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BackgroundThe 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.ResultsWe 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.ConclusionThe 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.

BackgroundAedes aegypti, the main vector of dengue, yellow fever, and other arboviruses thrives in tropical and subtropical areas around the globe putting half of the world's population at risk. Despite aggressive efforts to control the transmission of those viruses, an unacceptable number of cases occur every year, emphasizing the need to develop new control strategies. Proposals for vector control focused on population suppression could offer a feasible alternative method to reduce disease transmission. The induction of extreme male-biased sex ratios has been hypothesized to be able to suppress or collapse a population, with previous experiments showing that stable expression of the male determining factor Nix in A. aegypti is sufficient to convert females into fertile males.Methodology/principal findingsHere, we report on the conditional expression of Nix in transgenic A. aegypti under the control of the tetracycline-dependent (Tet-off) system, with the goal of establishing repressible sex distortion. A masculinization phenotype was observed in three of the seven transgenic lines with females exhibiting male-like long maxillary palps and most importantly, the masculinized females were unable to blood feed. Doxycycline treatment of the transgenic lines only partially restored the normal phenotype from the masculinized transgenic lines, while RT-qPCR analysis of early embryos or adults showed no correlation between the level of masculinization and ectopic Nix expression.Conclusions/significanceWhile the conditional expression of Nix produced intersex phenotypes, the level of expression was insufficient to program full conversion. Modifications that increase both the level of activation (no tet) and the level of repression (with tet) will be necessary, as such this study represents one step forward in the development of genetic strategies to control vector-borne diseases via sex ratio distortion.

Abstract 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.

Abstract Background The wide distribution of Aedes aegypti, the main vector of dengue and yellow fever viruses, currently puts three billion people in the world at risk of infection with these viruses. Continuous transmission of these and other viruses despite aggressive efforts to prevent this emphasizes the need to develop new control strategies. Proposals to control disease transmission based on vector engineering, including both population suppression and population replacement, rely on the development of transgenes under the control of regulatory elements able to drive molecules in a specific tissue, time and strength. Methods Here we report the characterization of a promoter active in both the female germline and early zygote, derived from the transcription factor bZip1 in the mosquito Ae. aegypti, using transposon-based methods and RT-qPCR. Results We generated seven transgenic lines carrying AabZip1-reporter constructs and observed expression in both the ovary and early embryo. RT-qPCR analysis was performed to evaluate transcript expression patterns for each line, confirming that transgenic expression from the AabZip1 promoter largely recapitulated the endogenous expression pattern, albeit the strength of maternal expression appeared to be strongly influenced by chromosomal position. Conclusions This study provides a new regulatory sequence that can be useful for generating transgenic lines that can become a tool in vector control strategies.

Anopheles stephensi is a principal vector of urban malaria on the Indian subcontinent and an emerging model for molecular and genetic studies of mosquito biology. To enhance our understanding of female mosquito reproduction, and to develop new tools for basic research and for genetic strategies to control mosquito-borne infectious diseases, we identified 79 genes that displayed previtellogenic germline-specific expression based on RNA-Seq data generated from 11 life stage-specific and sex-specific samples. Analysis of this gene set provided insights into the biology and evolution of female reproduction. Promoters from two of these candidates, vitellogenin receptor and nanos, were used in independent transgenic cassettes for the expression of artificial microRNAs against suspected mosquito maternal-effect genes, discontinuous actin hexagon and myd88. We show these promoters have early germline-specific expression and demonstrate 73% and 42% knockdown of myd88 and discontinuous actin hexagon mRNA in ovaries 48 hr after blood meal, respectively. Additionally, we demonstrate maternal-specific delivery of mRNA and protein to progeny embryos. We discuss the application of this system of maternal delivery of mRNA/miRNA/protein in research on mosquito reproduction and embryonic development, and for the development of a gene drive system based on maternal-effect dominant embryonic arrest.

Anopheles stephensi is a principal vector of urban malaria on the Indian subcontinent and an emerging model for molecular and genetic studies of mosquito biology. To enhance our understanding of female mosquito reproduction, and to develop new tools for basic research and for genetic strategies to control mosquito-borne infectious diseases, we identified 79 genes that displayed previtellogenic germline-specific expression based on RNA-Seq data generated from 11 life stage-specific and sex-specific samples. Analysis of this gene set provided insights into the biology and evolution of female reproduction. Promoters from two of these candidates, vitellogenin receptor and nanos, were used in independent transgenic cassettes for the expression of artificial microRNAs against suspected mosquito maternal-effect genes, discontinuous actin hexagon and myd88. We show these promoters have early germline-specific expression and demonstrate 73% and 42% knockdown of myd88 and discontinuous actin hexagon mRNA in ovaries 48 hr after blood meal, respectively. Additionally, we demonstrate maternal-specific delivery of mRNA and protein to progeny embryos. We discuss the application of this system of maternal delivery of mRNA/miRNA/protein in research on mosquito reproduction and embryonic development, and for the development of a gene drive system based on maternal-effect dominant embryonic arrest.

Abstract Background The Asian malaria mosquito, Anopheles stephensi, is a major urban malaria vector in the Middle East and on the Indian subcontinent. Early zygotic transcription, which marks the maternal-to-zygotic transition, has not been systematically studied in An. stephensi or any other Anopheles mosquitoes. Improved understanding of early embryonic gene expression in An. stephensi will facilitate genetic and evolutionary studies and help with the development of novel control strategies for this important disease vector. Results We obtained RNA-seq data in biological triplicates from four early An. stephensi embryonic time points. Using these data, we identified 70 and 153 pure early zygotic genes (pEZGs) under stringent and relaxed conditions, respectively. We show that these pEZGs are enriched in functional groups related to DNA-binding transcription regulators, cell cycle modulators, proteases, transport, and cellular metabolism. On average these pEZGs are shorter and have less introns than other An. stephensi genes. Some of the pEZGs may arise de novo while others have clear non-pEZG paralogs. There is no or very limited overlap between An. stephensi pEZGs and Drosophila melanogaster or Aedes aegypti pEZGs. Interestingly, the upstream region of An. stephensi pEZGs lack significant enrichment of a previously reported TAGteam/VBRGGTA motif found in the regulatory region of pEZGs in D. melanogaster and Ae. aegypti. However, a GT-rich motif was found in An. stephensi pEZGs instead. Conclusions We have identified a number of pEZGs whose predicted functions and structures are consistent with their collective roles in the degradation of maternally deposited components, activation of the zygotic genome, cell division, and metabolism. The pEZGs appear to rapidly turn over within the Dipteran order and even within the Culicidae family. These pEZGs, and the shared regulatory motif, could provide the promoter or regulatory sequences to drive gene expression in the syncytial or early cellular blastoderm, a period when the developing embryo is accessible to genetic manipulation. In addition, these molecular resources may be used to achieve sex separation of mosquitoes for sterile insect technique.

The mosquito family Culicidae is divided into 2 subfamilies named the Culicinae and Anophelinae. Nix , the dominant male-determining factor, has only been found in the culicines Aedes aegypti and Aedes albopictus , 2 important arboviral vectors that belong to the subgenus Stegomyia. Here we performed sex-specific whole-genome sequencing and RNAseq of divergent mosquito species and explored additional male-inclusive datasets to investigate the distribution of Nix. Except for the Culex genus, Nix homologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from 3 highly divergent tribes comprising 4 genera, suggesting Nix originated at least 133 to 165 million years ago (MYA). Heterologous expression of 1 of 3 divergent Nix open reading frames (ORFs) in Ae. aegypti resulted in partial masculinization of genetic females as evidenced by morphology and doublesex splicing. Phylogenetic analysis suggests Nix is related to femaleless (fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes. Nix from all species has a conserved structure, including 3 RNA-recognition motifs (RRMs), as does fle. However, Nix has evolved at a much faster rate than fle. The RRM3 of both Nix and fle are distantly related to the single RRM of a widely distributed and conserved splicing factor transformer-2 (tra2). The RRM3-based phylogenetic analysis suggests this domain in Nix and fle may have evolved from tra2 or a tra2 -related gene in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex determination in mosquitoes and will inform broad applications of mosquito-control strategies based on manipulating sex ratios toward nonbiting males. [ABSTRACT FROM AUTHOR]

Good novelists often base their work on biographical and historical facts, but then transform those facts into the magic of fiction. In this essay, I show how Leslie Marmon Silko took factual raw materials and, through the alchemy of her creative artistry, transformed her novel Ceremony into something quite new and different. I consider first the real-life inspiration for several of the Laguna soldiers in Ceremony who fought in World War II. I then suggest a historical source for Silko’s information about the Bataan Death March and discuss some of the changes that Silko made in portraying the march in Ceremony. These changes help us to understand Silko’s purposes in creating the character of Tayo and the forces that put him in need of the ceremony that lies at the heart of the novel. I also show that Silko portrayed the Japanese with considerable sympathy.WAR VETERANSIn a letter to her friend James Wright on 16 June 1979, just two years after the publication of Ceremony, Silko wrote of having recently visited Laguna from her home in Tucson:I have just returned from a short visit at Laguna . . . I thought a great deal about two of my father’s first cousins, Jack and Les, both dead now—Les died while I was here in Tucson. He wasn’t old, but he was one of the men I was writing about when I wrote Ceremony. Les had been a football star at the U. of New Mexico for one semester before he was drafted. The local press called him “Squaw” because he was Laguna. He was over six feet tall and even these last years he was a strong man—except for what the liquor did. I suppose it might be because a good part of him became part of the main characters of the novel that I spent some time looking at the house he and his brother Jack had lived in. . . . I suppose Les will be remembered for being called “Squaw” in the Albuquerque Journal and for his car wrecks and brawls.

Leslie Marmon Silko’s Ceremony is a novel about a young man named Tayo who returns to the Laguna Reservation, probably in 1948, after horrifying experiences on the Pacific front in World War II and an unspecified period of time in a Veteran’s Administration hospital in California. He comes home a psychological mess after being released from a prisoner of war camp, probably in the Philippines. In one sense, Tayo’s story is straightforward enough. A man grows from confusion to clarity, from being lost to being found, from being muddleheaded to being clearheaded, from being alone to getting connected, from feeling afraid to feeling confident, from thinking of himself as a villain to realizing that he may just be a hero. Why, then, do so many first-time readers of Ceremony get so confused by this narrative? The central reason is that most of the novel takes place in the consciousness of Tayo, who, for the first hundred or so pages, has no idea who he is or where he is going.Part of Tayo’s problem is that he cannot sort things out properly; part of his madness is that he cannot distinguish past from present. And just as his mind is a jumble of events, the opening of the novel portrays unsorted, jumbled events. On the second page of the novel Tayo speaks of the tangle of events and memories in his mind:The memories were tangled with the present, tangled up like colored threads from old Grandma’s wicker sewing basket when he was a child, and he had carried them outside to play and they had spilled out of his arms into the summer weeds and rolled away in all directions, and then he had hurried to pick them up before Auntie found him. He could feel it inside his skull, the tension of little threads being pulled and how it was with tangled things, things tied together, and as he tried to pull them apart and rewind them into their places, they snagged and tangled even more.

We previously showed that Guy1, a primary signal expressed from the Y chromosome, is a strong candidate for a male-determining factor that confers female-specific lethality in Anopheles stephensi (Criscione et al., 2016). Here, we present evidence that Guy1 increases X gene expression in Guy1-transgenic females from two independent lines, providing a mechanism underlying the Guy1-conferred female lethality. The median level gene expression (MGE) of X-linked genes is significantly higher than autosomal genes in Guy1-transgenic females while there is no significant difference in MGE between X and autosomal genes in wild-type females. Furthermore, Guy1 significantly upregulates at least 40% of the 996 genes across the X chromosome in transgenic females. Guy1-conferred female-specific lethality is remarkably stable and completely penetrant. These findings indicate that Guy1 regulates dosage compensation in An. stephensi and components of dosage compensation may be explored to develop novel strategies to control mosquito-borne diseases.

Responding to a question directed to him by Francois Barbe Marbois, secretary of the French ligation in Philadelphia, regarding Indian monuments, Thomas Jefferson wrote of his investigations in opening an Indian burial mound, "I conjectured that in this barrow might have been a thousand skeletons." Although Jefferson may have been the first to dig into a burial mound, both Americans and Europeans were preoccupied with the study of racial differences between American Indians and Caucasians. The search for answers led, especially in the nineteenth century, to the collection of Indian remains and the development of American archaeology and physical anthropology. So any story about collecting Indian remains is also a narrative about these two sciences and the shifting, value-laden cultural context in which they developed.Historian of science Donna Haraway reminds us that physical anthropology is a political discourse about the body, with multiple representations; to a lesser extent, this description also may be attributed to archaeology. Accordingly, both physical anthropology and archaeology produce multiple narratives "adapted to engage in particular kinds of social struggles . . . ." Both fields create "contentious constituencies'' where, at times, certain stories or plots are privileged, while others are no longer acceptable.In recent writing, anthropologist Clifford Geertz holds similar views and acknowledges that anthropological stories-perhaps even text building itself-should be subjected to critical scrutiny. According to Geertz, the epistemological foundations of anthropology have been shaken “by a general loss of faith in received stories about the nature of representation,” and he speaks of “ethnological descriptions” as the ”describer’s descriptions, not those of the described.” The narratives produced by physical anthropology and archaeology also are the “describer’s descriptions” and are constructed out of the political and scientific discourse of an age. These narratives were considered necessary because the Indian had to be ”correctly” described and placed into the context of Western civilization. Such descriptions, however, reflected Western attitudes and Western conceptions of what it meant to be civilized. The gathering and interrogation of Indian remains-the work of nineteenth-century physical anthropology and archaeology- can be discussed only briefly here and focuses primarily on the gathering process.

Cellular heterogeneity is a hallmark of human neuroblastoma tumors and cell lines. Within a single neuroblastoma are cells from distinct neural crest lineages whose relative abundance is significant for prognosis. We postulate that a self-renewing multipotent tumor stem cell, which gives rise to diverse cell lineages, is the malignant progenitor of this cancer. To test this hypothesis, we have established 22 cloned, phenotypically homogeneous populations of the three major cell types from 17 neuroblastoma cell lines. In vitro, malignant neuroblastoma stem cells, termed 1-type (intermediate type), have distinct morphologic, biochemical, differentiative, and tumorigenic properties. I-type cells express features of both neuroblastic (N) cells (scant cytoplasm, neuritic processes, neurofilaments, pseudoganglia, and granin and neurotransmitter enzyme expression) and substrate-adherent (S) cells (extensive cytoplasm and vimentin and CD44 expression). Moreover, they show bidirectional differentiation to either N or S cells when induced by specific agents. I-type cells are significantly more malignant than N- or S-type cells, with four- to five-fold greater plating efficiencies in soft agar and six-fold higher tumorigenicity in athymic mice. Differences in malignant potential are unrelated to N-myc amplification/overexpression or the ability to digest and migrate through the extracellular matrix. Immunocytochemical analyses of a small series of tumors reveal that frequency of cells coexpressing N and S cell markers correlates with poor prognosis. Thus, I-type stem cells may be instrumental in the genesis and growth of tumors in the patient. Their unique biology deserves attention and further investigation.