Effects of ancient anthropogenic clam gardens on the growth, survival, and transcriptome of Pacific littleneck clams ( Leukoma staminea )

Clam gardens are habitat modifications established by coastal Indigenous Peoples of northwest North America to enhance intertidal clam habitat productivity to provide secure and reliable local food resources. These gardens were established long ago and are mostly unmaintained in present times. To determine whether unmaintained clam gardens still provide a more productive and beneficial habitat than unmodified clam beaches, Pacific littleneck clams ( Leukoma staminea ) were transplanted to unmaintained clam gardens or unmodified reference beaches and then evaluated for growth, survival, and transcriptomic signatures of the gill and digestive gland after 16 weeks. All beaches in the study were characterized for sediment grain-size distribution, carbonate concentration, and organic content to identify sediment characteristics that may differ between clam garden and reference sites, as well as potentially contribute to differences in clam productivity. Clam growth and survival, sediment grain-size distribution, and carbonate and organic content were not significantly affected by unmaintained clam gardens. Wide variations in survival, growth, and sediment characteristics were observed among beaches. Examining across beach location resulted in the identification of significant negative effects of small rocks (2.00 – 4.75 mm), and silt (< 63 µm) on both growth and survival. Sand (250 – 500 µm) had a significant positive influence on both growth and survival while fine sand (125 – 250 µm) had a significant positive effect only on growth. Coarse sand (0.50 – 1.00 mm) had a significant positive effect on survival, while very fine sand (63 – 125 µm), carbonate, and organic content all had significant negative effects. To evaluate transcriptomic effects, a de novo transcriptome for L. staminea was assembled. The final assembly contained 52,000 putative transcripts and those specific to each of the two tissues were identified. This revealed that similar functional categories were enriched in tissue-specific genes, but each tissue had its own transcripts comprising the categories. Transcriptomic analysis revealed differential expression in individuals from clam gardens, and although this effect was small in terms of the numbers of genes, specific response genes were identified consistently in both tissues. In summary, while the unmaintained clam gardens did not impact clam growth and survival over the 16 weeks of the study, it did greater influence on Pacific littleneck clam physiology, growth, and survival than the presence/absence of unmaintained clam gardens. mRNA enrichment with the NEB Next Poly(A) Magnetic Isolation Module (New England Biolabs) and cDNA synthesis was conducted using NEBNext RNA First Strand Synthesis and NEBNext Ultra Directional RNA Second Strand Synthesis Modules. Remaining library preparation used NEBNext Ultra II DNA Library Prep Kit for Illumina, with adapters and PCR primers (NEB). The remaining steps of library preparation were taken using the Quant-iT ™ PicoGreen ® dsDNA Assay Kit (Life Technologies) and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal Kit (Kapa Biosystems (Pty) Ltd.). Average fragment size was determined using a LabChip GX instrument (PerkinElmer). Libraries were run across four lanes of an Illumina HiSeq4000 PE 100 platform (Illumina) and the mean (± SD) read pairs per library (both tissues combined) was 54.4 ± 9.5 million. All library practices and shell-hash have on bivalve health. Transcripts related to stress response were identified by conducting differential expression analysis among beaches of largely differing survival. The characterized transcriptome provides a valuable resource for future transcriptomic studies investigating genetic and phenotypic responses of clams in different habitats and environmental conditions.