Minor impact of probiotic bacteria and egg white on Tenebrio molitor growth, microbial composition, and pathogen infection

Abstract

The industrial rearing of the yellow mealworm (Tenebrio molitor) for feed and food purposes on agricultural by-products may expose larvae and adults to entomopathogens used as biocontrol agents in crop production. Bacterial spores/toxins or fungal conidia from species such as Bacillus thuringiensis or Metarhizium brunneum could affect the survival and growth of insects. Therefore, the aim of this study was to investigate the potential benefits of a wheat bran diet supplemented with probiotic bacteria and dried egg white on larval development and survival and its effects on the gut microbiome composition. Two probiotic bacterial species, Pediococcus pentosaceus KVL B19-01 and Lactiplantibacillus plantarum WJB, were added to wheat bran feed with and without dried egg white, as an additional protein source, directly from neonate larval hatching until reaching a body mass of 20 mg. Subsequently, larvae from the various diets were exposed for 72 h to B. thuringiensis, M. brunneum, or their combination. Larval survival and growth were recorded for 14 days, and the bacterial microbiota composition was analyzed using 16S rDNA sequencing prior to pathogen exposure and on days 3 and 11 after inoculation with the pathogens. The results showed increased survival for T. molitor larvae reared on feed supplemented with P. pentosaceus in the case of co-infection. Larval growth was also impacted in the co-infection treatment. No significant impact of egg white or of P. pentosaceus on larval growth was recorded, while the addition of Lb. plantarum resulted in a minor increase in individual mass gain compared with infected larvae without the latter probiotic. On day 14, B. thuringiensis was no longer detected and the overall bacterial community composition of the larvae was similar in all treatments. On the other hand, the relative operational taxonomic unit (OTU) abundance was dependent on day, diet, and probiotic. Interestingly, P. pentosaceus was present throughout the experiments, while Lb. plantarum was not found at a detectable level, although its transient presence slightly improved larval performance. Overall, this study confirms the potential benefits of some probiotics during the development of T. molitor while underlining the complexity of the relationship between the host and its microbiome.

Keywords: Bacillus thuringiensis; Lactiplantibacillus plantarum; Pediococcus pentosaceus; entomopathogen; insect health; probiotics; yellow mealworm.

Figure 1

Tenebrio molitor experimental rearing system. Adults mate on a wheat bran (WB) diet or dried egg white and wheat bran (WE) diet supplemented with the selected probiotic species Lactiplantibacillus plantarum WJB (Lb) or Pediococcus pentosaceus KVL B19-01 (Pp) freeze dried in their live or deactivated (indicated by “t”) form.

Figure 2

Experimental setup of the inoculation of Tenebrio molitor larvae with the entomopathogens Bacillus thuringiensis sv. morrisoni biovar tenebrionis (btt) and Metarhizium brunneum KVL 12-30 (met) or with both pathogens (met_btt) and sampling for microbiota analysis. After 24 h of starvation (black line), the larvae were exposed to the entomopathogens or sterile water (c) with the free feeding method for 72 h (red lines), followed by 11 days of provision of uninfected feed [wheat bran with egg white (WE) or wheat bran (WB) diet] and water agar every 2 days (green lines).

Figure 3

Probability of survival (±95% confidence interval) of Tenebrio molitor larvae after 2 weeks of assay. Treatments were the presence in the diet of either live (Pediococcus pentosaceus Pp and Lactiplantibacillus plantarum Lb) or deactivated probiotic species (P. pentosaceus Ppt and Lb. plantarum Lbt) on the survival of T. molitor larvae fed the wheat bran (WB) diet or the wheat bran + egg white (WE) diet and infected with Bacillus thuringiensis (btt), Metarhizium brunneum (met), or both pathogens (met_btt). Each treatment is the mean of three biological replicates of groups of 20 larvae each. The addition of either live or deactivated P. pentosaceus resulted in a significantly higher survival of larvae fed WB in the co-infection conditions (met_btt) (Pp: p = 0.0415; Pp_t: p = 0.0227). *p ≤ 0.1; **p ≤ 0.05.

Figure 4

Effect of pathogens on the relative growth of Tenebrio molitor larvae reared on all treatments after 2 weeks of assay (14 days). Single infection with either pathogen [Bacillus thuringiensis sv. morrisoni biovar tenebrionis (btt) and Metarhizium brunneum (met)] had no significant impact on relative growth (btt: p = 0.84; met: p = 0.78) when compared to the control. Co-infection (met_btt) resulted in a significantly lower relative growth (p = 0.003) than the control. Bars show the estimated marginal means and black dots the single data points. Different letters denote significant differences.

Figure 5

Individual mean mass (IMM) of Tenebrio molitor larvae after 2 weeks of growth. Lactiplantibacillus plantarum deactivated (Lb_t) was the only treatment that had an effect on Metarhizium brunneum (met) and co-infected larvae (met_btt) fed wheat bran (WB; p = 0.038). The other treatments showed no significant effect on IMM (p > 0.05). **p ≤ 0.05.

Figure 6

Principal coordinate analysis based on the weighted UniFrac (PCoA) of larvae fed wheat bran (WB) (Left) and wheat bran and dried egg white (WE) (Right) given probiotic treatments [control (red), Lactiplantibacillus plantarum (yellow), Pediococcus pentosaceus (green), Lb. plantarum deactivated (blue), and P. pentosaceus deactivated (pink)] until day 0 (square) and then fed the control diet from day 3 (circle) and day 14 (triangle). Samples present a clustering on day 14, showing differences in the microbial community composition 14 days after treatment removal and in the homogeneity of the microbial community composition between the treatment groups within each day.

Figure 7

Alpha diversity indices of the microbiota operational taxonomic unit (OTU) data from Tenebrio molitor larvae grouped by probiotic treatments [no (control) (red), Lactiplantibacillus plantarum (yellow), Pediococcus pentosaceus (green), Lb. plantarum deactivated (blue), and P. pentosaceus deactivated (pink)] and infections [control, Bacillus thuringiensis sv. morrisoni biovar tenebrionis (btt), Metarhizium brunneum KVL 12-30 (met), and met_btt) on day 3. The box plot shows the OTU abundance between the grouped samples.