A single-pair method to screen Rickettsia-infected and uninfected whitefly Bemisia tabaci populations

Journal Pre-proof A single-pair method to screen Rickettsia-infected and uninfected whitefly Bemisia tabaci populations

Yuan Liu, Ze-Yun Fan, Xuan-An, Pei-Qiong Shi, Muhammad Z. Ahmed, Bao-Li Qiu PII:

S0167-7012(19)30781-X

DOI:

https://doi.org/10.1016/j.mimet.2019.105797

Reference:

MIMET 105797

To appear in:

Journal of Microbiological Methods

Received date:

7 September 2019

Revised date:

29 November 2019

Accepted date:

29 November 2019

Please cite this article as: Y. Liu, Z.-Y. Fan, Xuan-An, et al., A single-pair method to screen Rickettsia-infected and uninfected whitefly Bemisia tabaci populations, Journal of Microbiological Methods (2019), https://doi.org/10.1016/j.mimet.2019.105797

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© 2019 Published by Elsevier.

Journal Pre-proof Submitted to < Journal of Microbiological Methods>

A single-pair method to screen Rickettsia-infected and uninfected whitefly Bemisia tabaci populations Yuan Liua,b, Ze-Yun Fana,b, Xuan-Ana,b, Pei-Qiong Shic, Muhammad Z Ahmedd, Bao-Li Qiua,b,e,*

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[email protected] Key Laboratory of Bio-Pesticide Innovation and Application, Guangdong Province, Guangzhou 510640,

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China b

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Engineering Research Center of Pest Biocontrol, Ministry of Education and Guangdong Province, Guangzhou 510640, China

College of Agriculture, Guangdong Ocean University, Zhanjiang 524088, China

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Florida Department of Agriculture and Consumer Services, Division of Plant Industry, 1911 SW 34th Street, Gainesville, FL 32614-7100, USA

Department of Entomology, South China Agricultural University, Guangzhou 510640, China

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Corresponding author at: Department of Entomology, South China Agricultural University No.483,

Abstract

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Wushan Rd, Tianhe, Guangzhou 510640, China

Bacterial endosymbionts such as Rickettsia and Wolbachia play prominent roles in the development and behaviour of their insect hosts, such as whiteflies, aphids, psyllids and mealybugs. Accumulating studies have emphasized the importance of establishing experimental insect populations that are either lacking or bearing certain species of endosymbionts, because they are the basis in which to reveal the biological role of individual symbionts. In this study, using Rickettsia as an example, we explored a “single-pair screening”

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method to establish Rickettsia infected and uninfected populations of whitefly Bemisia tabaci MEAM1 for further experimental use. The original host population had a relatively low infection rate of Rickettsia (< 35%). When B. tabaci adults newly emerged, unmated males and females were randomly selected, and released into a leaf cage that covered a healthy plant leaf in order to oviposit F1 generation eggs. Following 6 days of oviposition, the parents were recaptured and used for PCR detection. The F1 progeny, for which

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parents were either Rickettsia positive or negative, were used to produce the F2 generation, and similarly in

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turn for the F3, F4 and F5 generations respectively; if the infection status of Rickettsia was consistent in the

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F1 to F5 generations, then the populations can be used as Rickettsia positive or negative lines for further

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maternal transmission in different generations.

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experiments. In addition, our phylogenetic analyses revealed that Rickettsia has high fidelity during the

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1. Introduction

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Keywords: Bemisia tabaci; endosymbionts; Rickettsia; maternal transmission; infection rate

Intracellular bacteria endosymbionts are ubiquitously found in a wide range of insects and arthropods (Stevens et al., 2001; Stouthamer et al., 1999). The interaction between bacteria and insects can be parasitic, symbiotic, or neutral (Bourtzis and Miller, 2006). These endosymbionts are mainly transmitted maternally from mother to offspring and can be categorized into two types: primary (obligate) and secondary (facultative) endosymbionts (Bing et al., 2013a; Bing et al., 2013b; Gnankiné et al., 2013). The primary endosymbionts, such as Portiera in whiteflies, Buchnera in aphids and Carsonella in psyllids, can supply essential amino acids and carotenoids lacking in their hosts’ diets (Baumann, 2005; Douglas, 1998),

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whereas secondary endosymbionts may have direct influences on the performance of their hosts (Kliot et al., 2014; Himler et al., 2011; Mahadav et al., 2008). In general, they substantially affect the physiology, ecology, reproduction and behaviour of their hosts in a variety of ways (Baumann et al., 2006; Bourtzis and Miller, 2006; O’Neill et al., 1997). Among the secondary (facultative) endosymbionts, Rickettsia (Alphaproteobacteria) is well known

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and is usually non-pathogenic in nature (Raoult and Roux, 1997). Rickettsia plays vital roles in promoting

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host fitness and female sex ratio (Himler et al., 2011), protecting their hosts against fungal pathogens

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(Ahmed et al., 2015; Oliver et al., 2003), assisting in escaping from natural enemy parasitism or predation

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(Kaltenpoth et al., 2010; Kaltenpoth et al., 2005; Currie et al., 2003a; Currie et al., 2003b), causing

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thelytokous parthenogenesis in the parasitoid wasp Pnigalio soemius (Giorgini et al., 2010), enhancing the ability of host insects to resist high temperature (Brumin et al., 2011), increasing susceptibility to some

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insecticides like imidacloprid, thiamethoxam and pyriproxyfen (Kontsedalov et al., 2010), increasing viral

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transmission efficacy (Kliot et al., 2014) and changing the colour of pea aphids, Acyrthosiphon pisum

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(Tsuchida et al., 2010). Rickettsia’s ability to modify host biology and physiology has therefore attracted increasing interest in its potential usage as a biological control agent for invertebrate pest management (Douglas, 2015). The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is one of the most important cosmopolitan polyphagous insects. It is a serious agricultural pest that affects over 500 crop species and has a worldwide distribution (Gill et al., 1938). Yield losses occur due to directly sucking phloem sap, honeydew contamination, and virus transmission, some of which are devastating (Karut et al., 2017). Bemisia tabaci is considered a cryptic species complex, and it harbors many bacterial symbionts such as

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Rickettsia, Hamiltonella, Cardinium, Hemipteriphilus, Arsenophonus, Wolbachia and Fritschea (Bing et al., 2013a; Gottlieb et al., 2006; Baumann, 2005; Everett et al., 2005). In order to evaluate the potential roles that individual symbionts play in complex systems consisting of insects and multiple endosymbiotic bacteria, establishing experimental insect populations with a controlled presence or absence of certain symbionts has proven to be a useful tool (Leonardo, 2010; Leonardo and

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Mondor, 2006; Sakurai et al., 2005; Tsuchida et al., 2004; Koga et al., 2003). Many researchers have tried to

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establish a symbiont-plus or symbiont-free population using a number of different methods (Koga et al.,

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2010). Yet, despite the advent of modern molecular and bioinformatic techniques, research of fastidious

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insect symbionts still faces numerous challenges (Douglas, 2014). Here, we used polymerase chain reaction

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(PCR) to identify infection status of endosymbionts, then calculated the infection rates of the endosymbionts in B. tabaci MEAM1 (Middle East Asia Minor 1) cryptic species from South China. Since

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Rickettsia has a relatively low infection rate (<35%) in natural populations, the “single-pair screening”

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method can easily establish stable Rickettsia positive and negative populations. Following on, we then

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tested for the presence or absence of Rickettsia in various life stages of the MEAM1 whitefly population development across five generations. Taken together, our study will not only provide an important reference for the screening of other symbiotic bacteria, but will also lay a foundation to uncover the function and transmission of symbionts.

2. Materials and methods 2.1. Host plants and insects

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The cotton plant (Gossypium hirsutum L. var. Lumianyan no. 32) was used to rear whiteflies. Cotton seeds were sown in plastic pots (12-cm diameter × 15-cm height) containing a soil–sand mixture (10% sand, 5% clay and 85% peat) in cages that were kept in an environmental chamber (20-35 °C, RH70-85%). All the plants were free of whiteflies and used for experiments at the 6-8 expanded leaf stage. The Bemisia tabaci (MEAM1 cryptic species) were initially acquired from eggplants (Solanum

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melongena) grown at the Engineering Technology Research Center of Pest Biocontrol, South China

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Agricultural University (SCAU), Guangzhou in 2015. Populations were then reared on cotton plants under

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standard laboratory conditions of 26 ± 2oC, 60% RH and a photoperiod of 14:10 (L:D) h. In addition,

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mitochondrial COI gene sequencing was used to maintain the purity of the B. tabaci populations (Qiu et al.,

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2009).

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2.2. PCR detection and DNA sequencing of endosymbionts in whitefly

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Total DNA was extracted from single whiteflies of collected populations following the method of Ahmed et

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al. (2010). Whitefly adults were individually homogenized in 2 μL STE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) using a 1.5 mL Eppendorf tube. Subsequently, another 14 μL lysis buffer and 0.5 μL proteinase K (10 mg/ml) were added into the tube. The mixture was then incubated at 56oC for 2-3 hours in a water bath and then at 96oC for 10 min. The samples were centrifuged briefly and then either used immediately for the PCR reactions or stored at -20oC for later use. All PCR reactions were run in a 25 μL buffer containing 1 μL of the template DNA lysate, 1 μL of each primer, 2.5 mM MgCl2, 200 mM for each dNTP and 1 unit of DNA Taq polymerase (Invitrogen, Guangzhou, China) (Shi et al., 2018). The primers used in this study were for the Rickettsia 16S rRNA gene (Gottlieb et

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al., 2006). The detection of other endosymbionts, including Portiera, Hamiltonella, Cardinium and Hemipteriphilus, was confirmed by using their genus-specific primers shown in Table 1(Bing et al., 2013a; Chiel et al., 2007; Gottlieb et al., 2006; Weeks et al., 2003; Zchori-Fein and Brown, 2002).

Table 1 PCR primers and associated program for endosymbiont detection in Bemisia tabaci MEAM1

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cryptic species. Primer sequence (5′-3′)

Rickettsia 16S r RNA

Rb-F:5'-GCTCAGAACGAACGCTATC-3'

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Annealing temperature

Symbiont

58°C

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Rb-R:5'-GAAGGAAAGCATCTCTGC-3' 28 F:5'-TGCAAGTCGAGCGGCATCAT-3'

16S rRNA

1098 R:5'-AAAGTTCCCGCCTTATGCGT-3'

Hamiltonella

Ham-F:5'-TGAGTAAAGTCTGGAATCTGG-3'

16S rRNA

Ham-R:5'- AGTTCAAGACCGCAACCTC-3'

Cardinium 16S rRNA

CFB-F:5'-GCGGTGTAAAATGAGCGTG-3'

Hemipteriphilus 16S rRNA

OLO-F:5'-GCTCAGAACGAACGCTRKC-3'

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Portiera

60°C

Product size (bp)

Reference

900

Gottlieb et al., 2006

1000-1100

Zchori-Fein and Brown, 2002

58°C

700

Chiel et al., 2007

57°C

400

Weeks et al., 2003

60°C

700

Bing et al., 2013a

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CFB-R:5'-ACCTMTTCTTAACTCAAGCCT-3'

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OLO-R:5'-TTCGCCACTGGTGTTCCTC-3'

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The amplified PCR products (5 μL) were electrophoresed in a 1.5% agarose gel containing Gold-View colourant in 1×TAE for 20 min at 120 mA and then photographed on a UV transilluminator. When bands with the expected size were visible on the gels, the remainder of the 20μL volumes of PCR products were sent to the Beijing Institute (BGI) for sequencing. The results were compared to known sequences using NCBI’ s BLAST algorithm. Finally, both the infection species and rates of endosymbionts were determined. Approximately 90-100 whitefly adults were randomly sampled and divided into three repeats for testing. The DNA of the primary endosymbiont Portiera was used as the positive control, and ddH2O was used as the negative control.

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2.3. Screening for Rickettsia positive and negative populations When the B. tabaci MEAM1 glasshouse populations were numerous enough, they were used in subsequent experiments. Population screening was conducted for single-pair purification using cotton as the host plants. Some whiteflies, which were newly emerged and not yet mated, were selected in order to identify their sex

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(females and males) under the stereomicroscope. Then, one pair of whiteflies were released into a leaf cage

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which was attached onto a clean cotton leaf to allow egg laying for 6 days. Following this, the parent adult

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whiteflies were recaptured from the cage and examined for the presence of Rickettsia using the

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Rickettsia-specific primers listed in Table 2 (16S rRNA, gltA and Pgt) (Caspi-Fluger et al., 2012; Gottlieb et

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al., 2006). The above steps were repeated to purify the population of B. tabaci MEAM1 until the F5 generation. DNA fragments from the whitefly parent adults, which were successfully amplified to about 900

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bp, 800 bp and 500 bp, were pooled to establish the Rickettsia-positive populations, while whitefly parent

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adults that tested negative for Rickettsia were pooled, and this population was treated as the

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Rickettsia-negative population. They had been established in order to ensure successive propagation on cotton. In addition, in order to ensure the purity of each population, approximately 100 adult whiteflies were selected to check the biotype and presence/absence of Rickettsia every month.

Table 2 The primers and protocols for Rickettsia PCR detection. Gene

Primer sequence (5′-3′)

Annealing temperature

Product size (bp)

Reference

16S r RNA

Rb-F:5'-GCTCAGAACGAACGCTATC-3'

58°C

900

Gottlieb et al.,

Rb-R:5'-GAAGGAAAGCATCTCTGC-3' gltA

gltA-F:5'-TCCTATGGCTATTATGCTTG-3' gltA-R:5'-CCTACTGTTCTTGCTGTGG-3'

2006 55°C

800

Caspi-Fluger et al., 2012

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Pgt-F1:5' -AGGTTTAGGCTAGTCTACACG-3'

52°C

500

Pgt-R1:5'-GTCTACGCACGATTGATG-3'

Caspi-Fluger et al., 2012

Pgt-F2:5'-ACTCATGAAATTATCGGCACAG-3' Pgt-R2:5'-GCATGAATTTGGCACTTAAGC-3'

2.4. Infection of Rickettsia at various stages of MEAM1 Bemisia tabaci development To examine the presence or absence of Rickettsia at various life stages of MEAM1 B. tabaci development,

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the DNA extraction of Rickettsia-positive whitefly eggs and nymphs (from 1st-4th instars), the diagnostic

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PCR, and the sequencing of DNA fragments were performed with essentially the same methods used for

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DNA extraction of eggs (about 10-15 eggs per test).

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whitefly adults, as described above. The only difference was that multiple individuals were used for each

2.5. Sequence alignment and phylogenetic analysis

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All the nucleotide sequences were edited and aligned using Lasergene v7.1 (DNASTAR, Inc., Madison,

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WI). The reference sequences were searched for homologous sequences of 16S rRNA gene in GenBank

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database using basic local alignment search tools. In addition, the Bayesian information criterion was used to select the best model and partitioning scheme in PartitionFinder v. 1.0.1 (Lanfear et al., 2012). Phylogenetic trees were generated based on the maximum likelihood (ML) method with 1,000 non-parametric bootstrap replications in RAxML (Stamatakis, 2006). Orientia tsutsugamushi was used as an outgroup.

2.6. Statistical analyses Infection comparisons of the endosymbionts in MEAM1 B. tabaci were analyzed using one-way analysis of

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variance (ANOVA), and means were compared using the Duncan test (SPSS17.0) at P<0.05. An infection chart was drawn with Sigmaplot 10.0.

3. Results 3.1. Detection of the endosymbiont species in MEAM1 Bemisia tabaci The PCR tests revealed that one primary endosymbiont, Portiera aleyrodidarum (16S rRNA, 1000-1100bp),

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and three species of secondary endosymbionts, Rickettsia (16S rRNA, about 900bp), Hamiltonella (16S

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rRNA, about 700bp), and Hemipteriphilus (16S rRNA, about 700bp) were found in MEAM1 B. tabaci (Fig.

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1).

Fig. 1. PCR detection of endosymbionts in Bemisia tabaci MEAM1 cryptic species. M, DNA marker; lane 1, positive control (Portiera 16S rRNA); lane 2, negative control (ddH2O); lane 3, Rickettsia; lane 4, Hamiltonella; lane 5, Cardinium; lane 6, Hemipteriphilus.

3.2. Infection rates of three endosymbionts in MEAM1 Bemisia tabaci

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Examination of individual adults revealed that all the whiteflies were infected with the primary endosymbiont Portiera aleyrodidarum (100%). The infection rates of Rickettsia, Hamiltonella and Hemipteriphilus in B. tabaci MEAM1 were 32.67 ± 2.03%, 91.67 ± 3.18%, and 94.33 ± 4.26% respectively (Fig. 2). The infection rate of Rickettsia was significantly lower than that of Hamiltonella and

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Hemipteriphilus (F2，8=112.75, P<0.01).

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Fig. 2. Infection rates of three endosymbionts in Bemisia tabaci MEAM1 cryptic species. Data are Mean ±SE of approximately 100 samples in three repeats. The different letters above the bars indicate

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significant differences between the endosymbionts according to Duncan’s test (P<0.05).

3.3. Screening for Rickettsia positive and negative populations In order to establish stable Rickettsia-positive and -negative populations of B. tabaci MEAM1, we tested for presence or absence of Rickettsia using three genes: 16S rRNA gene, gltA gene and Pgt gene. The PCR results for all five generations of whiteflies suggested that one Rickettsia-positive and one Rickettsia-negative population of B. tabaci MEAM1 were successfully established by using the “single-pair

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screening” method (Fig. 3).

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Fig. 3. Rickettsia PCR detection in the screening populations of Bemisia tabaci MEAM1 cryptic species. M, DNA marker; lane 1 positive control (Portiera 16S rRNA); lane 2 negative control (ddH2O);

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lane 3, Rickettsia 16S rRNA gene; lane 4, Rickettsia gltA gene; lane 5, Rickettsia Pgt gene. Panel A,

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Rickettsia positive population; Panel B, Rickettsia negative population.

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3.4. Infection of Rickettsia at various stages of MEAM1 Bemisia tabaci development

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PCR detection at different life stages of the B. tabaci MEAM1 population demonstrated that all the life stages that were examined (adults, 1st-4th instar nymphs, eggs) were infected with Rickettsia endosymbionts (Fig. 4).

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Fig. 4. Rickettsia detection in different life stages of Bemisia tabaci MEAM1 cryptic species. M, DNA marker, from top 2000, 1000, 750, 500, 250, 100bp; lane 1, positive control (Portiera 16S rRNA);

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lane 2 negative control (ddH2O); lanes 3-8, adults, 1st-4th instar nymphs, eggs respectively. Panel

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A, Rickettsia 16S rRNA gene; Panel B, Rickettsia gltA gene; C, Rickettsia Pgt gene.

3.5. Phylogenetic analysis of Rickettsia The results of the phylogenetic analysis revealed that Rickettsia had high fidelity during maternal transmission in F1-F5 generations of whitefly hosts; all of the Rickettsia samples were clustered into one branch belonging to the Bellii group (Fig. 5).

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Fig. 5. Maximum likelihood phylogenetic analysis of Rickettsia based on 16S rRNA gene. “R” represented Rickettsia endosymbionts in MEAM1 Bemisia tabaci. Rickettsia endosymbionts from F1-F5

group.

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generations of Rickettsia positive populations were all clustered into one branch within the Bellii

4. Discussion The majority of insect species have associations with endosymbiotic microorganisms (Weinert et al., 2015; Jeyaprakash and Hoy, 2000; Werren and Windsor, 2000). Bemisia tabaci carries at least eight species of endosymbionts (Shi et al., 2018; Shan et al., 2016). Previous studies have reported that the prevalence of the endosymbionts in insect hosts may be correlated with climate, ecology, food plants, and individual

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development (Toju and Fukatsu, 2011). For example, Rickettsia has become the dominant secondary endosymbiont of B. tabaci MEAM1 from Israel, the USA and China (Himler et al., 2011; Chiel et al., 2007; Gottlieb et al., 2006). In our current study, Rickettsia had a relatively low infection rate (< 35%), which fell within the range (12 to 84%) of other B. tabaci MEAM1 populations reported by Cass et al. (2015), which indicated that Rickettsia-positive and -negative populations of B. tabaci coexisted in nature.

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Many recent studies have revealed the essential role of bacterial symbionts in the life of their insect

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hosts (Douglas, 2015; Gerardo and Parker, 2014; Oliver and Martinez, 2014), and have shown that these

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bacterial symbionts are currently unculturable. In order to evaluate the potential roles that individual

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symbionts play in complex systems, establishing experimental insect populations with or without certain

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symbionts have proven to be a powerful tool (Leonardo, 2010; Leonardo and Mondor, 2006; Sakurai et al., 2005; Tsuchida et al., 2004; Koga et al., 2003). Most of the methods employed for this purpose entail curing

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by antibiotics, transfection by microinjection and high temperature treatment. However, there have been no

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2010).

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studies on optimizing the selectivity and efficacy of the symbiont elimination techniques (Koga et al.,

Several articles report that attempts to use antibiotic treatments to cure certain symbionts range from no success (Chiel et al., 2009), partial success (Zhong and Li, 2013; Ruan et al., 2006), to complete success (Su et al., 2013; Xue et al., 2012; Ahmed et al., 2010). Some of these studies have indicated that it is not feasible to selectively eliminate the facultative symbionts using antibiotics (rifampicin) without affecting the primary symbiont and establishment of host lines for experimental studies (Shan et al., 2016). Furthermore, rifampicin application may lead to high mortality in treated individuals, slow insect growth, inability for males to fertilize eggs, and produce a high male ratio in offspring (Wang et al., 2017). Transfection by

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microinjection can selectively transfer facultative symbiotic organisms into a recipient. However, this requires special instruments, and this artificially generated host-symbiotic combination often leads to unstable infection or considerable deleterious effects on host fitness (Russell and Moran, 2005; Koga et al., 2003; McGraw et al., 2002). In this study, based on the lower infection of Rickettsia in MEAM1 B. tabaci we set up a “single-pair screening” method to establish stable Rickettsia-positive and -negative populations

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from genetic perspectives. These can also provide technical support for studying the functional mechanism

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of secondary symbiotic bacteria.

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Our results showed that Rickettsia existed in all the developmental life stages of B. tabaci MEAM1

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Guangzhou population, which is consistent with the previous study of Gottlieb et al. (2006). Phylogenetic

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analyses showed that Rickettsia had high fidelity during their maternal transmission in different generations, and Rickettsia endosymbionts from F1-F5 generations of Rickettsia positive populations were all clustered

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into one branch within the Bellii group.

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5. Conclusions

Our study shows the infection rates of endosymbionts in B. tabaci MEAM1 collected from south China. Specifically, we set up a stable “single-pair screening” method to establish Rickettsia-positive and -negative experimental insect populations of B. tabaci MEAM1. This method can also be used in other endosymbiont population screening studies, as long as the endosymbionts’ infection rates are less than 100% in their arthropod hosts. Acknowledgements This research was funded by the National Natural Science Foundation of China (31672028), Guangdong

Journal Pre-proof Technological Innovation Strategy of Special Funds (2018B020205003), the Science and Technology Program of Guangzhou (201804020070), the Guangdong Science and Technology Innovation Leading Talent Program (2016TX03N273) to BLQ.

Author contribution We thank Dr. Andrew Cuthbertson (York UK) for his critical comments on the manuscript. The study was originally designed by BLQ; YL, ZYF, XA, PQS carried out the experiments; YL, PQS and MZA

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participated in data analysis; YL, BLQ and MZA wrote the paper. All authors gave their final approval for

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publication.

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Conflict of interest

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The authors declare no conflict of interest.

Data available

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The Pgt, gltA and 16S rRNA gene sequences of Rickettsia endosymbionts in whitefly Bemisia tabaci

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MEAM1 cryptic species were deposited in GenBank with accession numbers of KX645660-KX645662.

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Highlights Symbiotic bacteria play prominent roles in the development of their insect hosts.

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Availability of infected and uninfected hosts are the basis to reveal symbiont roles.

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Single-pair screening method was introduced to set up different symbiotic hosts.

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Whiteflies and endosymbiont Rickettsia were used as examples in this study.

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Rickettsia has high fidelity during the maternal transmission between generations.

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