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Escalating vector disease burdens pose significant global health risks, so innovative tools for targeting mosquitoes are critical. We engineered an antiviral strategy termed REAPER (vRNAExpressionActivatesPoisonousEffectorRibonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR Cas13 and its potent collateral activity. Akin to a stealthy Trojan Horse hiding in stealth awaiting the presence of its enemy, REAPER remains concealed within the mosquito until an infectious blood meal is up taken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13 mediated RNA targeting significantly reduces viral replication and its promiscuous collateral activity can even kill infected mosquitoes. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.One-Sentence SummaryEngineered Cas13-based antiviral sensor kills infected mosquitoes upon activation from arbovirus infection.

The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes.We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes.The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.

Enteral nutrition support plays a key role in minimizing malnutrition in critically-ill patients, and its provision is originally a nurse's responsibility. Thus, nurses need to have a sufficient knowledge and a positive attitude with regard to enteral nutrition. In Palestine, literature regarding nurses' knowledge, practices, and attitudes of enteral nutrition is rare.This study aimed to investigate Palestinian nurses' knowledge, practices, and attitudes regarding enteral nutrition.Interview based pre-tested questionnaire was used during data collection procedure. Cronbach Alpha was also used to determine the reliability of knowledge, attitude and practice questionnaire. Descriptive statistics, independent t-test, and ANOVA were performed.A total of 325 registered nurses were involved in the final analysis. The mean of the knowledge score is (9.6 ± 2.8) out of 20. It was confirmed that sociodemographic characteristics has no influence on nutrition knowledge score among nurses. The findings also reveal that certain aspects of enteral nutrition practices were consistent with the current guidelines such as flushing the tube and backrest elevation. On the other hand, certain practices showed inconsistency and differences with international guidelines such as checking gastric residual volume. Results also showed that nurses attitudes were positive towards enteral nutrition.It was found that nurses' have inadequate knowledge regarding enteral nutrition. The findings also showed that enteral nutrition practices among nurses were somewhat incongruent with best current evidence. However, it was noted that they have positive attitudes towards enteral nutrition. Promoting research utilization is highly needed as well as establishing evidence-based guidelines.

Simple Summary Mosquitoes from the family Culicidae are vectors of numerous human diseases such as dengue, Zika, and West Nile fevers, malaria, and lymphatic filariasis. The availability of high-quality genome assemblies for mosquitoes assists in identifying genes responsible for important epidemiological traits such as vector competence, insecticide resistance, and mosquito behavior and stimulates a better understanding of the genetic structure of mosquito populations. Thus, the development of genomic resources and tools is a necessary step for designing and implementing novel genome-based approaches to control mosquitoes. The emphasis of this study is the further development and optimization of a physical genome mapping approach. Physical mapping is placing genomic scaffolds to the chromosomes based on hybridization of the specific probes. This task is difficult for mosquitoes with large genome sizes which are enriched with repetitive DNA sequences and can be potentially misassembled. Here, we describe in detail a simple and robust technique for the physical mapping of such mosquito genomes. This method can be further used for physical genome mapping in other mosquitoes and insects. Abstract Long-read sequencing technologies have opened up new avenues of research on the mosquito genome biology, enabling scientists to better understand the remarkable abilities of vectors for transmitting pathogens. Although new genome mapping technologies such as Hi-C scaffolding and optical mapping may significantly improve the quality of genomes, only cytogenetic mapping, with the help of fluorescence in situ hybridization (FISH), connects genomic scaffolds to a particular chromosome and chromosome band. This mapping approach is important for creating and validating chromosome-scale genome assemblies for mosquitoes with repeat-rich genomes, which can potentially be misassembled. In this study, we describe a new gene-based physical mapping approach that was optimized using the newly assembled Aedes albopictus genome, which is enriched with transposable elements. To avoid amplification of the repetitive DNA, 15 protein-coding gene transcripts were used for the probe design. Instead of using genomic DNA, complementary DNA was utilized as a template for development of the PCR-amplified probes for FISH. All probes were successfully amplified and mapped to specific chromosome bands. The genome-unique probes allowed to perform unambiguous mapping of genomic scaffolds to chromosome regions. The method described in detail here can be used for physical genome mapping in other insects.

Background The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. Methods In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. Results The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. Conclusions This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

Background: The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes. Results: We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. Conclusion: The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.

Understanding the population structure and mechanisms of taxa diversification is important for organisms responsible for the transmission of human diseases. Two vectors of West Nile virus, Culex pipiens pipiens and Cx. p. molestus, exhibit epidemiologically important behavioral and physiological differences, but the whole-genome divergence between them was unexplored. The goal of this study is to better understand the level of genomic differentiation and population structures of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared the whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Principal Component, ADMIXTURE, and neighbor joining analyses of the nuclear genomes identified two major intercontinental, monophyletic clusters of Cx. p. pipiens and Cx. p. molestus. The level of genomic differentiation between the subspecies was uniform along chromosomes. The ADMIXTURE analysis determined signatures of admixture in Cx. p. pipens populations but not in Cx. p. molestus populations. Comparison of mitochondrial genomes among the specimens showed a paraphyletic origin of the major haplogroups between the subspecies but a monophyletic structure between the continents. Thus, our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units with monophyletic origin that have undergone incipient ecological speciation. Russian Science FoundationRussian Science Foundation (RSF) [19-14-00130] We thank Igor V. Sharakhov for the productive discussion of the manuscript, Dmitri A. Karagodin for the help with formatting figures, and Janet Webster for proofreading the text. This project was supported by the Russian Science Foundation grant No 19-14-00130 to MVS. The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Background The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes. Results We use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. Conclusion The AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.