meiosis - specific genes have been identified in several eukaryote pathogen genomes by phylogenomic analysis [ 5 ,  6 ] , with demonstrated functionality in giardia intestinalis   and trypanosoma brucei .
three functionally distinct , meiosis - specific proteins ( mnd1 , dmc1 , and hop1 ) were expressed in t.  b.  brucei strain j10 , implying that meiosis might be an integral part of the trypanosome s developmental cycle in the tsetse fly vector . to test this hypothesis and to obtain further information on the timing of meiosis
the endogenous loci of three genes expressed solely during  the prophase of meiosis i were modified so that each contained an n - terminal yfp tag ( yfp::mnd1 , yfp::dmc1 , yfp::hop1 ) .
we used the following trypanosomes : t.  b. brucei ( lister 427 ) , t.  b. rhodesiense ( 058 ) , t.  b. gambiense group 1 ( dal972 ) , and t.  b. gambiense group 2 ( th2 ) , the last three being human pathogens .
each strain expressed the meiosis - specific genes exactly as previously observed for t.  b. brucei j10 , confirming that the meiotic program takes place in the salivary glands ( sg ) during transmission of a clonal trypanosome strain ( figure  1 ) .
although successful crosses of t.  b.  brucei , t.  b. rhodesiense , and t.  b. gambiense group 2 have been reported previously , this is the first indication that t.  b. gambiense group 1 is capable of meiosis and , potentially , genetic exchange , despite lack of evidence of recombination in population genetics analyses [ 9 , 10 ] .
sex provides the opportunity for new pathogen strains to arise by recombination , a phenomenon already suspected in t.  b. rhodesiense , which has the capacity to become human infective by transfer of a single gene for human serum resistance ( sra ) .
trypanosomes expressing meiosis - specific genes were present in fly sg dissected between 14 and 38  days after infection ( figure  1c ) , with the highest frequency of expression around day 20 .
it follows that postmeiotic cells , including haploid gametes , should occur at the highest frequency at this time point . from measurements of nuclear dna contents of different life - cycle stages
, it has been assumed that t.  brucei is diploid throughout its developmental cycle [ 1315 ] .
but mating in trypanosomes is believed to involve haploid nuclei or cells because the pattern of inheritance in experimental crosses is largely mendelian ; moreover , triploid laboratory hybrids are often found , implying that fusion of haploid and diploid nuclei has taken place [ 3 , 17 , 18 ] .
the expression of meiosis - specific genes preceded cell fusion , and hence meiosis generates the cells that subsequently undergo fusion .
our initial search for haploid trypanosomes by flow cytometry of sg - derived trypanosomes was unsuccessful because cell numbers were too low and amounts of fly tissue and debris too great to distinguish signal from noise ; movie s1 ( available online ) illustrates the suboptimal nature of the material under analysis .
we therefore developed an alternative search strategy to find cells with gamete - like behavior in mixtures of sg - derived trypanosomes .
analysis of trypanosome crosses is facilitated by the incorporation of red fluorescent protein ( rfp ) or green fluorescent protein ( gfp ) into the parental clones , enabling hybrids to be identified by yellow fluorescence .
this system established that hybrid trypanosomes occur in the sg , not the midgut ( mg ) , of infected flies and are found as early as 13  days after flies are coinfected with the parental clones . here , rather than carrying out the cross in  vivo , we mixed the parental clones in  vitro using trypanosomes derived from the sg of flies separately infected with either red or green fluorescent cell lines .
trypanosomes were harvested from fly sg during the window of peak expression of meiosis - specific genes , and the ex  vivo mixtures were observed as living cells in microslides over the course of about an hour , and also after fixation on microscope slides .
twenty replicate experiments were carried out using mating - compatible pairs j10 rfp/1738 gfp or f1g2/f1r1 ( table s1a ) .
small clusters of two or more red and green fluorescent trypanosomes , as well as clusters of single color trypanosomes , were observed within 10  min of mixing the red and green fluorescent sg - derived parental cells ( figure  2 ; movies s2 and s3 ) .
we focused the search for gamete interactions on the red - green clusters because single - color clusters might also arise from cell division .
all red - green clusters contained at least one trypanosome with a short , wide body and long anterior flagellum .
these trypanosomes were observed to interact by intertwining their long flagella , often drawing the cell bodies into close proximity ( figure  2a ) .
these interactions are reminiscent of the recognition and attachment of gametes of the unicellular plant chlamydomonas reinhardtii via their paired flagella .
in the clusters , the trypanosomes were highly active , frequently changing position ( movies s2 and s3 ) .
these distinctive cell interactions had not been observed previously in our analysis of experimental crosses [ 3 , 18 , 20 ] . to rule out the possibility that the sg cells were 
sticky ,  we set up mixtures of mg- and sg - derived trypanosomes in all possible combinations ( table s1b ) .
the distinctive flagellar interactions were observed only among sg - derived trypanosomes and did not occur in mixtures of sg- and mg - derived trypanosomes or between mg - derived trypanosomes ; when red and green trypanosomes were found together in these mixtures , they were associated with debris or dead trypanosomes .
yellow fluorescent trypanosomes started to appear within 30  min of mixing the sg - derived parental lines ( figure  2b ) and were observed in 11 of 20 experiments ( table s1a ) .
the rapid appearance of yellow fluorescent trypanosomes signifies fusion of cell membranes and exchange of cytoplasm between red and green fluorescent cells rather than de novo synthesis of fluorescent proteins , which would take several hours .
these results demonstrate that clonal trypanosome populations derived from the sg already contain fusion - competent cells , ruling out the hypothesis that these cells are generated after recognition of nonself among trypanosomes of different genotypes .
the trypanosomes implicated as trypanosome gametes had a distinctive morphology , and dapi staining revealed either one kinetoplast and one nucleus ( 1k1n ) or two kinetoplasts and one nucleus ( 2k1n ) ( figure  2 ; movies s3 and s4 ) . in trypanosome biology
the closest is promastigote , which refers to a cell with an antenuclear kinetoplast and a flagellum that emerges anteriorly without connection to the cell body via an undulating membrane . in the following ,
we refer to these 1k1n and 2k1n cells collectively as promastigote - like ( pl ) cells .
both 1k1n and 2k1n pl cells displayed gamete - like cell - cell interactions , but we were unable to distinguish different behavior during mating .
the presence of two kinetoplasts in the 2k1n pl cells was confirmed by differential staining with dapi and propidium iodide ( pi )   ( figures 3a and 3b ) and also by visualization of the subcellular location of a yfp::p166 fusion construct ( figures 3c and s1a ) .
p166 is an intrinsic component of the tripartite attachment complex ( tac ) , the filamentous structure that physically attaches the kinetoplast to the basal body of the flagellum .
the intensity of fluorescence of p166 in the anterior tac was usually greater than that in the posterior tac ( figure  s1a ) , perhaps associated with the presence of a flagellum attached to the anterior kinetoplast .
the 2k1n pl cells had only one flagellum , although the two kinetoplasts were widely separated at the two poles of the cell on either side of the nucleus , and a new flagellum would normally be evident at this stage of cell division . to detect whether a second flagellum was present but not discernible by light microscopy
, we visualized the subcellular localization of pfr1 , a major structural protein of the paraflagellar rod ( pfr ) , using cells expressing the fusion yfp::pfr1 ( figures 3d and s1b ) .
of 26 2k1n pl cells examined , 23 ( 88% ) had only a single fluorescent flagellum , arising near the anterior kinetoplast .
the remaining three cells had two flagella , the posterior flagellum appearing as a tiny dot of fluorescence near the posterior kinetoplast ( figure  s1b ) .
the pfr extends from the point at which the flagellum emerges from the flagellar pocket to its distal tip , and thus pfr1 is absent from a transitional zone of the flagellum adjacent to the basal body .
we therefore can not rule out the presence in all 2k1n cells of a very short flagellum that does not extend beyond the flagellar pocket , as seen in leishmania amastigotes .
the above evidence on interactions and cytoplasmic fusion implicates pl cells as trypanosome gametes ; the crucial question is whether they are haploid , a defining feature of eukaryote gametes .
pl cells were present in small numbers in live preparations of sg spillout ( movies s1 and s5 ) , together with metacyclics and other unattached trypanosomes .
fixed cells were stained with both dapi and propidium iodide ( pi ) before measurement of total fluorescence intensity of the nucleus .
this provided two independent measures of nuclear dna content , because dapi and pi have different dna binding characteristics   ( although dna binding could in principle be affected by chromatin packing ) .
both 2k1n and 1k1n pl cells were haploid relative to metacyclics , the diploid , g1-arrested , mammal - infective stage also present in the sg   ( figure  4a ) .
the combined dapi and pi pixel intensities of pl cells peaked at 0.5 , compared to metacyclics at 1.0 ( 2c dna content ) ( figure  4a ) .
procyclics from the fly midgut had a major peak at 1.0 and a smaller peak at around 2.0 , consistent with the 2c and 4c peaks expected for this proliferating cell population ( figure  4a ) .
epimastigotes are also a proliferative insect stage , which are characteristically found attached to the sg epithelium via the flagellum , but here we examined free epimastigotes in the sg spillout ; these cells did not have the elongated posterior  nozzle  typical of attached epimastigotes .
the population shows nuclear dna content peaks at 1.0 ( 2c ) and 2.0 ( 4c ) like procyclics , but there is a noticeable shoulder of nuclei with dna content < 1.0 ( figure  4a ) .
reexamination of this population revealed ten cells apparently in division , as each had two nuclei ( either 3k2n or 2k2n ) , with nuclear dna contents in the haploid range .
we speculate that some of these are cells in reduction division ( meiosis ii ) , but they were difficult to distinguish from dividing pl cells , except by the extreme length of the flagellum ( figure  s2 ) . a total of 12 dividing pl cells was recorded among 600 cells examined ; because this was a clonal trypanosome population , this cell morphology is unlikely to arise by fusion of pl cells .
a small number of epimastigotes in meiosis i was identified by morphology ( 2k epimastigote with posterior nucleus and two short flagella ; figure  s1b ) ; these had nuclear dna contents consistent with 4c or greater ( mei , figure  4 ) .
meiotic cells are often found attached inside the sg ( figure  1b ) , explaining the small number of free meiotic cells observed .
pl cells frequently had a large , elongated anterior kinetoplast , which was highly conspicuous in dapi staining ( figure  2 ) , confirmed by measurement of kinetoplast dna ( kdna ) contents relative to the unit kinetoplast of metacyclics ( figure  4b ) .
twice the unit amount of kdna usually indicates a kinetoplast about to divide , suggesting a simple hypothesis for the generation of 2k1n pl cells by kinetoplast division in 1k1n pl cells .
however , this is contradicted by the fact that both 1k1n and 2k1n pl cells had enlarged kinetoplasts . alternatively
, 1k1n and 2k1n pl cells might arise by unequal division of a 3k1n cell , because both pl cell types were found in approximately equal numbers .
both 3k1n and 3k2n cells were observed ( e.g. , figure  s2e ) . in summary , we have shown that meiosis is a normal part of the developmental cycle of t.  brucei in the tsetse fly and have identified a novel haploid pl cell that displays the behavior expected of a gamete .
when pl cells of different strains are mixed , they readily form pairs or clusters and undergo cytoplasmic fusion .
we conclude that the haploid pl cells are trypanosome gametes , although formal proof will require the demonstration of exchange of nuclear and kinetoplast dna . until now ,
the extent and significance of sex in kinetoplastid parasites have been controversial , but this study reveals that t.  brucei is essentially a sexual organism .
hence , these results support the hypothesis that meiosis and sexual reproduction are ancestral and ubiquitous features of eukaryotes .
the following tsetse - transmissible strains of trypanosoma brucei subspecies were used : t.  b. brucei j10 ( mcro / zm/73/j10 clone 1 ) , 1738 ( movs / ke/70/eatro 1738 ) , lister 427 ( movs / ug/60/427 variant 3 ) ; t.  b.  rhodesiense 058 ( mhom / zm/74/058 clone
b ) ; t.  b. gambiense group 1 dal972 ( mhom / ci/86/dal972 clone 1 ) ; t.  b. gambiense group 2 ( mhom / ci/78/th2 ) .
the experimental cross of j10 and 1738 carrying cytoplasmically expressed genes for fluorescent proteins is described in   and produced hybrid progeny f1g2 and f1r1 .
procyclic form ( pf ) trypanosomes were grown in cunningham s medium ( cm )   supplemented with 10% v / v heat - inactivated fetal calf serum , 5  g / ml hemin , and 10  g / ml gentamycin at 27c .
fusion constructs of the yfp gene with the homologs of meiosis - specific genes ( dmc1 , tb09.211.1210 ; hop1 , tb10.70.1530 ; mnd1 , tb11.02.3380 ) and pfr1 ( tb927.3.4290 ) were described in .
p166 , a component of the tripartite attachment complex connecting the kinetoplast and basal body of the flagellum , was also tagged with yfp ( accession number fj407182 ) .
pfs were transfected by electroporation , antibiotic selected , and cloned as previously described . for analysis of expression of fusion proteins associated with meiosis ,
fly organs ( sg and alimentary tract from the proventriculus to the hindgut ) were dissected in a drop of pbs and examined for the presence of fluorescent trypanosomes using a dmrb microscope ( leica ) equipped with a retiga exi camera ( qimaging ) and volocity software ( perkinelmer ) .
cells were fixed in 2% w / v paraformaldehyde ( pfa ) at room temperature for 20  min and stained with dapi in vectashield mounting medium ( vector laboratories ) to visualize the nucleus and kinetoplast . for analysis of mating between red and green fluorescent trypanosomes ,
medium containing spilled - out trypanosomes was introduced into a microslide capillary for live imaging as above , together with hoechst live stain if required .
alternatively , preparations were spread on microscope slides using a cytospin after fixation in 2%  w / v pfa and were dapi stained and mounted as above .
 summaryin eukaryote pathogens , sex is an important driving force in spreading genes for drug resistance , pathogenicity , and virulence [ 1 ] . for the parasitic trypanosomes that cause african sleeping sickness , mating occurs during transmission by the tsetse vector [ 2 , 3 ] and involves meiosis [ 4 ] , but haploid gametes 
 have not yet been identified . here 
 , we show that meiosis is a normal part of development in the insect salivary glands for all subspecies of trypanosoma brucei , including the human pathogens . by observing insect - derived trypanosomes during the window of peak expression of meiosis - specific genes , we identified promastigote - like ( pl ) cells that interacted with each other via their flagella and underwent fusion , as visualized by the mixing of cytoplasmic red and green fluorescent proteins . 
 pl cells had a short , wide body , a very long anterior flagellum , and either one or two kinetoplasts , but only the anterior kinetoplast was associated with the flagellum . 
 measurement of nuclear dna contents showed that pl cells were haploid relative to diploid metacyclics . 
 trypanosomes are among the earliest diverging eukaryotes , and our results support the hypothesis that meiosis and sexual reproduction are ubiquitous in eukaryotes and likely to have been early innovations [ 5 ] . 