"Candidatus Intestinibacterium parameciiphilum"-member of the "Candidatus Paracaedibacteraceae" family (Alphaproteobacteria, Holosporales) inhabiting the ciliated protist Paramecium.

Protists frequently host diverse bacterial symbionts, in particular those affiliated with the order Holosporales (Alphaproteobacteria). All characterised members of this bacterial lineage have been retrieved in obligate association with a wide range of eukaryotes, especially multiple protist lineages (e.g. amoebozoans, ciliates, cercozoans, euglenids, and nucleariids), as well as some metazoans (especially arthropods and related ecdysozoans). While the genus Paramecium and other ciliates have been deeply investigated for the presence of symbionts, known members of the family "Candidatus Paracaedibacteraceae" (Holosporales) are currently underrepresented in such hosts. Herein, we report the description of "Candidatus Intestinibacterium parameciiphilum" within the family "Candidatus Paracaedibacteraceae", inhabiting the cytoplasm of Paramecium biaurelia. This novel bacterium is almost twice as big as its relative "Candidatus Intestinibacterium nucleariae" from the opisthokont Nuclearia and does not present a surrounding halo. Based on phylogenetic analyses of 16S rRNA gene sequences, we identified six further potential species-level lineages within the genus. Based on the provenance of the respective samples, we investigated the environmental distribution of the representatives of "Candidatus Intestinibacterium" species. Obtained results are consistent with an obligate endosymbiotic lifestyle, with protists, in particular freshwater ones, as hosts. Thus, available data suggest that association with freshwater protists could be the ancestral condition for the members of the "Candidatus Intestinibacterium" genus.

Bacterial communities associated with mushrooms in the Qinghai-Tibet Plateau are shaped by soil parameters.

Fungi capable of producing fruit bodies are essential food and medicine resources. Despite recent advances in the study of microbial communities in mycorrhizospheres, little is known about the bacterial communities contained in fruit bodies. Using high-throughput sequencing, we investigated the bacterial communities in four species of mushrooms located on the alpine meadow and saline-alkali soil of the Qinghai-Tibet Plateau (QTP). Proteobacteria (51.7% on average) and Actinobacteria (28.2% on average) were the dominant phyla in all of the sampled fairy ring fruit bodies, and Acidobacteria (27.5% on average) and Proteobacteria (25.7% on average) dominated their adjacent soils. For the Agria. Bitorquis, Actinobacteria was the dominant phylum in its fruit body (67.5% on average) and adjacent soils (65.9% on average). The alpha diversity (i.e., Chao1, Shannon, Richness, and Simpson indexes) of the bacterial communities in the fruit bodies were significantly lower than those in the soil samples. All of the fungi shared more than half of their bacterial phyla and 16.2% of their total operational taxonomic units (OTUs) with their adjacent soil. Moreover, NH4+ and pH were the key factors associated with bacterial communities in the fruit bodies and soils, respectively. These results indicate that the fungi tend to create a unique niche that selects for specific members of the bacterial community. Using culture-dependent methods, we also isolated 27 bacterial species belonging to three phyla and five classes from fruit bodies and soils. The strains isolated will be useful for future research on interactions between mushroom-forming fungi and their bacterial endosymbionts.

Bacterial communities in the gut of wild and mass-reared Zeugodacus cucurbitae and Bactrocera dorsalis revealed by metagenomic sequencing.

BACKGROUND: Insect pests belonging to genus Bactrocera sp. (Diptera: Tephritidae) pose major biotic stress on various fruits and vegetable crops around the world. Zeugodacus and Bactrocera sp. are associated with diverse bacterial communities which play an important role in the fitness of sterile insects. The wild populations of melon fly, Zeugodacus cucurbitae (Coquillett) and Oriental fruit fly, Bactrocera dorsalis (Hendel) were collected from pumpkin and mango fields, respectively. The laboratory populations of Z. cucurbitae and B. dorsalis were mass-reared on bottle gourd and sweet banana, respectively. Bacterial communities present in the gut of wild and mass-reared mature (~ 12 days old) and newly emerged (< 1 h after emergence) male and female adults of Z. cucurbitae and B. dorsalis were assessed. We used Illumina HiSeq next-generation sequencing of 16S rRNA gene to profile the gut bacterial communities of wild and mass-reared mature and newly emerged Z. cucurbitae and B. dorsalis adults. RESULTS: We found diverse bacterial composition in the gut of wild and mass-reared Z. cucurbitae (ZC) and B. dorsalis (BD) with varied relative abundance. Few taxonomic groups were common to both the species. The most dominant phyla in all samples of Z. cucurbitae and B. dorsalis adults were Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The phylum Proteobacteria occurred more in wild Z. cucurbitae (~ 87.72%) and B. dorsalis (~ 83.87%) as compared to mass-reared Z. cucurbitae (64.15%) and B. dorsalis (~ 80.96%). Higher relative abundance of Phylum Firmicutes was observed in mass-reared fruit fly than wild adults. Cyanobacteria/Chloroplast and Actinobacteria were also present with very low relative abundance in both wild as well as mass-reared melon fly and Oriental fruit fly. Enterobacteriaceae (61.21%) was dominant family in the gut of both wild and mass-reared adults. Providencia and Lactococcus were dominant genera with varied relative abundance in wild as well as in mass-reared mature and newly emerged fruit fly adults of both species. Some of the genera like Morganella and Serratia were only detected in mass-reared mature and newly emerged Z. cucurbitae and B. dorsalis adults. Principal Coordinate Analysis (PCoA) showed that fruit fly adult samples were grouped based on species and age of the adults while no grouping was observed on the basis of sex of the adult fruit fly. CONCLUSIONS: The gut bacterial communities associated with wild and mass-reared mature and newly emerged adults of Z. cucurbitae and B. dorsalis showed variation that depends on species and age of the insects. Understanding the gut microbiota of wild and mass-reared Z. cucurbitae and B. dorsalis using high throughput technology will help to illustrate microbial diversity and this information could be used to develop efficient mass-rearing protocols for successful implementation of sterile insect technique (SIT).

A new Cardinium group of bacteria found in Achipteria coleoptrata (Acari: Oribatida).

The understanding of the biology of arthropods requires an understanding of their bacterial associates. We determined the distribution of bacteria Wolbachia sp., Rickettsia sp., Cardinium sp., Spiroplasma sp., Arsenophonus sp., Hamiltonella sp., and Flavobacterium in oribatid mites (Acari: Oribatida). We identified Cardinium sp. in Achipteria coleoptrata. This is the first report of this bacterium in A. coleoptrata. Approximately 30% of the mite population was infected by Cardinium sp. The Cardinium 16S rDNA was examined for the presence of two sequences unique for this microorganism. One of them was noted in Cardinium sp. of A. coleoptrata. In the second sequence, we found nucleotide substitution in the 7th position: A instead of T. In our opinion, this demonstrated the unique nature of Cardinium sp. of A. coleoptrata. We also determined phylogenetic relationship between Cardinium sp., including the strain found in A. coleoptrata by studying the 16S rRNA and gyrB gene sequences. It revealed that Cardinium from A. coleoptrata did not cluster together with strains from groups A, B, C or D, and constituted a separate clade E. These observations make A. coleoptrata a unique Cardinium host in terms of the distinction of the strain.

Bacterial endosymbiont Cardinium cSfur genome sequence provides insights for understanding the symbiotic relationship in Sogatella furcifera host.

BACKGROUND: Sogatella furcifera is a migratory pest that damages rice plants and causes severe economic losses. Due to its ability to annually migrate long distances, S. furcifera has emerged as a major pest of rice in several Asian countries. Symbiotic relationships of inherited bacteria with terrestrial arthropods have significant implications. The genus Cardinium is present in many types of arthropods, where it influences some host characteristics. We present a report of a newly identified strain of the bacterial endosymbiont Cardinium cSfur in S. furcifera. RESULT: From the whole genome of S. furcifera previously sequenced by our laboratory, we assembled the whole genome sequence of Cardinium cSfur. The sequence comprised 1,103,593 bp with a GC content of 39.2%. The phylogenetic tree of the Bacteroides phylum to which Cardinium cSfur belongs suggests that Cardinium cSfur is closely related to the other strains (Cardinium cBtQ1 and cEper1) that are members of the Amoebophilaceae family. Genome comparison between the host-dependent endosymbiont including Cardinium cSfur and free-living bacteria revealed that the endosymbiont has a smaller genome size and lower GC content, and has lost some genes related to metabolism because of its special environment, which is similar to the genome pattern observed in other insect symbionts. Cardinium cSfur has limited metabolic capability, which makes it less contributive to metabolic and biosynthetic processes in its host. From our findings, we inferred that, to compensate for its limited metabolic capability, Cardinium cSfur harbors a relatively high proportion of transport proteins, which might act as the hub between it and its host. With its acquisition of the whole operon related to biotin synthesis and glycolysis related genes through HGT event, Cardinium cSfur seems to be undergoing changes while establishing a symbiotic relationship with its host. CONCLUSION: A novel bacterial endosymbiont strain (Cardinium cSfur) has been discovered. A genomic analysis of the endosymbiont in S. furcifera suggests that its genome has undergone certain changes to facilitate its settlement in the host. The envisaged potential reproduction manipulative ability of the new endosymbiont strain in its S. furcifera host has vital implications in designing eco-friendly approaches to combat the insect pest.

Incidence of Facultative Bacterial Endosymbionts in Spider Mites Associated with Local Environments and Host Plants.

Spider mites are frequently associated with multiple endosymbionts whose infection patterns often exhibit spatial and temporal variation. However, the association between endosymbiont prevalence and environmental factors remains unclear. Here, we surveyed endosymbionts in natural populations of the spider mite, Tetranychus truncatus, in China, screening 935 spider mites from 21 localities and 12 host plant species. Three facultative endosymbiont lineages, Wolbachia, Cardinium, and Spiroplasma, were detected at different infection frequencies (52.5%, 26.3%, and 8.6%, respectively). Multiple endosymbiont infections were observed in most local populations, and the incidence of individuals with the Wolbachia-Spiroplasma coinfection was higher than expected from the frequency of each infection within a population. Endosymbiont infection frequencies exhibited associations with environmental factors: Wolbachia infection rates increased at localities with higher annual mean temperatures, while Cardinium and Spiroplasma infection rates increased at localities from higher altitudes. Wolbachia was more common in mites from Lycopersicon esculentum and Glycine max compared to those from Zea mays This study highlights that host-endosymbiont interactions may be associated with environmental factors, including climate and other geographically linked factors, as well as the host's food plant.IMPORTANCE The aim of this study was to examine the incidence of endosymbiont distribution and the infection patterns in spider mites. The main findings are that multiple endosymbiont infections were more common than expected and that endosymbiont infection frequencies were associated with environmental factors. This work highlights that host-endosymbiont interactions need to be studied within an environmental and geographic context.

Cytological analysis of cytoplasmic incompatibility induced by Cardinium suggests convergent evolution with its distant cousin Wolbachia.

Cytoplasmic incompatibility (CI) is a conditional sterility in numerous arthropods that is caused by inherited, intracellular bacteria such as Wolbachia Matings between males carrying CI-inducing Wolbachia and uninfected females, or between males and females infected with different Wolbachia strains, result in progeny that die during very early embryogenesis. Multiple studies in diploid (Drosophila) and haplodiploid (Nasonia) insects have shown that CI-Wolbachia cause a failure of the paternally derived chromatin from resolving into distinct chromosomes. This leads to the formation of chromatin bridges and other mitotic defects as early as the first mitotic division, and to early mitotic arrest. It is currently unknown if CI-inducing symbionts other than Wolbachia affect similar cellular processes. Here, we investigated CI caused by an unrelated bacterium, Cardinium, which naturally infects a parasitic wasp, Encarsia suzannae CI crosses in this host-symbiont system resulted in early mitotic defects including asynchrony of paternal and maternal chromosome sets as they enter mitosis, chromatin bridges and improper chromosome segregation that spanned across multiple mitotic divisions, triggering embryonic death through accumulated aneuploidy. We highlight small differences with CI-Wolbachia, which could be due to the underlying CI mechanism or host-specific effects. Our results suggest a convergence of CI-related cellular phenotypes between these two unrelated symbionts.

Microbiome symbionts and diet diversity incur costs on the immune system of insect larvae.

Communities of symbiotic microorganisms that colonize the gastrointestinal tract play an important role in food digestion and protection against opportunistic microbes. Diet diversity increases the number of symbionts in the intestines, a benefit that is considered to impose no cost for the host organism. However, less is known about the possible immunological investments that hosts have to make in order to control the infections caused by symbiont populations that increase because of diet diversity. Using taxonomical composition analysis of the 16S rRNA V3 region, we show that enterococci are the dominating group of bacteria in the midgut of the larvae of the greater wax moth (Galleria mellonella). We found that the number of colony-forming units of enterococci and expressions of certain immunity-related antimicrobial peptide (AMP) genes such as Gallerimycin, Gloverin, 6-tox, Cecropin-D and Galiomicin increased in response to a more diverse diet, which in turn decreased the encapsulation response of the larvae. Treatment with antibiotics significantly lowered the expression of all AMP genes. Diet and antibiotic treatment interaction did not affect the expression of Gloverin and Galiomicin AMP genes, but significantly influenced the expression of Gallerimycin, 6-tox and Cecropin-D Taken together, our results suggest that diet diversity influences microbiome diversity and AMP gene expression, ultimately affecting an organism's capacity to mount an immune response. Elevated basal levels of immunity-related genes (Gloverin and Galiomicin) might act as a prophylactic against opportunistic infections and as a mechanism that controls the gut symbionts. This would indicate that a diverse diet imposes higher immunity costs on organisms.

Are aphid parasitoids locally adapted to the prevalence of defensive symbionts in their hosts?

BACKGROUND: Insect parasitoids are under strong selection to overcome their hosts' defences. In aphids, resistance to parasitoids is largely determined by the presence or absence of protective endosymbionts such as Hamiltonella defensa. Hence, parasitoids may become locally adapted to the prevalence of this endosymbiont in their host populations. To address this, we collected isofemale lines of the aphid parasitoid Lysiphlebus fabarum from 17 sites in Switzerland and France, at which we also estimated the frequency of infection with H. defensa as well as other bacterial endosymbionts in five important aphid host species. The parasitoids' ability to overcome H. defensa-mediated resistance was then quantified by estimating their parasitism success on a single aphid clone (Aphis fabae fabae) that was either uninfected or experimentally infected with one of three different isolates of H. defensa. RESULTS: The five aphid species (Aphis fabae fabae, A. f. cirsiiacanthoides, A. hederae, A. ruborum, A. urticata) differed strongly in the relative frequencies of infection with different bacterial endosymbionts, but there was also geographic variation in symbiont prevalence. Specifically, the frequency of infection with H. defensa ranged from 22 to 47 % when averaged across species. Parasitoids from sites with a high prevalence of H. defensa tended to be more infective on aphids possessing H. defensa, but this relationship was not significant, thus providing no conclusive evidence that L. fabarum is locally adapted to the occurrence of H. defensa. On the other hand, we observed a strong interaction between parasitoid line and H. defensa isolate on parasitism success, indicative of a high specificity of symbiont-conferred resistance. CONCLUSIONS: This study is the first, to our knowledge, to test for local adaptation of parasitoids to the frequency of defensive symbionts in their hosts. While it yielded useful information on the occurrence of facultative endosymbionts in several important host species of L. fabarum, it provided no clear evidence that parasitoids from sites with a high prevalence of H. defensa are better able to overcome H. defensa-conferred resistance. The strong genetic specificity in their interaction suggests that it may be more important for parasitoids to adapt to the particular strains of H. defensa in their host populations than to the general prevalence of this symbiont, and it highlights the important role symbionts can play in mediating host-parasitoid coevolution.

Surveys for maternally-inherited endosymbionts reveal novel and variable infections within solitary bee species.

Maternally-inherited bacteria can affect the fitness and population dynamics of their host insects; for solitary bees, such effects have the potential to influence bee efficacy as pollinators. We screened bee species for bacterial associates using 454-pyrosequencing (4 species) and diagnostic PCR (183 specimens across 29 species). The endosymbiont Wolbachia was abundant, infecting 18 species, including all specimens from the family Halictidae. Among commercially-supplied orchard bees (family Megachilidae), only 2/7 species were Wolbachia-infected, but one species showed variable infection among specimens. Two other maternally-inherited bacteria, Arsenophonus and Sodalis, were also detected, neither of which was fixed in infection frequency. Differential endosymbiont infection could potentially compromise fitness and reproductive compatibility among commercially redistributed pollinator populations.

The Susceptibility of Bemisia tabaci Mediterranean (MED) Species to Attack by a Parasitoid Wasp Changes between Two Whitefly Strains with Different Facultative Endosymbiotic Bacteria.

In this study, two strains of the mitochondrial lineage Q1 of Bemisia tabaci MED species, characterized by a different complement of facultative bacterial endosymbionts, were tested for their susceptibility to be attacked by the parasitoid wasp Eretmocerus mundus, a widespread natural enemy of B. tabaci. Notably, the BtHC strain infected with Hamiltonella and Cardinium was more resistant to parasitization than the BtHR strain infected with Hamiltonella and Rickettsia. The resistant phenotype consisted of fewer nymphs successfully parasitized (containing the parasitoid mature larva or pupa) and in a lower percentage of adult wasps emerging from parasitized nymphs. Interestingly, the resistance traits were not evident when E. mundus parasitism was compared between BtHC and BtHR using parasitoids originating from a colony maintained on BtHC. However, when we moved the parasitoid colony on BtHR and tested E. mundus after it was reared on BtHR for four and seven generations, we saw then that BtHC was less susceptible to parasitization than BtHR. On the other hand, we did not detect any difference in the parasitization of the BtHR strain between the three generations of E. mundus tested. Our findings showed that host strain is a factor affecting the ability of E. mundus to parasitize B. tabaci and lay the basis for further studies aimed at disentangling the role of the facultative endosymbiont Cardinium and of the genetic background in the resistance of B. tabaci MED to parasitoid attack. Furthermore, they highlight that counteradaptations to the variation of B. tabaci defence mechanisms may be rapidly selected in E. mundus to maximize the parasitoid fitness.

Endosymbiont Capture, a Repeated Process of Endosymbiont Transfer with Replacement in Trypanosomatids Angomonas spp.

Trypanosomatids of the subfamily Strigomonadinae bear permanent intracellular bacterial symbionts acquired by the common ancestor of these flagellates. However, the cospeciation pattern inherent to such relationships was revealed to be broken upon the description of Angomonas ambiguus, which is sister to A. desouzai, but bears an endosymbiont genetically close to that of A. deanei. Based on phylogenetic inferences, it was proposed that the bacterium from A. deanei had been horizontally transferred to A. ambiguus. Here, we sequenced the bacterial genomes from two A. ambiguus isolates, including a new one from Papua New Guinea, and compared them with the published genome of the A. deanei endosymbiont, revealing differences below the interspecific level. Our phylogenetic analyses confirmed that the endosymbionts of A. ambiguus were obtained from A. deanei and, in addition, demonstrated that this occurred more than once. We propose that coinfection of the same blowfly host and the phylogenetic relatedness of the trypanosomatids facilitate such transitions, whereas the drastic difference in the occurrence of the two trypanosomatid species determines the observed direction of this process. This phenomenon is analogous to organelle (mitochondrion/plastid) capture described in multicellular organisms and, thereafter, we name it endosymbiont capture.

Degradation of chlorpyriphos and polyethylene by endosymbiotic bacteria from citrus mealybug.

Chlorpyriphos is one of the major organophosphorus pesticides used widely to control a range of insect pests across several crops. This insecticide is hazardous to the environment and toxic to mammals, thus, it is essential to remove the same from the environment. Similarly, use of polythene is also increasing day by day. Therefore, it is highly important to identify ways to degrade chlorpyriphos and other pesticides from the environment. We studied the degradation of chlorpyriphos and polyethylene by Citrus mealybug (Planococcus citri) bacterial endosymbionts such as Bacillus licheniformis, Pseudomonas cereus, Pseudomonas putida and Bacillus subtilis. This investigation revealed that bacterial endosymbionts use the polythene as a source of carbon and solubilize them by their enzymatic machinery. The degradation of polyethylene by endosymbionts showed a significant reduction in weight of polyethylene sheet after 15, 30 and 45 days of treatment. The SEM images showed localized degradation of the polyethylene around the bacterial cells in the biofilm. Further, the tensile strength (percentage elongation) was significantly reduced after 45 days of incubation. The weight of paraffin wax showed significant reduction in B. cereus. A significant reduction in total amount of chlorpyriphos in soil was observed at an interval of 7, 14 and 21 days after treatment by the bacterial isolates. Among the bacteria, B. cereus and P. putida were found to be most effective. The results from this study show that endosymbionts can be significantly implicated in degrading chlorpyriphos and polyethylene from the environment.

Genome Analysis of a Verrucomicrobial Endosymbiont With a Tiny Genome Discovered in an Antarctic Lake.

Organic Lake in Antarctica is a marine-derived, cold (-13∘C), stratified (oxic-anoxic), hypersaline (>200 gl-1) system with unusual chemistry (very high levels of dimethylsulfide) that supports the growth of phylogenetically and metabolically diverse microorganisms. Symbionts are not well characterized in Antarctica. However, unicellular eukaryotes are often present in Antarctic lakes and theoretically could harbor endosymbionts. Here, we describe Candidatus Organicella extenuata, a member of the Verrucomicrobia with a highly reduced genome, recovered as a metagenome-assembled genome with genetic code 4 (UGA-to-Trp recoding) from Organic Lake. It is closely related to Candidatus Pinguicocccus supinus (163,218 bp, 205 genes), a newly described cytoplasmic endosymbiont of the freshwater ciliate Euplotes vanleeuwenhoeki (Serra et al., 2020). At 158,228 bp (encoding 194 genes), the genome of Ca. Organicella extenuata is among the smallest known bacterial genomes and similar to the genome of Ca. Pinguicoccus supinus (163,218 bp, 205 genes). Ca. Organicella extenuata retains a capacity for replication, transcription, translation, and protein-folding while lacking any capacity for the biosynthesis of amino acids or vitamins. Notably, the endosymbiont retains a capacity for fatty acid synthesis (type II) and iron-sulfur (Fe-S) cluster assembly. Metagenomic analysis of 150 new metagenomes from Organic Lake and more than 70 other Antarctic aquatic locations revealed a strong correlation in abundance between Ca. Organicella extenuata and a novel ciliate of the genus Euplotes. Like Ca. Pinguicoccus supinus, we infer that Ca. Organicella extenuata is an endosymbiont of Euplotes and hypothesize that both Ca. Organicella extenuata and Ca. Pinguicocccus supinus provide fatty acids and Fe-S clusters to their Euplotes host as the foundation of a mutualistic symbiosis. The discovery of Ca. Organicella extenuata as possessing genetic code 4 illustrates that in addition to identifying endosymbionts by sequencing known symbiotic communities and searching metagenome data using reference endosymbiont genomes, the potential exists to identify novel endosymbionts by searching for unusual coding parameters.

Arms race in a cell: genomic, transcriptomic, and proteomic insights into intracellular phage-bacteria interplay in deep-sea snail holobionts.

BACKGROUND: Deep-sea animals in hydrothermal vents often form endosymbioses with chemosynthetic bacteria. Endosymbionts serve essential biochemical and ecological functions, but the prokaryotic viruses (phages) that determine their fate are unknown. RESULTS: We conducted metagenomic analysis of a deep-sea vent snail. We assembled four genome bins for Caudovirales phages that had developed dual endosymbiosis with sulphur-oxidising bacteria (SOB) and methane-oxidising bacteria (MOB). Clustered regularly interspaced short palindromic repeat (CRISPR) spacer mapping, genome comparison, and transcriptomic profiling revealed that phages Bin1, Bin2, and Bin4 infected SOB and MOB. The observation of prophages in the snail endosymbionts and expression of the phage integrase gene suggested the presence of lysogenic infection, and the expression of phage structural protein and lysozyme genes indicated active lytic infection. Furthermore, SOB and MOB appear to employ adaptive CRISPR-Cas systems to target phage DNA. Additional expressed defence systems, such as innate restriction-modification systems and dormancy-inducing toxin-antitoxin systems, may co-function and form multiple lines for anti-viral defence. To counter host defence, phages Bin1, Bin2, and Bin3 appear to have evolved anti-restriction mechanisms and expressed methyltransferase genes that potentially counterbalance host restriction activity. In addition, the high-level expression of the auxiliary metabolic genes narGH, which encode nitrate reductase subunits, may promote ATP production, thereby benefiting phage DNA packaging for replication. CONCLUSIONS: This study provides new insights into phage-bacteria interplay in intracellular environments of a deep-sea vent snail. Video Abstract.

New Data on the Range Expansion of the Thaumetopoea pityocampa (Lepidoptera: Notodontidae) 'ENA clade' in Greece: The Role of Bacterial Endosymbionts.

The pine processionary moth, Thaumetopoea pityocampa (Denis and Schiffermüller), is an important insect in the Mediterranean region, as it defoliates pines and its urticating hairs can cause allergic reactions in humans and animals. Moreover, this species exhibits an interesting genetic structure as recently a distinct East-North African mtDNA lineage ('ENA clade') has been described. This clade has been recently detected in Greek populations where it has currently expanded its range by replacing the 'endemic' T. pityocampa lineages. Here, we report new data on the rapid spread of 'ENA clade' in the Greek island Evoia in only a few years. As the underlying mechanisms of the 'ENA clade' range expansion has not been studied so far, we screened T. pityocampa for an infection with the heritable bacterial endosymbionts Wolbachia (Bacteria: Anaplasmataceae), Cardinium (Bacteria: Bacteroidaceae), Rickettsia (Bacteria: Rickettsiaceae) and Spiroplasma (Bacteria: Spiroplasmataceae). These bacteria can manipulate the reproduction of infected hosts, something that could potentially explain the rapid spread of 'ENA clade' lineage. Therefore, we screened 28 individuals that exhibited T. pityocampa 'ENA clade' and 'endemic' T. pityocampa haplotypes from nine populations scattered all over Greece. None of them was infected with any of the four endosymbionts, suggesting that these bacteria do not cause reproductive manipulations in T. pityocampa lineages and, thus, other factors should be explored in future research efforts.

Chaperonin overproduction and metabolic erosion caused by mutation accumulation in Escherichia coli.

Bacterial cells adapting to a constant environment tend to accumulate mutations in portions of their genome that are not maintained by selection. This process has been observed in bacteria evolving under strong genetic drift, and especially in bacterial endosymbionts of insects. Here, we study this process in hypermutable Escherichia coli populations evolved through 250 single-cell bottlenecks on solid rich medium in a mutation accumulation experiment that emulates the evolution of bacterial endosymbionts. Using phenotype microarrays monitoring metabolic activity in 95 environments distinguished by their carbon sources, we observe how mutation accumulation has decreased the ability of cells to metabolize most carbon sources. We study if the chaperonin GroEL, which is naturally overproduced in bacterial endosymbionts, can ameliorate the process of metabolic erosion, because of its known ability to buffer destabilizing mutations in metabolic enzymes. Our results indicate that GroEL can slow down the negative phenotypic consequences of genome decay in some environments.

Ecological factors influencing the beneficial endosymbionts of the hemlock woolly adelgid (Hemiptera: Adelgidae).

Bacterial endosymbionts of sap-sucking insects provide their host with a number of beneficial qualities, including the supply of nutrition, defense against parasitoids, and protection from heat stress. Damage to these bacterial associates can therefore have a negative impact on the fitness of their insect host. We evaluated observational and experimental factors regarding the nonnative hemlock woolly adelgid (Adelges tsugae Annand) (Hemiptera: Adelgidae) to help understand the roles of its three recently identified symbionts, including under heat stress conditions. The prevalence of A. tsugae's facultative symbiont (Serratia symbiotica) was examined at different spatial scales to determine how variable infection rates are for this symbiont. There was no significant difference found in infection rates between adelgids on a tree, within a plot, or within a state. However, significantly more adelgids in Georgia (95%) had S. symbiotica compared to those in New York (68%). Microsatellite genotyping of the adelgids found that this difference was most likely not the result of a second introduction of A. tsugae into eastern North America. Comparison of S. symbiotica proportions between first and fourth instars showed that symbiont absence did not affect the ability of A. tsugae to survive aestivation. Evaluations of symbiont densities within each adelgid found that when S. symbiotica was absent, the density of obligate symbionts was significantly higher. Exposure to heat stress (32.5 °C) was not consistently correlated with changes in symbiont densities over a 4-d period. Overall, we have shown that symbiont prevalence and densities vary within the broad population of A. tsugae in eastern North America, with potentially significant effects upon the ecology of this important pest.

Variable Abundance and Distribution of Wolbachia and Cardinium Endosymbionts in Plant-Parasitic Nematode Field Populations.

The bacterial endosymbiont Wolbachia interacts with different invertebrate hosts, engaging in diverse symbiotic relationships. Wolbachia is often a reproductive parasite in arthropods, but an obligate mutualist in filarial nematodes. Wolbachia was recently discovered in plant-parasitic nematodes, and, is thus far known in just two genera Pratylenchus and Radopholus, yet the symbiont's function remains unknown. The occurrence of Wolbachia in these economically important plant pests offers an unexplored biocontrol strategy. However, development of Wolbachia-based biocontrol requires an improved understanding of symbiont-host functional interactions and the symbiont's prevalence among nematode field populations. This study used a molecular-genetic approach to assess the prevalence of a Wolbachia lineage (wPpe) in 32 field populations of Pratylenchus penetrans. Populations were examined from eight different plant species in Washington, Oregon, and California. Nematodes were also screened for the endosymbiotic bacterium Cardinium (cPpe) that was recently shown to co-infect P. penetrans. Results identified wPpe in 9/32 and cPpe in 1/32 of P. penetrans field populations analyzed. No co-infection was observed in field populations. Wolbachia was detected in nematodes from 4/8 plant-hosts examined (raspberry, strawberry, clover, and lily), and in all three states surveyed. Cardinium was detected in nematodes from mint in Washington. In the wPpe-infected P. penetrans populations collected from raspberry, the prevalence of wPpe infection ranged from 11 to 58%. This pattern is unlike that in filarial nematodes where Wolbachia is an obligate mutualist and occurs in 100% of the host. Further analysis of wPpe-infected populations revealed female-skewed sex ratios (up to 96%), with the degree of skew positively correlating with wPpe prevalence. Uninfected nematode populations had approximately equal numbers of males and females. Comparisons of 54 wPpe 16S ribosomal RNA sequences revealed high similarity across the geographic isolates, with 45 of 54 isolates being identical at this locus. The complete absence of wPpe among some populations and low prevalence in others suggest that this endosymbiont is not an obligate mutualist of P. penetrans. The observed sex ratio bias in wPpe-infected nematode populations is similar to that observed in arthropods where Wolbachia acts as a reproductive manipulator, raising the question of a similar role in plant-parasitic nematodes.

The first transcriptomes from field-collected individual whiteflies ( Bemisia tabaci, Hemiptera: Aleyrodidae): a case study of the endosymbiont composition.

Background:Bemisia tabaci species ( B. tabaci), or whiteflies, are the world's most devastating insect pests. They cause billions of dollars (US) of damage each year, and are leaving farmers in the developing world food insecure. Currently, all publically available transcriptome data for B. tabaci are generated from pooled samples, which can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technological limitations. Methods: In this study, we optimised a single whitefly RNA extraction procedure, and sequenced the transcriptome of four individual adult Sub-Saharan Africa 1 (SSA1) B. tabaci. Transcriptome sequencing resulted in 39-42 million raw reads. De novo assembly of trimmed reads yielded between 65,000-162,000 Contigs across B. tabaci transcriptomes. Results: Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLASTn searches on the four transcriptomes identified five endosymbionts; the primary endosymbiont Portieraaleyrodidarum and four secondary endosymbionts: Arsenophonus, Wolbachia, Rickettsia, and Cardinium spp. that were predominant across all four SSA1 B. tabaci samples with prevalence levels of between 54.1 to 75%. Amino acid alignments of the NusG gene of P. aleyrodidarum for the SSA1 B. tabaci transcriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of the NusG protein from P. aleyrodidarum in SSA1 with known NusG structures showed the deletion resulted in a shorter D loop. Conclusions: The use of field-collected specimens means time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. Our method is applicable to any small organism where RNA quantity has limited transcriptome studies.