Kevin A. Shufran

Mitochondrial DNA sequence divergence among Schizaphis graminum (Hemiptera: Aphididae) clones from cultivated and non-cultivated hosts: haplotype and host associations.

A 1.0 kb region of the mitochondrial cytochrome oxidase subunit I gene from the greenbug aphid, Schizaphis graminum (Rondani), was sequenced for 24 field collected clones from non-cultivated and cultivated hosts. Maximum likelihood, maximum parsimony and neighbour-joining phylogenies were estimated for these clones, plus 12 previously sequenced clones. All three tests produced trees with identical topologies and confirmed the presence of three clades within S. graminum. Clones showed no relationship between biotype and mtDNA haplotype. At least one biotype was found in all three clades, suggesting exchange among clades of genetic material conditioning for crop virulence, or the sharing of a common ancestor. However, there was a relationship between host and haplotype. Clade 1 was the most homogeneous and contained 12 of 16 clones collected from cultivated hosts and five of the six collected from johnsongrass, Sorghum halepense, a congener of cultivated sorghum, S. bicolor. Four of the six clones collected from Agropyron spp. were found in clade 2. Clade 3 contained two clones from wheat, Triticum aestivum, and four from non-cultivated hosts other than Agropyron spp. A partitioning of populations by mtDNA haplotype and host suggests the occurrence of host adapted races in Schizaphis graminum.

SPIDER PREDATION: SPECIES-SPECIFIC IDENTIFICATION OF GUT CONTENTS BY POLYMERASE CHAIN REACTION

Abstract We extend detection of arthropod predator gut contents by polymerase chain reaction (PCR), heretofore restricted to insect predators, to spiders. Single individuals of the corn lead aphid, Rhopalosiphum maidis, were detected in the guts of spiderlings of Oxyopes salticus up to 12 h after feeding; individuals of the congeneric bird cherry oat aphid, R. padi, were not detected. Unfed O. salticus and Misumenops sp. were also negative.

Dna Barcoding to Identify All Life Stages of Holocyclic Cereal Aphids (Hemiptera: Aphididae) on Wheat and Other Poaceae

ABSTRACT The cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA was sequenced in eight holocyclic monoecious aphids (Hemiptera: Aphididae) that occur on wheat, Triticum aestivum L.; barley, Hordeum vulgare L.; oat, Avena sativa L.; and sorghum, Sorghum bicolor (L.) Moench in the United States. The first 640 bp of the 5′ end were considered as a DNA barcoding technique for species identification. DNA barcoding successfully differentiated Schizaphis graminum (Rondani), Diruaphis noxia (Kurdjumov), Diruaphis tritici (Gillette), Diruaphis frequens (Walker), Diruaphis mexicana (McVicar Baker), Sipha flava (Forbes), Sipha elegans del Guercio, and Sitobion avenae (F.). In addition to the above-mentioned monoecious species, the common cereal aphids Rhopalosiphum padi (L.) and Rhopalosiphum maidis (Fitch) were included and successfully differentiated. DNA barcoding is a reliable alternative to traditional morphology in the identification of cereal aphids and their various life stages and morphs, including eggs. The application of DNA barcoding to aphid eggs found on grasses will be able to confirm whether D. noxia is now reproducing sexually in the United States after 20 yr of asexual reproduction.

Detection and differentiation of parasitoids (Hymenoptera: Aphidiidae and Aphelinidae) of the brown citrus aphid (Homoptera: Aphididae): Species-specific polymerase chain reaction amplification of 18S rDNA

Abstract The brown citrus aphid, Toxoptera citricida (Kirkaldy), is an important pest of Florida citriculture because it causes feeding damage to citrus and vectors citrus tristeza virus. Parasitoids recovered from brown citrus aphids in Florida include Lysiphlebus testaceipes (Cresson), Lipolexis scutellaris Mackauer, and Aphelinus gossypii Timberlake. Monitoring the levels of parasitism caused by each species is difficult because the parasitoids must be reared out or dissected from the aphid hosts. A simple and quick molecular approach was developed to detect and distinguish these parasitoids developing within the host aphid. Total genomic DNA was extracted from the brown citrus aphid and each of the three parasitoids and the 18S rRNA gene of each species was amplified by polymerase chain reaction (PCR). The PCR products were sequenced to obtain complete gene sequences for each species. The variable regions V2 of the genes were used to design species-specific primers for detecting and differentiating the three parasitoids. The species-specific PCR amplifications discriminated the parasitoid DNAs from each other and from the host DNA. Detection of L. testaceipes DNA within the host aphid was possible in 8% of samples during the first 2 h after parasitoid oviposition; in 66% of samples after 24 h; in 94% of samples after 48 h; and in 100% of samples after 72 h. The PCR approach described in this study provides earlier and more precise detection of parasitism and determination of species than rearing or dissection methods.

Genetic Similarities Among Geographic Isolates of Lysiphlebus testaceipes (Hymenoptera: Aphidiidae) Differing in Cold Temperature Tolerances

Abstract Lysiphlebus testaceipes (Cresson) is a solitary endoparasitoid of aphids and is the primary parasitoid attacking cereal aphids in the Great Plains, especially Schizaphis graminum (Rondani). In a previous study, it was found that a Lincoln, NE, isolate of L. testaceipes had a much higher survivorship at cold temperatures than isolates from Stillwater, OK, and Corpus Christi, TX. This suggested that the Nebraska isolate was locally adapted to the northern environment and perhaps genetically divergent from southern populations. We tested for genetic differentiation of the above isolates by sequencing portions of the COI and 16S mtDNA genes. We also examined a Florida isolate reared from Toxoptera citricida (Kirkaldy) and L. fabarum Marshall as an outgroup. The Great Plains isolates (Nebraska, Oklahoma, and Texas) were homogeneous with 0% and 0–0.2% sequence divergence in the COI and 16S gene fragments, respectively. The Florida isolate differed from the Great Plains isolates in nucleotide sequence by 1.4% (COI) and 0.5–0.7% (16S). Phylogenetic analysis placed the Florida isolate of L. testaceipes basal to the Great Plains isolates with L. fabarum, suggesting a possible species complex within L. testaceipes.

Molecular Markers for Identification of the Hyperparasitoids Dendrocerus carpenteri and Alloxysta xanthopsis in Lysiphlebus testaceipes Parasitizing Cereal Aphids

Polymerase chain reaction (PCR)-based molecular markers have been developed to detect the presence of primary parasitoids in cereal aphids and used to estimate primary parasitism rates. However, the presence of secondary parasitoids (hyperparasitoids) may lead to underestimates of primary parasitism rates based on PCR markers. This is because even though they kill the primary parasitoid, it’s DNA can still be amplified, leading to an erroneous interpretation of a positive result. Another issue with secondary parasitoids is that adults are extremely difficult to identify using morphological characters. Therefore, we developed species-specific molecular markers to detect hyperparasitoids. A 16S ribosomal RNA mitochondrial gene fragment was amplified by PCR and sequenced from two secondary parasitoid species, Dendrocerus carpenteri (Curtis) (Hymenoptera: Megaspilidae) and Alloxysta xanthopsis (Ashmead) (Hymenoptera: Charipidae), four geographic isolates of the primary parasitoid, Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae), and six aphid species common to cereal crops. Species-specific PCR primers were designed for each insect on the basis of these 16S rRNA gene sequences. Amplification of template DNA, followed by agarose gel electrophoresis, successfully distinguished D. carpenteri and A. xanthopsis from all four isolates of L. testaceipes and all six cereal aphid species in this laboratory test.

Host Race Evolution in Schizaphis graminum (Hemiptera: Aphididae): Nuclear Dna Sequences

ABSTRACT The greenbug aphid, Schizaphis graminum (Rondani) was introduced into the United States in the late 1880s, and quickly was established as a pest of wheat, oat, and barley. Sorghum was also a host, but it was not until 1968 that greenbug became a serious pest of it as well. The most effective control method is the planting of resistant varieties; however, the occurrence of greenbug biotypes has hampered the development and use of plant resistance as a management technique. Until the 1990s, the evolutionary status of greenbug biotypes was obscure. Four mtDNA cytochrome oxidase subunit I (COI) haplotypes were previously identified, suggesting that S. graminum sensu lato was comprised of host-adapted races. To elucidate the current evolutionary and taxonomic status of the greenbug and its biotypes, two nuclear genes and introns were sequenced; cytochrome c (CytC) and elongation factor 1-&agr; (EF1-&agr;). Phylogenetic analysis of CytC sequences were in complete agreement with COI sequences and demonstrated three distinct evolutionary lineages in S. graminum. EF1-&agr; DNA sequences were in partial agreement with COI and CytC sequences, and demonstrated two distinct evolutionary lineages. Host-adapted races in greenbug are sympatric and appear reproductively isolated. Agricultural biotypes in S. graminum likely arose by genetic recombination via meiosis during sexual reproduction within host-races. The 1968 greenbug outbreak on sorghum was the result of the introduction of a host race adapted to sorghum, and not selection by host resistance genes in crops.

Variation to Cause Host Injury Between Russian Wheat Aphid (Homoptera: Aphididae) Clones Virulent to Dn4 Wheat

Abstract Biotypes are infraspecific classifications based on biological rather than morphological characteristics. Cereal aphids are managed primarily by host plant resistance, and they often develop biotypes that injure or kill previously resistant plants. Although molecular genetic variation within aphid biotypes has been well documented, little is known about phenotypic variation, especially virulence or the biotype’s ability to cause injury to cultivars with specific resistance genes. Five clones (single maternal lineages) of Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), determined to be injurious to wheat, Triticum aestivum L., with the Dn4 gene, were evaluated on resistant and susceptible wheat and barley, Hordeum vulgare L., for their ability to cause chlorosis, reduction in plant height, and reduction in shoot dry weight. Variation to cause injury on resistant ‘Halt’ wheat, susceptible ‘Jagger’ wheat, and resistant ‘STARS-9301B’ barley was found among the Dn4 virulent clones. One clone caused up to 30.0 and 59.5% more reduction in plant height and shoot dry weight, respectively, on resistant Halt than other clones. It also caused up to 29.9 and 55.5% more reduction in plant height and shoot dry weight, respectively, on susceptible Jagger wheat. Although STARS-9301B barley exhibited an equal resistant response to feeding by all five clones based on chlorosis, two clones caused ≈20% more reduction in plant height and shoot dry weight than three other clones. The most injurious clones on wheat were not the most injurious clones on barley. This is the first report of variation to cause varying degrees of plant damage within an aphid biotype virulent to a single host resistance gene. A single aphid clone may not accurately represent the true virulent nature of a biotype population in the field.

Analysis of rILERS, an isoleucyl-tRNA synthetase gene associated with mupirocin production by Pseudomonas fluorescens NCIMB 10586.

Some strains of Pseudomonas fluorescens produce the antibiotic mupirocin, which functions as a competitive inhibitor of isoleucyl-tRNA synthetase (ILERS). Mupirocin-producing strains of P. fluorescens must overcome the inhibitory effects of the antibiotic to avoid self-suicide. However, it is not clear how P. fluorescens protects itself from the toxic effects of mupirocin. In this report, we describe a second gene encoding isoleucyl-tRNA synthetase ( rILERS ) in P. fluorescens that is associated with the mupirocin biosynthetic gene cluster. Random mutagenesis of the mupirocin-producing strain, P. fluorescens 10586, resulted in a mupirocin-defective mutant disrupted in a region with similarity to ILERS, the target site for mupirocin. The ILERS gene described in the present study was sequenced and shown to be encoded by a 3093 bp ORF, which is 264 bp larger than the ILERS gene previously identified in P. fluorescens 10586. rILERS from P. fluorescens is most closely related to prokaryotic or eukaryotic sources of ILERS that are resistant to mupirocin. Interestingly, the relatedness between rILERS and the ILERS previously described in P. fluorescens 10586 was low (24% similarity), which indicates that P. fluorescens contains two isoforms of isoleucyl-tRNA synthetase.