Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap "n" collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to ß-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance.

Anna Karenina as a promoter of microbial diversity in the cosmopolitan agricultural pest Zeugodacus cucurbitae (Diptera, Tephritidae).

Gut microbial communities are critical in determining the evolutive success of fruit fly phytophagous pests (Diptera, Tephritidae), facilitating their adaptation to suboptimal environmental conditions and to plant allelochemical defences. An important source of variation for the microbial diversity of fruit flies is represented by the crop on which larvae are feeding. However, a "crop effect" is not always the main driver of microbial patterns, and it is often observed in combination with other and less obvious processes. In this work, we aim at verifying if environmental stress and, by extension, changing environmental conditions, can promote microbial diversity in Zeugodacus cucurbitae (Coquillett), a cosmopolitan pest of cucurbit crops. With this objective, 16S rRNA metabarcoding was used to test differences in the microbial profiles of wild fly populations in a large experimental setup in Eastern Central Tanzania. The analysis of 2,973 unique ASV, which were assigned to 22 bacterial phyla, 221 families and 590 putative genera, show that microbial α diversity (as estimated by Abundance Coverage Estimator, Faith's Phylogenetic Diversity, Shannon-Weiner and the Inverse Simpson indexes) as well as ß microbial diversity (as estimated by Compositional Data analysis of ASVs and of aggregated genera) significantly change as the species gets closer to its altitudinal limits, in farms where pesticides and agrochemicals are used. Most importantly, the multivariate dispersion of microbial patterns is significantly higher in these stressful environmental conditions thus indicating that Anna Karenina effects contribute to the microbial diversity of Z. cucurbitae. The crop effect was comparably weaker and detected as non-consistent changes across the experimental sites. We speculate that the impressive adaptive potential of polyphagous fruit flies is, at least in part, related to the Anna Karenina principle, which promotes stochastic changes in the microbial diversity of fly populations exposed to suboptimal environmental conditions.

Sex-specific effects of the microbiota on adult carbohydrate intake and body composition in a polyphagous fly.

The microbiota influences hosts' health and fitness. However, the extent to which the microbiota affects host' foraging decisions and related life history traits remains to be fully understood. Our study explored the effects of microbiota manipulation on foraging preference and phenotypic traits of larval and adult stages of the polyphagous fruit fly Bactrocera tryoni, one of the main horticultural pests in Australia. We generated three treatments: control (non-treated microbiota), axenic (removed microbiota), and reinoculation (individuals which had their microbiota removed then re-introduced). Our results confirmed that axenic larvae and immature (i.e., newly emerged 0 day-old, sexually-immature) adults were lighter than control and reinoculated individuals. Interestingly, we found a sex-specific effect of the microbiota manipulation on carbohydrate intake and body composition of 10 day-old mature adults. Axenic males ate less carbohydrate, and had lower body weight and total body fat relative to control and reinoculated males. Conversely, axenic females ate more carbohydrate than control and reinoculated ones, although body weight and lipid reserves were similar across treatments. Axenic females produced fewer eggs than control and reinoculated females. Our findings corroborate the far-reaching effects of microbiota in insects found in previous studies and show, for the first time, a sex-specific effect of microbiota on feeding behaviour in flies. Our results underscore the dynamic relationship between the microbiota and the host with the reinoculation of microbes restoring some traits that were affected in axenic individuals.

Diversity of bacteria in different life stages and their impact on the development and reproduction of Zeugodacus tau (Diptera: Tephritidae).

Fruit flies usually harbor diverse communities of bacteria in their digestive systems, which are known to play a significant role in their fitness. However, little information is available on Zeugodacus tau, a polyphagous pest worldwide. This study reports the first extensive analysis of bacterial communities in different life stages and their effect on the development and reproduction of laboratory-reared Z. tau. Cultured bacteria were identified using the conventional method, and all bacteria were identified by high-throughput technologies (16S ribosomal RNA gene sequencing of V3-V4 region). A total of six bacterial phyla were identified in larvae, pupae, and male and female adult flies, which were distributed into 14 classes, 32 orders, 58 families and 96 genera. Proteobacteria was the most represented phylum in all the stages except larvae. Enterobacter, Klebsiella, Providencia, and Pseudomonas were identified by conventional and next-generation sequencing analysis in both male and female adult flies, and Enterobacter was found to be the main genus. After being fed with antibiotics from the first instar larvae, bacterial diversity changed markedly in the adult stage. Untreated flies laid eggs and needed 20 days before oviposition while the treated flies showed ovary development inhibited and were not able to lay eggs, probably due to the alteration of the microbiota. These findings provide the cornerstone for unexplored research on bacterial function in Z. tau, which will help to develop an environmentally friendly management technique for this kind of harmful insect.

Comparative genomics of Klebsiella michiganensis BD177 and related members of Klebsiella sp. reveal the symbiotic relationship with Bactrocera dorsalis.

BACKGROUND: Bactrocera dorsalis is a destructive polyphagous and highly invasive insect pest of tropical and subtropical species of fruit and vegetable crops. The sterile insect technique (SIT) has been used for decades to control insect pests of agricultural, veterinary, and human health importance. Irradiation of pupae in SIT can reduce the ecological fitness of the sterile insects. Our previous study has shown that a gut bacterial strain BD177 that could restore ecological fitness by promoting host food intake and metabolic activities. RESULTS: Using long-read sequence technologies, we assembled the complete genome of K. michiganensis BD177 strain. The complete genome of K. michiganensis BD177 comprises one circular chromosome and four plasmids with a GC content of 55.03%. The pan-genome analysis was performed on 119 genomes (strain BD177 genome and 118 out of 128 published Klebsiella sp. genomes since ten were discarded). The pan-genome includes a total of 49305 gene clusters, a small number of 858 core genes, and a high number of accessory (10566) genes. Pan-genome and average nucleotide identity (ANI) analysis showed that BD177 is more similar to the type strain K. michiganensis DSM2544, while away from the type strain K. oxytoca ATCC13182. Comparative genome analysis with 21 K. oxytoca and 12 K. michiganensis strains, identified 213 unique genes, several of them related to amino acid metabolism, metabolism of cofactors and vitamins, and xenobiotics biodegradation and metabolism in BD177 genome. CONCLUSIONS: Phylogenomics analysis reclassified strain BD177 as a member of the species K. michiganensis. Comparative genome analysis suggested that K. michiganensis BD177 has the strain-specific ability to provide three essential amino acids (phenylalanine, tryptophan and methionine) and two vitamins B (folate and riboflavin) to B. dorsalis. The clear classification status of BD177 strain and identification of unique genetic characteristics may contribute to expanding our understanding of the symbiotic relationship of gut microbiota and B. dorsalis.

Fruit host-dependent fungal communities in the microbiome of wild Queensland fruit fly larvae.

Bactrocera tryoni (Froggatt), the Queensland fruit fly (Qfly), is a highly polyphagous tephritid fly that is widespread in Eastern Australia. Qfly physiology is closely linked with its fungal associates, with particular relationship between Qfly nutrition and yeast or yeast-like fungi. Despite animal-associated fungi typically occurring in multi-species communities, Qfly studies have predominately involved the culture and characterisation of single fungal isolates. Further, only two studies have investigated the fungal communities associated with Qfly, and both have used culture-dependant techniques that overlook non-culturable fungi and hence under-represent, and provide a biased interpretation of, the overall fungal community. In order to explore a potentially hidden fungal diversity and complexity within the Qfly mycobiome, we used culture-independent, high-throughput Illumina sequencing techniques to comprehensively, and holistically characterized the fungal community of Qfly larvae and overcome the culture bias. We collected larvae from a range of fruit hosts along the east coast of Australia, and all had a mycobiome dominated by ascomycetes. The most abundant fungal taxa belonged to the genera Pichia (43%), Candida (20%), Hanseniaspora (10%), Zygosaccharomyces (11%) and Penicillium (7%). We also characterized the fungal communities of fruit hosts, and found a strong degree of overlap between larvae and fruit host communities, suggesting that these communities are intimately inter-connected. Our data suggests that larval fungal communities are acquired from surrounding fruit flesh. It is likely that the physiological benefits of Qfly exposure to fungal communities is primarily due to consumption of these fungi, not through syntrophy/symbiosis between fungi and insect 'host'.

Variations in the Bacterial Communities in Anastrepha obliqua (Diptera: Tephritidae) According to the Insect Life Stage and Host Plant.

Insects have established close relationships with a wide variety of microorganisms, which play a key role in insect ecology and evolution. Fruit flies in the Tephritidae family have economic importance at the global level, including species such as Anastrepha obliqua, which is an important pest in the neotropical region. Although several studies have been performed on the microbiota associated with fruit flies, there are still large gaps in our knowledge about the bacterial communities on the genus Anastrepha. During this study, we used high-throughput sequencing to characterize the bacterial communities of the polyphagous fly A. obliqua, and we evaluated the effect of the life stage (larvae and adults) and host plant (three plant species) on the structure of these communities. Our results show that the bacterial communities in A. obliqua appears to be structured according to the insect life stage and the host plant. The predominant genera belonging to the phylum Proteobacteria were Wolbachia and Enterobacter in both larvae and adults, and they displayed differences in abundance between them, with Wolbachia sp. being more abundant in larvae and Enterobacter sp. being more abundant in adults. Differences in the structures of the bacterial communities were also observed according to the host plant with higher abundance of Enterobacter and Acetobacter bacteria in mango and plum fruits. Based on our results, it can be hypothesized that the bacterial communities on A. obliqua reorganize according to the needs of these insects during their different life stages and could also play an important role in the establishment of this fly species on different host plants. This study represents the first approach to understanding microorganism-insect interactions in fruit flies in Colombia.

Potential of a fly gut microbiota incorporated gel-based larval diet for rearing Bactrocera dorsalis (Hendel).

BACKGROUND: The Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is an important polyphagous pest of horticultural produce. The sterile insect technique (SIT) is a proven control method against many insect pests, including fruit flies, under area-wide pest management programs. High quality mass-rearing process and the cost-effective production of sterile target species are important for SIT. Irradiation is reported to cause severe damage to the symbiotic community structure in the mid gut of fruit fly species, impairing SIT success. However, studies have found that target-specific manipulation of insect gut bacteria can positively impact the overall fitness of SIT-specific insects. RESULTS: Twelve bacterial genera were isolated and identified from B. dorsalis eggs, third instars larval gut and adults gut. The bacterial genera were Acinetobacter, Alcaligenes, Citrobacter, Pseudomonas, Proteus, and Stenotrophomonas, belonging to the Enterobacteriaceae family. Larval diet enrichment with the selected bacterial isolate, Proteus sp. was found to improve adult emergence, percentage of male, and survival under stress. However, no significant changes were recorded in B. dorsalis egg hatching, pupal yield, pupal weight, duration of the larval stage, or flight ability. CONCLUSIONS: These findings support the hypothesis that gut bacterial isolates can be used in conjunction with SIT. The newly developed gel-based larval diet incorporated with Proteus sp. isolates can be used for large-scale mass rearing of B. dorsalis in the SIT program.

The effect of diet and radiation on the bacterial symbiome of the melon fly, Zeugodacus cucurbitae (Coquillett).

BACKGROUND: Symbiotic bacteria contribute to a multitude of important biological functions such as nutrition and reproduction and affect multiple physiological factors like fitness and longevity in their insect hosts. The melon fly, Zeugodacus cucurbitae (Coquillett), is an important agricultural pest that affects a variety of cultivated plants belonging mostly to the Cucurbitaceae family. It is considered invasive and widespread in many parts of the world. Several approaches are currently being considered for the management of its populations including the environmentally friendly and effective sterile insect technique (SIT), as a component of an integrated pest management (IPM) strategy. In the present study, we examined the effect of diet and radiation on the bacterial symbiome of Z. cucurbitae flies with the use of Next Generation Sequencing technologies. RESULTS: Melon flies were reared on two diets at the larval stage, an artificial bran-based diet and on sweet gourd, which affected significantly the development of the bacterial profiles. Significant differentiation was also observed based on gender. The effect of radiation was mostly diet dependent, with irradiated melon flies reared on the bran diet exhibiting a significant reduction in species diversity and richness compared to their non-irradiated controls. Changes in the bacterial symbiome of the irradiated melon flies included a drastic reduction in the number of sequences affiliated with members of Citrobacter, Raoultella, and Enterobacteriaceae. At the same time, an increase was observed for members of Enterobacter, Providencia and Morganella. Interestingly, the irradiated male melon flies reared on sweet gourd showed a clear differentiation compared to their non-irradiated controls, namely a significant reduction in species richness and minor differences in the relative abundance for members of Enterobacter and Providencia. CONCLUSIONS: The two diets in conjunction with the irradiation affected significantly the formation of the bacterial symbiome. Melon flies reared on the bran-based artificial diet displayed significant changes in the bacterial symbiome upon irradiation, in all aspects, including species richness, diversity and composition. When reared on sweet gourd, significant changes occurred to male samples due to radiation, only in terms of species richness.

Next-Generation Sequencing reveals relationship between the larval microbiome and food substrate in the polyphagous Queensland fruit fly.

Insects typically host substantial microbial communities (the 'microbiome') that can serve as a vital source of nutrients and also acts as a modulator of immune function. While recent studies have shown that diet is an important influence on the gut microbiome, very little is known about the dynamics underpinning microbial acquisition from natural food sources. Here, we addressed this gap by comparing the microbiome of larvae of the polyphagous fruit fly Bactrocera tryoni ('Queensland fruit fly') that were collected from five different fruit types (sapodilla [from two different localities], hog plum, pomegranate, green apple, and quince) from North-east to South-east Australia. Using Next-Generation Sequencing on the Illumina MiSeq platform, we addressed two questions: (1) what bacterial communities are available to B. tryoni larvae from different host fruit; and (2) how does the microbiome vary between B. tryoni larvae and its host fruit? The abundant bacterial taxa were similar for B. tryoni larvae from different fruit despite significant differences in the overall microbial community compositions. Our study suggests that the bacterial community structure of B. tryoni larvae is related less to the host fruit (diet) microbiome and more to vertical transfer of the microbiome during egg laying. Our findings also suggest that geographic location may play a quite limited role in structuring of larval microbiomes. This is the first study to use Next-Generation Sequencing to analyze the microbiome of B. tryoni larvae together with the host fruit, an approach that has enabled greatly increased resolution of relationships between the insect's microbiome and that of the surrounding host tissues.

Two Gut-Associated Yeasts in a Tephritid Fruit Fly have Contrasting Effects on Adult Attraction and Larval Survival.

Yeast-insect interactions have been well characterized in drosophilid flies, but not in tephritid fruit flies, which include many highly polyphagous pest species that attack ripening fruits. Using the Queensland fruit fly (Bactrocera tryoni) as our model tephritid species, we identified yeast species present in the gut of wild-collected larvae and found two genera, Hanseniaspora and Pichia, were the dominant isolates. In behavioural trials using adult female B. tryoni, a fruit-agar substrate inoculated with Pichia kluyveri resulted in odour emissions that increased the attraction of flies, whereas inoculation with Hanseniaspora uvarum, produced odours that strongly deterred flies, and both yeasts led to decreased oviposition. Larval development trials showed that the fruit-agar substrate inoculated with the 'deterrent odour' yeast species, H. uvarum, resulted in significantly faster larval development and a greater number of adult flies, compared to a substrate inoculated with the 'attractive odour' yeast species, P. kluyveri, and a yeast free control substrate. GC-MS analysis of volatiles emitted by H. uvarum and P. kluyveri inoculated substrates revealed significant quantitative differences in ethyl-, isoamyl-, isobutyl-, and phenethyl- acetates, which may be responsible for the yeast-specific olfactory responses of adult flies. We discuss how our seemingly counterintuitive finding that female B. tryoni flies avoid a beneficial yeast fits well with our understanding of female choice of oviposition sites, and how the contrasting behavioural effects of H. uvarum and P. kluyveri raises interesting questions regarding the role of yeast-specific volatiles as cues to insect vectors. A better understanding of yeast-tephritid interactions could assist in the future management of tephritid fruit fly pests through the formulation of new "attract and kill" lures, and the development of probiotics for mass rearing of insects in sterile insect control programs.

High Diversity of Bacterial Communities in Developmental Stages of Bactrocera carambolae (Insecta: Tephritidae) Revealed by Illumina MiSeq Sequencing of 16S rRNA Gene.

Bactrocera carambolae is a highly polyphagous fruit pest of agricultural importance. This study reports the bacterial communities associated with the developmental stages of B. carambolae. The microbiota of the developmental stages were investigated by targeted 16S rRNA gene (V3-V4 region) sequencing using the Illumina MiSeq. At 97% similarity, there were 19 bacterial phyla and unassigned bacteria, comprising 39 classes, 86 orders, 159 families and 311 genera. The bacterial composition varied among the specimens of developmental stage and across developmental stages as well as exuviae. Four phyla of bacteria (with relative abundance of ≥1% in at least one specimen)-Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria-were recovered from the larva, pupa, adult stages and exuviae. Proteobacteria was the predominant phylum in all the developmental stages as well as the exuviae. Enterobacteriaceae (Proteobacteria) was the predominant family in the adult flies while the family [Weeksellaceae] (Bacteroidetes) was predominant in the larval and pupal stages. Among the genera occurring in more than one developmental stage of B. carambolae, Erwinia was more abundant in the larval stage, Halomonas more abundant in adult female, Stenotrophomonas more abundant in adult male, and Chryseobacterium more abundant in the larval and pupal stages. The results indicate transmission of bacteria OTUs from immatures to the newly emerged adults, and from exuviae to the environment.

The Microbiome of Field-Caught and Laboratory-Adapted Australian Tephritid Fruit Fly Species with Different Host Plant Use and Specialisation.

Tephritid fruit fly species display a diversity of host plant specialisation on a scale from monophagy to polyphagy. Furthermore, while some species prefer ripening fruit, a few are restricted to damaged or rotting fruit. Such a diversity of host plant use may be reflected in the microbial symbiont diversity of tephritids and their grade of dependency on their microbiomes. Here, we investigated the microbiome of six tephritid species from three genera, including species that are polyphagous pests (Bactrocera tryoni, Bactrocera neohumeralis, Bactrocera jarvisi, Ceratitis capitata) and a monophagous specialist (Bactrocera cacuminata). These were compared with the microbiome of a non-pestiferous but polyphagous tephritid species that is restricted to damaged or rotting fruit (Dirioxa pornia). The bacterial community associated with whole fruit flies was analysed by 16S ribosomal DNA (rDNA) amplicon pyrosequencing to detect potential drivers of taxonomic composition. Overall, the dominant bacterial families were Enterobacteriaceae and Acetobacteraceae (both Proteobacteria), and Streptococcaceae and Enterococcaceae (both Firmicutes). Comparisons across species and genera found different microbial composition in the three tephritid genera, but limited consistent differentiation between Bactrocera species. Within Bactrocera species, differentiation of microbial composition seemed to be influenced by the environment, possibly including their diets; beyond this, tephritid species identity or ecology also had an effect. The microbiome of D. pornia was most distinct from the other five species, which may be due to its ecologically different niche of rotting or damaged fruit, as opposed to ripening fruit favoured by the other species. Our study is the first amplicon pyrosequencing study to compare the microbiomes of tephritid species and thus delivers important information about the turnover of microbial diversity within and between fruit fly species and their potential application in pest management strategies.

The olive fly endosymbiont, "Candidatus Erwinia dacicola," switches from an intracellular existence to an extracellular existence during host insect development.

As polyphagous, holometabolous insects, tephritid fruit flies (Diptera: Tephritidae) provide a unique habitat for endosymbiotic bacteria, especially those microbes associated with the digestive system. Here we examine the endosymbiont of the olive fly [Bactrocera oleae (Rossi) (Diptera: Tephritidae)], a tephritid of great economic importance. "Candidatus Erwinia dacicola" was found in the digestive systems of all life stages of wild olive flies from the southwestern United States. PCR and microscopy demonstrated that "Ca. Erwinia dacicola" resided intracellularly in the gastric ceca of the larval midgut but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. "Ca. Erwinia dacicola" is one of the few nonpathogenic endosymbionts that transitions between intracellular and extracellular lifestyles during specific stages of the host's life cycle. Another unique feature of the olive fly endosymbiont is that unlike obligate endosymbionts of monophagous insects, "Ca. Erwinia dacicola" has a G+C nucleotide composition similar to those of closely related plant-pathogenic and free-living bacteria. These two characteristics of "Ca. Erwinia dacicola," the ability to transition between intracellular and extracellular lifestyles and a G+C nucleotide composition similar to those of free-living relatives, may facilitate survival in a changing environment during the development of a polyphagous, holometabolous host. We propose that insect-bacterial symbioses should be classified based on the environment that the host provides to the endosymbiont (the endosymbiont environment).

Wickerhamomyces queroliae sp. nov. and Candida jalapaonensis sp. nov., two yeast species isolated from Cerrado ecosystem in North Brazil.

Two novel yeast species, Wickerhamomyces queroliae sp. nov. and Candida jalapaonensis sp. nov., were isolated, respectively, from larvae of Anastrepha mucronata (Diptera: Tephritidae) collected from ripe fruit of Peritassa campestris ('Bacupari', Hippocrateaceae) and from flowers of Centropogon cornutus (Campanulaceae) in the Cerrado ecosystem of the state of Tocantins, Brazil. Analysis of the D1/D2 large-subunit rRNA gene sequences placed W. queroliae in the Wickerhamomyces clade near Wickerhamomyces ciferri and Candida silvicultrix. Candida jalapaonensis belongs to the Wickerhamiella clade and is related to Candida drosophilae. The type strain of Wickerhamomyces queroliae is UFMG-05-T200.1(T) (=CBS 10936(T)=NRRL Y-48478(T)) and the type strain of Candida jalapaonensis is UFMG-03-T210(T) (=CBS 10935(T)=NRRL Y-48477(T)).

Presence of specific symbiotic bacteria in flies of the subfamily Tephritinae (Diptera Tephritidae) and their phylogenetic relationships: proposal of 'Candidatus Stammerula tephritidis'.

The presence of symbiotic bacteria in flies belonging to the subfamily Tephritinae, which predominantly infest the flower heads of composite flowers (Asteraceae), was investigated. Twenty-five species of flies, collected mainly in northern Italy, were examined. The bacteria adhered to the midgut epithelium in a space external to the peritrophic membrane and therefore not in direct contact with the gut contents. Specific, unique and live, but unculturable bacteria were consistently found in the majority of the fly species and their presence was also shown to be persistent in flies reared under microbiologically controlled conditions and devoid of any residual culturable intestinal bacteria. Sequencing of the small subunit rRNA gene from the novel bacteria indicated that they belonged to the family Enterobacteriaceae. Three main strongly supported clades were delineated by phylogenetic trees, the first of which featured a coherent set of sequences displaying gene sequence similarities lower than 96 % compared with recognized taxa. The second and third clades featured cases with higher gene sequence similarities to culturable bacteria, including Erwinia persicina and Ewingella americana, respectively. Relative rate tests were supportive of a fast genetic evolution for the majority of the bacterial symbionts of the subfamily Tephritinae. In agreement with the interpretation suggested in 1929 after pioneering observations made by H. J. Stammer, a symbiotic relationship between the novel bacteria and the tephritid flies is postulated. The origin of this apparently polyphyletic relationship is discussed and a novel candidate organism is proposed for the first clade under the designation 'Candidatus Stammerula tephritidis'.