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Several insect lineages, including some fruit flies, have evolved mutualistic associations with primary symbiotic bacteria. Some species of Tephritinae, the most specialized subfamily of fruit flies (Diptera, Tephritidae) harbour co-evolved, vertically transmitted and non-culturable bacterial symbionts in their midgut, known as Candidatus Stammerula spp. (Enterobacteriaceae). While such associations have previously been reported in the Palearctic and Hawaiian Archipelago, their occurrence in Australasia had not been investigated. In this study we assessed the genetic diversity of eight Australian fruit fly’s species from six genera belonging to the Tephritini tribe using mitochondrial markers (16 S rRNA and COI–tRNALeu–COII genes) and compared their bacterial diversity using the 16 S rRNA gene. We detected the presence of specific symbiotic bacteria in all sampled species. Analysis of bacterial 16 S rRNA showed that, with one exception, all Australian symbionts clustered in a well-supported monophyletic clade with Ca. Stammerula detected in Palearctic and Hawaiian Tephritini. Distinct Stammerula lineages were identified in several taxa, while two species, Trupanea prolata and Spathulina acroleuca shared identical symbiont sequences and the same host plant. Notably, Australian and Palearctic Sphenella spp. harboured closely related symbionts. The cophylogenetic analysis revealed a substantial congruence between host and symbiont tree, supporting a history of cospeciation and suggesting biogeographic links between Australasian and Palearctic taxa. Overall, the results expand the geographic knowledge of Tephritini- Ca. Stammerula association and highlight a global pattern of co-diversification.

Fruit flies belonging to Tephritidae family are highly destructive agricultural pests, posing a significant threat to various fruits and vegetables grown in Bangladesh. A comprehensive year-round survey was conducted at Atomic Energy Research Establishment (AERE) campus located in the central region of Bangladesh. Three types of male lures (methyl eugenol, cue-lure and zingerone) were used to detect and assess the diversity of pest fruit fly species. A total of seventeen species of Tephritidae fruit flies were detected in this survey. The Bactrocera carambolae fruit fly has been discovered for the first time in our survey area, indicating spread of its range towards the north-west region from its previous detection sites (Chattogram and Sylhet Divisions) in Bangladesh. Among the detected pest species, we identified six abundant species: Bactrocera dorsalis , Zeugodacus cucurbitae , Zeugodacus tau , Bactrocera rubigina , Bactrocera zonata , and Dacus longicornis . The most abundant species was the polyphagous fruit pest B. dorsalis , comprising 76.83% of the total captured flies. The species Z. cucurbitae was the second most abundant, representing 13.82% of the total trapped flies. The fitted curve to survey data using Gaussian mixture model revealed the existence of overlapped subgroups in the temporal population distribution of B. dorsalis and Z. cucurbitae . In addition, our statistical analysis of the six abundant Tephritidae fruit fly species revealed correlation of population dynamics with several factors including temperature, rainfall, humidity, photoperiod, and fruiting time of host plant species in the selected area.

Phylogenomic analyses have revolutionized our understanding of evolutionary relationships, yet it is complicated by incongruence across the genome. Here, we reanalyzed a genomic dataset comprising 3170 orthologs, and evaluated three methods to identify reduced sets of loci that can accurately resolve evolutionary relationships among Anastrepha fruit flies. Previous phylogenetic analyses consistently revealed well-supported topologies for deeper evolutionary relationships, while more recent divergences, particularly within the A. fraterculus complex, exhibited high levels of phylogenetic incongruence due to gene flow, incomplete lineage sorting, or other evolutionary forces. Here, we explored strategies for selecting reduced subsets: number of informative sites per gene, site concordance factor above 60% for clades consistent to current taxonomy, and tip-to-root variation/bipartition support. Among the strategies tested, subsets based on concordance and evolutionary rate metrics produced topologies consistent with full dataset analyses, with reduced levels of discordance. These subsets maintained robust support for deeper relationships while increasing congruence at shallower nodes. Although genes in reduced subsets exhibited lower evolutionary rates, they had higher internode certainty, treeness, and coalescent times. These findings highlight the potential for carefully selected loci to improve phylogenetic resolution and mitigate conflicting signals. Our study offers practical approaches for refining phylogenomic analyses in systems with complex evolutionary histories.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata , and Zeugodacus cucurbitae , a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp − mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni . The conserved phenotype and independent nature of wp − mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests. The white pupae (wp) phenotype has been used for decades to selectively remove females of tephritid species in genetic sexing, but the determining gene is unknown. Here, the authors show that wp phenotype is produced by parallel mutations in a Major Facilitator Superfamily domain containing gene across multiple species.

Tephritidae is an economically important family among Diptera that also exhibits high diversity, biogeographical distribution, and different lifestyles. Despite the recent release of genomes and mitochondrial genome sequences of various species of the family, the evolutionary history of the group and the origin of host adaptation within it remain poorly understood. We undertook a whole-mitochondrial-genome study covering molecular variation at the mitochondrial level by analyzing 10 new mitochondrial genomes obtained from genomic data reported and downloaded from the SRA database from NCBI, analyzed in FastQC and assembled through MITGARD, and 44 mitogenomes available in the Organelle—Refseq database, in total representing 4 subfamilies, 9 tribes, 13 genera, and 54 species. We determined compositional asymmetry and codon usage patterns across the different subfamilies analyzed by using DNASp6 and CAICal. We found high evolutionary rates in the NADH genes, which could play an important role in the adaptation of species to different hosts and environmental variation. By using maximum likelihood phylogenetic reconstruction obtained by IQTREE and ModelFinder, and lifestyle and distribution data of the included species, we considered a generalist feature, explained as possible predominant adaptation in some members of the family. This study in Tephritidae tries to demonstrate possible patterns among molecular variability in mitogenomes, adaptations, and lifestyles. Our findings suggest that selection pressures on certain NADH genes may be linked to host specificity in some Tephritidae species, providing evolutionary insights into how molecular evolution drives ecological adaptation or biogeographical diversity, probably in response to changing environmental conditions and host–parasite co-evolution across taxa.

Background The Tephritidae family, commonly referred to as true fruit flies, comprises of a substantial group within order Diptera. Numerous species within this family are major agricultural pests, with a tendency to infest a wide array of fruits and vegetables in tropical and sub- tropical regions, leading to considerable damage and consequent reductions in the market value of the crops. Methods and results The current study was aimed to propose a promising solution to the menace posed by fruit flies by offering rapid, accurate and reliable species identification by using character-based DNA barcode methodology. The Tephritid specimens were collected from Cucurbitaceous plants of southern parts of West Bengal, India, and a total of eight species from Tephritidae family were obtained belonging to three genera, namely Bactrocera (Macquart, 1835), Dacus (Fabricius, 1805) and Zeugodacus (Hendel, 1927). Their morphological features were meticulously studied based on available literature, along with genetic analysis based on mitochondrial COI and ND1 gene sequences. A total of 30 uniquely variable sites at nucleotide position 42,48,51,60,66,72, 105,111,144,198,207,243, 273,297,307,318,345,357, 375,378,381,387,399,400, 402,436,444,450,453 and 460 in COI gene were discerned among Tephritid species in the present study. Conclusions The character-based DNA barcode holds the potential to differentiate closely related species of fruit flies and morphologically look-a-like ones. The novel method will be very significant in terms of rapid, precise and reliable species identification and might be extremely essential for early detection during pest outbreaks by facilitating timely intervention strategies to mitigate crop damage.

Background True fruit flies (Diptera: Tephritidae) are among the most destructive pests of fruit and vegetables worldwide and are on the top of quarantine pest lists. To respond effectively to a fruit fly invasion, we need to identify the species rapidly and reliably to understand its biological features and guide response decisions. Molecular techniques have been used to improve the diagnostic ability circumventing many difficulties of morphological identification. However, the commonly used Cytochrome Oxidase I ( COI ) gene lacks sufficient variation to distinguish species within Bactrocera species complexes. Here we conducted mitochondrial genome sequencing to identify additional genetic markers that could aid diagnosis of Bactrocera fruit fly species. Results We assembled 82 complete mitochondrial genomes from 16 Bactrocera species, including 13 species for which no mitochondrial genome data were previously available, as well as one species each from Dacus aneuvittatus , Dirioxa pornia and Zeugodacus gracilis . Phylogenetic analysis of the Tephritidae family confirmed the monophyly of the Bactrocera genus but could not properly resolve species within species complexes. Comparative mitochondrial genome analysis revealed that intergenic spacer and NADH dehydrogenase genes, specifically ND2 and ND6 , harbour enough variations for new specific real-time PCR assays. Based on these findings, six TaqMan-based real-time PCR assays targeting ND2 , COI , and CO3 genes were successfully designed and assessed for their specificity and sensitivity in detecting Bactrocera curvipennis , a member of the B. tryoni complex. Of these, one real-time PCR assay targeting the ND2 gene proved to be the most specific and sensitive. It detects B. curvipennis specifically at the level of 1 copy/µL of target DNA. Conclusions Mitochondrial sequence analysis and comparative studies indicate that mitochondrial genomes offer valuable genetic markers for accurate diagnosis of Bactrocera fruit flies. The successful development of the B. curvipennis real-time PCR assay highlights the importance of having additional genetic markers to advance the molecular diagnostics in economically important Bactrocera species.

Insect mitogenome organisation is highly conserved, yet, some insects, especially with parasitic life cycles, have rearranged mitogenomes. Furthermore, intraspecific mitochondrial diversity can be reduced by fitness-affecting bacterial endosymbionts like Wolbachia due to their maternal coinheritance with mitochondria. We have sequenced mitogenomes of the Wolbachia -infected endoparasitoid Dipterophagus daci (Strepsiptera: Halictophagidae) and four of its 22 known tephritid fruit fly host species using total genomic extracts of parasitised flies collected across > 700 km in Australia. This halictophagid mitogenome revealed extensive rearrangements relative to the four fly mitogenomes which exhibited the ancestral insect mitogenome pattern. Compared to the only four available other strepsipteran mitogenomes, the D. daci mitogenome had additional transpositions of one rRNA and two tRNA genes, and a single nucleotide frameshift deletion in nad5 requiring translational frameshifting or, alternatively, resulting in a large protein truncation. Dipterophagus daci displays an almost completely endoparasitic life cycle when compared to Strepsiptera that have maintained the ancestral state of free-living adults. Our results support the hypothesis that the transition to extreme endoparasitism evolved together with increased levels of mitogenome changes. Furthermore, intraspecific mitogenome diversity was substantially smaller in D. daci than the parasitised flies suggesting Wolbachia reduced mitochondrial diversity because of a role in D. daci fitness.

Background Insect gut microbiomes, including tephritid fruit flies, are shaped by multiple endogenous and environmental factors. While host species is a well-known driver of the gut microbiome of adult tephritids, the influence of sex is less clear. Our study evaluated the impacts of host sex and species influence the microbiome in laboratory-reared tephritids when controlled for location, time, and adult diet. We evaluated the gut microbiome of four lines of pest tephritid fruit fly adults ( Bactrocera dorsalis , Bactrocera latifrons , Ceratitis capitata , Zeugodacus cucurbitae ) using near full-length 16S rRNA sequencing with a PacBio Kinnex concatenation-based approach. We analyzed groups of males and females from each species at the same set of time, across four timepoints in a core insectary. Results Results demonstrate a clear impact of fruit fly species on the gut microbiome composition of the different fruit flies. Furthermore, for B. dorsalis , B. latifrons , and C. capitata , we saw an influence of sex on ASV composition. However, while there was a separation of samples between the sexes for each timepoint, there was no characteristic male or female microbiome in all cases. The use of near full-length 16S rRNA sequencing did not have a marked improvement in beta-diversity interpretation over V4 subunit, with most detected taxa matching those described from other tephritids, but did allow for improved taxonomic classification at the genus level. Conclusions Our results demonstrate that under laboratory conditions, different fruit fly species still exhibit distinct microbiomes. The impact of sex did have an impact on the gut microbiome of some species, but the magnitude of effect differed between hosts. This indicates that the sex has some impact on structuring the gut microbiome, but in a case-by-case basis. While full-length 16S rRNA sequencing affords improved classification, our study did not indicate an improvement over partial-fragments on beta-diversity metrics.

Abstract Fruit flies are important agricultural pests of cultivated fruit trees, which makes it necessary to conduct studies that analyze the population fluctuations of these insects, whether in agricultural or natural environments. This study investigated the diversity and population fluctuations of Anastrepha (Diptera: Tephritidae) in an Atlantic Forest fragment in Mata do Pau-Ferro State Park, Areia, Paraíba. Thirty-two McPhail traps containing food attractants were installed in closed and open canopy areas, positioned 2 and 4 m high, between February 2023 and January 2024. We evaluated the population fluctuations of the species and correlated their occurrence through Pearson's correlation analysis with the climatic factors of rainfall, temperature, and humidity. A total of 207 specimens were collected, with a predominance of females (87%), and ten species of Anastrepha were identified, of which Anastrepha obliqua (Macquart) was the most abundant (38.64%), followed by A. fraterculus (Wiedemann) (12.07%) and A. bahiensis Lima (11.12%). The highest number of species was recorded in the months of January and July. The closed canopy area presented greater species richness and abundance (66.82% of individuals). A. bahiensis was more captured between September and November in traps positioned at 4 meters. In the closed canopy, there was a positive correlation between A. obliqua, A. matertela Zucchi and A. barbiellinii Lima and temperature, whereas in the closed canopy, A. obliqua and A. bahiensis demonstrated a strong and significant positive correlation with precipitation. The results highlight the importance of systematic monitoring in natural areas to support integrated pest management in adjacent commercial orchards. Resumo As moscas-das-frutas são importantes pragas agrícolas de fruteiras cultivadas, o que torna necessário a realização de estudos que analisem a flutuação populacional desses insetos, seja em ambientes agrícolas ou naturais. Este estudo investigou a diversidade e a flutuação populacional de Anastrepha (Diptera: Tephritidae) em um fragmento de Mata Atlântica no Parque Estadual Mata do Pau-Ferro, Areia, Paraíba. Foram instaladas 32 armadilhas do tipo McPhail, contendo atrativos alimentares, em áreas de dossel fechado e aberto, posicionadas a 2 e 4 m de altura, entre fevereiro de 2023 e janeiro de 2024. Avaliamos a flutuação populacional das espécies e correlacionamos sua ocorrência, por meio da análise de correlação de Pearson, com os fatores climáticos de precipitação pluviométrica, temperatura e umidade. Foram coletados 207 espécimes, com predomínio de fêmeas (87%), sendo identificadas dez espécies de Anastrepha, das quais Anastrepha obliqua (Macquart) foi a mais abundante (38,64%), seguida por A. fraterculus (Wiedemann) (12,07%) e A. bahiensis Lima (11,12%). O maior número de espécies foi registrado nos meses de janeiro e julho. A área de dossel fechado apresentou maior riqueza e abundância de espécies (66,82% dos indivíduos). A. bahiensis demonstrou maior ocorrência entre setembro a novembro nas armadilhas posicionadas a 4 m. No dossel fechado houve correlação positiva entre A. obliqua, A. matertela Zucchi e A. barbiellinii Lima e a temperatura, enquanto no dossel fechado A. obliqua e A. bahiensis demonstraram correlação positiva forte e significativa com a precipitação. Os resultados destacam a importância do monitoramento sistemático em áreas naturais como subsídio ao manejo integrado de pragas em pomares comerciais adjacentes.