Prevalence and genetic diversity of endosymbiotic bacteria infecting cassava whiteflies in Africa

Background: Cassava provides over half of the dietary requirement for more than 200 million poor in Africa. In recent years, cassava has been affected by an epidemic of a virus disease called cassava brown streak disease (CBSD) that is spreading in much of eastern and central Africa, affecting food security and the economic development of the poor. The viruses that cause CBSD are transmitted by the insect vector whitefly (Bemisia tabaci), which have increased to very high numbers in some African countries. Strains of endosymbiotic bacteria infecting whiteflies have been reported to interact specifically with different whitefly populations with varied effects on its host biology and efficiency of virus transmission. The main aim of this study was therefore to investigate the prevalence and diversity of the secondary endosymbiotic bacteria infecting cassava whiteflies with a view to better understand their role on insect population dynamics and virus disease epidemics. Results: The genetic diversity of field-collected whitefly from Tanzania, Malawi, Uganda and Nigeria was determined by mitochondrial DNA based phylogeny and restriction fragment length polymorphism. Cassava in these countries was infected with five whitefly populations, and each one was infected with different endosymbiotic bacteria. Incidences of Arsenophonus, Rickettsia, Wolbachia and Cardinium varied amongst the populations. Wolbachia was the most predominant symbiont with infection levels varying from 21 to 97%. Infection levels of Arsenophonus varied from 17 to 64% and that of Rickettsia was 0 to 53%. Hamiltonella and Fritschea were absent in all the samples. Multiple locus sequence typing identified four different strains of Wolbachia infecting cassava whiteflies. A common strain of Wolbachia infected the whitefly population Sub-Saharan Africa 1-subgroup 1 (SSA1-SG1) and SSA1-SG2, while others were infected with different strains. Phylogeny based on 16S rDNA of Rickettsia and 23S rDNA of Arsenophonus also identified distinct strains. Conclusions: Genetically diverse bacteria infect cassava whiteflies in Africa with varied prevalence across different host populations, which may affect their whitefly biology. Further studies are required to investigate the role of endosymbionts to better understand the whitefly population dynamics

Fitness costs associated with infections of secondary endosymbionts in the cassava whitefly species Bemisia tabaci

We investigated the dual effects of bacterial infections and diseased cassava plants on the fitness and biology of the Bemisia tabaci infesting cassava in Africa. Isofemale B. tabaci colonies of sub-Saharan Africa 1-subgroup 3 (SSA1-SG3), infected with two secondary endosymbiotic bacteria Arsenophonus and Rickettsia (AR+) and those free of AR infections (AR-) were compared for fitness parameters on healthy and East African cassava mosaic virus-Uganda variant (EACMV-UG) infected cassava plants. The whitefly fecundity and nymph development was not affected by bacterial infections, or the infection of cassava by the virus. However, emergence of adults from nymphs was 50% and 17% higher by AR- on healthy and virus-infected plants, respectively than AR+ flies. Development time of adults also was 10 days longer in AR+ than AR-. The whiteflies were further compared for acquisition and retention of East African cassava mosaic virus-Uganda (EACMV-UG). Higher proportion of AR- acquired (91.8%) and retained (87.6%) the virus than AR+ (71.8%, 61.2%, respectively). Similarly, the AR- flies retained higher quantities of virus (~9 folds more) than AR+. These results indicated that bacteria-free whiteflies were superior and better transmitters of EACMV-UG, as they had higher adult emergence, quicker life-cycle and better virus retention abilities than those infected with bacteria

Fitness costs associated with infections of secondary endosymbionts in the cassava whitefly species Bemisia tabaci

We investigated the dual effects of bacterial infections and diseased cassava plants on the fitness and biology of the Bemisia tabaci infesting cassava in Africa. Isofemale B. tabaci colonies of sub-Saharan Africa 1-subgroup 3 (SSA1-SG3), infected with two secondary endosymbiotic bacteria Arsenophonus and Rickettsia (AR+) and those free of AR infections (AR-) were compared for fitness parameters on healthy and East African cassava mosaic virus-Uganda variant (EACMV-UG) infected cassava plants. The whitefly fecundity and nymph development was not affected by bacterial infections, or the infection of cassava by the virus. However, emergence of adults from nymphs was 50% and 17% higher by AR- on healthy and virus-infected plants, respectively than AR+ flies. Development time of adults also was 10 days longer in AR+ than AR-. The whiteflies were further compared for acquisition and retention of East African cassava mosaic virus-Uganda (EACMV-UG). Higher proportion of AR- acquired (91.8%) and retained (87.6%) the virus than AR+ (71.8%, 61.2%, respectively). Similarly, the AR- flies retained higher quantities of virus (~9 folds more) than AR+. These results indicated that bacteria-free whiteflies were superior and better transmitters of EACMV-UG, as they had higher adult emergence, quicker life-cycle and better virus retention abilities than those infected with bacteria

Transcriptional response of virus-infected cassava and identification of putative sources of resistance for cassava brown streak disease

Cassava (Manihot esculenta) is a major food staple in sub-Saharan Africa, which is severely affected by cassava brown streak disease (CBSD). The aim of this study was to identify resistance for CBSD as well as to understand the mechanism of putative resistance for providing effective control for the disease. Three cassava varieties; Kaleso, Kiroba and Albert were inoculated with cassava brown streak viruses by grafting and also using the natural insect vector the whitefly, Bemisia tabaci. Kaleso expressed mild or no disease symptoms and supported low concentrations of viruses, which is a characteristic of resistant plants. In comparison, Kiroba expressed severe leaf but milder root symptoms, while Albert was susceptible with severe symptoms both on leaves and roots. Real-time PCR was used to estimate virus concentrations in cassava varieties. Virus quantities were higher in Kiroba and Albert compared to Kaleso. The Illumina RNA-sequencing was used to further understand the genetic basis of resistance. More than 700 genes were uniquely overexpressed in Kaleso in response to virus infection compared to Albert. Surprisingly, none of them were similar to known resistant gene orthologs. Some of the overexpressed genes, however, belonged to the hormone signalling pathways and secondary metabolites, both of which are linked to plant resistance. These genes should be further characterised before confirming their role in resistance to CBSD

PAL1 gene of the phenylpropanoid pathway increases resistance to the Cassava brown streak virus in cassava

Background: The phenylalanine ammonia lyase genes play crucial role in plant response to biotic and abiotic stresses. In this study, we characterized the role of PAL genes in increasing resistance to the Cassava brown streak virus that causes the economically important cassava brown streak disease (CBSD) on cassava in Africa. Methods: The whole transcriptomes of eight cassava varieties differing in resistance to CBSD were obtained at 1, 5 and 8 weeks after CBSV infection. Results: Analysis of RNA-Seq data identified the overexpression of PAL1, PAL2, cinnamic acid and two chalcone synthase genes in CBSD-resistant cassava varieties, which was subsequently confirmed by RT-qPCR. The exogenous application of Acibenzolar-S-Methyl induced PAL1 gene expression to enhance resistance in the susceptible var. Kalawe. In contrast, the silencing of PAL1 by RNA interference led to increased susceptibility of the resistant var. Kaleso to CBSD. Conclusions: PAL1 gene of the phenylpropanoid pathway has a major role in inducing resistance to CBSD in cassava plants and its early induction is key for CBSD resistance

Characterization of transposable elements within the Bemisia tabaci species complex

Background: Whiteflies are agricultural pests that cause negative impacts globally to crop yields resulting at times in severe economic losses and food insecurity. The Bemisia tabaci whitefly species complex is the most damaging in terms of its broad crop host range and its ability to serve as vector for over 400 plant viruses. Genomes of whiteflies belonging to this species complex have provided valuable genomic data; however, transposable elements (TEs) within these genomes remain unexplored. This study provides the first accurate characterization of TE content within the B. tabaci species complex. Results: This study identified that an average of 40.61% of the genomes of three whitefly species (MEAM1, MEDQ, and SSA-ECA) consists of TEs. The majority of the TEs identified were DNA transposons (22.85% average) while SINEs (0.14% average) were the least represented. This study also compared the TE content of the three whitefly genomes with three other hemipteran genomes and found significantly more DNA transposons and less LINEs in the whitefly genomes. A total of 63 TE superfamilies were identified to be present across the three whitefly species (39 DNA transposons, six LTR, 16 LINE, and two SINE). The sequences of the identified TEs were clustered which generated 5766 TE clusters. A total of 2707 clusters were identified as uniquely found within the whitefly genomes while none of the generated clusters were from both whitefly and non-whitefly TE sequences. This study is the first to characterize TEs found within different B. tabaci species and has created a standardized annotation workflow that could be used to analyze future whitefly genomes. Conclusion: This study is the first to characterize the landscape of TEs within the B. tabaci whitefly species complex. The characterization of these elements within the three whitefly genomes shows that TEs occupy significant portions of B. tabaci genomes, with DNA transposons representing the vast majority. This study also identified TE superfamilies and clusters of TE sequences of potential interest, providing essential information, and a framework for future TE studies within this species complex

Understanding mosquito host choice behaviour: a new and cost-effective method of identifying the sex of human hosts in mosquito blood meals

Background: Mosquito-borne diseases are a global health problem, causing hundreds of thousands of deaths/year. Pathogens are transmitted by mosquitoes taking blood from an infected host and then feeding on a new host. Monitoring mosquito host-choice behaviour can help in many aspects of vector-borne disease control. Currently, it is possible to determine the host species and the individual human host using genotyping to match the blood profile of local inhabitants to the blood-meal found in mosquitoes. Epidemiological models generally assume biting behaviour is random, however, numerous studies have shown that certain individuals are more attractive to mosquitoes than others, due to e.g., genetic makeup and profiles of skin microbiota. Analysing blood-meals and illuminating host choice behaviour will help re-evaluate and optimise disease transmission models. Methods: We describe a new blood-meal assay that identifies the sex of the person a mosquito has bitten. The amelogenin locus (AMEL), a sex marker located on both X and Y chromosomes, was amplified by PCR in DNA extracted from Aedes aegypti and Anopheles coluzzii blood-meals. Results: Our results show that AMEL successfully amplifies up to 36 hours after a blood-meal in 63% of An. coluzzii and 80% of Ae. aegypti blood-meals, revealing the sex of humans that were fed on by individual mosquitoes, which enables further exploration of vector mosquito host preferences. This method was successfully tested in both Anopheles coluzzii and Aedes aegypti, important vectors of malaria and arboviruses, respectively. Conclusions: This method, developed with mosquitoes fed on volunteers, can be applied to field-caught mosquitoes where the host species, the biological sex of the human host and host diversity within blood-meals can be determined. Two important vector species were tested successfully in our laboratory experiments, demonstrating the potential of this technique to improve epidemiological models of vector-borne diseases. This viable and highly cost-effective approach has the capacity to improve our understanding of vector-borne disease transmission, specifically gender differences in exposure and attractiveness to mosquitoes. The data gathered from field-studies using our method will shape new transmission models and aid in the implementation of more effective and targeted vector control strategies by better understanding the drivers of vector-host interactions

A Dietary Test of Putative Deleterious Sterols for the Aphid Myzus persicae

The aphid Myzus persicae displays high mortality on tobacco plants bearing a transgene which results in the accumulation of the ketosteroids cholestan-3-one and cholest-4-en-3-one in the phloem sap. To test whether the ketosteroids are the basis of the plant resistance to the aphids, M. persicae were reared on chemically-defined diets with different steroid contents at 0.1-10 µg ml(-1). Relative to sterol-free diet and dietary supplements of the two ketosteroids and two phytosterols, dietary cholesterol significantly extended aphid lifespan and increased fecundity at one or more dietary concentrations tested. Median lifespan was 50% lower on the diet supplemented with cholest-4-en-3-one than on the cholesterol-supplemented diet. Aphid feeding rate did not vary significantly across the treatments, indicative of no anti-feedant effect of any sterol/steroid. Aphids reared on diets containing equal amounts of cholesterol and cholest-4-en-3-one showed fecundity equivalent to aphids on diets containing only cholesterol. Aphids were reared on diets that reproduced the relative steroid abundance in the phloem sap of the control and modified tobacco plants, and their performance on the two diet formulations was broadly equivalent. We conclude that, at the concentrations tested, plant ketosteroids support weaker aphid performance than cholesterol, but do not cause acute toxicity to the aphids. In plants, the ketosteroids may act synergistically with plant factors absent from artificial diets but are unlikely to be solely responsible for resistance of modified tobacco plants

Detection of cassava brown streak ipomoviruses in aphids collected from cassava plants

Cassava is an important staple food in Africa and a major source of carbohydrates for 800 million people globally. However, cassava suffers severe yield losses caused by many factors including pests and diseases. A devastating disease of cassava is cassava brown streak disease (CBSD) caused by the cassava brown streak ipomoviruses (CBSIs) (family Potyviridae), Cassava brown streak virus (CBSV), and Ugandan cassava brown streak virus (UCBSV). Spread of CBSD is mainly through planting infected stem cuttings used for propagation. Transmission of CBSIs by the insect vector (Bemisia tabaci) has been reported. However, experimental transmission efficiencies of CBSIs are usually low. Recent research has showed the occurrence of a DAG motif associated with aphid transmission in other potyviruses, within the coat protein gene of CBSV. Consequently this study aimed to explore the possibility that besides whiteflies, aphids may transmit CBSIs. Cassava plants were assessed during a survey for occurrence of CBSD and aphids as potential alternative CBSIs vectors. We collected aphids from CBSD-symptomatic and symptomless cassava plants within farmers' fields in Uganda during April–July 2020. The aphids were analyzed for the presence of CBSIs by reverse transcriptase-polymerase chain reaction (RT-PCR) and to determine aphid species using mitochondrial cytochrome oxidase (mtCOI) barcoding. Unusual aphid infestation of cassava plants was observed at 35 locations in nine districts across Uganda and on 11 other plant species within or adjacent to cassava fields. This is the first report of aphids infesting cassava in Uganda. Molecular analysis of the aphid confirmed presence of three different aphid species in the surveyed cassava fields, namely, Aphis solanella, Aphis fabae mordvilkoi, and Rhopalosiphum sp. mtCOI nucleotide sequences for the aphids in which CBSIs were detected are deposited with Genbank under accession numbers OP223337-40. Both UCBSV and CBSV were detected by RT-PCR in aphids collected from cassava fields with CBSD-affected plants. The CBSIs were detected in 14 aphid samples collected from 19 CBSD-symptomatic cassava plants. These results suggest the ability of aphids to acquire CBSIs, but transmission experiments are required on their vector potential

Differential transmission of Sri Lankan cassava mosaic virus by three cryptic species of the whitefly Bemisia tabaci complex

In recent years, Sri Lankan cassava mosaic virus (SLCMV), a begomovirus (genus Begmovirus, family Geminiviridae) causing cassava mosaic disease in Asia, poses serious threats to cassava cultivation in Asia. However, the transmission of SLCMV in the areas into which it has recently been introduced remain largely unexplored. Here we have compared the transmission efficiencies of SLCMV by three widely distributed whitefly species in Asia, and found that only Asia II 1 whiteflies were able to transmit this virus efficiently. The transmission efficiencies of SLCMV by different whitefly species were found to correlate positively with quantity of virus in whitefly whole body. Further, the viral transmission efficiency was found to be associated with varied ability of virus movement within different species of whiteflies. These findings provide detailed information regarding whitefly transmission of SLCMV, which will help to understand the spread of SLCMV in the field, and facilitate the prediction of virus epidemics