Application of 16S rDNA-DGGE and Plate Culture to Characterization of Bacterial Communities Associated with the Sawfly, Acantholyda erythrocephala (Hymenoptera, Pamphiliidae)

Abstract

Culture-based analysis was employed in parallel with PCR amplification of 16S rDNA, coupled with denaturing gradient gel electrophoresis (DGGE), to profile bacterial species associated with different developmental stages of the pine false webworm (PFW), Acantholyda erythrocephala, a sawfly pest responsible for incidents of severe defoliation in commercially important tree plantations in North America. Culture-based analysis revealed that Pseudomonas spp. along with Bacillus sphaericus and Arthrobacter sp. were the predominant components of the microflora of the internal organs and identified life-stage-specific associations including Photorhabdus temperata with egg and larval samples and a Janthinobacterium sp. with eonymphs. PCR-DGGE confirmed the predominance of Pseudomonas spp. and B. sphaericus in the majority of samples but did not detect Arthrobacter sp., P. temperate, or Janthinobacterium sp. In contrast, DGGE revealed the presence of a Chryseobacterium sp. as the predominant component of the PFW micoflora at all life stages, with the exception of adults. This species had been infrequently cultured, at low levels, from a limited number of samples and the existence of a possible relationship between this bacterium and the PFW had gone unnoticed using the culture-based approach. Our findings highlight the advantages of applying a dual approach to the study of microbe-insect associations and demonstrate that the benefits of one system can be used to overcome some of the limitations of the other.

Bacteria Associated with Copestylum (Diptera, Syrphidae) Larvae and Their Cactus Host Isolatocereus dumortieri

“…An example is the absence of bacteria from the genera Spiroplasma , Wolbachia and Bacillus , frequently found in association with other insects [27]; [34]; [35]; [36]; [30]; [29], but absent from both C. latum and C. limbipenne larva. The acetic acid bacteria Acetobacter , which has recently been described as a newly emerging symbiont of insects, is mostly absent from the CLAT and CLIM larvae, although it is relatively abundant in the cactus tissue colonized by CLAT.…”

The microbial flora of Aphis gossypii: Patterns across host plants and geographical space

“…Sequence band 2 is closely related to Pseudomonas. These soil microbes are common members of insect microflora (Zahner et al, 2008). The sequence corresponding to band 3 is closely related to a species of Acinetobacter.…”

The tsetse fly Glossina fuscipes fuscipes (Diptera: Glossina) harbours a surprising diversity of bacteria other than symbionts

“…Previous studies on insect microbiota have highlighted the need to combine culture dependent and culture independent methods since DNA based techniques also have limits (Forney et al 2004;Wintzingerode et al 1997;Baker et al 2003). For example, several or most of the bacterial species isolated on culture plates in insect microbiota screens were not identified with the culture independent methods used, which in theory should detect all bacteria present (Lindh et al 2005;Zahner et al 2008).…”