A novel RT-qPCR health assay reveals differential expression of stress and immunoregulatory genes between the seasonal migrations of humpback whales (Megaptera novaeangliae).

Health information is essential for the conservation management of whale species. However, assessing the health of free-ranging whales is challenging as samples are primarily limited to skin and blubber tissue. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers a method to measure health from blubber RNA, providing insights into energetic status, stress and immune activity. To identify changes in health, natural differences in baseline gene expression linked to an individual's sex, reproductive status and life-history stage must first be quantified. This study aimed to establish baseline gene expression indices of health in migrating humpback whales (Megaptera novaeangliae). To do this, we developed an assay to quantify seven health-related gene transcripts (Leptin, Leptin Receptor, Adiponectin, Aryl Hydrocarbon Receptor, Tumour Necrosis Factor-α, Interleukin-6, Heat Shock Protein-70) and used Bayesian mixed effect models to assess differential baseline expression based on sex, lactation status and migration stage (northbound to and southbound from the annual breeding grounds). Results showed no significant contribution of sex to differential baseline expression. However, lactating individuals exhibited downregulated AhR and HSP-70 compared to non-lactating conspecifics. Additionally, southbound individuals demonstrated significantly upregulated HSP-70 and downregulated TNF-alpha, suggesting a relationship between these inflammation-linked transcripts and migratory fasting. Our results suggest that baseline differences due to migratory stage and lactation status should be considered in health applications of this assay. Future monitoring efforts can use our baseline measurements to better understand how gene expression is tied to population-level impacts, such as reduced prey availability or migratory stressors.

Minke whale feeding rate limitations suggest constraints on the minimum body size for engulfment filtration feeding.

Bulk filter feeding has enabled gigantism throughout evolutionary history. The largest animals, extant rorqual whales, utilize intermittent engulfment filtration feeding (lunge feeding), which increases in efficiency with body size, enabling their gigantism. The smallest extant rorquals (7-10 m minke whales), however, still exhibit short-term foraging efficiencies several times greater than smaller non-filter-feeding cetaceans, raising the question of why smaller animals do not utilize this foraging modality. We collected 437 h of bio-logging data from 23 Antarctic minke whales (Balaenoptera bonaerensis) to test the relationship of feeding rates (λf) to body size. Here, we show that while ultra-high nighttime λf (mean ± s.d.: 165 ± 40 lunges h-1; max: 236 lunges h-1; mean depth: 28 ± 46 m) were indistinguishable from predictions from observations of larger species, daytime λf (mean depth: 72 ± 72 m) were only 25-40% of predicted rates. Both λf were near the maxima allowed by calculated biomechanical, physiological and environmental constraints, but these temporal constraints meant that maximum λf was below the expected λf for animals smaller than ~5 m-the length of weaned minke whales. Our findings suggest that minimum size for specific filter-feeding body plans may relate broadly to temporal restrictions on filtration rate and have implications for the evolution of filter feeding.

A mammalian messenger RNA sex determination method from humpback whale (Megaptera novaeangliae) blubber biopsies.

The large size of free-ranging mysticetes, such as humpback whales (Megaptera novaeangliae), make capture and release health assessments unfeasible for conservation research. However, individual energetic condition or reproductive health may be assessed from the gene expression of remotely biopsied tissue. To do this, researchers must reliably extract RNA and interpret gene expression measurements within the context of an individual's sex. Here, we outline an RNA extraction protocol from blubber tissue and describe a novel mammalian RNA sex determination method. Our method consists of a duplex reverse transcription-quantitative (real-time) polymerase chain reaction (RT-qPCR) with primer sets for a control gene (ACTB) and the X-chromosome inactivation gene (XIST). Products of each RT-qPCR had distinct melting temperature profiles based on the presence (female) or absence (male) of the XIST transcript. Using high-resolution melt analysis, reactions were sorted into one of two clusters (male/female) based on their melting profiles. We validated the XIST method by comparing results with a standard DNA-based method. With adequate quantities of RNA (minimum of approx. 9 ng µl-1), the XIST sex determination method shows 100% agreement with traditional DNA sex determination. Using the XIST method, future cetacean health studies can interpret gene expression within the context of an individual's sex, all from a single extraction.