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Ticks are increasingly important vectors of human and agricultural diseases. While many studies have focused on tick-borne bacteria, far less is known about tick-associated viruses and their roles in public health or tick physiology. To address this, we investigated patterns of bacterial and viral communities across two field populations of western black-legged ticks (Ixodes pacificus). Through metatranscriptomic analysis of 100 individual ticks, we quantified taxon prevalence, abundance, and co-occurrence with other members of the tick microbiome. In addition to commonly found tick-associated microbes, we assembled 11 novel RNA virus genomes from Rhabdoviridae, Chuviridae, Picornaviridae, Phenuiviridae, Reoviridae, Solemovidiae, Narnaviridae and two highly divergent RNA virus genomes lacking sequence similarity to any known viral families. We experimentally verified the presence of these in I. pacificus ticks across several life stages. We also unexpectedly identified numerous virus-like transcripts that are likely encoded by tick genomic DNA, and which are distinct from known endogenous viral element-mediated immunity pathways in invertebrates. Taken together, our work reveals that I. pacificus ticks carry a greater diversity of viruses than previously appreciated, in some cases resulting in evolutionarily acquired virus-like transcripts. Our findings highlight how pervasive and intimate tick-virus interactions are, with major implications for both the fundamental biology and vectorial capacity of I. pacificus ticks.ImportanceTicks are increasingly important vectors of disease, particularly in the United States where expanding tick ranges and intrusion into previously wild areas has resulted in increasing human exposure to ticks. Emerging human pathogens have been identified in ticks at an increasing rate, and yet little is known about the full community of microbes circulating in various tick species, a crucial first step to understanding how they interact with each and their tick host, as well as their ability to cause disease in humans. We investigated the bacterial and viral communities of the Western blacklegged tick in California and found 11 previously uncharacterized viruses circulating in this population.

Simple Summary: Scaphoideus titanus is the main transmission vector Flavescence doreé (FD) in Europe, causing significant damage to grapevine production. The causative agent of FD is the bacterium 'Candidatus Phytoplasma vitis'. However, little is known about the interaction of this plant pathogen with the bacterial communities within the insect host. In this study, we characterize the bacterial communities in S. titanus across different European populations and different life stages. We found significant differences in the microbial composition across different populations but did not observe any differences between nymphs and adults of the same population. The Nearctic leafhopper Scaphoideus titanus (Hemiptera: Cicadellidae) is the primary vector of 'Candidatus Phytoplasma vitis', the causative agent of Flavescence doreé in Europe. Although microorganisms play an important role in the ecology and behavior of insects, knowledge about the interaction between S. titanus and microbes is limited. In this study, we employed an amplicon metabarcoding approach for profiling the V4 region of the 16S rRNA gene to characterize the bacterial communities of S. titanus across several populations from four European localities. Additionally, we investigated changes in bacterial communities between nymphal and adult stages. In total, we identified 7,472 amplicon sequence variants (ASVs) in adults from the European populations. At the genus level, 'Candidatus Karelsulcia' and 'Candidatus Cardinium' were the most abundant genera, with both being present in every individual. While we found significant changes in the microbial composition of S. titanus across different European populations, no significant differences were observed between nymphal and adult stages. Our study reveals new insights into the microbial composition of S. titanus and highlights the role of geography in influencing its bacterial community. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the presence of SARS-CoV-2, yeasts, and bacteria in isolated free-living amoeba (FLA) from COVID-19 patients. Nasopharyngeal swabs (n = 60) were obtained from COVID-19 patients. After cultivation, morphological characterization, and RNA/DNA extraction, the presence of selected microorganisms was investigated. From 60 COVID-19 samples, 18 (30%) were positive for FLA. Acanthamoeba sp. Naegleria australiensis, Tetramitus sp. and Vermamoeba vermiformis were characterized in 12 (80%), 1 (6.66%), 2 (13.33%), and 7 (38.88%) of samples, respectively. SARS-CoV-2 RNA was not detected in FLA. Candida albicans, C. tropicalis, and C. parapsilosis were detected in (11/18; 61.11%), (3/18; 16.67%), and (3/18; 16.67%) of samples, respectively. Geotrichum candidum was detected in 10/18 (55.55%) of samples. Streptococcus spp. and Staphylococcus spp. were identified in 16/18 (88.88%) and 3/18 (16.67%), respectively. The presence of yeasts and bacteria signifies the possible role of FLA in distribution of secondary infections in susceptible patients. [ABSTRACT FROM AUTHOR]

Beet curly top virus (BCTV, curtovirus, geminiviridae) causes one of the most economically significant viral diseases in crops in the Western United States and is transmitted only by the beet leafhopper (Circulifer tenellus) in a non-propagative circulative manner. A better understanding of how this virus overcomes insect vector cellular barriers is essential to understanding virus–vector interactions. The distribution of BCTV in its beet leafhopper vector was investigated using immunofluorescence confocal laser scanning microscope analysis (iCLSM) on the whole-mount-dissected organs of leafhoppers. BCTV was localized in several lobes of the principal salivary glands, filter chamber, anterior midgut, and mid midgut, suggesting the occurrence of midgut and salivary gland barriers to BCTV transmission in its vector C. tenellus. This study also investigated the distribution of the chaperon GroEL homolog protein produced by primary endosymbiotic bacteria within the beet leafhopper, which is believed to indirectly affect viral transmission by enhancing insect immunity and resistance to viruses. GroEL was identified in leafhopper salivary glands lobes, the stylet, salivary canal, the filter chamber, and the Malpighian tubule. This is the first work to visualize the localization of a curtovirus within its beet leafhopper vector. Together, these results can help understand ssDNA virus–vector relationships, including cellular transmission barriers and other vector protein components. [ABSTRACT FROM AUTHOR]

BACKGROUND: Climate change has far‐reaching effects on food security and agriculture, affecting crop yields and food distribution. Agriculture relies heavily on water for irrigation and production, making it vulnerable to water scarcity. Additionally, climate change can affect crop pest insects, leading to increased global crop losses, particularly in cereals, an important component of the human diet. Aphids are major crop pests and have a symbiotic relationship with bacterial endosymbionts that can contribute to their success as pests under a climate change scenario. To test the effect of drought on aphids, we examined varying levels of water deficit and endosymbiont composition on the grain aphid (Sitobion avenae) performance on wheat under controlled laboratory conditions. We measured the intrinsic rate of population increase (rm), the body weight of adult aphids, and the pre‐reproductive period for different genotypes of the grain aphid (including Chilean superclones) under different irrigation regimes. We also analyzed the relative abundance of their endosymbionts under the different water treatments. RESULTS: Our findings revealed that water deficit affects each aphid genotype differently, impacting various traits. For instance, the body weight of adult aphids was notably affected by different water treatments, with aphids grown under intermediate water deficit (IW) being significantly bigger. The relative abundance of endosymbionts also varied among genotypes and water treatments—specifically Regiella insecticola had a noticeably higher abundance under IW (P < 0.05). CONCLUSION: This study provides valuable insights into the impact of water deficit on aphid performance and the role of endosymbionts in mitigating the effects of water deficit. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Abstract Background Insects act as disease vectors, spreading disease-causing organisms between plants and animals. There have been studies devoted to determining ways to control these pests. One of the most effective ways to accomplish this is to reduce their vector competency. This review article explains how these factors can reduce vector competency. Main body The major ways by which vector competence can be reduced were reviewed. Entomopathogens are organisms that cause disease in arthropods like insects, mites, and ticks. Wolbachia is a genus of intracellular bacteria that mostly infects arthropods, including a significant number of insects. It is one of the most frequent insect reproductive parasites that kill or severely disables insects. Entomopathogenic nematodes are a type of worm that attacks insects and kills them. Insect-specific viruses are a relatively new class of viruses with a variety of intriguing traits that could be used to better understand and possibly inhibit arbovirus transmission. Entomopathogenic fungi are a type of fungus that kills insects by attacking and infecting their insect hosts. Disrupting the environment and nutrition of insects could also help to reduce their ability to spread diseases to humans, animals, and plants. Conclusions Chemical control has been one of the most widely used methods for controlling disease vectors, but there have been reports of insect resistance, environmental degradation, and a variety of other side effects. Instead of chemical control, there are a variety of techniques that can be used, including targeting insects' endosymbionts (bacteria, viruses, fungi, nematodes, and protozoa), changing insects' nutrition, manipulating their environment, and many others. This paper discussed the alternative ways to reduce vectors’ competence without the use of synthetic chemical.

Simple Summary: Rice breeding programs have included traditional varieties and wild rice species as sources of resistance to planthoppers and leafhoppers, which are major insect pests of rice in Asia. However, herbivores can adapt to resistance (known as virulence adaptation), sometimes in as little as 2 years. Virulence adaptation is a major obstacle for deploying resistant varieties. This review assesses knowledge of the rates, consequences, and mechanisms of virulence adaptation and on possible strategies to delay adaptation in rice landscapes. The review indicates that virulence is a complex of responses to minor and major resistance factors. Of particular interest are adaptations to major rice resistance genes that are likely governed by corresponding herbivore virulence genes. Evidence suggests that pre-adapted individuals (called forerunners) with these virulence genes occur in planthopper populations at relatively low frequencies but can rapidly build up numbers in response to planted resistant rice. However, gradual shifts in virulence may also occur due to intra- and intergenerational improvements in the herbivores' abilities to detoxify plant defense toxins, or to confound defense response networks. These may involve gradual changes in the expression of genes involved in successful herbivore attacks and may be associated with the herbivores or their endosymbionts. Several research gaps remain. In particular, there is a need for increased attention to practical, ecologically based deployment strategies that delay adaptation. In recent decades, research on developing and deploying resistant rice has accelerated due to the availability of modern molecular tools and, in particular, advances in marker-assisted selection. However, progress in understanding virulence adaptation has been relatively slow. This review tracks patterns in virulence adaptation to resistance genes (particularly Bph1, bph2, Bph3, and bph4) and examines the nature of virulence based on selection experiments, responses by virulent populations to differential rice varieties (i.e., varieties with different resistance genes), and breeding experiments that interpret the genetic mechanisms underlying adaptation. The review proposes that varietal resistance is best regarded as a combination of minor and major resistance traits against which planthoppers develop partial or complete virulence through heritable improvements that are reversable or through evolutionary adaptation, respectively. Agronomic practices, deployment patterns, and herbivore population pressures determine the rates of adaptation, and there is growing evidence that pesticide detoxification mechanisms can accelerate virulence adaptation. Research to delay adaptation has mainly focused on gene pyramiding (i.e., including ≥ two major genes in a variety) and multilines (i.e., including ≥ two resistant varieties in a field or landscape); however, these strategies have not been adequately tested and, if not managed properly, could inadvertently accelerate adaptation compared to sequential deployment. Several research gaps remain and considerable improvements in research methods are required to better understand and manage virulence adaptation. [ABSTRACT FROM AUTHOR]

Spider mites are important pests of cotton, a vital fiber crop cultivated in sun-drenched regions. This study investigated the prevalence and distribution of spider mite species infesting cotton fields in Aydin province, Türkiye. Spider mites were found in a significant portion (89%) of the surveyed fields. The two most abundant mite species, were the red form (RF) of Tetranychus urticae (57.1%) and T. turkestani (39.3%). A minor presence of T. urticae green form (GF) (3.6%) was observed. Tetranychus urticae RF was more prevalent in coastal areas, while T. turkestani was more common inland. Field surveys demonstrated that areas with higher T. urticae RF prevalence exhibited more frequent spraying, potentially due to this species’ greater capacity to develop resistance compared to T. turkestani. This study also investigated the prevalence of Wolbachia, Rickettsia, Cardinium and Spiroplasma endosymbionts, which are linked with pesticide resistance due to their ability to degrade pesticides, in these mite populations. The study confirmed the presence of Wolbachia and Rickettsia endosymbionts in spider mite populations in Türkiye. However, no Cardinium or Spiroplasma were detected in any of the populations. Infection rates of the detected endosymbionts differed between T. urticae and T. turkestani. Future studies should investigate the resistance levels of these tetranychid mite species as well as the role of their endosymbiotic bacteria in resistance in cotton-growing areas. [ABSTRACT FROM AUTHOR]

Background: Insects act as disease vectors, spreading disease-causing organisms between plants and animals. There have been studies devoted to determining ways to control these pests. One of the most effective ways to accomplish this is to reduce their vector competency. This review article explains how these factors can reduce vector competency. Main body: The major ways by which vector competence can be reduced were reviewed. Entomopathogens are organisms that cause disease in arthropods like insects, mites, and ticks. Wolbachia is a genus of intracellular bacteria that mostly infects arthropods, including a significant number of insects. It is one of the most frequent insect reproductive parasites that kill or severely disables insects. Entomopathogenic nematodes are a type of worm that attacks insects and kills them. Insect-specific viruses are a relatively new class of viruses with a variety of intriguing traits that could be used to better understand and possibly inhibit arbovirus transmission. Entomopathogenic fungi are a type of fungus that kills insects by attacking and infecting their insect hosts. Disrupting the environment and nutrition of insects could also help to reduce their ability to spread diseases to humans, animals, and plants. Conclusions: Chemical control has been one of the most widely used methods for controlling disease vectors, but there have been reports of insect resistance, environmental degradation, and a variety of other side effects. Instead of chemical control, there are a variety of techniques that can be used, including targeting insects' endosymbionts (bacteria, viruses, fungi, nematodes, and protozoa), changing insects' nutrition, manipulating their environment, and many others. This paper discussed the alternative ways to reduce vectors' competence without the use of synthetic chemical. [ABSTRACT FROM AUTHOR]

The symbiosis between microorganisms and host arthropods can cause biological, physiological, and reproductive changes in the host population. The present study aimed to survey facultative symbionts of the genera Wolbachia , Arsenophonus , Cardinium , Rickettsia , and Nosema in Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) and Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) in the laboratory and evaluate the influence of infection on the fitness of these hosts. For this purpose, 16S rDNA primers were used to detect these facultative symbionts in the host species, and the hosts' biological and morphological features were evaluated for changes resulting from the infection caused by these microorganisms. The bacterial symbionts studied herein were not detected in the D. saccharalis samples analysed, but the endosymbiont Wolbachia was detected in C. flavipes and altered the biological and morphological aspects of this parasitoid insect. The results of this study may help to elucidate the role of Wolbachia in maintaining the quality of populations/lineages of C. flavipes. [ABSTRACT FROM AUTHOR]

Obligate intracellular endobacteria of the genus Wolbachia are widespread in arthropods and several filarial nematodes. Control programs for vector-borne diseases (dengue, Zika, malaria) and anti-filarial therapy with antibiotics are based on this important endosymbiont. Investigating Wolbachia, however, is impeded by the need for host cells. In this study, the requirements for Wolbachia wAlbB growth in a host cell-free in vitro culture system were characterized via qPCRs. A cell lysate fraction from Aedes albopictus C6/36 insect cells containing cell membranes and medium with fetal bovine serum were identified as requisite for cell-free replication of Wolbachia. Supplementation with the membrane fraction of insect cell lysate increased extracellular Wolbachia replication by 4.2-fold. Replication rates in the insect cell-free culture were lower compared to Wolbachia grown inside insect cells. However, the endobacteria were able to replicate for up to 12 days and to infect uninfected C6/36 cells. Cellfree Wolbachia treated with the lipid II biosynthesis inhibitor fosfomycin had an enlarged phenotype, seen previously for intracellular Wolbachia in C6/36 cells, indicating that the bacteria were unable to divide. In conclusion, we have developed a cell-free culture system in which Wolbachia replicate for up to 12 days, providing an in vitro tool to elucidate the biology of these endobacteria, e.g., cell division by using compounds that may not enter the C6/36 cells. A better understanding of Wolbachia biology, and in particular host-symbiont interactions, is key to the use of Wolbachia in vector control programs and to future drug development against filarial diseases. [ABSTRACT FROM AUTHOR]

A nematological survey was conducted between 2021 and 2022 in banana fields distributed in two major banana-producing regions in the western coast of Syria. As a result, six populations of Xiphinema spp. identified as X. diffusum, X. pachtaicum, X. simile, X. vuittenezi and X. zagrosense were isolated from the rhizosphere of banana plants (Musa sp.) and characterized using morphological and molecular data based on two rRNA genes (D2–D3 expansion segments of the 28S, and ITS fragments) and partial region of the cytochrome oxidase I gene (COI mtDNA) sequences. Moreover, the molecular identification of the endosymbionts of these populations was also performed using the complete 16S rDNA gene. The phylogenetic relationships of the recovered species of nematodes and respective endosymbionts were reconstructed. Candidatus Xiphinematobacter sp. (OR196969; OR196971) and Ca. Xiphinematobacter sp. (OR196970) were detected in X. diffusum and X. simile, respectively, and clustered together with other Ca. Xiphinematobacter sp.A and sp.I respectively. To our knowledge, this is the first report of X. diffusum, X. simile, X. vuittenezi and X. zagrosense parasitizing banana in Syria, extending the geographical distribution of these species within the Mediterranean Basin. [ABSTRACT FROM AUTHOR]

Microbe-microbe interactions - of central importance in biological systems - can be antagonistic or synergistic, acting to benefit or detriment of a host. Pederin-producing endosymbionts (PPE) are considered protective mutualists in their Paederus fuscipes (rove beetle) hosts but little is known about whether PPE affect other aspects of host fitness as well as host microbial diversity. We find strong deleterious costs of PPE infections on hosts related to an extended development time, smaller and shorter lifespan of females, as well as a lower hatching rates of progeny. 16S rRNA data reveal that PPE affect the microbiome of P. fuscipes depending on gender and organ, resulting in a decrease in the number of the potentially beneficial symbiotic bacteria Apibacter and the reproductive manipulator Wolbachia and an increase in the conditional pathogenic bacterium Bartonella. Predicted microbe functions related to metabolism, longevity, immunity and resistibility are enriched in uninfected females, potentially contributing to fitness costs of PPE infections. We further sequence and annotate the first complete genome of PPE and compare the pederin biosynthetic gene cluster (ped) with the pederin family biosynthetic gene cluster from other bacterial-eukaryote symbioses. This study highlights the fitness costs of PPE infections to P. fuscipes, even though the defensive pederin compound produced by PPE helps protect the host. The ecology and evolution of this mutualism is shaped by costs, not just the benefits they confer. Changes in the microbial community within infected rove beetles may mediate the negative impacts of PPE on fitness. [ABSTRACT FROM AUTHOR]

Interactions between eukaryotic hosts and their bacterial symbionts drive key ecological and evolutionary processes, from regulating ecosystems to the evolution of complex molecular machines and processes. Over time, endosymbionts generally evolve reduced genomes, and their relationship with their host tends to stabilize. However, host–bacteria relationships may be heavily influenced by environmental changes. Here, we review these effects on one of the most ancient and diverse endosymbiotic groups, formed by—among others— Legionellales, Francisellaceae , and Piscirickettsiaceae. This group is referred to as Deep-branching Intracellular Gammaproteobacteria (DIG), whose last common ancestor presumably emerged about 2 Ga ago. We show that DIGs are globally distributed, but generally at very low abundance, and are mainly identified in aquatic biomes. Most DIGs harbour a type IVB secretion system, critical for host-adaptation, but its structure and composition vary. Finally, we review the different types of microbial interactions that can occur in diverse environments, with direct or indirect effects on DIG populations. The increased use of omics technologies on environmental samples will allow a better understanding of host–bacterial interactions and help unravel the definition of DIGs as a group from an ecological, molecular, and evolutionary perspective. [ABSTRACT FROM AUTHOR]

Asexual lineages often exhibit broad distributions and can thrive in extreme habitats compared to their sexual counterparts. Several hypotheses can be proposed to explain this pattern. Asexual lineages could be versatile genotypes with wide environmental tolerance, enabling their dispersal and persistence across large geographic areas. Alternatively, asexual genotypes could be ecological specialists that thrive in specific environments and outcompete relative colonizing distantly related with similar conditions in the process. Several aphid species feature widespread obligate asexual lineages, commonly known as 'superclones'. Yet it is often unknown whether these clones are widespread ecological generalists or successful specialists. To explore these hypotheses, we examined climatic niche differentiation among six globally distributed obligate asexual lineages of the cosmopolitan aphid pest, Brachycaudus helichrysi. To ensure that we were investigating the aphid genotype niche and not a by‐product of their association with endosymbionts mediating thermal tolerance, we first verified that clones hosted similar endosymbiont communities. Subsequently, we conducted multivariate analyses on clone occurrence data on a worldwide scale. Our results revealed that, despite their global distribution, B. helichrysi superclones occupy different climatic niches. This study represents the first evidence that aphid superclone distribution can be mediated by distinctive ranges of climatic tolerance. [ABSTRACT FROM AUTHOR]

Endosymbiotic relationships have shaped eukaryotic life. As endosymbionts coevolve with their host, toward full integration as organelles, their genomes tend to shrink, with genes being completely lost or transferred to the host nucleus. Modern endosymbionts and organelles show diverse patterns of gene retention, and why some genes and not others are retained in these genomes is not fully understood. Recent bioinformatic study has explored hypothesized influences on these evolutionary processes, finding that hydrophobicity and amino acid chemistry predict patterns of gene retention, both in organelles across eukaryotes and in less mature endosymbiotic relationships. The exciting ongoing elucidation of endosymbiotic relationships affords an independent set of instances to test this theory. Here, we compare the properties of retained genes in the nitroplast, recently reported to be an integrated organelle, two related cyanobacterial endosymbionts that form "spheroid bodies" in their host cells, and a range of other endosymbionts, with free-living relatives of each. We find that in each case, the symbiont's genome encodes proteins with higher hydrophobicity and lower amino pKa than their free-living relative, supporting the data-derived model predicting the retention propensity of genes across endosymbiont and organelle genomes. [ABSTRACT FROM AUTHOR]

Characterization of the temperature effects on the abundance and richness of the microbiota of Lutzomyia longipalpis, insect vector of Leishmania infantum in America, is an aspect of pivotal importance to understand the interactions between temperature, bacteria, and Leishmania infection. We developed and used a customized device with a temperature gradient (21–34 °C) to assess the temperature preferences of wild females of Lu. longipalpis collected in a rural area (Ricaurte, Cundinamarca, Colombia). Each replicate consisted of 50 females exposed to the gradient for an hour. At the end of the exposure time, insects were collected and separated by the temperature ranges selected varying from 21 °C to 34 °C. They were organized in 17 pools from which total DNA extracts were obtained, and samples were subjected to 16S rRNA amplicon sequencing analyzes. The most abundant phyla across the different temperature ranges were Proteobacteria (17.22–90.73 %), Firmicutes (5.99–77.21 %) and Actinobacteria (1.56–59.85 %). Results also showed an abundance (30 % to 57.36 %) of Pseudomonas (mainly at temperatures of 21–29 °C and 34 °C) that decreased to 6.55 %-13.20 % at temperatures of 31–33 °C, while Bacillus increase its abundance to 67.24 % at 29–33 °C. Serratia also had a greater representation (49.79 %), specifically in sand flies recovered at 25–27 °C. No significant differences were found at α-diversity level when comparing richness using the Shannon-Wiener, Simpson, and Chao1 indices, while β-diversity differences were found using the Bray-Curtis index (F-value of 3.5073, p-value < 0.013, R-squared of 0,4889), especially in the groups of Lu. longipalpis associated at higher temperatures (29–33 °C). It was also possible to detect the presence of endosymbionts such as Spiroplasma and Arsenophonus in the range of 29–33 °C. Rickettsia was only detected in Lu. longipalpis sand flies recovered between 25–27 °C. It was possible to characterize Lu. longipalpis microbiota in response to intraspecific temperature preferences and observe changes in bacterial communities and endosymbionts at different ranges of said environmental variable, which may be important in its vector competence and environmental plasticity to adapt to new climate change scenarios.

Few studies have examined the genetic population structure of vector-borne microparasites in wildlife, making it unclear how much these systems can reveal about the movement of their associated hosts. This study examined the complex host–vector–microbe interactions in a system of bats, wingless ectoparasitic bat flies (Nycteribiidae), vector-borne microparasitic bacteria (Bartonella) and bacterial endosymbionts of flies (Enterobacterales) across an island chain in the Gulf of Guinea, West Africa. Limited population structure was found in bat flies and Enterobacterales symbionts compared to that of their hosts. Significant isolation by distance was observed in the dissimilarity of Bartonella communities detected in flies from sampled populations of Eidolon helvum bats. These patterns indicate that, while genetic dispersal of bats between islands is limited, some non-reproductive movements may lead to the dispersal of ectoparasites and associated microbes. This study deepens our knowledge of the phylogeography of African fruit bats, their ectoparasites and associated bacteria. The results presented could inform models of pathogen transmission in these bat populations and increase our theoretical understanding of community ecology in host–microbe systems.

Whiteflies in the Aleyrodidae family cause direct sap-sucking harm and responsible for increasing agricultural losses worldwide by spreading plant viruses. Whiteflies and their endosymbiotic bacteria, including Wolbachia and Cardinium, have complicated symbiotic relationships that affect host physiology, development, and immunity. Since resistance and environmental issues make traditional pest management methods more ineffective, innovative methods are needed to limit whitefly damage and virus spread. To disrupt the whitefly symbiosis, the use of camelid heavy-chain nanobodies offer novel pest control and disease prevention options. The nanobody (NB_An33) and GroEL protein sequences from Cardinium and Wolbachia endosymbionts were subjected to rigorous computational methodologies, including the pBLAST, ClustalW, MEGA, IntFOLD server, SWISS-MODEL, and HDOCK server. The phylogenetic analysis revealed that Cardinium and Wolbachia GroEL proteins belong to different clades. These proteins were given reliable three-dimensional structures using homology modelling, and the use of Ramachandran graphs confirmed their structural integrity. Additionally, the FunFOLD algorithm equipped IntFOLD server found several ligand binding sites in Wolbachia GroEL but none in Cardinium GroEL, highlighting the latter's special properties. The exceptional binding affinity of nanobodies, particularly the 1YC8 version, to Wolbachia GroEL proteins was shown by molecular docking investigations. The Wolbachia GroEL's enhanced ability to bind nanobodies suggests that it may be susceptible to disruption. This transdisciplinary computational study provides insights into complex biological interactions. It suggests how nanobodies may potentially disrupt symbiotic associations in whiteflies, offering new pest management methods. This strategy can become a game changer in controlling the spread of harmful plant viruses, which are supported by symbiotic partners of whiteflies. [ABSTRACT FROM AUTHOR]

Sap-feeding insects often maintain two or more nutritional endosymbionts that act in concert to produce compounds essential for insect survival. Many mealybugs have endosymbionts in a nested configuration: one or two bacterial species reside within the cytoplasm of another bacterium, and together, these bacteria have genomes that encode interdependent sets of genes needed to produce key nutritional molecules. Here, we show that the mealybug Pseudococcus viburni has three endosymbionts, one of which contributes only two unique genes that produce the host nutrition-related molecule chorismate. All three bacterial endosymbionts have tiny genomes, suggesting that they have been coevolving inside their insect host for millions of years. [ABSTRACT FROM AUTHOR]

In the present study, synthesis of silver nanobactericides was achieved from Curcuma longa L. and its endosymbiont for their antimicrobial activity. The nanobactericides exhibited notable antimicrobial potential against Escherichia coli and Staphylococcus aureus, as determined through broth dilution and minimum inhibitory concentration which ranged between 2.5 and 5 mg/mL. Characterization of nanobactericides revealed polydispersity with average size of 80 nm and crystalline nature showed distinct peaks. The Fourier transform infrared (FTIR) analysis revealed presence of exhibited peaks at 3362.24 cm⁻¹ (hydroxyl group), 1637.96 cm⁻¹ (carbonyl group), 1377.25 cm⁻¹ (alkane group), and 635.25 cm⁻¹ (alkyl group) which participated in the synthesis of nanobactericides. Endosymbiont crude extracts subjected to partial purification through thin-layer chromatography, and bioautography-guided fractionation identified an active compound at Rf 0.7 which showed clear zone of inhibition. The minimum inhibitory concentration of metabolite fraction was found to be 0.625 mg/mL against both the test pathogens. The dye degradation potential of nanobactericides was successful 81.27 % of degradation was achieved with safranin treated with silver nanobactericides from endosymbiont. Subsequently, nanobactericides synthesized from plants showed 59.88 % highest degradation with methylene blue. The profiling of metabolite was carried out with gas chromatography-mass spectrometry to identify as a novel metabolite, 1,2-benzenedicarboxylic acid, decyl octyl ester, showing antimicrobial potential against tested pathogens. The identified metabolite molecular formula was found to be C26H42O4 with molecular weight of 418. These promising results, especially for the scarcely reported compound, contribute to the understanding of plant-endosymbiont-synthesized nanobactericides with significant antimicrobial properties. The molecular analysis revealed the endosymbiont's affinity to Pseudomonas aeruginosa, and its sequence has been deposited in GenBank (Accession number OR984817). The study concludes with importance of nanobactericides from plant-endosymbiont consortium, highlighting their antimicrobial efficacy against human and phytopathogens.

ABSTRACT Facultative endosymbiotic bacteria, such as Wolbachia and Spiroplasma species, are commonly found in association with insects and can dramatically alter their host physiology. Many endosymbionts are defensive and protect their hosts against parasites or pathogens. Despite the widespread nature of defensive insect symbioses and their importance for the ecology and evolution of insects, the mechanisms of symbiont-mediated host protection remain poorly characterized. Here, we utilized the fruit fly Drosophila melanogaster and its facultative endosymbiont Spiroplasma poulsonii to characterize the mechanisms underlying symbiont-mediated host protection against bacterial and fungal pathogens. Our results indicate a variable effect of S. poulsonii on infection outcome, with endosymbiont-harboring flies being more resistant to Rhyzopus oryzae, Staphylococcus aureus, and Providencia alcalifaciens but more sensitive or as sensitive as endosymbiont-free flies to the infections with Pseudomonas species. Further focusing on the protective effect, we identified Transferrin-mediated iron sequestration induced by Spiroplasma as being crucial for the defense against R. oryzae and P. alcalifaciens. In the case of S. aureus, enhanced melanization in Spiroplasma-harboring flies plays a major role in protection. Both iron sequestration and melanization induced by Spiroplasma require the host immune sensor protease Persephone, suggesting a role of proteases secreted by the symbiont in the activation of host defense reactions. Hence, our work reveals a broader defensive range of Spiroplasma than previously appreciated and adds nutritional immunity and melanization to the defensive arsenal of symbionts.IMPORTANCEDefensive endosymbiotic bacteria conferring protection to their hosts against parasites and pathogens are widespread in insect populations. However, the mechanisms by which most symbionts confer protection are not fully understood. Here, we studied the mechanisms of protection against bacterial and fungal pathogens mediated by the Drosophila melanogaster endosymbiont Spiroplasma poulsonii. We demonstrate that besides the previously described protection against wasps and nematodes, Spiroplasma also confers increased resistance to pathogenic bacteria and fungi. We identified Spiroplasma-induced iron sequestration and melanization as key defense mechanisms. Our work broadens the known defense spectrum of Spiroplasma and reveals a previously unappreciated role of melanization and iron sequestration in endosymbiont-mediated host protection. We propose that the mechanisms we have identified here may be of broader significance and could apply to other endosymbionts, particularly to Wolbachia, and potentially explain their protective properties.

Obligate intracellular endobacteria of the genus Wolbachia are widespread in arthropods and several filarial nematodes. Control programs for vector-borne diseases (dengue, Zika, malaria) and anti-filarial therapy with antibiotics are based on this important endosymbiont. Investigating Wolbachia, however, is impeded by the need for host cells. In this study, the requirements for Wolbachia wAlbB growth in a host cell-free in vitro culture system were characterized via qPCRs. A cell lysate fraction from Aedes albopictus C6/36 insect cells containing cell membranes and medium with fetal bovine serum were identified as requisite for cell-free replication of Wolbachia. Supplementation with the membrane fraction of insect cell lysate increased extracellular Wolbachia replication by 4.2-fold. Replication rates in the insect cell-free culture were lower compared to Wolbachia grown inside insect cells. However, the endobacteria were able to replicate for up to 12 days and to infect uninfected C6/36 cells. Cell-free Wolbachia treated with the lipid II biosynthesis inhibitor fosfomycin had an enlarged phenotype, seen previously for intracellular Wolbachia in C6/36 cells, indicating that the bacteria were unable to divide. In conclusion, we have developed a cell-free culture system in which Wolbachia replicate for up to 12 days, providing an in vitro tool to elucidate the biology of these endobacteria, e.g., cell division by using compounds that may not enter the C6/36 cells. A better understanding of Wolbachia biology, and in particular host-symbiont interactions, is key to the use of Wolbachia in vector control programs and to future drug development against filarial diseases.

ABSTRACT Diaphorin is a polyketide produced by “Candidatus Profftella armatura” (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a devastating agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Physiological concentrations of diaphorin, which D. citri contains at levels as high as 2–20 mM, are inhibitory to various eukaryotes and Bacillus subtilis (Firmicutes: Bacilli) but promote the growth and metabolic activity of Escherichia coli (Gammaproteobacteria: Enterobacterales). Our previous study demonstrated that 5-mM diaphorin, which exhibits significant inhibitory and promoting effects on cultured B. subtilis and E. coli, respectively, inhibits in vitro gene expression utilizing purified B. subtilis and E. coli ribosomes. This suggested that the adverse effects of diaphorin on B. subtilis are partly due to its influence on gene expression. However, the result appeared inconsistent with the positive impact on E. coli. Moreover, the diaphorin concentration in bacterial cells, where genes are expressed in vivo, may be lower than in culture media. Therefore, the present study analyzed the effects of 50 and 500 µM of diaphorin on bacterial gene expression using the same analytical method. The result revealed that this concentration range of diaphorin, in contrast to 5-mM diaphorin, promotes the in vitro translation with the B. subtilis and E. coli ribosomes, suggesting that the positive effects of diaphorin on E. coli are due to its direct effects on translation. This study demonstrated for the first time that a pederin-type compound promotes gene expression, establishing a basis for utilizing its potential in pest management and industrial applications.IMPORTANCEThis study revealed that a limited concentration range of diaphorin, a secondary metabolite produced by a bacterial symbiont of an agricultural pest, promotes cell-free gene expression utilizing substrates and proteins purified from bacteria. The unique property of diaphorin, which is inhibitory to various eukaryotes and Bacillus subtilis but promotes the growth and metabolic activity of Escherichia coli, may affect the microbial flora of the pest insect, potentially influencing the transmission of devastating plant pathogens. Moreover, the activity may be exploited to improve the efficacy of industrial production by E. coli, which is often used to produce various important materials, including pharmaceuticals, enzymes, amino acids, and biofuels. This study elucidated a part of the mechanism by which the unique activity of diaphorin is expressed, constructing a foundation for applying the distinct property to pest management and industrial use.

Abstract Background Russian wheat aphid (Diuraphis noxia Kurd.) is a severe pest to wheat, and even though resistance varieties are available to curb this pest, they are becoming obsolete with the development of new virulent aphid populations. Unlike many other aphids, D noxia only harbours a single endosymbiont, Buchnera aphidicola. Considering the importance of Buchnera, this study aimed to elucidate commonalities and dissimilarities between various hosts, to better understand its distinctiveness within its symbiotic relationship with D. noxia. To do so, the genome of the D. noxia’s Buchnera was assembled and compared to those of other aphid species that feed on diverse host species. Results The overall importance of several features such as gene length and percentage GC content was found to be critical for the maintenance of Buchnera genes when compared to their closest free-living relative, Escherichia coli. Buchnera protein coding genes were found to have percentage GC contents that tended towards a mean of ~ 26% which had strong correlation to their identity to their E. coli homologs. Several SNPs were identified between different aphid populations and multiple isolates of Buchnera were confirmed in single aphids. Conclusions Establishing the strong correlation of percentage GC content of protein coding genes and gene identity will allow for identifying which genes will be lost in the continually shrinking Buchnera genome. This is also the first report of a parthenogenically reproducing aphid that hosts multiple Buchnera strains in a single aphid, raising questions regarding the benefits of maintaining multiple strains. We also found preliminary evidence for post-transcriptional regulation of Buchnera genes in the form of polyadenylation.

Hyalesthes obsoletus (Hemiptera: Fulgoromorpha: Cixiidae) is a pan-European polyphagous planthopper known as a significant vector of the plant pathogenic bacterium 'Candidatus Phytoplasma solani' (stolbur phytoplasma), which poses threats to various agricultural crops. A population of H. obsoletus associated with Convolvulus arvensis in Serbia was studied to investigate the presence and genetic diversity of Wolbachia, an endosymbiotic bacterium known for its promising biological control applications. Both insect-associated microorganisms, 'Ca. P. solani' and Wolbachia, were found in the assessed H. obsoletus population. The analyzed vector population had a 'Ca. P. solani' infection rate of 50%, while Wolbachia showed a high infection rate of 80%. Wolbachia presence displayed minimal variation across genders and was independent of individuals' phytoplasma-infection status. Genotyping of the identified 'Ca. P. solani' strains revealed four previously described stamp genotypes (Rqg50/St1, Rqg31/St2, STOL/St4 and M5/St28). Notably, a single novel Wolbachia ftsZ genotype, designated WHo1, was found in the assessed H. obsoletus population, providing a valuable insight into the genetic diversity of Wolbachia endosymbionts within the Cixiidae family. Phylogenetic analysis demonstrated intricate relationships between WHo1 and other Wolbachia strains infecting hosts from diverse hemipteran suborders. Although Wolbachia-based strategies show promise for phytoplasma vector control, further research is needed to elucidate its potential interactions with 'Ca. P. solani' and its effects on vector reproduction and fitness.

The Nearctic leafhopper Scaphoideus titanus (Hemiptera: Cicadellidae) is the primary vector of ‘Candidatus Phytoplasma vitis’, the causative agent of Flavescence doreé in Europe. Although microorganisms play an important role in the ecology and behavior of insects, knowledge about the interaction between S. titanus and microbes is limited. In this study, we employed an amplicon metabarcoding approach for profiling the V4 region of the 16S rRNA gene to characterize the bacterial communities of S. titanus across several populations from four European localities. Additionally, we investigated changes in bacterial communities between nymphal and adult stages. In total, we identified 7,472 amplicon sequence variants (ASVs) in adults from the European populations. At the genus level, ‘Candidatus Karelsulcia’ and ‘Candidatus Cardinium’ were the most abundant genera, with both being present in every individual. While we found significant changes in the microbial composition of S. titanus across different European populations, no significant differences were observed between nymphal and adult stages. Our study reveals new insights into the microbial composition of S. titanus and highlights the role of geography in influencing its bacterial community.

Beet curly top virus (BCTV, curtovirus, geminiviridae) causes one of the most economically significant viral diseases in crops in the Western United States and is transmitted only by the beet leafhopper (Circulifer tenellus) in a non-propagative circulative manner. A better understanding of how this virus overcomes insect vector cellular barriers is essential to understanding virus–vector interactions. The distribution of BCTV in its beet leafhopper vector was investigated using immunofluorescence confocal laser scanning microscope analysis (iCLSM) on the whole-mount-dissected organs of leafhoppers. BCTV was localized in several lobes of the principal salivary glands, filter chamber, anterior midgut, and mid midgut, suggesting the occurrence of midgut and salivary gland barriers to BCTV transmission in its vector C. tenellus. This study also investigated the distribution of the chaperon GroEL homolog protein produced by primary endosymbiotic bacteria within the beet leafhopper, which is believed to indirectly affect viral transmission by enhancing insect immunity and resistance to viruses. GroEL was identified in leafhopper salivary glands lobes, the stylet, salivary canal, the filter chamber, and the Malpighian tubule. This is the first work to visualize the localization of a curtovirus within its beet leafhopper vector. Together, these results can help understand ssDNA virus–vector relationships, including cellular transmission barriers and other vector protein components.

Aphids shelter several bacteria that benefit them in various ways. The associates having an obligatory relationship are non-culturable, while a few of facultative associates are culturable in insect cell lines, axenic media or standard microbiology media. In the present investigation, isolation, and characterization of the culturable bacterial associates of various aphid species, viz., Rhopalosiphum maidis, Rhopalosiphum padi, Sitobion avenae, Schizaphis graminum, and Lipaphis erysimi pseudobrassicae were carried out. A total of 42 isolates were isolated using different growth media, followed by their morphological, biochemical, and molecular characterization. The isolated culturable bacterial associates were found to belong to the genera Acinetobacter, Bacillus, Brevundimonas, Cytobacillus, Fictibacillus, Planococcus, Priestia, Pseudomonas, Staphylococcus, Sutcliffiella, and Tumebacillus which were grouped under seven families of four different orders of phyla Bacillota (Firmicutes) and Pseudomonata (Proteobacteria). Symbiont–entomopathogen interaction study was also conducted, in which the quantification of colony forming units of culturable bacterial associates of entomopathogenic fungal-treated aphids led us to the assumption that the bacterial load in aphid body can be altered by the application of entomopathogens. Whereas, the mycelial growth of entomopathogens Akanthomyces lecanii and Metarhizium anisopliae was found uninhibited by the bacterial associates obtained from Sitobion avenae and Rhopalosiphum padi. Analyzing persistent aphid microflora and their interactions with entomopathogens enhances our understanding of aphid resistance. It also fosters the development of innovative solutions for agricultural pest management, highlighting the intricate dynamics of symbiotic relationships in pest management strategies. [ABSTRACT FROM AUTHOR]

Obligate endosymbionts have a significant impact on the physiology and ecology of their insect hosts and consequently have played an important role in their diversification and evolution. Auchenorrhyncha is a sap-feeding insect group that includes cicadas, spittlebugs, leafhoppers, treehoppers, and planthoppers, some of which are well-studied vectors of plant pathogens causing important diseases. Here, we review the obligate symbiotic systems in Auchenorrhyncha. First, we address the diversity of obligate endosymbionts, illustrating the complex scenario characterised by replacements, new acquisitions, and loss of endosymbionts along evolution. Then, we describe the mechanisms that allowed maintaining these long-term associations. Also, we attend to the functional interdependence between host and obligate endosymbionts and how insect hosts support and regulate them. Moreover, we discuss current research that considers the disruption of host-endosymbionts associations as a novel strategy to control these sap-feeding insects. Finally, we suggest directions for further studies regarding obligate mutualistic relationships as well as other symbiotic systems that could be helpful in increasing the knowledge of the complex interactions between Auchenorrhyncha and their associated microbes. [ABSTRACT FROM AUTHOR]

Dicyemids (phylum Dicyemida) are the most common and most characteristic endosymbionts in the renal sacs of benthic cephalopod molluscs: octopuses and cuttlefishes. Typically, 2 or 3 dicyemid species are found in a single specimen of the host, and most dicyemids have high host specificity. Host-specific parasites are restricted to a limited range of host species by ecological barriers that impede dispersal and successful establishment; therefore, phylogenies of interacting groups are often congruent due to repeated co-speciation. Most frequently, however, host and parasite phylogenies are not congruent, which can be explained by processes such as host switching and other macro-evolutionary events. Here, the history of dicyemids and their host cephalopod associations were studied by comparing their phylogenies. Dicyemid species were collected from 8 decapodiform species and 12 octopodiform species in Japanese waters. Using whole mitochondrial cytochrome c oxidase subunit 1 (COI) sequences, a phylogeny of 37 dicyemid species, including 4 genera representing the family Dicyemidae, was reconstructed. Phylogenetic trees derived from analyses of COI genes consistently suggested that dicyemid species should be separated into 3 major clades and that the most common genera, Dicyema and Dicyemennea, are not monophyletic. Thus, morphological classification does not reflect the phylogenetic relationships of these 2 genera. Divergence (speciation) of dicyemid species seems to have occurred within a single host species. Possible host-switching events may have occurred between the Octopodiformes and Decapodiformes or within the Octopodiformes or the Decapodiformes. Therefore, the mechanism of dicyemid speciation may be a mixture of host switching and intra-host speciation. This is the first study in which the process of dicyemid diversification involving cephalopod hosts has been evaluated with a large number of dicyemid species and genera. [ABSTRACT FROM AUTHOR]

Simple Summary: The application of molecular methods in the studies of biological diversity revealed that there are many more species than we can recognise by their appearance. Species pairs or groups, which are difficult to recognise and are known as cryptic species, may differ in their ecological traits, place in trophic networks, and their functional role in ecosystems. It is important to separate cryptic species, particularly if we apply species composition or other diversity metrics for an assessment or monitoring of the ecosystem state or human pressures and impacts. In our study, we revealed a new cryptic species of aphid-hunting wasp that is virtually indistinguishable from a well-known one by morphology. These two species can be easily segregated using molecular characters, and presumably they differ also by nesting habitat. Upon exploring the mitotype diversity of the aphid-hunting wasp, Diodontus tristis, we revealed specimens with highly divergent mitotypes from two localities in Lithuania and nesting in clayey substrate, while the specimens with typical mitotypes were found nesting in sandy sites. The comparison of inter- and intra-specific distances and application of delimitation algorithms supported the species status of the clay-nesting populations. Using a set of DNA markers that included complete or partial sequences of six mitochondrial genes, three markers of ribosomal operon, two homeobox genes, and four other nuclear genes, we clarified the phylogenetic relationships of the new cryptic species. The endosymbiotic bacteria infestation was checked, considering the option that the divergent populations may represent clades isolated by Wolbachia infection; however, it did not demonstrate any specificity. We found only subtle morphological differences in the new clay-nesting species, D. argillicola sp. nov.; the discriminant analysis of morphometric measurements did not reliably segregate it as well. Thus, we provide the molecular characters of the cryptic species, which allow confident identification, its phylogenetic position within the genus, and an updated identification key for the D. tristis species group. [ABSTRACT FROM AUTHOR]

Ticks host different pathogens as endosymbiont and nonpathogenic microorganisms and play an important role in reproductive fitness and nutrient provision. However, the bacterial microbiomes of white-tailed deer ticks have received minimal attention. This study aimed to examine the bacterial microbiome of ticks collected from Odocoileus virginianus on the Mexico-United States border to assess differences in microbiome diversity in ticks of different species, sexes, and localities. Five different tick species were collected: Rhipicephalus microplus, Dermacentor nitens, Otobius megnini, Amblyomma cajennense, and A. maculatum. The tick microbiomes were analyzed using next-generation sequencing. Among all tick species, the most predominant phylum was Proteobacteria, followed by Actinobacteria and Firmicutes. The ticks from Tamaulipas and Nuevo León presented the highest bacterial species diversity. Acinetobacter johnsonii and A. lwoffii were the common bacterial species in the microbiome of all ticks, Coxiella were present in R. microplus, and Dermacentor nitens also exhibited a Francisella-like endosymbiont. The microbiome of most females in D. nitens was less diverse than that of males, whereas R. microplus occurs in females, suggesting that microbiome diversity is influenced by sex. In the bacterial communities of A. maculatum and O. megnini, Candidatus Midichloria massiliensis, and Candidatus Endoecteinascidia fumentensis were the most predominant endosymbionts. These results constitute the initial report on these bacteria, and this is also the first study to characterize the microbiome of O. megnini. [ABSTRACT FROM AUTHOR]

Recent metagenomic investigations of ticks have revealed that their microbiomes have a microbial diversity of endosymbionts and pathogens (pathobiome), which cause diseases in humans and other animals. Ticks are vectors of various diseases in cattle, so a more extensive study will elucidate their microbiome composition to propose new control and prevention strategies, such as anti-tick vaccines. This approach is promising and is based on identifying essential bacteria for the survival of the vector. Therefore, this work presents the metagenomic strategy to ticks for identifying the microbiota and the respective microbiome. [ABSTRACT FROM AUTHOR]

The European spruce bark beetle Ips typographus is a pest causing severe damage in forests dominated by Norway spruce in Europe. Microorganisms play essential roles in bark beetles, including nutrition, fitness as well as in overcoming host defenses. Here, we performed high-throughput 16S rRNA metabarcoding of I. typographus across different populations in Europe, to assess its bacterial community. We investigated four postglacial refugial areas in Europe and focused specifically on a current bark beetle hot spot in the Dolomites where we compared populations with different epidemiological phases (outbreaking vs. non-outbreaking) and across different seasons (pre-overwintering vs. overwintering). Our results show that the bacterial community structure varied among populations from the refugial areas and between different regions within the Dolomites. We found a significant difference in the bacterial community between pre-overwintering and overwintering individuals, but we did not find differences between epidemic and endemic populations. The prevalence of the genera Erwinia (which was present in all individuals) and Pseudoxanthomonas (present in almost all individuals) across all populations suggests that these taxa form the core bacterial community of I. typographus. Furthermore, several additional bacterial taxa occurred in all populations, but with variable frequencies. This study highlights a complex interaction of I. typographus and various bacterial taxa across different regions and ecological phases and provides new insights into the role of microorganisms in the biology of this important pest species. [ABSTRACT FROM AUTHOR]

ABSTRACT Ticks are increasingly important vectors of human and agricultural diseases. While many studies have focused on tick-borne bacteria, far less is known about tick-associated viruses and their roles in public health or tick physiology. To address this, we investigated patterns of bacterial and viral communities across two field populations of western black-legged ticks (Ixodes pacificus). Through metatranscriptomic analysis of 100 individual ticks, we quantified taxon prevalence, abundance, and co-occurrence with other members of the tick microbiome. In addition to commonly found tick-associated microbes, we assembled 11 novel RNA virus genomes from Rhabdoviridae, Chuviridae, Picornaviridae, Phenuiviridae, Reoviridae, Solemovidiae, Narnaviridae and two highly divergent RNA virus genomes lacking sequence similarity to any known viral families. We experimentally verified the presence of these in I. pacificus ticks across several life stages. We also unexpectedly identified numerous virus-like transcripts that are likely encoded by tick genomic DNA, and which are distinct from known endogenous viral element-mediated immunity pathways in invertebrates. Taken together, our work reveals that I. pacificus ticks carry a greater diversity of viruses than previously appreciated, in some cases resulting in evolutionarily acquired virus-like transcripts. Our findings highlight how pervasive and intimate tick–virus interactions are, with major implications for both the fundamental biology and vectorial capacity of I. pacificus ticks.IMPORTANCETicks are increasingly important vectors of disease, particularly in the United States where expanding tick ranges and intrusion into previously wild areas has resulted in increasing human exposure to ticks. Emerging human pathogens have been identified in ticks at an increasing rate, and yet little is known about the full community of microbes circulating in various tick species, a crucial first step to understanding how they interact with each and their tick host, as well as their ability to cause disease in humans. We investigated the bacterial and viral communities of the Western blacklegged tick in California and found 11 previously uncharacterized viruses circulating in this population.

ABSTRACT Bacterial endosymbionts of eukaryotic hosts typically experience massive genome reduction, but the underlying evolutionary processes are often obscured by the lack of free-living relatives. Endomicrobia, a family-level lineage of host-associated bacteria in the phylum Elusimicrobiota that comprises both free-living representatives and endosymbionts of termite gut flagellates, are an excellent model to study evolution of intracellular symbionts. We reconstructed 67 metagenome-assembled genomes (MAGs) of Endomicrobiaceae among more than 1,700 MAGs from the gut microbiota of a wide range of termites. Phylogenomic analysis confirmed a sister position of representatives from termites and ruminants, and allowed to propose eight new genera in the radiation of Endomicrobiaceae. Comparative genome analysis documented progressive genome erosion in the new genus Endomicrobiellum, which comprises all flagellate endosymbionts characterized to date. Massive gene losses were accompanied by the acquisition of new functions by horizontal gene transfer, which led to a shift from a glucose-based energy metabolism to one based on sugar phosphates. The breakdown of glycolysis and many anabolic pathways for amino acids and cofactors in several subgroups was compensated by the independent acquisition of new uptake systems, including an ATP/ADP antiporter, from other gut microbiota. The putative donors are mostly flagellate endosymbionts from other bacterial phyla, including several, hitherto unknown lineages of uncultured Alphaproteobacteria, documenting the importance of horizontal gene transfer in the convergent evolution of these intracellular symbioses. The loss of almost all biosynthetic capacities in some lineages of Endomicrobiellum suggests that their originally mutualistic relationship with flagellates is on its decline.IMPORTANCEUnicellular eukaryotes are frequently colonized by bacterial and archaeal symbionts. A prominent example are the cellulolytic gut flagellates of termites, which harbor diverse but host-specific bacterial symbionts that occur exclusively in termite guts. One of these lineages, the so-called Endomicrobia, comprises both free-living and endosymbiotic representatives, which offers the unique opportunity to study the evolutionary processes underpinning the transition from a free-living to an intracellular lifestyle. Our results revealed a progressive gene loss in energy metabolism and biosynthetic pathways, compensated by the acquisition of new functions via horizontal gene transfer from other gut bacteria, and suggest the eventual breakdown of an initially mutualistic symbiosis. Evidence for convergent evolution of unrelated endosymbionts reflects adaptations to the intracellular environment of termite gut flagellates.

ABSTRACT Insects frequently form heritable associations with beneficial bacteria that are vertically transmitted from parent to offspring. Long-term vertical transmission has repeatedly resulted in genome reduction and gene loss, rendering many such bacteria incapable of establishment in axenic culture. Among aphids, heritable endosymbionts often provide context-specific benefits to their hosts. Although these associations have large impacts on host phenotypes, experimental approaches are often limited by an inability to cultivate these microbes. Here, we report the axenic culture of Candidatus Fukatsuia symbiotica strain WIR, a heritable bacterial endosymbiont of the pea aphid, Acyrthosiphon pisum. Whole-genome sequencing revealed similar genomic features and high sequence similarity to previously described strains, suggesting that the cultivation techniques used here may be applicable to Ca. F. symbiotica strains from distantly related aphids. Microinjection of cultured Ca. F. symbiotica into uninfected aphids revealed that it can reinfect developing embryos and that infections are maintained in subsequent generations via transovarial maternal transmission. Artificially infected aphids exhibit phenotypic and life history traits similar to those observed for native infections. Our results show that Ca. F. symbiotica may be a useful tool for experimentally probing the molecular mechanisms underlying host-symbiont interactions in a heritable symbiosis.IMPORTANCEDiverse eukaryotic organisms form stable, symbiotic relationships with bacteria that provide benefits to their hosts. While these associations are often biologically important, they can be difficult to probe experimentally because intimately host-associated bacteria are difficult to access within host tissues, and most cannot be cultured. This is especially true for the intracellular, maternally inherited bacteria associated with many insects, including aphids. Here, we demonstrate that a pea aphid-associated strain of the heritable endosymbiont, Candidatus Fukatsuia symbiotica, can be grown outside of its host using standard microbiology techniques and can readily re-establish infection that is maintained across host generations. These artificial infections recapitulate the effects of native infections, making this host-symbiont pair a useful experimental system.

Novel bacterial isolates with the capabilities of lignin depolymerization, catabolism, or both, could be pertinent to lignocellulosic biofuel applications. In this study, we aimed to identify anaerobic bacteria that could address the economic challenges faced with microbial-mediated biotechnologies, such as the need for aeration and mixing. Using a consortium seeded from temperate forest soil and enriched under anoxic conditions with organosolv lignin as the sole carbon source, we successfully isolated a novel bacterium, designated 159R. Based on the 16S rRNA gene, the isolate belongs to the genus Sodalis in the family Bruguierivoracaceae. Whole-genome sequencing revealed a genome size of 6.38 Mbp and a GC content of 55 mol%. To resolve the phylogenetic position of 159R, its phylogeny was reconstructed using (i) 16S rRNA genes of its closest relatives, (ii) multilocus sequence analysis (MLSA) of 100 genes, (iii) 49 clusters of orthologous groups (COG) domains, and (iv) 400 conserved proteins. Isolate 159R was closely related to the deadwood associated Sodalis guild rather than the tsetse fly and other insect endosymbiont guilds. Estimated genome-sequence-based digital DNA-DNA hybridization (dDDH), genome percentage of conserved proteins (POCP), and an alignment analysis between 159R and the Sodalis clade species further supported that isolate 159R was part of the Sodalis genus and a strain of Sodalis ligni. We proposed the name Sodalis ligni str. 159R (=DSM 110549 = ATCC TSD-177). IMPORTANCE Currently, in the paper industry, paper mill pulping relies on unsustainable and costly processes to remove lignin from lignocellulosic material. A greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. Anaerobic bacteria are a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable by-products. We presented Sodalis ligni str. 159R and its characteristics as another example of potential mechanisms that can be developed for lignocellulosic applications.

Bacterial endosymbionts, in genera Wolbachia and Cardinium, infect various arthropods and some nematode groups. Manipulating these microbial symbionts presents a promising biocontrol strategy for managing disease-causing parasites. However, the diversity of Wolbachia and Cardinium in nematodes remains unclear. This study employed a genome skimming strategy to uncover their occurrence in plant-parasitic nematodes, analyzing 52 populations of 12 species. A metagenome analysis revealed varying endosymbiont genome content, leading to the categorization of strong, weak, and no evidence for endosymbiont genomes. Strong evidence for Wolbachia was found in five populations, and for Cardinium in one population, suggesting a limited occurrence. Strong Wolbachia evidence was noted in Pratylenchus penetrans and Radopholus similis from North/South America and Africa. Heterodera glycines from North America showed strong Cardinium evidence. Weak genomic evidence for Wolbachia was observed in Globodera pallida, Meloidogyne incognita, Rotylenchus reniformis, Pratylechus coffeae, Pratylenchus neglectus, and Pratylenchus thornei; for Cardinium was found in G. pallida, R. reniformis and P. neglectus; 27/52 populations exhibited no endosymbiont evidence. Wolbachia and Cardinium presence varied within nematode species, suggesting non-obligate mutualism. Wolbachia and Cardinium genomes differed among nematode species, indicating potential species-specific functionality. This study advances knowledge of plant-parasitic nematode–bacteria symbiosis, providing insights for downstream eco-friendly biocontrol strategies.

The degree of similarity between the microbiotas of host species often mirrors the phylogenetic proximity of the hosts. This pattern, referred to as phylosymbiosis, is widespread in animals and plants. While phylosymbiosis was initially interpreted as the signal of symbiotic transmission and coevolution between microbes and their hosts, it is now recognized that similar patterns can emerge even if the microbes are environmentally acquired. Distinguishing between these two scenarios, however, remains challenging. We recently developed HOME (host-microbiota evolution), a cophylogenetic model designed to detect vertically transmitted microbes and host switches from amplicon sequencing data. Here, we applied HOME to the microbiotas of Hawaiian spiders of the genus Ariamnes, which experienced a recent radiation on the archipelago. We demonstrate that although Hawaiian Ariamnes spiders display a significant phylosymbiosis, there is little evidence of microbial vertical transmission. Next, we performed simulations to validate the absence of transmitted microbes in Ariamnes spiders. We show that this is not due to a lack of detection power because of the low number of segregating sites or an effect of phylogenetically driven or geographically driven host switches. Ariamnes spiders and their associated microbes therefore provide an example of a pattern of phylosymbiosis likely emerging from processes other than vertical transmission. IMPORTANCE How host-associated microbiotas assemble and evolve is one of the outstanding questions of microbial ecology. Studies aiming at answering this question have repeatedly found a pattern of "phylosymbiosis," that is, a phylogenetic signal in the composition of host-associated microbiotas. While phylosymbiosis was often interpreted as evidence for vertical transmission and host-microbiota coevolution, simulations have now shown that it can emerge from other processes, including host filtering of environmentally acquired microbes. However, distinguishing the processes driving phylosymbiosis in nature remains challenging. We recently developed a cophylogenetic method that can detect vertical transmission. Here, we applied this method to the microbiotas of recently diverged spiders from the Hawaiian archipelago, which display a clear phylosymbiosis pattern. We found that none of the bacterial operational taxonomic units is vertically transmitted. We show with simulations that this result is not due to methodological artifacts. Thus, we provide a striking empirical example of phylosymbiosis emerging from processes other than vertical transmission.

In recent decades, research on developing and deploying resistant rice has accelerated due to the availability of modern molecular tools and, in particular, advances in marker-assisted selection. However, progress in understanding virulence adaptation has been relatively slow. This review tracks patterns in virulence adaptation to resistance genes (particularly Bph1, bph2, Bph3, and bph4) and examines the nature of virulence based on selection experiments, responses by virulent populations to differential rice varieties (i.e., varieties with different resistance genes), and breeding experiments that interpret the genetic mechanisms underlying adaptation. The review proposes that varietal resistance is best regarded as a combination of minor and major resistance traits against which planthoppers develop partial or complete virulence through heritable improvements that are reversable or through evolutionary adaptation, respectively. Agronomic practices, deployment patterns, and herbivore population pressures determine the rates of adaptation, and there is growing evidence that pesticide detoxification mechanisms can accelerate virulence adaptation. Research to delay adaptation has mainly focused on gene pyramiding (i.e., including ≥ two major genes in a variety) and multilines (i.e., including ≥ two resistant varieties in a field or landscape); however, these strategies have not been adequately tested and, if not managed properly, could inadvertently accelerate adaptation compared to sequential deployment. Several research gaps remain and considerable improvements in research methods are required to better understand and manage virulence adaptation.

The adaptation by planthoppers to feed and develop on resistant rice is a challenge for pest management in Asia. We conducted a series of manipulative experiments with the brown planthopper (Nilaparvata lugens (Stål)) on the resistant rice variety IR62 (BPH3/BPH32 genes) to assess behavioral and bionomic changes in planthoppers exhibiting virulence adaptation. We also examined the potential role of yeast-like symbionts (YLS) in virulence adaptation by assessing progeny fitness (survival × reproduction) following controlled matings between virulent males or females and avirulent males or females, and by manipulating YLS densities in progeny through heat treatment. We found virulence-adapted planthoppers developed faster, grew larger, had adults that survived for longer, had female-biased progeny, and produced more eggs than non-selected planthoppers on the resistant variety. However, feeding capacity-as revealed through honeydew composition-remained inefficient on IR62, even after 20+ generations of exposure to the resistant host. Virulence was derived from both the male and female parents; however, females contributed more than males to progeny virulence. We found that YLS are essential for normal planthopper development and densities are highest in virulent nymphs feeding on the resistant host; however, we found only weak evidence that YLS densities contributed more to virulence. Virulence against IR62 in the brown planthopper, therefore, involves a complex of traits that encompass a series of behavioral, physiological, and genetic mechanisms, some of which are determined only by the female parent.

Abstract Endosymbionts are known to have significant effects on their insect hosts, including nutrition, reproduction, and immunity. Insects gut microbiota is a critical component that affects their physiological and behavioral characteristics. The black cutworm (BCW), Agrotis ipsilon, is an economically important lepidopteran pest that has a diverse gut microbiome composed of nine species belonging to three phyla: Proteobacteria, Actinobacteria, and Firmicutes. This study was conducted to investigate the diversity of gut bacteria isolated from BCW larvae and moths and their effects on metabolism and pesticide degradation. The bacterial isolates were identified using the 16 S rRNA gene. The study showed that the gut microbiome composition significantly affected the metabolism of BCW larvae. Based on the screening results of synthesis of digestive enzymes and pesticide degradation, Brachybacterium conglomeratum and Glutamicibacter sp were selected to perform the remaining experiments as single isolates and consortium. The consortium-fed larvae showed high metabolic indices compared to antibiotic-fed larvae and the control. The gut bacteria were also shown to degrade three pesticide groups. Concerns regarding the health risk of chlorpyrifos have been raised due to its extensive use in agriculture. The isolated B. conglomeratum was more effective in chlorpyrifos degradation than the consortium. Furthermore, the study also examined the presence of sex related endosymbionts (Wolbachia, Spiroplasma, and Rickettsia) in the reproductive tissues of adults. The outcomes demonstrated that none of the examined endosymbionts existed. In conclusion, the study highlights the importance of the gut microbiome in insect physiology and behavior and its potential applications in biotechnology. It provides insights into developing eco-friendly pest control and bioremediation strategies using gut bacteria.

Water hyacinth, Pontederia crassipes, is a highly invasive plant native to South America and one of the most invasive aquatic plants in the world. For its control, the planthopper Megamelus scutellaris Berg (Hemiptera: Delphacidae), a phloem feeder also native to South America has been introduced to the USA and South Africa. Considering predicted climate change scenarios, understanding their impacts on biological control agents is crucial. An intriguing yet scarcely explored subject, is the effect of climatic changes on the obligate endosymbionts associated with sap-sucking feeders. Planthoppers establish an obligate relationship with yeast-like symbiotes (YLS), unicellular fungal microorganisms that play an important role in their development, providing missing nutrients in their diet. Considering that increased atmospheric CO2 affects plant chemical composition, this might have a direct impact on their insect host and on their number of YLS. We evaluated the effect of two different CO2 environments: current (cCO2 − 400 ppm) and elevated (eCO2 − 800 ppm) on the abundance of YLS (number of YLS cells/insect) of M. scutellaris, as well as the, age structure, sex, and weight of insects. Heavier females harbored more YLS under eCO2 which underscores the importance of the interaction of CO2 levels and insects’ weight in shaping the abundance of YLS. Additionally, there was a significant increase in the total abundance of insects for instars III to V and adults under eCO2 conditions. However, male number significantly exceeded that of females under both CO2 conditions. Our results suggest a potential positive impact of eCO2 on M. scutellaris populations, which could, in turn, enhance the control of P. crassipes.