Drosophila Host Research Articles

Temporal variability in host availability alters the outcome of competition between two parasitoid species.

Variability in the availability of resources through time is a common attribute in trophic interactions, but its effects on the fitness of different consumer species and on interspecific competition between them are not clearly understood. To investigate this, we allowed two parasitoid species, Trichopria drosophilae and Pachycrepoideus vindemiae, to exploit Drosophila host pupae under different temporal variability treatments, either on their own or simultaneously. When tested individually (in the absence of interspecific competition), both parasitoid species had lower fitness when hosts were exposed for a short duration at high density than when exposed for a long duration at low density. When both parasitoid species exploited hosts simultaneously, interspecific competition significantly decreased the number of offspring for both parasitoid species. The outcome of this interspecific competition depended on host temporal variability, with T. drosophilae or P. vindemiae dominating in short and long host exposure treatments, respectively. These results can be explained by the combination of host availability and egg load of female adult parasitoids. When abundant hosts are provided for a short period, the ample mature eggs of the proovigenic T. drosophilae enable them to exploit hosts more efficiently than P. vindemiae, which is synovigenic. However, P. vindemiae is an intrinsically superior competitor and dominates when multiparasitism occurs. Multiparasitism is more frequent when hosts are at low levels relative to the egg load of the parasitoids. Our results clearly demonstrate that resource temporal availability can alter the outcome of competition between consumers with different reproductive traits.

Host and venom evolution in parasitoid wasps: does independently adapting to the same host shape the evolution of the venom gland transcriptome?

BackgroundVenoms have repeatedly evolved over 100 occasions throughout the animal tree of life, making them excellent systems for exploring convergent evolutionary novelty. Growing evidence supports that venom evolution is predominantly driven by prey or host-related selection pressures, and the expression patterns of venom glands reflect adaptive evolution. However, it remains elusive whether the evolution of expression patterns in venom glands is likewise a convergent evolution driven by their prey/host species.ResultsWe utilized parasitoid wasps that had independently adapted to Drosophila hosts as models to investigate the convergent evolution of venom gland transcriptomes in 19 hymenopteran species spanning ~ 200 million years of evolution. Comparative transcriptome analysis reveals that the global expression patterns among the venom glands of Drosophila parasitoid wasps do not achieve higher similarity compared to non-Drosophila parasitoid wasps. Further evolutionary analyses of expression patterns at the single gene, orthogroup, and Gene Ontology (GO) term levels indicate that some orthogroups/GO terms show correlation with the Drosophila parasitoid wasps. However, these groups rarely include genes highly expressed in venom glands or putative venom genes in the Drosophila parasitoid wasps.ConclusionsOur study suggests that convergent evolution may not play a predominant force shaping gene expression levels in the venom gland of the Drosophila parasitoid wasps, offering novel insights into the co-evolution between venom and prey/host.

Coordinated molecular and ecological adaptations underlie a highly successful parasitoid.

The success of an organism depends on the molecular and ecological adaptations that promote its beneficial fitness. Parasitoids are valuable biocontrol agents for successfully managing agricultural pests, and they have evolved diversified strategies to adapt to both the physiological condition of hosts and the competition of other parasitoids. Here, we deconstructed the parasitic strategies in a highly successful parasitoid, Trichopria drosophilae, which parasitizes a broad range of Drosophila hosts, including the globally invasive species D. suzukii. We found that T. drosophilae had developed specialized venom proteins that arrest host development to obtain more nutrients via secreting tissue inhibitors of metalloproteinases (TIMPs), as well as a unique type of cell-teratocytes-that digest host tissues for feeding by releasing trypsin proteins. In addition to the molecular adaptations that optimize nutritional uptake, this pupal parasitoid has evolved ecologically adaptive strategies including the conditional tolerance of intraspecific competition to enhance parasitic success in older hosts and the obligate avoidance of interspecific competition with larval parasitoids. Our study not only demystifies how parasitoids weaponize themselves to colonize formidable hosts but also provided empirical evidence of the intricate coordination between the molecular and ecological adaptations that drive evolutionary success.

Comparative analysis of Wolbachia maternal transmission and localization in host ovaries

Many insects and other animals carry microbial endosymbionts that influence their reproduction and fitness. These relationships only persist if endosymbionts are reliably transmitted from one host generation to the next. Wolbachia are maternally transmitted endosymbionts found in most insect species, but transmission rates can vary across environments. Maternal transmission of wMel Wolbachia depends on temperature in natural Drosophila melanogaster hosts and in transinfected Aedes aegypti, where wMel is used to block pathogens that cause human disease. In D. melanogaster, wMel transmission declines in the cold as Wolbachia become less abundant in host ovaries and at the posterior pole plasm (the site of germline formation) in mature oocytes. Here, we assess how temperature affects maternal transmission and underlying patterns of Wolbachia localization across 10 Wolbachia strains diverged up to 50 million years—including strains closely related to wMel—and their natural Drosophila hosts. Many Wolbachia maintain high transmission rates across temperatures, despite highly variable (and sometimes low) levels of Wolbachia in the ovaries and at the developing germline in late-stage oocytes. Identifying strains like closely related wMel-like Wolbachia with stable transmission across variable environmental conditions may improve the efficacy of Wolbachia-based biocontrol efforts as they expand into globally diverse environments.

A specific innate immune response silences the virulence of Pseudomonas aeruginosa in a latent infection model in the Drosophila melanogaster host.

Microbial pathogenicity often depends on the route of infection. For instance, P. aeruginosa or S. marcescens cause acute systemic infections when low numbers of bacteria are injected into D. melanogaster flies whereas flies succumb much slower to the continuous ingestion of these pathogens, even though both manage to escape from the gut compartment and reach the hemocoel. Here, we have developed a latent P. aeruginosa infection model by feeding flies on the bacteria for a short period. The bacteria stably colonize internal tissues yet hardly cause any damage since latently-infected flies live almost as long as noninfected control flies. The apparently dormant bacteria display particular characteristics in terms of bacterial colony morphology, composition of the outer cell wall, and motility. The virulence of these bacteria can however be reactivated upon wounding the host. We show that melanization but not the cellular or the systemic humoral response is the predominant host defense that establishes latency and may coerce the bacteria to a dormant state. In addition, the lasting activation of the melanization responses in latently-infected flies provides a degree of protection to the host against a secondary fungal infection. Latent infection by an ingested pathogen protects against a variety of homologous or heterologous systemic secondary infectious challenges, a situation previously described for the endosymbiotic Wolbachia bacteria, a guard against viral infections.

Comparative analysis of Wolbachia maternal transmission and localization in host ovaries.

Many insects and other animals carry microbial endosymbionts that influence their reproduction and fitness. These relationships only persist if endosymbionts are reliably transmitted from one host generation to the next. Wolbachia are maternally transmitted endosymbionts found in most insect species, but transmission rates can vary across environments. Maternal transmission of wMel Wolbachia depends on temperature in natural Drosophila melanogaster hosts and in transinfected Aedes aegypti, where wMel is used to block pathogens that cause human disease. In D. melanogaster, wMel transmission declines in the cold as Wolbachia become less abundant in host ovaries and at the posterior pole plasm (the site of germline formation) in mature oocytes. Here, we assess how temperature affects maternal transmission and underlying patterns of Wolbachia localization across 10 Wolbachia strains diverged up to 50 million years-including strains closely related to wMel-and their natural Drosophila hosts. Many Wolbachia maintain high transmission rates across temperatures, despite highly variable (and sometimes low) levels of Wolbachia in the ovaries and at the developing germline in late-stage oocytes. Identifying strains like closely related wMel-like Wolbachia with stable transmission across variable environmental conditions may improve the efficacy of Wolbachia-based biocontrol efforts as they expand into globally diverse environments.

Transcriptomic Insights into Host Metabolism and Immunity Changes after Parasitization by Leptopilina myrica.

Parasitoids commonly manipulate their host's metabolism and immunity to facilitate their offspring survival, but the mechanisms remain poorly understood. Here, we deconstructed the manipulation strategy of a newly discovered parasitoid wasp, L. myrica, which parasitizes D. melanogaster. Using RNA-seq, we analyzed transcriptomes of L. myrica-parasitized and non-parasitized Drosophila host larvae. A total of 22.29 Gb and 23.85 Gb of clean reads were obtained from the two samples, respectively, and differential expression analysis identified 445 DEGs. Of them, 304 genes were upregulated and 141 genes were downregulated in parasitized hosts compared with non-parasitized larvae. Based on the functional annotations in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, we found that the genes involved in host nutrition metabolism were significantly upregulated, particularly in carbohydrate, amino acid, and lipid metabolism. We also identified 30 other metabolism-related DEGs, including hexokinase, fatty acid synthase, and UDP-glycosyltransferase (Ugt) genes. We observed that five Bomanin genes (Boms) and six antimicrobial peptides (AMPs) were upregulated. Moreover, a qRT-PCR analysis of 12 randomly selected DEGs confirmed the reproducibility and accuracy of the RNA-seq data. Our results provide a comprehensive transcriptomic analysis of how L. myrica manipulates its host, laying a solid foundation for studies on the regulatory mechanisms employed by parasitoid wasps in their hosts.

Facultatively ectoparasitic mites as vectors for entomopathogenic bacteria in Drosophila

Opportunistic bacterial infections are common in insect populations but there is little information on how they are acquired or transmitted. We tested the hypothesis that Macrocheles mites can transmit systemic bacterial infections between Drosophila hosts. We found that 24% of mites acquired detectable levels of bacteria after feeding on infected flies and 87% of infected mites passed bacteria to naïve recipient flies. The probability that a mite could pass Serratia from an infected donor fly to a naïve recipient fly was 27.1%. These data demonstrate that Macrocheles mites are capable of serving as vectors of bacterial infection between insects.

The Effect of Genotype Combinations of Wolbachia and Its Drosophila melanogaster Host on Fertility, Developmental Rate and Heat Stress Resistance of Flies.

The best-known effect of the intracellular bacterium Wolbachia is its mostly negative influence on the reproduction of the host. However, there is evidence of a positive influence of Wolbachia on the host's resistance to stress, pathogens, and viruses. Here, we analyzed the effects of two Wolbachia strains belonging to wMel and wMelCS genotypes on D. melanogaster traits, such as fertility, survival under acute heat stress, and developmental rate. We found that D. melanogaster lines under study differ significantly in the above-mentioned characteristics, both when the natural infection was preserved, and when it was eliminated. One of Wolbachia strains, wMel, did not affect any of the studied traits. Another strain, wMelPlus, had a significant effect on the development time. Moreover, this effect is observed not only in the line in which it was discovered but also in the one it was transferred to. When transferred to a new line, wMelPlus also caused changes in survival under heat stress. Thus, it could be concluded that Wolbachia-Drosophila interaction depends on the genotypes of both the host and the symbiont, but some Wolbachia effects could depend not on the genotypes, but on the fact of recent transfer of the symbiont.

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains.

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 ± 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 ± 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

Drosophila innate immunity suppresses the survival of xenografted mammalian tumor cells

Patient-derived xenograft (PDX) is an emerging tool established in immunodeficient vertebrate models to assess individualized treatments for cancer patients. Current xenograft models are deficient in adaptive immune systems. However, the precise role of the innate immunity in the xenograft models is unknown. With conserved signaling pathways and established genetic tools, Drosophila has contributed to the understanding of the mechanism of tumor growth as well as tumor–host interactions for decades, making it a promising candidate model for studying whether or not the hosts’ innate immunity can accommodate transplanted human tumor cells. Here we show initial observations that assess the behavior and impact of several human tumor cell lines when transplanted into Drosophila. We found that some injected cell lines persisted for a longer duration and reduced hosts’ lifespan. In particular, the human lung cancer cell line A549 were observed adjacent to the fly host tissues. We examined two factors that affect the survivability of cancer cells: (1) the optimal temperature of each cell line and (2) the innate immunity of Drosophila hosts. Especially, transplanted human tumor cells survived longer in immunodeficient flies, suggesting that the host innate immune system impedes the growth of xenografted cells. Our attempts for xenografting fly models thus provide necessary steps to overcome for establishing PDX cancer models using invertebrates.

Effects of Wolbachia on Transposable Element Expression Vary Between Drosophila melanogaster Host Genotypes.

Transposable elements (TEs) are repetitive DNA sequences capable of changing position in host genomes, thereby causing mutations. TE insertions typically have deleterious effects but they can also be beneficial. Increasing evidence of the contribution of TEs to adaptive evolution further raises interest in understanding what factors impact TE activity. Based on previous studies associating the bacterial endosymbiont Wolbachia with changes in the abundance of piRNAs, a mechanism for TE repression, and to transposition of specific TEs, we hypothesized that Wolbachia infection would interfere with TE activity. We tested this hypothesis by studying the expression of 14 TEs in a panel of 25 Drosophila melanogaster host genotypes, naturally infected with Wolbachia and annotated for TE insertions. The host genotypes differed significantly in Wolbachia titers inside individual flies, with broad-sense heritability around 20%, and in the number of TE insertions, which depended greatly on TE identity. By removing Wolbachia from the target host genotypes, we generated a panel of 25 pairs of Wolbachia-positive and Wolbachia-negative lines in which we quantified transcription levels for our target TEs. We found variation in TE expression that was dependent on Wolbachia status, TE identity, and host genotype. Comparing between pairs of Wolbachia-positive and Wolbachia-negative flies, we found that Wolbachia removal affected TE expression in 21.1% of the TE-genotype combinations tested, with up to 2.3 times differences in the median level of transcript. Our data show that Wolbachia can impact TE activity in host genomes, underscoring the importance this endosymbiont can have in the generation of genetic novelty in hosts.

P-element invasion fuels molecular adaptation in laboratory populations of Drosophila melanogaster.

Transposable elements (TEs) are mobile genetic parasites that frequently invade new host genomes through horizontal transfer. Invading TEs often exhibit a burst of transposition, followed by reduced transposition rates as repression evolves in the host. We recreated the horizontal transfer of P-element DNA transposons into a Drosophila melanogaster host and followed the expansion of TE copies and evolution of host repression in replicate laboratory populations reared at different temperatures. We observed that while populations maintained at high temperatures rapidly go extinct after TE invasion, those maintained at lower temperatures persist, allowing for TE spread and the evolution of host repression. We also surprisingly discovered that invaded populations experienced recurrent insertion of P-elements into a specific long non-coding RNA, lncRNA:CR43651, and that these insertion alleles are segregating at unusually high frequency in experimental populations, indicative of positive selection. We propose that, in addition to driving the evolution of repression, transpositional bursts of invading TEs can drive molecular adaptation.

New Wolbachia pipientis Genotype Increasing Heat Stress Resistance of Drosophila melanogaster Host Is Characterized by a Large Chromosomal Inversion.

The maternally transmitted endocellular bacteria Wolbachia is a well-known symbiont of insects, demonstrating both negative and positive effects on host fitness. The previously found Wolbachia strain wMelPlus is characterized by a positive effect on the stress-resistance of its host Drosophila melanogaster, under heat stress conditions. This investigation is dedicated to studying the genomic underpinnings of such an effect. We sequenced two closely related Wolbachia strains, wMelPlus and wMelCS112, assembled their complete genomes, and performed comparative genomic analysis engaging available Wolbachia genomes from the wMel and wMelCS groups. Despite the two strains under study sharing very close gene-composition, we discovered a large (>1/6 of total genome) chromosomal inversion in wMelPlus, spanning through the region that includes the area of the inversion earlier found in the wMel group of Wolbachia genotypes. A number of genes in unique inversion blocks of wMelPlus were identified that might be involved in the induction of a stress-resistant phenotype in the host. We hypothesize that such an inversion could rearrange established genetic regulatory-networks, causing the observed effects of such a complex fly phenotype as a modulation of heat stress resistance. Based on our findings, we propose that wMelPlus be distinguished as a separate genotype of the wMelCS group, named wMelCS3.

Monothiol Glutaredoxin Is Essential for Oxidative Stress Protection and Virulence in Pseudomonas aeruginosa.

Glutaredoxins (Grxs), ubiquitous redox enzymes belonging to the thioredoxin family, catalyze the reduction of thiol-disulfide exchange reactions in a glutathione-dependent manner. A Pseudomonas aeruginosa ΔgrxD mutant exhibited hypersensitivity to oxidative stress-generating agents, such as paraquat (PQ) and cumene hydroperoxide (CHP). In vitro studies showed that P. aeruginosa GrxD acts as an electron donor for organic hydroperoxide resistance enzyme (Ohr) during CHP degradation. The ectopic expression of iron-sulfur cluster ([Fe-S]) carrier proteins, including ErpA, IscA, and NfuA, complements the function of GrxD in the ΔgrxD mutant under PQ toxicity. Constitutively high expression of iscR, nfuA, tpx, and fprB was observed in the ΔgrxD mutant. These results suggest that GrxD functions as a [Fe-S] cluster carrier protein involved in [Fe-S] cluster maturation. Moreover, the ΔgrxD mutant demonstrates attenuated virulence in a Drosophila melanogaster host model. Altogether, the data shed light on the physiological role of GrxD in oxidative stress protection and virulence of the human pathogen, P. aeruginosa. IMPORTANCE Glutaredoxins (Grxs) are ubiquitous disulfide reductase enzymes. Monothiol Grxs, containing a CXXS motif, play an essential role in iron homeostasis and maturation of [Fe-S] cluster proteins in various organisms. We now establish that the human pathogen Pseudomonas aeruginosa GrxD is crucial for bacterial virulence, maturation of [Fe-S] clusters and facilitation of Ohr enzyme activity. GrxD contains a conserved signature monothiol motif (C29GFS), in which C29 is essential for its function in an oxidative stress protection. Our findings reveal the physiological roles of GrxD in oxidative stress protection and virulence of P. aeruginosa.

Multiple parasitoid species enhance top-down control, but parasitoid performance is context dependent.

Ecological communities are composed of many species, forming complex networks of interactions. Current environmental changes are altering the structure and species composition of ecological networks, which could modify interactions, either directly or indirectly. To predict changes in the functioning of communities, we need to understand whether species interactions are primarily driven by network structure (i.e. topology) or the specific identities of species (i.e. nodes). Yet, this partitioning of effects is challenging and thus rarely explored. Here we disentangled the influence of network structure and the identities of species on the outcome of consumer-resource interactions using a host-parasitoid system. We used four common community modules in host-parasitoid communities to represent network structure (i.e. host-parasitoid, exploitative competition, alternative host and a combination of exploitative competition and alternative host). We assembled nine different species combinations per community module in a laboratory experiment using a pool of three Drosophila hosts and three larval parasitoid species (Leptopilina sp., Ganaspis sp. and Asobara sp.). We compared host suppression and parasitoid performance across community modules and species assemblages to identify general effects linked to network structure and specific effects due to species community composition. We found that multiple parasitoid species enhanced host suppression due to sampling effect, weaker interspecific than intraspecific competition between parasitoids, and synergism. However, the effects of network structure on parasitoid performance were species specific and dependent on the identity of co-occurring species. Consequently, multiple parasitoid species generally strengthen top-down control, but the performance of the parasitoids depends on the identity of either the co-occurring parasitoid species, the alternative host species or both. Our results highlight the importance of preserving parasitoid diversity for ecosystem functioning and show that other effects depend on species community composition, and may therefore be altered by ongoing environmental changes.

CifB-transcript levels largely explain cytoplasmic incompatibility variation across divergent Wolbachia.

Divergent hosts often associate with intracellular microbes that influence their fitness. Maternally transmitted Wolbachia bacteria are the most common of these endosymbionts, due largely to cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-infected males. Closely related infections in females rescue CI, providing a relative fitness advantage that drives Wolbachia to high frequencies. One prophage-associated gene (cifA) governs rescue, and two contribute to CI (cifA and cifB), but CI strength ranges from very strong to very weak for unknown reasons. Here, we investigate CI-strength variation and its mechanistic underpinnings in a phylogenetic context across 20 million years (MY) of Wolbachia evolution in Drosophila hosts diverged up to 50 MY. These Wolbachia encode diverse Cif proteins (100% to 7.4% pairwise similarity), and AlphaFold structural analyses suggest that CifB sequence similarities do not predict structural similarities. We demonstrate that cifB-transcript levels in testes explain CI strength across all but two focal systems. Despite phylogenetic discordance among cifs and the bulk of the Wolbachia genome, closely related Wolbachia tend to cause similar CI strengths and transcribe cifB at similar levels. This indicates that other non-cif regions of the Wolbachia genome modulate cif-transcript levels. CI strength also increases with the length of the host’s larval life stage, presumably due to prolonged cif action. Our findings reveal that cifB-transcript levels largely explain CI strength, while highlighting other covariates. Elucidating CI’s mechanism contributes to our understanding of Wolbachia spread in natural systems and to improving the efficacy of CI-based biocontrol of arboviruses and agricultural pests globally.

Drosophila melanogaster hosts coevolving with Pseudomonas entomophila pathogen show sex-specific patterns of local adaptation

BackgroundIn spatially structured populations, local adaptation improves organisms’ fitness in their native environment. Hosts and pathogens can rapidly adapt to their local antagonist. Since males and females can differ in their immunocompetence, the patterns of local adaptation can be different between the sexes. However, there is little information about sex differences in local adaptation in host–pathogen systems.ResultsIn the current study, we experimentally coevolved four different replicate populations of Drosophila melanogaster (host) and Pseudomonas entomophila (pathogen) along with appropriate controls. We used the four host–pathogen coevolution populations to investigate the occurrence of local adaptation separately in males and females of the coevolving hosts. We also assessed local adaptation in pathogens. We set up a reciprocal infection experiment where we infected each of the four coevolving hosts with their local pathogen or non-local pathogens from the other three replicate populations. We found that overall, male and female hosts had better survivorship when infected with local pathogens, indicating that they were locally adapted. Interestingly, males were more susceptible to non-local pathogens compared to females. In addition, we found no fecundity cost in females infected with either local or non-local pathogens. We found no evidence of local adaptation among the pathogens.ConclusionOur study showed sex-specific adaptation in the coevolving hosts where female hosts had a broader response against allopatric coevolving pathogens with no cost in fecundity. Thus, our results might suggest a novel mechanism that can maintain variation in susceptibility in spatially structured populations.

In silico design and analyses of a multi-epitope vaccine against Crimean-Congo hemorrhagic fever virus through reverse vaccinology and immunoinformatics approaches

Crimean Congo Hemorrhagic Fever virus (CCHFV) is a deadly human pathogen that causes an emerging zoonotic disease with a broad geographic spread, especially in Africa, Asia, and Europe, and the second most common viral hemorrhagic fever and widely transmitted tick-borne viral disease. Following infection, the patients are presented with a variety of clinical manifestations and a fatality rate of 40%. Despite the high fatality rate, there are unmet clinical interventions, as no antiviral drugs or vaccines for CCHF have been approved. Immunoinformatics pipeline and reverse vaccinology were used in this study to design a multi-epitope vaccine that may elicit a protective humoral and cellular immune response against Crimean-Congo hemorrhagic fever virus infection. Three essential virulent and antigenic proteins (S, M, and L) were used to predict seven CTL and 18 HTL epitopes that were non-allergenic, antigenic, IFN-γ inducing, and non-toxic. The epitopes were connected using linkers and 50S ribosomal protein L7/L12 was used as an adjuvant and raised a multi-epitope vaccine (MEV) that is 567 amino acids long. Molecular docking and simulation of the predicted 3D structure of the MEV with the toll-like (TLR2, TLR3, and TLR4) receptors and major histocompatibility complex (MCH-I and MCH-II) indicate high interactions and stability of the complexes, MM-GBSA free binding energy calculation revealed a favourable protein–protein complex. Maximum MEV expression was achieved with a CAI value of 0.98 through in silico cloning in the Drosophila melanogaster host. According to the immune simulation, IgG1, T-helper cells, T-cytotoxic cells, INF-γ, and IL-2 were predicted to be significantly elevated. These robust computational analyses demonstrated that the proposed MEV is effective in preventing CCHFV infections. However, it is still necessary to conduct both in vitro and in vivo experiments to validate the potential of the vaccine.

Asparagine biosynthesis as a mechanism of increased host lethality induced by Serratia marcescens in simulated microgravity environments

While studies have shown an increase in pathogenicity in several microbes during spaceflight and after exposure to simulated microgravity, the mechanisms underlying these changes in phenotype are not understood across different pathogens, particularly in opportunistic pathogens. This study evaluates the mechanism for increased virulence of the opportunistic gram-negative bacterium, Serratia marcescens, in simulated microgravity. Low-shear modeled microgravity (LSMMG) is used in ground-based studies to simulate the effects of microgravity as experienced in spaceflight. Our previous findings showed that there was a significant increase in mortality rates of the Drosophila melanogaster host when infected with either spaceflight or LSMMG treated S. marcescens. Here, we report that LSMMG increases asparagine uptake and synthesis in S. marcescens and that the increased host lethality induced by LSMMG bacteria grown in rich media can be recapitulated in minimal media by adding only aspartate and glutamine, the substrates of asparagine biosynthesis. Interestingly, increased bacterial growth rate alone is not sufficient to contribute to maximal host lethality, since the addition of aspartate to minimal media caused an LSMMG-specific increase in bacterial growth rate that is comparable to that induced by the combination of aspartate and glutamine, but this increase in growth does not cause an equivalent rate of host mortality. However, the addition of both aspartate and glutamine cause both an increase in host mortality and an overexpression of asparagine pathway genes in a LSMMG-dependent manner. We also report that L-asparaginase-mediated breakdown of asparagine is an effective countermeasure for the increased host mortality caused by LSMMG-treated bacteria. This investigation underscores the importance of the asparagine utilization pathway by helping uncover molecular mechanisms that underlie increased mortality rates of a model host infected with microgravity-treated S. marcescens and provides a potential mitigation strategy.