Targeted knockdown of in vitro candidates does not alter Wolbachia density in vivo

Targeted knockdown of in vitro candidates does not alter Wolbachia density in vivo | 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 Targeted knockdown of in vitro candidates does not alter Wolbachia density in vivo View ORCID Profile Kimberley R. Dainty , View ORCID Profile Johanna M. Duyvestyn , View ORCID Profile Heather A. Flores doi: https://doi.org/10.1101/2025.02.16.638550 Kimberley R. Dainty 1 Institute of Vector-Borne Disease, Monash University , Melbourne, Victoria, Australia 2 Department of Microbiology, Monash University , Melbourne, Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kimberley R. Dainty Johanna M. Duyvestyn 1 Institute of Vector-Borne Disease, Monash University , Melbourne, Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Johanna M. Duyvestyn Heather A. Flores 1 Institute of Vector-Borne Disease, Monash University , Melbourne, Victoria, Australia 3 School of Biological Sciences, Monash University , Melbourne, Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Heather A. Flores For correspondence: heather.flores{at}monash.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The bacterial endosymbiont Wolbachia has emerged as an effective biocontrol method to reduce arbovirus transmission. Transinfection of w Mel Wolbachia from Drosophila melanogaster to Aedes aegypti results in the transfer of important Wolbachia -induced phenotypes including the reproductive modification, cytoplasmic incompatibility, and inhibition of viruses including dengue and chikungunya. However, the mechanisms underlying these critical traits as well other Wolbachia -host interactions are still not fully understood. Recently an in vitro genome wide RNAi screen was performed on w Mel-infected Drosophila S2 cells and identified large cohorts of host genes that alter w Mel density when targeted. If these findings can be replicated in vivo , this would provide a powerful tool for modulating w Mel density both systemically and in a tissue-specific manner allowing for interrogation of w Mel-host interactions. Here, we used the GAL4/UAS system to express RNAi molecules targeting host gene candidates previously identified to dysregulate w Mel density in vitro . We found systemic knockdown of two candidate D. melanogaster genes does not lead to w Mel density dysregulation. To explore the lack of consistency between our study and previous work, we also examined native tissue-specific density of w Mel in D. melanogaster . We show density is varied between tissues and find that individual tissue densities are not reliable linear predictors of other tissue densities. Our results demonstrate the complexities of implementing in vitro findings in systemic applications. Introduction In recent years, the use of Wolbachia has emerged as an effective biocontrol tool in multiple mosquito species, including Aedes aegypti . Transinfection of the Wolbachia strain, w Mel, from Drosophila melanogaster into Aedes aegypti results in the transfer of multiple w Mel-induced phenotypes including the reproductive modification of its host (cytoplasmic incompatibility - CI) and the inhibition of medically important arboviruses such as dengue, Zika, and chikungunya ( Walker et al. 2011 ; Hurk et al. 2012 ; Aliota, Walker, et al. 2016 ; Aliota, Peinado, et al. 2016 ; Tan et al. 2017 ; Carrington et al. 2018 ; Rocha et al. 2019 ; Pinto et al. 2021 ; Utarini et al. 2021 ). While the Wolbachia genes underlying CI have been identified, it is still unclear which host genes and pathways are necessary for CI. Additionally, the virus-inhibiting phenotype induced by w Mel and other Wolbachia strains is well documented in various Drosophila and mosquito species; however, the exact mechanism underpinning it is poorly understood. No study has found a single mechanism of large effect underlying virus blocking ( Lindsey et al. 2018 ). Furthermore, w Mel infection in Ae. aegypti is also linked to multiple fitness costs, including mild impacts on fecundity, hatch rate, and egg longevity ( Fraser et al. 2017 ; Ant et al. 2018 ; Allman et al. 2020 ). Elucidating the host genes involved in these w Mel-host interactions may provide insight into the stability of these critical w Mel-induced traits and identify ways to reduce the impact of w Mel on host fitness increasing the longevity of this biocontrol method. However, progress in this area has been hindered by the inability to genetically modify Wolbachia as well as the inability to easily modulate Wolbachia in its hosts. In vitro assays can be a powerful alternative when in vivo assays are not feasible. In particular, in vitro genome-wide RNAi screens can quickly screen large numbers of genes and identify potential candidate genes involved in a particular pathway or phenotype or interest. In Drosophila, genome-wide screens have been used to successfully identify candidate genes involved in a variety of pathways, including host pathogen interactions ( Cherry 2008 ), virus growth and replication ( Cherry 2008 ), cell growth and viability ( Boutros et al. 2004 ), and RNAi response ( Dorner et al. 2006 ). However, there are also several limitations to in vitro genome-wide RNAi screens. The in vitro system may not fully recapitulate the complexities of the system (e.g. tissue- or temporal-specific expression) and, of course, there is always the likelihood of off-target effects. Nonetheless, these screens can be a powerful first step to identifying genes and pathways of interest, which can be further interrogated. Previous work by Grobler et al. (2018) demonstrated that w Mel density levels can be dysregulated by way of host gene knockdown in vitro . A genome-wide RNAi screen in a D. melanogaster cell line revealed large cohorts of host genes that alter w Mel density when targeted including an enrichment of ribosomal genes which increased w Mel density when downregulated. Two genes associated with ribosomal biogenesis were validated in vivo by crossing w Mel-infected D. melanogaster to mutant allele stocks. As homozygous mutants were lethal, flies that were heterozygous for either ribosomal mutant were used, and heterozygotes showed a 1.2-3-fold increase in w Mel densities in germarium and egg chambers when measured by RNA FISH ( Grobler et al. 2018 ). If these findings can be further replicated in vivo with the identification of genes which cause both large up and downregulation of w Mel density, this would provide a novel way to modulate w Mel density within D. melanogaster . Combined with additional Drosophila tools, this could allow for modulation of w Mel both systemically and in a tissue-specific manner, providing a powerful tool to study Wolbachia -host interactions. Here, we used the GAL4/UAS system in Drosophila melanogaster to target host genes previously identified to dysregulate Wolbachia density when knocked-down in vitro . We successfully knocked down two target host genes with RNAi but find no significant changes in Wolbachia density. To further explore this, we analysed native tissue-specific density of w Mel in Drosophila melanogaster . We showed w Mel density is not uniform across tissues, and that individual w Mel tissue densities are not necessarily predictors of other tissue densities. Our study highlights the complexities of translating in vitro findings to a systemic in vivo approach. Experimental Procedures Drosophila melanogaster lines and rearing D. melanogaster lines were obtained from the Bloomington Drosophila Stock Centre, Dept Biology, Indiana University, Bloomington, IN, USA (Supplementary Table 1). For all experiments, flies were maintained on a semolina diet containing, per litre: 7.14 g potassium tartrate, 0.45 g calcium chloride, 4.76 g agar, 10.71 g yeast, 47.62 g dextrose, 23.81 g raw sugar, 59.52 g semolina, 3.56 mL Tegosept, and 3.57 mL propionic acid ( Henstridge et al. 2018 ), at 25°C and ambient humidity. RNAi candidate selection Previously published data by Grobler et al. (2018) was used to identify RNAi candidates to dysregulate w Mel Wolbachia density. Candidates that had no effect on host cell proliferation (as reported by Grobler et al. (2018) ) were ranked by largest degree of Wolbachia dysregulation, before being chosen depending on availability of D. melanogaster stocks at commercial stock centres with UAS-RNAi transgenes to the genes of interest. Genes chosen had significantly increased Wolbachia density with Robust Z scores (average Wolbachia per host cell normalised to the plate average) between 2.6 and 5 or had significantly decreased Wolbachia density with Robust Z scores between −3.8 and −7.7, as reported by Grobler et al. (2018) . Details regarding all D. melanogaster lines used in this study can be found in Supplementary Table 1. RNAi expression in D. melanogaster The GAL4/UAS system ( Brand and Perrimon 1993 ) was used to induce expression of the RNAi molecules. The Tubulin -GAL4 or daughterless -GAL4 promoters were used to drive the ubiquitous expression of RNAi molecules (Supplemental Table 1). Both of these driver strains were infected with w Mel- Wolbachia by crossing male flies from the GAL4 lines to w Mel-infected virgin females from a double balancer (CyO-GFP/IF; TM6B/MKRS) line (Supplementary Fig 1). Virgin females from each GAL4 driver were crossed with UAS-RNAi carrying males once, and progeny assessed for control and desired phenotypes. Control individuals carried the UAS-RNAi transgene, but no GAL4 transgene. Desired individuals carried both the UAS-RNAi and GAL4 transgenes. Control and desired offspring were obtained across multiple replicate vials to obtain enough offspring and minimise vial effects. Measurement of RNA expression by reverse transcription (RT) qPCR For each cross that resulted in viable offspring, both RNA and DNA were extracted from 23-24 control and desired offspring for each cross. Female flies were collected 7 days post emergence, after being allowed to freely mate. For each individual specimen, DNA and RNA were isolated simultaneously. Flies were homogenised in extraction buffer (10 mM Tris pH 8.2, 1mM EDTA, 50 mM NaCl, 25 ug/ml proteinase K), then incubated at 56 °C for 10 minutes, then 37 °C for 30 minutes. DNA and RNA was then extracted using the AllPrep DNA/RNA kit (QIAGEN) following manufacturer’s instructions, with the exception that DNA was eluted in a single 100 µL step. RNA was treated with RNase-Free Dnase Set (QIAGEN) on column. RNA was used to generate cDNA using First Strand cDNA Synthesis kit (K1612, Invitrogen by Thermo Fisher Scientific) according to the manufacturer’s instruction. qRT-PCR was performed using a LightCycler 480 II (Roche) using LightCycler 480 SYBR Green I Master (Roche) according to manufacturer’s protocol. RNA expression was calculated using candidate gene-specific primers relative to the reference D. melanogaster RpS17 gene (Supplementary Table 2) using the ΔΔCT method ( Livak and Schmittgen 2001 ). RNA expression for each individual knockdown cohort was normalised to the matching individual control cohort. Wolbachia density detection by qPCR The density of w Mel Wolbachia in desired offspring was compared to that of control offspring from each cross (N=23-24), using DNA extracted as described above. Total relative Wolbachia density was determined in whole individual female flies 7 days post emergence (allowed to freely mate), using qPCR with primers to amplify a fragment of the Wolbachia gene encoding wsp , and the reference D. melanogaster RpS17 gene (Supplementary Table 2). qPCR was performed for each sample using a LightCycler 480 II (Roche) using the QuantiNova Probe PCR Master Mix (QIAGEN) according to the manufacturer’s protocol. Wolbachia density was quantified relative to RpS17 using the ΔCT method. Tissue dissection 7-14-day old female flies (allowed to freely mate) were immobilised on CO 2 before being dissected in 1x phosphate buffered saline (PBS) solution. Dissected tissues were moved to fresh PBS to rinse away contaminating tissues and haemolymph and processed for DNA isolation. Individual tissues were homogenised and incubated in extraction buffer (10 mM Tris pH 8.2, 1mM EDTA, 50 mM NaCl, 25 ug/ml proteinase K) at 56 °C and 96 °C for 5 minutes each, before total DNA was isolated from individual tissues using the QIAamp 96 DNA QIAcube HT kit (Qiagen) following manufacturer’s instructions, except in the final step where DNA was eluted in 75 µL of AE buffer. The measurement of Wolbachia density in each tissue was performed as described above. Statistical analysis Statistical analysis was performed using GraphPad Prism version 9.0. Data normality was assessed by group using a Shapiro-Wilk normality test. Statistical significance was determined using the Mann Whitney test. Additional statistical analyses and information such as U values, sample sizes and medians can be found in Supplemental Table 3. Results Host gene knockdown causes no wMel density dysregulation Grobler et al. (2018) identified 1117 genes that, when targeted with RNAi in cell culture, resulted in a significant increase or decrease in Wolbachia density. We chose 14 of these candidates to knockdown to study in vivo (six predicted to upregulate and eight predicted to downregulate w Mel density). These candidates showed the highest magnitude of w Mel density dysregulation, had no effect on host cell proliferation in the Grobler et al. (2018) study, and had RNAi lines available from stock centres. These candidates did not include the two ribosomal biogenesis genes, RpL27a and RpS3 , which were validated in vivo by Grobler et al. (2018) . UAS- RpL27a could not be sourced, and the magnitude of dysregulation caused by RpS3 was lower than that of the chosen candidates. Additionally, studies that knocked down RpS3 with more widely expressed GAL4 drivers resulted in lethality of flies ( Perkins et al. 2015 ). Transgenic lines containing RNAi molecules targeting candidate genes that dysregulated w Mel density in vitro were assessed in the GAL4/UAS system using the ubiquitous driver, Tubulin -GAL4. Of the 14 crosses performed, seven were found to be lethal under ubiquitous Tubulin expression ( Figure 1 ). The crosses were repeated with a driver of lower ubiquitous expression, daughterless -GAL4 ( Scialo et al. 2016 ), but found to still be lethal. For the remaining seven crosses where progeny containing both the Tubulin -GAL4 and RNAi transgenes were obtained, RT-qPCR was performed. Four of the candidate genes showed no significant change in expression level, one ( dac ) unexpectedly showed significant increase in expression, and two ( CG9801 and su(r) ) showed a significant decrease in expression compared to controls ( Fig 1 ). We then measured the impact of gene expression modulation on w Mel density. The transgenic RNAi strain that caused an unexpected increase in target gene expression (RNAi targeting dac ) showed a significant increase in w Mel density ( Fig 2 ). The candidates that caused significant knockdown of the targeted genes, CG9801 and su(r) , did not cause dysregulation of w Mel density ( Figure 2 ). Two other genes showed small, but significant, changes in w Mel density, despite no significant change in target gene expression. The transgenic RNAi strain targeting yippee showed an increase in w Mel density compared to controls, whilst the strain targeting Pcf11 showed a decrease in w Mel density compared to controls. Download figure Open in new tab Figure 1. Expression of RNAi-targeted D. melanogaster genes. Knockdown of RNAi-targeted genes was assessed using qRT-PCR and significance determined using the ΔΔC t method. Data are the median and IQR of 23-24 flies. Blue bars represent genes identified to decrease Wolbachia density when downregulated, and orange bars represent genes identified to increase Wolbachia density when downregulated in Grobler et al. (2018) . Labels on the X axis indicate genotype of offspring from crosses. Bars labelled ‘knockdown’ represent offspring carrying both the Tubulin -GAL4 driver and the UAS-RNAi transgenes. Bars labelled ‘control’ represent offspring carrying only the UAS-RNAi transgene. GAL4-driven RNAi expression found to be lethal for offspring indicated under ‘Lethal’. Statistical significance was determined using the Mann Whitney test comparing each knockdown cohort to its matched control cohort (ns = not significant, *** P <0.001, **** P <0.0001). Download figure Open in new tab Figure 2. Wolbachia density in D. melanogaster targeted with RNAi. Density of w Mel Wolbachia in 7-day old female D. melanogaster targeted with RNAi against indicated genes and matched controls were determined by qPCR using primers to amplify a fragment of the Wolbachia wsp gene and the reference D. melanogaster RpS17 gene. The ΔC t method was used to calculate Wolbachia density. Data are the median and interquartile range of 23-24 flies. Blue bars represent genes suggested to decrease Wolbachia density when downregulated, and orange bars represent genes suggested to increase Wolbachia density when downregulated. Labels on the X axis indicate genotype of offspring from crosses. Bars labelled ‘knockdown’ represent offspring carrying both the Tubulin- GA4-driver and the UAS-RNAi transgenes. Bars labelled ‘control’ represent offspring carrying only the UAS-RNAi transgene. Bars labelled with a green circle indicate offspring with significant knockdown or upregulation of indicated genes ( Figure 1 ). Statistical significance was determined using the Mann Whitney test comparing each knockdown cohort to its matched control cohort (ns = not significant, * P <0.05, *** P <0.001, **** P <0.0001). wMel density varies substantially between tissues One potential explanation for host gene modulation not impacting w Mel density systemically, is that w Mel density may vary substantially across tissues and successful reduction in some tissues may be overwhelmed by less successful reduction in other tissues. To measure variation in w Mel density across tissues, 7-14-day old female flies were collected and the Malpighian tubules, salivary glands, ovaries, fat body, muscles, midgut, and brain were dissected. DNA was isolated from these tissues and w Mel density measured by qPCR. w Mel density was highly variable across tissue types, with some tissues having nearly one log higher w Mel density than others ( Fig 3A ). The Malpighian tubules and the salivary glands had higher median densities than the other tissues studied. However, the variation in density between individual flies in both tissues was also the largest. Interestingly, flies that had higher density in some tissues did not consistently have high density in other tissues, suggesting that high density is not uniform across tissue types ( Fig 3B ). Download figure Open in new tab Figure 3. Wolbachia density in D. melanogaster tissues. Density of w Mel Wolbachia in 7-14-day old female D. melanogaster was determined by qPCR using primers to amplify a fragment of the Wolbachia wsp gene and the reference D. melanogaster RpS17 gene. The ΔC t method was used to calculate Wolbachia density. A) Data are the median and interquartile range of 14 flies. B) Each coloured line and associated dot points represent the w Mel densities in tissues of an individual D. melanogaster . Discussion Many Wolbachia- induced phenotypes are influenced by endosymbiont density ( Lu et al. 2012 ; Osborne et al. 2012 ; Fraser et al. 2017 ). Thus, having the ability to modulate Wolbachia density in vivo may provide a powerful tool to study Wolbachia -host interactions. We, therefore, attempted to modulate w Mel density regulation in D. melanogaster genetically. We identified host candidate genes from the Grobler et al. (2018) in vitro study to knockdown in an in vivo systemic model. RNAi was used in the GAL4/UAS system to target these genes to dysregulate w Mel density. Despite our selection of candidates that did not impede cell proliferation in vitro , half of the candidates were lethal when ubiquitously knocked-down in vivo using two different Gal4 drivers. Of the remaining candidates, the majority were not significantly knocked-down when targeted with gene-specific RNAi molecules. However, we only assayed one RNAi line per gene. As RNAi lines can show significant variability in their targeting success due to both the RNAi sequence used and the location of the RNAi insertion ( Grill et al. 2023 ), it is possible that if additional RNAi lines were assayed, we would see more successful gene knock down. Two candidate genes, CG9801 and su(r) , did show significant knockdown. They did not, however, have the same impact systemically in D. melanogaster as was observed in the cell line. No perturbation to Wolbachia density was seen in the knockdown offspring compared to controls, suggesting ubiquitous targeting of these host genes individually does not systemically affect Wolbachia density in vivo . As discussed in multiple reviews and studies ( Yamamoto-Hino and Goto 2013 ; Heigwer et al. 2018 ; Grill et al. 2023 ), RNAi both in vitro and in vivo can give rise to false discoveries due to many technical reasons in addition to reasons unique to the biology of Wolbachia . First, it is possible that the level of gene knockdown achieved here was lower than that achieved in the Grobler et al., 2018 study, and thus not enough to dysregulate w Mel density. As the Grobler et al. 2018 study did not report gene expression levels post targeting, it is not possible to compare between the two studies. Second, it is possible that genes that regulate w Mel density in an in vitro embryonic cell line (S2 cells) are of lesser importance to in vivo due to lower overall expression in adults. This does not seem to be the case for CG9801 and su(r) as its expression in embryonic tissues is similar or higher than its expression in adult tissues ( Graveley et al. 2011 ). Finally, our study also did not assess if ubiquitous expression of the candidate RNAi molecules dysregulated w Mel densities variably among tissues. Multiple factors could be at play to cause this. First, expression of the target genes studied here vary across D. melanogaster tissues where w Mel is present. For example, the expression of CG9801 and su(r) are reported as very high and high respectively in the Malpighian tubules, but low and moderate, respectively, in the salivary glands ( Chintapalli et al. 2007 ). This may have led to w Mel dysregulation in some tissues being masked by no or less-dysregulated w Mel densities in other tissues. Second, as we have shown here, w Mel density varies substantially across different tissues which may interact further with variable host gene expression to mask w Mel density modulation. Ideally, host candidate genes with high ubiquitous expression across most tissues would be key targets for downregulation. However, many of these genes likely have important functions (e.g. ribosomal proteins) and their knockdown may be lethal. Our work here shows that w Mel density is highly variable across tissues. Wolbachia density variation across tissues has also been observed in other species of Drosophila as well as in mosquitoes ( Dobson et al. 1999 ; Osborne et al. 2012 ; Amuzu et al. 2015 ; Amuzu and McGraw 2016 ; Kaur et al. 2020 ). Moreover, the tissue localisation and density of Wolbachia can vary among strains and can change when Wolbachia are transinfected into novel hosts ( Osborne et al. 2009 ; Fraser et al. 2017 , 2020 ). The two tissues with the highest w Mel density in this study, the Malpighian tubules and salivary glands, have been shown to have high Wolbachia density in both w Mel-transinfected Ae. aegypti as well as other Drosophila species ( Moreira et al. 2009 ; Lu et al. 2012 ; Amuzu et al. 2015 ; Kaur et al. 2020 ). One notable between-species difference is the density of Wolbachia is consistently higher in mosquito ovaries than in Drosophila ovaries when compared to other tissue types ( Fraser et al. 2017 ; Kaur et al. 2020 ). Interestingly, in the two tissues that presented the highest w Mel density here, Malpighian tubules and salivary glands, density was also the most variable. This suggests that Wolbachia density may be more tightly regulated in some tissue types than others. Alternatively, Wolbachia may be less costly to the host in these tissues and thus be less tightly constrained. For example, it has been suggested that the high density of Wolbachia in the Malpighian tubules, excretory organs for insects, is due to the abundance of resources at this site ( FARIA and SUCENA 2013 ) such as electrolytes and organic solutes including amino acids ( Beyenbach et al. 2010 ), which have been shown to be beneficial for Wolbachia growth ( Caragata et al. 2014 ). This may indicate that these resources make it less costly to the host for Wolbachia to grow to high abundance here or may be more favourable for Wolbachia growth. Interestingly, w Mel tissue densities were found to not necessarily be linear predictors of other tissue densities. Individual flies were found to have high w Mel densities in particular tissues when compared to other flies, but low w Mel densities in other tissues compared to the same flies. Furthermore, individual flies did not always follow the tissue-specific density trends observed across the overall population ( Fig. 3 ). For example, some flies showed higher w Mel density in the salivary glands than the Malpighian tubules, or higher density in the fat body than the ovaries, even though the latter tissues had higher median densities across the population. These data showcase the variability of w Mel tissue propensities between individuals. Of note, the age of our flies was quite broad (7-14 days), and thus we cannot eliminate the possibility that age may interact with tissue-specific w Mel-density. Overall, our study supports previous work suggesting systemic w Mel density is not necessarily an appropriate predictor for studies interested in w Mel effects or interactions in specific tissues ( Osborne et al. 2012 ; Kaur et al. 2020 ). These findings should be considered during experimental design. Conclusions In conclusion, we found targeted knockdown of individual host genes found to dysregulate w Mel density in vitro did not transfer phenotypically in vivo . This study depicts the complexities of validating in vitro findings in vivo . We observed a large amount of lethality caused by gene knockdown despite choosing candidates that did not impact cell proliferation in vitro and found that when knocked-down, the majority of target genes did not show a significant decrease in gene expression. For the two genes where knockdown was achieved, no significant impact on Wolbachia density was observed, which may be due to the large variation observed in w Mel density across tissues. Further work is needed to understand whether individual host gene knockdown can disrupt regulation of w Mel density, or whether a more complex multigene approach is needed as the ability to modulate w Mel can provide a powerful took to dissect the role of w Mel in specific phenotypes. Perhaps focusing specifically on tissue-specific knock-downs in the future will allow for better success in the replication of the in vitro observations and/or alleviate issues of lethality associated with candidate gene knock-down. Acknowledgements We would like to acknowledge the contributions of Ritzel Gimeno and Limom Lim for technical assistance. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. References ↵ Aliota MT , Peinado SA , Velez ID and Osorio JE ( 2016 ) “ The wMel strain of Wolbachia Reduces Transmission of Zika virus by Aedes aegypti ,” Scientific Reports , 6 ( 1 ): 28792 , doi: 10.1038/srep28792 . 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Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Targeted knockdown of in vitro candidates does not alter Wolbachia density in vivo Kimberley R. Dainty , Johanna M. Duyvestyn , Heather A. Flores bioRxiv 2025.02.16.638550; doi: https://doi.org/10.1101/2025.02.16.638550 Share This Article: Copy Citation Tools Targeted knockdown of in vitro candidates does not alter Wolbachia density in vivo Kimberley R. Dainty , Johanna M. Duyvestyn , Heather A. 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