Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity.

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/µl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

Unsung heroes: fixing multifaceted sustainability challenges through insect biological control.

Insects are indispensable actors within global agri-food systems and ensure the delivery of myriad ecosystem services. A progressive decline in insect numbers - as inflicted by habitat loss, pollution or intensive agriculture - can jeopardize a sustained provisioning of those services. Though we routinely disregard how insects help meet multiple sustainable development challenges, a gradual insect decline can have grave, long-lasting consequences. Here, we describe how insect-mediated biological control not only defuses invasive pests and can reconstitute crop productivity, but equally delivers other positive social-ecological outcomes. Drawing upon the pan-tropical invasion of the cassava mealybug and its ensuing suppression by the monophagous parasitoid Anagyrus lopezi, we illuminate how biological control contributes to food security, poverty alleviation, human wellbeing and environmental preservation. Trans-disciplinary research and 'systems thinking' are needed to maximize the potential of these biodiversity-driven interventions, and thus reap the net positive spin-offs insects provide for farmers, the environment and human society.

An assessment of interspecific competition between two introduced parasitoids of Diaphorina citri (Hemiptera: Liviidae) on caged citrus plants.

Two parasitoids attacking nymphs of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Shafee, Alam & Agarwal) (Hymenoptera: Encyrtidae) are being released in California, USA in a classical biological control program. To evaluate the effect of multiple parasitoid species on D. citri mortality, we conducted mesocosm experiments under controlled conditions using a complete block design with 6 treatments (D. citri nymphs exposed to: no parasitoids; D. aligarhensis or T. radiata alone; D. aligarhensis or T. radiata released first (by 48 h); and both species released simultaneously). Parasitism of D. citri nymphs by T. radiata exceeded 60% and was unchanged when D. aligarhensis were present. Parasitism by D. aligarhensis was greatest when T. radiata was absent (∼28%) and was reduced in all treatments with T. radiata present (<3%). D. citri mortality and parasitoid-related mortality of D. citri was consistent across parasitoid treatments. Laboratory results suggest that competition between D. aligarhensis and T. radiata is asymmetric and favors T. radiata. It may be difficult for D. aligarhensis to contribute significantly to D. citri biological control where T. radiata is present. However, results reported here suggest that competition between T. radiata and D. aligarhensis is not likely to reduce parasitism by T. radiata or reduce parasitoid-induced mortality of D. citri.

A molecular diagnostic tool for the preliminary assessment of host-parasitoid associations in biological control programmes for a new invasive pest.

Evaluation of host-parasitoid associations can be tenuous using conventional methods. Molecular techniques are well placed to identify trophic links and resolve host-parasitoid associations. Establishment of the highly invasive brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), outside Asia has prompted interest in the use of egg parasitoids (Hymenoptera: Scelionidae) as biological control agents. However, little is known regarding their host ranges. To address this, a DNA barcoding approach was taken wherein general PCR primers for Scelionidae and Pentatomidae were developed to amplify and sequence >500-bp products within the DNA barcoding region of the cytochrome oxidase I (COI) gene that would permit the identification of key players in this association. Amplification of DNA from Pentatomidae and Scelionidae was consistent across a broad range of taxa within these families, and permitted the detection of Scelionidae eggs within H. halys 1 h following oviposition. In laboratory assays, amplification and sequencing of DNA from empty, parasitized eggs was successful for both host (100% success) and parasitoid (50% success). When applied to field-collected, empty egg masses, the primers permitted host identification in 50-100% of the eggs analysed, and yielded species-level identifications. Parasitoid identification success ranged from 33 to 67% among field-collected eggs, with genus-level identification for most specimens. The inability to obtain species-level identities for these individuals is due to the lack of coverage of this taxonomic group in public DNA sequence databases; this situation is likely to improve as more species are sequenced and recorded in these databases. These primers were able to detect and identify both pentatomid host and scelionid parasitoid in a hyperparasitized egg mass, thereby clarifying trophic links otherwise unresolved by conventional methodology.

Risk assessment of selected insecticides on Tamarixia triozae (Hymenoptera: Eulophidae), a parasitoid of Bactericera cockerelli (Hemiptera: Trizoidae).

Tamarixia triozae (Burks) (Hymenoptera: Eulophidae) is an important parasitoid of the potato or tomato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Trizoidae), a serious pest of potato (Solanum tuberosum L.), tomato (Solanum lycopersicum L.), and other solanaceous vegetables in many countries. To produce a marketable crop, insecticides are required when B. cockerelli populations reach economically damaging levels. We evaluated 11 commonly used insecticides for their effects on T. triozae. Glass-surface residues of spinetoram, imidacloprid-cyfluthrin, abamectin, and tolfenpyrad caused 100% mortality of T. triozae in 72 h, and the leaf residue of spinetoram was extremely toxic to T. triozae adults; even 15-d-old residues caused 100% mortality. Cyantraniliprole, fenpyroximate, pymetrozine, spirotetramat, spiromesifen, and chenopodium oil did not cause significant mortality in either glass surface or leaf-residue bioassays. Ingestion of spinetoram, abamectin, and imidacloprid+cyfluthrin (Leverage) by the adults resulted in 100% mortality in 12 h, and tolfenpyrad, 75.0% mortality in 12 h; whereas chenopodium oil and pymetrozine showed moderate effects on adult survival. Ingestion of abamectin, imidacloprid-cyfluthrin, and spinetoram killed all adults in the first day of treatment, whereas female adults in the treatment of pymetrozine lived 80.8 d, which was similar to those in the control. Ingestion of abamectin, imidacloprid-cyfluthrin, chenopodium oil, and spinetoram killed all male adults in the first day, whereas ingestion of other insecticides did not cause significant mortality, but reduced percent parasitism. Abamectin, imidacloprid-cyfluthrin, and spinetoram had the most deleterious effects on T. triozae, and have the least potential for use in integrated control programs using this parasitoid.

An economic comparison of biological and conventional control strategies for whiteflies (Homoptera: Aleyrodidae) in greenhouse poinsettias.

The objective of this study was to evaluate the costs of biologically controlling infestations of silverleaf whitefly, Bemisia argentifolii Bellows & Perring, in New England greenhouse operations on poinsettia, Euphorbia pulcherrima Wild, ex Koltz, using the parasitic wasp Encarsia formosa Gahan (Nile Delta strain). Partial budget analysis was used to compare costs for conventional verses biological control regimens. Four alternative whitefly control budgets are developed; two conventional chemical-based control budgets formulated with and without the use of imidacloprid, and two biological control budgets which demonstrate the impact of possibly greater pest monitoring efforts necessary to implement this type strategy successfully. The analysis shows that biological whitefly control costs were > 300% greater than conventional chemical-based control strategy costs. Most of this increase is caused by the higher costs of Encarsia formosa as the material control input. If monitoring costs are held constant across different strategies, labor costs actually decline for biological control. This is because of a significant reduction in the number of control applications made and the relatively lower cost of applying E. formosa. If more extensive monitoring efforts are required to implement biological control successfully, labor costs increase by 56% over the conventional pre-imidacloprid regimen. Based on these results, the authors conclude that cheaper and more reliable means of producing E. formosa must be developed before this strategy will become economically viable for commercial poinsettia greenhouse production.