Hemolymph exosomes inhibit Spiroplasma eriocheiris infection by promoting Tetraspanin-mediated hemocyte phagocytosis in crab.

Exosomes released from infected cells are thought to play an important role in the dissemination of pathogens, as well as in host-derived immune molecules during infection. As an intracellular pathogen, Spiroplasma eriocheiris is harmful to multiple crustaceans. However, the immune mechanism of exosomes during Spiroplasma infection has not been investigated. Here, we found exosomes derived from S. eriocheiris-infected crabs could facilitate phagocytosis and apoptosis of hemocytes, resulting in increased crab survival and suppression of Spiroplasma intracellular replication. Proteomic analysis revealed the altered abundance of EsTetraspanin may confer resistance to S. eriocheiris, possibly by mediating hemocyte phagocytosis in Eriocheir sinensis. Specifically, knockdown of EsTetraspanin in E. sinensis increased susceptibility to S. eriocheiris infection and displayed compromised phagocytic ability, whereas overexpression of EsTetraspanin in Drosophila S2 cells inhibited S. eriocheiris infection. Further, it was confirmed that intramuscular injection of recombinant LEL domain of EsTetraspanin reduced the mortality of S. eriocheiris-infected crabs. Blockade with anti-EsTetraspanin serum could exacerbate S. eriocheiris invasion of hemocytes and impair hemocyte phagocytic activity. Taken together, our findings prove for the first time that exosomes modulate phagocytosis to resist pathogenic infection in invertebrates, which is proposed to be mediated by exosomal Tetraspanin, supporting the development of preventative strategies against Spiroplasma infection.

Disproportionate investment in Spiralin B production limits in-host growth and favors the vertical transmission of Spiroplasma insect endosymbionts.

Insects frequently harbor endosymbionts, which are bacteria housed within host tissues. These associations are stably maintained over evolutionary timescales through vertical transmission of endosymbionts from host mothers to their offspring. Some endosymbionts manipulate host reproduction to facilitate spread within natural populations. Consequently, such infections have major impacts on insect physiology and evolution. However, technical hurdles have limited our understanding of the molecular mechanisms underlying such insect-endosymbiont interactions. Here, we investigate the nutritional interactions between endosymbiotic partners using the tractable insect Drosophila melanogaster and its natural endosymbiont Spiroplasma poulsonii. Using a combination of functional assays, metabolomics, and proteomics, we show that the abundance and amino acid composition of a single Spiroplasma membrane lectin, Spiralin B (SpiB), dictates the amino acid requirements of the endosymbiont and determines its proliferation within host tissues. Ectopically increasing SpiB levels in host tissues disrupts localization of endosymbionts in the fly egg chambers and decreases vertical transmission. We find that SpiB is likely to be required by the endosymbiont to enter host oocytes, which may explain the massive investment of S. poulsonii in SpiB synthesis. SpiB both permits vertical transmission of the symbiont and limits its growth in nutrient-limiting conditions for the host; therefore, a single protein plays a pivotal role in ensuring durability of the interaction in a variable environment.

Assembly properties of bacterial actin MreB involved in Spiroplasma swimming motility.

Bacterial actin MreB forms filaments composed of antiparallel double-stranded units. The wall-less helical bacterium Spiroplasma has five MreB homologs (MreB1-5), some of which are involved in an intracellular ribbon for driving the bacterium's swimming motility. Although the interaction between MreB units is important for understanding Spiroplasma swimming, the interaction modes of each ribbon component are unclear. Here, we examined the assembly properties of Spiroplasma eriocheiris MreB5 (SpeMreB5), one of the ribbon component proteins that forms sheets. Electron microscopy revealed that sheet formation was inhibited under acidic conditions and bundle structures were formed under acidic and neutral conditions with low ionic strength. We also used solution assays and identified four properties of SpeMreB5 bundles as follows: (I) bundle formation followed sheet formation; (II) electrostatic interactions were required for bundle formation; (III) the positively charged and unstructured C-terminal region contributed to promoting lateral interactions for bundle formation; and (IV) bundle formation required Mg2+ at neutral pH but was inhibited by divalent cations under acidic pH conditions. During these studies, we also characterized two aggregation modes of SpeMreB5 with distinct responses to ATP. These properties will shed light on SpeMreB5 assembly dynamics at the molecular level.

Rare Spiroplasma Bloodstream Infection in Patient after Surgery, China, 2022.

We report a case of Spiroplasma bloodstream infection in a patient in China who developed pulmonary infection, acute respiratory distress syndrome, sepsis, and septic shock after emergency surgery for type A aortic dissection. One organism closely related to Spiroplasma eriocheiris was isolated from blood culture and identified by whole-genome sequencing.

Male-killing toxin in a bacterial symbiont of Drosophila.

Several lineages of symbiotic bacteria in insects selfishly manipulate host reproduction to spread in a population 1 , often by distorting host sex ratios. Spiroplasma poulsonii2,3 is a helical and motile, Gram-positive symbiotic bacterium that resides in a wide range of Drosophila species 4 . A notable feature of S. poulsonii is male killing, whereby the sons of infected female hosts are selectively killed during development1,2. Although male killing caused by S. poulsonii has been studied since the 1950s, its underlying mechanism is unknown. Here we identify an S. poulsonii protein, designated Spaid, whose expression induces male killing. Overexpression of Spaid in D. melanogaster kills males but not females, and induces massive apoptosis and neural defects, recapitulating the pathology observed in S. poulsonii-infected male embryos5-11. Our data suggest that Spaid targets the dosage compensation machinery on the male X chromosome to mediate its effects. Spaid contains ankyrin repeats and a deubiquitinase domain, which are required for its subcellular localization and activity. Moreover, we found a laboratory mutant strain of S. poulsonii with reduced male-killing ability and a large deletion in the spaid locus. Our study has uncovered a bacterial protein that affects host cellular machinery in a sex-specific way, which is likely to be the long-searched-for factor responsible for S. poulsonii-induced male killing.

Spiroplasma infection as a cause of severe congenital keratouveitis, cataract and glaucoma.

BACKGROUND: Only seven cases of ocular Spiroplasma infection have been reported to date, all presenting as congenital cataracts with concomitant intraocular inflammation. We describe the first case of Spiroplasma infection initially presenting as a corneal infiltrate. CASE PRESENTATION: A 1-month-old girl was referred for a corneal infiltrate in the left eye. She presented in our hospital with unilateral keratouveitis. Examination showed a stromal corneal infiltrate and dense white keratic precipitates in the left eye. Herpetic keratouveitis was suspected and intravenous acyclovir therapy was initiated. Two weeks later, the inflammation in the left eye persisted and was also noticed in the right eye. Acute angle-closure glaucoma and a cataract with dilated iris vessels extending onto the anterior lens capsule developed in the left eye. The inflammation resolved after treatment with azithromycin. Iridectomy, synechiolysis and lensectomy were performed. Bacterial metagenomic sequencing (16 S rRNA) and transmission electron microscopy revealed Spiroplasma ixodetis species in lens aspirates and biopsy. Consequently, a diagnosis of bilateral Spiroplasma uveitis was made. CONCLUSIONS: In cases of congenital cataract with concomitant intraocular inflammation, Spiroplasma infection should be considered. The purpose of this case report is to raise awareness of congenital Spiroplasma infection as a cause of severe keratouveitis, cataract and angle-closure glaucoma in newborns. Performing molecular testing on lens aspirates is essential to confirm diagnosis. Systemic macrolides are suggested as the mainstay of treatment.

Integrated Information for Pathogenicity and Treatment of Spiroplasma.

Spiroplasma, belonging to the class Mollicutes, is a small, helical, motile bacterium lacking a cell wall. Its host range includes insects, plants, and aquatic crustaceans. Recently, a few human cases of Spiroplasma infection have been reported. The diseases caused by Spiroplasma have brought about serious economic losses and hindered the healthy development of agriculture. The pathogenesis of Spiroplasma involves the ability to adhere, such as through the terminal structure of Spiroplasma, colonization, and invasive enzymes. However, the exact pathogenic mechanism of Spiroplasma remains a mystery. Therefore, we systematically summarize all the information about Spiroplasma in this review article. This provides a reference for future studies on virulence factors and treatment strategies of Spiroplasma.

Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis.

Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their host's metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the fly's resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetse's viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetse's reproductive fitness. Using a Gff lab line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.

Diverse Molecular Mechanisms Underlying Microbe-Inducing Male Killing in the Moth Homona magnanima.

Male killing (MK) is a type of reproductive manipulation induced by microbes, where sons of infected mothers are killed during development. MK is a strategy that enhances the fitness of the microbes, and the underlying mechanisms and the process of their evolution have attracted substantial attention. Homona magnanima, a moth, harbors two embryonic MK bacteria, namely, Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), and a larval MK virus, Osugoroshi virus (OGV; Partitiviridae). However, whether the three distantly related male killers employ similar or different mechanisms to accomplish MK remains unknown. Here, we clarified the differential effects of the three male killers on the sex-determination cascades and development of H. magnanima males. Reverse transcription-PCR demonstrated that Wolbachia and Spiroplasma, but not OGVs, disrupted the sex-determination cascade of males by inducing female-type splice variants of doublesex (dsx), a downstream regulator of the sex-determining gene cascade. We also found that MK microbes altered host transcriptomes in different manners; Wolbachia impaired the host dosage compensation system, whereas Spiroplasma and OGVs did not. Moreover, Wolbachia and Spiroplasma, but not OGVs, triggered abnormal apoptosis in male embryos. These findings suggest that distantly related microbes employ distinct machineries to kill males of the identical host species, which would be the outcome of the convergent evolution. IMPORTANCE Many microbes induce male killing (MK) in various insect species. However, it is not well understood whether microbes adopt similar or different MK mechanisms. This gap in our knowledge is partly because different insect models have been examined for each MK microbe. Here, we compared three taxonomically distinct male killers (i.e., Wolbachia, Spiroplasma, and a partiti-like virus) that infect the same host. We provided evidence that microbes can cause MK through distinct mechanisms that differ in the expression of genes involved in sex determination, dosage compensation, and apoptosis. These results imply independent evolutionary scenarios for the acquisition of their MK ability.

Molecular detection of Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts in wild population of tsetse flies collected in Cameroon, Chad and Nigeria.

BACKGROUND: Tsetse flies are cyclical vectors of African trypanosomiasis (AT). The flies have established symbiotic associations with different bacteria that influence certain aspects of their physiology. Vector competence of tsetse flies for different trypanosome species is highly variable and is suggested to be affected by bacterial endosymbionts amongst other factors. Symbiotic interactions may provide an avenue for AT control. The current study provided prevalence of three tsetse symbionts in Glossina species from Cameroon, Chad and Nigeria. RESULTS: Tsetse flies were collected and dissected from five different locations. DNA was extracted and polymerase chain reaction used to detect presence of Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts, using species specific primers. A total of 848 tsetse samples were analysed: Glossina morsitans submorsitans (47.52%), Glossina palpalis palpalis (37.26%), Glossina fuscipes fuscipes (9.08%) and Glossina tachinoides (6.13%). Only 95 (11.20%) were infected with at least one of the three symbionts. Among infected flies, six (6.31%) had Wolbachia and Spiroplasma mixed infection. The overall symbiont prevalence was 0.88, 3.66 and 11.00% respectively, for Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts. Prevalence varied between countries and tsetse fly species. Neither Spiroplasma species nor S. glossinidius were detected in samples from Cameroon and Nigeria respectively. CONCLUSION: The present study revealed, for the first time, presence of Spiroplasma species infections in tsetse fly populations in Chad and Nigeria. These findings provide useful information on repertoire of bacterial flora of tsetse flies and incite more investigations to understand their implication in the vector competence of tsetse flies.

History matters: Thermal environment before but not during wasp attack determines the efficiency of symbiont-mediated protection.

The outcome of natural enemy attack in insects is commonly impacted by the presence of defensive microbial symbionts residing within the host. The thermal environment is a factor known to affect symbiont-mediated traits in insects. Lower temperatures, for instance, have been shown to reduce Spiroplasma-mediated protection in Drosophila. Our understanding of protective symbiosis requires a deeper understanding of environment-symbiont-protection links. Here, we dissect the effect of the thermal environment on Spiroplasma-mediated protection against Leptopilina boulardi in Drosophila melanogaster by examining the effect of temperature before, during and after wasp attack on fly survival and wasp success. We observed that the developmental temperature of the mothers of attacked larvae, but not the temperature of the attacked larvae themselves during or after wasp attack, strongly determines the protective influence of Spiroplasma. Cooler maternal environments were associated with weaker Spiroplasma protection of their progeny. The effect of developmental temperature on Spiroplasma-mediated protection is probably mediated by a reduction in Spiroplasma titre. These results indicate that historical thermal environment is a stronger determinant of protection than current environment. Furthermore, protection is a character with transgenerational nongenetic variation probably to produce complex short-term responses to selection. In addition, the cool sensitivity of the Spiroplasma-Drosophila symbioses contrasts with the more common failure of symbioses at elevated temperatures, indicating a need to understand the mechanistic basis of low temperature sensitivity on this symbiosis.

iTRAQ-based differential proteomic analysis of high- and low-virulence strains of Spiroplasma eriocheiris.

Spiroplasma eriocheiris is one of the major pathogenic bacteria in crustaceans, featuring high infectivity, rapid transmission, and an absence of effective control strategies, resulting in significant economic losses to the aquaculture industry. Research into virulence-related factors provides an important perspective to clarify how Spiroplasma eriocheiris is pathogenic to shrimps and crabs. Therefore, in this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was utilized to undertake a differential proteomic analysis of high- and low-virulence Spiroplasma eriocheiris strains at different growth phases. A total of 868 differentially expressed proteins (DEPs) were obtained, of which 31 novel proteins were identified by proteogenomic analysis. There were 62, 61, 175, and 235 DEPs between the log phase (YD) and non-log phase (YFD) of the high-virulence strain, between the log phase (CD) and non-log phase (CFD) of the low-virulence strain, between YD and CD, and between CFD and YFD, respectively. All the DEPs were compared with virulence protein databases (MvirDB and VFDB), and 68 virulence proteins of Spiroplasma eriocheiris were identified, of which 12 were involved in a total of 21 metabolic pathways, including motility, chemotaxis, growth, metabolism and virulence of the bacteria. The results of this study form the basis for further research into the molecular mechanism of virulence and physiological differences between high- and low-virulence strains of Spiroplasma eriocheiris, and provide a scientific basis for a detailed understanding of its pathogenesis.

Bend or Twist? What Plectonemes Reveal about the Mysterious Motility of Spiroplasma.

Spiroplasma is a unique, helical bacterium that lacks a cell wall and swims using propagating helix hand inversions. These deformations are likely driven by a set of cytoskeletal filaments, but how remains perplexing. Here, we probe the underlying mechanism using a model where either twist or bend drive spiroplasma's chirality inversions. We show that Spiroplasma should wrap into plectonemes at different values of the length and external viscosity, depending on the mechanism. Then, by experimentally measuring the bending modulus of Spiroplasma and if and when plectonemes form, we show that Spiroplasma's helix hand inversions are likely driven by bending.

Whole-chromosome hitchhiking driven by a male-killing endosymbiont.

Neo-sex chromosomes are found in many taxa, but the forces driving their emergence and spread are poorly understood. The female-specific neo-W chromosome of the African monarch (or queen) butterfly Danaus chrysippus presents an intriguing case study because it is restricted to a single 'contact zone' population, involves a putative colour patterning supergene, and co-occurs with infection by the male-killing endosymbiont Spiroplasma. We investigated the origin and evolution of this system using whole genome sequencing. We first identify the 'BC supergene', a broad region of suppressed recombination across nearly half a chromosome, which links two colour patterning loci. Association analysis suggests that the genes yellow and arrow in this region control the forewing colour pattern differences between D. chrysippus subspecies. We then show that the same chromosome has recently formed a neo-W that has spread through the contact zone within approximately 2,200 years. We also assembled the genome of the male-killing Spiroplasma, and find that it shows perfect genealogical congruence with the neo-W, suggesting that the neo-W has hitchhiked to high frequency as the male-killer has spread through the population. The complete absence of female crossing-over in the Lepidoptera causes whole-chromosome hitchhiking of a single neo-W haplotype, carrying a single allele of the BC supergene and dragging multiple non-synonymous mutations to high frequency. This has created a population of infected females that all carry the same recessive colour patterning allele, making the phenotypes of each successive generation highly dependent on uninfected male immigrants. Our findings show how hitchhiking can occur between the physically unlinked genomes of host and endosymbiont, with dramatic consequences.

Low Endosymbiont Incidence in Drosophila Species Across Peninsula Thailand.

Arthropods are known to harbor several endosymbionts, such as Cardinium, Rickettsia, Spiroplasma, and Wolbachia. Wolbachia, for example, are the most widespread known endosymbionts in the world, which are found in about half of all arthropod species. To increase their transmission, these endosymbionts must manipulate their hosts in several ways such as cytoplasmic incompatibility and male killing. In tropical regions, endosymbiont diversity has not been studied exhaustively. Here, we checked four endosymbionts, including Cardinium, Rickettsia, Spiroplasma, and Wolbachia, in eleven Drosophila species found in Thai Peninsula. The Wolbachia strain wRi-like was found in all populations of Drosophila ananassae and Drosophila simulans. Furthermore, we found two new strains, wMalA and wMalB, in two populations of Drosophila malerkotliana. Besides Wolbachia, we did not find any of the above endosymbionts in all fly species. This work reveals the hidden diversity of endosymbionts in Drosophila and is the first exhaustive study on Drosophila in the region.

The wild solitary bees Andrena vaga, Anthophora plumipes, Colletes cunicularius, and Osmia cornuta microbiota are host specific and dominated by endosymbionts and environmental microorganisms.

We characterized the microbial communities of the crop, midgut, hindgut, and ovaries of the wild solitary bees Andrena vaga, Anthophora plumipes, Colletes cunicularius, and Osmia cornuta through 16S rRNA gene and ITS2 amplicon sequencing and a large-scale isolation campaign. The bacterial communities of these bees were dominated by endosymbionts of the genera Wolbachia and Spiroplasma. Bacterial and yeast genera representing the remaining predominant taxa were linked to an environmental origin. While only a single sampling site was examined for Andrena vaga, Anthophora plumipes, and Colletes cunicularius, and two sampling sites for Osmia cornuta, the microbiota appeared to be host specific: bacterial, but not fungal, communities generally differed between the analyzed bee species, gut compartments and ovaries. This may suggest a selective process determined by floral and host traits. Many of the gut symbionts identified in the present study are characterized by metabolic versatility. Whether they exert similar functionalities within the bee gut and thus functional redundancy remains to be elucidated.

Microbiome of Zoophytophagous Biological Control Agent Nesidiocoris tenuis.

Many insects are associated with endosymbionts that influence the feeding, reproduction, and distribution of their hosts. Although the small green mirid, Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), a zoophytophagous predator that feeds on plants as well as arthropods, is a globally important biological control agent, its microbiome has not been sufficiently studied. In the present study, we assessed the microbiome variation in 96 N. tenuis individuals from 14 locations throughout Japan, based on amplicon sequencing of the 16S ribosomal RNA gene. Nine major bacteria associated with N. tenuis were identified: Rickettsia, two strains of Wolbachia, Spiroplasma, Providencia, Serratia, Pseudochrobactrum, Lactococcus, and Stenotrophomonas. Additionally, a diagnostic PCR analysis for three typical insect reproductive manipulators, Rickettsia, Wolbachia, and Spiroplasma, was performed on a larger sample size (n = 360) of N. tenuis individuals; the most prevalent symbiont was Rickettsia (69.7%), followed by Wolbachia (39.2%) and Spiroplasma (6.1%). Although some symbionts were co-infected, their prevalence did not exhibit any specific tendency, such as a high frequency in specific infection combinations. The infection frequency of Rickettsia was significantly correlated with latitude and temperature, while that of Wolbachia and Spiroplasma was significantly correlated with host plants. The predominance of these bacteria and the absence of obligate symbionts suggested that the N. tenuis microbiome is typical for predatory arthropods rather than sap-feeding insects. Rickettsia and Wolbachia were vertically transmitted rather than horizontally transmitted from the prey. The functional validation of each symbiont would be warranted to develop N. tenuis as a biological control agent.

Screening and functional analysis of three Spiroplasma eriocheiris glycosylated protein interactions with Macrobrachium nipponense C-type lectins.

N-glycosylation, one of the main protein posttranslational modifications (PTMs), plays an important role in the pathogenic process of pathogens through binding and invasion of host cells or regulating the internal environment of host cells to benefit their survival. However, N-glycosylation has remained mostly unexplored in Spiroplasma eriocheiris, a novel type of pathogen which has serious adverse effects on aquaculture. In most cases, N-glycoproteins can be detected and analyzed by lectins dependent on sugar recognition domains. In this study, three Macrobrachium nipponense C-type lectins, namely, MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3, were used to screen S. eriocheiris glycosylated proteins. First, qRT-PCR results showed that the expression levels of the three kinds of lectins were all significantly up-regulated in prawn hearts when the host was against S. eriocheiris infection. A bacterial binding assay showed that purified recombinant MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3 could directly bind to S. eriocheiris in vitro. Second, three S. eriocheiris glycosylated proteins, ATP synthase subunit beta (ATP beta), molecular chaperone Dnak (Dnak) and fructose bisphosphate aldolase (FBPA), were screened and identified using the three kinds of full-length C-type lectins. Far-Western blot and coimmunoprecipitation (CO-IP) further demonstrated that there were interactions between the three lectins with ATP beta, Dnak and FBPA. Furthermore, antibody neutralization assay results showed that pretreatment of S. eriocheiris with ATP beta, Dnak and FBPA antibodies could significantly block this pathogen infection. All the above studies showed that the glycosylated protein played a vital role in the process of S. eriocheiris infection.

Development and application of the MIRA and MIRA-LFD detection methods of Spiroplasma eriocheiris.

The tremor disease (TD) caused by Spiroplasma eriocheiris is the most destructive disease of the Chinese mitten crab, Eriocheir sinensis. This study attempts to construct Multienzyme Isothermal Rapid Amplification (MIRA), a quick and simple nucleic acid amplification method that operates at room temperature. Based on the gene sequences of S. eriocheiris, appropriate amplification primers were constructed and screened in this investigation. Both the relevant specific probe and the chosen specific amplification primers were designed and labeled. The MIRA and MIRA-LFD reaction conditions were then optimized. The result showed MIRA and MIRA-FFD could identify S. eriocheiris at 37 °C in 30 min and 15 min, respectively. To investigate the specificity of MIRA and MIRA-LFD, three Gram-negative bacteria (Bacillus subtilis, Bacillus thuringiensis, and Staphylococcus aureus), three Gram-positive bacteria (Escherichia coli, Aeromonas hydrophila, and Salmonella typhimurium) and S. eriocheiris were selected. The result showed MIRA and MIRA-LFD were highly specific to S. eriocheiris and did not react with other six pathogens. The sensitivities of PCR, MIRA, and MIRA-LFD were then evaluated. The result showed the detection limit of PCR is 1 ng/L whereas the detection limit of MIRA and MIRA-LFD is 10 pg/L. Finally, the established MIRA and MIRA-LFD detection methods had the advantages of being quick, sensitive, and specific for S. eriocheiris detection, as well as not requiring any specialized equipment.

Infection patterns of Harmonia axyridis (Coleoptera: Coccinellidae) by ectoparasitic microfungi and endosymbiotic bacteria.

The invasive alien ladybird Harmonia axyridis (Coleoptera: Coccinellidae) hosts a wide range of natural enemies. Many observations have been done in nature but experimental studies of interactions of multiple enemies on Ha. axyridis are rare. In light of this knowledge gap, we tested whether the host phenotype and presence of bacterial endosymbionts Spiroplasma and Wolbachia affected parasitism of Ha. axyridis by the ectoparasitic fungus Hesperomyces harmoniae (Ascomycota: Laboulbeniales). We collected 379 Ha. axyridis in the Czech Republic, processed specimens, including screening for He. harmoniae and a molecular assessment for bacteria, and calculated fecundity and hatchability of females. We found that high hatchability rate (71 %) was conditioned by high fecundity (20 eggs daily or more). The average parasite prevalence of He. harmoniae was 53 %, while the infection rate of Spiroplasma was 73 % in ladybirds that survived in winter conditions. Wolbachia was only present in 2 % of the analyzed ladybirds. Infection by either He. harmoniae or Spiroplasma did not differ among host color morphs. In the novemdecimsignata morph, younger individuals (with orange elytra) were more heavily parasitized compared to old ones (with red elytra). Fecundity and hatchability rate of females were unaffected by infection with either He. harmoniae or Spiroplasma. However, female ladybirds co-infected with He. harmoniae and Spiroplasma had a significantly lower fecundity and hatchability compared to females with only one or no symbiont.