Houston Toad Research Articles

Measuring interspecific admixture in endangered Houston toads (Bufo [= Anaxyrus] houstonensis) at a main recovery site in the Lost Pines ecosystem of Texas, USA

Measuring interspecific admixture in endangered Houston toads (Bufo [= Anaxyrus] houstonensis) at a main recovery site in the Lost Pines ecosystem of Texas, USA

Morphology and vocalization comparison of the Houston Toad and the Dwarf American Toad: implications for their historic range.

Documenting changes in the distribution and abundance of a given taxon requires historical data. In the absence of long-term monitoring data collected throughout the range of a taxon, conservation biologists often rely on preserved museum specimens to determine the past or present, putative geographic distribution. Distributional data for the Houston Toad (Anaxyrus houstonensis) has consistently been confounded by similarities with a sympatric congener, the Dwarf American Toad (A. americanus charlesmithi), both in monitoring data derived from chorusing surveys, and in historical data via museum specimens. In this case, misidentification can have unintended impacts on conservation efforts, where the Houston Toad is federally endangered, and the Dwarf American Toad is of least concern. Previously published reports have compared these two taxon on the basis of their male advertisement call and morphological appearance, often with the goal of using these characters to substantiate their taxonomic status prior to the advent of DNA sequencing technology. However, numerous studies report findings that contradict one another, and no consensus on the true differences or similarities can be drawn. Here, we use contemporary recordings of wild populations of each taxon to test for quantifiable differences in male advertisement call. Additionally, we quantitatively examine a subset of vouchered museum specimens representing each taxon to test previously reported differentiating morphometric characters used to distinguish among other Bufonids of East-Central Texas, USA. Finally, we assemble and qualitatively evaluate a database of photographs representing catalogued museum vouchers for each taxon to determine if their previously documented historic ranges may be larger than are currently accepted. Our findings reveal quantifiable differences between two allopatric congeners with respect to their male advertisement call, whereas we found similarities among their detailed morphology. Additionally, we report on the existence of additional, historically overlooked, museum records for the Houston Toad in the context of its putative historic range, and discuss errors associated with the curation of these specimens whose identity and nomenclature have not been consistent through time. These results bookend decades of disagreement regarding the morphology, voice, and historic distribution of these taxa, and alert practitioners of conservation efforts for the Houston Toad to previously unreported locations of occurrence.

Communicating Expectations: Informing Strategic Communications by Conservation Level Segmentation and Impression Management Preferences

In the United States, collaboration between landowners and endangered species conservation (ESC) organizations is required to accomplish conservation goals. However, collective action for ESC is stymied by governance legitimacy deficits. Strategic communication is a tool used to accomplish organizational goals that can restore legitimacy through communications that re-align governance institutions with societal notions of appropriateness. Strategic communication, though, has yet to be investigated in the context of landowners and ESC. To illustrate how strategic communication can increase ESC collaboration, we surveyed 956 owners in Houston toad range in Texas to elicit their preferences for stewardship and communication constructs. Findings revealed distinct segments of landowners based on their stewardship preferences that preferred different types of communications. If ESC agencies are to enhance legitimacy amongst an increasingly heterogeneous landowner audience, they will want to adopt landowner-centric governance and communication strategies that account for differences in landowner conservation values, ESC experiences, and organizational expectations.

COMPARISON OF SUBCUTANEOUS ALFAXALONE AND SUBCUTANEOUS ALFAXALONE-DEXMEDETOMIDINE FOR SEDATION IN THE HOUSTON TOAD (ANAXYRUS HOUSTONENSIS).

The Houston toad (Anaxyrus houstonensis), a primarily terrestrial amphibian of south-central Texas, has been listed as federally endangered since 1970. Sedation is an important tool for obtaining diagnostics and providing treatment in this species. This prospective, randomized, and blinded study compared the sedative effects of SC alfaxalone (Protocol A) at approximately 12 mg/kg (median [range] = 12.70 [12.09-13.95] mg/kg] to SC alfaxalone-dexmedetomidine (Protocol AD) at approximately 12 mg/kg (median [range] = 12.68 [12.16-13.56] mg/kg) and 0.1 mg/kg (median [range] = 0.1 [0.07-0.13] mg/kg), respectively, in adult Houston toads (n = 26). Toads from Protocol AD received atipamezole SC at approximately 1 mg/kg (median [range] = 0.96 [0.75-1.25] mg/kg) 45 min postinduction, whereas toads from Protocol A received the equivalent volume of SC sterile saline at the same time point. Heart rate, gular rate, and times to first effect, loss of righting reflex, ability to position for radiographs, loss of nociception, return of righting reflex, and full recovery were recorded. A significantly greater number of toads lost righting reflex, positioned for radiographs, and lost nociception with Protocol AD compared with Protocol A. Additionally, time to return of righting reflex and time to full recovery were significantly longer with Protocol AD than with Protocol A. The protocols did not differ significantly in time to first effect, time to radiographic positioning, or time to loss of nociception. Histologic examination of four toads euthanized during the study revealed acute injection site reactions from all administered drugs, including saline. No clinical adverse reactions were observed. This study demonstrates that the combination of SC alfaxalone and dexmedetomidine results in deeper sedation than SC alfaxalone alone, but also correlates with longer recovery times despite antagonist administration.

Using genome-wide data to ascertain taxonomic status and assess population genetic structure for Houston toads (Bufo [= Anaxyrus] houstonensis)

The Houston toad (Bufo [= Anaxyrus] houstonensis) is an endangered amphibian with a small geographic range. Land-use changes have primarily driven decline in B. houstonensis with population supplementation predominant among efforts to reduce its current extinction risk. However, there has been historic uncertainty regarding the evolutionary and conservation significance of B. houstonensis. To this end, we used 1170 genome-wide nuclear DNA markers to examine phylogenetic relationships between our focal taxon, representatives of the Nearctic B. americanus group, and B. nebulifer, a sympatric Middle American species. Phylogenetic analyses indicate B. houstonensis is a taxon that is distinct from B. americanus. We corroborated such genetic distinctiveness with an admixture analysis that provided support for recent reproductive isolation between B. americanus and B. houstonensis. However, ABBA-BABA tests for ancient admixture indicated historic gene flow between Nearctic species while no signal of historic gene flow was detected between Nearctic and Middle-American species. We used an admixture analysis to recognize four Management Units (MU) based on observed genetic differentiation within B. houstonensis and recommend captive propagation, population supplementation, and habitat restoration efforts specific to each MU. Our results re-affirm the evolutionary novelty of an endangered relict.

Quantification of chlamydiae in the endangered Houston toad (Anaxyrus houstonensis).

Two primer set/probe combinations targeting variable regions on the 23S rRNA gene were designed to detect and quantify chlamydiae in DNA extracted from brain swabs of the endangered Houston toad (Anaxyrus houstonensis) using SYBRGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Prevalence and abundance values for samples were generally different between SYBRGreen- and Taqman-based detection methods, with higher specificity observed for Taqman-based detection. Of the 314 samples analyzed, initial screening with SYBRGreen-based qPCR retrieved 138 positive samples, of which 52 were confirmed by Taqman-based analyses as chlamydiae. All of these samples were subsequently identified as C. pneumoniae by specific qPCR and confirmed by comparative sequence analyses of 23S rRNA gene amplicons. These results demonstrate the usefulness of our developed qPCR methods to screen for and verify prevalence of chlamydiae in DNA of brains swabs, and ultimately specifically identify and quantify chlamydiae and specifically C. pneumoniae in these samples.

Applying sperm collection and cryopreservation protocols developed in a model amphibian to three threatened anuran species targeted for biobanking management

The challenge of replicating environmental cues in amphibian captive assurance colonies has been shown to be one of the contributing factors leading to limited breeding success. To circumvent this challenge, assisted reproductive technologies (ART) have been developed in model organisms and can now be applied to at-risk species. This study tested the transferability of cryopreservation protocols developed in the Fowler's toad (Anaxyrus fowleri) to three threatened anurans, the Chiricahua leopard frog (Lithobates chiricahuensis), Puerto Rican crested toad (Peltophryne lemur), and Houston toad (Anaxyrus houstonensis). Males were administered human chorionic gonadotropin and/or gonadotropin releasing hormone-analog to elicit spermiation, and spermic urine was collected and analyzed for motility. Samples were cryopreserved with 10 % N,N-dimethylformamide + 10 % trehalose + 0.25 % bovine serum albumin and frozen at two rates, −32 to −45 °C/min and − 20 to −29 °C/min. Results show that sperm frozen at −32 to −45 °C/min recovered significantly higher post-thaw sperm motility than −20 to −29 °C/min for P. lemur (p = 0.04) and L. chiricahuensis (p = 0.03). When compared across species, L. chiricahuensis had significantly higher post-thaw motility at both freezing rates (p < 0.05) than the other species, yet there was no difference between sperm motility between the three bufonid species for either freezing rate (p > 0.06). These results indicate that transferability of a cryopreservation protocol between species is possible, and differences in sensitivity and response to cryopreservation may exist between genera.

Acute Bladder Snails, Physa acuta Draparnaud 1805, as ectosymbionts of the endangered Houston Toad, Bufo (Anaxyrus) houstonensis Sanders 1953

Acute Bladder Snails, Physa acuta Draparnaud 1805, as ectosymbionts of the endangered Houston Toad, Bufo (Anaxyrus) houstonensis Sanders 1953

Quantification of members of the Mycobacterium chelonae-abscessus complex in lesions of the endangered houston toad (Anaxyrus houstonensis)

Illumina-based 16S rRNA V3 amplicon sequencing of total DNA obtained from soft tissue lesions (joint granulomas) of the endangered Houston toad (Anaxyrus houstonensis) demonstrated that many reads represented members of the actinobacterial Mycobacterium chelonae-abscessus complex. In order to quantify members of this complex in those lesions, we designed three complex-specific primer set/probe combinations (sets I, II and III) targeting variable regions on the 23S rRNA gene for SybrGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Both SybrGreen- and Taqman-based analyses specifically detected members of the M. chelonae-abscessus complex in lesion samples, with numbers between 104 and 107 cells per 100-mg sample. Values within individual samples were generally comparable between SybrGreen- and Taqman-based detection methods and between all primer set/probe combinations, except for SybrGreen-based analyses of a few samples analyzed with primer set I that used a less specific forward primer. The development of highly specific detection and quantification methods for members of the M. chelonae-abscessus complex in lesion samples can enable group specific tracking of these organisms, particularly in captive or stewardship settings where source and transmission monitoring are valuable tools to husbandry and species conservation.

Association of Leeches with the Endangered Houston Toad

The prevalence of leech attachment on anuran taxa is largely uncharacterized. When parasitic, such relationships may regulate amphibian populations and function as an obstacle to the recovery of imperiled species. We evaluated the prevalence of leech attachment in a population of endangered Bufo (= Anaxyrus) houstonensis (Houston Toad) in Bastrop County, TX, during the spring of 2021. Of 191 toads examined, 2 (1%) were each infested with a single leech. Molecular analysis of the cytochrome c oxidase subunit I gene identified 1 of those leeches as Helobdella austinensis. This constitutes the first reported association of H. austinensis with a vertebrate host and extends the known range of H. austinensis ∼50 km eastward. Leech attachment in this Houston Toad population is rare and the nature of the association remains unclear.

Medical management of dermatitis‐associated morbidity and mortality in endangered Houston toads (Anaxyrus houstonensis)

AbstractThe Houston toad (Anaxyrus [Bufo] houstonensis) is protected under the U.S. Endangered Species Act and is endemic to east central Texas. Since 2007, the Houston Zoo Inc. participated in Houston toad recovery through captive breeding, re‐introduction and maintenance of a captive assurance colony. During 2012 and 2013, the zoo's Houston toad colony experienced increased morbidity and mortality attributed to dermatitis of various aetiologies. An 8‐month time period, from September 2012 through April 2013, was selected to evaluate disease aetiology, treatments administered and success of therapy. A total of 248 toad records were reviewed. During this time, 52 toads that died with dermatitis associated with a fungal or bacterial aetiology or a combination of both were included in the review. Toads that survived (n = 196) had been treated with either oral enrofloxacin (10 mg/kg once daily), itraconazole baths (0.01% solution for 10–15 minutes once daily), enhanced nutrition or a combination of these therapies.

Near-Infrared Spectroscopy (NIRS) as a Method for Biological Sex Discrimination in the Endangered Houston Toad (Anaxyrus houstonensis).

Biological sex is one of the more critically important physiological parameters needed for managing threatened animal species because it is crucial for informing several of the management decisions surrounding conservation breeding programs. Near-infrared spectroscopy (NIRS) is a non-invasive technology that has been recently applied in the field of wildlife science to evaluate various aspects of animal physiology and may have potential as an in vivo technique for determining biological sex in live amphibian species. This study investigated whether NIRS could be used as a rapid and non-invasive method for discriminating biological sex in the endangered Houston toad (Anaxyrus houstonensis). NIR spectra (N = 396) were collected from live A. houstonensis individuals (N = 132), and distinct spectral patterns between males and females were identified using chemometrics. Linear discriminant analysis (PCA-LDA) classified the spectra from each biological sex with accuracy ≥ 98% in the calibration and internal validation datasets and 94% in the external validation process. Through the use of NIRS, we have determined that unique spectral signatures can be holistically captured in the skin of male and female anurans, bringing to light the possibility of further application of this technique for juveniles and sexually monomorphic species, whose sex designation is important for breeding-related decisions.

Determinants influencing recruitment in the Houston toad programmatic safe harbor agreement

ABSTRACT The importance of private lands conservation (PLC) for endangered species continues to grow as habitat is increasingly lost or fragmented. In Texas, nearly 98% of all property is privately owned, rendering PLC critical for protecting endangered species. Despite case studies regarding factors leading to stewardship outcomes for endangered species, a knowledge gap exists regarding PLC for endangered species. To help address this gap, we situate findings from 30 key informant interviews and two focus groups within a novel stewardship framework to understand how PLC interventions for the endangered Houston toad (Anaxyrus houstonensis) appeal to landowners. Our approach revealed that local changes in conservation norms and networks can build relationships between resource-deficient landowners and practitioners that increase awareness and trust, combating barriers to PLC. Enhanced networks and cultural transformation facilitated by demographic changes and decentralized governance can allow stewardship obligations to supersede culturally embedded rural myths and attitudes of anti-government sentiment.

Optimizing the power of human performed audio surveys for monitoring the endangered Houston toad using automated recording devices.

Knowledge regarding the locations of populations of endangered species is a critical part of recovery and facilitates land use planning that avoids unnecessary impacts. Regulatory agencies often support the development of survey guidelines designed to standardize the methods and maximize the probability of detection, thereby avoiding incorrectly concluding a species is absent from a site. Here, using simulations with data collected using automated recording devices (ARDs) we evaluated the efficacy of the existing U.S. Fish and Wildlife Service’s survey requirements for the endangered Houston Toad (Anaxyrus houstonensis). We explored the effect of (1) increasing survey duration, (2) increasing the number of surveys, and (3) combinations of environmental conditions (e.g., temperature, humidity, rainfall) on the detection probability and the number of surveys needed to be 95% confident of absence. We found that increases in both the duration of the survey and the number of surveys conducted decreased the likelihood of incorrectly concluding the species was absent from the site, and that the number of surveys required to be 95% confident greatly exceeded the existing survey requirements. Targeting specific environmental conditions was also an effective way to decrease the number of surveys required but the infrequency in which these conditions occurred might make application difficult in some years. Overall, we suggest that the survey effort necessary to achieve confidence in the absence of Houston Toads at a site is more practically achievable with the use of ARDs, but this may not be suitable in all monitoring scenarios.

MYCOBACTERIA IN SKIN LESIONS AND THE HABITAT OF THE ENDANGERED HOUSTON TOAD (ANAXYRUS HOUSTONENSIS).

Head-starting of the federally endangered Houston toad (Anaxyrus houstonensis), that is, the release of egg strands, tadpoles, and metamorphic juveniles produced in captivity into the original breeding ponds, requires assessment of potential threats for the transmission of pathogens from captive to free-ranging toads. We used Illumina-based 16S rRNA V3 amplicon sequencing to investigate the community structure of bacteria from skin lesions of captive Houston toad and habitat (pond) samples. Proteobacteria, alone or together with Actinobacteria and, in some samples, Cyanobacteria represented virtually all reads in tissue lesion samples, whereas pond samples were much more diverse, with Acidobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Cyanobacteria, Firmicutes, Planctomycetes, Proteobacteria, and Verrucomicrobia present with little variation between samples. If present in lesions, Actinobacteria were largely represented by Mycobacteriaceae, and here mainly by one sequence identical to sequences of members of the Mycobacterium chelonae-abscessus complex. In pond samples, mycobacteria represented only a small portion of the actinobacteria, although at higher diversity with six distinct reads. Sequences for reads obtained from pond samples were identical to those representing the M. chelonae-abscessus complex, a group with Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium avium, a group with Mycobacterium vaccae, Mycobacterium fortuitum, Mycobacterium poriferae, and a group with Mycobacterium elephantis and Mycobacterium celeriflavum, whereas sequences of high similarity were detected for reads related to those of Mycobacterium holsaticum, Mycobacterium pallens, and Mycobacterium obuense, and Mycobacterium goodii. Our results indicated that lesions observed on the Houston toad in captivity are not the result of mycobacteria in every case, and that the presence of mycobacteria in the captive colony does not represent a novel pathogen threat to the wild populations because such bacteria are also seen in the natural pond habitats for the Houston toad.

Primary Hepatic Vascular Neoplasm in a Houston Toad (Anaxyrus houstonensis)

An 11-year-old captive, female Houston toad (Anaxyrus houstonensis) became lethargic and reluctant to move. This toad had been administered human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone (LHRH) annually as part of a hormone-assisted captive propagation programme for 8 years. Due to poor prognosis, euthanasia was elected. At necropsy, the liver was moderately enlarged, diffusely dark red and had a nodular superficial appearance. Microscopically, the hepatic nodules consisted of poorly demarcated, non-encapsulated, paucicellular infiltrative neoplastic growths. These consisted of spindle cells, similar to sinusoidal endothelial cells, that formed irregular blood-filled vascular channels resembling sinusoids separated by thin bands of collagen and reticulin fibres. Neoplastic cells dissected through and replaced hepatic cords and bile ducts. Based on the cytohistomorphological features and analogy with vascular neoplasms in other vertebrates, a diagnosis of low-grade angiosarcoma was made. To the best of our knowledge, this is the first record of primary hepatic vascular neoplasia in an amphibian. It is not known if the long-term administration of HCG and LHRH had any oncogenic role in this animal. Vascular neoplasia should be included in the differential diagnosis of hepatomegaly and non-specific lethargy and health decline in amphibian species.

Evaluating the effects of red imported fire ants (Solenopsis invicta) on juvenile Houston Toads (Bufo [=Anaxyrus] houstonensis) in Colorado County, TX.

The spread of invasive species is considered a major threat to biodiversity, second only to habitat loss. Red Imported Fire Ants (Solenopsis invicta) are a globally invasive species with negative impacts reported on native invertebrate and vertebrate species. Federally endangered Houston Toads (Bufo [=Anaxyrus] houstonensis), endemic to Texas, are among the vertebrates reportedly negatively impacted by Red Imported Fire Ants (RIFA). Threats posed by RIFA to Houston Toads needed to be explicitly characterized. Large-scale chemical treatments to suppress RIFA and facilitate brood survival in Attwater’s prairie-chickens (Tympanuchus cupido attwateri) at the Attwater Prairie Chicken National Wildlife Refuge (APCNWR) afforded us an opportunity to experimentally examine the influence of RIFA abundance on juvenile Houston Toad growth and survival. We also sought to examine whether juvenile Houston Toads could grow and survive in a vegetation type similar to a historic species locality. We conducted a terrestrial mesocosm experiment to test whether the application of bait-driven suppressant decreased counts of RIFA relative to untreated sites. We examined whether counts of native ant and non-ant native invertebrates were higher in response to potential decreases in RIFA. We compared growth and survival rates in juvenile Houston Toads among treated and untreated sites, expecting juvenile growth and survival to be higher in response to potentially decreased RIFA counts and increased native invertebrate counts. We saw lower counts of RIFA in treated prairies, but we also observed a decrease in native ant counts possibly due to chemical treatment. Therefore, the application of bait-driven suppressant may not affect RIFA alone. We saw no difference in counts of non-ant invertebrates among treated and untreated sites. Juvenile Houston Toads did not differ in growth and survival among treated and untreated sites. We recognize that the lack of a relationship between juvenile growth and survival with a treatment effect, and therefore RIFA abundance, may be limited to APCNWR. We encourage additional experimental studies to elucidate RIFA impacts at other sites. We extrapolated apparent survival estimates from our study to one year. These appear comparable to juvenile survivorship required in simulations for Houston Toad population persistence and on this basis, we recommend that APCNWR be re-evaluated as a reintroduction site for Houston Toads. We also recommend further studies to potentially broaden the regulatory definition of Houston Toad habitat beyond the current restrictive view of canopied forest alone. Such studies would need to examine the utility of native grasslands as dispersal corridors/upland habitat for juvenile Houston Toads. Our findings emphasize the utility of experimental studies in directly examining the influence of perceived threats to imperiled species and the role of such clarifications in adapting recovery efforts.

Chlamydia pneumoniae Polioencephalomyelitis and Ganglionitis in Captive Houston Toads (Anaxyrus houstonensis).

Chlamydia pneumoniae is a ubiquitous pathogen causing disease in humans, mammals, birds, reptiles, and amphibians. Since 2012, C. pneumoniae infection has caused neurologic disease and mortality in a breeding colony of endangered Houston toads (Anaxyrus houstonensis) at the Houston Zoo. The purpose of this report is to present the histopathologic and ultrastructural characteristics of C. pneumoniae infection in Houston toads. Fourteen cases were evaluated by histopathology and 1 case was evaluated by electron microscopy. The major histopathologic finding was necrotizing and histiocytic polioencephalomyelitis and ganglionitis. Bacteria formed intracytoplasmic inclusions within neurons but frequently extended into the surrounding tissue from necrotic cells. Ultrastructural evaluation showed the bacteria formed reticulate and elementary bodies characteristic of Chlamydia spp.

Development and Validation of Automated Detection Tools for Vocalizations of Rare and Endangered Anurans

AbstractFor many rare or endangered anurans, monitoring is achieved via auditory cues alone. Human-performed audio surveys are inherently biased, and may fail to detect animals when they are present. Automated audio recognition tools offer an alternative mode of observer-free monitoring. Few commercially available platforms for developing these tools exist, and little research has investigated whether these tools are effective at detecting rare vocalization events. We generated a recognizer for detecting the vocalization of the endangered Houston toad Anaxyrus houstonensis using SongScope© bioacoustics software. We developed our recognizer using a large sample of training data that included only the highest quality of recorded audio (i.e., low noise, no interfering vocalizations) divided into small, manageable batches. To track recognizer performance, we generated an independent set of test data through randomly sampling a large population of audio known to possess Houston toad vocalizations. We analyzed training data and test data recursively, using a criterion of zero tolerance for false-negative detections. For each step, we incorporated a new batch of training data into the recognizer. Once we included all training data, we manually verified recognizer performance against one full month (March 2014) of audio taken from a known breeding locality. The recognizer successfully identified 100% of all training data and 97.2% of all test data. However, there is a trade-off between reducing false-negative and increasing false-positive detections, which limited the usefulness of some features of SongScope. Methods of automated detection represent a means by which we may test the efficacy of the manual monitoring techniques currently in use. The ability to search any collection of audio recordings for Houston toad vocalizations has the potential to challenge the paradigms presently placed on monitoring for this species of conservation concern.

Leaping from Awareness to Action: Impacts of an Amphibian Educator Workshop

Abstract Where endangered species occur, recommendations call for conservation education programs that engage local educators; however, few studies have measured the effectiveness of implemented programs. We conducted a multipartner educator workshop for the endangered Houston Toad, Anaxyrus houstonensis, as one local example illustrating the broader issue of globally declining amphibians. We measured the effect of the workshop on participants' (n = 50) awareness/knowledge, values, beliefs, emotions, and intent to take action. We observed significant increases in awareness/knowledge and values regarding general amphibian declines and the focal species. The workshop significantly increased participants' belief that they had necessary resources to teach about the Houston Toad. Ninety-nine percent of participants agreed that they cared more about wild toads after meeting live ambassador toads. Postworkshop, we observed a 33% increase in use of amphibians or Houston Toads in participant learning settings. We ...