The Development of Cytogenetic Maps for Malaria Mosquitoes

Technical Note The Development of Cytogenetic Maps for Malaria Mosquitoes

Gleb N. Artemov 1, Vladimir N. Stegniy 1, Maria V. Sharakhova 1,2 and Igor V. Sharakhov 1,2,*

1 Laboratory of Ecology, Genetics and Environmental Protection, Tomsk State University, 36 Lenin Avenue, 634050 Tomsk, Russia; [email protected] (G.N.A.); [email protected] (V.N.S.); [email protected] (M.V.S.) 2 Department of Entomology, Fralin Life Science Institute, Institute and State University, Blacksburg, VA 24060, USA * Correspondence: [email protected]; Tel.: +1-540-231-7316

Received: 15 August 2018; Accepted: 13 September 2018; Published: 17 September 2018

Abstract: Anopheline mosquitoes are important vectors of human malaria. Next-generation sequencing opens new opportunities for studies of mosquito genomes to uncover the genetic basis of a Plasmodium transmission. Physical mapping of genome sequences to polytene chromosomes significantly improves reference assemblies. High-resolution cytogenetic maps are essential for anchoring genome sequences to chromosomes as well as for studying breakpoints of chromosome rearrangements and chromatin protein localization. Here we describe a detailed pipeline for the development of high-resolution cytogenetic maps using polytene chromosomes of malaria mosquitoes. We apply this workflow to the refinement of the cytogenetic map developed for Anopheles beklemishevi.

Keywords: cytogenetic map; chromosome map; malaria mosquitoes; Anopheles; polytene chromosomes; Anopheles beklemishevi; chromosome map development; photomap; high-pressure technique; chromosome straightening

1. Introduction Mosquito species within the Anopheles genus are the only vectors of human malaria, which caused approximately half a million deaths in 2017 [1]. There are 444 species of Anopheles that differ by the ability to transmit Plasmodium [2]. The genetic basis of such differences in vector status is still under investigation [3–5]. Whole-genome sequencing and the comparative study of the genomes are the most powerful approaches to better understanding of vector competence and its evolution [3,6]. However, genome sequencing alone cannot achieve chromosome-level assemblies, which are important for further genome analysis. Physical mapping of the genome can anchor scaffolds to polytene chromosomes using ﬂuorescence in situ hybridization (FISH) and can map up to 97% of a genome sequence to chromosomes [6,7]. Genome assemblies for 16 species of malaria vector mosquitoes have so far been obtained [6]. Physical mapping in ﬁve Anopheles species corrected mistakes in genome assemblies [7] as well as discovered a high rate of X chromosome evolution [6] and inter-arm rearrangements in pericentric regions [8]. High-resolution cytogenetic maps are required for physical genome mapping and studies of mosquito chromosome organization and evolution. The chromosome complement of Anopheles mosquitoes consists of three pairs of chromosomes: two sex chromosomes (XX in females and XY in males) and two pairs of submetacentric autosomes. Polytene chromosomes in Anopheles are developed in several cell types: salivary glands, Malpighian tubules, midgut and fat body of larvae, and Malpighian tubules and nurse cells of adults. Differences exist in the polytene chromosome quality among tissues and species, which depends on the degree of polyteny and chromatids conjugation. Polytene chromosomes in most tissues of Anopheles mosquitoes

Insects 2018, 9, 121; doi:10.3390/insects9030121 www.mdpi.com/journal/insects Insects 2018, 9, 121 2 of 15 are joined together in the chromocenter, except for ovarian nurse cell chromosomes of species from the Maculipennis group, where pericentromeric regions separate from each other [9]. The first cytogenetic maps of Anopheles polytene chromosomes were developed in the 1960s, and today there are about 100 cytogenetic maps available for different species [10]. The first cytogenetic maps were drawn, making them prone to a subjective interpretation of banding patterns. With the advance of micro-photography techniques, chromosome maps were built from chromosome photo images first on photocards and then using digital photos. Subjectivity is reduced by micro-photo techniques, but conventional photo-maps are usually based on only one chromosome spread. The best cytogenetic map can be developed from straightened chromosome images created by combining the most typical high-resolution regions into a single collage. This approach started with paper photo collages when chromosome images were cut from paper photos, arranged and glued on a paper sheet, and then photographed again [11–13]. Many details can be lost in a double-photography process. Chromosome staining can also reduce details in the banding pattern. This disadvantage has been overcome by phase-contrast microscopy, which is the most appropriate approach for detailed visualization of the polytene chromosome banding pattern [14–17]. A modern chromosome map development approach uses digital phase-contrast chromosome images to straighten and combine typical chromosome regions utilizing computer software. Using this approach, high-resolution chromosome maps for ten Anopheles species have been developed, including An. albimanus [7,17], An. funestus [14,18], An. stephensi [16], An. gambiae [15], An. darling [19,20], An. nili [21], An. sinensis [22], An. atroparvus [8,23], An. lesteri [24], An. beklemishevi [25], and An. sacharovi [26]. Some of these chromosome maps were used for physical mapping, thus, improving genome references for An. gambiae [15], An. stephensi [27], An. albimanus [7], An. atroparvus [8,23], and An. sinensis [28]. Here we describe in detail a pipeline for chromosome photomap development based on modern approaches. We apply it to the refinement of the cytogenetic map for An. beklemishevi published earlier [25].

2. Materials and Methods

2.1. Overview of the High-Resolution Cytogenetic Map Development The goal of high-resolution cytogenetic maps is to make chromosomes and chromosome regions easily identifiable. Toward this goal, we propose three major steps: (1) preparation of chromosome arms for straightening; (2) chromosome arms straightening, trimming, and processing (brightness and contrast adjustment, chromosome regions combination and substitution, etc.); and (3) dividing chromosomes by numbered and lettered regions. Processing the chromosome map development starts with squashed (or high-pressure squashed) chromosome preparations and microscopy of no less than 50 chromosome squashes. Only high-quality images of the chromosomes with typical banding patterns, chromosome edge profiles, and centromere and telomere ends are included in a chromosome map. These features are defined as typical if they are present in more than half of chromosome preparations. Chromosome straightening aimed to organize all chromosome bands in a parallel orientation to each other (a barcode-like pattern) and to align the chromosomes along the central axis. Chromosomes must preserve the native and typical (reproducible) morphology of a banding pattern, which is improved by straightening. Trimming of chromosome images reveals the shape of the chromosome, which is important for identifying some chromosome landmarks. No less than three best-straightened chromosomes must be combined in the final cytogenetic map. A chromosome map reflects the proportions of chromosome arms and position of landmarks relative to the chromosome ends. The average measurements of chromosome arm length and the distance from the telomere end to a landmark are calculated for the relative chromosome length adjustment and placement of a landmark to the proper position. The final step of the cytogenetic map development is to divide chromosomes by both numbered divisions and lettered subdivisions according to existing chromosome maps or by relying on a basic InsectsInsects2018 2018, ,9 9,, 121 x 3 3of of 15 15

principle of this procedure described below. The boundaries between divisions and subdivisions are principlealways placed of this to procedure the borders described between below. bands Theand boundariesinterbands. between divisions and subdivisions are always placed to the borders between bands and interbands. 2.2. Tissue Fixation and Chromosome Preparation 2.2. Tissue Fixation and Chromosome Preparation For polytene chromosome preparation, salivary gland cells of larvae or ovarian nurse cells of adultsFor of polytene An. beklemishevi chromosome, An. preparation,sacharovi and salivary An. quadrimaculatus gland cells of were larvae used. orovarian For salivary nurse glands cells of adultsfixation, of aAn. whole beklemishevi 4th instar, An.larvae sacharovi were directlyand An. put quadrimaculatusin Carnoy’s solutionwere (3 used. volumes For of salivary 100% ethanol glands fixation,and 1 volume a whole of 4th glacial instar acetic larvae acid). were directlyFor ovarian put innurse Carnoy’s cells solutionfixation, (3half-gravid volumes of females 100% ethanol were anddissected 1 volume and oftheir glacial ovaries acetic were acid). transferred For ovarian into nurse Carnoy’s cells fixation,solution. half-gravid Material was females stored were in Carnoy’s dissected ◦ andat − their20 °C. ovaries were transferred into Carnoy’s solution. Material was stored in Carnoy’s at −20 C. SalivarySalivary glandsglands oror 15–2015–20 ovarianovarian follicles were dissected and and put put onto onto a a slide slide containing containing a adrop drop ofof 50% 50% propionicpropionic acid for for 5 5 min. min. A A filter filter paper paper was was used used to remove to remove the liquid, the liquid, and a and new a drop new dropof 50% of 50%propionic propionic acid acidwas wasadded. added. The material The material was covered was covered by a coverslip, by a coverslip, then by then a piece by aof piecefilter ofpaper, filter paper,and squashed and squashed by tapping by tapping it with it with a pencil a pencil eras eraserer tip. tip. High-pressure High-pressure squashed squashed chromosome chromosome preparationspreparations were were prepared prepared followingfollowing protocolsprotocols adaptedadapted forfor An.An. gambiae [15,29].[15,29].

2.3.2.3. Microscopy, Microscopy, ChromosomeChromosome Measurement,Measurement, andand ImageImage Processing ChromosomesChromosomes werewere viewedviewed usingusing AxioImagerAxioImager A1 (Carl Zeiss, OPTEC, OPTEC, Novosibirsk, Novosibirsk, Russia) Russia) underunder phase-contrast phase-contrast with with a a 100 100×× objective.objective. High-resolutionHigh-resolution chromosomechromosome imagesimages werewere capturedcaptured byby a CCDa CCD camera camera MrC5 MrC5 (Carl (Carl Zeiss, Zeiss, OPTEC, OPTEC, Novosibirsk, Novosibirsk, Russia) Russia) and and AxioVision AxioVision 4.8.2 4.8.2 software software (Carl (Carl Zeiss, OPTEC,Zeiss, OPTEC, Novosibirsk, Novosibirsk, Russia) using Russia) a 2584 using× 1936 a pixels2584 multicolor× 1936 pixels camera multicolor mode. Chromosomes camera mode. were measuredChromosomes using were the “Measurement” measured using option the “Measurement” in the AxioVision option 4.8.2 in software the AxioVision package. 4.8.2 Chromosome software imagespackage. were Chromosome further manipulated images were and further modiﬁed manipu usinglated Photoshop and modified CS6 (Adobeusing Photoshop Systems Incorporated, CS6 (Adobe SanSystems Jose, CA,Incorporated, US). San Jose, CA, US).

2.4.2.4. Image Image AcquisitionAcquisition ChromosomeChromosome images images cancan bebe obtainedobtained with any available software software compatible compatible with with a a microscope microscope digitaldigital camera. camera. High-resolutionHigh-resolution imagesimages shouldshould bebe createdcreated usingusing a 100100×× objectiveobjective and and a a camera camera mode mode ofof no no less less than than 2584 2584× × 19361936 pixels.pixels. Shading, white ba balance,lance, and and exposure exposure must must be be adjusted adjusted prior prior to to photographingphotographing (Figure(Figure1 ).1). ItIt isis importantimportant toto avoidavoid over-contrastedover-contrasted (Figure(Figure1 1b)b) andand over-bleachedover-bleached imagesimages (Figure (Figure1 c),1c), which which will will be be impossible impossible to to correct correct during during furtherfurther steps.steps.

FigureFigure 1.1. LevelsLevels of of shadows shadows and and highlights highlights in inimag imageses of a of 3R a arm 3R armchromosome chromosome region region of An. of An.quadrimaculatus quadrimaculatus fromfrom ovarian ovarian nurse nursecells. ( cells.a) Appropriate (a) Appropriate levels of levelsshadows of shadowsand highlights; and highlights; (b) over- (bshadowed) over-shadowed levels; (c levels;) over-highlighted (c) over-highlighted levels. levels.

Usually,Usually, polytenepolytene chromosomes are are too too long long to tofit fitinto into thethe view view of the of objective the objective lens field lens at field 100× at 100magnification.× magnification. This Thiscan canbe a be challenge a challenge for for ch chromosomeromosome arm arm identification, identification, chromosome chromosome map map measurement,measurement, and and chromosome chromosome straightening. straightening. To To overcome overcome this this difficulty, difficulty, a set ofa set overlapping of overlapping images

Insects 2018, 9, 121 4 of 15 Insects 2018, 9, x 4 of 15 ofimages different of chromosomedifferent chromosome regions should regions be obtained should (Figure be obtained2a). In case (Figure the same 2a). chromosome In case the spreads same arechromosome not completely spreads ﬂat inare a preparation, not completely several flat images in a of preparation, the same chromosome several images spread withof the different same focuschromosome planes must spread be with obtained different (Figure focus2b). planes must be obtained (Figure 2b).

FigureFigure 2. ImagesImages merging merging scheme. scheme. (a) (Aa) chromosome A chromosome region region from from a single a single microscope microscope field; ﬁeld;(b) a (chromosomeb) a chromosome region region in different in different focal focalplanes. planes. Images Images 1 and 1 2 and are 2taken are taken from from two twodifferent different and andoverlapped overlapped microscope microscope fields ﬁelds (a) or (a) from or from two two different different focal focal planes planes (b). ( bMerged). Merged images images performed performed by bya “Merge a “Merge Layer” Layer” function function (a ()a or) or by by cutting cutting unfocused unfocused fragments fragments of of images images with with a a “Lasso “Lasso Tool” priorprior toto mergingmerging layerslayers ((bb).). AllAll manipulationmanipulation tooltool referencesreferences relaterelate toto PhotoshopPhotoshop CS6.CS6.

OnlyOnly imagesimages ofof chromosomeschromosomes (or(or regions)regions) withwith aa good-qualitygood-quality bandingbanding patternpattern shouldshould bebe chosenchosen forfor thethe map map development. development. Good-quality Good-quality chromosomes chromosomes (or regions) (or haveregions) the following have the 5 characteristics: following 5 (1)characteristics: chromosomes (1) are chromosomes well separated are from well each separa otherted and from do noteach overlap; other and (2) chromosomes do not overlap; are not(2) stretched;chromosomes (3) bordersare not stretched; between bands(3) borders and interbandsbetween bands are well-deﬁned;and interbands (4) are thin well-defined; bands are visible;(4) thin andbands (5) are chromosomes visible; and and (5) chromosome chromosomes landmarks and chro havemosome typical landmarks reproducible have structures typical reproducible and should bestructures well-polytenized. and should be well-polytenized. 2.5. Image Selection and Processing 2.5. Image Selection and Processing For chromosome map development, we usually have a set of images—some with a single For chromosome map development, we usually have a set of images—some with a single chromosome, others with 2 or several different chromosomes or even parts of chromosomes. If a chromosome, others with 2 or several different chromosomes or even parts of chromosomes. If a chromosome is on 2 or more images, these images must be merged prior to further processing. For this chromosome is on 2 or more images, these images must be merged prior to further processing. For purpose, copies of such images must be moved as new layers to a separate sheet (Figure2). For a this purpose, copies of such images must be moved as new layers to a separate sheet (Figure 2). For precise chromosome assembly without gaps, the images of the chromosome must overlap. Before a precise chromosome assembly without gaps, the images of the chromosome must overlap. Before merging the layers, the edges of the front layers must be smoothed with a smooth brush such as a merging the layers, the edges of the front layers must be smoothed with a smooth brush such as a “Smooth Tool” or an “Erase Tool” to keep the edge of the overlapping layer visible. “Smooth Tool” or an “Erase Tool” to keep the edge of the overlapping layer visible. If a whole chromosome is not in the same focus plane on a preparation, several images of different If a whole chromosome is not in the same focus plane on a preparation, several images of chromosome parts must be obtained. At this step, all images are moved to a new sheet and are different chromosome parts must be obtained. At this step, all images are moved to a new sheet and overlapped as in the previous case. Then, each in-focus image of the chromosome regions is preserved, are overlapped as in the previous case. Then, each in-focus image of the chromosome regions is while out-of-focus parts are removed. Undesirable image areas should be selected with the “Lasso preserved, while out-of-focus parts are removed. Undesirable image areas should be selected with Tool” or “Rectangular Marquee Tool” and deleted. The edges of images can be processed with “Smooth the “Lasso Tool” or “Rectangular Marquee Tool” and deleted. The edges of images can be processed Tool” or by “Eraser Tool” to achieve a gradual transition between the layers. with “Smooth Tool” or by “Eraser Tool” to achieve a gradual transition between the layers. After processing, the appropriate chromosome images must be cleaned of excess background After processing, the appropriate chromosome images must be cleaned of excess background areas and desaturated if they were colored. Chromosomes must be contoured by a “Lasso Tool” areas and desaturated if they were colored. Chromosomes must be contoured by a “Lasso Tool” and and indented from the chromosome edge by approximately half of the chromosome width so that indented from the chromosome edge by approximately half of the chromosome width so that each each chromosome is surrounded by a stripe of the background. This step is crucial for chromosome chromosome is surrounded by a stripe of the background. This step is crucial for chromosome straightening, as adjacent parts of chromosome edges cannot coincide and must be trimmed (see straightening, as adjacent parts of chromosome edges cannot coincide and must be trimmed (see below). A chromosome region selected by the “Lasso Tool” should be transposed to a new layer and below). A chromosome region selected by the “Lasso Tool” should be transposed to a new layer and

Insects 2018,, 9,, 121x 55 of 1515

moved to a new sheet where all chromosome arm images are stored. Some layers with chromosome movedimages towill a newbe used sheet as wheretemplates all chromosome for straightening arm imageschromosomes are stored. in the Some following layers steps. with chromosome images will be used as templates for straightening chromosomes in the following steps. 2.6. Chromosome Landmarks Layout and Work Area Preparation 2.6. Chromosome Landmarks Layout and Work Area Preparation Chromosome arms and regions are defined by regions with unusual structures—i.e., chromosomeChromosome landmarks. arms and Landmarks regions are deﬁnedare characteri by regionszed withby the unusual following structures—i.e., features: (1) chromosome irregular landmarks.banding pattern Landmarks (a batch of are juxtap characterizedosed bands by or the large following interband); features: (2) unusually (1) irregular wide bandingbands; (3) pattern bands (aof batchhigh density of juxtaposed (darker bands than othe or larger bands); interband); and, (4) (2) protrusions unusually and wide hollows bands; on (3) the bands chromosome of high density edge (darkerprofile (Figure than other 3). bands); and, (4) protrusions and hollows on the chromosome edge proﬁle (Figure3).

Figure 3.3. ExamplesExamples of of landmark landmark regions regions in chromosome in chromosome 2 of An. 2 sacharoviof An. sacharoviand 3R arm and of An.3R arm Beklemishevi of An.: 1—aBeklemishevi set of irregular: 1—a set bands; of irregular 2—a dark bands; single 2—a band; dark 3—a sing regionle band; with 3—a long region interbands; with long 4—a interbands; region with 4— a seta region of 3 dark with bands; a set of 5—a 3 dark protrusion; bands; 5—a 6—an protru indentation;sion; 6—an 7—an indentation; indentation 7—an surrounded indentation by two surrounded dark bands; and,by two 8—a dark large bands; interband and, 8—a restricted large by interband 2 bands. restricted by 2 bands.

Landmarks are important for identifying the chromosome arm and regions surroundingsurrounding landmarks. For chromosome map development, it is crucial to select landmark regions and to definedefine their positionposition relativerelative toto chromosomechromosome armarm ends.ends. Most often,often, peritelomericperitelomeric andand pericentromericpericentromeric regions are sufficientsufficient landmarkslandmarks forfor identificationidentification ofof chromosomechromosome arms.arms. InIn many cases, one or several landmarks withinwithin aa chromosome armarm cancan bebe selected.selected. For instance, this can be useful for determining the orientationorientation ofof inversions.inversions. TheThe position position of of such such “internal” “internal” landmark landmark regions regions is is calculated calculated relative relative to theto the telomeric telomeric end end in order in order to establish to establish its proper its proper location location on a chromosomeon a chromosome map. map. It is advisable It is advisable to make to measurementsmake measurements of the distanceof the distance between between the marginal the ma telomererginal telomere point to apoint certain to pointa certain on thepoint landmark on the regionlandmark using region no less using than no 3 differentless than high-quality 3 different high-quality unstretched chromosomeunstretched images.chromosome The average images. value The ofaverage the measurements value of the measurements and fitted 5% error and fitted will create 5% er aror frame will increate which a frame landmarks in which have landmarks to appear have on a chromosometo appear on a map chromosome (Figure4). map (Figure 4).

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Figure 4. A workﬂow of the work area layout of the 3L arm of An. sacharovi nurse cells polytene Figure 4. A workflow of the work area layout of the 3L arm of An. sacharovi nurse cells polytene chromosomes. (a) Templates of 3L arms, named chr1, chr2, chr3, Tel—telomere (black line), chromosomes. (a) Templates of 3L arms, named chr1, chr2, chr3, Tel—telomere (black line), Cen— Cen—centromerecentromere (red line), (red line),with the with “bird the ey “birde” landmark eye” landmark arrowed arrowedby orange by line; orange (b) table line; of (theb) tabledistances of the distancesbetween between telomere telomere and centromere and centromere ends (Tel-Cen), ends (Tel-Cen), and between and between telomere telomere end and end the and“bird the eye” “bird eye”landmark landmark (Tel-LM) (Tel-LM) of of3 arms 3 arms examples examples and and their their av averageerage value, value, with with 5% 5% error error margins, margins, in inmicrons microns andand pixels; pixels; (c) ( workc) work area area layout: layout: telomere telomere end end (Tel) (Tel) marked marked by by vertical vertical black black guideline, guideline, centromere centromere end (Cen)end and (Cen) “bird and eye” “bird landmark eye” landmark (LM) positions (LM) positions marked marked as colored as colored areas limitedareas limited by the by pairs the ofpairs vertical of lines,vertical where lines, the where centromere the centromere end and theend landmark and the landmark must appear. must appear.

ToTo layout layout a worka work area area where where a a chromosome chromosome mapmap will be developed, 3 3horizontal horizontal and and several several verticalvertical guidelines guidelines are are created created (Main (Main menu menu >> ViewView >> NewNew guide in Photoshop). Photoshop). Three Three horizontal horizontal guidelinesguidelines should should be be at at an an equal equal distance distance fromfrom eacheach other. The central central guideline guideline is is the the axis axis of ofa future a future straightenedstraightened chromosome; chromosome; the the other other 2 2 are are auxiliaryauxiliary guidelines,guidelines, situated situated at at middle middle distance distance from from the the chromosomechromosome axis axis to to the the chromosome chromosome edge. edge. TheseThese 33 horizontal guidelines guidelines fa facilitatecilitate the the establishment establishment of bandof band and and interband interband positions positions relative relative toto thethe chromosomechromosome axis axis during during chromosome chromosome straightening. straightening. TheThe ﬁrst first vertical vertical guide guide is is set set on on the the left left side side ofof thethe sheetsheet where the the telomere telomere end end will will appear. appear. On On the the rightright side side of of the the sheet, sheet, aa pair of of vertical vertical guidelines guidelines is placed is placed to mark to mark the borders the borders of a centromere of a centromere end. end.The The positions positions of these of these guidelines guidelines are calculated are calculated as average as average chromosome chromosome arm length arm ± length5% error± (Figure5% error (Figure4). The4). aim The of aim the of layout the layout is to predict is to predict the locati theons locations of landmarks of landmarks and a centromere and a centromere end on a end future on a futurechromosome chromosome map. map.

2.7.2.7. Chromosome Chromosome Straightening Straightening EvenEven the the best best chromosome chromosome preparationspreparations havehave minor curves curves that that should should be be straightened straightened to to produce chromosome maps. Usually curves do not deform the band shape. Instead, curves transform produce chromosome maps. Usually curves do not deform the band shape. Instead, curves transform

Insects 2018, 9, 121 7 of 15 Insects 2018, 9, x 7 of 15 thethe interbandinterband shape.shape. WhenWhen aa chromosomechromosome isis aligned,aligned, thethe distancedistance betweenbetween thethe adjacentadjacent bandsbands isis approximatelyapproximately the the same same as theas the full widthfull width of a chromosome.of a chromosome. When aWhen chromosome a chromosome is curved, is thecurved, distance the betweendistance bandsbetween on bands the major on the radius major of radius the curve of the becomes curve becomes bigger bigger and, on and, the on minor the minor radius radius of the of curve,the curve, becomes becomes smaller, smaller, than than those those in a in straight a straig chromosome.ht chromosome. The The natural natural distance distance between between bands bands is maintainedis maintained only only on theon the axial axial part part of theof the chromosome chromosome (Figure (Figure5). 5).

Figure 5. A principle of chromosome straightening. The distances between marginal points of the Figure 5. A principle of chromosome straightening. The distances between marginal points of the different bands (a, c) became equal to the distance between the axial points of the bands (b) after different bands (a, c) became equal to the distance between the axial points of the bands (b) after chromosome straightening. chromosome straightening. Thus, chromosome straightening is a procedure of adjusting the distance between the marginal Thus, chromosome straightening is a procedure of adjusting the distance between the marginal parts of chromosome bands to the distance between axial parts of the chromosome bands (Figure5). parts of chromosome bands to the distance between axial parts of the chromosome bands (Figure 5). All bands on the chromosome map have to be set perpendicular to the chromosome axis and set All bands on the chromosome map have to be set perpendicular to the chromosome axis and set to the native distance between them. Moreover, the central point of each band has to coincide with the to the native distance between them. Moreover, the central point of each band has to coincide with chromosome’s axis. To control parallelism between all bands and its position on the chromosome axis, the chromosome’s axis. To control parallelism between all bands and its position on the chromosome vertical guidelines are created (Figure6). axis, vertical guidelines are created (Figure 6). Chromosome straightening is performed from left to right (from the telomere to the centromere) Chromosome straightening is performed from left to right (from the telomere to the centromere) and consists of 4 steps: (1) cutting the layer with a band (with surrounding interbands and, sometimes, and consists of 4 steps: (1) cutting the layer with a band (with surrounding interbands and, other bands) from the template chromosome image; (2) adjusting the band to a vertical orientation and sometimes, other bands) from the template chromosome image; (2) adjusting the band to a vertical establishing it relative to the chromosome axis; (3) establishing a band on the proper distance relative orientation and establishing it relative to the chromosome axis; (3) establishing a band on the proper to the previous band; and, (4) retouching the edges of 2 adjacent layers (Figure6). The techniques of distance relative to the previous band; and, (4) retouching the edges of 2 adjacent layers (Figure 6). this process are described below. The techniques of this process are described below. 1. Using the “Lasso Tool” or “Rectangular Marquee Tool”, the selection, which includes current 1. Using the “Lasso Tool” or “Rectangular Marquee Tool”, the selection, which includes current (second) and previous (first) adjacent bands, is made on the template chromosome image. The copy of (second) and previous (first) adjacent bands, is made on the template chromosome image. The copy the new layer is created from the selection and transposed to the work area by “Move Tool.” of the new layer is created from the selection and transposed to the work area by “Move Tool.” 2. The second (current) band is aligned according to the vertical guideline to make it 2. The second (current) band is aligned according to the vertical guideline to make it perpendicular to the chromosome axis. This is performed by rotating the layer with the use the perpendicular to the chromosome axis. This is performed by rotating the layer with the use the transformation function. transformation function. 3. The layer with the second band is moved to the front and is overlapped with the first layer by 3. The layer with the second band is moved to the front and is overlapped with the first layer by using the “Move Tool”. The axial part of the first band copy on the second layer must coincide with the using the “Move Tool”. The axial part of the first band copy on the second layer must coincide with axial part of the first band. This placement is required in order to follow the native distance between the axial part of the first band. This placement is required in order to follow the native distance two adjacent bands. After that, some parts of the first layers can be covered by the second layer, as this between two adjacent bands. After that, some parts of the first layers can be covered by the second part of the second layer must be deleted. A new selection in the work area, which includes the first layer, as this part of the second layer must be deleted. A new selection in the work area, which band on the second layer and a region of the second layer which covers the first band on the first layer, includes the first band on the second layer and a region of the second layer which covers the first is made using the “Lasso Tool” or “Rectangular Marquee Tool”, and then deleted. band on the first layer, is made using the “Lasso Tool” or “Rectangular Marquee Tool”, and then deleted.

InsectsInsects2018 2018,,9 9,, 121 x 88 ofof 15 15

4. The left edge of the second layer is sharp and makes the transition between the first layer to the second4. The leftlayer edge visible. of the Tosecond conceal layer the transition, is sharp and the makesleft edge the of transition the second between layer must the ﬁrstbe processed layer to theusing second an “Eraser layer visible. Tool” with To conceal a smooth the border transition, brush the or left by edge the “Blur of the Tool.” second layer must be processed usingIteration an “Eraser of Tool”these withfour asteps smooth applied border to brusheach band or by from the “Blur left to Tool.” right in a curved chromosome regionIteration makes of it thesecompletely four steps straight. applied to each band from left to right in a curved chromosome region makes it completely straight.

Figure 6. A workflow of chromosome straightening; example for the region with two bands: a—selection,Figure 6. A whichworkflow includes of chromosome two adjacent straightening; bands, using example “Lasso Tool” for the or “Rectangularregion with Marqueetwo bands: Tool,” a— copyingselection, and which moving includes the new two layer adjacent with “Movebands, Tool”using to“Lasso the work Tool” field; or b—rotation“Rectangular and Marquee movement Tool,” of thecopying new layer and (orangemoving area)the new with layer “Move with tool”, “Move applying Tool” the to “Transform”the work field; function, b—rotation and adjustment and movement of the firstof the band new (blue layer line), (orange vertically, area) relativewith “Move to the tool”, vertical applying guideline; the the“Transform” distance between function, marginal and adjustment points of theof the band first is theband same (blue relative line), tovertically, the outer relative horizontal to the lines; vertical c—new guideline; selections the on distance the template between chromosome marginal image,points whichof the band include is the the same whole relative second to band the oute andr thehorizontal whole, orlines; at least c—new part, selections of the first on band the template (dotted bluechromosome line); copying image, selection which andinclude moving the whole the new second layer toba thend and work the field; whole, d—rotation or at least and part, movement of the first of theband second (dotted layer blue (green line); area) copying with “Moveselection tool”, and applyingmoving the “Transform” new layer function, to the work and adjustmentfield; d—rotation of the secondand movement band (magenta of the line),second vertically, layer (green relative area) to thewith vertical “Move guideline; tool”, applying the distance “Transform” between marginalfunction, pointsand adjustment of the band of is the the second same relative band (magenta to the outer line), horizontal vertically, lines; relative e—the to secondthe vertical layer guideline; is brought the to thedistance front andbetween overlapped marginal with points the firstof the layer band using is the “Move same Tool”;relative the to axialthe outer part ofhorizontal the first bandlines; one—the the secondsecond layerlayer coincidesis brought with to the the front axial and part overlapped of the first bandwith ofthe the first first layer layer using (varying “Move the Tool”; opacity the of axial the secondpart of layerthe first is useful); band on f—a the newsecond selection layer coincides in the work wi field,th the which axial part includes of the the first first band band of ofthe the first second layer layer(varying (blue the dotted opacity line) of the and second a region layer of theis useful); second f—a layer new which selection covers in the the firstwork band field, of which the first includes layer (bluethe first line) band using of “Lasso the second Tool” layer or “Rectangular (blue dotted Marquee line) and Tool”; a region g—deletion of the second of selection; layer which h—blurring covers thethe leftfirst border band of the first second layer layer, (blue using line) “Eraser using “Lasso Tool” with Tool” smooth or “Rectangular border brush Marquee or “Blur Tool”; Tool”, g—deletion to make a gradualof selection; transition h—blurring from the the first left layer border to theof the second second layer. layer, using “Eraser Tool” with smooth border brush or “Blur Tool”, to make a gradual transition from the first layer to the second layer.

Insects 2018, 9, 121 9 of 15

2.8. Chromosome Regions Combination and Substitution A perfect whole-arm chromosome image is almost impossible to obtain. After the straightening procedure, some regions of chromosomes have deficiencies that must be edited. We created at least 3 native view variants of straightening chromosome arms because different regions could have a different quality in each variant. Such regions are selected and combined to make the best result. Selected layers are copied using “Move Tool” in a new work area, where the finished variant of a chromosome arm will be developed. Centering, aligning, and retouching procedures are the same as described above. Centromere and telomere ends are important landmarks for a chromosome arm discrimination, and they should be thoroughly developed on the chromosome map. Because of their heterochromatic nature, these regions usually have no typical banding pattern. Similar to other regions, centromere and telomere ends can have the most typical variants. The telomere and the centromere ends are usually substituted from an appropriate template image that does not need straightening. In the chromosomes of nurse cells, homologues are often asynaptic at the centromere end. These homologues typically have different degrees of stretching and varying banding patterns and curves. Therefore, the centromere end cannot be transposed as a whole to a chromosome map. To resolve this problem, one homologue from a pair is processed and transposed to the chromosome map. Then, it is copied to a separate layer and flipped. In this case, the angle between homologues and the length of the asynaptic centromere region must have the most typical characteristics for this region.

2.9. Chromosome Trimming One of the most crucial steps of chromosome map development is the trimming of the background from the straightened chromosome arm. The chromosome edge profile, along with the banding pattern, is an important characteristic of chromosome and region identification, and often serves as a landmark. A straightened chromosome arm is surrounded by a background from the template chromosome images. These parts of the layers are deleted to uncover a chromosome edge profile. Usually, marginal points of the bands are at the bottom of the indentations, and the interbands (or puffs and diffuse heterochromatin) form protrusions. However, there are cases which depart from this rule, and they can also be important landmarks. The procedure of trimming must not change a landmark’s features. For this reason, landmarks, as well as the thinnest and the widest regions on the chromosome arm, must receive special attention. Following these observations, the sequence of protrusions and indentations on the chromosome edge profile is marked by a “Pen Tool”. These marginal points are then curved by a “Convert Point Tool” in accordance with the native shape of each chromosome region (Figure7). The “Path” needs to be transformed into the “Selection” using the “Make selection” function, then the selection needs to be inverted with the “Select inverse” function and the background must be deleted leaving layers containing the straightened chromosome.

2.10. Final Corrections Final corrections are performed on a separate sheet where all of the stretched chromosome arms are collected.

2.10.1. Brightness and Contrast Adjustment Brightness and contrast can be changed at the step of image processing, but the excess background can impede appropriate image edition. After trimming, only chromosome images will be processed. Brightness and contrast adjustments are important, not only for general image processing, but also for modifying landmark regions according to their native view. Usually desirable brightness and contrast values are achieved by adjusting tones in the levels menu (Image > Adjustments > Levels). Dragging the black and white points to the left and right edges Insects 2018, 9, 121 10 of 15 of the histogram palette darkens the shadows and brightens the highlights. Mid-tone points can be adjusted also, but over-shadowing and over-highlighting must be avoided (Figure1). Insects 2018, 9, x 10 of 15

Figure 7. Trimming of a chromosome arm map (the pericentromeric region of 3R arm of An. beklemishevi nurseFigure cells 7. asTrimming an example): of a a—makingchromosome the arm path, map using (the “Path pericentromeric Tool”, and marking region protuberances of 3R arm of and An. hollowsbeklemishevi of chromosome nurse cells edge as proﬁlean example): (red line); a—making b—smoothing the path, angles using using “Path “Convert Tool”, Point and Tool” marking (red line);protuberances and, c—making and hollows the selection of chromosome from the path edge and deletingprofile (red the backgroundline); b—smoothing area. angles using “Convert Point Tool” (red line); and, c—making the selection from the path and deleting the 2.10.2.background Relative Chromosome area. Length Position Adjustment Relative chromosome length and centromere indexes are very important karyotype characteristics, 2.10.2. Relative Chromosome Length Position Adjustment and a chromosome map must reﬂect them. To avoid differences in the levels of polyteny and chromatin condensation,Relative 20–30chromosome images oflength the chromosome and centromere sets must indexes be obtained. are very These important images are karyotype used for calculatingcharacteristics, relative and chromosome a chromosome lengths map and must centromere reflect indexes. them. To For avoid each chromosomedifferences in set, the the levels lengths of ofpolyteny arms are and measured, chromatin and condensation relative chromosome, 20–30 images lengths of the chromosome and centromere sets indexesmust be areobtained. calculated. These Theimages average are valuesused for of thesecalculating characteristics relative arechromosome used to adjust lengths straightened and centromere chromosome indexes. arms. For each chromosomeAfter being set, assembled, the lengths centromere of arms are ends measured have to, and be found relative within chromosome the frame, lengths which meansand centromere that the averageindexes chromosome are calculated. length The must average be within values a of± 5%these error. characteristics However, the are current used to lengths adjust of straightened individual chromosomechromosome maps arms. do not completely correspond to the calculated proportions. Using the shortest chromosomeAfter being arm assembled, as a standard, centromere new values ends for have each to chromosomebe found within arm the map frame, length which are calculated.means that Thethelengths average of chromosome each chromosome length arm must layer be are within adjusted a ±5% to the error. new lengthHowever, values the using current “Move lengths Tool”. of Minorindividual corrections chromosome to the width mapsof do the not chromosomes completely co arerrespond also occasionally to the calculated required. proportions. Using the shortest chromosome arm as a standard, new values for each chromosome arm map length are 2.10.3.calculated. Chromosome The lengths Map of Structure each chromosome arm layer are adjusted to the new length values using “MoveChromosome Tool”. Minor arm corrections maps are arranged to the width on a of canvas the chromosomes according to are these also rules: occasionally required.

2.10.3. Chromosome Map Structure Chromosome arm maps are arranged on a canvas according to these rules: 1. The telomere ends must be right oriented, and the centromere ends must be left oriented.

Insects 2018, 9, 121 11 of 15

Insects 2018, 9, x 11 of 15 1. The telomere ends must be right oriented, and the centromere ends must be left oriented. 2. 2. SexSex chromosomes chromosomes are are located located on on the the top top of ofth thee map. map. The The remaining remaining chromosome chromosome arms arms are are positionedpositioned according according to tothe the division division number, number, starting starting from from the the right right arm. arm. 3. All arms must be left-edge-justified. The distances between chromosome arms should be 3. All arms must be left-edge-justiﬁed. The distances between chromosome arms should be sufﬁcient sufficient to allow the numbering and lettering of divisions and subdivisions. to allow the numbering and lettering of divisions and subdivisions.

2.11.2.11. Chromosome Chromosome Regions Regions Division Division and and Nomenclature Nomenclature ToTo reference reference certain certain regions,regions, chromosomechromosome arms arms are are divided divided into into numbered numbered divisions divisions and lettered and letteredsubdivisions. subdivisions. Divisions Divisions (subdivisions) (subdivisions) should shou correspondld correspond to painted to painted or photo or photo maps maps of the of samethe samecell-type cell-type chromosomes chromosomes for succession.for succession. It may It may be difﬁcult be difficult in chromosome in chromosome map subdivisionsmap subdivisions to do to this dofor this different for different cell types cell becausetypes because of inter-tissue of inter-ti differencesssue differences in banding in banding patterns. patterns. However, However, it should it be shoulddone be for done divisions, for divisions, at least. at least. TheThe borders borders of ofdivisions divisions (subdivisions) (subdivisions) must must always always mark mark the the borders borders of ofbands bands and and interbands interbands (Figure(Figure 8).8).

FigureFigure 8. Chromosome 8. Chromosome division, division, numbering, numbering, and and lettering lettering (peritelomeric (peritelomeric region region of of3R 3R An.An. beklemishevi beklemishevi as asan an example). example).

TheThe result result of ofa aboundary boundary setting setting is isto todefine define the the band band or or interband interband to toa acertain certain division division (subdivision).(subdivision). Each Each division division (subdivision) (subdivision) contains contains at least at least one band. one band. The borders The borders of divisions of divisions and subdivisionsand subdivisions are indicated are indicated by vertical by verticallines. Each lines. border Each is border drawn is from drawn the fromchromosome the chromosome edge exactly edge oppositeexactly the opposite band-interband the band-interband border below border the below straightened the straightened chromosome chromosome image. image. Division Division lines are lines longerare longer than subdivision than subdivision lines (Figure lines (Figure 8). 8). ChromosomeChromosome names names are are aligned aligned with with the the axis axis of ofeach each chromosome chromosome arm arm and and are are placed placed near near the the telomeretelomere end. end. In In the the final final step, step, the the numbering numbering and letteringlettering ofof chromosomechromosome regions regions should should follow follow the thenomenclature nomenclature of of existing existing chromosome chromosome maps maps for fo certainr certain species. species. In In the the right right arms, arms, numeration numeration and andlettering lettering are are from from the the telomere telomere to to the the centromere centromere end end of of the the chromosome chromosome arm arm (from (from left left to to right) right) but, but,in thein the left left arms, arms, from from the centromerethe centromere to the to telomere the telomere ends (fromends (from right toright left). to This left). approach This approach provides providescontinuity continuity in numbering in numbering and lettering and lettering of regions of regions for the wholefor the chromosome. whole chromosome. 3. Results 3. Results Following the procedures described above and high-pressure techniques [15,29–31], we upgraded Following the procedures described above and high-pressure techniques [15,29–31], we the cytogenetic maps of two regions of nurse cell chromosomes of An. beklemishevi [29]: regions 6A–6B upgraded the cytogenetic maps of two regions of nurse cell chromosomes of An. beklemishevi [29]: in the 2R arm (a telomeric end) and regions 15A–16A in the 2L arm (a pericentromeric end). The main regions 6A–6B in the 2R arm (a telomeric end) and regions 15A–16A in the 2L arm (a pericentromeric result of the high-pressure application was the discovery of several minor bands, which are absent on end). The main result of the high-pressure application was the discovery of several minor bands, the non-high-pressure chromosome map: 5 bands in region 6A–6B and 11 bands in region 15A–16A which are absent on the non-high-pressure chromosome map: 5 bands in region 6A–6B and 11 bands (Figure9). in region 15A–16A (Figure 9). For example, two fine bands at the end of the peritelomeric region in the 2R arm became For example, two fine bands at the end of the peritelomeric region in the 2R arm became visible visible only after high-pressure squashing. Another important result was the visualization of only after high-pressure squashing. Another important result was the visualization of near-regular near-regular banding-like structure of the pericentric diffuse chromatin in region 15A of the 2L banding-like structure of the pericentric diffuse chromatin in region 15A of the 2L arm. After high- arm. After high-pressure squashing, the chromatin fibers and numerous aggregates corresponding to pressure squashing, the chromatin fibers and numerous aggregates corresponding to heterochromatic “bands” are revealed. heterochromatic “bands” are revealed.

Insects 2018, 9, 121 12 of 15 Insects 2018, 9, x 12 of 15

Figure 9. Comparison of the high-pressure (top) and non-high-pressure (bottom) maps for 6A–6B Figure 9. Comparison of the high-pressure (top) and non-high-pressure (bottom) maps for 6A–6B region in the 2R arm and 15A–16A in the 2L arm of nurse cell chromosomes of An. beklemishevi. The new region in the 2R arm and 15A–16A in the 2L arm of nurse cell chromosomes of An. beklemishevi. The bands in the high-pressure map are labeled by green circles. new bands in the high-pressure map are labeled by green circles. 4. Discussion 4. Discussion Here we describe the basic principles and detailed techniques of a high-resolution cytogenetic map’sHere development we describe for the malaria basic principles mosquitoes’ and polytenedetailed techniques chromosomes. of a high-resolution We used high-resolution cytogenetic digitalmap’s development images of the for squashed malaria mosquitoes’ preparation polytene of unstained chromosomes. polytene We chromosomes used high-resolution to develop digital the map.images Such of the images squashed preserve preparation fine details of ofunstained the chromosome polytene bandingchromosomes pattern. to Thedevelop major the principle map. Such of mapimages development preserve fine is to details reproduce of the the chromosome native structure banding of the pattern. polytene The chromosomes major principle and to of make map chromosomedevelopment armsis to and reproduce regions onthe thenative map structure easily identifiable. of the polytene In our chromosomes map-developing and approach, to make thischromosome principle is arms achieved and regions by straightening on the map chromosomes easily identifiable. and by payingIn our map-developing special attention toapproach, the position this andprinciple morphology is achieved of landmarks. by straightening The straightening chromosomes procedure and by is performedpaying special by aligning attention the to chromosome the position bandsand morphology sequentially, makingof landmarks. them parallel The straightenin and barcode-like.g procedure Landmarks is performed and their positions by aligning facilitate the identificationchromosome ofbands the chromosome sequentially, arms making and them surrounding parallel internal and barcode-like. regions. Chromosome Landmarks shapeand their is a verypositions important facilitate landmark, identification and our approachof the chromosome reflects the chromosome arms and surrounding edge profile. Thus,internal in definingregions. chromosomeChromosome regions, shape is an a investigatorvery important relies landmark, not only onand the our banding approach pattern reflects but the also chromosome on the sequence edge ofprofile. protrusions Thus, in and defining on the indentationschromosome ofregions, the chromosomes. an investigator relies not only on the banding pattern but alsoChromosomal on the sequence divisions of protrusions and nomenclature, and on inthe general, indentations are adopted of the fromchromosomes. the tradition for dipteran polyteneChromosomal chromosomes. divisions The divisions and nomenclature, are indicated in by ge numbers,neral, are andadopted subdivision from the names tradition are spelt for withdipteran capital polytene letters [chromosomes.31–33]. However, The wedivisions have changed are indicated the sequence by numbers, of subdivision and subdivision letters innames the left are autosomalspelt with armscapital to letters follow [31–33]. the sequence However, of numbers we have from changed centromeres the sequence to telomeres. of subdivision letters in the leftThe autosomal chromosomal arms position to follow of DNAthe sequence markers of after numbers FISH canfrom be centromeres identified using to telomeres. cytogenetic maps. Therefore,The chromosomal genome physical position mapping of DNA can anchormarkers sequencing after FISH scaffolds can be identified to polytene using chromosomes cytogenetic during maps. theTherefore, mapping genome procedure. physical Chromosome mapping mapscan anchor developed sequencing using thisscaffolds strategy to havepolytene been chromosomes successfully usedduring for the physical mapping mapping procedure. of the fiveChromosome anopheline maps genomes: developedAn. gambiaeusing this[15], strategyAn. stephensi have [been27], An.successfully albimanus used[7], An. for atroparvusphysical mapping[8,23], and of theAn. five sinensis anopheline[28]. genomes: An. gambiae [15], An. stephensi [27],The An. qualityalbimanus of [7], polytene An. atroparvus chromosomes [8,23], andand cytogeneticAn. sinensis [28]. maps determines the resolution of the physicalThe mapping. quality of Resolution polytene chromosomes is important for and identifying cytogenetic the maps direction determines of supercontigs the resolution mapped of bythe FISH,physical when mapping. the beginning Resolution and the is important end of each for supercontig identifying is labelledthe direction by different of supercontigs fluorochromes. mapped If the by structureFISH, when of the the polytene beginning chromosome and the end is of “friable”, each superc and theontig borders is labelled between by different bands and fluorochromes. interbands are If fuzzy,the structure DNA markers of the polytene of small chromosome supercontigs willis “friable”, overlap. and Sometimes the borders the between resolution bands of genome and interbands physical mappingare fuzzy, can DNA be improved markers byof small a high-pressure supercontigs technique will overlap. [15,29, 30Sometimes] which makes the resolution the banding of patterngenome clearerphysical and mapping many new can smallbe improved bands visible. by a high-pressure technique [15,29,30] which makes the banding pattern clearer and many new small bands visible.

Insects 2018, 9, 121 13 of 15

Here we applied a high-pressure technique to refine two regions on the chromosome map of An. beklemishevi. This approach makes visible an additional 45% of bands in two subdivisions of the 2R arm and 38% of bands in five subdivisions of the 2L arm. The high-pressure method reveals a previously invisible band-like structure in heterochromatic regions. The fan structure of fibrils in region 15A has been confirmed by FISH of 15A-specific micro-dissected DNA-probe with nurse cell chromosomes of An. beklemishevi [34]. The high-pressure technique is required to make a detailed map of heterochromatin regions of polytene chromosomes, which often have a diffuse structure [35]. Despite its advantages, the high-pressure technique can introduce deformation in the chromosome structure. Chromosome overstretching and band over-squashing are common outputs of this technique. The approach results in appropriate squashing of only some chromosomes on a preparation or some regions within a chromosome arm. Moreover, it can transform morphology of some regions to make it unnatural, as it was shown with a puffed region in 15C; after high-pressure squashing, it made protrusions, which are absent in regular-pressure chromosome preparations (Figure9). Thus, the high-pressure technique is not suitable for routine physical mapping. The high-pressure chromosome preparations may be used for improvement of specific chromosomal regions when precise mapping is required.

5. Conclusions High-resolution cytogenetic maps are essential for anchoring genome sequences to chromosomes as well as for studying chromosome organization and evolution. We developed a detailed pipeline for the construction of high-resolution cytogenetic maps and applied this workﬂow to the reﬁnement of the cytogenetic map for An. beklemishevi. The method presented here can be used to develop high-resolution cytogenetic maps for other Anopheles mosquitoes, as well as for other dipteran insects with polytene chromosomes.

Author Contributions: Conceptualization, G.N.A. and M.V.S.; Methodology, G.N.A.; Validation, V.N.S., M.V.S. and I.V.S.; Investigation, G.N.A.; Writing—Original Draft Preparation, G.N.A.; Writing—Review & Editing, I.V.S., M.V.S.; Visualization, G.N.A.; Supervision, I.V.S. Funding: This research was funded by the Russian Science Foundation grant No. 15-14-20011 to IVS. Acknowledgments: We thank Melissa Wade for proofreading the text. Conﬂicts of Interest: The authors declare no conﬂicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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