Ayyamperumal Jeyaprakash

Long PCR improves Wolbachia DNA amplification: wsp sequences found in 76% of sixty-three arthropod species.

Bacteria belonging to the genus Wolbachia are associated with a variety of reproductive anomalies in arthropods. Allele‐specific polymerase chain reaction (= Standard PCR) routinely has been used to amplify Wolbachia DNA from arthropods. While testing the two‐spotted spider mite Tetranychus urticae and other arthropods known to be infected with Wolbachia, Standard PCR frequently produced false negatives, perhaps because the DNA from the arthropod host interfered with amplification by Taq DNA polymerase. Long PCR, which uses two enzymes (Taq and Pwo), consistently amplified Wolbachia DNA and a sensitivity analysis indicated that Long PCR was approximately six orders of magnitude more sensitive than Standard PCR in amplifying plasmid DNA spiked into insect genomic DNA. A survey indicated that 76% of sixty‐two arthropod species and two subspecies in thirteen orders tested positive for the Wolbachia wsp sequence by Long PCR, which is considerably higher than the rate of 16.9% obtained previously for the ftsZ sequence using Standard PCR ( Werren, J.H., Windsor, D. and Gao, L. (1995a)Proc R Soc Lond B 262: 197–204). A subsample of Long PCR products from fourteen arthropod species and two subspecies were sequenced, both directly and after cloning. Two A‐ and eleven B‐Wolbachia strains were detected and their wsp sequences displayed a maximum of 23.7% sequence divergence at this locus. Two new groups (named Fus and Ten) were identified in addition to nineteen reported earlier ( Zhou, W., Rousset, F. and O’Neill, S.L. (1998)Proc R Soc Lond B 265: 1–7; van Meer, M.M.M., Witteveldt, J. and Stouthamer, R. (1999)Insect Mol Biol 8: 399–408), because they displayed more than 2.5% sequence divergence from other Wolbachia wsp sequences. PCR products from seventeen of twenty‐nine (59%) arthropod species analysed could not be sequenced directly due to apparent infection by multiple Wolbachia strains. The wsp sequences cloned from two such species (Plutella xylostella and Trichoplusia ni) indicated both A‐ and B‐Wolbachia were present in a single individual. Hence, superinfection also may be more widespread than the 1.2% incidence previously estimated.

Morphological and molecular characterization of a new microsporidian species from the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari, Phytoseiidae).

A new microsporidian species is described from the predatory mite Metaseiulus (formerly Typhlodromus or Galendromus) occidentalis (Nesbitt) (Acari, Phytoseiidae). The ultrastructure of this new species is presented together with the first molecular characterization for a microsporidium of mites. All stages of this new microsporidium are haplokaryotic and develop in direct contact with the host-cell cytoplasm. Sporogony is disporoblastic and spores are formed in eggs, immature stages, and adults of M. occidentalis. There are two morphological classes of spores, one with a short polar filament (3-5 coils) that measured 2.53 x 1.68 microm and one with a longer polar filament (8-9 coils) that measured 3.14 x 1.77 microm. Horizontal transmission of this new species occurs by cannibalism of eggs and other stages and perhaps involves the spores with the long polar filament. Spores with the short polar filament may play a role in autoinfection and vertical (transovarial) transmission that is highly efficient in transferring the microsporidium from adults to progeny. Analysis of the small subunit ribosomal DNA indicated that this species from M. occidentalis is most closely related to the Nosema/Vairimorpha clade of microsporidia. A conflict between the morphological and molecular data is discussed. The species is compared to previously described microsporidia of arachnids resulting in creation of Oligosporidium occidentalis n. sp. in the family Unikaryonidae.

Complete sequence of a mariner transposable element from the predatory mite Metaseiulus occidentalis isolated by an inverse PCR approach

Degenerate primers designed and synthesized based on two conserved regions of the mariner transposase open reading frame were used to amplify a 454 bp DNA fragment from M. occidentalis. Two inverse primers were then synthesized and used to amplify flanking genomic DNA fragments from M. occidentalis by a ligation‐mediated inverse PCR. The complete mariner element (Moc 1) was 1284 bp long, including the imperfect 28 bp inverted terminal repeat sequences, and shared 59% similarity to an active 1286 bp long D. mauritiana mariner element (Mos 1). Insertions, deletions and substitutions were observed in the Moc 1 sequence at several positions. No intact open reading frame was detected and the Moc 1 element is considered inactive. Stringent Southern blot hybridizations revealed at least twelve copies of mariner sequences similar to Moc 1 in the colonies tested.

HIGH-FIDELITY PCR ASSAY DISCRIMINATES BETWEEN IMMATURE LIPOLEXIS OREGMAE AND LYSIPHLEBUS TESTACEIPES (HYMENOPTERA: APHIDIIDAE) WITHIN THEIR APHID HOSTS

Abstract Species-specific molecular markers were developed to identify and distinguish between two parasitoids of the brown citrus aphid, Toxoptera citricida Kirkaldy, in Florida. PCR primers were developed for Lysiphlebus testaceipes Cresson and Lipolexis oregmae Gahan (= scutellaris Mackauer) with DNA sequences from the internal transcribed spacer (ITS) region between the 5.8S and 28S nuclear rRNA genes. With High-fidelity PCR, the L. testaceipes-specific primer produced a 520-bp band while that of L. oregmae resulted in a 270-bp band. Eggs of both parasitoids within their aphid hosts could be detected by 6 h after oviposition, but 100% detection rates only occurred after 24 h. A sensitivity analysis indicated that a parasitoid egg within a single aphid could be detected 100% of the time when combined with DNA from up to 36 unparasitized aphids. A single first instar parasitoid could be detected by High-fidelity PCR when the parasitized aphid was combined with up to 500 unparasitized aphids, indicating a high level of sensitivity. Species-specific primers detected both immature parasitoid species within aphids commonly found in citrus in Florida, including Aphis craccivora Koch, Aphis gossypii Glover, Aphis spiraecola Patch, Toxoptera aurantii Boyer and T. citricida. This High-fidelity PCR assay provides an efficient method to monitor establishment of L. oregmae in citrus groves in this classical biological control program in Florida.

A DNA extraction procedure that allows mite specimens to be slide mounted: phytoseiid species evaluated as a model

Four protocols for extracting DNA from mites, using phytoseiid species as exemplars, were evaluated to determine whether the DNA obtained could be used to amplify nuclear, mitochondrial or Random Amplified Polymorphic DNA (RAPD) markers from males, females and eggs. Protocol 3 was identified as the best and this allowed High-fidelity PCR (Hf-PCR) and Hf-RAPD PCR to be used successfully; it left behind the intact body of adult mites so they could be slide mounted for morphological analyses, although the eggs had to be pricked in order to yield sufficient DNA for amplifications. Protocol 3 involved soaking intact specimens in a GuSCN buffer and using a silica matrix, which binds nucleic acids, to yield DNA for amplification. The DNA isolated could be stored up to a month, indicating that the quality was good. This DNA extraction protocol will allow researchers to collect mites, store them in 95% ethanol, and subsequently extract sufficient DNA from single adults or eggs to provide diagnostic PCR products from both nuclear and mitochondrial DNA, yet leave the bodies intact for morphological analyses.

Molecular and field analyses of the fortuitous establishment of Lipolexis oregmae (Hymenoptera: Aphidiidae) in Jamaica as a natural enemy of the brown citrus aphid

Abstract The brown citrus aphid, Toxoptera citricida (Kirkaldy), is a serious pest of citrus because it efficiently transmits citrus tristeza closterovirus (CTV). The invasion of Jamaicas citrus by T. citricida resulted in serious economic losses and the importation and consideration was given to the establishment of Lipolexis oregmae Gahan (Hymenoptera: Aphidiidae) in a classical biological control program. Prior to introducing L. oregmae, we conducted a survey to determine the abundance of Lysiphlebus testaceipes Cresson (Hymenoptera: Aphidiidae) in T. citricida. A high-fidelity PCR analysis was conducted using Lysiphlebus- and Lipolexis-specific ITS2 rRNA primers on alcohol-preserved brown citrus aphids and the results were positive for both parasitoids. The fortuitous establishment of L. oregmae in Jamaica was surprising, and was confirmed by rearing adults from parasitized aphids and obtaining taxonomic confirmation based on morphology. In addition, its identity was confirmed molecularly by cloning and sequencing 16S, 12S, and COI sequences from the Florida colony of L. oregmae and from Jamaican specimens. The three DNA sequences were 100% identical for specimens from both sources. The phylogenetic analysis using 16S sequences indicated a close relationship of the Florida and Jamaica populations of L. oregmae with L. gracilis. During 2005, an island-wide survey of citrus in six parishes confirmed that both L. testaceipes and L. oregmae commonly parasitize T. citricida, but that L. oregmae was the more widely distributed and abundant parasitoid. The pathway by which L. oregmae might have entered Jamaica is discussed.

Genetic Characteristics of Bisexual and Female-Only Populations of Odontosema anastrephae (Hymenoptera: Figitidae)

ABSTRACT Odontosema anastrephae Borgmeier is a figitid parasitoid of Anastrepha fruit fly larvae infesting fallen fruit. It is of potential use in biological control as a complement to parasitoids that attack larvae infesting fruit still on the tree and to parasitoids that can only oviposit into larvae near the surface of the fruit, because Odontosema pursues larvae deep within the pulp. A newly discovered Mexican all-female (presumably thelytokous) population, provisionally referred to here as O. near anastrephae, appears to be morphologically indistinguishable from arrhenotokous individuals. Thelytokous reproduction can potentially lower costs in mass rearing facilities and increase parasitoid efficacy in the field. PCR amplification and sequencing of mitochondrial (COI) and nuclear (ITS2) genetic sequences suggested that these populations are genetically distinct, but no more so than often occurs among distinct populations within recognized species. In addition to the description of an all-female population of Odontosema, this study presents the first genetic sequence data for members of the genus Odontosema, enabling phylogenetic comparison between Odontosema and other figitid genera and the development of methods for the identification of Odontosema species by PCR. The implications of thelytoky for a cladistic definition of speciation, especially for newly diverging populations such as these, as well as the potential practical implications of our findings for fruit fly biological control, are discussed.

Real-Time PCR Reveals Endosymbiont Titer Fluctuations in Metaseiulus 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:/

Multiple Wolbachia strains in Apis mellifera capensis from South Africa

Eggs of the honeybee Apis mellifera capensis from South Africa were screened for Wolbachia using degenerate primers designed to amplify a segment of the wsp A gene sequences. This strategy resulted in the identification of two new strains (wCap-B2, and -A1) in addition to the one (wCap-B1) characterized earlier from A. m. capensis and A. m. scutellata. Strain-specific primers were designed and used to assay eggs from both A. m. capensis and A. m. scutellata. The wCap-B1 sequence was amplified consistently from both A. m. capensis and A. m. scutellata, but the wCap-B2 and -A1 sequences were amplified sporadically only from A. m. capensis. This indicates that the wCap-B1 strain could be present at a higher titer or that wCap-B2 and -A1 are present only in some individuals in the wider A. m. capensis population. The detection of these new Wolbachia strains suggests that additional investigations are required to determine the role of Wolbachia in the biology of A. m. capensis workers.ZusammenfassungArbeiterinnen der südafrikanischen Kapbiene sind in der Lage, Weibchen zu produzieren, während Arbeiterinnen aller anderen Subspezies der Honigbiene nur Männchen produzieren können. Dieses Merkmal erlaubt es Arbeiterinnen der Kapbiene, in andere Bienenvölker einzudringen und die Königin zu ersetzen, was letztendlich eine Reduktion der Honigproduktion zur Folge hat. Obwohl es inzwischen Erkenntnisse über die genetischen Grundlagen dieses Verhaltens gibt, kann nicht ausgeschlossen werden, dass hierbei auch Wolbachia-Bakterien eine Rolle spielen können. Frühere Untersuchungen mittels Universalprimern detektierten den Wolbachia-Stamm wCap-A1. Da aber erstens dieser Stamm sowohl in Kapals auch in Scutellata-Bienen gefunden wurde, und zweitens Universalprimer zwar Sequenzen der Mehrzahl aber nicht aller Wolbachia-Stämm amplifizieren können, entwickelten wir degenerierte Primer für die wsp A Gensequenzen aller A und B Referenzstämme (Tab. II), um zu sehen, ob in Kapbienen zusätzliche Stämme gefunden werden können. Die Amplifikation der wsp Sequenzen aus Eiern der Kapbiene mittels dieser degenerierten Primer ermöglichte die Identifizierung zweier zusätzlicher Stämme (wCap-B2 and −A1) (Abb. 1). Für diese wurden sodann spezifische Primer entwickelt (Tab. II), mit denen Eier aus verschiedenen Völkern untersucht werden konnten. Wir fanden, dass der wCap-B1 Stamm sowohl in Kapals auch in Scutellata-Völkern konstant präsent war, während die Stämme wCap-B2 und wCap-A1 vereinzelt in Kapbienen detektiert wurden (Tab. I). Es ist daher möglich, dass entweder der Titer für den wCap-B1 Stamm höher ist, oder dass die Stämme wCap-B2 und wCap-A1 nur in einzelnen Arbeiterinnen der Kapbiene vorkommen. Zusätzliche Untersuchungen sind notwendig, um der Frage nachzugehen, ob diese Wolbachia-Stämme im Verhalten der Arbeiterinnen der Kapbiene eine Rolle spielen.