Isolation and identification of bacteria associated with adult laboratory Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae).

From the guts of new and old colonies (female and male) of Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae), we identified a total of 18 different bacterial species belonging to the family Enterobacteriaceae, Pseudomonadaceae, Vibrionaceae, Micrococcaceae, Deinococcacea, Bacillaceae, and the genus Listeria. Enterobacter, Providencia, Serratia, and Staphylococcus spp. were the most frequently isolated genera, with Citrobacter, Streptococcus, Aerococcus, and Listeria found less frequently. We found Bacillus cereus, Enterobacter sakazakii, Providencia stuartii, and Pseudomonas aeruginosa only in the new colony, Aeromonas hydrophila and Klebsiella pneumoniae spp. pneumoniae only in the old colony. We also studied resistance/sensitivity to 12 antibiotics for six bacterial isolates such as Enterobacter cloacae, E. sakazakii, K. pneumoniae spp., Providencia rettgeri, P. aeruginosa, and Bacillus cereus. Isolates on the whole were resistant to penicillin and ampicillin (five of six isolates) and sensitive to rifampin and streptomycin (six of six isolates). Antibiotic resistance profiles might be useful characteristics for distinguishing among species and strains of these bacteria, probably having ecological significance with respect to intra- and inter-specific competition within host cadavers, and could have implications for the utility of these organisms for biological control, including the alternative control strategy, paratransgenesis.

Variation in the Bemisia tabaci s. l. species complex (Hemiptera: Aleyrodidae) and its natural enemies leading to successful biological control of Bemisia biotype B in the USA.

Parasitoids of the Bemisia tabaci (Gennadius) species complex collected in Spain and Thailand were evaluated as biological control agents of B. tabaci biotype B in cole crops in Texas, USA. Parasitoids were identified by morphological and RAPD-PCR analyses. The most abundant parasitoid from Spain was Eretmocerus mundus Mercet with apparent field parasitism of 39-44%. In Thailand, Encarsia formosa Gahan, E. transvena Timberlake, E. adrianae Lopez-Avila, Eretmocerus sp. 1 and sp. 2 emerged, with apparent field parasitism of 1-65%. Identification and molecular classification of B. tabaci associated with parasitoid collections and in the release site in Texas were accomplished using morphological traits and nucleotide sequence comparison of the mitochondrial cytochrome oxidase I gene (COI) (700-720 bp). Collections of B. tabaci from Thailand grouped separately from B types from Arizona and Florida and the target B type from Texas, USA, a cluster from India, and other New World B. tabaci. The Spanish B. tabaci host of E. mundus which was laboratory and field-tested to achieve biological control of the B type was most closely related to non-B type B. tabaci populations from Spain and Sudan, the latter which formed a second group within the larger clade that also contained the B type cluster. Laboratory tests indicated that E. mundus from Spain parasitized more B. tabaci type B than did Eretmocerus spp. native to Texas and other exotic parasitoids evaluated. Eretmocerus mundus from Spain also successfully parasitized B. tabaci type B when field-released in a 0.94 million ha test area in Texas, and has significantly enhanced control of B. tabaci type B in California, USA. In contrast, parasitoids from Thailand failed to establish in the field in Texas, collectively suggesting a positive correlation between the centres of diversity of compatible parasitoid-host complexes.

Improved cultivation systems for isolation of the colorado potato beetle spiroplasma.

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.

Effects of microbial floras on the distributions of five domestic Drosophila species across fruit resources.

The distributions of five Drosophila species and four components of the microflora have been compared across a total of 48 traps baited with four different fruit and vegetable substrates in two domestic compost heaps in Canberra (Australia). Large and consistent differences are found, both among the Drosophila and among the microbial classes, in their distributions across traps baited with different substrates. Moreover the distribution of each Drosophila species shows a unique set of strong associations with the microbial distributions. Thus the distributions of both D. simulans and D. melanogaster are found to be strongly negatively correlated with the abundance of bacteria while D. simulans is also strongly positively correlated with the titre of fermenter yeasts. D. immigrans is strongly positively correlated both with bacteria and with non-fermenter yeasts. D. hydei is positively correlated with nonfermentery yeasts and D. busckii is negatively correlated with fermenter yeasts. Moulds are the only microbial class not consistently associated with the distribution of any of the Drosophila species. The correlations with the other microbial classes are sufficient to explain the majority of the abundance differences of the Drosophila species among the trap types. It is therefore proposed that the clear partitioning of the fruit resources by the Drosophila is due to their differing primary interactions with the microflora.

Analysis of spatial and temporal variation in the community structure of yeasts associated with decayingOpuntia cactus.

The microbial structure within, between, and over time in decaying cladodes of the common prickly pearOpuntia stricta was studied at each of two separate localities. In general, the effective number of yeast species and yeast species diversity increased as the rot aged to the observed maximum time of 4 weeks. Yeast heterogeneity at the two localities differed in the mode of environmental influence, with spatial variability (among rots) most important at one and temporal variability (within rots over time) most important at the other. Differences in cactus density and quality (age) are most likely determinants of the differences in yeast community structure.

Allozyme genotypes of Drosophila buzzatii: feeding and oviposition preferences for microbial species, and habitat selection.

Mature, mated female D. buzzatii were given a choice of nine microbial communities actively growing on cactus homogenate in laboratory population cages, and tests were made to determine if flies of different genotypes (for seven allozyme loci) chose different microorganism species for either feeding or oviposition. Variation in feeding preferences was determined from assays of electrophoretic genotypes and the ingested microorganism species of individual flies. Oviposition preference variation was analyzed indirectly by assaying the genotypes of individuals raised from eggs laid on different microorganisms. No significant evidence was found for differences in feeding preferences among adults of different genotypes. For oviposition preferences, there were significant microorganism-genotype associations for each of seven polymorphic loci. Analyses of the total electrophoretic genotype, rather than of individual loci, showed that the genotypes of eggs laid on the same microorganism species were more similar than those laid on different species. That is, females of different genotypes show habitat selection for oviposition sites, which would facilitate the maintenance of genetic polymorphisms.

Relevance of the ecology ofCitrus yeasts to the diet ofDrosophila.

A study of the yeast flora of necrotic oranges and associatedDrosophila yielded a total of 221 isolates composed ofKloeckera apiculata (75),Pichia fermentans (75),Pichia kluyveri (50),Torulopsis stellata (17),Hanseniaspora uvarum (2),P. membranaefaciens (1), andCandida vini (1). The yeast species of all samples of oranges and adultDrosophila were very similar; however, the speciesof Drosophila contained a higher proportion ofP. fermentans and a lower proportion ofK. apiculata than was found in the rotting oranges.P. fermentans was subsequently found more frequently on the surface of the necrotic tissue, where the flies feed, than was found internally. SinceP. fermentans characteristically produces a pellicle and pseudomycelium andK. apiculata does not, it is concluded that the growth characteristics of the yeasts are an important factor determining adultDrosophila diets.