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.

Blubber gene expression and cortisol concentrations reveal changing physiological stress in a Southern ocean sentinel species.

The health of migratory eastern Australian humpback whales (Megaptera novaeangliae) can reflect the condition of their remote polar foraging environments. This study used gene expression (LEP, LEPR, ADIQ, AhR, TNF-α, HSP-70), blubber hormone concentrations (cortisol, testosterone), and photogrammetric body condition to assess this sentinel species during a period of unprecedented changes to anthropogenic activity and natural processes. The results revealed higher cortisol concentrations in 2020 compared to 2021, suggesting a decline in physiological stress between years. Additionally, metabolic transcripts LEPR, and AhR, which is also linked to xenobiotic metabolism, were upregulated during the 2020 southbound migration. These differences suggest that one or more environmental stressors were reduced between 2020 and 2021, with upregulated AhR possibly indicating a Southern Ocean pollutant declined between the years. This research confirms a Southern Ocean-wide decrease in whale stress during the study period and informs efforts to identify key stressors on Antarctic marine ecosystems.

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.

Measuring physiological stress in Australian flying-fox populations.

Flying-foxes (pteropid bats) are the natural host of Hendra virus, a recently emerged zoonotic virus responsible for mortality or morbidity in horses and humans in Australia since 1994. Previous studies have suggested physiological and ecological risk factors for infection in flying-foxes, including physiological stress. However, little work has been done measuring and interpreting stress hormones in flying-foxes. Over a 12-month period, we collected pooled urine samples from underneath roosting flying-foxes, and urine and blood samples from captured individuals. Urine and plasma samples were assayed for cortisol using a commercially available enzyme immunoassay. We demonstrated a typical post-capture stress response in flying-foxes, established urine specific gravity as an attractive alternative to creatinine to correct urine concentration, and established population-level urinary cortisol ranges (and geometric means) for the four Australian species: Pteropus alecto 0.5-305.1 ng/mL (20.1 ng/mL); Pteropus conspicillatus 0.3-370.9 ng/mL (18.9 ng/mL); Pteropus poliocephalus 0.3-311.3 ng/mL (10.1 ng/mL); Pteropus scapulatus 5.2-205.4 ng/mL (40.7 ng/mL). Geometric means differed significantly except for P. alecto and P. conspicillatus. Our approach is methodologically robust, and has application both as a research or clinical tool for flying-foxes, and for other free-living colonial wildlife species.