Publications

4485

AbstractBackgroundThe symbiosis between the olive fruit fly,Bactrocera oleae, andCandidatusErwinia dacicola has been demonstrated as essential for the fly’s larval development and adult physiology. The mass rearing of the olive fruit fly has been hindered by several issues, including problems which could be related to the lack of the symbiont, presumably due to preservatives and antibiotics currently used during rearing under laboratory conditions. To better understand the mechanisms underlying symbiont removal or loss during the rearing of lab colonies of the olive fruit fly, we performed experiments that focused on bacterial transfer from wild female flies to their eggs. In this research, eggs laid by wild females were treated with propionic acid solution, which is often used as an antifungal agent, a mixture of sodium hypochlorite and Triton X, or water (as a control). The presence of the bacterial symbiont on eggs was evaluated by real-time PCR and scanning electron microscopy.ResultsDGGE analysis showed a clear band with the same migration behavior present in all DGGE profiles but with a decreasing intensity. Molecular analyses performed by real-time PCR showed a significant reduction inCa. E. dacicola abundance in eggs treated with propionic acid solution or a mixture of sodium hypochlorite and Triton X compared to those treated with water. In addition, the removal of bacteria from the surfaces of treated eggs was highlighted by scanning electron microscopy.ConclusionsThe results clearly indicate how the first phases of the colony-establishment process are important in maintaining the symbiont load in laboratory populations and suggest that the use of products with antimicrobial activity should be avoided. The results also suggest that alternative rearing procedures for the olive fruit fly should be investigated.

Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.

AbstractBackgroundThe olive fly,Bactrocera oleae,is the most important insect pest in olive production, causing economic damage to olive crops worldwide. In addition to extensive research onB. oleaecontrol methods, scientists have devoted much effort in the last century to understanding olive fly endosymbiosis with a bacterium eventually identified asCandidatusErwinia dacicola. This bacterium plays a relevant role in olive fly fitness. It is vertically transmitted, and it benefits both larvae and adults in wild populations; however, the endosymbiont is not present in lab colonies, probably due to the antibiotics and preservatives required for the preparation of artificial diets. Endosymbiont transfer from wildB. oleaepopulations to laboratory-reared ones allows olive fly mass-rearing, thus producing more competitive flies for future Sterile Insect Technique (SIT) applications.ResultsWe tested the hypothesis thatCa. E. dacicola might be transmitted from wild, naturally symbiotic adults to laboratory-reared flies. Several trials have been performed with different contamination sources ofCa. E. dacicola, such as ripe olives and gelled water contaminated by wild flies, wax domes containing eggs laid by wild females, cages dirtied by faeces dropped by wild flies and matings between lab and wild adults. PCR-DGGE, performed with the primer set 63F-GC/518R, demonstrated that the transfer of the endosymbiont from wild flies to lab-reared ones occurred only in the case of cohabitation.ConclusionsCohabitation of symbiotic wild flies and non-symbiotic lab flies allows the transfer ofCa. E. dacicola through adults. Moreover, PCR-DGGE performed with the primer set 63F-GC/518R was shown to be a consistent method for screeningCa. E. dacicola, also showing the potential to distinguish between the two haplotypes (htA and htB). This study represents the first successful attempt at horizontal transfer ofCa. E. dacicola and the first step in acquiring a better understanding of the endosymbiont physiology and its relationship with the olive fly. Our research also represents a starting point for the development of a laboratory symbiotic olive fly colony, improving perspectives for future applications of the Sterile Insect Technique.

Abstract Background Microbial communities recurrently establish metabolic associations resulting in increased fitness and ability to perform complex tasks, such as xenobiotic degradation. In a previous study, we have described a sulfonamide-degrading consortium consisting of a novel low-abundant actinobacterium, named strain GP, and Achromobacter denitrificans PR1. However, we found that strain GP was unable to grow independently and could not be further purified. Results Previous studies suggested that strain GP might represent a new putative species within the Leucobacter genus (16S rRNA gene similarity < 97%). In this study, we found that average nucleotide identity (ANI) with other Leucobacter spp. ranged between 76.8 and 82.1%, further corroborating the affiliation of strain GP to a new provisional species. The average amino acid identity (AAI) and percentage of conserved genes (POCP) values were near the lower edge of the genus delimitation thresholds (65 and 55%, respectively). Phylogenetic analysis of core genes between strain GP and Leucobacter spp. corroborated these findings. Comparative genomic analysis indicates that strain GP may have lost genes related to tetrapyrrole biosynthesis and thiol transporters, both crucial for the correct assembly of cytochromes and aerobic growth. However, supplying exogenous heme and catalase was insufficient to abolish the dependent phenotype. The actinobacterium harbors at least two copies of a novel genetic element containing a sulfonamide monooxygenase (sadA) flanked by a single IS1380 family transposase. Additionally, two homologs of sadB (4-aminophenol monooxygenase) were identified in the metagenome-assembled draft genome of strain GP, but these were not located in the vicinity of sadA nor of mobile or integrative elements. Conclusions Comparative genomics of the genus Leucobacter suggested the absence of some genes encoding for important metabolic traits in strain GP. Nevertheless, although media and culture conditions were tailored to supply its potential metabolic needs, these conditions were insufficient to isolate the PR1-dependent actinobacterium further. This study gives important insights regarding strain GP metabolism; however, gene expression and functional studies are necessary to characterize and further isolate strain GP. Based on our data, we propose to classify strain GP in a provisional new species within the genus Leucobacter, ‘Candidatus Leucobacter sulfamidivorax‘.

Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) Diaphorina citri, the principal vector of ‘Candidatus Liberibacter asiaticus’ (Las). Las is the pathogen associated with huanglongbing (HLB), the Asian form of which is the most devastating disease of Citrus species and cultivars (Rutaceae: Aurantioideae). M. paniculata is a common ornamental and S. glutinosa is grown as an ornamental, a citrus rootstock, and a hedgerow fence plant. Because of the uncertain status of these plants as reservoirs of Las, a series of cross-inoculation bioassays were carried out in different environments, using infected Valencia sweet orange (Citrus × aurantium) infected shoot tops as a source of inoculum and D. citri nymphs and adults reared on M. paniculata and S. glutinosa to inoculate pathogen-free Valencia orange plantlets. In contrast to sweet orange, Las was more unevenly distributed and reached much lower titers in M. paniculata and S. glutinosa. Infections in M. paniculata and S. glutinosa were also transient. Very few insects that successfully acquired Las from M. paniculata and S. glutinosa were able to transmit the pathogen to healthy citrus. Transmission rates were low from M. paniculata (1.0%) and S. glutinosa (2.0%) and occurred only in a controlled environment highly favorable to Las and ACP using 10-day-old adults that completed their life cycle on Las-positive plants. Our study showed that in HLB-endemic areas, M. paniculata and S. glutinosa can be deemed as epidemiologically dead-end hosts for Las and are not important alternative hosts of the pathogen for transmission to citrus. However, under a combination of conditions highly favorable to Las infection and transmission and in the absence of effective quarantine procedures, these plants could eventually serve as carriers of Las to regions currently free from HLB.