Jesse H. de León

Identifying the predator complex of Homalodisca vitripennis (Hemiptera: Cicadellidae): a comparative study of the efficacy of an ELISA and PCR gut content assay

A growing number of ecologists are using molecular gut content assays to qualitatively measure predation. The two most popular gut content assays are immunoassays employing pest-specific monoclonal antibodies (mAb) and polymerase chain reaction (PCR) assays employing pest-specific DNA. Here, we present results from the first study to simultaneously use both methods to identify predators of the glassy winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). A total of 1,229 arthropod predators, representing 30 taxa, were collected from urban landscapes in central California and assayed first by means of enzyme-linked immunosorbent assay (ELISA) using a GWSS egg-specific mAb and then by PCR using a GWSS-specific DNA marker that amplifies a 197-base pair fragment of its cytochrome oxidase gene (subunit I). The gut content analyses revealed that GWSS remains were present in 15.5% of the predators examined, with 18% of the spiders and 11% of the insect predators testing positive. Common spider predators included members of the Salticidae, Clubionidae, Anyphaenidae, Miturgidae, and Corinnidae families. Common insect predators included lacewings (Neuroptera: Chrysopidae), praying mantis (Mantodea: Mantidae), ants (Hymenoptera: Formicidae), assassin bugs (Hemiptera: Reduviidae), and damsel bugs (Hemiptera: Nabidae). Comparison of the two assays indicated that they were not equally effective at detecting GWSS remains in predator guts. The advantages of combining the attributes of both types of assays to more precisely assess field predation and the pros and cons of each assay for mass-screening predators are discussed.

Development of molecular diagnostic markers for sharpshooters Homalodisca coagulata and Homalodisca liturata for use in predator gut content examinations

To aid in identifying key predators of Proconiini sharpshooter species present in California, we developed and tested molecular diagnostic markers for the glassy‐winged sharpshooter, Homalodisca coagulata (Say), and smoke‐tree sharpshooter, Homalodisca liturata (Ball) (Homoptera: Cicadellidae). Two different types of markers were compared, those targeting single‐copy sequence characterized amplified regions (SCAR) and mitochondrial markers targeting the multicopy cytochrome oxidase subunit genes I (COI) and II (COII). A total of six markers were developed, two SCAR and four mitochondrial COI or COII markers. Specificity assays demonstrated that SCAR marker HcF5/HcR7 was H. coagulata specific and HcF6/HcR9 was H. coagulata/H. liturata specific. COI (HcCOI‐F/R) and COII (HcCOII‐F4/R4) markers were H. coagulata specific, COII (G/S‐COII‐F/R) marker was H. coagulata/H. liturata specific, and lastly, COII marker (Hl‐COII‐F/R) was H. liturata specific. Sensitivity assays using genomic DNA showed the COI marker to be the most sensitive marker with a detection limit of 6 pg of DNA. This marker was 66‐fold more sensitive than marker Hl‐COII‐F/R that showed a detection limit of 400 pg of DNA. In addition, the COI marker was 4.2‐fold more sensitive than the COII marker. In predator gut assays, the COI and COII markers demonstrated significantly higher detection efficiency than the SCAR markers. Furthermore, the COI marker demonstrated slightly higher detection efficiency over the COII marker. Lastly, we describe the inclusion of an internal control (28S amplification) for predation studies performing predator gut analyses utilizing the polymerase chain reaction (PCR). This control was critical in order to monitor reactions for PCR failures, PCR inhibitors, and for the presence of DNA.

Population Genetic Structure of Homalodisca coagulata (Homoptera: Cicadellidae), the Vector of the Bacterium Xylella fastidiosa Causing Pierce’s Disease in Grapevines

Abstract In the current study, inter-simple sequence repeat (ISSR) primers (p-13 and p-15) were used to estimate the population genetic structure of the sharpshooter Homalodisca coagulata (Say) (Homopera: Cicadellidae). Eighteen populations from throughout the United States and a population from Tahiti, French Polynesia, were analyzed. Populations were arbitrarily assigned to three regions: southeastern, southwestern, and western. Exact tests for population differentiation indicated highly significant differences in marker frequencies among the 18 populations with both primers. Analyses of molecular variance also indicated significant geographic structuring with both primers. A dendrogram based on Reynolds coancestry distance performed with p-15 clustered the U.S. populations into two main groups. The southeastern populations were grouped into one cluster and the southwestern and western populations into a second cluster. Within the western region, dendrograms produced with p-13 and p-15 showed in both cases that two populations (Edison and Bakersfield) clustered as outliers. The average divergence (D) among all populations was 0.099. Divergence values of 0.254, 0.103, and 0.102 were observed when comparing Bakersfield and the southeastern, southwestern, and western populations, respectively. Within the western region, D values for Bakersfield were 1.8- (p-13) and 2.4-fold (p-15) higher than the D of the western populations. The present results suggest that a subset of insects in California may have their origins in the southwestern region (Texas); furthermore, these results are suggestive of more than one founding event in California and/or biotypes or geographic races.

Detection of DNA Polymorphisms in Homalodisca coagulata (Homoptera: Cicadellidae) by Polymerase Chain Reaction-Based DNA Fingerprinting Methods

Abstract DNA polymorphisms were detected in Homalodisca coagulata (Say) (Homoptera: Cicadellidae) with the following DNA fingerprinting methods: inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) and primer pair-ISSR-PCR (pp-ISSR-PCR), randomly amplified microsatellite polymophisms (RAMP), selective amplification of microsatellite polymorphic loci (SAMPL), and primer pair-random amplification of polymorphic DNA-polymerase chain reaction (pp-RAPD-PCR). But first, a small-scale DNA fingerprinting screening procedure was initiated with these methods with a few individual insects to estimate the most sensitive and efficient method(s). In total, 205 polymorphic markers were generated with the four methods. The efficiency ratio estimated the following order for each method: 1) pp-ISSR-PCR and ISSR-PCR, 2) RAMP, 3) pp-RAPD-PCR, and 4) SAMPL. The screening efficiency ratio estimated that pp-ISSR-PCR and ISSR-PCR were the most efficient methods. DNA polymorphisms were detected in a natural population of 10–30 insects. The number of polymorphic loci ranged from five (pp-RAPD-PCR reaction 6) to 32 (ISSR-PCR primer 13), and the percentage of polymorphic loci was 100% for most primers tested. DNA fingerprinting methods tested were able to detect geographic variation in populations of H. coagulata from Bakersfield and Riverside, CA, and Weslaco, TX. Dendrograms based on Nei’s genetic distance showed that H. coagulata from Bakersfield and Riverside formed a cluster separate from Weslaco in three DNA fingerprinting reactions tested incorporating simple sequence repeats. DNA fingerprinting methods tested were also able to distinguish between three Homalodisca sharpshooters: H. coagulata, Homalodisca insolita (Walker), and Homalodisca liturata (Ball). The present results confirmed the utility of the DNA fingerprinting screening procedure and demonstrated, for the first time, genetic variation in natural populations of glassy-winged sharpshooters by PCR-based DNA fingerprinting methods.

Molecular Markers Discriminate Closely Related Species Encarsia diaspidicola and Encarsia berlesei (Hymenoptera: Aphelinidae): Biocontrol Candidate Agents for White Peach Scale in Hawaii

ABSTRACT 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.

First Report of a Proconiine Sharpshooter, Anacuerna centrolinea (Hemiptera: Cicadellidae), in Chile, with Notes on Its Biology, Host Plants, and Egg Parasitoids

Abstract The first representative of the leafhopper tribe Proconiini (subfamily Cicadellinae), Anacuerna centrolinea (Melichar) is reported herein from the Tarapacá region in northern Chile. This species was discovered at high elevation (≈4,000 m) in the course of a survey conducted in South America by the USDA–ARS for the neoclassical biological control program against the glassy-winged sharpshooter, Homalodisca coagulata (Say) in California. New data are given on the biology and host plants of A. centrolinea. Information also is provided on its egg parasitoid, Gonatocerus tuberculifemur (Ogloblin) (Hymenoptera: Mymaridae) which also was a first record from Chile. This discovery encourages further exploration for leafhopper egg parasitoids in northern and central regions of Chile to identify new perspective biological control agents that are more adapted to Mediterranean climate (winters and wet summers), which are similar to California climate. In addition, it is possible that G. tuberculifemur may be a good candidate for the biological control of the recently discovered H. coagulata in Easter Island, Chile.

Host range of Gonatocerus sp. near tuberculifemur ‘Clade 1’ in Argentina, an egg parasitoid newly associated to the glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae), and candidate for its biological control in California, USA

The South American egg parasitoid Gonatocerus sp. near tuberculifemur “Clade 1” (G. sp. “Clade 1”) (Hymenoptera: Mymaridae) is a new association of the glassy-winged sharpshooter (GWSS), Homalodiscavitripennis (Germar) (Cicadellidae) and a candidate for its biological control in California, USA. In Argentina, G. sp. “Clade 1” was screened in the laboratory (no-choice tests) and in the field (multiple choice tests) against eggs of 32 Auchenorrhyncha host species and other four potential hosts unrelated to sharpshooters. In no-choice assays, it parasitized only eggs within the leafhopper tribe Proconiini. In contrast, in the long term field tests, it emerged not only from eggs of the Proconiini but also from two species of Cicadellini at low numbers (five wasps out of 698 exposed eggs). Two interpretations arise from the results: (1) Host associations of G. sp. “Clade 1” are restricted to the Proconiini whereas field parasitization of the Cicadellini species were false positive, or (2) G. sp. “Clade 1” parasitizes also some Cicadellini species and its rejection in the laboratory was a false negative. Both interpretations are discussed. Insect motivation could be the explanation for the negative results in the no-choice tests. On the other hand, in the more natural field situations, the host selection process and oviposition behavior should not have been affected and host range would be more realistic. The parasitism of the Cicadellini species would be indicative of a potential non-target effect on the sharpshooters in the USA.