A new technique for studying the stylet tracks of homopteran insects in hand-sectioned plant tissue using light or epifluorescence microscopy.

Homopteran insects, such as aphids, psyllids and scales, inject a proteinaceous salivary sheath into their host plant tissue during feeding. This sheath, also referred to as a stylet track, remains in the tissue after the stylets are withdrawn, and is useful for studying plant resistance to insects and plant virus transmission. We describe a new method for studying stylet tracks. Hand microtome sectioned plant material was fixed and cleared in ethanol. The stylet tracks were stained with acid fuchsin and counterstained with aniline blue or fast green. The acid fuchsin stained stylet tracks were pink to red under light microscopy, and orange under TRITC epifluorescence. Stylet tracks in unstained sections autofluoresced under DAPI epifluorescence. This new technique is significantly faster and less complex than previous techniques, and permitted visualization of stylet tracks with light or epifluorescence microscopy within 1 hr of collecting fresh plant material. The technique was also applicable to a broad range of homopterans and plant taxa and provided excellent photomicrographs.

Secondary endosymbionts of psyllids have been acquired multiple times.

Previous studies have established that psyllids (Hemiptera, Psylloidea) contain primary endosymbionts, designated as Carsonella ruddii, which cospeciate with the psyllid host. This association appears to be the consequence of a single infection of a psyllid ancestor with a bacterium. Some psyllids may have additional secondary (S-) endosymbionts. We have cloned and sequenced the 16S-23S ribosomal RNA genes of seven representative psyllid S-endosymbionts. Comparison of the S-endosymbiont phylogenetic trees with those of C. ruddii indicates a lack of congruence, a finding consistent with multiple infections of psyllids with different precursors of the S-endosymbionts and/or possible horizontal transmission. Additional comparisons indicate that the S-endosymbionts are related to members of the Enterobacteriaceae as well as to several other endosymbionts and insect-associated bacteria.

Cospeciation of psyllids and their primary prokaryotic endosymbionts.

Psyllids are plant sap-feeding insects that harbor prokaryotic endosymbionts in specialized cells within the body cavity. Four-kilobase DNA fragments containing 16S and 23S ribosomal DNA (rDNA) were amplified from the primary (P) endosymbiont of 32 species of psyllids representing three psyllid families and eight subfamilies. In addition, 0.54-kb fragments of the psyllid nuclear gene wingless were also amplified from 26 species. Phylogenetic trees derived from 16S-23S rDNA and from the host wingless gene are very similar, and tests of compatibility of the data sets show no significant conflict between host and endosymbiont phylogenies. This result is consistent with a single infection of a shared psyllid ancestor and subsequent cospeciation of the host and the endosymbiont. In addition, the phylogenies based on DNA sequences generally agreed with psyllid taxonomy based on morphology. The 3' end of the 16S rDNA of the P endosymbionts differs from that of other members of the domain Bacteria in the lack of a sequence complementary to the mRNA ribosome binding site. The rate of sequence change in the 16S-23S rDNA of the psyllid P endosymbiont was considerably higher than that of other bacteria, including other fast-evolving insect endosymbionts. The lineage consisting of the P endosymbionts of psyllids was given the designation Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.).