Local adaptation to hummingbirds and bees in Salvia stachydifolia: insights into pollinator shifts in a Southern Andean sage.

BACKGROUND AND AIMS: Differences among populations in pollinator assemblages can lead to local adaptation mosaics in which plants evolve different floral morphologies and attractive traits. Mountain habitats may promote local adaptation because of differences in environmental conditions with altitude, causing changes in pollinators, and because mountaintops can act as isolated habitats. We studied if the differences in floral shape, size and nectar traits in Salvia stachydifolia can be attributed to variations in the relative contribution of hummingbirds and insects. METHODS: We studied eight populations of S. stachydifolia in natural and under common garden conditions, to assess whether population differences have a genetic component. We recorded pollinators, their behaviour and visitation rates, and characterized pollinator assemblages. In addition, we measured nectar volume and concentration, and collected flowers to describe floral shape and size variation using geometric morphometric methods. We then applied an unsupervised learning algorithm to identify ecotypes based on morphometric traits. Finally, we explored whether populations with different pollinator assemblages had different climatic and/or elevation preferences. KEY RESULTS: We found that variation in the identity of the main pollinators was associated with differences among populations in all traits, as expected under a local adaptation scenario. These differences persisted in the common garden, suggesting that they were not due to phenotypic plasticity. We found S. stachydifolia populations were pollinated either by bees, by hummingbirds or had mixed pollination. We identified two ecotypes that correspond to the identity of the main pollinator guilds, irrespective of climate or altitude. CONCLUSIONS: Variation in S. stachydifolia floral traits did not follow any evident association with bioclimatic factors, suggesting that populations may have diverged as the product of historical isolation on mountaintops. We suggest that differences among populations point to incipient speciation and an ongoing pollinator shift.

Fragility of nocturnal interactions: Pollination intensity increases with distance to light pollution sources but decreases with increasing environmental suitability.

Light pollution represents a widespread long-established human-made disturbance and an important threat to nocturnal pollination. Distance from the niche centroid where optimal environmental conditions join may be related to species sensitivity to habitat change. We estimated the environmental suitability of the plant species Erythrostemon gilliesii and of its guild of hawkmoth pollinators. We considered the overlap of suitability maps of both partners as the environmental suitability of the interaction. We used a three-year record of ten E. gilliesii populations to calculate pollination intensity as the number of individuals that received pollen per population. In addition, for each population, we measured the distance to the high light pollution source around a buffer of 15 km radius. Finally, we predicted pollination intensity values for environmental suitability ranging from 0 to 1, and distance to high light pollution sources ranging from 0 to 56 Km. Pollination intensity decreased along an axis of increasing environmental suitability and increased with distance to sources of light pollution. The highest values of pollination intensity were observed at greatest distances to sources of light pollution and where environmental suitability of the interaction was lowest. The prediction model evidenced that, when environmental suitability was lowest, pollination intensity increased with distance to sources of high light pollution. However, when environmental suitability was intermediate or high, pollination intensity decreased away and until 28 km from the sources of high light pollution. Beyond 28 km from the sources of high light pollution, pollination intensity remained low and constant. Populations under conditions of low environmental suitability might be more likely to respond to disturbances that affect pollinators than populations under conditions of high environmental suitability.

Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression.

Haploidentical Natural Killer (NK) cells have been shown as an effective and safe alternative for the treatment of haematological malignancies with poor prognosis for which traditional therapies are ineffective. In contrast to haematological cancer cells, that mainly grow as single suspension cells, solid carcinomas are characterised by a tridimensional (3D) architecture that provide specific surviving advantages and resistance against chemo- and radiotherapy. However, little is known about the impact of 3D growth on solid cancer immunotherapy especially adoptive NK cell transfer. We have recently developed a protocol to activate ex vivo human primary NK cells using B lymphoblastic cell lines, which generates NK cells able to overcome chemoresistance in haematological cancer cells. Here we have analysed the activity of these allogeneic NK cells against colorectal (CRC) human cell lines growing in 3D spheroid culture and correlated with the expression of some of the main ligands regulating NK cell activity. Our results indicate that activated NK cells efficiently kill colorectal tumour cell spheroids in both 2D and 3D cultures. Notably, although 3D CRC cell cultures favoured the expression of the inhibitory immune checkpoint PD-L1, it did not correlate with increased resistance to NK cells. Finally, we have analysed in detail the infiltration of NK cells in 3D spheroids by microscopy and found that at low NK cell density, cell death is not observed although NK cells are able to infiltrate into the spheroid. In contrast, higher densities promote tumoural cell death before infiltration can be detected. These findings show that highly dense activated human primary NK cells efficiently kill colorectal carcinoma cells growing in 3D cultures independently of PD-L1 expression and suggest that the use of allogeneic activated NK cells could be beneficial for the treatment of colorectal carcinoma.