Detection of B chromosomes in interphase hemolymph nuclei from living specimens of the grasshopper <i>Eyprepocnemis plorans</i>

Abstract

The two most important evolutionary properties of B chromosomes are their transmission rate (which suggests their selfishness when significantly higher than 0.5) and their net effects on carrier fitness (usually negative for parasitic Bs). The study of transmission rate unavoidably requires the analysis of many controlled crosses in order to accurately measure population average transmission rate. Therefore, getting a marker closely associated to B presence is of crucial importance to alleviate the load of performing many useless crosses between lacking B individuals. After investigating several cytogenetic techniques on several tissues that may be sampled without drastically damaging live specimens of the grasshopper Eyprepocnemis plorans, we report here the excellent results provided by the CMA₃ fluorescence and C-banding techniques applied to hemocyte nuclei. These cells may be easily obtained from both males and females and provide information on B presence even during the interphase stage. The two cytogenetic techniques take advantage of the heterochromatic nature of the B chromosomes, so that Bs made predominantly of ribosomal DNA are revealed by CMA₃ as bright bodies in the interphase hemocytes, and Bs mostly made of satellite DNA are visualized by C-banding as intensely stained bodies in these cells.

A Single, Recent Origin of the Accessory B Chromosome of the Grasshopper Eyprepocnemis plorans

“…To date, B chromosome presence in E. plorans could only be determined by cytogenetic techniques, usually requiring animal sacrifice, since they are based on gonad analysis. The introduction of testis biopsy analysis (Ló pez-Leó n et al 1993b) permitted determining B chromosome presence in living males, and the development of a technique for determining B chromosome presence in interphase nuclei of hemocytes permitted analyzing B presence even in living females (Cabrero et al 2006). A PCR test for B-specific SCAR marker is applicable to live, dead, and preserved collection specimens.…”

The U2 snDNA Is a Useful Marker for B Chromosome Detection and Frequency Estimation in the Grasshopper <b><i>Abracris flavolineata</i></b>

“…2 c). In a previous study in the E. plorans grasshopper, Cabrero et al [2006] suggested that the hemolymph is the best material to analyze living grasshoppers for the presence of B chromosomes using C-banding and fluorochrome staining. Here, we demonstrate that this type of cell is also useful for FISH experiments, allowing a reliable identification of the B chromosome and scoring the number of B chromosomes per nucleus.…”

“…Each animal was numbered and maintained in plastic boxes until the experiments were performed. A drop of hemolymph was obtained from the pleura of the thorax in the base of the third pair of legs using a needle [Cabrero et al, 2006]. The hemolymph was aspirated using a pipette and spread onto a clean slide.…”

“…This analysis is difficult due to the need for the maintenance of living animals after determining the presence of B chromosomes. To facilitate this analysis and thus the possibility of performing controlled crosses, it is necessary to develop markers to detect B chromosomes in living animals without reducing the animals' reproductive success [Cabrero et al, 2006]. …”

“…Here, we describe a strategy to determine the presence of B chromosomes in live animals without compromising their life and reproductive capacity, as previously used in the grasshopper Eyprepocnemis plorans [Cabrero et al, 2006]. Our assay is based on obtaining hemolymph cells followed by FISH using U2 snDNA as a probe.…”