Rodriguez, S. M. | bioRxiv Science

bioRxiv2026

Optimizing DNA extraction from environmentally degraded bone samples for molecular identification of cetacean species

Molecular identification of cetacean bone remains can be limited by DNA degradation and the presence of PCR inhibitors. Here, we present an optimized DNA extraction protocol based on a total demineralization method for environmentally exposed cetacean bones. The protocol uses 100 mg of bone powder, 24 h digestion with EDTA, N-lauroylsarcosine, and proteinase K, followed by a modified silica-column purification. Nine environmentally degraded bone samples representing eight individuals were processed. DNA concentrations ranged from 7.3 to 57.1 ng/uL (mean SD = 25.91- 13.91 ng/uL). The mitochondrial cytochrome b gene was successfully amplified from all samples using conventional PCR, and five samples (55.6%) yielded sequences suitable for downstream analysis. BLASTn identified Balaenoptera physalus as the closest database match for all recovered sequences, and phylogenetic analysis further supported their association with B. physalus reference sequences. These results demonstrate that the proposed protocol provides a practical approach for recovering amplifiable and molecularly informative mitochondrial DNA from environmentally degraded cetacean bone material, facilitating molecular identification from challenging skeletal remains.

genetics↗

bioRxiv2025

Balancing sample sizes in parasitology: A novel experimental infection method using faecal parasite eggs and aquatic intermediate hosts.

Balancing sample sizes between infected and uninfected hosts is a common challenge in ecological parasitology, particularly when dealing with intermediate hosts involved in complex life cycles. In these life cycles, common in nature, the intermediate hosts accumulate parasites across their ontogeny, making it difficult to obtain similar numbers of naturally infected and uninfected individuals across full host body-size spectra. Here, we present a standardized and repeatable method to experimentally infect and re-infect intermediate hosts using parasite eggs derived directly from the fresh faeces of definitive hosts. We tested this methodology in a marine host-parasite system involving the acanthocephalan Profilicollis altmani, the mole crab Emerita analoga, and the grey gull Leucophaeus modestus. Crabs were raised in controlled laboratory conditions from early juvenile stages, ensuring no prior infections, and exposed to parasite eggs through filtered faecal suspensions. Larval development was tracked across two inoculation events. The first larval stage (acanthellae) appeared six days after inoculation and matured into cystacanths within the following week. After the first exposure, 55% of inoculated hosts were infected; after the second, infection prevalence reached 100%. Larger crabs acquired more parasites than smaller ones, likely due to higher filtration rates. The method closely mimics natural faecal-oral transmission while avoiding the need to isolate or handle parasite eggs directly. It also overcomes the common issue of parasite overdispersion in natural populations by enabling controlled infections across host size classes and experimental replicates. This technique is particularly useful in species with opaque cuticles, where internal parasites cannot be visually identified in vivo. Additionally, it can be extended beyond acanthocephalans to other helminth taxa (e.g., nematodes, cestodes, and digeneans) whose eggs are also shed in vertebrate faeces. Our results establish a reliable, ecologically relevant protocol for experimental parasitology in marine and freshwater systems.

ecology↗