Discovery of the first sea turtle adenovirus and turtle associated circoviruses.

Turtles are an evolutionarily unique and morphologically distinctive order of reptiles, and many species are globally endangered. Although a high diversity of adenoviruses in scaled reptiles is well-documented, turtle adenoviruses remain largely understudied. To investigate their molecular diversity, we focused on the identification and characterisation of adenoviruses in turtle-derived organ, swab and egg samples. Since reptile circoviruses have been scarcely reported and no turtle circoviruses have been documented to date, we also screened our samples for circoviruses. Host-virus coevolution is a common feature of these viral families, so we aimed to investigate possible signs of this as well. Two screening projects were conducted: one on Brazilian samples collected from animals in their natural habitat, and the other on Hungarian pet shop samples. Nested PCR systems were used for the detection of adeno- and circoviruses and purified PCR products were Sanger sequenced. Phylogenetic trees for the viruses were reconstructed based on the adenoviral DNA polymerase and hexon genes, circoviral Rep genes, and for the turtle hosts based on mitochondrial cytochrome b amino acid sequences. During the screening, testadeno-, siadeno-, and circovirus strains were detected. The circovirus strains were classified into the genus Circovirus, exhibiting significant evolutionary divergence but forming a monophyletic clade within a group of fish circoviruses. The phylogenetic tree of turtles reflected their taxonomic relationships, showing a deep bifurcation between suborders and distinct monophyletic clades corresponding to families. A similar clustering pattern was observed among the testadenovirus strains in their phylogenetic tree. As a result, this screening of turtle samples revealed at least three new testadenoviruses, including the first sea turtle adenovirus, evidence of coevolution between testadenoviruses and their hosts, and the first turtle associated circoviruses. These findings underscore the need for further research on viruses in turtles, and more broadly in reptiles, to better understand their viral diversity and the evolutionary processes shaping host-virus interactions.

Comparison of four different protocols using levobupivacaine for spinal anaesthesia in red-footed tortoises (Chelonoidis carbonarius).

Background: The popularity of tortoises kept in captivity is increasing and has caused concern regarding the necessity to establish safe and straightforward anaesthesia for those reptiles. Aim: This study aimed to compare four protocols using levobupivacaine in spinal anaesthesia for the blockade of the caudal neuraxis of red-footed tortoises (Chelonoidis carbonarius). Methods: Twenty-four tortoises were randomly assigned into four groups: G1, levobupivacaine 0.75% (1.15 mg kg-1); G2, levobupivacaine 0.37% (1.15 mg kg-1); G3, levobupivacaine 0.75% (2.3 mg kg-1); and G4, levobupivacaine 0.75% (0.1 ml 5 cm-1 of straight carapace length). Tortoises were evaluated for respiratory rate, muscle relaxation, response to hindlimb or tail pinch, and cloacal reflex. Results: A 1.15 mg kg-1 dose of levobupivacaine 0.37% appears adequate for shorter procedures, whereas a 1.15 mg kg-1 dose of levobupivacaine 0.75% should be appropriate for longer procedures in red-footed tortoises. Conclusion: Our results are the first to show the effects of levobupivacaine on spinal anaesthesia in reptiles. Weight-based doses presented more intense and more homogeneous effects than carapace length-based doses in red-footed tortoises. Spinal anaesthesia of red-footed tortoises was safe and effective with any of the weight-based protocols.

Shell biomechanics suggests an aquatic palaeoecology at the dawn of turtle evolution.

The turtle shell is a remarkable structure that has intrigued not only evolutionary biologists but also engineering and material scientists because of its multi-scale complexity and various functions. Although protection is its most apparent role, the carapace and plastron are also related to many physiological functions and their shape influences hydrodynamics and self-righting ability. As such, analysing the functional morphology of the shell could help understanding the ecology of Triassic stem-turtles, which will contribute to the century-long debate on the evolutionary origins of turtles. Here, we used 3D imaging techniques to digitize the shells of two of the earliest stem-turtle taxa, Proganochelys and Proterochersis, and submitted their models to biomechanical and shape analyses. We analysed the strength performance under five predation scenarios and tested the function of two morphological traits found in stem-turtles, the epiplastral processes and an attached pelvic girdle. The latter, also present in the crown-lineage of side-necked turtles, has been suggested to increase load-bearing capacity of the shell or to improve swimming in pleurodires. Our results do not confirm the shell-strengthening hypothesis and, together with the results of our shape analyses, suggest that at least one of the first stem-turtles (Proterochersis) was an aquatic animal.

Lung mechanics in juvenile and adult Chelonoidis carbonarius (Spix, 1824).

Testudines possess a rigid shell that influences the mechanics of the respiratory system. We studied respiratory mechanics in the terrestrial red-footed tortoise Chelonoidis carbonarius (Cryptodira), comparing juvenile individuals with a less ossified and more flexible carapace to adults with a well-ossified rigid shell. Combined with these ontogenetic differences, we analyzed respiratory system mechanics with animals in a supine and a prone position, as well as in the isolated lungs, to evaluate the impact of the viscera onto breathing mechanics. To do so, we used established protocols to measure pulmonary volumes (i.e., resting, VLr; and maximum, VLm), static (Cstat) and dynamic (Cdyn) compliances, and the work of breathing (W). We observed that isolated lungs displayed increased VLr, VLm, Cstat,Cdyn and decreased W. Additionally, pulmonary volumes, compliances, and W were affected by evaluated positions, such as a smaller VLr in a supine position. Cdyn and W showed a volume dependency while frequency had less influence on these variables. At similar levels of ventilation, juveniles showed a lower W than adults when standardized by body mass, but similar W when standardized by VLr. Clear ontogenetic changes could be observed in breathing mechanics between juvenile and adult C. carbonarius. While these differences might largely be explained by variations in shell ossifications, other explanations such as differences in visceral proportions or developmental degree of the post-pulmonary septum should also be taken into account.

Epidemiological survey of gastrointestinal helminths and protozoa in Testudines from Sardinia, Italy.

The order Testudines comprises some of the most endangered groups of vertebrates. Under specific circumstances, infectious and parasitic diseases may affect the survival and fecundity of these animals, potentially threatening Testudines populations. In Sardinia, besides the three species of tortoises present in the wild (Testudo hermanni, Testudo graeca and Testudo marginata), many others are kept as pets. However, epidemiological studies on these animals have not been conducted so far. Thus, the aim of the work was to investigate the presence of gastrointestinal parasites in captive and wild tortoises of Sardinia, Italy, with particular regard to those of zoonotic importance. For the 215 animals examined (n = 36 wild caught and n = 179 private-owned), fecal samples were collected and processed by flotation and modified Ziehl-Neelsen technique. An overall prevalence of 81.4% for endoparasites was detected, with oxyurids being the most prevalent (74.4%), followed by Nyctotherus spp. (18.6%), Cryptosporidium spp. (12.6%), Angusticaecum spp. (2.8%), strongyles (0.9%), Balantidium spp. (0.9%), coccidia (0.9%), cestodes (0.5%), and Giardia spp. (0.5%). Data suggest that tortoises are affected by a great variety of endoparasites, and further molecular analysis are required to assess the impact of Cryptosporidium and Giardia species in these hosts. Therefore, regular health screenings are of importance for the management of these animals and for preventing emerging infectious diseases.

Micronucleus test in reptiles: Current and future perspectives.

Micronucleus (MN) cell counting emerged in 1973-1975 as a valid alternative for characterizing chromosomal damage caused by different agents. It was first described in mammals, but its application was rapidly extended to other vertebrates, mainly fish. However, it was not until 28 years later that this test was implemented in studies on reptiles. Nowadays, reptiles are found to be excellent non-target species from environmental contamination exposure and MN test has become a fundamental tool for analyzing genotoxic effects induced by various xenobiotics. In this article we provide an updated review of the application of the MN test in reptile species, from an ecotoxicological perspective. Therefore, we present (I) a bibliometric analysis of the available research on genotoxic-induced MN formation in reptile species; (II) the use of reptiles as sentinel organisms in ecotoxicological studies; and (III) the strength and weakness of the application of the MN test in this group. With this review, we aim to provide a comprehensive view on the use of the MN test in ecotoxicology and to encourage further studies involving reptile species.

Blood Fluke Infection (Spirorchidiasis) and Systemic Granulomatous Inflammation: A Case Study of Green Sea Turtles (Chelonia mydas) on Jeju Island, South Korea.

Despite the precarious state of marine turtles as a highly endangered species, our understanding of their diseases remains limited. This case report presents a detailed pathological investigation of spirorchiidiasis, a blood fluke infection that poses a substantial threat to marine turtles. This retrospective study examined three cases of spirorchiid-infected sea turtles, specifically, green sea turtles stranded on Jeju Island, South Korea. Premortem examination of the three spirorchiid-infected green sea turtles demonstrated nonspecific clinical symptoms; blood analysis revealed dehydration, malnutrition, and anemia. Computed tomography scans provided insights into severe pulmonary and extrapulmonary manifestations, including the mass present in the joint region. Post-mortem examinations consistently indicated severe lung lesions and systemic manifestations, with histopathological examination confirming the presence of spirorchiid ova across various organs. Despite the global prevalence of spirorchiidiasis in sea turtles, disease severity varies regionally. This report provides a detailed demonstration of the pathology of spirorchiidiasis in sea turtles from Northeast Asia.

Postcranial anomalies of Eocene freshwater pleurodiran and cryptodiran turtles from the Spanish Duero Basin.

Testudines are one of the best-represented taxonomic groups among the Paleogene taxa of the Duero Basin (Castile and Leon Autonomous Community, central Spain). Among them, Neochelys (Podocnemidide) and Allaeochelys (Carettochelyidae) are most abundant, allowing the population to be assessed for osteological anomalies. The abundance of postcranial remains of both taxa allows us to identify several individuals with potential anomalies, mostly in their shells. Some of them have already been described in previous studies, but most of them are still unpublished. The objective of this study is to analyze in detail the anomalous Neochelys and Allaeochelys remains. As a result, different categories of causal agents (such as bacteria, fungi, parasites, or trauma) have been identified as potential producers of the anomalies in these freshwater turtles. Information regarding the pathogenesis and healing stages of some of these anomalies is provided.

A bothremydid turtle (Pleurodira) from the middle Cenomanian of Vale de Figueira (Belas, Portugal).

An unpublished turtle shell from the middle Cenomanian of Vale de Figueira, near Belas (Lisbon District, Portugal), is recognized by us as collected in 1880 under the direction of Carlos Ribeiro. No turtle remains from that region had so far been figured, described or discussed from a systematic point of view. The specimen corresponds to a partial but articulated shell. It is attributed to Pleurodira and, more specifically, to Bothremydidae. Only one pre-Campanian turtle specimen was previously identified at the infrafamilial level in the Cretaceous record of Portugal. It was a partial shell from the middle Cenomanian of Nazaré (Leiria District) attributed to the bothremydid Algorachelus peregrina, a species defined in a Spanish synchronous locality (Algora, in Central Spain). Several anatomical regions in the specimen from Vale de Figueira were not preserved in the partial carapace from Nazaré, and differences in the morphology of some elements are recognized between both shells. However, the individual studied here is also ascribed to Algorachelus peregrina, these differences being justified by intraspecific variability. Therefore, the specimen represents the second evidence on the species in Portugal, being the only one recognized for the Lisbon District.

The latest freshwater giants: a new Peltocephalus (Pleurodira: Podocnemididae) turtle from the Late Pleistocene of the Brazilian Amazon.

Overkill of large mammals is recognized as a key driver of Pleistocene megafaunal extinctions in the Americas and Australia. While this phenomenon primarily affected mega-mammals, its impact on large Quaternary reptiles has been debated. Freshwater turtles, due to the scarcity of giant forms in the Quaternary record, have been largely neglected in such discussions. Here we present a new giant podocnemidid turtle, Peltocephalus maturin sp. nov., from the Late Pleistocene Rio Madeira Formation in the Brazilian Amazon, that challenges this assumption. Morphological and phylogenetic analyses of the holotype, a massive partial lower jaw, reveal close affinities to extant Amazonian species and suggest an omnivorous diet. Body size regressions indicate Pe. maturin possibly reached about 180 cm in carapace length and is among the largest freshwater turtles ever found. This finding presents the latest known occurrence of giant freshwater turtles, hinting at coexistence with early human inhabitants in the Amazon.

Traces of oil in sea turtle feces.

In 2019, an oil spill hit the Brazilian Northeast coast causing impact to several ecosystems, including sea turtles' breeding and feeding areas. This study aimed to investigate whether sea turtles were impacted by this oil disaster, correlating the oil found inside feces with a sandy-oiled sample collected on the beach some days after the accident. The fecal samples were collected in the upper mid-littoral reef areas during three consecutive days in February 2020. The results suggested that sea turtles consumed algae contaminated by petroleum. Hydrocarbons composition of oil inside feces was similar to the sandy-oiled sample, suggesting they were the same. Lighter aliphatic and polycyclic aromatic compounds were missing, indicating both sandy-oiled and oil inside the feces had experienced significant evaporation prior to collection. Although the long-term damage is still unknown, the data are novel and relevant to support future research and alert authorities about the risks to sea turtles.

Survey of parasites in sea turtles rescued off the coast of Santa Catarina and Paraná, Brazil (2020-2022) and their relationship with marine pollution.

The identification of parasite fauna can contribute to the assessment of the health status of sea turtles, and thus make it possible to develop effective management and treatment strategies aimed at conserving these animals. The present study aimed to investigate the parasite fauna in sea turtles rescued in six sections of the coast of Santa Catarina and Paraná (Brazil) from January 2020 to December 2022 through the recording of exam reports available in the Information System of Aquatic Biota Monitoring. In total, records of 334 sea turtles were analyzed during this period, of which 219 (65.6 %) were infected with at least one parasite. Chelonia mydas was the most frequent species with 86.5 % of rescues and presented the highest species richness. At least 43 species belonging to 36 different genera have been recorded parasitizing sea turtles. The most common helminths were Cricocephalus albus, Metacetabulum invaginatum, Pronocephalus obliquus, with 12.3 %, 8.7 % and 8.4 %, respectively, while protozoa of the genus Entamoeba were the most prevalent in the turtles analyzed (8.1 %). An interesting finding was the unprecedented finding of Hymenolepis sp. eggs in faeces of some turtle species. The results showed that the parasite fauna was quite diverse, with several species known to be capable of harming the physical health and well-being of sea turtles. In view of this, it is necessary to adopt strategies for monitoring the health of the different rescued species with a view to conserving sea turtles in Brazilian territory.

Gonadal Morphology of Stillborn Hawksbill Sea Turtle (Eretmochelys imbricata) Hatchlings.

This study aimed to investigate sexual dimorphism in stillborn hawksbill sea turtles (Eretmochelys imbricata) through gonadal morphological characterizations. Macroscopic, light microscopy, and transmission electron analyses were performed for 30 gonad-mesonephros complexes. Female gonads were spindle-shaped and present a translucent whitish appearance with a grainy texture. Male gonads were approximately ovoid with a smooth opaque white surface. A primary sexual difference concerns different marrow structures, with females presenting organized cellularity featuring oocytes, lacunae, and blood vessels, while males presented a distinct organizational medulla pattern marked by testicular cords extending throughout the gonad length. Ultrastructurally, female's stroma presented interstitial cells and an abundant cytoplasm rich in electrodense droplets and large oval germline cells, with a conspicuous and noncentral nucleus. Males, on the other hand, presented testicular cord cells containing small amounts of heterochromatin and approximately triangular apical and basal cytoplasms with an evident nucleolus characteristic of support cells. Additionally, there were cells with a large spherical nucleus compared with the cell size and a relatively scarce cytoplasm, identified as gonocytes. These findings indicate that macroscopic, microscopic, and ultrastructural evaluations are effective and reliable techniques for the sexual identification of stillborn E. imbricata hatchlings.

The first full body diffusible iodine-based contrast-enhanced computed tomography dataset and teaching materials for a member of the Testudines.

Diffusible iodine-based contrast-enhanced Computed Tomography (diceCT) is now a widely used technique for imaging metazoan soft anatomy. Turtles present a particular challenge for anatomists; gross dissection is inherently destructive and irreversible, whereas their near complete shell of bony plates, covered with keratinous scutes, presents a barrier for iodine diffusion and significantly increases contrast-enhanced CT preparation time. Consequently, a complete dataset visualizing the internal soft anatomy of turtles at high resolution and in three dimensions has not yet been successfully achieved. Here we outline a novel method that augments traditional diceCT preparation with an iodine injection technique to acquire the first full body contrast-enhanced dataset for the Testudines. We show this approach to be an effective method of staining the soft tissues inside the shell. The resulting datasets were processed to produce anatomical 3D models that can be used in teaching and research. As diceCT becomes a widely employed method for nondestructively documenting the internal soft anatomy of alcohol preserved museum specimens, we hope that methods applicable to the more challenging of these, such as turtles, will contribute toward the growing stock of digital anatomy in online repositories.

Blood recovery of wild Mekong snail-eating turtles (Malayemys subtrijuga Schlegel and Müller, 1845) in captivity from leech infestation.

Blood cell counts are valuable diagnostic tools for assessing the health status of chelonians, however, reference standards for healthy blood parameters in various turtle species are lacking. In this study, forty wild female Malayemys subtrijuga were captured from ponds in Kasetsart University, and transported to laboratory for recuperating in captivity. All turtles were infected with a single leech species, Placobdelloides siamensis, with a mean of 513.7 ± 164.9 individuals per turtle, and exhibited penetrating and lesion wounds from leech infestations on both their skin and shell. Subsequently, they were cleaned and treated to eliminate ecto- and endoparasites before the recuperation period began. The turtles did not exhibit significantly differences in weight, carapace length (CL), red blood cell count (RCC), and white blood cell count (WCC) with a mean of 654.2 ± 199.9 g, 15.0 ± 2.5 cm, 327,080 ± 70,156 cells/mm3, and 73,340 ± 15,859 cells/mm3, respectively, during the initial records (week 0). However, after being maintained for 17 weeks, their health significantly improved in term of their blood parameters (RCC and WCC) and weight, except CL which remained unchanged, with a mean of 491,470 ± 16,169 cells/mm3, 18,790 ± 1496 cells/mm3, and 738.9 ± 191.5 g, respectively. Therefore, the health status obtained in this study can be used as a reference for blood parameters, weight, and recuperation period for the treatment of ill wild M. subtrijuga in captivity or as part of conservation management programs for turtles.

Detection of Hemopathogens in Chelonoidis carbonaria: Microscopic, Molecular, Hematological, and Clinical Biochemistry Aspects.

Background: The growing contact between men and wild animals, caused by the increase in the population in urban centers and the destruction of the habitat of these animals, has been leading to a greater circulation of pathogens between humans and wildlife. Chelonoidis carbonaria, a tortoise found throughout South America, is one of the animals most rescued from animal trafficking and illegal breeding. Considering this situation, this study aimed to verify the occurrence of hemoparasites in C. carbonaria. Materials and Methods: Blood samples from 73 C. carbonaria were collected from animals located in (1) a rural commercial breeding unit, (2) an urban zoo, and (3) a center of rescued animal screening. Genomic DNA was extracted from these animals and used in PCRs to detect specific genomic fragments of haemogregarines (i.e., Hepatozoon and Hemolivia), and members of the Anaplasmataceae Family (i.e., Ehrlichia sp. and Anaplasma sp.). Blood samples were screened for hemopathogens by direct microscopy and were used for hematological assays, and serum samples were analyzed to determine the concentration of serum components. Results: It was found that 34.2% of the tortoises presented Sauroplasma sp. in their blood samples; these animals showed clinical biochemistry changes that indicate altered liver function. Two zoo animals were positive for Ehrlichia sp. in PCR, and also presented clinical biochemistry and hematological changes. Conclusion: The present project is pioneer in the detection of Ehrlichia sp. in C. carbonaria, and was able to identify changes in clinical biochemistry that can be a result of the infection by hemopathogens in this species.

Mutagenicity, hepatotoxicity, and neurotoxicity of glyphosate and fipronil commercial formulations in Amazon turtles neonates (Podocnemis expansa).

Pesticides are considered one of the main causes of the population decline of reptiles worldwide, with freshwater turtles being particularly susceptible to aquatic contamination. In this context, we investigated the potential mutagenic, hepatotoxic, and neurotoxic effects in neonates of Podocnemis expansa exposed to substrate contaminated with different concentrations of glyphosate and/or fipronil during embryonic development. Eggs collected from the natural environment were artificially incubated in sand moistened with pure water, water added with glyphosate Atar 48® at concentrations of 65 and 6500 µg/L (groups G1 and G2, respectively), water added with fipronil Regent® 800WG at 4 and 400 µg/L (groups F1 and F2, respectively) and, water added with the combination of 65 µg/L glyphosate and 4 µg/L fipronil or with 6500 µg/L glyphosate and 400 µg/L fipronil (groups GF1 and GF2, respectively). For mutagenicity analysis, we evaluated the frequency of micronuclei (MN) and other erythrocyte nuclear abnormalities (ENAs), while for evaluation of hepatotoxicity and neurotoxicity, livers and encephalon were analyzed for histopathological alterations. Exposure to pesticides, alone or in combination, increased the frequency of erythrocyte nuclear abnormalities, particularly blebbed nuclei, moved nuclei, and notched nuclei. Individuals exposed to fipronil exhibited congestion and inflammatory infiltrate in their liver tissue, while, in the encephalon, congestion, and necrosis were present. Our study confirms that the incubation of eggs in substrate polluted with glyphosate and fipronil causes histopathological damage and mutagenic alteration in P. expansa, highlighting the importance of using different biomarkers to evaluate the ecotoxicological effects of these pesticides, especially in oviparous animals.

Molecular Phylogenies of Leeches and Haemoparasites Infecting Freshwater Turtles in Aquatic Ecosystems of Northern Africa Suggest Phylogenetic Congruence between Placobdella costata Sensu Lato and Haemogregarina stepanowi Sensu Lato.

Haemogregarines are blood parasites with a life-cycle involving a vertebrate as the intermediate host and an invertebrate as the definitive host and vector. Extensive phylogenetic investigations based on 18S-rRNA gene sequences have shown that Haemogregarina stepanowi (Apicomplexa: Haemogregarinidae) is able to infest a large diversity of freshwater turtle species, including the European pond turtle Emys orbicularis, the Sicilian pond turtle Emys trinacris, the Caspian turtle Mauremys caspica, the Mediterranean pond turtle Mauremys leprosa, and the Western Caspian turtle Mauremys rivulata, among others. From the same molecular markers, H. stepanowi is further considered to be a complex of cryptic species predisposed to infect the same host species. While Placobdella costata is known to be the unique vector of H. stepanowi, it is only recently that independent lineages within P. costata have been illustrated-suggesting the presence of at least five unique leech species across Western Europe. The aims of our study were therefore to investigate from mitochondrial markers (COI) the genetic diversity within haemogregarines and leeches infecting freshwater turtles of the Maghreb, in order to identify processes of parasite speciation. We showed that H. stepanowi consists of at least five cryptic species in the Maghreb, while two Placobella species were identified in the same area. Although an Eastern-Western speciation pattern was apparent for both leeches and haemogregarines, we cannot make definitive conclusions regarding co-speciation patterns between parasites and vectors. However, we cannot reject the hypothesis of a very strict host-parasite specificity within leeches.

Assessing spatial distribution, genetic variants, and virulence of pathogen Mycoplasma agassizii in threatened Mojave desert tortoises.

Mojave desert tortoises (Gopherus agassizii), a threatened species under the US Endangered Species Act, are long-lived reptiles that experience a chronic respiratory disease. The virulence of primary etiologic agent, Mycoplasma agassizii, remains poorly understood, but it exhibits temporal and geographic variability in causing disease outbreaks in host tortoises. Multiple attempts to culture and characterize the diversity of M. agassizii have had minimal success, even though this opportunistic pathogen chronically persists in nearly every population of Mojave desert tortoises. The current geographic range and the molecular mechanisms of virulence of the type-strain, PS6T, are unknown, and the bacterium is thought to have low-to-moderate virulence. We designed a quantitative polymerase chain reaction (qPCR) targeting three putative virulence genes annotated on the PS6T genome as exo-α-sialidases, enzymes which facilitate growth in many bacterial pathogens. We tested 140 M. agassizii-positive DNA samples collected from 2010 to 2012 across the range of Mojave desert tortoises. We found evidence of multiple-strain infections within hosts. We also found the prevalence of these sialidase-encoding genes to be highest in tortoise populations surrounding southern Nevada, the area from which PS6T was originally isolated. We found a general pattern of loss or reduced presence of sialidase among strains, even within a single host. However, in samples that were positive for any of the putative sialidase genes, one particular gene (528), was positively associated with bacterial loads of M. agassizii and may act as a growth factor for the bacterium. Our results suggest three evolutionary patterns: (1) high levels of variation, possibly due to neutral changes and chronic persistence, (2) a trade-off between moderate virulence and transmission, and (3) selection against virulence in environmental conditions known to be physiologically stressful to the host. Our approach of quantifying genetic variation via qPCR represents a useful model of studying host-pathogen dynamics.