Broad Ultrastructural and Transcriptomic Changes Underlie the Multinucleated Giant Hemocyte Mediated Innate Immune Response against Parasitoids

Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously, we showed that circulating MGHs have high motility and the interaction with the parasitoid rapidly triggers encapsulation. However, structural and molecular mechanisms behind these processes remained elusive. Here, we used detailed ultrastructural analysis and live cell imaging of MGHs to study encapsulation in <i>Drosophila ananassae</i> after parasitoid wasp infection. We found dynamic structural changes, mainly driven by the formation of diverse vesicular systems and newly developed complex intracytoplasmic membrane structures, and abundant generation of giant cell exosomes in MGHs. In addition, we used RNA sequencing to study the transcriptomic profile of MGHs and activated plasmatocytes 72 h after infection, as well as the uninduced blood cells. This revealed that differentiation of MGHs was accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts related to vesicular function, cytoskeletal organization, and adhesion were enriched in MGHs. In addition, several orphan genes encoding for hemolysin-like proteins, pore-forming toxins of prokaryotic origin, were expressed at high level, which may be important for parasitoid elimination. Our results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response.

Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes

The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

Comparison of long read sequencing technologies in resolving bacteria and fly genomes

ABSTRACTBackgroundThe newest generation of DNA sequencing technology is highlighted by the ability to sequence reads hundreds of kilobases in length, and the increased availability of long read data has democratized the genome sequencing and assembly process. PacBio and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. Released in 2019, the PacBio Sequel II platform advertises substantial enhancements over previous PacBio systems.ResultsWe used whole-genome sequencing data produced by two PacBio platforms (Sequel II and RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. Sequel II assemblies had higher contiguity and consensus accuracy relative to other methods, even after accounting for differences in sequencing throughput. ONT RAPID libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assemblies or combined ONT and Sequel II libraries for eukaryotic genome assemblies. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs.ConclusionsThe ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

Studies on Mating Preference and Productivity in Drosophila ananassae and D. pallidosa and Their Hybrids

During speciation different kinds of reproductive barriers originate to preclude gene flow between diverging populations. Reproductive isolation or barriers to gene flow can be categorized by the temporal nature of their effect: pre-zygotic barriers occur before fertilization and post-zygotic barriers occur after fertilization. In this study, we studied each components of reproductive isolation between D. ananassae and D. pallidosa, including both pre-zygotic and post-zygotic barriers. Because it might be possible that by dissecting these barriers one can get the answers of many unresolved questions related to the process of speciation of these two sibling species. We reported premating isolation because females of both D. ananassae and D. pallidosa were more discriminative for mating against the alien males rather than conspecific males, and this discrimination was much stronger in case of D. ananassae females for being the ancestral and cosmopolitan species. We also did not find any decrease in the production of progeny or viable offspring in comparison to conspecific males, indicating a lack of postmating prezygotic isolating barriers. Further, there is no complete lack of intrinsic post-zygotic isolation between these species or not complete presence of post-zygotic isolation, as both the hybrid sons were producing less number of progeny in comparison to all the crosses but it is near to significant but not significant and this is contrasting to the results of a previous study.

Evidence for Initiation of Post-Zygotic Reproductive Isolation between Drosophila ananassae and D. pallidosa as Indicated by Reduction in the Fertility of Hybrid Males

There are several barriers to preclude the gene flow between diverging populations. On the basis of their temporal nature, these can be broadly categorized into two forms: pre- and post-zygotic. Post-zygotic reproductive isolation can manifest in the form of reductions in hybrid fertility. Keeping this fact in view, in the present study, we studied sterility in hybrids of D. ananassae and D. pallidosa. Surprisingly a distinguishable pattern of infertility was found in the hybrids. This pattern, referred to as Haldane&rsquo;s rule, is often observed in hybrids of recently diverged populations or species. Reduction in the fertility of hybrids provides the clue of incipient kind of post-zygotic reproductive isolation in these two sibling species. This is the first report of hybrid sterility in this species pair. However, hybrid sterility is not very prominent especially when compared to that of other species pairs with the similar divergence time. Thus, on the basis of our results, we conclude that either sexual isolation between these sibling species is sufficient and does not require the aid of post-zygotic isolation to preclude gene flow or rate of divergence between D. ananassae and D. pallidosa is very slow in comparison to other species pair or even races of some species.