Exploitation of the Medfly Gut Microbiota for the Enhancement of Sterile Insect Technique: Use of Enterobacter sp. in Larval Diet-Based Probiotic Applications

Abstract

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a pest of worldwide substantial economic importance, as well as a Tephritidae model for sterile insect technique (SIT) applications. The latter is partially due to the development and utilization of genetic sexing strains (GSS) for this species, such as the Vienna 8 strain, which is currently used in mass rearing facilities worldwide. Improving the performance of such a strain both in mass rearing facilities and in the field could significantly enhance the efficacy of SIT and reduce operational costs. Recent studies have suggested that the manipulation of gut symbionts can have a significant positive effect on the overall fitness of insect strains. We used culture-based approaches to isolate and characterize gut-associated bacterial species of the Vienna 8 strain under mass rearing conditions. We also exploited one of the isolated bacterial species, Enterobacter sp., as dietary supplement (probiotic) to the larval diet, and we assessed its effects on fitness parameters under the standard operating procedures used in SIT operational programs. Probiotic application of Enterobacter sp. resulted in improvement of both pupal and adult productivity, as well as reduced rearing duration, particularly for males, without affecting pupal weight, sex ratio, male mating competitiveness, flight ability and longevity under starvation.

Fig 1. Dendrogram based on 16S rRNA gene sequences.

Enterobacter sp., Providencia sp. and Acinetobacter sp. isolated in this study (Cc_G) and gut bacterial species/strains reported in previous Tephritidae studies were used. Analysis was performed using MEGA 6.0 software. Cc_G_26 is the isolate used as probiotics in the present study. The evolutionary history was inferred using the Neighbor-Joining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site (see scale bar). There were a total of 1135 positions in the final dataset.

Fig 2. Pupae (A) and adult (B) recovery rates.

i) irrespective of the Enterobacter sp. concentration, or ii) considering the three different Enterobacter sp. concentrations as different treatments. Columns marked on top with the same letter are not significantly different (P>0.05)

Fig 3. Developmental times of immature stages at 22°C.

(A) egg to pupa duration, (B) pupal stage duration, (C) total duration of immature stages. The top and bottom of the box are the 25th and 75th percentiles (Q(0.25) and Q(0.75), respectively). The size of the box (Interquartile Range-IQR) is defined as IQR = Q(0.75)-Q(0.25). The bold line in the box represents the median.

Fig 4. Mating competitiveness of Enterobacter sp. treated males (Relative Index).

The mating competitiveness tests were performed in accordance to [38].