A CLas negative colony of ACP was initially collected from CLas-free Murraya exotica L. growing in the ornamental landscape of South China Agricultural University (SCAU, Guangzhou, China) in May 2014. Then it was reared on potted M. exotica in a greenhouse at SCAU. M. exotica plants were pruned regularly to promote the growth flushes necessary to stimulate ACP oviposition. The ACP populations were periodically (at least once a month) tested to ensure the colony was CLas-free using nested quantitative PCR detection according to the method described by Coy et al.30.
The parasitoid T. radiata used in the current study was initially collected from ACP hosts on M. exotica plants in the above-mentioned location during June 2015. Its population was maintained in rearing cages (60 × 60 × 60 cm) using a CLas-free ACP-M. exotica rearing system under laboratory conditions (26 ± 1 °C, RH 80 ± 10% with L:D = 14:10 photoperiods in insect incubators).
CLas-free and CLas-infected plants of Citrus reticulata Blanco cv. Shatangju were used in the current study. Both plant types were obtained from The Citrus Research Institute of Zhaoqing University (Guangdong, China). The CLas-infected plants were inoculated by shoot grafting. All plants were approximately 4-year old and 1.2−1.5 m in height, separated in nylon net greenhouses (70 mesh per inch2) at two different locations about 2.2 km apart in SCAU. Again, nested qPCR detection was performed periodically (at least once a month) to test for the presence or absence of CLas in the citrus plants according to the method described by Coy et al.30.
Acquisition and persistence of CLas in Tamarixia radiata
When new shoots of CLas-infected C. reticulata plants were grown to 5–8 cm, 20 pairs of 1 week-old ACP adults were introduced into one nylon bag covering one fresh shoot to lay eggs for 48 h. When the progeny of ACP developed through to 4th or 5th instar nymph (CLas-donor ACP), which are the stages preferred by T. radiata parasitoids, 150 of the ACP nymphs were randomly selected and the remaining ones were removed. Following this, 10 pairs of 3-day old T. radiata adults, randomly selected from the population that has been tested to be CLas-free, were introduced into the nylon bag in order to parasitize the 4th or 5th instar ACP nymphs for 48 h before being recaptured. Then the potentially parasitized ACP nymphs together with the citrus plants were cultured in a plant growth chamber (Jiangnan Instrument Company, RXZ-500D, at 26 ± 1 °C, 60 ± 2% RH and 14:10 h L:D photoperiod of 3,000 lx illumination).
When the progeny of T. radiata (considered F0 generation) developed to 3-day egg, 1st to 4th instar larvae, pupae, and adult stages respectively, they were identified and collected with the assistance of a stereomicroscope. DNA of each stage sample was extracted using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) for CLas qPCR detection and titer quantification. Thirty eggs, 20 individuals of 1st or 2nd instar, 10 individuals of 3rd or 4th instar larvae or pupa, as well as three individuals of female or male adults were subsequently ground together to represent each life stage in qPCR, and each stage qPCR detection was repeated three times.
The primers used for CLas qPCR detection were LJ900 primers, (F5′-GCCGTTTTAAC ACAAAAGATGAATATC-3′, R5′-ATAAATCAATTTGTTCTAGTTTAC GAC-3′), and 18S rRNA gene of T. radiata (F5′-AAACGGCTACCACATCCA-3′, R5′-ACCAGACT TGCCCTC CA-3′)31 was used as an internal control for DNA normalization and quantification. In order to normalize the qPCR values, each qPCR reaction was performed in three independent runs using SYBR Premix Ex Taq (Takara, Dalian, China) in Bio-Rad CFX Connect™ Real-Time PCR Detection System, with a protocol of initial denaturation at 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s and 72 °C for 30 s.
To monitor the CLas persistence in T. radiata, newly emerged female adults of T. radiata (considered F1 generation) were collected from the above experiment and fed with 20% honey water. After 1, 5, 10, and 15 days, 10 parasitoids were recaptured, subsequently ground for DNA extraction and CLas titer detection and quantification using qPCR. The protocol of DNA extraction and qPCR reaction was the same as above, and qPCR quantification was repeated three times for each treatment.
Localization patterns of CLas in Tamarixia radiata
Localization patterns of CLas in different instars of T. radiata
Fluorescent in situ hybridization (FISH) was used to visualize the distribution of CLas in T. radiata exposed to CLas positive ACP, following the method of Gottlieb et al.32 with a slight modification. Eggs and different larval instars of T. radiata were collected and fixed in Carnoy’s solution (chloroform-ethanol-glacial acetic acid [6:3:1,vol/vol] formamide) overnight at 4 °C. After fixation, the samples were washed three times in 50% ethanol with 1× phosphate buffered saline (PBS) for 5 min. Then the samples were decolorized in 6% H2O2 in ethanol for 12 h, after which they were hybridized overnight in 1 ml hybridization buffer (20 mM Tris-HCl pH 8.0, 0.9 M NaCl, 0.01% sodium dodecyl sulfate, 30% formamide) containing 10 pmol of fluorescent probes/ml in a 37 °C water bath under dark conditions. The CLas probe used for FISH was 5′-Cy3-GCCTCGCGACTTCGCAACCCAT-3′. Finally, the stained T. radiata samples were washed three times in a washing buffer (0.3 M NaCl, 0.03 M sodium citrate, 0.01% sodium dodecyl sulfate, 10 min per time). After the samples were whole mounted and stained, the slides were observed and photographed using a Nikon eclipse Ti-U inverted microscope. For each stage sample, approximately 20 individuals were examined to confirm the results.
Localization patterns of CLas in different organs of T. radiata
Different organs (gut, fat body, ovary, poison sac, salivary glands, spermatheca, and chest muscle) were dissected from newly emerged adults of T. radiata in 1× phosphate buffered saline (PBS) under a stereomicroscope using a depression microscope slide and a fine anatomical needle. After a sufficient number of each tissue sample was collected (20 or more), the tissues were washed three times with 1 × PBS, followed by the fixation, decolorization, and hybridization procedures as outlined above, except that this time of decolorization was 2 h. After hybridization, nuclei in the different organs were counterstained with DAPI (0.1 mg/ml in 1 × PBS) for 10 min, then the samples were transferred to slides, mounted whole in hybridization buffer, and viewed using confocal microscopy (Nikon, Japan).
Maternal transmission of CLas between Tamarixia generations
Five groups of experiments were used to clarify whether CLas can be transmitted vertically between different T. radiata generations. In the first group, 60 pairs of newly emerged T. radiata adults from the CLas-infected ACP colony (potential CLas-acquired parasitoid adults, F0 generation) were introduced into 60 nylon bags (one female per cage). Each bag covered one fresh citrus plant shoot with one marked CLas-free 4th instar nymph of ACP, the parasitoid females were given 24 h to oviposit, then transferred to another four groups successively to oviposit with intervals of 24 h before they were recaptured for CLas-PCR detection (58/60 and 56/60 T. radiata females and males respectively were CLas-infected). Only the progeny (F1 generation) in which parasitoid parents were both CLas-infected continued to be investigated.
When the F1 progeny of CLas-infected parasitoid females developed to egg, larval, pupal, and adult stages respectively, they were collected and divided into two groups; in one group samples were used for the qPCR detection of the CLas titer, and the other group was used for the FISH visualization of CLas. The qPCR and FISH analysis protocols of CLas as well as the number of tested individuals were the same as previously outlined. Each stage was repeated three times.
CLas detection in T. radiata-inoculated ACP
Quantitative PCR detection of CLas
Approximately 60 newly emerged parasitoid adult females from CLas-infected ACP hosts (potential CLas-acquired parasitoid adults) were collected using an aspirator. They were first starved for 5 h, then released into finger tubes (diameter 6 mm × length 30 mm); one female per tube containing one 4th instar nymph of CLas-free ACP (this was treated as one experimental replicate). The probing behavior of the parasitoids was observed under a stereomicroscope, after which the parasitoids were recaptured for CLas PCR detection (similar to the above experiment, approximately 95% were CLas-infected). Only those 4th instar ACP nymphs, probed for egg-laying by a CLas-infected parasitoid but survived from the probing (the averaged proportion of such samples was 5.36 ± 0.47% and were 100% CLas infected), were transferred onto fresh CLas-free M. exotica shoots to complete their development (hereafter referred as “T. radiata-inoculated ACP”). The experiment was repeated in 32 parallel replicates (Supplementary Table 1), in which 103 T. radiata-inoculated ACP nymphs were finally obtained.
Following the above, thirty T. radiata-inoculated ACP nymphs were collected when they developed into 5th instar nymphs (the stage when infection proliferation might have just begun since the infection was introduced at the 4th instar). In addition, thirty 8-day old adults that developed from the T. radiata-inoculated ACP nymphs were also collected. This was because the results in Wu et al.28 revealed that the proportion of CLas-infected ACP individuals exceeds 90% at the 12th day after infection acquisition, while ACP takes 4 days to develop into an adult from 5th instar stage. Their alimentary canals and salivary glands were dissected under a stereomicroscope using the methods of Ammar et al.33, and hemolymphs were collected with a 10 μl pipette tip using the method of Killiny et al.34. The DNA of the alimentary canals, salivary glands and hemolymphs were extracted using TIANamp Micro DNA Kit (Tiangen, Beijing, China), and the relative titers of CLas in each tissue of ACP nymphs and adults were detected by qPCR with of LJ900. The β-actin gene of ACP (F 5′-CCCTGGACTTTGAACAGGAA-3′; R 5′-CTCGTGGATACCGCAAGATT-3′) was selected as an internal control for data normalization and quantification35. For each sample, qPCR detection was repeated three times.
FISH visualization of CLas
The alimentary canals and salivary glands of 5th instar nymphs and 8-day old adults of T. radiata-inoculated ACP were dissected as described above, and the distribution of CLas was visualized by FISH and confocal microscopy. The alimentary canals and salivary glands of CLas-infected ACP nymphs and adults (collected from CLas-infected citrus plants) were used as a positive control, and five to ten samples were detected by FISH for each tissue.
CLas transmission from T. radiata-inoculated ACP to citrus plants
According to the above experimental results, if the CLas could be detected in the salivary glands of the 8-day old ACP adults (T. radiata-inoculated ACP), 30 more of these adults were randomly selected to inoculate on fresh shoots of CLas-free citrus. ACP adults that acquired CLas from plants and CLas-free ACP adults were used as positive and negative controls respectively.
After 20, 30, 40, and 50 days of feeding samples of the citrus leaves fed on by T. radiata-inoculated ACP (named as CLas-recipient citrus leaves), fed on by ACP that acquired CLas from plants (positive control), and fed on by CLas-free ACP (negative control) were cut (1 cm2). Their DNAs were extracted using DNAsecure Plant Kit (Tiangen, Beijing, China). The infections of CLas in these plants were detected by nested PCR based on the methods of Jagoueix et al.36 and Deng et al.37. The experiment was repeated in six plants for each of 20, 30, 40, and 50 days feeding duration, and the infection rates of CLas were calculated.
Localization of CLas in citrus plants fed on by T. radiata-inoculated ACP
In order to further confirm the infection of CLas in the recipient citrus leaves, FISH was used to visualize the localization of CLas. According to the above experimental results, after being fed on for 50 days by the T. radiata-inoculated ACP adults, citrus leaf sections containing the midrib were cross-sliced in 30 µ sections using a cryostat (CM1950, Leica, Germany). The leaf samples were prepared for FISH vitalization according to the protocol described by Gottlieb et al.32. Citrus leaves from the plant that had been fed on by ACP adults that acquired CLas from plants and CLas-free ACP adults were used as positive and negative controls, respectively. Five to 10 leaf samples were detected by FISH for each treatment.
Phylogenetic analysis of CLas bacteria in different ACP populations and citrus plants
To assess the identity of the CLas bacteria in CLas donor ACP, CLas vectored parasitoids, T. radiata-inoculated ACP and recipient citrus leaves, the outer membrane protein gene (omp) of CLas was PCR amplified with the primers HP1asinv (5′-GATGATAGG TGCATAAAAGTACAGAAG-3′) and Lp1c (5′-AATACCCTTATGGGATACAAAAA-3′) following the procedure described in Bastianel et al.38. Then the PCR products were sent for sequencing after visualizing the expected bands on 1% agarose gels.
All the DNA sequences of CLas omp gene were edited and aligned manually using Clustal X1.8339 in Mega 640. The best model and partitioning scheme were chosen using the Bayesian information criterion in PartitionFinder v.1.0.141. Phylogenetic analysis was undertaken using a maximum likelihood (ML) method with 1000 non-parametric bootstrap replications in RAxML42. Escherichia coli was used as an outgroup.
Statistics and reproducibility
Taking 18S rRNA gene of T. radiata and the β-actin gene of ACP as housekeeping genes, the relative titers of CLas in different stages and different tissues of T. radiata and ACP were calculated using the method of 2[−ΔΔct 43. For the parallel experiments that had more than three replicates the differences were compared using analysis of variance (ANOVA) with SPSS 18.0 at a significance level α = 0.05; while for CLas titer, two-sample comparison between genders of Tamarixia adults analysis was performed using paired t-test. Fluorescent pictures were processed using Photoshop CS5 software.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Source: Ecology - nature.com