Addressing a future pandemic: how can non-biological complex drugs prepare us for antimicrobial resistance threats?

Loss of effective antibiotics through antimicrobial resistance (AMR) is one of the greatest threats to human health. By 2050, the annual death rate resulting from AMR infections is predicted to have climbed from 1.27 million per annum in 2019, up to 10 million per annum. It is therefore imperative to preserve the effectiveness of both existing and future antibiotics, such that they continue to save lives. One way to conserve the use of existing antibiotics and build further contingency against resistant strains is to develop alternatives. Non-biological complex drugs (NBCDs) are an emerging class of therapeutics that show multi-mechanistic antimicrobial activity and hold great promise as next generation antimicrobial agents. We critically outline the focal advancements for each key material class, including antimicrobial polymer materials, carbon nanomaterials, and inorganic nanomaterials, and highlight the potential for the development of antimicrobial resistance against each class. Finally, we outline remaining challenges for their clinical translation, including the need for specific regulatory pathways to be established in order to allow for more efficient clinical approval and adoption of these new technologies.

A high-throughput amplicon sequencing approach for population-wide species diversity and composition survey.

Management of agricultural pests requires an understanding of pest species diversity, their interactions with beneficial insects and spatial-temporal patterns of pest abundance. Invasive and agriculturally important insect pests can build up very high populations, especially in cropping landscapes. Traditionally, sampling effort for species identification involves small sample sizes and is labour intensive. Here, we describe a multiprimer high throughput sequencing (HTS) metabarcoding method and associated analytical workflow for a rapid, intensive, high-volume survey of pest species compositions. We demonstrate our method using the taxonomically challenging Bemisia pest cryptic species complex as examples. The whiteflies Bemisia including the"tabaci" species are agriculturally important capable of vectoring diverse plant viruses that cause diseases and crop losses. Our multiprimer metabarcoding HTS amplicon approach simultaneously process high volumes of whitefly individuals, with efficiency to detect rare (i.e., 1%) test-species, while our improved whitefly primers for metabarcoding also detected beneficial hymenopteran parasitoid species from whitefly nymphs. Field-testing our redesigned Bemisia metabarcoding primer sets across the Tanzania, Uganda and Malawi cassava cultivation landscapes, we identified the sub-Saharan Africa 1 Bemisia putative species as the dominant pest species, with other cryptic Bemisia species being detected at various abundances. We also provide evidence that Bemisia species compositions can be affected by host crops and sampling techniques that target either nymphs or adults. Our multiprimer HTS metabarcoding method incorporated two overlapping amplicons of 472 bp and 518 bp that spanned the entire 657 bp 3' barcoding region for Bemisia, and is particularly suitable to molecular diagnostic surveys of this highly cryptic insect pest species complex that also typically exhibited high population densities in heavy crop infestation episodes. Our approach can be adopted to understand species biodiversity across landscapes, with broad implications for improving transboundary biosecurity preparedness, thus contributing to molecular ecological knowledge and the development of control strategies for high-density, cryptic, pest-species complexes.

Progressing Antimicrobial Resistance Sensing Technologies across Human, Animal, and Environmental Health Domains.

The spread of antimicrobial resistance (AMR) is a rapidly growing threat to humankind on both regional and global scales. As countries worldwide prepare to embrace a One Health approach to AMR management, which is one that recognizes the interconnectivity between human, animal, and environmental health, increasing attention is being paid to identifying and monitoring key contributing factors and critical control points. Presently, AMR sensing technologies have significantly progressed phenotypic antimicrobial susceptibility testing (AST) and genotypic antimicrobial resistance gene (ARG) detection in human healthcare. For effective AMR management, an evolution of innovative sensing technologies is needed for tackling the unique challenges of interconnected AMR across various and different health domains. This review comprehensively discusses the modern state-of-play for innovative commercial and emerging AMR sensing technologies, including sequencing, microfluidic, and miniaturized point-of-need platforms. With a unique view toward the future of One Health, we also provide our perspectives and outlook on the constantly changing landscape of AMR sensing technologies beyond the human health domain.

Releasing incompatible males drives strong suppression across populations of wild and Wolbachia-carrying Aedes aegypti in Australia.

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the "Debug" Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and wMel-Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable.

Improving climate suitability for Bemisia tabaci in East Africa is correlated with increased prevalence of whiteflies and cassava diseases.

Projected climate changes are thought to promote emerging infectious diseases, though to date, evidence linking climate changes and such diseases in plants has not been available. Cassava is perhaps the most important crop in Africa for smallholder farmers. Since the late 1990's there have been reports from East and Central Africa of pandemics of begomoviruses in cassava linked to high abundances of whitefly species within the Bemisia tabaci complex. We used CLIMEX, a process-oriented climatic niche model, to explore if this pandemic was linked to recent historical climatic changes. The climatic niche model was corroborated with independent observed field abundance of B. tabaci in Uganda over a 13-year time-series, and with the probability of occurrence of B. tabaci over 2 years across the African study area. Throughout a 39-year climate time-series spanning the period during which the pandemics emerged, the modelled climatic conditions for B. tabaci improved significantly in the areas where the pandemics had been reported and were constant or decreased elsewhere. This is the first reported case where observed historical climate changes have been attributed to the increase in abundance of an insect pest, contributing to a crop disease pandemic.

Standardized molecular diagnostic tool for the identification of cryptic species within the Bemisia tabaci complex.

BACKGROUND: The whitefly Bemisia tabaci complex harbours over 40 cryptic species that have been placed in 11 phylogenetically distinct clades based on the molecular characterization of partial mitochondrial DNA COI (mtCOI) gene region. Four cryptic species are currently within the invasive clade, i.e. MED, MEAM1, MEAM2 and IO. Correct identification of these species is a critical step towards implementing reliable measures for plant biosecurity and border protection; however, no standardized B. tabaci-specific primers are currently available which has caused inconsistencies in the species identification processes. RESULTS: We report three sets of polymerase chain reaction (PCR) primers developed to amplify the mtCOI region which can be used for genotyping MED, MEAM1 and IO species, and tested these primers on 91 MED, 35 MEAM1 and five IO individuals. PCR and sequencing of amplicons identified a total of 21, six and one haplotypes in MED, MEAM1 and IO respectively, of which six haplotypes were new to the B. tabaci database. CONCLUSION: These primer pairs enabled standardization and robust molecular species identification via mtCOI screening of the targeted invasive cryptic species and will improve quarantine decisions. Use of this diagnostic tool could be extended to other species within the complex. © 2017 Society of Chemical Industry.

The Trouble with MEAM2: Implications of Pseudogenes on Species Delimitation in the Globally Invasive Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species Complex.

Molecular species identification using suboptimal PCR primers can over-estimate species diversity due to coamplification of nuclear mitochondrial (NUMT) DNA/pseudogenes. For the agriculturally important whitefly Bemisia tabaci cryptic pest species complex, species identification depends primarily on characterization of the mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene. The lack of robust PCR primers for the mtDNA COI gene can undermine correct species identification which in turn compromises management strategies. This problem is identified in the B. tabaci Africa/Middle East/Asia Minor clade which comprises the globally invasive Mediterranean (MED) and Middle East Asia Minor I (MEAM1) species, Middle East Asia Minor 2 (MEAM2), and the Indian Ocean (IO) species. Initially identified from the Indian Ocean island of Réunion, MEAM2 has since been reported from Japan, Peru, Turkey and Iraq. We identified MEAM2 individuals from a Peruvian population via Sanger sequencing of the mtDNA COI gene. In attempting to characterize the MEAM2 mitogenome, we instead characterized mitogenomes of MEAM1. We also report on the mitogenomes of MED, AUS, and IO thereby increasing genomic resources for members of this complex. Gene synteny (i.e., same gene composition and orientation) was observed with published B. tabaci cryptic species mitogenomes. Pseudogene fragments matching MEAM2 partial mtDNA COI gene exhibited low frequency single nucleotide polymorphisms that matched low copy number DNA fragments (<3%) of MEAM1 genomes, whereas presence of internal stop codons, loss of expected stop codons and poor primer annealing sites, all suggested MEAM2 as a pseudogene artifact and so not a real species.

Global threat to agriculture from invasive species.

Invasive species present significant threats to global agriculture, although how the magnitude and distribution of the threats vary between countries and regions remains unclear. Here, we present an analysis of almost 1,300 known invasive insect pests and pathogens, calculating the total potential cost of these species invading each of 124 countries of the world, as well as determining which countries present the greatest threat to the rest of the world given their trading partners and incumbent pool of invasive species. We find that countries vary in terms of potential threat from invasive species and also their role as potential sources, with apparently similar countries sometimes varying markedly depending on specifics of agricultural commodities and trade patterns. Overall, the biggest agricultural producers (China and the United States) could experience the greatest absolute cost from further species invasions. However, developing countries, in particular, Sub-Saharan African countries, appear most vulnerable in relative terms. Furthermore, China and the United States represent the greatest potential sources of invasive species for the rest of the world. The analysis reveals considerable scope for ongoing redistribution of known invasive pests and highlights the need for international cooperation to slow their spread.

Phosphine resistance in Australian Cryptolestes species (Coleoptera: Laemophloeidae): perspectives from mitochondrial DNA cytochrome oxidase I analysis.

BACKGROUND: The flat grain beetle (FGB) species Cryptolestes ferrugineus, C. pusillus, C. pusilloides and C. turcicus are major stored-product pests worldwide, of which the first three are present in Australia. C. ferrugineus is also a species with high phosphine resistance status in various countries. Morphological identification of Cryptolestes species is difficult and represents an additional barrier to effective management of phosphine resistance in FGBs. RESULT: Mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene characterisation enabled differentiation of the four major FGB pest species through direct sequence comparison, and enabled the development of a PCR-RFLP method for rapid species differentiation. We detected two mtDNA haplotypes (Cunk-01, 02) present at low frequencies with an average nucleotide divergence rate of 0.079 ± 0.011 (SE) from C. pusillus. This nucleotide divergence rate is similar to that between C. ferrugineus and C. pusilloides (0.088 ± 0.012). Male and female genitalia morphologies of the Cunk-02 individuals indicated they were consistent with C. pusillus, yet DNA sequence analyses suggested species-level divergence. The mtDNA COI gene of phosphine-bioassayed, lab-reared F1 generation survivors supported the presence of strong phosphine resistance in C. ferrugineus, but unexpectedly also in C. pusilloides and C. pusillus F1 survivors. CONCLUSION: We demonstrated the utility of molecular DNA techniques for differentiating closely related insect species, and its usefulness in assisting the management of pest insect species. The likely presence of a cryptic C. pusillus species in Australia and the possible development of strong phosphine resistance in Australian FGB pest species require further investigation. © 2014 Society of Chemical Industry.

Bees at war: interspecific battles and nest usurpation in stingless bees.

We provide the first evidence for interspecific warfare in bees, a spectacular natural phenomenon that involves a series of aerial battles and leads to thousands of fatalities from both attacking and defending colonies. Molecular analysis of fights at a hive of the Australian stingless bee Tetragonula carbonaria revealed that the attack was launched by a related species, Tetragonula hockingsi, which has only recently extended its habitat into southeastern Queensland. Following a succession of attacks by the same T. hockingsi colony over a 4-month period, the defending T. carbonaria colony was defeated and the hive usurped, with the invading colony installing a new queen. We complemented our direct observations with a 5-year study of more than 260 Tetragonula hives and found interspecific hive changes, which were likely to be usurpation events, occurring in 46 hives over this period. We discuss how fighting swarms and hive usurpation fit with theoretical predictions on the evolution of fatal fighting and highlight the many unexplained features of these battles that warrant further study.

Wind-borne dispersal of a parasitoid: the process, the model, and its validation.

The aphelinid parasitoid Eretmocerus hayati Zolnerowich & Rose (Hymenoptera: Aphelinidae) was recently released in Australia as a biocontrol agent against the crop pest Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). It was found that the parasitoid can spread over several kilometers in a single generation and continue laying eggs for over a fortnight. A simple wind-advection model was fitted to emergence data from a first release between Fassifern and Kalbar, Queensland, and its predictive ability was tested against the second release near Carnarvon, Western Australia. The fitting of the model was used to develop several hypotheses about the dispersal of E. hayati, which were validated by the second release: E. hayati flies in the same direction as the wind to a distance proportional to the wind speed; this wind-borne flight takes place at any time during daylight hours; a flight is attempted every day after emergence unless there are high wind conditions during that day; and the high wind condition that will delay flight is wind speeds in excess of ≍2 m/s. This model of E. hayati dispersal may be contrasted with previous models fitted for Eretmocerus species, for which dispersal was dominated by diffusion processes, and parasitoid spread was constrained to the scales of tens and hundreds of meters.

Is agriculture driving the diversification of the Bemisia tabaci species complex (Hemiptera: Sternorrhyncha: Aleyrodidae)?: Dating, diversification and biogeographic evidence revealed.

BACKGROUND: Humans and insect herbivores are competing for the same food crops and have been for thousands of years. Despite considerable advances in crop pest management, losses due to insects remain considerable. The global homogenisation of agriculture has supported the range expansion of numerous insect pests and has been driven in part by human-assisted dispersal supported through rapid global trade and low-cost air passenger transport. One of these pests, is the whitefly, Bemisia tabaci, a cryptic species complex that contains some of the world's most damaging pests of agriculture. The complex shows considerable genetic diversity and strong phylogeographic relationships. One consequence of the considerable impact that members of the B. tabaci complex have on agriculture, is the view that human activity, particularly in relation to agricultural practices, such as use of insecticides, has driven the diversification found within the species complex. This has been particularly so in the case of two members of the complex, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED), which have become globally distributed invasive species. An alternative hypothesis is that diversification is due to paleogeographic and paleoclimatological changes. RESULTS: The idea that human activity is driving speciation within the B. tabaci complex has never been tested, but the increased interest in fossil whiteflies and the growth in molecular data have enabled us to apply a relaxed molecular clock and so estimate divergence dates for the major lineages within the B. tabaci species complex. The divergence estimates do not support the view that human activity has been a major driver of diversification. CONCLUSIONS: Our analysis suggests that the major lineages within the complex arose approximately 60-30 mya and the highly invasive MED and MEAM1 split from the rest of the species complex around 12 mya well before the evolution of Homo sapiens and agriculture. Furthermore, the divergence dates coincide with a period of global diversification that occurred broadly across the plant and animal kingdoms and was most likely associated with major climatic and tectonic events.

The initial dispersal and spread of an intentional invader at three spatial scales.

The way an invasion progresses through space is a theme of interest common to invasion ecology and biological pest control. Models and mark-release studies of arthropods have been used extensively to extend and inform invasion processes of establishment and spread. However, the extremely common single-scale approach of monitoring initial spread leads to misinterpretation of rate and mode. Using the intentional release of a novel biological control agent (a parasitic hymenoptera, Eretmocerus hayati Zolnerowich & Rose (Hymenoptera: Aphelinidae), we studied its initial dispersal and spread at three different spatial scales, the local scale (tens of metres), field scale (hundreds of metres) and landscape scale (kilometres) around the release point. We fit models to each observed spread pattern at each spatial scale. We show that E. hayati exhibits stratified dispersal; moving further, faster and by a different mechanism than would have been concluded with a single local-scale post-release sampling design. In fact, interpretation of each scale independent of other scales gave three different models of dispersal, and three different impressions of the dominant dispersal mechanisms. Our findings demonstrate that using a single-scale approach may lead to quite erroneous conclusions, hence the necessity of using a multiple-scale hierarchical sampling design for inferring spread and the dominant dispersal mechanism of either human intended or unintended invasions.

Evidence for horizontal transmission of secondary endosymbionts in the Bemisia tabaci cryptic species complex.

Bemisia tabaci (Hemiptera: Aleyrodidae) is a globally distributed pest composed of at least 34 morphologically indistinguishable cryptic species. At least seven species of endosymbiont have been found infecting some or all members of the complex. The origin(s) of the associations between specific endosymbionts and their whitefly hosts is unknown. Infection is normally vertical, but horizontal transmission does occur and is one way for new infections to be introduced into individuals. The relationships between the different members of the cryptic species complex and the endosymbionts have not been well explored. In this study, the phylogenies of different cryptic species of the host with those of their endosymbionts were compared. Of particular interest was whether there was evidence for both coevolution and horizontal transmission. Congruence was observed for the primary endosymbiont, Portiera aleyrodidarum, and partial incongruence in the case of two secondary endosymbionts, Arsenophonus and Cardinium and incongruence for a third, Wolbachia. The patterns observed for the primary endosymbiont supported cospeciation with the host while the patterns for the secondary endosymbionts, and especially Wolbachia showed evidence of host shifts and extinctions through horizontal transmission rather than cospeciation. Of particular note is the observation of several very recent host shift events in China between exotic invader and indigenous members of the complex. These shifts were from indigenous members of the complex to the invader as well as from the invader to indigenous relatives.

Will the real Bemisia tabaci please stand up?

Since Panayiotis Gennadius first identified the whitefly, Aleyrodes tabaci in 1889, there have been numerous revisions of the taxonomy of what has since become one of the world's most damaging insect pests. Most of the taxonomic revisions have been based on synonymising different species under the name Bemisia tabaci. It is now considered that there is sufficient biological, behavioural and molecular genetic data to support its being a cryptic species complex composed of at least 34 morphologically indistinguishable species. The first step in revising the taxonomy of this complex involves matching the A. tabaci collected in 1889 to one of the members of the species complex using molecular genetic data. To do this we extracted and then amplified a 496 bp fragment from the 3' end of the mitochondrial DNA cytochrome oxidase one (mtCOI) gene belonging to a single whitefly taken from Gennadius' original 1889 collection. The sequence identity of this 123 year-old specimen enabled unambiguous assignment to a single haplotype known from 13 Mediterranean locations across Greece and Tunisia. This enabled us to unambiguously assign the Gennadius A. tabaci to the member of the B. tabaci cryptic species complex known as Mediterranean or as it is commonly, but erroneously referred to, as the 'Q-biotype'. Mediterranean is therefore the real B. tabaci. This study demonstrates the importance of matching museum syntypes with known species to assist in the delimitation of cryptic species based on the organism's biology and molecular genetic data. This study is the first step towards the reclassification of B. tabaci which is central to an improved understanding how best to manage this globally important agricultural and horticultural insect pest complex.

Inactivation of Wolbachia reveals its biological roles in whitefly host.

BACKGROUND: The whitefly Bemisia tabaci is cryptic species complex composed of numerous species. Individual species from the complex harbor a diversity of bacterial endosymbionts including Wolbachia. However, while Wolbachia is known to have a number of different roles, its role in B. tabaci is unclear. Here, the antibiotic rifampicin is used to selectively eliminate Wolbachia from B. tabaci so as to enable its roles in whitefly development and reproduction to be explored. The indirect effects of Wolbachia elimination on the biology of Encarsia bimaculata, a dominant parasitoid of B. tabaci in South China, were also investigated. METHODOLOGY/PRINCIPAL FINDING: qRT-PCR and FISH were used to show that after 48 h exposure to 1.0 mg/ml rifampicin, Wolbachia was completely inactivated from B. tabaci Mediterranean (MED) without any significant impact on either the primary symbiont, Portiera aleyrodidarum or any of the other secondary endosymbionts present. For B. tabaci MED, Wolbachia was shown to be associated with decreased juvenile development time, increased likelihood that nymphs completed development, increased adult life span and increased percentage of female progeny. Inactivation was associated with a significant decrease in the body size of the 4(th) instar which leads us to speculate as to whether Wolbachia may have a nutrient supplementation role. The reduction in nymph body size has consequences for its parasitoid, E. bimaculata. The elimination of Wolbachia lead to a marked increase in the proportion of parasitoid eggs that completed their development, but the reduced size of the whitefly host was also associated with a significant reduction in the size of the emerging parasitoid adult and this was in turn associated with a marked reduction in adult parasitoid longevity. CONCLUSIONS/SIGNIFICANCE: Wolbachia increases the fitness of the whitefly host and provides some protection against parasitization. These observations add to our understanding of the roles played by bacterial endosymbionts.

Predicting the benefits of banana bunchy top virus exclusion from commercial plantations in Australia.

Benefit cost analysis is a tried and tested analytical framework that can clearly communicate likely net changes in producer welfare from investment decisions to diverse stakeholder audiences. However, in a plant biosecurity context, it is often difficult to predict policy benefits over time due to complex biophysical interactions between invasive species, their hosts, and the environment. In this paper, we demonstrate how a break-even style benefit cost analysis remains highly relevant to biosecurity decision-makers using the example of banana bunchy top virus, a plant pathogen targeted for eradication from banana growing regions of Australia. We develop an analytical approach using a stratified diffusion spread model to simulate the likely benefits of exclusion of this virus from commercial banana plantations over time relative to a nil management scenario in which no surveillance or containment activities take place. Using Monte Carlo simulation to generate a range of possible future incursion scenarios, we predict the exclusion benefits of the disease will avoid Aus$15.9-27.0 million in annual losses for the banana industry. For these exclusion benefits to be reduced to zero would require a bunchy top re-establishment event in commercial banana plantations three years in every four. Sensitivity analysis indicates that exclusion benefits can be greatly enhanced through improvements in disease surveillance and incursion response.

Predicting invasive fungal pathogens using invasive pest assemblages: testing model predictions in a virtual world.

Predicting future species invasions presents significant challenges to researchers and government agencies. Simply considering the vast number of potential species that could invade an area can be insurmountable. One method, recently suggested, which can analyse large datasets of invasive species simultaneously is that of a self organising map (SOM), a form of artificial neural network which can rank species by establishment likelihood. We used this method to analyse the worldwide distribution of 486 fungal pathogens and then validated the method by creating a virtual world of invasive species in which to test the SOM. This novel validation method allowed us to test SOM's ability to rank those species that can establish above those that can't. Overall, we found the SOM highly effective, having on average, a 96-98% success rate (depending on the virtual world parameters). We also found that regions with fewer species present (i.e. 1-10 species) were more difficult for the SOM to generate an accurately ranked list, with success rates varying from 100% correct down to 0% correct. However, we were able to combine the numbers of species present in a region with clustering patterns in the SOM, to further refine confidence in lists generated from these sparsely populated regions. We then used the results from the virtual world to determine confidences for lists generated from the fungal pathogen dataset. Specifically, for lists generated for Australia and its states and territories, the reliability scores were between 84-98%. We conclude that a SOM analysis is a reliable method for analysing a large dataset of potential invasive species and could be used by biosecurity agencies around the world resulting in a better overall assessment of invasion risk.

Biosecurity and yield improvement technologies are strategic complements in the fight against food insecurity.

The delivery of food security via continued crop yield improvement alone is not an effective food security strategy, and must be supported by pre- and post-border biosecurity policies to guard against perverse outcomes. In the wake of the green revolution, yield gains have been in steady decline, while post-harvest crop losses have increased as a result of insufficiently resourced and uncoordinated efforts to control spoilage throughout global transport and storage networks. This paper focuses on the role that biosecurity is set to play in future food security by preventing both pre- and post-harvest losses, thereby protecting crop yield. We model biosecurity as a food security technology that may complement conventional yield improvement policies if the gains in global farm profits are sufficient to offset the costs of implementation and maintenance. Using phytosanitary measures that slow global spread of the Ug99 strain of wheat stem rust as an example of pre-border biosecurity risk mitigation and combining it with post-border surveillance and invasive alien species control efforts, we estimate global farm profitability may be improved by over US$4.5 billion per annum.

Genetic identity of the Bemisia tabaci species complex and association with high cotton leaf curl disease (CLCuD) incidence in Pakistan.

BACKGROUND: The cotton whitefly, Bemisia tabaci (Gennadius), is a cryptic species complex, and members of the complex have become serious pests in Pakistan because of their feeding and their ability to transmit cotton leaf curl virus (CLCuV). Here, an analysis was made of the identity of B. tabaci collected from cotton and a range of non-cotton hosts in the cotton-growing zones in Punjab and Sindh, the main cotton-producing provinces of Pakistan, using a portion of the mitochondrial cytochrome oxidase 1 gene. The geographic distribution of the different members of the complex was then compared with the incidence of CLCuD. RESULTS: Using the Dinsdale nomenclature, the results revealed three putative species, Asia 1, Asia II 1 and Middle East-Asia Minor 1. Asia II 1 (also referred to in the literature as biotypes K, P, PCG-1, PK1, SY and ZHJ2) was only recorded from Punjab cotton plants, whereas Asia 1 (also referred to in the literature as biotypes H, M, NA and PCG-2) was found in both Sindh and Punjab. Middle East-Asia Minor 1 (commonly known as biotype B and B2) was found only in Sindh. Moreover, Asia II 1 was associated with high incidences of CLCuD, whereas regions where Middle East-Asia Minor 1 was present had a lower incidence. Phylogenetic analysis showed that the Middle East-Asia Minor 1 population in Sindh formed a distinct genetic subgroup within the putative species, suggesting that the Sindh province of Pakistan may form part of its home range. So far, no individuals from the putative species Mediterranean (commonly known as biotypes Q, J and L) have been found in Pakistan. CONCLUSIONS: The capacity to manage pests and disease effectively relies on knowledge of the identity of the agents causing the damage. In the case of CLCuD in Pakistan, this knowledge has been obscured to some extent because of the inconsistent approach to identifying and distinguishing the different B. tabaci associated with CLCuD. The situation has now been clarified, and a strong association between disease incidence and vector identity and abundance has been shown. Given this advance, future research can now focus on factors that influence the capacity of different vector species to transmit the viruses that cause CLCuD, the reason for differences in vector abundance and the lack of geographic overlap between the cryptic vector species. This knowledge will contribute to the development of improved methods with which to manage the disease in Pakistan.