Ultraspiracle and caste differentiation in Apis mellifera

Author(s)

Angel Roberto Barchuk

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2004-09-27

Examining board members

Zila Luz Paulino Simoes; Carlos Alberto Garofalo; Margareth de Lara Capurro Guimaraes; Gonçalo Amarante Guimarães Pereira; Ricardo Guelerman Pinheiro Ramos

• Advisor: Zila Luz Paulino Simoes

Abstract

In addition to their general roles in insect development and reproduction, juvenile hormone (JH) and ecdysone (20E) coordinate specific processes in social insects. They are directly involved in the differentiation of the alternative caste phenotypes, and, particularly in the honey bee, Apis mellifera, they modulate behavioral development in adult workers. In the present study, we have cloned and sequenced a cDNA encoding Apis mellifera Ultraspiracle (AmUsp), and analyzed its responses to both morphogenetic hormones, JH and 20E. Amt/sp seems to be a single copy gene that produces two transcripts (~4 and 5kb) that are differentially expressed in the different tissues and organs of a bee. In its amino acid sequence, AmUsp shows greater similarity to its orthologs from the vertebrate-crab-locust group than to the dipteran-lepidopteran group. These characteristics and its rapid up-regulation by JH suggest that some of the Apis mellifera Usp functions may depend on ligand binding. To investigate possible target genes regulated by AmUsp, we have used an iRNA technique to down-regulate usp expression during pupal development of Apis mellifera workers. This allowed us to test the response of ftz-f1, juvenile hormone esterase (jhe), pro-phenoloxidase (proPo) and vitellogenin (vg) genes to decreased levels of usp expression. While vg expression levels were not clearly altered in usp knock-down pupae (kd), the proPo and the genes were down regulated. Interestingly, ftz-f1 (an orphan nuclear receptor) also turned out to be down regulated in kd pupae, indicating Usp protein participation in controlling the expression of these genes. A low JH titer during the final stages of larval development has been shown to trigger programmed cell death in the ovaries of worker larvae to the point that only a few ovarioles remain to form the functional reproductive system of these females. Hence, we determined the AmUsp expression pattern in the ovaries of queen and worker honeybees during the late stages of larval development. RT-PCR analyses showed that usp expression closely follows the haemolymph JH titer, and that the higher levels of usp expression in L4 queens reflect the higher titers of this hormone. Immunocytochemistry experiments with a mAb raised against a dipteran Usp protein revealed stained structures only in the region of cluster formation in ovarioles of worker larvae preparing for metamorphosis. This region corresponds to the germ cell center where processes of cell death and proliferation have been shown to occur. The pattern of usp expression suggests its participation in these processes, either alone or by heterodimerization with other transcription factors. These reports and the known positive response of usp to high levels of JH allow us to start building a conceptual model for the gene expression network promoted by JH during honey bee development and caste differentiation. (AU)