Molecular identification and detection of Rickettsia endosymbiont in the green leafhopper: Cicadella viridis (Hemiptera: Cicadellidae)

    Molecular identification and localization of a Rickettsia endosymbiont in the green leafhopper: Cicadella viridis (Hemiptera: Cicadellidae) Qi-Xian Lian, Jian-Feng Liu, Mao-Fa Yang, Chang Han PII: DOI: Reference:

S1226-8615(15)30049-2 doi: 10.1016/j.aspen.2016.05.010 ASPEN 808

To appear in:

Journal of Asia-Pacific Entomology

Received date: Revised date: Accepted date:

20 July 2015 19 May 2016 22 May 2016

Please cite this article as: Lian, Qi-Xian, Liu, Jian-Feng, Yang, Mao-Fa, Han, Chang, Molecular identification and localization of a Rickettsia endosymbiont in the green leafhopper: Cicadella viridis (Hemiptera: Cicadellidae), Journal of Asia-Pacific Entomology (2016), doi: 10.1016/j.aspen.2016.05.010

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ACCEPTED MANUSCRIPT Molecular identification and localization of a Rickettsia endosymbiont in the green leafhopper: Cicadella viridis

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(Hemiptera: Cicadellidae)

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Qi-Xian Liana,b, Jian-Feng Liu a, Mao-Fa Yanga*1, Chang Hana a Institute of Entomology, College of Agriculture, Guizhou University, Guizhou 550025, P. R. China b Department of Chemical Biology, Xingyi Normal University for Nationalities, Xingyi, Guizhou 562400, P. R. China

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Abstract

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The leafhopper Cicadella viridis (Hemiptera: Cicadellidae), a sap-sucking insect, is a notorious pest of fruit trees and seedlings. They harbor “Candidatus sulcia merii” which is an obligatory symbiotic bacterium housed in a special organ called the

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bacteriome. In this study universal eubacterial primers 27F and 1513R were used to

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amplify approximately 1462 bp of symbiotic bacteria 16S rRNA gene from C. viridis. Cloning, sequencing, and phylogenetic analysis targeting the 16S rRNA gene revealed

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the presence of bacteria stably associated with C. viridis, and a Rickettsia endosymbiont was detected for the first time in this species. Rickettsia endosymbiont localization was studied using quantitative PCR which showed that it was found in all developmental stages. It was also co-localized in the bacteriomes, ovaries, testes, guts, and salivary glands. By an antibiotic treatment, the antibiotic-treated insects exhibited lower Rickettsia infection density than the untreated.

Keywords: Rickettsia endosymbiont; Cicadella viridis; localization; 16S rRNA gene

INTRODUCTION Cicadella viridis (Hemiptera: Cicadellidae) is a widespread leafhopper. It is a *Corresponding author. Tel.:+86 851 83850050; fax: +86 851 83856206. E-mail address:[email protected] (M.F. Yang)

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ACCEPTED MANUSCRIPT sap-sucking insect and a notorious pest of fruit trees and seedlings. Adult and nymphal insects may feed on the stems and leaves of the plants. In addition to the direct damage caused by feeding on the plant phloem and the secretion of honeydew,

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C. viridis is a known vector of plant viruses. C. viridis is a multivoltine species whose

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development requires five nymphal stages. As with other insect vectors of diseases, there is increasing interest in studying the microbial community associated with C.

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viridis, with the aim of detecting microorganisms that might be exploited for alternative control strategies. An important factor in the ecological and evolutionary

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success of insects is their endosymbiotic microflora. Endosymbiotic microorganisms can be scattered throughout different organs/tissues (e.g. the ovary). These

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endosymbionts are vertically transmitted to the offspring and this process generally occurs via transovarial transmission. The different insect endosymbionts are probably

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involved in different metabolic pathways, and there is strong evidence that these

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microorganisms contribute to the nutrition, growth, and fertility of the insect hosts (Ishikawa, 2003). Furthermore, bacteria that manipulate host reproduction are another

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group of microorganisms that have probably had a strong impact on insect evolution (Bandi, et al., 2001).

The genus Rickettsia is a phylogenetically well-defined bacterial group that belongs to the order Rickettsiales in the class α-Proteobacteria (Weisburg, et al., 1989; Roux, et al., 1995; Stothard, et al., 1995). Many species have medical importance as they are pathogens of humans and other vertebrates. Pathogenic Rickettsia species infect their hosts through blood-feeding arthropods, including lice, fleas, ticks, and mites (Parola, et al., 2005; Walker and Ismail. 2008). Although some Rickettsia species occasionally infect humans and other vertebrates, and a few species are obligatorily associated with vertebrates, it is believed that the primary hosts of most Rickettsia species are arthropods and other invertebrates (Dasch, et al., 1992; Kikuchi, et al., 2002). The Rickettsia bacterium is often detected in host populations, and can be regarded as a facultative endosymbiotic associate (Dasch, et al., 1992). So far, many Rickettsia symbionts have been identified from a wide variety of invertebrates, such as ladybird 2

ACCEPTED MANUSCRIPT beetles (Werren, et al., 1994; Hurst, et al., 1996; von der Schulenburg, et al., 2001), aphids (Chen, et al., 1996; Sakura, et al., 2005; Lukasik, et al., 2013), leafhoppers (Davis, et al., 1998; Noda, et al., 2012; Ishii, et al., 2013), a bruchid beetle (Fukatsu,

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and Shimada, 1999), ticks (Noda, 1997; Benson, et al., 2004), leeches (Kikuchi, et al.,

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2002), and others.

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In this paper, we report, for the first time, that a Rickettsial endosymbiont was detected in C. viridis and was characterized by molecular phylogenetic analysis based

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on its 16S rRNA gene sequence. We further studied its localization at various stages of C. viridis development and in different organs.

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MATERIALS AND METHODS

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Leafhopper collection

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During June to October, 2013/2014, C. viridis adult individuals were collected from rice fields in Huaxi region, Guiyang, China. They were kept in 95% ethanol (EtOH) at

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–20°C until needed. DNA extraction

Five males and five females from rice fields were selected for individual DNA extraction using a TIANamp Genomic DNA Kit (Tiangen Biotech (Beijing) Co., Ltd). An individual leafhopper was placed in a 1.5 ml microfuge tube and crushed by grinding with a disposable pestle. The DNA extraction was carried out according to the manufacturer’s instructions. Purified DNA samples were resuspended in 100 µl TE buffer and stored at –20oC until needed. DNA PCR, cloning, and sequencing Universal eubacterial primers 27F (5’-AGA GTT TGA TCM TGG CTC AG-3’) and 1513R (5’-ACG GYT ACC TTG TTA CGA CTT-3') (Weisburg, et al, 1991) were used to amplify approximately 1500 bps of 16S rDNA using DNA samples purified from the adult individuals as templates. The reaction mixes contained 2.5 µl 3

ACCEPTED MANUSCRIPT of buffer (Takara Biotechnology (Dalian) Co., Ltd, China ) with MgCl2, 2 µl of DNTP mixture (10 Mm), 0.5 µl of each primer, 0.5 µl of Taq DNA polymerase (Takara), 1 µl of DNA template, and 18 µl of ddH2O in a total volume of 25 µl. The PCR cycle

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included a denaturation step (95°C, 5 min), followed by 30 rounds of denaturation

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(95°C, 30s), annealing (63.5°C, 30s), extension (72°C, 1 min), and then a final extension step at 72°C for 10 min (Wangkeeree, et al., 2012). The PCR products were

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analyzed by gel electrophoresis and the positives were cloned into the pMD® 18-T Vector (Takara) following the manufacturer’s instructions. Ten recombinant plasmid

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clones were randomly selected from each leafhopper DNA template for sequencing analysis. Nucleotide sequencing was carried out at Sangon Biotech (Beijing) Co., Ltd.

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The Rickettsia sp. sequences obtained were compared with other 16S rRNA gene sequences by doing a nucleotide BLAST search of the National Center for

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Phylogenetic analysis

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Biotechnology Information (NCBI) database.

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Almost all the Rickettsia sp. sequences from C. viridis were analyzed for phylogenetic relationships with other selected sequences in the NCBI database, including closely related sequences and unrelated sequences (the accession numbers are given in Figure 1). Multiple alignments of the nucleotide sequences were generated using the Mega 6.05 program under the default settings with a gap opening and a gap extension (Tamura et al., 2013). The best-fit model of sequence evolution was first selected from the 88 standard nucleotide models found in JMODELTEST (Posada, 2008). TrN+G best substitution models were used for Bayesian (BA) and maximum-likelihood (ML) analysis using the programs within the MrBayes 3.1.2 (Ronquist, et al., 2003) and PAUP4.0 (Swofford, 1998) programs, respectively. Bootstrap tests were conducted using 1,000 resamplings for ML analysis. Posterior probabilities were calculated for each node and were used for statistical evaluation in the BA analysis. The trees were displayed by using the FigTree v1.4 program. Prevalence of the Rickettsia endosymbiont in C. viridis 4

ACCEPTED MANUSCRIPT The alignment of the Rickettsia 16S rRNA gene sequence with related bacterial sequences was used to design primers that specifically targeted a fragment of the new sequence. During the process of designing primers, regions where variability was high

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were chosen, and the BLAST program was used to examine the number of

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mismatches that candidate primers had with all other known 16S rRNA gene sequences, in order to avoid potential amplification from other bacteria. The forward

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and reverse primers were RickendoCV-F1 (CTGGC TCAGA ACGAA CGCTA) and RickendoCV-R2 (ACTAA ACCGC CTACG CACTC), respectively. They amplified a

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466bp fragment of the newly discovered sequence. A total of 44 C. viridis individuals were analyzed by PCR as described above. The cycling conditions were as follows:

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94°C for 3 min, 30 cycles of 94°C for 30s, 60°C for 30 s, 72°C for 45 s, and 10 min at 72°C.

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Localization of Rickettsia endosymbiont in dissected organs

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Three females and three males were dissected with forceps under a light

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microscope. The dissected organs/tissues were ovaries, bacteriomes, guts, and pairs of salivary glands (ovaries, bacteriomes from three females; testes from three males; guts, and pairs of salivary glands from three females/males). Isolated organs/tissues were individually washed several times with fresh phosphate-buffered saline to minimize possible microbial contamination and were immediately subjected to DNA extraction by using DNeasy Blood & Tissue Kit (QIAGEN, Germany). Each sample was tested for the selected bacterial endosymbiont by a quantitative PCR technique by using the TaqMan PCR in terms of 16S rRNA gene copies. Quantitative PCR amplifications were as described below. Rickettsia endosymbiont detection at each life stage of C. viridis In May, 2014, laboratory strains of C. viridis originating from rice fields in Huaxi region, Guiyang, China, were maintained in a cage on rice seedlings stock until they produced eggs and nymphs. Some of the eggs and nymphs were collected and kept in 95% ethanol at –20°C until needed for DNA extraction, and some were transferred to 5

ACCEPTED MANUSCRIPT a new plant. A number of adults from the first generation were transferred to a new plant and maintained until they produced the second generation of offspring. The eggs and nymphs of the second generation were collected and preserved in 95% ethanol at

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–20°C until needed for DNA extraction. DNA extraction from single insects was

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conducted using a DNA extraction by using DNeasy Blood & Tissue Kit (QIAGEN, Germany), and the presence of the target Rickettsia endosymbiont was assessed using

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PCR and specific primers, as described above. At the same time, infection densities of Rickettsia endosymbiont were measured by a quantitative PCR technique by using the

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TaqMan PCR in terms of 16S rRNA gene copies. Quantitative PCR amplifications

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were as described below. Antibiotic treatment

Laboratory strains of C. viridis were maintained in a cage on rice seedlings stock

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until they produced eggs and nymphs. Administration of antibiotic was performed as

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described previously (Noda, et al., 2001; Narita, et al., 2007) and appropriately modified. One-day nymphs were fed continuously 12h with the artificial diet

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containing 0.05% antibiotics (rifampicin/ tetracycline hydrochloride/ Penicillin G potassium) and transferred to rice seedlings which were sprayed containing 0.1% antibiotics (12h) . For a control treatment, nymphs were fed with continuously 12h with the artificial diet and transferred to rice seedlings which were treated with distilled water instead of the antibiotics. Following, these treated insects were collected at 5d, 10d, 15d, 20d, 33-35d (male), 35-37d (female), respectively. An individual specimen was subjected to DNA extraction by using DNeasy Blood & Tissue Kit (QIAGEN, Germany). Rickettsia titers in the DNA samples were measured by a quantitative PCR technique by using the TaqMan PCR. Quantitative PCR amplifications were as described below. Quantitative PCR Infection densities of Rickettsia endosymbiont were measured by a quantitative PCR technique in terms of 16S rRNA gene copies from the host insect. Quantitative PCR was performed by using TaqMan PCR and a LightCycler®480 (Roche). 6

ACCEPTED MANUSCRIPT Rickettsia was quantified in terms of 16S rRNA gene copies by using primers RickendoCV-F3 (CGTAG GCGGT TTAGT AAGTT GG) and RickendoCV-R4 (GGTAT CTAAT CCTGT TTGCT CCC) and a probe RickendoCV-P (5'-FAM- ACC TCG GAA

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TTG CTT TCA AA A CTA C -BHQ-3').

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Genbank accession numbers

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Representatives of the 16S rRNA bacterial genes sequenced from C. viridis were submitted to the GenBank database; the accession numbers are from KT340612 to

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KT340626 for Rickettsia endosymbionts of C. viridis.

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RESULTS

16S rRNA bacterial gene sequencing and BLAST search

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The DNA of 10 individual leafhoppers was extracted, and PCR was performed

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using universal primers (27F-1513R) for the bacterial 16S rRNA gene, followed by cloning, sequencing, and a BLAST search. These individuals examined harbored

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Rickettsia symbiont in addition to the essential symbiont Sulcia and Sodalis. In these clones, twenty-four clones (4clones of female, 20clones of male, (Table 1)) were classed as Rickettsia, Rickettsiaceae, Rickettsiales, Alphaproteobacteria, Proteobacteria. All the sequences, which were between 1 461 and 1 463 bp in length, showed high similarity, on average, 99%, with the pea aphid Acyrthosiphon pisum-associated microorganism 16S rRNA gene (GenBank accession no.:AB196668) (Sakurai, et al., 2005). These sequences were amplified from nine tested leafhoppers. The data suggested that they were stably associated with C. viridis. Molecular phylogenetic analysis of Rickettsia endosymbionts Molecular phylogenetic analyses were performed on 16S rRNA gene sequences obtained from Rickettsia endosymbiont of C. viridis. The 16S rRNA gene sequences of the phylogenetic tree were from three different groups of the Rickettsia genus, namely, the typhus group, the spotted fever group, and the Rickettsia bellii group (Stothard, et al., 1994; Roux, et al., 1997; Blanc, et al., 2007). The phylogenetic tree 7

ACCEPTED MANUSCRIPT was generated by the Bayesian (BA) and maximum-likelihood (ML) methods. The Rickettsia endosymbiont sequences were ordered in the Rickettsia bellii group with 1/95(BA/ML) bootstrap value support within the group, and were most closely related

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to the bacterium associated with the pea aphid Acyrthosiphon pisum (Fig. 1).

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Prevalence of Rickettsia endosymbiont in C. viridis

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A collection of 44 individuals (including 10 cloned individuals) of C. viridis, recovered in different years from Huaxi, was screened by specific PCR assays to

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evaluate the prevalence of Rickettsia endosymbiont in populations of C. viridis. Rickettsia endosymbionts were found in field-collected individuals (field infection

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rate, 98%). Of the 22 females and 22 individual males examined, 21 females and all males were found to be positive (Table 2).

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Localization of Rickettsia endosymbionts in C. viridis

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Six adults (three females and three males) were dissected and quantitative PCR analyses for Rickettsia 16S rRNA gene copies were performed with DNA extracted

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from the different organs/tissues (ovaries, bacteriomes from three females; testes from three males; guts, and pairs of salivary glands from three females/males ). Rickettsia endosymbionts were detected in dissected organs of the leafhopper (the ovaries, testes, bacteriomes, guts, and salivary glands). Rickettsia was the most abundant in the ovary and the lowest density in the slivary gland of male (Fig. 2). Prevalence and infection density of the Rickettsia endosymbiont at different development stages of C. viridis In 2014, the leafhoppers were reared on rice seed stocks that tested negative for the presence of the Rickettsia endosymbiont. Insects from each stage were collected and DNA was extracted from single individuals. Rickettsia endosymbiont was detected at all developmental stages of the subsequent first and second generations by specific PCR. The rates of infection were 100% in eggs, all nymph stages of the first and second generation from the first to fifth nymphal instars, and in both female and male adults (Table 3). 8

ACCEPTED MANUSCRIPT Rickettsia 16S rRNA gene copy number per insect from different developmental stages of C. viridis was determined using a quantitative TaqMan-PCR approach. The infection density of Rickettsia unsteadily increased from egg to the fifth instar, which

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was the lowest in the third instar and the highest in the fifth instar. (Fig. 3).

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Infection density of Rickettsia between antibiotic-treated and untreated insects By using a quantitative PCR technique, we investigated the copy number of the

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Rickettsia symbiotic bacteria in the antibiotic-treated leafhoppers and the untreated leafhoppers. The antibiotic-treated insects exhibited lower infection density than the

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untreated insects. The differences were statistically significant among different antibiotic (rifampicin/ tetracycline hydrochloride/ Penicillin G potassium)

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(generalized linear model after Bonferroni correction; P< 0.05) (Table 4).

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DISCUSSION

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Our results clearly demonstrate that the Rickettsia endosynbiont of C. viridis resides within the α-Proteobacteria, and belongs to the genus Rickettsia. All members

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of Rickettsia are intracellular parasites of arthropods, and many are arthropod-vectored disease agents in vertebrates (Raoult and Roux, 1997). This genus has been classified into three groups, namely, the typhus group, the spotted fever group and the Rickettsia bellii group (Stothard, et al., 1994; Roux, et al., 1997; Blanc, et al., 2007). The spotted fever group members, which contain the species type R. rickettsii, are causative agents of spotted fever and are vectored by ticks. The typhus group members, R. typhi and R. prowazekii, are vectored by fleas, and cause murine typhus and epidemic typhus, respectively. Orientia tsutsugamushi (Tamura, et al., 1995), previously assigned to the genus Rickettsia, is the causative agent of scrub typhus and is transmitted by mites. The agent of California murine typhus, the ELB bacterium, was isolated from cat fleas (Adams, et al., 1990). The previously described AB bacterium is associated with male deaths of a predaceous ladybird beetle, Adalia bipunctata (Werren, et al., 1994). We analyzed the 16S rRNA gene sequences of the Rickettsia endosymbiont in C. 9

ACCEPTED MANUSCRIPT viridis, and it showed the highest level of similarity with the aphid endosymbiont R. bellii (99% nucleotide similarity for 16S rRNA gene sequences,). Phylogenetic analyses based on 16S rRNA gene sequences placed the Rickettsia endosymbiont of C.

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viridis in a well-supported basal clade that includes R. bellii and the symbionts of

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non-blood-feeding arthropods. The Bayesian maximum-likelihood analyses

C. viridis was placed in the R. bellii group.

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successfully resolved the phylogeny of Rickettsia and the Rickettsia endosymbiont of

The Rickettsia endosymbiont was widely distributed within the insect body of C.

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viridi, colonizing different organs. Interestingly, in the gonads, the endosymbiont was detected in both the oocytes and testes, which suggests a venereal transmission from

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male to female, as reported previously for beneficial symbionts in aphids (Moran, et al., 2006) and for the acetic acid bacteria Asaia sp. in Anopheles stephensi (Damiani,

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et al., 2008, Favia, et al., 2007).

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Rickettsia bacteria are facultative symbionts, but in the booklouse, Liposcelis bostrychophila, the association is strictly obligate and Rickettsia has an essential role

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in oocyte development (Perotti, et al., 2006; Yusuf, et al., 2004). Facultative symbiotic Rickettsia strains have been reported to negatively affect some aspects of host fitness, causing reductions in body weight, fecundity, and longevity in the pea aphid (Chen, et al., 2000; Sakurai, et al., 2005; Simon, et al., 2007), reductions in the viability of some blood-feeding arthropod vectors (Azad Beard, 1998; Niebylski, et al., 1999), and increased susceptibility to insecticides in the sweet potato whitefly (Kontsedalov, 2008). There is also evidence that Rickettsia has positive effects on host fitness, such as a larger body size in infected leeches (Kikuchi and Fukatsu, 2005) and a possible role in the oogenesis of a bark beetle (Zchori-Fein, et al., 2006). Finally, facultative symbiotic Rickettsia can be reproductive parasites of insects. Rickettsia strains are the causal agents of male killing (infected male embryos die) in some ladybirds (von der Schulenburg, et al., 2001; Werren, et al., 1994) and buprestid leaf-mining beetles (Lawson, et al., 2001). They are also the cause of thelytokous parthenogenesis (in which mothers produce only daughters from unfertilized eggs) in 10

ACCEPTED MANUSCRIPT a parasitoid wasp (Hagimori, et al., 2006). We investigated infection density of the Rickettsia endosymbiont in terms of 16S rRNA gene copy number at different development stages of C. viridis. Rickettsia was determined at each life stage, which

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illustrated that Rickettsia endosymbiont is stably associated with C. viridis. By

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antibiotic-treatment, the antibiotic-treated insects exhibited lower infection density of Rickettsia endosymbiont than the untreated insects. Although we do not know the role

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of this microorganism in C. viridis biology, this specific Rickettsia distribution may

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inform future studies.

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ACKNOWLEDGMENTS

We thank De-Yan GE (Institute of Zoology, Chinese Academy of Science) and Mao-Fu Li (Beijing Academy of Agricultural Sciences) for their help during the

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preparation of this work. We also need to thank undergraduate students Ting Luo,

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Guang Wu, Xiao-Yun Zhou for helping us observing some experiments. This project was supported by the National Natural Science Foundation of China (30770253), the

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National Specialized Research Fund for Basic Science of the Ministry of Science and Technology of China (2006FY120100), Program for New Century Excellent Talents in University (NCET-07- 0221), the Program of Science and Technology Innovation Talents Team, Guizhou Province (20144001), and the Provincial Outstanding Graduate Program for Agricultural Entomology and Pest Control (ZYRC-(2013)010).

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TABLE 1 No. of positive clones of Rickettsia endosymbionts in leafhopper C. viridis.

TABLE 2 Infection frequency of Rickettsia endosymbiont, in a natural population of C.

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TABLE3 Results of PCR amplification of DNA from Rickettsia endosymbionts at different developmental stages of the leafhopper C. viridis.

TABLE 4 Rickettsia 16S rRNA gene copies in the antibiotic-treated leafhoppers and the untreated leafhoppers.

FIGURE 1 Molecular phylogenetic analysis of the Rickettsia endosymbiont from Cicadella viridis based on the 16S ribosomal RNA gene sequence. A Bayesian (BA) phylogeny is shown. The maximum likelihood (ML) analysis gave substantially the same results. Statistical support values > 60 % are shown at the nodes in the order of BA/ML. The numbers in brackets are accession numbers Three major groups in the genus Rickettsia, “spotted fever group,” “typhus group,” and “bellii group,” are indicated on the right hand side.

FIGURE 2 Rickettsia densities in terms of 16S rRNA gene copies in the different organs/tiisues of C. viridis. The organs/tissues were dissected from three insects (ovaries, bacteriomes from three females; testes from three males; guts, and pairs of salivary glands from three females/males). Means and standard error are shown. 16

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FIGURE 3 Rickettsia densities in terms of 16S rRNA gene copies at the developmental stages of C. viridis.

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Means and standard error are shown.

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Figure 1

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Figure 2

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Figure 3

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ACCEPTED MANUSCRIPT The tables of the article (ASPEN 808)

Collection date

Male and Female

1

29/Jun/2013

M

11

2

13/Jul/2013

M

11

3

3

13/Jul/2013

F

12

1

4

3/Aug/2013

M

9

3

5

3/Aug/2013

F

9

1

6

20/Aug/2013

M

11

6

7

20/Aug/2013

F

11

1

8

10/Sep/2013

M

6

3

9

10/Sep/2013

F

12

1

10

1/Oct/2013

F

10

0

102(53F/49M)

24(4F/20M)

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Total

Total no. of clones

No. of positive clones

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Sample code

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TABLE 1 No. of positive clones of Rickettsia endosymbionts in leafhopper C. viridis

5

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F, female leafhopper; M, male leafhopper

TABLE 2 Infection frequency of Rickettsia endosymbiont, in a natural population of C. viridis Yr 2013 2014

generation

No. tested

No. positive

Infection rate (%)

First generation Second generation First generation Second generation

5F/5M 5F/5M 6F/6M 6F/6M 22F/22M

5F/5M 4F/5M 6F/6M 6F/6M 21F/22M

100F/100M 80F/100M 100F/100M 100F/100M 95F/100M

Total F, female leafhopper; M, male leafhopper

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ACCEPTED MANUSCRIPT TABLE 3 Results of PCR amplification of DNA from Rickettsia endosymbionts at different developmental stages of the leafhopper C. viridis No. positive/no. tested (%) First generation

Second generation

Egg

10/10 (100)

10/10 (100)

Nymph 1

10/10 (100)

10/10 (100)

Nymph 2

10/10 (100)

10/10 (100)

Nymph 3

10/10 (100)

10/10 (100)

Nymph4

10/10 (100)

Nymph 5

10/10 (100)

Female

6/6 (100)

Male

6/6(100)

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Stage

10/10 (100)

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10/10 (100)

6/6 (100)

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6/6 (100)

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TABLE 4 Rickettsia 16S rRNA gene copies in the antibiotic-treated leafhoppers and the untreated leafhoppers. 5d

10d

15d

20d

33-35d (M)

35-37d (F)

Untreated

7866.67±341.97a

9416.67±117.95a

10093.33±457.58a

12366.67±290.59a

42500.00±1552.42a

43333.33±1369.10a

Penicillin G potassium

7423.33±676.49a

9356.67±98.21a

9893.33±206.67a

10120.00±349.48b

29866.67±1316.98b

33066.67±2867.25b

Terramycin hydrochloride

4230.00±124.23b

5150.00±246.64b

6220.00±223.68b

9703.33±148.36b

14200.00±665.83c

14300.00±208.17c

Rifampicin

1156.67±26.03c

2506.67±106.51c

6040.00±112.69b

7226.67±54.87c

15833.33±384.42c

16567.00±233.00c

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Control and Antibiotic

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