Protein Content and Composition in Leiolepis rubritaeniata from Northeastern Thailand

Abstract

Background and Objectives: The genus Leiolepis (family Agamidae), commonly known as butterfly lizards, is widely distributed across Southeast Asia and typically inhabits dry, open areas with sandy soils and sparse vegetation. Among the ten recognized species, L. rubritaeniata is notable for its distinctive red-striped flanks and occurrence in northeastern Thailand. Although previous studies have explored its taxonomy, distribution, and behavior, limited information is available on its physiological and nutritional biology, particularly regarding protein content and amino acid composition. Proteins are essential macromolecules involved in tissue development, immune response, metabolism, and energy balance, and reptiles living in seasonal environments may develop adaptations to cope with fluctuating food availability. In some rural communities, agamid lizards are also consumed as supplementary sources of protein, making their nutritional value relevant from both ecological and ethnobiological perspectives. Despite this importance, most reptilian nutrition studies have focused on snakes and turtles, leaving small lizard species such as L. rubritaeniata understudied. This research aims to confirm the species identity of L. rubritaeniata specimens collected from Udon Thani Province using morphological and ND2 gene sequence analyses and to determine and compare the total protein content and amino acid composition in the body and tail tissues of male and female individuals. The findings will provide baseline data that support further studies in reptilian nutritional ecology, physiological adaptation, and conservation planning.

Methodology: Specimens of L. rubritaeniata were collected from Kham Khok Sung and Bayao Subdistricts in Wang Sam Mo District, Udon Thani Province, using snares and excavation methods. Following euthanasia, liver tissues were preserved in 95% ethanol at –20 °C for DNA analysis. For protein and amino acid composition analysis, muscle samples were collected separately from two regions: (1) the body, comprising the head, neck, trunk and limbs, and (2) the tail, consisting exclusively of tail muscle. All samples were promptly processed for analysis. All specimens were deposited in the Khon Kaen Vertebrate Collection (KKUC), and the study received ethical approval from the Institutional Animal Care and Use Committee of Khon Kaen University (Ref. no. 660201.2.11/318 [50]). Species identification was based on morphology and mitochondrial ND2 gene sequences, amplified using METF6 and CO1R1 primers and sequenced by Pacific Science. Sequences were aligned in SeaView and phylogenetic trees constructed using Maximum Parsimony in PAUP* and Bayesian Inference in MrBayes, with model selection via MrModelTest. Pairwise sequence divergences were calculated in MEGA X. Morphological identification included snout-vent length (SVL), tail length, scale and pore counts, and coloration traits. Crude protein was analyzed in triplicate using the Kjeldahl method with a Gerhardt KT 8s system, and protein content was calculated from nitrogen percentage following Horwitz and Latimer’s formula. For amino acid composition, hydrolyzed samples were analyzed using ion-exchange chromatography and an automatic amino acid analyzer (SCION Artemis 6000), with detection via post-column derivatization and ninhydrin reaction.

Main Results: Molecular analyses based on the mitochondrial ND2 gene confirmed that the Leiolepis specimens from Udon Thani Province belong to L. rubritaeniata. Phylogenetic analyses using Maximum Parsimony and Bayesian Inference revealed that the new sequences (PV746783 and PV746784) clustered strongly with published L. rubritaeniata sequences in GenBank, supported by minimal genetic distances and clear separation from L. reevesii and L. glaurung. Morphologically, the Udon Thani Province specimens closely resembled L. rubritaeniata in key traits such as size, scalation, and coloration patterns, including diagnostic flank markings. Although slightly larger, their body measurements, labial counts, and femoral pore numbers fell within the species' intraspecific variation. Crude protein analysis showed significantly higher protein content in the tail than in the body in both sexes, confirmed by paired t-tests (p < 0.05), while no significant sex-based differences were observed. Amino acid profiles showed that female tail tissue contained the highest total amino acid content, with glutamic acid, aspartic acid, and lysine being the most abundant. Cysteine was consistently the least abundant across all samples. These molecular, morphological, and nutritional findings provide robust evidence for the identification of the specimens as L. rubritaeniata and highlight notable tissue-specific differences in protein quantity and amino acid composition, with possible biological and ecological significance.

Conclusion: This study confirms the identification of L. rubritaeniata specimens from Udon Thani Province, northeastern Thailand, through both molecular and morphological analyses. Mitochondrial ND2 gene sequencing placed the specimens firmly within the L. rubritaeniata clade with strong nodal support, and their external morphological traits were consistent with those of known L. rubritaeniata populations. Crude protein analysis revealed that tail tissues contained significantly higher protein levels than body tissues in both sexes, while no significant differences were found between males and females for either body or tail protein content. Amino acid profiling showed that glutamic acid, aspartic acid, and lysine were the most abundant amino acids across all samples, with the highest total amino acid content observed in female tail tissues. These findings provide evidence for the taxonomic status of L. rubritaeniata in Udon Thani and also offer new insights into its nutritional composition, particularly the higher protein and amino acid content in tail tissues, which may have implications for both biological understanding and potential utilization in reptiles and amphibians.