Aphid DNA detection by molecular gut content analysis in a natural enemy consuming honeydew

The introduction of insectary and banker plants is widely used in agroecosystems to improve the survival and reproduction of natural enemies of pest arthropods. Natural enemies that are attracted to and/or maintained on insectary plants need to move from the insectary plants to crop plants to suppress pest populations effectively. In many cases, however, direct evidence supporting the migration of natural enemies is lacking. To investigate the movement of Orius bugs (Hemiptera: Anthocoridae), which are efficient zoophytophagous predators used in biological control, we performed gut content analyses based on DNA markers. Specific PCR primers were designed to detect the DNA of the insectary plant, Scaevola aemula. We also carried out gut content analyses using prey insect DNA markers to estimate Orius bugs’ predation spectrum. DNA of the insectary plant was detected in the abdomen of Orius bugs collected on eggplant leaves, suggesting that Orius bugs had consumed food resources from the insectary plants and relocated to the crop plants. DNA of three prey insect species—two thrips species, Thrips palmi and Frankliniella intonsa, and cotton aphid, Aphis gossypii—was detected in Orius bugs in the eggplant field, indicating that Orius bugs preyed on them.

Gut content analysis is a useful tool when studying arthropod predator‐prey interactions. We used polymerase chain reaction (PCR) technique to examine how detection of prey DNA in the gut content of predators was influenced by digestion time and temperature. Such knowledge is critical before applying PCR‐based gut content analysis to field collected predators. Larvae of the two‐spotted ladybeetle (Adalia bipunctata L.) were fed with the bird cherry‐oat aphid (Rhopalosiphum padi L.) at either 21°C or 14°C. After consuming one aphid, the predators were allowed to digest the prey for a range of time periods up to 24 hours. The influence of temperature on A. bipunctata feeding behavior was also recorded. From the fed larvae, total DNA was extracted and PCR reactions with R. padi specific primers were run. The number of A. bipunctata that tested positive for R. padi DNA was negatively related to the length of digestion time. Temperature influenced larval feeding behavior but did not have a significant effect on R. padi DNA detection. After pooling the data from both temperature treatments we estimated the time point when R. padi DNA could be amplified from 50% of the fed A. bipunctata by PCR to be 4.87 hours. With such a rapid decrease in prey DNA detection success, positive PCR reactions will most likely be the result of predation events occurring shortly before capture. If a defined digestion temperature range has proven not to influence prey detection, PCR data obtained from predators collected within that particular range can be interpreted in the same way.

Aphids (Hemiptera: Aphididae) are pests of a vast array of crops globally, contributing to significant yield loss annually. Biological control, utilizing natural enemies to suppress focal pest species, has often been integrated into aphid management practices. To ensure that arthropod predators are consuming the target aphid, and ultimately have an impact on pest population dynamics, it is important to gather information on consumption patterns in space and time. Molecular gut content analysis can be used to assess the strength of trophic linkages between predators and prey by detecting the presence of species-specific DNA (including aphid DNA) in their gut. However, many generalist predators readily consume aphid honeydew as an additional nutritive resource beyond direct consumption of prey. This raises the important question of whether aphid honeydew is responsible for false-positive predation events being recorded using DNA-based gut content analysis, should honeydew contain detectable quantit...

Aphis fabae Scopoli and Myzus persicae Sulzer (Hemiptera: Aphididae) are economically important pests of sugar beet. Natural enemies have potential for pest regulation. However their contribution to the control of the two sugar beet aphids is poorly understood. To elucidate the trophic relationships, we selected three aphid predators for molecular gut content analysis: two generalists Cantharis lateralis L. (Coleoptera: Cantharidae) and Coccinella septempunctata L. (Coleoptera: Coccinellidae), and the specialist Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) larvae. The first two species were chosen for their abundance, the latter for its role as a specialist aphid predator. Predators were collected in sugar beet fields, and qPCR was used to detect A. fabae and M. persicae remains in their gut. The DNA half-life detectability was assessed in a feeding assay and was found to be between 8 h and 33 h for the three predators. In the field, the most abundant aphid was A. fabae, with densities of over 14 aphids per plant, while M. persicae densities were over 100 times lower. In total 75% of the predators tested positive for A. fabae DNA. Despite the low densities of M. persicae in the field, 28% of the predators tested positive for this aphid species, indicating a preference for M. persicae. Based on the findings of the gut content analysis and predator densities, we assessed the total predation potential of three of the predators for the two years studied. We found that generalist predators contributed to 93% of the total predation for A. fabae, while the specialist predator C. carnea played more of a role in the predation of M. persicae, where it accounted for 23% of the total predation. While A. fabae densities exceeded predator capacity, M. persicae densities remained below total predation potential. These findings suggest that the three predators contribute to aphid control in sugar beet and help prevent outbreaks of M. persicae, a particularly serious pest.

Molecular gut content analysis via diagnostic PCR or high-throughput sequencing (metabarcoding) of consumers allows unravelling of feeding interactions in a wide range of animals. This is of particular advantage for analyzing the diet of small invertebrates living in opaque habitats such as the soil. Due to their small body size, which complicates dissection, microarthropods are subjected to whole-body DNA extraction-step before their gut content is screened for DNA of their food. This poses the problem that body surface contaminants, such as fungal spores may be incorrectly identified as ingested food particles for fungivorous species. We investigated the effectiveness of ten methods for body surface decontamination in litter-dwelling oribatid mites using Steganacarus magnus as model species. Furthermore, we tested for potential adverse effects of the decontamination techniques on the molecular detection of ingested prey organisms. Prior to decontamination, oribatid mites were fed with an oversupply of nematodes (Plectus sp.) and postmortem contaminated with fungal spores (Chaetomium globosum). We used diagnostic PCR with primers specific for C. globosum and Plectus sp. to detect contaminants and prey, respectively. The results suggest that chlorine bleach (sodium hypochloride, NaClO, 5%) is most efficient in removing fungal surface contamination without significantly affecting the detection of prey DNA in the gut. Based on these results, we provide a standard protocol for efficient body surface decontamination allowing to trace the prey spectrum of microarthropods using molecular gut content analysis.

The nematode-based food-chain of a temperate deciduous forest

The soil system comprises a high diversity of coexisting species interacting in close association. Generally, soil animals are assumed to be trophic generalists feeding on resources of even different trophic levels. The complex structure of soil habitats hampers locating specific resources and this results in feeding on a broad range of resources. It has been assumed that most decomposers rely on labile resources of high nutritional value, such as high quality litter and microorganisms. However, there is increasing evidence that recalcitrant carbon sources being physically and chemically stabilised in soil aggregates are mobilised by certain decomposer groups. However, the soil system with its multitude of trophic interactions and ways of resource utilisation by consumers is still poorly understood. The aim of this thesis was to examine utilisation of food resources and energy channels of important key invertebrate taxa as representatives of different trophic levels in the soil food web. We aimed at identifying trophic niches of closely related species and the driving factors for its variations. Classical techniques, such as microscopic identification of gut contents and feeding experiments, examining trophic interactions and energy flows in soil food webs are increasingly replaced by novel molecular techniques. We used stable isotope, fatty acid and molecular gut content analyses providing reliable time-integrated and indirect methods to unravel trophic linkages between cryptic organisms in opaque soil systems. In Chapter 2 We investigated feeding strategies of two centipede species to identify their trophic niches and variations therein with forest type. In combination with body size measurements, we conducted stable isotope and fatty acid analyses on centipedes allowing insight into trophic levels and the utilisation of basal resources. The results indicated centipedes to occupy distinct trophic niches with the smaller species relying more on bacterial resources and root-derived carbon as compared to the larger species. Differences in trophic niches were more pronounced in coniferous than in beech forests. The results suggest that habitat structure and body size may act as determinants for variations in trophic niches of soil arthropod predators. Complementing the study in Chapter 2, in Chapter 3 we investigated centipede gut contents of the same individuals for three prey taxa (collembolans, dipterans and earthworms) applying specific PCR assays. In this approach insight into mechanisms was deepened by including a second region, the Schorfheide-Chorin, and factors, such as prey abundance, litter mass and soil pH in the analyses. The results indicated that forest type did not affect prey choice, but factors representing habitat characteristics, such as litter mass and soil pH, were responsible for differences. Similar to Chapter 2 the results suggest body size and habitat structure to act as important factors in trophic niche differentiation. In Chapter 4 we investigated the use of carbon resources of ecologically different earthworm species being abundant decomposers in soil and preferred prey of centipedes (Chapters 2 and 3). Using compound-specific 13C stable isotope analysis of fatty acids, we related earthworm 13C signatures to resources of different stability. Endogeic earthworm species, in contrast to epigeic species, were found to predominantly assimilate recalcitrant carbon sources. Results also pointed to the utilisation of stable resources associated with clay attached to soil aggregates. This study revealed specific stable soil carbon pools to essentially contribute to nutrition of endogeic earthworm species. In Chapter 5 we combined fatty acid and stable isotope analyses to investigate trophic niches of ecologically different collembolan species and their association with litter and root resources. Furthermore, we investigated feeding interactions by relating collembolan fatty acids to that of litter-dwelling microorganisms, which are regarded as major food resource. Results indicate that collembolans occupy several trophic niches acting as decomposers of litter- and root-derived resources as well as as predators. Moreover, we found collembolan fatty acid patterns to not correspond with that of microorganisms suggesting that they feed on specific microsites in soil keeping their trophic niche constant. This thesis allowed insight into soil animals as generalistic species with distinct trophic niches. The three soil animal taxa occupying different trophic levels were documented to differ in strategies of resource utilisation and variation therein. Furthermore, the studies challenge the view of fixed energy channels with definite associated animal groups in soil food webs. Overall, this thesis considerably contributes to the understanding of soil food web structure and functioning. Using novel techniques it presented insights into trophic interactions on different trophic levels in an elegant way.

Halyomorpha halys (Stål, 1855) is an invasive agricultural pest in North America and Europe. Most of the information on H. halys predators in invaded areas comes from North America. This work focused on the molecular identification of arthropod predator species capable of feeding on H. halys in northern Italy. Predatory arthropods were collected in the field in four urban parks using the tree‐beating technique. A real‐time PCR workflow was applied to detect H. halys DNA from the gut content of predators. Of the 190 predator individuals analysed, 46 were positive for H. halys DNA and belonged to 10 insect taxa (1 Dermaptera, 3 Coleoptera, 2 Hemiptera and 4 Orthoptera) and six arachnid taxa (2 Opiliones and 6 Araneae). The integration of gut content analysis with laboratory bioassays and field observations allows the identification of a greater number of predators and therefore a better understanding of how the invaded ecosystem is responding to the introduction of a new species, given that samples are taken from the invaded environment itself. Therefore, the gut content analysis provides essential elements for conservation biocontrol in integrated pest management programmes.

Generalist predators play an important role in many terrestrial systems, especially within agricultural settings, and ants (Hymenoptera: Formicidae) often constitute important linkages of these food webs, as they are abundant and influential in these ecosystems. Molecular gut content analysis provides a means of delineating food web linkages of ants based on the presence of prey DNA within their guts. Although this method can provide insight, its use on ants has been limited, potentially due to inhibition when amplifying gut content DNA. We designed a series of experiments to determine those ant organs responsible for inhibition and identified variation in inhibition among three species (Tetramorium caespitum (L.), Solenopsis invicta Buren, and Camponotus floridanus (Buckley)). No body segment, other than the gaster, caused significant inhibition. Following dissection, we determined that within the gaster, the digestive tract and crop cause significant levels of inhibition. We found significant differences in the frequency of inhibition between the three species tested, with inhibition most evident in T. caespitum The most effective method to prevent inhibition before DNA extraction was to exude crop contents and crop structures onto UV-sterilized tissue. However, if extracted samples exhibit inhibition, addition of bovine serum albumin to PCR reagents will overcome this problem. These methods will circumvent gut content inhibition within selected species of ants, thereby allowing more detailed and reliable studies of ant food webs. As little is known about the prevalence of this inhibition in other species, it is recommended that the protocols in this study are used until otherwise shown to be unnecessary.

The relationship between phytophagous insects and plants is a central aspect of food webs and ecosystem functioning. The introduction of new species into an environment can have significant impacts on the food web of a native ecosystem. In many cases, there is a lack of knowledge on the biology and feeding behavior of invasive species prior their introduction and in the invaded regions. Gut content analyses of insects have provided valuable information on the host spectrum of insects. However, current approaches are time‐consuming and costly. Here, we describe a new molecular gut content analysis (GCA) approach using the Oxford Nanopore (ONT) Flongle sequencing platform to characterize the plant DNA present in the gut of the highly polyphagous insect species Halyomorpha halys. We demonstrate that this technique efficiently amplifies and correctly identifies plant DNA in a mock community. We performed a feeding experiment to determine the sensitivity of this approach and to assess how long the plant DNA can be detected. All plants used in the feeding experiment were correctly identified and detected after 56 days. Surprisingly, we also detected various plant genera that were not included in the feeding experiment and thus were likely ingested months before the experiment. Our study suggests that the GCA using the ONT Flongle sequencing platform represents a rapid and cost‐efficient diagnosis of the dietary preferences, host range, and the diversity of consumed plant species of pest insects with high precision.

The pupae of Bactrocera oleae (Diptera: Tephritidae) complete their development during autumn and winter in the soil, rather than in the drupe, resulting susceptible to edaphic predators. Environmentally friendly methods to control this olive pest involve the identification of its natural enemies. This study evaluated the role of Ocypus olens (Coleoptera: Staphylinidae) in the predation of B. oleae pupae, by means of molecular gut content analysis. Modified dry pitfall traps were used to collect live specimens from low-input olive orchards in Tuscany (Italy). Sampling was fine-tuned with a degree-day model estimating the presence of pest pupae in the soil. PCR analyses carried out on field-collected specimens demonstrated that O. olens is a predator of B. oleae, at least during autumn. These results are consistent with predictions of the degree-day model. Knowledge on species composition, traits and complementarity of the natural enemies of B. oleae pupae needs further investigation to advance conservation biological control strategies.

Unveiling soil food web links: New PCR assays for detection of prey DNA in the gut of soil arthropod predators

Resource overlap and infrequent predation on key pests show vulnerability in cotton biological control services

The Hibiscus mealybug, Nipaecoccus viridis (Newstead), has recently established in Florida citrus and become a pest of concern given secondary pest outbreaks associated with management of citrus greening disease. Chemical controls used to manage other citrus arthropod pests are not as effective against N. viridis due to its waxy secretions, clumping behavior, and induced cellular changes to host plant tissue which increase microhabitats. Populations of this mealybug pest are regulated by natural enemies in its native region, but it remains unclear if resident natural enemies in Florida citrus could similarly suppress N. viridis populations. This investigation: 1) established species-specific primers for N. viridis based on the mitochondrial gene Cytochrome-oxidase 1 (COI), 2) determined duration of N. viridis DNA detectability in a known predator, the mealybug destroyer (Cryptolaemus montrouzieri Mulsant), by using identified primers in molecular gut content analysis, and 3) screened field-collected predators for the presence of N. viridis DNA. The detection rate of N. viridis DNA was >50% at 36 h after adult C. montrouzieri feeding but DNA was no longer detectable by 72 h after feeding. Field-collected predators were largely comprised of spiders, lacewings, and C. montrouzieri. Spiders, beetles (primarily C. montrouzieri), and juvenile lacewings were the most abundant predators of N. viridis, with 17.8, 43.5, and 58.3 of field-collected samples testing positive for N. viridis DNA, respectively. Our results indicate that Florida citrus groves are hosts to abundant predators of N. viridis and encourage the incorporation of conservation or augmentative biological control for management of this pest.

The search for effective biological control agents without harmful non-target effects has been constrained by the use of impractical (field direct observation) or imprecise (cage experiments) methods. While advances in the DNA sequencing methods, more specifically the development of high-throughput sequencing (HTS), have been quickly incorporated in biodiversity surveys, they have been slow to be adopted to determine arthropod prey range, predation rate and food web structure, and critical information to evaluate the effectiveness and safety of a biological control agent candidate. The lack of knowledge on how HTS methods could be applied by ecological entomologists constitutes part of the problem, although the lack of expertise and the high cost of the analysis also are important limiting factors. In this review, we describe how the latest HTS methods of metabarcoding and Lazaro, a method to identify prey by mapping unassembled shotgun reads, can serve biological control research, showing both their power and limitations. We explain how they work to determine prey range and also how their data can be used to estimate predation rates and subsequently be translated into food webs of natural enemy and prey populations helping to elucidate their role in the community. We present a brief history of prey detection through molecular gut content analysis and also the attempts to develop a more precise formula to estimate predation rates, a problem that still remains. We focused on arthropods in agricultural ecosystems, but most of what is covered here can be applied to natural systems and non-arthropod biological control candidates as well.