Real-time PCR (Polymerase Chain Reaction) can estimate Wolbachia endosymbiont population density in mosquitoes (Wiwatanaratanabutr & Kittayapong 2009). The COS (Carbaryl-OP-Sulfur resistant) colony of the phytoseiid Metaseiulus (=Typhlodromus or Galendromus) occidentalis (Nesbitt) harbors several endosymbionts, including Cardinium, Wolbachia, Enterobacter, and Bacteroidetes in their gut and reproductive tissues (Johanowicz & Hoy 1996; Hoy & Jeyaprakash 2005). Johanowicz & Hoy (1998) observed mating incompatibility when females from a heat-treated (33°C) COS colony (a treatment presumed to eliminate Wolbachia) was crossed with males from a room-temperature (23°C) COS colony containing Wolbachia, resulting in the production of shriveled eggs that did not hatch. It was not clear whether the Wolbachia population was reduced or completely eliminated from the heat-treated mites because a standard PCR protocol was used, which is relatively insensitive compared to high-fidelity PCR (Jeyaprakash & Hoy 2000). In this paper, we estimate the endosymbiont population density in COS colonies of M. occidentalis held under different temperatures in the laboratory using real-time PCR.
One colony of M. occidentalis was maintained at room-temperature (23–25°C) and 2 colonies initiated from this line were maintained at 32 and 34°C for More than 1 year in growth chambers prior to experiments. Female mites (100) were isolated from each colony and their genomic DNA extracted by Puregene and QIAGEN methods (Jeyaprakash & Hoy 2007), and resuspended in 50 µL of sterile water. Four different plasmids carrying endosymbiont 16S rRNA sequences; pAJ233 (Cardinium), pAJ234 (Bacteroidetes), pAJ235 (Wolbachia) and pAJ239 (Enterobacter), were extracted and their yield estimated with a BioPhotometer Plus (Eppendorf AG, Hamburg, Germany). Species-specific primers were designed with Primer 3 software ( http://frodo.wi.mit.edu/primer3/) and a probe was designed with Primer X software (Applied Biosystems, Foster City, CA) from the variable regions for each species (Table 1). Real-time PCR was performed in a 20-µL reaction volume containing genomic DNA from 2 females (1 µL) or serially-diluted plasmid DNA (100 µg to 1 fg) or a no DNA control (1 µL), forward and reverse primers (400 pM), probe (100 pM) and TaqMan master mix (10 µL). Two linked profiles: (1) one cycle of 50°C for 2 min and 95°C for 10 min, and (2) 60 cycles consisting of denaturation at 95°C for 15 s, annealing at 59°C (Cardinium) or 57°C (Wolbachia or Enterobacter or Bacteroidetes) for 30 s and extension at 72°C for 30 s, were used. Each treatment was replicated 3 times. The starting copy number of all plasmid DNA dilutions used in the real-time PCR was obtained with an open-source software ( http://molbiol.edu.ru/eng/scripts/h01_07.html). A standard regression curve was generated with the Ct-value (Number of PCR cycles required for sample fluorescence to reach the threshold level) obtained from all 9 serial plasmid DNA dilutions. The Ct-value obtained from the 2 female mites was then used in the regression analysis to estimate the copy number of bacterial cells present, which was divided by 2 to obtain the copy numbers in a single female.
TABLE 1.
LIST OF SPECIES-SPECIFIC PRIMERS AND PROBES DESIGNED FOR REAL-TIME PCR.
The titer of Cardinium and Wolbachia was much higher than that of the gut endosymbionts Enterobacter and Bacteroidetes in the room-temperature M. occidentalis COS colony (Table 2). The Cardinium, Wolbachia and Enterobacter titer was reduced, but not eliminated, in both heattreated colonies. By contrast, Bacteroidetes had a higher titer in the heat-treated colonies, but their density was below detectable levels (1 or 2) in the room-temperature colony, and could not be reliably amplified by real-time PCR (Table 2).
We think that the titer of Wolbachia or Cardinium could play a role in mating compatibility when making crosses between heat-treated (uninfected) females and room-temperature (infected) males. However, it is not clear which endosymbiont actually causes mating incompatibility in the COS colony because this requires generating colonies infected with only Cardinium or Wolbachia. Weeks & Stouthamer (2004) reported that Wolbachia infection in another colony of M. occidentalis is lost when fed a Wolbachia-free two-spotted spider mite diet. During the summer of 2009 we determined, by high-fidelity PCR, that the two-spotted spider mite culture in our greenhouse had inadvertently lost Wolbachia after being heated to 40–42°C for several months (3–6) due to a maintenance problem and, subsequently, the 23–25, 32 and 34°C COS colonies that were fed this Wolbachia-free spider mite diet were determined to have lost Wolbachia (data not shown), suggesting that the Wolbachia in these colonies of M. occidentalis was obtained from their spider mite prey.
TABLE 2.
MEAN NUMBER (S.D.) OF ENDOSYMBIONTS ESTIMATED PER ADULT FEMALE METASEIULUS OCCIDENTALIS MAINTAINED AT 3 DIFFERENT TEMPERATURES1.
To determine whether the Wolbachia-free COS colonies reared at 23–25 and 34°C exhibited incompatibility several inbred lines were generated. They displayed no fluctuation in Cardinium and Enterobacter titers (Table 2). A total of 7 or 8 reciprocal single-pair and control crosses were performed on 3 separate dates. Equal numbers of reciprocal and control crosses were successful and produced unshriveled eggs that subsequently hatched, suggesting that Cardinium is not involved in causing cytoplasmic incompatibility in the COS colony at these titers (Table 2) and that Wolbachia might be involved. It appears that this COS colony is obtaining Wolbachia by feeding on its Wolbachia-infected prey. These results are consistent with earlier data indicating that there are no differences in 16S, ftz and wsp sequences from Wolbachia in the COS and T. urticae colonies (Hoy & Jeyaprakash 2005).
The standard PCR protocol previously used by Johanowicz & Hoy (1996) we now know is not sufficiently sensitive to be certain that endosymbionts truly are lacking (Jeyaprakash & Hoy 2000) and should not be used when comparing heat-treated and room-temperature colonies. Real-time PCR is an efficient tool to quantify endosymbiont titer based on this study and that by Wiwatanaratanabutr & Kittayapong (2009). This research was supported in part by the Davies, Fischer and Eckes Endowment in Biological Control to M. A. Hoy.
SUMMARY
Real-time PCR amplification of 16S rRNA sequences from 4 M. occidentalis endosymbionts; Cardinium, Wolbachia, Enterobacter, and Bacteroidetes, provided an estimate of their titer in colonies reared under 3 temperatures. This work also revealed, for the first time, that the Wolbachia and Cardinium titer were reduced in heat-treated COS colonies, but not completely eliminated. However, feeding M. occidentalis colonies the Wolbachia-free two-spotted spider mite diet did eliminate Wolbachia from this predator. No cytoplasmic incompatibility was detected when crosses were made between males reared at room-temperature (23–25°C) containing Cardinium (and lacking Wolbachia) and females reared at 34°C containing a much lower titer of Cardinium (and lacking Wolbachia), indicating that Wolbachia may be involved in causing previously observed incompatibilities rather than Cardinium.
