In order to standardize dual‐fluorescence DNA flow cytometry using cytokeratin (CK) antibodies, normal colonic mucosa and tumor tissue were sampled from 308 colorectal surgical specimens. Fresh colon specimens were processed directly and stored frozen until dissociation. The samples were divided into aliquots for manual dissociation with tweezers and scalpel, and parallel dissociation with an automated disaggregation device (Medimachine, DAKO Diagnostika GmbH, Hamburg, Germany). An indirect immunofluorescence method with anti‐cytokeratin antibodies and propidiumiodide was applied and measured on a single‐laser flow cytometer (FACScan, Becton Dickinson [BDI], Heidelberg, Germany). Evaluation with CellFit (BDI) or MultiPlus (Phoenix Flow Systems, San Diego, CA) showed that dual‐parameter fluorescence propidiumiodide (DNA staining) and fluorescein‐isothiocyanate (cytokeratin labeling) provides a reasonable staining method for DNA analysis of epithelial cells. No significant differences in coefficient of variation in CK‐gated versus ungated cells could be observed. Normal colon mucosa served as a reliable internal, diploid DNA control. Medimachine dissociation led to a significantly higher gain of cytokeratin‐positive cells compared to percentage of cytokeratin‐positive cells after manual tissue disaggregation. Cytokeratin gating led to a clear‐cut separation of S‐phase fractions within the respective ploidy groups, irrespective of manual or automated dissociation. The S‐phase fraction increased significantly from normal tissue to diploid and nondiploid tumors. In general, automated tissue preparation with the Medimachine allows simple cell‐isolation for dual DNA/CK‐flow cytometric measurement, improving the gain of CK‐positive cells, and facilitating a standardized DNA analysis. Cytometry (Comm. Clin. Cytometry) 38:184–191, 1999.  © 1999 Wiley‐Liss, Inc.

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Doublesex (Dsx) has a conserved function in controlling sexual morphological differences in insects, but our knowledge of its role in regulating sexual behaviour is primarily limited to Drosophila . Here, we show with the parasitoid wasp Nasonia vitripennis that males whose Dsx gene had been silenced ( NvDsx -i) underwent a three-level pheromonal feminization: (i) NvDsx -i males were no longer able to attract females from a distance, owing to drastically reduced titres of the long-range sex pheromone; (ii) NvDsx -i males were courted by wild-type males as though they were females, which correlated with a lower abundance of alkenes in their cuticular hydrocarbon (CHC) profiles. Supplementation with realistic amounts of synthetic ( Z )-9-hentriacontene ( Z 9C31), the most significantly reduced alkene in NvDsx -i males, to NvDsx -i males interrupted courtship by wild-type conspecific males. Supplementation of female CHC profiles with Z 9C31 reduced courtship and mating attempts by wild-type males. These results prove that Z 9C31 is crucial for sex discrimination in N. vitripennis ; and (iii) Nvdsx -i males were hampered in eliciting female receptivity and thus experienced severely reduced mating success, suggesting that they are unable to produce the to-date unidentified oral aphrodisiac pheromone reported in N. vitripennis males. We conclude that Dsx is a multi-level key regulator of pheromone-mediated sexual communication in N. vitripennis .

The transcription factor Doublesex (Dsx) has a conserved function in controlling sexual morphological differences in insects, but our knowledge on its role in regulating sexual behavior is widely limited to Drosophila. Here, we show in the parasitoid wasp Nasonia vitripennis that males whose Dsx gene had been silenced by RNA interference (NvDsx-i) underwent a three-level pheromonal feminization: (1) NvDsx-i males were no longer able to attract females from a distance, owing to drastically reduced titers of the abdominal long-range sex pheromone. (2) NvDsx-i males were courted by wild-type males like females which correlated with a lower abundance of alkenes in their cuticular hydrocarbon (CHC) profiles. Supplementation of NvDsx-i male CHC profiles with realistic amounts of synthetic (Z)-9-hentriacontene (Z9C31), the most significantly reduced alkene in NvDsx-i males, interrupted courtship by wild-type conspecific males. Supplementation of female CHC profiles with Z9C31 reduced courtship and mating attempts by wild-type males. These results prove that Z9C31 is crucial for sex discrimination in Nasonia. (3) Nvdsx-i males were hampered in eliciting female receptivity during courtship and thus experienced severely reduced mating success, suggesting that they are unable to produce the hitherto unidentified oral aphrodisiac pheromone reported in N. vitripennis males. We conclude that Dsx is a multi-level key regulator of pheromone-mediated sexual communication in N. vitripennis. Silencing Dsx by RNA interference provides a new avenue for unraveling the molecular mechanisms underlying the pheromone-mediated sexual communication in insects.