Single-cell transcriptome sequencing reveals that Wolbachia induces gene expression changes in Drosophila ovary cells to favor its own maternal transmission.

Wolbachia is an obligate endosymbiont that is maternally inherited and widely distributed in arthropods and nematodes. It remains in the mature eggs of female hosts over generations through multiple strategies and manipulates the reproduction system of the host to enhance its spreading efficiency. However, the transmission of Wolbachia within the host's ovaries and its effects on ovarian cells during oogenesis, have not been extensively studied. We used single-cell RNA sequencing to comparatively analyze cell-typing and gene expression in Drosophila ovaries infected and uninfected with Wolbachia. Our findings indicate that Wolbachia significantly affects the transcription of host genes involved in the extracellular matrix, cytoskeleton organization, and cytomembrane mobility in multiple cell types, which may make host ovarian cells more conducive for the transmission of Wolbachia from extracellular to intracellular. Moreover, the genes nos and orb, which are related to the synthesis of ribonucleoprotein complexes, are specifically upregulated in early germline cells of ovaries infected with Wolbachia, revealing that Wolbachia can increase the possibility of its localization to the host oocytes by enhancing the binding with host ribonucleoprotein-complex processing bodies (P-bodies). All these findings provide novel insights into the maternal transmission of Wolbachia between host ovarian cells.IMPORTANCEWolbachia, an obligate endosymbiont in arthropods, can manipulate the reproduction system of the host to enhance its maternal transmission and reside in the host's eggs for generations. Herein, we performed single-cell RNA sequencing of ovaries from Drosophila melanogaster and observed the effects of Wolbachia (strain wMel) infection on different cell types to discuss the potential mechanism associated with the transmission and retention of Wolbachia within the ovaries of female hosts. It was found that the transcriptions of multiple genes in the ovary samples infected with Wolbachia are significantly altered, which possibly favors the maternal transmission of Wolbachia. Meanwhile, we also discovered that Wolbachia may flexibly regulate the expression level of specific host genes according to their needs rather than rigidly changing the expression level in one direction to achieve a more suitable living environment in the host's ovarian cells. Our findings contribute to a further understanding of the maternal transmission and possible universal effects of Wolbachia within the host.

The gender-specific impact of starvation on mitotypes diversity in adults of Drosophila melanogaster.

In animals, starvation can increase the level of reactive oxygen species (ROS) in some tissues. Mitochondrial DNA (mtDNA) is more vulnerable to being attacked by ROS due to the lack of histone protection, leading to oxidative damage. However, whether starvation is associated with the genetic diversity of mtDNA remains unclear. Here, by using adult individuals of Drosophila melanogaster under three different feeding treatments (starvation, with the provision of only water, and normal feeding), based on the high-throughput sequencing results of the PCR amplicons of the partial sequences of the mitochondrial gene cytochrome c oxidase subunit I (mt-cox1), no significant difference in the mean number of mitochondrial haplotypes and the mean genetic distance of haplotypes within individuals were identified between the three treatment groups. Coupled with the low proportion of heterogeneous mt-cox1 sequences within each individual, it suggested that starvation had a limited impact on mitotype genetic diversity and mitochondrial function. Nevertheless, starvation could significantly increase the sequence number of haplotypes containing specific mutations, and for males with higher levels of mitochondrial heteroplasmy than females in the normal feeding group, starvation could further increase their mitochondrial heteroplasmy.

Impact of mitotype diversity on metabarcoding biodiversity estimations in Insecta and Arachnida using different sample preparation strategies.

DNA barcoding and metabarcoding have been increasingly used in species delimitation and species diversity assessment, respectively, and the molecular markers used in animals are mainly derived from mitochondrial DNA. It is well known that the phenomenon of multiple mitochondrial haplotypes within the same specimen (hereafter referred to as "mitotype diversity") may have a negative impact on the proper assessment of biodiversity by metabarcoding. However, few studies have focused on the incidence of this phenomenon and its effects on metabarcoding results using different sample preparation strategies, such as mock community construction using pooled high-throughput sequencing (HTS) data, DNA-pooling and Tissue-pooling. In this study, we investigated mitotype diversity and its influence on metabarcoding based on 398 specimens from 66 species of Insecta and 82 specimens from 16 species of Arachnida by HTS of the mitochondrial cox1 gene fragment. The results revealed that mitotype diversity was common in the studied taxa and significantly increased the number of operational taxonomic units (OTUs) using the three sample preparation strategies. The results also showed that the bioinformatics pipeline based on authentic amplicon sequence variants was more reliable than the pipeline based on OTUs. Regarding the sample preparation strategies of DNA-pooling and Tissue-pooling commonly used in metabarcoding, our results revealed that their results of metabarcoding were quite similar, and the Tissue-pooling strategy was therefore preferred because of its simplicity. Our study calls for additional attention to the interference of mitotype diversity on the results of DNA metabarcoding in biodiversity assessment.

Distribution and Evolution of the Bacteriophage WO and Its Antagonism With Wolbachia.

The symbiosis system comprising eukaryotic hosts, intracellular bacterium Wolbachia, and temperate bacteriophages WO is widely spread through nearly half the number of arthropod species. The relationships between the three components of the system are extremely intricate. Even though the bacteriophage WO can have diverse influences on the ecology and evolution of Wolbachia, little is known about the distribution and evolution of the phages. To the best of our knowledge, this study is the first to report that in infected fig wasps (Ceratosolen solmsi, Kradibia gibbosae, and Wiebesia pumilae), the genomes of all the Wolbachia strains had only one cryptic WO prophage, which contained defects in the genomic structural modules. This phenomenon was contrary to the widely accepted understanding that Wolbachia with cryptic prophages usually possesses at least one intact WO prophage consisting of gene sequences of the head, baseplate, and tail modules, through which the prophage could form intact virions. In addition to the genetic structure features, the phylogenetic relationships of WO and Wolbachia also revealed that bacteriophage WO can horizontally spread among a certain genus or a group of insect hosts, nearly free from the restriction of the affiliation of Wolbachia. Combined with the vertical transmission along with Wolbachia, the wide spread of WO phages can be explained. Furthermore, the gender preference and functional module preference for transcriptional activity of the genes in cryptic WOs implied the antagonized coevolutionary pattern between WO prophages and their Wolbachia hosts.

Tracking the Distribution and Burst of Nuclear Mitochondrial DNA Sequences (NUMTs) in Fig Wasp Genomes.

Mitochondrial DNA sequences can be transferred into the nuclear genome, giving rise to nuclear mitochondrial DNA sequences (NUMTs). NUMTs have been described in numerous eukaryotes. However, the studies on the distribution of NUMTs and its influencing factors are still inadequate and even controversial. Previous studies have suggested that Hymenoptera may be a group rich in NUMTs, in which we selected 11 species of fig wasps (Chalcidoidea, Hymenoptera) to analyze the distribution and evolution of NUMTs at the genomic level. The results showed that the contents of NUMTs varied greatly in these species, and bursts of NUMTs existed in some species or lineages. Further detailed analyses showed that the large number of NUMTs might be related to the large genomes; NUMTs tended to be inserted into unstable regions of the genomes; and the inserted NUMTs might also be affected by transposable elements (TEs) in the neighbors, leading to fragmentations and duplications, followed by bursts of NUMTs. In summary, our results suggest that a variety of genomic environmental factors can determine the insertion and post-insertion fate of NUMTs, resulting in their species- or lineage-specific distribution patterns, and that studying the evolution of NUMTs can provide good evidence and theoretical basis for exploring the dynamics of exogenous DNA entering into the nuclear genome.