1. The bottom-up factors that determine parasitoid host use are an important area of research in insect ecology. Host size is likely to be a primary cue for foraging parasitoids due to its potential influence on offspring development time, the risk of multiparasitism, and host immunocompetence. Host size is mediated in part by host-plant traits that influence herbivore growth and potentially affect a herbivore's quality as a host for parasitoids.2. Here, we tested how caterpillar host size and host plant species influence adult fly parasitoid size and whether host size influences wasp parasitoid sex allocation. We measured the hind tibia lengths and determined the sex of wasp and fly parasitoids reared from 11 common host species of polyphagous caterpillars (Limacodidae) that were in turn reared on foliage of seven different host plant species.3. We also tested how host caterpillar species, host caterpillar size, and host and parasitoid phenology affect how the parasitoid community partitions host resources. We found evidence that parasitoids primarily partition their shared hosts based on size, but not by host species or phenology. One index of specialisation (d ) supports our observation that these parasitoids are quite generalised within the Limacodidae. In general, wasps were reared from caterpillars collected in early instars, while flies were reared from caterpillars collected in late instars. Furthermore, for at least one species of solitary wasp, host size influenced sex allocation of offspring by ovipositing females.4. Host-plant quality indirectly affected the size attained by a tachinid fly parasitoid through its direct effects on the size and performance of the caterpillar host. The host plants that resulted in the highest caterpillar host performance in the absence of enemies also yielded the largest parasitoid flies, which suggests that host plant quality can cascade up to influence the third trophic level.

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.

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.

Summary1. Natural sunlight gradients occur on multiple scales in space and time. However, the direct (via habitat) and indirect effects (via plant quality) of light environment are often confounded, obscuring the relative importance of each in influencing herbivore responses. 2. Potted saplings of Quercus alba (white oak, Fagaceae) were grown in either full sun or full shade from budburst to leaf hardening to manipulate leaf phenotype (creating sun or shade leaves), then placed in either sunny light gaps or adjacent shaded forest understorey habitats. This two-way factorial design isolated the effects of sunlight level during leaf expansion from light environment late in the growing season on leaf phenology, leaf traits associated with host plant quality for herbivores, herbivore density and folivory. 3. Sunlight level during leaf expansion and hardening had strong and persistent effects on Q. alba leaf phenology and phenotype. Shade saplings had later budburst (c. 4 days), and fewer but larger leaves, resulting in greater total leaf area compared with sun leaf saplings. Shade leaves had higher water content, specific leaf area and nitrogen content, and lower toughness, carbon content, C/N ratios and concentrations of hydrolysable and condensed tannins than sun leaves. 4. Despite the apparent higher quality of shade leaves, forest habitat better predicted damage by folivores than leaf type, suggesting that the direct effects of light environment predominate for herbivory. Potted saplings of both leaf types placed in the shaded understorey suffered almost two times more folivory, on average, than saplings in sunny light gaps, despite more than three times higher mid-season herbivore density on sun leaf saplings relative to shade leaf saplings. 5. Taken together, these results suggest that both leaf phenotype and forest habitat, two factors frequently confounded in nature, have significant but distinct effects on leaf quality and herbivory. These findings have implications for plant-herbivore interactions following disturbances such as treefalls, when shade leaves may be present in sunny habitats, and may help explain patterns of herbivory in understorey plants with early leaf flushing phenology relative to the canopy, when sun leaves are present in the shaded understorey.

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1 . 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 2 . 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 3 . They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4,5 . Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6,7 , the mosquitoes Aedes aegypti and Anopheles gambiae 8,9 and the tobacco hornworm, Manduca sexta 10,11 . 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 12 .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 13 . Video LinkThe video component of this article can be found at http://www.jove.com/video/4173/ Protocol 1. Material Preparation 1. Prepare needles (1-2 μm tip and 3-4 mm taper) using borosilicate glass capillary tubes and a micropipette puller. Instrument settings: ramp: 561; velocity: 20; heat: 560; delay: 1; pull: 100; pressure: 500. 2. Prepare the collection solution: 60% of Grace's Medium (GM) supplemented with 10% of Fetal Bovine Serum (FBS) and 20% of Anticoagulant Buffer (98 mM NaOH, 186 mM NaCI, 1.7 mM EDTA and 41 mM citric acid, pH 4.5). The above solution is prepared fresh under sterile conditions and kept on ice at all times. 3. Prepare the incubation buffer solution: 90% of GM supplemented with 10% of FBS. This solution is also prepared fresh and kept on ice at all times. 4. Prepare a total of at least 15 μl × the number of insects for both the collection a...

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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.

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.