Microbiome changes in Akanthomyces attenuatus JEF-147-infected two-spotted spider mites.

The two-spotted spider mite (Tetranychus urticae Koch) is an agriculturally serious polyphagous pest that has acquired strong resistance against acaricides because of its short life cycle and continuous exposure to acaricides. As an alternative, mite-pathogenic fungi with different modes of action could be used to control the mites. The spider mite has symbiotic microorganisms that could be involved in the physiological and ecological adaptations to biotic stresses. In this study, mite-pathogenic fungi were used to control female adults, and the microbiomes changes in the fungus-infected mites were analyzed. The acaricidal activity of 77 fungal isolates was tested, and Akanthomyces attenuatus JEF-147 exhibited the highest acaricidal activity. Subsequently a dose-response assay and morphological characterization was undertaken For microbiome analysis in female adults infected with A. attenuatus JEF-147, 16S rDNA and ITS1 were sequenced using Illumina Miseq. Infected mite showed a higher Shannon index in bacterial diversity but lower index in fungal diversity. In beta diversity using principal component analysis, JEF-147-treated mites were significantly different from non-treated controls in both bacteria and fungi. Particularly in bacterial abundance, arthropod defense-related Rickettsia increased, but arthropod reproduction-associated Wolbachia decreased. The change in major bacterial abundance in the infected mites could be explained by a trade-off between reproduction and immunity against the early stage of fungal attack. In fungal abundance, Akanthomyces showed up as expected. Foremost, this work reports microbiome changes in a fungus-infected mite and suggests a possible trade-off in mites against fungal pathogens. Future studies will focus on gene-based investigations related to this topic.

Auto-dissemination of Cordyceps fumosorosea amongst adult females of the two-spotted spider mite.

Tetranychus urticae is an important pest worldwide. The auto-dissemination of spores of entomopathogenic fungi from an infected individual to conspecifics may be important for controlling pests that can build high populations. The current study was carried out to determine the auto-dissemination of the entomopathogenic fungus Cordyceps fumosorosea strain PFs-1 (Priority®) between T. urticae females. The study consisted of four experiments. First, the efficacy of entomopathogenic fungus bioassays was assessed in Petri dishes (experiment 1) and on potted bean plants (experiment 2). In the auto-dissemination trials (experiments 3 and 4, in Petri dishes and on potted plants, respectively), contaminated adult females (1-5) were released among uncontaminated females (10 individuals). All experiments were carried out separately, and observations were made on days 3, 5, and 7. In exp. 1, the control was different from Priority on all observation days. In exp. 2, the average number of surviving individuals in the control was significantly higher than in the Priority treatment. In the auto-dissemination experiments, as the number of contaminated individuals increased, the mortality rate of uncontaminated individuals also increased, in exp. 3 (Petri dishes) on all observation days, and in exp. 4 (potted plants) only on days 5 and 7. The median lethal time (LT50) decreased as the number of individuals contaminated with Priority increased in both Petri dish and pot trials. Consequently, the effectiveness of biological control may increase with the occurrence of indirect contamination from infected to uncontaminated individuals.

High susceptibility of Tetranychus merganser (Acari: Tetranychidae), an emergent pest of the tropical crop Carica papaya, towards Metarhizium anisopliae s.l. and Beauveria bassiana strains.

Background: The mite Tetranychus merganser is considered to be an emerging pest of various crops in tropical countries. It is one of the most detrimental pests in the papaya orchards of some regions of México. The current field control of Tetranychus spp. involves the extensive use of chemicals that have some degree of toxicity to humans or the environment and may cause selective resistance. The use of biological alternatives such as parasitoids and mite predators have limited effectiveness. In order to find effective but non-toxic alternatives for mite pest management, bio-products that are able to be mass produced and applied to large production areas have been sought, including the entomopathogen fungi. B. bassiana and M. anisopliae s.l. are the fungi most extensively used for the biological control of insect pests. Although they do not cause natural epizootic diseases in mites, there are reports that show that they infect T. urticae, and should be evaluated for use in the biological control of papaya's mite pests. Methods: A T. merganser colony was established and the susceptibility of adult females to 30 entomopathogenic fungi strains was evaluated under laboratory conditions with an in vitro mass screening bioassay. Ten strains of Metarhizium anisopliae sensu lato (s.l.), eleven of Beauveria bassiana, nine of Lecanicillium sp. and one of Hirsutella thompsonii var. sinematosa were tested. The infectivity of adult females was evaluated calculating the percentage of mortality. To calculate the LC50 and LT50 of the most virulent strains, a bioassay was performed using serial concentrations (1×104-1×108 conidia/mL) for each strain. Strains showing ability to infect eggs laid were evaluated with a novel egg-infectivity bioassay. The internal transcribed spacer (ITS) region of the more lethal strains were sequenced. Results: T. merganser and T. urticae were found in orchards of Carica papaya (Maradol variety and Tainung hybrid) in Campeche, México. All tested strains of M. anisopliae s.l. and B. bassiana were infectious to the adult female of T. merganser at a concentration of 1×108 conidia/mL. Six strains of M. anisopliae (Ma002, Ma003, Ma004, Ma005, Ma014 and Ma034) caused 100% mortality, and one of B. bassiana (Bb016) caused 95% mortality. The most virulent was Ma034, with an LC50 of 1.73×106 conidia/mL followed by Ma005 and Ma003. Ma005 and Ma034 were the fastest strains to reach LT50,achieving this in less than 3.7 days. Additionally, Ma034 and Ma014 strains were infectious to more than 70% of the eggs. Conclusions: T. merganser and T. urticae are present in the papaya orchards of Campeche, México. The high susceptibility of T. merganser adult females and eggs toward several M. anisopliae s.l. or B. bassiana strains suggests that these fungi are a viable alternative to control this emergent pest. The most virulent strain, Ma034, was also infective to eggs, and is the most promising to be tested in the field.

Wolbachia affects reproduction in the spider mite Tetranychus truncatus (Acari: Tetranychidae) by regulating chorion protein S38-like and Rop.

Wolbachia-induced reproductive regulation in hosts has been used to control pest populations, but little is known about the molecular mechanism underlying Wolbachia regulation of host genes. Here, reproductive regulation by Wolbachia in the spider mite Tetranychus truncatus was studied at the molecular level. Infection with Wolbachia resulted in decreasing oviposition and cytoplasmic incompatibility in T. truncatus. Further RNA-seq revealed genes regulated by Wolbachia in T. truncatus. Real-time quantitative polymerase chain reaction (qPCR) showed that genes, including chorion protein S38-like and Rop were down-regulated by Wolbachia. RNA interference (RNAi) of chorion protein S38-like and Rop in Wolbachia-uninfected T. truncatus decreased oviposition, which was consistent with Wolbachia-induced oviposition decrease. Interestingly, suppressing Rop in Wolbachia-infected T. truncatus led to increased Wolbachia titres in eggs; however, this did not occur after RNAi of chorion protein S38-like. This is the first study to show that chorion protein S38-like and Rop facilitate Wolbachia-mediated changes in T. truncatus fertility. In addition, RNAi of Rop turned the body colour of Wolbachia-uninfected T. truncatus black, which indicates that the role of Rop is not limited to the reproductive regulation of T. truncatus.

Mode of entry of secondary metabolites of the bacteria Xenorhabdus szentirmaii and X. nematophila into Tetranychus urticae, and their toxicity to the predatory mites Phytoseiulus persimilis and Neoseiulus californicus.

The bacterial metabolites in supernatants of Xenorhabdus species have acaricidal activity, but this mode of entry into mites has not yet been elucidated. Herein, we report on the possible mode of entry of Xenorhabdus szentirmaii and Xenorhabdus nematophila supernatants into Tetranychus urticae (Acari: Tetranychidae) adult females. We also assessed the toxicity of the supernatants against the developmental stages of the predatory mites, Phytoseiulus persimilis and Neoseiulus californicus (Acari: Phytoseiidae). Experiments were conducted at 25 ± 1 °C, 70 ± 5% relative humidity, and 16:8h light:dark conditions. Our data showed that the bioactive acaricidal compound is most effective (86.5 to 89% mortality) when the entire integument of T. urticae comes in contact with it compared to contact of the ventral side only (26.5-34%). Against P. persimilis and N. californicus at 6 days post-application (dpa), the eggs were not affected by the X. szentirmaii or X. nematophila supernatant, whereas mortality of the mobile stages (larva, protonymph, deutonymph, adult) was 18.5% to 39.2%. Overall, the predatory mites were less affected by the bacterial metabolites than T. urticae. We hypothesize that the differences in morphology such as longer legs and thicker cuticle, as well as the diet of the predatory mites, reduce the contact of the body parts to the supernatant-treated surfaces. We need to isolate, identify, and characterize the X. szentirmaii and X. nematophila metabolite(s) and demonstrate efficacy to pestiferous mites and safety to plants, non-target organisms and the environment before it can be used as an acaricide.

Endosymbiont diversity in natural populations of Tetranychus mites is rapidly lost under laboratory conditions.

Although the diversity of bacterial endosymbionts in arthropods is well documented, whether and how such diversity is maintained remains an open question. We investigated the temporal changes occurring in the prevalence and composition of endosymbionts after transferring natural populations of Tetranychus spider mites from the field to the laboratory. These populations, belonging to three different Tetranychus species (T. urticae, T. ludeni and T. evansi) carried variable infection frequencies of Wolbachia, Cardinium, and Rickettsia. We report a rapid change of the infection status of these populations after only 6 months of laboratory rearing, with an apparent loss of Rickettsia and Cardinium, while Wolbachia apparently either reached fixation or was lost. We show that Wolbachia had variable effects on host longevity and fecundity, and induced variable levels of cytoplasmic incompatibility (CI) in each fully infected population, despite no sequence divergence in the markers used and full CI rescue between all populations. This suggests that such effects are largely dependent upon the host genotype. Subsequently, we used these data to parameterize a theoretical model for the invasion of CI-inducing symbionts in haplodiploids, which shows that symbiont effects are sufficient to explain their dynamics in the laboratory. This further suggests that symbiont diversity and prevalence in the field are likely maintained by environmental heterogeneity, which is reduced in the laboratory. Overall, this study highlights the lability of endosymbiont infections and draws attention to the limitations of laboratory studies to understand host-symbiont interactions in natural populations.

Variation in the microbiome of the spider mite Tetranychus truncatus with sex, instar and endosymbiont infection.

Most arthropod-associated bacterial communities play a crucial role in host functional traits, whose structure could be dominated by endosymbionts. The spider mite Tetranychus truncatus is a notorious agricultural pest harboring various endosymbionts, yet the effects of endosymbionts on spider mite microbiota remain largely unknown. Here, using deep sequencing of the 16S rRNA gene, we characterized the microbiota of male and female T. truncatus with different endosymbionts (Wolbachia and Spiroplasma) across different developmental stages. Although the spider mite microbiota composition varied across the different developmental stages, Proteobacteria were the most dominant bacteria harbored in all samples. Positive relationships among related operational taxonomic units dominated the significant coassociation networks among bacteria. Moreover, the spider mites coinfected with Wolbachia and Spiroplasma had a significantly higher daily fecundity and juvenile survival rate than the singly infected or uninfected spider mites. The possible function of spider-mite associated bacteria was discussed. Our results highlight the dynamics of spider mite microbiotas across different life stages, and the potential role of endosymbionts in shaping the microbiota of spider mites and improving host fitness.

Transcriptome of Tetranychus urticae embryos reveals insights into Wolbachia-induced cytoplasmic incompatibility.

The endosymbiont Wolbachia is known for manipulating host reproduction in selfish ways. However, the molecular mechanisms have not yet been investigated in embryos. Here, we found that Wolbachia had no effect on the number of deposited eggs in Tetranychus urticae Koch (Acari: Tetranychidae) but caused two types of reproductive manipulation: killing uninfected female embryos via cytoplasmic incompatibility (CI) and increasing the hatching ratio of infected female embryos. RNA sequencing analyses showed that 145 genes were differentially expressed between Wolbachia-infected (WI) and Wolbachia-uninfected (WU) embryos. Wolbachia infection down-regulated messenger RNA (mRNA) expression of glutathione S-transferase that could buffer oxidative stress. In addition, 1613 and 294 genes were identified as CI-specific up-/down-regulated genes. Compared to WU and WI embryos, embryos of CI cross strongly expressed genes involved in transcription, translation, tissue morphogenesis, DNA damage and mRNA surveillance. In contrast, most of the genes associated with energy production and metabolism were down-regulated in the CI embryos compared to the WU and WI embryos, which provides some clues as to the cause of death of CI embryos. These results identify several genes that could be candidates for explaining Wolbachia-induced CI. Our data form a basis to help elucidate the molecular consequences of CI in embryos.

The microbiota in spider mite feces potentially reflects intestinal bacterial communities in the host.

Microorganisms provide many physiological functions to herbivorous hosts. Spider mites (genus Tetranychus) are important agricultural pests throughout the world; however, the composition of the spider mite microbial community, especially gut microbiome, remains unclear. Here, we investigated the bacterial community in five spider mite species and their associated feces by deep sequencing of the 16S rRNA gene. The composition of the bacterial community was significantly different among the five prevalent spider mite species, and some bacterial symbionts showed host-species specificity. Moreover, the abundance of the bacterial community in spider mite feces was significantly higher than that in the corresponding spider mite samples. However, Flavobacterium was detected in all samples, and represent a "core microbiome". Remarkably, the maternally inherited endosymbiont Wolbachia was detected in both spider mite and feces. Overall, these results offer insight into the complex community of symbionts in spider mites, and give a new direction for future studies.

Wolbachia dominate Spiroplasma in the co-infected spider mite Tetranychus truncatus.

Wolbachia and Spiroplasma are both maternally inherited endosymbionts in arthropods, and they can co-infect the same species. However, how they interact with each other in the same host is not clear. Here we investigate a co-infected Tetranychus truncatus spider mite strain that shares the same genetic background with singly infected and uninfected strains to detect the impacts of the two symbionts on their host. We found that Wolbachia-infected and Spiroplasma-infected mites can suffer significant fitness costs involving decreased fecundity, although with no effect on lifespan or development. Wolbachia induced incomplete cytoplasmic incompatibility in T. truncatus both in singly infected and doubly infected strains, resulting in female killing. In both females and males of the co-infected spider mite strain, Wolbachia density was higher than Spiroplasma density. Transcriptome analysis of female adults showed that the most differentially expressed genes were found between the co-infected strain and both the singly infected Spiroplasma strain and uninfected strain. The Wolbachia strain had the fewest differentially expressed genes compared with the co-infected strain, consistent with the higher density of Wolbachia in the co-infected strain. Wolbachia, therefore, appears to have a competitive advantage in host mites over Spiroplasma and is likely maintained in populations by cytoplasmic incompatibility despite having deleterious fitness effects.

Co-infection of Wolbachia and Spiroplasma in spider mite Tetranychus truncatus increases male fitness.

Wolbachia and Spiroplasma are intracellular bacteria that are of great interest to entomologists, because of their ability to alter insect host biology in multiple ways. In the spider mite Tetranychus truncatus, co-infection of Wolbachia and Spiroplasma can induce cytoplasmic incompatibility (CI) and fitness costs; however, little is known about the effect of co-infection at the genetic level and the molecular mechanisms underlying CI. In this study, we explored the influence of the two symbionts on male mite host fitness and used RNA sequencing to generate the transcriptomes of T. truncatus with four different types of infection. In total, we found symbiont-infected lines had a higher hatch proportion than the uninfected line, and the development time of the uninfected line was longer than that of the other lines. Co-infection changed the expression of many genes related to digestion detoxification, reproduction, immunity and oxidation reduction. Our results indicate that co-infection of Wolbachia and Spiroplasma confers multiple effects on their hosts, and helps illuminate the complex interactions between endosymbionts and arthropods.

A change in the bacterial community of spider mites decreases fecundity on multiple host plants.

Bacterial symbionts may influence the fitness of their herbivore hosts, but such effects have been poorly studied across most invertebrate groups. The spider mite, Tetranychus truncatus, is a polyphagous agricultural pest harboring various bacterial symbionts whose function is largely unknown. Here, by using a high-throughput 16S rRNA amplicon sequencing approach, we characterized the bacterial diversity and community composition of spider mites fed on five host plants after communities were modified following tetracycline exposure. We demonstrated that spider mite bacterial diversity and community composition were significantly affected by host plants and antibiotics. In particular, the abundance of the maternally inherited endosymbionts Wolbachia and Spiroplasma significantly differed among spider mites that were reared on different plant species and were completely removed by antibiotics. There was an overall tendency for daily fecundity to be lower in the mites with reduced bacterial diversity following the antibiotic treatment. Our data suggest that host plants and antibiotics can shape spider mite bacterial communities and that bacterial symbionts improve mite performance.

Acaricidal effect of cell-free supernatants from Xenorhabdus and Photorhabdus bacteria against Tetranychus urticae (Acari: Tetranychidae).

The effects of secondary metabolites produced by the following symbiotic bacteria, Xenorhabdus szentirmaii, X. nematophila, X. bovienii, X. cabanillasii, Photorhabdus luminescens and P. temperata, associated with entomopathogenic nematodes, were investigated against various developmental stages of Tetranychus urticae (Acari: Tetranychidae) using cell-free bacterial supernatants in Petri dishes. In addition, the effects of the most active bacterial supernatant(s) found in Petri dish experiments were tested on T. urticae in pot experiments. All studies were conducted at 25 ±â€¯1 °C temperature, 70 ±â€¯5% relative humidity and a light cycle of 16 h in a climate room. The result of the Petri dish experiments showed that the supernatants had little or no effect on the egg stage, as less than 4% mortality was recorded. Depending on the bacterial supernatant, mortality in the other stages was 46-97% for larvae, 30-96% for protonymphs, 41-92% for deutonymphs, 92-100% for adult males and 46-93% for adult females. Control mortalities ranged from 1-7% for larvae, 2-9% for protonymphs, 4-10% for deutonymphs, 6-10% for adult males and 4-8% for adult females. Among supernatants tested, X. szentirmaii and X. nematophila were the most efficacious with mortality greater than 90% on the mobile stages of T. urticae. According to the results from pot experiments, the supernatants of X. szentirmaii and X. nematophila, singularly and in combination, significantly reduced the T. urticae population. However, the mixture of X. szentirmaii and X. nematophila supernatants did not increase efficiency to reduce T. urticae population compared to each supernatant alone. Further studies are warranted to find the active compound(s) in the supernatants of X. szentirmaii or and X. nematophila and assess whether the supernatant(s) has the potential of being a practical and economical control agent for T. urticae.

Pathogenicity of Metarhizium anisopliae and Beauveria bassiana fungi to Tetranychus ludeni (Acari: Tetranychidae) / Patogenicidade de fungos Metarhizium anisopliae e Beauveria bassiana ao Tetranychus ludeni (Acari: Tetranychidae)

The use of acaricides is the main control method for Tetranychus ludeni (Acari: Tetranychidae) in horticultural crops. This mite has been recorded causing damage to sweet potato (Ipomoea batatas L.). The use of pathogenic fungi is an alternative to chemical control. The objective was to evaluate the pathogenicity of the fungi Metarhizium anisopliae (Meetch) Sorok, and Beauveria bassiana (Bals.) Vuill. to phytophagous mite T. ludeni in sweet potato plants. Conidial suspensions of M. anisopliae and B. bassiana, at concentrations of 106 and 107 con.mL-1 were applied to sweet potato leaves. After 24 hours, five females of T. ludeni newly emerged were released on the leaves. The bioassay was arranged in a completely randomized design with factorial arrangement 2 × 2 (two species of fungi and two concentrations) plus the control (distilled water), with 10 repetitions per treatment. The evaluation consisted of observing of three biological parameters of the mite: mortality, oviposition, and repellency, after 24, 48, 72, and 96 hours of contact with the fungi. The isolates of M. anisopliae cause high mortality rates of T. ludeni in laboratory. Beauveria bassiana has the potential to suppress future generations of mite, reducing its oviposition rate. Repellency behavior was not observed.(AU)

O uso de acaricidas é o principal método de controle de Tetranychus ludeni (Acari: Tetranychidae) em cultivos hortícolas. Esse ácaro foi registrado causando danos em batata-doce (Ipomoea batatas (L.). A utilização de fungos patogênicos é uma alternativa ao controle químico. O objetivo foi avaliar a patogenicidade dos fungos Metarhizium anisopliae (Meetch) Sorok. e Beauveria bassiana (Bals.) Vuill. ao ácaro fitófago T. ludeni em batata-doce. Suspensões conidiais de M. anisopliae e B. bassiana, nas concentrações de 106 e 107 con.mL-1, foram aplicadas sobre folhas de batata-doce. Após 24 horas, cinco fêmeas recém-emergidas foram liberadas sobre as folhas. O bioensaio foi inteiramente randomizado, com arranjo fatorial 2 × 2 (duas espécies de fungos e duas concentrações) e controle (água destilada), com 10 replicações por tratamento. A avaliação consistiu na observação de três parâmetros biológicos do ácaro: mortalidade, oviposição e repelência, após 24, 48, 72 e 96 horas de contato com os fungos. Os isolados de M. anisopliae causam altas taxas de mortalidade de T. ludeni em laboratório. Beauveria bassiana tem potencial para suprimir futuras gerações do ácaro, reduzindo a taxa de oviposição. Comportamento de repelência nos ácaros não foi observado.(AU)

Wolbachia both aids and hampers the performance of spider mites on different host plants.

In the last few decades, many studies have revealed the potential role of arthropod bacterial endosymbionts in shaping the host range of generalist herbivores and their performance on different host plants, which, in turn, might affect endosymbiont distribution in herbivore populations. We tested this by measuring the prevalence of endosymbionts in natural populations of the generalist spider mite Tetranychus urticae on different host plants. Focusing on Wolbachia, we then analysed how symbionts affected mite life-history traits on the same host plants in the laboratory. Overall, the prevalences of Cardinium and Rickettsia were low, whereas that of Wolbachia was high, with the highest values on bean and eggplant and the lowest on morning glory, tomato and zuchini. Although most mite life-history traits were affected by the plant species only, Wolbachia infection was detrimental for the egg-hatching rate on morning glory and zucchini, and led to a more female-biased sex ratio on morning glory and eggplant. These results suggest that endosymbionts may affect the host range of polyphagous herbivores, both by aiding and hampering their performance, depending on the host plant and on the life-history trait that affects performance the most. Conversely, endosymbiont spread may be facilitated or hindered by the plants on which infected herbivores occur.

Performance of Metarhizium anisopliae-treated foam in combination with Phytoseiulus longipes Evans against Tetranychus evansi Baker & Pritchard (Acari: Tetranychidae).

BACKGROUND: Tetranychus evansi (Te) is an exotic pest of solanaceous crops in Africa. The predatory mite Phytoseiulus longipes (Pl) and the fungus Metarhizium anisopliae (Ma) are potential biocontrol agents of Te. The present study investigated the efficacy of fungus-treated foam placed above or below the third Te-infested tomato leaf. The persistence of fungus-treated foam and the performance of Pl with and without fungus-treated foam were evaluated. RESULTS: The fungus-treated foam was effective when Te infestation was below the third tomato leaf as no damage was recorded on any of the upper tomato leaves up to 30 days post-treatment. However, in the control treatments, the infestation increased considerably from 9 ± 0.3% to 100 ± 0% (mean ± standard error) at 15 days post-treatment. The reuse of the fungus-treated foam at 15, 30 and 45 days post-treatment resulted in 19 ± 1.4%, 25 ± 1.2% and 54 ± 2.1%, respectively, infestation by Te. The fungus-treated foam and Pl alone were efficient, but there was no benefit to combining them for use against Te. CONCLUSION: The fungus-treated foam is an effective method to optimise the use of Ma in screenhouse conditions. These two control agents could be integrated in an integrated pest management strategy for crop protection. However, these results need to be confirmed in large field trials. © 2018 Society of Chemical Industry.

Pathogenic differences of the entomopathogenic fungus Isaria cateniannulata to the spider mite Tetranychus urticae (Trombidiformes: Tetranychidae) and its predator Euseius nicholsi (Mesostigmata: Phytoseiidae).

Isaria cateniannulata and Euseius nicholsi are two important biological control agents currently being used in many areas of China to control a variety of pests. In order to determine the possibility of a concomitant application with the two agents in a biocontrol program involving the two-spotted spider mite, Tetranychus urticae, we quantified the pathogenicity of a strain of I. cateniannulata (08XS-1) against females of both T. urticae and E. nicholsi. We observed the infection process using scanning electron microscopy and fluorescence microscopy to distinguish differences in fungal performance. The female mites were infected by I. cateniannulata at 2 × 107 conidia/ml. The mortality of T. urticae was 100% when treated with submerged conidia and 92% when treated with aerial conidia (spray), and that of E. nicholsi was 4.2 and 6.7%, correspondingly. Following infection with aerial or submerged conidia, mated E. nicholsi females displayed no significant differences between treatments and control, indicating the fungus had no obvious effect on their vitality and fertility. This demonstrates that I. cateniannulata is safe to E. nicholsi when used to control T. urticae. The two types of propagules of I. cateniannulata are readily produced by common culture, and the submerged conidia, because of their substantially higher mortality, are preferable to the aerial conidia. Our results indicate that I. cateniannulata and E. nicholsi are viable candidates to be concomitantly applied in the biocontrol programs of T. urticae.

Identification of spider-mite species and their endosymbionts using multiplex PCR.

Spider mites of the genus Tetranychidae are severe crop pests. In the Mediterranean a few species coexist, but they are difficult to identify based on morphological characters. Additionally, spider mites often harbour several species of endosymbiotic bacteria, which may affect the biology of their hosts. Here, we propose novel, cost-effective, multiplex diagnostic methods allowing a quick identification of spider-mite species as well as of the endosymbionts they carry. First, we developed, and successfully multiplexed in a single PCR, primers to identify Tetranychus urticae, T. evansi and T. ludeni, some of the most common tetranychids found in southwest Europe. Moreover, we demonstrated that this method allows detecting multiple species in a single pool, even at low frequencies (up to 1/100), and can be used on entire mites without DNA extraction. Second, we developed another set of primers to detect spider-mite endosymbionts, namely Wolbachia, Cardinium and Rickettsia in a multiplex PCR, along with a generalist spider-mite primer to control for potential failure of DNA amplification in each PCR. Overall, our method represents a simple, cost-effective and reliable method to identify spider-mite species and their symbionts in natural field populations, as well as to detect contaminations in laboratory rearings. This method may easily be extended to other species.

Exploring virulence of new and less studied species of Metarhizium spp. from Brazil for two-spotted spider mite control.

The two-spotted spider mite Tetranychus urticae is an important pest of strawberry crops in Brazil and many other countries. Focus for biocontrol studies involving entomopathogenic fungi has been on three species from the genus Metarhizium: M. anisopliae sensu stricto (s.s.), M. brunneum and M. robertsii. Also, the species Beauveria bassiana has been studied for spider mite control and one isolate (ESALQPL63) is commercially available in Brazil. New and undescribed Metarhizium species have been found recently in Brazil and provide a pool of isolates with potential for biocontrol in Brazil and probably also elsewhere. The mortality of adult females of T. urticae when exposed to four new Brazilian species of Metarhizium was compared to the mortality when exposed to M. anisopliae s.s., M. brunneum, M. pingshaense, M. robertsii and Beauveria bassiana ESALQPL63. Fungal suspensions were sprayed onto mites at 107 conidia/mL with 0.05% Tween 80 in laboratory bio-assays. We measured total mortality and percentage sporulating cadavers 10 days after exposure and calculated median lethal time (LT50). The lowest LT50 (4.0 ± 0.17) was observed for mites treated with Metarhizium sp. Indet. 1 (ESALQ1638), which also performed well with respect to mortality after 10 days and capacity to sporulate from cadavers. Among the other little studied species tested, M. pingshaense (ESALQ3069 and ESALQ3222) and Metarhizium Indet. 2 (ESALQ1476) performed well and were comparable to B. bassiana (ESALQPL63). The new Metarhizium isolates and species thus showed potential for biological control.

Endosymbiont diversity and prevalence in herbivorous spider mite populations in South-Western Europe.

Bacterial endosymbionts are known as important players of the evolutionary ecology of their hosts. However, their distribution, prevalence and diversity are still largely unexplored. To this aim, we investigated infections by the most common bacterial reproductive manipulators in herbivorous spider mites of South-Western Europe. Across 16 populations belonging to three Tetranychus species, Wolbachia was the most prevalent (ca. 61%), followed by Cardinium (12%-15%), while only few individuals were infected by Rickettsia (0.9%-3%), and none carried Arsenophonus or Spiroplasma. These endosymbionts are here reported for the first time in Tetranychus evansi and Tetranychus ludeni, and showed variable infection frequencies between and within species, with several cases of coinfections. Moreover, Cardinium was more prevalent in Wolbachia-infected individuals, which suggests facilitation between these symbionts. Finally, sequence comparisons revealed no variation of the Wolbachia wsp and Rickettsia gtlA genes, but some diversity of the Cardinium 16S rRNA, both between and within populations of the three mite species. Some of the Cardinium sequences identified belonged to distantly-related clades, and the lack of association between these sequences and spider mite mitotypes suggests repeated host switching of Cardinium. Overall, our results reveal a complex community of symbionts in this system, opening the path for future studies.