Morphogenesis of integument in Apis mellifera: building the adult exoskeleton

Author(s)

Moysés Elias Neto

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC)
• Defense date: 2008-02-25

Examining board members

Marcia Maria Gentile Bitondi; Cláudio Roberto Simon; Chao Yun Irene Yan

• Advisor: Marcia Maria Gentile Bitondi

Abstract

The evolutionary success of the insects is to a large extent due to the structural and mechanical properties of the integument, which is made up of an outer cuticle layer and the subjacent epidermis. As an effective interface between the insect soft body and the environment, the integument performs all the functions of a skin and of an exoskeleton. It not only supports the insect, but gives it its shape, means of locomotion, and provides protection against desiccation, besides being involved in defense strategies towards predators and pathogenic agents. Building and maturation of the adult exoskeleton include complex biochemical pathways where the enzymes Laccases (E.C. 1.10.3.2) may have a key role. Laccases have been characterized mainly in fungi and bacteria. In insects, the function of these enzymes has been linked to cuticle tanning (pigmentation and sclerotization) and stabilization of the protein-based exoskeleton. It was our aim to identify and investigate the function and regulation of the gene, Amlac 2, which encodes the enzyme Laccase 2 in the honeybee, Apis mellifera. Semi-quantitative RT-PCR analyses evidenced that Amlac 2 is highly expressed in the integument of pharate adults in correlation with cuticle pigmentation and sclerotization. Transcription increases in thoracic, abdominal and wing integuments immediately after pupal-imaginal apolysis, and remains abundant all through pharate adult development. Consistent with the different degree of sclerotization in cuticle areas recovering distinct body parts, the increase in the levels of Amlac2 transcripts occurs later in abdominal than in thoracic and wing integuments. A comparative approach using honeybee workers, queens and drones also revealed caste and sex-specific patterns of adult integument differentiation. Post-transcriptional Amlac2 gene silencing resulted in abnormalities in cuticle structure, melanization and sclerotization, as revealed by histological analyses, and drastically affected the adult molt. Such results clearly indicate a critical role of Laccase 2 in the differentiation of the adult exoskeleton in the honeybee. Experiments using a ligature to prevent the increase in ecdysteroid titer in abdomen resulted in inhibition of Amlac 2 transcription and severely impaired cuticular differentiation. These results strongly indicate that Amlac 2 expression is controlled by ecdysteroids, and has a crucial role in the differentiation and maturation of the adult cuticle. Moreover, a radioimmunoassay using hemolymph from ligated abdomens suggested the existence of an alternative source of ecdysteroids, in addition to prothoracic glands, thus leading us to propose a new endocrine model for differentiation of the adult honeybee. (AU)

• FAPESP's process: 05/03301-5 - Amlac 2 and integument differentiation in castes and sex of Apis mellifera: building the adult exoskeleton
• Grantee: Moysés Elias Neto
• Support Opportunities: Scholarships in Brazil - Master