positive shift. However, as the size of the circles increases and increases in color (trypanosome abundance), PC1 values exhibit a negative shift.

Fig. 5

The relationship between temperature and the proportion of band neutrophils in adult male hellbenders across the 8-month paternal care period. The line represents model predictions from the top ranking model using AICc, while the points represent observed values.

Fig. 6

Seasonal differences in the proportion of toxic neutrophils across the 8-month parental care period of hellbenders. Box plots represent the distribution of model predictions from the top-ranking models using AICc while the points represent raw data. Letters in superscript indicate statistical differences in the proportion of toxic neutrophils across the parental care period (Supplemental Table 2). Sampling day represents repeated capture intervals corresponding to offspring development in which oviposition represents nest initiation (Day ∼0), mid-embryonic represents mid-embryonic development (Day ∼30), hatching represents larval hatching (Day ∼60), and emergence represents spring larval emergence (Day ∼200).

Fig. 7

The relationship between parasitic infection and fate of hellbenders nests. (A) The distribution of nest outcomes in response to the abundance of leeches during oviposition. (B) The distribution of nest outcomes in response to the abundance of trypanosomes during oviposition. Box plots represent the relative distribution of parasite abundance within the categorized nest outcome and points indicate raw data. Trypanosome abundance represents the total number of trypanosomes observed within 50 random fields of view on buffy coat smears. Cannibalism represents the nest fate category in which nest failure was confirmed as whole-clutch cannibalism. While all categories of nest outcomes are expressed in the figure, statistical analyses were performed separately to evaluate whether parasite infection influenced the likelihood of success versus failure and success versus whole-clutch cannibalism.