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

Abstract

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.

Near infrared spectroscopy for the identification of live anurans: Towards rapid and automated identification of species in the field

“…Considering that light can pass through superficial tissues, information about internal organs, stomach and intestine contents and the presence of pregnant females 51 may be present in the spectral signatures. These sources of physiological variation were not controlled in the spectrum of each individual (even if of the same species), which may be a limitation of our study.…”

Enhancing predictive performance for spectroscopic studies in wildlife science through a multi-model approach: A case study for species classification of live amphibians

“…NIR reflectance spectra ( N = 1661) were collected with an ASD FieldSpec 3 IndicoPro (Malvern Panalytical, ASD Analytical Spectral Devices Inc., Boulder, CO, USA). Spectra were collected using a 2 cm diameter low-powered plant contact probe (as per [ 19 ]; Fig 5 ) from the cloacal region of eleven amphibian species, seven of which were anuran species (frogs or toads) including the boreal toad ( Anaxyrus boreas , N = 53 individuals), Fowler’s toad ( Anaxyrus fowleri , N = 47), Houston toad ( Anaxyrus houstonensis , N = 133), Puerto Rican crested toad ( Peltophryne lemur , N = 95), Chiricahua leopard frog ( Lithobates chiricahuensis , N = 77), southern leopard frog ( Lithobates sphenocephalus , N = 26), and dusky gopher frog ( Lithobates sevosa , N = 69), and the remaining four species were caudates (salamanders) including the spotted salamander ( Ambystoma maculatum , N = 33), tiger salamander ( Ambystoma tigrinum , N = 38), Chinese giant salamander ( Andrias davidianus , N = 48), and the eastern hellbender salamander ( Cryptobranchus alleganiensis , N = 49). All spectra were collected across the wavelength range of 350–2500 nm (resolution of 1.4 nm for the 350–1000 nm range and 2 nm for the 1000–2500 nm range), where each spectral replicate was comprised of 50 scans then averaged into a single spectrum (integration time approximately 34 ms).…”

“…While such multi-model approaches are routinely conducted to benchmark and evaluate candidate models in fields such as engineering, medicine, and agriculture [ 4 , 5 , 7 , 8 , 10 – 18 ], the majority of spectroscopic-based studies in wildlife sciences (61/65 studies; see Methods ) have applied a single-algorithm approach for model calibration and testing ( Table 1 ). PLS models have been applied to answer a diverse set of questions across many different sampling types, such as species discrimination from dried blood traces between Bengal tigers and Indian leopards, and quantification of dietary components from the feces of black bears [ 19 , 20 ]. However, it may be that different sample types and associated questions can be better addressed through the exploration of other algorithms [ 21 ].…”

“…The ability to hold, restrain, and scan amphibians in less than the minute needed to obtain spectra, makes them a valuable vertebrate class for studying how NIR spectroscopy can be used to obtain information on whole, live animals. Recent NIR studies on live amphibians have demonstrated promising results for evaluating and categorizing physiological factors such as biological sex, species, and reproductive status [ 19 , 36 , 37 , 77 ]. For example, taxonomic classification that previously required genetic sequencing or specialized field biologists to distinguish closely related anurans has been facilitated with NIR spectroscopy [ 35 ].…”

Current State of Conservation Physiology for Amphibians: Major Research Topics and Physiological Parameters

“…These include techniques such as X-ray imaging, blood biochemistry analysis, immunological and hormonal analyses, and histological examination. Chen et al (2022b) proposed a technique using near-infrared spectroscopy to determine biological sex [88], while Lazcano et al (2021) proposed the use of MRI techniques for brain research on amphibians [89]. Furthermore, studies that determine the genetic range of a population in conjunction with its physiological state are also suggested to specify management ranges [90].…”