Molecular prey identification in Central European piscivores

Diet analysis is an important aspect when investigating the ecology of fish‐eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time‐consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two‐step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection of fish DNA in dietary samples. This approach encompasses 78 fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. All targeted taxa were successfully amplified from 25 template molecules, and each assay was specific when tested against a wide range of invertebrates and vertebrates inhabiting aquatic environments. The applicability of the multiplex PCR system was evaluated in a feeding trial, wherein it outperformed morphological prey analysis regarding species‐specific prey identification in faeces of Eurasian otters. Additionally, a wide spectrum of fish species was detected in field‐collected faecal samples and regurgitated pellets of Common Kingfishers and Great Cormorants, demonstrating the broad applicability of the approach. In conclusion, this multiplex PCR system provides an efficient, easy to use and cost‐effective tool for assessing the trophic ecology of piscivores in Central Europe. Furthermore, the multiplex PCRs and the primers described therein will be applicable wherever DNA of the targeted fish species needs to be detected at high sensitivity and specificity.     

Prey selection by linyphiid spiders: molecular tracking of the effects of alternative prey on rates of aphid consumption in the field

A molecular approach, using aphid-specific monoclonal antibodies, was used to test the hypothesis that alternative prey can affect predation on aphids by linyphiid spiders. These spiders locate their webs in cereal crops within microsites where prey density is high. Previous work demonstrated that of two subfamilies of Linyphiidae, one, the Linyphiinae, is web-dependent and makes its webs at sites where they were more likely to intercept flying insects plus those (principally aphids) falling from the crop above. The other, the Erigoninae, is less web-dependent, making its webs at ground level at sites with higher densities of ground-living detritivores, especially Collembola. The guts of the spiders were analysed to detect aphid proteins using enzyme-linked immunosorbent assay (ELISA). Female spiders were consuming more aphid than males of both subfamilies and female Linyphiinae were, as predicted, eating more aphid than female Erigoninae. Rates of predation on aphids by Linyphiinae were related to aphid density and were not affected by the availability of alternative prey. However, predation by the Erigoninae on aphids was significantly affected by Collembola density. Itinerant Linyphiinae, caught away from their webs, contained the same concentration of aphid in their guts as web-owners. However, nonweb-owning Erigoninae, living away from Collembola aggregations at web-sites, contained significantly higher concentrations of aphid. For both subfamilies there was evidence of a disproportionate increase in predation on aphids once Collembola populations had declined. It was concluded that nonaphid prey, by helping to maintain spiders in the field, can significantly affect predation on aphids.     

Electrophoretic analysis of the prey spectrum of Teretrius nigrescens (Lewis) (Col., Histeridae), a predator of Prostephanus truncatus (Horn) (Col., Bostrichidae), in Mexico, Honduras, and Benin

Abstract: Extensive collections of Teretrius nigrescens in Mexico, Honduras, and Benin, were electrophoretically analysed to elucidate the prey spectrum of the predator. Both polyacrylamide gel electrophoresis and cellulose acetate electrophoresis were used. Beetles were sampled with pheromone traps, using the synthetic aggregation pheromone of Prostephanus truncatus , and directly from farmers' maize stores. The proportion of electrophoretically detected prey protein from adult T. nigrescens in pheromone traps was low: of the 1108 specimens analysed, only in 34 cases, prey protein could be clearly identified. More frequently, prey protein was detected in adult T. nigrescens sampled in maize stores in Mexico, Honduras, and Benin, with 87 samples showing distinct prey bands of the total 1214 predators analysed. Of the 241 T. nigrescens larvae sampled in maize stores in Benin, 136 showed distinct bands of prey protein. In all samples, P. truncatus was the most frequently detected prey species. The second most often identified prey species was Sitophilus zeamais . The results are discussed with regard to various methods for prey spectrum analysis and specifically the biology of T. nigrescens .     

Selectivity underlies the dissociation between seasonal prey availability and prey consumption in a generalist predator

Generalist predators are capable of selective foraging, but are predicted to feed in close proportion to prey availability to maximize energetic intake especially when overall prey availability is low. By extension, they are also expected to feed in a more frequency‐dependent manner during winter compared to the more favourable foraging conditions during spring, summer and fall seasons. For 18 months, we observed the foraging patterns of forest‐dwelling wolf spiders from the genus Schizocosa (Araneae: Lycosidae) using PCR‐based gut‐content analysis and simultaneously monitored the activity densities of two common prey: springtails (Collembola) and flies (Diptera). Rates of prey detection within spider guts relative to rates of prey collected in traps were estimated using Roualdes’ c_st model and compared using various linear contrasts to make inferences pertaining to seasonal prey selectivity. Results indicated spiders foraged selectively over the course of the study, contrary to predictions derived from optimal foraging theory. Even during winter, with overall low prey densities, the relative rates of predation compared to available prey differed significantly over time and by prey group. Moreover, these spiders appeared to diversify their diets; the least abundant prey group was consistently overrepresented in the diet within a given season. We suggest that foraging in generalist predators is not necessarily restricted to frequency dependency during winter. In fact, foraging motives other than energy maximization, such as a more nutrient‐focused strategy, may also be optimal for generalist predators during prey‐scarce winters.     

Quantitative prey species detection in predator guts across multiple trophic levels by mapping unassembled shotgun reads

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.     

Land cover diversity increases predator aggregation and consumption of prey

A lower diversity of land cover types is purported to decrease arthropod diversity in agroecosystems and is dependent on patterns of land use and fragmentation. Ants, important providers of ecosystem services such as biological control, are susceptible to landscape‐level changes. We determined the relationships between land cover diversity and fragmentation on the within‐field spatial associations of ants to pests and resulting predation events by combining mapping and molecular tools. Increased land cover diversity and decreased fragmentation increased ant abundance, spatial association to pests and predation. Land cover diversity and fragmentation were more explanatory than land cover types. Even so, specific land cover types, such as deciduous forest, influenced ant and pest diversity more so than abundance. These results indicate that geospatial techniques and molecular gut content analysis can be combined to determine the role of land use in influencing predator–prey interactions and resulting predation events in agroecosystems.     

Differential antigen decay rates during digestion of molluscan prey by carabid predators

Carabid beetles (Coleoptera: Carabidae) were fed upon slugs (Mollusca: Pulmonata) in the laboratory, and their crop contents analysed for mollusc remains, using an enzyme-linked immunosorbent assay (ELISA) with an anti-Arion ater (L.) haemolymph antiserum. Crop weight loss and antigenic recognition of prey proteins were examined as separate variables in a series of validatory experiments. Two model predators,Abax parallelepipedus Piller and Mitterpacher andPterostichus madidus F., were fed upon two species of pest slugs,Deroceras reticulatum (Müller) andArion hortensis Férussac. The fitting of regression equations to the transformed antigenic response data allowed the ‘half-life’ and detection period to be calculated for each predator-prey combination. Following a one hour feeding interval, the half-life of the antigenic response toD. reticulatum remains was almost twice as long inP. madidus as that inA. parallelepipedus, and the detection period more than 2.5 times as long. However, covariant analysis showed that there was a significant difference between predator species in the rate at which detectability declined, but not in the rate of crop weight loss. WhenA. parallelepipedus was allowed to feed uponA. hortensis for eight hours, prey remains were still detectable at the end of the experiment, 13 days after feeding. Calibration of the differential rates of antigen decay and crop weight loss could potentially be used to calculate the size of the original meal, but only if prey species, and the time since feeding, can be determined. Potential solutions to these problems are discussed.     

Detection and decay rates of prey and prey symbionts in the gut of a predator through metagenomics

DNA methods are useful to identify ingested prey items from the gut of predators, but reliable detection is hampered by low amounts of degraded DNA. PCR‐based methods can retrieve minute amounts of starting material but suffer from amplification biases and cross‐reactions with the predator and related species genomes. Here, we use PCR‐free direct shotgun sequencing of total DNA isolated from the gut of the harlequin ladybird Harmonia axyridis at five time points after feeding on a single pea aphid Acyrthosiphon pisum. Sequence reads were matched to three reference databases: Insecta mitogenomes of 587 species, including H. axyridis sequenced here; A. pisum nuclear genome scaffolds; and scaffolds and complete genomes of 13 potential bacterial symbionts. Immediately after feeding, multicopy mtDNA of A. pisum was detected in tens of reads, while hundreds of matches to nuclear scaffolds were detected. Aphid nuclear DNA and mtDNA decayed at similar rates (0.281 and 0.11 h^?1 respectively), and the detectability periods were 32.7 and 23.1 h. Metagenomic sequencing also revealed thousands of reads of the obligate Buchnera aphidicola and facultative Regiella insecticola aphid symbionts, which showed exponential decay rates significantly faster than aphid DNA (0.694 and 0.80 h^?1, respectively). However, the facultative aphid symbionts Hamiltonella defensa, Arsenophonus spp. and Serratia symbiotica showed an unexpected temporary increase in population size by 1–2 orders of magnitude in the predator guts before declining. Metagenomics is a powerful tool that can reveal complex relationships and the dynamics of interactions among predators, prey and their symbionts.     

Prey DNA detection success following digestion by intraguild predators: influence of prey and predator species

Intraguild predation (IGP) has been increasingly recognized as an important interaction in ecological systems over the past two decades, and remarkable insights have been gained into its nature and prevalence. We have developed a technique using molecular gut-content analysis to compare the rate of IGP between closely related species of coccinellid beetles (lady beetles or ladybirds), which had been previously known to prey upon one another. We first developed PCR primers for each of four lady beetle species: Harmonia axyridis, Coccinella septempunctata, Coleomegilla maculata and Propylea quatuordecimpunctata. We next determined the prey DNA detection success over time (DS(50) ) for each combination of interacting species following a meal. We found that DS(50) values varied greatly between predator-prey combinations, ranging from 5.2 to 19.3 h. As a result, general patterns of detection times based upon predator or prey species alone are not discernable. We used the DS(50) values to correct field data to demonstrate the importance of compensation for detection times that are specific to particular predator-prey combinations.     

Molecular diagnostics reveal spiders that exploit prey vibrational signals used in sexual communication

Vibrational signalling is a widespread form of animal communication and, in the form of sexual communication, has been generally regarded as inherently short‐range and a private communication channel, free from eavesdropping by generalist predators. A combination of fieldwork and laboratory experiments was used to test the hypothesis that predators can intercept and exploit such signals. First, we developed and characterized PCR primers specific for leafhoppers of the genus Aphrodes and specifically for the species Aphrodes makarovi. Spiders were collected from sites where leafhoppers were present and screened with these primers to establish which spider species were significant predators of this species during the mating period of these leafhoppers. Analysis using PCR of the gut contents of tangle‐web spiders, Enoplognatha ovata (Theridiidae), showed that they consume leafhoppers in the field at a greater rate when signalling adults were present than when nymphs were dominant, suggesting that the spiders were using these vibrations signals to find their prey. Playback and microcosm experiments then showed that E. ovata can use the vibrational signals of male leafhoppers as a cue during foraging and, as a result, killed significantly more male than female A. makarovi. Our results show, for the first time, that arthropod predators can exploit prey vibrational communication to obtain information about prey availability and use this information to locate and capture prey. This may be a widespread mechanism for prey location, one that is likely to be a major unrecognized driver of evolution in both predators and prey.     

Species on the menu of a generalist predator, the eastern red bat (Lasiurus borealis): using a molecular approach to detect arthropod prey

One of the most difficult interactions to observe in nature is the relationship between a predator and its prey. When direct observations are impossible, we rely on morphological classification of prey remains, although this is particularly challenging among generalist predators whose faeces contain mixed and degraded prey fragments. In this investigation, we used a polymerase chain reaction and sequence-based technique to identify prey fragments in the guano of the generalist insectivore, the eastern red bat ( Lasiurus borealis ), and evaluate several hypotheses about prey selection and prey defences. The interaction between bats and insects is of significant evolutionary interest because of the adaptive nature of insect hearing against echolocation. However, measuring the successes of predator tactics or particular prey defences is limited because we cannot normally identify these digested prey fragments beyond order or family. Using a molecular approach, we recovered sequences from 89% of the fragments tested, and through comparison to a reference database of sequences, we were able to identify 127 different species of prey. Our results indicate that despite the robust jaws of L. borealis , most prey taxa were softer-bodied Lepidoptera. Surprisingly, more than 60% of the prey species were tympanate, with ears thought to afford protection against these echolocating bats. Moths of the family Arctiidae, which employ multiple defensive strategies, were not detected as a significant dietary component. Our results provide an unprecedented level of detail for the study of predator–prey relationships in bats and demonstrate the advantages which molecular tools can provide in investigations of complex ecological systems and food-web relationships.     

Predator gut state and prey detectability using electrophoretic analysis of gut contents

Abstract. I. An investigation of the effects of position of food in the gut (gut state), time since feeding, meal size and environmental temperature on the detection of prey using the electrophoretic separation of proteins with esterase activity was undertaken. The waterbug Notonecta glauca Linn (Hemiptera/Heteroptera) was used as the model predator.
2. Seven easily recognizable gut states can be identified at different times since commencement of a meal.
3. Specific prey esterase 'fingerprint' bands can be detected whilst food remains in the foregut.
4. Meal size and environmental temperature (affecting digestion and assimilation rates) both affect the rate of filling and emptying of the foregut, and hence the time period over which prey proteins can be detected by electrophoresis following a meal. Small meals and high temperatures lead to quite rapid changes in gut state with time and thus reduce the time period over which prey can be identified from gut contents (20 h). Large meals and low temperatures extend this period (48 h).
5. Predator gut state is the most important parameter in prey detectability from gut contents, and from a visual examination of the gut state it is immediately evident whether or not prey detection by electrophoresis will be possible.
6. There is further evidence in support of a twocompartment food processing model for Nofoonecru , and the significance of this type of processing in relation to gut content analysis of sucking predators is discussed.     

Molecular detection of invertebrate prey in vertebrate diets: trophic ecology of Caribbean island lizards

Understanding community assembly and population dynamics frequently requires detailed knowledge of food web structure. For many consumers, obtaining precise information about diet composition has traditionally required sacrificing animals or other highly invasive procedures, generating tension between maintaining intact study populations and knowing what they eat. We developed 16S mitochondrial DNA sequencing methods to identify arthropods in the diets of generalist vertebrate predators without requiring a blocking primer. We demonstrate the utility of these methods for a common Caribbean lizard that has been intensively studied in the context of small island food webs: Anolis sagrei (a semi‐arboreal ‘trunk‐ground’ anole ecomorph). Novel PCR primers were identified in silico and tested in vitro. Illumina sequencing successfully characterized the arthropod component of 168 faecal DNA samples collected during three field trips spanning 12 months, revealing 217 molecular operational taxonomic units (mOTUs) from at least nine arthropod orders (including Araneae, Blattodea, Coleoptera, Hemiptera, Hymenoptera, Isoptera, Lepidoptera and Orthoptera). Three mOTUs (one beetle, one cockroach and one ant) were particularly frequent, occurring in ≥50% of samples, but the majority of mOTUs were infrequent (180, or 83%, occurred in ≤5% of samples). Species accumulation curves showed that dietary richness and composition were similar between size‐dimorphic sexes; however, female lizards had greater per‐sample dietary richness than males. Overall diet composition (but not richness) was significantly different across seasons, and we found more pronounced interindividual variation in December than in May. These methods will be generally useful in characterizing the diets of diverse insectivorous vertebrates.     

Sequence information and preparation of resolved by; two-dimensional gel electrophoresis: no longer just spots on gels

In 1975 O'Farrell described, in detail, a procedure to separate proteins by polyacrylamide gel electrophoresis in two dimensions. This powerful new technique relied on two characteristics of proteins: charge and molecular mass. In the first dimension, proteins were separated on the basis of net charge in a pH gradient by isoelectric focusing, and in the second dimension the proteins were further fractionated exclusively on the basis of their molecular mass by SDS gel electrophoresis. Since two-dimensional gel electrophoresis (2DGE) has a resolving power of at least 20 fold greater than one-dimensional electrophoresis, it has found wide spread application in modern biological research. However, beyond the detection of a given protein, 2DGE provides little additional information about a specific protein other than molecular mass, isoelectric point, and approximate relative abundance. In recent years, the development of new technologies have made it possible to directly obtain sequence information, and produce specific antisera for proteins resolved by 2DGE. These new technological developments serve to further increase the power and utility of 2DGE in the analysis of proteins of importance to plant physiology.     

The significance of facultative scavenging in generalist predator nutrition: detecting decayed prey in the guts of predators using PCR

Gut-content analyses using molecular techniques are an effective approach to quantifying predator-prey interactions. Predation is often assumed but scavenging is an equally likely route by which animal DNA enters the gut of a predator/scavenger. We used PCR (polymerase chain reaction) to detect scavenged material in predator gut homogenates. The rates at which DNA in decaying slugs (Mollusca: Pulmonata) and aphids (Homoptera: Aphididae) became undetectable were estimated. The detectability of DNA from both carrion types in the guts of the generalist predator Pterostichus melanarius (Coleoptera: Carabidae) was then determined. The effects of carrion age and weight, as well as beetle sex, on detection periods, were quantified. Laboratory trials measured prey preference of beetles between live and decaying prey. Further experiments measured, for the first time, feeding by P. melanarius on dead slugs and aphids directly in the field. In both field and laboratory, P. melanarius preferentially fed on dead prey if available, but preference changed as the prey became increasingly decayed. Disappearance rates for slug carrion in wheat fields and grasslands were estimated and P. melanarius was identified as the main scavenger. Comparison of the retention time for dead slugs in the field, with the detection period for decaying slug material in the guts of the predators, showed that PCR-based techniques are not able to distinguish between predated and scavenged food items. This could potentially lead to overestimation of the impact of predation on slugs (and other prey) by carabids. Possible implications of facultative scavenging by invertebrate predators for biocontrol and food-web research are discussed.     

卡他莫拉菌UspA1蛋白多克隆抗体的制备及鉴定

为制备抗卡他莫拉菌(Moraxella catarrhalis,Mc)表面蛋白UspA1胞外结构域的多克隆抗体(PcAb),对UspA1蛋白进行生物信息学分析,获取胞外结构域中抗原表位最为丰富的肽段,找到其对应的基因序列并引入大肠杆菌偏好性密码子,对其优化后化学合成全基因序列。将该基因序列按常规方法克隆入表达载体p ET-28a(+)后表达重组UspA1-His融合蛋白并纯化。以该纯化抗原免疫新西兰大白兔,经4次免疫后,用Protein A亲和层析柱从抗血清中纯化出抗UspA1-His融合蛋白PcAbIgG。经免疫荧光法、酶联免疫吸附法及Western blotting鉴定,抗UspA1-His融合蛋白PcAb能特异性识别UspA1蛋白的表面暴露区。该多抗的制备为下一步建立卡他莫拉菌快速检测技术奠定了基础。     

抗吡虫啉单克隆抗体的制备及鉴定

为制备灵敏度高,特异性强的抗吡虫啉单克隆抗体,建立经济、快速、准确的吡虫啉残留免疫学分析方法,采用分子模拟技术分析吡虫啉及其类似农药的电荷分布后,选择1-[6-(2-羧乙硫基-3-吡啶)甲基]-N-硝基-2-咪唑啉亚胺 (H1) 作为免疫半抗原,1-(6-氯-3-吡啶甲基)-3-羧丙基-N-硝基-2-咪唑啉亚胺 (H2) 作为包被半抗原,利用NHS酯法将H1和H2分别与牛血清蛋白 (BSA) 和卵清蛋白 (OVA) 偶联合成免疫原与包被原。免疫BALB/c小鼠后,采用常规杂交瘤技术共获得2株稳定分泌抗吡虫