Peter E. Dunn

Fate of bacteria injected into naive and immunized larvae of the tobacco hornworm Manduca sexta

Abstract The susceptibility of Manduca sexta larvae to infection by several Gram-negative bacteria has been examined. Injected Escherichia coli D31 caused no mortality at any dose tested. Serratia marcescens and Pseudomonas aeruginosa ATCC 9027 were moderately pathogenic and caused low mortality at long times after injection of doses exceeding 10 6 bacteria/insect. P. aeruginosa P11-1 was quite virulent, killing all injected insects at doses exceeding 10 3 bacteria/insect. The period between treatment with P11-1 and death was dose-dependent. The fate of injected viable cells of P. aeruginosa ATCC 9027 and P11-1 and of E. coli D31 was examined in naive (nonimmunized) larvae of M. sexta (bacterial clearance). The concentrations of viable cells of all three bacteria were reduced significantly during the first hour following injection at all doses tested. This initial reduction in concentration was followed by a strain and dose-dependent multiphasic outcome. Immediately after the initial reduction there was a period of no change in bacterial concentration followed by either complete elimination of viable bacteria or bacterial multiplication and host death. The fate of viable cells of P. aeruginosa (P11-1 and ATCC 9027) was also examined in larvae immunized with bacterins of both strains of P. aeruginosa and of E. coli D31. In these immunized insects, the initial reduction in concentration of viable bacteria of all strains was followed by subsequent reductions to low levels of bacteria which were maintained for at least 24 hr. Viable cells of E. coli D31 were comparable to homologous bacterin as an immunogen against P. aeruginosa P11-1. It is hypothesized that the initial reduction in bacterial concentration observed with all strains was a result of nodule formation at or near the injection site. Subsequent control of bacterial multiplication in naive and immunized larvae may reflect efficient phagocytosis by hemocytes and susceptibility of bacteria to induced humoral bactericidal factors. The pathogenicity of P. aeruginosa P11-1 in naive insects may be due to relative resistance of this strain to phagocytosis resulting in multiplication prior to the appearance of bactericidal factors and the accumulation of cytotoxins excreted by the bacterium.

Soluble peptidoglycan fragments stimulate antibacterial protein synthesis by fat body from larvae of Manduca sexta

Both hemocytes and fat body from larvae of Manduca sexta, which have been injected with inducers of antibacterial protein synthesis, contain immunoreactive lysozyme. However, fat body is a richer source and has been demonstrated to synthesize and release lysozyme and cecropin-like peptides (bactericidins) in vitro. Fat body secretion of lysozyme and bactericidins is stimulated by addition of soluble peptidoglycan fragments to culture medium. The rate of lysozyme secretion by fat body varies as a function of peptidoglycan inducer concentration. These data are consistent with the hypothesis that, in vivo, bacteria must be phagocytized and partially degraded (processed) by hemocytes to generate a signal (peptidoglycan) that subsequently induces antibacterial protein synthesis by fat body.

Structure and induction of a lysozyme gene from the tabacco hornworm, manduca sexta

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.

Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa

Abstract The time course of clearance of an injected dose of 106 CFU ml−1 hemolymph of Pseudomonas aeruginosa ATCC 9027 in larvae of the tobacco hornworm, Manduca sexta, has been examined in detail. The clearance process has been subdivided into three stages during which the rates of reduction in concentration of circulating viable bacteria were clearly different. Contributions of hemocyte reactions to bacterial clearance were examined during stages I and II. During stage I (0–2 hr postinoculation (PI), nodule formation produced a dramatic reduction in circulating bacteria by entrapping over 90% of the injected dose in the first 30 min. Phagocytosis of bacteria by circulating hemocytes and subsequent intracellular digestion contributed significantly to reductions in circulating bacteria during stage II (2–8 hr PI). Viable cells of the virulent P. aeruginosa P11-1 were trapped in nodules as efficiently as the less virulent 9027 during the first 30 min after injection into M. sexta. Bacteria of strain P11-1 were also phagocytosed by hemocytes during stage II, however, phagocytosed bacteria were observed less frequently in P11-1-treated insects and intracellular digestion of these bacteria was only rarely observed. The increased virulence of P11-1 in larvae of M. sexta may be due to less efficient phagocytosis by circulating hemocytes and to insensitivity of this strain to killing reactions in nodules and following phagocytosis.

Antibacterial proteins in the midgut of Manduca sexta during metamorphosis

Abstract 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.

Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria

A technique for the collection of stable hemolymph from larvae of Manduca sexta has been developed. The method avoids the cell clumping and melanization reactions commonly encountered with insect hemolymph by minimizing contact between hemocytes and surfaces which provoke defensive or repair responses. The circulating hemocyte population of second-day, fifth-instar larvae (2dL5) of M. sexta consisted of 4.5 ± 2.5 × 106 cells/ml (n = 15, range 2–7 × 106 cells/ml) and contained five cell types: prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. Two strains of Pseudomonas aeruginosa which differ in pathogenicity (P11-1 and 9027) and Escherichia coli D31 grew well at 26°C in cell-free hemolymph prepared from naive (nonimmunized) 2dL5 M. sexta. When viable cells of any of the three bacteria were injected into M. sexta larvae, changes in both the total hemocyte count (THC) and differential hemocyte count were observed. Viable bacteria were not required to produce these changes since formalin-killed cells of P. aeruginosa 9027 produced a qualitatively and quantitatively similar response. Following injection of bacteria, the THC increased, reaching a maximal level at 1 hr postinjection, and remained elevated for at least 4 hr after injection. While prohemocytes, plasmatocytes, granulocytes, and spherulocytes all increased in number, 80% of the increased cell population at 1 hr postinjection of bacteria were the latter two cell types. Granulocytes and spherulocytes are cells with recognized defensive capabilities. The increased numbers of these cells in circulation soon after injection of bacteria may confer an advantage on M. sexta larvae in dealing with bacterial infections. This could explain in part the unusual resistance of M. sexta to certain bacterial pathogens.

Host search behaviour of neonate western corn rootworm (Diabrotica virgifera virgifera)

Abstract Paths made by neonate western corn rootworm larvae in an arena were analysed to determine host-finding behaviour. Larvae shifted from long distance ranging behaviour to localized search behaviour after 5 min of contact with the roots of maize and wheat, but not contact with oats, giant foxtail, or soybean. This shift from ranging to localized search is initiated by contact cues, because exposure to germinating maize plant volatiles for 10 min or for 1 h did not result in a similar switch. After time, unrewarded larvae shifted back to ranging behaviour. Response to contact cues dominated over response to volatile cues because immediately after contact with maize roots, larvae were no longer attracted to carbon dioxide.

Plasmatocyte depletion in larvae of Manduca sexta following injection of bacteria

Injection of bacteria and bacterial cell walls into larvae of the tobacco hornworm elicits a rapid, specific depletion of plasmatocytes from circulation. Plasmatocyte depletion in response to injection of bacteria is dose dependent with a threshold for response between 10(3)-10(4) bacteria per insect and a maximal depletion at greater than 10(7) bacteria per insect. Injection of saline, latex beads that are phagocytized, or fragments of peptidoglycan that elicit antibacterial protein synthesis do not affect plasmatocyte abundance.

Effect of parasitism by Cotesia congregata on the susceptibility of Manduca sexta larvae to bacterial infection

Eggs and larvae of the braconid Cotesia congregata are not encapsulated within the hemocoel of their habitual host, Manduca sexta. Experiments were performed to evaluate the status of antibacterial defensive responses in M. sexta larvae parasitized by this gregarious endoparasitoid. Previous investigations have shown that immunologically naive, nonparasitized M. sexta larvae are resistant to infection by Pseudomonas aeruginosa. Studies using naive, parasitized larvae demonstrated that inoculation with 10 colony forming units of P. aeruginosa 9027, one hour after oviposition, resulted in greater than 90% host mortality. Examination of the fate of P. aeruginosa and Escherichia coli injected into parasitized larvae suggested that the cellular antibacterial defenses of the host, nodule formation, and phagocytosis, were impaired.

Differential inhibition by Bacillus thuringiensis δ endotoxin of leucine and aspartic acid uptake into BBMV from midgut of Manduca sexta

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.