Candidatus Cardinium Hertigii Research Articles

Ecological Factors Associated with the Distribution of Bemisia tabaci Cryptic Species and Their Facultative Endosymbionts

The sweetpotato whitefly, Bemisia tabaci species complex, comprises at least 44 morphologically indistinguishable cryptic species, whose endosymbiont infection patterns often varied at the spatial and temporal dimension. However, the effects of ecological factors (e.g., climatic or geographical factors) on the distribution of whitefly and the infection frequencies of their endosymbionts have not been fully elucidated. We, here, analyzed the associations between ecological factors and the distribution of whitefly and their three facultative endosymbionts (Candidatus Cardinium hertigii, Candidatus Hamiltonella defensa, and Rickettsia sp.) by screening 665 individuals collected from 29 geographical localities across China. The study identified eight B. tabaci species via mitochondrial cytochrome oxidase I (mtCOI) gene sequence alignment: two invasive species, MED (66.9%) and MEAM1 (12.2%), and six native cryptic species (20.9%), which differed in distribution patterns, ecological niches, and high suitability areas. The infection frequencies of the three endosymbionts in different cryptic species were distinct and multiple infections were relatively common in B. tabaci MED populations. Furthermore, the annual mean temperature positively affected Cardinium sp. and Rickettsia sp. infection frequencies in B. tabaci MED but negatively affected the quantitative distribution of B. tabaci MED, which indicates that Cardinium sp. and Rickettsia sp. maybe play a crucial role in the thermotolerance of B. tabaci MED, although the host whitefly per se exhibits no resistance to high temperature. Our findings revealed the complex effects of ecological factors on the expansion of the invasive whitefly.

Bt protein hasten entomopathogenic fungi-induced death of nontarget pest whitefly by suppressing protective symbionts

The risk assessment of Bacillus thuringiensis (Bt) crops on nontarget pests has received much attention. Despite the knowledge of various beneficial bacterial symbionts in pests, whether Bt proteins affect these symbionts and subsequently alter the pest's ecology remains largely unknown. The whitefly Bemisia tabaci is one of the most serious nontarget pests in Bt cotton. Here, we explored the Bt Cry1Ac protein-induced changes in whitefly symbiont abundance and the subsequent effects on whitefly response against a naturally prevalent entomopathogenic fungus Cordyceps javanica. The obligate symbiont ‘Candidatus Portiera aleyrodidarum’ (hereafter P. aleyrodidarum) as well as facultative symbionts ‘Candidatus Hamiltonella defensa’ (hereafter H. defensa), ‘Candidatus Cardinium hertigii’ (hereafter C. hertigii) and ‘Candidatus Rickettsia bellii’ (hereafter R. bellii) dominate the microbial community of whiteflies. The Bt exposure had no effects on H. defensa infected (H) and H. defensa-C. hertigii doubly infected (HC) whiteflies, but decreased the total copy number of symbionts as well as the R. bellii proportion in H. defensa-C. hertigii- R. bellii triply infected whiteflies (HCR). C. javanica caused whitefly adults 100 % mortality within 8 days. Without Bt protein exposure, HCR whiteflies survived significantly longer than H and HC whiteflies sprayed by C. javanica, suggesting that R. bellii confers protection. However, in Bt-exposed groups, C. javanica generated synchronous death of H, HC and HCR whiteflies. Specifically, in H and HC whiteflies, Bt protein-exposure showed no significant difference in progress of death caused by C. javanica. But in HCR whiteflies, Bt exposure hastened death induced by C. javanica, suppressing the R. bellii-conferred protection. This is the first report revealing that Bt protein altered symbiont community conferred adverse effects on nontarget pests, providing a new perspective for Bt risk assessment and biocontrol strategies of nontarget pests.

Identifying potential candidate Culicoides spp. for the study of interactions with Candidatus Cardinium hertigii.

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors responsible for the transmission of several viruses of veterinary importance. Previous screens of Culicoides have described the presence of the endosymbiont Candidatus Cardinium hertigii (Bacteroidetes). However, any impacts of this microbe on vectorial capacity, akin to those conferred by Wolbachia in mosquitoes, are yet to be uncovered and await a suitable system to study Cardinium-midge interactions. To identify potential candidate species to investigate these interactions, accurate knowledge of the distribution of the endosymbiont within Culicoides populations is needed. We used conventional and nested PCR assays to screen Cardinium infection in 337 individuals of 25 Culicoides species from both Palearctic and Afrotropical regions. Infections were observed in several vector species including C. imicola and the Pulicaris complex (C. pulicaris, C.bysta, C. newsteadi and C. punctatus) with varying prevalence. Phylogenetic analysis based on the Gyrase B gene grouped all new isolates within 'group C' of the genus, a clade that has to date been exclusively described in Culicoides. Through a comparison of our results with previous screens, we suggest C. imicola and C. sonorensis represent good candidates for onward study of Cardinium-midge interactions.

Effect of Cardinium Infection on the Probing Behavior of Bemisia tabaci (Hemiptera: Aleyrodidae) MED.

Facultative endosymbionts can affect the growth, physiology, and behavior of their arthropod hosts. There are several endosymbionts in the invasive whitefly Bemisia tabaci Mediterranean (MED, Q biotype) that influence host fitness by altering stylet probing behavior. We investigated the probing behavior of B. tabaci MED infected with the facultative endosymbiont Candidatus Cardinium hertigii (Cardinium (Sphingobacteriales: Flexibacteraceae)). We generated genetically similar Cardinium-infected (C*+) and uninfected (C-) clonal sublines and analyzed the probing behavior of newly emerged adult on cotton (Malvales: Malvaceae), Gossypium hirsutum L., using electropenetrography (EPG). The C- subline demonstrated a longer duration of E2 (2.81-fold) and more events of E2 (2.22-fold) than the C*+ subline, indicating a greater level of sustained ingestion of plant phloem. These findings provide insight into the fitness costs (fitness of a particular genotype is lower than the average fitness of the population) of the Cardinium-infected B. tabaci.

Genome Sequence of the Soybean Cyst Nematode (Heterodera glycines) Endosymbiont "Candidatus Cardinium hertigii" Strain cHgTN10.

In this study, we present the genome sequence of the "Candidatus Cardinium hertigii" strain cHgTN10, an endosymbiotic bacterium of the plant-parasitic nematode Heterodera glycines This is the first genome assembly reported for an endosymbiont directly sequenced from a tylenchid nematode.

Detection of Low-Level Cardinium and Wolbachia Infections in Culicoides.

Bacterial endosymbionts have been identified as potentially useful biological control agents for a range of invertebrate vectors of disease. Previous studies of Culicoides (Diptera: Ceratopogonidae) species using conventional PCR assays have provided evidence of Wolbachia (1/33) and Cardinium (8/33) infections. Here, we screened 20 species of Culicoides for Wolbachia and Cardinium, utilizing a combination of conventional PCR and more sensitive quantitative PCR (qPCR) assays. Low levels of Cardinium DNA were detected in females of all but one of the Culicoides species screened, and low levels of Wolbachia were detected in females of 9 of the 20 Culicoides species. Sequence analysis based on partial 16S rRNA gene and gyrB sequences identified "Candidatus Cardinium hertigii" from group C, which has previously been identified in Culicoides from Japan, Israel, and the United Kingdom. Wolbachia strains detected in this study showed 98 to 99% sequence identity to Wolbachia previously detected from Culicoides based on the 16S rRNA gene, whereas a strain with a novel wsp sequence was identified in Culicoides narrabeenensis. Cardinium isolates grouped to geographical regions independent of the host Culicoides species, suggesting possible geographical barriers to Cardinium movement. Screening also identified Asaia bacteria in Culicoides. These findings point to a diversity of low-level endosymbiont infections in Culicoides, providing candidates for further characterization and highlighting the widespread occurrence of these endosymbionts in this insect group.

First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus, important Palaearctic vectors of bluetongue virus

Heritable bacteria have been highlighted as important components of vector biology, acting as required symbionts with an anabolic role, altering competence for disease transmission, and affecting patterns of gene flow by altering cross compatibility. In this paper, we tested eight U.K. species of Culicoides (Diptera: Ceratopogonidae) midge for the presence of five genera of endosymbiotic bacteria: Cardinium (Bacteroidales: Bacteroidaceae); Wolbachia (Rickettsiales: Rickettsiaceae); Spiroplasma (Entomoplasmatales: Spiroplasmataceae); Arsenophonus (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Cardinium spp. were detected in both sexes of Culicoides pulicaris and Culicoides punctatus, two known vectors of bluetongue virus. Cardinium spp. were not detected in any other species, including the Culicoides obsoletus group, the main vector of bluetongue and Schmallenberg viruses in northern Europe. The other endosymbionts were not detected in any Culicoides species. The Cardinium strain detected in U.K. Culicoides species is very closely related to the Candidatus Cardinium hertigii group C, previously identified in Culicoides species in Asia. Further, we infer that the symbiont is not a sex ratio distorter and shows geographic variation in prevalence within a species. Despite its detection in several species of Culicoides that vector arboviruses worldwide, the absence of Cardinium in the C. obsoletus group suggests that infections of these symbionts may not be necessary to the arboviral vector competence of biting midges.

Assessment of Survival and Body Size Variation of Culicoides imicola (Diptera: Ceratopogonidae) as Functions of “Candidatus Cardinium” (Bacteroidetes) Infection Status

"Candidatus Cardinium hertigii" (Bacteroidetes) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of "Candidatus Cardinium" infection was documented as moderate, with no detectable sex bias. We therefore investigated whether "Candidatus Cardinium" affects important fitness parameters, such as survival and body size, in Culicoides imicola, a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account "Candidatus Cardinium" infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with "Candidatus Cardinium" infection data. The findings revealed no difference in survival of Culicoides infected with "Candidatus Cardinium" and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for "Candidatus Cardinium"-infected versus uninfected midges. In aggregate, these findings support our conclusion that "Candidatus Cardinium" does not have an overt effect on the survival and size of adult C. imicola midges. "Candidatus Cardinium" may affect immature stages or may alter adult reproductive performance.

Prevalence of the symbiont Cardinium in Culicoides (Diptera: Ceratopogonidae) vector species is associated with land surface temperature

Prevalence of infection by bacterial symbionts may reflect their interactions with the host and has been shown to be correlated with environmental factors. Yet, it is still unclear whether infection by symbionts is determined by environmental factors affecting the early or imago stage of the host. Here, we identified and localized the symbiont Candidatus Cardinium hertigii (Bacteroidetes) in sympatric Culicoides biting midge species, examined its abundance, and studied its association with environmental factors. The prevalence of adult infection differed, with 50.7% from C. imicola, 31.4% from C. oxystoma, and 0% from C. schultzei gp., although phylogenetic analyses showed that Cardinium in these species is almost identical. In addition, prevalence of infection differed between climate regions, with lowest prevalence in the arid region and highest prevalence in the Mediterranean region. Multivariate linear regression analysis of Cardinium prevalence together with climatic and satellite imagery data-derived environmental variables revealed that infection prevalence is significantly associated with land surface temperature and explained up to 89.7% of infection prevalence variability. These findings suggest that the observed variation of Cardinium infection of the imago stage of Culicoides may be influenced by environmental conditions during the latter's early developmental stages.

Detection and Identification of Species-Specific Bacteria Associated with Synanthropic Mites

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2 × 10(2) to 1.4 × 10(3) per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of "Candidatus Cardinium hertigii" and as a separate novel cluster.

Bacterial Endosymbiont Localization in Hyalesthes obsoletus , the Insect Vector of Bois Noir in Vitis vinifera

One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by "Candidatus Phytoplasma solani" and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the "Candidatus Sulcia muelleri" cluster in the Bacteroidetes and in the "Candidatus Purcelliella pentastirinorum" group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name "Candidatus Vidania fulgoroideae." Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and "Candidatus Cardinium hertigii." Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As "Ca. Sulcia" is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between "Ca. Sulcia" and "Ca. Purcelliella," "Ca. Vidania," or both.

Prevalence of Cardinium Bacteria in Planthoppers and Spider Mites and Taxonomic Revision of “ Candidatus Cardinium hertigii” Based on Detection of a New Cardinium Group from Biting Midges

Cardinium bacteria, members of the phylum Cytophaga-Flavobacterium-Bacteroides (CFB), are intracellular bacteria in arthropods that are capable of inducing reproductive abnormalities in their hosts, which include parasitic wasps, mites, and spiders. A high frequency of Cardinium infection was detected in planthoppers (27 out of 57 species were infected). A high frequency of Cardinium infection was also found in spider mites (9 out of 22 species were infected). Frequencies of double infection by Cardinium and Wolbachia bacteria (Alphaproteobacteria capable of manipulating reproduction of their hosts) were disproportionately high in planthoppers but not in spider mites. A new group of bacteria, phylogenetically closely related to but distinct from previously described Cardinium bacteria (based on 16S rRNA and gyrB genes) was found in 4 out of 25 species of Culicoides biting midges. These bacteria possessed a microfilament-like structure that is a morphological feature previously found in Cardinium and Paenicardinium. The bacteria close to the genus Cardinium consist of at least three groups, A, B, and C. Group A is present in various species of arthropods and was previously referred to as "Candidatus Cardinium hertigii," group B is present in plant parasitic nematodes and was previously referred to as "Candidatus Paenicardinium endonii," and group C is present in Culicoides biting midges. On the basis of morphological and molecular data, we propose that the nomenclature of these three groups be integrated into a single species, "Candidatus Cardinium hertigii."

Bacterial taxa associated with the hematophagous mite Dermanyssus gallinae detected by 16S rRNA PCR amplification and TTGE fingerprinting

Dermanyssus gallinae (Arthropoda, Mesostigmata) is suspected to be involved in the transmission of a wide variety of pathogens, but nothing is known about its associated non-pathogenic bacterial community. To address this question, we examined the composition of bacterial communities in D. gallinae collected from standard poultry farms in Brittany, France. Genetic fingerprints of bacterial communities were generated by temporal temperature gradient gel electrophoresis (TTGE) separation of individual polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments, followed by DNA sequence analysis. Most of the sequences belonged to the Proteobacteria and Firmicute phyla, with a majority of sequences corresponding to the Enterobacteriales order and the Staphylococcus genus. By using statistical analysis, we showed differences in biodiversity between poultry farms. We also determined the major phylotypes that compose the characteristic microbiota associated with D. gallinae. Saprophytes, opportunistic pathogens and pathogenic agents such as Pasteurella multocida, Erysipelothrix rhusiopathiae and sequences close to the genus Aerococcus were identified. Endosymbionts such as Schineria sp., Spiroplasma sp. Anistosticta, “ Candidatus Cardinium hertigii” and Rickettsiella sp. were also present in the subdominant bacterial community. Identification of potential targets within the symbiont community may be considered in the future as a means of ectoparasite control.

Distribution of the endosymbiotic bacterium Cardinium in Chinese populations of the carmine spider mite Tetranychus cinnabarinus (Acari: Tetranychidae)

Abstract: ‘Candidatus Cardinium hertigii (Cardinium)’ from the Bacteroidetes group is a symbiont that has recently received attention as a reproductive manipulator of its arthropod hosts. Though the spatial distribution of symbionts depends heavily on that of their hosts, evidence has shown that dispersal limitations also have effect on some micro‐organisms. In order to estimate the distribution of Cardinium in Chinese populations of the carmine spider mite, twenty seven geographical populations of Tetranychus cinnabarinus were collected and screened, using Cardinium‐specific primers for the 16S ribosomal DNA. Six geographical populations collected between 30° and 36°N tested positive for the presence of Cardinium, whereas it could not be detected in populations collected between 19°–29°N and 37°–46°N, suggesting that the symbiont is not evenly distributed, comparing with a wider distribution of Wolbachia previously reported in the same populations. In addition, the infection rates of Cardinium seem to increase from the south to the north of China. All the six Cardinium‐infected populations were found to be doubly infected with Wolbachia. Phylogenetic analysis based on the 16S rDNA gene sequence of Cardinium shows a close relationship between Cardinium in Chinese carmine spider mite and symbionts found in other species in the Tetranychidae.

‘Candidatus Paenicardinium endonii’, an endosymbiont of the plant-parasitic nematode Heterodera glycines (Nemata: Tylenchida), affiliated to the phylum Bacteroidetes

Bacteria-like endosymbionts of females of the plant-parasitic nematodes Globodera rostochiensis and Heterodera goettingiana and juveniles of Heterodera glycines were first observed during transmission electron microscopy (TEM) studies conducted in the 1970s. These organisms were characterized as being rod-shaped, ranging in size from 0.3 to 0.5 microm in diameter and 1.8 to 3 microm in length and containing structures labelled as striated inclusion bodies or tubular structures. A population of H. glycines was obtained from the soybean field where infected nematodes were first discovered in order to conduct TEM studies of females and males and to determine the phylogenetic position of the H. glycines endosymbiont among bacteria by studying the 16S rRNA and gyrB gene sequences. The bacterium was observed in the pseudocoelom and intestine of juveniles, females and males, in hypodermal chords of juveniles and males, in ovary walls and in oocytes and spermatozoa. The bacterium was polymorphic, measuring 0.4-0.8 x 2.5-4.5 microm, and many specimens contained an array of microfilament-like structures similar to those observed in "Candidatus Cardinium hertigii", the endosymbiont of Encarsia spp. wasps. Phylogenetic analysis of the 16S rRNA and gyrB genes of the H. glycines-infecting bacterium revealed 93 % and 81 % sequence identity, respectively, to the homologous genes in "Candidatus C. hertigii". Thus, the name "Candidatus Paenicardinium endonii" is proposed for the bacterial endosymbiont of the plant-parasitic nematode H. glycines.

A Novel Bacteroidetes Symbiont Is Localized in Scaphoideus titanus , the Insect Vector of Flavescence Dorée in Vitis vinifera

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, "Candidatus Phytoplasma vitis," which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the "Candidatus Cardinium hertigii" group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of "Ca. Cardinium hertigii." This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of "Ca. Phytoplasma vitis" were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and "Ca. Phytoplasma vitis" have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.

Characterization of a 'Bacteroidetes' symbiont in Encarsia wasps (Hymenoptera: Aphelinidae): proposal of 'Candidatus Cardinium hertigii'.

Previously, analysis of 16S rDNA sequences placed a newly discovered lineage of bacterial symbionts of arthropods in the 'Bacteroidetes'. This symbiont lineage is associated with a number of diverse host reproductive manipulations, including induction of parthenogenesis in several Encarsia parasitoid wasps (Hymenoptera: Aphelinidae). In this study, electron microscopy and phylogenetic analysis of the 16S rRNA and gyrB genes of symbionts from Encarsia hispida and Encarsia pergandiella are used to describe and further characterize these bacteria. Phylogenetic analyses based on these two genes showed that the Encarsia symbionts are allied with the Cytophaga aurantiaca lineage within the 'Bacteroidetes', with their closest described relative being the acanthamoeba symbiont 'Candidatus Amoebophilus asiaticus'. The Encarsia symbionts share 97 % 16S rDNA sequence similarity with Brevipalpus mite and Ixodes tick symbionts and 88 % sequence similarity with 'Candidatus A. asiaticus'. Electron microscopy revealed that many of the bacteria found in the ovaries of the two Encarsia species contained a regular, brush-like array of microfilament-like structures that appear to be characteristic of the symbiont. Finally, the role of this bacterium in parthenogenesis induction in E. hispida was confirmed. Based on phylogenetic analyses and electron microscopy, classification of the symbionts from Encarsia as 'Candidatus Cardinium hertigii' is proposed.