Submitted to: Archives of Insect Biochemistry and Physiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/17/2003
Publication Date: 5/1/2004
Citation: Loeb, M.J., Coronel, N.A., Natsukawa, D., Takeda, M. 2004. Immunological identification of heliothis virescens midgut cells in vitro and in vivo with antibodies to midgut differentiation factors: reflections on the functions of midgut cell products . Archives of Insect Biochemistry and Physiology. Interpretive Summary: The tubular midgut is the second largest organ in a caterpillar, and is responsible for digesting the food needed to make the caterpillar grow. Toxins and viruses that are used as pesticides are eaten, and also enter the caterpillar through the midgut. Therefore, it is important to understand how the midgut works in order to design more efficient pesticides. The midgut is primarily made up of a layer of mature working cells that digest food and keep the salt composition of the gut and blood constant. The round stem cells at the bases of the mature cells multiply before a caterpillar molts to the next stage, soon changing into more mature working cells of the gut as they make the midgut larger to fit the next size caterpillar. We culture stem cells from midguts of the tobacco budworm caterpillar, a pest that destroys cotton and many vegetable crops. In past work, we identified 4 growth factors, called midgut differentiation factors (MDFs), which induce midgut stem cells to transform to mature gut cells. In this work we used antibodies to the growth factors to find to which midgut cells they attached to in order to find more about their true functions in the gut. We discovered that they all attach to one type of mature cell, the columnar cell. Material that attaches to MDFs 2 and 3 seems to be released from the columnar cells to the inside of the gut, and, in the case of MDF-4, to both the inside and to the blood side of the midgut. This indicates that the midgut factors are hormones that may work outside the midgut cells as well as inside the cells. However, only MDF-4 attached to stem cells and developing cells, and caused a decrease in the number of differentiated cells when added to live cultures, indicating that it is the main differentiation factor. MDFs 1-3 may affect differentiation indirectly. This work may be help scientists develop new pesticides that will prevent the MDFs from working inside the insects.
Technical Abstract: Antibodies to the peptides that induce differentiation of midgut larval stem cells, the midgut differentiating factors MDF-2, MDF-3 and MDF-4, bind to columnar cells in midgut cultures and in intact midgut and of H. virescens, similar to anti- MDF-1. Antibodies to MDF-2 and MDF-3 also stained droplets in the midgut lumen, suggesting that columnar cells may also release cytokines to the lumen. Antibody to MDF-4 exhibited similar staining patterns but also recognized stem and differentiating cells, the presumed targets of peptides that regulate stem cell differentiation. Antibody to MDF-4 also stained one type of endocrine cell in midgut cultures and in sections of midgut, as well as the secretion released both to the midgut lumen as well as the hemolymph. Antibodies to the MDFs 1, 2 and 3, incubated with cultures of midgut cells, did not appear to prevent differentiation of the stem cells in the cultures but affected viability of the mature cells, reflected by increased apoptosis and doubling of the number of differentiating cells compared to controls. Only antibody to MDF-4 induced temporary necrosis and inhibition of population recovery, and may be the true differentiation factor. The other MDFs may have additional functions beyond regulation of midgut stem cell differentiation in vivo.