DIGE based quantitation of Atta columbica ant male seminal fluid exposed to queen spermatheca fluid.

ABSTRACT: Polyandrous ant queens are inseminated early in their life and store sperm mixtures for a potential reproductive life of decades. However, they cannot re-mate later in life and are thus expected to control the loss of viable sperm because their life-time reproductive success is ultimately limited by the viability of sperm obtained in their single reproductive event. In the leaf-cutting ant Atta colombica, we have previously shown that sperm survival is lowered when getting in contact with seminal fluid of other males, and remains stable when in contact with secretions of the queen sperm storage organ (spermatheca). Here we aim to resolve the main protein-level interactions that mediate sperm competition dynamics and sperm preservation. We used artificial insemination and DIGE-based proteomics to identify proteomic changes when seminal fluid is exposed to spermathecal fluid. Seminal fluid was collected from mature males during field season experiments in Panama, by gently squeezing males’ abdomen until the ejaculate came out. Ejaculates from several males were kept together in ice and then centrifuged for 10 minutes at 13,500 g, the supernatant collected in a separate tube and then centrifuged again. The resulting supernatant was then transferred to another tube and stored at -20° C until further use. Four biological replicates of SF were collected from 4 colonies, each consisting of 400 µl SF from approximately 350 males. From each biological replicate three 100 µl aliquots were retrieved, and assigned to one of the following treatments: (1) un-inseminated controls, (2) inseminated SF retrieved after 30 min, and (3) inseminated SF retrieved after 12 h (Fig 1). Next, each of those aliquots was used to artificially inseminate 10 virgin queens (10 µl of aliquot per queen), for a total of 80 queens inseminated. Queens were allowed to recover for 30 min or 12 h before dissecting them to obtain the spermathecal content. All these spermathecal contents were then pooled per treatment and replicate, resulting in a total of 12 samples (3 treatments – un-inseminated SF, inseminated SF retrieved after 30 min inside the queen, and inseminated SF retrieved after 12 hours inside the queen - with 4 biological replicates). Proteins were precipitated in each sample by adding 4 volumes of ice-cold acetone for 4 hours at -20°C and centrifuged at 14,000 g for 10 minutes, after which supernatants were discarded and the protein pellets were stored at -80°C until further use. All of those 12 samples entered the DIGE analysis.

INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS

ORGANISM(S): Atta Columbica

TISSUE(S): Seminal Vesicle Fluid

SUBMITTER: Julia Grassl  

LAB HEAD: Prof Boris Baer

PROVIDER: PXD011320 | Pride | 2019-01-03

REPOSITORIES: Pride