Department of Entomology

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Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology
Martin-Martin, Ines; Leon, Paola Carolina Valenzuela; Amo, Laura; Shrivastava, Gaurav; Iniguez, Eva; Aryan, Azadeh; Brooks, Steven; Kojin, Bianca B.; Williams, Adeline E.; Bolland, Silvia; Ackerman, Hans; Adelman, Zach N.; Calvo, Eric (Cell Press, 2022-04-12)
Saliva from mosquitoes contains vasodilators that antagonize vasoconstrictors produced at the bite site. Sialokinin is a vasodilator present in the saliva of Aedes aegypti. Here, we investigate its function and describe its mechanism of action during blood feeding. Sialokinin induces nitric oxide release similar to substance P. Sialokinin-KO mosquitoes produce lower blood perfusion than parental mosquitoes at the bite site during probing and have significantly longer probing times, which result in lower blood feeding success. In contrast, there is no difference in feeding between KO and parental mosquitoes when using artificial membrane feeders or mice that are treated with a substance P receptor antagonist, confirming that sialokinin interferes with host hemostasis via NK1R signaling. While sialokinin-KO saliva does not affect virus infection in vitro, it stimulates macrophages and inhibits leukocyte recruitment in vivo. This work highlights the biological functionality of salivary proteins in blood feeding.
Bed Bugs and Infectious Disease: A Case for the Arboviruses
Adelman, Zach N.; Miller, Dini M.; Myles, Kevin M. (PLOS, 2013-08-01)
Bed bug infestations (Cimicidae; Cimex lectularius) have been increasing worldwide over the last few decades [1,2]. Several factors have been posited to explain this resurgence, including widespread insecticide resistance, human population growth, and increased international travel [1]. Clinically, reactions to bed bug bites vary from unapparent, to small (,5 mm) maculopapular lesions, to large wheals (2–6 cm); other reactions include bullous rashes, dermatitis, and asthma [1,3]. However, in the developed world the psychological, social, and economic impacts of bed bugs may be the most troubling aspects of the resurgence [2]. While the bed bug invasion cuts across economic lines, those with sufficient resources are able to clear the infestations, while those without may have to live with their bed bugs into the foreseeable future [2,4].
Cooler Temperatures Destabilize RNA Interference and Increase Susceptibility of Disease Vector Mosquitoes to Viral Infection
Adelman, Zach N.; Anderson, Michelle A. E.; Wiley, Michael R.; Murreddu, Marta G.; Samuel, Glady Hazitha; Morazzani, Elaine M.; Myles, Kevin M. (PLOS, 2013-05)
Background: The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus), exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi) pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery. Methodology/Principal Findings: We utilized transgenic "sensor'' strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor'' strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2) or Argonaute-2 (AGO-2). We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18 degrees C as compared with 28 degrees C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA) trigger for RNAi, showed no change in EGFP expression when reared at 18 degrees C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses. Conclusions/Significance: This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.
Deep Sequencing of Pyrethroid-Resistant Bed Bugs Reveals Multiple Mechanisms of Resistance within a Single Population
Adelman, Zach N.; Kilcullen, Kathleen A.; Koganemaru, Reina; Anderson, Michelle A. E.; Anderson, Troy D.; Miller, Dini M. (PLOS, 2011-10-19)
A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD50 bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.
Double Subgenomic Alphaviruses Expressing Multiple Fluorescent Proteins Using a Rhopalosiphum padi Virus Internal Ribosome Entry Site Element
Wiley, Michael R.; Roberts, Lisa O.; Adelman, Zach N.; Myles, Kevin M. (PLOS, 2010-11-10)
Double subgenomic Sindbis virus (dsSINV) vectors are widely used for the expression of proteins, peptides, and RNA sequences. These recombinant RNA viruses permit high level expression of a heterologous sequence in a wide range of animals, tissues, and cells. However, the alphavirus genome structure and replication strategy is not readily amenable to the expression of more than one heterologous sequence. The Rhopalosiphum padi virus (RhPV) genome contains two internal ribosome entry site (IRES) elements that mediate cap-independent translation of the virus nonstructural and structural proteins. Most IRES elements that have been characterized function only in mammalian cells but previous work has shown that the IRES element present in the 5′ untranslated region (UTR) of the RhPV genome functions efficiently in mammalian, insect, and plant systems. To determine if the 5′ RhPV IRES element could be used to express more than one heterologous sequence from a dsSINV vector, RhPV 5′ IRES sequences were placed between genes for two different fluorescent marker proteins in the dsSINV, TE/3′2J/mcs. While mammalian and insect cells infected with recombinant viruses containing the RhPV sequences expressed both fluorescent marker proteins, only single marker proteins were routinely observed in cells infected with dsSINV vectors in which the RhPV IRES had been replaced by a luciferase fragment, an antisense RhPV IRES, or no intergenic sequence. Thus, we report development of a versatile tool for the expression of multiple sequences in diverse cell types.
Germline excision of transgenes in Aedes aegypti by homing endonucleases
Aryan, Azadeh; Anderson, Michelle A. E.; Myles, Kevin M.; Adelman, Zach N. (Nature Publishing Group, 2013-04)
Aedes (Ae.) aegypti is the primary vector for dengue viruses (serotypes1-4) and chikungunya virus. Homing endonucleases (HEs) are ancient selfish elements that catalyze double-stranded DNA breaks (DSB) in a highly specific manner. In this report, we show that the HEs Y2-I-AniI, I-CreI and I-SceI are all capable of catalyzing the excision of genomic segments from the Ae. aegypti genome in a heritable manner. Y2-I-AniI demonstrated the highest efficiency at two independent genomic targets, with 20-40% of Y2-I-AniI-treated individuals producing offspring that had lost the target transgene. HE-induced DSBs were found to be repaired via the single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways in a manner dependent on the availability of direct repeat sequences in the transgene. These results support the development of HE-based gene editing and gene drive strategies in Ae. aegypti, and confirm the utility of HEs in the manipulation and modification of transgenes in this important vector.
The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes
Haac, Mary Etna; Anderson, Michelle A. E.; Eggleston, Heather; Myles, Kevin M.; Adelman, Zach N. (2015-04-20)
Aedes aegypti mosquitoes vector several arboviruses of global health significance, including dengue viruses and chikungunya virus. RNA interference (RNAi) plays an important role in antiviral immunity, gene regulation and protection from transposable elements. Double-stranded RNA binding proteins (dsRBPs) are important for efficient RNAi; in Drosophila functional specialization of the miRNA, endo-siRNA and exo-siRNA pathway is aided by the dsRBPs Loquacious (Loqs-PB, Loqs-PD) and R2D2, respectively. However, this functional specialization has not been investigated in other dipterans. We were unable to detect Loqs-PD in Ae. aegypti; analysis of other dipteran genomes demonstrated that this isoform is not conserved outside of Drosophila. Overexpression experiments and small RNA sequencing following depletion of each dsRBP revealed that R2D2 and Loqs-PA cooperate non-redundantly in siRNA production, and that these proteins exhibit an inhibitory effect on miRNA levels. Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production. Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer. We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways.
Identification of Candidate Iron Transporters From the ZIP/ZnT Gene Families in the Mosquito Aedes aegypti
Tsujimoto, Hitoshi; Anderson, Michelle A. E.; Myles, Kevin M.; Adelman, Zach N. (Frontiers, 2018-04-12)
Mosquito-transmitted viral pathogens, such as dengue and Zika, afflict tens of thousands of people every year. These viruses are transmitted during the blood-feeding process that is required for mosquito reproduction, the most important vector being Aedes aegypti. While vertebrate blood is rich in protein, its high iron content is potentially toxic to mosquitoes. Although iron transport and sequestration are essential in the reproduction of vector mosquitoes, we discovered that culicine mosquitoes lack homologs of the common iron transporter NRAMP. Using a novel cell-based screen, we identified two ZIP and one ZnT genes as candidate iron transporters in the mosquito A. aegypti, the vector of dengue, Zika, and chikungunya. We determined the organ-specific expression pattern of these genes at critical time points in early reproduction. The result indicates modulation of these genes upon blood feeding, especially a ZIP13 homolog that is highly up-regulated after blood feeding, suggesting its importance in iron mobilization during blood digestion and reproduction. Gene silencing resulted in differential iron accumulation in the midgut and ovaries. This study sets a foundation for further investigation of iron transport and control strategies of this viral vector.
Insights into the Preservation of the Homomorphic Sex-Determining Chromosome of Aedes aegypti from the Discovery of a Male-Biased Gene Tightly Linked to the M-Locus
Hall, Andrew Brantley; Timoshevskiy, Vladimir A.; Sharakhova, Maria V.; Jiang, Xiaofang; Basu, Sanjay; Anderson, Michelle A. E.; Hu, Wanqi; Sharakhov, Igor V.; Adelman, Zach N.; Tu, Zhijian Jake (Oxford University Press, 2014-01-01)
The preservation of a homomorphic sex-determining chromosome in some organisms without transformation into a heteromorphic sex chromosome is a long-standing enigma in evolutionary biology. A dominant sex-determining locus (or M-locus) in an undifferentiated homomorphic chromosome confers the male phenotype in the yellow fever mosquito Aedes aegypti. Genetic evidence suggests that the M-locus is in a nonrecombining region. However, the molecular nature of the M-locus has not been characterized. Using a recently developed approach based on Illumina sequencing of male and female genomic DNA, we identified a novel gene, myo-sex, that is present almost exclusively in the male genome but can sporadically be found in the female genome due to recombination. For simplicity, we define sequences that are primarily found in the male genome as male-biased. Fluorescence in situ hybridization (FISH) on A. aegypti chromosomes demonstrated that the myo-sex probe localized to region 1q21, the established location of theM-locus.Myo-sex is a duplicated myosin heavy chain gene that is highly expressed in the pupa and adult male.Myo-sex shares 83% nucleotide identity and 97% amino acid identity with its closest autosomal paralog, consistent with ancient duplication followed by strong purifying selection. Compared with males, myo-sex is expressed at very low levels in the females that acquired it, indicating that myo-sexmay be sexually antagonistic. This study establishes a framework to discover male-biased sequences within a homomorphic sex-determining chromosome and offers new insights into the evolutionary forces that have impeded the expansion of the nonrecombining M-locus in A. aegypti.
Mosquito-Borne Viruses and Suppressors of Invertebrate Antiviral RNA Silencing
O'Neal, Scott T.; Samuel, Glady Hazitha; Adelman, Zach N.; Myles, Kevin M. (MDPI, 2014-11-11)
The natural maintenance cycles of many mosquito-borne viruses require establishment of persistent non-lethal infections in the invertebrate host. While the mechanisms by which this occurs are not well understood, antiviral responses directed by small RNAs are important in modulating the pathogenesis of viral infections in disease vector mosquitoes. In yet another example of an evolutionary arms race between host and pathogen, some plant and insect viruses have evolved to encode suppressors of RNA silencing (VSRs). Whether or not mosquito-borne viral pathogens encode VSRs has been the subject of debate. While at first there would seem to be little evolutionary benefit to mosquito-borne viruses encoding proteins or sequences that strongly interfere with RNA silencing, we present here a model explaining how the expression of VSRs by these viruses in the vector might be compatible with the establishment of persistence. We also discuss the challenges associated with interrogating these viruses for the presence of suppressor proteins or sequences, as well as the candidates that have been identified in the genomes of mosquito-borne pathogens thus far.
Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight
Aryan, Azadeh; Anderson, Michelle A. E.; Biedler, James K.; Qi, Yumin; Overcash, Justin M.; Naumenko, Anastasia N.; Sharakhova, Maria V.; Mao, Chunhong; Adelman, Zach N.; Tu, Zhijian Jake (NAS, 2020-06-12)
A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti. Nix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sexconversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus,was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.
Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9
Martin-Martin, Ines; Aryan, Azadeh; Meneses, Claudio; Adelman, Zach N.; Calvo, Eric (PLOS, 2018-09)
Background Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis. Methodology/Principal findings Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this nonmodel organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed. Conclusion and significance The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions.
Unique features of a global human ectoparasite identified through sequencing of the bed bug genome
Benoit, Joshua B.; Adelman, Zach N.; Reinhardt, Klaus; Dolan, Amanda M.; Poelchau, Monica; Jennings, Emily C.; Szuter, Elise M.; Hagan, Richard W.; Gujar, Hemant; Shukla, Jayendra Nath; Zhu, Fang; Mohan, M.; Nelson, David R.; Rosendale, Andrew J.; Derst, Christian; Resnik, Valentina; Wernig, Sebastian; Menegazzi, Pamela; Wegener, Christian; Peschel, Nicolai; Hendershot, Jacob M.; Blenau, Wolfgang; Predel, Reinhard; Johnston, Paul R.; Ioannidis, Panagiotis; Waterhouse, Robert M.; Nauen, Ralf; Schorn, Corinna; Ott, Mark-Christoph; Maiwald, Frank; Johnston, J. Spencer; Gondhalekar, Ameya D.; Scharf, Michael E.; Peterson, Brittany F.; Raje, Kapil R.; Hottel, Benjamin A.; Armisen, David; Crumiere, Antonin Jean Johan; Refki, Peter Nagui; Santos, Maria Emilia; Sghaier, Essia; Viala, Severine; Khila, Abderrahman; Ahn, Seung-Joon; Childers, Christopher; Lee, Chien-Yueh; Lin, Han; Hughes, Daniel S. T.; Duncan, Elizabeth J.; Murali, Shwetha C.; Qu, Jiaxin; Dugan, Shannon; Lee, Sandra L.; Chao, Hsu; Dinh, Huyen; Han, Yi; Doddapaneni, Harshavardhan; Worley, Kim C.; Muzny, Donna M.; Wheeler, David; Panfilio, Kristen A.; Jentzsch, Iris M. Vargas; Vargo, Edward L.; Booth, Warren; Friedrich, Markus; Weirauch, Matthew T.; Anderson, Michelle A. E.; Jones, Jeffery W.; Mittapalli, Omprakash; Zhao, Chaoyang; Zhou, Jing-Jiang; Evans, Jay D.; Attardo, Geoffrey M.; Robertson, Hugh M.; Zdobnov, Evgeny M.; Ribeiro, Jose M. C.; Gibbs, Richard A.; Werren, John H.; Palli, Subba R.; Schal, Coby; Richards, Stephen (Nature, 2016-02-02)
The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite

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