Rapid PCR-based method for herbivore dietary evaluation using plant-specific primers

Polyphagous pests cause significant economic loss worldwide through feeding damage on various cash crops. However, their diets in agricultural landscapes remain largely unexplored. Pest dietary evaluation in agricultural fields is a challenging task currently approached through visual observation of plant feeding and microscopic identification of semi-digested plant material in pest’s guts. While molecular gut content analysis using metabarcoding approaches using universal primers (e.g., rbcl and trnL) have been successful in evaluating polyphagous pest diet, this method is relatively costly and time-consuming. Hence, there is a need for a rapid, specific, sensitive, and cost-effective method to screen for crops in the gut of pests. This is the first study to develop plant-specific primers that target various regions of their genomes, designed using a whole plant genome sequence. We selected Verticillium wilt disease resistance protein (VE-1) and pathogenesis related protein-coding genes 1–5 (PR-1-5) as our targets and designed species-specific primers for 14 important crops in the agroecosystems. Using amplicon sizes ranging from 115 to 407 bp, we developed two multiplex primer mixes that can separate nine and five plant species per PCR reaction, respectively. These two designed primer mixes provide a rapid, sensitive and specific route for polyphagous pest dietary evaluation in agroecosystems. This work will enable future research to rapidly expand our knowledge on the diet preference and range of crops that pests consume in various agroecosystems, which will help in the redesign and development of new crop rotation regimes to minimize polyphagous pest pressure and damage on crops.

Tachinid flies (Diptera: Tachinidae) reared from deciduous plant-feeding lepidopteran larvae at Hokkaido University Tomakomai Forest (Japan), with descriptions of three new species

Seven species of tachinid flies reared from herbivorous lepidopterans collected from the deciduous forest canopy at Hokkaido University Tomakomai Experimental Forest (0.2 ha plot), Hokkaido, Japan, are recorded and described: Blepharomyia brevicornis sp. nov. ex Erannis golda Djakonov (Geometridae), Catocala lara Bremer and Orthosia odiosa (Butler) (both Noctuidae); Ctenophorinia grisea Mesnil ex Himeropteryx miraculosa Staudinger (Notodontidae); Blepharipa carbonata (Mesnil) ex Marumba jankowskii (Oberthür) (Sphingidae); Cyzenis equifacialis sp. nov. ex Faristenia geminisignella Ponomarenko (Gelechiidae), Archips crataegana (Hübner), A. nigricauda Walshingham, Epinotia exquisitana Christoph and Pseudohedya gradana Christo (all Tortricidae); Cyzenis tetrasetosa sp. nov. ex Lomographa simplicior (Butler), Operophtera brunnea Nakajima and O. relegata Prout (Geometridae); Eulasiona zimini Mesnil ex Archips crataegana (Hübner), Pseudohedya gradana (Christoph), Rhopobota naevana (Hübner) and Rhopobota sp. (all Tortricidae); Panzeria sp. (nr. japonica Shima) ex Erannis golda Djakonov (Geometridae). The genus Eulasiona Townsend is moved to the subfamily Tachininae from its present position in the subfamily Dexiinae, and the female and puparium of Eulasiona zimini Mesnil are described for the first time. The parasitization rates and life habits of these tachinids are briefly discussed.

Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology

High-throughput sequencing platforms are increasingly being used for targeted amplicon sequencing because they enable cost-effective sequencing of large sample sets. For meaningful interpretation of targeted amplicon sequencing data and comparison between studies, it is critical that bioinformatic analyses do not introduce artefacts and rely on detailed protocols to ensure that all methods are properly performed and documented. The analysis of large sample sets and the use of predefined indexes create challenges, such as adjusting the sequencing depth across samples and taking sequencing errors or index hopping into account. However, the potential biases these factors introduce to high-throughput amplicon sequencing data sets and how they may be overcome have rarely been addressed. On the example of a nested metabarcoding analysis of 1920 carabid beetle regurgitates to assess plant feeding, we investigated: (i) the variation in sequencing depth of individually tagged samples and the effect of library preparation on the data output; (ii) the influence of sequencing errors within index regions and its consequences for demultiplexing; and (iii) the effect of index hopping. Our results demonstrate that despite library quantification, large variation in read counts and sequencing depth occurred among samples and that the sequencing error rate in bioinformatic software is essential for accurate adapter/primer trimming and demultiplexing. Moreover, setting an index hopping threshold to avoid incorrect assignment of samples is highly recommended.

Nematodofauna of potato tubers in the Republic of Moldova

According to the multiannual researches, it was observed that, in the conditions of the Republic of Moldova, the Nematodofauna of potato tubers (Solanum tuberosum) of different varieties, collected from deposits, is specific, with the dominance of saprophytes due to the spread of bacteriosis and fungal infections. In the potato tubers, sick with dithylenchosis (phases 4, 5), the presence of 30 spec8es of nematoodes was identified, belonging to 24 genera, 14 families, 5 orders, 2 classes: 1. Class Secernentea, Orders Aphelenchida, Rhabditida and Tylenchida; 2. Class Adenophorea, Orders Dorylaimida and Plectida. Most species (19 species) are saprophytes of the Class Secernentea, Order Rhabditida with the dominance of the Superfamilies Cephaloboidea and Diplogasteroidea. According to the trophic-ecological group, the species detected in the potatoes tubers are included in all 5 groups: plant feeding, hiphal feeding, bacterial feeding, animal predation, omnivorous. In all researched varieties, both in frequency (100%) and density (thousands of individuals/gram of infested tissue) distinguished species Ditylenchus destructor Thorne 1945, Order Tylenchida, Family Anguinidae, which is also the main parasite in the culture of Solanum tuberosum in the Republic of Moldova, capable of causing dithylenhosis, as well as epiphytotics – mass infestation of potatoes in deposits

Dental microwear texture gradients in guinea pigs reveal that material properties of the diet affect chewing behaviour

Dental microwear texture analysis (DMTA) is widely used for diet inferences in extant and extinct vertebrates. Often, a reference tooth position is analysed in extant specimens, while isolated teeth are lumped together in fossil datasets. It is therefore important to test whether DMT is tooth position specific, and if so, what the causes for wear differences are. Here we present results from controlled feeding experiments with 72 guinea pigs, which either received fresh or dried natural plant diets of different phytolith content (lucerne, grass, bamboo), or pelleted diets with and without mineral abrasives (frequently encountered by herbivorous mammals in natural habitats). We test for gradients in dental microwear texture along the upper cheek tooth row. Regardless of abrasive content, pelleted diets display an increase in surface roughness along the tooth row, indicating that posterior tooth positions experience more wear compared to anterior teeth. Guinea pigs feedings on plants of low phytolith content and low abrasiveness (fresh and dry lucerne, fresh grass) show almost no DMT differences between tooth positions, while individuals feeding on more abrasive plants (dry grass, fresh and dry bamboo) show a gradient of decreasing surface roughness along the tooth row. We suggest that plant feeding involves continuous intake and comminution by grinding, resulting in posterior tooth positions mainly processing food already partly comminuted and moistened. Pelleted diets require crushing, which exerts higher loads, especially on posterior tooth positions, where bite forces are highest. These differences in chewing behaviour result in opposing wear gradients for plant versus pelleted diets.

Development of a chimeric odour blend for attracting gravid malaria vectors

Background Odour-based tools targeting gravid malaria vectors may complement existing intervention strategies. Anopheles arabiensis are attracted to, and stimulated to oviposit by, natural and synthetic odours of wild and domesticated grasses associated with mosquito breeding sites. While such synthetic odour lures may be used for vector control, these may have limited efficacy when placed in direct competition with the natural source. In this study, workflows developed for plant-feeding pests was used to design and evaluate a chimeric odour blend based on shared attractive compounds found in domesticated grass odours. Methods Variants of a synthetic odour blend, composed of shared bioactive compounds previously identified in domesticated grasses, was evaluated sequentially in a two-choice olfactometer to identify a ratio-optimized attractive blend for malaria vectors. During this process, blends with ratios that were significantly more attractive than the previously identified synthetic rice blend were compared to determine which was most attractive in the two-choice olfactometer. To determine whether all volatile components of the most attractive blend were necessary for maximal attraction, subtractive assays were then conducted, in which individual components were removed for the most attractive blend, to define the final composition of the chimeric blend. Binary logistic regression models were used to determine significance in all two-choice assays. The chimeric blend was then assessed under field conditions in malaria endemic villages in Ethiopia, to assess the effect of dose, trap type, and placement relative to ground level. Field data were analyzed both descriptively and using a Welch-corrected t-test. Results A ratio-optimized chimeric blend was identified that significantly attracted gravid An. arabiensis under laboratory conditions. In the field, trap captures of An. arabiensis and Anopheles pharoensis were dependent on the presence of the lure, trap type (CDC, BG Sentinel and Suna traps), placement relevant to ground level, with low release rates generally luring more mosquitoes. Conclusions The workflow designed for the development of chimeric lures provides an innovative strategy to target odour-mediated behaviours. The chimeric lure identified here can be used in existing trapping systems, and be customized to increase sustainability, in line with goals of the Global Vector Control Response Group.

Effects of plant species diversity on nematode community composition and diversity in a long-term biodiversity experiment

Diversity loss has been shown to change the soil community; however, little is known about long-term consequences and underlying mechanisms. Here, we investigated how nematode communities are affected by plant species richness and whether this is driven by resource quantity or quality in 15-year-old plant communities of a long-term grassland biodiversity experiment. We extracted nematodes from 93 experimental plots differing in plant species richness, and measured above- and belowground plant biomass production and soil organic carbon concentrations (Corg) as proxies for resource quantity, as well as C/Nleaf ratio and specific root length (SRL) as proxies for resource quality. We found that nematode community composition and diversity significantly differed among plant species richness levels. This was mostly due to positive plant diversity effects on the abundance and genus richness of bacterial-feeding, omnivorous, and predatory nematodes, which benefited from higher shoot mass and soil Corg in species-rich plant communities, suggesting control via resource quantity. In contrast, plant-feeding nematodes were negatively influenced by shoot mass, probably due to higher top–down control by predators, and were positively related to SRL and C/Nleaf, indicating control via resource quality. The decrease of the grazing pressure ratio (plant feeders per root mass) with plant species richness indicated a higher accumulation of plant-feeding nematodes in species-poor plant communities. Our results, therefore, support the hypothesis that soil-borne pathogens accumulate in low-diversity communities over time, while soil mutualists (bacterial-feeding, omnivorous, predatory nematodes) increase in abundance and richness in high-diversity plant communities, which may contribute to the widely-observed positive plant diversity–productivity relationship.

Methanogenesis in the digestive tract of the tropical millipedes Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae)

Methanogens represent the final decomposition guild in anaerobic degradation of organic matter, occurring in digestive tract of various invertebrates. However, factors determining their community structure and activity in distinct gut sections is still debated. In this study, we focused on the tropical millipede species Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae), which release considerable amounts of methane. We aimed to characterize relationships between physicochemical parameters, methane production rates, and methanogen community structure in the two major gut sections, midgut and hindgut. Microsensor measurements revealed that both sections were strictly anoxic, with reducing conditions prevailing in both millipedes. Hydrogen concentration culminated in anterior hindgut of E. pulchripes. In both species, the intestinal pH was significantly higher in the hindgut than in the midgut. An accumulation of acetate and formate in the gut indicated bacterial fermentation activities in the digestive tract of both species. Phylogenetic analysis of 16S rRNA genes showed a prevalence of Methanobrevibacter spp. (Methanobacteriales), accompanied by a small fraction of so far unclassified “Methanomethylophilaceae” (Methanomassiliicoccales), in both species, which suggests that methanogenesis is mostly hydrogenotrophic. We conclude that anoxic conditions, negative redox potential, and bacterial production of hydrogen and formate promotes the gut colonization by methanogens. The higher activities of methanogens in the hindgut are explained by the higher pH of this compartment and their association with ciliates, which are restricted to this compartment and present an additional source of methanogenic substrates. Importance Methane (CH4) is the second most important atmospheric greenhouse gas after CO2 and is believed to account for 17% of global warming. Methanogens are a diverse group of archaea and can be found in various anoxic habitats including digestive tract of plant-feeding animals. Termites, cockroaches, the larvae of scarab beetles, and millipedes are the only arthropods known to host methanogens and emit large amounts of methane. Millipedes are ranked third most important detritivores after termites and earthworms, and they are considered keystone species in many terrestrial ecosystems. Both methane producing and non-emitting species have been observed in millipedes, but what limits their methanogenic potential is not known. In the present study, we show that physico-chemical gut conditions and the distribution of symbiotic ciliates are important factors determining CH4 emission in millipedes. We also find great similarities to other methane-emitting arthropods, which might be associated with their similar plant-feeding habits.