Mi­cro­bial Match­mak­ers

by Merry Youle

Back in 1983, re­searchers at Yale bor­rowed a mi­cro­bi­o­log­i­cal tac­tic to study evo­lu­tion at a "sin­gle gene lo­cus" in a mul­ti­cel­lu­lar an­i­mal — the use of al­tered growth me­dia to se­lect for nu­tri­tional mu­tants. They con­fronted pop­u­la­tions of Drosophila pseudoob­scura with a growth medium con­tain­ing ei­ther mal­tose or starch as the sole car­bo­hy­drate source and ob­served in­her­ited changes to the α‑amylase al­lozyme fre­quen­cies and the enzyme's lo­ca­tion within the gut in the "starch flies." In­ter­est­ing, use­ful, but not rev­o­lu­tion­ary.

A mat­ing pair of Drosophila melanogaster. Source

A few years later a Yale Ph.D. stu­dent work­ing with the same flies re­ported some­thing un­ex­pected. Look­ing at pop­u­la­tions that had been reared on the mal­tose or starch medium for a year, she found that starch flies pre­ferred to mate with starch flies rather than mal­tose flies, and like­wise the mal­tose flies pre­ferred mal­tose mates. In her pa­per she con­cluded that the new be­hav­ior was a pleiotropic by-prod­uct of the adap­tion to the dif­fer­ent me­dia.

And so the mat­ter rested for about two decades un­til re­vis­ited by a group of five re­searchers from Is­rael and one from Maine, with some most in­trigu­ing — and mi­cro­bial — in­sights. (You knew that there had to be a mi­crobe lurk­ing some­where. Oth­er­wise I wouldn't be writ­ing about fly mat­ing be­hav­ior here.) In their re­cent PNAS pa­per they present ev­i­dence that the flies raised on dif­fer­ent food sources har­bor dif­fer­ent pop­u­la­tions of bac­te­r­ial com­men­sals, and that it's these mi­crobes that are the match­mak­ers.

The ex­per­i­men­tal pro­ce­dure. A pop­u­la­tion of flies was di­vided, reared for a num­ber of gene­rations on ei­ther starch or CMY me­dia, grown sep­a­rately for one gene­ration on CMY medium, and then tested for mat­ing pre­fer­ence. Mul­ti­ple-choice mat­ing tests were per­formed in wells con­tain­ing four flies: one male and one fe­male starch-reared and one male and one fe­male CMY-reared. Source

These re­searchers used an in­bred lab strain of wild type D. melanogaster reared on the stan­dard corn­meal-mo­lasses-yeast agar medium (CMY). For their ex­per­i­ments, they di­vided the flies into two groups, one reared on CMY and the other on a starch medium for the spec­i­fied num­ber of gen­er­a­tions. All flies were then grown for one more gen­er­a­tion in CMY to re­move any di­rect ef­fects from the me­dia it­self be­fore be­ing tested for their mat­ing pref­er­ence. Again there were more ho­mogamic ("like with like") mat­ings — the CMY flies pre­fer­ring CMY flies, starch flies pre­fer­ring starch flies — thus ex­tend­ing the ear­lier re­sults to a dif­fer­ent Drosophila species reared on a dif­fer­ent pair of growth me­dia.

But the re­searchers didn't stop here. Next they tested to see how quickly the mat­ing pref­er­ences arose af­ter trans­fer to the dif­fer­ent me­dia. The an­swer was af­ter only two gen­er­a­tions, the ear­li­est time­point they tested. This al­most in­stan­ta­neous adap­ta­tion by a mul­ti­cel­lu­lar an­i­mal brought to mind an ear­lier ob­ser­va­tion by Eu­gene Rosenberg's lab (one of the au­thors of this Drosophila pa­per) when study­ing a coral (Oculina patag­o­nica) found along Israel's Mediter­ranean coast. These corals, which had pre­vi­ously been bleached each sum­mer by the pathogen Vib­rio shiloi, in 2004 sud­denly be­came re­sis­tant to in­fec­tion. In a sub­se­quent pa­per, Ilana Zil­ber-Rosen­berg and Eu­gene Rosen­berg noted that all mul­ti­cel­lu­lar plants and an­i­mals carry a com­plex mi­cro­biota that can po­ten­tially en­able them to re­spond rapidly to chang­ing con­di­tions. When con­fronted with a new pathogen, a new her­bi­cide, or some other en­vi­ron­men­tal shift, it is the holo­biont — the host plus sym­bionts — that is acted on by nat­ural se­lec­tion. Adap­ta­tion can re­sult from changes to the genomes of the host or any sym­biont, by the ac­qui­si­tion of new sym­bionts or by a shift in the rel­a­tive abun­dance of ex­ist­ing ones.

Re­sults for mat­ing pref­er­ence tests af­ter grow­ing sepa­rately for 11 gen­er­a­tions on starch or CMY medium. Source

Re­turn­ing to the Drosophila ex­per­i­ments, the re­sults sug­gest that the rapid ac­qui­si­tion of new mat­ing pref­er­ences might be due to a change in the fly's mi­cro­biota. Any ev­i­dence for this? Starch and CMY flies reared in the pres­ence of an­tibi­otics ("cured" flies) ex­hibit ran­dom mat­ing. Fur­ther­more, 16S rRNA analy­sis showed a marked dif­fer­ence in the mi­cro­biota as­so­ci­ated with the CMY flies ver­sus the starch flies. One species — Lac­to­bacil­lus plan­tarum — that made up 3% of the com­mu­nity in the CMY flies in­creased to 26% in the starch flies (vi­able count of 2.3 × 10⁵ per fly). This is not too sur­pris­ing since chang­ing an animal's diet is known to al­ter the mi­cro­biota and L. plan­tarum makes an ac­tive α‑amylase. More ev­i­dence? In­fect­ing cured flies with L. plan­tarum re­stores their mat­ing pref­er­ences. In­fec­tion with other cul­tur­able bac­te­ria iso­lated from starch flies — ei­ther in­di­vid­u­ally or as a mix­ture of 41 strains — had no ef­fect. The Wol­bachia en­dosym­bionts don't seem to be in­volved as both groups of flies har­bor the same Wol­bachia strain, and in­fect­ing cured flies with Wol­bachia does not re­store the mat­ing pref­er­ences.

Some ques­tions re­main here, not the least of which is how these com­men­sal mi­crobes af­fect mate se­lec­tion What comes to mind is that they may al­ter the fly sex pheromones. Drosophila and many other dipter­ans carry long-chain hy­dro­car­bon pheromones on their cu­ti­cle (termed cu­tic­u­lar hy­dro­car­bons, or CHs). Other flies per­ceive these com­pounds upon con­tact or at a short dis­tance. Some 56 CHs were re­cently char­ac­ter­ized in D. melanogaster, some of which are gen­der-spe­cific or are al­tered upon mat­ing. The Rosen­berg group did an ini­tial screen­ing of the CHs present in their CMY and starch flies, look­ing for dif­fer­ences that could be at­trib­uted to the al­tered mi­cro­biota. In­deed, there were dif­fer­ences for sev­eral CHs be­tween the CMY and starch flies, and these dif­fer­ences were re­duced by an­tibi­otic cur­ing. It will be in­ter­est­ing to hear whether the L. plan­tarum make some of the CHs them­selves, or how they might al­ter CH syn­the­sis by the host.

Mat­ing pref­er­ence (as­sor­ta­tive mat­ing) is one way to pro­duce re­pro­duc­tive iso­la­tion with­out phys­i­cal sep­a­ra­tion, a mech­a­nism re­quired for sym­patric spe­ci­a­tion. I find it pleas­ing that such a large evo­lu­tion­ary ef­fect ap­pears to be dri­ven by a Small Thing.

Ref­er­ence

Sharon G, Se­gal D, Ringo JM, Hefetz A, Zil­ber-Rosen­berg I, Rosen­berg E. (2010). Com­men­sal bac­te­ria play a role in mat­ing pref­er­ence of Drosophila melanogaster. Proc Natl Acad Sci USA. PMID 21041648