The battle of Cardinium and Wolbachia in double-infected mite cultures

The battle of Cardinium and Wolbachia in double-infected mite cultures | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The battle of Cardinium and Wolbachia in double-infected mite cultures Jan Hubert, Eliza Glowska, Stano Pekar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4806277/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Apr, 2025 Read the published version in Journal of Stored Products Research → Version 1 posted You are reading this latest preprint version Abstract Cultures of the stored product mite Tyrophagus putrescentiae are often single-infected by one of intracellular bacteria Cardinium or Wolbachia . No naturally occurring multi-infected Cardinium and Wolbachia a T. putrescentiae mites are known. Under laboratory conditions, we mixed two single-infected mite cultures of each endosymbiont to obtain four multi-infected cultures and to test the intraspecific competition among individuals under the influence of Cardinium and Wolbachia . The mite population growth, as a fitness indicator, and endosymbiont prevalence were estimated repeatedly during five months, repeated in three separate experiments. The fitness of four single-infected populations varied significantly. The proportion of infected individuals was higher in the two Cardinium infected populations than in the two Wolbachia infected populations. The fitness of multi-infected populations fluctuated over time in all three experiments. After six months of experiments, Cardinium infected individuals prevailed in 50% of the populations, the Wolbachia infected individuals prevailed in 41.7%, and asymbiotic individuals in 8.3% of populations (N = 12). The fitness of two multi-infected populations was like the fitness of single-infected populations. In one of them, the proportion of Cardinium or Wolbachia infected individuals was, on average, similar during the experiment. At the same time, symbionts disappeared, resulting in the majority of asymbiotic individuals in the second population. No correlation between symbionts proportion and mite fitness was found. The results indicated that the outcome of the battle between Cardinium and Wolbachia is strongly stochastic at the population level. Mite Cardinium Wolbachia Genome Gene expression Interaction Figures Figure 1 Figure 2 Figure 3 Introduction Many arthropods are hosts of intracellular bacteria, such as Wolbachia and Cardinium (Breeuwer et al. 2012 ; Schneider et al. 2012 ). These bacteria are maternally transmitted and possess mechanisms manipulating the host behavior to achieve successful distribution in the host populations. Best known manipulations include male killing, feminization, thelytokous parthenogenesis, and cytoplasmic incompatibility (CI), and result in an increased proportion of infected females in the host population (Perlman et al. 2008 ; Ros and Breeuwer 2009 ). Mites are also frequently infected by intracellular bacteria. For example, Tetranychus populations (N = 16) were mostly infected by Wolbachia (ca. 61%), followed by Cardinium (12–15%), and Rickettsia (0.9–3%) (Zele et al. 2018a ). Mite individuals might be infected by one to several intracellular bacteria (Brown et al. 2018 ; Duron et al. 2008 ; Gottlieb et al. 2008 ; Zele et al. 2018a , b ; Zhu et al. 2012 ; Zytynska 2019 ). The double infections of Cardinium and Wolbachia have been reported in several Bryobia (Ros et al. 2012 ) and Tetranychus (Gotoh et al. 2007 ; Ros et al. 2012 ; Zhu et al. 2012 ) species. The co-infection by Cardinium and Wolbachia led to various interactions that were usually interpreted only via correlations in the context of their occurrence (Mathe-Hubert et al. 2019 ; Zytynska 2019 ). However, a few studies revealed the co-infection effects (Brinker et al. 2019 ; Bustamante-Brito et al. 2019 ). Mite co-infected with multiple symbiont taxa represent a complex system with many interactions between these symbionts and the host. Such systems alter host physiological responses and survival (Bing et al. 2019 ). It opens a question whether the double-infected populations are stable or just transient stadia of the Wolbachia and Cardinium battle in the mite hosts. Stored product mite Tyrophagus putrescentiae (Schrank, 1781) cultures are infected either by Wolbachia or Cardinium , and the infections appear stable when cultures are maintained in the laboratory (Erban et al. 2016 ; Hubert et al. 2021a ). No double-infection ( Cardinium + Wolbachia ) occurred on individual and population levels (Hubert et al. 2021a ). However, artificial mixing of single-infected Cardinium and Wolbachia cultures resulted in double-infected culture, where Cardinium and Wolbachia co-occurred on both individual and population levels (Hubert et al. 2021b ). In the experiment, the presence of Cardinium was negatively correlated with the presence of Wolbachia (Hubert et al. 2021b ). It indicated competition between these bacteria, but the mechanism involved has not been known in mites. In the previous experiment, we prepared the mixed Cardinium / Wolbachia mite cultures from parental cultures infected by Cardinium or Wolbachia , only. The mixed cultures had lower abundances of Wolbachia , while the abundance of Cardinium did not change, and double-infected cultures had lower population growth than parental single-infected cultures (Hubert et al. 2021b ). It indicated that Cardinium inhibits the growth of Wolbachia in mixed cultures (Hubert et al. 2021b ). The experiment was done only with two cultures thus we could not generalize the observed pattern. Here, we used to more cultures of a different origin Cardinium or Wolbachia . By combining all four populations we obtained four artificial cultures, which were further run in three different long-term experiments. We measured the fitness of all populations and estimated prevalence of bacteria using PCR with taxa-specific primers. Materials and methods Single-infected cultures For the experiments, four cultures of T. putrescentiae that were infected with either Cardinium (c1 and c2) or Wolbachia (w1 and w2) were used; for a detailed description, see (Table 1 ). The cultures were maintained at the Crop Research Institute in Prague, Czechia. The mites were kept in IWAKI 70 mL tissue culture flasks with a surface area of 25 cm 2 . These flasks were placed in Secador desiccators by Bel-Art Products, which maintained a relative humidity of 85% through a saturated KCl solution. The desiccators were kept in darkness under controlled conditions of humidity (75% RH) and temperature (25 ± 1°C). The mites were fed a diet called SPMd, which consisted of wheat germ and Mauripan-dried yeast extract ( Saccharomyces cerevisiae ) in a 10:1 w/w proportion. The diet was mill-powdered, sieved (mesh size, 500 µm), and heated to 70°C for 0.5 h before being fed to the mites. Table 1 The list of single-infected cultures of Tyrophagus putrescentiae used in the experiment. The origin of the cultures, symbionts and rearing diet are provided. The symbiont profile in mite bacterila microbiome is provided based on the mean (± standard deviation) values from the data published previsouly (Hubert et al. 2021a ). Name ID Population Collector Year Diet Site Symbionts c1 5L laboratory E. Zdarkova 1996 SPMd grain, Bustehrad, Czechia Cardinium 45 ± 3% w1 5N dog J. Hubert 2007 F food producing factory, St. Louis, Missouri, USA Wolbachia 62 ± 6 % w2 5P Phillips T. W. Phillips 2014 SPMd laboratory strain, Manhattan, Kansas, USA Wolbachia 60 ± 2 % c2 5S ham A. Sala 2013 SPMd food-producing factory, Cesena, Italy Cardinium 53 ± 4% Legend: SPMd – stored product mite diet, F – dog kernel, Purina Pro Plan FOCUS Adult Sensitive Skin & Stomach Salmon & Rice Formula Dry Dog Food. Double-infected cultures To create mixed cultures, we transferred 10 unsexed adults from a Cardinium -infected culture (c1 or c2) and another 10 from a Wolbachia -infected culture (w1 or w2) into a new flask. We made sure to have c1w1, c1w2, c2w1, and c2w2 in each flask, with 10 mites in every combination in 6 replicates. Each replicate was carried out in a separate flask that contained 0.3 g of SPMd. The flasks containing double-infected mites were stored in desiccators under the same conditions as used for mite rearing. Every culture was renewed monthly by transferring around 5,000 live mites from the cap or surface of the flask into a new flask containing 0.3 g of SPMd. The remaining mites in the parent flask were used for growth tests and DNA extraction. For DNA extraction, the mites were collected from the flask caps and surface, transferred into 70% ethanol, and stored in a freezer at − 40°C before extraction. The mites were re-transferred into a new flask every month. This experimental was repeated three times. DNA extraction For DNA extraction, mites were collected after 2, 3, 4, and 5 months of incubation and mite samples were processed. We pooled all mites from the treatment and experimental time altogether and then we selected 30 mites. We followed the procedure below to extract DNA from a single mite, 30 mites were randomly collected from 6 chambers and placed into ethanol. Then ethanol was dried, and mites were transferred singly into a 0.2 mL thin wall tube (Thermo Scientific™, cat no: AB0620) that contained 25 µL of DEP-25 START‐Blue reagent (cat no: D226). The tube was then heated to 95°C for 20 minutes using a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). After heating, the tube was cooled to room temperature and 25 µl of DEP-25 STOP solution was added and mixed by vortexing. The samples were stored in a deep freezer prior to PCR analyses. PCR reaction PCR reactions were carried out using the master mix EmeraldAmp (catalogue number: RR310A, Takara Bio). The master mix contained an optimized buffer, PCR enzyme, dNTP mixture, gel loading dye (green), and a density reagent in a 2X premix format. Subsequently, ddH2O and primers were added to the mix. The amplification process was carried out using the C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The detection of Wolbachia WpF (5’-TTGTAGCCTGCTATGGTA-3’) and WpR (5’-GAATAGGTATGATTTTCA-3’) primers (O’Neill et al. 1992 ) was used with the following amplification profile: initial denaturation at 94°C for 5 minutes, followed by 35 cycles of 95°C for 60 seconds, 52°C for 60 seconds, and 72°C for 60 seconds. The final extension was done at 72°C for 5 minutes. For the detection of Cardinium , we used the Card4 (5’-CTTAACGCTAGAACTGCGA-3’) and Card6 (5’-TCAAGCTCTACCAACTCC-3’) primers (Kopecky et al. 2013 ) and conducted amplification with the following protocol: initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 50 seconds, annealing at 56°C for 50 seconds, extension at 72°C for 60 seconds, and final extension at 72°C for 10 minutes. The reaction mixture contained 2 µL of DNA, 12.5 µL of EmeraldAmp master mix, 8.5 µL of ddH2O, and 1 µL of each 10 µM primer. We used a negative control with DNA replaced by ddH2O and a positive control with cloned DNA previously obtained by amplification of mite extracts using universal bacterial primers (F27 and 1492R) (Lane 1991 ). The PCR products were observed on a 1% gel using the GeneSnap (Syngene InGenius LHR2 Gel Imaging System; cat. no: 316616). For preparing the 1% gel, 1.5 g of agarose (Lonza SeaKem® LE 500 g, cat no: 50004, Lonza, USA) was mixed with 150 mL of buffer (ROTIPHORESE® Buffer 50 x TAE, cat no: R.CL86.2, Carl Roth, Germany). The agarose was dissolved in hot a buffer and then cooled down under constant stirring. After that, 8 µL of SYBR® Safe DNA Gel Stain (cat no: S33102, Invitrogen, USA) was added to the solution. The diluted SYBR® Safe DNA Gel Stain was made by using 10 µL of SYBR® Safe DNA Gel Stain and 90 µL of dimethyl sulfoxide - DMSO. The size of the products was measured using a 50 bp ladder (Generuler 50bp, cat no: SM0373, ThermoFisher Scientific). The amplification process was successful when the PCR products were visible and were the expected size. The asymbiotic mite individuals were identified based on the presence of the product from universal bacterial primers and the absence of the product from Cardinium and/or Wolbachia primers. Mite growth test The population growth of mites in the original double-infected stock cultures was recorded at two-month intervals. The first growth test was established after 2 months of incubation. Mites were collected from the plugs and surface of rearing flasks and then transferred to separate Petri dishes. The controls consisted of a single-infected population. Ten unsexed adult mites were moved from a Petri dish to new flasks that contained 0.01 ± 0.005 grams of SPMd. The flasks were kept under controlled conditions. After 21 days, the experiment ended, and mites were counted using a dissection microscope (Hubert et al. 2016 ). Statistical analyses For the data analyses, we used a measure of population fitness, which was calculated as the intrinsic rate of population increase ( r ) assuming exponential population growth as the time was short and resources were not limited. We used the formula for density-independent continuous population growth: N t = N 0 e rt , where N t is the final mite density, N 0 is the initial mite density, and t = 21 days (the duration of mite population growth). The intrinsic rates had a distribution not different from the normal ; thus, differences among populations were studied using a general linear model (LM). To test for consistency (repeatability) in the rate in the same population mixtures grown in three experiments, we used linear mixed-effect models (LME) from the nlme package (Pinheiro and Bates 2000 ). The intrinsic rate was the response variable, and population type and time were explanatory variables. The experiment was a random effect. We calculated the intraclass correlation coefficient (ICC) from the variance estimated as a measure of consistency. The prevalence of endosymbionts in single-infected populations were analyzed with Generalized linear models with binomial error structure (GLM-b) (Pekar and Brabec 2016 ). The prevalences (after applying an angular transformation to approach normal distribution) in mixture populations were compared among population types and time using LME in order to estimate consistency in prevalence using ICC. Eventually, the effect of prevalence on the fitness of populations was studied using LM. All analyses were performed in R (R Development Core Team 2023). Results Fitness The single-infected cultures of T. putrescentiae reached different numbers after 21 days so their fitness was significantly different (LM, F 3,20 = 27.6, P < 0.001, Fig. 1 A). One population of those infected by Cardinium (c2) and one from those by Wolbachia (w1) had significantly higher fitness than the other two. The four multi-infected cultures showed significantly different rates of population increase (LME, F 6,202 = 2.7, P = 0.015). After one month, all four cultures increased at a similar rate (Fig. 1 B). After three months, the population growth increased at lower rates, but only for c1w2, which was significantly lower than after one month. After five months, all cultures increased at significantly different rates: the c2w1 and c2w2 had higher fitness than c1w2 and c1w1. These two cultures had fitness that was not significantly different from single-infected cultures (w2 and c2). The low intraclass correlation coefficient (0.19) shows that the three experiments had low consistency. Prevalence of endosymbionts The prevalence of endosymbionts in single-infected cultures was significantly different between bacteria species (GLM, χ 2 2 = 89.4, P < 0.0001): prevalence was higher in Cardinium infected cultures than in Wolbachia infected cultures (Fig. 2 A); among cultures (GLM, χ 2 3 = 50.0, P < 0.0001): c1 and w1 had higher prevalence than c2 and w2; and between experiments (GLM, χ 2 1 = 14.3, P = 0.0002): the first experiment had generally higher prevalence than the second experiment. In the double-infected cultures, the prevalence of the two bacteria varied significantly among cultures (LME, F 6,154 = 2.42, P = 0.028). In all cultures, the prevalence of both endosymbionts fluctuated over six months but not significantly (LME, F 1,142 = 0.3, P = 0.61). The consistency of the prevalence in the three experiments was nearly zero (ICC < 0.01). After six months, the Cardinium -infected individuals prevailed in 50% of the mite cultures, the Wolbachia -infected individuals prevailed in 41.7%, and asymbiotic individuals in 8.3% of cultures (N = 12). In the c1w1 culture on average, Cardinium infected individuals always prevailed over Wolbachia infected individuals, which decreased, so there were 2.6times more Cardinium infected individuals at the end. In the c1w2 culture, on average, Cardinium infected individuals gradually decreased over Wolbachia infected individuals, which increased, so at the end, there were 11-times more Wolbachia infected individuals. In the c2w1 culture, on average, Cardinium- infected individuals always prevailed over Wolbachia -infected individuals, which did not change, so in the end, there were almost 2 times more Cardinium infected. In the c2w2 culture, on average, both Cardinium infected and Wolbachia infected individuals decreased so that, in the end, the majority were asymbiotic individuals. Few individuals in the mixed culture were double-infected by both Wolbachia and Cardinium . The frequency of their occurrence was not explained by the time (GLM-qb, F 1,52 = 13.0, P = 0.08) or the experiment (GLM-qb, F 2,53 = 4.0, P = 0.60) but by the culture type (GLM-qb, F 3,49 = 47.1, P = 0.014): there were significantly more double-infected individuals in the c2w1 than in other cultures (Fig. 3 ). By combining data on fitness and prevalence, we found that neither occurrence of Wolbachia- infected individuals (LM, F 1.34 < 0.1, P = 0.97), Cardinium -infected individuals (LM, F 1.34 = 1.4, P = 0.25) nor asymbiotic individuals (LM, F 1.34 = 2.9, P = 0.097) affected the fitness of cultures after 21 days. Discussion The study showed that the multiple infections on the population level in T. putrescentiae are unstable, and the mixing of parental cultures resulted in a decrease of fitness in the initial time of experiments. Only two double infected cultures achieved a similar population growth rate as parental single infected cultures. The process of infection is stochastic (i.e. genetic drift (Jansen et al. 2008 )) and can result in all possible situations, i.e. Cardinium or Wolbachia wins. Still also both symbionts can lose and the mite culture switch to asymbiotic. The mean generation time of T. putrescentiae was found to be 17 days at conditions used (Sanchez-Ramos and Castanera 2005 ), suggesting that in our study there were approximately ten generations. Although the cultures were multiple-infected, double-infected individuals were rare based on our data. This situation differs from Tetranychus spider mites where the field population showed variability in intracellular symbiont ( Cardinium , Rickettsia , and Wolbachia ) prevalence (Zele et al. 2018a ). However, the laboratory experiment showed apparent loss of Rickettsia and Cardinium , but not Wolbachia , during 6 months of experiments (15 generations) (Zele et al. 2020 ). The natural prevalence of symbionts in Tetranychus populations (N = 16) decreased from Wolbachia (61%), followed by Cardinium (12–15%) to Rickettsia (0.9–3%) (Zele et al. 2018a ). In T. putrescentiae (N = 9), both Cardinium and Wolbachia infect the same proportion (22%), and the rest of the cultures (33%) are without intracellular symbionts (Hubert et al. 2021a ). In double-infected Tetranychus piercei , the males had suppressed Wolbachia while accelerating the growth of Cardinium , while single and double-infected females had the same level of Cardinium , indicating competition between symbionts (Zhu et al. 2012 ). However, in T. putrescentiae , it is a different situation due to the low proportion of double-infected individuals, but the competition is still possible at a population level, and the low number of double-infected individuals should be the result of such competition. The indirect evidence of the competition is the decrease in population growth of all multiple-infected cultures compared to parental cultures, which still exist in some cultures during the experiment. It is currently unknown whether cytoplasmic incompatibility (CI) exists for T. putrescentiae infections. Both Cardinium and Wolbachia are well documented to induce CI in Tetranychus and Bryoba mites (Breeuwer 1997 ; Gotoh et al. 1995 , 2007 ). However, CI induced by Wolbachia differed among Tetranychus populations (Zele et al. 2020 ). In the double-infected parasitoid wasp Encarsia inaron , Wolbachia caused CI, whereas Cardinium did not. In the contrast, T. urticae double-infection of Wolbachia and Cardinium induced strong CI (Xie et al. 2016 ). In this study, the decreases in fitness should be the result of CI in multi-infected cultures at the beginning of experiments. Alternatively, the subsequent effect on mite physiology and microbiome can explain the population decrease. For example, Eurema hecabe , growth rates evaluated by development time were slower in progenies of Wolbachia double-infected mothers than in those of single-infected mothers (Li et al. 2022 ). Cardinium and combined Cardinium + Wolbachia infections led to a reduction in bacterial diversity, alteration of bacterial community structure, and metabolic changes, which had negative fitness effects on the host (Li et al. 2022 ). The study revealed different processes in competition between symbionts in Tetranychus mites. The genetic difference of symbionts should explain the difference mentioned above in the studies. In this study we used atypical intracellular symbionts, i.e. Wolbachia is a member of a novel supergroup Q of mite symbionts (unpublished data, GenBank Acess No JAUEMM01 and GIJY01), while Wolbachia from Tetranychus AH2012_wTtru (NZ_CP099592) is similar to insect Wolbachia (e.g. Wolbachia symbiont of Bemisia tabaci (NZ_CP016430). The genome analyses of Cardinium (JAUEML01) revealed that it forms a separate cluster of symbionts of the planthopper Sogatella furcifera (cSFur) (Zeng et al. 2018 ). In addition, the subsequent genome analysis revealed that both intracellular bacteria are identical in T. putrescentiae cultures (unpublished data). Whether genome differences are responsible for the different effects of symbionts on the host is the further direction of studies. The success of mites is based on the spreading via insects, animals including humans, and almost exponential growth under suitable conditions (Aspaly et al. 2007 ). In T. putrescentiae , 39 allergen groups have been identified (Zhou et al. 2023 ). The previous study showed that T. putrescentiae cultures with distinct microbiomes differ in the expression of allergens; namely Wolbachia inhabited cultures associated allergens belonging to groups no. 4, 6, 7, 13, 20, and 36, while asymbiotic culture-associated allergens from groups no. 5, 28, 30, and 35 (Hubert et al. 2019 ). Here, we found that the mites and their physiological features are determined by intracellular factors, providing a high degree of variability during the growth of the mite population in stored food. It means that symbionts determine allergen production in terms of the allergen production due to different mite populations’ growth and by different expressions as the response of mite-influenced immune/regulatory pathways. The stochastic models of symbiont infection are based on the low initial numbers of mites that establish the population (Jansen et al. 2008 ; Zele et al. 2020 ). The faunistic analyses of the stored grain fulfil the low initial population of T. putrescetnaie ; i.e. 14% of samples (N = 514) with density lower than 1 ind.g − 1 grain, while 1% of samples with 5 and more individuals g − 1 grain (Stejskal and Hubert 2008 ). It means that natural conditions favour the stochastic symbiont selection model. From a practical point of view, T. putrecentiae is an important pest of stored products infecting cereals, dried ham and sausages, cheeses and dog food (Olivry and Mueller 2019 ; Robertson 1961 ; Stejskal et al. 2015 ; Zhang et al. 2018 ). Population density is the key to pest importance, and Tyrophagus mites can reach massive biomass. Such contaminated food can avoid anaphylactic shock (Sanchez-Borges et al. 2005 ). There is an effort to establish a model of the population growth based on the abiotic condition in the stored food (Pekar and Zdarkova 2004 ; Sanchez-Ramos and Castanera 2001 , 2005 ). Declarations Funding The study was supported by the project of the Czech Science Foundation GF22-15841K. Author Contribution JH and EG designed and organized experiments; SP provided statistical analyses; all authors wrote the manuscript. Acknowledgements The authors are obligated to Eliska Tresnakova, Marta Nesvorna, and Martin Markovic for technical help. References Aspaly G, Stejskal V, Pekar S, Hubert J (2007) Temperature-dependent population growth of three species of stored product mites (Acari: Acaridida). Exp Appl Acarol 42(1):37–46. https://doi.org/10.1007/s10493-007-9074-1 Bing X-L, Zhao D-S, Hong X-Y (2019) Bacterial reproductive manipulators in rice planthoppers. Arch Insect Biochem Physiol 101(2):e21548. https://doi.org/10.1002/arch.21548 Breeuwer H, Ros VID, Groot TVM (2012) Cardinium : the next addition to the family of reproductive parasites. In: Zchori-Fein E, Bourtzis K (eds) Manipulative tenants: bacteria associated with arthropods. 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Springer, New York, NY. https://doi.org/10.1007/b98882 R Core Team (2023) R: a language and environment for statistical computing, version 4.3.1. R Foundation for Statistical Computing, Vienna. http://www.R-project.org . Accessed 23 November 2023 Robertson PL (1961) A morphological study of variation in Tyrophagus (Acarina), with particular reference to populations infesting cheese. Bull Entomol Res 52(3):501–529. https://doi.org/10.1017/S0007485300055565 Ros VID, Breeuwer JAJ (2009) The effects of, and interactions between, Cardinium and Wolbachia in the doubly infected spider mite Bryobia sarothamni . Heredity 102(4):413–422. https://doi.org/10.1038/hdy.2009.4 Ros VID, Fleming VM, Feil EJ, Breeuwer JAJ (2012) Diversity and recombination in Wolbachia and Cardinium from Bryobia spider mites. BMC Microbiol 12(Suppl 1):S13. https://doi.org/10.1186/1471-2180-12-S1-S13 Sanchez-Borges M, Suarez-Chacon R, Capriles-Hulett A, Caballero-Fonseca F (2005) An update on oral anaphylaxis from mite ingestion. Ann Allergy Asthma Immunol 94(2):216–221. https://doi.org/10.1016/S1081-1206(10)61298-7 Sanchez-Ramos I, Castanera P (2001) Development and survival of Tyrophagus putrescentiae (Acari: Acaridae) at constant temperatures. Environ Entomol 30(6):1082–1089. https://doi.org/10.1603/0046-225X-30.6.1082 Sanchez-Ramos I, Castanera P (2005) Effect of temperature on reproductive parameters and longevity of Tyrophagus putrescentiae (Acari: Acaridae). Exp Appl Acarol 36(1–2):93–105. https://doi.org/10.1007/s10493-005-0506-5 Schneider D, Miller WJ, Riegler M (2012) Arthropods shopping for Wolbachia . In: Zchori-Fein E, Bourtzis K (eds) Manipulative tenants: bacteria associated with arthropods. CRC, Boca Raton, FL, pp 149–174. https://doi.org/10.1201/b11008-13 Stejskal V, Hubert J (2008) Risk of occupational allergy to stored grain arthropods and false pest-risk perception in Czech grain stores. Ann Agric Environ Med 15(1):29–35 Stejskal V, Hubert J, Aulicky R, Kucerova Z (2015) Overview of present and past and pest-associated risks in stored food and feed products: European perspective. J Stored Prod Res 64:122–132. https://doi.org/10.1016/j.jspr.2014.12.006 Xie R-r, Sun J-t, Xue X-f, Hong X-Y (2016) Cytoplasmic incompatibility and fitness benefits in the two-spotted spider mite Tetranychus urticae (red form) doubly infected with Wolbachia and Cardinium . Syst Appl Acarol 21(9):1161–1173. https://doi.org/10.11158/saa.21.9.1 Zele F, Santos I, Matos M, Weill M, Vavre F, Magalhaes S (2020) Endosymbiont diversity in natural populations of Tetranychus mites is rapidly lost under laboratory conditions. Heredity 124(4):603–617. https://doi.org/10.1038/s41437-020-0297-9 Zele F, Santos I, Olivieri I, Weill M, Duron O, Magalhaes S (2018a) Endosymbiont diversity and prevalence in herbivorous spider mite populations in South-Western Europe. FEMS Microbiol Ecol 94(4):fiy015. https://doi.org/10.1093/femsec/fiy015 Zele F, Weill M, Magalhaes S (2018b) Identification of spider-mite species and their endosymbionts using multiplex PCR. Exp Appl Acarol 74(2):123–138. https://doi.org/10.1007/s10493-018-0224-4 Zeng Z, Fu Y, Guo D, Wu Y, Ajayi OE, Wu Q (2018) Bacterial endosymbiont Cardinium cSfur genome sequence provides insights for understanding the symbiotic relationship in Sogatella furcifera host. BMC Genomics 19(1):688. https://doi.org/10.1186/s12864-018-5078-y Zhang X, Hendrix JD, Campbell YL, Phillips TW, Goddard J, Cheng W-H, Kim T, Wu T-L, Schilling MW (2018) Biology and integrated pest management of Tyrophagus putrescentiae (Schrank) infesting dry cured hams. J Stored Prod Res 79:16–28. https://doi.org/10.1016/j.jspr.2018.08.001 Zhou Y, Klimov PB, Gu X, Yu Z, Cui X, Li Q, Pan R, Yuan C, Cai F, Cui Y (2023) Chromosome-level genomic assembly and allergome inference reveal novel allergens in Tyrophagus putrescentiae . Allergy 78(6):1691–1695. https://doi.org/10.1111/all.15656 Zhu L-Y, Zhang K-J, Zhang Y-K, Ge C, Gotoh T, Hong X-Y (2012) Wolbachia strengthens Cardinium -induced cytoplasmic incompatibility in the spider mite Tetranychus piercei McGregor. Curr Microbiol 65(5):516–523. https://doi.org/10.1007/s00284-012-0190-8 Zytynska SE (2019) Cohabitation and roommate bias of symbiotic bacteria in insect hosts. Mol Ecol 28(24):5199–5202. https://doi.org/10.1111/mec.15295 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4806277","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":339689678,"identity":"b764428c-2b0e-4585-be0d-b154ab2cfb50","order_by":0,"name":"Jan Hubert","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYPACCwYGCSD1AYjZ2InTIgFGjDNAWphJ0cLMA2IT0sLffvbZgw+/JBK3S/cYf7b5tU2ej5mB8cPHHDzGn0k3N5zZJ5G4c84ZM+ncvtuGbcwMzJIzt+HWYsCQxibN2yORuOFGjhlzbs9tRqAWNmZefFr4n8G1GH+27LltT1iLBNAWnh9gLQbSDD9uJxLUInHjGbvhzAYJ450z0sokextuJ7cxMzbj9Qt/fxrbgw9/bGS3SyRv/vDjz23b+e3NBz98xKMFCNgYGNsYHDeAmEAGiGzAqx6sheEPg70BmP2HkOJRMApGwSgYiQAAfaNO8tgGB1MAAAAASUVORK5CYII=","orcid":"","institution":"Crop Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Jan","middleName":"","lastName":"Hubert","suffix":""},{"id":339689679,"identity":"83ab1418-314a-4bcd-bf12-4a1f7af90d38","order_by":1,"name":"Eliza Glowska","email":"","orcid":"","institution":"Adam Mickiewicz University in Poznan","correspondingAuthor":false,"prefix":"","firstName":"Eliza","middleName":"","lastName":"Glowska","suffix":""},{"id":339689681,"identity":"edc83b7f-aab4-4bbe-bac8-fae6d43d5895","order_by":2,"name":"Stano Pekar","email":"","orcid":"","institution":"Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Stano","middleName":"","lastName":"Pekar","suffix":""}],"badges":[],"createdAt":"2024-07-26 07:51:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4806277/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4806277/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.jspr.2025.102663","type":"published","date":"2025-04-23T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63015616,"identity":"dfd42e1a-2c0e-4044-b69a-856da6505fd6","added_by":"auto","created_at":"2024-08-22 06:40:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23052,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the intrinsic rate of \u003cem\u003eTyrophagus putrescentiae\u003c/em\u003e population growth (\u003cem\u003er\u003c/em\u003e) among four single-infected cultures (\u003cstrong\u003eA\u003c/strong\u003e) and among four double-infected cultures over three months (\u003cstrong\u003eB\u003c/strong\u003e). Horizontal lines are estimated means, grey boxes are 95% confidence intervals.\u003c/p\u003e","description":"","filename":"fig114.png","url":"https://assets-eu.researchsquare.com/files/rs-4806277/v1/3e5003799960ff381365192d.png"},{"id":63016404,"identity":"096da217-26e2-4ae7-8ab4-fa44dbd4d1b2","added_by":"auto","created_at":"2024-08-22 06:48:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34290,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the prevalence of \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e in the single-infected mite \u003cem\u003eTyrophagus putrescentiae\u003c/em\u003e cultures (2 replicates) and in the double-infected mite culture over six months.\u003c/p\u003e","description":"","filename":"fig215.png","url":"https://assets-eu.researchsquare.com/files/rs-4806277/v1/4638d19a04643a69b4410083.png"},{"id":63015622,"identity":"77db4b30-cdfe-43bc-a14d-5be6116d1d58","added_by":"auto","created_at":"2024-08-22 06:40:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9274,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the relative frequency of double-infected individuals of \u003cem\u003eTyrophagus putrescentiae\u003c/em\u003e in four mixed populations. Horizontal lines are estimated means, grey boxes are 95% confidence intervals.\u003c/p\u003e","description":"","filename":"fig313.png","url":"https://assets-eu.researchsquare.com/files/rs-4806277/v1/83bcb1905c6ebdb9b3846754.png"},{"id":95946915,"identity":"d443bcfc-5914-40b4-a3d7-6a510302a366","added_by":"auto","created_at":"2025-11-14 17:56:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":681632,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4806277/v1/c1aeb06f-46b9-4b7d-a92e-17944bb3e51e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The battle of Cardinium and Wolbachia in double-infected mite cultures","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMany arthropods are hosts of intracellular bacteria, such as \u003cem\u003eWolbachia\u003c/em\u003e and \u003cem\u003eCardinium\u003c/em\u003e (Breeuwer et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Schneider et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These bacteria are maternally transmitted and possess mechanisms manipulating the host behavior to achieve successful distribution in the host populations. Best known manipulations include male killing, feminization, thelytokous parthenogenesis, and cytoplasmic incompatibility (CI), and result in an increased proportion of infected females in the host population (Perlman et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ros and Breeuwer \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMites are also frequently infected by intracellular bacteria. For example, \u003cem\u003eTetranychus\u003c/em\u003e populations (N\u0026thinsp;=\u0026thinsp;16) were mostly infected by \u003cem\u003eWolbachia\u003c/em\u003e (ca. 61%), followed by \u003cem\u003eCardinium\u003c/em\u003e (12\u0026ndash;15%), and \u003cem\u003eRickettsia\u003c/em\u003e (0.9\u0026ndash;3%) (Zele et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). Mite individuals might be infected by one to several intracellular bacteria (Brown et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Duron et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Gottlieb et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zele et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zytynska \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The double infections of \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e have been reported in several \u003cem\u003eBryobia\u003c/em\u003e (Ros et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and \u003cem\u003eTetranychus\u003c/em\u003e (Gotoh et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ros et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) species. The co-infection by \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e led to various interactions that were usually interpreted only via correlations in the context of their occurrence (Mathe-Hubert et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zytynska \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, a few studies revealed the co-infection effects (Brinker et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bustamante-Brito et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMite co-infected with multiple symbiont taxa represent a complex system with many interactions between these symbionts and the host. Such systems alter host physiological responses and survival (Bing et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It opens a question whether the double-infected populations are stable or just transient stadia of the \u003cem\u003eWolbachia\u003c/em\u003e and \u003cem\u003eCardinium\u003c/em\u003e battle in the mite hosts.\u003c/p\u003e \u003cp\u003eStored product mite \u003cem\u003eTyrophagus putrescentiae\u003c/em\u003e (Schrank, 1781) cultures are infected either by \u003cem\u003eWolbachia\u003c/em\u003e or \u003cem\u003eCardinium\u003c/em\u003e, and the infections appear stable when cultures are maintained in the laboratory (Erban et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hubert et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). No double-infection (\u003cem\u003eCardinium\u0026thinsp;+\u0026thinsp;Wolbachia\u003c/em\u003e) occurred on individual and population levels (Hubert et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). However, artificial mixing of single-infected \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e cultures resulted in double-infected culture, where \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e co-occurred on both individual and population levels (Hubert et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). In the experiment, the presence of \u003cem\u003eCardinium\u003c/em\u003e was negatively correlated with the presence of \u003cem\u003eWolbachia\u003c/em\u003e (Hubert et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). It indicated competition between these bacteria, but the mechanism involved has not been known in mites.\u003c/p\u003e \u003cp\u003eIn the previous experiment, we prepared the mixed \u003cem\u003eCardinium\u003c/em\u003e/\u003cem\u003eWolbachia\u003c/em\u003e mite cultures from parental cultures infected by \u003cem\u003eCardinium\u003c/em\u003e or \u003cem\u003eWolbachia\u003c/em\u003e, only. The mixed cultures had lower abundances of \u003cem\u003eWolbachia\u003c/em\u003e, while the abundance of \u003cem\u003eCardinium\u003c/em\u003e did not change, and double-infected cultures had lower population growth than parental single-infected cultures (Hubert et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). It indicated that \u003cem\u003eCardinium\u003c/em\u003e inhibits the growth of \u003cem\u003eWolbachia\u003c/em\u003e in mixed cultures (Hubert et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). The experiment was done only with two cultures thus we could not generalize the observed pattern.\u003c/p\u003e \u003cp\u003eHere, we used to more cultures of a different origin \u003cem\u003eCardinium\u003c/em\u003e or \u003cem\u003eWolbachia\u003c/em\u003e. By combining all four populations we obtained four artificial cultures, which were further run in three different long-term experiments. We measured the fitness of all populations and estimated prevalence of bacteria using PCR with taxa-specific primers.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSingle-infected cultures\u003c/h2\u003e \u003cp\u003eFor the experiments, four cultures of \u003cem\u003eT. putrescentiae\u003c/em\u003e that were infected with either \u003cem\u003eCardinium\u003c/em\u003e (c1 and c2) or \u003cem\u003eWolbachia\u003c/em\u003e (w1 and w2) were used; for a detailed description, see (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The cultures were maintained at the Crop Research Institute in Prague, Czechia. The mites were kept in IWAKI 70 mL tissue culture flasks with a surface area of 25 cm\u003csup\u003e2\u003c/sup\u003e. These flasks were placed in Secador desiccators by Bel-Art Products, which maintained a relative humidity of 85% through a saturated KCl solution. The desiccators were kept in darkness under controlled conditions of humidity (75% RH) and temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C). The mites were fed a diet called SPMd, which consisted of wheat germ and Mauripan-dried yeast extract (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e) in a 10:1 w/w proportion. The diet was mill-powdered, sieved (mesh size, 500 \u0026micro;m), and heated to 70\u0026deg;C for 0.5 h before being fed to the mites.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe list of single-infected cultures of \u003cem\u003eTyrophagus putrescentiae\u003c/em\u003e used in the experiment. The origin of the cultures, symbionts and rearing diet are provided. The symbiont profile in mite bacterila microbiome is provided based on the mean (\u0026plusmn;\u0026thinsp;standard deviation) values from the data published previsouly (Hubert et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCollector\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSymbionts\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elaboratory\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eE. Zdarkova\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSPMd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003egrain, Bustehrad, Czechia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eCardinium\u003c/em\u003e 45\u0026thinsp;\u0026plusmn;\u0026thinsp;3%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ew1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edog\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJ. Hubert\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efood producing factory, St. Louis, Missouri, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eWolbachia\u003c/em\u003e 62\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003cem\u003e%\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ew2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhillips\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT. W. Phillips\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSPMd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003elaboratory strain, Manhattan, Kansas, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eWolbachia\u003c/em\u003e 60\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003cem\u003e%\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eham\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA. Sala\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSPMd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efood-producing factory, Cesena, Italy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eCardinium\u003c/em\u003e 53\u0026thinsp;\u0026plusmn;\u0026thinsp;4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eLegend: SPMd \u0026ndash; stored product mite diet, F \u0026ndash; dog kernel, Purina Pro Plan FOCUS Adult Sensitive Skin \u0026amp; Stomach Salmon \u0026amp; Rice Formula Dry Dog Food.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDouble-infected cultures\u003c/h2\u003e \u003cp\u003eTo create mixed cultures, we transferred 10 unsexed adults from a \u003cem\u003eCardinium\u003c/em\u003e-infected culture (c1 or c2) and another 10 from a \u003cem\u003eWolbachia\u003c/em\u003e-infected culture (w1 or w2) into a new flask. We made sure to have c1w1, c1w2, c2w1, and c2w2 in each flask, with 10 mites in every combination in 6 replicates. Each replicate was carried out in a separate flask that contained 0.3 g of SPMd.\u003c/p\u003e \u003cp\u003eThe flasks containing double-infected mites were stored in desiccators under the same conditions as used for mite rearing. Every culture was renewed monthly by transferring around 5,000 live mites from the cap or surface of the flask into a new flask containing 0.3 g of SPMd. The remaining mites in the parent flask were used for growth tests and DNA extraction. For DNA extraction, the mites were collected from the flask caps and surface, transferred into 70% ethanol, and stored in a freezer at \u0026minus;\u0026thinsp;40\u0026deg;C before extraction. The mites were re-transferred into a new flask every month. This experimental was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction\u003c/h2\u003e \u003cp\u003eFor DNA extraction, mites were collected after 2, 3, 4, and 5 months of incubation and mite samples were processed. We pooled all mites from the treatment and experimental time altogether and then we selected 30 mites. We followed the procedure below to extract DNA from a single mite, 30 mites were randomly collected from 6 chambers and placed into ethanol. Then ethanol was dried, and mites were transferred singly into a 0.2 mL thin wall tube (Thermo Scientific\u0026trade;, cat no: AB0620) that contained 25 \u0026micro;L of DEP-25 START‐Blue reagent (cat no: D226). The tube was then heated to 95\u0026deg;C for 20 minutes using a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). After heating, the tube was cooled to room temperature and 25 \u0026micro;l of DEP-25 STOP solution was added and mixed by vortexing. The samples were stored in a deep freezer prior to PCR analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePCR reaction\u003c/h2\u003e \u003cp\u003ePCR reactions were carried out using the master mix EmeraldAmp (catalogue number: RR310A, Takara Bio). The master mix contained an optimized buffer, PCR enzyme, dNTP mixture, gel loading dye (green), and a density reagent in a 2X premix format. Subsequently, ddH2O and primers were added to the mix. The amplification process was carried out using the C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003cp\u003eThe detection of \u003cem\u003eWolbachia\u003c/em\u003e WpF (5\u0026rsquo;-TTGTAGCCTGCTATGGTA-3\u0026rsquo;) and WpR (5\u0026rsquo;-GAATAGGTATGATTTTCA-3\u0026rsquo;) primers (O\u0026rsquo;Neill et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) was used with the following amplification profile: initial denaturation at 94\u0026deg;C for 5 minutes, followed by 35 cycles of 95\u0026deg;C for 60 seconds, 52\u0026deg;C for 60 seconds, and 72\u0026deg;C for 60 seconds. The final extension was done at 72\u0026deg;C for 5 minutes.\u003c/p\u003e \u003cp\u003eFor the detection of \u003cem\u003eCardinium\u003c/em\u003e, we used the Card4 (5\u0026rsquo;-CTTAACGCTAGAACTGCGA-3\u0026rsquo;) and Card6 (5\u0026rsquo;-TCAAGCTCTACCAACTCC-3\u0026rsquo;) primers (Kopecky et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and conducted amplification with the following protocol: initial denaturation at 94\u0026deg;C for 5 minutes, followed by 35 cycles of denaturation at 94\u0026deg;C for 50 seconds, annealing at 56\u0026deg;C for 50 seconds, extension at 72\u0026deg;C for 60 seconds, and final extension at 72\u0026deg;C for 10 minutes. The reaction mixture contained 2 \u0026micro;L of DNA, 12.5 \u0026micro;L of EmeraldAmp master mix, 8.5 \u0026micro;L of ddH2O, and 1 \u0026micro;L of each 10 \u0026micro;M primer. We used a negative control with DNA replaced by ddH2O and a positive control with cloned DNA previously obtained by amplification of mite extracts using universal bacterial primers (F27 and 1492R) (Lane \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe PCR products were observed on a 1% gel using the GeneSnap (Syngene InGenius LHR2 Gel Imaging System; cat. no: 316616). For preparing the 1% gel, 1.5 g of agarose (Lonza SeaKem\u0026reg; LE 500 g, cat no: 50004, Lonza, USA) was mixed with 150 mL of buffer (ROTIPHORESE\u0026reg; Buffer 50 x TAE, cat no: R.CL86.2, Carl Roth, Germany). The agarose was dissolved in hot a buffer and then cooled down under constant stirring. After that, 8 \u0026micro;L of SYBR\u0026reg; Safe DNA Gel Stain (cat no: S33102, Invitrogen, USA) was added to the solution. The diluted SYBR\u0026reg; Safe DNA Gel Stain was made by using 10 \u0026micro;L of SYBR\u0026reg; Safe DNA Gel Stain and 90 \u0026micro;L of dimethyl sulfoxide - DMSO. The size of the products was measured using a 50 bp ladder (Generuler 50bp, cat no: SM0373, ThermoFisher Scientific). The amplification process was successful when the PCR products were visible and were the expected size. The asymbiotic mite individuals were identified based on the presence of the product from universal bacterial primers and the absence of the product from \u003cem\u003eCardinium\u003c/em\u003e and/or \u003cem\u003eWolbachia\u003c/em\u003e primers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMite growth test\u003c/h2\u003e \u003cp\u003eThe population growth of mites in the original double-infected stock cultures was recorded at two-month intervals. The first growth test was established after 2 months of incubation. Mites were collected from the plugs and surface of rearing flasks and then transferred to separate Petri dishes. The controls consisted of a single-infected population. Ten unsexed adult mites were moved from a Petri dish to new flasks that contained 0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 grams of SPMd. The flasks were kept under controlled conditions. After 21 days, the experiment ended, and mites were counted using a dissection microscope (Hubert et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eFor the data analyses, we used a measure of population fitness, which was calculated as the intrinsic rate of population increase (\u003cem\u003er\u003c/em\u003e) assuming exponential population growth as the time was short and resources were not limited. We used the formula for density-independent continuous population growth: \u003cem\u003eN\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e = \u003cem\u003eN\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003ee\u003csup\u003e\u003cem\u003ert\u003c/em\u003e\u003c/sup\u003e, where \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003et\u003c/em\u003e\u003c/sub\u003e is the final mite density, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e is the initial mite density, and \u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21 days (the duration of mite population growth). The intrinsic rates had a distribution not different from the normal ; thus, differences among populations were studied using a general linear model (LM). To test for consistency (repeatability) in the rate in the same population mixtures grown in three experiments, we used linear mixed-effect models (LME) from the nlme package (Pinheiro and Bates \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The intrinsic rate was the response variable, and population type and time were explanatory variables. The experiment was a random effect. We calculated the intraclass correlation coefficient (ICC) from the variance estimated as a measure of consistency. The prevalence of endosymbionts in single-infected populations were analyzed with Generalized linear models with binomial error structure (GLM-b) (Pekar and Brabec \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The prevalences (after applying an angular transformation to approach normal distribution) in mixture populations were compared among population types and time using LME in order to estimate consistency in prevalence using ICC. Eventually, the effect of prevalence on the fitness of populations was studied using LM. All analyses were performed in R (R Development Core Team 2023).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFitness\u003c/h2\u003e \u003cp\u003eThe single-infected cultures of \u003cem\u003eT. putrescentiae\u003c/em\u003e reached different numbers after 21 days so their fitness was significantly different (LM, F\u003csub\u003e3,20\u003c/sub\u003e = 27.6, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). One population of those infected by \u003cem\u003eCardinium\u003c/em\u003e (c2) and one from those by \u003cem\u003eWolbachia\u003c/em\u003e (w1) had significantly higher fitness than the other two.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe four multi-infected cultures showed significantly different rates of population increase (LME, F\u003csub\u003e6,202\u003c/sub\u003e = 2.7, P\u0026thinsp;=\u0026thinsp;0.015). After one month, all four cultures increased at a similar rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). After three months, the population growth increased at lower rates, but only for c1w2, which was significantly lower than after one month. After five months, all cultures increased at significantly different rates: the c2w1 and c2w2 had higher fitness than c1w2 and c1w1. These two cultures had fitness that was not significantly different from single-infected cultures (w2 and c2). The low intraclass correlation coefficient (0.19) shows that the three experiments had low consistency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePrevalence of endosymbionts\u003c/h2\u003e \u003cp\u003eThe prevalence of endosymbionts in single-infected cultures was significantly different between bacteria species (GLM, χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;89.4, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001): prevalence was higher in \u003cem\u003eCardinium\u003c/em\u003e infected cultures than in \u003cem\u003eWolbachia\u003c/em\u003e infected cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA); among cultures (GLM, χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;50.0, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001): c1 and w1 had higher prevalence than c2 and w2; and between experiments (GLM, χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;14.3, P\u0026thinsp;=\u0026thinsp;0.0002): the first experiment had generally higher prevalence than the second experiment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the double-infected cultures, the prevalence of the two bacteria varied significantly among cultures (LME, F\u003csub\u003e6,154\u003c/sub\u003e = 2.42, P\u0026thinsp;=\u0026thinsp;0.028). In all cultures, the prevalence of both endosymbionts fluctuated over six months but not significantly (LME, F\u003csub\u003e1,142\u003c/sub\u003e = 0.3, P\u0026thinsp;=\u0026thinsp;0.61). The consistency of the prevalence in the three experiments was nearly zero (ICC\u0026thinsp;\u0026lt;\u0026thinsp;0.01). After six months, the \u003cem\u003eCardinium\u003c/em\u003e-infected individuals prevailed in 50% of the mite cultures, the \u003cem\u003eWolbachia\u003c/em\u003e-infected individuals prevailed in 41.7%, and asymbiotic individuals in 8.3% of cultures (N\u0026thinsp;=\u0026thinsp;12). In the c1w1 culture on average, \u003cem\u003eCardinium\u003c/em\u003e infected individuals always prevailed over \u003cem\u003eWolbachia\u003c/em\u003e infected individuals, which decreased, so there were 2.6times more \u003cem\u003eCardinium\u003c/em\u003e infected individuals at the end. In the c1w2 culture, on average, \u003cem\u003eCardinium\u003c/em\u003e infected individuals gradually decreased over \u003cem\u003eWolbachia\u003c/em\u003e infected individuals, which increased, so at the end, there were 11-times more \u003cem\u003eWolbachia\u003c/em\u003e infected individuals. In the c2w1 culture, on average, \u003cem\u003eCardinium-\u003c/em\u003einfected individuals always prevailed over \u003cem\u003eWolbachia\u003c/em\u003e-infected individuals, which did not change, so in the end, there were almost 2 times more \u003cem\u003eCardinium\u003c/em\u003e infected. In the c2w2 culture, on average, both \u003cem\u003eCardinium\u003c/em\u003e infected and \u003cem\u003eWolbachia\u003c/em\u003e infected individuals decreased so that, in the end, the majority were asymbiotic individuals.\u003c/p\u003e \u003cp\u003eFew individuals in the mixed culture were double-infected by both \u003cem\u003eWolbachia\u003c/em\u003e and \u003cem\u003eCardinium\u003c/em\u003e. The frequency of their occurrence was not explained by the time (GLM-qb, F\u003csub\u003e1,52\u003c/sub\u003e = 13.0, P\u0026thinsp;=\u0026thinsp;0.08) or the experiment (GLM-qb, F\u003csub\u003e2,53\u003c/sub\u003e = 4.0, P\u0026thinsp;=\u0026thinsp;0.60) but by the culture type (GLM-qb, F\u003csub\u003e3,49\u003c/sub\u003e = 47.1, P\u0026thinsp;=\u0026thinsp;0.014): there were significantly more double-infected individuals in the c2w1 than in other cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy combining data on fitness and prevalence, we found that neither occurrence of \u003cem\u003eWolbachia-\u003c/em\u003einfected individuals (LM, F\u003csub\u003e1.34\u003c/sub\u003e \u0026lt; 0.1, P\u0026thinsp;=\u0026thinsp;0.97), \u003cem\u003eCardinium\u003c/em\u003e-infected individuals (LM, F\u003csub\u003e1.34\u003c/sub\u003e = 1.4, P\u0026thinsp;=\u0026thinsp;0.25) nor asymbiotic individuals (LM, F\u003csub\u003e1.34\u003c/sub\u003e = 2.9, P\u0026thinsp;=\u0026thinsp;0.097) affected the fitness of cultures after 21 days.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study showed that the multiple infections on the population level in \u003cem\u003eT. putrescentiae\u003c/em\u003e are unstable, and the mixing of parental cultures resulted in a decrease of fitness in the initial time of experiments. Only two double infected cultures achieved a similar population growth rate as parental single infected cultures. The process of infection is stochastic (i.e. genetic drift (Jansen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e)) and can result in all possible situations, i.e. \u003cem\u003eCardinium\u003c/em\u003e or \u003cem\u003eWolbachia\u003c/em\u003e wins. Still also both symbionts can lose and the mite culture switch to asymbiotic. The mean generation time of \u003cem\u003eT. putrescentiae\u003c/em\u003e was found to be 17 days at conditions used (Sanchez-Ramos and Castanera \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), suggesting that in our study there were approximately ten generations. Although the cultures were multiple-infected, double-infected individuals were rare based on our data.\u003c/p\u003e \u003cp\u003eThis situation differs from \u003cem\u003eTetranychus\u003c/em\u003e spider mites where the field population showed variability in intracellular symbiont (\u003cem\u003eCardinium\u003c/em\u003e, \u003cem\u003eRickettsia\u003c/em\u003e, and \u003cem\u003eWolbachia\u003c/em\u003e) prevalence (Zele et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). However, the laboratory experiment showed apparent loss of \u003cem\u003eRickettsia\u003c/em\u003e and \u003cem\u003eCardinium\u003c/em\u003e, but not \u003cem\u003eWolbachia\u003c/em\u003e, during 6 months of experiments (15 generations) (Zele et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The natural prevalence of symbionts in \u003cem\u003eTetranychus\u003c/em\u003e populations (N\u0026thinsp;=\u0026thinsp;16) decreased from \u003cem\u003eWolbachia\u003c/em\u003e (61%), followed by \u003cem\u003eCardinium\u003c/em\u003e (12\u0026ndash;15%) to \u003cem\u003eRickettsia\u003c/em\u003e (0.9\u0026ndash;3%) (Zele et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). In \u003cem\u003eT. putrescentiae\u003c/em\u003e (N\u0026thinsp;=\u0026thinsp;9), both \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e infect the same proportion (22%), and the rest of the cultures (33%) are without intracellular symbionts (Hubert et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). In double-infected \u003cem\u003eTetranychus piercei\u003c/em\u003e, the males had suppressed \u003cem\u003eWolbachia\u003c/em\u003e while accelerating the growth of \u003cem\u003eCardinium\u003c/em\u003e, while single and double-infected females had the same level of \u003cem\u003eCardinium\u003c/em\u003e, indicating competition between symbionts (Zhu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, in \u003cem\u003eT. putrescentiae\u003c/em\u003e, it is a different situation due to the low proportion of double-infected individuals, but the competition is still possible at a population level, and the low number of double-infected individuals should be the result of such competition. The indirect evidence of the competition is the decrease in population growth of all multiple-infected cultures compared to parental cultures, which still exist in some cultures during the experiment.\u003c/p\u003e \u003cp\u003eIt is currently unknown whether cytoplasmic incompatibility (CI) exists for \u003cem\u003eT. putrescentiae\u003c/em\u003e infections. Both \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e are well documented to induce CI in \u003cem\u003eTetranychus\u003c/em\u003e and \u003cem\u003eBryoba\u003c/em\u003e mites (Breeuwer \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Gotoh et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, CI induced by \u003cem\u003eWolbachia\u003c/em\u003e differed among \u003cem\u003eTetranychus\u003c/em\u003e populations (Zele et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the double-infected parasitoid wasp \u003cem\u003eEncarsia inaron\u003c/em\u003e, \u003cem\u003eWolbachia\u003c/em\u003e caused CI, whereas \u003cem\u003eCardinium\u003c/em\u003e did not. In the contrast, \u003cem\u003eT. urticae\u003c/em\u003e double-infection of \u003cem\u003eWolbachia\u003c/em\u003e and \u003cem\u003eCardinium\u003c/em\u003e induced strong CI (Xie et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this study, the decreases in fitness should be the result of CI in multi-infected cultures at the beginning of experiments. Alternatively, the subsequent effect on mite physiology and microbiome can explain the population decrease. For example, \u003cem\u003eEurema hecabe\u003c/em\u003e, growth rates evaluated by development time were slower in progenies of \u003cem\u003eWolbachia\u003c/em\u003e double-infected mothers than in those of single-infected mothers (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eCardinium\u003c/em\u003e and combined \u003cem\u003eCardinium\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eWolbachia\u003c/em\u003e infections led to a reduction in bacterial diversity, alteration of bacterial community structure, and metabolic changes, which had negative fitness effects on the host (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe study revealed different processes in competition between symbionts in \u003cem\u003eTetranychus\u003c/em\u003e mites. The genetic difference of symbionts should explain the difference mentioned above in the studies. In this study we used atypical intracellular symbionts, i.e. \u003cem\u003eWolbachia\u003c/em\u003e is a member of a novel supergroup Q of mite symbionts (unpublished data, GenBank Acess No JAUEMM01 and GIJY01), while \u003cem\u003eWolbachia\u003c/em\u003e from \u003cem\u003eTetranychus\u003c/em\u003e AH2012_wTtru (NZ_CP099592) is similar to insect \u003cem\u003eWolbachia\u003c/em\u003e (e.g. \u003cem\u003eWolbachia\u003c/em\u003e symbiont of \u003cem\u003eBemisia tabaci\u003c/em\u003e (NZ_CP016430). The genome analyses of \u003cem\u003eCardinium\u003c/em\u003e (JAUEML01) revealed that it forms a separate cluster of symbionts of the planthopper \u003cem\u003eSogatella furcifera\u003c/em\u003e (cSFur) (Zeng et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition, the subsequent genome analysis revealed that both intracellular bacteria are identical in \u003cem\u003eT. putrescentiae\u003c/em\u003e cultures (unpublished data). Whether genome differences are responsible for the different effects of symbionts on the host is the further direction of studies.\u003c/p\u003e \u003cp\u003eThe success of mites is based on the spreading via insects, animals including humans, and almost exponential growth under suitable conditions (Aspaly et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In \u003cem\u003eT. putrescentiae\u003c/em\u003e, 39 allergen groups have been identified (Zhou et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The previous study showed that \u003cem\u003eT. putrescentiae\u003c/em\u003e cultures with distinct microbiomes differ in the expression of allergens; namely \u003cem\u003eWolbachia\u003c/em\u003e inhabited cultures associated allergens belonging to groups no. 4, 6, 7, 13, 20, and 36, while asymbiotic culture-associated allergens from groups no. 5, 28, 30, and 35 (Hubert et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Here, we found that the mites and their physiological features are determined by intracellular factors, providing a high degree of variability during the growth of the mite population in stored food. It means that symbionts determine allergen production in terms of the allergen production due to different mite populations\u0026rsquo; growth and by different expressions as the response of mite-influenced immune/regulatory pathways.\u003c/p\u003e \u003cp\u003eThe stochastic models of symbiont infection are based on the low initial numbers of mites that establish the population (Jansen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zele et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The faunistic analyses of the stored grain fulfil the low initial population of \u003cem\u003eT. putrescetnaie\u003c/em\u003e; i.e. 14% of samples (N\u0026thinsp;=\u0026thinsp;514) with density lower than 1 ind.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e grain, while 1% of samples with 5 and more individuals g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e grain (Stejskal and Hubert \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It means that natural conditions favour the stochastic symbiont selection model. From a practical point of view, \u003cem\u003eT. putrecentiae\u003c/em\u003e is an important pest of stored products infecting cereals, dried ham and sausages, cheeses and dog food (Olivry and Mueller \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Robertson \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1961\u003c/span\u003e; Stejskal et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Population density is the key to pest importance, and \u003cem\u003eTyrophagus\u003c/em\u003e mites can reach massive biomass. Such contaminated food can avoid anaphylactic shock (Sanchez-Borges et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). There is an effort to establish a model of the population growth based on the abiotic condition in the stored food (Pekar and Zdarkova \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Sanchez-Ramos and Castanera \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was supported by the project of the Czech Science Foundation GF22-15841K.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJH and EG designed and organized experiments; SP provided statistical analyses; all authors wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors are obligated to Eliska Tresnakova, Marta Nesvorna, and Martin Markovic for technical help.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAspaly G, Stejskal V, Pekar S, Hubert J (2007) Temperature-dependent population growth of three species of stored product mites (Acari: Acaridida). 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Mol Ecol 28(24):5199\u0026ndash;5202. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mec.15295\u003c/span\u003e\u003cspan address=\"10.1111/mec.15295\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mite, Cardinium, Wolbachia, Genome, Gene expression, Interaction","lastPublishedDoi":"10.21203/rs.3.rs-4806277/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4806277/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCultures of the stored product mite \u003cem\u003eTyrophagus putrescentiae\u003c/em\u003e are often single-infected by one of intracellular bacteria \u003cem\u003eCardinium\u003c/em\u003e or \u003cem\u003eWolbachia\u003c/em\u003e. No naturally occurring multi-infected \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia a T. putrescentiae\u003c/em\u003e mites are known. Under laboratory conditions, we mixed two single-infected mite cultures of each endosymbiont to obtain four multi-infected cultures and to test the intraspecific competition among individuals under the influence of \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e. The mite population growth, as a fitness indicator, and endosymbiont prevalence were estimated repeatedly during five months, repeated in three separate experiments. The fitness of four single-infected populations varied significantly. The proportion of infected individuals was higher in the two \u003cem\u003eCardinium\u003c/em\u003e infected populations than in the two \u003cem\u003eWolbachia\u003c/em\u003e infected populations. The fitness of multi-infected populations fluctuated over time in all three experiments. After six months of experiments, \u003cem\u003eCardinium\u003c/em\u003e infected individuals prevailed in 50% of the populations, the \u003cem\u003eWolbachia\u003c/em\u003e infected individuals prevailed in 41.7%, and asymbiotic individuals in 8.3% of populations (N\u0026thinsp;=\u0026thinsp;12). The fitness of two multi-infected populations was like the fitness of single-infected populations. In one of them, the proportion of \u003cem\u003eCardinium\u003c/em\u003e or \u003cem\u003eWolbachia\u003c/em\u003e infected individuals was, on average, similar during the experiment. At the same time, symbionts disappeared, resulting in the majority of asymbiotic individuals in the second population. No correlation between symbionts proportion and mite fitness was found. The results indicated that the outcome of the battle between \u003cem\u003eCardinium\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e is strongly stochastic at the population level.\u003c/p\u003e","manuscriptTitle":"The battle of Cardinium and Wolbachia in double-infected mite cultures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 06:40:09","doi":"10.21203/rs.3.rs-4806277/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"03b9f335-0665-4f36-bef7-bb87f60879b6","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-14T17:56:40+00:00","versionOfRecord":{"articleIdentity":"rs-4806277","link":"https://doi.org/10.1016/j.jspr.2025.102663","journal":{"identity":"journal-of-stored-products-research","isVorOnly":true,"title":"Journal of Stored Products Research"},"publishedOn":"2025-04-23 00:00:00","publishedOnDateReadable":"April 23rd, 2025"},"versionCreatedAt":"2024-08-22 06:40:09","video":"","vorDoi":"10.1016/j.jspr.2025.102663","vorDoiUrl":"https://doi.org/10.1016/j.jspr.2025.102663","workflowStages":[]},"version":"v1","identity":"rs-4806277","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4806277","identity":"rs-4806277","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}