Susana Pallarés

The comparative osmoregulatory ability of two water beetle genera whose species span the fresh-hypersaline gradient in inland waters (Coleoptera: Dytiscidae, Hydrophilidae).

A better knowledge of the physiological basis of salinity tolerance is essential to understanding the ecology and evolutionary history of organisms that have colonized inland saline waters. Coleoptera are amongst the most diverse macroinvertebrates in inland waters, including saline habitats; however, the osmoregulatory strategies they employ to deal with osmotic stress remain unexplored. Survival and haemolymph osmotic concentration at different salinities were examined in adults of eight aquatic beetle species which inhabit different parts of the fresh—hypersaline gradient. Studied species belong to two unrelated genera which have invaded saline waters independently from freshwater ancestors; Nebrioporus (Dytiscidae) and Enochrus (Hydrophilidae). Their osmoregulatory strategy (osmoconformity or osmoregulation) was identified and osmotic capacity (the osmotic gradient between the animal’s haemolymph and the external medium) was compared between species pairs co-habiting similar salinities in nature. We show that osmoregulatory capacity, rather than osmoconformity, has evolved independently in these different lineages. All species hyperegulated their haemolymph osmotic concentration in diluted waters; those living in fresh or low-salinity waters were unable to hyporegulate and survive in hyperosmotic media (> 340 mosmol kg-1). In contrast, the species which inhabit the hypo-hypersaline habitats were effective hyporegulators, maintaining their haemolymph osmolality within narrow limits (ca. 300 mosmol kg-1) across a wide range of external concentrations. The hypersaline species N. ceresyi and E. jesusarribasi tolerated conductivities up to 140 and 180 mS cm-1, respectively, and maintained osmotic gradients over 3500 mosmol kg-1, comparable to those of the most effective insect osmoregulators known to date. Syntopic species of both genera showed similar osmotic capacities and in general, osmotic responses correlated well with upper salinity levels occupied by individual species in nature. Therefore, osmoregulatory capacity may mediate habitat segregation amongst congeners across the salinity gradient.

Aquatic insects dealing with dehydration: do desiccation resistance traits differ in species with contrasting habitat preferences?

Background Desiccation resistance shapes the distribution of terrestrial insects at multiple spatial scales. However, responses to drying stress have been poorly studied in aquatic groups, despite their potential role in constraining their distribution and diversification, particularly in arid and semi-arid regions. Methods We examined desiccation resistance in adults of four congeneric water beetle species (Enochrus, family Hydrophilidae) with contrasting habitat specificity (lentic vs. lotic systems and different salinity optima from fresh- to hypersaline waters). We measured survival, recovery capacity and key traits related to desiccation resistance (fresh mass, % water content, % cuticle content and water loss rate) under controlled exposure to desiccation, and explored their variability within and between species. Results Meso- and hypersaline species were more resistant to desiccation than freshwater and hyposaline ones, showing significantly lower water loss rates and higher water content. No clear patterns in desiccation resistance traits were observed between lotic and lentic species. Intraspecifically, water loss rate was positively related to specimens’ initial % water content, but not to fresh mass or % cuticle content, suggesting that the dynamic mechanism controlling water loss is mainly regulated by the amount of body water available. Discussion Our results support previous hypotheses suggesting that the evolution of desiccation resistance is associated with the colonization of saline habitats by aquatic beetles. The interespecific patterns observed in Enochrus also suggest that freshwater species may be more vulnerable than saline ones to drought intensification expected under climate change in semi-arid regions such as the Mediterranean Basin.

Cuticle hydrocarbons in saline aquatic beetles

Hydrocarbons are the principal component of insect cuticle and play an important role in maintaining water balance. Cuticular impermeability could be an adaptative response to salinity and desiccation in aquatic insects; however, cuticular hydrocarbons have been poorly explored in this group and there are no previous data on saline species. We characterized cuticular hydrocarbons of adults and larvae of two saline aquatic beetles, namely Nebrioporus baeticus (Dytiscidae) and Enochrus jesusarribasi (Hydrophilidae), using a gas chromatograph coupled to a mass spectrometer. The CHC profile of adults of both species, characterized by a high abundance of branched alkanes and low of unsaturated alkenes, seems to be more similar to that of some terrestrial beetles (e.g., desert Tenebrionidae) compared with other aquatic Coleoptera (freshwater Dytiscidae). Adults of E. jesusarribasi had longer chain compounds than N. baeticus, in agreement with their higher resistance to salinity and desiccation. The more permeable cuticle of larvae was characterized by a lower diversity in compounds, shorter carbon chain length and a higher proportion of unsaturated hydrocarbons compared with that of the adults. These results suggest that osmotic stress on aquatic insects could exert a selection pressure on CHC profile similar to aridity in terrestrial species.

Limited thermal acclimation capacity in cave beetles

Thermal tolerance is a key vulnerability factor for species that cannot cope with changing conditions by behavioural adjustments or dispersal, such as subterranean species. Previous studies of thermal tolerance in cave beetles suggest that these species may have lost some of the thermoregulatory mechanisms common in temperate insects, and appear to have a very limited thermal acclimation ability. However, it might be expected that both thermal tolerance and acclimation ability should be related with the degree of specialization to deep subterranean environments, being more limited in highly specialized species. To test this hypothesis, we use an experimental approach to determine the acclimation capacity of cave beetles within the tribe Leptodirini (family Leiodidae) with different degrees of specialization to the deep subterranean environment. For this, we acclimate groups of individuals at 1. a temperature close to their upper thermal limit (20oC) or 2. a control temperature (approximately that of the cave in which they were found) for 2 or 10 days (shortvs. long-term acclimation). ‡ § | ¶ # ¤ ‡

The chicken or the egg? Adaptation to desiccation and salinity tolerance in a lineage of water beetles

Transitions from fresh to saline habitats are restricted to a handful of insect lineages, as the colonization of saline waters requires specialized mechanisms to deal with osmotic stress. Previous studies have suggested that tolerance to salinity and desiccation could be mechanistically and evolutionarily linked, but the temporal sequence of these adaptations is not well established for individual lineages. We combined molecular, physiological and ecological data to explore the evolution of desiccation resistance, hyporegulation ability (i.e., the ability to osmoregulate in hyperosmotic media) and habitat transitions in the water beetle genus Enochrus subgenus Lumetus (Hydrophilidae). We tested whether enhanced desiccation resistance evolved before increases in hyporegulation ability or vice versa, or whether the two mechanisms evolved in parallel. The most recent ancestor of Lumetus was inferred to have high desiccation resistance and moderate hyporegulation ability. There were repeated shifts between habitats with differing levels of salinity in the radiation of the group, those to the most saline habitats generally occurring more rapidly than those to less saline ones. Significant and accelerated changes in hyporegulation ability evolved in parallel with smaller and more progressive increases in desiccation resistance across the phylogeny, associated with the colonization of meso‐ and hypersaline waters during global aridification events. All species with high hyporegulation ability were also desiccation‐resistant, but not vice versa. Overall, results are consistent with the hypothesis that desiccation resistance mechanisms evolved first and provided the physiological basis for the development of hyporegulation ability, allowing these insects to colonize and diversify across meso‐ and hypersaline habitats.