Impact and Persistence of Serratia marcescens in Tenebrio molitor Larvae and Feed under Optimal and Stressed Mass Rearing Conditions

Abstract

Industrial insect mass rearing aims to produce quality insects under safe sanitary conditions which can be compromised by pathogens and abiotic stressors. Therefore, knowledge on pathogen persistence, virulence and means of detection is of importance. This study focuses on the opportunistic pathogen Serratia marcescens (Sm) as a possible candidate to reveal sanitary issues in Tenebrio molitor (Tm) breeding. A screening test was performed to assess the impact of abiotic stressors (starvation, density and sieving) in presence and absence of Sm. Two Sm detection methods were conducted, and the kinetics of Sm persistence were investigated. Our results show that (i) the presence of Sm had a low but significant effect on Tm mortality, (ii) a short temporary starvation period had a negative impact on larval growth, (iii) the detection of Sm by q-PCR was sensitive but less convenient than a specific Sm growth media, (iv) the kinetics of persistence showed that Sm declined but survived for nine days in the feed and in the feces for three weeks. Both the relatively low virulence and the persistence in the environment suggest that Sm could be used as an indicator for the sanitary status of mealworm production.

Keywords: Serratia marcescens; food safety; insect for food and feed; microbial persistence; qPCR; yellow mealworm.

Figure 1

Experimental design. The figure shows the time schedule of experimental setup: when the feed was delivered, the data collections and when stresses were applied. The experiment lasted for 24 days.

Figure 2

Impact of starvation and density on Individual Mean Mass increase. The IMM relative gain is expressed as a percentage. In this figure, LD and HD stand for low and high larval density, while LS and HS stand for no-starvation and starvation, respectively.

Figure 3

(A,B) respectively, represent FCR (Feed Conversion Ratio) and the relative mass gain expressed as a percentage and as a function of starvation after 24 days.

Figure 4

Larval mortality after 24 days, “Control” corresponds to the cups without the addition of Serratia to the feed while “Serratia” indicates the cups in which the bacterial culture was added to the feed at the beginning of the experiment.

Figure 5

(A) The qPCR standard curve, necessary to perform the quantitative analysis, was made with the positive control supplied in the kit. The number of Ct was plotted on the graph. Knowing the number of copies of the positive control, the standard curve was created, and the number of copies of an unknown sample could be plotted. The lower the Ct, the higher the number of bacteria. (B) The numbers refer to the quantity of Sm bacteria extrapolated from qPCR analysis on DNA extracted from different Tm samples and the quantity of Sm in an overnight culture used to inoculate wheat bran consumed by Tm.

Figure 6

Quantity of Serratia detected (on erythritol plates) during 9 days from 80 mg bran inoculated with 6 × 10⁹ CFU Sm culture and stored at 30 °C. Mean of 3 batches SEM (standard error of the mean).

Figure 7

The kinetics of the presence of Serratia marcescens (Sm) in Tm feces. The numbers are Sm CFU’s (on erythritol medium) from 2 mg of feces, according to time after consumption (0–24 h) of Sm-contaminated wheat bran. The experiment lasted for 27 days but feces were collected from day 2 (24–48 h) with a certain interval until no more feces were excreted (48–50 h) and again after consumption of feed without Sm during hours 50–56 h. Although no feed was added, feces and Sm were still collected from day 8 to day 27 (19 d) h (hours), d (days).