High-fidelity Polymerase Chain Reaction Research Articles

Replication of the Venezuelan Equine Encephalitis Vaccine from a Synthetic PCR Fragment.

There is no approved human vaccine for Venezuelan equine encephalitis (VEE), a life-threatening disease caused by the VEE virus (VEEV). In previous studies, plasmid DNA encoding the full-length RNA genome of the VEE V4020 vaccine was used for the preparation of experimental live virus VEE vaccines in the plasmid-transfected cell culture. Here, we used the high-fidelity polymerase chain reaction (PCR) to prepare synthetic, transcriptionally active PCR (TAP) fragments encoding the V4020 genome. TAP fragment initiated the replication of the V4020 live virus vaccine in TAP fragment-transfected cells. A transfection of less than 1 ug of TAP fragment resulted in the replication of the V4020 vaccine virus in CHO cells. We conclude that not only plasmid DNA but also synthetic PCR-generated DNA fragments can be used for the manufacturing of live vaccines for VEEV and, potentially, other viruses.

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

High Prevalence of Group III-Like Mutations Among BLPACR and First Report of Haemophilus influenzae ST95 Isolated from Blood in China.

We aimed to evaluate antibiotic resistance and molecular epidemiological characteristics of non-invasive Haemophilus influenzae (H. influenzae) from pneumonia patients and analyze the whole genome of one invasive H. influenzae isolated from blood in pediatric patients. Antibiotic susceptibility was tested using the turbidimetric method. β-lactamase-producing and serotyping genes were evaluated via multiplex polymerase chain reaction (PCR), and ftsI was amplified using high-fidelity PCR. Lastly, whole genome sequencing (WGS) was conducted using Illumina HiSeq and PacBio sequencing technology. We observed that the ampicillin (AMP) and amoxicillin/clavulanate (AMC) resistance rates of non-invasive H. influenzae were as high as 99.06% (after adjustment) and 49.53%, respectively. The β-lactamase gene of 106 AMP-resistant strains was blaTEM-1 . Group III-like mutation accounted for 71.15% of β-lactamase-positive, AMC-resistant (BLPACR) strain mutants. The novel Asn-526→His mutation was present in one β-lactamase-negative AMP-susceptible (BLNAS) strain. Non-invasive H. influenzae strains all belonged to non-typeable H. influenzae (NTHi). In contrast, the invasive H. influenzae 108 isolated from blood in China belonged to H. influenzae type b (Hib). It belonged to sequence typing ST95 and exhibited sensitivity to all 11 antibiotics. Three prophages were identified, and the capb loci of the H. influenzae strain 108 revealed regions I-III exist in duplicate; however, complete deletion of IS1016 was only present in one of the copies. Non-invasive H. influenzae NTHi with β-lactamase-positive was highly prevalent. Notably, group III-like mutations had increased prevalence among BLPACR strains. H. influenzae belonging to Hib and ST95 was first reported to cause sepsis in China.

One-Day TALEN Assembly Protocol and a Dual-Tagging System for Genome Editing.

This study developed a new rapid transcription activator-like effector nuclease (TALEN) preparation protocol by thoroughly redesigning the widely used Golden Gate TALEN and TAL Effector Kit 2.0. The new protocol can be used to prepare any custom 18-bp binding TALENs in just one day (about 12 h), more rapidly than CRISPR. This protocol used a set of linear monomers, a final TALE-FokI backbone plasmid, and a pipeline to assemble the ready-to-use TALEN expression plasmid, which were all newly developed for this study. The set of linear monomers can be easily produced and reproduced by high-fidelity polymerase chain reaction (PCR) amplification in a 96-well plate using a pair of universal primers. Most important of all, our rapid TALEN construction pipeline can easily obtain many positive colonies with high efficiency (over 80%). By preparing five pairs of TALENs targeting five NF-κB genes (RELA, RELB, CREL,NFKB1, and NFKB2) and editing these genes in different cell lines (293T, HepG2, and PANC1), this study demonstrated that the new protocol has high efficiency, reproducibility, reliability, and applicability. Moreover, this study showed that the fabricated TALEN has much higher editing efficiency than CRISPR. Finally, this study developed a dual-tagging system for simultaneously tagging target proteins and successfully edited cells with a streptavidin-binding peptide (SBP) or AviTag via homology-directed repair, which could have wide applications in protein (antigen) preparation, immunoprecipitation, and a transcription factor chromatin immunoprecipitation assay.

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

First Report of Grapevine Red Blotch Virus in Mexico

Grapevine red blotch virus (GRBV) (Al Rwahnih et al. 2013) is a member of the recently recognized genus Grablovirus in the family Geminiviridae (Varsani et al. 2017). GRBV is the causal agent of grapevine red blotch disease (Yepes et al. 2018), affecting viticulture in North America and some other regions around the world (reviewed in Sudarshana et al. [2015]). In this study, we sampled grapevine plants displaying red blotch symptoms in the major wine producing region of Mexico: Ensenada, Baja California, in 2016 and 2017. A total of 46 samples from cultivars Vitis vinifera L. ‘Pinot Noir’ and ‘Merlot’ were tested in 2016 by end-point polymerase chain reaction (PCR) using primers GRBV-V2F (5′-ATGGGTTAGGGGATGAGGCT-3′) and GRBV-V1R (5′-CGGCAATGACTCCTGCGGCT-3′). Results showed amplification of the expected 798-bp product from 31 samples. One of these amplicons was cloned, sequenced, and compared with reported sequences through a BLASTn search. The fragment had high nucleotide sequence identity (98%) with GRBV isolate BCRB5 from Canada (GenBank accession no. KX234092). The presence of GRBV was corroborated using the AmplifyRP Acceler8 kit (Agdia, Elkhart, IN), which detects the GRBV genome by isothermal recombinase polymerase amplification (RPA). In 2017 symptomatic V. vinifera ‘Nebbiolo’ plants were tested by multiplex PCR to amplify a region of the replicase-associated protein (Rep) gene with primers Repfor/Reprev (expected product 318 bp) and a fragment from the coat protein (CP) gene with primers CPfor/CPrev (expected product 257 bp) (Krenz et al. 2014). Amplicons from a positive sample were cleaned using a GenJet PCR purification kit (Thermo Fisher Scientific, Waltham, MA) and analyzed by Sanger sequencing. Both Rep and CP fragments shared >99% nucleotide sequence identity with GRBV isolates ONRB7 (KY316025) and ONRB4 (KY316022) from Canada (Poojari et al. 2017). To gain a deeper insight into the phylogeny of local GRBV isolates, we sequenced the full-length viral genome of 2016 and 2017 representative isolates. Each genome was amplified by high-fidelity PCR in two fragments, combining primers CPfor/Reprev (expected product 1.8 kb) and Repfor/CPrev (expected product 1.9 kb). After Sanger sequencing, the genomes were assembled in the GeneStudio sequence analysis software version 2.2.0.0 (GeneStudio, Suwanee, GA). A phylogenetic tree constructed after comparing the local isolates GRBV-Gpe-JGB (MH557096) and GRBV-Gpe-JCT (MH557095) to GRBV isolates from Canada and the United States grouped the Baja Californian isolates with clade 1 isolates. To our best knowledge, this is the first report of GRBV in Mexico.

Hiding Secret Messages in Living Cells in the Age of Next-Generation Sequencing

Hiding secret messages (SMs) based on DNA technologies plays a critical role in DNA computing. In recent years, scientists have hidden SMs at a specific site in the plasmids of various living cells. Unfortunately, this strategy was proposed without considering the attack of next-generation sequencing (NGS). Nowadays, NGS, which is a revolutionary massively parallel sequencing technique, is developing rapidly. With the help of NGS, sequencing completely unknown genomes is becoming popular in various laboratories throughout the world. The ability to sequence completely unknown genomes is a major threat to existing hiding strategies in living cells. To prevent against the attack of NGS, this paper proposes a scheme to hide SMs in living cells (SHSM). Compared with previous studies, the main contribution of SHSM is changing the specific single hiding site into random multiple hiding sites through the application of seamless clustered regularly interspaced short palindromic repeats/CRISPR-associated9 (CRISPR/Cas9) technique. In SHSM, SMs are hidden in living cells with different puzzling messages at different sites every time to prevent against the attack of NGS. To read the SM, high-fidelity polymerase chain reaction (PCR) and Sanger sequencing are used. A hash function is used to ensure the integrity of the message. These measures mean that an attacker would not be able to determine the location of the SM using NGS. The feasibility of SHSM was validated by a wet-lab experiment, and the security was demonstrated by the system’s entropy. The author hopes this study will bring attention to the threat of NGS, and advance the development of hiding SMs in living cells.

Molecular Detection of the Laurel Wilt Fungus, Raffaelea lauricola

The laurel wilt disease fungus, Raffaelea lauricola, is killing redbay trees, spreading rapidly in the U.S. southeastern coastal plain forest, and posing a serious threat to the avocado industry in Florida. A molecular tool is urgently required to facilitate detection of this pathogen. The 5' region of the large ribosomal RNA (28S) gene is highly variable among Raffaelea spp. and ideal for this purpose but amplification of this sequence from R. lauricola has been difficult. Different amplification conditions were tested and a high-fidelity polymerase chain reaction (PCR) procedure utilizing a dNTP mix containing 7-deaza-dGTP was found to reliably amplify 28S sequences from R. lauricola. Sequencing the amplified products or cloned inserts also turned out to be difficult and required using a custom-blended sequencing mix containing 1 M betaine, 5% dimethyl sulfoxide, and dGTP-BigDye v3.1. Three GC-rich stem and loop or cruciform secondary structures were discovered, which may have interfered with amplification. This improved protocol made it possible to partially characterize the internal transcribed spacers sequence from R. lauricola, which also has interfering secondary structures. A TaqMan real-time PCR assay was designed using the species-specific 28S sequences and this allowed detection of R. lauricola from wood tissues or cultures. Wood tissues from symptomatic redbay, avocado, and sassafras trees in Florida were screened using this TaqMan assay and several were found to test positive for R. lauricola. Results were further confirmed by performing Koch's postulates for avocado specimens collected from commercial grooves.

Formation of para-Bombay phenotype caused by homozygous or heterozygous mutation of FUT1 gene

This study was aimed to explore the molecular mechanisms for para-Bombay phenotype formation. The H antigen of these individuals were identified by serological techniques. The full coding region of alpha (1, 2) fucosyltransferase (FUT1) gene of these individuals was amplified by high-fidelity polymerase chain reaction (PCR). PCR product was identified by TOPO cloning sequencing. Analysis and comparison were used to explore the mechanisms of para-bombay phenotype formation in individuals. The results indicated that the full coding region of FUT1 DNA was successfully amplified by PCR and gel electrophoresis. DNA sequencing and analysis found that h1 (547-552delAG) existed in one chromosome and h4 (35C > T) existed in the other chromosome of NO.1 individual. Meantime, h1 (547-552delAG) was found in two chromosomes of NO.2 and NO.3 individual. It also means that FUT1 gene of NO.1 individual was h1h4 heterozygote, FUT1 gene of NO.2 and NO.3 individuals were h1h1 homozygote. It is concluded that homozygous and heterozygous mutation of FUT1 gene can lead to the formation of para-Bombay phenotype.

Translation of branched-chain aminotransferase-1 transcripts is impaired in cells haploinsufficient for ribosomal protein genes

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells.

Generation of Helper Plasmids Encoding Mutant Adeno-associated Virus Type 2 Capsid Proteins with Increased Resistance against Proteasomal Degradation.

Adeno-associated virus type 2 (AAV2) vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV2 vector helper plasmids encoding capsid proteins with single, double or triple Y→F mutations. A one-step, high-fidelity polymerase chain reaction (PCR) cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV2 cap genes encoding VP1 mutants with Y→F substitutions in residues 444, 500 or 730. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y444500F mutant), subcloning (Y444730F and Y500730F mutants) or a combination of both techniques (Y444500730F mutant). Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV2 cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV2 vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping. PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV2 vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.

A Comparison of Standard and High-Fidelity PCR: Evaluating Quantification and Detection of Pathogen DNA in the Presence of Orchid Host Tissue.

The polymerase chain reaction (PCR) has been used with increasing frequency for detecting and identifying plant pathogens. Although PCR is sensitive, research has shown that amplification of target microbial DNA from within another organism, such as an arthropod or plant, can be inhibited by the presence of host DNA. In this study, the sensitivity of standard and high-fidelity PCR, which incorporates a second DNA polymerase with proofreading ability, to detect and amplify DNA from the fungal pathogen Pseudocercospora odontoglossi while in the presence of Cattleya orchid DNA, was compared. Different dilutions of plasmids containing internal transcribed spacer (ITS)1, 5.8S, and ITS2 rDNA from P. odontoglossi were spiked with Cattleya orchid plant DNA. The high-fidelity PCR could detect and amplify as few as 207 plasmids containing the fungal DNA, whereas the standard PCR required over 200 million copies. The high-fidelity PCR was more efficient than conventional PCR in detecting Sclerotium rolfsii and a Dickeya sp. from freshly inoculated orchid plants, demonstrating its increased sensitivity in early detection of fungal and bacterial pathogens that are difficult to discriminate early in disease development.

Detection of Low-Level K65R Variants in Nucleoside Reverse Transcriptase Inhibitor–Naive Chronic and Acute HIV-1 Subtype C Infections

To substantiate reports of greater emergence of the K65R nucleoside reverse transcriptase inhibitor (NRTI) mutation in human immunodeficiency virus type 1 (HIV-1) subtype C, we examined natural low-level K65R expression in subtype C relative to subtypes B and AE. We used allele-specific polymerase chain reaction to screen HIV-1 amplified by reverse-transcription high-fidelity polymerase chain reaction from subtype C-infected South African women and infants and CRF01(subtype AE) from Thailand; all subjects were NRTI naive. We found low-level K65R of unknown clinical significance in NRTI-naive subtype C-infected women and infants at frequencies above the natural occurrence in subtypes B and AE. The frequent appearance of subtype C frameshift deletions at codon 65 supports a propensity for transcription error in this region.

Real-Time PCR Reveals Endosymbiont Titer Fluctuations inMetaseiulus occidentalis(Acari: Phytoseiidae) Colonies Held at Different Temperatures

Real-time PCR (Polymerase Chain Reaction) can estimate Wolbachia endosymbiont population density in mosquitoes (Wiwatanaratanabutr & Kittayapong 2009). The COS (?arbaryl-OP-Sul fur resistant) colony of the phytoseiid Metaseiulus (=Typhlodromus or Gcdendromus) occidentalis (Nesbitt) harbors several endosymbionts, includ ing Cardinium, Wolbachia, Enterobacter, and Bacteroidetes in their gut and reproductive tis sues (Johanowicz & Hoy 1996; Hoy & Jeyaprakash 2005). Johanowicz & Hoy (1998) ob served 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 pro duction 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 pro tocol was used, which is relatively insensitive compared to high-fidelity PCR (Jeyaprakash & Hoy 2000). In this paper, we estimate the endo symbiont population density in COS colonies of M. occidentalis held under different tempera tures in the laboratory using real-time PCR. One colony of M. occidentalis was maintained at room-temperature (23-25?C) and 2 colonies ini tiated 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 pL 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 de signed with Primer 3 software (http:// frodo.wi.mit.edu/primer3/) and a probe was de signed with Primer X software (Applied Biosys tems, Foster City, CA) from the variable regions for each species (Table 1). Real-time PCR was per formed in a 20-pL reaction volume containing ge nomic DNA from 2 females (1 pL) or serially-di luted plasmid DNA (100 pg to 1 fg) or a no DNA control (1 pL), forward and reverse primers (400 pM), probe (100 pM) and TaqMan master mix (10 pL). Two linked profiles: (1) one cycle of 50?C for 2 min and 95?C for 10 min, and (2) 60 cycles consist ing of denaturation at 95?C for 15 s, annealing at 59?C (Cardinium) or 57?C (Wolbachia or Entero bacter or Bacteroidetes) for 30 s and extension at 72?C for 30 s, were used. Each treatment was rep l cated 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:/

Microbial Diversity Associated with the Fruit-Piercing and Blood-Feeding Moth Calyptra thalictri (Lepidoptera: Noctuidae)

Abstract Previous inventories of the diversity of lepidopteran symbionts have been limited to Eubacteria. We conducted a microbial survey of Calyptra thalictri Borkhausen (Lepidoptera: Noctuidae) by using polymerase chain reaction (PCR) primers for 16S rRNA sequences for Eubacteria, and primers for Archaea, fungi including yeast-like organisms, Microsporidia, and Wolbachia. Heads and abdomens of adult males of this fruit-piercing and blood-feeding moth were assayed separately. High-fidelity PCR and subsequent DNA analyses indicated that at least five microorganisms belonging to the α-, β-, and γ-Proteobacteria were present. Two eubacterial sequences, related to a Klebsiella sp. and a Sinorhizobium sp., were detected in the abdomens of all nine individuals sampled, and three additional sequences, two related to species in the genus Alcaligenes and one related to a Rhizobium sp., were found in some of the abdominal samples, suggesting all five could be associated with abdominal structures. No Archaea, fungi including yeast-like organisms, Microsporidia, or Wolbachia were detected. These results document the first microbial associates in a fruit-piercing and blood-feeding moth.

Molecular Survey of Endosymbionts in Florida Populations of Diaphorina citri (Hemiptera: Psyllidae) and Its Parasitoids Tamarixia radiata (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae)

A molecular survey of endosymbionts was conducted in Florida populations of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and its parasitoids Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Shafee, Alam and Agarwal) (Hymenoptera: Encyrtidae). Using a high-fidelity polymerase chain reaction (PCR), we detected 3 eubacterial species each in D. citri (the primary symbiont Candidatus Carsonella rudii, a secondary symbiont related to Oxalobacter and Herbaspirillum species, and Wolbachia), and T. radiata (Caulobacter sp., Methylobacterium sp. and a bacterium in the family Alcaligenaceae), whereas only 1 species was identified in D. aligarhensis (Wolbachia). Each eubacterial symbiont of D. citri was detected in the eggs, which is indicative of transovarial transmission. However, none of the eubacterial symbionts of T. radiata were detected in the eggs, suggesting that they are only transient associates. Stable horizontal transfer of these eubacterial species likely has not occurred between the host and its parasitoids because each insect hosts a different complement of Eubacteria. For example, different strains of Wolbachia were detected in D. citri and D. aligarhensis, based on evidence from the 16S rRNA and wsp gene sequences. Also, the orf7 gene of the bacteriophage WO associated with Wolbachia was detected in D. citri but not in D. aligarhensis. No endosymbiotic Archaea, Helicosporidia, Microsporidia, Fungi, or Yeast-like symbionts were detected with PCR in these populations of D. citri or its parasitoids.

The complete mitochondrial genome of rock carp Procypris rabaudi (Cypriniformes: Cyprinidae) and phylogenetic implications

Rock carp, Procypris rabaudi (Tchang), is an endemic fish species in China. We sequenced the complete mitochondrial genome of it by high-fidelity polymerase chain reaction with conserved primers and primer walking sequencing method. The complete mitochondrial genome of rock carp is 16595 bp in length and contains 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and one control region, with an identical order to that of most other vertebrates. The origin of L-strand replication (OL) in rock carp mitochondrion is located in a cluster of five tRNA genes (WANCY region) with 35 nucleotides in length. The control region is located between the tRNA-Pro and tRNA-Phe genes and is 943 bp in length. Three conserved sequence blocks (CSB), an extended termination associated sequence (ETAS), an AT-repeat microsatellite sequence and a putative promoter sequence for H-strand transcription (HSP) were identified within this region. The microsatellite sequence has a very low variation, with only one repeat alteration in 50 checked individuals (from 12 to 13 repeats). The phylogenetic analysis for rock carp was performed with Bayesian and Maximum likelihood (ML) methods based on the concatenated nucleotide sequence of 12 protein-coding genes on the heavy strand. The result suggested that traditional taxonomic barbines possibly originated more early than cyprininaes; rock carp was placed at the position between barbines and cyprininaes, while has a closer relationship with cyprininaes than barbines.

HotStart-IT ® : A Novel Hot Start PCR Method Based on Primer Sequestration

HotStart-IT ^® : A Novel Hot Start PCR Method Based on Primer Sequestration

Removal of Fungal Contaminants and their DNA from the Surface of Diaphorina citri (hemiptera: psyllidae) Prior to a Molecular Survey of Endosymbionts

biology (Dale & Moran 2006), and molecular sur veys to detect arthropod-associated microbes have bypassed the necessity of culturing them in vitro for identification (Darby & Welburn 2006). To date, no consistent method has been developed to eliminate microbial DNA from the external surfaces of arthropods prior to conducting molec ular surveys of endosymbionts. This is problem atic because resolving the relationships between a host and its endosymbionts can be complicated by polymerase chain reaction (PCR) amplification of DNA from contaminating external microbes (Thornhill et al. 2008). A variety of techniques that may kill microbes on arthropod surfaces have been reported, including washing with ace tone (Fukatsu & Nikoh 1998), ethanol (Davidson et al. 1994; Van Borm et al. 2002), bleach (David son et al. 1994), formaldehyde (Duperchy & Zim mermann 2003), exposure to ultraviolet light (Van Borm et al. 2002), radiation (Duperchy & Zimmermann 2003) and antibiotic treatment (Duperchy & Zimmermann 2003). However, the effectiveness of these methods for eliminating DNA from external microbes so that it is not am plified in subsequent PCRs has not been evalu ated. DNA from contaminating external fungi should be eliminated, if possible, prior to deter mining if there are endosymbiotic fungi associ ated with a laboratory colony of the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Examples of fungal endosymbionts have been reported in association with other hemipteran insects, including aphids and plan thoppers (Noda et al. 1994; Fukatsu & Ishikawa 1995; Hongoh & Ishikawa 2000). A laboratory col ony of D. citri (Skelley & Hoy 2004) was used to evaluate a variety of solutions (Table 1) for their ability to kill surface-inhabiting fungi and to eliminate their DNA so that they could not be de tected using a high-fidelity PCR protocol. Adult D. citri in this colony support an abundance of exter nal fungal growth, primarily on the ventral por tion of their abdomens. This is due to being reared 702 Florida Entomologist 91(4) December 2008

Wolbachia-Associated Thelytoky in Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), A Parasitoid of the Asian Citrus Psyllid

Wolbachia is an obligate intracellular ct-pro teobacterium associated with arthropods and nematodes (O'Neill et al. 1997; Bazzochi et al. 2000). Wolbachia is transovarially transmitted by females to their progeny, and infections often are associated with reproductive anomalies in their host (O'Neill et al. 1997). In parasitoids, Wol bachia can cause cytoplasmic incompatibility (Stouthamer et al. 1999), thelytoky (parthenogen esis) (Stouthamer et al. 1990), and alter aspects of fecundity (Grenier et al. 2002). In Florida, colonies of the thelytokous endo parasitoid Diaphorencyrtus aligarhensis (Hymen optera: Encyrtidae) (Shafee, Alam and Agarwal) and the arrhenotokous ectoparasitoid Tamarixia radiata Waterston (Hymenoptera: Eulophidae) were imported from Taiwan and Vietnam, respec tively, and released in a classical biological control program against the Asian citrus psyllid, Diapho rina citri Kuwayama (Hemiptera: Psyllidae) (Hoy & Nguyen 1998, 2000; Hoy et al. 1999; Skelley & Hoy 2004). Worldwide, these parasitoids have a significant impact on reducing populations of D. citri, which is the most economically important citrus pest in regions where it vectors citrus greening disease (Chien 1995; Halbert & Manju nath 2004). Jeyaprakash and Hoy (2000) detected Wolba chia in the imported population of D. aligarhen sis. We hypothesized that Wolbachia causes thely toky in D. aligarhensis, and this was tested by at tempting to eliminate Wolbachia with antibiotics following the previous work of Stouthamer et al. (1990). This research is important because D. aligarhensis populations are low in Florida, and this could be due to its low reproductive rate (Skelley & Hoy 2004) which may be influenced by Wolbachia. A laboratory colony of D. aligarhensis was maintained as follows. Ten small citrus trees (20 50 cm tall) grown in 15.2-cm diameter pots were pruned each week, fertilized with Peter's 20-20-20 (N-P-K) water-soluble fertilizer (United Indus tries, St. Louis, MO), and placed in wooden framed mesh cages (0.76 m x 0.91 m x 1.11 m) in a greenhouse at 20-32?C with a 16L:8D photope riod. Adult female psyllids oviposited on the new growth (flush) produced by the trees. Adult D. ali garhensis were aspirated and released into the cages when immature D. citri reached the first or second instar. After emergence, adult D. aligar hensis were fed pure clover honey smeared on small strips of Kimwipes (Kimberly-Clark, Roswell, GA) and used to initiate the next gener ation. During a 4-year rearing period, all D. ali garhensis observed in this colony were females (J. Meyer, personal observation). A preliminary toxicity test indicated that 10 mg/mL tetracycline + honey did not negatively in fluence longevity in adult female D. aligarhensis, so this dosage was adopted for this experiment. For 3 consecutive generations, 50 newly-emerged female D. aligarhensis were administered pure clover honey + 10 mg/mL tetracycline hydro chloride (Sigma Chemical Co., St. Louis, MO) (Stouthamer et al. 1990) for 24 h at 70-75% RH, 24-25?C with a 16L:8D photoperiod. Treated par asitoids were released into a separate cage and maintained as described above. After the third generation, approximately 60 adult male D. ali garhensis were observed and collected. Female and male D. aligarhensis were placed on a glass slide and submerged in 95% EtOH or Euparal mounting medium (BioQuip, Rancho Domingez, CA) for photography with the Auto Montage Pro system with software ver. 5.02 (Syn optics, Frederick, MD). Morphological differences were observed between female and male D. ali garhensis (Fig. 1). The male abdomen was small and all black, but the female abdomen was larger and was yellowish and black (Fig. 1A, D). Both the geniculate antennae (Fig. IB, E) and genitalia (Fig. 1C, F) of female and male D. aligarhensis were structurally distinguishable. The antennae of male D. aligarhensis in an arrhenotokous pop ulation from Asia (Shafee et al. 1975) were simi lar to those observed in male D. aligarhensis pro duced here. Molecular analyses were used to determine if Wolbachia was eliminated from male D. aligarhen sis. DNA was isolated from each of 3 individual fe male and male D. aligarhensis with PUREGENE reagents (Gentra Systems, Minneapolis, MN) ac cording to the manufacturer's protocol. A 25-uL high-fidelity polymerase chain reaction (PCR) was conducted according to Hoy et al. (2001) to detect the wsp gene of Wolbachia with the primers wsp 81F (5'-TGGTCCAATAAGTGATGAAGAAAC-3') and wsp 691R (5'-AAAAATTAAACGCTACTCCA 3') (Braig et al. 1998). For a DNA template control, the mitochondrial cytochrome c oxidase I gene (COI) was amplified with the primers CI-J1632