Journal of Insect Physiology 48 (2002) 835–843 www.elsevier.com/locate/jinsphys A ribosome-free extract from cultured cells improves recovery of polysomes from the mosquito fat body: analysis of vitellogenin and ribosomal protein rpL34 gene expression Ling Ling Niu 1 , Ann M. Fallon * Department of Entomology, University of Minnesota, 1980 Folwell Avenue, St. Paul, MN 55108, USA Received 1 April 2002; accepted 14 June 2002 Abstract We have examined the association of ribosomal protein rpL34 mRNA with polysomes in Aedes albopictus C7–10 cells in culture using a simple, two-step sucrose gradient. In growing cells, 40–50% of the ribosomes were engaged on polysomes. This proportion could be increased to 80% when metabolism was stimulated by refeeding the cells with fresh medium. Conversely, ribosomes shifted off polysomes when cells were starved with phosphate-buffered saline or cell lysates were treated with puromycin. When similar approaches were used with fat body from blood-fed female Aedes aegypti mosquitoes, we were unable to obtain the polysome fraction that contained vitellogenin mRNA, which is abundantly translated after a blood meal. Addition of post-mitochondrial supernatant from fat body to polysomes from cultured cells shifted the polysome profile towards smaller polysomes and monosomes, in a dose-dependent fashion. Disruption of fat body tissue in a post-ribosomal supernatant from refed cells improved the recovery of polysomes, demonstrating both the engagement of vitellogenin mRNA on polysomes and the mobilization of rpL34 from messenger- ribonuceloprotein particles onto polysomes in blood-fed mosquitoes. These observations suggested that ribonucleases remain active when polysomes are prepared from mosquito fat body, and that cell culture supernatant contains a ribonuclease inhibitor.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Mosquito; Polysomes; Vitellogenin; Fat body; Translational control 1. Introduction Coordination of gene expression during mosquito reproduction likely involves regulatory processes that affect both transcription and translation of essential gene products. We have been particularly interested in two interrelated aspects of fat body metabolism: (1) pro- duction of ribosomes, which provide an essential compo- nent of the protein synthetic machinery; and (2) engage- ment of ribosomes in synthesis of vitellogenin (Vg) in the fat body of blood-fed mosquitoes. Both ultra- structural (Raikhel and Lea, 1983) and biochemical (Hotchkin and Fallon, 1987; Niu and Fallon, 2000) data indicate that ribosome content in mosquito fat body * Corresponding author. Tel.: +1-612-625-3728; fax: +1-612-625- 5299. E-mail address: fallo002@tc.umn.edu (A.M. Fallon). 1 Present address: Animal Health and Biomedical Sciences, Univer- sity of Wisconsin, Madison, WI 53706, USA. 0022-1910/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII:S0022-1910(02)00129-4 increases approximately three-fold after the blood meal. This increase in protein synthetic machinery presumably supports the massive synthesis of Vg protein that accompanies digestion of the blood meal (Raikhel, 1992). After a blood meal, expression of the rRNA and Vg genes is regulated at the level of transcription, but measurable transcription of ribosomal protein (rp) mRNAs does not occur (Niu and Fallon, 2000). Recently, we have shown that after the blood meal, ribo- some assembly involves translation of pre-existing rp mRNA, which accumulates in the fat body during the first few days after eclosion. We suggest that this accumulation of rp mRNAs to relatively high levels is an important component of the poorly defined events that contribute to reproductive competence in anautogenous mosquitoes. We hypothesize that in competent fat body of non- blood-fed female Aedes aegypti, rp mRNAs are stored as messenger- ribonucleoprotein particles (mRNPs), from which the mRNA is rapidly recruited into polysomes