Jikradia olitoria ([Hemiptera]:[Cicadellidae]) Transmits the Sequevar NAGYIIIß Phytoplasma Strain Associated with North American Grapevine Yellows in Artificial Feeding Assays.

North American Grapevine Yellows (NAGY) is a destructive disease of grapevines caused by phytoplasmas, wall-less bacteria that are insect-transmitted and found in plant phloem tissues. Although the disease was recognized in vineyards in the eastern United States since the 1980s, the identities of vectors remain unknown. The objectives of this study were to survey potential phytoplasma vector insects inhabiting Virginia vineyards that expressed NAGY symptoms and to evaluate their ability to transmit phytoplasmas associated with NAGY. Phytoplasmas were identified as 'Candidatus Phytoplasma pruni'-related NAGYIIIß strains and 'Ca. Phytoplasma asteris'-related NAGYI-B strains. To determine the identities of the potential vectors, artificial feeding solution was used to evaluate the ability of leafhopper species to release phytoplasmas during feeding and phytoplasma strains were identified using molecular tools. Out of 49 insect species screened, Jikradia olitoria was the only insect that released phytoplasmas into the feeding solutions; all phytoplasmas, thus, detected were identified as NAGYIIIß strains by nucleotide sequencing of three different genomic regions. No NAGYI-B strain was detected. To our knowledge, this is the first evidence of a potential insect vector of a specific phytoplasma associated with NAGY disease, and it is the first report of J. olitoria being a putative vector of a plant pathogenic phytoplasma.

Direct and indirect effects of light environment generate ecological trade-offs in herbivore performance and parasitism.

A variety of ecological factors influence host use by parasitoids, including both abiotic and biotic factors. Light environment is one important abiotic parameter that differs among habitats and influences a suite of plant nutritional and resistance traits that in turn affect herbivore performance. However, the extent to which these bottom-up effects "cascade up" to affect higher trophic levels and the relative importance of direct and indirect effects of sunlight on tritrophic interactions are unclear. The objective of this study was to test how light environment (light gap vs. shaded forest understory) and leaf type (sun vs. shade leaves) affect the performance and incidence of parasitism of two species of moth larvae, Euclea delphinii and Acharia stimulea (Limacodidae). We manipulated the leaf phenotype of potted white oak saplings by growing them in either full sun or full shade throughout leaf expansion to produce sun and shade leaves, respectively. These saplings were then placed in light gap and adjacent shaded understory habitats in the forest in a full-factorial design, and stocked with sentinel larvae that were exposed to parasitism ("exposed" experiments). We reared additional larvae in sleeve cages (protected from parasitism) to isolate light environment and leaf phenotype treatment effects on larval performance in the absence of enemies ("bagged" experiments). In the exposed experiments, light environment strongly affected the likelihood of parasitism, while leaf phenotype did not. Euclea delphinii larvae were up to 6.6 times more likely to be parasitized in light gaps than in shaded understory habitats. This pattern was consistent for both tachinid fly and wasp parasitoids across two separate experiments. However, the larval performance of both species in the bagged experiments was maximized in the shade-habitat/sun-leaf treatment, a habitat/leaf-type combination that occurs infrequently in nature. Taken together, our results suggest that the direct effects of light environment on the incidence of parasitism supersede any indirect effects resulting from altered leaf quality and reveal inherent ecological trade-offs for herbivores confronted with choosing between sunny (high leaf quality, harsh environment, high parasitism) and shaded (reduced leaf quality less harsh environment, reduced parasitism) habitats.

Differential pollinator effectiveness and importance in a milkweed (Asclepias, Apocynaceae) hybrid zone.

PREMISE OF THE STUDY: Exceptions to the ideal of complete reproductive isolation between species are commonly encountered in diverse plant, animal, and fungal groups, but often the causative ecological processes are poorly understood. In flowering plants, the outcome of hybridization depends in part on the effectiveness of pollinators in interspecific pollen transport. In the Asclepias exaltata and A. syriaca (Apocynaceae) hybrid zone in Shenandoah National Park, Virginia, extensive introgression has been documented. The objectives of this study were to (1) determine the extent of pollinator overlap among A. exaltata, A. syriaca, and their hybrids and (2) identify the insect taxa responsible for hybridization and introgression. METHODS: We observed focal plants of parental species and hybrids to measure visitation rate, visit duration, and per-visit pollinia removal and deposition, and we calculated pollinator effectiveness and importance. KEY RESULTS: Visitation rates varied significantly between the 2 yr of the study. Overall, Apis mellifera, Bombus sp., and Epargyreus clarus were the most important pollinators. However, Bombus sp. was the only visitor that was observed to both remove and insert pollinia for both parent species as well as hybrids. CONCLUSIONS: We conclude that Bombus may be a key agent of hybridization and introgression in these sympatric milkweed populations, and hybrids are neither preferred nor selected against by pollinators. Thus, we have identified a potential mechanism for how hybrids act as bridges to gene flow between A. exaltata and A. syriaca. These results provide insights into the breakdown of prezygotic isolating mechanisms.

Intraplant movement of generalist slug caterpillars (Limacodidae: Lepidoptera): effects of host plant and light environment.

Insect herbivores frequently move about on their host plants to obtain food, avoid enemies and competitors, and cope with changing environmental conditions. Although numerous plant traits influence the movement of specialist herbivores, few studies have examined movement responses of generalist herbivores to the variable ecological conditions associated with feeding and living on an array of host plants. We tested whether the movement patterns of two generalist caterpillars (Euclea delphinii Boisduval and Acharia stimulea Clemens, Limacodidae) differed on six different host tree species over 10 d. Because these tree species vary in the range of light environments in which they commonly grow, we also compared the movement responses of E. delphinii caterpillars to two contrasting light environments, sun and shade. For both caterpillar species, multiple measures of movement varied significantly among host tree species. In early censuses, movement rates and distances were highest on red oak and black cherry and lowest on white oak. Site fidelity was greatest on white oak and lowest on black cherry. Movement of both caterpillar species varied inversely with mean predator density on five of the six host trees. Other ecological predictors (e.g., leaf size and the density of other herbivores) were unrelated to movement. Light environment altered behavior such that caterpillars in the shade moved and fed more often, and moved greater distances, than caterpillars in the sun. Although the mechanism(s) promoting or inhibiting movement under these different conditions requires further study, the consequences of increased movement for caterpillar development and mortality from natural enemies are discussed.

Facilitative effects of group feeding on performance of the saddleback caterpillar (Lepidoptera: Limacodidae).

Gregarious feeding by insect herbivores is a widely observed, yet poorly understood, behavioral adaptation. Previous research has tested the importance of group feeding for predator deterrence, noting the ubiquity of aposematism among group-feeding insects, but few studies have examined the role of feeding facilitation for aggregates of insect herbivores. We tested the hypothesis that group feeding has facilitative effects on performance of the saddleback caterpillar, Acharia stimulea Clemens, a generalist herbivore of deciduous trees. In an understory forest setting, we reared caterpillars alone or in groups on two different host plants, white oak (Quercus alba L.) and American beech (Fagus grandifolia Ehrlich), and recorded multiple measures of insect performance during regular field censuses. As predicted, A. stimulea caterpillars feeding in groups on white oak had increased relative growth rates compared with caterpillars feeding alone, and the magnitude of this facilitative effect varied among censuses, conferring benefits both early and late in development. By contrast, no facilitative effects of group feeding were detected on beech, suggesting that the benefits of facilitative feeding may be host specific. On both hosts, caterpillar development time was slightly faster for group-feeding cohorts compared with their solitary counterparts. Because early instar caterpillars are particularly vulnerable to predation and parasitism, even modest increases in growth rates and reductions in development time may decrease exposure time to enemies during these vulnerable stages. On both hosts, group feeding also reduced the trade-off between individual development time and cocoon mass, suggesting that feeding efficiency is improved in group feeders relative to solitary caterpillars.

Hemocyte density increases with developmental stage in an immune-challenged forest caterpillar.

The cellular arm of the insect immune response is mediated by the activity of hemocytes. While hemocytes have been well-characterized morphologically and functionally in model insects, few studies have characterized the hemocytes of non-model insects. Further, the role of ontogeny in mediating immune response is not well understood in non-model invertebrate systems. The goals of the current study were to (1) determine the effects of caterpillar size (and age) on hemocyte density in naïve caterpillars and caterpillars challenged with non-pathogenic bacteria, and (2) characterize the hemocyte activity and diversity of cell types present in two forest caterpillars: Euclea delphinii and Lithacodes fasciola (Limacodidae). We found that although early and late instar (small and large size, respectively) naïve caterpillars had similar constitutive hemocyte densities in both species, late instar Lithacodes caterpillars injected with non-pathogenic E. coli produced more than a twofold greater density of hemocytes than those in early instars. We also found that both caterpillar species contained plasmatocytes, granulocytes and oenocytoids, all of which are found in other lepidopteran species, but lacked spherulocytes. Granulocytes and plasmatocytes were found to be strongly phagocytic in both species, but granulocytes exhibited a higher phagocytic activity than plasmatocytes. Our results strongly suggest that for at least one measure of immunological response, the production of hemocytes in response to infection, response magnitudes can increase over ontogeny. While the underlying raison d' être for this improvement remains unclear, these findings may be useful in explaining natural patterns of stage-dependent parasitism and pathogen infection.

A simple protocol for extracting hemocytes from wild caterpillars.

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster, the mosquitoes Aedes aegypti and Anopheles gambiae and the tobacco hornworm, Manduca sexta. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild. Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms.