Efforts to characterize insect antibacterial mechanisms have focused on defenses deployed via the hemolymph to eliminate bacteria in the body cavity. Little attention has been directed to defensive mechanisms targeted to the cuticular surfaces of the insect, or the alimentary canal, which are the primary sources of bacterial infection, While studying the synthesis of hemolymph antibacterial proteins in tobacco hornworm larvae, we discovered two antibacterial responses targeted to the midgut lumen: a ''malaise syndrome'' elicited concurrently with the synthesis of hemolymph antibacterial proteins following hemocoelic infection and a prophylactic mechanism apparently regulated by the endocrine signals that initiate metamorphosis, During metamorphosis, the lepidopteran midgut is restructured and the peritrophic membrane is lost, exposing the midgut epithelium to the natural bacterial flora of the gut lumen, During this period, differentiating pupal midgut epithelial cells synthesize and release into the lumen a cocktail of potent antibacterial proteins including lysozyme, bactericidal activity against Escherichia coli, hemolin, and phenoloxidase. Progress in the characterization of these proteins is reviewed and a potential similarity of the metamorphosis-associated, prophylactic response to the ''malaise syndrome'' is discussed.

The cell populations that compose all multicellular organisms share the need to sense their environment and survey features of their cellular neighbors. These surveillance functions serve to ensure appropriate cell sorting and cellular association during embryonic development and metamorphosis, trigger repair processes that eliminate damaged or dysfunctional cells, and detect the presence of foreign (nonself) cells and elicit appropriate responses to eliminate the intruders. Collectively, the surveillance and effector mechanisms that serve the latter repair and defense functions are referred to as the organism's immune system.

Lysozyme is hypothesized to play a central role in initiating and maintaining the antibacterial defense response of Manduca sexta. We isolated a cDNA clone encoding a M. sexta lysozyme. Results of Northern blot analyses using this cDNA as a probe indicated that the abundance of lysozyme transcripts increased in seven tissues following treatment with peptidoglycan, with the highest level of accumulation occurring in the fat body. An analysis of the kinetics of accumulation of the transcripts in the fat body demonstrated low levels of transcripts in the naive larvae which increased rapidly after treatment and remained elevated over several days. A genomic fragment containing a lysozyme gene was also isolated and the nucleotide sequence and transcription start site of the gene was determined.

Larvae of the tobacco hornworm, Manduca sexta, respond to intrahemocoelic injection of bacteria or bacterial cell wall peptidoglycan with induced synthesis of a suite of antibacterial proteins. Previous studies have demonstrated peptidoglycan regulation of the synthesis of these antibacterial proteins. In addition to eliciting enhanced synthesis of antibacterial proteins, peptidoglycan fragments also elicit a "malaise syndrome" characterized by decreased feeding and growth, delayed metamorphosis, and altered excretion. We speculate that these symptoms may be components of a mechanism to flush out and sterilize the midgut lumen, one of the primary sources of bacterial infection in insects. Studies of naive larvae have demonstrated the accumulation of lysozyme in the differentiating pupal midgut epithelium and release of lysozyme into the pupal midgut lumen after the larval midgut epithelium has been sloughed off. These observations have been extended by the identification of potent bactericidal activity against E. coli and immunoreactive hemolin, together with lysozyme, in the lumen of the newly differentiated pupal midgut.

The unidirectional transport of proline by midgut epithelium cells of the tobacco hornworm, Manduca sexta, was investigated in brush border membrane vesicles. Both K.(+)-stimulated and K(+)-insensitive transport pathways were identified. Analyses of K(+)-dependent proline transport revealed 1:1 ratio of K+ to proline, a Km of 13 mM for K+ and a decrease in both Km (from 18 mM to 3 mM) and Vmax (from 37 nmol/mg protein/min to 10 nmol/mg protein/min) for proline in the presence of a K+ gradient. The profiles of cis-inhibition by other amino acids demonstrated that proline is transported into midgut cells by a transport system that is shared by other neutral amino acids.

The unidirectional transport of leucine and aspartic acid by midgut epithelium cells of the tobacco ornworm, Manduca sexta, was investigated using brush border membrane vesicles. Both K+-stimulated and K+-insensitive transport pathways for these two amino acids were identified. Kinetic analyses and profiles of cis-inhibition by other amino acids demonstrated that leucine and aspartic acid are transported by separate pathways and exhibit different sensitivity to potassium ion.

1. Compared to crude homogenates, Manduca sexta brush border membrane vesicles (BBMV) had increased specific activity of marker enzymes leucine aminopeptidase and alkaline phosphatase while Leptinotarsa decemlineata BBMV had increased leucine aminopeptidase specific activity and decreased alkaline phosphatase specific activity. 2. L. decemlineata BBMV were impermeable to a proton gradient while M. sexta BBMV sustained an alkali cation gradient. 3. Both K+-stimulated and K+-independent unidirectional leucine transport pathways were identified in M. sexta BBMV while only cation-independent unidirectional leucine transport was identified in L. decemlineata BBMV.

Low levels of lysozyme were found in the midgut epithelium of the tobacco hornworm, Manduca sexta, during the early part of the fifth larval stadium. This was observed in control insects as well as in bacterially challenged insects. No lysozyme was detected in the gut contents of either group of insects which were actively eating or in the early stages of metamorphosis. However, high levels of lysozyme activity were detected in homogenates of midgut tissue collected from insects later in the stadium. Immunocytochemical studies demonstrated that lysozyme accumulates in large apical vacuoles in regenerative cells of the midgut during the larval-pupal molt. These cells, initially scattered basally throughout the larval midgut epithelium, multiply and form a continuous cell layer underneath the larval midgut cells. At the larval/pupal ecdysis the larval midgut epithelium is sloughed off and the regenerative cells, now forming the single cell layer of the midgut, release the contents of their vacuoles into the midgut lumen. This release results in high lysozyme activity in the lumen of the pupal midgut and is thought to confer protection from bacterial infection. This is the first indication that the lysozyme gene may be developmentally regulated in a specific tissue in the absence of a bacterial infection.

Pre-incubation of brush border membrane vesicles (BBMV) isolated from the midgut of Manduca sexta with activated Bacillus thuringiensis delta endotoxin for a short period resulted in differential inhibition of K(+)-dependent transport of leucine relative to the effect on K(+)-dependent transport of aspartic acid. The difference in I1/2 (5 fold greater for aspartic acid than for leucine) is interpreted as the result of enhanced binding of the B. thuringiensis delta endotoxin to the leucine transport system.

The pericardial cell-heart complex (pericardial complex) of fifth instar Manduca sexta larvae has been shown to contain, to synthesize and to release lysozyme. Lysozyme activity was present in homogenates of pericardial complex. Immunocytochemical analysis demonstrated that lysozyme in the pericardial complex was located in pericardial cells. Injection of peptidoglycan elicitors, which markedly increase levels of hemolymph lysozyme, also elevated lysozyme activity in homogenates of pericardial complex, but only moderately. Lysozyme synthesis in the pericardial complex was demonstrated in vitro by the incorporation of [3H]leucine into immunoprecipitable lysozyme. This tissue did exhibit an increase in the release of a variety of newly synthesized proteins but not a selective increase in the synthesis and release of lysozyme after peptidoglycan stimulation.