Polymerase chain reaction (PCR) products corresponding to 803 bp of the cytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNA COI-II) were deduced to consist of multiple haplotypes in three Sitobion species. We investigated the molecular basis of these observations. PCR products were cloned, and six clones from one individual per species were sequenced. In each individual, one sequence was found commonly, but also two or three divergent sequences were seen. The divergent sequences were shown to be nonmitochondrial by sequencing from purified mtDNA and Southern blotting experiments. All seven nonmitochondrial clones sequenced to completion were unique. Nonmitochondrial sequences have a high proportion of unique sites, and very few characters are shared between nonmitochondrial clones to the exclusion of mtDNA. From these data, we infer that fragments of mtDNA have been transposed separately (probably into aphid chromosomes), at a frequency only known to be equalled in humans. The transposition phenomenon appears to occur infrequently or not at all in closely related genera and other aphids investigated. Patterns of nucleotide substitution in mtDNA inferred over a parsimony tree are very different from those in transposed sequences. Compared with mtDNA, nonmitochondrial sequences have less codon position bias, more even exchanges between A, G, C and T, and a higher proportion of nonsynonymous replacements. Although these data are consistent with the transposed sequences being under less constraint than mtDNA, changes in the nonmitochondrial sequences are not random: there remains significant position bias, and probable excesses of synonymous replacements and of conservative inferred amino acid replacements. We conclude that a proportion of the inferred change in the nonmitochondrial sequences occurred before transposition. We believe that Sitobion aphids (and other species exhibiting mtDNA transposition) may be important for studying the molecular evolution of mtDNA and pseudogenes. However, our data highlight the need to establish the true evolutionary relationships between sequences in comparative investigations.

Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae).

Coccinellids have been widely used in biological control for over a century, and the methods for using these predators have remained virtually unchanged. The causes for the relatively low rates of establishment of coccinellids in importation biological control have not been examined for most species. Augmentative releases of several coccinellid species are well documented and effective; however, ineffective species continue to be used because of ease of collection. For most agricultural systems, conservation techniques for coccinellids are lacking, even though they are abundant in these habitats. Evaluation techniques are available, but quantitative assessments of the efficacy of coccinellids have not been done for most species in most agricultural crops. Greater emphasis is needed on evaluation, predator specificity, understanding colonization of new environments, and assessment of community-level interactions to maximize the use of coccinellids in biological control.

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Predaceous Coccinellidae in biological control.

Aphids, which constitute one of the most important groups of agricultural pests, ingest nutrients from sieve tubes, the photoassimilate transport conduits in plants. Aphids are able to successfully puncture sieve tubes with their piercing mouthparts (stylets) and ingest phloem sap without eliciting the sieve tubes' normal occlusion response to injury. Occlusion mechanisms are calcium-triggered and may be prevented by chemical constituents in aphid saliva injected into sieve tubes before and during feeding. We recorded aphid feeding behavior with the electrical penetration graph (EPG) technique and then experimentally induced sieve tube plugging. Initiation of sieve tube occlusion caused a change in aphid behavior from phloem sap ingestion to secretion of watery saliva. Direct proof of "unplugging" properties of aphid saliva was provided by the effect of aphid saliva on forisomes. Forisomes are proteinaceous inclusions in sieve tubes of legumes that show calcium-regulated changes in conformation between a contracted state (below calcium threshold) that does not occlude the sieve tubes and a dispersed state (above calcium threshold) that occludes the sieve tubes. We demonstrated in vitro that aphid saliva induces dispersed forisomes to revert back to the nonplugging contracted state. Labeling Western-blotted saliva proteins with 45Ca2+ or ruthenium red inferred the presence of calcium-binding domains. These results demonstrate that aphid saliva has the ability to prevent sieve tube plugging by molecular interactions between salivary proteins and calcium. This provides aphids with access to a continuous flow of phloem sap and is a critical adaptation instrumental in the evolutionary success of aphids.

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Molecular sabotage of plant defense by aphid saliva

Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the gypsy moth (Lymantria dispar L.), whose gut is highly alkaline, by using traditional culturing and culture-independent methods. We also examined the effects of diet on microbial composition. Analysis of individual third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the γ-Proteobacteria and low G+C gram-positive divisions and that the cultured members represented more than half of the phylotypes identified. Less frequently detected taxa included members of the α-Proteobacterium, Actinobacterium, and Cytophaga/Flexibacter/Bacteroides divisions. The 16S rRNA gene sequences from 7 of the 15 cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that an Enterococcus species and culture-independent analysis indicated that an Entbacter sp. were both present in all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among individual insects. These results provide the first comprehensive description of the microbial diversity of a lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut bacterial community.

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Census of the Bacterial Community of the Gypsy Moth Larval Midgut by Using Culturing and Culture-Independent Methods

Aphid salivation in sieve elements and phloem sap ingestion were linked to waveforms in the Electrical Penetration Graph (EPG). Non-viruliferous Rhopalosiphum padi (L.) (Hemiptera, Aphididae) on barley yellow dwarf virus (BYDV) infected wheat could acquire the virus, which was used as an indication for phloem sap ingestion, whereas virus inoculation by viruliferous aphids on healthy plants was associated with salivation in sieve elements or other phloem cells. Probing was monitored and the waveforms recorded were related to ELISA results of test plants. The EPG patterns A, B, and C are indicative of the stylet pathway phase, whereas patterns E1 and E2 reflect the phloem (sieve element) phase with an unknown activity (E1) or with ingestion and concurrent salivation (E2). Aphids showing pathway and E1 rarely acquired virus, suggesting that little or no phloem sap ingestion can occur during these patterns, whereas those showing additionally pattern E2 did so substantially, indicating phloem sap ingestion. The main pattern related to virus inoculation was E1, although some aphids were able to inoculate plants during pathway. Pattern El clearly reflects the most important salivation into sieve elements. Pattern E2 had no clear contribution to virus inoculation, supporting the present hypothesis that during this pattern the saliva is mixed with the phloem sap in the single canal at the stylet tips and ingested immediately, without reaching the plant tissue. Sustained sap ingestion did not affect virus inoculation. So, BYDV inoculation mainly occurs during the first period of a sieve element puncture which is always formed by E1. Implications on persistent virus transmission are discussed.

Aphid activities during sieve element punctures.

Aphids: Their Biology, Natural Enemies and Control Edited by A. K. Minks and P. Harrewijin. pp. 312. In World Crop Pests, vol. 2C. Series Editor-in-Chief W. Helle. xvi + 312 pp. Amsterdam: Elsevier, 1989. Hard cover ISBN 0-444-42799-6

M. J. Chacko

Papers

Aphids: Their Biology, Natural Enemies and Control Edited by A. K. Minks and P. Harrewijin. pp. 312. In World Crop Pests, vol. 2C. Series Editor-in-Chief W. Helle. xvi + 312 pp. Amsterdam: Elsevier, 1989. Hard cover ISBN 0-444-42799-6