Genome analyses of four Wolbachia strains and associated mitochondria of Rhagoletis cerasi expose cumulative modularity of cytoplasmic incompatibility factors and cytoplasmic hitchhiking across host populations

Background The endosymbiont Wolbachia can manipulate arthropod reproduction and invade host populations by inducing cytoplasmic incompatibility (CI). Some host species are coinfected with multiple Wolbachia strains which may have sequentially invaded host populations by expressing different types of modular CI factor (cif) genes. The tephritid fruit fly Rhagoletis cerasi is a model for CI and Wolbachia population dynamics. It is associated with at least four Wolbachia strains in various combinations, with demonstrated (wCer2, wCer4), predicted (wCer1) or unknown (wCer5) CI phenotypes. Results We sequenced and assembled the draft genomes of the Wolbachia strains wCer1, wCer4 and wCer5, and compared these with the previously sequenced genome of wCer2 which currently invades R. cerasi populations. We found complete cif gene pairs in all strains: four pairs in wCer2 (three Type I; one Type V), two pairs in wCer1 (both Type I) and wCer4 (one Type I; one Type V), and one pair in wCer5 (Type IV). Wolbachia genome variant analyses across geographically and genetically distant host populations revealed the largest diversity of single nucleotide polymorphisms (SNPs) in wCer5, followed by wCer1 and then wCer2, indicative of their different lengths of host associations. Furthermore, mitogenome analyses of the Wolbachia genome-sequenced individuals in combination with SNP data from six European countries revealed polymorphic mitogenome sites that displayed reduced diversity in individuals infected with wCer2 compared to those without. Conclusions Coinfections with Wolbachia are common in arthropods and affect options for Wolbachia-based management strategies of pest and vector species already infected by Wolbachia. Our analyses of Wolbachia genomes of a host naturally coinfected by several strains unravelled signatures of the evolutionary dynamics in both Wolbachia and host mitochondrial genomes as a consequence of repeated invasions. Invasion of already infected populations by new Wolbachia strains requires new sets of functionally different cif genes and thereby may select for a cumulative modularity of cif gene diversity in invading strains. Furthermore, we demonstrated at the mitogenomic scale that repeated CI-driven Wolbachia invasions of hosts result in reduced mitochondrial diversity and hitchhiking effects. Already resident Wolbachia strains may experience similar cytoplasmic hitchhiking effects caused by the invading Wolbachia strain.

MONITORING OF RHAGOLETIS CERASI L. THROUGH THE DECIS TRAP AT IAȘI-ROMANIA

Rhagoletis cerasi (L.) is the main agent of damage to sweet cherry plantations. Found in all sweet cherry plantations, it is important for an integrated phytosanitary protection of the crop. The population dynamics in the Iasi area was monitored at different time intervals. The climatic conditions of the year and the biological reserve favored the appearance and development of the pest studied. The first catches were recorded on 12 May and the highest number of catches was recorded on 17 June 2020. Adult monitoring Rhagoletis cerasi (L.) was performed using “Decis Trap”, an attractive trap containing ammonium carbonate, which attracts by its orange color and fights with the active substance deltamethrin providing a duration of protection of 5 months. The appearance and dynamics of pests have directly influenced the phytosanitary protection program. The observations were made during the vegetation period of the cherry plantation within the Research and Development Station for fruit growing in Iași in 2020.

Using the rDNA Internal Transcribed Spacer 1 to Identify the Invasive Pest Rhagoletis cerasi (Diptera: Tephritidae) in North America

The cherry-infesting fruit fly Rhagoletis cerasi Loew is a significant commercial pest in Europe that has recently invaded North America. To date, it has been trapped only in Canada and northwestern counties of New York. It has the potential to spread further and threaten production and movement of cherry commodities. Timely diagnosis of the pest will facilitate surveys and quick response to new detections. Adult morphology of the pest is distinct from other flies in North America. However, when flies are significantly damaged on traps or the immature life stages are found in fruits, molecular methods of identification are important to confirm presence and host-use records. Other than DNA sequencing of genes from flies which takes over a day to complete, there are no timely methods of molecular identification for this pest. In this study, we report the first sequence record of the internal transcribed spacer 1 (ITS1) from R. cerasi and develop two diagnostic tests for the pest based on ITS1 differences among species in North America. The tests use loop-mediated isothermal amplification (LAMP) and multiplex, conventional polymerase chain reaction (mcPCR) technologies that target the same region of the R. cerasi ITS1 sequence. Both tests performed well when tested against collections of R. cerasi from North America and Europe, generating Diagnostic Sensitivity estimates of 98.4–99.5%. Likewise, the tests had relatively high estimates of Diagnostic Specificity (97.8–100%) when tested against Rhagoletis Loew species present in North America that also use cherry as a developmental host.

BIOLOGICAL TOOLS FOR CONTROLLING THE MAIN PESTS OF SWEET CHERRY

The paper presents the data collected from an ecological plot of sweet cherries with an area of 0.5 ha, where different methods of monitoring and combating the main pests were tested: European cherry fruit fly - Rhagoletis cerasi L. and black cherry aphid - Myzus cerasi F. The study took place during the years 2019-2020. European cherry fruit fly monitoring - Rhagoletis cerasi L. was performed with both atraCERAS optical panels and Decis Trap devices, with both a warning and control role, the results obtained highlighting a similar flight dynamics. The control of the European cherry fruit fly, carried out by direct control with the help of Decis Trap devices, the dose of 100 traps / ha, proved to be effective, the Abbott effectiveness indicator registering values between 83.4 - 90.5% in 2019, respectively 81.6 - 87.8% in 2020, depending on the studied variety. To control the black cherry aphid - Myzus cerasi F., the product Prev-Am was mainly used, based on orange oil, which was used with good results for the first attacks observed.

Wolbachia-Mitochondrial DNA Associations in Transitional Populations of Rhagoletis cerasi

The endosymbiont Wolbachia can manipulate arthropod host reproduction by inducing cytoplasmic incompatibility (CI), which results in embryonic mortality when infected males mate with uninfected females. A CI-driven invasion of Wolbachia can result in a selective sweep of associated mitochondrial haplotype. The co-inheritance of Wolbachia and host mitochondrial DNA can therefore provide significant information on the dynamics of an ongoing Wolbachia invasion. Therefore, transition zones (i.e., regions where a Wolbachia strain is currently spreading from infected to uninfected populations) represent an ideal area to investigate the relationship between Wolbachia and host mitochondrial haplotype. Here, we studied Wolbachia-mitochondrial haplotype associations in the European cherry fruit fly, Rhagoletis cerasi, in two transition zones in the Czech Republic and Hungary, where the CI-inducing strain wCer2 is currently spreading. The wCer2-infection status of 881 individuals was compared with the two known R. cerasi mitochondrial haplotypes, HT1 and HT2. In accordance with previous studies, wCer2-uninfected individuals were associated with HT1, and wCer2-infected individuals were mainly associated with HT2. We found misassociations only within the transition zones, where HT2 flies were wCer2-uninfected, suggesting the occurrence of imperfect maternal transmission. We did not find any HT1 flies that were wCer2-infected, suggesting that Wolbachia was not acquired horizontally. Our study provides new insights into the dynamics of the early phase of a Wolbachia invasion.

Effects of photoperiod and relative humidity on diapause termination and post-winter development of Rhagoletis cerasi pupae

The European cherry fruit fly, Rhagoletis cerasi (Diptera: Tephritidae), is a univoltine species that undergoes obligatory summer–winter diapause at pupal stage in the soil (2–5 cm) beneath host trees. To study the effects of photoperiod and relative humidity on diapause termination and post-winter developmental duration of R. cerasi, pupae collected from Dossenheim (Germany) were exposed to different photoperiod or relative humidity regimes during a chilling period ranging from 2 to 8.5 months. Specifically, pupae were exposed to four photoperiod regimes: (a) light conditions (24L:00D), (b) dark conditions (00L:24D), (c) short photoperiod (08L:16D) and (d) long photoperiod (16L:08D), as well as to three relative humidity regimes: (a) low (40% RH), (b) medium (60% RH) and (c) high (70–80% RH). Data revealed that relative humidity is not a significant predictor of diapause termination, but it affects the post-winter developmental period. Higher relative humidity promotes post-winter pupae development. On the other hand, photoperiod significantly affected both diapause termination and post-winter development of R. cerasi pupae. Light conditions (24L:00D) accelerate adult emergence, particularly for females. Regardless of the photoperiod (24L:00D, 00L:24D, 08L:16D), rates of adult emergence were high (>75%) for chilling intervals longer than 6.5 months. Nonetheless, exposure to a long day photoperiod (16L:08D), during chilling, dramatically reduced the proportion of adult emergence following 6 months exposure to chilling. Our findings broaden the understanding of factors regulating diapause responses in European cherry fruit fly, local adaptation and synchronization of adult emergence with the ripening period of major hosts.