Genomic Assessment of the Contribution of the Wolbachia Endosymbiont of Eurosta solidaginis to Gall Induction.

We explored the genome of the Wolbachia strain, wEsol, symbiotic with the plant-gall-inducing fly Eurosta solidaginis with the goal of determining if wEsol contributes to gall induction by its insect host. Gall induction by insects has been hypothesized to involve the secretion of the phytohormones cytokinin and auxin and/or proteinaceous effectors to stimulate cell division and growth in the host plant. We sequenced the metagenome of E. solidaginis and wEsol and assembled and annotated the genome of wEsol. The wEsol genome has an assembled length of 1.66 Mbp and contains 1878 protein-coding genes. The wEsol genome is replete with proteins encoded by mobile genetic elements and shows evidence of seven different prophages. We also detected evidence of multiple small insertions of wEsol genes into the genome of the host insect. Our characterization of the genome of wEsol indicates that it is compromised in the synthesis of dimethylallyl pyrophosphate (DMAPP) and S-adenosyl L-methionine (SAM), which are precursors required for the synthesis of cytokinins and methylthiolated cytokinins. wEsol is also incapable of synthesizing tryptophan, and its genome contains no enzymes in any of the known pathways for the synthesis of indole-3-acetic acid (IAA) from tryptophan. wEsol must steal DMAPP and L-methionine from its host and therefore is unlikely to provide cytokinin and auxin to its insect host for use in gall induction. Furthermore, in spite of its large repertoire of predicted Type IV secreted effector proteins, these effectors are more likely to contribute to the acquisition of nutrients and the manipulation of the host's cellular environment to contribute to growth and reproduction of wEsol than to aid E. solidaginis in manipulating its host plant. Combined with earlier work that shows that wEsol is absent from the salivary glands of E. solidaginis, our results suggest that wEsol does not contribute to gall induction by its host.

Abscisic Acid: A Potential Secreted Effector Synthesized by Phytophagous Insects for Host-Plant Manipulation.

Abscisic acid (ABA) is an isoprenoid-derived plant signaling molecule involved in a wide variety of plant processes, including facets of growth and development as well as responses to abiotic and biotic stress. ABA had previously been reported in a wide variety of animals, including insects and humans. We used high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-(ESI)-MS/MS) to examine concentrations of ABA in 17 species of phytophagous insects, including gall- and non-gall-inducing species from all insect orders with species known to induce plant galls: Thysanoptera, Hemiptera, Lepidoptera, Coleoptera, Diptera, and Hymenoptera. We found ABA in insect species in all six orders, in both gall-inducing and non-gall-inducing species, with no tendency for gall-inducing insects to have higher concentrations. The concentrations of ABA in insects often markedly exceeded those typically found in plants, suggesting it is highly improbable that insects obtain all their ABA from their host plant via consumption and sequestration. As a follow-up, we used immunohistochemistry to determine that ABA localizes to the salivary glands in the larvae of the gall-inducing Eurosta solidaginis (Diptera: Tephritidae). The high concentrations of ABA, combined with its localization to salivary glands, suggest that insects are synthesizing and secreting ABA to manipulate their host plants. The pervasiveness of ABA among both gall- and non-gall-inducing insects and our current knowledge of the role of ABA in plant processes suggest that insects are using ABA to manipulate source-sink mechanisms of nutrient allocation or to suppress host-plant defenses. ABA joins the triumvirate of phytohormones, along with cytokinins (CKs) and indole-3-acetic acid (IAA), that are abundant, widespread, and localized to glandular organs in insects and used to manipulate host plants.

The Localization of Phytohormones within the Gall-inducing Insect Eurosta solidaginis (Diptera: Tephritidae).

The phytohormone production hypothesis suggests that organisms, including insects, induce galls by producing and secreting plant growth hormones. Auxins and cytokinins are classes of phytohormones that induce cell growth and cell division, which could contribute to the plant tissue proliferation which constitutes the covering gall. Bacteria, symbiotic with insects, may also play a part in gall induction by insects through the synthesis of phytohormones or other effectors. Past studies have shown that concentrations of cytokinins and auxins in gall-inducing insects are higher than in their host plants. However, these analyses have involved whole-body extractions. Using immunolocalization of cytokinin and auxin, in the gall inducing stage of Eurosta solidaginis, we found both phytohormones to localize almost exclusively to the salivary glands. Co-localization of phytohormone label with a nucleic acid stain in the salivary glands revealed the absence of Wolbachia sp., the bacterial symbiont of E. solidaginis, which suggests that phytohormone production is symbiont independent. Our findings are consistent with the hypothesis that phytohormones are synthesized in and secreted from the salivary glands of E. solidaginis into host-plant tissues for the purpose of manipulating the host plant.

Cytokinins Are Abundant and Widespread Among Insect Species.

Cytokinins (CKs) are a class of compounds that have long been thought to be exclusively plant growth regulators. Interestingly, some species of phytopathogenic bacteria and fungi have been shown to, and gall-inducing insects have been hypothesized to, produce CKs and use them to manipulate their host plants. We used high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-MS/MS) to examine concentrations of a wide range of CKs in 17 species of phytophagous insects, including gall- and non-gall-inducing species from all six orders of Insecta that contain species known to induce galls: Thysanoptera, Hemiptera, Lepidoptera, Coleoptera, Diptera, and Hymenoptera. We found CKs in all six orders of insects, and they were not associated exclusively with gall-inducing species. We detected 24 different CK analytes, varying in their chemical structure and biological activity. Isoprenoid precursor nucleotide and riboside forms of trans-zeatin (tZ) and isopentenyladenine (iP) were most abundant and widespread across the surveyed insect species. Notably, the observed concentrations of CKs often markedly exceeded those reported in plants suggesting that insects are synthesizing CKs rather than obtaining them from the host plant via tissue consumption, compound sequestration, and bioaccumulation. These findings support insect-derived CKs as means for gall-inducing insects to manipulate their host plant to facilitate cell proliferation, and for both gall- and non-gall-inducing insects to modify nutrient flux and plant defenses during herbivory. Furthermore, wide distribution of CKs across phytophagous insects, including non-gall-inducing species, suggests that insect-borne CKs could be involved in manipulation of source-sink mechanisms of nutrient allocation to sustain the feeding site and altering plant defensive responses, rather than solely gall induction. Given the absence of any evidence for genes in the de novo CK biosynthesis pathway in insects, we postulate that the tRNA-ipt pathway is responsible for CK production. However, the unusually high concentrations of CKs in insects, and the tendency toward dominance of their CK profiles by tZ and iP suggest that the tRNA-ipt pathway functions differently and substantially more efficiently in insects than in plants.

Beetles in bamboo forests: community structure in a heterogeneous landscape of southwestern Amazonia.

Amazonian bamboo forests dominated by large woody bamboo plants in the genus Guadua cover approximately 180,000 km2 and represent a key resource for many organisms. In southwestern Amazonia, native bamboo forests differ in structure, biodiversity, and growth dynamics from other forest types in the region. However, with the exception of a few species in which habitat specialization or a strong habitat association has been demonstrated, little is known about how bamboo forests influence animal community structure. In an effort to understand more about the animal assemblages associated with Amazonian bamboo forests, we characterized the structure of ground-dwelling beetle assemblages living in bamboo forests and adjacent terra firme forests in a lowland rainforest site in Peru. We conducted intensive pitfall trap surveys in 13 bamboo habitat patches and 13 adjacent terra firme habitat patches to determine if there were differences in the abundance and richness of beetle species in these two habitat types. Additionally, given that southwestern Amazonia experiences distinct dry and wet seasons, we conducted our study during the dry and wet season of one year to account for differences in seasonality. We found a distinct beetle assemblage associated with each forest type, and identified a set of dominant species that significantly contributed to the distinctness in beetle community structure between bamboo and terra firme forest. The terra firme forest had a greater number of rare species than the bamboo forest. Several beetle species exhibited a strong association with the bamboo forest, including a large species of Scarabaeidae that appears to be specializing on bamboo. We also found marked differences in beetle assemblages between dry and wet seasons. Our results support the prediction that beetle community structure in bamboo forest differs from that of terra firme in terms of species richness, abundance, and composition. Bamboo-associated animal communities require more exploration and study, and must be included in regional conservation plans seeking to protect entire animal communities in southwestern Amazonia.

Estimating species - area relationships by modeling abundance and frequency subject to incomplete sampling.

Models and data used to describe species-area relationships confound sampling with ecological process as they fail to acknowledge that estimates of species richness arise due to sampling. This compromises our ability to make ecological inferences from and about species-area relationships. We develop and illustrate hierarchical community models of abundance and frequency to estimate species richness. The models we propose separate sampling from ecological processes by explicitly accounting for the fact that sampled patches are seldom completely covered by sampling plots and that individuals present in the sampling plots are imperfectly detected. We propose a multispecies abundance model in which community assembly is treated as the summation of an ensemble of species-level Poisson processes and estimate patch-level species richness as a derived parameter. We use sampling process models appropriate for specific survey methods. We propose a multispecies frequency model that treats the number of plots in which a species occurs as a binomial process. We illustrate these models using data collected in surveys of early-successional bird species and plants in young forest plantation patches. Results indicate that only mature forest plant species deviated from the constant density hypothesis, but the null model suggested that the deviations were too small to alter the form of species-area relationships. Nevertheless, results from simulations clearly show that the aggregate pattern of individual species density-area relationships and occurrence probability-area relationships can alter the form of species-area relationships. The plant community model estimated that only half of the species present in the regional species pool were encountered during the survey. The modeling framework we propose explicitly accounts for sampling processes so that ecological processes can be examined free of sampling artefacts. Our modeling approach is extensible and could be applied to a variety of study designs and allows the inclusion of additional environmental covariates.

Estimating the Effects of Habitat and Biological Interactions in an Avian Community.

We used repeated sightings of individual birds encountered in community-level surveys to investigate the relative roles of habitat and biological interactions in determining the distribution and abundance of each species. To analyze these data, we developed a multispecies N-mixture model that allowed estimation of both positive and negative correlations between abundances of different species while also estimating the effects of habitat and the effects of errors in detection of each species. Using a combination of single- and multispecies N-mixture modeling, we examined for each species whether our measures of habitat were sufficient to account for the variation in encounter histories of individual birds or whether other habitat variables or interactions with other species needed to be considered. In the community that we studied, habitat appeared to be more influential than biological interactions in determining the distribution and abundance of most avian species. Our results lend support to the hypothesis that abundances of forest specialists are negatively affected by forest fragmentation. Our results also suggest that many species were associated with particular types of vegetation as measured by structural attributes of the forests. The abundances of 6 of the 73 species observed in our study were strongly correlated. These species included large birds (American Crow (Corvus brachyrhynchos) and Red-winged Blackbird (Agelaius phoeniceus)) that forage on the ground in open habitats and small birds (Red-eyed Vireo (Vireo olivaceus), House Wren (Troglodytes aedon), Hooded Warbler (Setophaga citrina), and Prairie Warbler (Setophaga discolor)) that are associated with dense shrub cover. Species abundances were positively correlated within each size group and negatively correlated between groups. Except for the American Crow, which preys on eggs and nestlings of small song birds, none of the other 5 species is known to display direct interactions, so we suspect that the correlations may have been associated with species-specific responses to habitat components not adequately measured by our covariates.

Estimating abundances of interacting species using morphological traits, foraging guilds, and habitat.

We developed a statistical model to estimate the abundances of potentially interacting species encountered while conducting point-count surveys at a set of ecologically relevant locations - as in a metacommunity of species. In the model we assume that abundances of species with similar traits (e.g., body size) are potentially correlated and that these correlations, when present, may exist among all species or only among functionally related species (such as members of the same foraging guild). We also assume that species-specific abundances vary among locations owing to systematic and stochastic sources of heterogeneity. For example, if abundances differ among locations due to differences in habitat, then measures of habitat may be included in the model as covariates. Naturally, the quantitative effects of these covariates are assumed to differ among species. Our model also accounts for the effects of detectability on the observed counts of each species. This aspect of the model is especially important for rare or uncommon species that may be difficult to detect in community-level surveys. Estimating the detectability of each species requires sampling locations to be surveyed repeatedly using different observers or different visits of a single observer. As an illustration, we fitted models to species-specific counts of birds obtained while sampling an avian community during the breeding season. In the analysis we examined whether species abundances appeared to be correlated due to similarities in morphological measures (body mass, beak length, tarsus length, wing length, tail length) and whether these correlations existed among all species or only among species of the same foraging guild. We also used the model to estimate the effects of forested area on species abundances and the effects of sound power output (as measured by body size) on species detection probabilities.

The checkered history of checkerboard distributions.

To address the idea that the process of interspecific competition can be inferred from data on geographical distribution alone and that evidence from geographical distribution implies an important role for interspecific competition in shaping ecological communities, we reexamine the occurrence of "true checkerboard" distributions among the land and freshwater birds in three Melanesian archipelagoes: Vanuatu, the Bismarck Archipelago, and the Solomon Islands. We use the most recently published distributional records and explicitly include the geography of the distributions of species within each archipelago. We use the overlap of convex hulls to estimate the overlap in the geographic range for each pair of species in each of these archipelagoes. We define a "true checkerboard" to consist of a pair of species with exclusive island-by-island distributions, but that have overlapping geographical ranges. To avoid the "dilution effect," we follow Diamond and Gilpin in focusing only on congeneric and within-guild species pairs as potential competitors. Few, if any, "true checkerboards" exist in these archipelagoes that could possibly have been influenced by competitive interactions, and even "true checkerboards" can arise for reasons other than interspecific competition. The similarity between related species pairs (congeneric and within-guild pairs) and unrelated species pairs in their deviation from expectation of the number of islands shared and the overlap of their geographic ranges indicates that these are not distinct statistical populations, but rather a single population of species pairs. Our result, which is based on an examination of the distributional data alone, is consistent with the interpretation that, in these avifaunas, the distributions of congeneric, within-guild, and unrelated species pairs are shaped by a common set of biological and physical environmental processes.

Detecting insect pollinator declines on regional and global scales.

Recently there has been considerable concern about declines in bee communities in agricultural and natural habitats. The value of pollination to agriculture, provided primarily by bees, is >$200 billion/year worldwide, and in natural ecosystems it is thought to be even greater. However, no monitoring program exists to accurately detect declines in abundance of insect pollinators; thus, it is difficult to quantify the status of bee communities or estimate the extent of declines. We used data from 11 multiyear studies of bee communities to devise a program to monitor pollinators at regional, national, or international scales. In these studies, 7 different methods for sampling bees were used and bees were sampled on 3 different continents. We estimated that a monitoring program with 200-250 sampling locations each sampled twice over 5 years would provide sufficient power to detect small (2-5%) annual declines in the number of species and in total abundance and would cost U.S.$2,000,000. To detect declines as small as 1% annually over the same period would require >300 sampling locations. Given the role of pollinators in food security and ecosystem function, we recommend establishment of integrated regional and international monitoring programs to detect changes in pollinator communities.

Automating identification of avian vocalizations using time-frequency information extracted from the Gabor transform.

Based on the Gabor transform, a metric is developed and applied to automatically identify bird species from a sample of 568 digital recordings of songs/calls from 67 species of birds. The Gabor frequency-amplitude spectrum and the Gabor time-amplitude profile are proposed as a means to characterize the frequency and time patterns of a bird song. An approach based on template matching where unknown song clips are compared to a library of known song clips is used. After adding noise to simulate the background environment and using an adaptive high-pass filter to de-noise the recordings, the successful identification rate exceeded 93% even at signal-to-noise ratios as low as 5 dB. Bird species whose songs/calls were dominated by low frequencies were more difficult to identify than species whose songs were dominated by higher frequencies. The results suggest that automated identification may be practical if comprehensive libraries of recordings that encompass the vocal variation within species can be assembled.

Factors affecting the reproductive success of the crab spider Misumenoides formosipes: the covariance between juvenile and adult traits.

To examine the importance of covariance between stages in traits related to foraging, we quantified the relationships between reproductive success and sizerelated variability in weight gain in juvenile and adult instars of the crab spider Misumenoides formosipes (Araneae: Thomisidae). Prereproductive weight and fecundity are both highly correlated with carapace width, a linear measure of size which does not change within an instar. In field populations, adult females with larger carapaces gain more weight and are more likely to reproduce than females with smaller carapaces. The growth rate of spiders fed ad libitum in the laboratory is unrelated to size, suggesting that size-related differences in the field are due to variation in prey-capture success. Adult females with a carapace width less than 3.4 mm comprised 22% of the population, but were never found to reproduce. Of the individuals that did reproduce, a 17% increase in carapace width resulted in a 100% increase in fecundity. Juvenile stages must be examined to understand adult foraging and reproductive success, because the net weight gained by juvenile instars determines adult size. The final weight gained by spiders in the antepenultimate and penultimate instars explained nearly all the variation in carapace width in the penultimate and adult instars, respectively. We found that constraints on foraging in late juvenile stages are different from the adult stage. Penultimate foraging behavior differs from that of adults, because of constraints on foraging in the period preceding ecdysis. Additionally, in both late juvenile instars, carapace width had little or no effect on the final weight gained within the instar suggesting that factors that affect foraging are different between the juvenile and adult stages. These analyses stress the fact that to fully understand the effects of foraging on reproductive success, we must examine stage-specific constraints throughout an organism's life history.