Do whales accumulate mtDNA damage in their skin as they age and does pigmentation confer protection against UV-induced mtDNA damage?

Using quantitative real time PCR methodology (qPCR), we screened for mtDNA damage from the skin of three whale species (fin, sperm and blue whales; Fig. 1A) with different skin colour (Fig. 1A–C) and sea surface behaviour. The level of blue whale mtDNA damage was inversely predicted by melanin density of individual samples (t 11 = −2.65, p = 0.02; see details in Table S1A; Fig. 2C), which suggests that greater pigmented individuals accumulate lower levels of damage. In addition, the abundance of melanin was also inversely related to the level of microscopic lesions (cytoplasmic vacuolation and intracellular oedema, methodology described previously8)) for whales across the three species studied (t 95 = −2.11, p = 0.04 and t 95 = −4.19, p<0.0001 respectively; see details in Table S2; Fig. 2A and B). Furthermore, as predicted under the assumption that mtDNA damage accumulates over time within the cell (aided by compromised mtDNA repair mechanisms10), the amount of whale skin mtDNA lesions increased with age for blue whales (t 11 = 2.24, p = 0.05; see details in Table S1A; Fig. 2E).

Figure 1 The contrasting skin colours of the three studied species. (A) Photograph showing, from top to bottom, a blue whale (pale grey skin colour, the lightest species), a sperm whale (dark grey skin colour) and a fin whale (black skin colour, the darkest species). This photograph was taken by DG. (B) Differences in density of melanocytes (thick bars) and melanin (thin bars) amongst the three studied species (n = 53, n = 17, n = 45 for blue, sperm and fin whales respectively). (C) Association between melanin abundance and melanocyte counts in whales. Grey dots correspond to blue whales, black dots to fin whales and crosses to sperm whales. The counting area was determined as previously described (see8). Briefly, the number of melanocytes per 100 arbitrary units was determined in triplicate along the entire epidermal ridge. The number of epidermal ridges to count was established a priori based on a cumulative curve8. Full size image

Figure 2 Melanin abundance, HSP70 expression and age influence sensitivity to UV-induced damages in whales. Relationship between whale melanin abundance and skin lesions: (A) intracellular oedema (n = 39 and n = 66 for absence and presence, respectively) (B) cytoplasmic vacuolation (n = 9, n = 23, n = 41, n = 35 for vacuolation levels of 0, 1, 2 and 3, respectively; see8) and (C) blue whale mitochondrial DNA lesions. (D) Inverse correlation between mitochondrial DNA (mtDNA) lesion density and HSP70 expression levels (ΔCt). (E) Direct correlation between mtDNA lesion density and individual minimum age of blue whales (calculated by taking into account the first year of observation reported for a particular individual in the Gulf of California; individuals with a minimum age of 1 were excluded; individuals that were observed in the Gulf of California on the year they were born and as such their exact age was known, were included). Bars = ± 95% CI. Full size image

Melanin abundance was not only dependent on the quantity of melanocytes (F 1,40 = 19.83, p<0.0001; Fig. 1C) but also on the transcriptional activity of TYR and P53 (F 2,40 = 6.43, p = 0.004 and F 1,40 = 4.06, p = 0.05, respectively), genes known to be involved in the process of melanogenesis23,24,25,26. The abundance of melanocytes8 and of melanin was highest for fin whales, the darkest of the species studied here (t 95 = 3.99, p = 0.0001 and t 95 = 3.42, p<0.001 when compared with sperm and blue whale, respectively; see details in Table S2; Fig. 1B and Fig. S1A). Intriguingly, both blue and sperm whales shared comparable levels of melanin (t 95 = −0.93, p = 0.35; see details in Table S2), although melanocytes were more prevalent in sperm whales than in blue whales8, suggesting that melanin production capacity is restricted in sperm whales compared to blue whales.

The capacity of whales to respond to UV and the interspecies differences in the molecular mechanisms used in this process.

To accomplish this we tested three hypotheses. Firstly, we predicted that UV-induced damage would lead to changes in the expression of genes involved in genotoxic stress pathways in the studied whale species. Secondly, species with different skin colour and sea surface behaviour (time spent at the sea surface) might use different cellular mechanisms to protect themselves from UV exposure. Thirdly, whales would expectedly modulate UV-counteractive molecular mechanisms in response to the seasonal increase in UV levels.

As part of the process in addressing the first two hypotheses, the level of HSP70, an indicator of cellular stress15,16, was measured using qPCR. Transcription of HSP70 was significantly higher in sperm whales when compared with fin and blue whales (t 30 = 4.57, p = 0.0001 and t 30 = 3.75, p<0.001 respectively; see details in Table S3B; Fig. S1B). In addition, HSP70 expression and mtDNA lesions were found to be inversely related across species (t 15 = −4.26, p<0.001, see details in Table S1B; Fig. 2D). To complete the process of addressing the two hypotheses, the expression of KIN, a cell cycle control protein up-regulated by UV was determined19,20. The results show that KIN expression was also higher in sperm whales (t 33 = 3.12, p = 0.004 and t 33 = 3.95, p<0.0005; see details in Table S3A; Fig. S1B).

To address the third hypothesis of seasonal changes in stress response, we found that when studying temporal changes in gene expression levels, the transcription levels of P53 and HSP70 formed an ascending curve between February and May, peaking in March/April (LR = 11.93, p<0.01 and LR = 8.96, p = 0.03, respectively; see details in Table S3; Fig. 3A). Following the seasonal increase in UV radiation that reached the Gulf of California (Fig. 3B), blue whales increased melanocyte abundance (F 1,47 = 3.07, p = 0.04; see details in Table S4; Fig. 3C), suggesting that individuals of this species are capable of modulating their degree of pigmentation. Although statistically non-significant, melanin density appeared to follow the same increasing trend over months suggesting tanning ability (see details in Table S4; Fig. 3C). Intriguingly, we did not observe analogous changes in the pigmentation of the (comparatively darker) fin whale (Fig. 3D).

Figure 3 Monthly fluctuations in gene expression and pigmentation levels follow seasonal variation in UV. (A) Monthly differences in mean expression levels of P53 and HSP70 genes in blue and fin whales (data pooled; expressed as ΔCt, y axis inverted; n = 11, 13, 12 and 6 for February, March, April and May, respectively). Sperm whales were not included as they were sampled exclusively in May. (B) UV index recorded between January and June over the Gulf of California, Mexico (data average records for 26°–28°N and 109°–112°W) for the years 2007 (red), 2008 (blue) and 2009 (green). Calculation (a simply function of total column ozone and the solar zenith angle) was conducted under local noon and clear sky conditions and does not consider cloud or aerosol effects. Observation years extend from 1979–2010 (32-year running average shown by a thick black line). The lower and upper thin black lines show the minimum and maximum value observed, respectively. The grey shading shows the probability distribution function (i.e., 80% of the observations are within the light grey shading, while 40% are within the dark shading). Plot obtained using total ozone observations from the Total Ozone Mapping Spectrometer (TOMS) and Ozone Monitoring Instrument (OMI). This figure was constructed by Eric Nash and Paul Newman from NASA Goddard Space Flight Center. (C) Monthly variation of blue whale melanocyte and melanin abundance during 2007 (n = 3, 13, 7 and 3 for February, March, April and May, respectively). (D) Monthly variation of fin whale melanocyte and melanin abundance during 2008 (n = 6, 3 and 2 for February, March and April, respectively). Bars ± 95% CI. Full size image

Another, non-exclusive, hypothesis for the observed differences in pigmentation plasticity amongst species might entail their distinct migratory behaviour. While fin whales are year-long residents of the Gulf of California27, blue whales migrate annually from higher to lower latitudes28, where levels and intensity of UV are greater29. Consequently, when blue whales arrive at the Gulf of California they are exposed suddenly to higher levels of UV. It is possible that the higher prevalence of microscopic lesions14 and mtDNA lesions that occur at the beginning of the season (Fig. 4A, Table S1A) reflects the time needed for UV acclimatization to occur30, especially as blue whale pigmentation seems to then increase gradually (Fig. 4B).