Relative Abundance of Carsonella ruddii (Gamma Proteobacterium) in Females and Males of Cacopsylla pyricola (Hemiptera: Psyllidae) and Bactericera cockerelli (Hemiptera: Triozidae).

Carsonella ruddii (Gamma Proteobacterium) is an obligate bacterial endosymbiont of psyllids that produces essential amino acids that are lacking in the insect's diet. Accurate estimations of Carsonella populations are important to studies of Carsonella-psyllid interactions and to developing ways to target Carsonella for control of psyllid pests including pear psylla, Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae) and potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae). We used two methods, namely fluorescence in situ hybridization and quantitative polymerase chain reaction (qPCR), to estimate relative abundance of Carsonella in bacteriocytes and whole bodies of psyllids, respectively. Using these two methods, we compared Carsonella populations between female and male insects. Estimations using fluorescence in situ hybridization indicated that Carsonella was more abundant in bacteriocytes of female C. pyricola than in those of males, but Carsonella abundance in bacteriocytes did not differ between sexes of B. cockerelli. Analyses by qPCR using whole-body specimens indicated Carsonella was more abundant in females than in males of both psyllids. Neither fluorescence in situ hybridization nor qPCR indicated that Carsonella populations differed in abundance among adults of different ages (0-3 wk after adult eclosion). Using fluorescence in situ hybridization, Carsonella was observed in ovarioles of newly emerged females and formed an aggregation in the posterior end of mature oocytes. Results of our study indicate that female psyllids harbor greater populations of Carsonella than do males and that sex should be controlled for in studies which require estimations of Carsonella populations. Published by Oxford University Press on behalf of the Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US.

Psyllids (Hemiptera: Pyslloidea) are phloem-feeding insects comprising eight Hemipteran families (Burckhardt and Ouvrard 2012). Several psyllid species are major agricultural pests, including pear psylla, Cacopsylla pyricola (Förster) (Hemiptera: Psyllidae), and potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae). C. pyricola is a pest of cultivated pear, Pyrus communis L. (Rosaceae) because it produces abundant honeydew which causes fruit russetting, provides and medium for the growth of sooty mold and increases labor costs during harvest (Westigard and Zwick 1972). B. cockerelli is a pest of Solanaceous crops including potato and tomato and is the vector of “Candidatus Liberibacter solanacearum,” the pathogen associated with zebra chip disease of potato (Munyaneza 2012). Both psyllids are primarily controlled using insecticides, but current research efforts seek to develop ecologically based strategies to manage these pests.

All psyllids (Hemiptera: Psylloidea) have an obligate relationship with the bacterial endosymbiont, Carsonella ruddii (Gamma Proteobacterium) (Thao et al. 2000). Carsonella is transovarially transmitted and is housed within specialized host cells called bacteriocytes which form aggregates called bacteriomes (Chang and Musgrave 1969, Thao et al. 2000). This endosymbiont produces essential amino acids that are lacking in the insect’s diet, and elimination of this endosymbiont using antibiotics leads to death of the insect (Baumann 2005, Nakabachi et al. 2006). Because Carsonella is vital to the survival of psyllids, novel control strategies for psyllid pests including C. pyricola and B. cockerelli could be developed by targeting this endosymbiont (Rio et al. 2004, Douglas 2007, Crotti et al. 2012).

Paramount to discovering ways to manipulate Carsonella is the ability to obtain accurate estimations of Carsonella populations. A recent study by Cooper and Horton (2014) indicated that bacteriomes of female psyllids were larger than those of males, suggesting that females may harbor more Carsonella or other bacteriome-associated endosymbionts than do males. If so, then insect sex represents an important source of biological variation that must be accounted for when estimating Carsonella populations in psyllids. The objectives of our study were to 1) develop methods using fluorescence in situ hybridization and quantitative polymerase chain reaction (qPCR) to estimate Carsonella populations in C. pyricola and B. cockerelli, 2) compare Carsonella populations between female and male C. pyricola of varying ages (fifth-instar nymph to 21-d-old adult), and 3) document in both psyllid species the presence of Carsonella in varying ages of oocytes.

Materials and Methods

Insects

C. pyricola adults (150–200) were collected from pear trees located at the USDA experimental farm near Moxee, WA. To facilitate oviposition, psyllids were confined to six potted pear trees using a mesh cage for 36 h at 25°C with a photoperiod of 16:8 (L:D) h. The development of the resulting eggs and nymphs was closely monitored. Twelve-fifth instars (6 per sex) were collected and frozen for qPCR analysis of Carsonella. Adults (n = 150) that had emerged within a 24-h period were transferred to new pear trees and used for experiments. Each week for 4 wk (week 0, week 1, week 2, and week 3), 10 adults (five per sex) were collected for fluorescence in situ hybridization and 6 adults (three per sex) were collected for qPCR analysis.

Newly molted B. cockerelli adults were obtained from a laboratory colony and were maintained on potato “Russet Burbank” for 5 d. The colony was originally established from psyllids of the western haplotype collected near Prosser, WA, in summer 2012. These assays were limited to 5-d-old adults to simplify the experimental design. Female B. cockerelli of this age contain mature oocytes. The 5-d-old adults were collected for fluorescence in situ hybridization (5 per sex) and qPCR (10 per sex). The experiment on B. cockerelli was conducted twice (repetition) with different cohorts of insects.

For both psyllids, adults collected for fluorescence in situ hybridization were immediately dissected and processed as described in “Carsonella Densities in Individual Bacteriocytes” subsection. Adults collected for qPCR were stored in −20°C until they were processed as described in the “Carsonella titers in whole insects” subsection.

Carsonella Densities Within Individual Bacteriocytes

Fluorescence in situ hybridization was performed using methods similar to those described by Cooper et al. (2014). Each psyllid was mounted ventral side facing up on a glass microscope slide using double-sided tape. A drop of phosphate-buffered saline was placed over the insect and held in place by cohesion. Using two number 5 forceps (D’Outils Dumont SA, Montignez Switzerland), bacteriomes and fat body were removed from each psyllid and were transferred to a positively charged microscope slide (Tissue Tack, Polysciences Inc., Warrington, PA). After the dissected tissues air dried, the slides were maintained for 3 min on a slide warmer set at 50°C to adhere the tissues to the slides. Samples were fixed in Carnoy’s solution for 1 h, briefly rinsed in 100% ethanol, and washed three times for 20 min in hybridization buffer consisting of 20 mM Tric-HCL (pH 8.0), 0.9 M NaCl, 0.01% sodium dodecyl sulfate, and 30% formamide. Samples were hybridized overnight with 250 picomol/ml of High Performance Liquid Chromatography-purified oligonucleotide probe labeled with Alexa Fluor 488 on the 5’-end (Invitrogen, Carlsbad, CA). The probe sequence was GCT GCC TTC CTT GAA AGT (Fukatsu and Nikoh 1998). No probe controls (n = 2, one per sex) were included to ensure the lack of interfering autofluorescence. Because bacteriocytes of all pear psylla and potato psyllid harbor Carsonella, fat body cells hybridized with the probe were used as negative controls (no Carsonella). During probe hybridization, samples were kept under humid conditions within an incubator maintained at 25°C. After hybridization, samples were briefly washed in hybridization buffer, followed by two washes for 20 min in hybridization buffer, and one 20-min wash in Tris-buffered saline. Carsonella was observed at 400 × using a fluorescence microscope (Zeiss Axioskop 40 FL, Carl Zeiss USA, Thornwood, NY) with Zeiss filter-set 10 (excitation wavelength = 450–490 nm, beam splitter = 510 nm, and emission wavelength = 515–565 nm). Each sample was photographed using a DP25 camera mounted to the microscope and operated using the CellSens software (Olympus America Inc., Central Valley, PA). All photographs were captured with an ISO of 200 and an exposure time of 80 ms. The mean intensity of fluorescence was estimated from four to nine bacteriocytes from each insect using ImageJ software (http://imagej.nih.gov.ij, last accessed 2 March 2015) (Burgess et al. 2010, Schneider et al. 2012).

Fluorescence intensity values associated with Carsonella densities within individual bacteriocytes were analyzed using the GLIMMIX procedure of SAS 9.3 (SAS Institute 2012; Cary, NC). For analysis of Carsonella densities in C. pyricola, sex, age, and the sex by age interaction were included as the fixed effects, and insect (sex age) was included as the random variable. Because age was not a factor in analysis of Carsonella in B. cockerelli, sex was included as the fixed effect, and insect (sex) was included as the random variable. For each analysis, data were examined for heterogeneity of variance and nonnormality of errors by inspecting residual and normal quantile–quantile plots, respectively. Based on these plots, data were modeled assuming a Gaussian distribution using the DIST=G option of the MODEL statement.

Carsonella Titers in Whole Psyllids

DNA was extracted from all samples using a modified cetyltrimethlyammonium bromide (CTAB) method (Zhang et al. 1998, Crosslin et al. 2011). The insects were ground in 500 µl of CTAB buffer using a micropestle in a 1.5 ml microfuge tube. The samples were incubated in 65°C for 30 min and then maintained at room temperature for 3 min before adding 500 µl of ice-cold chloroform. After centrifugation at 16,000 × g for 10 min, the aqueous layer was transferred to 0.6 volume of isopropanol containing 1 µl of glycogen. Samples were held on ice for 20 min to precipitate DNA. After centrifugation at 16,000 × g for 10 min, the resulting pellet was washed twice with ice-cold 70% ethanol, air-dried, and dissolved in 50 µl of nuclease-free water. Samples used for qPCR were precipitated in isopropanol for a second time and dissolved in 50 µl of nuclease-free water.

To design qPCR primers, a region of 16S of Carsonella was first amplified using DNA from a group of five field-collected C. pyricola or five B. cockerelli obtained from the colony. Approximately 930-bp region of 16S was amplified using the universal primers for eubacteria, 27F-AGA GTT TGA TCM TGG CTC AG and 1494R-TAC GGC TAC CTT GTT ACG AC according to Weisburg et al. (1991). The excised PCR products were purified using GenElute minus EtBr spin columns (Sigma, St. Louis, MO) and cloned using a TOPO TA cloning kit with TOP10 Escherichia coli chemically competent cells (Invitrogen, Carlsbad, CA). Plasmid DNA was extracted from selected colonies using the QIAprep spin mini prep kit (Qiagen, Valencia, CA), and DNA clones were sequenced by MC Laboratories (MC Lab, San Francisco, CA). Sequences were analyzed using BLASTn (Altschul et al. 1990) function on the NCBI website (http://www.ncbi.nlm.nih.gov/, last accessed 2 March 2015). Sequences identified as Carsonella (GenBank accessions KR045611 and KR045612) were used to design qPCR primers for Carsonella of C. pyricola and B. cockerelli using Primer3Plus software (Thornton and Basu 2011). The Carsonella-specific PCR primers were CarF-AAG AAG AGA TTA GAA TTT C and CarR-AAA TAG TTG ACA TCG TTT AC. To confirm the target specificity of this primer set, conventional PCR was performed in 10 µl reactions using Invitrogen Amplitaq Gold 360 PCR Master Mix, 5 µM forward and reverse primers, and DNA isolated from five C. pyricola or five B. cockerelli adults. The thermal-cycling conditions were 95°C for 10 min, 40 cycles for 95°C of 30 s, 60°C for 30 s, and 72°C for 35 s, followed by 72°C for 7 min. The resulting 170-bp PCR products from each psyllid species were cloned, sequenced, and analyzed as described above (three clones per adult).

The quantity and quality of DNA from experimental insects (see “Insects” subsection) was analyzed using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA); none of the samples exhibited a 260/280 ratio above 1.7. Real-time qPCR was performed on an Applied Biosystems 79HT real-time PCR system (Applied Biosystems, Waltham, MA). Each 20 µl reaction included 10 µl of SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA), 1 µl of each primer (final concentration of 250 nM), 10 ng/µl template DNA, and nuclease-free water. The PCR conditions were 40 cycles of 95°C for 15 s and 60°C for 1 min. Three technical replications were included for each sample, and each technical replication was performed on a separate plate. A 10-fold serial dilution from 20 to 0.04 ng/µl of vector+16 S insert was used to determine the relationship between copies of 16S of Caronella and CT. The concentration of copies of 16S of Carsonella was estimated according to Whelan et al. (2003), and linear regression (PROC REG; SAS Institute 2012, Cary, NC) was used to model the relationship between relative abundance of Carsonella and CT. The resulting linear regression model was used to estimate the number of copies of 16S of Carsonella present in each experimental insect from the mean CT values. The efficiency of the qPCR reactions was determined using the equation, efficiency = 10(−1/slope) − 1. To estimate repeatability of the assay, the coefficient of variation of the average CT values of technical replications calculated as the ratio of the standard deviation to mean.

Carsonella titers within whole insects were compared among insect sexes and ages using the GLIMMIX procedure of SAS 9.3. For each analysis, the log of the estimated number of copies of 16S of Carsonella was included as the dependent variable. Sex, age, and the sex by age interactions were included as the fixed effects in analysis of Carsonella in C. pyricola. For analysis of Carsonella titers in B. cockerelli, sex was included as the fixed effect, and repetition was included as a random variable. For each analysis, data were examined for heterogeneity of variance and nonnormality of errors by inspecting residual and normal quantile-quantile plots, respectively. Based on these plots, data were modeled assuming a Gaussian distribution using the DIST=G option of the MODEL statement.

Carsonella in Developing Oocytes

We used fluorescence in situ hybridization to observe Carsonella in the oocytes of female C. pyricola and B. cockerelli. Five females of each psyllid species were dissected, and the ovaries and developing oocytes were transferred to a microscope slide. Because of difficulties washing probe from psyllid sperm (Cooper et al. 2014), spermatophores were removed from the reproductive organs of mated females before transferring the tissues to microscope slides. At least two females of each species were newly emerged (unsclerotized) and unmated. Fluorescence in situ hybridization was performed as described in “Carsonella Densities in Individual Bacteriocytes” subsection. Each sample was photographed at 200 or 400× with an ISO of 200 and an exposure time between 80 and 100 ms.

Results

Fluorescence was not associated with fat body cells hybridized with the 16S probe designed to bind to Carsonella suggesting the probe did not bind to nontarget DNA (data not shown because photographs taken with the standardized settings of an ISO of 200 and exposure time of 80 ms appeared black). In addition, fluorescence was not associated with fat body cells or bacteriocytes hybridized without the probe indicating a lack of interfering autofluorescence. However, the cytoplasm of bacteriocytes of both C. pyricola and B. cockerelli fluoresced bright-green indicating the presence of Carsonella (Fig. 1A inset).

Fig. 1.

(A) Comparison of estimated intensity of fluorescence associated with Carsonella in bacteriocytes of female and male C. pyricola. Inset shows observed Carsonella (green fluorescence) in bacteriocytes of insects. (B) Comparison of the number of copies of 16S from Carsonella in whole bodies of female and male C. pyricola. Error bars represent standard errors.

In most cases, the bacteriocytes of female C. pyricola fluoresced visibly brighter than those of males suggesting a greater concentration of Carsonella (Fig. 1A inset). Analysis of the fluorescence-intensity values obtained from ImageJ confirmed these visual observations. Fluorescence intensity for bacteriocytes of females was significantly greater than that of males (Fig. 1A; F = 19.2; df = 1, 22; P < 0.001). Fluorescence intensity did not change with insect age (F = 2.4; df = 3, 22; P = 0.095), and the lack of a significant sex × week interaction (F = 1.1; df = 3, 22; P = 0.391) indicated that the differences between sexes in fluorescence intensity were consistent among the different insect ages (newly emerged adults, 1-wk-old adults, 2-wk-old adults, and 3-wk-old adults).

In contrast with our findings on C. pyricola, we did not observe visible differences in fluorescence of bacteriocytes of female and male B. cockerelli (Fig. 2A; F = 0.4; df = 1, 17; P = 0.549).

Fig. 2.

(A) Comparison of estimated intensity of fluorescence associated with Carsonella in bacteriocytes of female and male B. cockerelli. (B) Comparison of the number of copies of 16S from Carsonella in whole bodies of female and male B. cockerelli. Error bars represent standard errors.

Linear regression analysis revealed a strong relationship between DNA dilutions and CT values associated with the amplification of 16S of Carsonella of C. pyricola (R2 = 0.998) and B. cockerelli (R2 = 0.993). The estimated efficiency of qPCR amplification for 16S of Carsonella was 71% for C. pyricola and 83% for B. cockerelli. The coefficient of variation was 0.014 for both species, providing evidence that the qPCR assay was highly repeatable.

Analysis of the estimated number of 16S copies of Carsonella of C. pyricola indicated that Carsonella was more abundant in females than in males (Fig. 1B; F = 9.3; df = 1, 22; P = 0.006). We did not observe a significant effect for age (F = 1.0; df = 4, 22; P = 0.421) and the lack of a significant sex × age interaction (F = 1.1; df = 4, 22; P = 0.400) indicated that the observed effects of sex on Carsonella populations were consistent among the different insect ages (fifth instars, newly emerged adults, 1-wk-old adults, 2-wk-old adults, and 3-wk-old adults). Analysis of Carsonella titers in 1-wk-old B. cockerelli also indicated that females harbored significantly more Carsonella than did males (Fig. 2B; F = 10.6; df = 1, 35.1; P = 0.003).

Carsonella was present in both previtellogenic and vitellogenic ovarioles of C. pyricola and B. cockerelli (Fig. 3), including ovarioles of newly emerged (unsclerotized) females (Fig. 3A). Carsonella was distributed throughout the ovarioles but formed a honeycomb pattern around previtellogenic oocytes and appeared more aggregated near the posterior end of the mature oocyte (Fig. 3B and C).

Fig. 3.

(A) Observation of Carsonella in previtellogenic oocytes of a newly emerged female C. pyricola. (B) Carsonella in a vitellogenic oocyte of a mature C. pyricola female. (C) Carsonella in previtellogenic (white arrows) and vitellogenic (red arrows) oocytes of a B. cockerelli female. Bars in (A) and (B) represent 20 µM, whereas the bar in (C) represents 50 µM.

Discussion

We previously reported that the bacteriomes of female psyllids were larger than those of males (Cooper and Horton 2014). Although inconclusive, staining of bacteriocytes with eosin/hemotoxcylin in that study suggested that bacteria densities were also higher in bacteriocytes of female psyllids. We developed two new methods to estimate and compare abundance of Carsonella in C. pyricola and B. cockerelli using florescence in situ hybridization and qPCR. Results of fluorescence in situ hybridization indicated that the bacteriocytes of female C. pyricola harbor greater densities of Carsonella than do those of males. Because female psyllids have larger bacteriomes that harbor more Carsonella than those of males, it is not surprising that Carsonella was also more abundant in whole bodies of female C. pyricola. We also compared the abundance of Carsonella between 5-d-old (reproductively mature) females and 5-d-old males of B. cockerelli using both techniques. We did not observe differences between B. cockerelli sexes in Carsonella densities within bacteriocytes, but we did observe significantly higher whole-body Carsonella titers in female B. cockerelli than in males using qPCR. Dossi et al. (2014) reported that compared with female Diaphorina citri (Hemiptera: Psyllidae), males have larger populations of endosymbionts including Carsonella. Therefore, our results indicating that females of C. pyricola and B. cockerelli harbor more Carsonella than do males may not be true for all psyllids.

The biological relevance of observed gender-specific variations in bacteriomes of psyllids (Cooper and Horton 2014) and the gender-specific variations in Carsonella populations is not known but may be associated with egg production. Carsonella produces essential amino acids that are lacking in the psyllid’s diet (Nakabachi et al. 2006), so larger populations of Carsonella may be necessary to satisfy the nutritional demands associated with metabolically expensive egg production. This explanation is consistent with previous reports that Carsonella proliferation corresponds with insect growth and ovarian development (Waku and Endo 1987, Dossi et al. 2014). Differences in Carsonella titers between female and male psyllids may also be due in part to the distribution of Carsonella outside the bacteriomes of females. Carsonella is vertically transmitted (Thao et al. 2001), and Carsonella appeared to be abundant in both previtellogenic and vitellogenic ovarioles of females using fluorescence in situ hybridization. Our observation of Carsonella in previtellogenic ovarioles of newly emerged females suggests that undeveloped ovarioles already harbor Carsonella prior to adult eclosion, which has been observed in the whitefly, Aleuorchiton aceris Modeer (Hemiptera: Aleyrodiniae) (Szklarzewicz and Moskal 2001).

Our observations of Carsonella in psyllid ovarioles revealed structural patterns that changed as ovarioles matured. Carsonella aggregations formed a honeycomb pattern in previtellogentic ovarioles of both psyllids species, but the cause of the pattern is unknown. Previous researchers showed that endosymbionts of psyllids colonize the hemocoel surrounding the ovarioles, the space between the follicular epithelium, and follicular cells, and form aggregations (peripheral maculae) near the posterior end of mature oocytes (Chang and Musgrave 1969, Waku and Endo 1987). Our observations using fluorescence in situ hybridization showed that Carsonella occurred throughout or surrounding follicular cells and developing oocytes consistent with the previous reports. Our observations also confirm the presence of peripheral maculae as previously documented using electron micrographs, but the role of these bacterial aggregations are not known (Chang and Musgrave 1969, Waku and Endo 1987).

Long-term goals of our research include exploring interactions among bacterial endosymbionts, psyllids, and their host plants. These long-term research goals require control of biological variation that may impede accurate measurements of Carsonella abundance. Previous studies that assessed Carsonella populations among psyllids did not control for insect sex and age (Nachappa et al. 2011, Alvarado et al. 2012, Arp et al. 2014). The combined results of our previously published study (Cooper and Horton 2014) and our current study clearly indicate that insect sex represents an important source of variability that must be considered in studies which rely on estimates of Carsonella populations. In addition, this report describes two new methods to estimate Carsonella abundance in psyllids by measuring Carsonella densities in individual bacteriocytes (fluorescence in situ hybridization) or Caronella titers in whole insects (qPCR). Although fluorescence in situ hybridization has been used previously to observe unculturable bacterial endosymbionts (Fukatsu and Nikoh 1998, Cooper et al. 2014), our study is the first to use relative intensity of fluorescence to estimate relative abundance of endosymbionts in insects.