Infection risk by oral contamination does not induce immune priming in the mealworm beetle (Tenebrio molitor) but triggers behavioral and physiological responses

Abstract

In invertebrates, immune priming is the ability of individuals to enhance their immune response based on prior immunological experiences. This adaptive-like immunity likely evolved due to the risk of repeated infections by parasites in the host's natural habitat. The expression of immune priming varies across host and pathogen species, as well as infection routes (oral or wounds), reflecting finely tuned evolutionary adjustments. Evidence from the mealworm beetle (Tenebrio molitor) suggests that Gram-positive bacterial pathogens play a significant role in immune priming after systemic infection. Despite the likelihood of oral infections by natural bacterial pathogens in T. molitor, it remains debated whether ingestion of contaminated food leads to systemic infection, and whether oral immune priming is possible is currently unknown. We first attempted to induce immune priming in both T. molitor larvae and adults by exposing them to food contaminated with living or dead Gram-positive and Gram-negative bacterial pathogens. We found that oral ingestion of living bacteria did not kill them, but septic wounds caused rapid mortality. Intriguingly, the consumption of either dead or living bacteria did not protect against reinfection, contrasting with injury-induced priming. We further examined the effects of infecting food with various living bacterial pathogens on variables such as food consumption, mass gain, and feces production in larvae. We found that larvae exposed to Gram-positive bacteria in their food ingested less food, gained less mass and/or produced more feces than larvae exposed to contaminated food with Gram-negative bacteria or control food. This suggests that oral contamination with Gram-positive bacteria induced both behavioral responses and peristalsis defense mechanisms, even though no immune priming was observed here. Considering that the oral route of infection neither caused the death of the insects nor induced priming, we propose that immune priming in T. molitor may have primarily evolved as a response to the infection risk associated with wounds rather than oral ingestion.

Keywords: Tenebrio molitor; bacteria; behavioral defense; entomopathogen; evolution of immune priming; gut immunity; oral infection.

Figure 1

Survival of T. molitor after a secondary septic wound occurring either 24 hours or 15 days after the feeding treatment among adults (A) and larvae (B), according to the bacteria species. Bc, Bacillus cereus. Sm, Serratia marcescens.

Figure 2

Survival of adult T. molitor after a secondary septic wound occurring either 24 hours (A) or 15 days (B) after the feeding treatment, regardless of the bacteria species used.

Figure 3

Survival of adult T. molitor after a secondary septic wound involving either Bacillus cereus (A) or Serratia marcescens (B), regardless of the timing of the secondary septic wound.

Figure 4

Survival of male (A) and female (B) T. molitor individuals after a secondary septic wound occurring either 24 hours or 15 days, depending on the bacteria species used (Bc, Bacillus cereus; Sm, Serratia marcescens).

Figure 5

Total quantity of food, in milligrams, eaten by larvae over the three-day feeding period. Treatment groups were compared to the control group. Bc, Bacillus cereus; Sa, Staphylococcus aureus; Sm, Serratia marcescens; Ec, Escherichia coli; ns, non-significant difference. *p < 0.05. Numbers in the bars are sample sizes.

Figure 6

Mass, in milligrams, of feces produced by larvae 24 hours after the end of the feeding period. Treatment groups were compared to the control group. ns, non-significant difference. *p < 0.05. **p < 0.01. N = 20 in each group.

Figure 7

Number of colony-forming units (CFU) from the selected feces samples. Thick lines are the medians, boxes are the upper and lower quartiles, whiskers are the upper and lower interquartile range. Treatment groups were compared to the group including Bacillus cereus. ns, non-significative difference. **p < 0.01. *** p < 0.001. N = 20 in each group.

Figure 8

Survival of T. molitor larvae to an infection, whether or not individuals were fed with the same bacteria used for infection. (A) Escherichia coli. (B) Staphylococcus aureus. (C) Bacillus cereus. (D) Serratia marcescens.