Integrated assessment of chemical pollution and its impacts on wild
populations of Crocodylus moreletii in the Mexican Yucatan Peninsula
Mauricio González-Jáuregui1, Asela Marisol Buenfil Rojas2, Teresa Álvarez-Legorreta3,José António L. Barão-Nóbrega4, Sergio E. Padilla-Paz1, Hisato Iwata2,5, Kei Nomiyama2,5
1 Autonomous University of Campeche – CEDESU
2 Ehime University
3 El Colegio de la Frontera Sur – Unidad Chetumal
4 Operation Wallacea
5 LaMer

Purposes
The proposed study will measure levels of metals and trace elements (V, Cr, Mn,Co, Cu, Zn, Se, As, Rb, Sr, Mo, Ag, Cd, Sn, Sb, Cs, Ba, Hg, Tl, Pb, and Bi) andPOPs (e.g., PCBs, dichlorodiphenyltrichloroethane (DDTs), poly-brominateddiphenyl ethers (PBDEs), and perfluoroalkyl substances (PFASs)) in bloodplasma, caudal scutes, and claw samples of 11 wild C. moreletii populations
within the YP (n = 10 per site).
Simultaneously, we will also reveal transcriptome (~10,000 transcripts) andmetabolome profiles (hundreds of lipids, carbohydrates, amino acids, andhormones) in these samples. Bioinformatic approaches will be applied todetermine the biomarkers and the biological pathways/networks affected bycontaminants [1].

Materials and Methods
Our proposed plan is being conducted in two teams: Fieldwork in Mexico and Lab
work in Japan. For the fieldwork in Mexico, crocodile capture and collection ofsamples was executed following our well-established protocols, targeting
reproductive adults (>1.0 m total length) [2, 3]. Blood samples (whole andplasma) were drawn from all captured individuals prior to their body size
measurement and then caudal scutes and claws were obtained [2, 3]. To
determine health condition, blood smears were prepared for blood cell counts.
Eight fieldwork campaigns for sample collection were conducted from July 2021
– July 2023. The previous plan only envisioned six campaigns from July 2021 –November 2022; however, the number of samples was below 70%. Thus, twomore campaigns were conducted in 2023 to achieve the number of samples
expected, and simultaneously, a postdoctoral collaboration from a researcher

4 / 9from Northern Mexico allowed not only to achieve the targeted number but to get12% more samples than expected in the original plan. Blood counts in Mexico
are still in progress.
Samples were preserved frozen and transcriptome samples were preserved inRNAlater® solution (Invitrogen™) until their arrival at the laboratory of theAutonomous University of Campeche (CEDESU). The second batch of samples
exportation permission is currently under issuance for their subsequent
exportation to the host researcher’s laboratory at Ehime University, Japan.
For the Lab work in Japan, the first batch of samples of blood samples, claws andscutes were digested in a microwave system, and metals and trace elements
were determined by HG-AAS, ICP-MS, and CV-AAS [4]. POPs in blood plasmaand scutes were determined by GC-MS [1]. Transcriptome profiles in soft tissuesof caudal scutes were successfully determined by next-generation RNA
sequencing (RNA-seq) using Illumina HiSeq 2500 system [1]. Metabolites will bedetermined in blood plasma by LC coupled with Q-TOF-MS [5-7]. Differences incontaminant concentrations between populations will be tested through leftcensored data analyses [8], followed by post-hoc analyses. Relationships
between contaminant levels, metabolites and transcriptome profiles will beevaluated using class coinertia analysis (CIA) and factor analysis for multiple
testing (FAMT) [1]. For mRNAs and metabolites that exhibit relationships withcontaminant levels, enrichment analysis of transcription factors, pathways,
networks, and diseases will be performed using databases (KEGG, Reactomeand DiseaseComps) and bioinformatics analysis tools (DAVID, STRING,Cytoscape and TfactS).
Results
By July 2023, a total of 125 individuals were captured and 858 samples wereobtained for this project. We observed 29.5% of the captured crocodiles areabove their “optimal” weight, while 70.4% of them are below their “optimal” weight.
Metal and POPs concentrations have been determined in the first batch ofsamples (figs. 1 and 2) and 16 libraries of good quality for transcriptome analysis
were successfully generated (figs. 3 and 4). Currently we are processing thetranscripts by two bioinformatic approaches: mapping genes by using a genomereference (Alligator misssissippiensis) or de novo analysis to generate our mRNAreference based on our libraries results. Depending on the best mapping
numbers, we will perform the rest of the analysis and subsequently apply
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mixOmics to understand how contaminants might be related to transcriptome andmetabolome results.

Figure 1. DDTs and PCBs linear relationships in blood plasma of C. moreletii from 2021.

Figure 2. Heatmap analysis of trace elements in caudal scutes of C. moreletii collected in
the Yucatan Peninsula, Mexico 2021.

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Figure 3. RNA extracts quality control for caudal scutes biopsies of C. moreletii collected inthe Yucatan Peninsula, Mexico 2021.

Figure 4. FastQC summary of the library generated for sample CBR18_1.

Future Challenges
1) We expected to receive 585 samples from the second batch this year. However,exportation is getting relatively problematic due to bureaucratic issues(Presidential elections). We are working our best to collaborate with the Mexican
government to go through the procedure as soon as possible.
2) After receiving the second batch, we still need to consider enough budget togenerate 95 libraries of RNA, based on the number of samples collected for7 / 9transcriptome.
3) After getting the best bioinformatics approach for the mappings and we needto select the best approach for the integration of all the datasets.

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