Richard L. Roehrdanz

An improved primer for PCR amplification of mitochondrial DNA in a variety of insect species

A PCR primer from the mitochondrial COi gene is described that enhances the amplification of the COI‐COJI region of insect mtDNA. When used in conjunction with a primer from the COII gene identified by R. Crozier, a 1600–1700 bp segment is amplified in nine species of insects representing the orders Lepidop‐tera, Diptera, Coleoptera and Hymenoptera.

Genome of the Asian longhorned beetle (Anoplophora glabripennis), a globally significant invasive species, reveals key functional and evolutionary innovations at the beetle-plant interface

BackgroundRelatively little is known about the genomic basis and evolution of wood-feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle.ResultsThe Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates.ConclusionsAmplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants.

Amplification of complete insect mitochondrial genome in two easy pieces

The entire insect mitochondrial genome has been amplified in just two PCR reactions. ‘Universal’ insect primers and techniques to optimize amplification of long PCR products were used to obtain segments of the mitochondrial genomes of Helicoverpa zea and Heliothis virescens (Lepidoptera) ranging in size from 3.7 kb to 14 kb or about 25% to over 85% of a typical 16 kb mtDNA. Overlapping pairs of fragments contain the complete mtDNA sequence.

Intraspecific genetic variability in mitochondrial DNA of the screwworm fly (Cochliomyia hominivorax).

Mitochondrial DNA variability has been analyzed in the primary screwworm fly (Cochliomyia hominivorax) using restriction endonuclease fragment patterns and restriction site mapping. A total of 30 different screwworm lines originating from Texas to Costa Rica and the Island of Jamaica was examined using 15 restriction endonucleases. Eleven of the restriction enzymes revealed polymorphism and yielded 16 mitochondrial genotypes or haplotypes. Two of the haplotypes were widely distributed, haplotype 1 being found scattered across southern Mexico and haplotype 2 along the west coast of Mexico. Haplotype 1 also appeared paired with several other haplotypes in mixed lines that were most likely the result of collecting an egg mass to which more than one female had contributed or to some form of contamination by haplotype 1 after introduction into the laboratory. These lines became fixed before single insects were examined and thus it is impossible to rule out heteroplasmy. The other 14 haplotypes were found in only a single locale and 12 of these were found in only one line. The average sequence diversity among 27 mainland lines was about 0.5%. The two Jamaican lines and one east coast mainland line differed from the others by >2%. The pattern of geographical distribution, a small number of apparently recurring haplotypes and a substantial number (75%) of the haplotypes unique, bears similarities to patterns observed in other insects such asDrosophila. The high frequency of unique genotypes in southern Mexico suggests a population with a very reduced gene flow, which may have had a positive effect on the sterile male release control program.

Genetic variation in geographical populations of western and Mexican corn rootworm

Genetic variation in the nuclear rDNA ITS1 region of western corn rootworm, Diabrotica virgifera virgifera (WCR), and Mexican corn rootworm, D. v. zeae (MCR) was studied. Two sites were detected which differentiated WCR and MCR in the 642‐base sequence. Polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) analysis of the first internal transcribed spacer region (ITS1) sequence revealed no variation within or among the twelve WCR and two MCR populations. PCR‐RFLP of 75% of the mitochondrial DNA genome detected one significant polymorphic site out of the approximately 190 restriction sizes observed in WCR. The polymorphism did not differentiate geographical populations of WCR and is not diagnostic for the subspecies. The low levels of variation observed in WCR suggests either high levels of gene flow or a recent geographical expansion from a relatively small base. Gene flow would facilitate the rapid spread of traits that could compromise control programmes, such as insecticide resistance or behavioural modifications. The minimal genetic differentiation between WCR and MCR raises questions about the evolutionary history of these subspecies and how the distinct phenotypes are maintained.

Genetic Differentiation of Southeastern Boll Weevil and Thurberia Weevil Populations of Anthonomus grandis (Coleoptera: Curculionidae) Using Mitochondrial DNA

Abstract The southeastern boll weevil, the Mexican boll weevil, and the thurberia weevil are considered to be morphologically similar but behaviorally different variants of the same species, Anthonomus grandis Boheman. A polymerase chain reaction (PCR)-amplified 9.2-kb section of the mitochondrial DNA was cleaved with restriction enzymes. RFLPs of weevils from three cotton growing locations in Texas and one in northeastern Mexico were compared with thurberia weevil from three sites in Arizona. Six haplotypes were observed in the Texas/Mexico collections and 12 haplotypes were found among the thurberia weevil. There were no shared haplotypes between these two groups. Polymorphism was observed within the weevil types. The three thurberia weevil locations exhibit some geographic isolation and exhibit differences in both the haplotypes present and the relative frequencies of the haplotypes. Only one haplotype was recovered at all three Arizona sites. The Texas/Mexico samples showed less genetic variability with the northern most site having the lowest polymorphism. 52/53 of these weevils appear to be genetically southeastern boll weevil. Two haplotypes were shared by all four of these populations and comprised 72% of the insects examined. The range of genetic distances between haplotypes was <0.001–0.022. The Mexican boll weevil was not explicitly examined; however, three individuals were discovered that appear to represent a genetically distinct third population. One was from Mexico and the other two were from a thurberia weevil site. These three individuals may represent the Mexican boll weevil. The results include apparent diagnostic restriction fragment differences between the thurberia weevil and the southeastern boll weevil that could be used to help determine whether future weevils found in Arizona or California cotton are thurberia weevil, southeastern boll weevil, or another population of weevils.

Reproductive compatibility and mitochondrial DNA restriction site analysis of New World screwworm, Cochliomyia hominivorax, from North Africa and Central America

Abstract. The reproductive compatibility of New World screwworms, Cochliomyia hominivorax (Coquerel), from North Africa and a strain being mass produced for the Mexican eradication programme was examined to assess the feasibility of using flies from the Mexican screwworm mass production facility for a sterile insect technique eradication programme in North Africa. Males from the production strain mated randomly with females from North Africa and from the production strain when both were present. Neither strain of males discriminated between cuticular extracts of North African and production strain females containing a contact sex pheromone. Interstrain crosses between North African flies and production flies were fertile and produced fertile progeny. Chromosome morphology did not differ significantly between the two strains and homologue pairing was normal in hybrid meiotic and polytene nuclei. Mitochondrial DNA restriction site analyses indicated that the genetic divergence of the North African strain from Mexican and Central American strains was within the range of the diversity observed in Central American, Mexican and Caribbean populations. Test results indicate that New World screwworms from North Africa are reproductively compatible with the strain currently being mass produced in Mexico. Mating barriers should not impede the progress of an eradication programme using the sterile insect technique in North Africa with sterile screwworms from the Mexican mass production facility.

Histone and ribosomal RNA repetitive gene clusters of the boll weevil are linked in a tandem array

Histones are the major protein component of chromatin structure. The histone family is made up of a quintet of proteins, four core histones (H2A, H2B, H3 & H4) and the linker histones (H1). Spacers are found between the coding regions. Among insects this quintet of genes is usually clustered and the clusters are tandemly repeated. Ribosomal DNA contains a cluster of the rRNA sequences 18S, 5.8S and 28S. The rRNA genes are separated by the spacers ITS1, ITS2 and IGS. This cluster is also tandemly repeated. We found that the ribosomal RNA repeat unit of at least two species of Anthonomine weevils, Anthonomus grandis and Anthonomus texanus (Coleoptera: Curculionidae), is interspersed with a block containing the histone gene quintet. The histone genes are situated between the rRNA 18S and 28S genes in what is known as the intergenic spacer region (IGS). The complete reiterated Anthonomus grandis histone‐ribosomal sequence is 16 248 bp.

Mitochondrial DNA and ITS1 Differentiation in Geographical Populations of Northern Corn Rootworm, Diabrotica barberi (Coleoptera: Chrysomelidae): Identification of Distinct Genetic Populations

Abstract Genetic variation of mitochondrial DNA (mtDNA) and the nuclear ribosomal spacer, ITS1, in local and dispersed geographical populations of northern corn rootworm, Diabrotica barberi Smith & Lawrence was examined. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for mtDNA and DNA sequencing plus PCR-RFLP analysis was used for ITS1. Insects were collected in 10 states ranging from Pennsylvania to the Great Plains. Sequencing of the ITS1 amplicon revealed three potential polymorphic sites, one of which altered a restriction site for the restriction enzyme BclI. PCR-RFLP analysis with BclI detected three genotypes. Many beetles had heterogeneity at the nucleotide site recognized by BclI, which was supported by DNA sequence data. There appears to be a phylogeographic pattern of ITS1 genetic diversity. Eastern populations were homogeneous for one genotype, populations from central and northern locations had two genotypes, and western populations were composed of all three genotypes. The mtDNA had 58 haplotypes that displayed a strong east-west geographical partition. The region of overlap occurred in a few counties of east-central Illinois. Populations to the east had less variability than those to the west. A network of restriction site changes and trees based on genetic distance measurements of the mtDNA produced two distinct clades. One clade contained all the eastern haplotypes along with a group of haplotypes from the northern Great Plains. The other clade included the remaining western haplotypes. Possible reasons for this population structure including expansion from different glacial relicts, historic host plant differences, and endosymbiont driven reproductive incompatibilities are discussed.

Multiplex Polymerase Chain Reaction Method for Differentiating Western and Northern Corn Rootworm Larvae (Coleoptera: Chrysomelidae)

Western corn rootworm, Diabrotica virgifera virgifera LeConte, and northern corn rootworm, D. barberi Smith and Lawrence, are sympatric species and serious pests of corn cultivation in North America. Comparison of nucleotide sequence of mitochondrial cytochrome oxidase I and II was used to design polymerase chain reaction (PCR) primers that discriminate immature stages of the two species based on differences in amplicon size. Multiplex PCR can be used to give a positive test for each species in a single amplification reaction. This provides a method to identify field caught larvae and facilitates investigations of larval interaction and competition between the species.