Although she may not look it, the malaria-transmitting female mosquito Anopheles gambiae is a fussy insect; she won't breed in any old water and likes to lay her eggs in clean freshwater. However, some of her relatives are less picky and have evolved the ability to breed in saltwater. This switch is not easy, as Nora Besansky, from the University of Notre Dame, USA explains: ‘When the external environment is a lot saltier than they are, [the mosquitoes] have to regulate ion balance by actively pumping out ions or matching the salinity of the environment.’ While A. gambiae mosquitoes may not breed in saltwater, in recent years they have started to breed in water high in other ions, such as ammonia. As this trait might be related to salt tolerance, Besansky and her team wanted to identify the genetic basis of salt tolerance in close relatives (p. ).
Explaining her ultimate plan, Besansky says, ‘The way you genetically dissect a trait is by crossing organisms that differ in that trait and then follow the segregation of genetic markers associated with the trait in extended pedigrees.’ However, the team first needed to determine what concentration of salt would kill intolerant, but not tolerant, mosquitoes. ‘We exposed colonies of the freshwater- and saltwater-[tolerant] species to different doses of salt and then monitored their survival’, explains Besansky. ‘In acute assays we limited exposure to 24 h at a late developmental stage of the larvae, whereas in chronic exposures the different doses were kept constant from hatching up to the emergence of the adult.’
Besansky admits that the experiment took a lot of patience; her post doc Bradley White, and undergraduate student Peter Kundert, spent hours gently prodding over 50,000 larvae from three different mosquito species to see whether they reacted and were alive. The work paid off, and the team found that 15.85 g NaCl l−1 was enough salt to kill off all larvae from two freshwater species, A. gambiae and Anopheles coluzzii, in both chronic and acute exposures.
However, although the salt-tolerant Anopheles merus larvae survived without problems during the chronic exposure, the team noticed they weren't tolerant during acute exposures. Prior to acute exposure all the larvae were kept in freshwater, and the team wondered whether A. merus larvae need earlier exposure to salt to gain tolerance. Sure enough, the team found that larvae only became tolerant if they had been exposed to salt within 24 h of hatching. Besansky knew that in a very distant relative, early exposure to saltwater was crucial for altering the tissue location of a Na+/K+-ATPase (an ion pump, which may help when dealing with salty water). Teaming up with Paul Linser's group at University of Florida, USA, the team found a similar switch occurring in A. merus, although what controls this location switch is unknown.
Next, the team decided to see how offspring with A. merus and A. coluzzii parents would fare, and found that their tolerance was in-between that of their parents. ‘We think this means that there are multiple factors that control the genetics of this trait, and it tells us that the factors conferring salt tolerance are not dominant, otherwise the offspring would have looked like the saltwater-tolerant parent’, says Besansky. The team also found that offspring whose mothers were saltwater tolerant did better than those with a freshwater-loving mum. Besansky suspects that this means that some of the tolerance may be inherited through the female X chromosome. An extensive breeding program now lies ahead of Besansky and her team to pinpoint the exact genetic basis of salt tolerance, but the hunt has begun.
