Cultivable gut bacteria from Anastrepha fraterculus

SALGUEIRO J.; NUSSENBAUM A.L.; JUÁREZ M. L.; VERA M. T.; BOURTZIS, K.; CLADERA, J.; LANZAVECCHIA, S.B.; SEGURA, D.

Congreso; 10th International Symposium on Fruit Flies of Economic Importance; 2018

Background: Previous studies, mainly conducted in Ceratitis capitata, have demonstrated that the enrichment of larval diet with Enterobacteria isolated from adult guts improves hatching and pupation, acting as a true probiotic. In this study we gave the first steps towards the development of a probiotic to increase the efficiency of Anastrepha fraterculus artificial rearing. In this regards, we proposed: 1. to establish a collection of bacterial isolates fromintestinal tissue of lab and wild A. fraterculus adults; 2. to identify and characterize the isolates by means of molecular tools; 3. to evaluate the effect of adding some of these isolates into the artificial larval medium on early stages of development of A. fraterculus. Methods: Adult males of A. fraterculus from 3 different origins were collected; laboratory (LL), wild caught (WW) and adults derived from infested guava fruit (WL). The guts were extracted and pooled (5 guts), 3 replicates per origin. Bacterial isolates were selected based on morphological characteristics of their colonies and were identified through Colony-PCR and sequencing of 16S rRNA gene. Two isolates named WW1.13 and LL1.14, identified as Enterobacter, were tested as probiotics. Bacteria were grown in LB (W and L cultures respectively) and half of the volume of each culture was autoclaved (Wa and La cultures respectively). A. fraterculus eggs were transferred to artificial larval medium subjected to different treatments of LB-probiotic adding: L, La, W, Wa, and LB medium (m ? without bacteria) and a negative control using larval diet without LB medium (D). Variables recorded:% egg hatch, % pupation at day 10 after seeding, % egg-pupae recovery, pupal weight.Results: We obtained a total of 221 bacterial isolates from guts of LL, WW and WL flies. Themost representative genera were identified as Enterococcus sp., Providencia sp. andEnterobacter sp. obtained from WL flies; Kluyvera sp., Pseudomona sp., Klebsiella sp.,Enterobacter sp. from LL flies and Serratia sp., Enterococcus sp., Citrobacter sp., Erwinia,Pantoea sp., Kluyvera sp., Enterobacter sp in WW flies. Based on taxonomic groups identifiedwe observed the highest richness of cultivable gut bacteria in WW and the lowest in LL. Theresult of probiotic assay showed a lack of significant differences among origins for all thevariables recorded. Nonetheless, the percentage of egg-pupae recovery showed a trend to highervalues in La and W compared to the other treatments, and at the same time exhibited low %pupation at 10th day.Conclusion: Our approach allowed to establish a bacterial collection of 221 entries with nativeisolates obtained from A. fraterculus guts. This collection was taxonomically characterized andused to initiate probiotic assays. Although no effects were detected, a consolidate probioticbioassay was established, and further screening by means of bioassay and biochemicalcharacterization are currently being conducted.