Hemoglobin (Hb) synthesis in the subepidermal fat body cell during the development of Chironomus thummi was investigated by using positive diamino-benzidine (DAB) reaction as an indicator for the presence of Hb.The localization of DAB reaction products in the ER and Golgi cisternae is stage-specific. Active synthesis of Hb, as indicated by heavy deposits of reactive products, is interrupted during third-fourth larval ecdysis, resumes about the fourth day of the fourth instar, continues throughout the fourth instar and stops again shortly before the transition from the pharate pupa to the pharate adult a t which time the fat body shows numerous autophagic vacuoles. These results support the concept of the existence of specific switches in the production, release and degradation of Hb present in Chironomus.The dipteran Chironomus is noted for a n extensive polymorphism of its hemoglobins (Hbs) (Braunitzer and Braun, '65). Within a single species there are several molecular forms of this blood pigment which show both species-specificity and racial diversity (Thompson and English, '66; Thompson et al., '69). Equally striking is the developmental polymorphism, for during ontogeny new forms of Hb may appear while others decrease in concentration. Certain Hbs are specific for certain stages of development (Manwell, '66; Shrivastava and Loughton, '70; Wulker e t al., '69). I t has been proposed that the temporal variation of the Hb pattern results from the activation and inactivation of different gene loci a t specific stages of development (Manwell, '66). This possibility suggests that the system may be an ideal one for the study of genetic control of Hb biosynthesis during development.The fat body is generally considered to be a principal site of insect metabolic activity (Wigglesworth, '72; Wyatt, '751, including the synthesis of all the molecular forms of Hb in Chironomus (Bergtrom e t al., '76; Laufer e t al., '76; Schin e t al., '77a,b). One portion of this tissue, the subepidermal fat body, has been identified as the major site of Hb biosynthesis (Schin et al., '77a,b).In the present study we utilized a cytochemical approach in combination with electron microscopy to obtain detailed information on the developmental aspects of Hb synthesis in relation to the ultrastructure of fat body and to the molecular species of Hbs present during different developmental stages.
MATERIALS AND METHODSChironomus thummi were cultured on an iron-rich diet of nettle powder as described previously by Laufer and Wilson ('70). Individuals were staged by developmental characteristics such as size, head coloration, molts and other morphological criteria (Kroeger, '73; Travis and Schin, '77). Subepidermal fat body or "body wall" was dissected from larvae and pupae, and immediately processed for electron microscopic cytochemical localization of Hb. The term "body wall" used herein includes the epidermis, cuticle, intersegmental musculature and all of the adhering subepidermal fat body.
E M cytochemistry for H b localizationFollow...
