Gene editing of the ABC Transporter/White locus using CRISPR/Cas9-mediated mutagenesis in the Indian Meal Moth.

ATP binding cassette (ABC) proteins are involved in transport of substrates across membranes including eye pigments. Mutations of ABC transporter white, brown and scarlet genes of Drosophila and other insects result in visible eye color phenotypes. White locus was identified in a genome assembly of Plodia interpunctella and was found to extend for 16,670 bp comprising 13 exons. We report here recovery of heritable mutants in white in the Indian meal moth, P. interpunctella, using CRISPR/Cas9-mediated mutagenesis. A white eye strain of P. interpunctella c.737delC (Piw-/-) was previously isolated in 1986. Guide RNA (sgRNA) was designed for exon 1 (sgRNA242). Microinjection of Cas9/sgRNA242 complex into Plodia wild type eggs (≤20 min post oviposition) produced 156 viable larvae of which 81 eclosed as adults. Forty-five (56 %) adults displayed wild type phenotype, while 26 females (32 %) and 10 males (12 %) showed full or partial white eye phenotype. The 26 white eye females were mated with Piw-/- males and 21 matings resulted in F1 white eye progeny. Thirteen of the Piw-242 lines were established and sequencing showed indels at the CRISPR/Cas9 242AM site. Based on RT-PCR analysis, most white mutations resulted in suppressed levels of transcript. These results demonstrate the utility of CRISPR/Cas9 gene editing in Plodia which suggests this technology can be used to characterize the role of various genetic elements including those that encode novel targets or confer insecticide resistance mechanisms.

Frequency-specific modification of locomotor components by the white gene in Drosophila melanogaster adult flies.

The classic eye-color gene white (w) in Drosophila melanogaster (fruitfly) has unexpected behavioral consequences. How w affects locomotion of adult flies is largely unknown. Here, we show that a mutant allele (w1118 ) selectively increases locomotor components at relatively high frequencies (> 0.1 Hz). The w1118 flies had reduced transcripts of w+ from the 5' end of the gene. Male flies of w1118 walked continuously in circular arenas while the wildtype Canton-S walked intermittently. Through careful control of genetic and cytoplasmic backgrounds, we found that the w1118 locus was associated with continuous walking. w1118 -carrying male flies showed increased median values of path length per second (PPS) and 5-min path length compared with w+ -carrying males. Additionally, flies carrying 2-4 genomic copies of mini-white+ (mw+ ) in the w1118 background showed suppressed median PPSs and decreased 5-min path length compared with controls, and the suppression was dependent on the copy number of mw+ . Analysis of the time-series (i.e., PPSs over time) by Fourier transform indicated that w1118 was associated with increased locomotor components at relatively high frequencies (> 0.1 Hz). The addition of multiple genomic copies of mw+ (2-4 copies) suppressed the high-frequency components. Lastly, the downregulation of w+ in neurons but not glial cells resulted in increased high-frequency components. We concluded that mutation of w modified the locomotion in adult flies by selectively increasing high-frequency locomotor components.

CRISPR/Cas9 mediated disruption of the white gene leads to pigmentation deficiency and copulation failure in Drosophila suzukii.

The Spotted-wing Drosophila (Drosophila suzukii) is a devastating invasive pest of fruit crops. In D. melanogaster, the white (w) gene was associated with pigmentation and mating behavior, which are also important aspects to understand the invasion biology as well as to develop control strategies for D. suzukii. Here, we show that the generation of D. suzukii white-eyed mutants by CRISPR/Cas9 mutagenesis of the w gene resulted in the complete failure of copulation when w- males were individually paired with w- females in small circular arenas (diameter 0.7 cm) for 24 h. Further analysis showed that the mating defect was associated with w- males and could not be rectified by two years of inbreeding by crossing sibling w- females with w+ males, dim red illumination, male-female sexual training, changing to large arenas (diameter 3.5 cm), or different sex ratios. Profound pigmentation deficiency was detected in the compound eyes, ocelli, Malpighian tubules and testis sheaths in the w- flies. Specifically, testis imaging showed that w- males failed to deposit any pigments into pigment cells of the testis sheath, and produced smaller sperms and less seminal fluid compared to those from wildtype males. Together these observations suggest that the w gene plays an essential role in the regulation of sexual behavior and reproduction in D. suzukii. The similarities and differences in w gene function between D. suzukii and D. melanogaster in the context of pigmentation and mating behavior are discussed.

Targeted somatic mutagenesis through CRISPR/Cas9 ribonucleoprotein complexes in the olive fruit fly, Bactrocera oleae.

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo-we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene-specific single-guide RNAs. Gene disruption of Bo-we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo-we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.

The ABCs of CRISPR in Tephritidae: developing methods for inducing heritable mutations in the genera Anastrepha, Bactrocera and Ceratitis.

Tephritid fruit flies are destructive agricultural pests that are the targets of expensive population eradication and suppression efforts. Genetic pest management is one of the strategies for reducing or eliminating tephritid populations, relying upon the genetic manipulation of insects to render them sterile or capable of transmitting deleterious traits through gene drive. Currently, radiation, chemical mutagenesis, and transgenic techniques are employed to generate agents for genetic pest management, but new methods must be explored and developed for all tephritid pest species. Targeted mutagenesis induced by nonhomologous end join repair of clustered regularly interspaced short palindromic repeats and the CRISPR associated protein 9 (Cas9) (commonly known as CRISPR/Cas9) has been demonstrated to be an efficient method for creating knock-out mutants and can be utilized to create germline mutations in Tephritidae. In this paper, we describe detailed methods to knockout the white gene in three tephritid species in the genera Anastrepha, Bactrocera and Ceratitis, including the first demonstration of CRISPR/Cas9 induced mutations in the genus Anastrepha. Lastly, we discuss the variables in tephritid systems that directed method development as well as recommendations for performing injections in remote containment facilities with little molecular biology capabilities. These methods and recommendations combined can serve as a guide for others to use in pursuit of developing CRISPR/Cas9 methods in tephritid systems.

[The role of white gene and its mechanism during the formation of curly wing in Drosophila melanogaster.] / é»‘è ¹æžœè‡whiteåŸºå› åœ¨H2O2å½±å“ä¸‹å·ç¿ å½¢æˆä¸­çš„ä½œç”¨åŠæœºç†.

Curly wing is one of the most frequently used genetic markers in Drosophila melanogaster, but its molecular mechanism is remained unclear. Previous results have showed that physicochemical stimulation would affect the formation of the cruly wing. Our recent study found that H2O2 could not only affect the eclosion rate of D. melanogaster, but also induce the formation of curly wing. Here, we aimed to uncover the specific factors influencing the formation of curly wing in D. melanogaster via changing the concentration of H2O2 and the temperature as well as the time of H2O2 treatment. We measured the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX), in order to examine the effects of H2O2 on antioxidative capacity of D. melanogaster. The results showed that the eclosion rate of D. melanogaster was inversely correlated with the concentration of H2O2. The change of temperature, H2O2 concentration and the period of H2O2 treatment affected the degree of the curl and the proportion of the curly wing. The white mutant flies responded most significantly to these three conditions, the mini-white (white gene reverse mutation) flies could rescue the curly phenotype, and responded similarly to the wild type OR. H2O2 had effects on the formation of the curly wing which contained the Cy mutation, leading to increased rate of the curly wing. D. melanogaster treated with H2O2 would reduce the antioxidative capacity. Results from real-time quantitative PCR showed that H2O2 treatment resulted in a change in gene expression. The formation of curly wing was a complicated process, and H2O2 might act as a signaling molecule or indirectly affect certain factors in the formation of curly wing in D. melano-gaster. This process might share the same signaling pathway with the Cy mutant, or might be regulated by different modulating patterns.

Walking behavior in a circular arena modified by pulsed light stimulation in Drosophila melanogaster w1118 line.

The Drosophila melanogaster white-eyed w1118 line serves as a parental stock, allowing genetic recombination of any gene of interest along with a readily recognizable marker. w1118 flies display behavioral susceptibility to environmental stimulation such as light. It is of great importance to characterize the behavioral performance of w1118 flies because this would provide a baseline from which the effect of the gene of interest could be differentiated. Little work has been performed to characterize the walking behavior in adult w1118 flies. Here we show that pulsed light stimulation increased the regularity of walking trajectories of w1118 flies in circular arenas. We statistically modeled the distribution of distances to center and extracted the walking structures of w1118 flies. Pulsed light stimulation redistributed the time proportions for individual walking structures. Specifically, pulsed light stimulation reduced the episodes of crossing over the central region of the arena. An addition of four genomic copies of mini-white, a common marker gene for eye color, mimicked the effect of pulsed light stimulation in reducing crossing in a circular arena. The reducing effect of mini-white was copy-number-dependent. These findings highlight the rhythmic light stimulation-evoked modifications of walking behavior in w1118 flies and an unexpected behavioral consequence of mini-white in transgenic flies carrying w1118 isogenic background.

Persistent One-Way Walking in a Circular Arena in Drosophila melanogaster Canton-S Strain.

We describe persistent one-way walking of Drosophila melanogaster in a circular arena. Wild-type Canton-S adult flies walked in one direction, counter-clockwise or clockwise, for minutes, whereas white-eyed mutant [Formula: see text] changed directions frequently. Locomotion in the circular arena could be classified into four components: counter-clockwise walking, clockwise walking, nondirectional walking and pausing. Genetic analysis revealed that while wild-type genetic background was associated with reduced directional change and reduced numbers of one-way (including counter-clockwise and clockwise) and nondirectional walks, the white ([Formula: see text]) locus promoted persistent one-way walking by increasing the maximal duration of one-way episodes. The promoting effect of [Formula: see text] was further supported by the observations that (1) [Formula: see text] duplicated to the Y chromosome, (2) four genomic copies of mini-white inserted on the autosomes, and (3) pan-neuronal overexpression of the White protein increased the maximal duration of one-way episodes, and that RNAi knockdown of [Formula: see text] in the neurons decreased the maximal duration of one-way episodes. These results suggested a pleiotropic function of [Formula: see text] in promoting persistent one-way walking in the circular arena.

Timing of Locomotor Recovery from Anoxia Modulated by the white Gene in Drosophila.

Locomotor recovery from anoxia follows the restoration of disordered ion distributions and neuronal excitability. The time taken for locomotor recovery after 30 sec anoxia (around 10 min) is longer than the time for the propagation of action potentials to be restored (<1 min) in Drosophila wild type. We report here that the white (w) gene modulates the timing of locomotor recovery. Wild-type flies displayed fast and consistent recovery of locomotion from anoxia, whereas mutants of w showed significantly delayed and more variable recovery. Genetic analysis including serial backcrossing revealed a strong association between the w locus and the timing of locomotor recovery, and haplo-insufficient function of w(+) in promoting fast recovery. The locomotor recovery phenotype was independent of classic eye pigmentation, although both are associated with the w gene. Introducing up to four copies of mini-white (mw(+)) into w1118 was insufficient to promote fast and consistent locomotor recovery. However, flies carrying w(+) duplicated to the Y chromosome showed wild-type-like fast locomotor recovery. Furthermore, Knockdown of w by RNA interference (RNAi) in neurons but not glia delayed locomotor recovery, and specifically, knockdown of w in subsets of serotonin neurons was sufficient to delay the locomotor recovery. These data reveal an additional role for w in modulating the timing of locomotor recovery from anoxia.

Distinct population structure for co-occurring Anopheles goeldii and Anopheles triannulatus in Amazonian Brazil

To evaluate whether environmental heterogeneity contributes to the genetic heterogeneity in Anopheles triannulatus, larval habitat characteristics across the Brazilian states of Roraima and Pará and genetic sequences were examined. A comparison with Anopheles goeldii was utilised to determine whether high genetic diversity was unique to An. triannulatus. Student t test and analysis of variance found no differences in habitat characteristics between the species. Analysis of population structure of An. triannulatus and An. goeldii revealed distinct demographic histories in a largely overlapping geographic range. Cytochrome oxidase I sequence parsimony networks found geographic clustering for both species; however nuclear marker networks depicted An. triannulatus with a more complex history of fragmentation, secondary contact and recent divergence. Evidence of Pleistocene expansions suggests both species are more likely to be genetically structured by geographic and ecological barriers than demography. We hypothesise that niche partitioning is a driving force for diversity, particularly in An. triannulatus.

Molecular evidence for a single taxon, Anopheles nuneztovari s.l., from two endemic malaria regions in Colombia

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F ST) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study.

Systematic notes of anopheles konderi and its first record in paraná state, brazil

We analyzed nuclear (second internal transcribed spacer and white gene) and mitochondrial(cytochrome c oxidase subunit I) data from Anopheles konderi collected in the Amazonian states of Acre,Amapa´, and Rondoˆnia and the southern Brazilian state of Parana´ . This was the first record of An. konderi inthe state of Parana´ . We found a high degree of genetic divergence within the Amazonian region and supportfor An. konderi as a species complex, possibly consisting of 3 species...

A new mtDNA COI gene lineage closely related to Anopheles janconnae of the Albitarsis complex in the Caribbean region of Colombia

An understanding of the taxonomic status and vector distribution of anophelines is crucial in controlling malaria. Previous phylogenetic analyses have supported the description of six species of the Neotropical malaria vector Anopheles (Nyssorhynchus) albitarsis s.l. (Diptera: Culicidae): An. albitarsis, Anopheles deaneorum, Anopheles marajoara, Anopheles oryzalimnetes, Anopheles janconnae and An. albitarsis F. To evaluate the taxonomic status of An. albitarsis s.l. mosquitoes collected in various localities in the Colombian Caribbean region, specimens were analyzed using the complete mitochondrial DNA cytochrome oxidase I (COI) gene, the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) region and partial nuclear DNA white gene sequences. Phylogenetic analyses of the COI gene sequences detected a new lineage closely related to An. janconnae in the Caribbean region of Colombia and determined its position relative to the other members of the complex. However, the ITS2 and white gene sequences lacked sufficient resolution to support a new lineage closely related to An. janconnae or the An. janconnae clade. The possible involvement of this new lineage in malaria transmission in Colombia remains unknown, but its phylogenetic closeness to An. janconnae, which has been implicated in local malaria transmission in Brazil, is intriguing.

Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and a new record for Anopheles dunhami in the Brazilian Amazon

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) rDNA and partial sequences of the cytochrome coxidase subunit I (COI) mtDNA and white gene nDNA were obtained from specimens of Anopheles nuneztovari A collected in Macapá (state of Amapá), Óbidos, Prainha and Almeirim (state of Pará), Itacoatiara and Parintins (state of Amazonas), Brazil, and compared with previously published sequences of A. nuneztovari s.l. Results of the Bayesian phylogenetic analyses performed using either COI or combined ITS2, COI and white gene sequences suggest that An. nuneztovari B/C is distinct from specimens obtained in the Amazonas/Solimões River basin. Anopheles goeldii, currently in synonymy with An. nuneztovari, was described from individuals collected in Belterra (= Fordlândia) in the Tapajós River, state of Pará, Southern Amazonas River. Morphological comparisons of the characteristics of the male genitalia indicated that An. nuneztovari A and An. goeldii are similar but distinct from An. nuneztovariB/C by the apex of the aedeagus. In considering the results of the phylogenetic analyses and morphological comparisons, An. goeldii is resurrected from synonymy with An. nuneztovari. Additionally, Anopheles dunhamiis reported for the first time in Parintins. This species can be distinguished from An. goeldiiby characters of the male genitalia and molecular data.