Transcriptome Profiling of ‘Candidatus Liberibacter asiaticus’ in Citrus and Psyllids

‘Candidatus Liberibacter asiaticus’ (Las) is an emergent bacterial pathogen that is associated with the devastating citrus huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful, and dual-transcriptome analyses aiming to profile gene expression in both Las and its hosts have a low coverage of the Las genome because of the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, a lack of understanding of the Las transcriptome remains a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited high expression in citrus include transporters, ferritin, outer membrane porins, specific pilins, and genes involved in phage-related functions, cell wall modification, and stress responses. We also found 106 genes to be differentially expressed in citrus versus Asian citrus psyllids. Genes related to transcription or translation and resilience to host defense response were upregulated in citrus, whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. Finally, we determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

A Systematic Review of Zoonotic Enteric Parasites Carried by Flies, Cockroaches, and Dung Beetles

Filth flies, cockroaches, and dung beetles have been close neighbors with humans and animals throughout our joint histories. However, these insects can also serve as vectors for many zoonotic enteric parasites (ZEPs). Zoonoses by ZEPs remain a paramount public health threat due to our close contact with animals, combined with poor water, sanitation, and hygiene access, services, and behaviors in many global regions. Our objective in this systematic review was to determine which ZEPs have been documented in these vectors, to identify risk factors associated with their transmission, and to provide effectual One Health recommendations for curbing their spread. Using PRISMA guidelines, a total of 85 articles published from 1926 to 2021 were reviewed and included in this study. Qualitative analysis revealed that the most common parasites associated with these insects included, but were not limited to: Ascaris spp., Trichuris spp., Entamoeba spp., and Cryptosporidium spp. Additionally, prominent risk factors discovered in the review, such as poor household and community WASH services, unsafe food handling, and exposure to domestic animals and wildlife, significantly increase parasitic transmission and zoonoses. The risk of insect vector transmission in our shared environments makes it critically important to implement a One Health approach in reducing ZEP transmission.

The Occurrence of Malignancy in Trypanosoma brucei brucei by Rapid Passage in Mice

Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals.

Comprehensive Genomic Discovery of Non-Coding Transcriptional Enhancers in the African Malaria Vector Anopheles coluzzii

Almost all regulation of gene expression in eukaryotic genomes is mediated by the action of distant non-coding transcriptional enhancers upon proximal gene promoters. Enhancer locations cannot be accurately predicted bioinformatically because of the absence of a defined sequence code, and thus functional assays are required for their direct detection. Here we used a massively parallel reporter assay, Self-Transcribing Active Regulatory Region sequencing (STARR-seq), to generate the first comprehensive genome-wide map of enhancers in Anopheles coluzzii, a major African malaria vector in the Gambiae species complex. The screen was carried out by transfecting reporter libraries created from the genomic DNA of 60 wild A. coluzzii from Burkina Faso into A. coluzzii 4a3A cells, in order to functionally query enhancer activity of the natural population within the homologous cellular context. We report a catalog of 3,288 active genomic enhancers that were significant across three biological replicates, 74% of them located in intergenic and intronic regions. The STARR-seq enhancer screen is chromatin-free and thus detects inherent activity of a comprehensive catalog of enhancers that may be restricted in vivo to specific cell types or developmental stages. Testing of a validation panel of enhancer candidates using manual luciferase assays confirmed enhancer function in 26 of 28 (93%) of the candidates over a wide dynamic range of activity from two to at least 16-fold activity above baseline. The enhancers occupy only 0.7% of the genome, and display distinct composition features. The enhancer compartment is significantly enriched for 15 transcription factor binding site signatures, and displays divergence for specific dinucleotide repeats, as compared to matched non-enhancer genomic controls. The genome-wide catalog of A. coluzzii enhancers is publicly available in a simple searchable graphic format. This enhancer catalogue will be valuable in linking genetic and phenotypic variation, in identifying regulatory elements that could be employed in vector manipulation, and in better targeting of chromosome editing to minimize extraneous regulation influences on the introduced sequences.Importance: Understanding the role of the non-coding regulatory genome in complex disease phenotypes is essential, but even in well-characterized model organisms, identification of regulatory regions within the vast non-coding genome remains a challenge. We used a large-scale assay to generate a genome wide map of transcriptional enhancers. Such a catalogue for the important malaria vector, Anopheles coluzzii, will be an important research tool as the role of non-coding regulatory variation in differential susceptibility to malaria infection is explored and as a public resource for research on this important insect vector of disease.

Complete de novo assembly of Wolbachia endosymbiont of Diaphorina citri Kuwayama (Hemiptera: Liviidae) using long-read genome sequencing

Wolbachia, a gram-negative $$\mathrm{\alpha }$$ α -proteobacterium, is an endosymbiont found in some arthropods and nematodes. Diaphorina citri Kuwayama, the vector of ‘Candidatus Liberibacter asiaticus’ (CLas), are naturally infected with a strain of Wolbachia (wDi), which has been shown to colocalize with the bacteria pathogens CLas, the pathogen associated with huanglongbing (HLB) disease of citrus. The relationship between wDi and CLas is poorly understood in part because the complete genome of wDi has not been available. Using high-quality long-read PacBio circular consensus sequences, we present the largest complete circular wDi genome among supergroup-B members. The assembled circular chromosome is 1.52 megabases with 95.7% genome completeness with contamination of 1.45%, as assessed by checkM. We identified Insertion Sequences (ISs) and prophage genes scattered throughout the genomes. The proteins were annotated using Pfam, eggNOG, and COG that assigned unique domains and functions. The wDi genome was compared with previously sequenced Wolbachia genomes using pangenome and phylogenetic analyses. The availability of a complete circular chromosome of wDi will facilitate understanding of its role within the insect vector, which may assist in developing tools for disease management. This information also provides a baseline for understanding phylogenetic relationships among Wolbachia of other insect vectors.

Comparative plant transcriptome profiling of Arabidopsis and Camelina infested with Myzus persicae aphids acquiring circulative and non-circulative viruses reveals virus- and plant-specific alterations relevant to aphid feeding behavior and transmission

Background: Evidence accumulates that plant viruses alter host-plant traits in ways that modify their insect vectors' behavior. These alterations often enhance virus transmission, which has led to the hypothesis that these effects are manipulations caused by viral adaptation. However, the genetic basis of these indirect, plant-mediated effects on vectors and their dependence on the plant host and the mode of virus transmission is hardly known. Results: Transcriptome profiling of Arabidopsis thaliana and Camelina sativa plants infected with turnip yellows virus (TuYV) or cauliflower mosaic virus (CaMV) and infested with the common aphid vector Myzus persicae revealed strong virus- and host-specific differences in the gene expression patterns. CaMV infection caused more severe effects on the phenotype of both plant hosts than did TuYV infection, and the severity of symptoms correlated strongly with the proportion of differentially expressed genes, especially photosynthesis genes. Accordingly, CaMV infection modified aphid behavior and fecundity stronger than did infection with TuYV. Conclusions: Overall, infection with CaMV — relying on the non-circulative transmission mode — tends to have effects on metabolic pathways with strong potential implications for insect-vector / plant-host interactions (e.g. photosynthesis, jasmonic acid, ethylene and glucosinolate biosynthetic processes), while TuYV — using the circulative transmission mode — alters these pathways only weakly. These virus-induced deregulations of genes that are related to plant physiology and defense responses might impact aphid probing and feeding behavior on both infected host plants, with potentially distinct effects on virus transmission. Keywords: Caulimovirus, polerovirus, aphid vector, transmission, feeding behavior, insect-plant interactions, transcriptome profiling, RNA-seq.

The 2017 Dengue virus 1 outbreak in northern Vietnam was caused by a locally circulating virus group

Background Dengue virus (DENV) is a member of insect vector-borne viruses, and it causes dengue fever. Southeast Asia is the epi-center of dengue fever in the world. The characterization of the virus is essential to identify the transmission and evolution of DENV. Objectives In 2017, there was an outbreak of Dengue virus type 1 (DENV1) in northern Vietnam and the neighboring countries. To identify the genetic character of the outbreak virus in the area, we conducted whole-genome sequencing analysis on the samples positive for the DENV1 along with real-time PCR. Study design In total, 1026 blood samples were collected from patients with suspected dengue fever in Ha Nam and Hai Duong province, nearby areas of the capital of Vietnam. After screening by real-time PCR, 40 of DENV1 positive samples were subjected to whole-genome sequencing, and 28 complete coding sequences were obtained. Results All 28 sequences were genotype I of DENV1, which is dominant in the southeast and East Asian countries. The phylogenetic analysis of the E region showed that they fell into a single cluster with the reported sequences from Vietnam between 2009 and 2016, in which the isolates from other countries are very rare. Our results suggested that the 2017 outbreak in the area was caused by locally circulating viruses.

Identification of the Host Substratome of Leishmania-Secreted Casein Kinase 1 Using a SILAC-Based Quantitative Mass Spectrometry Assay

Leishmaniasis is a severe public health problem, caused by the protozoan Leishmania. This parasite has two developmental forms, extracellular promastigote in the insect vector and intracellular amastigote in the mammalian host where it resides inside the phagolysosome of macrophages. Little is known about the virulence factors that regulate host-pathogen interactions and particularly host signalling subversion. All the proteomes of Leishmania extracellular vesicles identified the presence of Leishmania casein kinase 1 (L-CK1.2), a signalling kinase. L-CK1.2 is essential for parasite survival and thus might be essential for host subversion. To get insights into the functions of L-CK1.2 in the macrophage, the systematic identification of its host substrates is crucial, we thus developed an easy method to identify substrates, combining phosphatase treatment, in vitro kinase assay and Stable Isotope Labelling with Amino acids in Cell (SILAC) culture-based mass spectrometry. Implementing this approach, we identified 225 host substrates as well as a potential novel phosphorylation motif for CK1. We confirmed experimentally the enrichment of our substratome in bona fide L-CK1.2 substrates and showed they were also phosphorylated by human CK1δ. L-CK1.2 substratome is enriched in biological processes such as “viral and symbiotic interaction,” “actin cytoskeleton organisation” and “apoptosis,” which are consistent with the host pathways modified by Leishmania upon infection, suggesting that L-CK1.2 might be the missing link. Overall, our results generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogens adapted for intracellular survival.

Understanding Citrus Greening Disease and Its Possible Management Strategies in Nepal

Huanglongbing (HLB), also known as citrus greening, is a devastating disease of citrus that has decimated several citrus orchards throughout the world. The disease is associated with three species of unculturable and phloem-limited bacteriae, Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. The most common species of bacteria found in Nepal is Candidatus Liberibacter asiaticus which is transmitted by an insect vector, Asian citrus psyllid (Diaphorina citri). This disease has been detected in several economically important citrus production areas of Nepal, which resulted in heavy yield loss. No cure for the disease has been discovered yet and it is essential to practice proper management strategies to maintain citrus health and sustain citrus production under HLB pressure. Several disease management approaches such as pathogen-free nursery establishment, use of disease tolerant rootstock cultivars, proper irrigation and nutrient supply, removal of HLB affected trees, and control of psyllid with frequent insecticide application are widely practiced throughout the world. This review article highlights the characteristics of the citrus greening disease and its insect vector and gives insights into their management techniques. Several technologically advanced options available to minimize the HLB infection might not be feasible currently in Nepal due to economic and topographic constraints. This article also aims to bring into focus the cost-effective methods that growers in Nepal can practice to mitigate the impact of HLB disease in their citrus orchards. Int. J. Appl. Sci. Biotechnol. Vol 9(4): 227-238.