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We report the complete genome sequences of two bacteriophages, Aussie and StopSmel, isolated from soil using the host Sinorhizobium meliloti NRRL L-50. The genomes are similar in length and gene content and share 76% nucleotide identity. Comparative analysis of Aussie and StopSmel identified core functional modules associated with Mu-like bacteriophages., Competing Interests: The authors declare no conflict of interest.

Protists in general comprise about one-third of the parasitic species infecting arthropod vectors, the role of free-living and epibiotic ciliates on mosquitoes have been insufficiently studied either due to their low pathogenicity or facultative parasites. Studies have shown that exposure of Paramecium ciliate protists, like Vorticella species, to first instar Culex nigripalpus Theobald, larvae delayed larval development and reduced biomass of emerged adults due to competition for food sources like bacteria and other microbes essential to mosquito growth and survival. Thus, we report on the capacity of a Vorticella sp. protist's ability to cross-infect host species and parasitize multiple mosquito larvae. The unique adapted behavior with the ability to remain on the exuviae in tree hole habitats provide a novel delivery system to develop products for target species-specific mosquitocides, larvicides, or viricides to be applied and sustained in aquatic systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The Diaprepes root weevil (DRW), Diaprepes abbreviatus, is a broadly polyphagous invasive pest of agriculture in the southern United States and the Caribbean. Its genome was sequenced, assembled, and annotated to study genomic correlates of specialized plant-feeding and invasiveness and to facilitate the development of new methods for DRW control. The 1.69 Gb D. abbreviatus genome assembly was distributed across 653 contigs, with an N50 of 7.8 Mb and the largest contig of 62 Mb. Most of the genome was comprised of repetitive sequences, with 66.17% in transposable elements, 5.75% in macrosatellites, and 2.06% in microsatellites. Most expected orthologous genes were present and fully assembled, with 99.5% of BUSCO genes present and 1.5% duplicated. One hundred and nine contigs (27.19 Mb) were identified as putative fragments of the X and Y sex chromosomes, and homology assessment with other beetle X chromosomes indicated a possible sex chromosome turnover event. Genome annotation identified 18,412 genes, including 43 putative horizontally transferred (HT) loci. Notably, 258 genes were identified from gene families known to encode plant cell wall degrading enzymes and invertases, including carbohydrate esterases, polysaccharide lyases, and glycoside hydrolases (GH). GH genes were unusually numerous, with 239 putative genes representing 19 GH families. Interestingly, several other beetle species with large numbers of GH genes are (like D. abbreviatus) successful invasive pests of agriculture or forestry., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Diaphorina citri is an insect vector of "Candidatus Liberibacter asiaticus" (CLas), the gram-negative bacterial pathogen associated with citrus greening disease. Control measures rely on pesticides with negative impacts on the environment, natural ecosystems, and human and animal health. In contrast, gene-targeting methods have the potential to specifically target the vector species and/or reduce pathogen transmission., Results: To improve the genomic resources needed for targeted pest control, we assembled a D. citri genome based on PacBio long reads followed by proximity ligation-based scaffolding. The 474-Mb genome has 13 chromosomal-length scaffolds. In total, 1,036 genes were manually curated as part of a community annotation project, composed primarily of undergraduate students. We also computationally identified a total of 1,015 putative transcription factors (TFs) and were able to infer motifs for 337 TFs (33%). In addition, we produced a genome-independent transcriptome and genomes for D. citri endosymbionts., Conclusions: Manual annotation provided more accurate gene models for use by researchers and provided an excellent training opportunity for students from multiple institutions. All resources are available on CitrusGreening.org and NCBI. The chromosomal-length D. citri genome assembly serves as a blueprint for the development of collaborative genomics projects for other medically and agriculturally significant insect vectors., (© The Author(s) 2024. Published by Oxford University Press GigaScience.)

Dried fruit beetle, Carpophilus hemipterus (Linnaeus, 1758) (Coleoptera: Nitidulidae), is a serious pest of ripened fresh fruit in the orchard and dried fruit in postprocessing storage. Despite the economic impact and widespread distribution of C. hemipterus, there is a lack of functional genomics research seeking to elucidate features of molecular physiology for improved pest management. Here, we report the characterization of the gene named Vermilion in C. hemipterus (ChVer) that encodes for tryptophan 2,3-dioxygenase. The Vermilion is frequently used as a visual marker for genomics approaches as tryptophan 2,3-dioxygenase is involved in the biosynthesis of eye coloration pigments in insects. We identified 1628 bp long full-length transcript of ChVer from transcriptomic database of C. hemipterus. The expression analysis among adult body parts revealed peak ChVer expression in head compared to thorax and abdomen, which is consistent with its role. Among the C. hemipterus developmental stages, peak ChVer expression was observed in first instar larva, second instar larva, and adult male stages, whereas the lowest levels of expression were seen in third instar larva, prepupa, and pupa. The nanoinjection of ChVer double-stranded RNA in larval C. hemipterus resulted in a significant reduction in ChVer transcript levels as well as caused a loss of eye color, that is, the white-eyed phenotype in adults. Characterization of visually traceable marker gene and robust RNA interference response seen in this study will enable genomics research is this important pest., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

The Gill's mealybug, Ferrisia gilli Gullan, (Hemiptera: Pseudococcidae) has emerged as a major pest of pistachio in California. Because F. gilli is only relatively recently described, there are no validated reference genes to normalize the expression data from real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in this species. We selected and validated 8 commonly used reference genes (RPS8, TBP, UBQE2, RPL7, RPL5, RPL40, RPLP1, and HEL) for expression stability in F. gilli. These genes were evaluated in 5 different geographical populations of F. gilli collected from organic and conventionally grown pistachio orchards. Candidate reference genes were also evaluated in F. gilli fed with 4 plant hosts: pistachio, almond, grapes, and lima beans. The stability of candidate genes was analyzed using 4 software algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Three genes RPS8, RPL40, and RPL7 encoding for ribosomal proteins were identified as the most stable across the treatments and thus were recommended for normalizing the qRT-PCR data. These findings will support resistance monitoring, molecular toxicology, and functional genomics research in F. gilli., (Published by Oxford University Press on behalf of Entomological Society of America 2023.)

Picorna-like viruses of the order Picornavirales are a poorly defined group of positive-sense, single-stranded RNA viruses that include numerous pathogens known to infect plants, animals, and insects. A new picorna-like viral species was isolated from the wild lime psyllid (WLP), Leuronota fagarae, in the state of Florida, USA, and labelled: Leuronota fagarae picorna-like virus isolate FL (LfPLV-FL). The virus was found to have homology to a picorna-like virus identified in the Asian Citrus Psyllid (ACP), Diaphorina citri, collected in the state of Florida. Computational analysis of RNA extracts from WLP adult heads identified a 10,006-nucleotide sequence encoding a 2,942 amino acid polyprotein with similar functional domain structure to polyproteins of both Dicistroviridae and Iflaviridae. Sequence comparisons of nucleic acid and amino acid translations of the conserved RNA-dependent RNA polymerase, along with the entire N-terminal nonstructural coding region, provided insight into an evolutionary relationship of LfPLV-FL to insect-infecting iflaviruses. Viruses belonging to the family Iflaviridae encode a polyprotein of around 3000 amino acids in length that is processed post-translationally to produce components necessary for replication. The classification of a novel picorna-like virus in L. fagarae, with evolutionary characteristics similar to picorna-like viruses infecting Bactericera cockerelli and D. citri, provides an opportunity to examine virus host specificity, as well as identify critical components of the virus' genome required for successful transmission, infection, and replication. This bioinformatic classification allows for further insight into a novel virus species, and aids in the research of a closely related virus of the invasive psyllid, D. citri, a major pest of Floridian citriculture. The potential use of viral pathogens as expression vectors to manage the spread D. citri is an area that requires additional research; however, it may bring forth an effective control strategy to reduce the transmission of Candidatus Liberibacter asiaticus (CLas), the causative agent of Huanglongbing (HLB)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Gill's mealybug, Ferrisia gilli (Gullan) (Hemiptera: Pseudococcidae), is a major pest of pistachio in California. Insecticide treatment is the primary control method and acetamiprid is widely used to control this pest. However, there have been numerous reports of control failures for F. gilli after field applications of recommended insecticides in recent years. The purpose of this study was to develop a method for routine monitoring of F. gilli susceptibility and quantify current levels of F. gilli susceptibility to acetamiprid. A leaf-dip bioassay method using lima bean leaves was established and baseline susceptibility responses of 5 field populations were determined. Lethal concentrations to kill 50% of population (LC50) for second instar nymphs at 48 h ranged from 0.367 to 2.398 µg(AI)ml-1 of acetamiprid. Similarly, lethal concentrations to kill 90% of population (LC90) for second instar nymphs at 48 h ranged from 2.887 to 10.752 µg(AI)ml-1 of acetamiprid. The F. gilli population collected from Hanford area showed up to 6.5-fold significantly decreased mortality to acetamiprid compared to other populations. The resistance identified in this study, although relatively low, indicates that there has been repeated pressure to select for acetamiprid resistance and resistance levels can further magnify if effective management steps are not taken. The baseline susceptibility established in this study can be used to investigate potential cause of recent acetamiprid failures against F. gilli. In the long-term, results of this study will support the development of resistance management strategies by monitoring shifts in the susceptibility of F. gilli populations., (Published by Oxford University Press on behalf of Entomological Society of America 2023.)

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system and RNA interference (RNAi)-based non-transgenic approaches are powerful technologies capable of revolutionizing plant research and breeding. In recent years, the use of these modern technologies has been explored in various sectors of agriculture, introducing or improving important agronomic traits in plant crops, such as increased yield, nutritional quality, abiotic- and, mostly, biotic-stress resistance. However, the limitations of each technique, public perception, and regulatory aspects are hindering its wide adoption for the development of new crop varieties or products. In an attempt to reverse these mishaps, scientists have been researching alternatives to increase the specificity, uptake, and stability of the CRISPR and RNAi system components in the target organism, as well as to reduce the chance of toxicity in nontarget organisms to minimize environmental risk, health problems, and regulatory issues. In this review, we discuss several aspects related to risk assessment, toxicity, and advances in the use of CRISPR/Cas and topical RNAi-based technologies in crop management and breeding. The present study also highlights the advantages and possible drawbacks of each technology, provides a brief overview of how to circumvent the off-target occurrence, the strategies to increase on-target specificity, the harm/benefits of association with nanotechnology, the public perception of the available techniques, worldwide regulatory frameworks regarding topical RNAi and CRISPR technologies, and, lastly, presents successful case studies of biotechnological solutions derived from both technologies, raising potential challenges to reach the market and being social and environmentally safe., Competing Interests: TC and HC were employed by the SEMPRE AgTech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Touzdjian Pinheiro Kohlrausch Távora, de Assis dos Santos Diniz, de Moraes Rêgo-Machado, Chagas Freitas, Barbosa Monteiro Arraes, Chumbinho de Andrade, Furtado, Osiro, Lima de Sousa, Cardoso, Márcia Mertz Henning, Abrão de Oliveira Molinari, Feingold, Hunter, Fátima Grossi de Sá, Kobayashi, Lima Nepomuceno, Santiago and Correa Molinari.)

Background: Huanglongbing, a devastating disease of citrus, is caused by the obligate, intracellular bacterium "Candidatus Liberibacter asiaticus" (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid. Development of transmission-blocking strategies to manage huanglongbing relies on knowledge of CLas and D. citri interactions at the molecular level. Prior transcriptome analyses of D. citri point to changes in psyllid biology due to CLas infection but have been hampered by incomplete versions of the D. citri genome, proper host plant controls, and/or a lack of a uniform data analysis approach. In this work, we present lessons learned from a quantitative transcriptome analysis of excised heads, salivary glands, midguts, and bacteriomes from CLas-positive and CLas-negative D. citri using the chromosomal length D. citri genome assembly., Results: Each organ had a unique transcriptome profile and response to CLas infection. Though most psyllids were infected with the bacterium, CLas-derived transcripts were not detected in all organs. By analyzing the midgut dataset using both the Diaci_v1.1 and v3.0 D. citri genomes, we showed that improved genome assembly led to significant and quantifiable differences in RNA-sequencing data interpretation., Conclusions: Our results support the hypothesis that future transcriptome studies on circulative, vector-borne pathogens should be conducted at the tissue-specific level using complete, chromosomal-length genome assemblies for the most accurate understanding of pathogen-induced changes in vector gene expression., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Hox genes and their cofactors are essential developmental genes specifying regional identity in animals. Hox genes have a conserved arrangement in clusters in the same order in which they specify identity along the anterior-posterior axis. A few insect species have breaks in the cluster, but these are exceptions. We annotated the 10 Hox genes of the Asian citrus psyllid Diaphorina citri , and found a split in its Hox cluster between the Deformed and Sex combs reduced genes - the first time a break at this position has been observed in an insect Hox cluster. We also annotated D. citri orthologs of the Hox cofactor genes homothorax, PKNOX and extradenticle and found an additional copy of extradenticle in D. citri that appears to be a retrogene. Expression data and sequence conservation suggest that the extradenticle retrogene may have retained the original extradenticle function and allowed divergence of the parental extradenticle gene., Competing Interests: The authors declare that they have no competing interests., (© The Author(s) 2022.)

The circadian rhythm involves multiple genes that generate an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the Earth's rotation on its axis. Here, we present the results of the manual curation of 27 genes that are associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect is the vector for the bacterial pathogen Candidatus Liberibacter asiaticus ( C Las), the causal agent of citrus greening disease (Huanglongbing). This disease severely affects citrus industries and has drastically decreased crop yields worldwide. Based on cry1 and cry2 identified in the psyllid genome, D. citri likely possesses a circadian model similar to the lepidopteran butterfly, Danaus plexippus . Manual annotation will improve the quality of circadian rhythm gene models, allowing the future development of molecular therapeutics, such as RNA interference or antisense technologies, to target these genes to disrupt the psyllid biology., Competing Interests: The authors declare that they have no competing interests., (© The Author(s) 2022.)

Chitinases are enzymes that digest the polysaccharide polymer chitin. During insect development, breakdown of chitin is an essential step in molting of the exoskeleton. Knockdown of chitinases required for molting is lethal to insects, making chitinase genes an interesting target for RNAi-based pest control methods. The Asian citrus psyllid, Diaphorina citri , carries the bacterium causing Huanglongbing, or citrus greening disease, a devastating citrus disease. We identified and annotated 12 chitinase family genes from D. citri as part of a community effort to create high-quality gene models to aid the design of interdictory molecules for pest control. We categorized the D. citri chitinases according to an established classification scheme and re-evaluated the classification of chitinases in other hemipterans. In addition to chitinases from known groups, we identified a novel class of chitinases present in D. citri and several related hemipterans that appears to be the result of horizontal gene transfer., Competing Interests: The authors declare that they have no competing interests., (© The Author(s) 2022.)

Ubiquitination is an ATP-dependent process that targets proteins for degradation by the proteasome. Here, we annotated 15 genes from the ubiquitin-proteasome pathway in the Asian citrus psyllid, Diaphorina citri . This psyllid vector has come to prominence in the last decade owing to its role in the transmission of the devastating bacterial pathogen, Candidatus Liberibacter asiaticus ( C Las). Infection of citrus crops by this pathogen causes Huanglongbing (HLB), or citrus greening disease, and results in the eventual death of citrus trees. The identification and correct annotation of these genes in D. citri will be useful for functional genomic studies to aid the development of RNAi-based management strategies aimed at reducing the spread of HLB. Investigating the effects of C Las infection on the expression of ubiquitin-proteasome pathway genes may provide new information about the role these genes play in the acquisition and transmission of C Las by D. citri ., Competing Interests: The authors declare that they have no competing interests., (© The Author(s) 2022.)

Aegle marmelos (L.) Correa belongs to the family Rutaceae is generally known as "bael fruit tree" occuring across the south Asian countries. The current investigation screened the main derivatives from crude ethanolic extracts of the Bael tree leaf and evaluated activity effects on the larvae and adults of Aedes aegypti (L.) Dengue vector mosquito and a non-target aquatic predator. The GC-MS results showed that the peak area was found to be profound in N-methyl-1-adamantaneacetamide (N-M 1a) followed by oleic acid (OA) with 63.08 and 11.43% respectively. The larvicidal activity against the fourth instar larvae and the crude Ex-Am showed prominent mortality rate (93.60%) at the maximum dosage of 100 ppm. The mortality rate of N-M 1a and OA was occurred at 10 ppm (97.73%) and 12 ppm (95.4%). The repellent activity was found to be prominent at crude Ex-Am (50 ppm) as compared to the pure compounds (N-m 1a and OA) with maximum protection time up to 210 min. The non-target screening of Ex-Am, N-M 1a, and OA on mosquito predator Tx. splendens showed that they are scarcely toxic even at the maximum dosage of 1000 ppm (34.13%), 100 ppm (27.3%), and 120 ppm (31.3%) respectively. Thus, the present investigation clearly proved that the crude Ex-Am and their major derivatives Nm 1-a and OA showed their acute larval toxicity as well as potential mosquito repellent against the dengue mosquito and eco-safety against the mosquito predator., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Citrus greening disease is caused by the pathogen Candidatus Liberibacter asiaticus and transmitted by the Asian citrus psyllid, Diaphorina citri . No curative treatment or significant prevention mechanism exists for this disease, which causes economic losses from reduced citrus production. A high-quality genome of D. citri is being manually annotated to provide accurate gene models to identify novel control targets and increase understanding of this pest. Here, we annotated 25 D. citri genes involved in glycolysis and gluconeogenesis, and seven in trehaloneogenesis. Comparative analysis showed that glycolysis genes in D. citri are highly conserved but copy numbers vary. Analysis of expression levels revealed upregulation of several enzymes in the glycolysis pathway in the thorax, consistent with the primary use of glucose by thoracic flight muscles. Manually annotating these core metabolic pathways provides accurate genomic foundation for developing gene-targeting therapeutics to control D. citri ., Competing Interests: The authors declare that they have no competing interests., (© The Author(s) 2022.)

The hemipteran insect Diaphorina citri , or Asian citrus psyllid, is a vector for Candidatus Liberibacter asiaticus ( C Las), the bacterium causing citrus greening disease, or Huanglongbing (HLB). Millions of citrus trees have been destroyed, and every grove in Florida, USA, has been directly affected by this disease. In eukaryotes, vacuolar-type ATP synthase (V-ATPase) is an abundant heterodimeric enzyme that serves the cell with essential compartment acidification through the active processes that transport protons across the membrane. Fifteen putative V-ATPase genes in the D. citri genome were manually curated. Comparative genomic analysis revealed that D. citri V-ATPase subunits share domains and motifs with other insects, including the V-ATPase-A superfamily domain. Phylogenetic analysis separates D. citri V-ATPase subunits into expected clades with orthologous sequences. Annotation of the D. citri genome is a critical step towards developing directed pest management strategies to reduce the spread of HLB throughout the citrus industry., Competing Interests: The authors declare that they have no competing interests., (© The Author(s) 2022.)

RNA interference (RNAi) comprises a natural mechanism of gene regulation and antiviral defense system in eukaryotic cells, and results in sequence-specific degradation of RNAs. Recent scientific studies demonstrate the feasibility of use RNAi-based strategies to control pest and pathogens in plants. A key step in developing RNAi-based products is a reliable method to appropriated screening of selected dsRNAs.Herein presented are a bioassay for screening dsRNAs to control the Asian citrus psyllid (ACP), Diaphorina citri, vector of citrus Huanglongbing (HLB) and other hemipterans. The RNAi feeding bioassay, called in plant system (iPS), uses vegetative new growth citrus flush to deliver double-strand RNA (dsRNA ) to ACP during natural feeding ., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)