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.     

Predation of cotton bollworm by green lacewings in the presence of cotton aphid as alternative prey on transgenic Bt and conventional cotton

Experiments were conducted in small arenas and on whole plants to explore the effect of cotton aphids, Aphis gossypii Glover (Hemiptera: Aphididae), as alternative prey on the predation of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) larvae by green lacewing larvae, Mallada signatus Schneider (Neuroptera: Chrysopidae). Transgenic Bt (Bollgard II^®) and conventional cotton plants were included to explore potential differences in the predator's performance on these cotton types. In small arenas, the presence of 20 aphids reduced predation on H. armigera larvae by 22% (from 5.5 to 3.3 of 10) by a single lacewing larva over a 24‐h period. The presence of H. armigera reduced predation on aphids by ca. 29% (from 16.8 to 11.0 of 20) over 24 h. On whole plants, the presence of alternative prey had no effect on the number of H. armigera larvae or aphids remaining after 3 days. The presence of H. armigera larvae alone, without the predator, caused a 24% reduction in the numbers of aphids on conventional, but not on Bt cotton plants. The combination of Bt cotton and lacewing larvae caused a 96.6% removal of early‐stage H. armigera larvae, a statistically significant increase over the addition of the proportions (91.6%) removed by each factor measured separately, providing evidence of synergism. These studies suggest that the presence of aphids as alternative prey would not necessarily disrupt the predation by green lacewing on larvae of H. armigera, especially on Bt cotton.     

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe‐based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra‐ and inter‐assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100‐fold more sensitive than the cPCR. No cross‐reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10¹¹ and 2·75 × 10⁸ target copies ml^?1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.     

Teasing apart crypsis and aposematism – evidence that disruptive coloration reduces predation on a noxious toad

Both cryptic and aposematic colour patterns can reduce predation risk to prey. These distinct strategies may not be mutually exclusive, because the impact of prey coloration depends on a predator's sensory system and cognition and on the environmental background. Determining whether prey signals are cryptic or aposematic is a prerequisite for understanding the ecological and evolutionary implications of predator–prey interactions. This study investigates whether coloration and pattern in an exceptionally polymorphic toad, Rhinella alata, from Barro Colorado Island, Panama reduces predation via background matching, disruptive coloration, and/or aposematic signaling. When clay model replicas of R. alata were placed on leaf litter, the model's dorsal pattern – but not its colour – affected attack rates by birds. When models were placed on white paper, patterned and un‐patterned replicas had similar attack rates by birds. These results indicate that dorsal patterns in R. alata are functionally cryptic and emphasize the potential effectiveness of disruptive coloration in a vertebrate taxon.     

Refining trophic dynamics through multi‐factor Bayesian mixing models: A case study of subterranean beetles

Food web dynamics are vital in shaping the functional ecology of ecosystems. However, trophic ecology is still in its infancy in groundwater ecosystems due to the cryptic nature of these environments. To unravel trophic interactions between subterranean biota, we applied an interdisciplinary Bayesian mixing model design (multi‐factor BMM) based on the integration of faunal C and N bulk tissue stable isotope data (δ¹³C and δ¹⁵N) with radiocarbon data (Δ¹⁴C), and prior information from metagenomic analyses. We further compared outcomes from multi‐factor BMM with a conventional isotope double proxy mixing model (SIA BMM), triple proxy (δ¹³C, δ¹⁵N, and Δ¹⁴C, multi‐proxy BMM), and double proxy combined with DNA prior information (SIA + DNA BMM) designs. Three species of subterranean beetles (Paroster macrosturtensis, Paroster mesosturtensis, and Paroster microsturtensis) and their main prey items Chiltoniidae amphipods (AM1: Scutachiltonia axfordi and AM2: Yilgarniella sturtensis), cyclopoids and harpacticoids from a calcrete in Western Australia were targeted. Diet estimations from stable isotope only models (SIA BMM) indicated homogeneous patterns with modest preferences for amphipods as prey items. Multi‐proxy BMM suggested increased—and species‐specific—predatory pressures on amphipods coupled with high rates of scavenging/predation on sister species. SIA + DNA BMM showed marked preferences for amphipods AM1 and AM2, and reduced interspecific scavenging/predation on Paroster species. Multi‐factorial BMM revealed the most precise estimations (lower overall SD and very marginal beetles' interspecific interactions), indicating consistent preferences for amphipods AM1 in all the beetles' diets. Incorporation of genetic priors allowed crucial refining of the feeding preferences, while integration of more expensive radiocarbon data as a third proxy (when combined with genetic data) produced more precise outcomes but close dietary reconstruction to that from SIA + DNA BMM. Further multidisciplinary modeling from other groundwater environments will help elucidate the potential behind these designs and bring light to the feeding ecology of one the most vital ecosystems worldwide.     

Studies of the effect of environmental factors on the rotifer predator–prey system in freshwater

The aim of this work is to evaluate the effect of environmental factors: temperature and photoperiod on the zooplankton predator–prey system. Rotifers, an important and cosmopolitan group of zooplankton in freshwater, were used in our study. We investigated the effect of temperature (20, 23, and 30°C) and of photoperiod (L:D = 12:0 and 0:12) on the predatory rotifer Asplanchna brightwelli consuming rotifer Brachionus calyciflorus as prey. Under A. brightwelli predation, populations of B. calyciflorus prey were consumed more slowly at 20 ± 1 and 30 ± 1°C as compared to 23 ± 1°C. Prey consumption by A. brightwelli increased from 0.63 ± 0.09 ind. predator⁻¹ at 20°C to a peak of 1.22 ± 0.12 ind. predator⁻¹ at 23°C, then decreased significantly to 0.93 ± 0.14 ind. predator⁻¹ at 30 ± 1°C. In addition, predation responded to temperature changing sensitively and rapidly. Statistical analysis showed that the prey consumption were significant different under altered temperature periods during 12 h. Photoperiod also significantly influenced the rate of A. brighwelli predation. B. calyciflorus suffered less predation in darkness than in light. The rate of prey consumption in light (1.06 ind. predator⁻¹) was twice the average of that in darkness (0.51 ind. predator⁻¹). Furthermore, predation rate varied under changing photoperiod but predators moved back into the light did not resume their original consumption rate. Our results demonstrate that whether the predation in rotifer successfully or not is strongly influenced by temperature and photoperiod.     

Nudibranch predation and dietary preference for the polyps of <Emphasis Type="Italic">Aurelia</Emphasis><Emphasis Type="Italic">labiata</Emphasis> (Cnidaria: Scyphozoa)

There is concern that jellyfish blooms may be increasing worldwide. Some factors controlling population size, such as temperature and food, often have been studied; however, the importance of predators is poorly known. Aeolid nudibranchs feed on cnidarians, but their predation on the benthic polyps of scyphozoan rarely has been documented. To understand the potential of nudibranchs to consume polyps, we tested several predation preference hypotheses with the generalist feeding nudibranch, Hermissenda crassicornis, and polyps of the common moon jellyfish, Aurelia labiata. Of the six prey species tested during feeding experiments, A. labiata polyps and the tunicate Distaplia occidentalis were significantly preferred. Nudibranch size, diurnal cycle, and ingestive conditioning did not significantly influence prey choice. Nudibranchs showed significant positive chemotaxis toward living polyps, hydroids, and tunicates, but not to sea anemones. Nudibranch chemotaxis was significantly more positive to polar extract of A. labiata than of D. occidentalis. Consumption of polyps was correlated with nudibranch size, with mean consumption by large nudibranchs (>0.92 g) of about 31 polyps h⁻¹. Three other nudibranch species also ate A. labiata polyps. Our results emphasize the potential importance of predation for controlling jellyfish benthic polyp populations and consequent jellyfish blooms.     

Coevolution of a marine gastropod predator and its dangerous bivalve prey

The fossil record of the interaction between the predatory whelk Sinistrofulgur and its dangerous hard‐shelled bivalve prey Mercenaria in the Plio‐Pleistocene of Florida was examined to evaluate the hypothesis that coevolution was a major driving force shaping the species interaction. Whelks use their shell lip to chip open the shell of their prey, often resulting in breakage to their own shells, as well as to their prey. Mercenaria evolved a larger shell in response to an intensifying level of whelk predation. Reciprocally, an increase in attack success (ratio of successful to unsuccessful attacks) and degree of stereotypy of attack position by the predator suggest reciprocal adaptation by Sinistrofulgur to increase efficiency in exploiting hard‐shelled prey. A decrease in prey effectiveness (ratio of unsuccessful to total whelk predation attempts) and an increase in the minimum boundary of a size refuge from whelk predation for Mercenaria may indicate that predator adaptation has outpaced prey antipredatory adaptation. Evolutionary size increase in Sinistrofulgur most likely occurred in response to prey adaptation to decrease the likelihood of feeding‐induced shell breakage and unsuccessful predation when encounters with damage‐inducing prey occur, coupled with (or reinforced by) an evolutionary response to the whelk's own predators. Predator adaptation to Mercenaria best explains temporal changes in whelk behaviour to decrease performance loss (shell breakage) associated with feeding on hard‐shelled prey; this behavioural change limits attacks on prey to when the whelk's shell lip is thickest and most resistant to breakage. Despite evidence of reciprocal adaptation between predator and prey, the contribution of Mercenaria to Sinistrofulgur evolution is likely only a component of the predator's response to dangerous bivalve prey. This study highlights the importance of understanding the interactions among several species in order to provide the appropriate context to test evolutionary hypotheses about any specific pair of species. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 409–436.     

Scaling up from individual behaviour of <Emphasis Type="Italic">Orius sauteri</Emphasis> foraging on <Emphasis Type="Italic">Thrips palmi</Emphasis> to its daily functional response

Functional responses of predators are generally measured under laboratory conditions at rather high prey densities. This is also true for the predation capability of the anthocorid predatory bug Orius sauteri (Poppius). To quantify the daily impact of one female Orius predator on its prey Thrips palmi Karny on greenhouse eggplants where the prey is present below the economic threshold density, we use its patch-leaving and feeding behaviour on eggplant leaves with different prey numbers and scale up to the larger spatio-temporal scale of the greenhouse and one foraging day by means of a simulation model. For this, we also use literature data on the distribution of T. palmi over eggplant leaves. The simulation results in a typical type II functional response for O. sauteri as a function of average T. palmi density: O. sauteri can find and eat approximately 10 prey items per day if T. palmi is present around its economic injury level. The daily mean number of prey eaten per O. sauteri predator, i.e., its predation capability, is highly sensitive to the actual baseline leaving tendency, the effect size of the presence of prey on the baseline leaving tendency and the effect size of the encounter rate with prey thereon.     

Within‐patch oviposition site shifts by spider mites in response to prior predation risks decrease predator patch exploitation

In egg‐laying animals with no post‐oviposition parental care, between‐ or within‐patch oviposition site selection can determine offspring survival. However, despite the accumulation of evidence supporting the substantial impact predators have on oviposition site selection, few studies have examined whether oviposition site shift within patches (“micro‐oviposition shift”) reduces predation risk to offspring. The benefits of prey micro‐oviposition shift are underestimated in environments where predators cannot disperse from prey patches. In this study, we examined micro‐oviposition shift by the herbivorous mite Tetranychus kanzawai in response to the predatory mite, Neoseiulus womersleyi, by testing its effects on predator patch exploitation in situations where predatory mites were free to disperse from prey patches. Adult T. kanzawai females construct three‐dimensional webs on leaf surfaces and usually lay eggs under the webs; however, females that have experienced predation risks, shift oviposition sites onto the webs even in the absence of current predation risks. We compared the predation of eggs on webs deposited by predator‐experienced females with those on leaf surfaces. Predatory mites left prey patches with more eggs unpredated when higher proportions of prey eggs were located on webs, and egg survival on webs was much higher than that on leaf surfaces. These results indicate that a micro‐oviposition shift by predator‐experienced T. kanzawai protects offspring from predation, suggesting adaptive learning and subsociality in this species. Conversely, fecundity and longevity of predator‐experienced T. kanzawai females were not reduced compared to those of predator‐naïve females; we could not detect any costs associated with the learned micro‐oviposition shift. Moreover, the previously experienced predation risks did not promote between‐patch dispersal of T. kanzawai females against subsequently encountered predators. Based on these results, the relationships of between‐patch oviposition site selection and micro‐oviposition shift are discussed.     

Predation rates of nemertean predators: the case of a rocky shore hoplonemertean feeding on amphipods

Estimates of the predation rates of benthic nemerteans are often based on observations of single individuals, and consequently they may not be representative for all members of a population of these predators. Herein we conducted controlled and repeatable laboratory experiments on the predation rate of the hoplonemertean Amphiporus nelsoni Sánchez 1973, which is common at exposed rocky shores along the central Chilean coast. During the austral fall (April, May 2000), nemerteans were observed in relatively high numbers crawling in the intertidal zone during early morning or late-afternoon low tides. When these nemerteans were offered living amphipods held by a forceps, they immediately attacked the amphipods and fed on them. In the laboratory experiments, nemerteans preferred the amphipod Hyale maroubrae Stebbing, 1899, which is also very common in the natural habitat of A. nelsoni. The nemerteans preyed to a higher extent on small males and non-ovigerous females than would have been expected from their abundance. We suggest that these (non-reproductive) stages of H. maroubrae are very mobile and therefore have a high likelihood of encounters with nemerteans. Predation rates reached maxima when nemerteans were provided prey densities of four or more of their preferred prey species, H. maroubrae, furthermore indicating that encounter rates with prey may affect predation rates. In long-term laboratory experiments, A. nelsoni consumed more amphipods during low tide conditions than during high tide conditions. Many nemerteans in the field prefer particular environmental conditions (e.g. nocturnal low tides), which restricts the time available for successful feeding. In the long-term experiment, predation rates of A. nelsoni never exceeded 0.5 amphipods nemertean^–1 d^–1. Maximum feeding events were 3 or 4 amphipods nemertean^–1 d^–1, but this only occurred during 10 out of a possible 2634 occasions. Nemerteans that had consumed 3 or 4 amphipods during 1 day, consumed substantially less prey during the following days. Towards the end of the long-term experiment, average predation rates decreased to 0.2 amphipods nemertean^–1 d^–1, corresponding to predation rates reported for other nemertean species (0.1–0.3 prey items nemertean^–1 d^–1). We suggest that predation rates from laboratory experiments represent maximum estimates that may not be directly transferable to field populations. Additionally, low predator–prey encounter rates with preferred prey in the field may further limit the predation impact of nemertean predators in natural habitats.     

Bioenergetics and growth in the ctenophore <Emphasis Type="Italic">Pleurobrachia pileus</Emphasis>

Knowledge of how energetic parameters relate to fluctuating factors in the natural habitat is necessary when evaluating the role of gelatinous zooplankton in the carbon flow of coastal waters. In laboratory experiments, we assessed feeding, respiration and growth of the ctenophore, Pleurobrachia pileus, and constructed carbon budgets. Clearance rates (F, l d⁻¹) of laboratory-reared Acartia tonsa as prey increased as a function of ctenophore polar length (L, mm) as F = 0.17L ^1.9. For ctenophores larger than about 11 mm, clearance rate was depressed in containers of 30–50 l volume. Clearance rates on field-collected prey were highest on the copepod, Centropages typicus, intermediate on the cladoceran, Evadne nordmanni and low on the copepods, Acartia clausi and Temora longicornis. Specific growth rates of 8–10 mm P. pileus increased with increasing prey concentrations to a maximum of 0.09 d⁻¹ attained at prey carbon densities of 40 and 100 μg C l⁻¹ of Artemia salina and A. tonsa, respectively. Weight-specific respiration rates increased hyperbolically with prey concentration. From experiments in which growth, ingestion and respiration were measured simultaneously, a carbon budget was constructed for individuals growing at maximum rates; from the measured parameters, the assimilation efficiency and net growth efficiency were estimated to be 22 and 37%, respectively. We conclude that the predation rates of P. pileus depend on ctenophore size, prey species, prey density and experimental container volume. Because the specific growth rates, respiration, assimilation and net growth efficiencies all were affected by food availability, knowledge of the ambient prey field is critical when evaluating the role of P. pileus in the carbon flow in coastal waters.     

Long‐range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Aims

An extra‐long‐range quantitative PCR (LR‐qPCR) method was developed for estimating genome damage to adenovirus 2 caused by UV irradiation. The objective was to use LR‐qPCR as a rapid method to determine adenovirus UV inactivation.

Methods

The LR‐qPCR consisted of two steps: a long‐range PCR (up to 10 kb fragment) and a real‐time, quantitative (q) PCR for quantifying the products of the first PCR. We evaluated LR‐qPCR with adenovirus irradiated with medium‐pressure (MP, polychromatic emission) and low‐pressure (LP, 254 nm) mercury vapour lamps and compared results with cell culture infectivity.

Results

Using LR‐qPCR, a fragment of 6 kb estimated DNA damage in a linear relationship to doses between 0 and 20 mJ cm^?2, and a 1‐kb fragment related linearly to doses between 20 and 100 mJ cm^?2. The LR‐qPCR results for the 6‐kb fragment were similar to infectivity assays results for adenovirus exposed to MP UV. For adenovirus irradiated with LP lamps, LR‐qPCR results for the shorter fragment size (1 kb) were similar to reduction in viral infectivity. No difference was observed between 10 and 6 kb LR‐qPCR results.

Conclusion

The LR‐qPCR can be used as a tool for estimating DNA damage caused by UV in adenovirus. The LR‐qPCR results were related to reduction in viral infectivity.

Significance and Impact of the Study

The use of LR‐qPCR to determine DNA damage and estimate inactivation of adenovirus 2 from UV disinfection allows for same‐day results compared with >7 days required for cell culture. This accelerates adenovirus inactivation results for the water industry where adenovirus is used as a representative virus for crediting UV systems. This PCR approach provides a framework that can be used for other viral viability assays using the inhibition of amplification of viral nucleic acid after pretreatments, such as propidium monoazide, and for cellular biology studies of DNA damage.     

Multiple intraguild predators reduce mortality risk of a mutual agricultural pest prey in simple,but not in complex,experimental settings

Multiple predator species that coexist with each other and their mutual prey can have combined effects on prey mortality that are similar to the sum of each predator's individual impact (linear effects), greater than the sum of each predator's individual impact (risk enhancement), or less than the sum of each predator's individual impact (risk reduction). Understanding multiple predator effects is important to determine the impact of predators on pest prey in agroecosystems. If two predators share the same broad spatial domain and hunting mode and engage in intraguild predation, then their combination is expected to result in risk reduction for a mutual prey. We tested this hypothesis using both additive and replacement experimental designs on two species of generalist wolf spider predators (Tasmanicosa leuckartii and Hogna crispipes) that hunt in the same domain, and a mutual insect prey (cotton bollworm Helicoverpa armigera). We used two types of enclosures: a small simple laboratory enclosure, and a larger more complex cotton plant enclosure. We found that in the small simple laboratory enclosures, the presence of two spiders led to risk reduction of Helicoverpa larva mortality as expected, but in larger more complex cotton plant enclosures the presence of both species resulted in linear effects rather than risk reduction on Helicoverpa mortality. Furthermore, intraguild predation did not change multiple predator effects in laboratory or plant enclosures. This study has implications for managing arthropod predators in agroecosystems; contrary to predictions of ecological frameworks, coexistence of predators that share the same hunting mode and hunting domain may not lead to risk reduction on a mutual prey in more complex environments, where encounters among predators can be lower. Conservation of multiple predators of a single guild can play an essential role on biological control of insect pests.     

Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

Environmental DNA (eDNA) analysis is a rapid, cost‐effective, non‐invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species‐specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and “metabarcoding” have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real‐time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using high‐throughput sequencing technology. With qPCR and a detection threshold of 1 of 12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4 of 12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species‐specific surveys.     

SPIKEPIPE: A metagenomic pipeline for the accurate quantification of eukaryotic species occurrences and intraspecific abundance change using DNA barcodes or mitogenomes

The accurate quantification of eukaryotic species abundances from bulk samples remains a key challenge for community ecology and environmental biomonitoring. We resolve this challenge by combining shotgun sequencing, mapping to reference DNA barcodes or to mitogenomes, and three correction factors: (a) a percent‐coverage threshold to filter out false positives, (b) an internal‐standard DNA spike‐in to correct for stochasticity during sequencing, and (c) technical replicates to correct for stochasticity across sequencing runs. The SPIKEPIPE pipeline achieves a strikingly high accuracy of intraspecific abundance estimates (in terms of DNA mass) from samples of known composition (mapping to barcodes R² = .93, mitogenomes R² = .95) and a high repeatability across environmental‐sample replicates (barcodes R² = .94, mitogenomes R² = .93). As proof of concept, we sequence arthropod samples from the High Arctic, systematically collected over 17 years, detecting changes in species richness, species‐specific abundances, and phenology. SPIKEPIPE provides cost‐efficient and reliable quantification of eukaryotic communities.     

A real‐time PCR assay for detection and quantification of Lactococcus garvieae

Aims: To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples. Methods and Results: A real‐time quantitative PCR (qPCR) assay with primers for CAU12F and CAU12R based on the 16S rRNA gene of L. garvieae was successfully established. The limit of detection for L. garvieae genomic DNA was 1 ng DNA in conventional PCR and 32 fg with a mean C_T value of 36·75 in qPCR. Quantification of L. garvieae vegetative cells was linear (R² = 0·99) over a 7‐log‐unit dynamic range down to ten L. garvieae cells. Conclusions: This method is highly specific, sensitive and reproducible for the detection of L. garvieae compared to gel‐based conventional PCR assays, thus providing precise quantification of L. garvieae in food and natural environments. Significance and Impact of the Study: This work provides efficient diagnostic and monitoring tools for the rapid identification of L. garvieae, an emerging pathogen in aquaculture and an occasional human pathogen from other members of the genus Lactobacillus.     

Spider predation on a mirid pest in Japanese rice fields

Spiders are common generalist predators, and understanding their potential in biological control is important for the development of integrated pest management programs. In this study, predation by three groups of spiders on the mirid bug Stenotus rubrovittatus (Hemiptera: Miridae) in rice paddies was investigated using DNA-based gut-content analysis. A laboratory feeding study revealed that the detection half-lives of bug DNA in the spider gut at 25 °C was 3.4 days for Lycosidae and 1.5 days for Tetragnathidae. Individual spider predation on the mirid bug was investigated by detecting DNA of prey in field-collected spiders. In total, 1199 spiders were assayed from three spider groups: Pirata subpiraticus (Lycosidae), Tetragnatha spp. (Tetra-gnathidae), and Pachygnatha clercki (Tetra-gnathidae), which each differ in their preferred microhabitat as well as their predatory habits. Detection rates of prey DNA in spiders increased significantly with the density of prey across all spider groups. P. subpiraticus and Tetragnatha spp. predation showed a better fit to a saturated response curve to increasing prey density, while P. clercki showed a simple linear relationship with prey density. Densities of alternative prey species did not affect the detection rates of mirids. These results suggest that predation on pests by generalist predators in an agroecosystem is affected not only by prey abundance but also by predator preference for specific prey. Predator preference is therefore an important factor to consider when estimating the role of natural enemies as biological control agents.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

Differential feeding by common heterotrophic protists on four Scrippsiella species of similar size

The dinoflagellate genus Scrippsiella is known to cause red tides. Mortality due to predation should be assessed in order to understand the population dynamics of Scrippsiella species. However, predation has been explored only in a few species. In this study, we examined feeding by common heterotrophic dinoflagellates Oxyrrhis marina, Gyrodinium dominans, Polykrikos kofoidii, Oblea rotunda, and Pfiesteria piscicida, and a ciliate Strombidinopsis sp., on four Scrippsiella species, of similar size, namely Scrippsiella acuminata, Scrippsiella donghaiensis, Scrippsiella lachrymosa, and Scrippsiella masanensis. All the heterotrophic protists tested could feed on all the four Scrippsiella species. However, the numerical and functional responses of P. kofoidii to the mean prey concentration were apparently different between the Scrippsiella species. With increasing prey concentration, the growth and ingestion rates of P. kofoidii on S. lachrymosa increased rapidly, and then saturated similar to those on S. acuminata, as previously reported, but those on S. donghaiensis continuously decreased. The cells of S. donghaiensis lysed P. kofoidii cells. In contrast, the growth and ingestion rates of P. kofoidii on S. masanensis were not significantly related to the prey concentration. At similarly high mean prey concentration, the growth and ingestion rates of G. dominans were significantly different between the four Scrippsiella species Therefore, differences in the growth and/or ingestion rates of G. dominans and P. kofoidii on the four Scrippsiella species might result in different ecological niches of both the predator and prey species.