Proteomic analysis of crocodile white blood cells reveals insights into the mechanism of the innate immune system.

Crocodiles have a particularly powerful innate immune system because their blood contains high levels of antimicrobial peptides. They can survive injuries that would be fatal to other animals, and they are rarely afflicted with diseases. To better understand the crocodile's innate immune response, proteomic analysis was performed on the white blood cells (WBC) of an Aeromonas hydrophila-infected crocodile. Levels of WBC and red blood cells (RBC) rapidly increased within 1 h. In WBC, there were 109 up-regulated differentially expressed proteins (DEP) that were up-regulated. Fifty-nine DEPs dramatically increased expression from 1 h after inoculation, whereas 50 up-regulated DEPs rose after 24 h. The most abundant DEPs mainly had two biological functions, 1) gene expression regulators, for example, zinc finger proteins and histone H1 family, and 2) cell mechanical forces such as actin cytoskeleton proteins and microtubule-binding proteins. This finding illustrates the characteristic effective innate immune response mechanism of crocodiles that might occur via boosted transcription machinery proteins to accelerate cytoskeletal protein production for induction of phagocytosis, along with the increment of trafficking proteins to transport essential molecules for combating pathogens. The findings of this study provide new insights into the mechanisms of the crocodile's innate immune system.

Preserving Pure Siamese Crocodile Populations: A Comprehensive Approach Using Multi-Genetic Tools.

Hybrids between the critically endangered Siamese crocodile (Crocodylus siamensis) and least-concern saltwater crocodile (C. porosus) in captive populations represent a serious challenge for conservation and reintroduction programs due to the impact of anthropogenic activities. A previous study used microsatellite and mitochondrial DNA data to establish the criteria for identifying species and their hybrids; however, the results may have been influenced by biased allelic frequencies and genetic drift within the examined population. To overcome these limitations and identify the true signals of selection, alternative DNA markers and a diverse set of populations should be employed. Therefore, this study used DArT sequencing to identify genome-wide single nucleotide polymorphisms (SNPs) in both species and confirm the genetic scenario of the parental species and their hybrids. A population of saltwater crocodiles from Australia was used to compare the distribution of species-diagnostic SNPs. Different analytical approaches were compared to diagnose the level of hybridization when an admixture was present, wherein three individuals had potential backcrossing. Approximately 17.00-26.00% of loci were conserved between the Siamese and saltwater crocodile genomes. Species-diagnostic SNP loci for Siamese and saltwater crocodiles were identified as 8051 loci and 1288 loci, respectively. To validate the species-diagnostic SNP loci, a PCR-based approach was used by selecting 20 SNP loci for PCR primer design, among which 3 loci were successfully able to differentiate the actual species and different hybridization levels. Mitochondrial and nuclear genetic information, including microsatellite genotyping and species-diagnostic DNA markers, were combined as a novel method that can compensate for the limitations of each method. This method enables conservation prioritization before release into the wild, thereby ensuring sustainable genetic integrity for long-term species survival through reintroduction and management programs.

Should the Identification Guidelines for Siamese Crocodiles Be Revised? Differing Post-Occipital Scute Scale Numbers Show Phenotypic Variation Does Not Result from Hybridization with Saltwater Crocodiles.

Populations of Siamese crocodiles (Crocodylus siamensis) have severely declined because of hunting and habitat fragmentation, necessitating a reintroduction plan involving commercial captive-bred populations. However, hybridization between Siamese and saltwater crocodiles (C. porosus) has occurred in captivity. Siamese crocodiles commonly have post-occipital scutes (P.O.) with 4-6 scales, but 2-6 P.O. scales were found in captives on Thai farms. Here, the genetic diversity and population structure of Siamese crocodiles with large P.O. variations and saltwater crocodiles were analyzed using mitochondrial DNA D-loop and microsatellite genotyping. Possible crocodile hybrids or phenotypic variations were ascertained by comparison with our previous library from the Siam Crocodile Bioresource Project. Siamese crocodiles with <4 P.O. scales in a row exhibit normal species-level phenotypic variation. This evidence encourages the revised description of Siamese crocodiles. Moreover, the STRUCTURE plot revealed large distinct gene pools, suggesting crocodiles in each farm were derived from distinct lineages. However, combining both genetic approaches provides evidence of introgression for several individual crocodiles, suggesting possible hybridization between Siamese and saltwater crocodiles. We proposed a schematic protocol with patterns observed in phenotypic and molecular data to screen hybrids. Identifying non-hybrid and hybrid individuals is important for long-term in situ/ex situ conservation.

High genetic diversity and demographic history of captive Siamese and Saltwater crocodiles suggest the first step toward the establishment of a breeding and reintroduction program in Thailand.

The Siamese crocodile (Crocodylus siamensis) and Saltwater crocodile (C. porosus) are two of the most endangered animals in Thailand. Their numbers have been reduced severely by hunting and habitat fragmentation. A reintroduction plan involving captive-bred populations that are used commercially is important and necessary as a conservation strategy to aid in the recovery of wild populations. Here, the genetic diversity and population structure of 69 individual crocodiles, mostly members of captive populations, were analyzed using both mitochondrial D-loop DNA and microsatellite markers. The overall haplotype diversity was 0.924-0.971 and the mean expected heterozygosity across 22 microsatellite loci was 0.578-0.701 for the two species. This agreed with the star-like shaped topology of the haplotype network, which suggests a high level of genetic diversity. The mean ratio of the number of alleles to the allelic range (M ratio) for the populations of both species was considerably lower than the threshold of 0.68, which was interpreted as indicative of a historical genetic bottleneck. Microsatellite markers provided evidence of introgression for three individual crocodiles, which suggest that hybridization might have occurred between C. siamensis and C. porosus. D-loop sequence analysis detected bi-directional hybridization between male and female individuals of the parent species. Therefore, identification of genetically non-hybrid and hybrid individuals is important for long-term conservation management. Relatedness values were low within the captive populations, which supported their genetic integrity and the viability of a breeding and reintroduction management plan. This work constitutes the first step in establishing an appropriate source population from a scientifically managed perspective for an in situ/ex situ conservation program and reintroduction of crocodile individuals to the wild in Thailand.

Bioactive compounds from crocodile (Crocodylus siamensis) white blood cells induced apoptotic cell death in hela cells.

Crocodile (Crocodylus siamensis) white blood cell extracts (WBCex) were examined for anticancer activity in HeLa cell lines using the MTT assay. The percentage viability of HeLa cells significantly deceased after treatment with WBCex in a dose- and time-dependent manner. The IC50 dose was suggested to be approximately 225 µg/mL protein. Apoptotic cell death occurred in a time-dependent manner based on investigation by flow cytometry using annexin V-FITC and PI staining. DAPI nucleic acid staining indicated increased chromatin condensation. Caspase-3, -8 and -9 activities also increased, suggesting the induction of the caspase-dependent apoptotic pathway. Furthermore, the mitochondrial membrane potential (ΔΨm ) of HeLa cells was lost as a result of increasing levels of Bax and reduced levels of Bcl-2, Bcl-XL, Bcl-Xs, and XIAP. The decreased ΔΨm led to the release of cytochrome c and the activation of caspase-9 and -3. Apoptosis-inducing factor translocated into the nuclei, and endonuclease G (Endo G) was released from the mitochondria. These results suggest that anticancer agents in WBCex can induce apoptosis in HeLa cells via both caspase-dependent and -independent pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 986-997, 2016.

Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.

Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD.

Inhibition of HeLa cells metastasis by bioactive compounds in crocodile (Crocodylus siamensis) white blood cells extract.

Matrix metalloproteinases (MMPs) play a key role in cancer progression, including cell invasion, metastasis, cell growth, apoptosis, angiogenesis, and cell adhesion. Thus, suppression of the MMPs activities is crucial for inhibiting cancer cells metastasis. Herein, bioactive agents from crocodile (Crocodylus siamensis) leukocyte extracts (WBCex) showed the anticancer activity with HeLa cells and inhibited the migration and invasion process by reducing gelatinases (MMP-2, MMP-9) activity and their protein levels. This mechanism is regulated via interfering Ras and p38 signal transduction. Moreover, disrupting VEGF and integrin-signaling cascade by bioactive agents are the predictable mechanisms that cause the decreasing of MMP-2 and MMP-9 activity. Hence, bioactive substances in WBCex may play the mode of action similar with MMPs inhibitor due to HeLa cell metastasis being suppressed in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1329-1336, 2016.

Antibacterial activity of plasma from crocodile (Crocodylus siamensis) against pathogenic bacteria.

BACKGROUND: The Siamese crocodile (Crocodylus siamensis) is a critically endangered species of freshwater crocodiles. Crocodilians live with opportunistic bacterial infection but normally suffer no adverse effects. They are not totally immune to microbial infection, but their resistance thereto is remarkably effective. In this study, crude and purified plasma extracted from the Siamese crocodile were examined for antibacterial activity against clinically isolated, human pathogenic bacterial strains and the related reference strains. METHODS: Crude plasma was prepared from whole blood of the Siamese crocodile by differential sedimentation. The crude plasma was examined for antibacterial activity by the liquid growth inhibition assay. The scanning electron microscopy was performed to confirm the effect of crude crocodile plasma on the cells of Salmonella typhi ATCC 11778. Effect of crude crocodile plasma on cell viability was tested by MTT assay. In addition, the plasma was purified by anion exchange column chromatography with DEAE-Toyopearl 650 M and the purified plasma was tested for antibacterial activity. RESULTS: Crude plasma was prepared from whole blood of the Siamese crocodile and exhibited substantial antibacterial activities of more than 40% growth inhibition against the six reference strains of Staphylococcus aureus, Salmonella typhi, Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus epidermidis, and the four clinical isolates of Staphylococcus epidermidis, Pseudomonas aeruginosa, Salmonella typhi, and Vibrio cholerae. Especially, more than 80% growth inhibition was found in the reference strains of Salmonella typhi, Vibrio cholerae, and Staphylococcus epidermidis and in the clinical isolates of Salmonella typhi and Vibrio cholerae. The effect of the crude plasma on bacterial cells of Salmonella typhi, a certain antibacterial material probably penetrates progressively into the cytoplasmic space, perturbing and damaging bacterial membranes. The effect of the crude plasma was not toxic by the yellow tetrazolium bromide (MTT) assay using a macrophage-like cell, RAW 264.7. The pooled four fractions, designated as fractions D1-D4, were obtained by column chromatography, and only fraction D1 showed growth inhibition in the reference strains and the clinical, human pathogenic isolates. CONCLUSIONS: The crude and purified plasma from the Siamese crocodile significantly showed antibacterial activity against pathogenic bacteria and reference strains by damage cell membrane of target bacterial cells. From the MTT assay, the Siamese crocodile plasma was not cytotoxic to the cells.

Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts.

Four novel antibacterial peptides, Leucrocin I-IV from Siamese crocodile white blood cell extracts were purified by reverse phase high performance liquid chromatography (RP-HPLC). Leucrocins exhibit strong antibacterial activity towards Staphylococcus epidermidis, Salmonella typhi and Vibrio cholerae. The peptides were 7-10 residues in length with different primary structure. The amino acid sequence of Leucrocin I is NGVQPKY with molecular mass around 806.99 Da and Leucrocin II is NAGSLLSGWG with molecular mass around 956.3 Da. Further, the interaction between peptides and bacterial membranes as part of their killing mechanism was studied by fluorescence and electron microscopy. The outer membrane and cytoplasmic membrane was the target of action of Leucrocins as assayed in model membrane by release of ß-galactosidase due to the membrane permeabilization. Finally, the hemolytic effect was tested against human red blood cell. Leucrocin I, III and IV showed less toxicity against human red blood cells than Leucrocin II.

Isolation and characterisation of crocosin, an antibacterial compound from crocodile (Crocodylus siamensis) plasma.

An antibacterial compound from crocodile blood was partially purified and functionally characterised. The freshwater crocodile (Crocodylus siamensis) plasma with antibacterial activity was partially purified by using a centrifugal concentrator and reverse phase high powered liquid chromatography, and designated as crocosin. Crocosin exhibits antibacterial activity toward Salmonella typhi and Staphylococcus aureus. Crocosin is thermostable and resistant to pronase digestion. The structure of crocosin analyzed by mass spectrometry contains repeating units of 94 and 136 m/z. Scanning electron microscopy indicates that crocosin probably penetrates progressively into cytoplasm space, perturbing and damaging bacterial membranes. Crocosin may provide an early defense mechanism toward bacterial infection in freshwater.