Non-canonical odor coding in the mosquito.

Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.

orco Mutagenesis Causes Loss of Antennal Lobe Glomeruli and Impaired Social Behavior in Ants.

Life inside ant colonies is orchestrated with diverse pheromones, but it is not clear how ants perceive these social signals. It has been proposed that pheromone perception in ants evolved via expansions in the numbers of odorant receptors (ORs) and antennal lobe glomeruli. Here, we generate the first mutant lines in the clonal raider ant, Ooceraea biroi, by disrupting orco, a gene required for the function of all ORs. We find that orco mutants exhibit severe deficiencies in social behavior and fitness, suggesting they are unable to perceive pheromones. Surprisingly, unlike in Drosophila melanogaster, orco mutant ants also lack most of the ∼500 antennal lobe glomeruli found in wild-type ants. These results illustrate that ORs are essential for ant social organization and raise the possibility that, similar to mammals, receptor function is required for the development and/or maintenance of the highly complex olfactory processing areas in the ant brain. VIDEO ABSTRACT.

Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans.

Multiple sensory cues emanating from humans are thought to guide blood-feeding female mosquitoes to a host. To determine the relative contribution of carbon dioxide (CO2) detection to mosquito host-seeking behavior, we mutated the AaegGr3 gene, a subunit of the heteromeric CO2 receptor in Aedes aegypti mosquitoes. Gr3 mutants lack electrophysiological and behavioral responses to CO2. These mutants also fail to show CO2-evoked responses to heat and lactic acid, a human-derived attractant, suggesting that CO2 can gate responses to other sensory stimuli. Whereas attraction of Gr3 mutants to live humans in a large semi-field environment was only slightly impaired, responses to an animal host were greatly reduced in a spatial-scale-dependent manner. Synergistic integration of heat and odor cues likely drive host-seeking behavior in the absence of CO2 detection. We reveal a networked series of interactions by which multimodal integration of CO2, human odor, and heat orchestrates mosquito attraction to humans.

Mosquito brains encode unique features of human odour to drive host seeking.

A globally invasive form of the mosquito Aedes aegypti specializes in biting humans, making it an efficient disease vector1. Host-seeking female mosquitoes strongly prefer human odour over the odour of animals2,3, but exactly how they distinguish between the two is not known. Vertebrate odours are complex blends of volatile chemicals with many shared components4-7, making discrimination an interesting sensory coding challenge. Here we show that human and animal odours evoke activity in distinct combinations of olfactory glomeruli within the Ae. aegypti antennal lobe. One glomerulus in particular is strongly activated by human odour but responds weakly, or not at all, to animal odour. This human-sensitive glomerulus is selectively tuned to the long-chain aldehydes decanal and undecanal, which we show are consistently enriched in human odour and which probably originate from unique human skin lipids. Using synthetic blends, we further demonstrate that signalling in the human-sensitive glomerulus significantly enhances long-range host-seeking behaviour in a wind tunnel, recapitulating preference for human over animal odours. Our research suggests that animal brains may distil complex odour stimuli of innate biological relevance into simple neural codes and reveals targets for the design of next-generation mosquito-control strategies.

Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

A single unidirectional piRNA cluster similar to the flamenco locus is the major source of EVE-derived transcription and small RNAs in Aedes aegypti mosquitoes.

Endogenous viral elements (EVEs) are found in many eukaryotic genomes. Despite considerable knowledge about genomic elements such as transposons (TEs) and retroviruses, we still lack information about nonretroviral EVEs. Aedes aegypti mosquitoes have a highly repetitive genome that is covered with EVEs. Here, we identified 129 nonretroviral EVEs in the AaegL5 version of the A. aegypti genome. These EVEs were significantly associated with TEs and preferentially located in repeat-rich clusters within intergenic regions. Genome-wide transcriptome analysis showed that most EVEs generated transcripts although only around 1.4% were sense RNAs. The majority of EVE transcription was antisense and correlated with the generation of EVE-derived small RNAs. A single genomic cluster of EVEs located in a 143 kb repetitive region in chromosome 2 contributed with 42% of antisense transcription and 45% of small RNAs derived from viral elements. This region was enriched for TE-EVE hybrids organized in the same coding strand. These generated a single long antisense transcript that correlated with the generation of phased primary PIWI-interacting RNAs (piRNAs). The putative promoter of this region had a conserved binding site for the transcription factor Cubitus interruptus, a key regulator of the flamenco locus in Drosophila melanogaster Here, we have identified a single unidirectional piRNA cluster in the A. aegypti genome that is the major source of EVE transcription fueling the generation of antisense small RNAs in mosquitoes. We propose that this region is a flamenco-like locus in A. aegypti due to its relatedness to the major unidirectional piRNA cluster in Drosophila melanogaster.

Racial disparities in non-recommendation of adjuvant chemotherapy in stage II-III ovarian cancer.

OBJECTIVES: To identify patient factors associated with not receiving a recommendation for adjuvant chemotherapy after primary surgery for ovarian cancer. METHODS: This retrospective cohort study used the National Cancer Database (NCDB) data from 2004 to 2015 to identify patients with stage II-III ovarian cancer who underwent primary surgery. Multivariate logistic regression analyses evaluated factors associated with notation in the NCDB that "chemotherapy was not recommended/administered because it was contraindicated due to patient risk factors (i.e., comorbid conditions, advanced age)." Survival data were assessed via Kaplan-Meier analyses. RESULTS: Of the 48,245 patients who met the inclusion criteria, 522 (1.08%) did not receive adjuvant chemotherapy because it was determined to be contraindicated. In multivariate analyses, independent predictors for not receiving a recommendation for adjuvant chemotherapy were age ≥ 70 years old (adjusted odds ratio, aOR = 2.43, p < 0.0001), non-zero Charlson-Deyo comorbidity scores (score 1, aOR = 1.41, p = 0.002; score ≥ 2, aOR = 2.57, p < 0.0001), and Black race (aOR = 2.12, p < 0.0001). For Black patients, recommendation against adjuvant chemotherapy occurred at a younger median age (64.5 years vs. 72 years) and was associated with lower 5-year survival (25.9% vs. 40.3%, p < 0.0001). CONCLUSIONS: Patients with ovarian cancer who underwent surgery but did not receive chemotherapy "because it was contraindicated due to patient risk factors" were older and had higher comorbidity scores. Even after controlling for these differences, Black patients were disproportionately not recommended for chemotherapy, which was associated with worse survival. Determining eligibility for adjuvant chemotherapy requires an individualized approach, and the possible influence of racial bias on risk estimation should be further investigated.

Molecular evidence of intertidal habitats selecting for repeated ice-binding protein evolution in invertebrates.

Ice-binding proteins (IBPs) have evolved independently in multiple taxonomic groups to improve their survival at sub-zero temperatures. Intertidal invertebrates in temperate and polar regions frequently encounter sub-zero temperatures, yet there is little information on IBPs in these organisms. We hypothesized that there are far more IBPs than are currently known and that the occurrence of freezing in the intertidal zone selects for these proteins. We compiled a list of genome-sequenced invertebrates across multiple habitats and a list of known IBP sequences and used BLAST to identify a wide array of putative IBPs in those invertebrates. We found that the probability of an invertebrate species having an IBP was significantly greater in intertidal species than in those primarily found in open ocean or freshwater habitats. These intertidal IBPs had high sequence similarity to fish and tick antifreeze glycoproteins and fish type II antifreeze proteins. Previously established classifiers based on machine learning techniques further predicted ice-binding activity in the majority of our newly identified putative IBPs. We investigated the potential evolutionary origin of one putative IBP from the hard-shelled mussel Mytilus coruscus and suggest that it arose through gene duplication and neofunctionalization. We show that IBPs likely readily evolve in response to freezing risk and that there is an array of uncharacterized IBPs, and highlight the need for broader laboratory-based surveys of the diversity of ice-binding activity across diverse taxonomic and ecological groups.

The Role of Sentinel Lymph Node Mapping in High-grade Endometrial Cancer.

OPINION STATEMENT: Total hysterectomy with lymph node assessment is the current standard-of-care for surgical staging in apparent early-stage endometrial cancer. Compared to the traditional complete pelvic lymphadenectomy with or without para-aortic lymphadenectomy, sentinel lymph node (SLN) mapping results in fewer surgical complications, decreased operative time, and lower rates of chronic lymphedema. The technique is endorsed by the National Comprehensive Cancer Network and the Society of Gynecologic Oncology guidelines, and over the past two decades the majority of gynecologic oncologists worldwide have adopted SLN mapping into their practice. However, as the results of the initial SLN studies were mostly based on low-grade tumors, adoption of the technique in high-grade tumors has been slower and more controversial. In this review, we discuss the most recent studies evaluating the SLN mapping in high-grade endometrial cancers. The results of these studies suggest that the SLN detection rate is acceptably high and the negative predictive value is sufficiently low to support the use of SLN mapping in high-grade endometrial tumors to replace complete lymphadenectomy. Validity of SLN mapping techniques does, however, require following a standard algorithm, and success depends also on surgeon expertise. Moreover, the impact of SLN mapping on overall survival in high-grade tumors requires future prospective randomized studies. Finally, a transition toward near-universal SLN mapping techniques for endometrial cancers could significantly impact on the adequacy of gynecologic oncology fellows' surgical training and competency in lymphadenectomy.

Incidence of Gynecologic Cancers in Women after Uterine Artery Embolization.

STUDY OBJECTIVE: To characterize the short-term incidence of gynecologic cancer after undergoing uterine artery embolization (UAE). DESIGN: Retrospective cohort study. SETTING: Commercial insurance claims database. PATIENTS: Total of 15 393 United States women aged 18 to 64 years who underwent UAE between 2007 and 2017. INTERVENTIONS: We used the IBM MarketScan (Armonk, NY) claims to identify adult women without previous gynecologic cancer diagnoses undergoing UAE between 2007 and 2017. Database queries identified women with any diagnostic or procedure codes related to gynecologic malignancies occurring in the first 3 years after UAE. A malignancy diagnosis was suggested by recurrent malignancy-related claims not linked exclusively to diagnostic testing (e.g., transvaginal ultrasound) and malignancy codes linked to tissue pathology claims. Incidence of malignancy diagnosis was calculated. Rates of endometrial sampling in the year before UAE were identified. MEASUREMENTS AND MAIN RESULTS: Thirty-one women undergoing UAE had gynecologic cancer diagnoses within 3 years of the procedure (22 of 31, 71% uterine cancers; 7 of 31, 23% ovarian cancers; and 2 of 31, 6% cervical cancers). On average, cancer diagnoses were made 1.1 ± 0.9 years after UAE. One in 497 women undergoing UAE was diagnosed with a gynecologic malignancy within 3 years, with an incidence of 1.1 malignancies per 1000 person-years. Cancer incidence increased with age at the time of UAE: short-term malignancy diagnoses were made in 1 in 377 women aged 45 to 54 years, and 1 in 79 women aged 55 to 64 years. In the year before UAE, 28% (4311 of 15 362) of women without cancer, and 23% (5 of 22) of women diagnosed with uterine cancer had preprocedural endometrial sampling. CONCLUSION: These data can inform risk/benefit counseling and shared decision-making regarding UAE and its alternatives. Short-term malignancies after UAE highlight the importance of preprocedure evaluation in symptomatic women and women with age-related risk.

How to turn an organism into a model organism in 10 'easy' steps.

Many of the major biological discoveries of the 20th century were made using just six species: Escherichia coli bacteria, Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast, Caenorhabditis elegans nematodes, Drosophila melanogaster flies and Mus musculus mice. Our molecular understanding of the cell division cycle, embryonic development, biological clocks and metabolism were all obtained through genetic analysis using these species. Yet the 'big 6' did not start out as genetic model organisms (hereafter 'model organisms'), so how did they mature into such powerful systems? First, these model organisms are abundant human commensals: they are the bacteria in our gut, the yeast in our beer and bread, the nematodes in our compost pile, the flies in our kitchen and the mice in our walls. Because of this, they are cheaply, easily and rapidly bred in the laboratory and in addition were amenable to genetic analysis. How and why should we add additional species to this roster? We argue that specialist species will reveal new secrets in important areas of biology and that with modern technological innovations like next-generation sequencing and CRISPR-Cas9 genome editing, the time is ripe to move beyond the big 6. In this review, we chart a 10-step path to this goal, using our own experience with the Aedes aegypti mosquito, which we built into a model organism for neurobiology in one decade. Insights into the biology of this deadly disease vector require that we work with the mosquito itself rather than modeling its biology in another species.

Modeling the Putative Ancient Distribution of Aedes togoi (Diptera: Culicidae).

The coastal rock pool mosquito, Aedes (Tanakius) togoi (Theobald) (Diptera: Culicidae), is found in coastal east Asia in climates ranging from subtropical to subarctic. However, a disjunct population in the Pacific Northwest of North America has an ambiguous heritage. Two potential models explain the presence of Ae. togoi in North America: ancient Beringian dispersal or modern anthropogenic introduction. Genetic studies have thus far proved inconclusive. Here we described the putative ancient distribution of Ae. togoi habitat in east Asia and examined the climatic feasibility of a Beringian introduction into North America using modern distribution records and ecological niche modeling of bioclimatic data from the last interglacial period (~120,000 BP), the last glacial maximum (~21,000 BP), and the mid-Holocene (~6000 BP). Our results suggest that suitable climatic conditions existed for Ae. togoi to arrive in North America through natural dispersal as well as to persist there until present times. Furthermore, we find that ancient distributions of suitable Ae. togoi habitat in east Asia may explain the genetic relationships between Ae. togoi populations identified in other studies. These findings indicate the utility of ecological niche modeling as a complementary tool for studying insect phylogeography.

The neurotranscriptome of the Aedes aegypti mosquito.

BACKGROUND: A complete genome sequence and the advent of genome editing open up non-traditional model organisms to mechanistic genetic studies. The mosquito Aedes aegypti is an important vector of infectious diseases such as dengue, chikungunya, and yellow fever and has a large and complex genome, which has slowed annotation efforts. We used comprehensive transcriptomic analysis of adult gene expression to improve the genome annotation and to provide a detailed tissue-specific catalogue of neural gene expression at different adult behavioral states. RESULTS: We carried out deep RNA sequencing across all major peripheral male and female sensory tissues, the brain and (female) ovary. Furthermore, we examined gene expression across three important phases of the female reproductive cycle, a remarkable example of behavioral switching in which a female mosquito alternates between obtaining blood-meals from humans and laying eggs. Using genome-guided alignments and de novo transcriptome assembly, our re-annotation includes 572 new putative protein-coding genes and updates to 13.5 and 50.3 % of existing transcripts within coding sequences and untranslated regions, respectively. Using this updated annotation, we detail gene expression in each tissue, identifying large numbers of transcripts regulated by blood-feeding and sexually dimorphic transcripts that may provide clues to the biology of male- and female-specific behaviors, such as mating and blood-feeding, which are areas of intensive study for those interested in vector control. CONCLUSIONS: This neurotranscriptome forms a strong foundation for the study of genes in the mosquito nervous system and investigation of sensory-driven behaviors and their regulation. Furthermore, understanding the molecular genetic basis of mosquito chemosensory behavior has important implications for vector control.

Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms.

Down Syndrome cell adhesion molecule (Dscam) genes encode neuronal cell recognition proteins of the immunoglobulin superfamily. In Drosophila, Dscam1 generates 19,008 different ectodomains by alternative splicing of three exon clusters, each encoding half or a complete variable immunoglobulin domain. Identical isoforms bind to each other, but rarely to isoforms differing at any one of the variable immunoglobulin domains. Binding between isoforms on opposing membranes promotes repulsion. Isoform diversity provides the molecular basis for neurite self-avoidance. Self-avoidance refers to the tendency of branches from the same neuron (self-branches) to selectively avoid one another. To ensure that repulsion is restricted to self-branches, different neurons express different sets of isoforms in a biased stochastic fashion. Genetic studies demonstrated that Dscam1 diversity has a profound role in wiring the fly brain. Here we show how many isoforms are required to provide an identification system that prevents non-self branches from inappropriately recognizing each other. Using homologous recombination, we generated mutant animals encoding 12, 24, 576 and 1,152 potential isoforms. Mutant animals with deletions encoding 4,752 and 14,256 isoforms were also analysed. Branching phenotypes were assessed in three classes of neurons. Branching patterns improved as the potential number of isoforms increased, and this was independent of the identity of the isoforms. Although branching defects in animals with 1,152 potential isoforms remained substantial, animals with 4,752 isoforms were indistinguishable from wild-type controls. Mathematical modelling studies were consistent with the experimental results that thousands of isoforms are necessary to ensure acquisition of unique Dscam1 identities in many neurons. We conclude that thousands of isoforms are essential to provide neurons with a robust discrimination mechanism to distinguish between self and non-self during self-avoidance.

Decoupling factors affecting plant diversity and cover on extensive green roofs.

Supplemental irrigation systems are often specified on green roofs to ensure plant cover and growth, both important components of green roof performance and aesthetics. Properties of the growing media environment too can alter the assemblage of plant species able to thrive. In this study we determine how plant cover, above ground biomass and species diversity are influenced by irrigation and growing media. Grass and forb vegetative cover and biomass were significantly greater in organic based growing media but there was no effect of supplemental irrigation, with two warm season grasses dominating in those treatments receiving no supplemental irrigation. On the other hand, plant diversity declined without irrigation in organic media, and having no irrigation in inorganic growing media resulted in almost a complete loss of cover. Sedum biomass was less in inorganic growing media treatments and species dominance shifted when growing media organic content increased. Our results demonstrate that supplemental irrigation is required to maintain plant diversity on an extensive green roof, but not necessarily plant cover or biomass. These results provide evidence that planting extensive green roofs with a mix of plant species can ensure the survival of some species; maintaining cover and biomass when supplemental irrigation is turned off to conserve water, or during extreme drought.

Postmenopausal mixed gestational trophoblastic neoplasia with complete response to immunotherapy - A case report.

•Mixed GTN.•GTN in a post-menopausal woman.•Treatment of GTN with immunotherapy.

Design and Validation of Guide RNAs for CRISPR-Cas9 Genome Editing in Mosquitoes.

CRISPR-Cas9 has revolutionized gene editing for traditional and nontraditional model organisms alike. This tool has opened the door to new mechanistic studies of basic mosquito biology as well as the development of novel vector control strategies based on CRISPR-Cas9, including gene drives that spread genetic elements in the population. Although the promise of the specificity, flexibility, and ease of deployment CRISPR is real, its implementation still requires empirical optimization for each new species of interest, as well as to each genomic target within a given species. Here, we provide an overview of designing and testing single-guide RNAs for the use of CRISPR-based gene editing tools.

Validating Single-Guide RNA for Aedes aegypti Gene Editing.

Creating transgenic mosquitoes allows for mechanistic studies of basic mosquito biology and the development of novel vector control strategies. CRISPR-Cas9 gene editing has revolutionized gene editing, including in mosquitoes. This protocol details part of the gene editing process of Aedes aegypti mosquitoes via CRISPR-Cas9, through testing and validating single-guide RNAs (sgRNAs). Gene editing activity varies depending on the sequence of sgRNAs used, so validation of sgRNA activity should be done before large-scale generation of mutants or transgenics. sgRNA is designed using online tools and synthesized in <1 h. Once mutants or transgenics are generated via embryo microinjection, sgRNA activity is validated by quick genotyping polymerase chain reaction (PCR) and DNA sequencing.

Automated Egg-Counting Approaches for Aedes aegypti Oviposition Experiments.

Egg-laying preferences of mosquitoes can reveal key neurosensory mechanisms informing the decision-making process for this critical behavior. A single blood meal results in a gravid female Aedes aegypti mosquito laying more than 100 eggs. Therefore, egg counting represents a potentially time-consuming and laborious component to behavioral assays, such as those that measure oviposition preference or fecundity. Automated algorithms that count eggs from images can dramatically reduce the time required for this step of data processing and analysis and increase reproducibility associated with having multiple human observers count the eggs. Here, we present two distinct approaches for counting melanized Ae. aegypti eggs laid on white filter paper.