Biology subjects

Heu, C. C.

Publications and source records attributed to Heu, C. C..

6 recordsLinked to original sources

bioRxiv2023

Sindbis virus is suppressed in the yellow fever mosquito Aedes aegypti by ATG-6/Beclin-1 mediated activation of autophagy

Autophagy is a critical modulator of pathogen invasion response in vertebrates and invertebrates. However, how it affects mosquito-borne viral pathogens that significantly burden public health remains underexplored. To address this gap, we use a genetic approach to activate macroautophagy/autophagy in the yellow fever mosquito (Aedes aegypti), infected with a recombinant Sindbis virus (SINV) expressing an autophagy activator. We first demonstrate a 17- amino acid peptide derived from the Ae. aegypti autophagy-related protein 6 (ATG-6/beclin-1-like protein) is sufficient to induce autophagy in C6/36 mosquito cells, as marked by lipidation of ATG- 8 and puncta formation. Next, we engineered a recombinant SINV expressing this bioactive beclin- 1-like peptide and used it to infect and induce autophagy in adult mosquitoes. We find that modulation of autophagy using this recombinant SINV negatively regulated production of infectious viruses. The results from this study improve our understanding of the role of autophagy in arboviruses in invertebrate hosts and also highlight the potential for the autophagy pathway to be exploited for arboviral control.

microbiology↗

bioRxiv2022

Chromosome-scale genome assembly of the pink bollworm, Pectinophora gossypiella, a global pest of cotton

The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is a major global pest of cotton. Current management practices include chemical insecticides, cultural strategies, sterile insect releases, and transgenic cotton producing crystalline (Cry) protein toxins of the bacterium Bacillus thuringiensis (Bt). These strategies have contributed to eradication of P. gossypiella from the cotton growing areas of the United States and northern Mexico. However, this pest has evolved resistance to Bt cotton in Asia, where it remains a critical pest, and the benefits of using transgenic Bt crops have been lost. A complete annotated reference genome is needed to improve global Bt resistance management of the pink bollworm. We generated the first chromosome-level genome assembly for pink bollworm from a Bt-susceptible laboratory strain (APHIS-S) using PacBio continuous long reads for contig generation, Illumina Hi-C for scaffolding, and Illumina whole-genome re-sequencing for error-correction. The psuedohaploid assembly consists of 29 autosomes and the Z sex chromosome. The assembly exceeds the minimum Earth BioGenome Project quality standards, has a low error-rate, is highly contiguous at both the contig and scaffold level (L/N50 of 18/8.26 MB and 14/16.44 MB, respectively), and complete, with 98.6% of lepidopteran single-copy orthologs represented without duplication. The genome was annotated with 50% repeat content and 14,107 protein-coding genes, further assigned to 41,666 functional annotations. This assembly represents the first publicly available complete annotated genome of pink bollworm and will serve as the foundation for advancing molecular genetics of this important pest species.

genomics↗

bioRxiv2022

Examination of rickettsial host range for shuttle vectors based on dnaA and parA genes from the pRM plasmid of Rickettsia monacensis

The genus Rickettsia encompasses a diverse group of obligate intracellular bacteria that are highly virulent disease agents of mankind as well as symbionts of arthropods. Native plasmids of Rickettsia amblyommatis (AaR/SC) have been used as models to construct shuttle vectors for genetic manipulation of several Rickettsia species. Here we report on the isolation of the complete plasmid (pRM658B) from Rickettsia monacensis (IrR/Munich) mutant Rmona658B and the construction of shuttle vectors based on pRM. To identify regions essential for replication, we made vectors containing the dnaA and parA genes of pRM with varying portions of the region surrounding these genes and a selection-reporter cassette conferring resistance to spectinomycin and expression of green fluorescent protein. Rickettsia amblyommatis (AaR/SC), R. monacensis (IrR/Munich), Rickettsia bellii (RML 369-C), Rickettsia parkeri (Tates Hell), and Rickettsia montanensis (M5/6) were successfully transformed with shuttle vectors containing pRM parA and dnaA. PCR assays targeting pRM regions not included in the vectors revealed that native pRM was retained in R. monacensis transformants. Determination of native pRM copy number using a plasmid-encoded gene (RM_p5) in comparison to chromosomal encoded gltA indicated reduced copy numbers in R. monacensis transformants. In transformed R. monacensis, native pRM and shuttle vectors with homologous parA and dnaA formed native plasmid-shuttle vector complexes. These studies provide insight on the maintenance of plasmids and shuttle vectors in rickettsiae. IMPORTANCEO_LIThis paper describes a new series of plasmid shuttle vectors for the transformation of rickettsiae. C_LIO_LIShuttle vectors based on parA and dnaA sequences of the plasmid pRM can be used to transform diverse rickettsia as well as its native host Rickettsia monacensis. C_LIO_LIOur results provide insight into the maintenance of rickettsial-based shuttle vectors in rickettsiae. C_LI

microbiology↗

bioRxiv2020

Everything you wanted to know about Mayaro virus but were afraid to ask: Characterization and lifecycle of Mayaro virus in vertebrate and invertebrate cellular backgrounds

Mayaro virus (MAYV) is an emerging new world alphavirus (genus Alphavirus, family Togaviridae) that causes acute multiphasic febrile illness, skin rash, polyarthritis, and occasional severe clinical phenotypes. The virus lifecycle alternates between invertebrate and vertebrate hosts. Here we characterize the replication features, cell entry, life cycle, and virus-related cell pathology of MAYV using vertebrate and invertebrate in vitro models. Electron dense clathrin-coated pits in infected cells, and reduced viral production in the presence of dynasore, ammonium chloride, and bafilomycin, indicates that viral entry occurs through pH-dependent endocytosis. Increase in FITC-dextran uptake (an indicator of macropinocytosis) in MAYV-infected cells, and dose-dependent infection inhibition by 5-(N-ethyl-N-isopropyl) amiloride (a macropinocytosis inhibitor), indicated that macropinocytosis is an additional entry mechanism of MAYV in vertebrate cells. Acutely infected vertebrate and invertebrate cells formed cytoplasmic or membrane-associated extracytoplasmic replication complexes. Mosquito cells showed modified hybrid cytoplasmic vesicles that supported virus replication, nucleocapsid production, and maturation. Mature virus particles were released from cells by both exocytosis and budding from the cell membrane. MAYV replication was cytopathic and associated with induction of apoptosis by the intrinsic pathway, and later by the extrinsic pathway in infected vertebrate cells. Given that MAYV is expanding its geographical existence as a potential public health problem, this study lays the foundation of biological understanding valuable for therapeutic and preventive interventions.

microbiology↗

bioRxiv2020

Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR-Cas9 ribonucleoprotein

CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel wasp Nasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes and whiteflies. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adult N. vitripennis females using either ReMOT control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. We demonstrate efficient delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing have been demonstrated. This approach will greatly facilitate CRISPR-applied genetic manipulation in this and other rising model organisms.

molecular biology↗

bioRxiv2020

CRISPR/Cas9-based genome editing in the silverleaf whitefly (Bemisia tabaci)

Bemisia tabaci cryptic species Middle East-Asia Minor I (MEAM1) is a serious agricultural polyphagous insect pest, and vector of numerous plant viruses, causing major worldwide economic losses. B. tabaci control is limited by lack of robust gene editing tools. Gene editing is difficult in B. tabaci due to small embryos that are technically challenging to inject, and which have high mortality post-injection. We developed a CRISPR/Cas9 gene editing protocol based on injection of vitellogenic adult females rather than embryos ("ReMOT Control"). We identified an ovary-targeting peptide ligand ("BtKV") that, when fused to Cas9 and injected into adult females, transduced the ribonucleoprotein complex to the germline, resulting in efficient, heritable editing of the offspring genome. In contrast to embryo injection, adult injection is easy and does not require specialized equipment. Development of easy-to-use gene editing protocols for B. tabaci will allow researchers to apply the power of reverse genetic approaches to this species and will lead to novel control methods for this devastating pest insect.

genetics↗