Insect Colonies

Small citrus trees (20-50 cm tall) were grown in 15.2-cm diameter pots to maintain colonies of D. citri and its 2 parasitoids in wooden-framed, mesh-covered cages (0.76 m × 0.91 m × 1.11 m) held in a greenhouse at 20-32°C with a 16L:8D photoperiod (Skelley & Hoy 2004). Ten trees were pruned each week, fertilized with Peter’s 20-20-20 (N-P-K) water-soluble fertilizer (United Industries, St. Louis, MO), and watered as necessary. Mated adult females of D. citri oviposited on the new growth (flush) that appeared approximately 2 weeks after the trees were pruned. Adult parasitoids were aspirated and separately released into the cages according to species when immature D. citri reached the first or second instar. Psyllid nymphs that were not attacked by the parasitoids and developed into adults were used to initiate the next generation.

DNA Extraction

Insects were treated with bleach prior to DNA extraction to remove DNA from external microbes that may contaminate the PCR (Meyer 2007). Treatment of insects was conducted in a plexiglass hood that was first washed with 70% EtOH, then with bleach (6% sodium hypochlorite), and DNA Away (Molecular BioProducts, San Diego, CA), according to the following protocol: (i) individual live insects were placed in sterile 15-mL centrifuge tubes containing 5 mL of the decontamination solution and vortexed vigorously for 1 min; (ii) the solution was discarded, leaving the insects in the tube; (iii) insects were rinsed 3× with 10 mL of sterile water, which was discarded after each rinse; (iv) insects were transferred to a sterile 1.5-mL centrifuge tube using a sterile pipette tip wetted with DNA extraction buffer prior to DNA extraction (Jeyaprakash & Hoy 2000). For the microbial surveys, 2 replicates of 10 newly emerged adult insects were pooled according to species before DNA extraction. DNA was extracted from insects by homogenization with a disposable blunt-ended sterile pipette tip with PUREGENE DNA isolation reagents (Gentra Systems, Minneapolis, MN) according to the manufacturer’s protocol. DNA pellets were air dried, resuspended in 10-50 μL sterile water, and stored at -70°C. Ten individual D. citri, T. radiata, and D. aligarhensis from the laboratory colonies and 10 individual D. citri and T. radiata collected from the field in Polk county, Florida (28°03.656’ N, 81°34.937’ W) were used to isolate DNA for microbe-specific PCR. These insects were processed as described above, and their DNA was resuspended in 10 μL of sterile water. No field-collected D. aligarhensis were included because populations of this parasitoid are scarce in Florida. Twenty eggs of D. citri and T. radiata collected from the laboratory colonies were pooled, surface decontaminated, and the DNA was isolated as described above and resuspended in 10 μL of sterile water. No DNA was isolated from eggs of D. aligarhensis because the endoparasitoid deposits eggs inside of D. citri nymphs, and attempts to dissect and recover these eggs were unsuccessful.

High Fidelity Polymerase Chain Reaction

A high-fidelity PCR assay was used that can detect as few as 100 copies of the target template 100% of the time and as few as 10 copies 50% of the time (Hoy et al. 2001). For the molecular survey of endosymbionts and microbe-specific PCRs, the 50-μL reaction contained 34 μL of sterile water, 5 μL of PCR buffer consisting of 50 mM Tris, pH 9.2, 16 mM ammonium sulfate and 1.75 mM MgCl2, 5 μL of 350 mM dNTPs, 2 μL each of the forward and reverse primers at a concentration of 800 pmol, and 1 μL of polymerase mix containing 1 unit of Pwo DNA polymerase and 5 units of Taq DNA polymerase (Roche Applied Science, Indianapolis, IN) Biochemicals) (Barnes 1994) to amplify 1 μL of template DNA. The primers and annealing temperatures used in the PCR for the survey of endosymbionts and microbe-specific analyses are listed in Table 1 (Hoy & Jeyaprakash 2005). Three linked profiles were used for the high-fidelity PCR that included (i) 1 cycle of denaturation at 94°C for 2 min; (ii) 10 cycles of denaturation at 94°C for 10 s, annealing (Table 1) for 30 s, and elongation at 68°C for 1 min; and (iii) 25 cycles of denaturation at 94°C for 10 s, annealing (Table 1) for 30 s, and extension at 68°C for 1 min plus an additional 20 s for each consecutive cycle (Hoy et al. 2001). Agarose gel electrophoresis (1% TAE gels) was used to separate PCR-amplified DNA, which was stained with ethidium bromide and visualized with UV light.

Cloning and Restriction Fragment Length Polymorphism Analysis

PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA), ligated into the pCR2.1 TOPO plasmid (Invitrogen, Carlsbad, CA), and cloned into competent E. coli cells (Bioline USA, Inc., Randolph, MA). Plasmid DNA was extracted from E. coli colonies with QIAGEN Plasmid Mini columns (Valencia, CA). The presence and size of the inserted DNA was confirmed by gel electrophoresis of plasmids digested with EcoRI for 2 h at 37°C. Plasmids digested with RsaI were used for the restriction fragment length polymorphism (RFLP) analyses (Jeyaprakash et al. 2003). Clones of interest were bidirectionally sequenced at the University of Florida Interdisciplinary Core Facility, Gainesville, FL.

Phylogenetic Analysis

Eubacterial DNA sequences obtained in the surveys were used in a BLAST search to identify related DNA sequences in GenBank with default settings. Retrieved sequences were aligned with CLUSTAL X v. 1.83 (Thompson et al. 1997), and manual adjustments of the alignment were conducted using MacClade 4.0 (Maddison & Maddison 2000). The outgroup taxon was the Gram-positive bacterium Bacillus subtilis. Maximum likelihood (ML) and maximum parsimony (MP) analyses were conducted using heuristic searches implemented in PAUP* 4.0b4a (Swofford 2001). For ML, the data matrix was subjected to the MODELTEST 3.7 program (Posada & Crandall 1998) to select the best-fit nucleotide substitution model for the alignment. The substitution rate-matrix parameters and shape parameter (alpha) were estimated via ML. The bootstrap method (100 replicates) was used to support each branch in the ML tree in PAUP*.

High-Fidelity PCR Amplification of Endosymbionts in D. citri and its Parasitoids

Results from the high-fidelity PCR assays for the endosymbionts of surface-decontaminated D. citri, T. radiata and D. aligarhensis indicate that relatively few endosymbionts were present (Table 2). Amplification of the eubacterial 16S rRNA gene was detected with template DNA from each insect, but no amplification products were detected in any of the insects based on the primers for Archaea, Fungi, Helicosporida, or Microsporidia. The orf7 gene from the bacteriophage WO of Wolbachia was amplified with DNA from D. citri, but not from D. aligarhensis or T. radiata as the template.

Endosymbionts of D. citri

The eubacterial 16S rRNA PCR products were cloned from 2 replicate high-fidelity PCR reactions, and a total of 15 clones were analyzed from each replicate by the RFLP technique. Analysis with EcoRI digests indicated that all clones from each replicate had an insert approximately 1.4-kb in length. Digests with RsaI resulted in 2 and 3 unique banding patterns in the RFLP analysis according to replicate, respectively. A clone representing each unique banding pattern in both replicates was sequenced (5 total sequences). Two pairs of these sequences were 100% identical between each replicate, respectively. These comparisons indicated that there were at least 3 types of Eubacteria associated with our laboratory colony of D. citri. The 3 16S rRNA sequences obtained from D. citri were used in a BLAST search to identify related sequences in GenBank.

The first sequence (1466 bp, GenBank accession EF433792) was 100% identical over a 1463-bp homologous region of the 16S rRNA gene of the secondary (syncytium) symbiont of D. citri from an Asian population (GenBank accession AB038368) (Subandiyah et al. 2000), which is related to b-proteobacteria species in the genera Oxalobacter and Herbaspirillum (Fig. 1).

The second sequence was 1429 bp (GenBank accession EF433793) and produced significant alignments to the 16S rRNA gene of species in the a-proteobacterial family Rickettsiaceae. This sequence was 99% identical (1177/1185 bp) to a 1185-bp homologous region of a partial 16S rRNA gene sequence from the endosymbiont Wolbachia of D. citri from an Asian population (GenBank accession ABO38370) (Subandiyah et al. 2000). Five of the 8 nucleotide differences between the 2 Wolbachia sequences were attributed to errors (bases designated “N”) in the sequences deposited by Subandiyah et al. (2000) which could have been due to their use of a standard PCR protocol because it does not include a proofreading polymerase.

The third sequence (1459 bp, GenBank accession EF450250) produced significant alignments to the 16S rRNA gene of a g-proteobacterial species and was 99% identical to a homologous region of the 16S rRNA gene sequence from the primary (mycetocyte) symbiont, Ca. C. rudii, of D. citri from an Asian population (GenBank accession AB038367) (Subandiyah et al. 2000).

Subandiyah et al. (2000) reported 2 additional eubacterial symbionts, Ca. L. asiaticus and Arsenophonus, associated with an Asian population of D. citri, but no clones containing sequences related to these symbionts were detected in this survey. To confirm that clones from these symbionts were not missed in our survey, the microbe-specific primers designed by Subandiyah et al. (2000) to amplify the 16S rRNA gene from Ca. L. asiaticus and Arsenophonus were tested on the 2 replicates of DNA samples isolated from the Florida population of D. citri and used in the molecular survey of endosymbionts. No positive PCR reactions were detected for Ca. L. asiaticus indicating that our laboratory colony of D. citri is free of the citrus greening pathogen. This confirmed prior results of Hoy et al. (2001) and was consistent with the findings of Meyer et al. (2007), in which none of 1793 and 179 field-collected D. citri adults and nymphs, respectively, tested positive for Ca. L. asiaticus. Three replicate PCR reactions with the Arsenophonus-specific primers did not produce amplification products of the expected size, and they produced non-specific products in the no-DNA control. Future research is needed to develop improved Arsenophonus-specific primers that do not produce artifacts in the PCR in order to confirm whether this symbiont is lacking in D. citri populations in Florida. Only 45% of the Asian population of D. citri that was analyzed had Arsenophonus, so this is not likely an essential endosymbiont and may be associated with the Asian environment.

To determine if there are multiple Wolbachia strains in the Florida population of D. citri, the Wolbachia wsp gene was amplified and 10 clones were sequenced. For each clone, an identical 539-bp sequence was obtained that was 100% identical to the wsp sequence previously reported from the Florida population of D. citri (GenBank accession AF217721) (Jeyaprakash & Hoy 2000). These results suggest that only a single Wolbachia strain is present in the laboratory population of D. citri.

A putative capsid protein gene (orf7) of the bacteriophage WO was PCR-amplified from DNA extracted from adult D. citri. In a BLAST search, the 409-bp orf7 sequence (GenBank accession EF444818) produced significant alignments to orf7 sequences of the bacteriophage WO of Wolbachia from multiple sources, and it was 99% identical (405/499 bp) to 2 deposited orf7 sequences associated with Wolbachia-infected Culex pipiens (GenBank accession AY505105, AY505101). The orf7 gene has been found in most, but not all Wolbachia-infected arthropods (Masui et al. 2000; Fuji et al. 2004; Gavotte et al. 2004; Braquart-Varnier et al. 2005; Hoy & Jeyaprakash 2005). Masui et al. (2000) and Braquart-Varnier et al. (2005) found that 100% of the Wolbachia strains tested had bacteriophage WO, and Gavotte et al. (2007) reported that 70% of the Wolbachia strains that were tested were positive for bacteriophage WO. Phylogenetic comparisons of the bacteriophage WO and host Wolbachia have suggested that the bacteriophage WO is horizontally transferred among different lineages of Wolbachia by unknown mechanisms (Masui et al. 2000; Gavotte et al. 2004, 2007).

Diagnostic, microbe-specific PCR was conducted to determine the proportion of laboratory and field-collected D. citri that were positive for each eubacterial species and for the bacteriophage WO. Amplification products of a portion of the 16S rRNA gene from the primary symbiont, Ca. C. ruddii, the secondary symbiont, Wolbachia and the bacteriophage WO of Wolbachia were detected in 100% of DNA samples extracted from adult D. citri from the laboratory colony (n = 10: 5 females, 5 males) and from 100% of the DNA isolated from psyllids collected from the field (n = 10: 5 females, 5 males). Both the primary and secondary symbionts were detected in 100% of D. citri in an Asian population by Subandiyah et al. (2000). The percentage of Wolbachia (100%) in the Florida population (Meyer et al. 2007) was higher than in the Asian population of D. citri (76%) (Subandiyah et al. 2000). This could reflect true differences in the endosymbiotic complex of D. citri, or could be related to the increased sensitivity of the high-fidelity PCR assay used in this study compared to the standard PCR protocol used by Subandiyah et al. (2000). Hoy et al. (2001) reported that the high-fidelity PCR assay was up to seven-fold more sensitive than standard PCR.

To determine the transmission mechanism of each symbiont, DNA isolated from D. citri eggs was used in a high-fidelity, microbe-specific PCR reaction. The primary symbiont, secondary symbiont, Wolbachia and the bacteriophage WO were detected both in samples that were not surface decontaminated with bleach and in samples that were bleach treated. This indicated that the symbiont DNA was present inside the eggs (transovarial transmission). Transovarially or maternally-transmitted symbionts often are obligatory to the host, such as in the case of Buchnera species that provide nutrients to their aphid hosts (Moran & Degnan 2006), or can manipulate their hosts’ reproductive system to their advantage, as does Wolbachia in a variety of arthropods (O’Neill et al. 1997).

Endoymbionts of T. radiata

Amplification products from the eubacterial 16S rRNA gene were cloned from each of 2 replicate high-fidelity PCR reactions with DNA from surface-decontaminated adult T. radiata. A total of 15 clones were analyzed from each replicate by RFLP technique. Analysis with EcoRI digests indicated that all clones from each replicate had an insert approximately 1.4 kb in length. Digests with RsaI resulted in a total of 2 and 3 unique banding patterns in the RFLP analysis according to replicate, respectively. A clone representing each unique banding pattern in both replicates was sequenced (5 total sequences). Two pairs of these sequences were 100% identical between each replicate, respectively. These comparisons indicated that there are at least 3 types of Eubacteria associated with the laboratory colony of T. radiata.

The three 16S rRNA sequences obtained from T. radiata were used in a BLAST search to identify related sequences in GenBank. The first sequence (1409 bp, GenBank accession EF433789) produced significant alignments to Caulobacter species of the a-proteobacterial family Caulobacteraceae. The sequence was 100% identical to the 16S rRNA gene sequence from Caulobacter sp. strain FWC41 (GenBank accession AJ227775) identified from activated sludge in a secondary treatment facility in Calgary, Alberta, Canada (Abraham et al. 1999). The sequence was also 100% identical to an uncultured eubacterial species (1283 bp, GenBank accession AY038618) from an “environmental sample”. Other reports have associated Caulobacter species with the gut of arthropods, including a millipede (Abraham et al. 1999), Acromyrmex leafcutter ants (Van Borm et al. 2002), and the mite Tetranychus urticae (Koch) (Hoy & Jeyaprakash 2005). Thus, it is unclear whether this microorganism is associated with the gut of T. radiata as a permanent or transient component.

The second sequence (1410 bp, GenBank accession EF433790) produced significant alignments to Methylobacterium species of the a-proteobacterial family Methylobacteriaceae. This sequence was 100% identical to homologous portions of the 16S rRNA gene sequence from Methylobacterium lusitanum strain NCIMB 13779 (1435 bp, GenBank accession AB175635) (Kato et al. 2005) and from a Methylobacterium sp. strain TNAU12 (1340 bp, GenBank EF116590) reported from the phyllosphere of soybean. Species belonging to the genus Methylobacterium have been reported in a wide variety of environments (Kato et al. 2005), but are not known to be endosymbionts of parastioids.

The third sequence (1457 bp: GenBank EF43379) produced significant alignments to the 16S rRNA gene sequence from b-proteobacterial species in the family Alcaligenaceae. This sequence was 99% identical to the homologous portion of the 16S rRNA gene sequence from an unidentified bacterium in the family Alcaligenaceae (1452/1454 bp) found on the epiphytic surfaces of rice (Hiraoka et al. 2006). Again, we have no reason to believe that this microorganism is a permanent endosymbiont of T. radiata.

In the diagnostic PCR assay, using microbe-specific primers (Table 1), the Caulobacter, Methylobacter and Alcaligenaceae species were detected in 70, 30 and 20% of the individual T. radiata collected from the laboratory colony (N = 10), respectively, and 20, 0 and 0% of the field collected specimens (N = 10), respectively. None of the species were detected in the bleach-treated or untreated eggs of T. radiata. It appears that the eubacterial species detected in T. radiata are likely acquired from the environment and are not transovarially transmitted, with the possible exception of the Caulobacter species. Despite the efforts to surface-sterilize the insects and to use newly emerged adult T. radiata prior to DNA extraction, it is possible that these Eubacteria were acquired when adult T. radiata chewed a hole in the dorsum of the mummified and potentially contaminated D. citri host. If so, these Eubacteria would be protected from the bleach treatment because they were located in the gut of T. radiata, and likely are facultative or transient associates.

Endosymbionts of D. aligarhensis

PCR amplification products for the eubacterial 16S rRNA gene were detected with template DNA from 2 separate DNA extractions of D. aligarhensis. Ten clones from each replicate were analyzed by RFLP technique. All clones had an approximate 1.4-kb insert as indicated by digestion with EcoRI. RsaI digests resulted in a single banding pattern for all 10 clones in both replicates, and a single clone from each replicate was sequenced. Both sequences were 1429 bp and 100% identical, suggesting that only one endosymbiotic eubacterial species is associated with the laboratory colony of D. aligarhensis.

A BLAST search was conducted with this sequence (GenBank accession EF433794) that produced significant alignments to multiple Wolbachia species in the a-proteobacterial family Rickettsiaceae. This sequence was different than the 16S rRNA sequence from Wolbachia obtained from the Florida population of D. citri (GenBank accession EF433793), and a sequence alignment indicated the sequences were 99% identical (1416/1429 bp). The different Wolbachia infections in D. citri and D. aligarhensis indicated that horizontal transfer of Wolbachia did not occur between these species, at least not recently.

To determine if D. aligarhensis had multiple strains of Wolbachia, the wsp gene was PCR-amplified and the amplification products were cloned. Ten clones were sequenced and the sequences were 100% identical in size (542 bp) and nucleotide composition to the wsp gene previously reported in our laboratory population of D. aligarhensis (Jeyaprakash & Hoy 2000). This suggested that only a single strain of Wolbachia was present in this population of D. aligarhensis. The wsp gene sequences of Wolbachia from D. citri and D. aligarhensis were different, further supporting the conclusion based on differences in the 16S rRNA gene sequences that these 2 Wolbachia strains are different. Although the sequences are different, the 2 Wolbachia strains are classified in the same “Super-Group B” of Wolbachia (Jeyaprakash & Hoy 2000).

Amplification products for the wsp gene of Wolbachia were detected in 100% of DNA samples extracted from adult female D. aligarhensis from our laboratory colony (n = 10). No field-collected specimens were available for analysis because populations of this parasitoid are scarce in Florida. No eggs were sampled to confirm the transmission of Wolbachia in the eggs of D. aligarhensis, due to difficulties obtaining eggs from the endoparasitoid, which deposits them inside D. citri nymphs. However, we speculate that Wolbachia is transmitted inside the egg of D. aligarhensis, based on the results from this study regarding Wolbachia transmission in D. citri and on other reports showing such a transmission mechanism (Kose & Karr 1995). Wolbachia infection in D. aligarhensis is correlated with thelytokous reproduction, as demonstrated by male production following tetracycline treatment of parental females (Meyer & Hoy 2007).

Despite conducting replicate high-fidelity PCR reactions, no amplification products were obtained for the orf7 gene of the bacteriophage WO of Wolbachia with D. aligarhensis DNA as the template. This was in contrast to the presence of the bacteriophage WO in D. citri and indicated that the Wolbachia species in D. aligarhensis either does not have bacteriophage WO or that the sequence representing the orf7 gene is not amplifiable with the primers used in this survey. This finding is not uncommon; Wolbachia strains lacking the bacteriophage WO have been reported from parasitoids in the genus Trichogramma and nematodes (Gavotte et al. 2007).

Phylogenetic Analysis

The eubacterial 16S rRNA gene sequences obtained from D. citri, T. radiata and D. aligarhensis were used for phylogenetic analyses. The GTR + G nucleotide substitution model (Yang et al. 1994) was selected by MODELTEST for the data matrix and used for maximum likelihood (ML) analysis. The topologies of the ML and maximum parsimony (MP) trees were similar, and each method grouped the taxa according to their expected classification within the gram-negative Eubacteria and separated them from the outgroup taxon, Bacillus subtilis, a gram-positive eubacterium. Fig. 1 depicts the tree constructed by ML analysis. Three major clades were produced that included taxa belonging to the a-, b-, and g-proteobacteria, respectively, and the separation of each major eubacterial division was strongly supported by the bootstrap method. The primary (mycetocyte) symbiont, Ca. C. ruddii of D. citri (Florida) was grouped in the g-proteobacteria in a clade including the primary symbiont from D. citri (Asia) and the primary symbiont of Cacopsylla brunneipennis (Edwards) (Hemiptera: Psyllidae). Thao et al. (2000) provided a detailed phylogenetic analysis of additional psyllid species and their associated primary endosymbionts. The secondary symbiont of D. citri (Florida) was grouped in the b-proteobacteria with the secondary symbiont of D. citri (Asia) and related Eubacteria in the genera Oxalobacter and Herbaspillium. These findings were consistent with the phylogenetic analysis conducted by Subandiyah et al. (2000). Wolbachia of D. citri (Florida) was grouped in the a-proteobacteria in a clade consisting of 3 Wolbachia species from D. citri (Asia), D. aligarhensis and Culex pipiens Linnaeus (Diptera: Culicidae), respectively. The Caulobacter and Methylobacter microbes found in T. radiata were clustered with other species in the a-proteobacteria, and the bacterium in the family Alcaligenaceae was grouped with the b-proteobacteria. Separation in the clades including the eubacterial symbionts of D. citri, T. radiata, and D. aligarhensis was strongly supported by the bootstrap values.

We concluded that the endosymbiotic communities in our laboratory population of D. citri and its parasitoids are relatively simple. A total of 3 eubacterial species each were detected in D. citri and T. radiata and only 1 species was detected in D. aligarhensis, but none of these insects harbored any detectable endosymbionts belonging to the other taxa surveyed. The eubacterial 16S rRNA sequences obtained from these insects were not identical when compared between species, indicating that stable horizontal transfer of these microorganisms likely has not occurred in this host-parasitoid system.

Potential Caveats of the PCR-based Survey

The microbes identified in this study were detected with the sensitive high-fidelity PCR protocol but were not isolated in pure culture or located in the insects by in situ hybridization (or an equivalent technique). It is possible that we underestimated the diversity of endosymbionts if the “universal” PCR primers failed to amplify all microbial sequences in a particular taxonomic group. The authors recognize that additional endosymbionts may be associated with these insects for which no PCR primers have been developed. In addition, there may be endosymbionts associated with wild populations of D. citri and its parasitoids in Florida that were not identified in this survey, which was based on laboratory insect colonies and only limited numbers of field-collected insects. The titer of the microbial DNA may influence the sensitivity of the PCR. For example, microbial DNA sequences that are very rare may not compete with abundant microbial DNA sequences or could be below the sensitivity of the high-fidelity PCR assay (Hoy et al. 2001). The random selection of clones, number of clones analyzed, choice of restriction enzymes, and analysis of clones potentially containing different sequences but yielding identical RFLP profiles also could contribute to an underestimation of the total diversity present.