Yellow shapes mosquito pigmentation and vector competence to Plasmodium berghei

Yellow shapes mosquito pigmentation and vector competence to Plasmodium berghei | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Yellow shapes mosquito pigmentation and vector competence to Plasmodium berghei View ORCID Profile Dennis Klug , Amandine Gautier , View ORCID Profile Eric Marois , View ORCID Profile Stéphanie Blandin doi: https://doi.org/10.1101/2025.09.24.678207 Dennis Klug 1 INSERM, CNRS, Université de Strasbourg, U1257, UPR9022, Institut de Biologie Moléculaire et Cellulaire , 67000 Strasbourg, France 2 Institute of Physiology and Pathophysiology, Department of Molecular Cell Physiology, Philipps University Marburg , Emil-Mannkopff-Strasse 2, 35037 Marburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dennis Klug For correspondence: klugd{at}staff.uni-marburg.de sblandin{at}unistra.fr Amandine Gautier 1 INSERM, CNRS, Université de Strasbourg, U1257, UPR9022, Institut de Biologie Moléculaire et Cellulaire , 67000 Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eric Marois 1 INSERM, CNRS, Université de Strasbourg, U1257, UPR9022, Institut de Biologie Moléculaire et Cellulaire , 67000 Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Eric Marois Stéphanie Blandin 1 INSERM, CNRS, Université de Strasbourg, U1257, UPR9022, Institut de Biologie Moléculaire et Cellulaire , 67000 Strasbourg, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stéphanie Blandin For correspondence: klugd{at}staff.uni-marburg.de sblandin{at}unistra.fr Abstract Full Text Info/History Metrics Preview PDF Abstract The insect-specific yellow gene, first described over a century ago, remains enigmatic despite its conserved role in pigmentation. In the malaria vector mosquito Anopheles coluzzii , yellow is strongly expressed during juvenile stages and in adult ovaries, mirroring pigmentation patterns that likely enhance desiccation resistance, camouflage, and reproductive fitness. To dissect its function, we generated a complete knockout allele yellow(-)KI by replacing the first exon with an EGFP reporter, and introgressed it into multiple genetic backgrounds. Loss of yellow abolished black cuticle pigmentation and delayed larval development but left adult lifespan unaffected. Developmental delay likely impaired mating success in competition with wild-type mosquitoes, hastening the decline of the X-linked yellow(-)KI allele. Notably, yellow(-)KI females showed a consistent twofold increase in susceptibility to the rodent malaria parasite Plasmodium berghei , independent of the complement factor TEP1 and melanization, whereas infection with P. falciparum was unchanged. Antibiotic treatment reversed the heightened P. berghei susceptibility, implicating altered gut microbiota as a mediator. Our findings reveal that yellow not only shapes pigmentation but also indirectly modulates vector competence through microbiota-dependent mechanisms, underscoring the complex interplay between cuticle biochemistry, microbial ecology, and pathogen transmission. Introduction Defects in cuticle or eye pigmentation have long served as powerful genetic markers in Drosophila melanogaster , providing visually striking, easily scorable phenotypes that facilitated some of the earliest genetic mapping efforts. Mutations in pigmentation genes were among the first identified in classical mutagenesis screens, as the resulting phenotypes were easy to detect and generally viable under laboratory conditions. The first null allele of yellow was described as early as 1911 by Edith Wallace ( Lindsley D.L., 1968 ). In yellow -deficient flies, black pigment is absent from the entire adult cuticle as well as from larval mouthparts and denticles ( Geyer and Corces, 1987 ; Wittkopp, True and Carroll, 2002 ; Hinaux et al ., 2018 ). Comparable phenotypes have been reported in Bombyx mori , Tribolium castaneum , Apis mellifera , Rhodnius prolixus and other insects, following either germline transgenesis or RNAi-mediated knockdown of yellow family genes ( Futahashi et al ., 2008 ; Noh et al ., 2015 ; Nie et al ., 2021 ; Berni et al ., 2022 ). In D. melanogaster , yellow is strongly expressed from ∼46 h after pupation in wing discs, thorax, and abdomen, tissues that later develop intense pigmentation, and in specific neuronal populations throughout the life cycle ( Hinaux et al ., 2018 ). This dual expression pattern underlies phenotypes that go beyond visible pigmentation. While reduced male mating success in yellow mutants was initially attributed to behavioural changes linked to neuronal expression ( Drapeau, Cyran, et al ., 2006 ), recent work demonstrated that decreased pigmentation weakens the stiffness of male sex combs, impairing their ability to grasp females during mating ( Massey et al ., 2019 ). Yellow belongs to a rapidly evolving insect-specific protein family, with up to 14 members in Anopheles gambiae and A. mellifera , but only eight in D. pseudoobscura . Such variation suggests lineage-specific diversification or loss under selective pressure ( Drapeau, Albert, et al ., 2006 ). While yellow -like proteins have also been identified in bacteria such as Deinococcus radiodurans ( Maleszka and Kucharski, 2000 ), phylogenetic analyses confirm that the insect family is distinct. Despite over a century of study, the biochemical role of Yellow remains unresolved ( Drapeau, 2003 ). Cuticle pigmentation derives from tyrosine, which is converted to L-Dopa by tyrosine hydroxylase (TH) and then to dopamine by L-Dopa decarboxylase (DDC) ( Fig. 1 ). Dopamine acts as the precursor for multiple pigmentation pathways, producing brown NBAD pigment, black eumelanin, yellow pheomelanin, or colourless sclerotization components. The cysteine peptidase Tan reverts NBAD to dopamine, and loss of tan reduces pigmentation, though less severely than yellow loss ( True et al ., 2005 ). Yellow is thought to act in the conversion of dopamine quinone to eumelanin, either directly or via dopachrome intermediates. Its sequence similarity to a dopachrome conversion enzyme in Aedes aegypti ( Johnson, Li and Christensen, 2001 ) led to the hypothesis that it encodes such an activity ( Wittkopp, True and Carroll, 2002 ). While dopachrome-converting activity has been demonstrated for Yellow-f and Yellow-f2, no biochemical evidence exists for Yellow itself ( Han et al ., 2002 ). Research in D. melanogaster has largely focused on Yellow’s role in pigmentation patterns, mate choice, and speciation. More recently, Yellow has also been linked to cuticle stiffness and eggshell permeability ( Farnesi et al ., 2017 ; Massey et al ., 2019 ; Noh et al ., 2021 ). However, whether altered pigment precursor homeostasis in non-cuticular tissues has systemic effects remains poorly understood. This is particularly relevant given that melanization is a key immune defence in insects, targeting parasites such as microfilariae in mosquitoes, parasitic wasps in Drosophila , and Plasmodium ookinetes during their passage through the mosquito midgut epithelium. Here, we demonstrate for the first time that Yellow influences antiparasitic defence in the malaria vector Anopheles coluzzii . Knockout of yellow increased susceptibility to the rodent malaria parasite Plasmodium berghei independently of the complement factor TEP1 and melanization capacity, and this effect could be reversed by antibiotic treatment, implicating the gut microbiota as a mediator. Our findings reveal that a gene classically associated with cuticle pigmentation can exert systemic effects on microbial ecology and vector–parasite interactions, highlighting an unanticipated link between insect pigmentation pathways and pathogen transmission. Download figure Open in new tab Figure 1. Cuticle pigmentation pathway in insects. Illustration modified from ( Pentzold et al ., 2018 ). In oenocytes, the amino acid tyrosine is converted by the enzyme tyrosin hydroxylase (TH) into L-Dopa and further decarboxylated by the L-Dopa decarboxylase (DDC) into dopamine. Dopamine is the precursor of multiple pigments. It can be converted into N-acetyl dopamine (NADA) through enzymatic activity of the arylalkylamine-N-acetyltransferase (aaNAT) which is believed to lead to the formation of mostly colorless components of the cuticle required for sclerotization ( Osanai-Futahashi et al ., 2012 ). Another branch of the pathway leads to the formation of N-ß-alanyl dopamine (NBAD) via conjugation of dopamine to ß-alanine by the ß-alanyl biogenic amid synthetase Ebony ( Richardt et al ., 2003 ). This enzymatic reaction can be reverted by the cysteine peptidase Tan keeping dopamine and NBAD in equilibrium ( Wagner et al ., 2007 ). While being secreted from epidermal cells to form the cuticle, NADA, NBAD and dopamine are oxidized by Laccase 2 (Lac2) into dopamine quinone and NBAD quinone, the latter being further processed to NBAD pigment and sclerotizing components of the cuticle. Dopamine quinone is either conjugated to cysteine to form pheomelanin, or further processed into dopachrome and 5,6-dihydroxyindole through activity of a dopachrome conversion enzyme (DCE) to form black eumelanin. Yellow is believed to act during both, or one of the last two steps of the eumelanin formation. L-dopa: L-dihydroxyphenylalanine; NADA: N-acetyl dopamine; NBAD: N-ß-alanyl dopamine; DCE: dopachrome conversion enzyme; Lac2: Laccase 2; aaNAT: arylalkylamine-N-acetyltransferase; DDC: L-dopa decarboxylase; ADC: aspartate decarboxylase; TH: tyrosine hydroxylase. Although Yellow, highlighted by a red square, is believed to act in the last steps of eumelanin synthesis, the exact function of Yellow is still unknown. Results Phenotypic characterization of yellow(-)KI mosquitoes and promoter trap activity To generate a yellow loss of function mutant in the malaria mosquito A. coluzzii, we targeted the first exon of yellow (AGAP000879) by CRISPR/Cas9 ( Dong et al ., 2018 ). A repair template was provided to replace the targeted sequence by a fluorescence cassette encoding EGFP under control of the artificial 3xP3 promoter ( Fig. 2A ) ( Klug et al ., 2022 ). 3xP3 is predominantly expressed in the eyes and ventral nervous chain of larval mosquito stages and enables efficient sorting of transgenic larvae. 3xP3 was flanked by lox sites in order to place egfp under the control of the endogenous yellow promoter upon Cre-mediated recombination. Yellow(-)KI mosquitoes were easily identified both through GFP expression and the complete absence of eumelanin as previously described in the fruit fly D. melanogaster ( Wittkopp, True and Carroll, 2002 ; Hinaux et al ., 2018 ) ( Fig. 2B ). Mutant mosquitoes displayed complete yellow body coloration and appeared more translucent, especially at the abdomen, allowing better imaging through the cuticle ( Klug et al ., 2022 ). Interestingly, the eggs of yellow(-)KI females were completely yellow, in contrast to the black coloration of wild type eggs ( Fig. 2C ). We noticed that, even without removing the 3xP3 promoter, egfp was expressed in other tissues than the eyes and nervous chain, where 3xP3 is restricted ( Fig. 2D , compares EGFP fluorescence in yellow(-)KI and sag(-)KI ). These results suggest that in yellow(-)KI , egfp expression is also influenced by the directly neighbouring endogenous yellow promoter. High levels of EGFP fluorescence were visible in developing yellow(-)KI larva, before hatching from eggs (not shown), as well as in all larval stages and in pupae ( Fig. 2D ). EGFP was expressed mainly in the head and at the anterior of each abdominal segment, resulting in a zebra-like appearance. This expression pattern matches that of a D. melanogaster line expressing a fluorescently labeled Yellow protein ( Hinaux et al ., 2018 ). However, in this line, the zebra-like expression pattern first appears in pupae while larvae display little yellow expression that is restricted to a subset of neurons, and possibly cells responsible for the development of the larval mouthparts ( Hinaux et al ., 2018 ). EGFP fluorescence in yellow(-)KI larval stages, especially in the abdomen, appears mostly controlled by the yellow promoter. Download figure Open in new tab Figure 2. Generation and characterization of the yellow(-)KI transgene. A) Genomic locus of AgYellow . The knockin (KI) transgene is composed of the EGFP cassette flanked by Lox sites. Upon insertion, exon 1, including the start codon, is replaced by the transgene, thus preventing transcription of yellow . Transgenic larvae were selected through expression of the fluorescent marker EGFP driven by the 3xP3 promoter. Note that the illustration is not drawn to scale. B) Changes in cuticular pigmentation of yellow(-)KI females and males compared to wild type. Note the complete absence of eumelanin in yellow(-)KI mosquitoes. C) yellow(-)KI eggs compared to wild type about 24 hours after oviposition (bright field). D) EGFP fluorescence in yellow(-)KI larvae (top images) at days 3 (d3), 6 (d6) and 9 (d9), and in pupae. EGFP fluorescence and bright field images are shown for the pupal stage. For yellow(-)KI a homozygous (hom) and a heterozygous (het) pupae surrounded by negatives are shown. The same developmental stages are shown for the sag(-)KI line in which egfp is expressed exclusively under the control of the 3xP3 promoter in juvenile stages, when the Saglin promoter is transcriptionally silent ( Klug et al ., 2023 ). Fluorescence signals in this line are concentrated in the head and eyes and become weaker with progressive development (red arrows). Fitness costs caused by the absence of Yellow To distinguish between transgenic and wild type siblings, we made use of flow cytometry based on the larval expression of the introduced fluorescence cassette ( Marois et al ., 2012 ). Initial analyses of the yellow(-)KI line revealed that the X-linked yellow gene undergoes dosage compensation, which is reflected by a lower fluorescence in heterozygous females as compared to transgenic males, although both of them carry a single copy of the yellow(-)KI cassette ( Fig. 3A,B ). Indeed, by sorting the L1 larvae of each population and rearing them separately to adulthood, we could define the population with the lowest EGFP intensity to be heterozygous females, while males displayed intermediate and homozygous females, the highest fluorescence intensity. To assess potential fitness costs associated with the absence of Yellow, we mixed an equal number of homozygous yellow(-)KI and wild type siblings, and we followed the proportion of each genotype over 6 generations ( Fig. 3C ). We observed a sharp decline of the proportions of transgenic larvae (homozygous, hemi- and heterozygous). After six generations, the wild type had almost entirely overtaken the cage population, indicating a high fitness cost for mosquitoes lacking yellow expression ( Fig. 3C ). To investigate at which stage of the life cycle the fitness of yellow(-)KI carriers was reduced, L1 larvae obtained from wild type and yellow(-)KI colonies were mixed in equal ratios and raised until pupation. New pupae were sorted each day according to their EGFP fluorescence and the proportion of wild type / yellow(-)KI was calculated ( Fig. 3D ). The first pupae were observed at day 7 after eggs hatched and appeared to be nearly exclusively wild type. The proportion of pupae homozygous for yellow(-)KI was always below 50% except on the last collection day (d10) when it reached its maximum (63%). These data indicate that pupation is delayed by ∼2 days in yellow(-)KI , and that a larger proportion of yellow(-)KI mosquitoes die during juvenile stages as compared to wild type. To identify the stages most sensitive to the lack of Yellow, we daily followed larval and pupal development in different yellow(-)KI and WT lines that were raised separately ( Fig. 3E ): yellow(-)KI , a double mutant yellow(-)KI;tan(-)KI lacking expression of yellow and tan , a yellow(-)KI-7b mutant homozygous for yellow(-)KI and the 7b transgene suppressing the expression of the major complement factor TEP1 ( Pompon and Levashina, 2015 ), and a yellow(-)aapp-DsRed line carrying yellow(-)KI together with a fluorescent reporter transgene driving expression of DsRed in the outer lobes of female salivary glands ( Klug et al ., 2022 ). The colonies yellow(-)KI and yellow(-)KI;tan(-)KI were generated in the Ngousso genetic background, while yellow(-)KI-7b and yellow(-)aapp-DsRed were hybrids of Ngousso and G3L , another frequently used wild type strain. Both yellow(-)KI and yellow(-)KI;tan(-)KI showed a strong decrease in the pupation rate, with only 25% larvae reaching the pupal stage for these two lines, vs. 75% for wild type controls. Interestingly, the pupation rate was higher for larvae from the two yellow(-)KI lines in the mixed Ngousso/G3L background, with yellow(-)aapp-DsRed displaying a similar pupation success as wild type ( Fig. 3E ). These results suggest that the fitness costs linked to Yellow loss vary depending on the genetic background and/or its degree of polymorphism. Of note, in both wild type and all yellow(-)KI lines, most losses occurred during larval development, followed by the last larval (L4) and the pupal stage ( Fig. 3E , S1 ). In yellow(-)KI ( Ngousso backgound) as well as in the two yellow lines in the Ngousso/G3L background, pupae with moulting defects were also detected ( Fig. S1 ). We further measured the life span of females homozygous for yellow(-)KI ( Fig. 3F ). For this, both genotypes at L1 larval stage were mixed in equal quantities and raised in synchrony. Four to five days after adult emergence, yellow(-)KI and wild type females were separated based on their fluorescence, placed in paper cups and kept under normal breeding conditions. The number of dead mosquitoes was monitored daily until no living mosquito remained. All mosquitoes died within 55 days with no difference in the life span of yellow(-)KI and wild type mosquitoes ( Fig. 3F ). Download figure Open in new tab Figure S1. Lethality and moulting defects at larval and pupal stages in yellow(-)KI mosquito lines. Example images of (1) a living yellow(-)KI pupa, (2) a dead pupa with an unusual elongated posture, (3) a dead L4 larva - those often appeared black, likely due to melanization, and of (4) a living pupa with moulting defect. The last larval moult is still attached to the tip of the pupal abdomen (red arrowhead). Dead younger larvae are often difficult to detect as they are rapidly consumed by siblings. Scale bar: 2mm. Download figure Open in new tab Figure 3. Absence of Yellow delays larval development but does not affect the life span of adult mosquitoes. A) The fitness of mosquitoes carrying yellow(-)KI was assessed in competition with wild type over six generations. Initially 50 males and 50 females homozygous for yellow(-)KI were mixed with 50 male and 50 female transgene-negative siblings. The distribution of different genotypes was monitored over five generations by COPAS analysis of more than 2000 L1 larvae per generation. The F1 generation was not analyzed because of the low number of larvae in this generation. The fact that heterozygous females are less fluorescent than yellow(-)KI hemizygous males made it possible to follow these two populations separately. B) COPAS plot of L1 larvae of the F3 generation following transgenesis. Larvae carrying the yellow(-)KI transgene form three populations; heterozygous females (lowest EGFP intensity), yellow(-)KI positive males (medium EGFP intensity) and females homozygous for yellow(-)KI (highest EGFP intensity). Larvae carrying no transgene form a population in the bottom left corner. Note that some larvae still inherited the Cas9:RFP transgene. As a result additional populations are visible carrying either solely Cas9:RFP or Cas9:RFP in combination with yellow(-)KI . C) Flow cytometry of L1 offspring from a mosquito colony homozygous for yellow(-)KI . Note that the only remaining populations are homozygous yellow(-)KI females and hemizygous males. D) Larvae negative or homozygous for the transgene yellow(-)KI were mixed in equal numbers and raised until pupation. Pupation started 7 days and ended 10 days after larvae hatched from eggs. Mean percentages of transgenic and wild type pupae from three independent experiments with 400 larvae per experiment and genotype are shown. E) Pupation rate of mosquitoes homozygous for yellow(-)KI only, or yellow(-)KI in combination with homozygous tan(-)KI , 7b or aapp-DsRed in comparison to wild type mosquitoes. The genetic background of the mosquito colonies is shown above the bars. For each experiment at least 800 larvae per colony were raised synchronously and the number of pupae was counted until day 11 after larvae hatched from eggs. Pupae, dead L4 larvae and pupae, as well as pupae with moulting defects were counted and the percentage in relation to the total population of seeded larvae was calculated. Means of 2 (wild type, yellow(-)KI and yellow(-)-aapp-DsRed ) or 3 ( yellow(-)KI;tan(-)KI and yellow(-)KI-7b ) experiments. F) Survival of homozygous yellow(-)KI females and wild type siblings kept on 10% sucrose solution. The number of dead mosquitoes was monitored daily. Shown is the mean and standard error of the mean from two independent experiments with >30 mosquitoes each. Data were tested for significance with the Log-rank (Mantel-Cox) test. ns: not significant. Impact of Yellow on infection with Plasmodium spp To investigate whether yellow(-)KI mosquitoes display an altered susceptibility to the rodent malaria parasite Plasmodium berghei , wild type ( Ngousso ) and yellow(-)KI mosquitoes raised as a mix were infected with high and low doses of P. berghei gametocytes ( Fig. 4A,B ). In both cases, yellow(-)KI females carried significantly higher oocyst numbers compared to wild type controls (medians in high gametocyte dose: 115 vs 68, and in low dose: 46 vs 27, respectively). To exclude that the increase in susceptibility was linked to the impaired fitness of the yellow(-)KI colony ( Fig. 3E ), we repeated infections with high and low doses of P. berghei gametocytes using the yellow(-)aapp-DsRed colony which displayed a similar pupation rate as wild type ( Fig. 3E ). Yellow(-)aapp-DsRed were also more susceptible to P. berghei infection than wild types, with more pronounced differences (medians in high gametocyte dose: 121 vs 20, and in low dose: 6 vs 21 in Yellow(-)aapp-DsRed vs WT , respectively, Fig. 4C,D ). In contrast, no significant difference in the parasite load could be detected between yellow(-)KI and wild type upon P. falciparum infection ( Fig. 4E ) . Albeit not statistically significant, yellow(-)KI females even appeared to be less infected than WT (medians: 7 vs 11, respectively), possibly indicating that the absence of Yellow has a negative effect on the infection with human malaria parasites as previously shown for the knockdown of Yellow-g in Anopheles dirus upon infection with P. vivax ( Mongkol et al ., 2021 ). Download figure Open in new tab Figure 4. The loss of yellow but not of tan increases parasite load upon infection with Plasmodium berghei but not with Plasmodium falciparum . For each experiment, WT and sibling control mosquitoes were infected with P. berghei ( PbGOMO or Pbfluo ) or P. falciparum ( PfNF54 ). The mosquito line ( yellow(-)KI , yellow; tan(-)KI , gray, yellow(-)KI + aapp-dsRed , red; WT controls, black) is indicated below the graphs and the Plasmodium species and strain, and infection regime (high or low dose) is depicted above each dataset. For each mosquito line and parasite regime, the number of oocysts per infected mosquito is plotted, with red lines indicating medians. The percentage of infected mosquitoes (prevalence) is shown in the bar graph as the mean ± SEM of N replicates. The number of biological replicates (N) is shown below the pictogram while the total number of analysed mosquitoes is shown below each genotype. Statistical significance was tested using Mann Whitney (MW) to compare parasite loads, and Fisher’s exact test (FT) for prevalences. A) MW: **p = 0.0015; FT: not significant (ns), p = 0.9206. B) MW: *p = 0.0245; FT: ns, p = 0.8374. C) MW: ***p = 0.0002; FT: ns, p = 1.0. D) MW : ***p = 0.0001; FT: ns, p = 0.7387. E) MT: ns, p = 0.7183; FT: ns, p= 1.0. F) MW: ns, p = 0.5361; FT: ns, p = 0.6417. We next investigated if other perturbations of the pigmentation pathways could affect mosquito susceptibility to P. berghei infection. For this, we used a loss-of-function mutant of the hydrolase Tan involved in the homeostasis of the two pigment precursors dopamine and NBAD ( Fig. 1 ). In contrast to yellow(-)KI mutants, tan(-)KI mosquitoes displayed similar parasite loads as wild type controls raised together upon infection with high doses of P. berghei gametocytes (medians: 70 vs 58, respectively, Fig. 4F ). Melanization response of yellow(-)KI mosquitoes against P. berghei oocysts Ookinetes that are killed upon traversal of the midgut epithelium are either lyzed or melanized, an immune response that is highly dependent on the mosquito genetic background ( Volz et al ., 2006 ). Melanization is triggered through the specific activation of prophenoloxidases (PPOs) in proximity of the parasite. In their activated form, PPOs oxidize monophenols and o-diphenols to o-quinones that are precursors of melanin ( Nappi and Christensen, 2005 ). Since Yellow is believed to affect the synthesis of the quinone dopachrome, a precursor of eumelanin, we hypothetized that in the absence of Yellow, this molecule is absent or treduced in its concentration, potentially altering pathogen melanization. Since melanizing activity in Ngousso and G3L is very low, we introgressed the yellow(-)KI transgene into the L3-5 line that is resistant to infection with P. berghei and displays a strong melanization response ( Collins et al ., 1986 ). Females carrying the yellow(-)KI transgene were backcrossed 6 times with L3-5 males ( Fig S2A ). Female yellow(-)KI carriers of the sixth generation (BC6) were crossed again with L3-5 males and allowed to lay eggs separately in single tubes to generate isofemale families. Each BC6 female was genotyped for TEP1 ( Fig. S2B ), and the progenies of 6 females homozygous for TEP1R were kept and mixed to ensure that all BC7 individuals were homozygous for TEP1R , an immune gene allele that is critical for the resistance and melanization phenotypes typical of L3-5 mosquitoes ( Zheng et al ., 1997 ). BC7 mosquitoes were intercrossed to establish a L3-5 colony with a floating yellow(-)KI allele. Subsequently wild type and yellow(-)KI-L3-5 females derived from this colony were infected with P. berghei and the numbers of live oocysts (EGFP-positive) and melanized ookinetes (black spots in brightfield) were counted 7-8 days after infection. Susceptible mosquitoes from Ngousso wild type were infected in parallel as a control, to confirm all infections were efficient and produced high and similar numbers of oocysts. Homozygous yellow(-)KI mosquitoes were fully resistant to P. berghei infection, as wild type L3-5 mosquitoes, and 100% of them also carried melanized ookinetes, however significantly fewer than wild types (medians: 67 vs 125, respectively) ( Fig. 5A,B ). These results indicate that, in the absence of Yellow, a larger proportion of killed ookinetes is eliminated through lysis instead of melanization. Still, melanized parasites in yellow(-)KI and WT appeared identical ( Fig. 5C ), suggesting that mosquitoes are able to melanize pathogens in the absence of Yellow. Download figure Open in new tab Figure S2. Introgression of the yellow(-)KI transgene into the P. berghei refractory mosquito strain L3-5 . A) Crossing scheme to introgress the yellow(-)KI transgene into the L3-5 genetic background. Females homozygous for yellow(-)KI were crossed with L3-5 males. In the next generation, heterozygous BC1 yellow(-)KI females were sorted and crossed again with L3-5 males. This crossing scheme was repeated for a total of seven times to yield a population of BC7 yellow(-)KI mosquitoes back crossed seven times to L3-5. B) During the last backcross, individual BC6 females were separated to lay eggs and genotyped in order to select progenies of homozygous TEP1R females only as founders of the yellow(-)KI-L3-5 line. Indeed, TEP1R homozygosity is an essential feature of the L3-5 phenotype. TEP1 was genotyped using primers EM175 and EM176 and the PCR product was digested with NcoI cutting the PCR product of TEP1S once (two bands at 783 and 151 bp) while leaving PCR product of TEP1R uncut (one band at 934 bp). Females 1, 2, 4, 6, 9 and 10 were homozygous for TEP1R and their BC7 progeny was intercrossed. A G3L mosquito homozygous for TEP1S was used as control. For female seven (marked with a red asterisk) no PCR product was obtained. Download figure Open in new tab Figure 5. yellow(-)KI does not affect resistance to P. berghei but reduces ookinete melanisation in L3-5 mosquitoes. A) Mosquitoes homozygous for yellow(-)KI and negative siblings of the resistant strain L3-5 , and mosquitoes from the susceptible Ngousso strain were infected with fluorescent P. berghei (PbGOMO). Numbers of live (EGFP-positive) and melanized parasites at 7-8 days post infection are plotted, with red bars indicating medians. Two independent biological replicates. Statistical significance was tested using Mann Whitney test: **p = 0.0025. B) Percentage of mosquitoes carrying live and melanized parasites (prevalence) from the same infections as in (A). Shown is the mean and the standard error of the mean (SEM). C) Midgut of an infected L3-5 mosquito homozygous for yellow(-)KI in comparison to a transgene-negative sibling (control) seven days post infection. Melanized parasites appear as black dots. Scale bar: 500 µm (left) and 100 µm (right, zoom). Elevated parasite counts in yellow(-)KI females are independent of the complement factor TEP1 A major resistance factor to Plasmodium spp. in mosquitoes of the A. gambiae complex is the thioesther protein TEP1 ( Blandin et al ., 2004 ). TEP1 is a structural homolog of the human complement factor C3 and believed to act in a similar fashion, inducing lysis, phagocytosis or melanization through binding to foreign surfaces ( Blandin, Marois and Levashina, 2008 ). To investigate whether increased P. berghei loads in yellow(-)KI mosquitoes could be due to a requirement of Yellow for TEP1 activity, we introgressed the 7b transgene in the yellow(-)KI line. 7b is an X-linked dominant transgene that triggers silencing of the endogenous TEP1 locus ( Pompon and Levashina, 2015 ). We reasoned that if the yellow(-)KI phenotype was dependent on TEP1, then 7b and yellow(-)KI;7b mosquitoes would display similar susceptibilities to P. berghei infections, else yellow(-)KI;7b would be more infected. Virgin homozygous 7b females and yellow(-)KI males were crossed and neonate F1 larvae were COPAS sorted for EGFP (marker of the yellow(-)KI transgene) and dsRed (marker of the 7b transgene). Since both 7b and yellow reside on the X chromosome, F1 mosquitoes carrying both transgenes were exclusively female. To select for recombinants harboring both transgenes on the same chromosome, F1 females were backcrossed to yellow(-)KI males and F2 offspring was again fluorescence sorted for dsRed and EGFP. Since male Anopheles mosquitoes possess only one X chromosome, F2 males positive for EGFP and DsRed had to be recombinants. These recombinant males were backcrossed to yellow(-)KI females and subsequent generations were fluorescence sorted for EGFP and DsRed until saturation. Of note, F1 males negative for yellow(-)KI but positive for 7b were backcrossed to 7b females to establish a separate colony with a similar genetic background as yellow(-)KI;7b . This colony served as control for all infection experiments with yellow(-)KI-7b . We first confirmed by Western Blot that TEP1 was depleted in 7b and yellow(-)KI;7b mosquitoes ( Fig. 6A ). It appeared to be expressed at similar levels in wild type and yellow(-)KI mosquitoes, suggesting that TEP1 expression and processing is not affected by Yellow depletion ( Fig. 6A ). Upon infection with P. berghei , 7b mosquitoes carried higher parasite loads than wild type mosquitoes infected in different experiments but in similar conditions, as expected in the absence of TEP1 (medians: 239 vs 42, respectively, Fig. 6B and 4B ). Still, yellow(-)KI-7b mosquitoes were even more infected than 7b mosquitoes (medians: 397 vs 239, respectively, Fig. 6B ), indicating that the increase in susceptibility to P. berghei in yellow(-)KI mosquitoes is independent of TEP1 function. This also indicates that the high parasite loads observed in the absence of TEP1 do not reflect a maximal reachable infection level, since it can be further increased by removal of additional factors such as Yellow. Download figure Open in new tab Figure 6. The antiparasitic effect of Yellow is independent of TEP1. A) Yellow(-)KI and yellow(-)KI-7b mosquitoes were infected with P. berghei ( PbGOMO ). Parasite load (oocysts per infected mosquito) with red lines indicating medians, and percentage of infected mosquitoes (prevalence, mean ± SEM) from three biological replicates. The number of analysed midguts is given below each genotype. Note the increased parasite burden in 7b mosquitoes compared to controls expressing TEP1 (see Fig. 4 ). Statistical significance was tested using Mann Whitney for parasite loads: ***p < 0.0001 and Fisher’s exact test for prevalences: not significant (ns), p = 1.0. B) Western blot on the hemolymph of yellow(-)KI-7b and 7b mosquitoes probed against TEP1. Wild type and yellow(-)KI mosquitoes were used as positive controls expressing TEP1. An Anti-PPO2 (prophenoloxidase 2) antibody was used as loading control. C) Model of RNAi mediated knockdown of TEP1 in 7b mosquitoes. A transgene composed of the tep1 open reading frame under control of the Hsp70 promoter from D. melanogaster causes a locus-specific effect leading to the transcription of a tep1 antisense RNA subsequently inducing knockdown of TEP1 expression. Antibiotic treatment of yellow(-)KI mosquitoes rescues the infection phenotype The increased parasite load in yellow(-)KI mosquitoes upon infection with P. berghei suggested a potential role of Yellow in the mosquito midgut where the first steps of parasite infection take place. Taking advantage of the fact that egfp is under control of the endogenous yellow promoter in the yellow(-)KI line, we examined whether yellow is expressed in this tissue. We did not detect EGFP fluorescence in yellow(-)KI mosquito midguts, including after blood feeding ( Fig. 7A ). This is coherent with our previous data showing that the yellow promoter activity is weak in adults and mostly restricted to the ovaries in females ( Klug et al ., 2022 ). Thus we hypothesized that the effect of Yellow on parasite development is indirect, either by influencing transcription and/or function of genes and/or proteins located downstream of Yellow, or through systemic metabolic changes induced by the absence of Yellow. Of note, the mosquito midgut microbiota composition influences the outcome of an infection and is regulated by a network of mosquito proteins for example FREP13 (fibrinogen-related protein 13) ( Chauhan et al ., 2020 ; Zaković et al ., 2025 ). We thus tested whether the effect of Yellow absence on parasite development could be mediated by the microbiota. For this, yellow(-)KI and control mosquitoes were bred as a mixture and emerging adults were treated with different antibiotics supplied in 10% sucrose solution ( Fig. 7B ). Treatment of mosquitoes with an antibiotic cocktail (AB cocktail) composed of Penicillin, Amphotericin B, Streptomycin and Gentamicin completely rescued the infection phenotype in yellow(-)KI mosquitoes that displayed similar parasite loads as WT (medians: 122 vs 142, respectively, Fig. 7C ). The same rescue was achieved upon treatment with streptomycin alone, an antibiotic acting on both Gram-negative and Gram-positive bacteria (median yellow(-)KI and WT: 78 and 57, respectively), but not when treated with Gentamicin alone, an antibiotic primarily acting on gram-negative bacteria ( Fig. 7C ). In this case, the phenotype was still present (median yellow(-)KI and WT: 167 and 85, respectively). Taken together, our results indicate that Yellow affects parasite development through its effect on the mosquito midgut microbiota, and that Yellow absence likely leads to the proliferation of Gram-positive bacteria that are beneficial, either directly or indirectly, to parasites. Download figure Open in new tab Figure 7. Differences in the oocyst burden between yellow(-)KI and control females disappear when treated with antibiotics. A) Midguts of A. stephensi ( As ) G12, A. stephensi ( As ) wild type (WT), and of A. coluzzii ( Ac ) yellow(-)KI , imaged to detect EGFP fluorescence 48 hours after blood feeding. The dashed white line highlights the shape of the midgut, while blood that is either in the intestinal lumen or has leaked into the surrounding area appears black. The anterior (at) or posterior (pt) end of the displayed guts are indicated within the images. G12 is a A. stephensi mosquito line expressing high levels of EGFP exclusively after blood feeding that was used here as positive control ( Nolan et al ., 2011 ). B) Juvenile stages of Y ellow(-)KI and wild type (WT) mosquitoes were bred together and allowed to emerge in the same cage. Adults were provided with cotton pads soaked with 10% sucrose solution supplemented with different antibiotics. They were infected with a high dose of P. berghei gametocytes seven to eight days after emergence. Oocysts were counted in the midguts on day 14-15 of life. The number of independent biological replicates (N) is given above each graph while the total number of dissected midguts (n) is given below. Statistical significance between yellow(-)KI and controls was tested using Mann Whitney (MW). The different antibiotics regimens are the following: C) streptomycin, penicillin, gentamicin and amphotericin B (AB cocktail). MW: not significant (ns), p = 0.1832; gentamicin. MW: *p = 0.0109; streptomycin. MW: ns, p = 0.2013. D) Activity profiles of gentamicin and streptomycin. Gentamicin targets predominantly Gram-negative bacteria while streptomycin targets both Gram-positive and Gram-negative bacteria. Discussion Although Yellow was discovered more than a century ago, its precise role in pigmentation remains unclear. Current insights into Yellow function are largely based on visual observations and phylogenetic analyses, with no in-depth biochemical characterization. Moreover, phylogenetic studies have so far been restricted to insects, the only group in which this protein family is uniquely present. The only known related proteins, the major royal jelly proteins (MRJPs), constitute the primary protein source in honey bee royal jelly and have been implicated in the determination of queen development ( Kamakura, 2011 ). While Yellow and MRJPs share some sequence similarity, available data to date suggest they perform distinct functions. We generated yellow(–)KI , a complete knockout allele of yellow , by inserting a reporter transgene into the first exon of the gene, and introgressed this allele into several genetic backgrounds to study its role in different mosquito life-history traits. In A. coluzzii (Ngousso background), the absence of Yellow significantly affected mosquito fitness, primarily through increased lethality and delayed development during juvenile stages. The resulting rapid elimination of the mutation in mixed populations may have been further exacerbated in our competition experiments with wild-type mosquitoes due to the experimental setup: most females—wild-type and mutant—likely mated with wild-type males, which emerged two days earlier than yellow(–)KI males and were thus ready to mate before their mutant counterparts. This would have accelerated the decline of the X-linked yellow(–)KI genotype in mixed cage populations. The increased lethality observed in some populations homozygous for the yellow(–)KI allele may stem from: (1) inbreeding effects due to the low number of founder transgenics, (2) susceptibility of specific genetic backgrounds to Yellow depletion, (3) increased susceptibility to bacterial proliferation in the culture water and (4) potential synergistic effects with other transgenes, notably tan(–)KI and 7b. Interestingly, in the yellow(–)KI-aapp-DsRed colony—a hybrid of Ngousso and G3L— although pupation was still delayed, both pupation rate and larval fitness were restored to wild type levels. These results suggest that the fitness impairment observed in competition experiments was largely due to delayed juvenile development in the absence of yellow expression, though additional effects on mating success, fertility and susceptibility to bacteria cannot be ruled out. In the yellow(–)KI transgene, the egfp reporter is under the direct control of the artificial 3xP3 promoter, positioned immediately downstream of the endogenous yellow promoter. Consequently, its expression reflects not only classical 3xP3-driven patterns but also endogenous yellow activity. A similar pattern was reported for the Sag(–)KI allele, where Sag(–)KI and Sag(–)EX (post-3xP3 excision) showed comparable egfp expression pattern in the salivary gland, albeit expression was weaker in Sag(–)EX ( Klug et al ., 2022 ). This suggests that in this genomic context, the 3xP3 promoter amplifies egfp expression from the endogenous promoter, in addition to driving its expression in the eyes and ventral nerve cord. In A. coluzzii , we detected strong egfp signals in eggs, all larval stages, and pupae, whereas activity in adults was reduced and largely restricted to the ovaries in females ( Klug et al ., 2022 ). This differs markedly from D. melanogaster , in which yellow is predominantly expressed 46 hours after pupation, with minimal larval expression restricted to a subset of neurons and cells involved in mouthpart development. Consistent with this, yellow knockout in D. melanogaster has not been reported to affect larval development. Differences in yellow expression align with pigmentation patterns: while mosquito eggs and larvae are pigmented, fly eggs and larvae are mostly white and transparent. These pigmentation differences likely reflect adaptations to distinct breeding environments. The strong ovarian activity of the yellow promoter in mosquitoes correlates with the pronounced melanization of eggs after deposition. In various mosquito species, eggs with lighter cuticles exhibit lower hatching rates, indicating that pigmentation contributes to desiccation resistance ( Farnesi et al ., 2017 ; Noh et al ., 2021 ). This is advantageous when oviposition occurs in small, ephemeral puddles prone to rapid drying. Similarly, the pigmented cuticle of mosquito larvae and pupae aligns with yellow activity and may serve as camouflage against predators. In contrast, fruit fly eggs are deposited in moist, decomposing material, where larvae quickly burrow, making both desiccation resistance and camouflage less critical. In adult D. melanogaster , yellow loss alters cuticle properties, and yellow -deficient males exhibit reduced sex comb stiffness, impairing mating success ( Massey et al ., 2019 ). Beyond visible phenotypes, yellow(–)KI mosquitoes displayed a consistent increase in susceptibility to P. berghei , with mutant mosquitoes carrying about twice as many oocysts as controls. This effect was independent of the complement factor TEP1 and the melanization response, as homozygous yellow(–)KI-L3-5 females still melanized most killed ookinetes. Interestingly, knockout of yellow increased parasite burden in mosquito lines with the TEP1^S allele (linked to Plasmodium susceptibility) ( Fig. 4 ) and in TEP1-depleted mosquitoes ( Fig. 6 ), but yellow(– ) KI mosquitoes expressing the TEP1R allele remained resistant ( Fig. 5 ). Remarkably, the heightened P. berghei infection in yellow(–)KI mosquitoes could be rescued by antibiotic treatment—either with a cocktail or with streptomycin alone (effective against both Gram-negative and Gram-positive bacteria). Gentamicin alone (targeting mainly Gram-negative bacteria) reduced but did not eliminate the difference between mutants and controls. These findings suggest that yellow(–)KI alters gut microbiota composition or abundance, thereby influencing susceptibility to P. berghei . Possible explanations include reduced overall microbial load, similar to antibiotic treatment, or shifts in microbiota composition affecting Plasmodium colonization. For example, changes in gut pH and tryptophan catabolism mediated by Asaia borogensis and Pseudomonas alcaligenes can impact Plasmodium oocyst numbers ( Wang et al ., 2021 ; Feng et al ., 2022 ), while different microbial communities correlate with opposite outcomes of P. falciparum infection ( Zaković et al ., 2025 ). Here, the lack of increased P. falciparum susceptibility suggests that microbiota changes resulting from Yellow depletion affect P. berghei and P. falciparum differently. Further research is needed to elucidate how a pigmentation gene like Yellow influences mosquito microbiota. The reduced parasite melanization observed in yellow(–)L3-5 females suggests alterations in melanin precursor concentrations and/or composition. Overall, our results indicate that in Anopheles , loss of Yellow may have two major effects: (1) systemic changes in pigmentation precursors, and (2) altered gut microbiota density and/or composition, possibly as a downstream consequence of (1), since yellow is not expressed in the gut. This underscores the complex interplay between genetic traits, physiology, gut microbiota, and pathogen susceptibility, and opens new avenues for exploring the role of Yellow in mosquito biology. View this table: View inline View popup Table 1: Key Resources Materials and methods Breeding conditions A. coluzzii (G3L, L3-5 and Ngousso ) mosquitoes were kept at standard conditions (27-28°C, 75-80% humidity, 12-hr/12-hr light/dark cycle). Larvae were hatched in deionized water and fed with finely pulverized fish food (TetraMin). After pupation, pupae were collected in small glass dishes and transferred into netted cages. Hatched mosquitoes were fed with 10% sugar solution ad libitum using the capillarity of a cord, dipping in a tube filled with sugar solution. To propagate colonies, four to seven day old mosquitoes were blood fed for 10-15 minutes on anesthetized mice. Two days later mosquitoes were offered a glass dish with wet filter paper to allow egg laying. L1 larvae hatched after two days and were raised as described above. Mosquito transgenesis The transgenesis of mosquitoes was performed following standard protocols ( Volohonsky et al ., 2015 ). Detailed information about the generation of Yellow(-)KI , Tan(-)KI and aapp-DsRed mosquitoes can be found in ( Klug et al ., 2022 ). Infection of mosquitoes with P. berghei CD1 mice were infected through intraperitoneal injection of a frozen stock of Plasmodium berghei ( PbANKA ) parasites, PbGOMO (Manzoni et al ., 2015), genetically engineered to constitutively express EGFP. Subsequently parasitemia was monitored by FACS (AccuriC6 SORP, Becton Dickinson). Once parasitemia reached 3-5 % mice were bled by cardiac puncture and infected blood passaged into naïve mice by intravenous injection into the tail vein. The number of injected mice depended on the number of mosquitoes planned to be infected while the number of transferred parasites depended on the planned infection date. Usually 2 x 10 7 parasites were transferred into two mice to achieve a parasitemia of 2-3% at day three post injection. Four to seven day old female mosquitoes were transferred into paper cups (approximately 50 females per cup) the day before infection. On the day of the infection infected mice were anaesthetized and placed on the cup to allow mosquitoes to feed for approximatively 15 minutes covered by a dark blanket. Subsequently mosquitoes were anaesthetized on ice and non-fed females were removed. Infected mosquitoes were kept at 21°C, 60% humidity with a 12 hours light, 12 hours dark cycle. To achieve different infection levels mice which had been intravenously infected with P. berghei were either fed two days (low infection regime) or three days (high infection regime) after passage. Introgression of yellow(-)KI into L3-5 Virgin homozygous yellow(-)KI females were crossed with virgin L3-5 males to obtain heterozygous F1 yellow(-)KI mosquitoes inheriting 50% of the genetic background of each parental strain ( Ngousso and L3-5). Virgin F1 females were crossed with L3-5 males to enrich for L3-5 specific genetic traits. Backcrossing was repeated for six generations while the yellow(-)KI transgene was selected by isolating only EGFP positive females through COPAS (Union Biometrica) sorting. In a last step of the introgression process, TEP1R homozygous females were isolated. This was necessary because this TEP1 allele contributes strongly to the highly refractory phenotype of L3-5 mosquitoes against Plasmodium spp. infection. To create a mosquito colony expressing exclusively TEP1R, females from backcross six with unknown TEP1 genotype were mated with L3-5 males (which are homozygous for TEP1R) and blood fed. After two days, females were isolated in single Drosophila breeding tubes containing a piece of wet filter paper to allow egg laying. The offspring of selected females that laid eggs was raised as described above and the genomic DNA of founder females was extracted and used for TEP1 genotyping by PCR with primers EM175 / EM176. The generated PCR product (934 bp) was digested with the restriction enzyme NcoI (Thermo Fisher Scientific) for one hour at 37 °C, before gel electrophoresis. NcoI cuts specifically TEP1S but not TEP1R resulting in one fragment (undigested PCR product; 934 bp) if mosquitoes are homozygous for TEP1R, three fragments (digested PCR fragments: 783 and 151 bp as well as the full-length fragment of 934 bp) if mosquitoes are heterozygous for TEP1R and TEP1S and two fragments (completely digested PCR product; 783 and 151 bp) if mosquitoes are homozygous for TEP1S. The offspring of females homozygous for TEP1R was self-crossed to generate the homozygous yellow(-)KI line in the L3-5 background. Generation of yellow(-)KI-7b and yellow(-)-aapp-DsRed mosquitoes Virgin 7b females and yellow(-)KI males were crossed and the F1 was COPAS sorted for EGFP (marker of the yellow(-)KI transgene) and dsRed (marker of the 7b transgene). Since the 7b transgene as well as yellow reside on the X chromosome, F1 mosquitoes heterozygous for both transgenes were exclusively female. To select for recombinants that harbour both transgenes on the same X chromosome, F1 females were backcrossed to yellow(-)KI males and the F2 offspring was fluorescence sorted for dsRed and EGFP. Since male Anopheles mosquitoes possess only one X chromosome, F2 males positive for EGFP and DsRed were recombinants possessing both transgenes on one X chromosome. Subsequently, recombinant males were backcrossed to yellow(-)KI females and following generations were fluorescence sorted for EGFP and DsRed until saturation. Of note, F1 males negative for EGFP (negative for yellow(-)KI ) but positive for 7b were backcrossed to 7b females to establish a separate colony with a comparable genetic background as yellow(-)KI-7b . This colony served as control for all infection experiments in which yellow(-)KI-7b was used. To confirm absence of TEP1 protein, hemolymph of yellow(-)KI-7b , yellow(-)KI and control females was probed with αTEP1 antibodies on western blots which revealed complete absence of TEP1 in yellow(-)KI-7b females while yellow(-)KI females expressed TEP1 in levels comparable to the control ( Fig. 6B ). To generate yellow(-)KI-aapp-DsRed mosquitoes, virgin yellow(-)KI females were crossed to virgin male aapp-DsRed mosquitoes. The F1 generation of this cross was self-crossed and F2 larvae were sorted using the COPAS to keep only EGFP fluorescent larvae (marker for yellow(-)KI ). EGFP positive larvae were treated with puromycin as described ( Volohonsky et al ., 2015 ). The selection process was repeated until EGFP reached saturation and lethality after puromycin treatment could no longer be observed. To exclude the presence of yellow(-)KI females heterozygous for aapp-DsRed , which would be sufficient to confer resistance to puromycin, 20 freshly mated and blood fed females were separated in single plastic vials for egg laying. The offspring of these females was raised and self-crossed to obtain the F2 generation without any selection. In case founder females were heterozygous for aapp-DsRed , yellow(-)KI females without aapp-DsRed must be present in the F2 generation. To exclude contamination with specimens lacking aapp-DsRed , living females of each colony were screened for red fluorescent salivary glands with a Nikon SMZ18 Stereomicroscope ( Klug et al ., 2022 ). Families with DsRed negative females were excluded from further breeding while colonies displaying only DsRed positive specimens were pooled to create a homozygous yellow(-)KI-aapp-DsRed line. Assessing differences in pupal development of yellow(-)KI and control mosquitoes Yellow(-)KI and control ( Ngousso ) mosquitoes were reared in synchrony as two separate colonies. Newly hatched L1 larvae of both colonies were sorted using COPAS in batches of 50 larvae each. Mixed cultures of control and yellow(-)KI larvae were seeded in equal proportions, with 50 larvae for each genotype. Larvae were reared according to standard conditions. From day 7 to day 10, all emerging pupae were collected once a day and screened for the presence of EGFP using a Nikon SMZ18 Stereomicroscope with the respective fluorescence filter. This was necessary to clearly distinguish control (negative for EGFP) from yellow(-)KI pupae (positive for EGFP). Estimating fitness of yellow(-)KI lines Yellow(-)KI , control ( Ngousso ), yellow(-)KI-7b , yellow(-)-app-DsRed and yellow(-)KI;tan(-)KI mosquitoes were reared in synchrony as separate homozygous colonies. Newly hatched L1 larvae of all colonies were sorted using COPAS in batches of 200 larvae each and seeded in separate pans. For each experiment (biological replicate) four pans (technical replicate) per genotype with 200 larvae each were reared. Larvae were grown in standard conditions. Between day 7 and day 10 emerging pupae, dead L4 larvae and dead pupae as well as living pupae with moulting defects were collected and counted. Experiments were terminated at day 11. The few remaining larvae were often small and appeared sick, so it was assumed that they would not continue to develop. Survival experiments Four to seven day old homozygous yellow(-)KI and control females were separated in “survival” cups and kept at 21°C, 60% humidity and 12 hours light, 12 hours dark cycle. Survival cups were made from normal and conic drinking cups made from paper. The bottom of normal drinking cups was replaced with a glued conic cup to create a bottom with a pointed end. Subsequently the tip of the conic cup was removed and the resulting hole was closed with cotton. Dead mosquitoes gathered at the tip of each cone and were easily removed by removing the cotton while limiting escape risk. “Survival cups” were placed in small glass dishes to keep them stable. Mosquitoes received 10% sugar solution ad libitum provided as soaked cotton pads covered by petri dishes on top of each cup. Cotton pads were moistened every two to three days and renewed every seven to ten days. Dead mosquitoes were counted and removed every day. Treatment of mosquitoes with antibiotics Yellow(-)KI and control ( Ngousso ) mosquitoes were reared synchronously as two separate colonies. Newly hatched F1 larvae of both genotypes were mixed to roughly 2/3 yellow(-)KI and 1/3 control larvae per pan. This was necessary to compensate for the higher mortality of the yellow(-)KI genotype and still obtain similar numbers of hatching yellow(-)KI and control females. Larvae were raised in standard conditions until pupation. Pupae were collected in small glass dishes and allowed to hatch in netted cages. From hatching onwards a 10% sucrose solution was provided supplemented either with an antibiotic (AB) cocktail (Gibco, Invitrogen Antibiotic-Antimycotic solution: 100 U/mL Penicillin, 100 µg/mL Streptomycin, 0,25 µg/mL Amphotericin-B; supplemented with 100 µg/mL Gentamicin (Sigma-Aldrich)) or exclusively with 100 µg/mL Streptomycin or 100 µg/mL Gentamicin. For treatment with Gentamicin and Streptomycin only, both chemicals (Sigma-Aldrich) were used at the indicated concentrations. The antibiotic treatment used is based on previous studies ( Chabanol et al ., 2020 ), but concentrations were slightly elevated as only adult animals were treated and not larval stages. Mosquitoes were infected 4-5 days after hatching as described before. Fluorescence imaging Adults ( Fig. 2B ), eggs ( Fig. 2C ), larvae ( Fig. 2D , S2 ), pupae ( Fig. 2D ) and midguts ( Fig. 5C ) were imaged using a Nikon SMZ18 Stereomicroscope with the respective filtersets for DsRed, EGFP and transmission light (no filter). The age and genotype of the specimens are indicated in the respective figures or in the figure legends, if applicable. RT-PCR For RNA isolation 20 male and 20 female mosquitoes of wild type ( Ngousso ) and tan(-)KI were anaesthetized on ice and transferred to cryovials. 5-10 ceramic beads and 600 µl Trizol were added and homogenization of whole mosquitoes was performed using a Precellys (Bertin Technologies). Samples were either stored at −80°C or directly processed to isolate RNA. For RNA isolation 40 µl of p-Bromoanisol (BAN) was added. Tubes were vortexed for approximately 15 seconds and centrifuged for 20 min at 4°C and 16 rcf. After centrifugation the supernatant was transferred into a clean plastic reaction tube and 400 µl of isopropanol was added. Samples were mixed by gently inverting tubes several times before being centrifuged for 10 min at 4°C and 16 rcf. Subsequently the supernatant was discarded and pellets were washed with 500 µl of 70% ethanol. Samples were centrifuged for 5 min at 4°C and 16 rcf and the supernatant was discarded. Pellets were allowed to air dry for 5-10 min before being resuspended in 100 µl nuclease free water. Resuspended RNA was measured with a NanoDrop (Thermo Fisher Scientific) and 1 µg of RNA per sample was used for subsequent cDNA generation. DNAse digestion and reverse transcription was performed with the iScript gDNA Clear cDNA Synthesis Kit (Biorad, Hercules, California, United States) according to manufacturer’s protocols. The presence of TAN- (primer DK158 and DK159) and TEP1-specific cDNA (primer DK136 and DK137) was verified by perfoming PCRs using Phusion polymerase with 40 amplification cycles. Western blotting Hemolymph was collected in denaturing protein loading buffer (Tris-HCl 0.35 M, SDS 10.3%, glycerol 36%, β-mercaptoethanol 5%, bromophenol blue 0.012%) by proboscis clipping from 10 mosquitoes ≥4 days after emergence. Hemolymph samples were denatured at 65°C for 5 min. Samples were separated by 7% SDS-PAGE. Protein membrane transfer, antibody incubations, and detection were carried out as previously described ( Levashina et al ., 2001 ). Ethics statement The experiments complied with Directive 2010/63/EU of the European Parliament concerning the protection of animals used for scientific research. Our animal facility is approved under license number I-67-482-2 by the veterinary authorities of the Bas-Rhin department (Direction Départementale de la Protection des Populations). Authorization for animal use in this project, as well as for the production and use of transgenic organisms (bacteria, mosquitoes, parasites), was granted by the French Ministry of Higher Education, Research and Innovation under permit numbers APAFIS#20562-2019050313288887 v3 and 3243. Software and statistics Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA). Data sets were either tested with a one-way ANOVA or a Student’s t test. A value of p<0.05 was considered significant. ChatGPT (GPT-5, OpenAI, San Francisco, CA, USA) was used to assist in refining the manuscript’s phrasing, spelling, and grammar. Funding This work was supported by the Laboratoire d’Excellence (LabEx) ParaFrap (grant LabEx ParaFrap ANR-11-LABX-0024 to SAB), by the Equipement d’Excellence (EquipEx) I2MC (grant ANR-11-EQPX-0022 to EM and SAB), by the ANR grant GDaMo (ANR-19CE35-0007 to EM) and by the ERC Starting Grant Malares (No260918 to SAB) and by CNRS, Inserm, and the University of Strasbourg. Additional funding was awarded to DK by the DFG as a postdoctoral fellowship (KL 3251/1-1). Competing interests The authors declare no competing interests. Acknowledgements We thank Nathalie Schallon and Amandine Gautier for their assistance in mosquito rearing and P. berghei infections, and Ludvine Ramolu for technical support. We are also grateful to Lionel Brice Feufack Donfack for help with P. falciparum infections. We further thank the Mosquito Immune Responses (MIR) team for their support in mosquito breeding and for counting dead mosquitoes during survival assays. 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Share Yellow shapes mosquito pigmentation and vector competence to Plasmodium berghei Dennis Klug , Amandine Gautier , Eric Marois , Stéphanie Blandin bioRxiv 2025.09.24.678207; doi: https://doi.org/10.1101/2025.09.24.678207 Share This Article: Copy Citation Tools Yellow shapes mosquito pigmentation and vector competence to Plasmodium berghei Dennis Klug , Amandine Gautier , Eric Marois , Stéphanie Blandin bioRxiv 2025.09.24.678207; doi: https://doi.org/10.1101/2025.09.24.678207 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17690) Bioengineering (13892) Bioinformatics (41935) Biophysics (21451) Cancer Biology (18587) Cell Biology (25499) Clinical Trials (138) Developmental Biology (13377) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24318) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88601) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15152) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)