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Quantifying species trophic interaction strengths is crucial for understanding community dynamics and has significant implications for pest management and species conservation. DNA-based methods to identify species interactions have revolutionized these efforts, but a significant limitation is the poor ability to quantify the strength of trophic interactions, that is the biomass or number of prey consumed. We present an improved pipeline, called Lazaro, to map unassembled shotgun reads to a comprehensive arthropod mitogenome database and show that the number of prey reads detected is quantitatively predicted from the prey biomass consumed, even for indirect predation. Two feeding bioassays were performed: starved coccinellid larvae consuming different numbers of aphids (Prey Quantity bioassay), and starved coccinellid larvae consuming a chrysopid larvae that had consumed aphids (Direct and Indirect Predation bioassay). Prey taxonomic assignment against a mitochondrial genome database had high accuracy (99.8% positive predictive value) and the number of prey reads was directly related to the number of prey consumed and inversely related to the elapsed time since consumption with high significance (r2  = .932, p = 4.92E-6). Aphids were detected up to 6 h after direct predation plus 3 h after indirect predation (9 h in total) and detection was related to the predator-specific decay rates. Lazaro enabled quantitative predictions of prey consumption across multiple trophic levels with high taxonomic resolution while eliminating all false positives, except for a few confirmed contaminants, and may be valuable for characterizing prey consumed by field-sampled predators. Moreover, Lazaro is readily applicable for species diversity determination from any degraded environmental DNA.

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BACKGROUND: A central challenge of DNA gut content analysis is to identify prey in a highly degraded DNA community. In this study, we evaluated prey detection using metabarcoding and a method of mapping unassembled shotgun reads (Lazaro). RESULTS: In a mock prey community, metabarcoding did not detect any prey, probably owing to primer choice and/or preferential predator DNA amplification, while Lazaro detected prey with accuracy 43-71%. Gut content analysis of field-collected arthropod epigeal predators (3 ants, 1 dermapteran, and 1 carabid) from agricultural habitats in Brazil (27 samples, 46-273 individuals per sample) revealed that 64% of the prey species detections by either method were not confirmed by melting curve analysis and 87% of the true prey were detected in common. We hypothesized that Lazaro would detect fewer true- and false-positive and more false-negative prey with greater taxonomic resolution than metabarcoding but found that the methods were similar in sensitivity, specificity, false discovery rate, false omission rate, and accuracy. There was a positive correlation between the relative prey DNA concentration in the samples and the number of prey reads detected by Lazaro, while this was inconsistent for metabarcoding. CONCLUSIONS: Metabarcoding and Lazaro had similar, but partially complementary, detection of prey in arthropod predator guts. However, while Lazaro was almost 2× more expensive, the number of reads was related to the amount of prey DNA, suggesting that Lazaro may provide quantitative prey information while metabarcoding did not.

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As environmental change persists, understanding resource use patterns is of value to predict the consequences of shifting trophic structures. While many sharks are opportunistic predators, some exhibit prey selectivity, putting them at higher risk compared to species with greater trophic plasticity. In the Gulf of Mexico (GOM), Clupeids and Sciaenids comprise 69% of blacktip shark (Carcharhinus limbatus) diets, which is consequential considering potential responses of these prey groups to disturbance and over harvesting. We assessed if blacktips exhibit selectivity for Clupeids and Sciaenids in the western GOM based on stomach contents from sharks in coastal Texas. Clupeids comprised <2% of diets, while striped mullet (Mugil cephalus) and red drum (Sciaenops ocellatus) comprised >70% of identifiable prey. Ontogenetic shifts from smaller (Clupeids, small Sciaenids) to larger, higher trophic level (Ariidae, Elasmobranchii) prey fits our understanding of foraging among coastal sharks, and suggests our regional understanding of blacktip trophic ecology may be limited by the sizes of sampled sharks. Observed increases in blacktip densities coupled with declines in prey (Mugilids, Sciaenids) is concerning if blacktips have limited diet plasticity. Yet GOM blacktips may be more generalized than previously thought, which is promising for conservation and management.

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We developed species-specific primers of five microcrustacean preys, Ceriodaphnia richardi, Diaphanosoma cf. brevireme, Daphnia gessneri, Simocephalus serrulatus, Thermocyclops decipiens and Mesocyclops sp., to analyze food-web interactions involving their two insect predators Rheumatobates crassifemur and Martarega uruguayensis distributed in a tropical shallow lake. We designed internal primers of the COI gene (177-282 bp), and tested them, by means of PCR, for specificity and sensitivity. In our tests for specificity, all primers successfully amplified the DNA target but were species-specific failing to amplify the biomarker from any of the other species tested, even in a mixed DNA sample, including predators' DNA. In tests for sensitivity, primers successfully amplified zooplankton biomarkers from low concentration of DNA extractions and also from digestive tract of predators, even after many hours of ingestion. This technique provides a framework as an efficient tool for evaluation of food-web research in natural aquatic environments, where it is impossible to observe if predation occurs. Furthermore, this technique provides an effective solution for the identification of zooplankton species from the predator's digestive tract, where morphological identification alone is sometimes difficult because predators do not consume the prey but feeds using extra-oral digestion, such is the case of heteropterans.

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Diaphorina citri Kuwayama is the vector of the pathogenic bacteria Candidatus Liberibacter spp., the causative agent of Huanglongbing (HLB), the most serious disease of citrus worldwide. Because predatory insects have been historically neglected in biological control programs for D. citri, the impact of generalist predators on D. citri population densities is little understood. A useful tool to evaluate the dynamics of predator-prey interactions is molecular analysis of predators' gut content. We constructed a specific molecular marker to detect D. citri DNA in the gut content of predator insects, for use in estimating the predation rate of field-collected predators in citrus orchards on D. citri. Bioassays of the DNA half-life detection time were carried out with two predatory species, the ladybird beetle Hippodamia convergens Guérin-Méneville and the lacewing Chrysoperla externa (Hagen). The D. citri DNA half-life detection time (DT50) was 6.11 h for H. convergens and 5.46 h for C. externa. One hundred and seven field-collected predators were used for gut-content analysis (52 larvae/adults of ladybirds and 55 larvae of lacewings). The assays showed that 17.3% of ladybirds but no lacewings tested positive for D. citri DNA. These results show that generalist predators can contribute to biological control of D. citri and should be considered for use in pest management programs in citrus orchards.

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Resumen: La determinación de los hábitos alimentarios permite la comprensión de muchos procesos ecológicos que se dan en los ecosistemas lóticos. El objetivo de este trabajo es: 1) conocer los hábitos alimentarios de ninfas de Claudioperla tigrina en un amplio gradiente altitudinal y latitudinal en Argentina; 2) asignar el grupo funcional trófico (GFT) de C. tigrina; 3) evaluar diferencias entre dietas de los sitios estudiados. Las ninfas estudiadas fueron recolectadas a lo largo de un amplio rango espacial en el noroeste de Argentina e incluyen diferentes estados del desarrollo (cuatro clases de tamaño). El material ingerido fue extraído de la parte anterior del tubo digestivo mediante disección ventral del tórax. Los perfiles dietarios fueron analizados a través de la estimación de parámetros de la distribución multinomial de Dirichlet. Se efectuaron ANOVA's para cada rubro alimentario en función de los sitios. Se utilizó la técnica Multidimensional Scaling (MDS) para identificar los sitios con perfiles alimentarios promedios similares. Se realizó un análisis de amplitud de nicho trófico para evaluar la diversificación de los recursos consumidos en cada sitio. El aparato bucal no muestra variaciones a lo largo del desarrollo ninfal, excepto una mayor esclerotización con la edad. Poseen palpos mandibulares y maxilares relativamente cortos y mandíbulas y maxilas fuertes, esclerozadas y dentadas. Las ninfas ingirieron más de un ítem alimentario, sugiriendo que se trata de una especie flexible en su alimentación. El ítem dominante en la dieta fue la MOPG, los signos de haber sido masticado indican hábitos trituradores. Se encontraron diferencias entre sitios para los ítems MOPF, invertebrados, algas y sedimento, pero no para MOPG. Se obtuvieron las correlaciones para cada eje del análisis MDS. Con el eje 1 se correlacionaron negativamente los sitios AP, LT, LI, C y M (Selva de Yungas y Pastizal de neblina) asociados a un mayor consumo de MOPF, en el lado positivo del eje se encontró el sitio P (Altos Andes) asociado a los ítems invertebrados y sedimento. Para el eje 2 se relacionaron positivamente IN (Pastizal neblina) y LR (Monte de Sierras y Bolsones) con la presencia de algas. Los resultados del análisis de amplitud de nicho revelaron diferencias en el uso de los recursos secundarios de la dieta en los sitios estudiados que puede deberse a la oferta ambiental en las ecorregiones consideradas. El GFT de C. tigrina es triturador/ recolector en Selva de Yungas y Pastizales de Neblina y triturador/ depredador en Altos Andes. La definición del GFT de C. tigrina y el establecimiento de su rol como procesador primario de materia orgánica representa un paso importante para futuros estudios de redes tróficas y otras metodologías basadas en los grupos funcionales.

Abstract: Dietary information gives insight into several ecological processes acting in lotic ecosystems. This work aimed: 1) to identify the dietary habits of Claudioperla tigrina immature stages along a wide altitudinal as well as latitudinal gradient in North Argentina; 2) to define the functional feeding group (FFG) of C. tigrina; 3) to evaluate differences in diet in the studied sites. Studied nymphs were collected from localities widely scattered in Northwestern Argentina and they fell into different developmental stages (four size classes). The ingested material was extracted from the foregut and midgut by using thorax ventral dissection. Dietary profiles were analyzed through the estimation of parameters associated with a Dirichletmultinomial distribution. ANOVA's were performed for each food item using sites as factor. Multidimensional Scaling was used to identify sites with similar dietary profiles. An analysis of food-niche breadth was also performed to evaluate the degree of dietary diversification for the resources consumed in each site. Mouthparts are similar across the different size classes, excepting the increasing sclerotization recorded with age. Mouthparts retained most of the typical chewing groundplan, showing relatively short labial and maxillar palps, and strong, sclerotized and denticulated mandibles and maxillae. Our results pointed out that the nymphs of C. tigrina always ingest two or more food items (CPOM, FPOM, invertebrates and algae), suggesting thus a flexible diet. The diet changed in relation to body size, while finer particles were consumed in the early stages, larger particles were ingested in final stages. Coarse particulate organic matter was the dominant food item, with signals of shredding during ingestion. Differences between sites for FPOM, invertebrates, algae and sediment were detected, but not for MOPG. Correlations were obtained for the first two axis of the MDS analysis. Sites AP, LT, LI, C and M (Yungas Rainforest and Humid Grassland) were negatively correlated with the axis 1 which was associated with increased consumption of FPOM. On the positive side of the axis the site P (High Andes) is associated with a greater proportion of invertebrates and sediment. The sites IN (Humid Grassland) and LR (Argentine Northwest Monte and Thistle of the Prepuna) were located at the positive domain of axis 2 which is in turn associated with a greater count of algae in the dietary contents. We found significant differences in the quantity of secondary items, and this is likely related with the resources environmental availability. The FFG of Claudioperla tigrina is primarily shredder/collector-gatherer in Yungas Rainforest and Humid Grassland shredder/predator in High Andes. FFG classification of C. tigrina and the definition of their role for organic matter processing is an important step for future studies based on functional groups such as analysis of food webs. Rev. Biol. Trop. 65 (3): 1174-1184. Epub 2017 September 01.