Molecular markers discriminate closely related species Encarsia diaspidicola and Encarsia berlesei (Hymenoptera: Aphelinidae): biocontrol candidate agents for white peach scale in Hawaii.

We genetically characterized Encarsia diapsidicola Silvestri and Encarsia berlesei Howard (Hymenoptera: Aphelinidae) by two molecular methods: phylogenetic analysis of the cytochrome oxidase subunit I gene (COI) and intersimple sequence repeat-polymerase chain reaction (ISSR-PCR) DNA fingerprinting. These two closely related endoparasitoids are candidate biological control agents for the white peach scale, Pseudaulacaspis pentagona Targioni-Tozetti (Hemiptera: Diaspididae), in Hawaii. We developed species-specific COI molecular markers that discriminated the two species, and we tested the utility of the E. diaspidicola-specific COI marker to detect parasitism of white peach scale. The COI sequence data uncovered 46-bp differences between the two Encarsia spp. The level of COI genetic divergence between the two species was 9.7%, and the two clustered into their own clade on a parismonious phylogram. ISSR-PCR readily discriminated the two Encarsia spp. because each was observed with fixed species-specific banding patterns. The COI molecular markers were specific for each species because cross-reactivity was not observed with nontarget species. The E. diaspidicola-specific COI markers were successful at detecting parasitism of white peach scale by E. diaspidicola by 24 h. Both molecular marker types successfully discriminated the two Encarsia spp., whereas the COI markers will be useful as tools to assess levels of parasitism in the field and to study competitive interactions between parasitoids.

Analysis of pMA67, a predicted rolling-circle replicating, mobilizable, tetracycline-resistance plasmid from the honey bee pathogen, Paenibacillus larvae.

This work characterizes a recently discovered natural tetracycline-resistance plasmid called pMA67 from Paenibacillus larvae--a Gram-positive bacterial pathogen of honey bees. We provide evidence that pMA67 replicates by the rolling-circle mechanism, and sequence comparisons place it in the pMV158 family of rolling-circle replicons. The plasmid contains predicted rep, cop, and rnaII genes for control of replication initiating at a predicted double-strand origin. The plasmid has an ssoT single-strand origin, which is efficient enough to allow only very small amounts of the single-stranded DNA intermediate to accumulate. The overall efficiency of replication is sufficient to render the plasmid segregationally stable without selection in P. larvae and in Bacillus megaterium, but not in Escherichia coli. The plasmid is expected to be mobilizable due to the presence of a mob gene and an oriT site. The plasmid contains a tetL gene, whose predicted amino acid sequence implies a relatively ancient divergence from all previously known plasmid-encoded tetL genes. We confirm that the tetL gene alone is sufficient for conferring resistance to tetracyclines. Sequence comparisons, mostly with the well-characterized pMV158, allow us to predict promoters, DNA and RNA secondary structures, DNA and protein motifs, and other elements.

Genetic differentiation among geographic populations of Gonatocerus ashmeadi, the predominant egg parasitoid of the glassy-winged sharpshooter, Homalodisca coagulata.

The aim of genetically comparing different populations of the same species of natural enemies is to identify the strain that is most adapted to the environment where it will be released. In the present study, Inter-Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) was utilized to estimate the population genetic structure of Gonatocerus ashmeadi (Girault) (Hymenoptera: Mymaridae), the predominant egg parasitoid of Homalodisca coagulata (Say) (Homoptera:Cicadellidae), the glassy-winged sharpshooter. Six populations from throughout the U.S. and a population from Argentina identified as near G. ashmeadi were analyzed. Four populations (California; San Antonio, Texas; Weslaco, Texas [WTX-2]; and Florida) were field collected and two (Louisiana and Weslaco, Texas [WTX-1]) were reared. Three ISSR-PCR reactions were pooled to generate 41 polymorphic markers among the six U.S. populations. Nei's expected heterozygosity values (h), including the reared population from Louisiana, were high (9.01-14.3%) for all populations, except for a reared population from WTX-1 (2.9%). The total genetic diversity value (Ht) for the field populations was high (23%). Interestingly, the Florida population that was collected from one egg mass (siblings) generated the greatest number of polymorphic markers (20) and was observed with the highest gene diversity value (14.3%). All populations, except WTX-2 generated population-specific markers. Comparison of genetic differentiation estimates, which evaluate the degree of genetic subdivision, demonstrated good agreement between G(ST) and theta values, 0.38 and 0.50, respectively for field populations, and 0.44 and 0.50, respectively for all populations. Genetic divergence (D) indicated that the WTX-1 population was the most differentiated. Average D results from the Argentina population support the taxonomic data that it is a different species. The present results estimate the population genetic structure of G. ashmeadi, demonstrating genetic divergence and restricted gene flow (Nm = 0.83) among populations. These results are of interest to the Pierce's disease/glassy-winged sharpshooter biological control program because the key to successful biological control may not be in another species, but instead in different geographic races or biotypes.

Molecular distinction between populations of Gonatocerus morrilli, egg parasitoids of the glassy-winged sharpshooter from Texas and California: do cryptic species exist?

Two molecular methods were utilized to distinguish geographic populations of Gonatocerus morrilli (Howard) from Texas and California and to test the possibility that this species could exist as a species-complex. Inter-Simple Sequence Repeat-Polymerase Chain Reactions (ISSR-PCR) were performed with a 5'-anchored ISSR primer. Twenty-five markers were generated with four populations (40 individuals) of G. morrilli. Twenty-three were polymorphic and the percentage of polymorphic loci was 92%. Most markers could be considered diagnostic since there was no band sharing between the Texas and California populations. Such differences typically are not found unless the populations are reproductively isolated. Exact tests for population differentiation indicated significant differences in marker frequencies among the populations. Comparison of other genetic differentiation estimates, which evaluate the degree of genetic subdivision, demonstrated excellent agreement between GST and theta values, 0.92 and 0.94, respectively, indicating that about 92 to 94% of the variance was distributed among populations. The average genetic divergence (D), as measured by genetic distance, was extremely high (Nei = 0.82 and Reynolds = 2.79). A dendrogram based on Nei's genetic distance separated the Texas and California populations into two clusters, respectively. Amplification of the Internal Transcribed Spacer-1 (ITS-1) region showed no size differences, whereas the ITS-2 DNA fragment varied in size between the two geographic populations. The ITS-2 fragment sizes were about 865 and 1099 base pairs for the California and Texas populations, respectively. The present study using the two molecular methods provides novel data critical to the glassy-winged sharpshooter/Pierce's disease biological control program in California.